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Title:
PYRIMIDINE NUCLEOTIDE PRECURSORS FOR TREATMENT OF SYSTEMIC INFLAMMATION AND INFLAMMATORY HEPATITIS
Document Type and Number:
WIPO Patent Application WO/1994/013687
Kind Code:
A1
Abstract:
Pyrimidine nucleotide precursors including acyl derivatives of cytidine, uridine, and orotate, and uridine phosphorylase inhibitors, and their use in enhancing resistance to sepsis or systemic inflammation are disclosed.

Inventors:
VON BORSTEL REID WARREN
BAMAT MICHAEL KEVIN
HILTBRAND BRADLEY M
Application Number:
PCT/US1993/011531
Publication Date:
June 23, 1994
Filing Date:
December 01, 1993
Export Citation:
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Assignee:
PRO NEURON INC (US)
International Classes:
A61K45/00; A61K31/00; A61K31/505; A61K31/513; A61K31/515; A61K31/70; A61K31/7042; A61K31/7052; A61K31/706; A61K31/7064; A61K31/7068; A61K31/7072; A61K31/7076; A61K31/708; A61K31/715; A61K38/00; A61K45/06; A61P1/16; A61P17/00; A61P29/00; A61P31/00; A61P31/04; A61P35/00; A61P37/02; A61P37/06; A61P39/02; A61P43/00; C07H19/06; C07D239/54; C07H19/10; C07H; (IPC1-7): C07H19/067; C07H19/167; C07H19/06; A61K31/70
Foreign References:
US4874602A1989-10-17
US3868451A1975-02-25
US4757139A1988-07-12
GB1297398A1972-11-22
JPS60174797A1985-09-09
Other References:
Journal of Pharmaceutical Sciences, Volume 76, No. 2, issued February 1987, MARTIN et al., "Synthesis and Antiviral Activity of Various Esters of 9-((1,3-Dihydroxy-2-propoxy)methyl)guanine" , pages 180-183, see esp. page 180, column 1, paragraph 1 and Discussion on page 182.
Biochemical Pharmacology, Vol. 15, issued 1966, CASIDA et al., "3',5'-Diesters of 5-Fluoro-2'-Deoxyuridine and Thymidine: Hydrolysis by Esterases in Human, Mouse, and Insect Tissue", pages 627-644, see esp. pages 627-628.
Biochemical Pharmacology, Vol. 28, issued 1979, ENSMINGER et al., "Thymidine 5'-O-Pivaloate: A Prodrug Derivative of Thymidine with Potential Applications in High-Dose Methotrexate Therapy", pages 1541-1545, see esp. page 1545, last two paragraphs.
Cancer Research, Vol. 42, issued October 1982, MARTIN et al., "High-Dose 5-Fluorouracil with Delayed Uridine "Rescue" in Mice", pages 3964-3970, see esp. page 3970, second column.
CERRA et al., "Molecular and Cellular Mechanisms of Septic Shock: Role of Monokines in Altering Hepatic Metabolism in Sepsis", published 1989 by Alan R. Liss, Inc., pages 265-277.
Gastroenterology, Vol. 40, issued June 1961, SHAFER et al., "Uridine Metabolism in Chronic Liver Disease", pages 782-784, see entire document.
Aust. N.Z.J. Med., Vol. 3, issued 1973, W.J. O'SULLIVAN, "Orotic Acid", pages 417-422, see entire document.
See also references of EP 0679160A4
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Claims:
WHAT IS CLAIMED IS:
1. A method for treating or preventing tiεsue damage due to systemic inflammatory response syndrome comprising administering to an animal a therapeutically effective amount of a pyrimidine nucleotide precursor.
2. A method for treating or preventing sepsis comprising administering to an animal a therapeutically effective amount of a pyrimidine nucleotide precursor.
3. A method as in claim 2 wherein said pyrimidine nucleotide precursor iε uridine, cytidine, orotic acid, or an acyl derivative of uridine, cytidine, or orotic acid, or a pharmaceutically acceptable salt thereof.
4. A method as in claim 3 wherein said acyl derivative of uridine is triacetyluridine.
5. A method as in claim 2 wherein said animal is a human.
6. A method as in claim 2 further comprising administering an inhibitor of uridine phosphorylaεe.
7. A method for treating or preventing sepsis comprising administering to an animal a therapeutically effective amount of an inhibitor of uridine phosphorylase.
8. A method for reducing toxicity of a therapeutic cytokine or inflammatory stimuluε comprising administering to an animal a therapeutically effective amount & of a pyrimidine nucleotide precursor prior to, during, or after administration of said cytokine or said stimulus.
9. A method as in claim 8 wherein said pyrimidine nucleotide precursor iε uridine, cytidine, orotic acid, or an acyl derivative of uridine, cytidine, or orotic acid, or a pharmaceutically acceptable salt thereof.
10. A method aε in claim 9 wherein said acyl derivative of uridine is triacetyluridine.
11. A method as in claim 8 wherein said cytokine or said stimuluε iε selected from the group consiεting of interleukin 1, interleukin2, interleukin 6, tumor necroεiε factor, endotoxin, fungal polyεaccharideε, and doubleεtranded RNA.
12. A method aε in claim 8 further compriεing the εtep of ad iniεtering an inhibitor of uridine phoεphorylaεe.
13. A method for reducing toxicity of a therapeutic cytokine or inflammatory εtimuluε comprising administering to an animal a therapeutically effective amount of an inhibitor of uridine phosphorylase prior to, during, or after administering said cytokine or said stimuluε.
14. A method aε in claim 13 wherein said cytokine or εaid stimulus is selected from the group consiεting of interleukin 1, interleukin2, interleukin 6, tumor necroεiε factor, endotoxin, fungal polyεaccharideε, and doubleεtranded RNA. ?^.
15. A method for treating cancer compriεing adminiεtering to an animal a therapeutically effective amount of a therapeutic cytokine or inflammatory εtimuluε and a therapeutically effective amount of a pyrimidine nucleotide precurεor prior to, during, or after administration of said cytokine or said stimulus.
16. A method as in claim 15 wherein said pyrimidine nucleotide precurεor iε uridine, cytidine, orotic acid, or an acyl derivative of uridine, cytidine, or orotic acid, or a pharmaceutically acceptable εalt thereof.
17. A method aε in claim 16 wherein said acyl derivative of uridine is triacetyluridine.
18. A method aε in claim 15 wherein εaid cytokine or said stimulus iε εelected from the group conεiεting of interleukin 1, interleukin2, interleukin 6, tumor necroεiε factor, endotoxin, fungal polyεaccharideε, and doubleεtranded RNA.
19. A method aε in claim 15 further compriεing the εtep of adminiεtering an inhibitor of uridine phoεphorylaεe.
20. A method for treating cancer comprising administering to an animal a therapeutically effective amount of a therapeutic cytokine or inflammatory stimulus and a therapeutically effective amount of an inhibitor of uridine phosphorylaεe prior to, during, or after adminiεtering said cytokine or εaid stimuluε. 7^ .
21. A method as in claim 20 wherein said cytokine or said stimuluε iε selected from the group consiεting of interleukin 1, interleukin2, interleukin 6, tumor necrosis factor, endotoxin, fungal polysaccharides, and doubleεtranded RNA.
22. A method for treating or preventing inflammatory hepatitis comprising administering to an animal a therapeutically effective amount of an acyl derivative of uridine, cytidine or orotic acid, or a pharmaceutically acceptable salt thereof.
23. A method as in claim 22 wherein said acyl derivative of uridine is triacetyluridine.
24. A method as in claim 22 including the further step of adminiεtering an inhibitor of uridine phoεphorylaεe.
25. A method for treating or preventing inflammatory hepatitiε compriεing adminiεtering to an animal a therapeutically effective amount of an inhibitor of uridine phosphorylase.
26. A method for treating or preventing inflammatory hepatitis comprising adminiεtering to an animal a therapeutically effective amount of uridine or cytidine.
27. A method aε in claim 26 wherein from 2 to 40 gramε of uridine or cytidine are adminiεtered per day.
28. A method for treating or preventing hepatic damage in an animal receiving total parenteral nutrition 7£ comprising administering intravenously to said animal a therapeutically effective amount of a pyrimidine nucleotide precurεor.
29. A method aε in claim 28 wherein said pyrimidine nucleotide precursor is uridine, cytidine, orotic acid, or an acyl derivative of uridine, cytidine, or orotic acid, or a pharmaceutically acceptable salt thereof.
30. A method as in claim 28 wherein from 2 to 40 grams of said pyrimidine nucleotide precursor are adminiεtered per day.
31. A method as in claim 28 including the further step of administering an inhibitor of uridine phosphorylaεe.
32. A method for treating or preventing hepatic damage in an animal receiving total parenteral nutrition compriεing adminiεtering to said animal an inhibitor of uridine phosphorylaεe.
33. A method for treating or preventing hepatic damage in an animal receiving a liver tranεplant comprising administering to said animal a therapeutically effective amount of a pyrimidine nucleotide precurεor.
34. A method aε in claim 33 wherein said pyrimidine nucleotide precurεor iε uridine, cytidine, orotic acid, or an acyl derivative of uridine, cytidine, or orotic acid, or a pharmaceutically acceptable εalt thereof.
35. A method as in claim 33 wherein from 2 to 40 grams of said pyrimidine nucleotide precursor are administered per day.
36. A method as in claim 33 including the further step of administering an inhibitor of uridine phosphorylaεe.
37. A method for treating or preventing hepatic damage in an animal receiving a liver tranεplant compriεing adminiεtering to εaid animal an inhibitor of uridine phoεphorylaεe.
38. A compoεition compriεing: a) an acyl derivative of a pyrimidine nucleotide precurεor and; b) an inhibitor of uridine phoεphorylase.
39. A composition comprising: a) an acyl derivative of a pyrimidine nucleotide precursor and; b) a purine nucleotide precurεor.
40. A compoεition aε in claim 39 where said pyrimidine nucleotide precursor iε uridine, cytidine, or orotate.
41. A compoεition aε in claim 39 where said purine nucleotide precursor is inosine, adenosine, or an acyl derivative of inoεine or adenoεine.
Description:
Title of the Invention

