Aminothiol reduction of ischemia-reperfusion-induced cell death

Abstract

Members of the PrC-210 family of aminothiols, including PrC-211 and PrC-252, are shown to be highly effective in reducing ischemia-reperfusion injury in two preclinical models, including kidney transplant and myocardial infarct. Compositions and methods employing members of the PrC-210 family of aminothiols are disclosed for suppressing ischemia-reperfusion-induced cell and organ toxicities in a number of settings, significantly including organ transplant and myocardial infarct.

Claims

1. A method of reducing an ischemia-reperfusion injury (IRI) in a subject, comprising administering to said subject an effective amount of a compound PrC-210, having the following structure ##STR00009##  or a pharmaceutically acid addition salt thereof.

2. A method for reducing cell death in a tissue of a subject having suffered from an ischemia-reperfusion injury (IRI), wherein a compound PrC-210 of the formula ##STR00010##  or a pharmaceutically acid addition salt thereof, is administered to the subject.

3. A method for reducing apoptosis in a tissue of a subject having suffered from an ischemia-reperfusion injury (IRI), wherein a compound PrC-210 of the formula ##STR00011##  or a pharmaceutically acid addition salt thereof, is administered to the subject.

4. A method for reducing caspase activity in a tissue of a subject having suffered from an ischemia-reperfusion injury (IRI), wherein a compound PrC-210 of the formula ##STR00012##  or a pharmaceutically acid addition salt thereof, is administered to the subject.

5. The method according to claim 1, wherein the compound PrC-210 or pharmaceutically acceptable acid addition salt thereof is administered during or before ischemia-reperfusion injury (IRI).

6. The method according to claim 1, wherein the compound PrC-210 or pharmaceutically acceptable acid addition salt thereof, is administered to a subject having a myocardial infarction or a stroke.

7. The method according to claim 1, wherein the compound PrC-210 or pharmaceutically acceptable acid addition salt thereof, is administered to a subject at risk for at least one of myocardial infarction and stroke.

8. The method according to claim 1, wherein the ischemia-reperfusion injury (IRI) occurs in a kidney of said subject.

9. The method according to claim 1, wherein the compound PrC-210 or pharmaceutically acceptable acid addition salt thereof, is administered to a subject undergoing surgery.

10. The method according to claim 9, wherein the surgery is coronary bypass surgery.

11. The method according to claim 1, wherein the compound PrC-210 or pharmaceutically acceptable acid addition salt thereof, is administered to said subject before, during or after the ischemia-reperfusion injury (IRI).

12. The method according to claim 1, wherein the compound PrC-210 or pharmaceutically acceptable acid addition salt thereof, is administered systemically at an effective time before, during or after an ischemia-reperfusion injury (IRI).

13. A method for reducing an ischemia-reperfusion injury (IRI) in a transplant organ, wherein a compound PrC-210 of the formula ##STR00013##  or pharmaceutically acceptable acid addition salt thereof, is administered to a transplant organ.

14. A method for reducing an ischemia-reperfusion injury (IRI) in a transplant organ, wherein a compound PrC-210 of the formula ##STR00014##  or pharmaceutically acceptable acid addition salt thereof, is administered to a donor of the transplant organ before organ removal from said donor.

15. A method for reducing an ischemia-reperfusion injury (IRI) in a transplant organ, wherein a compound PrC-210 of the formula ##STR00015##  or pharmaceutically acceptable acid addition salt thereof, is administered to a donor of the transplant organ during organ removal from the donor.

16. A method for reducing an ischemia-reperfusion injury (IRI) in a transplant organ, wherein a compound PrC-210 of the formula ##STR00016##  or pharmaceutically acceptable acid addition salt thereof is administered to a recipient of the transplant organ before or after organ transplantation.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a schematic for one of the proposed mechanisms of protection conferred by PrC-210 against ischemia-reperfusion injury to cells.

(2) FIG. 2 shows the efficacy of PrC-210 versus 12 commonly cited “antioxidants” in their dose-dependent ability to suppress hydroxyl radical (—OH)-induced nicking of pUC19 plasmid DNA when added to the reaction.

(3) FIG. 3 shows complete prevention of .OH-induced pUC19 plasmid damage when PrC-210 is added to the reaction mixture as little as 30 seconds before generating the .OH insult.

