TREATMENT OF ENTERAL FEEDING INTOLERANCE AND MUSCLE WASTING TECHNICAL FIELD

[0001] This application relates generally to methods, formulations, and systems for improving outcomes of seriously ill patients typically treated in an intensive care unit, including for prevention, diagnosis and treatment of Enteral Feeding Intolerance and conditions characterized by loss of muscle mass. RELATED APPLICATIONS

[0002] This application claims priority to provisional application 62/335,010, filed May 11, 2016, entitled“Treatment of Enteral Feeding Intolerance,” provisional application No. 62/425,899, filed November 23, 2016, entitled“Treatment of Disorders Characterized By Decreased Muscle Mass,” and provisional application No. 62/457,008, filed February 9, 2017, entitled“Measuring Skeletal Muscle Mass,” the content of each of which is incorporated by reference herein in its entirety and for all purposes. BACKGROUND OF THE INVENTION

[0003] Ulimorelin is a selective agonist of the ghrelin/growth hormone secretagogue receptor (GHSR), a G protein-coupled receptor. The secreted hormone ghrelin is the natural ligand of the GHSR. Endogenous ghrelin blood levels are pulsatile and rise sharply prior to eating and decline post-prandially (e.g., about every four to six hours) around the time of, or in anticipation of, thrice daily meals. FIGURE 1 shows average plasma ghrelin concentrations over a 24 hour period in 10 human subjects who consumed breakfast, lunch, and dinner during the analysis period (adapted from Cummings et al., 2001. A Preprandial Rise in Plasma Ghrelin Levels Suggests a Role in Meal Initiation in Humans, Diabetes 50:1714– 1719). [0004] Ulimorelin is bound in plasma by Alpha-1 Acid Glycoprotein (AAGP; also known as orosomucoid), an acute phase reactant protein (see Wargin et al., 2009, Clin Drug Invest 29:409-418). Plasma levels of AAGP increase in various disease states (acute illness; infection; various types of cancer; cardiovascular disease; central nervous system disorders; diseases of the kidney, liver, and lung; chronic inflammatory diseases; and the like) (see Boucher et al., 2006, Crit Care Clin 22:255– 271; Taguchi et al., Chapter 6: Molecular Aspects of Human Alpha-1 Acid Glycoprotein - Structure and Function, from Acute Phase Proteins, edited by Sabina Janciauskiene, InTech, 2013; Israili and Dayton, 2001, Drug Metabolism Reviews, 33:161–235; and Gruys et al., 2005, J Zhejiang Univ SCI 6B (11):1045-1056).

[0005] AAGP levels are also reportedly higher in obese individuals and in patients with injury, trauma, and severe burns, and in recipients of bone marrow and organ transplants (see Israili and Dayton, supra; Eap et al., 1990, Clin. Pharmacol. Ther. 47:338– 346; Benedek et al., 1984, Br. J. Clin. Pharmacol. 18:941–946; Bloedow et al., 1986, J. Clin. Pharmacol. 26:147–151; Macfie et al., 1992, 69, 447–450; Wilkinson, 1983, Drug Metab. Rev. 14:427–65; Raynes, 1982, 36:77–86.; Booker et al., Br. J. Anaesth. 1996, 76, 365–368; and Comments in Br. J. Anaesth. 1996, 77, 130). In addition, elderly patients with acute illness and those with cachexia of chronic disease also reportedly have elevated AAGP levels (Israili and Dayton, supra; Lyngbye and Kroll, Clin. Chem.1971, 17, 495500; and Verbeeck et al., Eur. J. Clin. Pharmacol. 1984, 27, 91–97). The levels of AAGP reportedly rise after surgery, peaking at 3 to 4 days postoperatively, and then decline to baseline values after 2 to 4 weeks (Israili and Dayton, supra; Hanada et al., Int J Clin Pharmacol Ther 2011;49(7):415-421; Comments in Br. J. Anaesth.1996, 77, 130; Garfinkel et al., Ann. Intern. Med.1987, 107, 48–50; and Jungbluth et al., J. Pharm. Sci.1989, 78, 807–811). However, in some patients, the acute-phase response is reportedly either incomplete or absent. Moreover, lower than normal levels of AAGP in plasma have reportedly been found in patients with pancreatic cancer, hepatic cirrhosis, hepatitis, hyperthyroidism, and malnutrition (Israili and Dayton, supra; Trautner et al., Scand. J. Urol. Nephrol. 1980, 14, 143–149; Pacifici et al., Ther. Drug. Monit.1986, 8, 259–263; and O’Connor and Feely, 1987, Clin. Pharmacokinet.13:345–64). SUMMARY OF THE INVENTION

[0006] Patients admitted to hospital intensive care units (ICUs) have very serious, generally life-threatening, conditions, diseases, or injuries. In addition to the primary medical condition, secondary conditions often arise as a result of the ICU stay itself. Two examples of such secondary conditions are Enteral Feeding Intolerance (EFI) and loss of skeletal muscle mass.

[0007] Virtually all ICU patients experience loss of skeletal muscle mass during their ICU stay, while many also enter the ICU with inadequate muscle mass. See Puthucheary et al., 2013, JAMA 310(15):1591-1600). In some cases, this loss of skeletal muscle mass is a component or cause of ICU-acquired weakness (ICU-AW), a physiologic and functional syndrome that commonly includes muscle wasting and impairment of muscle function as both etiology and manifestation, and which may be observed during or after discharge from the ICU. See Walsh et al., 2014, Clin Chest Med. 35:811-26. The second ICU-related condition, Enteral Feeding Intolerance, occurs in patients receiving enteral feeding (in which nutrition is provided through a feeding tube into the stomach or small bowel).

[0008] Ulimorelin is indicated for preventing or reducing loss of muscle mass in critically ill patients and/or for restoring lost muscle. In one approach a therapeutically effective amount of ulimorelin is administered intravenously three times per day to increase muscle mass in a patient or promote or accelerate the recovery of lost muscle or muscle mass. In one aspect, ulimorelin therapy is administered according to the methods disclosed herein to ICU patients with EFI. In one aspect, ulimorelin is administered according to the methods disclosed herein to ICU patients receiving enteral feeding but not diagnosed with EFI, to prevent or reduce loss of muscle mass, and to prevent or reduce risk of developing EFI. In one aspect, ulimorelin is administered according to the methods disclosed herein to ICU patients diagnosed as at risk of loss of muscle mass but neither diagnosed with EFI nor receiving enteral feeding. In one aspect, ulimorelin is administered to a patient whose feeding is being provided both enterally and parenterally. In one aspect ulimorelin is administered to a patient receiving parenteral nutrition, or total parenteral nutrition. In one aspect, ulimorelin is administered according to the methods disclosed herein to patients not in the ICU but who have experienced a loss of muscle mass and/or are at risk for such loss, e.g. at risk for muscle wasting. In some embodiments the patients have been discharged from the ICU and suffer from a condition related to the ICU stay. In some embodiments the patient is at risk of loss of muscle mass, or has experienced such loss, for reasons unrelated to an ICU stay.

[0009] Ulimorelin is indicated for the treatment of enteral feeding intolerance (EFI) in critically ill patients. In another embodiment, ulimorelin is indicated, in accordance with the invention, for the treatment of gastroparesis and/or delayed and/or impaired gastric emptying in critically ill patients with intolerance to enteral feedings. In another embodiment, ulimorelin is indicated, in accordance with the invention, for the treatment of gastroparesis and/or delayed and/or impaired gastric emptying in critically ill patients receiving enteral feedings. In another embodiment, ulimorelin is indicated, in accordance with the invention, for the treatment of gastroparesis and/or delayed and/or impaired gastric emptying in critically ill patients. In another embodiment, ulimorelin is indicated, in accordance with the invention, for increasing muscle mass in a patient, slowing the rate of loss of muscle mass in a patient, and/or treating a disease or disorder characterized by decreased muscle mass in a patient.

[0010] For convenience, loss of skeletal muscle mass during an ICU stay, inadequate muscle mass at the time of admittance into the ICU, or ICU-AW experienced following time in an intensive care unit, to the extent the ICU-AW is related to deficiencies in muscle, are sometimes referred to herein as“ICU-muscle conditions”,“muscle wasting”, and/or“muscle loss.” ICU-muscle conditions and Enteral Feeding Intolerance are sometimes referred to herein as“ICU-related conditions.” Diseases and disorders characterized by decreased muscle mass may be known as myopenia (see Fearon et al., 2011, J Cachexia Sarcopenia Muscle 2(1):1-3, incorporated by reference herein), are numerous, and include, without limitation, ICU-muscle conditions, cancer, sarcopenia, chronic kidney disease (CKD), congestive heart failure (CHF), and Chronic Obstructive Pulmonary Disease (COPD), as well as other conditions described herein (e.g., in Section 10, infra). BRIEF DESCRIPTION OF THE FIGURES

[0011] Figure 1 is graphically presented data showing that endogenous ghrelin blood levels are pulsatile, rising sharply prior to eating thrice daily and declining post-prandially. Plasma samples were collected and tested at various time points. Peaks were evident at 0800, 1200, and 1730, as indicated by dashed lines. Adapted from: Cummings et al., 2001, “A Preprandial Rise in Plasma Ghrelin Levels Suggests a Role in Meal Initiation in Humans” Diabetes 50:1714–19.

[0012] Figure 2 is graphically presented data illustrating total and free plasma ulimorelin concentrations and showing the short half-life of free ulimorelin relative to that of total plasma ulimorelin. Half lives were calculated based on data from the Tranzyme Thorough QT (TQT) study, a study in which plasma samples obtained from healthy volunteers following a 30 minute IV infusion of 600 μg/kg ulimorelin were analyzed.

[0013] Figure 3 shows results from two clinical studies (LP101-CL-101 and LP101-CL- 102) demonstrating acceleration of gastric emptying after administration of ulimorelin to healthy volunteers.

[0014] Figure 4 shows pooled gastric emptying results from the LP101-CL-101 and LP101-CL-102 studies.

[0015] Figure 5 and Figure 6 show Emax plots from the LP101-CL-101 and LP101-CL- 102 studies, individually and pooled, respectively, demonstrating the relationship between ulimorelin Cmaxfree and improvement in the time for 50% liquid gastric emptying (^t50) relative to baseline on Day 1 and Day 4 of the studies, as measured by scintigraphic imaging.

[0016] Figure 7 shows plasma concentration time curves for total and free ulimorelin at doses of 150, 300, 600, 900, and 1200 μg/kg IV on Day 1 from the LP101-CL-101 study, for the period from dosing through 8 hours, the defined dosing interval.

[0017] Figure 8 shows plasma concentration time curves for total and free ulimorelin at the 600, 900, and 1200 μg/kg IV single doses administered in the LP101-CL-101 study, for the period from dose administration through 96 hours. The longer 96 hour period is shown to illustrate the terminal phase, and thus half-life, of free and total drug.

[0018] Figure 9 shows a log-normal plot of AAGP levels measured in samples from healthy and ICU populations. Healthy population data are from the LP101-CL-101 and LP101-CL-102 studies; both individual and pooled data are shown. ICU data are from the REDOXS and RE-ENERGIZE studies.

[0019] Figure 10 shows Cmaxfree and Ctroughfree relative to MEC (a prospectively defined target Minimum Efficacious Concentration) in HV’s receiving 600 μg/kg ulimorelin IV Q8H. Samples for C _troughfree were collected at the t = 0 time point immediately prior to the first dose of the day, starting on Day 2. Samples for Cmaxfree were collected at the end of the infusion (0.5 hour post the start of the first daily infusion) on Days 4 through 7.

[0020] Figure 11 shows C _troughfree relative to MEC in healthy volunteers (HV) receiving 80, 150, 300, and 600 μg/kg ulimorelin IV Q8H. Samples were collected at the t = 0 time point immediately prior to the first dose of the day starting on Day 2.

[0021] Figure 12 shows Cpfree relative to MEC in HV’s receiving 80, 150, 300, and 600 μg/kg ulimorelin IV Q8H. Samples were collected at the t = 6 hour time point (six hours after initiation of administration of the first dose of the day) on Days 1 and 7.

[0022] Figure 13 shows the calculated point estimate (mean, median, and geometric mean) distribution of steady state Cmaxfree predicted for ICU patients receiving 600 μg/kg ulimorelin IV Q8H.

[0023] Figure 14 shows the relationship between heart rate and Cmaxfree in healthy subjects following a single IV administration of ulimorelin at doses of 600, 900, and 1200 μg/kg IV.

[0024] Figure 15 shows the Growth Hormone peak levels in healthy volunteers following administration of ulimorelin or placebo. Data were pooled from the LP101-CL-101 and LP101-CL-102 studies. DETAILED DESCRIPTION OF THE INVENTION

1.0 Definitions, Abbreviations, and Conventions

[0025] Ulimorelin (CAS Number 842131-33-3) has the structure shown below:

[0026] Ulimorelin drug formulations referred to herein and in the scientific literature include“TZP-101” (a designation used in studies conducted by Tranzyme Pharma) and “LP101” (a designation used in studies conducted by Lyric Pharmaceuticals, Inc.). Ulimorelin quantities referenced herein (e.g., 600 μg) refer to an amount of ulimorelin free base or an equivalent molar amount of an ulimorelin salt, solvate, hydrate, enantiomer or combination thereof or other form thereof (e.g., ulimorelin hydrochloride monohydrate). In the context of a dose of ulimorelin administered to a patient, reference to“μg/kg” means“μg/kg patient body weight.”

[0027] As used herein, the term“dosage regimen” refers to the, quantity, route of administration, and frequency of administration for provision of a drug to a subject, e.g., a healthy volunteer or patient in a clinical trial, or to a patient for therapeutic benefit (e.g.,

[0028] As used herein,“QD” and“TID” have their usual meaning in the art and refer to once-daily and three-times-per-day administration, respectively. Except as otherwise indicated, TID administration means three administrations of an equal amount of drug (i.e., one-third the total daily drug dose). In many embodiments of the present invention, TID administration is Q8H administration, i.e., each administration will follow the prior administration by about 8 hours (e.g., 8 hours +/- 1 hour). The 8 hour time frame may be measured from the beginning of an administration period. For example, on a Q8H schedule, a 30-min IV infusion initiated at 0800 h and ending at 0830 h is followed by an infusion beginning at 1600 h (about eight hours after the initiation of the prior administration).

[0029] As used herein,“ICU” (intensive care unit) refers to any hospital setting where care is provided to critically or severely ill patients. For example and without limitation, a“burn unit” or any isolation ward (e.g., post-transplant) is an“ICU” for purposes of the invention.

[0030] As used herein,“IV” means“intravenous.”

[0031] As used herein,“IV TID” refers to intravenous administration of ulimorelin on a TID schedule.

[0032] As used herein, ranges (e.g., 600-900) are inclusive of the end points.

[0033] As used herein, the“geometric mean” is similar to the arithmetic mean the difference being that the former is intended to indicate the central tendency for log-normal distributions and uses the product of a set of numbers as opposed to their sum, which is used to generate the arithmetic mean. [0034] As used herein, reference to a“physician” refers to medical doctors and any other medical professionals operating under a physician’s direction. In some embodiments, “physician” encompasses the umbrella entity (e.g., hospital) administering and setting practice guidelines and policies for an ICU.

[0035] As used herein, references to a“first dose” and“second dose,” and the like, are intended to convey a temporal relationship of administrations of ulimorelin, in which the second dose is administered after the first dose (usually with no intervening administration of the same drug).

[0036] “Cmax” means“maximal plasma concentration (of ulimorelin)”;“Cmaxfree” means“maximal plasma concentration of free ulimorelin;”“Cpfree” means“plasma concentration of free ulimorelin.”“Ctroughfree” means“minimal plasma concentration of free ulimorelin;“Tmax” means the time Cmax (generally Cmaxfree) is reached (sometimes expressed as minutes after drug administration is initiated);”SAD” means“single ascending dose”;“MAD” means“multiple ascending dose;”“HV” refers to“healthy volunteer.”

[0037] As used herein, a "therapeutically effective amount" of a drug (e.g., ulimorelin) is an amount of a drug that, when administered to a subject with a medical condition (e.g., Enteral Feeding Intolerance and/or ICU-muscle conditions), will have the intended therapeutic effect, e.g., alleviation, amelioration, or elimination of one or more manifestations of the condition in the subject. The full therapeutic effect does not occur by administration of one dose, and generally occurs after administration of a series of doses. Thus, a therapeutically effective amount may be administered in more than one administration. As "therapeutically effective amount" of a drug (e.g., ulimorelin) may also be administered to a patient at risk of a medical condition to reduce the likelihood the patient will develop manifestations of the condition.

[0038] As used herein,“critically ill patients”,“ICU patients” and“patients in the ICU” are used interchangeably.

[0039] As used herein,“loss of muscle mass” includes or refers to“loss of skeletal muscle mass” unless otherwise clear from context.

[0040] As used herein the terms“standard dosing protocol”“standard protocol” and “single dosing protocol” are used interchangeably. 2.0 Introduction

[0041] In accordance with the invention, ulimorelin is administered to ICU patients to prevent or treat Enteral Feeding Intolerance and/or to prevent or treat muscle wasting or ICU-muscle conditions. In one aspect, ulimorelin is administered to ICU patients intravenously three times per day. Although the pharmacokinetics of ulimorelin are complex including due to AAGP binding, the inventors have determined, unexpectedly, that the great majority of ICU patients may be efficaciously treated using a standard dosing protocol (e.g., treatment with 600 ulimorelin TID delivered by IV infusion), without the need to measure (and without prior knowledge of) patients’ plasma AAGP levels. In one embodiment, patients entering the ICU begin ulimorelin treatment at the time of admittance, prior to the onset of, or progression of, loss of skeletal muscle mass, and prior to or concurrently with initiation of enteral feeding. In another embodiment, ulimorelin treatment is begun 24 hours or more after admittance to the ICU, e.g. after a diagnosis of EFI or upon a physician determining that the patient is at risk for developing EPI or muscle wasting or both.

[0042] The discoveries that TID IV dosing provides optimal benefit to EFI patients and other patients at risk of developing or suffering from ICU-related conditions and that a standard ICU dosing protocol can be used for ulimorelin despite variable and unpredictable AAGP levels (as conceptualized in the discussion of “AAGP Multiples” herein) were first made in the context of studies focused on prevention and treatment of EFI and the promotility and anabolic effects of ulimorelin, as described in Section 2.1 below. The inventors also recognized that this same dosing regimen would provide therapeutic benefit to ICU patients not diagnosed with EFI (and not receiving enteral feeding) but at risk of loss of skeletal muscle mass. That is, ulimorelin, administered according to the methods described herein, has anabolic and other effects that make it ideally suited for the treatment for any patient at risk of muscle loss (muscle wasting) for which therapeutic intervention is warranted. In this therapeutic context as well, the inventive“AAGP multiples” rationale applies, as it is generally applicable to the entire ICU population including patients receiving enteral feeding and at risk of developing or diagnosed with EFI and patients not receiving enteral feeding or not diagnosed with EFI but at risk of muscle loss or muscle wasting. [0043] The inventors also realized that ulimorelin bound to AAGP does not contribute to the thrice-daily signaling event resulting from IV TID administration of ulimorelin and thus that the only drug half-life relevant to dosing frequency for achieving three promotility signals per day is the free drug half-life. The relatively short half-life of free ulimorelin and the fact that the gastric emptying resulting from ulimorelin administration is a Cmaxfree driven event make the Q8H IV dosing regimen of the present invention critical for achieving optimal therapeutic efficacy for treatment and prevention of EFI.

[0044] Without intending to be bound by a particular mechanism, at the doses used in the present invention, only a small fraction of plasma ulimorelin is“free ulimorelin.” The inventors appreciated that ulimorelin in the bound fraction does not contribute to ulimorelin efficacy, as it by definition does not contribute meaningfully to Cmaxfree, and that, even when bound ulimorelin becomes unbound, thereby contributing quantitatively to the free fraction, the rate of release is too slow to contribute to the desired therapeutic effect in the context of treating EFI or muscle wasting efficaciously. This is because Cmaxfree itself is reached very rapidly after IV dosing (e.g., at the end of the 30 minute infusion or the end of a 2 minute bolus injection) and is characterized by a narrow peak. That is, Tmax occurs at the time the infusion or bolus injection is completed (i.e., Tmax for a 30 minute infusion is 30 minutes measured from start of infusion). As a result, essentially the only ulimorelin that contributes to Cmaxfree is in the free ulimorelin fraction present initially after dosing. Previously bound (i.e. newly unbound) free drug is not relevant to exerting a physiologically meaningful promotility effect because the majority of such bound drug becomes free at a time after Cmaxfree has been achieved (i.e., after Tmax). Drug that subsequently becomes free does so too late to contribute to this initial free Cmax level and is cleared rapidly. Also see Section 3.0, below, entitled”The Pharmacodynamic Effects of Ulimorelin Are Driven By the Free Drug Cmax.”

2.1 Enteral Feeding Intolerance

[0045] Ulimorelin may be administered according to the methods described herein, to treat patients with Enteral Feeding Intolerance (EFI). In one approach, ulimorelin is administered by IV infusion three times per day at a dose of about 600 μg per kg body weight to about 900 μg per kg body weight. In one approach, ulimorelin is administered by IV bolus injection three times per day at a dose of about 190 μg per kg body weight to about 450 μg per kg body weight (e.g., 275-425 μg per kg body weight). Surprisingly, the inventors have determined that a standard dosing protocol (e.g., 600 μg per kg TID for consecutive days) can be safely practiced without the need to measure (and without prior knowledge of) patients’ plasma AAGP levels and is effective for most patients likely to be dosed.

[0046] EFI is a condition related to enteral tube feeding, a method used to provide nutrition to patients with serious illnesses, such as patients admitted to Intensive Care Units (ICU). A common complication of enteral tube feeding is Enteral Feeding Intolerance (EFI) caused by gastric or gastrointestinal dysmotility. One non-limiting example of such dysmotility is gastroparesis. Another example is impaired gastric emptying. Another example is delayed gastric emptying. EFI has a reported prevalence of approximately 30% among ICU patients receiving enteral feeding and is associated with poor patient outcomes. See Gungabissoon et al., Journal of Parenteral and Enteral Nutrition, 2014 Mar 17). There are no satisfactory treatments for enteral feeding intolerance.

[0047] Enteral feeding Intolerance (EFI) caused by gastric dysmotility and/or gastroparesis and/or delayed and/or impaired gastric emptying is a serious condition in patients with critical illness admitted to Intensive Care Units (ICU) and similar critical care facilities, limiting the ability of the caregiver to administer nutrition to such patients. Food is provided to many ICU patients via nasogastric tube or similar device (“enteral feeding”). Patients with EFI cannot tolerate (benefit from) such feedings to the degree necessary to meet their target feeding goals. Malnutrition in the ICU, especially protein malnutrition, is associated with poorer long term outcomes, including increased mortality (see Malnutrition and Outcomes, Kenneth B. Christopher, M.D., International Symposium on Intensive Care and Emergency Medicine, Brussels, Belgium, March 18-21, 2014). EFI may be diagnosed in a patient using methods and criteria known to physicians including, without limitation, methods described in Section 10, below.

[0048] Clinical evidence of improved outcomes associated with the administration of nutrition in the ICU was established in the ACCEPT study, a randomized controlled prospective trial of 462 evaluable patients that showed that improved ICU nutrition (enabled by protocolized treatment algorithms) resulted in shortened hospital stay (p = 0.003) and reduced mortality (trend, p = 0.058) (see Multicentre, Cluster-Randomized Clinical Trial Of Algorithms For Critical-Care Enteral And Parenteral Therapy (ACCEPT), Martin et al., CMAJ, JAN.20, 2004; 170 (2)). Similarly, in a prospective observational cohort study of 113 ICU patients in a tertiary referral hospital, a higher provision of protein and amino acids was associated with lower mortality (Allingstrup et al., 2012, Clinical Nutrition 31:462e468, incorporated herein by reference).

