AU759824B2 - Treatment of ischemia reperfusion injury and treatment of cellular dysfunction including arrhythmia and heart failure subsequent to myocardial infarction - Google Patents

Description

a-'

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant: UNIVERSITY OF MANITOBA Invention Title: TREATMENT OF ISCHEMIA REPERFUSION INJURY AND TREATMENT OF CELLULAR DYSFUNCTION INCLUDING ARRHYTHMIA AND HEART FAILURE SUBSEQUENT TO MYOCARDIAL INFARCTION The following statement is a full description of this invention, including the best method of performing it known to me/us: oo

S

S

S

SoS S S.o.

5 0

S

OS..

S S

S

0

S

S 0 o S 05* TREATN[ENT OF ISCIIEMA REPERFUSION INJURY AND 'EREATMNT OF CELLULAR DYSFUNCTION INCLUJDING ARRHYTEIU1A AND) HEART FAILURE SUBSEQUENT TO MYOCARDIAL, INFARCTION FIELD2 QF THE This invention relates to treating ischernia reperfusion injuries in different organs and to treating cellular dysfunction including arrhythmia ana heart failure subsequent to myocardial infarction.

Isehernia is defined by an organ or a part of the body failing to receive a sufficient blood supply. An organ that is deprived of a blood supply is said to be hypoxic. An organ will become hypoxic erven when the blood supply temporarily ceases, such as during a surgical procedure or during temporary artery blockage. 'When blood flow resumes to an organ after temporary cessation, this is known as ischemic reperflusion of the organ. Ischemic reperfiision to an organ may lead to injury of the organ by producing structural and functional abnormalities in the tissue of the organ.

Conditions observed with ischemnia reperfusion injury include neutrophil infiltration, hemorrhage, edemia, and necrosis.

One example of an ischemia reperfusion injury is myocardial infarction.

Myocardial infarction arises from the interruption of blood supply to the nmyocardium (the muscular wall of the heart). This interruption of blood supply leads to the development of an infarct, blood-deprived region of the myocardiumn and, ultirnately, to the loss of function of the myocardiumn or heart failure.

The ischemnia reperfiision injury is believed to arise from the generation of excess oxidative free radicals by the blood as it reperfuses the ischernic organs and to 30 disturbance in the cellular stores of adenosine triphosphate For example, mnyocardial infarction is associated with a disturbance in AT? and the occurrence of intracellular calcium overload.

2 SUMMARY OF THE INVENTION The present invention relates to the discovery that pyridoxal-5'-phosphate (PLP and also called P-5P) can be used to alleviate ischemia reperfusion injuries in an organ and to treat arrhythmia and contractile dysfunction subsequent to myocardial infarction.

PLP, is, chemically, 3hydroxy-2-methyl-5-[(phosphonooxy)methyl]-4-pyridinecarboxaldehyde, of chemical formula: N CH 3

II

HO--P--O--CH

2

OH

OH CHO PLP is a derivative of vitamin B 6 (pyridoxine hydrochloride) and has potent B 6 activity. Mammals produce PLP by phosphorylating and oxidizing vitamin B 6 The phosphorylation of vitamin B 6 is accomplished by pyridoxal kinase. PLP 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 20 with concentrated phosphoric acid followed by oxidation.

The biological role of PLP includes acting as a coenzyme and as an antagonist. PLP is a coenzyme at the glycogen phosporylase level (glycogenolysis) and at the transamination level in the malate aspartate shuttle (glycolysis and glycogenolysis). Further, PLP is an antagonist of a purinergic receptor, thereby affecting ATP binding. To date, PLP has been therapeutically used as an i enzyme cofactor vitamin.

The present invention includes methods for 30. treating ischemia-related conditions. In one aspect, the STR/ invention includes a method for alleviating ischemia H:\suzanet\Keep\Speci\94210-98.1 SPECIdoc 13/02/03 3 reperfusion injury in mammals that includes administering to the mammal a therapeutic amount of a compound selected from the group consisting of pyridoxine, pyridoxal, and pyridoxamine.

In another aspect there is provided a method of treating arrhythmia subsequent to myocardial infarction in mammals comprising: administering to said mammal a therapeutic amount of a compound selected from the group consisting of: pyridoxal-5'-phosphate, pyridoxine, pyridoxal, and pyridoxamine.

In a further aspect there is provided a method of treating contractile dysfunction subsequent to myocardial infarction in mammals comprising: administering to said mammal a therapeutic amount of a compound selected from the group consisting of: pyridoxine, pyridoxal, and pyridoxamine.

In a still further aspect there is provided a method of treating heart failure subsequent to myocardial infarction in mammals comprising: administering to said mammal a therapeutic amount of a compound selected from the group consisting of: pyridoxine, pyridoxal, and pyridoxamine.

SDESCRIPTION OF THE INVENTION 25 The present invention provides methods for treatment of ischemia-related conditions, such as ischemia Sreperfusion injury and cellular dysfunction. The invention generally is directed to administering pharmaceutical compositions containing a therapeutic amount of at least one compound derived from vitamin Be.

In accordance with the present invention, it has been found that PLP can be used in the treatment of ischemia reperfusion injuries and cellular dysfunction.

Examples of cellular dysfunction include arrhythmia and heart dysfunction subsequent to myocardial infarction.

"Treatment" and "treating" as used herein include T7\ preventing, inhibiting, alleviating, and healing the H:\suzannet\Keep\Speci\94210-98.1 SPECIdoc 13/02/03 3a ischemia-related conditions or symptoms thereof affecting mammalian organs and tissues. For instance, a composition of the present invention can be administered prior to ischemia to prevent, inhibit, or protect against ischemia reperfusion injuries and cellular dysfunction of organs and tissues. Alternatively, a composition of the invention can be administered during or following ischemia (including during or following reperfusion) to alleviate or heal ischemia reperfusion injuries and cellular dysfunction of organs and tissues.

