AU9421098A - Treatment of ischemia reperfusion injury and treatment of cellular dysfunction including arrhythmia and heart failure subsequent to myocardial infarction - Google Patents

Description

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: o 0 00 0 000 S00. 0 *0 0* 0 *p 00 119 TREATME~NT OF ISCHIIA REPERFUSION INJURY AND TREATMENT OF CELLULAR DYSFUNCTION INCLUDING ARRHYUJMJA AND HEART FAILURE~ SUB3SEQUENTr TO MYOCARDL INFARCTION FIEDQE E NETO This invention relates to treating ischemjia reperfusion injuries in different organs and to treating cellular dysfunction including arrhythmia anid heart failure subsequent to myocardial infarction.

Iseheujia is defined by an organ or a part of the body failing to receive a sufficient blood supply. An organ thar is deprived of a blood supply is said to be hypoxic. An organ will become hypoxic even 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 ischemnic reperfusion of the organ. Ischexnic reperfusion 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 ischemia, reperfusion injury include neutrophil infiltration, 20 hemnorrhage, edema, and necrosis.

One example of an ischemia reperfusion, injury is myocardial infarction.

Myocardial infarction arises from the interruption of blood supply to the mnyocardium 4000 (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, ultimately, to the loss of function of the myocardiumn or heart failure.

p. The ischernia reperfiusion injury is believed to arise from the generation of excess oxidative free radicals by the blood as it reperfuses the ischeniic organs and to 0: 30 disturbance in the cellular stores of adenosine triphosphate (ATP). For example, 0 myocardial infarction is associated with a disturbance in AT? and the occurrence of ixntracellular calcium overload.

SUMAYOFT

MUMVNTO

The present invention relates to the discovery that pyridoxal-S'-phosphat

(PLP

and also called P-5P) can be used to treat ischemia reperhusion injuries in an organ and to treat arrhythmia and contractile dysfunction subsequent to miyocardia[ infarction.

PLP, is, chemnically, 3-hydroxy-2-methyl-Sf(p~hosphoniooxy) methylJ- 4 -pyridine7-carboxaldehyde, of chemical formula: N C4 HO-P-0-4 OH orI CIR2 PLP is a derivative of vitamin Br, (pyridoxine hydrochloride) and has potent Bis activity. Mammals produce PL? by phosphorylating and oxidizing vitamin B 6 The phosphorylation of vitamin B36 is accomplished by pynidoxcal kinase. PLP can be chemically synthesized in a anber of ways, for example, by the action of AT? on pyridoxal, by the action of phosphorus oxychioride on pyridoxal in aqueous solution, 1S and by phosphorylation of pyridoxarnine with concentrated phosphoric acid followed by so&* 06*0 0oxidation- The biological role of PL? includes acting as a coenzyme and as an antagonist- PLP is a coenizyme at the glycogen phosphorylase level (glycogenolysis) and at the 20 transamnination 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 enzyme cofactor vitamin.

0**The present invention includes methods and compositions for treating ischemiarelated conditions. In one aspect, the invention includes a method for treating ischemia 5 reperfusion injury and cellular dysfunction in mammals that includes administering to the mammrai a therapeutic amount of a compound selected from the group consisting of V" pyridoxal-5'-phosphate, pyridoxine, pyridoxal, and pyridoxamine.

.0 3 In another aspect, the invention is directed to a pharmaceutical composition that includes a phannacentically acceptable carrier and a therapeutic amount of a compound selected from the group consisting of pyridoxai-5'-phosphate, pyridoxine, pyridoxal, and pyridoxarmine for treating ischemia reperfusion injury and cellular dysfunction.

