WO1995003054A1

WO1995003054A1 - Methods of treating apoptosis and associated conditions

Info

Publication number: WO1995003054A1
Application number: PCT/US1994/008268
Authority: WO
Inventors: L. David Tomei
Original assignee: Lxr Biotechnology Inc.
Priority date: 1993-07-23
Filing date: 1994-07-22
Publication date: 1995-02-02
Also published as: KR960703597A; EP0711168A4; BR9407145A; CA2167805A1; EP0711168A1; JPH09503749A; AU7370594A

Abstract

Methods are presented to prevent or delay apoptosis by administering a therapeutically effective amount of a physiologically acceptable dioxopiperazine.

Description

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METHODS OF TREATING APOPTOSIS AND

ASSOCIATED CONDITIONS

Field of the Invention

The present invention is directed to methods to treat medical conditions associated with acute, undesirable apoptotic cell death in tissues and organs.

Background of the Invention

There are two known types of cell death, necrosis and apoptosis. Necrosis is the traumatic destruction of cells leading to the release into the surrounding tissues of enzymes and intact DNA, which is then broken into random fragments. Unlike necrosis, apoptosis is a normal physiologic process that determines individual cell death and ultimate deletion of the cell from tissue. Apoptosis is an atraumatic, orderly, predictable dismantling of cells in which they shrink within their membranes while still in place in the tissue, and their DNA is packaged into nucleosomes for safe removal by white blood cells. Apoptotic cell death is characterized by cellular shrinkage, chromatin condensation, cytoplasmic blebbing, increased membrane permeability and interchromosomal DNA cleavage. Gerschenson et al. (1992) FASEB J. 6:2450-2455; and Cohen and Duke (1992) Ann. Rev. Immunol. 10:267-293. For review see, Apoptosis: The Molecular Basis of Cell Death, Tomei and Cope eds. Current Communications in Cell and Molecular Biology 3 , Cold Spring Harbor Laboratory Press, New York, 1991; and Apoptosis II The Molecular Basis of Apoptosis and Disease, Tomei and Cope eds. Current Communications in Cell and Molecular Biology 8, Cold Spring Harbor Laboratory Press, New York, 1994.

Apoptosis is involved in a variety of normal and pathogenic biological events and can be induced by a number of unrelated stimuli. Changes in the biological regulation of apoptosis also occur during aging and are responsible for many of the conditions and diseases related to aging. Recent studies of apoptosis have implied that a common metabolic pathway leading to cell death may be initiated by a wide variety of signals, including changes in hormone levels, serum growth factor deprivation, chemotherapeutic agents, and ionizing radiation. Wyllie (1980) Nature, 284:555-556; Kanter et al. (1984) Biochem. Biophys. Res. Commun., 118:392-3999; Duke and Cohen (1986) Lymphokine Res., 5:289-299; Tomei et al . (1988) Biochem. Biophys. Res. Commun., 155:324- 331; and Kruman et al . (1991) J. Cell. Physiol . , 148:267- 273. Agents that affect the biological control of apoptosis thus have therapeutic utility in a wide variety of conditions.

All publications cited herein, both supra and infra are hereby incorporated by reference.

Summary of the Invention The present invention is directed to methods of reversibly inhibiting apoptotic cell death associated with acute medical conditions. The methods comprise administering to a subject an amount of a therapeutically effective dioxopiperazine such as dexrazoxane, (DZR, ICRF187) sufficient to prevent or delay the appearance of apoptotic cell death.

Brief Description of the Drawing(s)

Figure 1 is a graph depicting the effects of dioxopiperazine on apoptosis as determined by the C3H- 10 model. The X axis of Figure 1 is presented in the log bis (dioxopiperazine) concentration. The results are presented in Table 4 and discussed in Example 3.

