TITLE

se of Pyπdoxal-5'-phosphate for Improving the Immune Response and Treatment and Prevention of Post-operative Infections

FIELD OF INVENTION

The present invention relates to a method of improving immune response and/or resistance to an infection in a patient following surgery.

BACKGROUND

Post-operative infection is a well-known phenomenon which has been shown to complicate recovery and thereby lead to increased length of hospitalization (Lazar et al, 1995). While measures have been instituted to limit post-operative infections, this still remains a significant problem. Post-operative infections, such as those following coronary artery bypass graft (CABG) surgery, are not confined to the wound site but may affect other sites in the body as well, hi order to deal with the insurgence of microorganisms, the immune system produces increased levels of white blood cells. High white blood cell counts, which can be an indicator of infection, are also strongly correlated with cardiovascular events, stroke and mortality. A lower than normal white blood cell count may indicate a depressed immune system that is unable to respond to infection.

Pyridoxal 5 '-phosphate has been shown to be effective in preventing necrosis and damage after PCI (Kandzari et al, 2003) and coronary artery bypass graft surgery (Tardif et al, 2007). Additionally, pyridoxal 5 '-phosphate may also provide protection against inflammation which can occur as a result of infection, injury or tissue destruction. Previous studies have indicated that pyridoxal 5 '-phosphate levels are decreased in the plasma during a systemic inflammatory response, such as is seen in critically or chronically ill patients (Talwar et al, 2003), or those with rheumatoid arthritis (Roubenoff et al, 1995).

Pyridoxal 5 '-phosphate's primary mechanism of action is through purinergic P2- receptor antagonism (Wang et al, 1999, Trezise et al, 1994). Extracellular ATP is released from platelets and nerve endings during various stress conditions such as ischemia or reperfusion, and acts on purinergic P2-receptors on cardiomyocytes to stimulate an

increase in intracellular calcium. The resulting calcium overload leads to cell damage and death. By inhibiting the binding of ATP to the P2-receptors and thereby decreasing calcium flux into cardiomyocytes, pyridoxal 5 '-phosphate has been shown to prevent damage due to ischemia and ischemia-reperfusion injury in both preclinical (Kandzari et al, 2005) and clinical (Kandzari et al, 2003) studies, hi addition to reducing the amount of damage in terms of the number of large MIs, P2-receptor blockage with pyridoxal 5'- phosphate also reduces the sequelae of ischemia and ischemia-reperfusion injury, such as atrial fibrillation due to dysregulated cellular calcium handling.

A further benefit of pyridoxal 5 '-phosphate is reduced infection and inflammation. hi inflammatory cells, P2Z/P2X ₇ receptor activation stimulates the processing and release of pro-inflammatory cytokines, including IL- 1/3, a key mediator of host response to infection and a primary cause of inflammation (Ferrari et al, 1997). Through P2Z/P2X ₇ receptor blockade, pyridoxal 5 '-phosphate reduces the processing and release of IL- 1/3, thereby hindering initiation of the inflammatory cascade, hi vitro experiments have demonstrated that treatment with pyridoxal 5 '-phosphate significantly reduces ATP- induced IL-1/3 processing and release in THP-I cells by 80.0% (p < 0.01) (MC6021-RD- 04014 internal document). Furthermore, IL-IjS stimulates the synthesis of C-reactive protein (CRP), an acute phase reactant (Yeh, 2005). CRP has been demonstrated to play a direct role in atherosclerosis and is now recognized as a predictive entity for cardiovascular risk (Yeh, 2005). Therefore, a reduction in CRP levels would indicate a reduction in inflammation and a reduced risk for cardiovascular disease. It has been demonstrated that plasma pyridoxal 5 '-phosphate levels are inversely correlated with C- reactive protein (CRP, a marker for inflammation) in patients with a systemic inflammatory response (Chiang et al, 2003) as well as in patients with stroke (Kelly et al, 2004). CRP levels were measured in the Phase II MATCHED (pyridoxal 5 '-phosphate and ACE Therapeutic Combination for Hypertensive Diabetics) study. CRP levels were significantly reduced from baseline over 8 weeks by 34.5% (p = 0.0037) and over 16 weeks by 50% (p = 0.0059) in the MATCHED study. Low plasma pyridoxal 5 '-phosphate levels have also been associated with other markers for inflammation, such as tumor necrosis factor-α, in patients with rheumatoid arthritis (Roubenoff et al, 1995). Many epidemiological studies have linked low plasma pyridoxal 5 '-phosphate levels to cardiovascular disease, leading several authors to suggest that low plasma pyridoxal 5'-