DE-SCRIPDOQcF JIME INV TON The present invention provides compositions and methods for treatment of ischemnia-related conditions, such as ischemia reperfusion 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 B 6 In accordance with the present invention, it has been found that PLP can be used in the treatment of ischemia reperbusion injuries and cellular dysfunction. Examples of cellular dysfunction include arrhythmia and heart dysfunction subsequent to myocardial infarction. "iTreatment"f and rtreatingil as used herein include preventing, inhibiting, alleviating, and healing the ischentia-related conditions or symptoms thereof affecting marrunalian organs and tissues. For instance, a composition of the present invention can be administered prior to ischernijato prevent, inhibit, or protect against ischemnia 20 reperfusion injuries and cellular dysfunction of organs and tissues. Alternatively, a 0*0 composition of the invention can be administered during or following isclaernia (including during or following reperfirsion) to aLleviate or heal ischexnia reperfusion injuries and cellular dysfunction of organs and tissues.

Other pharmaceutical compounds derived from vitamin B 6 suitable for treatment 0 0of ischemia-related conditions include pyridoxine, pyridoxal, and pyridoxamiae. One skilled in the art would appreciate that these derivatives would have nearly identical **Soo:effects as PLP after being adjusted for metabolic and molecular weight differences.

0& 30 In one aspect, the invention is directed to a method of treating ischemnia 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, pyridoxai, and pyridoxamine- Cellular dysfunction, 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 patients 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 ofa 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 i' *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 giv' immediately Prior to surgery and then continuously for up to one week following surgery. After hospitalization, a human can be administered an anteral dose of PLP for a period determined suitable by a physician, usually, for example, not to exceed 8 to 12 months.

SU'Mlarly, a human may be administered an enteral dose of PLP beginning with the onset of symptoms of ischernia-related conditions through the surgical procedure.

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

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

In yet another aspect of the invention, a method of preventing or treating a particular cellular dysfunction known as arrhythimia of the heart in mammals includes administering to the mammal a therapeutic amount of a compound selected from the 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 dysfunclion that is treated is heart failure resulting from miyocardial infarction.

A pharmaceutical composition of the present invention is directed to a composition suitable for the'treatment of ischernia reperfusion injury and cellular C C. 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 of pyridoxal-S t -phosphate, pyridoxine, pyridoxal, and pyridoxamine.

A

E~g. 30 pharmiaceutically acceptable carrier includes, but is not limited to, physiological saline, rigrphosphate buffered saline, and other carriers known in the art. Pharmaceutia 9 woo 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 mnimized and the performance of the compounid is not canceled or inhibited to such an extent that treatrmt is ineffective. Preferably, the compound selected is PLP.

The pharmaceutical compositions may be administered enteraily and par-enterally. Parenteral administration includes subcutaneous, intramuscular, intradennal. intramammary, intravenous, anid 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 ruixing PLP with a in** *.*pharmaceutically acceptable solution, for example, buffered aqueous saline solution at -owes: 20 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 Pb? 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 sterile, refrigerated conditions. Furthermore, because PLP is light sensitive, the PLP C solution should be stored in containers suitable for protecting the PLP solution from the ligt, uchasamber-colored vils or btls S 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.

-7 According to the vitamin J3 6 homocysteine theory, a relative deficiency of vitamin B 6 leads to an accumulation Of ho0MOCYSteine. Homocysteine is an atherogenic amino ac-id. Homocysteirje accumulation results in vascular endothelium damage, platelet function derangements, and arteriosclerosis. vjtari B 6 is known to help prevent the negative effects of homocysteine accumulation. Because PLP is the coenzyme required for catabolism 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 hoiuocysteine by PLP administered according to the present invention.

t0 An alternative to the vitamin B 6 homocysteine theory is the ATP theory. In response to ischemnia, excess ATP is made available. Because AT affects cardiomyocytes and the contraction of vascular smooth muscle, extracelluar ATP exerts significant influence on cardiovascular fuinction. For instance, ATP modulates ionic currents, calcium homeostasis, and excitation-contraction coupling in atrial and ventricular rnyocytes. We have shown that P12 depresses the ATP-induced increase in intracellular calcium. Thus, PLP may serve as an endogenous antagonist of AT? receptor (P2X) and buffer the action of exogenously released ATP ox) cardiomyocytes and vascular myocytes.