Detailed Description of the Invention

The present invention is to methods of ameliorating or preventing apoptosis. The method comprises administering to a subject an amount of a therapeutically effective dioxopiperazine sufficient to prevent or delay apoptosis. The dioxopiperazines are not administered to ameliorate the cardiac damage caused by certain anti-neoplastic agents, but rather are administered alone or with any other, non-anti- neoplastic, or pharmaceutically acceptable substances. Use of the term dioxopiperazine herein refers to any of a broad range of compounds. The particular dioxopiperazine utilized will depend on the particular indication. Generally, those dioxopiperazines exhibiting anti-apoptotic activity and lacking substantial toxic side effects will be suitable for use in the present invention. More preferably, those dioxopiperazines exhibiting pharmacologic effects similar to the bis (dioxopiperazine) s 2,3; 2,5; and 2,6 bis (dioxopiperazine) s are suitable for use in the present invention. More preferably, 2,3; 2,5; and 2,6 bis (dioxopiperazine) s are suitable for use in the present invention. Most preferably, 2,6 piperazinedione 4,4' -(1- methyl-1, 2-ethanediyl) bis-, (S) (+) [chemical formula C₁₁H₁₆N₄0₄] is suitable for use in the present invention. In the case of 2,6 bis (dioxopiperazine) s, the ÷enantiomer (ICRF-187) is preferred. Also suitable for use are various pro-drugs described in U.S. Patent No. 4,755,619.

Dioxopiperazines include a wide variety of compounds. The known therapeutic efficacy of these compounds is limited as demonstrated by the FDA approval for limited clinical use. For instance, 2,6 bis (dioxopiperazine) (dexrazoxane) and related compounds have been found to have weak anti-neoplastic effects and also to have been of use in treatment of psoriasis. For review see itiak and Wei (1988) . For descriptions of known indications and pharmaceutical compositions of dioxopiperazines see United States Patent Nos . 4,275,063; 4,755,619; 4,902,714; 5,149,710; 3,941,790; and U.K. Patent No. 1,374,979. Both neoplasias and psoriasis involve uncontrolled cellular proliferation.

Additionally, 2,6 bis (dioxopiperazine) has been found to ameliorate the cardiomyopathies associated with administration of a variety of chemotherapeutic agents. See e.g. Carlson (1992) Oncology, 6:95-100, 104, 107- 1000. For reviews of these compounds and their known physiological effects see, Herman et al . , (1982) Advances in Pharmacology and Chemotherapy, 19:249-290; and Witiak and Wei (1990) Progress in Drug Research, 35:249-363. Notwithstanding the history of the ability of dioxopiperazines to exert anti-neoplastic effects, it has now been found that dioxopiperazines are effective at preventing or delaying apoptosis.

Apoptosis is a normal cellular event which can also be induced by pathological conditions and a variety of biological insults such as transient lack of oxygen, trauma, or exposure to damaging radiation. Enhanced apoptosis levels are involved in a wide variety of conditions including, but not limited to, cardiomyopathies, infarctions, cancer regression, immunoregulation, viral diseases, anemia, neurological disorders and neurodegenerative diseases, diarrhea and dysentery, muscular wastage, kidney failure, diabetes, hair loss, rejection of organ transplants, failure of cell transplants and prostate involution. The methods described herein are suitable for use in any of these indications. As used herein, cardiomyopathies are not those induced by anti-neoplastic agents, particularly those induced by anthracyclines . A viral disease that causes severe apoptosis is AIDS (acquired immunodeficiency syndrome) . The present invention is thus particularly suitable for use in the treatment of AIDS.

In general, those dioxopiperazines that are physiologically acceptable and exert an anti-apoptotic effect are suitable for use in the present invention. Dioxopiperazines are said to exert an anti-apoptotic effect if they are found to do so d-n vivo and/or if they test positively in any anti-apoptosis assay. .In vivo indicia of anti-apoptotic effects include, but are not limited to reduction of apoptotic cell death in the myocardium, brain, and kidney following either transient ischemia brought on by reduction of adequate blood perfusion or exposure to blood-borne toxic substances. Suitable in vitro assays include but are not limited to the assay described in co-pending United States patent application Serial No. 08/056,439 which is incorporated herein by reference. The assay is further described in the following examples.