Other pharmaceutical compounds derived from vitamin B 6 suitable for treatment of ischemia-related conditions include pyridoxine, pyridoxal, and pyridoxamine. One skilled in the art would appreciate that these derivatives would have nearly identical effects as PLP after being adjusted for metabolic and molecular weight differences.

In one aspect, the invention is directed to a method of treating ischemia reperfusion injury and cellular dysfunction in mammals comprising administering to the mammal a therapeutic amount of a compound selected from the group consisting of pyridoxine, pyridoxal, and pyridoxamine. Cellular •dysfunction *i r H:\suzannet\Keep\Speci\94210-98.1 SPECI.doc 13/02/03 may include an arrhythmia of the heart or heart failure resulting from myocardial infarction. A "therapeutic amount" as used herein includes a prophylactic amount, for example, an amount effective for preventing or protecting against ischemia-related conditions, and amounts effective for alleviating or healing ischemia-related conditions.

Administering a therapeutic amount of a compound for treating ischemia reperfusion injury and cellular dysfunction preferably is in the range of about 1-50 mg/kg of a patient's body weight, more preferably in the range of about 5-25 mg/kg of a patient's body weight, per daily dose. The compound may be administered for periods of short and long duration- Although some individual situations may warrant to the contrary, short-term administration of doses larger than 25 mg/kg of a patient's body weight is preferred to long-term administration. For instance, as described in the Examples, the compound may be administered in an amount up to 50 mg/kg of a patient's body weight for a short term, for example, 21 days without noticeable side effects. In this same vein when long-term administration (such as months or years) is required, the suggested dose should be no more than 25 mg/kg of a patient's body weight A therapeutic amount of the compound for treating ischemia-related conditions 20 can be administered before, during, or following ischemia (including during or following reperfusion), as well as continually for some period spanning from pre- to post-ischemia. For example, the compound may be administered prior to heart procedures, including bypass surgery, thrombolysis, and angioplasty, and prior to any other procedures that require blood flow be interrupted and then resumed. Additionally, the compound may be taken on a regular basis to protect against cellular dysfunction arising from arrhythmia and heart failure.

As an illustration, administration to a human of a pharmaceutical composition *containing PLP will be described. When a human is presented for a heart procedure, for 30 example, bypass surgery, thrombolysis, or angioplasty, or for a procedure requiring interruption of blood flow, an aqueous solution comprising PLP in a therapeutic amount can be given intravenously, immediately prior to surgery and then throughout a patient's hospitalization. Alternatively, the pharmaceutical composition comprising PLP can be given immediately prior to surgery and then continuously for up to one week following surgery. After hospitalization, a human can be administered an enteral dose of PLP for a period determined suitable by a physician, usually, for example, not to exceed 8 to 12 months.

Similarly, a human may be administered an enteral dose of PLP beginning with the onset of symptoms of ischemia-related conditions through the surgical procedure.

Furthermore, a human at risk for arrhythmia or heart failure may be administered a regular enteral dose of PLP to protect against cellular dysfunction.

In a preferred aspect of the invention, a method of treating ischemia reperfusion injury and cellular dysfunction in mammals includes administering to the mammal a therapeutic amount of PLP for treating the ischemia reperfusion injury and cellular dysfunction. In another aspect, the compound administered may be pyridoxine, pyridoxal, or pyridoxamine.

In yet another aspect of the invention, a method of preventing or treating a particular cellular dysfunction known as arrhythmia of the heart in mammals includes administering to the mammal a therapeutic amount of a compound selected from the 20 group consisting of pyridoxal-5'-phosphate, pyridoxine, pyridoxal, or pyridoxamine for treating arrhythmia of the heart. In still another aspect of the invention, the cellular dysfunction that is treated is heart failure resulting from myocardial infarction.

A pharmaceutical composition of the present invention is directed to a composition suitable for the treatment of ischemia reperfusion injury and cellular dysfunction. Examples of cellular dysfunction include arrhythmia of the heart and heart failure arising from myocardial infarction. The pharmaceutical composition comprises a pharmaceutically acceptable carrier and a compound selected from the group consisting ofpyridoxal-5'-phosphate, pyridoxine, pyridoxal, and pyridoxamine. A S 30 pharmaceutically acceptable carrier includes, but is not limited to, physiological saline, ringers, phosphate buffered saline, and other carriers known in the art. Pharmaceutical compositions may also include stabilizers, anti-oxidants, colorants, and diluents.

Pharmaceutically acceptable carriers and additives are chosen such that side effects from the pharmaceutical compound are minimized and the performance of the compound is not canceled or inhibited to such an extent that treatment is ineffective. Preferably, the compound selected is PLP.

The pharmaceutical compositions may be administered enterally and parenterally. Parenteral administration includes subcutaneous, intramuscular, intradermal, intramammary, intravenous, and other administrative methods known in the art. Enteral administration includes solution, tablets, sustained release capsules, enteric coated capsules, and syrups. When administered, the pharmaceutical composition should be at or near body temperature.

Methods of preparing pharmaceutical compositions containing a pharmaceutically acceptable carrier and a therapeutic compound selected from PLP, pyridoxine, pyridoxal, and pyridoxamine are known to those of skill in the art. As an illustration, a method of preparing a pharmaceutical composition containing PLP will be described.

Generally, a PLP solution may be prepared by simply mixing PLP with a pharmaceutically acceptable solution, for example, buffered aqueous saline solution at an acidic or alkaline pH (because PLP is essentially insoluble in water, alcohol, and ether), at a temperature of at least room temperature and under sterile conditions.