0 :It has been found, according to the present invention, that PLP, pyridoxine, pyridoxal, and pyridoxarnine appropriately admnistered can have prcviously unexpected, highly beneficial effects on ischemia 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.

see**# DESCRTODN OF CLMNCAL EXPE~fiRENTs Example I In. Vitro Isehemia geigrfbion in isolated R~at Hearts =a W0mmn ofi-eft Ventricular Develone~d Pressure Male Sprague--Dawley rats (200-250 g) were sacrificed by decapitation, and their hearts were rapidly removed and perfused according to the Langendorif procedure at a constant flow of 10 mli/min using the Kreb's-Heinsleit buffer (K-H buffer) oxygenated with 95% 02 and 5% C0) 2 PH 7A4. After equilibration, a Langendorf Perfusion apparatus using K-H buffer was used to study the effect of PLP on ischenaa reperfusion.

After an equilibration period of 15 min, total ischemia was induced by stopping the perfusion for 30 min while the hea"t were kept at constant humidity and temperature of 371C. In ischemic-Teperfused hearts, perfusion with normal K-H buffer was reinstated for 60 mini after 30 m~in of global ischemia. The hearts were electrically stimulated (Phipps and Bird stimulator) at 300 beats/min via a square wave of 1.5 ms duration at twice the threshold voltage. Trhe left ventricular developed pressure *9e(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 mmn Hg at the beginning of each experiment, and the balloon .9:.was connected to pressure transducer (model 105ORP-BJOPAC SYSTEMS INC.) 9 Data was recorded on-line through analogue-digital interface (MP 100, BIOPAC SYSTEMS INC.) and stored and processed with "Acknowledge 3.01 for Windows" .9(130PAC SYSTEM INC.). In experiments where the effect of the $09 phosphate (PL.P) were studied, the hearts were perfused with PU' (15 jiM) K-H buffer for 10 mini before inducing ischemnia. This delivery of PLP (15 tiM) in the K-H buffer was continued throughout the reperfusion period in these experiments.

The left ventricular developed pressure (LVDP) reflects the contractile activity of the heart, Once the heart was reperfused aiier ischemia, it tends to become arrhythmic.

There is a time lapse before the heart stabilizes into a normal mode of rhythmr.

The results of these experiments are shown below in Table 1. The control group comprised 13 animals, the PLP--treated group comprised 6 animals. All values in the Table are percentage of pre-ischernic values, Global ischemia resulted in a decline of left ventricular developed pressure (LYDP). Reperfision of the isehemic 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 hand, about 80% recovery of depressions in LVDP was evident upon reperfuion of the hearts with PEP 10 mini before inducing ischeinia. Also the time to 50% recovery (time taken to teach half of the maximum contractile force recovery or) reperfuision) was reduced in treated hearts.

9 So 0 *0*9e to) TABLE 1 Parameters [Control jTreated Tim~e to re gular rhythm (rnii) 18.3*5.0 5.3*2.1 LVDP 30 min recovery) 30±8.6 78.2*9.2 LVDP 60 min recovery) 44.2±--9.3 84.7:L6.3 Time to 50% recovery (Mlin) 39.3j=8.1 16.0:b4.6 Example 2 IsoLatim wof Membrane Preparation and Determini no dvv Cyclase Activit At the end of each perfusion/reperfusion period, the heart was removed from the cannula and the crude membranes were prepared by the method used previously by Sethiet al., 1. Cardiac 1Faiur. 1(5) (1995) and Sethi et al., Am. J. PhMso. 272 (1997).

Briefly, the hearts were minced and then homogenized in 50 mM Tris-HCI, pH 7.5 mu/g tissue) with a PT-20 polytron (JBrinkmrt Instruments, Westbury, NY, USA), twice for 20s each at a setting of S. The resulting homogenate was centrifuged at 1000 x g for &eve. 10 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-HCL, pH a. 4 7.4 and used for various biochemical assays.