[0049] Gastric dysmotility is also problematic in certain settings outside the ICU where patients are generally in better health and a variety of promotility agents have been studied in these settings. While ulimorelin has been extensively studied in humans it has not been approved for any human therapeutic use. Functional activity, as measured by gastric motility and pharmacodynamic (PD) responses, was observed with ulimorelin administered as a 30 min intravenous (IV) infusion once daily in Phase 2 studies in diabetic gastroparesis patients (see Ejskjaer et al., 2009, Aliment Pharmacol Ther 29:1179–87) and shortened time to first bowel movement in a Phase 2 study of patients with post-operative ileus following once daily dosing (QD) for up to 7 days (see Dis Colon Rectum 2010; 53: 126–134)). In these Phase 2 studies, it was observed that ulimorelin both accelerated gastric emptying of solid and liquid food (10 patients with diabetic gastroparesis) and accelerated recovery of gastrointestinal (GI) function in subjects who underwent partial large bowel resection (168 patients with post-operative ileus). An additional Phase 2 study of diabetic gastroparesis patients showed improvements in GCSI Loss of Appetite and Vomiting scores (see Ejskjaer et al., 2010, Neurogastroenterol Motil 22:1069–e281). Unfortunately, these initial results failed to generalize as they did not reproduce in two larger prospective randomized, double- blinded, controlled pivotal Phase 3 trials of a postoperative ileus patient population in which ulimorelin was administered once daily for up to 7 days. In these larger trials, ulimorelin failed to achieve the target clinical endpoint of GI motility in patients who have undergone partial bowel resection.

[0050] Critically ill patients with EFI in the ICU are generally more ill than those studied in the ulimorelin Phase 3 trials described above. Given their serious medical condition and a general recognition of the importance of providing enteral feeding to them, these patients are often prescribed a medication in an attempt to restore gastric motility and emptying, even though current medications are unsatisfactory. Current medication choices are limited, as there are no drugs approved by the Food and Drug Administration (FDA) or its European counterpart the European Medicines Agency (EMEA) for this clinical indication. Drugs in common usage“off label” for this condition include metoclopramide and erythromycin, and to a much lesser degree, alvimopan (Entereg£) and methylnaltrexone (Relistor£). While physicians may seek to treat using up to 5-7 day courses and possibly with additional repeated courses with these medications, clinical studies demonstrate that not only is the maximal efficacy of both metoclopramide and erythromycin limited in resolving excessive gastric residual volume (GRV), but also the duration of any such efficacy is short lived, typically shorter than seven days and sometimes as little as one to two days (see Nguyen et al., 2007, Crit Care Med 35:2). Among other safety concerns, metoclopramide has a“Black Box” warning from the US FDA for CNS toxicity, and use of erythromycin, an antibiotic, for a non-infectious purpose may lead to bacterial resistance. Both of these safety issues are undesirable in the EFI intent-to-treat population.

[0051] In one aspect, the invention provides a method of treating EFI in a patient in need of treatment, comprising intravenously administering to the patient a therapeutically effective dose of ulimorelin three times daily for at least one day and usually two or more consecutive days. In one embodiment the patient receives ulimorelin treatment for at least three consecutive days, or for at least 5 consecutive days.

[0052] In one aspect, the invention provides a method of treating EFI in a patient in need of treatment, comprising administering to the patient a therapeutically effective dose of about 600 μg per kg body weight ulimorelin to about 900 μg per kg body weight, by IV infusion three times daily for one day or for two or more consecutive days.

[0053] In one aspect, the invention provides a method of treating EFI in a patient in need of treatment, comprising administering to the patient a therapeutically effective dose of about 600 μg per kg body weight ulimorelin to about 900 μg per kg body weight, by 30 minute IV infusion three times daily for one day or for two or more consecutive days.

[0054] In one aspect, the invention provides a method of treating EFI in a patient in need of treatment, comprising administering ulimorelin to the patient at a total daily dose of 1800 μg to 2700 μg per kg patient body weight, wherein the ulimorelin is administered by three times per day IV infusion of one-third the daily dose for one or more consecutive days.

[0055] In one aspect, the invention provides a method of treating EFI in a patient in need of treatment, comprising intravenous administration to the patient of ulimorelin three times per day for one day or two or more consecutive days, wherein each administration comprises one-third of a total daily dose, and wherein each administration results in a ulimorelin Cmaxfree in the range of 0.5-125 ng/mL. The intravenous administration may be by IV infusion or IV bolus injection. In some embodiments the duration of the bolus injection is 30 seconds to 3 minutes. In some embodiments the duration of the bolus injection is less than 2 minutes.

[0056] In one aspect, the invention provides a method of treating EFI in a patient in need of treatment, comprising administering to the patient a therapeutically effective dose of about 190 μg per kg body weight ulimorelin to 450 μg per kg body weight, by IV bolus injection three times per day for one day or for two or more consecutive days.

[0057] In one aspect, the invention provides a method of treating EFI in a patient in need of treatment, comprising administering ulimorelin to the patient at a total daily dose of 570 μg to 1350 μg per kg patient body weight, wherein the ulimorelin is administered by three times per day IV bolus injection of one-third the daily dose for one or more consecutive days.

[0058] In one aspect, the invention provides a method of treating EFI in a patient in need of treatment, comprising administering to the patient ulimorelin three times per day for one or more days, wherein the administering comprises: (i) administering ulimorelin at a first dose, (ii) monitoring the patient’s heart rate (HR) to determine whether the HR slows by at least a predetermined threshold reduction at about Tmax, and (iii) administering a second dose of ulimorelin, wherein, depending on the result in (ii), the second dose is the same as, lower than, or higher than, the first dose.

[0059] The present invention arises, in part, from several insights and surprising discoveries by the inventors relevant to ulimorelin pharmacokinetics and pharmacodynamics. By application of these discoveries and other insights to the problem of EFI, the inventors have identified new and effective preventative and treatment methods for EFI and for prevention and treatment of muscle wasting/muscle loss. Unexpectedly, the inventors have determined that a standard dosing protocol can be adopted by an ICU and used to treat most EFI patients likely to be seen there. The dosing protocol can be safely practiced without the need to measure (and without prior knowledge of) the ICU patient’s plasma AAGP level, ensuring that patients in need of treatment receive it quickly and without the need for testing procedures using a non-routine assay, as AAGP assays are typically not run on a daily basis in most hospitals. Thus, the invention provides for the first time a practical and effective treatment for EFI, and so can be used to treat critically ill patients in an ICU for whom there is no therapy currently approved.

[0060] For example and without limitation, exemplary discoveries and insights are summarized in the paragraphs below. This summary is provided for clarity and it is to be understood by the reader that the order of discussion below and herein generally is not intended to convey a particular sequence or hierarchy of discoveries and insights, or that the invention necessarily arises from any single one, particular combination, or all of or no other than the elements listed below. For avoidance of doubt, discoveries and insights of the inventors are not limited to those called out in this summary.

[0061] The inventors’ insights and discoveries include, then, for ease of discussion, first, that three discrete drug-induced gastric promotility signaling events per day are desirable for optimal therapeutic benefit when using ulimorelin to restore an ICU EFI patient’s ability to meet feeding goals typically prescribed for them. As discussed below, TID promotility signaling in EFI patients will lead to gastric emptying in a manner that best enables a patient to reach or stay at targeted feeding rates for typically-targeted enteral feeding prescription.

[0062] Second, the inventors recognized that the promotility signaling resulting from ulimorelin binding to the ghrelin/growth hormone secretagogue receptor is a Cmax-driven event. As such, drug effect is optimized by the drug rapidly reaching high occupancy of this receptor at each TID administration, resulting in a stronger, more discrete signal than otherwise possible, and leading to the optimal coordinated physical responses required for efficient gastric emptying.

[0063] Without intending to be bound by a particular mechanism, the inventors’ insights and discoveries also include that the requirement for three discrete drug-induced gastric promotility signaling events per day relates to and arises out of the capacity of the human stomach and the volume of enteral nutrition required by an ICU patient. Enteral feeding in the ICU is typically given by continuous feeding at maximal (targeted) infusion rates of 80 to 100 mL/hour (for a total of 640 to 800 mL over eight hours). In instances where feeding is given by bolus, the amount of volume provided over each eight hour period typically will not exceed this same 640-800 mL amount. In instances where feeding rates are calculated based on a volume-based feeding (VBF) protocol, feeding rates may temporarily exceed 100 mL/hour, e.g. for periods usually no longer than approximately 6 hours. This may be done, for example, to compensate for a period when feedings were held due to minor surgical procedure, CT-scan or the like, such that a“catch-up” period of more rapid feeding is desired. Even so, the targeted total daily food intake generally remains the same as for sites that do not practice VBF, both for any given day and for the totality of a patient’s ICU days. Catch-up periods typically occur only on select days. As such, while it is possible that a 640 to 800 mL feeding volume may be exceeded over a given eight hour period under a VBF protocol, on average this will not be the case. Meanwhile, the stomach can hold generally about 1 liter of food (see Sherwood, L., 1997, Human physiology: from cells to systems. 3 ^rd Ed. Belmont, CA: Wadsworth Pub. Co.). The inventors appreciated that promotility signaling, particularly with ghrelin receptor agonists, less frequent than three times per day will not induce sufficient stomach emptying to allow the patient to tolerate typically prescribed target feeding goals.

[0064] EFI, in many settings, is diagnosed by a patient having an excessive gastric residual volume (GRV). GRV is typically checked after discrete periods of enteral feeding (often 4– 8 hours) and represents the amount of provided food that remains in the stomach rather than having been propelled into the small intestine, i.e. the volume of residual food. Depending on practice, GRV’s may be deemed excessive by some physicians if greater than a figure as low as 200 mL, and by others if greater than 500 mL, or a value in between these figures. Excessive GRV’s are evidence that the innate promotility signaling in a patient has not resulted in adequate gastric emptying to support reaching feeding goals, such that administration of a promotility drug would be beneficial. Thus, a dosing regimen of a drug effective at promoting gastric emptying offering three signaling events per day will provide optimal frequency of gastric emptying events to ensure for most patients that the physician targeted volume of food given throughout the day will not exceed the innate capacity of the stomach.

[0065] The inventors appreciated that once-daily IV dosing of any drug, including ulimorelin, results in only one Cmax event per day and that such once-daily dosing and the resulting single daily signaling event cannot provide the optimal three events for the patients with ICU-related conditions contemplated herein. The inventors additionally appreciated that yet more frequent promotility signaling (e.g., more than three signaling events per day) would not further contribute meaningfully to achievement of patient feeding goals. Moreover, the inventors believe that if more frequent drug dosing were required, such extra dosing would increase risk and inconvenience without increasing benefit. Risks would include, for example, potential side effects and/or tachyphylaxis. Convenience would decrease for the physician or other caregiver due to the logistics of more dosing events.

[0066] Third, without intending to be bound by a particular mechanism, the inventors realized that ulimorelin bound to AAGP does not contribute to the thrice-daily signaling event resulting from IV TID administration of ulimorelin. Without intending to be bound by a particular mechanism, the only drug half-life relevant to ulimorelin dosing frequency needed to achieve three promotility signals per day is the free drug half-life. The relatively short half-life of free ulimorelin and the fact that the gastric emptying resulting from ulimorelin administration is a Cmaxfree driven event make the Q8H IV dosing regimen of the present invention critical for achieving optimal therapeutic efficacy.

[0067] As reported in the scientific literature, most plasma ulimorelin is bound to AAGP (see FIGURE 2 and TABLE 1). At the doses used in the present invention only a small fraction of plasma ulimorelin is“free ulimorelin.” The inventors appreciated that ulimorelin in the bound fraction does not contribute to ulimorelin efficacy, as it by definition does not contribute meaningfully to Cmaxfree, and that, even if/when bound ulimorelin becomes unbound, thereby contributing quantitatively to the free fraction, the rate of release is too slow to contribute to the desired therapeutic effect. This is because Cmaxfree itself is reached very rapidly after IV dosing (e.g., at the end of the 30 minute infusion) and is characterized by a narrow peak. That is, Tmax occurs at the time infusion or bolus injection is completed (i.e., Tmax for a 30 minute infusion is 30 minutes measured from start of infusion). As a result, essentially the only ulimorelin that contributes to Cmaxfree is in the free ulimorelin fraction present initially upon dosing. Previously bound (i.e. newly unbound) free drug is not relevant to exerting a physiologically meaningful promotility effect, because the majority of such drug becomes free at a time after Cmaxfree has been achieved (i.e., after Tmax). Drug that subsequently becomes free does so too late to contribute to this Tmax (the Cmaxfree level after a dosing is completed) and the free drug is cleared relatively rapidly.

[0068] Prior human dosing in studies of the promotility effect of ulimorelin to treat upper and lower GI motility disorders has been done using once per day (QD) dosing. Ulimorelin was known to have a long total drug half-life (reported to be 15-20 hours), and such QD dosing is typical for a drug with a long half-life, indicating total drug half-life guided the selection of dose frequency in prior studies. However, QD dosing, whatever the half-life, cannot drive three Cmax-driven events per day and so would not be adequate for efficacious treatment of EFI, because it can only provide one Cmax-driven event. To achieve three distinct Cmaxfree events per day for a given drug, three doses per day are required.

[0069] Fourth, the inventors conducted Phase 1 clinical trials to assess tolerability and gastric emptying in healthy volunteers (HV) receiving ulimorelin. In the SAD (single ascending dose) phase, HV received single doses of 600, 900, or 1200 μg/kg ulimorelin, which doses were determined to be safe and well tolerated. In the MAD (multiple ascending dose) phases, HV received total daily doses of 240, 450, 900, or 1,800 μg/kg (in each case administered as three equal doses IV TID), and the tolerability and effect on gastric emptying at these doses were determined. Each of the tested TID doses was safe and resulted in significant increases in gastric emptying relative to placebo. Information generated in these human trials demonstrated that ulimorelin administered IV TID has a broad therapeutic window.

[0070] Fifth, in the same Phase 1 clinical trials, the inventors determined that gastric emptying activity with ulimorelin is seen with a measured ulimorelin Cmaxfree of at least about 0.3 ng/mL on Day 1 and higher values on later days, and that the dosing regimens disclosed herein produce free drug concentrations above these thresholds in humans.

[0071] Sixth, the inventors measured AAGP levels in healthy subjects, arriving at a normal value of 53 mg/dL +/- 12 (mean +/- SD) using an assay that was validated for the purpose. Notably, this AAGP level is lower than“normal” values given in several published reports. The inventors also determined, for the first time, the distribution of AAGP levels in ICU patient populations representative of the target populations of interest for this invention, e.g. patients who may develop, and be treated for, EFI or muscle wasting or loss or the prevention thereof. The inventors compared normal (healthy subject) AAGP levels to the range of AAGP levels in ICU patients (representative of the EFI population), and, in an innovative approach, determined“AAGP Multiples” for EFI patients and demonstrated administration protocols according to the invention should be safe and effective in the majority of EFI patients based on such analyses.

[0072] Using the AAGP Multiples approach, the inventors extrapolated from the efficacious drug exposures determined in studies of the HV population to the intended EFI patient population, accounting for the differences in each of these population’s AAGP levels and thereby ensuring suitable safety and efficacy of ulimorelin drug use in the latter. They determined that, unexpectedly, the great majority of EFI patients may be efficaciously treated with ulimorelin administered by IV infusion TID at a total daily dose of from 1800- 2700 μg per kg patient body weight, regardless of a patient’s AAGP levels (as reflected by the AAGP Multiple but regardless of whether the AAGP level has been measured or the Multiple calculated). These doses have an exceptionally and unexpectedly high likelihood to result in Cmaxfree’s with each dose that are all at an efficacious level in the intended EFI patient population. As such, and importantly, an ICU adopting this invention may rely on a standard dosing protocol for EFI patients, notwithstanding the broad spectrum of AAGP levels in the ICU patient population. Advantageously, the dosing protocol does not require prior or ongoing measurement of patients’ AAGP levels.

[0073] Seventh, the inventors determined that a dose-related reduction in heart rate observed starting at an ulimorelin Cmaxfree of about 30 ng/mL (e.g., as measured in healthy subjects receiving the 600 μg/kg, 900 μg/kg, or 1200 μg/kg single doses) can produce a desirable net reduction in sympathetic tone in some subjects. Significantly, the inventors recognized that the observed reduction in HR could be used as a marker for the level of free drug in a patient with an unknown free drug level and/or an unknown AAGP level, and proposed step-up and step-down approaches in which a standard dose of drug is administered initially, and then the dose is raised, lowered, or maintained at the same level based on the degree of reduction in HR.

[0074] These and other elements are discussed in additional detail in the following sections after a brief discussion of another important application of the invention, the preservation of skeletal muscle mass in patients at risk of or suffering from a muscle wasting condition. [0075] Also see commonly owned International Application PCT/US15/60222, filed November 11, 2015, and published as WO2016/077498 on May 19, 2016, which claims priority to provisional application No. 62/078,888, filed November 12, 2014, the content of each of which is incorporated by reference herein in its entirety and for all purposes.

2.2 Preserving Skeletal Muscle Mass

[0076] Ulimorelin may be administered according to the methods described herein, to treat patients in need of therapy to prevent or reduce loss of muscle mass or to promote or accelerate the recovery of lost muscle or muscle mass, e.g. for conditions associated with muscle wasting. In one embodiment, ulimorelin is administered by IV infusion three times per day at a dose of about 600 μg per kg body weight to about 900 μg per kg body weight to such a patient. In one embodiment, ulimorelin is administered by IV bolus injection three times per day at a dose of about 190 μg per kg body weight to about 450 μg per kg body weight (e.g., 275-425 μg per kg body weight). As with the EFI indication, a standard dosing protocol, e.g., at 600 ug/kg TID can be safely practiced without the need to measure (and without prior knowledge of) patients’ plasma AAGP levels and is effective for most patients likely to be dosed.

[0077] Ulimorelin, administered according to the methods described herein, has anti-catabolic and pro-metabolic (collectively, anabolic) effects and is a suitable treatment for any patient at risk of muscle loss for which therapeutic intervention is warranted. Ulimorelin therapy is used to prevent or reduce loss of muscle mass or to promote or accelerate the recovery of lost muscle or muscle mass. A number of conditions, e.g., as described in Section 12, infra, result in loss of muscle mass.

[0078] In various aspects, ulimorelin is administered according to any administration method described herein, including administration regimens disclosed as useful for treatment of EFI to patients in the ICU (e.g., by IV infusion three times per day at a dose of about 600 μg per kg body weight to about 900 μg per kg body weight; or by IV bolus injection three times per day at a dose of about 190 μg per kg body weight to about 450 μg per kg body weight). Patients with, or at risk of, loss of muscle mass include ICU patients receiving enteral feeding (with or without EFI), ICU patients not receiving enteral feeding, patients not in the ICU but with ICU-related loss of muscle mass, and patients at risk of loss of muscle mass for reasons unrelated to an ICU stay. 3.0 The Pharmacodynamic Effects of Ulimorelin Are Driven By the Free Drug Cmax

[0079] At clinically relevant ulimorelin levels, ~99% of ulimorelin in plasma is bound by AAGP (see Wargin et al., 2009, supra). Also see TABLE 1 (adapted from data provided in Study Report ANA-07-0012-R0 (Tranzyme) and FIGURE 2).

[0080] TABLE 1

In Vitro Ulimorelin Binding to Total Plasma Proteins, Purified Human Alpha 1-Acid

Glycoprotein (1000 μg/mL AAGP), and Albumin (4% w/v HSA).

[0081] The effective half-life of free ulimorelin (unbound to AAGP), approximately 2 hours, is significantly shorter than that of total plasma ulimorelin, as shown in TABLE 2. Also see FIGURE 2, showing pharmacokinetic parameters calculated using data from the TQT Study showing a terminal half-life of approx 4 hours. Also see FIGURES 7 and 8, showing pharmacokinetic parameters determined in healthy volunteers in Lyric Pharmaceuticals’ LP101-CL-101 and LP101-CL-102 studies (discussed in Examples).

[0082] As a consequence of the short half-life of free ulimorelin, IV TID dosing achieves three daily pulses in which Cmaxfree is reached rapidly (e.g., at the end of a 30 min infusion period) after which plasma levels of free drug (Cpfree) drop rapidly. [0083] TABLE 2

Mean Total and Free Pharmacokinetic Parameters in Healthy Subjects Following a 30 Minute

IV Infusion of Ulimorelin

h)

Geometric mean Cmax, and mean t1/2, are presented; SD: single dose; na: not available;—:

not applicable; TZP-101-CL-006 dosed for 5 days. [0084] A consequence of the pharmacokinetics of ulimorelin, as recognized by the inventors, is that TID administration at the doses disclosed herein, enhances the anabolic effects of ulimorelin in ICU patients, in addition to its promotility effects. In clinical trials in which ulimorelin was administered using protocols described herein multiple infusions of ulimorelin Q8H in healthy volunteers resulted in Cmaxfree approximately 2.4-fold greater than that reached after the first infusion, an increase reached almost fully following the second infusion. See Examples, below in Section 19. In ICU patients, similar or even greater free fraction accumulation (increase) was observed (based on preliminary clinical study data) compared to that seen in these healthy volunteers.

[0085] Without intending to be bound by a particular mechanism, several factors influence the Cmax of total ulimorelin (Cmaxtotal, including both AAGP-bound ulimorelin and free ulimorelin) and the Cmax of free ulimorelin (Cmaxfree). In the case of Cmaxtotal, the accumulation of the drug to steady state concentrations (the state wherein a given drug level reached, such as Cmax, is the same for each dose) is dependent upon the t _1/2 of the drug and the dosing interval. The longer the t _1/2 and the shorter the dosing interval, the greater the accumulation, and thus the greater each of the various steady state concentrations. However, in the case of ulimorelin, only free ulimorelin is pharmacologically active and only Cmaxfree drives ulimorelin’s pharmacology. AAGP binding reduces concentration of free drug, a factor which is complicated by the lack of predictability of AAGP levels, especially in ICU patients. Thus, the Cmaxfree and pharmacokinetics of free ulimorelin are influenced by AAGP levels (e.g., the binding capacity of the AAGP“pool”), total drug and free drug concentrations, and clearance of free drug.

[0086] According to these principles it is believed that when administered QD (a 24 h dosing interval), the concentration of total ulimorelin just prior to administration will have declined by about 50% relative to the Cmaxtotal achieved by the prior administration (t _1/2 for total drug is about 22 h). This reduction serves to free up part of the AAGP drug binding “pool” to bind to newly administered ulimorelin molecules. It is believed that in healthy volunteers with normal AAGP levels and with once daily dosing, the binding capacity of the AAGP pool is already great enough that upon a“next” dose, enough of the newly dosed drug binds to AAGP that the resulting free (unbound) fraction is not significantly greater than that from the“prior” dose; as such, no significant accumulation of free drug occurs and free Cmaxfree is similar on all dosing days. In ICU patients with elevated AAGP, the AAGP binding pool capacity is often even greater, such that with once daily dosing effectively no accumulation of free drug (no increase in Cmaxfree compared with prior levels) is expected.

[0087] Using TID dosing, only about 1/6th of total drug has cleared by the time a “next” dose occurs, meaning that the AAGP pool remains much more saturated with already-bound ulimorelin relative to QD dosing. With more drug already bound to AAGP, less unbound AAGP is available to bind newly dosed ulimorelin molecules and more of the newly administered ulimorelin molecules remain in the free, pharmacologically active) state. Over the first few dosings, the system reaches a state where the ulimorelin-AAGP binding pool transiently becomes fully or almost fully saturated after a given dose. And over these first few dosings, resulting Cmaxfree levels are meaningfully higher than those reached from the initial dose. This rise in Cmaxfree levels is an important difference from the case with once daily dosing, wherein all Cmaxfree's are roughly the same. TID ulimorelin dosing data collected to date indicates that Cmaxfree achieves steady state by about the fourth dose (first dose on day 2). 4.0 IV TID Administration of Ulimorelin

[0088] As discussed above in §2, the inventors recognized that three discrete drug- induced gastric promotility signaling events per day are required to provide the EFI patient with optimal nutrition. As also discussed above, the inventors appreciated that intravenous administration of ulimorelin TID at the doses described herein optimizes the promotility effects of the drug (including acceleration of normal gastric emptying and/or restoration of otherwise impaired or delayed gastric emptying in a patient). IV TID dosing achieves three daily pulses in which Cmaxfree is reached rapidly (e.g. at the end of a 30 min infusion period). After reaching Cmaxfree, plasma levels of free drug (Cpfree) drop rapidly. Without intending to be bound by a particular mechanism, it is believed that the thrice-daily rapid rise and rapid fall of free ulimorelin levels in plasma results in more effective gastric emptying sustained over the course of treatment than would be the case for a drug with a longer half-life.