phosphate levels may be an independent cardiovascular risk factor (Robinson et al, 1995; Serfontein et al, 1985; Vermaak et al, 1986; Chasan-Taber et al, 1996; Folsom et al, 1998; Robinson et al, 1998).

Pyridoxal 5 '-phosphate (3-hydroxy-2-methyl-5-[(phosphonooxy)methyl]-4- pyridine-carboxaldehyde) is a naturally occurring metabolite of pyridoxine (Vitamin B6) and is formed in mammalian cells by phosphorylation and oxidation reactions

Pyridoxal 5 '-phosphate can be chemically synthesized in a number of ways, for example, by the action of ATP on pyridoxal, by the action of phosphorus oxychloride on pyridoxal in aqueous solution, and by phosphorylation of pyridoxamine with concentrated phosphoric acid followed by oxidation.

Pyridoxal 5 '-phosphate and other, related compounds such as pyridoxine, pyridoxal and pyridoxamine, have been previously shown to be effective in the treatment of hypertrophy, hypertension, congestive heart failure, ischemic heart disease, ischemia reperfusion injuries in an organ and to treat arrhythmia and contractile dysfunction subsequent to myocardial infarction

SUMMARY OF INVENTION

The present invention provides a method of improving the immune response and/or resistance to an infection in a patient following a surgical procedure comprising administration of a therapeutically effective amount of pyridoxal 5 '-phosphate or a pharmaceutically acceptable salt thereof to said patient.

In an embodiment of the invention, the pyridoxal 5 '-phosphate or pharmaceutically acceptable salt thereof is administered to the patient prior to the surgical procedure.

In an embodiment of the invention, the therapeutically effective amount of the pyridoxal 5 '-phosphate or pharmaceutically acceptable salt thereof is administered between 1 and 24 hours prior to the surgical procedure.

In an embodiment of the invention, the therapeutically effective amount of the pyridoxal 5 '-phosphate or pharmaceutically acceptable salt thereof is administered between 3 and 10 hours prior to the surgical procedure.

In an embodiment of the invention, the pyridoxal 5 '-phosphate or pharmaceutically acceptable salt thereof is administered to the patient subsequent to the surgical procedure.

In an embodiment of the invention, the therapeutically effective amount of pyridoxal 5 '-phosphate or a pharmaceutically acceptable salt thereof is administered for between 1 and 30 days following the surgical procedure.

hi an embodiment of the invention, the pyridoxal 5 '-phosphate or pharmaceutically acceptable salt thereof is administered to the patient before and subsequent to the surgical procedure.

hi an embodiment of the invention, the therapeutically effective amount of pyridoxal 5 '-phosphate or pharmaceutically acceptable salt thereof is between 0.5 and 50 mg/kg of body weight per day.

hi an embodiment of the invention, the therapeutically effective amount of pyridoxal 5 '-phosphate or pharmaceutically acceptable salt thereof is between 1 and 15 mg/kg of body weight per day.

hi an embodiment of the invention, the therapeutically effective amount of pyridoxal 5'-phosphate or pharmaceutically acceptable salt thereof is about 250 mg per day.

In an embodiment of the invention, the therapeutically effective amount of pyridoxal 5 '-phosphate or pharmaceutically acceptable salt thereof is about 5 mg per day administered intravenously.

In an embodiment of the invention, the therapeutically effective amount of pyridoxal 5 '-phosphate or pharmaceutically acceptable salt is administered orally, parenterally or intravenously.

hi an embodiment of the invention, the method further comprises administering a therapeutically effective amount of an antibiotic.