(4) FIG. 4 shows the effects of PrC-210 on renal caspase and renal function 24 hr after mouse renal I-R injury. (A) Experimental design; all groups underwent 30 min of left (LT) ischemia (clamp) and right (RT) nephrectomy followed by 24 hr of reperfusion. Aminothiol (PrC-210, PrC-211 or PrC-252) was administered as a single intraperitoneal (IP) injection 20 min before LT kidney clamp. Serum and LT kidney were harvested at 24 hr. (B) Kidney tissue supernatant caspase activity was measured as described in Example 3. Linearity of the caspase assay conditions over 60 min were first established. (C) BUN levels were determined on serum harvested 24 hr post-clamp.

(5) FIG. 5 shows the dose-dependent PrC-210 suppression of left kidney caspase activity 24 hr post-clamp. PrC-210 doses were administered as a single IP injection 20 min before clamp, and caspase activity was measured 24 hr later. PrC-210 doses are indicated as fractions of the IP maximum tolerated dose (MTD) determined previously on wild-type mice, which is 504 ug/gm body weight (b.w.).

(6) FIG. 6 shows the ability of each of the indicated aminothiols (and their structures) to reduce the level of kidney caspase 24 hr after the 30 min I-R insult to the kidney. A 0.24 MTD dose of each aminothiol was administered as a single intraperitoneal injection to mice 20 min before the 30 min clamp of the left kidney was initiated. Kidneys were harvested 24 hr later and assayed for caspase activity.

(7) FIG. 7 shows significant reduction of cardiac muscle death in mice that receive a systemic dose of PrC-210 10 min before the left coronary artery ligation and release (after 40 min) in this mouse myocardial infarction ischemia-reperfusion model. Staining of live tissue in the hearts was done 24 hr after the coronary artery ligation-release I-R insult to the heart.

(8) FIG. 8 shows a significant reduction of hydrogen peroxide (H.sub.2O.sub.2)-induced cardiac myocyte cell death by coincident addition of PrC-210 to the myocyte tissue culture medium.

(9) FIG. 9 shows significant PrC-210 suppression of γ-H2AX foci (i.e., ROS-induced DNA double-strand breaks) in X-ray-irradiated human blood lymphocytes. PrC-210 at the indicated concentrations (0 to 23 mM) was added to human whole blood samples 2 hr before the 100 mGy irradiation to the whole blood samples.

(10) FIG. 10 (left panel) shows that amifostine induces retch and emesis responses in a ferret model; (right panel) shows that PrC-210 does not induce retch and emesis responses in a ferret model.

(11) FIG. 11 shows that PrC-210 does not cause hypotension side effects. The left top panels show recorded blood pressure after administration of amifostine. The left bottom panels show recorded blood pressure after administration of PrC-210.

(12) FIG. 12 shows mouse plasma levels of the active PrC-210 thiol form at various times after the molecule was administered either by (A) a single intraperitoneal injection or (B) a single oral gavage delivery to the mouse stomach.

EXAMPLES

Example 1

(13) During the development of embodiments of the technology provided herein, experiments were conducted demonstrating that pUC19 plasmid can be separated on an agarose gel to show both the supercoiled form (Supercoil) and the nicked (Nicked) form (FIG. 2, Panel A). As shown in the results, when one or more bonds within the supercoiled plasmid DNA backbone are broken by chemical attack from the reactive oxygen species (ROS) generated during x-irradiation to the plasmid DNA, the supercoiled plasmid DNA is changed into a nicked form and can be separated from the supercoiled plasmid DNA on the agarose gel during electrophoresis. In this Example, the ROS generated during x-irradiation are simply mimicking the same ROS species that are formed during a standard ischemia and reperfusion cycle. ROS-attack on the naked plasmid DNA results in a relaxed plasmid form that migrates more slowly on the gel. In this Example, the indicated small molecules were individually added to plasmid tubes 15 min prior to x-irradiation to determine what protection, if any, they conferred against the bolus of ROS generated during x-irradiation. 15 min after test molecules were added to pUC19 plasmid incubations, tubes received 90 Gy of radiation over 30 min. (A) Aliquots of incubations were then electrophoresed and quantitative imaging of ethidium bromide stained gels was done. Intensities of lower (supercoiled) and upper (nicked) bands were quantified using Image J software. (B) Band intensities were plotted using Graphpad Prism software. (C) Structures of some of the tested molecules are shown. Addition of PrC-210, WR-1065 or cysteamine to the incubations each conferred dose-dependent suppression of the ROS-induced damage to the plasmid DNA.