It has now been found that apoptosis is responsible for reperfusion damage due to coronary artery obstruction; spinal/head trauma and concomitant severe paralysis; and reperfusion damage due to other insults such as frostbite. Dioxopiperazines are particularly useful in treating these apoptosis-associated conditions. Dioxopiperazines are also suitable for use in treating any indication previously thought to be treatable by superoxide dismutase (SOD) or antioxidant agents.

Ischemic cellular damage following coronary artery obstruction may result in acute myocardial infarction (AMI) . AMIs generally are caused by a sudden insufficiency of oxygen-rich blood supply due to emboli, thrombi, or low pressure that eventually lead to the appearance of macroscopic areas of focal necrosis; the heart, brain, spleen, kidney, intestine, lung and testes are likely to be affected. Until recently, it was thought that the cell death associated with infarcts is directly due to the ischemia. It has now been found that apoptosis occurs to tissue in the regions of ischemia and upon reperfusion of oxygen-rich blood to the area; thus, dioxopiperazines agents are effective if administered at the onset of acute ischemia, during reperfusion with oxygen-rich blood, or shortly thereafter.

It has also been found that apoptotic cells are not removed from the heart as rapidly as they are from many other tissues of the body, in part because macrophages do not circulate as freely in the heart muscle tissue as they do in other parts of the body. As a result, apoptotic cells accumulate in the heart tissue and soon become a core of necrotic cells. This secondary necrosis then leads rapidly to inflammation and further damage and the heart cannot continue to function. If the heart does survive, there will be a permanent scar that will impair the ability of the heart. This secondary necrosis has now been found to occur between one and six hours following a heart attack. Dioxopiperazines are thus suitable for use in preventing this permanent impairment of heart function or cessation of heart function. The invention thus encompasses administration of therapeutically effective amounts of dioxopiperazines following a heart attack and preferably between one and six hours following a heart attack.

The present invention is thus suitable for use in treating and thus ameliorating cardiac conditions unrelated to the cardiomyopathies caused by anti- neoplastic agents. These cardiac conditions include, but are not limited to, development of and damage subsequent to, myocardial infarction injury or other acute events of the heart such as in post-cardiopulmonary bypass when the attempt is made to restart the heart . In the case of myocardial infarction, effective amounts of dioxopiperazines can be administered intravenously at the first indication of coronary arterial insufficiency often associated with onset of acute chest pain or angina pectoris. The dioxopiperazines may also be administered after occurrence of the infarct . Preferably, administration of an effective amount of dioxopiperazines occurs within six hours of a heart attack. More preferably, administration occurs within one hour of a heart attack. Other suitable methods of administration include but are not limited to intraperitoneal and direct administration to the heart.

Methods of administration of dioxopiperazines are known in the art and need not be described in detail . In all types of administration, a therapeutically effective amount of a dioxopiperazine is suspended or solubilized in an isotonic, physiologically acceptable, sterile liquid including, but not limited to, isotonic saline. In intravenous and intraperitoneal administration, the dioxopiperazine is introduced by methods known in the art including but not limited to injection into a central venous line, infusion via a pump or direct intravenous injection. Direct administration to the heart includes but is not limited to direct intracardiac injection. Devices for such direct injection are known in the art; for instance, the Aboject cardiac syringe.

The effective concentration of dioxopiperazine will depend on the particular dioxopiperazine utilized and the method of administration. For instance/ when administered intravenously, the dioxopiperazine 054

- 8 - concentration will be lower than when administered intracardiac. The total amount of dioxopiperazine administered to the patient may be greater over time when administered intravenously so as to attain an effective concentration at the site of the infarct. An effective concentration for amelioration of cardiotoxic side effects of various chemotherapeutics is 500- mg/m² 2,6 bis(dioxopiperazine) ; thus this is a preferred dose. A broad range of doses may be effective, typically the range is from the concentration required to exert anti- apoptotic effects up to the concentration at which side effects begin to occur. Determination of the appropriate dosage is within the skill of one in the art.