[0089] On binding of ulimorelin to the ghrelin/growth hormone secretagogue receptor (GHSR), the receptor-hormone pair is internalized into the cell, triggering an intracellular cascade that leads ultimately to gastrointestinal tract muscle activity. Without intending to be bound by a particular mechanism, when an effective dose of ulimorelin is administered IV TID as provided by the present invention, there is a rapid rise to an ulimorelin Cmaxfree which is believed to result in high occupancy of cell surface GHSR receptors.

[0090] Optimally the ulimorelin administration results in a Cmaxfree of 2.5 ng/mL or higher, although, in some patients, at a given physiological state, a Cmaxfree as low as about 0.3 ng/mL (first dose on first day, for example) will be sufficient to achieve the intended biological response. Without intending to be bound by a particular mechanism, it is believed that rapidly achieving a Cmaxfree of sufficient magnitude results in rapid binding and subsequent internalization of cell-surface GHSR. Without intending to be bound by a particular mechanism, it is believed that because the effective half-life of the active-form of ulimorelin is sufficiently short, there is sufficient time in the IV TID dosing interval for GHSR to recycle back to the cell surface where the receptors remain generally unoccupied in the absence of significant levels of ligand, such that the number of available unoccupied cell- surface receptors is adequate at the time of the next ulimorelin administration. Without intending to be bound by a particular mechanism, it is believed that the cycle of rapid high occupancy, receptor internalization, and receptor recycling in the absence of significant levels of ligand, results in a stronger, more discrete signal than otherwise possible, leading to the coordinated physical responses required for efficient gastric emptying.

[0091] FIGURE 10 shows steady state Cmaxfree and Ctroughfree in HV receiving 600 μg/kg ulimorelin IV TID as described in EXAMPLE 3, below. As shown in the figure, Ctroughfree generally covaried with Cmaxfree. Seven (7) of eight (8) study subjects had Ctroughfree values that were less than 2.5 ng/mL at all time points measured. One subject had a Ctroughfree greater than 2.5 ng/mL on Days 5 and 6 at t = 0 hr (3.16 and 2.92 ng/mL) but not on Day 7, at which time Ctroughfree was less than 2.5 ng/mL. As shown in FIGURE 11, at all other TID dose levels (80 μg/kg - 300 μg/kg IV TID) Ctroughfree was always below 2.5 ng/mL in all subjects. It will be appreciated that the elevated AAGP levels characteristic of the majority of ICU patients (e.g., EFI patients and muscle patients) during the majority of their time in the ICU will cause these patients’ Ctroughfree’s to almost always be below 2.5 ng/mL, because the elevated AAGP level will result in a generalized lowering of all Cpfree values. As such, and as confirmed in FIGURE 11, Ctroughfree may almost always be expected to fall below the MEC at a dose of 600 μg/kg ulimorelin IV TID administered in the ICU. Without wishing to be bound by theory, it will also be appreciated by those of skill in the art that typically Cmaxfree and Ctroughfree will directly correlate such that the higher the maximal value, the higher the trough value and vice versa. For patients dosed with ulimorelin who reach Cmaxfree values in the higher ranges, far above 2.5 ng/mL, Ctroughfree may likewise remain above 2.5 ng/mL; even so, the broad delta between the two values (i.e. Cmaxfree minus Ctroughfree), a difference which itself traces back to an effect of the short t1/2 of free ulimorelin, will increase the likelihood that receptor recycling in the time between TID doses has been sufficient to enable a subsequent dose to offer clinical benefit.

[0092] FIGURE 12 shows Cpfree in healthy volunteers (HV) 6 hours after initiation of administration of ulimorelin IV Q8H at dose levels ranging from 80– 600 μg/kg ulimorelin. The HV data shown in FIGURE 12 demonstrate further that for most ICU patients, not only may Cpfree be expected to fall to below the MEC prior to a subsequent dose, but that it will do so at least two hours prior to a subsequent dose, providing time for receptor recycling in the absence of significant levels of ligand, for dosing at a Q8H frequency. As shown in FIGURE 12, on both Day 1 and on Day 7 (a time after steady state is reached), for all doses 80– 600 μg/kg ulimorelin IV TID, Cpfree falls below MEC six (6) hours post (initiation of) dosing, with rare exception. Once again, given the effect of AAGP elevation on Cpfree, it may be expected that for ICU patients (e.g., EFI and muscle wasting patients), the large majority will reach a Cpfree below MEC at least two hours prior to a subsequent dose. The data in FIGURE 12 teach, when factoring in the elevated AAGP levels of ICU patients, that in many ICU patients (e.g., EFI and muscle wasting patients), Cpfree will fall below 1 ng/ml at least two hours prior to a subsequent dose. This is seen at all doses 80– 600 μg/kg ulimorelin IV TID on Day 1, and in all HV’s studied at doses 80– 300 μg/kg, and most HV’s studied at the dose of 600 μg/kg, ulimorelin IV TID on Day 7 (representative of steady state).

[0093] Based on the measurements made in HV, the effect on Cpfree of AAGP elevation in ICU patients (e.g., EFI patients as well as other ICU patients in the intent to treat population), may be calculated. FIGURE 13 shows the distribution of the point estimate (mean, median and geometric mean) at steady state C _maxfree predicted to occur in ICU patients receiving 600 μg/kg ulimorelin IV Q8H. The large majority will reach a Cmaxfree above MEC and, as well, a similarly large majority will reach Cpfree below MEC at least two hours prior to a subsequent dose. As described in EXAMPLE 4, infra, preliminary data from a Phase 2 clinical trial indicate that Cmaxfree values in EFI patients treated with 600 μg/kg IV TID are in close agreement with the predicted steady state Cmaxfree distribution curve shown in FIGURE 13.

[0094] In certain embodiments of the invention, after an administration of a dose of ulimorelin to a patient, the ulimorelin Cpfree falls below 2.5 ng/mL prior to next administration of ulimorelin to the patient or falls below 1 ng/mL prior to next administration of ulimorelin to the patient. In some embodiments, the ulimorelin Cpfree falls below 2.5 ng/mL within 6 hours of the initiation of administration of the dose or falls below 1 ng/mL within 6 hours of the initiation of administration of the dose. 5.0 A Wide Range of Ulimorelin Doses Are Tolerated; Administration of Ulimorelin IV TID Increases Gastric Emptying Rate in HVs

[0095] As described in EXAMPLE 3, below, Phase 1 clinical trials were carried out to assess tolerability and gastric emptying in healthy volunteers (HV) receiving ulimorelin. In the SAD phase, HV received single doses of either 600, 900, or 1200 μg/kg, all of which were safe and well tolerated. In the MAD phases, HV received daily doses of either 240, 450, 900, or 1,800 μg/kg (in each case administered as three equal doses TID) and the tolerability and effect on gastric emptying at these doses were determined.

[0096] Gastric emptying (GE), as measured by the time it takes to empty 25% (T25%) and 50% (T50%) of the stomach contents, was determined in HV receiving 80, 150, 300, and 600 μg/kg TID. GE was improved (accelerated) at each of the tested TID doses, showing significant increases in gastric emptying relative to placebo. See FIGURE 3 and FIGURE 4.

[0097] FIGURE 5 and FIGURE 6 show the relationship of Cmaxfree to gastric emptying (^T50%), and supports the conclusions that robust activity occurs at Cmaxfree levels of about 0.5 to 1 ng/mL on Day 1 (EC ₅₀ = 0.62 ng/mL), and 2.5 ng/mL or more on Day 4 (EC ₅₀ = 1.1 ng/mL). In FIGURE 6 three non-responders were excluded on Day 4. As shown in FIGURE 5 and FIGURE 6, there was a maximum effect of about 50% improvement in gastric emptying. Achieving an ulimorelin Cmaxfree of at least 0.3 ng/mL should provide significant acceleration and/or improvement in gastric emptying in most patients on the first day of administration. Maintaining achievement of this Cmaxfree level in subsequent dosing intervals, or achieving a higher Cmaxfree level (e.g., 2.5 ng/ml or higher), should provide significant acceleration and/or improvement in gastric emptying in most patients for six days or longer. Preferably, an ulimorelin Cmaxfree of at least 2.5 ng/mL, at least 5 ng/mL, or at least 10 ng/mL is obtained following each dosing.

[0098] FIGURE 7 and FIGURE 8 show the relationship, in healthy individuals, between a single dose of ulimorelin and the plasma concentration over time of free ulimorelin (Cpfree) and total ulimorelin (Cptotal). TABLE 3 shows Cmaxfree following a single dose of ulimorelin. [0099] TABLE 3

Mean Cmaxfree Following Single or Initial Dose Ulimorelin Administration to Healthy

Volunteers

[0100] As described in EXAMPLE 3, below, IV administration of ulimorelin in normal volunteers resulted in Cmaxfree levels in the range of about 0.3 ng/mL (Cmaxfree for an 80 μg/kg dose) to about 125 ng/mL (as measured at the end of the 30 minute infusion in a single subject receiving a single 1200 μg/kg dose where the mean for that cohort was about 75 ng/mL). Administration of 600 μg/kg IV TID resulted in a steady state mean Cmaxfree (at steady state) of about 37. TABLE 3 shows the mean Cmaxfree following various single doses, or initial doses, of ulimorelin.

[0101] After consecutive daily TID administration to HV, ulimorelin Cmaxfree levels generally rise relative to those observed on Day 1. By Day 4, higher levels were required in HV to achieve a given degree of acceleration and/or improvement in gastric emptying compared with Day 1, as shown by the combination of increased EC50 (see, e.g., FIGURE 6) and reduced maximal gastric emptying improvement statistics (see, e.g., FIGURE 3). While there was the appearance of slight down-regulation of the gastric emptying effect between Days 1 and 4, the maximal effects of drug were relatively similar. In particular, gastric emptying was similar on Day 4 and Day 6 at 600 μg/kg TID (LP101-CL-102), confirming that the pharmacodynamic response on Day 4 was preserved and that equilibrium had been established. The preservation of prokinetic effects through Day 6 suggested that tachyphylaxis was not occurring at the ghrelin receptor at the doses and exposures tested. [0102] The increase in EC50 and decrease in maximal GE in HV likely results from normal counter-regulatory mechanisms that will be more predominant in healthy volunteers (with a normally functioning GI tract) and absent or less pronounced in EFI patients. Without intending to be bound by a particular mechanism, the increase in EC50 and decrease in maximal GE improvement noted in HV is more likely to be less pronounced in patients than in these HV. Based on this, it is expected that the activity and EC50 of a Day 1 dose in a patient will likely be more uniform through multiple administrations of ulimorelin (e.g., at least through and after TID administration for 4 days) than was observed in HV.

[0103] Subjects generally have lower Cmaxfree values on first dosing than at steady state, and the latter is reached for Cmaxfree around the fourth dose (i.e. first dose on Day 2), while for Ctroughfree steady state generally is reached around Day 5, as the pharmacokinetics for Ctroughfree are dependent on both free and total drug concentrations and steady state for total drug is generally not reached until this time.

[0104] In some embodiments of the invention, the patient’s steady state Ctroughfree during treatment is below 2.5 ng/mL during the course of treatment. In one embodiment, the invention provides a method for increasing muscle mass in a patient, slowing the rate of loss of muscle mass in a patient, and/or treating a disease or disorder characterized by decreased muscle mass in a patient and/or treating EFI by ulimorelin intravenously to the patient three times per day for at least two consecutive days wherethe administration results in a ulimorelin Cmaxfree in the range of 2.5-125 ng/mL, and results in a Cpfree below 2.5 ng/mL, optionally below 1 ng/mL, prior to the subsequent administration of ulimorelin to the patient.

[0105] When ulimorelin is administered to HV intravenously on a TID schedule, significant acceleration of gastric emptying is achieved with doses that result in Cmaxfree above about 0.5 ng/mL. An ulimorelin dose that resulted in Cmaxfree of 125 ng/mL was tolerated (safe) in the healthy volunteer study. Based on these and other data, the inventors conclude that IV TID doses that result in a Cmaxfree of from about 0.5 ng/mL to 125 ng/mL may be used in EFI and muscle wasting patients.

[0106] The inventors have determined that reaching Cmaxfree of 2.5 ng/mL at steady state is a desirable target for individual EFI patients receiving ulimorelin therapy. The minimum efficacious concentration (MEC) was prospectively set at 2.5 ng/mL, equivalent to the mean free Cmax on Day 4 at 150 μg/kg Q8H, the initial dose at which the maximal prokinetic effects (^t50) of drug were realized under steady state conditions in HV studies. Although prokinetic activity was observed at doses as low as 80 μg/kg and free Cmax below 1 ng/mL in these studies, MEC embodies a target that predicts that the maximal effects of ulimorelin will be achieved in the intended patient population.

[0107] In some embodiments, enteral feeding intolerance (EFI) may be treated by intravenous administration of ulimorelin three times per day for one or more consecutive days, wherein each administration comprises one-third of a total daily dose, and each administration results in an ulimorelin Cmaxfree in the range of 0.5-125 ng/mL. As noted above, it is believed that significant benefit is achieved when ulimorelin administration results in a Cmaxfree of 2.5 ng/mL or higher, although, in some patients, at a given physiological state, a Cmaxfree as low as about 0.3 ng/mL will be sufficient to achieve the intended biological response. In some embodiments the dose and mode of administration (e.g., infusion or bolus injection, for a specified period) is selected to result in an ulimorelin Cmaxfree of at least 2.5 ng/mL, alternatively at least 5 ng/mL, and often at least at least 10 ng/mL. In some embodiments the dose and mode of administration is selected to result in an ulimorelin Cmaxfree in the range of 1-100 ng/mL, sometimes in the range of 2.5-100 ng/mL, sometimes in the range of 2.5-75 ng/mL, sometimes in the range of 2.5-50 ng/mL, sometimes in the range of 2.5-40 ng/mL sometimes in the range of 5-100 ng/mL, sometimes in the range of 5-75 ng/mL, sometimes in the range of 5-50 ng/mL, sometimes in the range of 10-100 ng/mL, and sometimes in the range of 10-75 ng/mL. In some embodiments the dose and mode of administration results in an ulimorelin Cmaxfree in the range from 2.5 ng/mL to 50 ng/mL, alternatively from 5 ng/mL to 50 ng/mL, and sometimes from 10 ng/mL to 50 ng/mL.

[0108] In some embodiments the dose and mode of administration is selected to result in a Cmaxfree in the range of 1 ng/mL-100 ng/mL. In some embodiments the dose and mode of administration is selected to result in a particular ulimorelin Cpfree profile in which the Cmaxfree is in the range of 1 ng/mL-10 ng/mL in a substantial portion of the intent to treat population (e.g., >25%), is in the range of 10 ng/mL-50 ng/mL in a substantial portion of the intent to treat population and/or is in the range of 50 ng/mL-100 ng/mL in a substantial portion of the intent to treat population. In some embodiments the dose and mode of administration is selected to result in a particular ulimorelin Cpfree profile, e.g., in which ulimorelin Cmaxfree is in the range of 5-100 ng/mL or in the range of 5-50 ng/mL, and is followed by a drop to less than 5 ng/mL prior to the subsequent ulimorelin administration. In some embodiments the dose and mode of administration is selected to result in an ulimorelin Cmaxfree in the range of 5-100 ng/mL or in the range of 5-50 ng/mL, and the ulimorelin Cpfree drops to less than 2.5 ng/mL prior to the subsequent ulimorelin administration. In some embodiments the dose and mode of administration is selected to result in an ulimorelin Cmaxfree in the range of 5-100 ng/mL or in the range of 5-50 ng/mL, and the ulimorelin Cpfree drops to less than 1 ng/mL prior to the subsequent ulimorelin administration. 6.0 AAGP Levels in ICU and Healthy Populations; Effect of AAGP on Cmaxfree; AAGP

Multiples

[0109] As described in EXAMPLE 1, the inventors measured AAGP levels in healthy (“normal”) subjects to determine the baseline against which AAGP levels in other populations could be compared. A wide variety of AAGP values for healthy subjects have been reported. AAGP levels in the target (ICU) population were also determined, as described in EXAMPLE 2. The inventors compared the AAGP levels in healthy subjects with the spectrum of AAGP levels in ICU populations. The inventors’ understanding of this relationship contributed to the identification of the treatment regimen of the invention that confers therapeutic benefit to a majority of EFI and muscle wasting patients by administering ulimorelin.

6.1 AAGP Levels in Healthy Subjects

[0110] As shown in EXAMPLE 1, below, the range of AAGP values in a study of healthy volunteers was 32-88 mg/dL, with mean and standard deviation of approximately 53 +/- 11.8 mg/dL (LP101-CL-101 = 50.8 +/- 10.6 mg/dL; LP101-CL-102 = 59.6 +/- 12.9 mg/dL). See FIGURE 9. All AAGP samples were measured using a validated assay with calibration standards assigned using CRM470, an international secondary reference material.

[0111] The mean AAGP value of ~53 mg/dL determined for healthy individuals was unexpectedly low, in view of several reports in the scientific literature describing higher “normal” AAGP levels in healthy subjects. For example, AAGP ranges from about 55-140 mg/dL have been reported for healthy subjects (see, e.g., Israili and Dayton, Drug Metabolism Reviews, 33(2), 161–235 (2001); Kremer et al., Pharmacol. Rev.1988, 40, 1–47; Lentner, C. DOCUMENTA GEIGY SCIENTIFIC TABLES, PHYSICAL CHEMISTRY, BLOOD, SOMATOMETRIC DATA, 8TH ED., Ciba Geigy Corporation: West Caldwell, NJ, 1984: Vol. 3, 135–137, 140–142; Schmeling et al., Exp. Clin. Immunogenet, 1986, 13, 78–83.159; Lyngbye and Kroll, Clin. Chem. 1971, 17, 495–500.160; Blain et al., Br. J. Clin. Pharmacol. 1985, 20, 500–502.161; and Israili et al., [abstract]. Fed. Proc. 1985, 44, 1124). The literature accompanying the commercially available AAGP assay available from Randox Laboratories Ltd. (“Randox Assay;”“Alpha-1-Acid Glycoprotein (AGT) RX Series” (www.randox.com), which was used for the AAGP assay results reported herein, reports a normal AAGP range of 50–120 mg/dL. Given that, in the healthy volunteer population tested using the Randox Assay (as described in EXAMPLE 1), approximately 75% of the subjects had values between 40-70 mg/dL, and the range of values was 32 mg/dL to 88 mg/dL, it is not clear that a value as high as 120 mg/dL is normal. In summary, the“normal” AAGP level determined by the inventors is low relative to many published values.

6.2 AAGP Levels in ICU Subjects

[0112] As described in EXAMPLE 2, AAGP levels were assayed in 233 plasma samples from more than 92 ICU and Burn Unit patients (jointly referred to as“patient population”). Importantly, the assay used to determine AAGP levels in patients was the same assay used to determine the healthy subject baseline. The mean AAGP level in the patient population was 141 mg/dL with a standard deviation of approximately 58 mg/dL. See FIGURE 9. Measured AAGP levels in patients ranged from 44 mg/dL (a baseline reading) to 390 mg/dL (a Day 4 reading). If one uses the mean value of 50.8 mg/dL (the mean HV value determined in the LP101-CL-101 study) as the healthy subject baseline, this represents an almost 9-fold variation of patient AAGP relative to healthy baseline (44/50.8 = 0.86; 390/50.8 = 7.68; 7.68/0.86 = 8.9). As discussed below, free ulimorelin concentration is inversely correlated with AAGP level. The wide variation of AAGP levels in the patient population should therefore result in a similar variation in free ulimorelin concentration at a given ulimorelin dose, indicating to an artisan of ordinary skill in the art that administration of a standard dose to the patient population will result in broadly different plasma levels of the active (free) form of the drug from patient to patient, in particular the Cmaxfree levels. In addition, the AAGP levels in an individual ICU patient may change significantly during the ICU stay, indicating to an artisan of ordinary skill in the art that administration of a standard dose to an individual patient could result in broadly different Cpfree and Cmaxfree levels in the patient over time.

[0113] Initial data from an ongoing clinical study of an ICU patient population with diagnosed EFI (the LP101-CL-201 study; see EXAMPLE 4, below) confirm the predictive accuracy of these evaluations. The first 17 ICU EFI patients enrolled in the clinical study have measured AAGP values ranging from 34– 315 mg/dL and a mean of 165 mg/dL as measured in 95 samples drawn from Study Days -1 to 5. These values are very similar to the range and mean of the patients assessed in the study described in EXAMPLE 2.

6.3 Overcoming Problems of Elevated and Diverse AAGP Levels on Cmaxfree

6.3.1 AAGP Guided Dosing is Time Consuming

[0114] AAGP levels dictate how much of a given ulimorelin dose remains in unbound (free) form– the form that exerts the desired pharmacologic effect for EFI and other conditions. The higher the AAGP level, the lower the proportion of total drug administered that will be in free form at a given dose level and the lower Cmaxfree will be (with the opposite of these true as well). Put differently, to achieve a given target ulimorelin Cmaxfree, a higher ulimorelin dose must be given to an ICU patient with an elevated AAGP level than is required for an ICU patient with a less elevated AAGP level, or for a healthy individual with a normal AAGP level. Although a physician can, guided by this disclosure, measure AAGP levels and calculate an individual dose for each patient, doing so is not optimal practice in the ICU setting. EFI patients are critically ill and need immediate treatment, because they are in need of nutrition yet unable to advance to their target feeding rate. By the time EFI is diagnosed, by definition, desired nutrition has not been received. Similarly, ICU patients at risk of, or suffering from, loss of muscle mass have an increased risk of near-term mortality. These patients can lose as much as 1 kg of such mass per day. Accordingly, treatment should begin as soon as practicable, but if an AAGP test were required to be run before a dose could be administered safely or efficaciously, then vital treatment would be delayed. Moreover, if for each patient the AAGP test had to be repeated over the course of an ICU stay given that individual patient AAGP levels may change significantly over the course of treatment, then multiple such similar delays might be encountered. Moreover, single and repeated AAGP tests add to medical and operational costs.

[0115] The methods of the present invention provide safe and efficacious dosing without requiring AAGP levels to be checked in advance, enabling the provision of nutrition to EFI patients or the initiation of treatment for ICU-muscle conditions more rapidly.

6.3.2 Use of AAGP Multiples to Guide Dosing

[0116] In one aspect, the present invention provides a standard dosage regimen that can be used to treat the majority of ICU patients (e.g., EFI patients and patients in need of treatment to mitigate muscle loss) effectively, despite the variability of AAGP levels in the target population and the unpredictability of such levels in any individual patient. Using the methods of the invention, physicians can administer doses to the patient population with confidence that the dose will be safe and effective (e.g., resulting in Cmaxfree of at least about 0.5 ng/mL and typically 2.5 ng/mL or higher, but not exceeding 125 ng/mL and typically not exceeding 50 ng/mL) without having to measure AAGP levels. It will be appreciated, however, that nothing in the present invention is intended to preclude measurement of AAGP levels if desired by the physician, and the guidance provided herein ensures that such individually tailored dosing will be as safe and as effective as practicable.

[0117] An additional contribution by the inventors is the concept and quantitation of “AAGP Multiple.” An AAGP Multiple provides a means for comparing AAGP values from prospective or actual patient groups, such as the ICU patients in the REDOXS and RE- ENERGIZE studies described in EXAMPLE 2, AAGP values from healthy volunteers (e.g., as described in EXAMPLE 1), for purposes of assessing likely efficacious dosing of ulimorelin or other AAGP-binding drugs for EFI or other ICU-related conditions. The AAGP Multiple is determined by calculating the ratio of a given patient’s AAGP level (in this illustration, using the levels from the REDOXS and RE-ENERGIZE studies) at a given time to a derived value for a normal AAGP level (in this illustration, using the levels from the HVs as described in EXAMPLE 1), i.e., using the patient’s AAGP level as numerator and the normal level value as denominator. A distribution of AAGP Multiples for a given population may be obtained by determining AAGP Multiples for patients representative of that population (and a suitable HV or other“normal” population). [0118] Thus, the percentage of patients from the two ICU studies that fall into a specified range of AAGP Multiples (as shown below in TABLE 4) should be similar to that for the general ICU intent to treat populations, including those with EFI and muscle wasting conditions. Accordingly, the distribution of AAGP Multiples obtained using samples intended to mirror the intent to treat population can be used to predict the likelihood that Cmaxfree for an EFI or muscle loss patient from a given ulimorelin dose will be different from that typically seen in a healthy normal person who receives the same dose, and by how much.