Adenylyl cyclase activity is increased during ischemia reperfusion leading to arrhytbmias and damage to the myocardium duc to increased cAMP levels and 20 increased calcium entry. Treatment with EL? partially reverses this increased enzyme activity to control levels.

The adenylyl cyclase activity was determined by measuring the formation of [zx- 5:50: 3Pj cAMP [oL-"P ATP as described by Sethi et al., U Tnless otherwise indicated, the incubation assay medium contained 50 mnM glycyiglycine (pH mM Mg ATP, (a-V2P] ATP (1 -1,5 x 106 cpm), 5 mM MgC 12 (in excess of the ATP s~ concentration), 100 mM NaC1, 0.5 mM cAMP, 0.1 mM EGTA, 0.5 mM 3--isobutyl-1-

(I

methyixanthine, 10 U/mi adenosine deaminase, and an AT? regenerating system comprsing of 2 mM creatine phosphate and 0. 1 mg creatine kinase/in i a final volume of 200 p4. IncubatiOns were initiated by the addition of membrane (30-70 ug) to the reaction mixture, which had equilibrated for 3 miun at 3 7 0 C. The incubation time was mini at 37C and the reaction was terninated by the addition of 0.6 ml 120 mM zinc acetate containing 0.5 mM unlabelled cAMP. The [a-5 2 P] cAMP formed during the reaction was determnined upon coprecipitation of other nucleotides with NaCO 3 by the addition of 0.5 ml 144 mM Na 2

CO

3 and subsequent chromatography- The unlabelled cAMP served to monitor the recovery of 3 2 P] cAMP by measuring absorbency at 259 nm. tinder 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 (ischeinia reperfusion), the membrane preparation was from hearts in which, after a 20 minute stabilizing period ischemia was induced for 30 minutes followed by *eve 60 minutes reperfusion with normal K-H buffer. In a group denoted PIZ, the preparation was from hearts in which, after a 20 minute stabilizing period, the hearts 0*S 20 were perfused with 15 gim PLP plus normal K-H buffer for 10 minutes, followed by .:ischemnia induced for 30 minutes followed by 60 minutes reperfusion with normal K-H eve:buffer. Note that PLP was present all through the reperflision period.

0 0 The results are shown in Table 2. They are from n7-6 experiments- I -N TABLE 2 Effect of various stimulants on adenylyl cyclase activity in rat heat crude membrane preparations from control ischemnia reperfusion and treated group (PR).

Adenyl Cyclase Activity pmol cAMP/mg protein/lO min Group JBasalI jNaF(5 mM) 1 Forskolin Gpp(NH~p (30 gKM Control J296±32 23431 18 JJ423:402 1123*98 IR 529±21* 3490±d76* 2192-+111* 1865 :8 t* PR J391d81# 2960±132# j1804*129# 1492*l01# P< 0. 05, signi~ficantly different from Control and PR group.

P< 0, 05, significantly different from Control and D? group.

Example 3 In vivo CoroMar Artery Ligatinn Myocardial infarction was produced in male Sprague- Dawley rats (200-250 g) by occlusion of the left coronary artery as described by Sethi et at., s Rats were anesthetized with 1-5% isoflurane in 100% 02 (2L flow rate). The skin was incised 15 along the left sterna border and the 4th rib was cut proximal to the sternum and a retractor inserted. The pericardial sac was opened and the heart externalized. The left be anterior descending coronary artery was ligated approximately 2mnm from its origin on the aort4 using a 6-0 silk suture. The heart was then repositioned in the chest and the U* 20incision closed via purse-string sutures- Sham operated rats underwent identical treartneat except that the artery was not ligated. Mortality due to surgery was less than Unless indicated in the text, the experimental animals showing infarct size >300/u0 *-beef#:of the left ventricle weire used in this study. All animals were allowed to recover, received food and water ad libitum, and were maintained for a period of 21 days for Electrocardiogram (ECGx), hemodynarnic and histological assessment.

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 S The animals prepared as described in Example 3 were anesthetized with an Sinjection 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 (AcqKnowlcdge 3.1 30 Harvard, Montreal, Canada).