Administration of the dioxopiperazine should be maintained for a length of time effective to prevent apoptosis. Generally, apoptosis is initiated by acute lack of oxygen in the active normal myocardium. Administration of the dioxopiperazine should thus be initiated at the time of oxygen deficit or shortly thereafter. Delaying the onset of apoptosis for 24 to 72 hours will allow reperfusion of the affected myocardium to occur without concomitant cell death.

The dioxopiperazines may be administered alone or in combination with other, non-anti-neoplastic, therapeutic agents provided there are no toxic cross- reactions. For instance, in treating myocardial infarction, dioxopiperazines can be administered in conjunction with tissue plasminogen activator (t-PA) , urokinase, or streptokinase. In the case of other apoptotic indications, dioxopiperazines may be administered in conjunction with a variety of other therapeutic agents including but not limited to, anti- inflammatory agents; anti-oxidants, SOD inhibitors, immunosuppressants, and antiviral agents. The following examples are intended to illustrate, but not limit, the invention.

Example 1 Cell Culture Technigues

The following is a detailed description of the anti-apoptosis drug screening assay as performed using C3H-10T1/2 clone 8 mouse embryonic cells.

The' cells are obtained at the lowest available serial passage level preferably less than level 11. The phenotypic characteristics of the cells are verified as meeting the following criteria. Standard cell culture techniques are used.

1. Confirmation of a mean doubling time of 22 (±2 ) hours under standard conditions of basal Eagle's growth medium supplemented with 10% (v/v) fetal bovine serum in plastic culture flasks maintained at 37° C in a humidified atmosphere of 5% C0₂.

2. Cloning efficiency determined to be 25% (±2 ) at densities of 200 cells/20 cm² under standard growth conditions.

3. Saturation cell density confirmed to be 5 x 10⁵ (±2 x 10⁵) cells/20 cm² plastic petri dish under standard growth conditions. 4. At saturation cell density, it is confirmed that more than 98% of cells are in the G_ phase of the cell cycle.

5. The morphology of exponentially proliferating cultures is radically changed at saturation density such that the spindle shaped cells having extensive overlapping and lack of parallel orientation during exponential proliferation changes to wide, flat epithelioid monolayer without distinct intercellular demarcation and no overlapping. 054

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6. Cells are sensitive to malignant transformation by chemical carcinogens, typically 3- methyl cholanthrene, or ultraviolet irradiation yielding transformed foci. 7. Cells do not form a fibrosarcoma tumor when injected at levels of 10^s cells subcutaneously in the suprascapular region of syngeneic animals, whereas, following malignant transformation in vi tro, tumors are observed under similar conditions. The cells are then cultured in containers that are typically 60 mm diameter plastic petri dishes specially prepared for mammalian cell culture and are commonly available from several commercial sources.

Cells for replicate culture are obtained from stock cultures which are confirmed to be in exponential phase proliferation and not in post-confluent saturation density to ensure that cells are not arrested in the G phase of the cell cycle.

The cells are seeded onto each plate in a volume of 5 ml complete growth medium in which are suspended a standardized number of cells. The standardized number should be not less than 10³ but not greater than 10⁴ cells.

Special care must be taken to ensure that cells are uniformly distributed over the surface of each culture dish in order to prevent clustering of cells during subsequent growth which results in non-uniform increases in cell densities and premature saturation densities in limited regions on each dish. Premature saturation density in limited regions results in serious errors in assay results.

Growth medium is renewed each 48 hours. When cell density reaches approximately 70% uniformly across each dish surface, which typically corresponds to a density of 1 x 10^s to 3 x 10⁵ cells/dish, the complete growth medium is removed by aspiration and replaced with fresh serum-free growth medium.

Drugs or agents to be assayed can be premixed into the serum-free medium, or added in appropriately small volumes immediately after the medium change.