In a further embodiment of the invention, the antibiotic is selected from a group consisting of: beta-lactams (penicillins and cephalosporins), vancomycins, bacitracins, macrolides (erythromycins), lincosamides (clindomycin), chloramphenicols, tetracyclines,

aminoglycosides (gentamicins), amphotericins, cefazolins, clindamycins, mupirocins, sulfonamides and trimethoprim, rifampicins, metronidazoles, quinolones, novobiocins, polymixins, gramicidins, pharmaceutical salts thereof, and combinations thereof.

In an embodiment of the invention, the patient has or is undergoing a cardiovascular surgical procedure.

In an embodiment of the invention, the patient has or is undergoing a coronary artery bypass graft or a percutaneous coronary intervention.

In another aspect, the present invention provides a use of a therapeutically effective amount of pyridoxal 5 '-phosphate or a pharmaceutically acceptable salt thereof for improvement of the immune response and/or resistance to an infection in a patient following a surgical procedure.

hi an embodiment of the invention, the use comprises administration of the therapeutically effective amount of pyridoxal 5 '-phosphate or pharmaceutically acceptable salt thereof to the patient prior to the surgical procedure.

hi an embodiment of the invention, the use comprises administration of a therapeutically effective amount of pyridoxal 5 '-phosphate or pharmaceutically acceptable salt between 1 and 24 hours prior to the surgical procedure.

In an embodiment of the invention, the use comprises administration of a therapeutically effective amount of pyridoxal 5 '-phosphate or pharmaceutically acceptable salt between 3 and 10 hours prior to the surgical procedure.

hi an embodiment of the invention, the use comprises administration of the therapeutically effective amount of the pyridoxal 5 '-phosphate or pharmaceutically acceptable salt thereof to the patient subsequent to the surgical procedure.

hi an embodiment of the invention, the use comprises administration of the therapeutically effective amount of pyridoxal 5 '-phosphate or a pharmaceutically acceptable salt thereof to the patient between 1 and 30 days following the surgical procedure.

In an embodiment of the invention, the use comprises administration of the therapeutically effective amount of the pyridoxal 5 '-phosphate or pharmaceutically acceptable salt thereof to the patient before and subsequent to the surgical procedure.

In an embodiment of the invention, the therapeutically effective amount of pyridoxal 5 '-phosphate or pharmaceutically acceptable salt thereof is between 0.5 and 50 mg/kg of body weight per day.

In an embodiment of the invention, the therapeutically effective amount of pyridoxal 5 '-phosphate or pharmaceutically acceptable salt thereof is between 1 and 15 mg/kg of body weight per day.

In an embodiment of the invention, the therapeutically effective amount of pyridoxal 5 '-phosphate or pharmaceutically acceptable salt thereof is about 250 mg per day.

In an embodiment of the invention, the therapeutically effective amount of pyridoxal 5 '-phosphate or pharmaceutically acceptable salt thereof is about 5 mg per day administered intravenously.

In an embodiment of the invention, the administration of the therapeutically effective amount of pyridoxal 5 '-phosphate or pharmaceutically acceptable salt is oral administration, parenteral administration or intravenous administration.

In an embodiment of the invention, the use further comprises administration of a therapeutically effective amount of an antibiotic.

In an embodiment of the invention, the antibiotic is selected from a group consisting of: beta-lactams (penicillins and cephalosporins), vancomycins, bacitracins, macrolides (erythromycins), lincosamides (clindomycin), chloramphenicols, tetracyclines, aminoglycosides (gentamicins), amphotericins, cefazolins, clindamycins, mupirocins, sulfonamides and trimethoprim, rifampicins, metronidazoles, quinolones, novobiocins, polymixins, gramicidins, pharmaceutical salts thereof, and combinations thereof.

In an embodiment of the invention, the patient has or is undergoing a cardiovascular surgical procedure.

In an embodiment of the invention, wherein the patient has or is undergoing a coronary artery bypass graft or a percutaneous coronary intervention.

DETAILED DESCRIPTION

Major surgeries affect the response of the immune system and increase the risk of post-operative infection in patients having undergone surgery. The present inventors have previously disclosed the usefulness of pyridoxal-5' phosphate for reducing the incidence and severity of cardiovascular complications following cardiovascular surgical procedures, for example, the incidence and severity of restenosis in individuals having undergone a coronary bypass graft for the treatment of atherosclerosis (see, for example,

PCT/CA2005/001471). The present inventors have now discovered that pyridoxal-5' phosphate is also useful for reducing the incidence and severity of infections following such surgical interventions. Pyridoxal-5' phosphate has now been shown to improve the immune response and/or resistance to an infection in patients following a surgical procedure.

