Field of the Invention

This invention relates generally to compositions and methods for treating or preventing short bowel syndrome (SBS), or conditions involving colonic resection, small bowel resection, and massive small bowel resection.

Background of the Invention

Short bowel syndrome (SBS) is defined as the spectrum of malabsorption which occurs following the surgical resection of a major portion of the small intestine. The syndrome is characterized by a malabsorption of nutrients. SBS affects people of all ages, from premature infants to adults. There are many conditions for which surgical removal of a significant length of small bowel is indicated, leading to SBS, including but not limited to necrotizing enterocolitis (NEC), abdominal wall defects, jejunal ileal atresia, mid-gut volvulus, and inflammatory bowel disease.

In infants, SBS can be caused by many events, for example after surgical intervention for necrotizing enterocolitis (NEC) or volvulus. SBS results in decreased nutrient absorption which may lead to decreased growth, failure to thrive, and serious developmental deficits and mortality (Buchman, A. L., et. al., 1994; Strong, S. A., et. al., 1999). Further, children may go on to develop end-stage liver disease secondary to cholestasis induced by long-term total parenteral nutrition.

In adults, SBS can be caused by many factors, for example, surgical intervention for Crohn's disease and other maladies, and also results in decreased nutrient absorption, and sometimes deficits in certain essential nutrients. This occurs when the distal ileum is removed. The distal ileum expresses the mechanisms for the absorption of certain essential nutrients (for example vitamin B 12 and other fat-soluble vitamins) and absorption of bile salts. In adults, SBS is less life threatening than in infants, but can result in serious life style changes, for example chronic diarrhea, chronic dehydration, cramping, dependency on total parenteral nutrition (TPN), infection, and reliance on exogenous supplements such as vitamin B12.

A number of investigators have demonstrated a role for EGF in intestinal adaptation following massive small bowel resection (Chaet, M. S., et. al., 1994; Dunn, J. C, et. al., 1997; O'Loughlin, E. V., et. al., 1994; Sham, J., et. al., 2002; Shin, C. K, et. al., 1998; Thompson, J. S., 1999). EGF treatment augments the mucosal hyperplasia in response to massive small bowel resection (Chaet, M. S., et. al., 1994; Goodlad, R. A., et. al., 1988; Shin, C. E., et. al. 1998) and increases glucose absorption (Hardin, J. A., et. al., 1997; O'Loughlin, E. V., et. al., 1994) and digestive enzyme expression (Dunn, J. C, et. al., 1997; O'Loughlin, E. V., et. al., 1994) in remnant intestine. The major source of endogenous production of EGF is from the salivary glands. Adaptation after small bowel resection is attenuated by sialoadenectomy, and this can be reversed with exogenous EGF (Helmrath, M. A., et. al., 1998). EGF receptor expression is increased in remnant bowel (Avissar,

N. E., et. al., 2000; Warner, B. W., et. al., 1997). Adaptation is impaired in animals with a defective EGF receptor (Helmrath, M. A., et. al., 1997) and intestinal overexpression of EGF enhances adaptation following small bowel resection (Erwin, C. R., et. al., 1999). Furthermore, EGF treatment has been shown to inhibit the rate of enterocyte apoptosis following massive small bowel resection, an effect that may contribute to the ability of EGF to maintain barrier integrity (Stern, L. E., et. al., 2000). Finally, in a recent pilot human clinical trial, 5 pediatric SBS patients with <50% normal bowel length were treated with oral recombinant human EGF. EGF treatment resulted in increased 3-O-methyl glucose absorption and feed tolerance and all patients gained weight. No adverse events were linked with treatment. (Sigalet, D., et. al., 2005). Plasma L-arginine and L-citrulline, the main source of endogenous arginine production, are decreased in pediatric patients that are unable to adapt following massive small bowel resection (Wasa, M., et al., 1999), and in adult and pediatric patients on total parenteral nutrition (TPN) with SBS compared to patients on TPN without SBS (Wasa, M., et. al., 1999). L-citrulline is primarily produced by the intestinal breakdown of glutamine, and a decrease in L-citrulline production (Chen, K., et. al., 1996; Dejong, C. H., et. al., 1998), intestinal glutamine uptake (Chen, K., et. al., 1996) and renal citrulline-to-arginine conversion has been reported in rats with massive small bowel resection (Dejong, C. H., et. al., 1998). Resected rats on an arginine deficient diet demonstrate weight loss, negative nitrogen balance and markedly reduced muscle arginine concentrations suggesting arginine becomes an essential amino acid following massive small bowel resection (Wakabayashi, Y., et. al., 1994).