S' Myocardial infarction for 3 weeks produced a progressive increase in left ventricular end diastolic pressure VEP) without a changes in either heart rate of 4 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 relaxation (-dP/dt) were sign~ificantly depressed in the infarcted animals. The elevation in LVEDP anid depression in both +dPldt and -dP/dt were partially prevented upon treating the infarcted animals with PLP for 3 weeks, The results are given below., in Tables 3 and 4.

Data are expressed as mean*SE of 10 animals. All measurements were made using a Miller microcatheter; the catheter was inserted int the left ventricle via cannulation of the right carotid artery. L VSP, left ventricular systolic pressure;- L VEDJ, left ventricular end- diastolic pressure: +dP/dt, rate of conlraction; dP/dt rate of relaxation, Animals were randomly divided into fow- 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.

1s 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 JSham Shamn Ml PrD HR~batsTflO)1 Drug HRbasri) 376:L18 398-122 405*22 475±16 LVSP (mm Hg) 126±7 122±6 128±6 123*-6 LVEDP (mm Hg) 2.2+±0.2 1.9±0.09 12.2+-0.9* 5.7:f-O.9# +dP/dt (rom Hg/s) 5899*302 5772±312 2654±1 11* 42Tf2±223# -d~~ldt (m 54928 401*297 2348±99* 3998*179# 0. 05) signofcantly differene from the sham control and the PrD group.

4 0. 05) sigificantly different from sham control group and MI group.

~e00 0 *000

S

tOOe.e 6 0 t

'S

0100 0 000 S 00 0 00 00 S 0* 6S 00 0 0 000000 0 a *0000~ 0 00 0p 0S6 00 0

S

0 TABLE 4 A later confirmation of hemodynarnic parameters of rates with myocardial infarction with or without PLP treatment for 21 days starting at 1 hour after and 2 days before coron~ay artery ligation.

Paaees Sa' Sham 1MI P PP2 Parameters~~ Sa+Drug JII_ HR 381±18 396±22 402*22 378±16 381±10 (ba/min) LVSP 124±7 122*6 124j=6 127±6 129*b5 (mim Hg) LVEDP 2.2±0.2 1.9±0.09 12.2-0.9* 5.2±0.81'# (mm. Hg) +dPldt 599±302 5772-±312 2654±1 11* 4272;k223*# 4199+-219*# (mm, Hg/s) -dP/dt 5469±:284 5401±297 23484-99* 3998±179t-# 3918±177** (mm Hg/s) 05) sign iflcantly differe ni from th e Aham and ha m dr ug gro up- 0, 05) significan ly different fro m MI gro up.

There were three groups of rats, 20 each: (MI) untreated coronary litigated, (PPI) orally PLP ancP daily starting at 1 hour after ligation, (PP2) orally PLP once daily starting at 2 days befo~re ligation.

Example 5 EleWtrcardigrm (ECO.) Reworinzs Six lead HI, III, aVr, aVf, aVI) ECG recordings were made from rats in all groups (sham operated, corory artery ligated sham operated with drug treatment, coronary artery ligated with drug treatment 2 days before ligation, coronary artery 000 0000: 0000 0 0e @0000 066 0 0

/G

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 III three leads at I to 21 days after coronary artery ligation in untreated rats. Treatment with PLP attenuated the degree of ST segment elevation/depression following occlusion, 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- 0 17 ST segment changes in rats with myocardial infarction (MU) with or without PLP treatment for 21 days starting at 2 days before coronaxy artery ligation (PrD).

ST segment (mV) Group ]7Day 14 Day 121 Day Leadl II MIf 0. 1 7;L.02* 0.17±0.02* 0. 15±+0.01* PrD 0.08±0.01 j0.05±0.01 0.03±00 .1 Lead II NG 0.15;L001* 0.15=0-01* 0.14:±0.01* PrD 0.07±0O.01 }0.04±0L.01 0,031-0.01 Lead MI AG 0A8*0.02* 0. 17-h0-02* 0 -1 4±-001* PrD 0.06±0.01 j0.04±0.01 0.02±0.01 e*.