The methods of improving the immune response and/or resistance to an infection in a patient following a surgical procedure according to the invention comprise the administration of a therapeutically effective amount of pyridoxal-5' phosphate or a pharmaceutically acceptable salt to a patient in need thereof. Animal and human studies show that pyridoxal-5' phosphate is easily tolerated and has low toxicity. With respect to post-operative patients suffering cardiovascular disease, such as atherosclerosis, pyridoxal-5' phosphate treatment in addition to reducing the incidence and severity of post-operative infections, is also effective for modulating a number of factors underlying the development and progression of cardiovascular disease including hypercholesterolemia, lipid oxidation, vascular cell proliferation, hyperhomocysteinemia, vascular cell apoptosis, inflammation, and thrombosis. Pyridoxal-5' phosphate is useful for the treating the underlying cardiovascular condition and preventing the incidence and severity of post-operative infections, and thus fewer classes of drugs are required in order to treat post-operative cardiovascular patients as compared to current combination therapies. In some circumstances it may be desirable to administer pyridoxal-5' phosphate

in conjunction with an antibiotic in order to more effectively treat the patient. Given orally, pyridoxal-5' phosphate does not inhibit hepatic cytochrome enzymes. Therefore combination therapies comprising pyridoxal-5' phosphate have lower incidences of adverse drug-drug interactions.

In contrast to current combination therapies, the present invention provides methods of treatment that are safe, effective for ameliorating multiple risk factors, conducive to patient compliance, and low cost. Pyridoxal-5' phosphate provides a simple alternative to currently available combination therapies which require the administration of multiple classes of drugs which are often quite costly, especially with respect to patients undergoing cardiovascular surgery who benefit not only from the immunostimulatory benefits of pyridoxal-5' phosphate treatment but also the cardiovascular benefits associated with pyridoxal-5' phosphate treatment. As only one drug, namely pyridoxal-5' phosphate, needs to be administered, the ease of carrying out the methods of treatment according the invention is much greater as compared to prior art combination therapies and hence the likelihood of patient compliance is greatly increased.

hi one aspect, the present invention provides a method of improving the immune response and/or resistance to an infection in a patient following a surgical procedure comprising administration of a therapeutically effective amount of pyridoxal 5 '-phosphate or a pharmaceutically acceptable salt thereof to said patient.

As used herein, "improving the immune response and/or resistance to an infection" includes the initiation or enhancement of one or more immunological mechanisms effective for preventing the onset of an infection or for reducing the severity of an existing infection.

As used herein, "infection" includes the pathological state resulting from the invasion of the body by an infectious agent, including but not limited to: bacterium, fungi, and viruses.

As used herein the term "surgical procedure" refers to any medical treatment of disease, injury, and deformity by operation or manipulation. Examples of surgical procedure include but are not limited to: cardiovascular procedures such as coronary artery bypass graft, percutaneous coronary interventions, carotid endarterectomy; gynecological

and obstetric procedures such as hysterectomy or cesarean section; bariatric procedures such as gastric bypass; dermatologic and cosmetic procedures such as skin grafts or liposuction, orthopedic procedures such as hip replacement or knee replacement; or procedures involving the removal in whole or part, of tissue and/or organs such as cholecystectomy, hemorrhoidectomy, partial colectomy, tonsillectomy, mastectomy, appendectomy gastrectomy, and prostatectomy.

In a preferred embodiment of the invention, the patient being treated will be or has undergone a cardiovascular surgerical procedure, and more preferably the patient is or has undergone a coronary artery bypass graft (CABG) or a percutaneous coronary intervention (PCI).