Example 2

(14) These experiments show the agarose gel separation (FIG. 3) of supercoiled and nicked/.OH-damaged forms of pUC19 plasmid DNA after exposure of plasmid DNA to a 60 sec pulse of an .OH generator (H.sub.2O.sub.2+UV light; Floyd et al., J Biochem Biophys Methods 1984; 10:221-235). Supercoiled DNA was incubated with water (lanes a, b) or 20 mM PrC-210 (lanes c-h) for the indicated times and then exposed for 1 min to the .OH generator. Aliquots of each reaction were electrophoresed, stained with EtBr and digitally imaged. Three replicate reactions and gels were done, and band intensities were quantified using Image J software. P value for comparison of supercoiled band intensities in lanes a vs g is indicated. Addition of PrC-210 as little as 30 seconds before the .OH insult conferred complete suppression of ROS DNA damage.

Example 3

(15) Experiments were conducted demonstrating (FIG. 4) that administering a single IP injection of PrC-210 (0.24 MTD=0.116 mg/gm body weight) caused an 84% reduction in the level of kidney caspase 24 hr after the I-R insult to the kidney, and a like reduction in serum blood urea nitrogen (BUN) level. (A) Experimental design; all groups underwent 30 min of left (LT) ischemia (clamp) and right (RT) nephrectomy followed by 24 hr of reperfusion. PrC-210 was administered as a single intraperitoneal injection 20 min before LT kidney clamp. Serum and LT kidney were harvested at 24 hr. (B) Kidney supernate caspase activity was measured as follows: Caspase 3 and 7 activity in kidney homogenate supernates was determined using the Apo-ONE fluorescent substrate (Promega, Madison, Wis.). Briefly, thawed kidneys were mixed with an 8-fold excess of lysis buffer containing 50 mM Na HEPES, pH 7.4, 100 mM NaCl, 1 mM EDTA, 10 mM DTT, 10% glycerol and homogenized at 4° C. for 30 sec with a stainless steel blade homogenizer (5,000 rpm). The kidney homogenate was then centrifuged at 4° C. at 16,000×g in an Eppendorf 5418 microfuge for 20 min. The resultant supernates were immediately frozen and stored at −70° C. Supernate protein was measured by the Bradford method using bovine serum albumin standards. The caspase assay was performed as follows: 38 μg of supernate protein diluted to a total volume of 50 ul with the above lysis buffer, was mixed with 50 μL of the Apo-ONE substrate in the well of a black 96 well plate to initiate the 60 min reaction. Plates were shaken at 200 rpm at 37° C. for 60 min. The DEVD caspase substrate peptide cleavage was measured using a BMG Clariostar fluorescent plate reader at an excitation wavelength of 499 nm and an emission wavelength of 521 nm. A caspase standard was included for each experiment. (C) BUN levels were determined on serum harvested 24 hr post-clamp as a functional indicator of kidney health over the 24 hr following the I-R insult.

Example 4

(16) Experiments were conducted demonstrating (FIG. 5) that administering single IP injections of PrC-210, at a range of three doses (0.105 MTD, 0.152 MTD, 0.230 MTD=0.053, 0.077, 0.116 mg/gm body weight, respectively) caused a PrC-210 dose-dependent reduction in the level of kidney caspase measured 24 hr after the I-R insult to the kidney.

Example 5

(17) Experiments were conducted demonstrating (FIG. 6) that administering a single IP injection of PrC-210, PrC-211 or PrC-252 at their respective 0.24 MTD doses (PrC-210 MTD=504 ug/gm body weight; PrC-211 MTD=500 ug/gm body weight; PrC-252 MTD=287 ug/gm body weight) caused highly significant reductions in the level of kidney caspase measured 24 hr after the I-R insult to the kidney.