S

*5 S 0 0* 0*Oe 0 S. 0 0S 0 *0

S

*0 0 0 @000-..

00

S

S

9 5 *5 0. 05 compared to control and treated group.

TABLE 6 ST segn~nt changes in rats with myocardial infarction (NU with or witout PLP treatment for 7 days starting at I hour after coronary artery ligation (PrD).

-0 ST segment (mV) Day Leadl II

.Z-

NU 0.19d*0.02* 0.19*0.02* 0. 16±0.0 1 PrD j0.13±0.01 0.09±0.01 j0.07±0.01191 LeadI 0.20-+0-01 f0. 19±0. 01 0.17±0O.01* PrD j0. 14;L0.01 0.10±0.01 10,07:0.01 MI0.20±0.02* 0.18+-0.01* 0.15=L0.01"' PrD 0.13±0.01 0.08A±0.01 0.04d±0.01

C

0

S

S. C 0O 9000 5.5.

S 0 0@ C

S*

C S

S.

e.g.

C

50 0 0 0**9@e OS C Ce

S

S

CSS

C

C S 0* P<O. 05 compared to Control and treated group.

OThtnval and Mortality-Follgowijag Mocardial IfarotiQn The QT interval on the surface electrocardiogrm is an indirect measure of the ventricular action potential duration and its prolongation is often associated with the occurrence of malignant ventricular arrhythmias 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 rernainin$ after subsequent weeks were used for ECQT estimations. Values are M M mean±SE. 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 (rusec) in mnyocardial 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 PrD 313±21 456±22*# 43 7±23# 410±2 1*# MI3 2± 756 2w5 2±6-J 5 6±9- P< 0- 05 compared to control.

P< 0,.05 compared with MI group.

9 9.S** 0 9. 0 9 9* S 990 9 9*00 0 0e 0 9* 99 9.

9 9* .9 0

S

0 *09*9* 0 0* 0.S 0* 9 S 99 15 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 inf etion with or without PLP treatment for up to 21 days startin at 2 days before cclronary artery ligation.

Group Control I Day 3 Day 7 Day 21 Day Nff 302±*17 601+17* 571±18* 522±1-26* 506:±29* PPI 313:L23 530+25*# 486±15** 4571-23*# 410:±21# PP 162 41±33** 495:631*# 452-19*# 401±17* 0. 05 compared to controL 25 0-05 compared with MI group.

I

There were three groups of rats, 20 each*: (MI) untreated coronary litigated,

(PPI)

orally PLP once daily, (PP2) orally PLP twice daily. n--20, values mean;ESE- Conn~ol values taken before induction of mnyocardial infarction.

Time dependent changes of QT. interval (msec) in myocardial infarction with or without PLP treatment for up to 21 days starting at 1 hour after coronary artery ligation..

Grou control 1 Day 3 Day 7 Day 21 Day MT 322*1I7 594±22* 562±18* 540:*20* 53+22 PPJ 310±21 516±21 05±13# 430±lf:I j4# 404:118*# PP2- 311114 535.+23*# 484-q21-# 421±26*# 397±119*# 0. 05 compared to control 05 compared with MI group.

15 There were three groups of rats, 20 each: (Ml) untreated coronary litigated,

(PI~P)

orally PLP once daily, (MP) orally PLP twice daily. n--20, values are mean*SE.

Control values were taken before induction of myocardial 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 25 and these ame caused primarily by lethal ventricular arrhythmias. In the present study, *ee*..mortality was highest in the first 48 hours afLer coronary ligation in both untreated and treated raks, however, mortality was significantly less in treated animals. This decreased mortality was accompanied by several imlproved ECG findings suggesting an anfiarrhYthnc action of PLP (decreased incidences of pathological Q-waves and

PYCS).