PYRIMIDINE NUCLEOTIDE PRECURSORS

FOR TREATMENT OF SYSTEMIC INFLAMMATION

AND INFLAMMATORY HEPATITIS

Field of the Invention

This invention relates generally to pyri idine nucleotide precursors including acyl derivatives of cytidine, uridine and orotate, and to the prophylactic and therapeutic uses of these compounds. The invention also relates to the administration of these compounds, alone or in combinations, with or without other agents, to animals. These compounds are capable of enhancing resistance of an animal to bacterial endotoxin and other inflammatory stimuli, and inflammatory mediators.

Background of the Invention

Sepsis, also referred to as sepsis syndrome, is a consequence of serious infection by bacteria, fungi, or viruses. Sepsis accounts for tens of thousands of deaths in the United States every year; it is a leading cause of death of patients in surgical intensive care units.

Sepsis is an inflammatory disorder in which endogenous cytokines and other bioactive molecules, produced or released in response to an inflammatory stimulus such as bacterial endotoxin (a component of the cell wall of gram-negative bacteria), cause various symptoms including fever, neutropenia, blood coagulation disorders, hypotension, shock, and organ damage.

Sepsis (or in its more severe form, septic shock), is one example of a broader class of disease called the "Systemic Inflammatory Response Syndrome" (SIRS), which is an organism's reaction to inflammatory stimuli such as endotoxin (which can be present in the bloodstream without bactere ia, e.g. due to leakage of endotoxin from gram-negative bacteria into the circulation from a localized infection or from the intestine); SIRS can also be triggered by gram-positive bacteria, fungi, viruses, and can also be a consequence of autoimmune disorders or administration of therapeutic inflammatory cytokines.

Current treatment of SIRS involves circulatory and respiratory support, but does not directly address improvement of tissue resistance to inflammatory stimuli such as endotoxin, or inflammatory mediators.

Monoclonal antibodies for neutralizing endotoxins or mediators of its physiologic effects are under development. However, it is expensive or impractical to use antibodies as prophylaxis in susceptible patients, prior to the onset of symptoms of endotoxin poisoning. Moreover, it is difficult to determine which patients are likely to benefit from antibody treatment, since the time required to culture and identify

infectious organisms often exceeds the time limit for implementation of effective therapy. Similar problems have been encountered in attempts to use receptor antagonists of specific inflammatory meidators like interleukin-1.

Endotoxin toxicity is in part mediated by endogenous cytokines and other bioactive molecules released from macrophageε, Kupffer cells (sessile macrophages in the liver) and other cell types in response to endotoxin. Among the most significant of these mediators are tumor necrosis factor (TNF) and interleukin-1 (IL-1). Others include platelet activating factor (PAF) , interleukin-6, and leukotrienes and other arachidonic acid derivatives. Administration of these cytokines or mediators results in symptoms similar to at least some of those elicited by endotoxin. Agents or pathological conditions other than bacterial endotoxin can result in elevated production or activity of (or sensitivity to) TNF or

IL-1, resulting in tissue damage. Such conditions include infection with gram-positive bacteria, viruses or fungi, or liver damage. Inflammatory cytokines can produce tissue damage if present in excess, but when elicited in moderate amounts, they are important in the defense against infectious organisms or viruses. For example, antibodies to TNF can reduce toxicity of an administered dose of endotoxin (by blocking the negative effects of TNF elicited by the endotoxin), but can have a deleterious effect in the case of some bacterial infections, converting a sublethal state of infection into an overwhelming lethal infection (Havell, J

Immunol. f 1987, 139:4225-4231; Echtenacher et al., ^

Immunol.. 1990 145:3762-3766). Thus, there are inherent

problems with strategies for treating sepsis syndrome or SIRS with agents which directly inactivate inflammatory cytokines.

The liver is a major site for clearance or detoxification of endotoxin (Farrar and Corwin, Ann. N.Y. Acad. Sci.. 1966 133:668-684) and inflammatory proteins like TNF; conversely, the liver is susceptible to damage by endotoxin and its mediators. Liver damage from many originating causes (e.g. carbon tetrachloride, choline deficiency, viral infection, Reye's syndrome, alcohol) is in part mediated by bacterial endotoxin or mediators elicited by endotoxin even when symptoms of systemic sepsis are not present (Nolan, Gastroenterology, 1975, 69:1346-1356; Nolan, Hepatoloσy f 1989, 10:887-891). Hepatic toxicity is dose-limiting in patients receiving intentional injections of endotoxin for possible efficacy in treating cancer (Engelhardt et al.. Cancer Research, 1991, 51:2524-2530). The liver has been reported to be the first vital organ displaying pathological alterations in septic shock (Kang et al., J. Histoche . Cytochem.. 1988 36:665-678). Moreover, hepatic dysfunction occurs in the early stages of sepsis and may initiate sequential organ failure (Wang et al.. Arch. Surσ.. 1991, 126:219-224)

The liver is important in regulating the sensitivity of an animal to endotoxin. Various treatments which impair liver function or metabolism, such as poisoning with lead acetate, cycloheximide, Actinomycin D or galactosamine can increase the sensitivity of animals to endotoxin or TNF, in some cases by several orders of magnitude.

9

Galactosamine-induced liver damage is unique in that it is readily reversible during a period before cell death occurs. Galactosamine selectively depletes hepatic uridine nucleotides, by locking them into UDP-hexosamines that are not converted back into free nucleotides. This can lead to liver damage if the depletion of uridine nucleotides is sufficiently prolonged, due to impairment of RNA and protein synthesis. The biochemical deficiency induced by galactosamine is readily reversed by administration of uridine, which replenishes the uridine nucleotides trapped by the galactosamine. Thus, administration of uridine shortly before or after administration of galactosamine attenuates galactosamine- induced hepatic damage and consequently restores sensitivity to endotoxin toward normal values (Galanos et al., PNAS f 1979, 76:5939-5943).

Similarly, endotoxin hypersensitivity in mice deliberately treated with the rodent hepatotoxin TCDD was partially reversed by administration of uridine (Rosenthal et al.. Toxicology, 1989 56:?.39-251).

However, in contrast to these situations wherein uridine partially restored experimentally-reduced resistance to endotoxin, uridine was reported to have no protective effect in normal mice challenged with endotoxin (Markley et al., J. Trauma 1970, 10:598-607), i.e., it did not result in greater- than-nor al resistance to endotoxin.