Example 6

(18) Experiments were conducted demonstrating that administering two IP injections of PrC-210 (0.252 mg/gm body weight, and 30 min later 0.05 mg/gm body weight) to mice in which the left coronary artery had been intentionally ligated (see FIG. 7) caused an average 36% reduction in the percentage of the total cardiac muscle that was stained as dead-tissue 24 hr after release of the 40 min artery ligation. The degree of cardiac muscle tissue-death caused by the surgically-induced “infarct” (40 min artery ligation) in PrC-210-injected mice was significantly less (P=0.0143) than the degree of cardiac muscle tissue-death in the mice that were injected with saline as a control.

Example 7

(19) Experiments were conducted demonstrating that addition of PrC-210 (2.3 mM) to tissue culture medium in the culture wells containing primary neonate cardiac myocytes (30,000 cells/96 well) from 3-day old mice, conferred a highly significant reduction in the myocyte cell death that was induced by adding increasing concentrations of H.sub.2O.sub.2 to the cells (see FIG. 8). In this example, addition of PrC-210 to the media either 15 min or 30 secs before the addition of H.sub.2O.sub.2 showed the same outcome in which ˜80% of the H.sub.2O.sub.2-induced cell death was eliminated.

Example 8

(20) These experiments show highly significant PrC-210 suppression of γ-H2AX foci formation (indication of DNA double-stranded breaks) in x-ray irradiated human blood lymphocytes (FIG. 9). PrC-210 at the indicated concentrations (0 to 23 mM) was added to human whole blood samples 2 hr before the 100 mGy irradiation of the samples. Insets: Immunostaining of γ-H2AX foci (green) in human lymphocytes; nuclei were stained with 4,6-diamidino-2-phenylindole; whole blood samples were irradiated with 100 mGy x-ray 2 hr after receiving either no drug (100 mGy) or 23 mM PrC-210 (100 mGy+PrC-210).

Example 9

(21) Experiments were conducted demonstrating (Table 3) that when rats received a single IP injection or four topical applications of the indicated aminothiols 30 min before irradiation, and then received a single x-ray dose of 17.2 Gy to a defined rectangle area of skin (1.5×3.0 cm) on their dorsal backs, radiation dermatitis was reduced. 13 days following drug application and irradiation, the severity of x-ray-induced radiodermatitis within the irradiated skin area was scored. Either dermato-topical or intraperitoneal administration of these aminothiol ROS-scavenger molecules to the rats conferred 100% suppression of radiodermatitis induced by x-ray-generated ROS during irradiation of the rat skin.

(22) TABLE-US-00002 TABLE 3 Topical (or IP) aminothiol prevention of radiation-dermatitis (2) (3) Drug Ionizing Radiation-ROS- Molecule (1) Application Induced Dermatitis Name MW Drug Dose Route n (% Clear Skin.sup.a) Vehicle — — Topical 12    0% PrC-210 148  370 mM (50:30:20).sup.b Topical 10 100 1200 mM (0:90:10).sup.b 4 100  200 ug/g b.w. IP 2 100 PrC-211 120 1400 mM (50:30:20).sup.b Topical 3  55 2200 mM (0:90:10).sup.b 100  320 ug/g b.w. IP 3  87 PrC-252 105 Expt. 1  300 mM Topical 3  10  600 mM 3  45  900 mM 3  57 1800 mM 3  68 Expt. 2  450 mM Topical 3  70  181 ug/g b.w. IP 2 100 Amifostine 214  100 mM Topical 4  0 .sup.aPercentage of irradiated skin that is clear of any scab material 13 days following 17.3 Gy radiation dose to a 1.5 cm × 3.0 cm rectangle on rat's dorsal back .sup.b(ethanol:propylene glycol:water)

Example 10

(23) Experiments were conducted demonstrating that when ferrets, which had the same retch/emesis response as humans, received a subcutaneous ferret equivalent dose of the mouse 0.5 MTD dose of amifostine, that all four ferrets (data from 062 and 089 are shown here) responded with significant bouts of both retching and emesis (FIG. 10, left panel). This replicates the high incidence of nausea/emesis reported in human cases who received amifostine at the human equivalent dose of the mouse 0.5 MTD dose of amifostine.