In an embodiment of the invention, the patient is administered a therapeutically effective amount of pyridoxal-5' phosphate or pharmaceutically acceptable salt thereof, between 1 and 24 hours prior to undergoing the surgical procedure and more preferably, 3 and 10 hours prior to the procedure. In a further embodiment of the invention, the method comprises the further step of administering a therapeutically effective amount of pyridoxal-5' phosphate or a pharmaceutically acceptable salt thereof, following the surgical procedure. The duration of post-operative treatment with pyridoxal-5' phosphate will depend on a particular patient's need. In some circumstances, long term treatment and even indefinite treatment may be desirable. In other circumstances, short term treatment may be desirable. In a preferred embodiment, pyridoxal-5' phosphate or a pharmaceutically acceptable salt thereof is administered between 1 and 30 days following the surgical procedure. In a further preferred embodiment, pyridoxal-5' phosphate or a pharmaceutically acceptable salt thereof is administered for at least 30 days following the surgical procedure.

In some circumstances, it may be desirable to co-administer an antibiotic before and/or following the surgical procedure. The term "antibiotic" as used herein refers to any compound known to one of ordinary skill in the art that will inhibit the growth of, or kill, a microbial agent, and in particular bacteria. The term "antibiotic" includes, but is not limited to, beta-lactams (penicillins and cephalosporins), vancomycins, bacitracins, macrolides (erythromycins), lincosamides (clindomycin), chloramphenicols, tetracyclines, aminoglycosides (gentamicins), amphotericins, cefazolins, clindamycins, mupirocins,

sulfonamides and trimethoprim, rifampicins, metronidazoles, quinolones, novobiocins, polymixins, gramicidins or any salts or variants thereof. The antibiotic used will depend on the type of bacterial infection selected by information know in the art.

The methods of treatment according to the invention comprise the administration of a therapeutically effective amount of pyridoxal 5 '-phosphate or a pharmaceutically acceptable salt thereof. By an "effective amount" or a "therapeutically effective amount" of a pharmacologically active agent is meant a nontoxic but sufficient amount of the drug or agent to provide the desired effect, hi a combination therapy of the present invention, an "effective amount" of one component of the combination is the amount of that compound that is effective to provide the desired effect when used in combination with the other components of the combination. The amount that is "effective" will vary from subject to subject, depending on the age and general condition of the individual, the particular active agent or agents, and the like. Thus, it is not always possible to specify an exact "effective amount." However, an appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

The precise dose for any of the pharmaceutical compositions of the present invention will depend on a number of factors which will be apparent to those skilled in the art and in light of the disclosure herein. In particular these factors include: the identity of the compounds to be administered, the formulation, the route of administration employed, the patient's gender, age, and weight, and the severity of the condition being treated. Methods for determining dosage and toxicity are well known in the art with studies generally beginning in animals and then in humans if no significant animal toxicity is observed. For example, in instances where the pharmaceutical compositions are used to treat an infection, the appropriateness of the dosage can be assessed by monitoring the severity of the infection using conventional methods known in the art. Where the dose provided does not cause levels of the infectious agent to decline to normal or tolerable levels, following at least three to fourteen days of treatment, the dose can be increased. The patient should be monitored for signs of adverse drug reactions and toxicity.

Generally, the therapeutically effective amount of pyridoxal-5' phosphate is between 0.1 and 100 mg/kg of body weight per day. In an embodiment of the invention, the preferred therapeutically effective amount of pyridoxal-5' phosphate is between 0.5

1 λ

and 50 mg/body weight per day. In a further embodiment of the invention, the preferred therapeutically effective amount of pyridoxal-5' phosphate is between 1 and 25 mg/kg body weight per day. In yet another embodiment of the invention, the therapeutically effective amount of pyridoxal-5' phosphate is preferably between 1 and 15 mg/kg body weight per day.

Pyridoxal-5' phosphate or its pharmaceutically acceptable salt may be administered to a patient in need thereof by any suitable route. Preferably, the methods of treatment of the according to the invention comprise the oral administration of pyridoxal- 5' phosphate or a pharmaceutically salt thereof. Preferred oral dosage forms contain a therapeutically effective unit dose suitable for a once-daily oral administration. Typically, the unit dosage for pyridoxal-5' phosphate will be between 100, 250, 300, 500, 750 and lOOO mg/day.