Preferably, the PLP solution is prepared immediately prior to administration to the mammal. However, if the PLP solution is prepared at a time more than immediately prior to the administration to the mammal, the prepared solution should be stored under 25 sterile, refrigerated conditions. Furthermore, because PLP is light sensitive, the PLP solution should be stored in containers suitable for protecting the PLP solution from the light, such as amber-colored vials or bottles.

Although it is not intended that this invention should be limited to any particular 30 mechanism or theory of action, the following is offered as a tentative explanation for better understanding of the invention as a whole.

S

AO

A

S..

A

*5 9.

A S *5

A

A

S

According to the vitamin B 6 homocysteine theory, a relative deficiency of vitamin B 6 leads to an accumulation of homocysteine. Homnocysteine is an atherogenic, amino acid. Homocysteirie accumulation results in vascular endothelium damage, platelet fuinction derangements, and arteriosclerosis Vitamin Br, is known to help prevent the negative effects of homnocysteine accumulation.' Because PLP is the coezym reuird fo -aaol of homocysteine, it has been suggested that vitamin

B

6 's therapeutic ability is related to an increased formation of PLP. Thus, the beneficial effects of PLP in treating ischemia-related conditions may arise from the catabolism of homocysteine by PLP administered according to the present invention.

An alternative to the vitamin B, 6 homocysteine theory is the AT? theory- In response to ischemia, excess ATP is made available. Because ATP? affects cardiomyocytes and the contraction of vascular smooth muscle, extracelluar ATP exerts significant influence on cardiovascular function. For insiance,'ATP modulates ionic currents, calcium homeostasis, and excitation-contraction coupling in atrial and ventricular mnyocytes. We have shown that PLP depresses the AlT-induced increase in intracellular calcium. Thus, PU' may serve as an endogenous antagonist of ATP receptor (P7X) and buffer the action of exogenously released ATP oD cardiomyocytes and vascular myocytes.

It has been found, according to the present invention, that PU', pyridoxine, pyridoxal, and pyridoxamine appropriately administered can have previously unexpeeted highly beneficial effects on ischemnia reperfusion injuries in mammals and in treatment of heart dysfunction subsequent to coronary occlusion. For illustrative purposes, the beneficial effect of administering PLP is demonstrated in the specific examples detailed below. The Examples describe both in vitro and in vivo experiments.

0 400: 00 DESCRIPTION OF CLMNCAL EXPERMS Exam~ple I h3nYitro IscheMia Rooerfsion in bsolated Rat-Hearts and-M-eaguremeiit of Jef -tentricular Developd Pressure Male Sprague-Dawley rats (200-250 g) were sacrificed by decapitation, and their hearts were rapidly removed and perfused according to the Langeridorff procedure at a constant flow of 10 m/Umin using the Kreb's-Heinsleit buffer (K-H buffer) oxygenated with 95% 02 and 5% C0 2 pH 7.4. After equilibration, a Langendorf Perfusioni apparatus using K-H buffer was used to study the effect of PLP on ischemia repedfusion.

After an equilibration period of 15 min, total ischemia was induced by stopping the perfusion for 30 min while the hearts were kept at constant humidity and temperature of 371C. In iscliemic-Teperfused hearts, perfusion with normal K-H buffer was reinstated for 60 min after 30 muin of global ischernia. The hearts were electrically stimulated (Phipps and Bird stimulator) at 300 beats/mn via a square wave of 1.5 ms duration at twice the threshold voltage. The left ventricular developed pressure (LVDP), the rate of change in developed pressure (+dP/dt) and the rate of change in relaxation(-dP/dt) were measured by using a water filled latex balloon inserted into the :left ventricle. The volume of the balloon as adjusted at the left ventricular end-diastolic pressure (LVEDP) of 10 mm Hg at the beginning of each experiment 7 and the balloon 00:. *was connected to pressure transducer (model 1050BP-BJOPAC SYSTEMS INC.).

CC 0Data was recorded on-line through analogue-digital interface (MP 100, B3IOPAC SYSTEMS I.NC.) and stored and processed -with "Acknowledge 3.01 for Windows" (BIO0PAC SYSTEM INC.). In experiments where the effect of the phosphate (PEA') were studied, The hearts were perfused with PLP (15 jiM) K-H buffer for 10 min before inducing ischemaia. This delivery of PLP (15 fiM) M' the K-H C buffer was continued throughout the reperfusion period in these experiments.

0~0* 00 The left ventricular developed pressure (LVDP) reflects the contractile activity of the heart.

Once the heart was reperfused after ischemia, it tends to become arrhythmic.

There is a time lapse before the heart stabilizes into a nonmaJ mode of rhythm.

The results of these expecriments are showvn below in Table 1. The control group comprised 13 animals, the PLP--treated group c omprised 6 animals, All values in the Table are percentage of pre-ischemic values.

Global ischemia resulted in a decline of left ventricular developed pressure (LVDP). Reperiizsion of the ischemic heart was found to induce a slow recovery of changes in LVDP. These parameters showed about 40% recovery over a 60 min' reperfusion period- On the other band, about 80% recovery of depressions in LVDP was evident upon-reperfuion of the hearts with PU' 10 min before inducing ischemiL- Also the time to 50% recovery (time taken to reach half of the maximum contractile force recovery on rep erfusion) was reduced in treated hearts.

.ow ow...