Studies examining the role of arginine in intestinal adaptation following resection have produced conflicting results. Supplementation of elemental diets with L-arginine increased structural adaptation in rats with massive small bowel resection (Hebiguchi, T., et. al., 1997). Camli et al., (2005) reported an increase in weight gain and intestinal adaptation following intraperitoneal arginine administration in a model of SBS. They also noted an increase in serum, saliva and urinary EGF following arginine administration and concluded that the beneficial effect of arginine may be due to upregulation of EGF levels. Intraperitoneal L-arginine administration was reported to increase weight gain, cellular proliferation and structural adaptation in rats with a 90% small bowel resection (Ozturk, H., et. al., 2002). Oral feeding of ornithine alpha-ketoglutarate, a precursor of glutamine and arginine, to rats following massive small bowel resection has been reported to increase intestinal structural adaptation, ornithine decarboxylase activity and ornithine content and plasma and muscle glutamine levels (Dumas, F., et. al., 1998). Conversely, in a rat model of SBS, animals given L-arginine in their drinking water demonstrated decreased jejunal and ileal weight, and decreased structural adaptation of remnant bowel with increased rates of enterocyte apoptosis compared to untreated SBS rats (Sukhotnik et. al., 2003). Subcutaneous arginine administration in a

rat model of SBS reduced intestinal protein synthesis and the uptake of glutamine indicating a slowing of the adaptive process although intestinal permeability was improved in rats given arginine (Welters, C. F., et. al., 1999). Parenteral administration of arginine impaired intestinal adaptation compared with untreated resected animals in a model of SBS (Sukhotnik et al., 2005). Administration of glutamine supplemented TPN to rats with massive small bowel resection significantly increased gut glutamine uptake, mucosal glutaminase activity and mucosal DNA and protein content but resulted in decreased circulating L-arginine levels compared to controls (Chen, K. ₅ et. al., 1996). Finally, stable SBS patients with a mean time from intestinal resection of 46 months, on an arginine-free diet for five days showed a significant decrease in plasma arginine, ornithine and hydroxyproline levels and alterations in urinary output of orotic acid and nitrogen compounds but had no adverse clinical events (Pita et al., 2004).

Current treatment options for SBS, however, remain limited. Special diets are prescribed as well as medications to slow peristalsis and to reduce stomach acids. Cases of SBS, which don't resolve in time, can require lifelong treatment. In severe cases, a liquid diet administered intravenously or a small intestine transplant operation are considered.

Summary of the Invention

An aspect of the invention is to provide a composition comprising an EGF receptor agonist and L- arginine, a bioequivalent of L-arginine, or an NO-donor wherein the ratio of the agonist and L- arginine, bioequivalent thereof, or NO-donor is between 1:454000000 and 1:1 (mole:mole), or between 1:45400000 and about 1:4500 (mole:mole), or between 1:4540000 and 1:45000 (mole:mole) for the treatment or prevention of short bowel syndrome. The composition may be in solid, lyophilized or solution form. The solution may be suitable for oral delivery to a human, for example for enteral delivery to a human. The solution may be suitable for intravenous administration. The EGF receptor agonist may be EGF and it may be synthetic, optionally manufactured by chemical synthesis or recombinantly, or derived from natural sources. The composition may comprise L-arginine. The composition may be used in increasing weight gain in a person having short bowel syndrome or at risk for short bowel syndrome. The composition may be used in increasing intestinal absorptive surface area in a person having short bowel syndrome or at risk for short bowel syndrome. The ratio of the EGF receptor agonist and L-arginine, bioequivalent thereof, or NO-donor may be 1 : 100,000 (mole:mole).

Another aspect of the invention is to provide a unit dose comprising L-arginine, a bioequivalent thereof, or an NO-donor and an EGF receptor agonist suitable for the oral administration to a subject at risk for, or diagnosed with, short bowel syndrome upon dissolution with a pharmaceutically acceptable liquid. The liquid may be selected from the group consisting of water, saline, infant formula, buffered solution, expressed breast milk, other suitable carriers, and combinations thereof. The ratio of the agonist and L-arginine, bioequivalent thereof, or an NO-donor may be between

1:454000000 and 1:1 (molermole), or between 1:45400000 and about 1:4500 (mole:mole), or between 1 :4540000 and 1 :45000 (mole:mole). The EGF receptor agonist may be EGF and the unit dose may comprise L-arginine.

Another aspect of the invention is to provide a unit dose comprising L-arginine, a bioequivalent thereof, or an NO-donor and an EGF receptor agonist suitable for the intravenous administration to a subject at risk for or diagnosed with short bowel syndrome upon dissolution with a pharmaceutically acceptable solution. The ratio of the agonist and L-arginine, bioequivalent thereof, or an NO-donor may be between 1:454000000 and 1:1 (mole:mole), or between 1:45400000 and about 1:4500

(mole:mole), or between 1:4540000 and 1:45000 (mole:mole). The EGF receptor agonist may be EGF and the unit dose may comprise L-arginine.