Patients who are unable to swallow an oral dose following surgery may receive an intravenous dose of pyridoxal-5' phosphate or a pharmaceutically acceptable salt thereof. Preferred intravenous dosage forms contain a therapeutically effective unit dose suitable for daily bolus dose administration. Typically, the intravenous unit dosage will be 5 mg/day.

Although the invention has been described with reference to illustrative embodiments, it is to be understood that the invention is not limited to these precise embodiments, and that various changes and modification are to be intended to be encompassed in the appended claims.

EXAMPLES

Example One - Pyridoxal 5'-phosphate Treatment Reduces the Incidence of Infection in Patients Following Coronary Artery Bypass Graft Surgery

A randomized, double-blind placebo-controlled, dose-ranging, parallel-arm multi- center study was undertaken, on high-risk patients undergoing coronary artery bypass graft (CABG) surgery with cardiopulmonary bypass.

Patients were identified as "high risk" if they had two or more of the following risk factors:

• Age greater than 65 years

• Current smoker

• History of diabetes mellitus requiring treatment other than diet

• Evidence of left ventricular dysfunction or congestive heart failure

• History of a previous non-disabling stroke, transient ischemic attack, or carotid endarterectomy

• Urgent CABG intervention defined as the need to stay in the hospital (although the patient may be operated on within a normal scheduling routine).

• History of myocardial infarction that occurred more than 48 hours but less than 6 weeks prior to CABG surgery

• Prior peripheral artery surgery or angioplasty

• Moderate renal dysfunction defined as creatinine > 133 micromol/L (1.5 mg/dl), but < 250 micromol/L (2.8 mg/dl)

• Presence of at least one asymptomatic carotid artery stenosis (>50%) either in one or two carotid arteries.

Approximately 900 high risk pre-CABG patients in 40 different treatment centers in North America were screened and randomized to three groups of approximately 300 patients each, prior to their bypass surgery, as follows. Patients were placed in a control group (placebo), treated with 250 mg/day of pyridoxal 5 '-phosphate, (250 mg/day), or treated with 750 mg/day of pyridoxal 5'-phosphate (750 mg/day).

The first dose of study medication was administered at 3-10 hours prior to CABG surgery. In the event of surgery delay or rescheduling, a second pre-operative dose of pyridoxal 5 '-phosphate was administered so that all patients received study medication 3- 10 hours before surgery. Treatment continued for 30 days after surgery (post operative

day (POD) 30). Patients received follow-up evaluations up to and including POD 4, on POD 30 and on POD 90.

Patients were measured for combined incidence of cardiovascular death, nonfatal myocardial infarction (MI), and nonfatal cerebral infarction up to and including POD 30. Patients were also measured for nonfatal myocardial infarction alone.

All deaths without an identifiable non-cardiovascular cause were considered of cardiovascular origin.

Cerebral infarction (stroke) was defined clinically as any new sudden onset focal neurological deficit lasting at least 24 hours as assessed by a neurologist and after neuroimaging (computed tomography [CT] Brain Scan or magnetic resonance imaging [MRI]) has excluded an intracerebral hemorrhage. All patients suspected clinically of having a stroke or transient ischemic attack (TIA) received a neurological examination (conducted by a neurologist or internist with expertise in cerebral vascular disease) within 24 hours of onset of symptoms. All patients suspected clinically of having a stroke were submitted to cerebral imaging.

When determining whether a patient had myocardial infarction, the following definitions were used:

A peak creatine kinase - myocardial band (CK-MB) above a certain threshold. Since different experts and different prior art clinical trials used different cut- offs for this threshold, three different thresholds were used to determine whether a patient had myocardial infarction. For Experiment IA, the determination of whether the patient had myocardial infarction was made using a cut off threshold of 100 ng/ml. Thus, in Experiment IA, patients with peak CK-MB of 100 ng/ml or greater on days up to and including POD 4 were considered to have myocardial infarction. For Experiment IB, a cut off threshold of 70 ng/ml was used. For Experiment 1C, a cut off threshold of 50 ng/ml was used;

A new q-wave evidence of myocardial infarction along with CK-MB of 35 ng/ml or above on days up to and including POD 4;

A peak CK-MB of 5x ULN (25 ng/ml) or above occurring after POD 4;

A new q-wave evidence of myocardial infarction that was not present at POD 4; or

A q-wave or non-q-wave myocardial infarction as identified by the investigator and confirmed by the Clinical Endpoint Committee.