&*oo of 0 I0 TABLE I Parameters 1Control [Treated Time to regular rhythm (min) 18.3*5-0 5.3*2.1 LVDP 30 mini recovery) 30±8.6 78.2*9.2 LVDP 60 mini recovery) 44.2±9.3 84.7:L6.3 Time to 50% recovery (Min) 39.3±8.1 16-0*4.6 Example 2 Iso LatiomsfMembrane Preparation and Determingtio of Adenvl Cvclase Activty At. the end of each perfusion/reperfus ion period, the heart was removed from the cannula and the crude membranes were prepared by the method used previously by Sethiet al., J. Cardiac Failue 1(5) (1995) and Sethi et al., Am. J. Physil. 272 (1997).

Briefly, the hearts were minced and then homogenized in 50 mM Txis-HCI, pH 7.5 mi/g tissue) with a PT-20 polytron (Brinkman Insftruments, Westbury, NY, USA), twice for 20s each at a setting of S. The resulting homogenate was centrifuged at 1000 x g for min and the pellet was discarded. The supernatant was centrifuged at 3048000 X g for 25 min. The resulting pellet was resuspended and centrifuged twice in the same buffer and at the same speed; the final pellet was resuspended in 50 mM Tris-ICL, pH 7.4 and used for various biochemical assays.

Poo* Adenylyl cyclase activity is increased during ischemia. reperfusion leading to arrhytbniias and damage to the myocardium due to increased cAMP levels and 20 increased calcium entry. Treatment with PLI' partially reverses this increased enzyme 9 *5 activity to control levels.

**Goo:The adenylyl cyclase activity was determined by measuring the formation of [a- '2PJ cAMP I AT? as described by Sethi et aL, s Unless otherwise indicated, the incubation assay medium contained 50 MM glycyiglycine (PH mM Mg ATP, (a- 3 2 P] ATP (1-1,5 x 106 cpm), 5 MM MgC1 2 (in excess of the ATP concentration), 100 mM NaCl, 0.5 mM cAMP, 0. 1 mM EGTA, 0.5 mM 3--isobutyl-lmethyixanthine, 10 U/mi, adenosine deaminase, and an AT? regenerating system comprising of 2 mM creatine phosphate and 0.1 rmg creation kinase/mi in a flnal volume of 200 gJ. Incubations were initiated by the addition of membrane (30-70 ug) to the reaction mixture, which had equilibrated for 3 min at 37 0 C. The incubation time was mini at 3 7'C and the reaction was terminated by the addition of 0. 6 ml 120 mM zinc acetate containing 0.5 mM unlabe lled cAMP. The [a- 3 2 P] cAMP forned during the reaction was determined upon coprecipirati on of other nucleotides with NaCO 3 by the addition of 0.5 rmd 144 mM Na 2

CO

3 and subsequent chromatography. The unlabelled cAMP served to monitor the recovery of [a- 32 p] cAMP by measuring absorbenoy at 259 nm. Under the assay conditions used, the adenylyl cyclase activity was linear with respect to protein concentration and time of incubation, In a control Group C, the membrane preparation prepared as described in Example 2 was from hearts which, after a 20 minute stabilizing period, were perfused with normal K-H buffer or normal K-H buffer plus PLP for 90 minutes. In a group denoted ER (ischemia reperfusion), the membrane preparation was from hearts in which, after a 20 minute stabilizing period, isehemnia was induced for 30 minutes followed by minutes reperfusian with normal K-H buffer. In a group denoted PR, the Gov* preparation was from hearts in which, after a 20 minute stabilizing period, the hearts :20 wer perfused with 15 gra PLP plus normal K-H buffer for 10 inutes, followed by 00. ischemia induced for 30 minutes followed by 60 minutes reperfusion with normal K-H @go:buffer. Note that PLP was present all through the reperfizsion period.

The results are shown in Table 2. They are from n=-6 experiments- TABLE 2 Effect of various stimulants on adenylyl cyclase activity in rat heart crude membrane preparations from control ischemia reperfusiori and treated group (PR).

Adenyl Cyclase Activity pmol CAMP/mg protein/lO min Group Basal NaF (5 mM) Forskolin OGPP(NHp (100pJM) (0g Control 296±32 2343d: I18 1423LI02 1123*98 IR 529±21* 3490Wd76* 219Z+111* 1865:F81'P PR 391d:18# 2960±132# 1804:L129# 1492*l01O# P< 0. 05, significantly differenf from Control and PR group.

P<.05, significantly different from Control and 11? group.

Example 3 In vivoQ Coronary Artery Li!to Myocardia] in~farction was produced in male Sprague- Dawley rats (200-250 g) by occlusion of the left coronary artery as described by Sethi et al., Rats were anesthetized with 1-5% isoflurane in 100% 02 (2L flow rate). The skin was incised *415 along the left sterna border and the 4th rib was cut proximnal to the stemrnm and a ago* retractor inserted. The pericardial sac was opened and the heart externalized. The left anterior descending coronary artery was ligated approximately 2 mm from its origin on the aort4 wsing a 6-0 silk suture, The heart was then repositioned in the chest and the incision closed via purse-string sutures. Sham operated rats underwent identical 20 treaumeat except that the artery was ntligated. Mortality due to surgery was less tUm Unless indicated in the text, the experimental animals showing infarct size >3011o of the left ventricle were used in this study. AU animnals were allowed to recover, a received food and water ad libitum, and were maintained for a period of 21 days for Electrocardiogram (ECO), hemodynamic and histological assessment.

*%too 13 Occlusion of the coronary artery in rats has been shown to produce myocardial cell damage which results in scar formation in the left ventricle and heart dysfunction.

While the complete healing of the scar occurs within 3 weeks of the coronary occlusion, mild, moderate and severe stages of congestive heart failure have been reported to occur at 4, 8 and 16 weeks after ligation. Accordingly, the contractile dysfunction seen at 3 weeks after the coronary occlusion in rats is due to acute ischemic changes.