[0119] To illustrate this more readily for the artisan of ordinary skill, one can define ranges of AAGP Multiples, such as, for example, 1 to <2. Such ranges may be standalone and so predict the likelihood that a given patient in a population will have an AAGP level in that particular range (e.g., a range of 1 to <2 or a range of 2 to <3), or, more usefully for purposes of understanding and appreciating the benefits of the present invention, cumulative, e.g., 1 to <3 or 1 to <4. If the value for normal AAGP level is set at 50 mg/dL (as determined approximately in the LP101-CL-101 trial), then TABLE 4 provides the cumulative percent of the ICU population with AAGP values in the specified range.

[0120] AAGP levels dictate how much of a given ulimorelin dose remains in unbound (free) form– the form that exerts the desired pharmacologic effect for EFI and for muscle loss (prevention or therapy). The higher the AAGP, or stated differently but with the same implication, the higher the AAGP Multiple, the lower the proportion of total drug administered that will be in free form at a given dose level (i.e., therapeutically active), and in particular, the lower Cmaxfree will be (with the opposite of these true as well).

[0121] TABLE 4

Cumulative Percent of Patient Population With AAGP Levels in Each AAGP Multiple Range

[0122] In TABLE 4, the various ratios shown that fall into each resulting cutoff range correlate with the likelihood that any given ICU patient will receive therapeutic benefit from ulimorelin at a particular dose. For example, given, from TABLE 4, that 62% of patients studied had AAGP Multiples in a cumulative range less than 3, the treating physician can infer that the likelihood that any specific patient under his or her care has an AAGP level at a given time within this same cumulative AAGP Multiple range is also about 62%. (Ranges of AAGP Multiples in TABLE 4 may also be represented as, from row 1 to row 6, <1; 1 to <2; 1 to <3; 1 to <4; 1 to <5; 1 to <6; <1 to >6.)

[0123] Data collected and analyzed as above combined with the Cmaxfree data (presented in EXAMPLE 3) from healthy volunteers receiving ulimorelin show that administration of a 600 μg/kg dose IV TID (total daily dose of 1800 μg/kg) to patients with AAGP Multiples at least as high as 4 (encompassing 87% of patients in the target population) will result in a Cmaxfree high enough to provide robust improvement in GE (compare to effect of 150 μg/kg dose IV TID in HV’s FIGURE 4). Administration of a 900 μg/kg dose IV TID (total daily dose of 2700 μg/kg) would provide similar benefit to patients with AAGP Multiples as high as 6 (encompassing 99% of patients in the target population). Moreover, this analysis in combination with safety data show that, at the doses described herein as efficacious in the treatment of EFI, e.g., 600 μg/kg to 900 μg/kg, any patient with a lower AAGP Multiple would not, upon receiving such a dose, be expected to experience Cmaxfree levels high enough to create a safety concern.

[0124] TABLE 5

Relationship of AAGP Multiple to Equivalent Ulimorelin IV TID Dose for ICU EFI Patients

Versus Healthy Volunteers

[0125] As illustrated in TABLE 5, if the patient’s AAGP Multiple is 4, then to achieve approximately the same Cmaxfree achieved by a dose of 150 μg/kg in a person with normal AAGP, the adjusted patient dose would be 150 μg/kg x 4, or 600 μg/kg. It will be appreciated that such calculations are population based, as they are derived from mean HV Cmaxfree calculations. Furthermore, acceleration of gastric emptying of healthy volunteers versus placebo was demonstrated at statistical significance at a dose of 80 μg/kg TID, although greater activity was achieved at higher doses. The activity at 150 μg/kg TID was deemed to correlate with a plateauing of GE acceleration activity. As such, while an expectation of robust improvement in GE may be calculated to occur in 87% of patients (at a dose of 600 μg/kg IV TID) by the methodology described herein, it is expected that an even larger percentage of ICU patients will have meaningful improvement in GE, even if not maximal improvement, at that dose. Meanwhile, when describing the response to therapy of an individual patient and not a population, it will be recognized that variability in responsiveness due to innate physiology of a particular patient may influence the degree of that patient’s specific gastric emptying response, as may changes in AAGP levels in a patient over the course of an ICU stay.

[0126] Given that a dose of 600 μg/kg IV TID should generally produce a Cmaxfree in an ICU patient above the minimally efficacious concentration (while also being safe to administer) for any AAGP value within the AAGP Multiple Range 1-4, and a dose of 900 μg/kg IV TID range should generally produce a Cmaxfree above the minimally efficacious concentration (while also being safe to administer) for any AAGP value within the AAGP Multiple Range 1– 6, the physician practicing in accordance with the invention will know there is a high likelihood of achieving an efficacious and safe Cmaxfree when administering such doses, without the need to know the specific patient’s AAGP level.

[0127] Those of skill in the art, upon contemplation of this discussion and the data and analysis presented herein, may appreciate that, prior to the present invention, it was impossible to predict with any confidence what Cmaxfree values would result from administering ulimorelin to a particular ICU patient, even if that artisan were focused on Cmaxfree, simply because of the variability of AAGP levels in the population and in an individual patient throughout an ICU stay. The present invention solves that seemingly intractable problem. 7.0 ICU Protocols

[0128] It will be appreciated from the foregoing that one advantage of the discoveries described herein is that a hospital ICU or physician may rely on a standard dosing protocol (sometimes called a “standard protocol”) for ulimorelin treatment notwithstanding the broad spectrum of AAGP levels in the ICU patient population. As used herein a standard dosing protocol encompasses IV TID administration of ulimorelin at a specified dose(s) to a defined ICU patient population, such as an ICU patient population described in §10.1, infra. Surprisingly and advantageously, the inventors have discovered that a standard protocol can be safely practiced without the need to measure (and without prior knowledge of) the ICU patient’s plasma AAGP level. This ensures that patients in need of treatment receive it quickly and without the need for inconvenient and time consuming testing procedures, and simplifying physician prescribing practice. The ability to apply a standard protocol in the ICU has a variety of benefits, some of which are discussed by Chang et al., 2012, Critical Care 16:306, incorporated herein by reference. These include reducing unnecessary variability in care, improved patient outcomes, effecting more rapid translation of clinical research into clinical practice, more streamlined care of critically ill patients, and cost reduction.

[0129] Thus, in one embodiment most or nearly all patients receiving ulimorelin treatment in a particular ICU will be treated with IV TID ulimorelin, and most or nearly all patients may be treated, at least initially, with a standard dose, e.g., 600 μg/kg ulimorelin IV TID as a 30 minute infusion, as described herein. Examples of standard doses for infusion are 600 μg/kg, 750 μg/kg, and 900 μg/kg. For example, in one embodiment most or nearly all ICU patients receiving ulimorelin treatment in a particular ICU will be treated with IV TID ulimorelin, and most or nearly all ICU (e.g., EFI) patients may be treated, at least initially, with a standard dose, e.g., 600 μg/kg ulimorelin IV TID as a 30 minute infusion, as described herein, or with a standard dose of about 190 μg per kg body weight to about 450 μg per kg body weight by bolus administration, as described herein. As another example, the ICU standard protocol may apply to particular patient populations such as a population identified in Section 10 below (e.g., all patients, EFI patients, burn patients, and the like)

[0130] In one example, the ICU standard protocol for treating EFI is 600 μg/kg ulimorelin IV TID as a 30 minute infusion, for at least one day, usually at least 2 days, and sometimes at least 3, 4, 5, of 6 days or longer. In one example, the ICU standard protocol for treating EFI is 750 μg/kg ulimorelin IV TID as a 30 minute infusion, for at least one day, usually at least 2 days, and sometimes at least 3, 4, 5, of 6 days or longer. In one example, the ICU standard protocol for treating EFI is 900 μg/kg ulimorelin IV TID as a 30 minute infusion, for at least one day, usually at least 2 days, and sometimes at least 3, 4, 5, of 6 days or longer. In some embodiments the standard protocol is a dose selected from 600 μg/kg, 650 μg/kg, 700 μg/kg, 750 μg/kg, 800 μg/kg, 850 μg/kg, or 900 μg/kg administered by IV infusion. In one approach, ulimorelin is administered by IV bolus injection three times per day at a dose of about 190 μg per kg body weight to about 450 μg per kg body weight (e.g., 275-425 μg per kg body weight). In some embodiments the standard protocol is a dose selected from 250 μg/kg, 275 μg/kg, 300 μg/kg, 325 μg/kg, 350 μg/kg, 375 μg/kg, 400 μg/kg, 425 μg/kg, or 450 μg/kg administered by bolus injection. In one approach, ulimorelin IV TID is administered as a bolus injection, for at least one day, usually at least 2 days, and sometimes at least 3, 4, 5, or 6 days or longer. In some embodiments the duration of the ulimorelin bolus injection is 30 seconds to 3 minutes. In some embodiments the duration of the ulimorelin bolus injection is less than 2 minutes. The selection of parameters of the standard protocol may take into account factors such as approvals or guidelines of drug regulatory agencies. In one approach the aforementioned dose regimens are used to prevent or reverse loss of muscle mass.

[0131] In one approach, the ICU standard protocol for treating EFI is IV bolus injection three times per day at a dose of about 190 μg per kg body weight to about 450 μg per kg body weight, for at least one day, usually at least 2 days, and sometimes at least 3, 4, 5, or 6 days or longer. In one approach, the ICU standard protocol for treating EFI is 600 μg/kg ulimorelin IV TID as a 30 minute infusion, for at least one day, usually at least 2 days, and sometimes at least 3, 4, 5, or 6 days or longer. In one approach, the ICU standard protocol for treating EFI is 190-300 μg/kg ulimorelin IV TID as a bolus injection for at least one day, usually at least 2 days, and sometimes at least 3, 4, 5, or 6 days or longer. In one approach, the ICU standard protocol for treating EFI is 220-550 ulimorelin IV TID as a bolus injection, for at least one day, usually at least 2 days, and sometimes at least 3, 4, 5, or 6 days or longer. In one approach, the ICU standard protocol for treating EFI is 250-450 μg/kg ulimorelin IV TID as a bolus injection, for at least one day, usually at least 2 days, and sometimes at least 3, 4, 5, or 6 days or longer. In some embodiments the duration of the ulimorelin bolus injection is 30 seconds to 3 minutes. In some embodiments the duration of the ulimorelin bolus injection is less than 2 minutes. The selection of parameters of the standard protocol may take into account factors such as approvals or guidelines of drug regulatory agencies. In one approach, any of the aforementioned dose regimens is used to prevent or reverse loss of muscle mass.

[0132] In some embodiments, the patient’s AAGP levels are not known at the time of first administration of ulimorelin. In some embodiments, a physician order for an assay to determine the patient’s AAGP levels has been submitted (e.g. to a hospital laboratory) after ICU admission but the laboratory result is not known at the time of first administration. In one embodiments the ICU patient’s AAGP level is not measured during the patient’s stay in the ICU. In some embodiments the ICU patient’s AAGP level is measured only after the patient has received at least one administration of ulimorelin. In some embodiments the patient’s AAGP level is not determined prior to the second administration of ulimorelin. In some embodiments the patient’s AAGP level is not determined prior to the third administration of ulimorelin. In some embodiments the patient’s AAGP level is not determined prior to the fourth administration of ulimorelin.

[0133] In some embodiments, the ICU standard protocol includes a step-up or step- down dosing step. In some embodiments, the ICU standard protocol may include AAGP- Based Dose Selection, as described in Section 13, below. In these cases, the standard protocol may comprise administration of ulimorelin at more than one dose (e.g., initial dosing at 600 ug/kg and step-up dosing at 900 ug/kg).

[0134] In a related embodiment, the ICU standard protocol is to administer ulimorelin by bolus injection where the standard dose is a specified dose is within a range set forth in Table 6, below (see also, Section 9).

[0135] Thus, in one aspect, the invention provides a method of treating a population of EFI or muscle wasting patients in an ICU by administering ulimorelin IV TID as described herein, wherein at least 50%, usually at least 75%, and often at least 90% of the patients in need of treatment or prophylaxis for EFI or for prophylaxis against muscle loss or recovery therefrom receive the same standard treatment according to the invention. In one aspect, the invention provides a method of treating a population of EFI or muscle loss patients in an ICU by administering ulimorelin IV TID as described herein, wherein at least 50%, at least 75%, or at least 90% of the patients receiving ulimorelin treatment for EFI or muscle loss receive the same standard treatment according to the invention. Without limitation, exemplary standard treatments are three times per day (i) administration of 600– 900 μg ulimorelin per kg patient body weight (μg/kg) to the patient by 30 minute intravenous infusion, (ii) administration of 600 μg ulimorelin per kg patient body weight (μg/kg) to the patient by 30 minute intravenous infusion, (iii) administration of 750 μg ulimorelin per kg patient body weight (μg/kg) to the patient by 30 minute intravenous infusion, (iv) administration of 900 μg ulimorelin per kg patient body weight (μg/kg) to the patient by 30 minute intravenous infusion; (v) administration of 220-550 μg ulimorelin per kg patient body weight (μg/kg) by bolus injection; (vi) a step-up and/or step-down regimen as described above. In one approach, ulimorelin is administered by IV bolus injection three times per day at a dose of about 190 μg per kg body weight to about 450 μg per kg body weight (e.g.275- 425 μg per kg body weight). In one approach, ulimorelin IV TID is administered as a bolus injection, for at least one day, usually at least 2 days, and sometimes at least 3, 4, 5, or 6 days or longer. In some embodiments the duration of the ulimorelin bolus injection is 30 seconds to 3 minutes. In some embodiments the duration of the ulimorelin bolus injection is less than 2 minutes.

[0136] It will be appreciated by the reader that one aspect of the present invention is administration of ulimorelin to a patient in need of treatment by a route and dosage described in this Section 7. That is, individual patients may be treated according to ICU protocols described herein. 8.0 Reduction in Heart Rate

[0137] Ulimorelin administration was associated with a dose-dependent reduction in heart rate. See EXAMPLE 3 and FIGURE 14. As illustrated in FIGURE 14, plasma levels of approximately 30 ng/mL– 125 ng/mL correlated with a reduction in heart rate. Only one occurrence of an HR reduction in the LP101-CL-101 and LP101-CL-102 study population met the criteria for an adverse event (bradycardia). This was seen in an HV who entered the study with a HR of 45 and who had transient reduction to 39 post-dosing with no reduction in blood pressure. In view of this, and because of the associated benefits in some instances of a slowing of HR, in some embodiments of the invention, ulimorelin is administered to achieve a target ulimorelin Cmaxfree in the range 30 ng/mL to 125 ng/mL, or 50 ng/mL to 100 ng/mL.

[0138] The potential minor reduction in HR associated with an ulimorelin Cmaxfree higher than about 30 ng/mL reflects, without intending to be bound by theory, an increase in vagal tone or, conversely, a reduction in sympathetic tone, or both, and is expected to provide clinical benefit to the EFI population. Whether increased parasympathetic tone or decreased sympathetic tone or both, all of these may be equated to decreased“net sympathetic tone.”

8.1 Administration of Ulimorelin to ICU Patients at a Dose High Enough to Cause Mild Decrease of Heart Rate

[0139] Excess sympathetic tone is common in critically ill patients. While not wishing to be bound by theory, the effects of ulimorelin on heart rate, are exerted, at least in part, via increased vagal (parasympathetic) activity (tone) and/or decreased sympathetic tone (collectively“decreased net sympathetic tone”). In a patient for whom the treating physician suspects, or has evidence of, a sympathetic tone that is unhealthy, excessive and/or otherwise may be deemed deleterious to the patient, the physician may elect to derive benefit from the effect of ulimorelin, at high enough doses, to cause a decrease in sympathetic tone as evidenced by a mild decrease of heart rate and to continue dosing or, indeed, to aim to dose from the outset, at levels that will cause such decrease. While not wishing to be bound by theory, slowing of heart rate in such patients is a sign of decreased net sympathetic tone. Without wishing to be bound by theory, decreased net sympathetic tone will lead to decreased inflammatory-mediated catabolism and/or decreased resting energy expenditure. For certain patients such decreases may occur at levels of drug lower than those causing decreased heart rate, while for others these benefits (decreased inflammatory-mediated catabolism and/or decreased resting energy expenditure) may occur at drug levels which are the same as those that cause decreased heart rate.

[0140] Thus, for some ICU (e.g. EFI) patients, dosing ulimorelin (in some cases at the high end of the therapeutic range, e.g., 750 μg/kg to 900 μg/kg TID), can provide a significant anti-catabolic benefit for patients requiring anti-catabolic therapy or otherwise benefitting from a lowering of their resting energy expenditure. In one approach to achieve these benefits, ulimorelin is administered at a level that results in a Cmaxfree value in the range of 50 ng/mL or higher, such as 50 ng/mL to 125 ng/mL.

8.2 Monitoring HR to Assess the Patient’s Response to an Ulimorelin Dose

[0141] The present invention offers the physician a way to follow the patient’s clinical course to assess whether a prescribed ulimorelin dose is sufficient, too high, or too low. In some cases, the physician may infer or learn about a patient’s AAGP level by following HR at Tmax (the time the drug achieves Cmaxfree) without actually assaying the patient’s AAGP level. In general, Tmax will be at or close to the end of or just after the infusion or bolus injection period (as measured from the start of bolus injection or infusion). For a given dose of ulimorelin (e.g., 900 μg/kg), an ICU patient with a very low AAGP, for example, will be more likely to exhibit reduced HR, or may exhibit an HR reduction of a larger magnitude, than a patient with a higher level of AAGP. 8.2.1 Step-Up Dosing

[0142] In one aspect, the invention provides a method of treating EFI in a patient in need of treatment, comprising administering ulimorelin to the patient three times per day for one or more days, wherein the administering comprises: (i) administering ulimorelin to the patient at a first dose, (ii) monitoring the patient’s heart rate (HR) to determine whether the HR slows by at least a predetermined targeted threshold reduction at about Tmax; and then, (iii) if the patient’s HR does not slow by at least the predetermined threshold administering to the patient a second dose of ulimorelin that is higher than the first dose, and if the patient’s HR has slowed by at least the predetermined threshold administering a second dose that is the same as or lower than the first dose. In one aspect, any of the aforementioned dose regimens can be used in accordance with the invention in patients being treated for muscle wasting, e.g. to prevent or reverse loss of muscle mass.

[0143] Again, for these embodiments, Tmax is generally achieved at the end of the infusion or bolus injection period. For purposes of the invention measurements made at Tmax are made at the end of the infusion or injection period (or equivalently, made using a plasma sample or other patient sample obtained at the end of the infusion or injection period). In some embodiments Tmax is determined within 1 minute or within 30 seconds after the end of the administration period.

[0144] In some embodiments, for illustration and not limitation, the first dose is 600-750 μg/kg (e.g., 600, 650, 700, or 750 μg/kg). In some embodiments the second dose is in the range 750-900 μg/kg (e.g., 750, 800, 850, or 900 μg/kg). Thus, in some embodiments, the first dose is 600 μg/kg, and the second dose is selected from 750, 800, 850, or 900 μg/kg. In some embodiments, the first dose is 650 μg/kg, and the second dose is selected from 750, 800, 850, or 900 μg/kg. In some embodiments, the first dose is 700 μg /kg, and the second dose is selected from 750, 800, 850, or 900 μg/kg.

[0145] In some embodiments, for illustration and not limitation, the first dose is in the range 250-300 μg/kg administered as a bolus and the second dose is in the range 400- 450 μg/kg administered by bolus injection. In some embodiments, the first dose is in the range 190-300 μg/kg administered as a bolus and the second dose is in the range 325-450 μg/kg administered by bolus injection. [0146] In some embodiments, the predetermined threshold reduction is an HR reduction by more than about 20% of the patient’s baseline HR (such as a reduction in the range 20% to 30% of the baseline), sometimes by more than about 10% of the baseline HR (such as a reduction in the range from 10% to 20% of the baseline). As used herein, the patient’s HR baseline is the patient’s HR after admission to the ICU but prior to administration of the first ulimorelin dose. For example, baseline may be determined within a 1, 2, or 4 hour period prior to administration of the first dose of ulimorelin. If a physician is monitoring a treated patient’s HR to evaluate the effect of ulimorelin on HR, then a baseline may be determined prior to any dose for which the physician wishes to monitor HR effects.

[0147] In some embodiments, the predetermined threshold reduction is a reduction by more than 20 heart beats per minute (bpm) relative to baseline, sometimes by more than 10 bpm relative to baseline, and sometimes by more than 5 bpm relative to baseline.

[0148] In some embodiments, the predetermined threshold reduction is a reduction to a HR below 80 bpm, sometimes below 100 bpm, and sometimes below 120 bpm.

[0149] While the general application of this method will be to ensure that the patient receives the highest safe dose possible (for maximal benefit in decreased net sympathetic tone), it will be recognized, in view of the disclosure herein, that in some embodiments (e.g. for special“at risk” groups of patients, for example) the threshold may be zero or very low, meaning essentially no slowing in HR is desired.

[0150] In one aspect of the invention, any of the aforementioned dose regimens are used to prevent or reverse loss of muscle mass.

8.2.2 Step-Down Dosing

[0151] In one aspect, the invention provides a method of treating EFI in a patient in need of treatment, comprising administering ulimorelin to the patient three times per day for one or more days, wherein the administering comprises: (i) administering ulimorelin to the patient at a first dose of 750-900 μg per kg patient body weight, (ii) monitoring the patient’s heart rate (HR) to determine whether the HR slows by at least a predetermined threshold amount at about Tmax; and then, (iii) if the patient’s HR slows by at least the predetermined threshold, administering to the patient a second dose of ulimorelin that is lower than the first dose, and if the patient’s HR does not slow by at least the predetermined threshold, administering a second dose that is the same as the first dose, or at least higher than the first dose. In some embodiments, the“first” dose may be a first elevated dose given to a patient during the course of treatment for EFI or other condition. In one aspect, any of the aforementioned dose regimens can be used in accordance with the invention in patients being treated for muscle wasting, e.g. to prevent or reverse loss of muscle mass.

[0152] In some embodiments, for illustration and not limitation, the first dose is in the range 750-900 μg/kg (e.g., 750, 800, 850, or 900 μg/kg). In some embodiments the second dose is 600-750 μg/kg (e.g., 600, 650, 700, or 750 μg/kg). Thus, in some embodiments the first dose is 750 μg/kg and the second dose is selected from 600, 650, or 700 μg/kg. In some embodiments the first dose is 800 μg/kg and the second dose is selected from 600, 650, 700, or 750 μg/kg. In some embodiments, the first dose is 850 μg/kg and the second dose is selected from 600, 650, 700, or 750 μg/kg. In some embodiments, the first dose is 900 μg/kg and the second dose is selected from 600, 650, 700, 750 or 800 μg/kg.

[0153] In some embodiments, for illustration and not limitation, the first dose is in the range 400-450 μg/kg administered as a bolus and the second dose is in the range 250- 300 μg/kg administered by bolus injection. In some embodiments, the first dose is in the range 325-450 μg/kg administered as a bolus and the second dose is in the range 190-300 μg/kg administered by bolus injection.

[0154] In one approach any of the aforementioned dose regimens are used to prevent or reverse loss of muscle mass.

[0155] The predetermined threshold reduction in HR reduction may be as described above in the description of the step-up approach. 9.0 Intravenous Administration

[0156] In accordance with the methods of the invention, ulimorelin is administered intravenously (IV administration), resulting in a rapid rise to a target Cmaxfree to provide the desired therapeutic benefit to ICU patients, e.g. prevention or treatment of EFI or muscle wasting. IV administration may, without limitation, be by infusion or by bolus injection, both of which are well known to physicians and medical providers, and are briefly described below. 9.1 Administration by Infusion

[0157] In one approach, ulimorelin is delivered (administered) three times per day by infusion. Administration of drugs by infusion is well known and the medical provider guided by this disclosure will be able to select particular infusion parameters to meet the needs of individual patients. In one approach, ulimorelin is administered by IV infusion over a 30 minute period to reach the desired Cmaxfree rapidly so as to provide therapeutic benefit to EFI and/or muscle wasting patients. Any conventional infusion practice may be used, including, without limitation, use of IV bags or use of syringe pumps.