Another aspect of the invention is to provide a unit dose comprising L-arginine, a bioequivalent thereof, or an NO-donor and an EGF receptor agonist suitable for the parenteral administration to a subject at risk for, or diagnosed with, short bowel syndrome upon dissolution with a pharmaceutically acceptable solution. The liquid may be selected from the group consisting of water, saline, infant formula, buffered solution, expressed breast milk, other suitable carriers, and combinations thereof. The ratio of the agonist and L-arginine, bioequivalent thereof, or an NO- donor may be between 1:454000000 and 1:1 (mole:mole), or between 1:45400000 and about 1:4500 (mole:mole), or between 1:4540000 and 1:45000 (mole:mole). The EGF receptor agonist may be EGF and trie unit dose may comprise L-arginine. The unit doses of the invention may be used in increasing weight gain in a person having short bowel syndrome or at risk for short bowel syndrome. The unit doses may also be used in increasing intestinal absorptive surface area in a person having short bowel syndrome or at risk for short bowel syndrome.

Another aspect of the present invention is to provide a method of treating a patient diagnosed with short bowel syndrome or at risk of short bowel syndrome, the method comprising administering an EGF receptor agonist and L-arginine, a bioequivalent thereof, or an NO-donor to a patient in need thereof. The patient may have undergone a resection of the small bowel or will undergo a resection of the small bowel.

Another aspect of the present invention is to provide a method of treating or preventing short bowel syndrome in a patient in need thereof, the method comprising enterally administering to the patient an EGF receptor agonist and L-arginine, a bioequivalent thereof, or an NO-donor, preferably, at least once daily. The EGF receptor agonist and L-arginine, a bioequivalent thereof, or an NO-donor may be administered together in a mixture, for example at least once daily. The EGF receptor agonist may be EGF. The EGF may be synthesized recombinantly or by a synthetic chemical process. The method may comprise L-arginine.

The methods of the present invention may be used in increasing weight gain in a person having short bowel syndrome or at risk for short bowel syndrome. The methods may also be used in increasing intestinal absorptive surface area in a person having short bowel syndrome or at risk for short bowel syndrome. Another aspect of the invention is to provide a kit comprising therapeutic amounts of an EGF receptor agonist and L-arginine, a bioequivalent thereof, or an NO-donor, and instructions for use in the treatment or prevention of short bowel syndrome. The EGF receptor agonist and L-arginine, a bioequivalent thereof, or NO-donor may be supplied combined in solid form. The kit may also include instructions for the step of dissolving the solid form in a solution suitable for oral administration, for example for intravenous administration. The agonist and L-arginine, a bioequivalent thereof, or an NO-donor may be supplied separately. They may be in the form of a solid or a solution. The instructions may include a step of mixing the agonist and L-arginine, a bioequivalent thereof, or an NO-donor before administration.

The kits may be used in increasing weight gain in a person having short bowel syndrome or at risk for short bowel syndrome. The kits may also be used in increasing intestinal absorptive surface area in a person having short bowel syndrome or at risk for short bowel syndrome.

Another aspect of the invention is to provide a method of treating or preventing SBS in a patient, the method comprising: (i) administering to the patient subcutaneously, or intravenously a composition comprising an EGF receptor agonist and L-arginine, a bioequivalent thereof, or an NO-donor over a first treatment period; and (ii) administering to the patient enterally a composition comprising an EGF receptor agonist and L-arginine, a bioequivalent thereof, or an NO-donor over a second treatment period, the second period being subsequent to the first period. The second period may be initiated following resolution of ileus. The enteric administration may include administration by enema or oral administration. The second period may include two phases, a first phase in which the administration is oral, and a second phase in which the administration is by an enema. For example, the administration during the first period may be by intravenous administration and the administration during the second period may be by oral route and done 4 to 6 days after resection.

Another aspect of the invention is to provide a method of treating SBS in a patient or treating a patient at risk of SBS comprising delivering an EGF receptor agonist to the intestinal tract of the patient and increasing the in vivo generation of NO within the intestinal tract of the patient. Increasing the in vivo generation of NO may include administering a substrate of nitric oxide synthase to the patient, for example administering an NO-donor. The patient may have had a surgical resection of a major portion of the small intestine.

The methods of the present invention may be used in increasing weight gain in a person having short bowel syndrome or at risk for short bowel syndrome. The methods may also be used in increasing

intestinal absorptive surface area in a person having short bowel syndrome or at risk for short bowel syndrome.