The rats were housed in clear cages in a temperature and humidity controlled room, on a 12 hour light-dark cycle. Food and water were supplied ad libitum. Rats at random were divided into five groups: sham operated, coronary artery ligated without treatment, sham operated with PLP treatment, coronary artery ligated with PLP treatment (25 mg/kg body weight orally by gastric gauge) two days before surgery, and coronary artery ligated with PLP treatment (25 mg/kg body weight) one hour after surgery. These animals were used in all the studies below. For EKG studies, these animals were used as their controls before surgery, so that before surgery was done on these animals EKG traces were taken which were then used as controls for the same animals after surgery.

Example 4 Hemodynamic Changes The animals prepared as described in Example 3 were anesthetized with an injection, of cocktail of ketamine hydrochloride (60 mg/kg) and xylazine (10 mg/kg).

**The right carotid artery was exposed, and cannulated with a microtip pressure transducer (model PR-249, Millar Instruments, Houston, TX). The catheter was advanced carefully through the lumen of the carotid artery until the tip of the transducer entered the left ventricle. The catheter was secured with a silk ligature around the artery. The hemodynamic parameters such as left ventricular systolic pressure (LVLSP), left ventricular end diastolic pressure (LVEDP), rate of contraction (+dP/dt), and rate of relaxation (-dP/dt) were recorded on a computer system (AcqKnowledge 3.1 30 Harvard, Montreal, Canada).

Myocardial infarction for 3 weeks produced a progressive increase in left ventricular end diastolic pressure (LVEDP) without any changes in either heart rate of left Ventricular systolic pressure (LVSP). Furthermore, both rate of force of contraction (+dPfdt) and rate Of force of rela)ation (-dP/dt) were signifiantly depressed in the infarted animals. The elevation in LVEDP and depression in both +dP/dt and -dP/dt were partially prevented uipon treating the infarcted animals with PLP for 3 weeks.

The results are given below, in Tables 3 arid 4.

Data are expressed as mean±SE of 10 animals. All measurements were made using a Miller microcatheter; the catheter was inserted info the left ventricle via cannulat ion of the right carotid artery. L VS'P, left ventricular systolic pressure; L VEDP, left ventricular end- diastolic pressure dPdt, rate of contraction; -dP/dt rate of relaxation. Animals were randomly divided into four groups: Sham, Sham Drug treated, Drug treated starting at 2 days before ligation (PrD) for up to 21 days and coronary ligated group Treatment group was given PLP (25 mg/kg body wt) orally by gastric gauge once a day.

is TABLE 3 Hemodynamic parameters of rates with myocardial infarction with or without PLP treatment for 21 days starting at 2 days before coronary artery ligation (PrD).

Parameters Sham Sham Ml PrD Drug HR(beatslmin) 376d-18 398+-22 405*22 475-±16 LVSP (xum Hg) 126±7 122-+6 128±6 123:F6 LVEDP (mm Hg) 2.2 ±0.2 1.9±0.09 12.2±0O.9* 5.7±0.9# +dP/dt (mm HS/s) 5899*302 5772±312 2654±1 11* 4272-+223# -dP/dt (rpmnHg/s) 5469--284 5401*297 2348±99* 3998*179# 0.05) sign ificantly difrent from the sham controlI and the Pr!) gro up.

0. 05) s ign ifican tly diffe re nt fro m sh am co ntrolI gro up and MI gro up.

0 0 0:0 0 *000 .96 TABLE4 A later confirmation of hemnodynamic parameters of rates with myocardial infarction with or without PLP treatment for 21 days starting at 1 hour after and 2 days before coronazy arery ligation.

Parameters ham S+Dru PP2 ru HR 381±18 396±+22 402*+22 378±06 381±10 (beats/mini) LVSP 124±7 122*6 124±6 127±6 129*5L (mm Hg) LVEDP 2.2±0.2 1.9±0.09 12.2:i*0.9* 5.2±0O.8*# (MM Hg) +dPldt 5899d:302 5772-+312 2654±1 11* 4272+223"'# 4199±L2l9*# (mm Is) -dP/dt 5469d:284 5401±297 2348±99* 3998±179"# 3918±177*# (mm Hg/a) 0. 05) s ign ific antly differe nitfr om th e sham an d Aha m dr ug gro up.

0.065) s ign ifican ty diffe ren I fro m Mt gr oup.

There were three groups of rats, 20 each: (MI) untreated coronary litigated, (PPI) orally PLP oncp daily starting at I hour after ligation, (PP2) orally Pb? once daily starting at 2 days before ligation.

Example 5 Electroardiogim (EGG) Recordirws Six lead HI, m, aVr, aVf, aVI) ECG recordings were made from rats in all groups (sham operated, coronary artery ligated sham operated with drug treatment, 20 coronary artery ligated with drug treatment 2 days before ligation, coronary artery S. S 0 5 0050 0000 S 0O S

SS

0 0 *5 ese.

0O S. S 0 0

S

0 @00 000 S 0 0 0@ ligation with drug treatment within 1 hour of ligation) prior to coronary artery ligation and at 1, 3, 7, 14 and 21 days after occlusion. Surface ECG's were recorded under isoflurane anesthesia using a model EC-60 Cardiac and Respiratory monitor (Silogic International Limited, The ST segment abnormality was defined as depression or elevation of at least 1 mm from the base line that persisted for 1 min. The magnitude of the ST segment shift was measured 60 ms after the J point in all.of the six leads. The QT interval was measured by standard criteria and then corrected for heart rate using Bazett's formula (QT, QT/square root of RR interval). The longest QT interval of all lead was measured from onset of the Q-wave until termination of the T wave. Onset of the R wave was used if Q waves were not present. The R-R interval immediately preceding the QT interval measurement was used to correct for heart rate. Pathological Q-waves were defined as a negative deflection, at least 25 uV in amplitude, preceding the R-wave.