Uridine, cytidine, and orotate have been tested for effects on liver function in hepatic disorders and in experimental models, with mixed results. Shafer and

S

Isselbacher (Gastroenterology 1961, 40:782-784) reported that daily intravenous infusion of 25 to 100 milligrams of cytidine and uridine, for 3 to 7 days, to patients with hepatic cirrhosis had no effect on clinical status. Orotic acid added to rat diet in a concentration of 1 percent results in fatty infiltration of the liver (von Euler et al, J. Biol. Chem.. 1963, 238:2464-2469); orotic acid administered by intraperitoneal injection reduced liver damage in rats treated with carbon tetrachloride, dichloroethane, DDT, and 9,10- dimethyl-l,2-benzanthracene (Pates et al., Farmakol Toksikol. , 1968, 31:717-719). Lysine-orotate potentiated the toxicity of hepatotoxic extracts from the mushroom Amanita Phalloides; sodium orotate and orotic acid had no effect on Amanita extract toxicity (Halacheva et al., Toxicon, 1988, 26:571-576). Orotic acid has been administered clinically to humans for treatment of neonatal hyperbilirubinemia and for improving recovery from myocardial infarction (O'Sullivan, Aust. N.Z. J. Med.. 1973, 3:417-422). Orotate is not well absorbed after oral administration, in part due to poor solubility.

Clinical trials involving the administration of uridine

(e.g. for the purpose of attenuating host toxicity of the antineoplastic drug 5-fluorouracil) have been complicated due to the biological properties of uridine itself. Uridine is poorly absorbed after oral administration; diarrhea is dose limiting in humans (van Groeningen et al.. Proceedings of the

AACR, 1987, 28:195). Parenteral administration of uridine requires use of a central venous catheter (with consequent discomfort and risk of infection), since phlebitis was a

problem in early clinical trials when uridine was administered via a brachial venous catheter (van Groeningen et al. Cancer Treat Rep.. 1986, 70:745-50).

Administration of acyl derivatives of uridine and cytidine, which are readily absorbed from the gut into the bloodstream, and which are then hydrolyzed to yield free uridine or cytidine in the circulation, overcome the problem of poor oral absorption of the free nucleosides (US Patent Applications Serial No. 438,493, 115,929, and 903,107, hereby incorporated by reference).

Objects of the Invention

It is a primary object of the invention to provide therapeutic and prophylactic agents which are effective in improving survival and in preventing tissue damage from systemic inflammatory response syndrome, including sepsis.

It is a primary object of this invention to provide a family of compounds which effectively enhance resistance to systemic inflammation. Administration of these compounds to an animal before, during or after exposure to endotoxin or other inflammatory stimuli, prevents or treats the effects of systemic inflammation.

It is a further object of this invention to provide a family of compounds for the treatment of a variety of disorders involving inflammatory stimuli or inflammatory cytokines in their etiology.

O 94/136 7

It is a further object of this invention to provide a family of compounds to improve survival or physiological functions in animals subjected to endotoxin poisoning or other systemic inflammatory disorders.

It is a further object of the invention to provide a family of compounds to treat or prevent inflammatory hepatitis.

It is a further object of the invention to provide compounds which can be administered orally or parenterally.

Summary Of The Invention

These and other objects of the invention are achieved by precursors of pyri idine nucleotides such as orotic acid or its salts, uridine, cytidine, or prodrug derivatives of these agents including acyl derivatives or phosphate esters, which can be administered to animals, including mammals such as humans. The administration of these compounds alone, or in combination, is useful in treatment or prevention of consequences of systemic inflammation. Systemic inflammation is caused by infection with bacteria, fungi, or viruses, constituents of bacteria, fungi or viruses, e.g. endotoxin, polysaccharides or viral proteins respectively, by inflammatory mediators, or as a consequence of autoimmune disorders.

Thus, the compounds of the invention, alone or in combination, are useful in the treatment and prevention of sepsis or toxic effects of inflammatory cytokines; are useful as prophylactic agents in patients at risk of sepsis e.g.

patients undergoing surgical procedures, or afflicted with serious burns or wounds, or immunocompromised as a consequence of chemotherapy for cancer or other diseases.

An important aspect of this invention is the discovery that pyrimidine nucleotide precursors such as orotate, uridine, or cytidine, and acyl derivatives of such compounds, have unexpected therapeutic properties.

One embodiment of the invention involves the use of the compounds and compositions of the invention in treatment and prevention of toxicity encountered during therapeutic administration of inflammatory cytokines, e.g. for treatment of cancer.

One embodiment of the invention involves the use of the compounds and compositions of the invention in treatment and prevention of inflammatory hepatitis.

Compounds of the Invention

The compounds useful in enhancing resistance to inflammatory stimuli or inflammatory mediators have the following structures:

In all cases except where indicated, letters and letters with subscripts symbolizing variable substituents in the chemical structures of the compounds of the invention are applicable only to the structure immediately preceding the description of the symbol.

(1) Uridine or an acyl derivative of uridine having the formula:

wherein R., R 2 , R 3 and R 4 are the same or different and each is hydrogen or an acyl radical of a metabolite, or a pharmaceutically acceptable salt thereof.

(2) Cytidine or an acyl derivative of cytidine having the formula;

wherein R 2 , R 2 , R 3 and R 4 are the same or different and each is hydrogen or an acyl radical of a metabolite or a pharmaceutically acceptable salt thereof.

(3) An acyl derivative of uridine having the formula:

/ *

wherein R x , R 2 , and R 3 are the same, or different, and each is hydrogen or an acyl radical of a. an unbranched fatty acid with 5 to 22 carbon atoms, b. an amino acid selected from the group consisting of glycine, the L forms of alanine, valine, leucine, isoleucine, tyrosine, proline, hydroxyproline, serine, threonine, cystine, cysteine, aspartic acid, glutamic acid, arginine, lysine, histidine, carnitine and ornithine, c. a dicarboxylic acid having 3-22 carbon atoms, d. a carboxylic acid selected from one or more of the group consisting of glycolic acid, pyruvic acid, lactic acid, enolpyruvic acid, lipoic acid, pantothenic acid, acetoacetic acid, p-aminobenzoic acid, betahydroxybutyric acid, orotic acid, and creatine.

(4) An acyl derivative of cytidine having the formula:

//

94/13687

wherein R α , R 2 , R 3 , and R 4 are the same, or different, and each is hydrogen or an acyl radical of a. an unbranched fatty acid with 5 to 22 carbon atoms, b. an amino acid selected from the group consisting of glycine, the L forms of phenylalanine, alanine, valine, leucine, isoleucine, tyrosine, proline, hydroxyproline, serine, threonine, cystine, cysteine, aspartic acid, glutamic acid, arginine, lysine, histidine carnitine and ornithine, c. a dicarboxylic acid having 3-22 carbon atoms, d. a carboxylic acid selected from one or more of the group consisting of glycolic acid, pyruvic acid, lactic acid, enolpyruvic acid, lipoic acid, pantothenic acid, acetoacetic acid, p-aminobenzoic acid, betahydroxybutyric acid, orotic acid, and creatine.

(5) An acyl derivative of uridine having the formula:

/Z

wherein at least one of R 2 , R 2 , or R 3 is a hydrocarbyloxycarbonyl moiety containing 2-26 carbon atoms and the remaining R substituents are independently a hydrocarbyloxycarbonyl or hydrocarbylcarbonyl moiety or H or phosphate.

(6) An acyl derivative of cytidine having the formula:

wherein at least one of R χ/ R 2 , R 3 or R 4 is a hydrocarbyloxycarbonyl moiety containing 2-26 carbon atoms and the remaining R substituents are independently a hydrocarbyloxycarbonyl or hydrocarbylcarbonyl moiety or H or phosphate.

(7) Orotic acid or salts thereof:

Pharmaceutically-acceptable salts of orotic acid include those in which the cationic component of the salt is sodium, potassium, a basic amino acid such as arginine or lysine, methylglucamine, choline, or any other substantially nontoxic water soluble cation with a molecular weight less than about 1000 daltons.

8) Alcohol-substituted orotate derivatives:

wherein R. is a radical of an alcohol containing 1 to 20 carbon atoms joined to orotate via an ester linkage.

/ -

Also encompassed by the invention are the pharmaceutically acceptable salts of the above-noted compounds.

Advantageous compounds of the invention are short-chain fatty acid esters of uridine or cytidine. Particularly advantageous compounds are triacetyluridine, triacetylcytidine or salts of orotic acid.

Inhibitors of uridine phosphorylase

As an alternative or adjunct to the above-noted pyrimidine nucleotide precursors, the following compounds are useful in the invention. These agents elevate tissue uridine nucleotide levels by inhibiting catabolism of endogenous or exogenous uridine. Co-administration of uridine phosphorylase inhibitors with pyrimidine nucleotide precursors reduces the amount of nucleotide precursor required to obtain therapeutic benefit.