Typically, in order to ensure statistical reliability, each agent or specific treatment is performed on at least four replicate cultures and appropriate controls are also incorporated.

After a standardized period of incubation at 37°C under humidified 5% C0₂ atmosphere of between 3 hours and 72 hours, typically 24 hours, each plate is prepared for measurement of responses. The following measurements are performed:

1. All non-adherent or loosely adherent cells are removed from the culture dish and measured by appropriate techniques typically counting by electric particle counting instrument. 2. The remaining adherent cells are exposed to a buffered (typically pH 7.3) balanced salt solution such as Hanks Balanced Salt Solution containing a standardized concentration of the enzyme trypsin. The trypsin concentration is typically 0.1 mg/ml but can be between 1 and 0.001 mg/ml, typically in a volume of 1 ml.

Each culture is incubated either at ambient temperature or 37°C on a rocking platform to ensure uniform distribution of the trypsin reagent over the culture surface. After a standardized period of typically 10 minutes, the released cells are removed from each dish, and measured by the same means described above, typically electronic particle counting. This measurement is referred to as the serum deprivation release or SDR count and typically contains at least 98% apoptotic cells. The remaining adherent cells in each dish are then released by exposure to a buffered solution containing a calcium ion chelating agent typically EDTA typically at a concentration of 2 mg/ml. This measurement is referred to as the proteinase sensitive or PS count and typically contains the cells that would have otherwise died by apoptosis in the absence o-f an effective inhibitor.

The final cells remaining adhered to the solid support are then immediately dispersed and removed from the dish for measurement by the same means used in previous measurements, typically electronic particle counting. This measurement is referred to as the proteinase resistant or PR count and typically contains cells that express the property of resistance to proteinase-induced shape change which has been identified to be a critical specific expression related to control of apoptosis.

Each cell count is typically performed in duplicate on each of four replicate dishes for each experimental treatment group and control.

Final data are initially expressed as the mean cell number and standard deviation of the mean according to conventional statistical data-analysis. The statistical analysis is performed as described in Daniel, Biostatistics : A Foundation in the Health Sciences, 2ed., John Wiley and Sons (1978) . The results obtained are shown in Table 1. The numbers in Table 1 printed in bold print represent the numbers used in Table 2 as described in Example 2. In Table 1, the data are reported as cells/plate x 10,000. The abbreviations used are: SD, standard deviation; PR, proteinase resistant cells; and PS, proteinase sensitive. OJ OJ N-3 H UI o in σ on

Table 1 : Bovine serum screening test C3H-10T1/2 cells

Apoptotic +/- SD % control PR +/- SD % control PS + /- SD % control Total % control