Rats intended for operating on, were randomly divided into four groups, each Sham, Sbam Drug treated, Drug treated starting aT 2 days before ligation (NU 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 hemodynarnie changes, they were considered as one group. The results are showng below in Tables 10, 11, 12 arnd 13, IABLE-1 Mortality in rats with myocardial infarction with or without PLP treatment for 21 days starting at 2 days before coronary artery ligation (PrD).

IF-No. of animnals 999 9 *99999 9 9. 0 15 0909 0 *9*9 weee 9* 0e 9 9 @9 99 9 9 9*99*9 9 9 @90996 9

S.

9 .9.

.5 9 9 9 99 Mortality MI jPrD Oanthe Ist day301 On the 2nd day 10 On the 3rdday 5 0 Within 2:1 days 45 Sign iflcantly 0.05S) difere ntfro m th e MI gro up. Sh am gro up had no moarrality.

At tha 21 st day, 3 animals from the MI group appeared very sick arnd may not have survived another week, Later coirmation and expantsion of mortality in rats with myocardial infarction wit or without PLP treatment for 21 days starting at 2 days before coronary artery ligation.

I

No. of animals (Yo) Morait Mi PP1 JPP2 I-On the Ist day 30 20 On the 2nd day 10 On the:3rd day 5 0 Within 21days(% 45 25"* *Significantly 0.05) different from the Mjgroup.

untreated coronary litigated, (PPl) orally PLP once daily, (Pp2) orally pEp twice daily.I E ,n a second, similar test, rats intended for operating on, were randomly divided into four groups, 20 each: Shame, Sham Drug treated, drug treated starting at I hour after ligation (PrD) for up to 7 days and coronary ligated group Since the shown and shamh drug group had no differences in regards to mortaity anid other hemodyi~amic 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 PEP treatmnent for 7 days starting at 1 houtr after coronary artery ligatiou (PrJ) *9* 9 *99** 9 .9 9 .9 9*g9 9 9eOS *99e 9 9 9* S 9 9 9999 99 9. 9 9 99999 9 9 @99999 9 .9 9 999 9. 9

S

.9 No. of animals Mortality MI Jr On the1$ day 25 On the 2nd day 15 On the Nd day 5 0 Within 7 'days 45 *SigntficantlY 0. 05) dif/erent from the Mrgroup. Sham group had no mortality.

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

No. of animals(% Mortality wM JPPI PP2 On thelst day 30 20 16 On the 2adday 10 8 8 On the 3kdday 5 0 0 Within 2.1 days 45 28* 24* *Signi/icantfy 0. 05) different from the Mif gro up.

9000 0 *0@0 000.00

S

S. S 0 00 SeeS

S

*055 S 0e C 9* 0 09 *5 09 00 S. 0

C

@000e0 .00 @00 @0

S

050 *5 0 C 5 *9 There were three groups of rats: 20 rats, untreated coronary litigated, (PP1), rats, orally PLP once daly, (PP2), 25 rats, orally PLP twice daily.

Antiarrythmic Action of PLP Reveald by ECG's The ECGs of the animals in the previously reported tests for mortality rate showed s everal findings indicating an andtiarythmic action of PLP- One of these is a decreased incidence of pathological Q-waves.

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

TALEI4 General Characteristics and pathological wave appearance of rats with mnyocardial 25 infarctiogi with or without PLP treatment f'or up to 21 days, starting at 1 hour after coronary artery ligation.

Parameter sham Sham NU PPI 2 Body wt.(g) 321±3 312±4 332±+5 342±7 34-1 Q wave appearance 58 27* 38* within 21 days(% (Pathological) Infarct size 43 21* 23* of LV) *Significantly differenz from the MIf group.

There were three groups of rats, 20 each: (MI) untreated coronary litigated, (PP 1) orally PLP once daily, (MP) orally PU' twice daily. n--20, values are mean*+SE.

Sham group was given saline- 10 General Characteristics and pathological "EQ" wave appearance of rats with miyocardial infarction with or without PLP treatment for up to 21 days, 2 days after coronary artery ligation.