Another aspect of the present invention is to provide a use of an EGF receptor agonist and L- arginine, a bioequivalent of L-arginine, or an NO-donor for treating or preventing short bowel syndrome.

Another aspect of the present invention is to provide a use of an EGF receptor agonist and L- arginine, a bioequivalent of L-arginine, or an NO-donor for increasing weight gain in a person having short bowel syndrome or at risk for short bowel syndrome.

Another aspect of the present invention is to provide a use of an EGF receptor agonist and L- arginine, a bioequivalent of L-arginine, or an NO-donor for increasing intestinal absorptive surface area in a person having short bowel syndrome or at risk for short bowel syndrome.

Another aspect of the present invention is to provide a use of an EGF receptor agonist and L- arginine, a bioequivalent of L-arginine, or an NO-donor for preparing a medicament for treating or preventing short bowel syndrome. Another aspect of the present invention is to provide a use of an EGF receptor agonist and L- arginine, a bioequivalent of L-arginine, or an NO-donor for preparing a medicament for increasing weight gain in a person having short bowel syndrome or at risk for short bowel syndrome.

Another aspect of the present invention is to provide a use of an EGF receptor agonist and L- arginine, a bioequivalent of L-arginine, or an NO-donor for preparing a medicament for increasing intestinal absorptive surface area in a person having short bowel syndrome or at risk for short bowel syndrome.

These and other aspects of the present invention will become evident upon reference to the following detailed description and attached figures. In addition, various references are set forth below which describe in more detail certain procedures, devices or compositions. All references mentioned herein are incorporated herein by reference as though each document were reproduced herein in its entirety. Applicant reserves the right, at its discretion, to incorporate directly any or all of such a document during pendency of this application.

Brief Description of the Drawings

Figure 1 is a graph showing weight loss expressed as the percentage of the animal's original weight for the five test groups of rats .

Figure 2 is a bar graph showing intestinal width upon autopsy of SBS animals which were untreated (SBS), treated with arginine (arg), treated with EGF (EGF), treated with EGF and arginine (EGF- arginine) and animals not having SBS (control).

Figures 3 a, 3b and 3 c are bar graphs showing the gross intestinal morphology on autopsy describing intestinal length, intestinal wet weight and intestinal wet weight/length respectively.

Figures 4a and 4b are bar graphs describing villus height and crypt depth in jejunal tissue in control, SBS, arg, EGF and EGF-arg treated animals. Detailed Description of the Invention

Prior to setting forth the invention, it may be helpful to an understanding thereof to set forth definitions of certain terms that will be used hereinafter.

"Comprising" as used herein means contains, includes, or is composed of certain elements, but is not limited to only those elements. It is open ended. For example, the term, "a pharmaceutical composition comprising EGF and L-arginine" means any pharmaceutical composition containing EGF and L-arginine, wherein the composition may also contain other elements, for example, another active ingredient, flavourings, adjuvants and the like.

"Short bowel syndrome" or SBS or short gut syndrome as used herein means a gastrointestinal syndrome characterized by symptoms resulting from the malabsorption of nutrients such as abdominal pain, diarrhea, fluid retention, unintended weight loss, and extreme fatigue following the surgical resection of a significant length of small bowel. Accordingly, as used herein, the term "short bowel syndrome" also includes short gut syndrome and massive small bowel resection. Intestinal hormonal reflexes and feedback loops can be disrupted leading to an increase in the volume of proximal gastric and small bowel secretions and altered motility patterns. Water, sodium and magnesium losses can lead to electrolyte disturbances. Certain specific absorptive functions may also be impaired which are unique to certain parts of the intestine, such as the absorption of vitamin B 12, bile salts and other fat soluble vitamins by the ileum.

"EGF receptor agonist" as used herein means any molecule which will produce a biochemical effect when bound to any of the erbB(l-4) receptors, particularly the erbBl receptor, such that any or all of the following effects occur: intestinal glucose transport is increased, the apical surface of the enterocytes (cells lining the lumen of the small intestine) are altered, the colonization and translocation of pathogenic organisms across mucosal surfaces is inhibited, and gut maturation is induced. The molecule is preferably epidermal growth factor. It may otherwise be an antibody, small molecule, protein, peptide, peptidic analogues, or peptidomimetic. "Epidermal growth factor" or EGF as used herein is a 53-amino acid protein known to be synthesized in the duodenum and salivary glands of normal humans, and expressed in human breast milk. The amino acid sequence of human EGF is:

Asn Ser Asp Ser GIu Cys Pro Leu Ser His Asp GIy Tyr Cys Leu His Asp GIy VaI Cys Met Tyr lie GIu Ala Leu Asp Lys Tyr Ala Cys Asn Cys VaI VaI GIy Tyr lie GIy GIu Arg Cys GIn Tyr Arg Asp Leu Lys Trp Trp GIu Leu Arg (Boonstra et al. 1995).