ST Segment Changes ST-segment depression reflecting subendocardial hypoperfusion is the most common ECG manifestation of ischemia, and ST segment deviation can be used as a noninvasive marker of the perfusion status of the heart. Electrodes positioned directly 20 over the injured zone typically record ST segment elevation whereas those in opposite areas of the torso detect "reciprocal" ST segment depression. In the present study, ST segment depression was recorded in lead I and ST segment elevation in leads II and HI three leads at 1 to 21 days after coronary artery ligation in untreated rats. Treatment with PLP attenuated the degree of ST segment elevation/depression following occlusioh, and accelerated recovery of the ST segment. The results are shown below in Tables 5 and 6. In Table 5, the values for ST segment deviation recorded prior to the occlusion (control) for all the three leads for MI and PrD group were 0.01, 0.02, 0.01 and 0.015, 0.02 and 0.01 respectively. In Table 6, the values for ST segment deviation recorded prior to the occlusion (control) for all the three leads for MI and PrD group 30 were 0.02, 0.02, 0.01 and 0.01, 0.009 and 0.015 respectively.

S.*

17

TAL

ST segment changes in rats with myocardial infarction (N41) with or witout PLP treatment for 21 days starting at 2 days before coronary artery ligation (PrD).

ST segment (mV) Group I7Day J14Day [21Day Leadl I Na 0. 1710,02* 0.17±0.02* 0. 15±+0.01* PrD 0.08±0.01 j0.05±0.01 10.03±0.01 Lead IIt Nff 0.154:0.01* 0,154:0-01* 0.14EO01* PrD 0.0710.01 0.041:0.01 j0.03+.0.01 Leadji M M 0.180.02* 0.17;L0.02* 0-1 4±0.0 1 PrD 0.06±0.01 0.0410.01 0.02±0.01, .90.

9 99*9 0 9@90@S

S

S. S 0 S

S.

S

0@ SO *000 0 0 0O 0 0@ 5 0 S0

S.

0 S 0S S P< 0. 05 compared to control and treated group.

TABLE 6 ST segm~nt changes in rats with myocardial infarction (MIf with or without PLP treatment for 7 days starting at I hour after coronary artery ligation (PrD).

0 0 0. S so ST segment (mV) OM3-P

P-

Lead I Nff 0.19*0.02* 0.19±0.02* 01±.l~ PrD 0.13A:0.01 0.09±0.01 0.07±0.01 Leadi 1I NU 0.20±001* 0.19±-0.01* 0.17±f-0.01* PrD 0.14:0.01 J0.10:0.01 0.07;L0.01 Leadff M MI 0.20±0.02't 0.18±0.01* 0.15-±0.01* PiD 0.13±0.01 J0.08±0.01 0.04±+0.01

SC.

C

S

C e.g.

S

CS 0 C C *5

CCC.

CCC.

OCOC

CO

S*

S

C@

CCC.

C.

OC P<O. 05 compared to control and treated group.

OQlntWryal arnd Mortality FoLqmoWi Myocardial nafar-fin The QT interval on the surface electrocardioga is an indirect measure of the ventricular action potential duration and its prolongation is often associated with the occurrence of malignanit ventricular arrhyffinLias in patients. A long QT interval on the 10 ECG is associated with a higher risk of sudden cardiac death following myocardial infarction. The data indicates that QT interval prolongation occurred by 1 day and then gradually declined from 3-21 days which coincided with the period of highest mortality following ligation in untreated rats. Treatment with PLP attenuated the QT prolongation and also accelerated the time course of recovery of the QT interval 15 following coronary occlusion.

The results are given below in Tables 7, 8 and 9.

Myocardial infarction was induced by coronary ligation. All the animals 20 remnainin~g after subsequent weeks were used for ECG estimations. Values are @6CC,.

S

@0

S

CCC

CCC S S

CC

nmean;ESE. Treated animals were given PLP (25 mg/kg) orally once or-twice a day.

Control values were taken before the induction of myocardial infarction.

TABLE 7 Time dependent changes of QT, interval (nisec) in myocardial infarction. with or without PLP treatment for up to 21 days starting at 2 days before coronary artery ligation (PrD).

SGroup Control 7 Day 14 Day 21 Day MIf 302-+17 {563±32* 522-+26* 10:L9 PrD 3137L21 456±:22*# 437±23*# J 410±21.*# P< 0. 05 compared to controL.

P< 0.05 compared with W group.

900 0 0 09 0S 09 99.9 There were two groups of rats, 20 each: (MI) untreated coronary litigated, (PrD) PLP orally once daily.

20 Later confirmation and expansion of time dependent changes of QT, interval (msec) in myocardial infarction with or without PLP treatment for up to 21 days starting at 2 days before cdronary artery ligation- Group Control I Day 3 Day 7 Day 21 Day MIf 302*17 601+±17* 571±18* 522±26* 5064±29* FF1 313123 530.+25*# 486±+15*# 4571 23 410+_21# PP2 316± 24- 541±33*# 495±31*# 452+19*# 401±17* SP< 0. 05 compared to controL.

P< 0.05f compared with Mlf group.

.9 0.

There w=r three groups of rats, 20 each: (Wl untreated coronary litigated, (PPI) orally PU' once daily, (PP2) orally PLP twice daily. values mne nSE.onrl values taken before induction of mnyocardial infarction.

IAL

Time dep~endent changes of QT,. interval (tnsec) in myocardial infarction with, or without PLP treatment for up to 21 days starting at 1 hour after coronary artery ligatio.