Pii -i hmSham NE PPI P1 E Paramet r r g_ _J

P

Bodw~)330.+8 322±5 331+7 332±:9 334±10 Q wave @ppearance 62 37* 39* within 21 days(% (Pathological) Infarct silze -43 27* 32* (of LV) *Significantly (P 05O) different from the MI gro up.

C..

S C**C

C

0S C 00 *0

CS,.

S

e.g.

CC..

C C C. S *0 C S C. 0

C

0*C*SC

S

0 .6.0e 0

S.

S

*0e S. C

'C

There were three groups of rats, .20 each: (Wl untreated coronary litigated, (PpI) injected PLP once daily, (PP2) injected PLP twice daily. n-20, values are MflaW±SE- 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 axtery ligation.

S

S. S

SO

*0

OSSS

0

*SS*

Oe 9* 5 0O 5 5 9 0@5q 0*

S

0 0e*e*0 0 0 es OS.

0S 0. 0 9* 0* TABLE 7 Effect of treatment with ?LP (starting at I hour after ligation continued for 7 days) on the incidenace of preventricular contraction (PVC) following coronary artery ligation.

PVC Incidence Croup] I Day 3 Day 7 Day M7 14 14 21 rD11* 3*V Significantly dferentftGM the AdY groUP.

As those skiled in the art would realize these preferred described details and compounids and methods can be subjected to substantial variation, mnodification, change, it

I

alteration, and substitution without affecting or modifying the function of the desc ribe emnbodrients.

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 frs"aF andi "the" include plural referents ueless the contenat 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 wordr such as "comprising" and "comprises", means "including, but not limited to", and is not intended to exclude other additives, components, integers, or steps.

0 S0.

0 *0 0 0

Claims (6)

1. A method of treating ischemnia reperfusion injury and cellular dysfuntion in manunals comprising: administering to said mammal a therapeutic amount of a compound selected from the group consisting of py-ridoxal-5'-phosphate, pyridoxine, pyridoxal, and pyridoxarninie.

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

3. The method of claim 1, wherein said therapeutic amount is in a range of about

5-25 mg/kg of a patient' s body weight. 4. The method of claim 1, wherein said compound is administered enterally or parenterally. The maethod of claim 1, wherein'said compound is

6. A method of treating ischeniia reperfusion injury in maimmals comprising, administering to said mammal a therapeutic amount of a compound selected from the :e *group consisting of pyridoxal-S'-phospiate, pyridoxine, pyridoxal, and pyridoxamine.

99.57. A method of treating cellular dysfunction in mammals comprising: 9. S administering to said mrnual a therapeutic amount of a compound selected from the group crnsisting of pyridoxal-5--phosphate, pyridoxine. pyridoxal, and pyridoxarnine. A, pharmaceutical composition comprising: a pharmaceutically acceptable carrier and therapeutic amount of a compound selected from the group consisting of pyridoxine, pyridoxal, and pyridoxamine. 9. The pharmaceutical composition of claim 8, wherein said compound is 9 455pyridoxal-5'-phosphate, A t 28 The pharmaceutical composition of claim 8, wherein said pharmaceutical composition is in a form suitable for enteral or parenteral administration. 11. 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 ischemia-related conditions. 12. Use according to claim 11, in which the condition is ischemia reperfusion injury or ischemia-related cellular dysfunction. 13. Use according to claim 11 or claim 12, in which the compound is present in a range of about 1 to 50 mg/kg of a patient's body weight. 14. Use according to claim 12, in which the compound is present in a range of about 5 to 25 mg/kg of a patient's 20 body weight. e a, 15. Use according to any one of claims 11 to 13 in which the compound is administered enterally or parentally. 16. Use according to any one of claims 11 to 14 in which the compound is 0 Dated this 27th day of November 1998 30 UNIVERSITY OF MANITOBA By their Patent Attorneys r*g. GRIFFITH HACK S" h Fellows Institute of Patent Attorneys of Australia Attorneys of Australia H:\Glenys\Keep\uni.mnitobadoc 27/11/98 I