Examples of inhibitors of uridine phosphorylase include but are not limited to 5-benzyl barbiturate or 5-benzylidene barbiturate derivatives including 5-benzyl barbiturate, 5- benzyloxybenzyl barbiturate, 5-benzyloxybenzyl-l-[(1- hydroxy-2-ethoxy)methyl] barbiturate, 5- benzy1oxybenzy1acety1-1-[(1-hydroxy-2-ethoxy)methyl] barbiturate, and 5-methoxybenzylacetylacyclobarbiturate,

2,2'-anhydro-5-ethyluridine, and acyclouridine compounds, particularly 5-benzyl substituted acyclouridine congeners including but not limited to benzylacyclouridine, benzyloxy- benzylacyclouridine, aminomethyl-benzylacyclouridine, -ST

aminomethylbenzyloxybenzylacyclouridine, hydroxymethyl- benzylacyclouridine, and hydroxymethyl-benzyloxybenzyl- acyclouridine. See also WO 89/09603 and WO 91/16315, hereby incorporated by reference.

Detailed Description of the Invention

The subject invention relates to pyrimidine nucleotide precursors including acyl derivatives of cytidine, uridine, and orotate, and the use of these compounds and/or uridine phosphorylase inhibitors for treating or preventing pathological consequences of endotoxin and other inflammatory stimuli or mediators in animals, including humans.

The invention disclosed herein involves methods for enhancing the resistance of an animal to inflammatory stimuli and mediators. Examples presented below demonstrate both prophylaxis and treatment of toxicity due to endotoxin and other inflammatory stimuli. The method of the invention can be used in conjunction with other methods for treating or preventing sepsis or systemic inflammation.

A. Definitions

The term "pyrimidine nucleotide precursor" as used herein refers to a compound which is converted to a pyrimidine nucleotide following administration to an animal. This includes especially cytidine, uridine, or orotic acid, or prodrugs (including acyl derivatives) of these compounds.

The term "acyl derivative" as used herein means a derivative of a pyrimidine nucleoside in which a substantially

nontoxic organic acyl substituent derived from a carboxylic acid is attached to one or more of the free hydroxyl groups of the ribose moiety of the oxypurine nucleoside with an ester linkage and/or where such a substituent is attached to the amine substituent on the purine ring of cytidine, with an amide linkage. Such acyl substituents are derived from carboxylic acids which include, but are not limited to, compounds selected from the group consisting of a fatty acid, an amino acid, nicotinic acid, dicarboxylic acids, lactic acid, p-aminobenzoic acid and orotic acid. Advantageous acyl substituents are compounds which are normally present in the body, either as dietary constituents or aε intermediary metabolites.

The term "pharmaceutically acceptable salts" as used herein means salts with pharmaceutically acceptable acid addition salts of the derivatives, which include, but are not limited to, sulfuric, hydrochloric, or phosphoric acids.

The term "coadministered" means that at least two of the compounds of the invention are administered during a time frame wherein the respective periods of pharmacological activity overlap.

The term "amino acids" as used herein includes, but is not limited to, glycine, the L forms of alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, proline, hydroxyproline, serine, threonine, cysteine, cystine, methionine, tryptophan, aspartic acid, glutamic acid, arginine, lysine, histidine, ornithine, hydroxylysine, carnitine, and other naturally occurring amino acids.

/7

The term "fatty acids" as used herein means aliphatic carboxylic acids having 2-22 carbon atoms. Such fatty acids may be saturated, partially saturated or polyunsaturated.

The term "dicarboxylic acids" as used herein means fatty acids with a second carboxylic acid substituent.

The term "therapeutically effective amount" as used herein refers to that amount which provides therapeutic effects for a given condition and administration regimen.

The term "sepsis" as used herein is a systemic inflammatory disorder in which endogenous cytokines and other bioactive molecules, produced or released in response to an inflammatory stimulus such as bacterial endotoxin (a component of the cell wall of gram-negative bacteria), cause various symptoms including fever, neutropenia, blood coagulation disorders, hypotension, shock, and organ damage.

The term "inflammatory stimulus" as used herein means an exogenous agent which triggers an inflammatory response in an animal. Examples of inflammatory stimuli include bacteria, fungi, viruses, nonviable fragments or components of bacteria (such as endotoxin), fungi or viruses, or agents which trigger allergic or anaphylactic responses. In the case of autoimmune disorders, endogenous elements of a patient's tissues, e.g. particular cellular proteins function as inflammatory stimuli.

The term "mediator" as used herein means endogenous or exogenous (e.g. recombinant polypeptides) bioactive compounds, proteins, or polypeptides that typically mediate the biological effects of" endotoxin or other inflammatory stimuli

such as fungal polysaccharides. Examples of such agents include but are not limited to tumor necrosis factor (TNF), interleukin-1 (IL-1), interleukin-6 (IL-6), plasminogen activator inhibitor (PAI), leukotrienes, elements of the complement cascade, nitric oxide, or platelet-activating factor.

B. Compounds of the Invention

A primary feature of the present invention is the unexpected discovery that uridine and other pyrimidine nucleotide precursors do in fact protect otherwise normal animals (e.g. animal models in which the organism has not received a clinically-irrelevant hepatotoxic sensitizing agent like galactosamine or TCDD) from toxicity due to bacterial endotoxin and other inflammatory stimuli which produce tissue damage through elicitation of endogenous inflammatory mediators.

Tissue uridine nucleotide levels can be increased by administration of several precursors. Uridine and cytidine are incorporated into cellular nucleotide pools by phosphorylation at the 5' position; cytidine and uridine nucleotides are interconvertible through enzymatic amination and de-amination reactions. Orotic acid is a key intermediate in de novo biosynthesis of pyrimidine nucleotides. Incorporation of orotic acid into nucleotide pools requires cellular phosphoribosyl pyrophosphate (PRPP). Alternatively (or in addition to provision of exogenous nucleotide precursors), availability of uridine to tissues is increased

/* "

by administration of compounds which inhibit uridine phosphorylase, the first enzyme in the pathway for degradation of uridine. The compounds of the invention useful in enhancing resistance to endotoxin or inflammatory mediators include uridine, cytidine, orotate, prodrug forms of these pyrimidine nucleotide precursors, particularly acyl derivatives and phosphate esters, and inhibitors of the enzyme uridine phosphorylase. Compounds of the invention have the following structures:

In all cases except where indicated, letters and letters with subscripts symbolizing variable substituents in the chemical structures of the compounds of the invention are applicable only to the structure immediately preceding the description of the symbol.

(1) An acyl derivative of uridine having the formula:

wherein R x , R 2 , R 3 and R 4 are the same or different and each is hydrogen or an acyl radical of a metabolite, provided that at least one of said R substituents is not hydrogen, or a pharmaceutically acceptable salt thereof.

(2) An acyl derivative of cytidine having the formula:

wherein R x , R 2 , R 3 and R 4 are the same or different and each is hydrogen or an acyl radical of a,metabolite, provided that at least one of said R substituents is not hydrogen, or a pharmaceutically acceptable salt thereof.

The compounds of the invention useful in enhancing resistance to endotoxin include:

(3) An acyl derivative of uridine having the formula:

wherein R. , R 2 , and R 3 are the same, or different, and each is hydrogen or an acyl radical of a. an unbranched fatty acid with 5 to 22 carbon atoms, b. an amino acid selected from the group consisting of glycine, the L forms of alanine, valine, leucine, isoleucine, tyrosine, proline, hydroxyproline, serine, threonine, cystine, cysteine, aspartic acid, glutamic acid, arginine, lysine, histidine, carnitine and ornithine, c. a dicarboxylic acid having 3-22 carbon atoms, d. a carboxylic acid selected from one or more of the group consisting of glycolic acid, pyruvic acid, lactic acid, enolpyruvic acid, lipoic acid, pantothenic acid, acetoacetic acid, p-aminobenzoic acid, betahydroxybutyric acid, orotic acid, and creatine.