[0158] Infusion temperature is typically room temperature. The infused product typically has a pH of about 4.5– 5.5, depending on the amount of diluent added. The cannula for infusion may vary according to the patient and to preferences of the physician, but for adults is typically 20 gauge or larger. Phlebitis, should it occur with a given infusion, can be prevented by good clinical practice or by infusion through a central line, or by a cannula engineered to stabilize the cannula site or to reduce mechanical venous irritation. Ulimorelin drug product is typically stored at controlled room temperature, and the prepared infused product is often stored under refrigeration for up to 24 hours and then allowed to come to room temperature for an hour prior to administration, and has a pH of about 4.5– 5.5 (the approximate pH of the infusate used in EXAMPLE 3).

9.2 Administration By Bolus Injection

[0159] In some embodiments, ulimorelin is administered by IV bolus injection rather than by infusion. Bolus injection may be used, for example, to save cost and/or quantity of drug used, or to reduce volume of infusate, or to increase nursing convenience, or to achieve Tmax more rapidly.

[0160] Bolus injection differs from IV infusion principally in length of time required to administer the drug to the patient. For IV infusion, drug is administered over a longer period (e.g., 30 min). For bolus injection, drug is administered over a shorter period (e.g., 3 min or less). In practice, ulimorelin for infusion is often diluted into a pharmaceutically acceptable diluent and administered using an IV bag, while ulimorelin is usually administered by bolus injection using a syringe, e.g., using a pump or pushed directly into an IV line port, i.e. by hand. For purposes of this description, ulimorelin bolus injections are completed in less than 3 minutes (from first drug into the patient’s bloodstream to end of administration). Bolus injections are usually completed within 2 minutes or less (e.g., 30 seconds to 2 minutes), and sometimes within 1 minute or less.

[0161] As described in this disclosure, 600-900 μg/kg TID administered by IV infusion is efficacious for treating EFI in the vast majority of ICU patients. These doses will result in a ulimorelin Cmaxfree (measured at the end of infusion) in the range 0.5 ng/mL to 125 ng/mL. When administered by bolus injection, as defined herein, a lower ulimorelin dose is required to achieve the same Cmaxfree, compared to a 30 minute infusion.

[0162] The preferred bolus injection dose may be determined empirically, for example by administering ulimorelin to healthy volunteers and determining which ulimorelin doses given under bolus injection conditions (e.g., 3 min bolus injection) result in the same Cmax (specifically, Cmaxfree) in the HV as observed following administration by (30 min) IV infusion of, for example, 600 μg/kg ulimorelin. Exemplary bolus injection doses may also be estimated based on pharmacokinetic principles, as shown in TABLE 6.

TABLE 6

Comparison of Exemplary Bolus Injection Doses and Infusion Doses

[0163] The bolus injection doses in TABLE 6 that are predicted to be equivalent to 600, 750, and 900 μg/kg infusion doses were determined using compartmental modeling techniques in Phoenix WinNonLin Version 6.4.

[0164] As described herein, when administered by IV infusion according to the invention, a 30 minute infusion time is used. In principle, other infusion times may be used, and, generally speaking, the longer the infusion time, the higher the dose required to reach the target Cmaxfree achieved by a 30 min infusion at 600 μg/kg TID. Those of skill in the art will appreciate that pharmacokinetic modeling, as well as or in addition to empiric determinations such as described above, may be used to derive effective doses for other infusion times (for example, between 15 and 30 minutes), where the purpose of the calculation is to select a dose that ensures that the target, or target range, of Cmaxfree is reached. 10.0 Intent-To-Treat Populations

[0165] The methods of the invention provide benefit to multiple patient populations. As discussed above, ulimorelin administered intravenously TID according to the methods disclosed herein, can be used to prevent or treat two ICU related conditions (EFI and muscle wasting/loss of muscle mass). Moreover, advantageously, as disclosed herein, the great majority of ICU patients may be efficaciously treated using a standard dosing protocol without the need to measure (and without prior knowledge of) patients’ plasma AAGP levels (see e.g. Section 7 of this disclosure). In addition, ulimorelin treatment according to the invention is beneficial to certain populations not in the ICU, especially patients who have been discharged from the ICU and have developed ICU-AW.

10.1 Patients in the ICU

[0166] In one aspect, ulimorelin is administered according to the methods disclosed herein to ICU patients. In one embodiment, ulimorelin is administered according to the invention to many or most patients entering the ICU. In some embodiments ulimorelin is administered to a patient or patients within a specified subpopulation. For example and not limitation, patient subpopulations believed to benefit particularly from ulimorelin administration are described in this section and elsewhere in this specification.

[0167] In some embodiments, ulimorelin is administered to most or all ICU admittees as a preventative treatment based primarily on their presence in the ICU. In other embodiments, one or more subset(s) of ICU admittees are identified as most likely to benefit from treatment with ulimorelin, and most or all ICU admittees in the subset(s) are treated with TID ulimorelin. For example, in some embodiments ulimorelin is administered to ICU patients with an ICU-related condition. In some embodiments, the hospital ICU or physician may administer ulimorelin to patients with particular conditions or characteristics, as discussed below.

[0168] In one embodiment, ulimorelin is administered according to the methods disclosed herein to ICU patients diagnosed with EFI. [0169] In one embodiment, ulimorelin is administered according to the methods disclosed herein to ICU patients receiving enteral feeding but not diagnosed with EFI, to prevent or reduce the risk of developing EFI. Ulimorelin may be administered concurrently with the initiation of enteral feeding, after initiation of enteral feeding, prior to the initiation of enteral feeding in a patient for whom enteral feeding is indicated or prescribed but not yet initiated, or, to a patient whose enteral feeding has been stopped or paused, for example but without limitation, due to EFI.

[0170] In one embodiment, ulimorelin is administered according to the methods disclosed herein to ICU patients expected to benefit from ulimorelin treatment, who are not receiving enteral feeding, and for whom enteral feeding is not indicated or prescribed. ICU patients expected to benefit from ulimorelin treatment include any patient at risk of muscle loss, or who has experienced muscle loss, or who would benefit from reduced loss of muscle mass or by recovery of lost muscle or muscle mass.

[0171] In some embodiments, ulimorelin is administered to ICU patients expected to have lengthy stays in the ICU. In this context, patients expected to be in the ICU for at least 3 days, or for at least 4 days, or for at least 5 days. Examples of patients expected to have lengthy stays in the ICU include, without limitation, burn patients, sepsis patients, acute respiratory distress syndrome (ARDS) patients, high-risk surgery patients, and others. Shorter stay patients may include, e.g., low-risk cardiac patients, and those not expected to live beyond 2-3 days.

[0172] In some embodiments, ulimorelin is administered to ICU patients with organ failure. Examples of organ failure that may indicate ulimorelin treatment include cardiac failure, renal system failure, hepatic system failure, pulmonary system failure, and gastrointestinal system failure (e.g., motility failure as in EFI).

[0173] In some embodiments, ulimorelin is administered to ICU patients with multiple organ failures. A clinical correlation between multiple organ failures (i.e., 2-3, or 4- 6, failed organs) and muscle wasting was previously reported by Puthucheary et al., 2013.

[0174] ICU patients with pulmonary failure are expected to receive particular benefit from ulimorelin treatment. In particular embodiments ulimorelin is administered to ICU patients who are intubated and mechanically ventilated. Also, ulimorelin may be administered to ICU patients with renal failure. [0175] In some embodiments, ulimorelin is administered to patients undergoing dialysis.

[0176] In some embodiments, ulimorelin is administered according to the methods disclosed herein to ICU patients receiving enteral feeding but not diagnosed with EFI, to prevent or reduce loss of muscle mass, and/or to reduce risk of developing EFI.

[0177] In some embodiments, ulimorelin is administered to a patient whose feeding is being provided both enterally and parenterally. In one aspect, ulimorelin is administered to a patient receiving parenteral nutrition, or total parenteral nutrition.

[0178] In some embodiments, ulimorelin is administered according to the methods disclosed herein, to ICU patients not diagnosed with EFI and not receiving enteral feeding, but at risk of loss of muscle mass. In this setting, ICU patients not receiving enteral feeding include patients who have not received enteral feeding at any time during their stay at the ICU and those who have received it but not recently.

[0179] In some embodiments, ICU patients are assessed to determine which among them may particularly benefit from a therapeutic or prophylactic intervention. As one example, a recent study in the ICU suggested that patients determined to be at relatively high nutritional risk may particularly benefit from superior protein feeding with regard to their mortality outcomes. (Compher et al., 2017, CCM 45(2):156-163, incorporated by reference herein). Patients at relatively high nutritional risk may particularly benefit from treatment with ulimorelin according to methods taught herein.

[0180] In some embodiments, ulimorelin is administered to ICU patients who show signs of malnutrition at the time of ICU admission or during the ICU stay and/or are deemed to be at high nutritional risk. See Malnutrition and Outcomes, Kenneth B. Christopher, M.D., supra. As discussed in Allingstrup et al., 2012, Clinical Nutrition 31:462e468, a higher provision of protein and amino acids was associated with lower mortality in patients.

[0181] In some approaches, ulimorelin is administered to ICU patients with low muscle mass. A recent study of ICU patients suggested that such patients with low skeletal muscle mass around the time of ICU admission are especially likely to benefit from improved feeding (specifically enteral protein feeding) with resulting reduced 6-month mortality risk. Such patients may be, for example, ones who are unable to receive adequate amounts of enteral protein feeding due to EFI but when treated according to the methods of the current invention become able to receive targeted amounts of enteral protein feeding (see Looijaard et al., 2017, JPEN. V41(2)).

[0182] In one aspect, ulimorelin is administered to any ICU patient at risk of losing muscle mass, in need of treatment to mitigate muscle loss, or in need of treatment to regain lost muscle. Generally, such conditions are referred to herein as“muscle wasting”.

[0183] In one embodiment, ulimorelin therapy is initiated in patients at the time of admittance to the ICU (e.g., within 24 h of admittance). In one embodiment, ulimorelin therapy is initiated after admittance to the ICU.

[0184] It is contemplated that medical professionals and hospital ICUs may develop treatment protocols based on combinations of criteria provided here. For example, in one embodiment, ulimorelin is administered to ICU patients who are admitted with low muscle mass and are expected to have a lengthy stay in the ICU.

10.1.1 Treatment for any Patient at Risk of Losing Muscle Mass

[0185] Ulimorelin administered according to the methods described herein is a suitable treatment for any patient at risk of muscle loss, or who has experienced same, and for which therapeutic intervention is warranted. Ulimorelin therapy is used to prevent or reduce loss of muscle mass or to promote or accelerate the recovery of lost muscle or muscle mass and so is generally useful for treating all muscle wasting conditions and prevention of the same. For the purposes of the invention described herein, and without wishing to be bound by theory, the therapeutic intervention will have a beneficial impact preferentially, if not entirely, on skeletal muscle tissue.

[0186] While various normal ranges have been published in men and women, a study using total body MRI reported that in healthy individuals aged 18– 88 (Janssen, Journal of Applied Physiology Published 1 July 2000 Vol. 89 no. 1, 81-88) approximately 30 percent of the female body and approximately 40 percent of the male body mass is made up of skeletal muscle, though with age this figure generally decreases. For example, a normal 175 pound (~80 kg) man has about 70 pounds (~32 kg) of such muscle mass. Puthucheary et al. (2013, JAMA 310(15):1591-1600) showed that, using the cross sectional area of rectus femoris muscle as a proxy for total body skeletal muscle, on average, critically ill patients lost approximately 10% of their muscle mass over the period starting after the first full day after ICU admission through the fifth full day post-admission, and the rate of loss persisted in an essentially linear manner for the first ten days of an ICU stay (ninth study day) at which point muscle mass loss was approximately 18%. Patients with multi-organ failure lost, on average, 22% of their muscle mass over this period. While these critically ill patients may not have had a normal amount of muscle at their starting point, i.e. the time of ICU admission, compared with that seen in a healthy individual, one may estimate that the amount of muscle mass lost over these ten days in an exemplary 80 kg patient was roughly 6 kg or higher. Most physicians would agree that reducing this loss of muscle mass would be beneficial in the vast majority of, if not all, critically ill patients.

[0187] It is anticipated that some patients being treated with TID IV ulimorelin who are at risk for significant muscle loss (e.g. similar to that described in the Puthucheary study) will lose no more than about 15% of their skeletal muscle mass over a ten day duration of therapy (i.e. approximately three quarters of the 18 - 22% figure seen with standard of care) while others will lose no more than about 10% of their skeletal muscle mass over a ten day duration of therapy (i.e. approximately half of the 18 - 22% figure seen with standard of care), while yet others will lose no more than about 5% (i.e. approximately one quarter of the 18 - 22% figure). In an exemplary 80 kg patient, these percentages equate to preservation of 1– 2 kg, 3– 4 kg, or 5 - 6 kg of skeletal muscle mass, respectively.

[0188] Those of skill in the art will appreciate that preservation of all pre-ICU baseline muscle mass is important given the difficulty of regaining any lost muscle mass. While the relative benefits over standard of care illustrated by these exemplary numbers are particularly substantial, even less preservation of muscle mass could provide clinically meaningful benefit. Furthermore, certain muscles have particular importance for clinically meaningful events, including ones that do not themselves have a numerically large muscle mass. One such example (and there are others) of particular importance relates to the diaphragm, where the need to preserve diaphragm muscle mass given that muscle’s enabling role in weaning a patient from a mechanical ventilator, an event that typically enables, or at least facilitates, discharge from an ICU.

[0189] It is anticipated that in some patients being treated with TID IV ulimorelin an increase in muscle mass is observed and can be measured after initiation of the treatment. For example, in some patients an increase in muscle mass by at least 5%, at least 10%, or at least 15% compared to baseline (e.g., weight at the time of admission to the ICU) is observed.

10.1.2 EFI Intent-To-Treat Population

[0190] In one aspect, the present invention is directed to treating patients for whom their physicians would like to provide nutrition by enteral tube feeding. Typically, these patients have been admitted to intensive care units (ICU) and have been placed on enteral tube feeding. Typically, the patients suffer from enteral feeding intolerance (e.g., impaired and/or delayed gastric emptying) or the physician is concerned that EFI will develop if not prevented. Those of skill in the art will appreciate that, while this aspect and these embodiments of the invention have been discussed with reference to treating EFI in the ICU, they are equally applicable to patients in need of treatment for EFI but not in the ICU.

[0191] In various embodiments the EFI patient has one of more of the following characteristics: (i) is an adult; (ii) is unconscious at the time of treatment; (iii) is unconscious at the initiation of a course of treatment; (iv) is mechanically ventilated; (v) is sedated; (vi) receives a general liquid feeding formula delivered as a continuous drip; (vii) receives a general liquid feeding formula delivered in boluses; (viii) receives feeding at rates according to a volume based feeding protocol; (ix) has been diagnosed with EFI based on a determination of GRV; (x) has been diagnosed with EFI based on a determination of clinical signs and/or symptoms; (xi) suffers from neurologic or head trauma; (xii) suffers from malnutrition; (xiii) suffers from burn; (xiv) has low muscle mass; and/or (xv) is in a catabolic state deemed unhealthy by a treating physician in addition to suffering from EFI.

[0192] As an illustrative example of all of the various embodiments of the invention in which the patient receiving benefit is on a nasogastric feeding tube or similar feeding device and the treating physician desires to treat EFI, the following is a description of a typical patient who could benefit by treatment in accordance with the invention. Such a typical patient may be admitted to an intensive care unit and be, as is frequently the case, unable to eat on his or her own. This can be due to many different causes, including, as non- limiting examples, sedation, such as from medically-induced coma or sedating pain medication, delirium, or, the presence of an endotracheal tube for the provision of mechanical ventilation. In typical practice, within the first 1 to 72 hours, or at a time when deemed acceptable by caregivers, a nasogastric or similar tube will be inserted into such a patient, through which a prescribed liquid feeding formula will be delivered, either as a continuous drip, in boluses, or as an intermittent continuous drip possibly with boluses as well.

[0193] Typically, after a certain amount of food has been infused and typically after 4 to 8 hours if by continuous drip, a study will be conducted to determine the GRV resulting from the amount provided, for which feeding will be interrupted. In some embodiments, a certain amount of time, such as approximately 5 - 20 minutes, will be allowed to elapse, and then gastric residual contents will be aspirated by syringe or similar method. Should the GRV (or other indicator of EFI) be deemed excessive in the caregiver’s judgment, typically in excess of an amount between 200 to 500 mL or more, as a non-limiting example, then feeding will be suspended or the feeding infusion rate will be reduced.

[0194] A certain amount of time will be allowed to elapse, and then typically the GRV (or other indicator of EFI) will be rechecked, with feeding having been restarted at the same or lower infusion rate in between, or with feeding on hold in between assessments, or both. If GRV (or other indicator of EFI) is deemed sufficiently low (i.e., below or on the low end of the threshold in use, typically within the range above, for example), then feeding will be restarted or increased. After a caregiver-determined number of GRV (or other indicator of EFI) readings are deemed excessively high, but often either one or two, the patient will be declared intolerant to enteral feeding, i.e., the patient may benefit from treatment of EFI, as provided herein.

10.1.3 Patients in need of treatment of gastroparesis and/or delayed and/or impaired gastric emptying, whether diagnosed with EFI or not, and whether receiving nutrition by enteral tube feeding or not

[0195] It will be recognized that ulimorelin administered IV TID in accordance with the invention provides benefit to critically ill patients in need of treatment of gastroparesis and/or delayed and/or impaired gastric emptying, whether diagnosed with EFI or not, and whether receiving nutrition by enteral tube feeding or not. Accordingly, TID administration of ulimorelin as described herein may be used to benefit the ICU population and the enterally fed population, as well the EFI population. Delayed gastric emptying was found in almost 50% of mechanically ventilated patients and up to 85% in certain diagnostic groups, including patients with polytrauma, traumatic brain injury, and sepsis. The highest rates of ICU EFI were observed among critically ill patients with cardiovascular, GI, and sepsis admission categories. Many ICU patients may suffer from incipient disease or undiagnosed (i.e., not yet diagnosed disease). That is, in one aspect, administration of ulimorelin as described herein provides benefit to the ICU population (i.e. critically ill patients). In one aspect, administration of ulimorelin as described herein provides benefit to the enteral feeding population (i.e., patients receiving nutrition by enteral feeding). In various embodiments, the methods of the invention are practiced to provide nourishment to patients in need thereof and otherwise diagnosed with polytrauma, traumatic brain injury, and/or sepsis. In other embodiments, the methods of the invention are practiced to prevent patients with delayed or impaired gastric emptying, or the reasonable potential therefor, from developing frank EFI.

10.1.4 Additional Beneficial Effects of Ulimorelin Treatment in EFI

[0196] Administration of ulimorelin as described herein stimulates and restores gastric motility and emptying, and also has anti-catabolic and pro-metabolic (collectively, anabolic) effects. In treating EFI, the treating physician may desire both the promotility effects of the drug and the anabolic benefit. These combined features, which are particularly beneficial for critically ill patients, as provided for ulimorelin herein, provide prokinetic and anabolic benefit to patients, and such treatment is thereby differentiated from any therapeutic that offers only one or the other benefit. While the invention is not to be limited in terms of putative mechanisms of action, some of these benefits are mediated through hormones such as growth hormone (see EXAMPLE 3, below), while other benefits may follow more generally merely from the provision of more protein or nutrition resulting from administration of ulimorelin as described herein, or others from the reduction of inflammation that leads to a catabolic state and loss of lean body mass. Some benefits may follow from the reduction of inflammation that leads to a catabolic state and loss of skeletal muscle mass.

[0197] While the invention is not to be limited in terms of putative mechanisms of action, some of these benefits are mediated through hormones such as growth hormone (see EXAMPLE 3, below), while other benefits may follow more generally merely from the provision of more protein or nutrition resulting from administration of ulimorelin as described herein, or others from the reduction of inflammation that leads to a catabolic state and loss of muscle mass. This treatment can, in effect, both provide more calories and protein to patients in need of such treatment and promote beneficial use of said nutrition to promote augmentation, or decreased loss, of muscle, lean body mass, and/or weight. This treatment can, in effect, decrease loss of muscle, lean body mass, and/or weight by leading to a reduction in resting energy expenditure.

[0198] Treatment with ulimorelin in accordance with the invention can provide beneficial outcomes in patients, and these benefits include, without limitation, one or more or all of the following, including as compared to patients not treated with ulimorelin: provision of more protein and calories via the enteral route; improved beneficial protein turnover; increased lean body mass (LBM); increased muscle; increased ventilator-free days; reductions in frequency of re-intubation episodes, reductions in aspiration rate, reductions in use of parenteral feeding; reductions in duration of critical care unit stay, reductions in duration of hospitalization, reductions in hospital-acquired infections, and/or reductions in near term mortality (such as 30- and 60-day) or longer term mortality (such as 6-month); and improvement in various measures of muscle strength and/or functional measures during hospitalization and/or measures of functional status post hospital discharge, including those self-reported by patients and those by their caretakers, such as Activities of Daily Living (ADLs) and Quality Of Life (QOL) or other tests of strength or functional status such as the Medical Research Council Scale (MRC).

10.1.5 Administration To Patients Receiving Enteral Feeding But Who Are Not Suffering From And/Or Have Not Been Diagnosed With EFI

[0199] In some embodiments, ulimorelin is administered, as described herein, to ICU patients receiving enteral feeding but who are not suffering from and/or have not been diagnosed with enteral feeding intolerance (EFI). Prophylactic administration will reduce the likelihood an at risk patient will develop EFI. In some embodiments, the patient for whom prophylactic treatment initiated is unconscious at the time treatment is initiated. In some embodiments, patients for whom prophylactic treatment is initiated are mechanically ventilated. 10.1.6 Administration to Patients for Whom EFI Is Resolved or Enteral

feedings Have Been Stopped

[0200] In other embodiments of the invention, after the EFI is resolved or enteral feedings have been stopped, the physician may continue administration of ulimorelin at the doses provided herein to provide continuing anabolic benefit to the patient. As demonstrated in the examples below, ulimorelin dosed in accordance with the invention to normal subjects causes spikes in the levels of growth hormone, which should have a beneficial therapeutic effect in patients in a catabolic state requiring treatment. As demonstrated in the examples below and discussed elsewhere herein, ulimorelin dosed in accordance with the invention to normal subjects causes, in some of them, decreased heart rate. While not to be bound by theory of mechanism, this slowing of HR is believed to be a vagally-mediated effect by which net sympathetic tone is reduced, which should have a beneficial therapeutic effect in patients in a catabolic state requiring treatment.

10.1.7 Administration to ICU Patients In Need Of Treatment To Mitigate

Muscle Loss

[0201] As described herein, administration of ulimorelin is indicated for ICU patients with EFI at risk of skeletal muscle loss, ICU patients without EFI (e.g., patients not receiving enteral feeding or those receiving enteral feeding without symptoms or manifestations of intolerance) at risk of skeletal muscle loss, patients at risk of skeletal muscle loss following discharge from the ICU, and patients at risk of skeletal muscle loss for reasons unrelated to an ICU stay. Such patients are safely and effectively treated using TID administration of ulimorelin according to the invention, e.g., by infusion or bolus injection. In one embodiment, ulimorelin is administered at a total daily dose of 570 μg to 1350 μg per kg patient body weight, wherein the ulimorelin is administered by three times per day bolus injection of one-third the daily dose for one day, or for two or more consecutive days. In one embodiment, ulimorelin is administered at a total daily dose of 1800 μg to 2700 μg per kg patient body weight, wherein the ulimorelin is administered by three times per day 30 minute intravenous infusion of one-third the daily dose for one day, or for two or more consecutive days. 10.2 Non-ICU Patients

[0202] In one aspect, ulimorelin is administered according to the methods disclosed herein to patients not in the ICU but who have experienced a loss of muscle mass and/or are at risk for such loss. In some embodiments, the patients have been discharged from the ICU and suffer from a condition related to the ICU stay. For example, ICU-acquired muscle weakness (ICU-AW) syndrome is a common complication of critical illness and injury (Friedrich et al., 2015, Physiol Rev 95:1025-1109; Puthucheary et al., 2103, JAMA 310(15):1591-1600). The associated skeletal muscle wasting is a negative prognostic factor for survival and the degree of skeletal muscle loss is associated with long-term outcomes (Hermans et al., 2014, AJRCCM 190(4)410-420; dos Santos et al., 2016, AJRCCM 194(7):1- 61). In some embodiments, the patient is at risk of loss of muscle mass for reasons unrelated to an ICU stay.