Adverse Event Reporting

Information regarding adverse events (AEs) was collected from the time the patient signed the informed consent form through and including the POD 90 assessment. Each patient was observed and queried in a nonspecific fashion at each visit during the study for any new or continuing symptoms since the previous visit. The following AEs were excluded from collection unless they are of a severity or frequency greater than would be expected for this post-surgery patient population:

Expected incision pain, parethesia, and dysesthiasias requiring analgesics or narcotics; edema extremities (due to vein harvesting) or mild post pump systemic edema; alteration in appetite, including anorexia or mild nausea/vomiting; alteration in bowel habits, including constipation, flatulence and mild diarrhea; mild degrees of insomnia, confusion or anxiety requiring treatment with sedatives, anxiolytics or hypnotics; mild hypokalemia or hypocalcemia requiring modest replacement; mildly decreased hgb/hct due to interop blood loss/dilution (i.e. loss of >5g/l hgb or requiring >2 U PRBCs or loss of more than 100 cc/day blood for 2 consecutive days); mild post thoracotomy pleural effusion and/or atelectisis; low grade, self limited fever (<=101F); hypotension (SBP<90mmHg) during bypass or mild htn without clinical significance.

Serious adverse events (SAEs) that met the criteria of cardiovascular mortality, myocardial infarction or cerebral infarction (stroke) were not reported as SAEs in the trial since they will be collected as study endpoints and adjudicated the an independent Clinical Endpoint Committee.

All other serious and non-serious adverse events were recoded on the appropriate case report form (CRF) and included the onset, duration, severity, relationship to study drug, and the management of the event.

Serious Adverse Events

Serious adverse events were those that met any of the following ICH criteria:

Fatal or immediately life-threatening;

Results in persistent or significant disability/incapacity;

Requires or prolongs inpatient hospitalization;

Is a congenital anomaly/birth defect in the offspring of the patient;

Is a cancer

Is an overdose (intentional or accidental)

Is judged to be medically significant (including laboratory abnormalities)

A medically significant AE is a medical event that may not be immediately life- threatening or result in death or hospitalization but may jeopardize the patient or require intervention to prevent one of the outcomes listed above.

AEs and SAEs events were stratified by two distinct time intervals: 1) randomization to POD 4 and 2) POD 4 to end of trial. Within each of these 2 time intervals, AEs and SAEs related to infections and inflammations were listed and summarized by two classifications,!) system organ class (Infections and Infestations) and 2) infections and inflammations.

Results

During the first time interval, randomization to POD 4, there was no significant difference in the distribution of AEs and SAEs amongst the groups. There were a similar number of adverse events in the infections and inflammations category for each treatment group (Placebo = 16, pyridoxal 5 '-phosphate 250 = 20, and pyridoxal 5 '-phosphate 750 = 15 adverse events). AE 's in the all infections and infestations category were not significantly different in the 3 treatment groups (Placebo = 9, pyridoxal 5 '-phosphate 250 = 13, and pyridoxal 5'-phosphate 750 = 8). This may be due to the small number of AEs and SAEs seen during this relatively short time period.

Table 1 - Adverse Events Reported from Randomization to POD 4

Table 2 - Serious Adverse Events from Randomization to POD 4

During the second period, POD 4 to end of trial, there was a significant reduction in the number of AEs and SAEs (Table 3 and Table 4, respectively). Firstly, a statistical analysis of the total AEs classified as "infections and inflammation" revealed a significant reduction in the AEs in the 750 mg pyridoxal 5 '-phosphate group (p = 0.0435) and a reduced number of AEs in the 250 mg pyridoxal 5 '-phosphate group that approached significance (p = 0.0665). An analysis of the SAEs classified as "infections and inflammation" revealed a significant reduction for both the 250 mg (p = 0.0139) and 750 mg (p = 0.0001) pyridoxal 5'-phosphate Secondly, an examination of AEs classified as system organ class (infections and infestations) found a reduction in the number of AEs in both the 250 mg pyridoxal 5 '-phosphate (p = 0.1565) and 750 mg pyridoxal 5 '-phosphate (p = 0.1086) treatment groups. An analysis of SAEs classified by system organ class (infections and infestations) found that the 750 mg pyridoxal 5 '-phosphate group had a significant reduction in SAEs (p = 0.0006) and the 250 mg pyridoxal 5 '-phosphate group had a reduction in the number of SAEs (p = 0.0704).