Group Control 1 Day 3 Day 7 Day 21 Day MI 3 22±l7 594±22* 562*18~ 540:f2O* 503+-2* PPI 3l0*2l 516*21 505±13# 430±lI'*# 404-k:8*# PP2 1311114 535*23*# 484:L21-# 421*126*# 397±19*# 0. 05 compared to control 0. 05 compared with MI group.

15 There were three groups of rats, 20 each; (MI) untreated coronary litigated, (PPI) orally PLP once daily, (PP2) orally PLP twice daily. a--20, values are mean*'SE.

Control values were taken before induction of mnyocardial infarction.

Accordingly the mortality rate was also significantly less in the PLP treated group.

Mortality Rates Most early deaths after myocardial infarction occur within the first few hours 000 25 and these are caused primarily by lethal ventricular arrhythmias. In the present study, mortality was highest in the first 48 hours after coronary ligation in both untreated and treated rats, however, mortality was significantly less in treated animals. This decreased mortality was accompanied by several improved ECG findings suggesting an

*I

antiarrhythmic action of PLP (decreased incidences of pathological Q-waves and

PVCS).

Rats intended for operating on, were randomly divided into four groups, each Sham, Sham Drug treated, Drug treated starting at 2 days before ligation (MI Drug) for up to 21 days and Coronary ligated Since the sham and sham drug group had no differences in regards to mortality and other hemodynamic changes, they were considered as one group. The results are shown below in Tables 10, 11, 12 and 13.

TABLEJO

Mortality in rats with myocardial infarction with or without PLP treatment for 21 days starting at 2 days before coronary artery ligation (PrD).

No. of animals 15 0* o*eo 5 *oo*

S°

20

S

ooooo* 0 0o Mortality MI PrD On the 1st day 30 On the 2nd day 10 On the 3td day 5 0 Within 2:1 days 45 Significantly (P<0.05) different from the MI group. Sham group had no mortality.

At the 21st day, 3 animals from the MI group appeared very sick and may not have survived another week.

TABLE 11 Later confirmation and expansion of mortality in rats with myocardial infarction with or without PLP treatment for 21 days starting at 2 days before coronary artery ligation, No. of animals Mortality MI PPI PP2 On the 1st day 30 20 On the 2nd day 10 5 On the 3rd day 5 0 Within 21 days 45 25* Significantly (P<0.05) different from the MI group.

(MI) untreated coronary litigated, (PPI) orally PLP once daily, (PP2) orally PLP twice daily.

In a second, similar test, rats intended for operating on, were randomly divided into four groups, 20 each: Sham, Sham Drug treated, drug treated starting at I hour after ligation (PrD) for up to 7 days and coronary ligated group Since the sham and shari drug group had no differences in regards to mortality and other hemodynarnic changes, they were considered as one group.

The results are shown below in Table 12.

15 TABLE 12 Mortality in rats with myocardial infarction with or without PLP treatment for 7 days starting at 1 hour after coronary artery ligation (PrD).

9 o 9 9 9 9. 9o 9 9 9* 9 @9 9 0* 9 *90 9 9 99 No. of animals Mortality MI PrD On the Is day 25 On the 2nd day 15 On the 3id day 5 0 Within 7'days 45 Significantly 0.05) different from the Mjgroup, Sham group had no mortalicy.

Later confirmation and expansion of mortality in rats with myocardial infarction with or without PLP treatment for 21 days starting at I hour after coronary artery ligation.

No. of animals Mortality Nff PPI PP2 On thelIst day 30 20 16 On the 2nd day 10 8 8 On the 3kdday 5 0 0 Within 2 days 45 28* 24* Significantly 0. 05) diffren1 from the WI g'roup.

.e.

0 0

SO

0S *Oe* 0 S 0S 0 *5 .0.9 0 0 S@ S There were three groups of rats: (Wl, 20 rats, untreated coronary litigated, (PPL), rats, orally PLP once daily, (PP2), 25 rats, orally PLP twice daily.

AntianytlMic Acaign of PLP Revealedl-h ECG's The EGs of the animals in the previously reported tests for mortality rate showed several finidings indicating an antiarrythmric action of PLP. One of these is a decreased incidence of pathological Q--waves.

20 These reisults are shown in Tables 14 and 15 below.

TALEI4 General Characteristics and pathologcal wave appearance of rats with mnyocardial 25 infarctiopn with or without PLP treaunent for up to 21 days, starting at 1 hour after coronary artery ligation.

00 0 *ee 0 5 @0 There were three groups of rats, 20 each: (MI) untreated coronary litigated, (PPI 1) orally PLP once daily, (PP2) orally PLP twice daily. n-20, values are mean±SE.

Sham group was given saline.

CCC...

e

S

OC*S

e g.

C C

C

C.

C

C

OC

0 10 General Characteristics and pathological wave appearance of rats with myocardial infarction with or without PLP treatment for up to 21 days, 2 days after coronary artery ligation.

Parameters Sham Sham MI PP PP2 7 Drug Body wt. 330A8 322-5 331±7 332:9 334L10 Qwave appearance 62 37* 39* within 21 days (Pathological) Infarct size 43 27* 32* ofLV) Signficantly 0.05) different from the MI group.

2g.' There were three groups of rats, 20 each: (Nil) untreated coronary litigated, (PPI) injected PLP once daily, (PP2) injected PLP twice daily. n-20, values are mean:FSE.

Sham group was given saline.

Another such finding is a decreased incidence of preventricular contraction (PVC) following coronary artery ligation- These results are shown in Tables 16 and 17 below.

Effect of treatment with PLP (starting at 2 days before ligation continued for 21 days) on the incidence of preventricular contraction (PVC) following coronary artery ligation.