(24) The FIG. 10, right panel shows that when 10 ferrets received a subcutaneous ferret equivalent dose of the mouse 0.5 MTD dose of PrC-210, that none of the 10 ferrets (data from 123 and A43 are shown here) responded with any discernible retching or emesis responses.

(25) As a positive control, with two weeks rest after the amifostine or PrC-210 challenge dose, all ferrets received a single challenge dose of loperamide, a known emetogen, and each of the 14 ferrets responded with strong retch and emesis responses. These data are shown as insets in FIG. 10.

Example 11

(26) Experiments were conducted demonstrating that when rats with arterial catheters to measure blood pressure received a single IP dose of the rat equivalent of the mouse 0.5 MTD dose of amifostine that an immediate and irreversible drop in blood pressure occurred, and that a challenge dose of IP epinephrine had no discernible effect upon blood pressure (FIG. 11). The chronic hypotension in the rats over the recording period was associated with a discernible ocular phenotype/toxicity.

(27) Catheterized rats that received a single IP dose of the rat equivalent of the mouse 0.5 MTD dose of PrC-210 showed no reduction in blood pressure, and a challenge dose of IP epinephrine caused a robust increase in blood pressure.

Example 12

(28) Experiments were conducted demonstrating that PrC-210 lacked the noxious odor (i.e., sulfurous odor) associated with conventional thiol compounds. Test subjects were exposed to a solution comprising PrC-210 at the upper limit of what an approximate single human dose of PrC-210 was contemplated and a dilution series of 2-mercaptoethanol (2-ME). Each subject assigned a “smell score” to the PrC-210 by comparing the smell of the PrC-210 with the 2-ME dilutions; the smell score denotes the 2-ME dilution having a sulfurous thiol smell that most closely matched the sulfurous thiol smell of the single human dose of PrC-210. One subject assigned a smell score of 8 and the other subject assigned a smell score of 7, corresponding to 1:18,750 and 1:93,750 dilutions of 2-ME. These results show that PrC-210 at a concentration of approximately a single maximum human dose has a thiol odor that is 56,250-fold lower than 2-ME (e.g., 93,750−18,750=75,000; 75,000±2=37,500; 37,500+18,750=56,250). A 56,250-fold dilution of 2-ME is nearly odor free.

(29) TABLE-US-00003 TABLE 4 2-ME Reviewer 1 Reviewer 2 Mean 2-ME Fold- PrC-210 PrC-210 PrC-210 Vial Dilution Smell Score.sup.A Smell Score.sup.A Smell Score 7.5 1 1 ∴PrC-210 single 2 5 maximum dose 3 25 has thiol odor that 4 125 is 56,250-fold 5 625 lower than 2-ME 6 3,125 i.e., 93,750 − 18,750 = 7 18,750 7 75,000 ÷ 2 = 8 93,750 8 37,500 + 18,750 = 9 468,750 56,250-fold dilution 10 2,343,750 [this is nearly odor free] .sup.Ai.e., the 2-ME (2-mercaptoethanol) vial whose “thiol odor” was scored the same as the “thiol odor” from the vial of PrC-210 which contained what was calculated to be an upper limit of what a single, human PrC-210 dose might be.

Example 13

(30) An example of a Cardioplegia Solution that is commonly used to flush a human heart, and in the process stop the heart from beating, prior to surgical manipulation of the un-beating heart in e.g., coronary bypass or valve repair surgery, includes (per liter of solution): 110 mmol sodium 16 mmol magnesium 160 mmol chloride 16 mmol potassium 1.2 mmol calcium sufficient sodium bicarbonate to achieve a pH of 7.4-7.8

Example 14

(31) An example of an organ preservation solution, here “Belzer U W Cold Storage Solution,” invented at the University of Wisconsin, which is commonly used at 4° C. to flush and maintain organs removed from donors prior to implant in the organ recipient, includes:

(32) TABLE-US-00004 BELZER UW ® COLD STORAGE SOLUTION INGREDIENT G/L MMOL/L Hydroxyethyl starch(Pentafraction) 50.0 NA Lactobionic acid (as Lactone) 35.83 105 Potassium dihydrogen phosphate 3.4 25 Magnesium sulfate heptahydrate 1.23 5 Raffinose pentahydrate 17.83 30 Adenosine 1.34 5 Allopurinol 0.136 1 Total Glutathione 0.922 3 Pottassium hydroxide* 5.61 100 Sodium hydroxide/Hydrochloric acid (adjust to pH 7.4) Water for injection q.s.