A further examination of the results for the POD 4 to the end of trial revealed that the 750 mg pyridoxal 5 '-phosphate treatment group had a reduction in AEs sepsis, pneumonia, post-procedural site wound infection and wound infection while 250 pyridoxal 5 '-phosphate treatment group had a reduction in AE sepsis NOS compared to placebo (Tables 3 and 4).. An further examination of the SAEs revealed that both the 250 and 750 mg pyridoxal 5 '-phosphate treatment groups had a reduction of pneumonia NOS and

sepsis, and the 750 mg treatment group had a reduction in wound infection and post- procedural site wound infection.

Table 3 - Adverse Events from POD 4 to End of Trial

Table 4 - Serious Adverse Events from POD 4 to End of Trial

Conclusions

Cardiac patients treated with pyridoxal 5 '-phosphate following a coronary artery bypass graft showed a significant lowered incidence of post-operative infections as compared to patients treated with the placebo.

Example Two - Pyridoxal 5'-phosphate Treatment Decreases White Blood Cell Counts in Diabetic Patients

A 14- week, single-blind, forced, dose-escalation study of the efficacy and safety of pyridoxal 5'-phosphate administered once daily over a dose range of 250 mg to 750 mg, for the treatment of mild to moderate hypertension, in patients with coexisting diabetes mellitus was completed.

This design supported the following efficacy comparisons:

• Pyridoxal 5 '-phosphate 250 mg OD versus Placebo Lead-in, Placebo Lead- Out, Other doses,

• Pyridoxal 5'-phosphate 500 mg OD versus Placebo Lead-in, Placebo Lead- Out, Other doses,

• Pyridoxal 5 '-phosphate 750 mg OD versus Placebo Lead-in, Placebo Lead- Out, Other doses,

• Dose Response, 0 (placebo), pyridoxal 5 '-phosphate 250 mg, 500 mg, and 750 mg

• Pyridoxal 5 '-phosphate efficacy in diabetics

All patients completed a fixed four-week placebo lead-in, to qualify for initiation of active treatment. Eligible patients then began treatment with 250 mg/day of pyridoxal 5 '-phosphate. This dose was administered orally on a daily basis for the next two (2) weeks, subject to dose-limiting side-effects, after which patients will be force-titrated to 500 mg/day of pyridoxal 5'-phosphate. Again, after two (2) weeks of treatment at the 500 mg/day dose level and subject to dose-limiting side-effects all patients were force-titrated to 750 mg/day of pyridoxal 5'-phosphate and maintained at this dose level in the absence

of dose-limiting side-effects for the next two (2) weeks. Thereafter, and for the final four (4) weeks of the study all patients were treated with placebo medication. Total study duration for any one patient was 14 weeks with twelve (12) clinic visits.

Laboratory Assessment

Samples of blood and urine were collected at baseline and end of treatment and the following tests were performed

Complete Blood Count (CBC) with differential; platelet estimate plus PT and PTT at screening and end of treatment.

Blood chemistries (SMA20) including total bilirubin, creatinine, SGOT, SGPT, alkaline phosphatase, BUN, serum glucose, total protein, electrolytes (including sodium, potassium, and chloride), and blood lipids (including total cholesterol, & triglycerides), folic acid, and serum homocysteine levels.

Standard qualitative urinalysis.

A CBC test includes the measurement of the number of white blood cells (WBCs).

WBC counts were significantly decreased by 9.57 % (p= 0.014), using >6.0 thousand cells/mcL as a cut off (where WBC counts >6.0 confer greater cardiovascular risk)

Conclusions

The administration of pyridoxal 5 '-phosphate improves the body's immune response which is indicated by a reduction in WBC.

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