(4) An acyl derivatives of cytidine having the formula:

wherein R α , R 2 , R 3 , and R 4 are the same, or different, and each is hydrogen or an acyl radical of a. an unbranched fatty acid with 5 to 22 carbon atoms, b. an amino acid selected from the group consisting of glycine, the L forms of phenylalanine, alanine, valine,

leucine, isoleucine, tyrosine, proline, hydroxyproline, serine, threonine, cystine, cysteine, aspartic acid, glutamic acid, arginine, lysine, histidine carnitine and ornithine, c. a dicarboxylic acid having 3-22 carbon atoms, d. a carboxylic acid selected from one or more of the group consisting of glycolic acid, pyruvic acid, lactic acid, enolpyruvic acid, lipoic acid, pantothenic acid, acetoacetic acid, p-aminobenzoic acid, betahydroxybutyric acid, orotic acid, and creatine.

(5) An acyl derivative of uridine having the formula:

wherein at least one of R x , R 2 , or R 3 is a hydrocarbyloxycarbonyl moiety containing 2-26 carbon atoms and the remaining R substituents are independently a hydrocarbyloxycarbonyl or hydrocarbylcarbonyl moiety or H or phosphate.

(6) An acyl derivative of cytidine having the formula:

wherein at least one of R x , R 2 , R 3 or R 4 is a hydrocarbyloxycarbonyl moiety containing 2-26 carbon atoms and the remaining R substituents are independently a hydrocarbyloxycarbonyl or hydrocarbylcarbonyl moiety or H or phosphate.

(7) Orotic acid or salts thereof:

Pharmaceutically-acceptable salts of orotic acid include those in which the cationic component of the salt is sodium, potassium, a basic amino acid such as arginine or lysine, methylglucamine, choline, or any other substantially nontoxic water soluble cation with a molecular weight less than about 1000 daltons.

»A

8) Alcohol-substituted orotate derivatives:

wherein R is a radical of an alcohol containing 1 to 20 carbon atoms joined to orotate via an ester linkage.

Also encompassed by the invention are the pharmaceutically acceptable salts of the above-noted compounds.

Advantageous compounds of the invention are short-chain fatty acid esters of uridine or cytidine. Particularly advantageous compounds are triacetyluridine or triacetylcytidine.

Inhibitors of uridine phosphorylase

Examples of inhibitors of uridine phosphorylase include but are not limited to 5-benzyl barbiturate or 5-benzylidene barbiturate derivatives including 5-benzyl barbiturate, 5- benzyloxybenzyl barbiturate, 5-benzyloxybenzyl-l-[ (1- hydroxy-2-ethoxy)methyl] barbiturate, 5- benzyloxybenzylacetyl-l-[(l-hydroxy-2-ethoxy)methyl] barbiturate, and 5-methoxybenzylacetylacyclobarbiturate, 2,2'-anhydro-5-ethyluridine, and acyclouridine compounds,

particularly 5-benzyl substituted acyclouridine congeners including but not limited to benzylacyclouridine, benzyloxy- benzylacyclouridine, a inomethyl-benzylacyclouridine, aminomethylbenzyloxybenzylacyclouridine, hydroxymethyl- benzylacyclouridine, and hydroxymethyl-benzyloxybenzyl- acyclouridine. See also WO 89/09603 and WO 91/16315, hereby incorporated by reference.

Compositions of the Invention

In one embodiment of the invention, novel pharmaceutical compositions comprise as an active agent one or more pyrimidine nucleotide precursors selected from the group comprised of uridine, cytidine or orotic acid or its salts, and acyl derivatives of these pyrimidine nucleotide precursors, together with a pharmaceutically acceptable carrier.

The compositions, depending on the intended use and route of administration, are manufactured in the form of a liquid, a suspension, a tablet, a capsule, a dragee, an injectable solution, or a suppository (see discussion of formulation below) .

In another embodiment of the invention, the composition comprises at least one pyrimidine nucleotide precursor and an agent which inhibits the degradation of uridine, such as an inhibitor of the enzyme uridine phosphorylase. Examples of inhibitors of uridine phosphorylase include but are not limited to 5-benzyl barbiturate or 5-benzylidene barbiturate derivatives including 5-benzyl barbiturate, 5-benzyloxybenzyl

barbiturate, 5-benzyloxybenzyl-l-[ (l-hydroxy-2-ethoxy)methyl] barbiturate, 5-benzyloxybenzylacetyl-l-[ (l-hydroxy-2- ethoxy) ethyl] barbiturate, and 5-methoxybenzylacetyl- acyclobarbiturate, 2,2'-anhydro-5-ethyluridine, and acyclouridine compounds, particularly 5-benzyl substituted acyclouridine congeners including but not limited to benzylacyclouridine, benzyloxybenzylacyclouridine, aminomethyl-benzylacyclouridine, aminomethylbenzyloxybenzyl- acyclouridine, hydroxymethyl-benzylacyclouridine, and hydroxymethyl-benzyloxybenzylacyclouridine. See also US 5,077,280 and WO 91/16315, hereby incorporated by reference. Furthermore, it is within the scope of the invention to utilize an inhibitor of uridine phosphorylase alone, without coadministration of a pyrimidine nucleotide precursor, for the purpose of improving tissue resistance to endotoxin or inflammatory mediators.

In another embodiment, the compounds of the invention include in addition to one or more compounds of the invention, and at least one of the following compounds which are also useful for treating endotoxin toxicity or sepsis: Antibodies or other proteins which bind to endotoxin, TNF or IL-1; Polymyxin B conjugated to a polymeric support matrix (in order to reduce Polymyxin B toxicity while taking advantage of its capacity to bind and inactivate endotoxin); antagonists of IL-1 or TNF receptors; antibiotics; inhibitors of the arachidonic acid cascade; arginine or ornithine; corticosteroids; glucose; ATP; a purine nucleotide precursor including inosine, adenosine, or an acyl derivative thereof; cyclic AMP or acyl derivatives thereof.

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In another embodiment of the invention, the composition comprises at least one compound of the invention and an antibacterial, antifungal, or antiviral compound.

Therapeutic Uses of the Compounds and Compositions of the Invention

The compounds, compositions, and methods of the invention are useful to enhance resistance to endotoxin or other inflammatory stimuli or mediators in animals. The compounds include pyrimidine nucleotide precursors as well as compounds which inhibit enzymatic degradation of uridine.

The compounds of the invention are useful in treating humans; however, the invention is not intended to be so limited, it being within the contemplation of the invention to treat all animals that experience a beneficial effect from the administration of the active compounds of the invention.

A primary feature of the invention is the discovery that administration of uridine nucleotide precursors results in supra-normal resistance to toxic or lethal effects of endotoxin or other inflammatory stimuli or mediators in vivo.

The invention is furthermore embodied in the oral or systemic administration of a pharmaceutical compound or composition containing pyrimidine nucleotide precursors (or their prodrugs) and/or agents which inhibit uridine catabolism, for the purpose of enhancing resistance to endotoxin, other inflammatory stimuli, or their mediators.

SIRS, Sepsis and septic shock

The compounds, compositions, and methods of the invention are useful for reducing tissue damage due to systemic inflammatory response syndrome (SIRS), including sepsis, triggered by bacterial (both gram-positive and gram-negative), viral, fungal, or parasitic (e.g. malaria) organisms. All of these types of infective organisms stimulate the formation or release of endogenous inflammatory mediators, resulting in tissue damage.

The compounds of the invention are administered to patients with symptoms of sepsis, e.g. fever, neutropenia, hypotension, etc., or proph lactically to patients at risk for sepsis, e.g. surgical patients, patients with serious burns or wounds, or patients with urinary tract catheters.

The compounds, compositions, and methods of the invention are optionally administered in conjunction with other agents which are useful in treating sepsis, including but not limited to one or more of the following: Antibodies or other proteins which bind to endotoxin, TNF or IL-1; Polymyxin B conjugated to a polymeric support matrix (in order to reduce Polymyxin B toxicity while taking advantage of its capacity to bind and inactivate endotoxin); antagonists of IL-1 or TNF receptors; antibiotics; inhibitors of the arachidonic acid cascade; leukotriene antagonists arginine or ornithine; corticosteroids; glucose; ATP; inosine; cyclic AMP or acyl derivatives thereof. The compounds of the invention are administered either before, after, or during exposure of the animal or patient to one or more of these other agents.