10.2.1 Non-ICU Patients In Need Of Treatment To Mitigate Muscle Loss

[0203] Elevated AAGP levels have been reported in patients with injury and trauma, and in elderly patients with acute illness or with cachexia of chronic disease. Administration of ulimorelin according to the methods of the invention (e.g., by IV infusion three times per day at a dose of about 600 μg per kg body weight to about 900 μg per kg body weight; or by IV bolus injection three times per day at a dose of about 190 μg per kg body weight to about 450 μg per kg body weight) is indicated for these and other such conditions as described in this Section 10 and elsewhere that result in loss of muscle mass.

[0204] In one aspect, ulimorelin therapy is provided to neonates and other infant patients, both with and without diagnosed EFI and/or to prevent muscle wasting and augment muscle development. In one aspect, ulimorelin therapy is provided to pediatric patients, including, without limitation, those with short-bowel syndrome.

[0205] For patients in need of treatment to preserve or increase muscle mass, TID administration of ulimorelin is believed most efficacious. However, therapeutic benefit, albeit reduced and suboptimal, still can be obtained in accordance with this aspect of the invention, even if the patient receives an administration of ulimorelin at a dose described herein as infrequently as every other day (qOD) or three times a week (for example, for patients undergoing chronic dialysis). For all of these indications, including treating EFI, preventing muscle loss in a critical care setting and treating muscle loss either during or after the patient is in a critical care setting, the anti-catabolic effect of ulimorelin will be particularly effective if one achieves maximum plasma concentrations (Cmax) rapidly, followed by a rapid decline.

10.2.2 Conditions For Which Ulimorelin Administration to Prevent or Reduce Loss of Muscle Mass or to Promote or Accelerate the Recovery of Lost Muscle Mass is Beneficial

[0206] Certain diseases and conditions increase risk of loss of muscle mass and are referred to herein as conditions associated with muscle wasting. In one aspect, ulimorelin therapy is provided to patients suffering from acute respiratory distress syndrome (ARDS). Survivors of acute respiratory distress syndrome are exemplary of those many ICU patients who often sustain muscle wasting and functional impairment resulting from and related to intensive care unit (ICU)-acquired weakness (ICU-AW), a disability which may persist for months or even years after ICU discharge. See Walsh et al., 2014, Clin Chest Med. 35:811- 26. In one aspect ulimorelin therapy is provided to patients suffering from emaciation. In one aspect, ulimorelin therapy is provided to patients suffering from anorexia. In one aspect ulimorelin therapy is provided to a patient suffering from pneumonia or other infection. In one aspect, ulimorelin therapy is provided to patients to treat cachexia (e.g., cachexia associated with cancer, chronic obstructive pulmonary disease (COPD), heart failure, malnutrition, post-operative status with complications, pancreatitis, HIV-infection, burn, tuberculosis, and end stage renal disease (ESRD)). In one aspect, ulimorelin therapy is provided to patients receiving dialysis for chronic renal failure. Chronic renal failure patients undergoing chronic dialysis, virtually all of whom have permanent IV access enabling convenient administration of an IV therapeutic despite being generally ambulatory, often suffer from anorexia and some degree of cachexia, and have suboptimal muscle mass. In one aspect, ulimorelin therapy is provided to elderly patients.

[0207] In one aspect, ulimorelin therapy is provided to patients with expected long duration of post-illness/injury recovery and rehabilitation (e.g., patients with hip fractures or patients undergoing high-risk surgery).

[0208] Ulimorelin treatment is indicated for patients in need of anabolic stimulation as a result of loss of muscle mass due to deprivation of calories and/or protein and/or due to systemic inflammation caused by critical illness such as, for example but without limitation, trauma, sepsis, cardiopulmonary disease, neoplasm, pneumonia and/or other serious infection, surgery, and/or gastrointestinal disease. Patients that benefit may also be at risk due to impaired growth hormone and/or other aberrant hormonal secretion, and/or excess net sympathetic tone, among other reasons.

[0209] Other conditions associated with loss of muscle mass for which ulimorelin treatment provides benefit, include corticosteroid use, denervation, limb immobilization, burn, neuromuscular disorders, sarcopenia of aging, arthritis, muscle wasting associated with HIV therapy, disorders involving mobility disability (e.g., arthritis, chronic lung disease); neuromuscular diseases (e.g., stroke, amyotrophic lateral sclerosis); rehabilitation after trauma, surgery (including hip-replacement surgery); recovery from catabolic illnesses such as infectious or neoplastic conditions; metabolic or hormonal disorders (e.g., diabetes mellitus, hypogonadal states, thyroid disease).

[0210] In addition,“muscle wasting” is common in patients confined to bed, due to inactivity and without regard to the effects of specific diseases, and is particularly common in patients in intensive care units. Thus, ulimorelin therapy provides benefit to bedridden patients, including patients being fed enterally, with or without diagnosed EFI, as well as patients not being fed enterally, such as patients receiving parenteral nutrition (or, alternatively, patients able to self-feed), without regard to the presence or absence of specific disease status. 11.0 Diagnosis of EFI

[0211] Those of skill in the art will appreciate that various physicians will determine the need for therapeutic intervention for EFI in a variety of ways, and the ways listed herein are illustrative and not exhaustive. For example, many physicians might take only a single GRV reading before diagnosing EFI and taking corrective action. Moreover, there are a variety of ways an individual physician may make a diagnosis of EFI without taking any GRV measurement, such as the observation of abdominal distension or vomiting, use of paracetamol absorption kinetic testing, use of scintigraphy, and others. The particular diagnostic methodology will often take into account the patient status; thus, for example, while GRV measurements are suitable for comatose patients, other methods, e.g., scintigraphy, are more appropriately done in on patients who are ambulatory. Those skilled in the art will appreciate that despite the variability in physician practice patterns and preferences with regard to making an EFI diagnosis, the underlying motility disorder, impaired and/or delayed gastric emptying and/or gastroparesis, is common to all such patients and as such the therapeutic promotility benefit of ulimorelin will apply generally to any patient diagnosed with EFI howsoever professionally diagnosed by a physician.

[0212] In particular, GRV measurements provide information about how much food remains in the stomach, but another way to diagnose EFI involves gastric emptying measurements made with scintigraphy (see Abell et al., 2008, Am. J. Gastroenterol.103:753- 763, incorporated herein by reference). Scintigraphy was used to measure gastric emptying in the HV study reported in EXAMPLE 3 but is not ideal for clinical practice in the ICU population because of difficulty implementing it in that setting.

[0213] Thus, the diagnosis of EFI may be made with or without use of gastric residual volume (GRV) measurement, including on clinical signs and/or symptoms alone, such as vomiting, distended abdomen, or others of similar GI relevance; or inferred from other measurements, such as gastric emptying scintigraphy or paracetamol absorption kinetic testing. 12.0 Ulimorelin Formulations and Dosage Forms

[0214] In various embodiments the invention relates to the use of an ulimorelin drug product concentrate provided for therapeutic use, including uses according to methods described herein. In some embodiments, the drug is provided as a solution (e.g., with a pharmaceutically acceptable excipient) in glass vials. For example, ulimorelin may be provided as a solution with buffered 5% dextrose in water for injection. The solution may be provided in a vial (e.g., a glass vial) labeled for use in accordance with the invention. The aforementioned ulimorelin solutions may be referred to as a“unit dose form.” It will be recognized that a single administration of the drug may use only a portion of the content of a vial, or, conversely, may use drug from more than one vial. It will be recognized that, in some cases, the unit dose form solution may be diluted, for example into an IV bag, prior to administration.

[0215] In various embodiments, the drug product may be provided at various concentrations, and may be provided in various sized containers (vials). Useful vial sizes include, without limitation, approximately 10 ml vials and 20 mL vials (referring to vial fill volumes). Larger volume unit dose forms (vials) and packaging providing higher fill volumes at, for example, 20 mL, 25 mL, 50 mL, and 100 mL per vial volume and corresponding fill amounts, or values in between these amounts, may be used. The drug product may be provided at various concentrations, such as 2 mg/mL, 3 mg/mL, 4 mg/mL, or 5 mg/mL. In some embodiments, the drug product is prepared using ulimorelin HCL monohydrate or other salt, in which case the ulimorelin concentration refers to the equivalent molar amount of the free base. In accordance with the invention, formulations of other ulimorelin salts are provided that may be used when more concentrated drug solutions (greater than 5 mg/ml) are desired. Alternate, higher solubility, therapeutically effective ulimorelin salts suitable for use in these embodiments of the invention include, without limitation, the succinate and malate salts and the invention provides drug products for such salts and the formulations containing them. It will be appreciated that ulimorelin may be provided in vials filled with solution and closed with rubber stoppers and flip-off aluminum seals.

[0216] In some embodiments, the drug product is the“2 mg/ml LP101 (Ulimorelin) Concentrate For Dilution For IV Infusion” formulation described in the Examples, below, and the drug product presentation (or“drug product intermediate” in some situations) is a clear vial filled with 10 mL (10.5 mL) of solution closed with rubber stoppers and flip-off aluminum seals. In some embodiments the product diluted, as needed, to the desired concentration.

[0217] Once the dose is determined, the formulation for administration is prepared. For example, to achieve a 600 μg/kg dose in a 70 kg patient requiring treatment for EFI, 21 mL of ulimorelin drug product concentrate (as described above at 2 mg/mL) can be diluted typically into 50 mL but optionally up to about 100 mL 5% dextrose in water (D5W), which is then administered as constant rate infusion over 30 minutes. Those of skill in the art will appreciate that in certain instances a certain amount of infusate will remain in IV tubing, by way of non-limiting example, such that to achieve a particular desired volume of infusate into the patient the actual volume of the formulation for administration that may be prepared may be augmented by an“overfill” amount and thereby exceed the actual amount delivered. [0218] Likewise, for a 900 μg/kg dose level, 31.5 mL of ulimorelin drug product concentrate is diluted, typically into 50 mL but optionally up to about 100 mL D5W which is then administered at a constant rate infusion over 30 minutes. Those skilled in the art will appreciate that the selection of 50 mL or 100 mL or, indeed, another volume of total infusate (including lower volumes, e.g., as low as 10 to 15 mL, some of which low volumes may be for doses that are given“neat”) may be determined by other factors, including, without limitation, a patient’s general fluid requirements or restrictions, as well as body weight, and the physician may use appropriate clinical judgment to decide the preferred total volume.

[0219] Individual physician (including any other medical professional operating under the physician’s direction) practice can vary widely without departing from the scope of the invention. As but illustrative examples, the physician may choose to use a different volume, e.g., 50 mL vs 100 mL or any other amount; to use no diluent (i.e., to use a“neat” administration); or to use a different diluent such as Lactated Ringer’s or Normal Saline.

[0220] Suitable infusion concentrations may be, for example, in the range of 0.15 mg/mL to 1.00 mg/mL. In alternate embodiments, suitable infusion concentrations may be in the range of 1.00 mg/mL– 2.00 mg/mL. In alternate embodiments, suitable infusion concentrations may be in the range of 2.00 mg/ml– 5.00 mg/ml. For illustration and not limitation, exemplary infusate drug concentrations include about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, and about 5.0 mg/mL.

[0221] Appropriate infusion rates may be readily determined by the physician. Typically, the infusion rate is in the range of 0.33 to 3.33 mL/min. One exemplary infusion rate is 1.67 mL/min for a total of 30 minutes. It will be recognized that, when administered by infusion, the infusion rate, final (e.g., diluted) drug concentration, and infusate volume may be varied to result in administration of a desired dose (e.g., 600 μg/kg) in a desired infusion period (generally 30 minutes) using a desired infusate volume. 13.0 AAGP-Based Dose Selection

[0222] In one aspect, the invention provides a method of treating EFI in a patient in need of treatment, comprising administering ulimorelin to the patient three times per day for one but typically, two, three, or more days, wherein the administering comprises: (i) administering ulimorelin to the patient at a first dose of 600 μg/kg IV TID and, prior to administration of a second dose, determining the patient’s AAGP level, and (ii) if the AAGP level is determined to be equal to or greater than a threshold level, then changing one or more subsequent doses to a higher dose (e.g., 750 μg/kg or 900 μg/kg) IV TID. The threshold value will be set by the physician or ICU but generally will be at least at the high end of the“normal” range and will typically be higher. Examples of suitable thresholds above the high end of the normal range include threshold values of 175-200 mg/dL and higher. In some embodiments the threshold value is in the range of 175 mg/dL to 250 mg/dL (e.g., 175, 200, 225 or 250 mg/dL).

[0223] In a related aspect, if the physician had elected to begin dosing at 900 μg/kg IV TID but without checking an AAGP, the physician may check AAGP levels after the initiation of treatment (e.g., after the first dose and before the second dose; or after any dose but before a subsequent dose) and, if the AAGP level is below the threshold value (e.g., 200 mg/dL), then the dose is lowered to 600 μg/kg IV TID.

[0224] In a related aspect, if the dose has been raised or lowered in this manner, the physician may recheck the AAGP level at any time (e.g. day 3, 4, 5, 6, or 7 of treatment). As one non-limiting example among several possible scenarios, if the AAGP level has fallen below the threshold value (e.g., 200 mg/dL), then the physician may lower the dose from 900 to 600 μg/kg IV TID.

[0225] In some embodiments the step of determining the patient’s AAGP level is carried out after the patient is admitted into the ICU and before the first dose is administered. 14.0 Determination of Body Weight

[0226] In application of the methods and products of the invention, the body weight of a patient in need of treatment for EFI is measured or estimated according to established medical practice for administering drugs based on body weight. Such measurements are made according to medical norms. In most cases, to calculate the actual dose for each administration of drug to a particular patient, measured or estimated dry body weight is used. In some cases, the reported or pre-ICU weight of the patient is used. [0227] For most patients dry body weight will be the same as usual (i.e. pre-morbid) body weight. For patients who have been fluid resuscitated (i.e. given fluid to maintain blood pressure during periods of hemodynamic instability according to customary medical practice) prior to or during the present ICU admission, the term dry body weight is used to clarify that extra patient weight attributable to resuscitation-specific fluids generally should not be included. Dry body weight reflects the patient’s weight prior to fluid resuscitation and is the actual body weight minus the weight of extra fluid deemed by a physician to be due to the resuscitation. Often such extra fluid will manifest as extravascular tissue fluid and may be approximated based on physical examination and other methods.

[0228] In summary, a typical application of the weight-based dosing methods and products of the invention will involve a determination of the body weight of a patient in need of treatment for EFI, and this determination will be made by measurement or estimation, and the measured or estimated weight will be used to calculate the actual dose for each administration of drug. In some cases (e.g., patients with obesity, emaciation, fluid overload), the physician may use“ideal body weight” or pre-morbid body weight for this determination. 15.0 Measuring AAGP Levels

[0229] Levels of AAGP are routinely measured by physicians for other purposes (as a prognostic or management factor for certain disorders, such as inflammatory bowel disease; see Kjeldsen et al., Scan. J. Gastroenterol. 1997, 32, 933–941; Gupta et al., Journal of Medical Microbiology (2010), 59, 400–407). Assays to determine AAGP are readily available commercially at facilities that routinely analyze clinical samples using technologies such as ELISA or nephelometry (see Genway Biotech, Human Orosomucoid (Alpha-1-Acid Glycoprotein) ELISA Quantitation Kit (http://www.genwaybio.com); Randox, Alpha-1-Acid Glycoprotein (AGT) RX Series (www. randox.com)). Unless otherwise apparent from context, AAGP levels discussed herein refer to the plasma or serum concentration.

[0230] In accordance with this invention, AAGP levels, if measured during a course of treatment with ulimorelin as described herein, can be measured at any time, i.e., immediately or shortly before the first administration of ulimorelin, concurrent with initial administration, or after the first (or any subsequent) administration (i.e., testing is“during administration” or concurrent with administration). In some embodiments measurements will be made using an assay with CRM470 calibrators. 16.0 Ulimorelin Assays

[0231] Free ulimorelin levels in plasma can be determined by any suitable analytical method. In one approach, free ulimorelin levels in plasma are determined by first dialyzing the plasma samples using a Rapid Equilibrium Device (RED) in 96 well-plate format. The plasma containing chamber (bound) and buffer containing chamber (free) are then diluted with buffer and plasma (1:1), respectively, and extracted and analyzed by LC-MS/MS. Quantitative concentrations are measured from high and low range standard curves in equivalent matrix for the bound and free, respectively. Appropriate quality controls are included for both concentration ranges.

[0232] For Lyric Pharmaceuticals-sponsored studies described herein, assays for plasma free and total ulimorelin levels were conducted at LGC (Cambridge, U.K.). Total plasma levels can be determined using standard bioanalytical LC-MS/MS methods. Briefly, free drug is measured by first dialyzing the plasma samples using a Rapid Equilibrium Device (RED) in 96 well-plate format. The plasma containing chamber (bound) and buffer containing chamber (free) are then diluted with buffer and plasma (1:1), respectively, and extracted and analyzed by LC-MS/MS. Quantitative concentrations are measured from high and low range standard curves in equivalent matrix for the bound and free, respectively. Appropriate quality controls are included for both concentration ranges. Other commercial laboratories, including Tandem Laboratories (Salt Lake City, Utah), also perform such assays. 17.0 Measurement Of Muscle Mass And Function

[0233] Benefits of the present invention (e.g., improvements in muscle metabolism, mass and function in patients treated with ulimorelin according to the invention) may be measured by various methods known in the art. Increases in muscle mass or a reduced rate of muscle loss may be observed, relative to a baseline. In addition, a reduced rate of loss of muscle may be observed in an individual by making measurements at multiple time points. Increases in muscle mass or a reduced rate or absolute level of decrease relative to reference values characteristic of changes in muscle mass in similar patients not treated with ulimorelin, including but not limited to those patients treated with a placebo or with clinical standard of care measures, may be observed.

[0234] The clinician generally will be able to assess the effect of ulimorelin treatment by appropriate monitoring to track muscle anabolism, catabolism, metabolism, and/or mass. Such monitoring may include, for example and without limitation, measurement of any indicator of muscle anabolism/catabolism. The clinician generally will be able to assess the effect of ulimorelin treatment based on, for example, the ability of a patient under his or her care to be weaned successfully from a ventilator, or to perform tasks requiring a minimal amount of effective muscle strength, such as picking up a remote control or telephone, or tasks requiring more strength, such as sitting up in bed, lifting an extremity, standing or climbing stairs, or walking a given distance or walking a distance over a given amount of time.

[0235] Quantitative assays for muscle metabolism and mass are also known, and determination of muscle mass (e.g., changes in muscle mass) is well within in the ability of the medical professional. However, the present invention also offers new methods for measuring muscle mass that are ideally suited for patients in an ICU. Thus, in an aspect of the invention, muscle mass is determined using a novel method in which D3-creatine dilution (or other heavy isotope dilution) following intravenous (IV) administration, as described in Section 18, infra, is used to determine muscle mass by inference. This method is particularly suitable when measuring muscle mass of patients in an ICU, because, for example, no patient assistance or mobility is needed for the determination. In a less optimal approach for ICU patients, the D3-creatine may be delivered orally (see Clark, et al., 2014, J Appl Physiol 116:1605-1613, incorporated by reference herein).

[0236] In another approach, the cross sectional area of rectus femoris muscle serves as a proxy for total body skeletal muscle, and muscle loss can be determined through serial ultrasound measurement of the rectus femoris cross-sectional area (see Puthucheary et al., 2013, JAMA 310(15):1591-1600, incorporated by reference herein).

[0237] In another approach, the muscle protein fractional synthesis rate (MPFSR) in skeletal muscle tissue is tested as described below in Example 5. Also see Berg, et al., 2013, Critical Care 17:R158 (doi:10.1186/cc12837); Bornø et al. 2014, Mass Spec. 49(8)674–80; Shankaran, et al., 2015, each of which is incorporated by reference herein.

[0238] Muscle synthesis, volume, function and metabolism also may be measured by various other art known methods, including: (a) ultrasound of the quadriceps and/or diaphragm; (b) cross-sectional CT scan at Lumbar 3 (CT-L3); (c) preservation or augmentation of skeletal muscle mass as measured by D3-creatine dilution and/or by 24 hour urinary creatinine excretion; (d) skeletal muscle protein degradation by 24 hour urinary 3-methyhistidine (3-me-His) excretion; (e) whole body N balance by 24 hour urinary total nitrogen (N) or urea nitrogen excretion; and (f) plasma muscle protein fractional synthesis rate (PPFSR) by measuring plasma levels of circulating skeletal muscle-derived proteins, such as creatine kinase M-type (CK-M) and carbonic anhydrase-3.

[0239] Still other useful assays for muscle mass or function are described in US Pat. Pub. US 20150072985, incorporated herein by reference, and include 1) muscle strength test (MST); 2) physical performance test (PPT); 3) muscle cross section area (CSA) measurement (via magnetic resonance imaging [MRI]); 4) muscle tissue biomarker measurement (muscle biopsy); and 5) muscle fiber size. Diaphragm thickness is a well accepted proxy for diaphragm muscle mass and may be assessed by standard medical ultrasound techniques.

[0240] To assess the effects of ulimorelin treatment, changes in lean body mass (LBM) also can also be measured. LBM is a commonly used proxy in clinical study of the effect of therapeutic or prophylactic interventions on muscle, and generally speaking, the therapeutic effect of ulimorelin on LBM and muscle mass will track together. However, in one aspect, the present invention provides methods for using ulimorelin to preserve or slow the loss of muscle, in patients with or without EFI, and for whom following LBM alone may be misleading. As discussed above, the ulimorelin treatments disclosed herein benefit patients with conditions that are typically treated in an Intensive Care Unit. These patients may have fluid shifts, edema, other derangements of fluid balance, as well as other relevant medical conditions, that may confound the interpretation of a measurement of LBM.

[0241] Non-limiting examples of patients for whom LBM measurements may not be reliable include patients with enlarged or deranged organ size or mass, which can occur for any of a number of medical reasons, one of which may be clinically significant visceral edema (e.g. pulmonary edema), as well as patients who have peripheral (limb) edema, as well as patients with cancer experiencing substantial changes to the size of a cancerous mass due to advancement of disease or response to therapy. Thus, the pathologies of these patients and other pathologic reasons can lead to a change in LBM that does not correlate directly and linearly with changes in muscle mass, as would be the case typically expected in a healthy individual or a patient without such fluid or other derangements. For example, if a patient treated with ulimorelin entered the ICU with significant edema and this condition improved during the ICU stay, that patient’s overall numerical LBM would go down significantly, even while the typically seen reduction in muscle mass was mitigated by therapeutic drug effect. See Example 10 below.

[0242] Skeletal muscle is a subset of total body muscle, as the latter also includes smooth muscle. For the purposes of the invention described herein, and without wishing to be bound by theory, the therapeutic benefit of the intervention will primarily impact, if not entirely be limited to, skeletal muscle tissue. Furthermore, the methodologies described herein for measuring muscle mass, such as, but not limited to, the D3-Creatine methodology and ultrasound-based techniques, are intended to measure skeletal muscle mass.

[0243] Skeletal muscles enable key human capabilities such as movement, which includes not only locomotion but also breathing, as the diaphragm is regarded as a skeletal muscle rather than smooth muscle. As well, chest wall skeletal muscle plays a key role in breathing. As such, a treatment that has a beneficial impact on skeletal muscle will offer significant benefit to patients and to the health care system overall, given the amount of money spent to assist and support, as well as the complications often seen in, patients who survive acute illness but cannot wean from ventilators, patients who become, or remain, weak or frail, patients who have limited or no mobility, patients who are unable to feed, bathe or clothe themselves, patients who are unable to ambulate, or patients who remain in or experience any one or more such life-changing and societally expensive states. 18.0 Intravenous (IV) D3-Creatine Dilution Test

[0244] In addition to methods described above in Section 17, muscle mass may be determined using a novel method, referred to as the“IV D3-Creatine Dilution Test.” The IV D3-Creatine Dilution Test is a method to measure accurately skeletal muscle mass by heavy isotope dilution following intravenous (IV) administration. In a preferred embodiment the heavy isotope is D3-creatine. The test is generally applicable to any ICU patient at any ICU facility and, more generally, to any patient or subject for whom one desires to measure skeletal muscle mass and for whom IV administration of a diagnostic agent is an option. For example, the IV D3-Creatine Dilution Test measures skeletal body mass in ICU patients and other critically ill patients including, by way of non-limiting examples, comatose and/or intubated patients.