The protein used in the experiments described herein had the foregoing sequence. The EGF used in the present invention is a recombinant human EGF (>97% purity) obtained from Protein Express (Higeta-Shoyu Co. Ltd., Japan). Non-human EGF sequences which act as EGF in humans are also contemplated. Species variants of EGF are thus also included, such as described for mouse, rat and pig (Jorgensen, P. E., et. al., 1998; Nexo, E., et. al., 1985; Pascall, J. C, et. al., 1991; and Simpson, R. J., et. al., 1985), or bovine EGF as cited in U.S. patent application 20030059802, or so-called supra-agonistic chimeras of different EGF receptor ligands (Lenferink, A. E., et. al., 2000). This definition also refers to a polypeptide having substantially the same sequence and activity as purified native epidermal growth factor. This includes recombinantly and chemically synthesized peptides or proteins. This term also refers to proteins varying from the native sequence by substitution with other amino acids or deletion of one or more amino acids, as long as the EGF biological activity is substantially preserved. The definition also includes fragments, peptidic analogues, peptidomimetics of EGF, and acylated forms as in US Pat. No. 5,070,188 as long as the EGF biological activity is substantially preserved. EGF biological activity can be screened by a receptor binding assay, and confirmed using any of the methods indicated above in connection with receptor agonists. Thus, for example, a human EGF protein in which the methionine (Met) at position 21 is replaced with isoleucine (He) falls within the scope of "EGF." Such a protein is denoted hEGF-I ₂ i generally, and is generally denoted rhEGF-I ₂ i if prepared recombinantly (chemically synthesized hEGF is included in the term "hEGF"). Similarly, hEGF having the Asp at position 11 replaced with GIu is generally denoted hEGF-Eπ. Some EGF proteins truncated near the carboxy terminal retain their biological activity, and are generally denoted with a subscript indicating the last peptide residue retained. Thus, EGF lacking the last 2 of its normal 53 peptides is generally indicated as EGF _5I . Proteins having an amino acid deletion, for example wherein Trp ₄₉ is absent, are generally denoted with the term "del" (or .DELTA.) and a subscript indicating the position, without altering the numbering of the remaining amino acids. Thus, if Trp ₄₉ were deleted, the resulting protein would be indicated EGF-.DELTA. ₄₉ . Insertions, increasing the chain length, are generally indicated as substitutions substituting 2 or more amino acids for one, e.g., rhEGF-L/Gis indicates insertion of GIy after the natural Leui ₅ . Finally, an EGF of the invention where HiSi ₆ has been replaced by another amino acid, with or without other modifications, is generally denoted generically by EGF- Xi ₆ . Muteins of EGF, as described for example in United States Patent No. 6,191,106 (Mullenbach et al), which issued February 20, 2001 also fall within this definition provided they have the requisite EGF activity.

"L-Arginine" as used herein means the semiessential amino acid (2-amino-5-guanidinovaleric acid) and its salts, e.g., acid addition salts suitable for administration to a mammal. A bioequivalent of L- arginine is a compound which, like L-arginine, is a substrate of nitric oxide synthase which generates NO in vivo, or may be converted to a substrate of nitric oxide synthase such as L-citrulline via the arginine-citrulline cycle or enzymes of the urea cycle. The rate-limiting enzyme in

endogenous L-arginine production is argininosuccinate synthase. The main site of endogenous L- arginine production is the kidney, which converts L-citrulline to L-arginine (Boger, R. H., et. al., 2001). Glutamine is converted to L-citrulline in the small intestine (Pita, A. M. _; et. al., 2003), and ornithine alpha-ketoglutarate is a precursor of glutamine (Dumas, F., et. al., 1998). A compound, typically a small organic molecule, capable of donating an NO molecule, an "NO donor" when administered in vivo may also be administered. Such compounds include, but are not limited to, S- nitroso-N-acetyl-penicillamine (SNAP), 3-morpholinosydnonimine (SIN-I), sodium nitroprusside (SNP) 4-phenyl-3-furoxancarbonitrile (PFC), glyceryl trinitrate (GTN), and isosorbide dinitrate (ISDN) (Feelisch, M., 1998; Pacher, P., et. ah, 2003; ZeIl, R., et. al., 2003). NO production can be measured in vitro by the Griess assay (Marion, R. _: M., et. al., 2003), or by chemiluminescent detection (Kikuchi, K., et. al., 1993; Kojima, H., et. al., 1998), or in vivo by the use of manometry and electronic nitric oxide sensors (Snygg, J., et. al.,2003; Levine, D. Z., et. al., 2001). A commercial assay for in vitro detection of NO is available from Cayman Chemicals (Ann Arbor, Michigan). Treatment or prevention of short bowel syndrome Materials and Methods