2^

For treatment or prevention of tissue damage due to sepsis, doses of the compounds of the invention ranging from about 0.5 to about 40 grams per day, advantageously 3 to 30 grams per day, are administered, depending on the therapeutic response and the condition of the patient. In patients with serious sepsis syndrome, the compounds of the invention are typically administered in liquid or suspension form via a nasogaεtric tube, especially if such a tube is already in place for delivery of nutrient suspensions or other enteral nutrition products. Patients with less serious illness typically receive compounds of the invention in either liquid form, or in capsules or tablets.. Patients who do not tolerate oral administration of the compounds of the invention (e.g. patients on total parenteral nutrition due to gastrointestinal tract damage) receive compounds of the invention that are sufficiently water soluble, such as uridine itself, by intravenous infusion.

Following an episode of shock, trauma or sepsis, patients often enter into a persistent state of hypermetaboliεm which can lead to multiple organ failure, usually beginning with hepatic failure. The hypermetabolic phase is due to the influence of endotoxin and its mediators on metabolic regulation (Cerra et al., in Molecular and Cellular Mechanisms Of Septic Shock, 265-277, Alan R. Liss, 1989). Hypermetabolism-organ failure is one of the leading causes of mortality among surgical intensive care patientε. Aε demonεtrated in the Exampleε, the compounds of the invention are effective in reducing tissue damage and improving survival in animals subjected to endotoxin or other inducerε of sepsis

__

and organ failure. The compounds, compositions, and methods of the invention are useful in the treatment of patientε at riεk for hyper etabolic organ failure.

A serious consequence of sepsiε is a propensity toward coagulation disorderε, especially disseminated intravascular coagulation (DIC). In DIC, both blood coagulation and fibrinolyεis are activated, so that blood clotting factors are rapidly consumed and aggregates of thrombin form in the circulation. DIC can result in either (or both) hemorrhage or thrombus formation. The liver iε the primary site for syntheεis of clotting factors and for clearing micro- aggregates of thrombin from the circulation. The protective and therapeutic effects of the compounds, compositions, and methods of the invention attenuate sepsis-induced alterations in blood coagulation (see Example 11).

Reduction of toxicity of therapeutic cytokines

Many of the biological effects of endotoxin and other inflammatory εtimuli are mediated by the release of endogenous bioactive molecules (mediators) from target cellε, particularly acrophageε and Kupffer cells (sessile macrophages in the liver). Evidence for thiε iε that macrophageε in the C3H/HEJ εtrain of mice are genetically non-responsive to endotoxin (in terms of releasing cytokines upon exposure to endotoxin), and endotoxin is relatively non¬ toxic in this strain. These mice are however sensitive to bioactive peptides normally released from macrophages, e.g. tumor necrosiε factor (TNF), and toxicity of LPS iε restored by transplantation of normal macrophages. TNF is generally

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held to be a primary mediator of endotoxin toxicity, but interleukin-1 (IL-1) and other agents also participate in the expresεion of endotoxin toxicity and sepsis..

Compounds, compositions, and methods of the invention are thus useful in modifying biological effects of inflammatory cytokineε, whether produced endogenously (especially from macrophages) , or introduced into the body from exogenous sources (e.g. polypeptides produced by reco binant DNA and fermentation technology) .

Various inflammatory cytokineε and even endotoxin itεelf have potential therapeutic applicationε. Tumor necroεis factor, as suggeεted by itε name, can deεtroy tumorε and εynergizeε with interferon-alpha in inhibiting viral infectionε. Thuε, TNF, and even bacterial endotoxin itεelf (which elicits the release of endogenous TNF), have been administered to patients for the treatment of cancer. Clasεeε of inflammatory cytokineε with both therapeutic activity and toxicity which limitε their clinical use include TNF, interleukins and interferonε. The compoundε, compositionε and methodε of the invention are uεeful in preventing or treating toxicity which occurε during therapeutic administration of such cytokines as well as inflammatory εtimuli.

When endotoxin iε adminiεtered to cancer patients by intravenous infusion, hepatic toxicity limitε the dose of endotoxin which can be administered (Engelhardt R et al.. Cancer. Reε. 1991 51:2524-30). In non-hepatic cancerε, protection of the liver from endotoxin permits administration of higher doses of endotoxin in order to maximize its

antitu or efficacy. Endotoxin also has immunostimulant properties. The compounds of the invention are thus useful for improving the therapeutic index of endotoxin, endotoxin analogs or derivatives (e.g. Lipid A, Lipid X, Monophosphoryl Lipid A, etc.) or their mediators. Hepatic toxicity is also dose-limiting during intentional administration of TNF to humans (Kimura et al.,Cancer Che other. Pharmacol. 1987, 20:223-229). Inflammatory stimuli of yeast or fungal origin, εuch aε the polysaccharides glucan or lentinan are also used therapeutically as immunomodulators for treatment of infections or cancer (Seljelid, Scand. J. Immunol. 1989, 29:181-92; Bowers et al., J. Surg. Res. 1989;47:183-8). Double-εtranded RNA, such polyinoεine-polycytidine, alεo haε therapeutic activity aε an inflammatory εtimulus for treatment of cancer or infections.

The inflammatory peptide Interleukin-1 (IL-1), which mediates some actionε of endotoxin, εimilarly haε important therapeutic potential (e.g. in restoring he atopoiesiε after damage cauεed by cancer chemotherapy) , but its use is limited by toxic side effectε which may be attenuated by utilization of the compoundε, compositions, and methods of the invention.

Interleukin-2 (IL-2) iε uεed clinically for treatment of several varieties of cancer; it also has potential activity as an immunomodulator in treatment of various infections and in modulating the responεe to vaccines. Hepatic toxicity in response to IL-2 is not uncommon in patients receiving therapeutic doseε of IL-2 for cancer treatment (Vienε et al., J. Immunother. 1992 11:218-24). In an experimental model of

94/13687

autoimmune hepatitis induced by administration of concanavalin A to mice, hepatic damage iε reported to be related to elevated production of endogenous IL-2 (Tiegε et al., J. Clin. Invest. 1992 90:196-203); as de onεtrated in Example 10, compounds, compositions, and methods of the invention are effective in attenuating hepatic damage in this model. The compounds, compositions, and methods of the invention are useful in reducing εide effects when administered in conjunction with IL-2; furthermore, the compounds, compositions, and methods of the inventions are useful in treating autoimmune hepatitis.

Interleukin 6, which has therapeutic potential in improving blood platelet production, induces hepatic TNF receptors, thus increasing tiεεue sensitivity to TNF. The compounds, compositions, and methods of the invention are thuε uεeful for uεe in combination with IL-6 or εimilar agentε which affect tiεεue εenεtivity to, or production of, TNF (Van Bladel et al.. Cytokine. 1991 3:149-54).

The combination of a particular therapeutic cytokine and a pyrimidine nucleotide precursorε and/or a uridine phoεphorylase inhibitor is used for treatment of the disorderε for which the particular therapeutic cytokine iε known to be effective. For example, interleukin 2 iε uεed for treatment of renal cancer, colon cancer, melanoma, lymphoma, leukemia and other neoplaεtic conditionε. TNF has antitumor efficacy against a variety of cancer types, but its use in therapy has heretofore been limited by its toxicity, (Ki ura et al.. Cancer. Chemother. Pharmacol. 1987; 20:223-9). Endotoxin haε

εhown εignificant antitumor efficacy (Engelhardt R et al., Cancer. Reε. 1991 51:2524-30).

For prevention or treatment of toxicity due to adminiεtration of therapeutic cytokines, approximately 0.5 to 40 grams of a pyrimidine nucleotide precurεor iε administered daily for one to several days, depending on the duration of the cytokine treatment. The pyrimidine nucleotide precursorε are adminiεtered before, during, or after administration of the therapeutic cytokine. The therapeutic cytokines are administered in the particular doses and regimens already establiεhed for experimental and clinical treatment of various forms of cancer, except that increased doseε of cytokines may be tolerated when the pyrimidine nucleotide precursors of the invention are administered, as would be determined in simple dose-eεcalation studies for each cytokine or inflammatory stimulus.

Inflammatory Hepatitis: Liver diεorderε involving endotoxin or mediators

The liver is susceptible to damage by endotoxin or its mediators, particularly when liver function is impaired. Liver damage from many originating causes (e.g. choline deficiency, Reye's εyndrome, or alcohol) which either increaεe hepatic εenεitivity to endotoxin or inhibit endotoxin clearance, iε in part mediated by bacterial endotoxin (normally preεent in the portal circulation due to leakage of small amounts from the intestine into the bloodstream) or mediators elicited by endotoxin (Nolan, Gastroenterology, 1975 69:1346-1356; Nolan, Hepatoloσy 1989 10:887-91). Hepatic

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toxicity is dose-_imiting in patients receiving intentional injections of endotoxin for possible efficacy in treating cancer (Engelhardt et al.. Cancer Research, 1991 51:2524-2530).