[0245] The IV D3-Creatine Dilution Test has advantages over current generally accepted methods for measuring skeletal muscle mass, lean body mass (LBM), a surrogate for muscle mass that is valid in certain, but not all, clinical settings, or other surrogates of muscle mass, such as whole body nitrogen. Other commonly used methods are more difficult to carry out than the present assay in ICU patients. The present method does not require moving the patient to a scanner (such as full body MRI or CT scan at lumbar 3 (CT- L3)), and does not require physiological adherence to biological assumptions that permit translating the measurement to muscle mass (such as D3-creatine dilution following oral administration, heavy water dilution, bioelectrical impedance (BIA), or dual-energy x-ray absorptiometry (DEXA)). The methods of the present invention have special advantages over prior art methods to assess muscle mass that require quantitative oral administration of a compound(s), as such methods cannot be reliable if such reliability is dependent on delivery of compound(s) to a stomach that is known not to have normal motility or emptying, such as is the case specifically for ICU EFI patients in particular and a concern for ICU patients generally.

[0246] For the purposes of the present assay, and in particular to avoid confounding results as might be obtained using the prior art method in which the D3-creatine is administered orally, intravenous administration of an isotopically-labeled creatine (typically D3-creatine, but other isotopes 2-3 atomic mass units (amu) or more greater in mass than creatine may also be used) ensures the entire administered dose is bioavailable for uptake into muscle in patients such that reliable results are obtained. Creatine is synthesized in vivo, primarily by the liver and kidneys, to a degree sufficient to sustain a more or less constant ratio of creatine to muscle mass even in the absence of dietary intake. Blood levels rise and fall depending on the availability of creatine in the diet. [0247] IV administration of D3-creatine will produce an overall blood exposure similar to orally administered D3-creatine, assuming such oral administration is to a healthy individual with a normally-functioning gastrointestinal tract, as oral bioavailability in such a person is essentially 100%. At 30 mg, a slow injection or infusion will ensure that maximum blood levels remain low, as even if administered as a rapid bolus, the maximum concentration in blood for a typical person could only be about 0.005 mg/mL (30 mg/5500 mL blood). In addition to patients with gastric dysmotility as described above, patients suitable for testing in accordance with the invention include, without limitation, those who may not be able to swallow on their own, who may be unconscious, who may be on a feeding tube but have uncertain gastrointestinal function, and/or who suffer from enteral feeding intolerance.

[0248] The specific data obtained via the methodology presented herein regarding a patient’s then-current skeletal muscle mass can assist a physician in determining (a) to what degree a patient may benefit from an intervention, such as a pharmacologic agent, intended to mitigate loss of muscle during an ICU stay and/or to help the patient more quickly or fully (or both) to recover such lost muscle, and/or (b) what to expect and how best to plan for and optimize the patient’s trajectory towards recovery post-ICU and/or his or her risk of muscle-loss-related morbidity and/or mortality, both during and after an ICU stay. Similarly, and as will be appreciated by those skilled in the art, patient assessment and management of this sort may be performed by a physician for non-ICU patients and, in fact, for any patient for whom the physician has reason to assess or monitor muscle mass and in whom an intravenous route of administration of D3-creatine is required or beneficial.

[0249] Generally, then, the invention provides a method for determining skeletal muscle mass in a subject, said method comprising (1) administering a dose of isotopically labeled creatine (having at least +2 amu greater molecular weight than unlabeled creatine and typically at least +3 amu) intravenously to a test subject, (2) collecting a urine sample at least 48 hours and less than 5 days after said administration step (optionally one can acquire a sample immediately prior to administration, but such pre-testing is not required for a first test, only for repeat testing), (3) quantitatively measuring the isotopically-labeled-creatine degradation product (isotopically-labeled creatinine) as well as endogenous creatine and creatinine in said urine sample, (4) calculating a corrected dose of isotopically labeled creatine by adjusting for renally excreted material using the creatine to creatinine ratio, (5) determining the creatine pool size by dividing the corrected dose by the enrichment ratio, (6) converting the creatine pool size to muscle mass by dividing by 4.3 mg creatine per gram of muscle.

[0250] Typically, the measurements will be made by mass spectroscopy, and the subjects will include patients suffering from a disorder of gastric motility, including gastroparesis, or from an impairment of gastrointestinal absorption. Such subjects, then, can be viewed as subjects suffering from diseases of gastric motility or gastrointestinal absorption or both, and such subjects can also include subjects suffering from diseases of small bowel motility or absorption. The methods of the invention are generally applicable to any subject where a determination of muscle mass is desired but oral administration of a test agent is not possible or contraindicated or where other methods of determining muscle mass are impractical, including but not limited to comatose patients, vomiting patients, and non-compliant patients, e.g. patients suffering from a debilitating mental illness.

[0251] In one embodiment of the present invention, the steps are (1) intravenous administration of heavy isotopically labeled creatine (D3-creatine; e.g. substitution of 3 hydrogen atoms with 3 deuterium atoms; +3 atomic mass units) to a test subject, (2) collection of one or more spot urine sample(s) prior to dosing and again >=48 hour post dose from the test subject, (3) quantitative measurement of the D3-creatine degradation product D3-creatinine together with endogenous creatine and creatinine in the sample(s) via bioanalytical assay, and (4) calculation of muscle mass by first dividing the D3-creatine dose, corrected for any lost D3-creatine by renal excretion, by the measured D3-creatinine enrichment ratio to determine the creatine pool size, and then converting the creatine pool size to muscle mass via the known constant ratio of 4.3 mg creatine per gram of muscle.

[0252] There is no restriction on the number of discrete IV D3-creatine dilution tests that may be conducted in an individual patient, as long as they are spaced at least 48 hours apart (so that the previous dose achieves steady state) and a predose urine sample is collected for determining predose isotopic enrichment in association with each subsequent test (predose enrichment is negligible before the first test and therefore the predose urine sample in this instance is not critical, though it is best practice to collect and measure it). [0253] It will be appreciated by those skilled in the art that this method relies on the dilution of heavy isotopically labeled creatine, in the exemplified embodiment D3-creatine, and that any heavy isotopically labeled creatine can be used in accordance with the teachings herein to achieve the same objective of providing a reliable measure of muscle mass. D3-creatine is a non-limiting example, as it is readily available commercially, isotopically stable, nonradioactive, and related bioanalytical assay technology has been previously developed. Any heavy isotope, typically at least 2 or more mass units greater than the endogenous creatine molecule (to avoid mass spectroscopic interference), may be used as long as it may be synthesized and a bioanalytical method adapted for its detection, including, as non-limiting examples, 13C, 15N, 2H (where deuterium, D, and 2H are synonymous) or combinations thereof.

[0254] The accuracy of this method derives from the following: (1) creatinine excretion can be used as a direct measure of skeletal muscle mass, (2) approximately 98% of an individual’s creatine pool is contained in skeletal muscle, (3) the average concentration of creatine per mass of muscle is relatively constant at 4.3 mg/g (regardless of diet), (4) creatine is irreversibly and nonenzymatically degraded to creatinine at a constant rate in vivo (~1.7% per day) and creatinine is excreted into urine at a constant rate (Kreisberg et al., 1970, J App. Physiol 28(3):264–267; Heymsfield et al., 1983, Am J Clin Nutr 37(3):478–494; Wang et al., 1996, Am J Clin Nutr 63(6):863–869). Therefore, in accordance with the invention, the creatinine enrichment in urine is used to determine the creatine pool size and ultimately, the mass of skeletal muscle, using an IV administered isotopically-labeled test agent (e.g. D3-creatine).

[0255] The advantages of the present invention can be appreciated by comparison and contrast with prior art methods. In one such method, all urine excreted over a 24 hour period (24-hour urine) is collected to determine the amount of creatinine in urine, and that amount is then correlated to muscle mass; however, such collection is cumbersome and prone to a lack of adherence and collection errors, which can lead to inaccurate determinations of muscle mass. The method is also inaccurate in the very old or young, in sick or injured patients, and in body builders. The amount of creatinine in urine is also influenced by other factors, such as disease (e.g. diabetes, renal function) and medications (e.g. ACE inhibitors, angiotensin II receptor antagonists) that make it less accurate and more variable in ICU patients. Furthermore, in ICU patients, urine samples are often needed for other tests, resulting in an incomplete 24-hour urine sample. While potentially elegant in its simplicity, the burden for implementation of measuring muscle mass based on a 24-hour urine collection lies in the success of the sample collection, which turns out to be exceedingly difficult to control in a hospital setting, especially in an ICU, where the priority is on patient care rather than sample collection.

[0256] The current invention circumvents this otherwise onerous sample collection requirement by instead requiring use of a dose of D3-creatine known to be accurately delivered and fully bioavailable, followed by the collection of a single urine sample approximately 2 to 3 days later (once isotopic steady state is achieved). This sample collection is neither volume nor time sensitive (i.e., a spot urine sample is sufficient). For one skilled in the art, it is easy to appreciate that administration of a single dose of test article, followed by a single spot urine sample collection, is much easier to achieve in a hospital setting than the required collection of the entirety of a patient’s urine output over an exact 24 hour period.

[0257] The collected spot urine sample (which can be a dry or wet urine sample) is then analyzed by a quantitative LC-MS/MS bioanalytical method to measure creatine, D3- creatinine (if the test agent used is D3-creatine), and endogenous creatinine, at minimum, for the calculation of creatine pool size and, ultimately, skeletal muscle mass via calculations discussed below.

[0258] One remarkable aspect of this aspect of this invention is that the methodology is not only easy to execute and accurate, but also economical, and as such, should be readily amenable to inclusion in a standard clinical laboratory battery of assays, such as those typically used in patient care and subject to such regulatory oversight as Clinical Laboratory Improvement Amendments of 1988 (CLIA) certification.

[0259] IV administration in accordance with the invention not only enables dosing to patients in need of this test, but also ensures that an accurate amount of D3-creatine is delivered to the systemic circulation for uptake by muscle, an amount which must be known to determine accurately muscle mass by a creatinine-based method. D3-creatine (C4H6D3N3O2) is commercially available; for example, as a microbiological and pyrogen tested material from Cambridge Isotope Laboratories, Inc. (CIL) as item number DLM-1302- MPT-PK at a chemical purity of 97% and label purity 98%. To be used in patient study or care, the material should be GMP grade material, as can be achieved during formulation via an initial GMP repurification step.

[0260] D3-creatine can be delivered at a dose at least as low as 30 mg and up to 1000 mg, but as 30 mg is sufficient to measure with certainty the levels of isotopically labeled and endogenous analytes, 30 mg D3-creatine is the most appropriate dose for IV administration in accordance with the invention. Creatine is soluble in water at approximately 13 mg/mL and is relatively stable at low temperatures and neutral pH. Suitable methodology used for formulating D3-creatine solution for IV administration in accordance with the invention include aqueous formulation (e.g. water, buffer, saline, 5% dextrose in water (D5W)), and such formulations are suitable for frozen storage conditions.

[0261] Typically, for the methods of the invention, the dose is administered as a slow bolus injection over approximately 3 minutes or as an IV infusion over 3 to 30 minutes. The duration of the administration is not critical: it is instead the known amount administered that is critical.

18.1 Spot Urine Sample Collection

[0262] The spot urine sample may be collected as early as 48 hours post D3-creatine administration, as isotopic steady state is achieved relatively quickly and levels remain quantifiable for >120 hours (Clark et al., 2014, J Appl Physiol 116:1605-1613). The urine sample may be collected as an aliquot of urine or by immersing an absorptive material, such as a strip of filter paper, into a sample of urine for later extraction. The timing of sample acquisition and volume of the sample are not crucial to success, once isotopic steady state has been achieved and is continuing (e.g., sample acquisition will take place at least 48 hours after administration and typically within 3 to 5 days and not longer).

18.2 Bioanalysis

[0263] While the invention is not to be restricted to any particular method of analysis, endogenous creatine and creatinine and labeled creatinine (e.g. D3-creatinine in some embodiments) must be quantitatively analyzed to calculate skeletal muscle mass using the methods of the invention. Therefore, a successful bioanalytical method accounts for endogenous material in blank matrix (creatine and creatinine may be present in human urine) and measures both the very low concentrations of isotopically labeled analyte and the approximately 1000-fold higher concentrations of endogenous analyte.

[0264] There are numerous existing assays that measure creatinine, including GC– MS assays, which require derivatization, LC–MS/MS assays, and spectrophotometric analysis using the Jaffé reaction, that can be adapted for use in this invention by application of ordinary skill. As well, alternate fit-for-purpose approaches for LC-MS/MS analysis have been developed that are applicable.

[0265] Known LC-MS/MS bioanalytical methods exist for D3-creatine and for a combination of D3-creatinine and endogenous creatinine (Leonard et al., 2014, Bioanalysis:6(6):745-759). Similar validated LC-MS/MS bioanalytical methods exist. All are suitable for use in the invention. These methods entail measuring endogenous creatinine in human urine by LC–MS/MS using a surrogate analyte, D3-creatinine, for the standard curve (SC) and quality controls (QCs). The concentration of creatinine in the biological sample is measured by using the concentration of the creatinine M+2 isotope and dividing this concentration by a dilution factor, where the dilution factor is the ratio of the concentration of creatinine M+2 to the concentration of creatinine M+0 in the biological sample. The M+2 isotope peak area is less intense than the M+0 isotope peak area, permitting detection of a signal in the same concentration range as D3-creatine without dilution (Leonard et al., 2014, Bioanalysis:6(6):745-759).

[0266] Another suitable method entails measuring endogenous analytes in human urine by LC–MS/MS using a surrogate analyte, the D3-counterpart, for either creatine or creatinine, using two sets of standard curves and quality controls, one at a low concentration for the D3-labeled analyte and one at a high concentration for the endogenous analyte, where the main peak (M+0) is measured. In this scenario the high range SC, QCs, and samples require dilution prior to analysis to avoid saturating the detector.

[0267] Yet another suitable method entails using a surrogate matrix (artificial urine) in place of blank human urine to construct the SC and QCs, to avoid contamination by endogenous analytes. In this method, high range SC and QCs could be used for the analysis of endogenous creatine and creatinine. Artificial urine can be prepared in a laboratory or purchased as a formulated product. An example of artificial urine (available from Pickering Laboratories (Mountain View CA) is: Sodium Chloride - 6.773 g/L; Sodium Phosphate Monobasic, Dihydrate - 1.33 g/L; Potassium Chloride - 6.065 g/; Calcium Chloride Dihydrate - 0.883 g/L; Sodium Citrate Dihydrate - 0.584 g/L; Sodium Phosphate Dibasic - 0.435 g/L; Sodium Sulfate - 2.431 g/L; Magnesium Sulfate, Heptahydrate - 0.731 g/L; Ammonium Chloride - 2.322 g/L; and Pro-Clean - 0.3 mL/L. The levels of creatine and creatinine in this “blank human urine” can be determined and subtracted from the SC and QCs.

[0268] The invention relies on isotopic dilution to measure the amount of an endogenous molecule, whereby a known amount of a tracer molecule (in one embodiment, D3-creatine) is administered, and the ratio of the tracer to endogenous molecule is measured to determine the endogenous molecule’s concentration. Of principle importance then, is to know the exact dose administered, and therefore, the key concerns are the dosing and the sampling, and this method precisely controls dosing by administering the test agent, which in the illustrated method is D3-creatine, by the IV route.

[0269] In this aspect of the invention, the dilution of D3-creatinine, converted from D3-creatine at a constant rate, is measured. The constant rate of degradation of creatine to creatinine means that the degradation of D3-creatine to D3-creatinine also occurs, so that the dilution of D3-creatinine into endogenous creatinine can be used to calculate the creatine pool size via the following mathematical equations (see Clark et al., 2014, J Appl Physiol 116:1605-1613; Leonard et al., 2014, Bioanalysis 6(6):745-759; Hellerstein et al., 2016, Experimental Biology Poster):

(1) Creatine Pool Size (mg) = [131.1/134.1]*[D3-creatine Dose (mg)– D3-creatine Dose Excreted in Urine (mg)] ÷ Enrichment Ratio,

where the amount of the D3-creatine dose excreted in urine can be derived from an empirically determined linear relationship to the ratio of endogenous creatine and creatinine;

(2) Ln(D3-creatinine Dose Excreted in Urine) = 1.2086*Ln(creatine/creatinine) + 0.7516,

in which the constants (slope and y-intercept) may be updated as additional clinical data becomes available; and

(3) Ln(D3-creatinine Dose Excreted in Urine) = 1.2897*Ln(creatine/creatinine) + 1.0283, and where the Enrichment Ratio is defined as:

(4) Enrichment Ratio = D3-creatinine ÷ (creatinine + D3-creatinine).

Finally, the creatine pool is converted to muscle mass by this equation:

(5) Creatine Pool Size (mg) ÷ 4.3 mg/g = Muscle Mass (g).

18.3 D3-Creatinine (D3C) IV Formulations

[0270] Creatine is a naturally occurring nutrient found in meat, and is readily available as an oral supplement taken as a solid or aqueous solution. Di- ¹⁵ N-creatine has been administered to subjects by IV infusion over 8 hours at a concentration of about 1% in a solution of D5W (Crim et al., 1976, J Nutr 106:471–481). Creatine phosphate (1000 mg), closely related in structure, has been delivered to human subjects intravenously (Hurlow et al., 1982, Br. J. Clin. Pharmac 13:803-806). Neither study reported any safety issues. However, D3-creatine has never been manufactured previously for intravenous administration. According to the present invention, D3-creatine is provided in a form suitable for IV administration. In one approach D3-creatine is provided as a sterile, pyrogen free aqueous solution (e.g. water, buffer, saline, 5% dextrose in water (D5W)) suitable for frozen storage. In one approach D3-creatine is provided in vials suitable for injection, e.g., a vial with a rubber stopper. In one approach D3-creatine is provided as a single dose, e.g., in a 10-mL injection vial. In one approach, the single dose is 30 mg in a volume of approximately 3mL solution. A solution comprising 30 mg D3C in 3.2 mL solution may be provided (e.g., in a 10 mL vial) and approximately 3.0 may be administered to the patient. Table 7 provides illustrative D3-creatine formulations of the invention suitable for use in this method of the invention.

[0271] TABLE 7

19.0 Examples

[0272] The invention, having been described in summary and detail above is now illustrated, without limitation, in the following examples.

[0273] Example 1 describes how the“normal” range of AAGP levels in healthy individuals was determined from samples collected from healthy volunteers (HV).

[0274] Example 2 describes how ICU patient samples were analyzed to estimate an expected range of AAGP in an ICU population.

[0275] Example 3 describes clinical trials that have been conducted to demonstrate that the methods of the invention are safe and efficacious in the treatment of EFI. The example also discusses results from administering ulimorelin to HVs (shown to have normal AAGP levels) at doses within ranges specified herein.

[0276] Example 4 describes a Phase 2 study in which patients with EFI are treated with ulimorelin.

[0277] Example 5 describes a protocol for a clinical trial to determine the effects of IV ulimorelin on skeletal muscle and muscle protein metabolism in patients with acute respiratory distress syndrome (ARDS).

[0278] Example 6 is an example describing prevention of muscle loss in a patient receiving enteral feeding.

[0279] Example 7 is an example describing treatment of a patient with pre-existing muscle loss. [0280] Example 8 is an example describing treatment of chronic muscle loss in a patient.

[0281] Example 9 is an example describing treatment of acute muscle loss in a patient.

[0282] Example 10 is an example describing prevention and treatment of muscle loss in a patient with clinically significant edema.

[0283] Example 11 is an example describing treatment of EFI to treat existing, and prevent further, muscle loss in a patient.

[0284] Example 12 is an example describing measuring muscle mass in a subject using an IV D3-creatine dilution test.

[0285] Example 13 is an example describing clinical study using an IV D3-creatine dilution test.

[0286] The results and accompanying discussion in the Example and Figure legends above are applicable to all aspects of this disclosure and demonstrate that treatment of ulimorelin in accordance with the invention provides therapeutic benefit by improving gastric emptying, reducing and treating loss of muscle mass, ensuring the Cmaxfree is in the desired therapeutic window, and increasing GH levels.

19.1 Example 1 - AAGP Levels in Healthy Individuals

[0287] AAGP levels were determined from samples collected from healthy volunteers enrolled in the Phase 1 studies at two different sites (described below in EXAMPLE 3). Samples from Day 1 and Day 7 were analyzed. Not all subjects provided samples at all time points. Over 90 samples from 51 subjects were tested. AAGP levels were measured using a validated method employing the Randox assay kit (Catalog number AG2472) and an AU640 analyzer (LGC method MW13766).

[0288] The range of AAGP values in healthy volunteers at the UK site was 32.4 - 77.2 mg/dL, with mean and standard deviation of approximately 50.8 +/- 10.6 mg/dL. The range of AAGP values in healthy volunteers at the US site was 34.7 - 88.1 mg/dL with mean and standard deviation of approximately 59.6 +/- 12.9 mg/dL. See FIGURE 9. As illustrated in FIGURES 10 and 11, the mean, high and low AAGP values and distribution were similar in both populations. As discussed above, these values differ from previously published values. 19.2 Example 2 - AAGP Levels In ICU and Other Critically Ill Patients

[0289] As discussed in the Background section, supra, plasma levels of AAGP increase in various disease states including acute illness, infection, various types of cancer, cardiovascular disease, surgery and chronic inflammatory diseases. In this example, analyses of AAGP levels in various ICU patient populations were carried out. To assess plasma levels of AAGP in ICU patients, serum and plasma samples were obtained and analyzed that had been collected in two prior clinical studies of ICU patients: The REDOXS study (see N Engl J Med 2013; 368:1489-1497) and the RE-ENERGIZE study. A total of 233 samples from 92 patients were analyzed. The AAGP level distribution in ICU patients is shown in FIGURE 9.

[0290] One hundred fifty-three (153) plasma samples (from 63 patients) collected during a prior clinical study of ICU patients with heterogeneous and broadly representative admitting diagnoses (REDOXS study; see N Engl J Med 2013; 368:1489-1497) were analyzed to assess the variability of AAGP levels in this sample set. The REDOXS study samples were from ICU patients with admitting diagnoses including, among others, pneumonia (infectious and aspiration), neoplasm, drug overdose, trauma, various gastrointestinal disorders, sepsis, cardiopulmonary illness (e.g., cardiogenic shock, cardiomyopathy, acute myocardial infarction, respiratory arrest, and, congestive heart failure), as well post-operative patients who had undergone various types of surgeries. In addition to the REDOXS study, approximately 80 plasma samples (from 29 patients) collected during a prior clinical study of burn patients in the ICU (the RE-ENERGIZE study) were analyzed in a second study to assess the variability of AAGP levels in this specific patient treatment population. These ICU patients are representative of those in the intent to treat patient treatment population, although patient samples were included regardless of whether the patient was receiving enteral feeding, for the methods of the present invention.

[0291] Samples analyzed for AAGP level in the REDOXS study were drawn at study entry (typically shortly after ICU admission, i.e.,“baseline”), as well as day 4 and day 7. Samples from the RE-ENERGIZE study were taken at days 4, 7, 14, and 21. Some samples (and therefore data) for various time points were missing for patients in both studies. All samples were analyzed in a commercial laboratory (LGC, UK) using a validated method based on Randox Laboratory, Ltd.’s alpha-1-acid-glycoprotein (AGT) kit (Cat. No. AG2472). [0292] Results: To assess variability and trends in AAGP levels in these patient samples, the data were analyzed to determine high and low values as well as whether an individual patient’s AAGP levels varied in a predictable fashion during their ICU stay. The data are graphically summarized in FIGURE 9 (upper right panel). From all patient samples at all time points measured, the lowest AAGP level was 44 mg/dL, from a sample obtained at the baseline time point. The next lowest were two readings of 45 mg/dL and 46 mg/dL, both from samples drawn at day 7. The lowest reading from a Day 4 sample was 56. From all samples at all time points, the highest AAGP level was 390 mg/dL, a Day 4 reading. The highest baseline reading was 291 mg/dL. The highest reading from a Day 7 sample was 360 mg/dL. The variability of readings was striking and unpredictable with regard both to extent and timing of elevated readings, when seen, as well as with regard to the presence of unexpectedly low values that were closer to values seen in healthy volunteers (see EXAMPLE 1). Generally, however, the majority of patients in each of the distinct admitting patient groups (i.e., post-surgical, sepsis, neoplasm, as examples) had elevated AAGP levels relative to healthy volunteers.