Animal model of disease

Sprague-Dawley rats (250-30Og) were housed initially for 7 days to allow acclimatization, and sham gavaged once per day for four days prior to surgery. Experimental design

Animals were randomized into 5 groups: 1) resected and gavaged daily with vehicle (0.9% sterile saline) (SBS), 2) resected and gavaged daily with 1.5 mmoles Arginine /kg/day (Arg), 3) resected and gavaged daily with EGF 100 ug/kg/day (EGF), 4) resected and gavaged daily with EGF 100 ug/kg/day and 1.5 mmoles Arginine /kg/day (EGF- Arg), and 5) controls gavaged daily with vehicle (Con). The EGF- Arg treatment is about 1:100,000 mole:mole. The L-arginine used in this study was the hydrochloride salt of the amino acid with a molecular weight of 210.7 rather than pure L- arginine with a molecular weight of 174.2. After an overnight fast, surgeries were performed under halothane anesthesia as previously described with 25 cm of remnant small intestine retained (Sham, J., et. al., 2002). Briefly, after laparotomy 25 cm of jejunum extending distally from the ligament of Treitz was measured, the bowel divided at that point and the entire distal bowel removed to the mid portion of the ascending colon just below the right colic artery and an anastomosis performed. Animals were gavaged daily between 9-10 am with study compound in 1 cc of vehicle for 10 days. The EGF used in the present invention is a recombinant human EGF obtained from Protein Express™ (Higeta-Shoyu Co. Ltd., Japan).

Measurements

After resection the animals were allowed water for the remainder of the postoperative day, and then dry food ad lib to a maximum of 20 g/day with pair feeding starting on the second day postoperative. Animals and the food in each animal's cage were weighed daily, and food intake quantified. Animals were given 20 grams of regular chow per day. Each set of controls (resected and non resected) plus the three treatment groups were followed as a set of five. If one animal in a group ate less than the 20 grams, then the following days the entire set of five got the weight that the animal ate plus 10%. The number of animals in each group was as follows: n=10 for argmine, EGF and EGF plus argmine groups, n=9 for SBS (untreated), n=8 for controls. Animals were fasted overnight pπor to sacrifice on day ten. There was a high mortality rate amongst the animals with SBS (final n = 3 for the untreated SBS animals, n = 6 for the Arg and EGF+Arg groups, n = 7 for the EGF treated group, n = 8 for controls). After their usual morning dose of study compound, all animals were injected mtrapentoneally with bromodeoxyuridme (BRDU) (50 mg/kg) along with oral gavage with 2 ml of Ensure™. One hour after BRDU injection the animals were anesthetized with halothane and the bowel was removed and rinsed. Intestinal length was determined using a standardized weight and the weight and diameter of the jejunal remnant measured. A 2 cm segment of tissue was taken 2 cm below the ligament of Treitz and fixed for histology and BRDU assessment.

Statistical analysis Results are reported as mean ± SEM. Statistical analysis was performed by analysis of variance (ANOVA) with Tukey post-tests. P<0.05 was considered significant.

Results

The surgical procedure resulted in severe SBS with significant weight loss and mortality in the resected groups. For this reason, animal weights were analyzed out to day five postsurgery pπor to significant mortality in the experimental groups. As shown in Figure 1, weight loss was expressed as a percentage of original weight and data points were plotted and the slope of each growth curve obtained and compared. Weight loss was significantly greater in all treatment groups and in the untreated SBS group compared to the control group (p<.001). Furthermore, weight loss was significantly less in animals receiving combination EGF-arginine compared to the untreated SBS group (ρ<0.05). Weight loss did not differ between treatment groups or between untreated SBS animals and animals treated with either EGF or arginme alone. Treatment with either EGF or argmine alone did not have a statistically significant effect, whereas treatment with the combination of both EGF and arginine resulted in statistically significant less weight loss. Accordingly, animals treated with the combination treatment of EGF and arginme gained more weight than animals treated with EGF or arginme alone.

Intestinal width (as a measurement of intestinal diameter) is shown in Figure 2. Intestinal width was significantly increased in animals treated with combined EGF-arginine compared to control animals (p<0.05). Intestinal width did not differ between any of the other groups. Accordingly, treatment with either EGF or arginine alone did not have a statistically significant effect on the intestinal width, whereas treatment with the combination of both EGF and Arginine resulted in statistically greater intestinal width. An increase in intestinal width, where the length of the intestine remains the same, results in an overall increase in intestinal absorptive surface area.