As is demonstrated in the Exampleε below, the compoundε, compositions, and methods of the invention significantly reduce hepatic damage induced by endotoxin and other inflammatory stimuli and mediators. The compounds, compositionε, and methods of the invention are useful in treating, preventing, or attenuating liver damage in a large variety of conditions in which hepatoxicity due to endotoxin or othe inflammatory stimuli or mediators are implicated in their etiology (whether or not systemic sepεis syndrome is present). Conditions in which damage to the liver by endotoxin or its mediatorε (e.g. TNF) are implicated include but are not limited to the following disease states:

A. Reye's syndrome

Reye's εyndrome iε characterized by rapid hepatic failure and iε moεt commonly found in children as a complication of influenza and other viral infections; aspirin may be a risk factor. The etiology of Reye's syndrome is believed to involve endotoxin or inflammatory mediators. Endotoxemia is found in most or all patients with Reye's syndrome; an animal model for Reye's syndrome involves treating rats with a combination of endotoxin and aspirin (Kilpatrick et al.. Metabolism. 1989, 38:73-7).

B. Alcoholic liver damage

Endotoxin and TNF contribute to hepatic proble ε aεεociated with expoεure to alcohol. (Nolan JP, Hepatology 1989 10:887-91; Arai M, , Nakano S, Okuno F, et al. Hepatology 1989; 9:846-851; McClain CJ and Cohen DA, Hepatology 1989; 9:349-351)

C. Fulminant hepatitis

Tumor necrosis factor is implicated in the etiology and progression of fulminant hepatitis, which can rapidly lead to hepatic failure and death (Aderka, et al., Med Hypotheseε. 1988 27:193-6)

D. Viral hepatitis

Endotoxin contributes to hepatocyte damage occurring during viral hepatitis. Viral hepatitis reduceε the LD B0 of endotoxin in animal models, and exclusion of endogenous endotoxin from experimental animals (by colectomy or by using axenic rodents) reduces the hepatic damage cauεed by a viral challenge. (Gut et al., J. Infect. Diεease., 1984, 149:621). In some caseε of hepatitis, immune or inflammatory responses to hepatic viral infection mediated by T lymphocytes or macrophages contributes to liver damage. In either situation, the compounds, compositionε, and methodε of the invention are uεeful for treating hepatic damage related to viral infection.

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E. Parasitic infections

Hepatic damage and morbidity which occurε during malaria infection iε mediated in part by TNF (Clark et al.. Am. J. Pathol. 1987, 129:192-9).

F. Hepatic damage during total parenteral nutrition

Hepatic complicationε are common in patientε receiving total parental nutrition (TPN) and who have no underlying liver diεeaεe. Pappo et al. (J. Surg. Reε. , 1991, 51:106-12) reported that endotoxin (LPS) or derived from the overgrowth of inteεtinal gram-negative bacteria iε reεponεible for TPN- associated hepatic steatoεiε, and that bowel decontamination and specific anti-LPS activity of polymyxin B will reduce fatty infiltration of the liver during TPN. Polymyxin B, which binds to and inactivates LPS, is toxic in humans, but served to demonstrate that hepatopathy observed during TPN iε in fact mediated in part by endotoxin or TNF. Therefore, incluεion of compounds of the invention in TPN solutionε is useful for reducing TPN-induced liver damage.

G. ead poisoning

Lead poisoning can dramatically increase sensitivity to endotoxin. Lead-induced interference with hepatic metabolism is implicated in the effect of lead on endotoxin toxicity (Taki et al., Eur. Surg. Res., 1985, 17:140-9).

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H. Partial hepatectomy

Following partial hepatectomy (e.g. for removal of cancerous tisεue), morbidity and mortality from hepatic failure iε not uncommon. Liver tiεεue undergoing regeneration after partial hepatectomy in animals is hypersensitive to the deleterious effects of endotoxin and mediators (Shirai et al., Aσta Pathol. Jpn.. 1987, 37:1127-1134).

I. Postanesthetic hepatitis

Inhalation anesthetics such as halothane can induce hepatitis, particularly if hepatic bloodflow is also impaired. Endotoxin is implicated in the etiology of postanesthetic hepatitis (Lomanto et al., nesth. Analg. , 1972, 51:264-270); the compounds of the invention are thus useful for administration to patients (prophylactically, therapeutically, or both) undergoing inhalation anestheεia for preventing and treating hepatitiε. Trauma itself may contribute to postaneεthetic hepatitis. Trauma furthermore often induces translocation of bacteria and endotoxin from the gut into other tisεueε via the bloodstream. Surgery patients are among the groups most susceptible to endotoxin poisoning (due to infection). Therefore, treatment of surgical patients with pyrimidine nucleotide precursorε (before, during, or after surgery) εignificantly improveε their resistance to endotoxin poisoning.

J. Cholestatic hepatitis

Hepatic injury due to bile duct obstruction or intrahepatic cholestasis is in part due to enterally-derived endotoxin. (Shibayama Y, 1989, J. Pathol. 159:335-9).

K. Liver transplantation

In patients receiving liver transplants, the presence of high endotoxin levels preoperatively and at the end of the anhepatic period iε aεεociated with graft failure and a high mortality. Patientε with primary nonfunction of their tranεplantε typically have severe endotoxe ia. Endotoxe ia could be a cause rather than an effect of perioperative complications and graft loss (Yokoyama et al, 1989, Transplant Proc. 21:3833-41). In a clinical situation, an animal, such as a human, receives a compound of the invention enterally or parenterally after a transplant, in doεeε ranging from about 2 to about 40 gramε per day, advantageouεly divided into one to about four doεeε. The donor liver can also be perfused with a compound of the invention, advantageously uridine, cytidine, orotic acids or saltε or acyl derivativeε thereof, prior to or during implantation in the recipient.

Endotoxinε and inflammatory mediators are also involved in other hepatic disorderε; the diverεity of the specific examples discuεsed above serve to indicate that the compounds, compositions, and methods of the invention are useful in treating or preventing a broad variety of liver diseases.

For treatment of inflammatory hepatitis, 0.5 to 40 grams (advantageouεly 3 to 30 gramε) of a pyrimidine nucleotide

precursor are administered daily, advantageously divided into one to about four doseε. The duration of the treatment regimen dependε on improvement of clinical symptoms; acute inflammatory liver disorderε will typically require a shorter course of treatment than chronic degenerative conditionε.

Other disorders

As is demonstrated in Examples 2, 4-6, and 9, the compounds of the invention protect tisεueε other than liver, e.g. muscle, as indicated by serum creatine phosphokinaεe (CPK) levels in animals treated with endotoxin or the fungal inflammatory agent zymosan. Serijm CPK activity iε elevated aε a conεequence of damage to εkeletal or heart muεcle.

Cachexia, a syndrome of weight losε, tiεsue wasting and misutilization of nutrients is a common complication in patients with cancer. TNF is implicated in the initiation and maintenance of cachectic states; "Cachectin" is a synonym for TNF. The compounds, compositions, and methods of the invention are useful for treating patients with cachexia.

Veterinary applications

In horses and other large animals, there iε a common syndrome known as laminitiε, which is one consequence of endotoxin from the gut entering into the systemic circulation (often after the animal overeats carbohydrate-rich foods, changing the bacterial populations in the gut). The compounds, compositions, and methods of the invention, since they attenuate tisεue damage due to endotoxin, are uεeful in

treating or preventing laminitis and other effects of endotoxin poisoning in animals.

Administration and Formulation of Compounds and Compositions of the Invention

The compounds and compoεitionε of the invention are ad iniεtered orally, by parenteral injection, intravenouεly, or by other meanε, depending on the condition being treated and the εtatus of the patient.

The compounds and compoεitions of the invention are administered chronically, intermittently, or acutely as needed. In the case of an event which involves endotoxin toxicity or syεtemic inflammatory syndrome, the compounds and compositions are administered prior to, during, or after such event.