Example 15

(33) Experiments were conducted demonstrating (FIG. 12) that when mice received a single IP injection of the 0.5 MTD IP PrC-210 dose (252 ug/gm body weight) or a single oral gavage dose of the 0.5 MTD Oral PrC-210 dose ((900 ug/gm body weight) discernible plasma levels of the active form PrC-210 thiol were measurable for extended periods afterward. Plasma concentrations of 1-3 mM PrC-210 thiol were associated with complete suppression of radiation-induced death that otherwise occurred in 100% of the vehicle-treated and irradiated mice.

Items of the Invention

(34) 1. A method of reducing or preventing ischemia-reperfusion cell death in cells which are affected by an ischemic event comprising contacting the cells with a compound having the following structure

(35) ##STR00005##
wherein A=—CH.sub.2NHR′ and B=—CH.sub.2NHR or A=—NRR′ and B=H; and
wherein R and R′ are independently selected from H, alkyl, and heteroalkyl,
with the proviso that R and R′ are not both H if B=H. 2. The method of item 1 wherein the compound is a compound having the following structure:

(36) ##STR00006## wherein R and R′ are independently selected from H, alkyl, and heteroalkyl. 3. The method of item 1 wherein the compound comprises a structure according to:

(37) ##STR00007## wherein R and R′ are independently selected from H, alkyl, and heteroalkyl, with the proviso that R and R′ are not both H. 4. The method of item 1 wherein the compound is selected from PrC-210, PrC-211, and PrC-252:

(38) ##STR00008## 5. The method of item 1 wherein the reduction or prevention of cell death is by reduction or prevention of apoptosis. 6. The method of item 1 wherein caspase activity is reduced in cells contacted with the compound when compared to cells not contacted with the compound. 7. The method of any of the preceding items further comprising scavenging reactive oxygen species. 8. The method of any of the preceding items further providing protection to the cells' DNA against a reactive oxygen species. 9. The method of item 1 wherein the cells are part of a transplanted organ. 10. The method of item 9 wherein the cells are contacted with the compound before implantation into a recipient. 11. The method of item 9 wherein the compound is part of an organ preservation solution or a solution used for flushing an organ. 12. The method of item 9 wherein the compound is administered to the donor before and during organ removal. 13. The method of item 1 wherein systemic administration of the compound to a subject reduces or prevents ischemia-reperfusion cell death in the subject. 14. The method of item 13 wherein the compound protects the subject from ischemia-reperfusion organ toxicity. 15. The method of item 13 wherein the compound prevents ischemia-reperfusion cell death before and during re-perfusion. 16. The method of item 13 wherein the subject is a transplant recipient. 17. The method of item 13 wherein the subject has suffered a heart attack or is at risk of suffering a heart attack. 18. The method of item 13 wherein the subject has suffered a stroke or is in risk of suffering a stroke. 19. The method of item 13 wherein an effective amount of the compound is administered systemically at an effective time before, during or after the ischemia-reperfusion event. 20. The method of any of the preceding items, wherein the compound is in a form of an acid-addition salt. 21. The method of item 1 wherein the cells are part of a heart perfused with a cardioplegia solution as part of the surgical manipulation of the heart. 22. The method of item 1 in which the compound is added to any flush solution that is used to protect an organ from IR injury. 23. An organ perfusion solution wherein a compound as defined in item 1 is present in a concentration of from about 1 to about 100 millimolar. 24. A unit dose of a compound as defined in item 1 that constitutes a crystalline or lyophilized powder form of an acid salt of the compound in an air-evacuated vial with a penetrable septum that enables liquid reconstitution for addition to an organ preservation solution, cardioplegia solution or to an IV bag to achieve a final concentration of the compound of 1-100 mM. 25. A cardioplegia solution wherein a compound as defined in item 1 is present in a concentration of from about 1 to about 100 millimolar. 26. A dry tablet or capsule form of a compound as defined in item 1 that enables oral delivery to a patient to achieve blood plasma concentrations of 0.5-5 mM.

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