Figure 3 shows the effect of the various treatments on gross intestinal parameters in animals following sacrifice. Intestinal wet weight per length was significantly increased in all treatment groups and the untreated SBS group compared to controls due to a significant increase in intestinal weight in all groups (p<0.01). Intestinal length did not differ between any of the groups. While there was a trend for increased intestinal wet weight in the combined EGF-arginine group compared to the untreated SBS and the EGF and arginine groups, this did not reach statistical significance. Although statistically significant results were not realized, there was a general trend for the EGF and arginine treatment to outperform both the EGF treatment and the arginine treatment.

Figure 4 shows villus height and crypt depth in jejunal tissue obtained from Control, SBS, arginine, EGF and EGF-arginine treated animals. Villus height was significantly increased in EGF treated animals compared to controls (p<0.05). Villus height did differ between any of the other groups. Crypt depth was significantly increased in the EGF (p<0.05), EGF-arginine and untreated SBS groups (p<0.01) compared to controls. Crypt depth did not differ between arginine treated animals and controls or between any of the resected groups.

Conclusions

Combined EGF-arginine administration was significantly more efficacious in the treatment of SBS than administration of EGF or arginine alone. The combination EGF-arginine treatment resulted in an increased weight gain and increased intestinal absorptive surface area in resected animals compared to EGF or arginine given alone. Weight gain is a primary end-point for this condition and shows the clinical benefit of the treatment. Increased intestinal absorptive surface area is a secondary end-point and is required to achieve the primary end-point. This study demonstrates significant improvement in both primary and secondary end-points for this condition.

While EGF and arginine alone were shown to provide some protection against weight loss, only the combined treatment was statistically significant compared to untreated SBS control animals. In addition, only the combined treatment improved intestinal absorptive surface area. Accordingly, the present invention establishes the feasibility of treating short bowel syndrome with the combination EGF and arginine. It clearly shows that the combination works in practice by increasing weight gain and increasing the intestinal absorptive surface area of patients suffering from SBS. Prior to this

invention, studies using arginine alone to treat SBS were contradictory in that some studies showed benefits while other studies indicated it was detrimental. In addition, other studies have indicated that glucagon-like peptide π, bombesin, insulin-like growth factor 1 Octreotide™, natural conjugated bile acids, low fat diets and glutamine may have beneficial and/or detrimental effects. However, the present invention clearly establishes that the combination of EGF and arginine is more efficacious in the treatment of SBS than administration of EGF or arginine alone.

Pharmaceutical compositions for the treatment or prevention of short bowel syndrome

Treatment or prevention of short bowel syndrome involves an EGF receptor agonist, and L-arginine or a bioequivalent thereof. SBS develops as subjects suffer malabsorption after surgery. Therefore, stimulating absorption following surgery should be preventative.

The relative amounts of EGF receptor agonist and L-arginine are conveniently determined on a molar basis. A bioequivalent of L-arginine, if capable of producing, on a molar basis, a greater amount of NO than L-arginine as a substrate of nitric oxide synthase, would be determined on a mole-equivalent basis of the amount of NO the bioequivalent is capable of producing. Similarly, the amount of NO released by an NO-donor would be determined on a mole-equivalent basis. Accordingly, a method of treating or preventing SBS is provided by delivering an EGF-receptor agonist to the intestinal tract of the patient and increasing the in vivo generation of NO within the intestinal tract of the patient. This may include administration of a substrate of nitric oxide synthase or administration of an NO-donor.

A treatment of the present invention would require administration by an appropriate route. In order to administer the agents to be biologically available to the free luminal side of epithelial cells of the intestine, an oral route of administration would be most preferred. As such, a unit dose of the two agents would be provided in a suitable container for ready opening and delivery to and mixture with a pharmaceutically acceptable solution such as a dietary product. A suitable amount of the components of the present invention could thus be provided in a powdered or granular form to be added directly to milk or other food. Subjects requiring total parenteral nutrition (TPN) may benefit from receiving these components, either as a separate formula or as components of the TPN composition. Alternatively, the components may be provided in a solution form suitable to be immediately or upon dilution with a suitable solution consumed by the patient. The preceding solutions may be administered to the patient via an orogastric tube.

Examples of suitable dietary products include water, saline, buffered solutions, oral rehydration solutions, infant formula, and expressed breast milk, other suitable carriers, or combinations thereof. Any solution suitable for oral administration may be used. Additives may be added which act as bystander proteins (i.e. nonactive protein "filler"), which protects the EGF from enzymatic

degradation by pancreatic proteases (Playford et al., 1993). For example, casein (a milk protein) has been used for this experimentally (Playford et al., 1993). Other approaches may involve administering with a protease inhibitor to preserve EGF structure and activity.