Lot #1 Control 22.6 2.6 100.0 17.7 3.6 100.0 20.4 3.3 100.0 60.7 100.0

1 nM TPA 19.8 0.6 87.7 27.8 4.4 157.1 16.7 2.6 81.9 64.3 105.9

10 nM TPA 13.1 1.5 58.0 48.1 4.4 271.8 15.9 1.2 77.9 77.1 127.0

Lot #2 Control 13.6 0.5 100.0 17.2 2.1 100.0 20.2 1.8 100.0 51.0 100.0

1 nM TPA 10.4 1.6 76.5 23.5 3.3 136.6 15.0 1.8 74.3 48.9 95.9

10 nM TPA 6.0 0.4 44.1 35.6 2.1 207.0 14.2 1.3 70.3 55.8 109.4

Lot #3 Control 30.8 2.0 100.0 16.7 5.1 100.0 25.9 5.2 100.0 73.4 100.0

1 nM TPA 25.4 1.3 82.5 26.3 4.5 157.5 22.3 2.8 86.1 74.0 100.8

10 nM TPA 15.4 1.5 50.0 49.5 1.8 296.4 13.3 1.2 51.4 78.1 106.4

Lot #4 Control 35.9 1.9 100.0 20.5 1.0 100.0 34.6 0.9 100.0 90.9 100.0

10 nM TPA 21.6 4.9 60.2 42.6 6.5 207.8 45.0 3.4 130.1 1 10.1 121.1

50 nM TPA 16.8 1.4 46.8 47.4 4.2 231.2 45.9 4.1 132.7 109.1 120.0

Lot #5 Control 49.1 6.0 100.0 20.5 4.2 100.0 49.6 2.5 100.0 1 19.2 100.0

10 nM TPA 30.7 5.1 62.5 53.9 2.5 262.9 47.0 4.9 94.8 131.6 1 10.4

50 nM TPA 16.7 2.4 34.0 55.6 6.1 271.2 52.2 3.2 105.2 124.5 104.3

Initial data-analyses for assay reliability should reveal that variations among replicate culture dishes for any of the three measurements should not be greater than 7% standard deviation. Data can be normalized by expressing the individual counts of SDR, PS, and PR as ratios to the corresponding value determined in control dishes.

A preferred positive result is typically dependent upon a statistically significant reduction of SDR cells in combination with a statistically significant increase in PR cells. However, putative apoptosis modulating agents can produce either reduction of SDR cells or increased PR cells and should be considered to be positive and warranting further consideration. Agents that produce either increased SDR or decreased total cell counts (i.e., SDR + PS + PR) should be considered to be potentially cytotoxic at the concentrations applied. A negative outcome would be failure to observe changes in either SDR or PR counts at concentrations determined to be non-toxic.

Example 2 Bovine Serum Screening Test The purpose of screening the bovine serum used as a supplement to the synthetic portion of the cell culture growth medium is to determine the best manufacturer's production batch in terms of optimal assay performance. This aspect of the assay is counter¬ intuitive in as much as conventional serum screening tests used by those skilled in the art are based primarily on determining the relative ability of various production batches of animal sera to maintain high viability or survival rates of various reference cell cultures. Contrary to that rationale, the apoptosis assay serum screening test determines the relative ability of various serum production batches to yield apoptotic, or dying and dead, cells upon withdrawal from the growth medium.

As an example, Tables 1 and 2 present typical data obtained in the process of screening 5 different bovine serum production batches obtained from a commercial source. These are listed as Lots 1 through 5. In order to distinguish between necrotic and apoptotic cells, cells are treated with TPA. Two independent variables to be measured are the number of apoptotic^" cells in the untreated control cultures compared with replicate cultures treated with TPA. These variables are the released cell or Apoptotic count, and the proteinase- resistant or PR cell count as shown in Table 1. Results were evaluated as follows: Apoptotic and PR responses are expressed in terms of a ratio or percentage of the corresponding response obtained in untreated replicate cultures. A desirable response would be simultaneous maximal reduction of Apoptotic cell count and enhancement of PR cell count. Therefore, the responses are then ranked according to each lot's respective response for each variable as shown in Table 2.

Table 2: Analysis of results of screening data Activity determined at 10 nM TPA

serum lot # rank Suppression of apoptosis rank Induction of prot-R total ranking

1 3 58.0 2 272 5

2 1 44.1 5 207 6

X 3 2 50.0 1 296 3

4 4 60.2 4 208 8

5 5 62.5 3 263 8

The results shown in Table 2 indicate that Lot #3 ranked second in suppression of apoptotic cells, and first in induction of trypsin resistance. This sum of 3 was then determined to be the best of the five lots evaluated and it was then selected for use in the peptide assay described in Example 3.

Example 3 Dioxopiperazine Anti-apoptosis Assay Utilizing the methods described in Examples 1 and 2, the dioxopiperazine, 2,6 piperazinedione 4,4' -(1- methyl-1, 2-ethanediyl) bis-, (S) (+) was tested in the C3H-10T^ assay to determine the anti-apoptosis activity. The assay was performed as described above with the concentrations of dioxopiperazine indicated in Tables 3 and 4. The results obtained are presented in Tables 3 and 4; and Figure 1.