[0293] The trends in the data generally demonstrated that ICU patients had markedly higher AAGP levels than healthy volunteers and that certain patients, particularly those with persistent medical illness, e.g., infectious disease, and those patients recovering from surgery, as examples, might reasonably be expected to have significantly elevated levels. For post-surgical patients, peak levels were typically seen in the Day 4 or Day 7 samples, consistent with the expectation that, as an acute phase reactant, the AAGP level should increase after an inflammatory triggering event (like surgery) and remain high or even increase for so long as the inflammatory condition exists.

19.3 Example 3 - Clinical Study Results in Healthy Volunteers

[0294] This example describes two Phase 1 studies in healthy volunteers (LP101-CL- 101 and LP101-CL-102) in which safety, PK, and pharmacodynamics (PD) of single and multiple (Q8H) doses of ulimorelin were evaluated. Healthy male and female volunteers aged 18 to 55 years participated in the trial. The objectives of the trial were to evaluate the safety, tolerability, and PK of single and multiple ascending IV doses of ulimorelin at higher doses than in prior studies; evaluate the PD of single and multiple ascending IV doses of ulimorelin, assessed by change in gastric emptying and growth hormone levels; and, explore the relationship between AAGP levels and total/free ulimorelin plasma concentrations. Liquid gastric emptying was studied to mimic tube-fed conditions in the ICU.

[0295] In the LP101-CL-101 trial, three cohorts were enrolled in a crossover design from the SAD to the MAD part of the study. The doses studied were SAD: 600, 900, and 1200 μg/kg and MAD: 80, 150, and 300 μg/kg Q8H for seven days. In the LP101-CL-102 trial, the dose studied was 600 ʅg/kg Q8H for seven days. The following endpoints were measured: safety, PK (total and free ulimorelin concentrations), AAGP levels, and PD. PD endpoints measured were gastric emptying by scintigraphic imaging (during the MAD part only), paracetamol absorption (LP101-CL-102 only), growth hormone levels, and reduction in HR (LP101-CL-102 only).

[0296] The ulimorelin used was LP101 labeled as“2 mg/ml LP101 (Ulimorelin) Concentrate For Dilution For Infusion.” The stock product was a sterile, pyrogen-free solution of ulimorelin hydrochloride monohydrate (equivalent to 2 mg/mL of ulimorelin free base) in water for injection buffered to pH 4.5 with 10 mM acetate buffer, and containing dextrose for tonicity adjustment. The product was diluted into 5% dextrose in an IV bag under aseptic conditions for administration. All infusions occurred over 30 min, with volumes and concentrations adjusted to permit achieving the nominal dose for each subject in each dose group.

[0297] Dose limiting toxicity was not achieved, but protocol-defied dose escalation stopping criteria as defined as a moderate AE in the same SOC (system organ class) in at least 2 subjects, specifically infusion site irritation, was achieved at 300 μg/kg Q8H. No cases of infusion site irritation were noted at the 600 μg/kg Q8H dose studied in the LP101- CL-102 study.

19.3.1 Study Results

[0298] LP101-CL-101 was a Phase 1 study in Nottingham UK, in which 39 subjects received ulimorelin or placebo 30-minute IV infusion, with ulimorelin doses of 600, 900 and 1200 μg/kg as a single dose and 80, 150, and 300 ʅg/kg Q8H for 7 days.

[0299] LP101-CL-102 was a follow on study in Lexington, Kentucky in which 12 subjects received ulimorelin or placebo 30-minute IV infusion at 600 ʅg/kg Q8H for 7 days.

[0300] Gastric emptying, as measured by the improvement in time relative to baseline it takes to empty 25% (^t25, aka GET25) and 50% (^t50, aka GET50) of the stomach contents, was improved at all MAD doses tested (ranging from 80 μg/kg to 600 μg/kg Q8H). FIGURE 3 shows the results of the LP101-CL-101 and LP101-CL-102 studies individually. FIGURE 4 shows pooled results for both studies.

[0301] FIGURES 5 and 6 show Emax plots for LP101-CL-101 and LP101-CL-102, showing the relationship between ulimorelin free Cmax and improvement in the time for 50% liquid gastric emptying (^t50) on Day 1 and Day 4 measured by scintigraphic imaging.

[0302] As shown in FIGURE 6, EC ₅₀ (concentration to achieve 50% of maximal effect) was observed at 0.62 ng/mL Cmaxfree to 1.1 ng/mL Cmaxfree, with 49% and 35% improvements of t ₅₀ at E _max (maximal effect) on Days 1 and 4, respectively. As shown in FIGURE 4 the prokinetic effect of 600 μg/kg Q8H ulimorelin was preserved through Day 6 LP101-CL-102 study.

[0303] Although the two studies were carried out in different countries, by different CRO’s, and using different scintigraphic scanning machinery, several metrics“justify” presuming consistency (see EXAMPLE 4, below). The population (healthy volunteers) and enteral formula were the same in both studies.

19.3.2 Consistency of Results from Phase I Trials in Different Countries

[0304] Studies have been done to confirm that data from the LP101-CL-101 and LP101-CL-102 clinical trials could be combined notwithstanding that they were conducted on healthy populations in different countries, by different contract research organizations and using different scintigraphic scanning machinery. A variety of metrics evidence the consistency between the two studies including (i) overlapping GE response in placebos and to drug (overlapping exposure response based on free concentration/Cmax); (ii) AAGP level distribution in HV’s in the US study was slightly right-shifted compared to the UK study but otherwise nearly identical; the slightly higher AAGP Levels in US vs UK study corresponds to slightly lower free exposures.

19.3.3 Dose-Related Reduction in Heart Rate

[0305] While single doses of 600, 900, and 1200 μg/kg and multiple doses of 80, 150, 300, and 600 μg/kg were safe and well tolerated in healthy volunteer studies, a dose-related reduction in heart rate (HR) was observed at doses starting at 600 μg/kg. See FIGURE 14. One episode of reduction in HR in the studies met the criteria for an adverse event (AE). [0306] No changes in vital signs were observed at the 80, 150, and 300 μg/kg Q8H doses. Mean decreases in sinus HR of -14% and -23% were observed at the 900 and 1200 μg/kg doses representing the exaggerated and expected pharmacologic impact of up- regulated vagal tone. Corresponding mean plasma Cmaxfree levels in healthy volunteers at these doses were 42 ng/mL and 74 ng/mL, respectively. At 600 μg/kg IV Q8H, a pattern of reduced HR was not observed, either on Day 1 or with repeat Q8H dosing at steady state, despite achieving Cmaxfree levels at steady state comparable to the 900 μg/kg single dose. The slowing of HR, when observed, corresponded to the peak free concentration at the end of the 30-minute infusion, was short-lived and predictable, and reversed with the rapid decline in concentration following the end of the infusion. No changes in blood pressure (BP) were observed, and no intervention was required in any subject.

19.3.4 Ulimorelin Effect on Growth Hormone Levels

[0307] In healthy adults non-stimulated resting Growth Hormone (GH) levels vary between 0 and 0.5 μg/L, and the mean spontaneous GH peak in healthy adults is approximately 3 μg/L. In healthy volunteers in the LP101-CL-101 and LP101-CL-102 trials, GH spiked following administration of ulimorelin. See FIGURE 15. As shown, levels on Day 1 were meaningfully greater than those seen in placebo subjects at any time point (pooled placebo mean GH levels were 2.2 μg/L and ranged from about 0.1 to 11 μg/L). Over time, the spikes in GH levels diminished but remained measureable and within or slightly greater than the expected physiological range of the assay (0-0.8 μg/L for males and 0-8 μg/L for females). Placebo peaks (if any) were random while ulimorelin peaks corresponded to drug administration with Tmax for peaks most often at 1 hr post dose.

19.4 Example 4– Phase 2 Study In EFI Patients

[0308] A Phase 2, multicenter, randomized, double-blind, comparator-controlled study of the efficacy, safety, and pharmacokinetics of intravenous ulimorelin in patients with enteral feeding intolerance (LP101-CL-201) was initiated.

19.4.1 Clinical Protocol Synopsis

[0309] The principal inclusion criteria for the study included the following (i) men and non-pregnant women aged 18 years and above; (ii) intubated and mechanically ventilated in the ICU; (iii) receiving continuous nasogastric, orogastric, or percutaneous gastric tube feeding; (iv) a 12-Fr or larger nasogastric, orogastric, or percutaneous gastric feeding tube, with its distal tip at least 10 cm below the gastroesophageal junction and visible in the stomach on a routine radiographic examination within 24 hours of screening; (v) enteral feeding intolerance, defined as a GRV of ш 500 mL on one or more measurements [N.B., a follow-on GRV < 500 mL prior to randomization does not exclude study participation]; (vi) expected to remain intubated, mechanically ventilated, and receiving nasogastric feeding for at least 72 hours.

[0310] According to the trial protocol, participants are randomized to receive either 600 ulimorelin TID by IV infusion (a total daily dose of 1800 ug ulimorelin per kg body weight)(active patients) or metoclopramide 10 mg TID by IV infusion (comparator patients), in both cases daily for 5 days.

19.4.1.1 Interim Cmaxfree PK Results

[0311] Preliminary patient data from the LP101-CL-201 Study was obtained. Preliminary analysis of PK data indicate:

[0312] On Study Day 1, the mean AAGP level was 166 mg/dL (37.7-277) (n=17 active and comparator patients) and the measured geometric mean ulimorelin Cmaxfree was 3.09 ng/mL (1.02-8.32) (n=15 active patients).

[0313] On Study Day 4, the mean AAGP level was 164 mg/dL (39.6-294) (n=15 active and comparator patients) and the measured geometric mean ulimorelin Cmaxfree was 15.2 ng/mL (1.73-86.4) (n=13 active patients).

[0314] These initial Day 1 and Day 4 Cmaxfree values are in close agreement with the predicted steady state C _maxfree distribution curve shown in FIGURE 13.

19.5 Example 5 - Treatment of Patients to Prevent or Slow Loss of Muscle Mass

[0315] This example provides a protocol for a clinical trial for a single or multi- center, randomized, double-blind study of the effects of IV ulimorelin on skeletal muscle and muscle protein metabolism in patients with acute respiratory distress syndrome (ARDS) or other medical condition for which an ICU stay of at least approximately ten days to two weeks or more may be typically expected.

[0316] As such, the study is intended to demonstrate the therapeutic benefit of administering ulimorelin in accordance with the methods of the invention not just to ARDS patients but to any patient at risk of muscle loss for which therapeutic intervention is warranted. [0317] A total of 20 evaluable patients, randomized 1:1 to ulimorelin or placebo, are deemed sufficient to meet the efficacy objectives of the study, providing approximately 10 evaluable patients per treatment arm. Given that up to 50% of patients who are randomized may not be evaluable, a total of approximately 40 patients will be enrolled.

[0318] For ulimorelin, study drug is provided as Ulimorelin Injection (2 mg/mL) or as a similar drug product or drug product intermediate at concentration up to 4 or 5 mg/mL, diluted in D5W or similar diluent for delivery of 50 mL, or placebo (D5W) 50 mL, administered intravenously (IV) over 30 minutes every 8 hours (Q8H) for 10 days. Patients will be randomized 1:1 in permuted block sizes of 4 to receive either ulimorelin (600 μg/kg), or placebo, Q8H.

[0319] ARDS patients that have been ventilated for approximately 3 to 5 days, are expected to remain intubated for an additional 10 days, and, meet study inclusion/exclusion criteria may enter the study. Study procedures will be performed after informed consent (by proxy if the patient is unable to provide valid informed consent) is obtained. Throughout the study, patients will receive treatment for their underlying medical conditions according to the local standard of care. D3-creatine (30 mg) will be administered IV or NG on Days 1 and 8 for the assessment of muscle mass. 50 mL of 70% D ₂ O will be administered NG or IV TID on Days 1– 6 and BID Days 7– 10 for MPFSR and PPFSR assessments. Pre-treatment and post-treatment muscle mass will be determined by a spot urine collection for D3-creatinine on Days 3 and 10 and by 24-hour urinary creatinine collections on Days 1, 2, 5, 6, 9, and 10. Microbiopsy or similar sampling of the vastus lateralis or similar muscle will be obtained on days 5 and 10 for MPFSR. Blood samples will be taken simultaneously for PPFSR of circulating muscle proteins. Spot urine collections will be obtained for isotope background rates and equilibrium and Days 1 (predose), 4, 6, 8, and 10. N-balance and rate of muscle protein degradation (3-MeHis excretion) will be assessed from 24-hr urine collections taken on days 1, 2, 5, 6, 9, and 10. Ultrasound (US) of the quadriceps and diaphragm muscle cross- sectional area will be performed on Day 1, 5, and Day 10. Blood samples for plasma ulimorelin levels will be collected minimally on Days 1, 5, and 10 at predose and at 0.5 and 24 hr post any daily infusion; the predose samples will be split for the determination of alpha-1 acid glycoprotein (AAGP) levels. [0320] Dietary intake of protein and calories will be measured daily. Feedings will target a minimum protein intake of 1.2 g/kg/day with no upper limit and may be provided by any combination of enteral or parenteral routes of administration. To optimize and standardize nutrition in the context of a clinical study it is the goal that patients who receive enteral nutrition will be fed according to a volume-based feeding protocol, although the study is generally intended to mirror clinical standard-of-care conditions in current use in order to highlight the benefit of the therapeutic intervention versus typical practice and as such it is possible that not all sites may use such a protocol given that there are other feeding protocols commonly in use and it is the intent of the invention that the therapeutic benefits accruing from the methods of the invention are not limited to settings in which volume-based feeding is employed.

[0321] Patients will be followed for AE’s and new pulmonary infections for 3 additional days for follow up (through Day 13). Patients who prematurely terminate the study will undergo early termination procedures. The duration of mechanical ventilation, ICU and hospital stay, and 30-day mortality will be recorded from the patient’s record.

[0322] Admissible patients for the study can include those meeting these criteria: age ш 50 y; moderate to severe ARDS (Berlin Definition); mechanically ventilated for at least 72 hours in ICU and less than 5 days; receiving enteral or parenteral nutrition; and expected to be ventilated for 10 or more days.

[0323] Twenty (20) evaluable patients (10 in the ulimorelin arm and 10 in the placebo arm) are required to provide 80% power with a two-sided alpha of 0.05 to demonstrate the superiority of ulimorelin versus placebo with respect to the primary endpoint (muscle protein fractional synthesis rate (MPFSR)). This estimate assumes a within group standard deviation (SD) of 0.25%/hr, with no change in MPFSR in the placebo group, and a 0.3%/hr increase with ulimorelin treatment due to increased enteral delivery of protein, enhanced growth hormone, and insulin-like growth factor-1 (IGF-1) secretion, and/or reduced systemic inflammation. An evaluable patient is defined as completing both 10 days of treatment with study drug and the vastus lateralis muscle biopsy on Day 10.

[0324] The primary endpoint for this study is skeletal muscle MPFSR on Day 10 in ulimorelin treated patients compared to placebo, with secondary endpoints of: changes in muscle mass by US of the quadriceps and diaphragm, analyzed separately, on Day 10 compared to baseline (Day 1); changes in muscle mass by US of the quadriceps and diaphragm, analyzed separately, on Day 5 compared to baseline (Day 1); change in muscle mass by D3-creatine dilution on Day 10 compared to baseline (Day 3); changes in muscle mass by mean 24-hour urinary creatinine excretion on Days 9 and 10 compared to baseline (mean of Days 1 and 2); changes in muscle mass by mean 24-hour urinary creatinine excretion on Days 5 and 6 compared to baseline (mean of Days 1 and 2); skeletal muscle protein degradation by mean 24-hour urinary 3-me-His excretion on Days 9 and 10 compared to baseline (mean of Days 1 and 2) and mean 24-hour urinary 3-me-His excretion on Days 5 and 6 compared to baseline (mean of Days 1 and 2); whole body N balance by mean 24-hour urinary N excretion on Days 9 and 10 compared to baseline (mean of Days 1- 2) and mean 24-hour urinary N excretion on Days 5 and 6 compared to baseline (mean of Days 1-2).

[0325] Other indicia of efficacy that may be evaluated as exploratory endpoints include improved dietary intake of protein and calories; reduced occurrence of pulmonary infections; reduced duration of mechanical ventilation, ICU, and hospital stay; reduced all cause 30-day mortality; and (diminished) use of prokinetic agents and treatments and small bowel feedings.

19.6 Example 6 - Prevention of Muscle Loss In A Patient On Enteral feeding (EF)

[0326] Patient 1, an adult male weighing 50 kg, whose pre-morbid weight was 60 kg, arrives in the ICU after having a neurosurgical procedure to remove a metastatic brain lesion and is placed on enteral feeding (EF) with a nasogastric tube by continuous drip. While no indication of EFI exists, the patient is expected to have a complicated post-operative course and 7– 10 day ICU stay. The physician orders that the patient be placed on IV ulimorelin dosed at a dose of 300 ug/kg TID by IV bolus injection. Treatment is continued until the patient is released from the ICU.

19.7 Example 7 - Treatment of Pre-Existing of Muscle Loss

[0327] Patient 2, an emaciated adult female residing in a nursing home and weighing 40 kg, arrives in the ICU after having experienced pneumonia, and is placed on Total Parenteral Nutrition (TPN). The physician orders that the patient be placed on IV ulimorelin at a dose of 750 ug/kg TID by IV infusion over 30 min. She gains 2 kg body weight over the next five days and treatment is continued until ICU discharge on day 6. 19.8 Example 8 - Treatment of Chronic Muscle Loss

[0328] Patient 3, an adult male weighing 50 kg, is receiving dialysis for chronic renal failure three times per week (MWF). The patient has difficulty ambulating due to proximal leg muscle weakness and reports poor appetite. His physician orders IV ulimorelin dosed at a dose of 350 ug/kg IV bolus injection into the patient’s AV fistula at the end of each dialysis treatment.

19.9 Example 9 - Treatment of Acute Muscle Loss

[0329] Patient 4, an 82 year old previously healthy woman is admitted during flu season to an ICU with viral pneumonia. Her condition worsens and on day 2 she is intubated, at which time an NGT is inserted. Two days later enteral feeding is initiated at a rate of 40 mL/hour. Though she tolerates the feeds the rate is never increased. Ten days later she is extubated and discharged from ICU to the hospital wards, but while there and continuing to recover from the infection/infiltration, is observed to have lost significant weight, lean body mass and muscle mass. When told she“needs to eat”, she reports that she has no appetite. Her physician prescribes ulimorelin to be administered at a dose of 600 ug/kg IV TID. After two days of dosing, the patient reports an approved appetite but complains about the injections, which are reduced to BID the next day, then to QD once per day until the day of the patient’s discharge, when treatment is stopped.

19.10 Example 10 - Prevention and Treatment of Muscle Loss in Patient with

Clinically Significant Edema

[0330] Patient 5, a 60 year old man presenting with cirrhosis, a distended abdomen and mental status changes including delirium and combativeness is admitted to the ICU where he is observed to have significant edema and fluid retention. A CT scan confirms portal hypertension and profound ascites. The patient is sedated and an NGT is inserted and enteral feeding begun. Feedings are tolerated and advanced without evidence of EFI but the patient becomes septic and has a protracted course over the next ten days during which time feeding are often held. During this time the patient’s ascites are tapped resulting in a decrease in total body weight of 5 kg. Diuretics result in a further weight decrease of 5 kg consistent with a reduction in peripheral edema. The patient survives and feedings are resumed successfully on day 14 at which time the physician notes that the patient’s body weight is 16 kg less than on admission, consistent with both the drainage of large amounts of fluid and with significant additional loss of muscle mass above and beyond the changes in lean body mass. To begin to address this loss of muscle mass the physician orders treatment with ulimorelin TID at a dose of 900 ug/kg, reasoning that the patient’s underlying serious chronic illness has resulted in significantly elevated AAGP levels. The patient’s heart rate is noted to decrease from 100 to 80 at the end of the first infusion which the physician deems desirable both to improve cardiac output and to decrease resting energy expenditure, and the dose is maintained at 900 ug/kg. Treatment continues for 15 days, 6 of which are while the patient is in the ICU, during which time his edema is successfully managed and his total body weight falls by a further 1 kg. During the remaining 9 days on the hospital floor the dose is lowered to 600 ug/kg upon the noting of a decrease in heart rate below 70 at the end of his first post-ICU dose. This decrease was felt by the physician to be excessive and was deemed to be due to higher free drug exposures consistent with a partially normalized AAGP resulting from the overall improvement in the patient’s health. The patient gains 5 kg while also receiving physical therapy, half of which gain is deemed to be from renewed edema (due to weaning from diuretics) but the rest deemed to be from a desirable increase in muscle mass.

19.11 Example 11 - Treatment of EFI to Treat Existing, and Prevent Further, Muscle Loss

[0331] Patient 6, an obese adult male weighing 140 kg known to have difficulty ambulating and generalized weakness arrives in the ICU after coronary bypass surgery complicated by difficulty weaning off a ventilator post-operatively. He is started on enteral feedings but unacceptably high GRV’s are noted when the feeding rate is advanced beyond 40 mL/hour. Over the next three days he receives only 30% of his prescribed daily protein feeding target. Due to other patient complications ICU staff do not address the patient’s EFI until day 4 at which time the physician orders that the patient be placed on IV ulimorelin at a dose of 750 ug/kg TID by IV infusion over 30 min. Feeding is successfully advanced to the target of 120 mL/hr and protein feeding goals are met. Treatment is continued until ICU discharge on day 9.

19.12 Example 12 - Measuring Muscle Mass Using An IV D3-Creatine Dilution Test

[0332] A 65 year old male ICU patient, with a BMI of 32 (approximately 200 lbs and 5 ft 6 in), is sedated and intubated and on enteral nutrition, but is not able to achieve feeding goals due to enteral feeding intolerance. The patient is admitted to the ICU due to myocardial infarction after cardiac surgery two days prior, necessitating resuscitation. The treating physician is concerned about the patient’s known greater risk of mortality and of prolonged ICU stay, as well as risk for poor long-term outcomes, if underfed, and orders supplemental parenteral nutrition. However, parenteral nutrition carries risk, so to inform and optimize its use the treating physician orders an initial IV D3-creatine dilution test to assess muscle mass and a follow-up assessment at seven-day intervals to follow changes in muscle mass in the patient. The physician uses this information both to support adjustments in protein target and to assess the patient’s prognosis for recovery. The initial IV D3-creatine dilution test result indicates the patient to have a muscle mass of 24.8 kg, or approximately 27% of usual body weight, which is low for his age. This muscle mass reading is determined from the following measured data: a creatine/creatinine ratio of 0.020, indicating only about 0.5 mg of the 30 mg dose of D3-creatine lost in urine (thus corrected dose of 29.5 mg) and a D3-creatinine enrichment ratio of 0.00027. After 7 days, the patient has lost more muscle mass, with a reading now at 23.0 kg (e.g., similar losses in urine indicated by a creatine to creatinine ratio of 0.024 (corrected dose of 29.3 mg) and a D3-creatinine enrichment of 0.00029), which indicates an approximately 7% decline in muscle mass. While this magnitude of loss might be less than expected for an ICU patient in this condition, considering the low muscle mass of the patient overall, the physician decides to increase the protein targets provided by parenteral nutrition by 50% and continue to monitor the patient’s muscle mass over time. The physician also prescribes ulimorelin, a therapeutic agent with anabolic properties.

19.13 Example 13 - Clinical Study Using An IV D3-Creatine Dilution Test

[0333] A clinical study on the effect of a pro-metabolic therapeutic intervention designed to mitigate loss of muscle mass is conducted in ICU patients who have been ventilated for 3 to 5 days, who are expected to remain intubated for an additional 10 days, and who meet study inclusion/exclusion criteria. Throughout the study, patients receive treatment for their underlying medical conditions according to the local standard of care. D3-creatine 30 mg is administered IV on Days 1 and 10 for the assessment of muscle mass. Baseline and post-treatment muscle mass is determined by a spot urine collection for D3- creatinine on Days 3 and 12. Spot urine collections are also obtained for isotope background rates on Days 1 and Day 10 prior to D3-creatine administration for the determination of background D3-creatine and D3-creatinine levels.

***

[0334] The present invention may be embodied in other specific forms without departing from its structures, methods, or other essential characteristics as broadly described herein and claimed hereinafter. The described embodiments and examples are to be considered in all respects only as illustrative, and not restrictive. The scope of the invention is, therefore, indicated by the appended claims, rather than by the foregoing description. All changes that come within the meaning and range of equivalency of the claims are to be embraced within their scope.