Alternatively, a treatment of the invention may be administered orally, enterally, parenterally, intravenously, subcutaneously, nasally, or by enema. It may be possible to choose an intravenous route for an initial period of treatment as, for example, when initially treating SBS, during which period some patients are incapable of receiving an orally administered treatment. The composition may be prepared as a spray, solution, suspension, colloid, concentrate, powder, granules, tablets, pressed tablets, capsules (included coated and uncoated tablets or capsules), suppository and the like. Delayed release or controlled release formulations are also included.

The formulations may include additives such as viscosity adjusting agents, osmosity adjusting agents, buffers, pH adjusting agents, flavorings, stabilizers, colorings, preservatives and the like where required

A unit dose would be a dosage suitable for administration in a single administration, i.e , a single feeding of the patient A unit dose of L-argmme administered for instance as a hydrochloride salt of L-Argmme thus includes from about 20 mg/kg/day (.09 mmol/kg/day) to about 2000 mg/kg/day (9 mmol/kg/day), or more preferably from about 100 mg/kg/day (.45 mmol/kg/day) to about 1000 mg/kg/day (4.5 mmol/kg/day), or more preferably from about 200 mg/kg/day (0.9 mmol/kg/day) to about 500 mg/kg/day (24 mmol/kg/day) of L-argmme, or more preferably from about 250 mg/kg/day (1.2 mmol/kg/day) to about 400 mg/kg/day (1.9 mmol/kg/day), or more preferably from about 300 mg/kg/day (1.4 mmol/kg/day) to about 350 mg/kg/day (1 6 mmol/kg/day) and from about 0.2 ug/kg/day (0.032 nmol/kg/day) to about 2 mg/kg/day (0.32 umol/kg/day), or more preferably from about 1 ug/kg/day (0.16 nmol/kg/day) to about 1 mg/kg/day (0.16 mmol/kg/day), or more preferably from about 2 ug/kg/day (0.32 nmol/kg/day) to about 0.2 mg/kg/day (32 nmol/kg/day) of EGF receptor agonist. In this specification "ug" means microgram, "umol" means micromole, etc. Typically, the L-argimne-EGF receptor agonist ratio would be between about 1:454000000 mol EGF receptor agonist/mol L-argmme and about 1:1 mol EGF receptor agonist/mol L-argimne, more likely between 1-45400000 mol EGF receptor agomst/mol L-argmme and about 1:4500 mol EGF receptor agonist/mol L-argmme, more likely between 1 '4540000 mol EGF receptor agonist/mol L- argmme and 1 45000 mol EGF receptor agonist/mol L-argmme It is contemplated that treatment would be administered probably at least once a day, 3 or 4 times per day, or even continuously. Intermittent doses could be administered by any convenient route, e g , bolus infusion, oral preparations discussed elsewhere herein, etc , subcutaneously, or by continuous i v drip. More continual administration would more typically be by i v drip or controlled release implants

A treatment of this invention could also be used to prophylactically treat a person at risk for developing SBS. A person at such πsk would include a patient who has just undergone surgical resection of a major portion of the small intestine. Such treatment may begin as soon as feasible after the surgery, possibly after the completion of surgery Following resection, the bowel typically starts to work after 3 to 4 days It may thus be possible to begin oral treatment starting 4 to 6 days after surgery. If immediate treatment post-surgery is indicated then the treatment may be administered intravenously, possibly followed by oral treatment once bowel function resumes. Accordingly the method of treatment would include a first treatment period followed by a second treatment period.

Generally speaking, EGF is prepared by a synthetic process, being manufactured by conventional biotechnological or chemical techniques Of course, EGF might be obtained from a natural source.

Preferably, the combination of factors of the invention would be provided as a single mixture and administered together, but they could be provided in a kit in separate compartments and mixed for administration, or administered separately Both may be biologically available to the luminal side of epithelial cells of the intestine

When prepared for mixture with a liquid, as for delivery with water, the unit dose could have a solubility enhancer incorporated thereinto.

It may well be that the effectiveness of a dosage would be increased by use of a coated composition, one that would not dissolve until it reached the intestine. Reference may be made to "Remington's Pharmaceutical Sciences", edited by Gennaro (Mack Publishing Company, 19th Ed., 1995). Pharmaceutically acceptable salts of the active agents (e g., acid addition salts of L-argmine) may be prepared using standard procedures known to those skilled in the art of synthetic organic chemistry and described (March, J 1992) As well, it may be desirable to include a suitable pharmaceutically acceptable carrier such as those used conventionally with peptide-based drugs, such as diluents, excipients and the like

The product would of course be provided in sealed sterile packaging Typically the EGF or equivalent polypeptide is provided as a lyophihzed matenal

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