Table 3. Affects of bis (dioxopiperazine) on Apoptosis in C3H-10T1/2 Model

Treatment Mean s.d. sem % x 100 x 100 x 100 Suppression

Control none 36.72 0.61 0.35 0.0 bis (dio 19 μM 31.28 3.22 1.86 14.8 xopiper azine)

1.9 μM 27.96 3.00 1.73 23.9

0.19 μM 30.00 1.67 0.96 18.3

19 nM 30.94 2.62 1.51 15.7

1.9 nM 31.40 2.54 1.46 14.5

0.19 nM 33.82 2.34 1.35 7.9

19 pM 35.57 0.68 0.39 3.1 n = 3 replicate cultures.

Duplicate cell counts performed on each of triplicate cultures

24 hour serum deprivation.

24 well multiwell plates.

Table 4.

Log[bis (dioxopipe Control -10.721 -9.721 -8.721 -7.721 -6.721 -5.721 -4.721 razine)

Concentration]

% Inhibition 0.0 3.1 7.9 14.5 15.7 18.3 23.9 14.8

10 n = 3 replicate cultures 24 hour serum deprivation 24 well multiwell plates

15

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25

Claims

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1. A method of delaying or preventing apoptosis comprising administering to a patient undergoing apoptosis a therapeutically effective, physiologically acceptable, dioxopiperazine in an amount 0 sufficient to prevent or delay apoptotic cell death.-

2. The method according to Claim 1, wherein the apoptosis is induced by an acute pathological event .

5 3. The method according to Claim 2, wherein the apoptotic event is selected from the group consisting of cardiomyopathies, infarctions, cancer regression, immunoregulation, viral diseases, anemia, neurological disorders, neurodegenerative diseases, diarrhea and o dysentery, diabetes, muscular wastage, kidney failure, renal toxicity, prostate involution, rejection of organ transplants and failure of cell transplants.

4. The method according to claim 3, wherein 5 the viral disease is caused by human immunodeficiency virus.

5. The method according to Claim 3, wherein the apoptotic event is associated with myocardial 0 infarction.

6. The method according to Claim 2, wherein the acute pathological event is transient ischemia and

5 O 95/03054

-20- the apoptosis is occurring in the myocardium, brain or 5 kidney.

7. The method according to Claim 1, wherein the apoptosis is associated with reperfusion damage due to coronary artery obstruction, spinal/head trauma and ₀ concomitant severe paralysis and reperfusion damage due to other physiologic insults.

8. The method according to Claim 1, wherein the dioxopiperazine is selected from the group consisting _5 of 2,3; 2,5; and 2,6 bis(dioxopiperazine) .

9. The method according to Claim 1, wherein the apoptotic event is associated with acute myocardial infarction and the dioxopiperazine is a 2,6 ₀ bis(dioxopiperazine) .

10. The method according to Claim 9, wherein the 2,6 bis(dioxopiperazine) is 2,6 piperazinedione 4,4'- (1-methyl-1,2-ethanediyl) bis-, (S) (+) . 5

11. A method of treating apoptosis following myocardial infarction comprising administering to a subject a therapeutically effective, physiologically acceptable dioxopiperazine in an amount sufficient to 0 prevent or delay apoptotic cell death.

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12. The method according to claim 11, wherein the dioxopiperazine is administered at the first indication of coronary artery insufficiency.

13. The method according to claim 11, wherein the dioxopiperazine is administered at least one hour following a heart attack.

14. The method according to claim 11, wherein the dioxopiperazine is administered within six hours following a heart attack.

15. The method according to Claim 11, wherein the dioxopiperazine is selected from the group consisting of 2,3; 2,5; and 2,6 bis (dioxopiperazine) .

16- The method according to Claim 15, wherein the 2,6 bis (dioxopiperazine) is 2,6 piperazinedione 4,4'- (1-methyl-1,2-ethanediyl) bis-, (S) (+) .