PVC Incidence Group 7 Day 14Day I21Day IMf 23 23 18 PrD 2* 3* 3* *Signifinantly 0.05)differenf from the M group.

C

g S* S 0O *g e.g.

gee.

C C C. C C CC C C S C CC go..

C.

C. C

C

C CCC CC C C

C

CCC...

C.

C C g e.g

C

C C C

CC

TABLE 17 Effect of treatment with PL.? (starting at I hour after ligation continued for 7 days) on the incidmnce of preventricular contraction (PVC) following coronary artery ligation.

PVC Encidence Group I Day [3 DW -]7-Day MI '14 14 2 PrD I* 1* 3 Significantly (P<0.05) differentfrom the M'I gro up.

As those silled in the art would realize these preferred described details and compounds and methods can be subjected to substantial variation, modifieation, change, .9 alteration, and substitution without affecting or modifying the function of the described embodiments.

Although embodiments of the invention have been described above, it is not limited thereto, and it will be apparent to persons skilled in the art that numerous modifications and variations form part of the present invention insofar as they do not depart from the spirit, nature and scope of the claimed and described invention.

It must be noted that, as used in this specification and the appended claims, the singular forms and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a composition containing "a compound" includes a mixture of two or more compounds.

Throughout the description and claims of this specification, the word "comprise" variations of the word, such as "comprising" and "comprises", means "including, but not limited to", and is not intended to exclude other additives, components, integers, or steps.

0* *0 99 o

S

Claims (28)

1. A method of alleviating ischemia reperfusion injury in mammals comprising: administering to said mammal a therapeutic amount of a compound selected from the group consisting of pyridoxal-5'-phosphate, pyridoxine, pyridoxal, and pyridoxamine.

2. The method of claim 1, wherein said therapeutic amount is in a range of about 1-50 mg/kg of a patient's body weight.

3. The method of claim 1 or claim 2, wherein said therapeutic amount is in a range of about 5-25 mg/kg of a patient's body weight.

4. The method of any one of claims 1 to 3, wherein said compound is administered enterally or parenterally.

5. The method of any one of claims 1 to 4, wherein said compound is

6. A method of treating arrhythmia subsequent to •myocardial infarction in mammals comprising: administering i 25 to said mammal a therapeutic amount of a compound selected from the group consisting of: pyridoxine, pyridoxal, and pyridoxamine.

7. The method of claim 6, wherein said therapeutic amount is in a range of about 1-50 mg/kg of a patient's body weight.

8. The method of claim 6 or claim 7, wherein said therapeutic amount is in a range of about 5-25 mg/kg of a patient's body weight. TT ^S iriz TS H:\suzannet\Keep\Speci\94210-98.1 SPECI.doc 13/02/03 28

9. The method of any one of claims 6 to 8, wherein said compound is administered enterally or parenterally.

The method of any one of claims 6 to 9, wherein said compound is

11. A method of treating contractile dysfunction subsequent to myocardial infarction in mammals comprising: administering to said mammal a therapeutic amount of a compound selected from the group consisting of: pyridoxal- pyridoxine, pyridoxal, and pyridoxamine.

12. The method of claim 11, wherein said therapeutic amount is in a range of about 1-50 mg/kg of a patient's body weight.

13. The method of claim 11 or claim 12, wherein said therapeutic amount is in a range of about 5-25 mg/kg of a patient's body weight.

14. The method of any one of claims 11 to 13, wherein said compound is administered enterally or S. parenterally. 25

15. The method of any one of claims 11 to 14, wherein said compound is

16. A method of treating heart failure subsequent to myocardial infarction in mammals comprising: administering to said mammal a therapeutic amount of a compound selected from the group consisting of: pyridoxine, pyridoxal, and pyridoxamine.

17. The method of claim 16, wherein said therapeutic amount is in a range of about 1-50 mg/kg of a patient's body weight. H:\suzannet\Keep\Speci\94210-98.1 SPECI.doc 13/02/03 29

18. The method of claim 16 or claim 17, wherein said therapeutic amount is in a range of about 5-25 mg/kg of a patient's body weight.

19. The method of any one of claims 16 to 18, wherein said compound is administered enterally or parenterally.

The method of any one of claims 16 to 19, wherein said compound is

21. Use of a compound selected from the group consisting of pyridoxal-5'-phosphate, pyridoxine, pyridoxal and pyridoxamine for the manufacture of a medicament for the injury alleviation of ischemia reperfusion.

22. Use of a compound selected from the group consisting of pyridoxal-5'-phosphate, pyridoxine, pyridoxal and pyridoxamine for the manufacture of a medicament for the treatment of arrhythmia subsequent to myocardial infarction.

Use of a compound selected from the group .consisting of pyridoxal-5'-phosphate, pyridoxine, pyridoxal 25 and pyridoxamine for the manufacture of a medicament for the treatment of contractile dysfunction subsequent to myocardial infarction.

24. Use according to any one of claims 21 to 23, in which the compound is present in a range of about 1 to mg/kg of a patient's body weight.

Use according to any one of claims 21 to 24, in which the compound is present in a range of about 5 to mg/kg of a patient's body weight. o H:\suzannet\Keep\Speci\94210-98.1 SPECI.doc 13/02/03 30

26. Use according to any one of claims 21 to 25 in which the compound is administered enterally or parenterally.

27. Use according to any one of claims 21 to 26 in which the compound is

28. Methods of alleviating ischemia reperfusion injury or treating arrhythmia contractile dysfunction or heart failure subsequent to myocardial infarction, substantially as hereinbefore described with reference to any one of the examples. Dated this 13th day of February 2003 UNIVERSITY OF MANITOBA By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia go o 0. s H:\suzannet\Keep\Speci\94210-98.1 SPECI.doc 13/02/03