The pharmacologically active compounds optionally are combined with suitable pharmaceutically acceptable carriers comprising excipients and auxiliarieε which facilitate proceεεing of the active compoundε. These are administered as tablets, dragees, capsules, and suppositories. The compositions are administered for example orally, rectally, vaginally, or released through the buccal pouch of the mouth, and may be applied in solution form by injection, orally or by topical administration. The compositions may contain from about 0.1 to 99 percent, preferably from about 50 to 90 percent of the active compound(s) , together with the excipient(s). ^

For parenteral administration by injection or intravenous infusion, the active compounds are εuspended or disεolved in aqueouε medium εuch aε sterile water or εaline solution. Injectable solutions or suεpenεions optionally contain a surfactant agent such as polyoxyethylenesorbitan esters, sorbitan esterε, polyoxyethylene etherε, or εolubilizing agentε like propylene glycol or ethanol. The εolution typically containε 1 to 25% of the active compoundε.

Suitable excipientε include fillerε εuch aε sugars, for example lactose, sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphateε, for example tricalcium phosphate or calcium hydrogen phosphate, as well as binderε εuch aε εtarch paεte, using, for example, maize starch, wheat starch, rice starch or potato starch, gelatin, tragacanth, methyl cellu-lose, hydroxypropylmethyl celluloεe, sodium carboxymethyl celluloεe and/or polyvinyl pyrrolidone.

Auxiliarieε include flow-regulating agentε and lubricantε, for example, εilica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate and/or polyethylene glycol. Dragee cores are provided with suitable coatings which, if desired, are resistant to gastric juices. For this purpose, concentrated εugar εolutions are used, which optionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations such aε acetylcelluloεe phthalate or hydroxypropylmethylcelluloεe phthalate are uεed.

Dyestuffs or pigments are optionally added to the tablets or dragee coatings, for example, for identification or in order to characterize different compound doses.

The pharmaceutical preparations of the present invention are manufactured in a manner which is itself known, for example, by means of conventional mixing, granulating, dragee-making, dissolving, or lyophilizing processes. Thus, pharmaceutical preparations for oral use are obtained by combining the active compound(ε) with εolid excipientε, optionally grinding the resulting mixture and processing the mixture of granules, after adding suitable auxiliaries, if desired or necesεary, to obtain tablets or dragee cores.

Other pharmaceutical preparations which are useful for oral delivery include push-fit capsuleε made of gelatin, aε well aε εoft-εealed capεuleε made of gelatin and a plasticizer such as glycerol or εorbitol. The puεh-fit capsules contain the active compound(s) in the form of granuleε which optionally are mixed with fillerε such as lactose, binders such as starches and/or lubricants such as talc or magnesium stearate, and, optionally stabilizers. In soft capsules, the active compounds are preferably dissolved or suspended in suitable liquids such as fatty oils, liquid paraffin, or polyethylene glycols. In addition, stabilizers optionally are added. Other formulations for oral administration include solutions, suεpenεions, or e ulsionε. In particular, a liquid form suitable for administration via an enteral catheter, e.g. a nasogaεtric tube, iε advantageouε, particularly for bedridden or unconscious patients.

Pharmaceutical preparations which are used rectally include, for example, suppoεitories which consiεt of a combination of active compounds with a suppoεitory baεe. Suitable suppository baseε are, for example, natural or synthetic triglycerideε, paraffin hydrocarbonε , polyethylene glycolε or higher alkanols. In addition, gelatin rectal capsules which consiεt of a combination of the active com- poundε with a baεe are uεeful. Baεe materialε include, for example, liquid triglycerideε, polyethylene glycols, or paraffin hydrocarbonε.

Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water soluble form, for example, water soluble εaltε. In addition, εuεpenεionε of the active compoundε aε appropriate in oily injection εuεpenεions are administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or tri-glycerides. Aqueous injection suspensionε optionally include εubεtances which increase the viεcoεity of the εuεpension which include, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suεpenεion optionally containε εtabilizerε.

Synthe is of the Compounds of the Invention

Acylated derivatives of pyrimidine nucleosideε are εynthesized by reacting a pyrimidine nucleoside or congener with an activated carboxylic acid. An activated carboxylic acid is one that has been treated with appropriate reagents to

render its carboxylate carbon more susceptible to nucleophilic attack than iε the case in the original carboxylic acid. Examples of useful activated carboxylic acids for synthesis of the compounds of the invention are acid chlorides, acid anhydrides, n-hydroxysuccinimide esters, or carboxylic acids activated with BOP-DC. Carboxylic acids may also be linked to pyrimidine nucleosideε or congeners with coupling reagents like dicyclohexylcarbodiimide (DCC).

During preparation of the acyl compounds of the invention, when the acid source of the desired acyl derivative has groups which interfere with the acylation reactions, e.g., hydroxyl or amino groupε, theεe groupε are blocked with protecting groups, e.g., t-butyldimethylsilyl ethers or t-BOC groups, reεpectively, before preparation of the anhydride. For example, lactic acid iε converted to 2-t-butyldimethyl- siloxypropionic acid with t-butyldimethylchlorosilane, followed by hydrolysis of the resulting silyl ester with aqueous base. The anhydride iε formed by reacting the protected acid with DCC. With amino acidε, the N-t-BOC derivative is prepared, using standard techniques, which is then converted to the anhydride with DCC. With acids containing more than one carboxylate group (e.g., succinic, fumaric, or adipic acid) the acid anhydride of the desired dicarboxylic acid is reacted with a pyrimidine nucleoside in pyridine or pyridine plus dimethylformamide or dimethylacetamide.

Amino acids are coupled to the exocyclic amino groups of cytidine, and to hydroxyl groups on the aldose moiety of

pyrimidine nucleosides or their congeners, by standard methodε using DCC in a suitable solvent, particularly a mixture of (i) methylene chloride and (ii) dimethylacetamide or dimethylformamide.

Carbylόxycarbonyl derivatives of non-methylated pyrimidine nucleoεides are prepared by reacting the nucleoside with the appropriate carbylchloroformate in a εolvent such as pyridine or pyridine plus dimethylformamide under anhydrous conditions. The εolvent iε removed under vacuum, and the reεidue is purified by column chromatography.

It will be obvious to the person skilled in the art that other methods of syntheεiε can be uεed to prepare the compoundε of the invention.

The following examples are illustrative, but not limiting of the methods and compositions of the present invention. Other suitable modifications and adaptations of a variety of conditions and parameters normally encountered in clinical therapy which are obvious to those εkilled in the art are within the spirit and scope of this invention.

EXAMPLES

Example 1: Tr acetyluridine and uridine improve survival in mice treated with killed E. Coli

Purpose:

Sepsis εyndrome can be initiated by gram-negative bacteria even if they are not alive, since the primary trigger is endotoxin, a component of the bacterial cell wall. The purpoεe of this study was to determine the effect of oral triacetyluridine and parenteral uridine on survival of mice treated with a lethal dose of killed E. Coli bacteria

Methods:

Eighteen female Balb/C mice (eight weeks old) were divided into groups of six animals each. All mice received 500 micrograms of an acetone powder of E. Coli (serotype 0111:B4) suspended by sonication in 0.2 ml of saline. Mice in one group received uridine (2000 mg/kg in 0.2 ml saline) by i.p. injection two hours prior to administration of the E. Coli. Another group of mice received triacetyluridine (6000 mg/kg in a vehicle of 1:1 corn oil/water containing 2.5 %

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Tween 80) by oral intubation. Survival was monitored for one week.

A. n = 6 E. Coli (Control)

B. n = 6 E. Coli (Control) + Urd i.p.

C. n = 6 E. Coli (Control) + TAU p.o.

-Results:

Animals in the Control group appeared to be in shock and were hypothermic 18 hours after administration of the E. Coli powder. Animalε in the treated groupε were active and maintaining body temperature, although their coats were scruffy throughout the firεt 48 h'ourε of the obεervation period. Animalε εurviving 48 hourε recovered completely. All of the mice treated only with E. Coli died within 48 hours. All mice treated with either uridine or triacetyluridine survived adminiεtration of killed E. Coli.

Example 2: Dose-response study of uridine in protection of tissues from endotoxin damage

Purpose:

The purpose of this study waε to determine the doεe- reεponεe characteristicε for uridine in prevention of inflammatory tiεεue damage cauεed by endotoxin (LPS).

Methods:

Female Balb/C mice (eight weeks old) were divided into six groups of six animals each. One group of animals remained untreated to provide basal values for serum chemistry indiceε

of tissue damage. Mice in the remaining five groups received 100 micrograms of Salmonella Typhimurium endotoxin by i.p. injection in a volume 0.2 ml saline. Two hourε prior to endotoxin adminiεtration, the five groupε of mice received uridine in doses of 0, 500, 1000, 2000 and 4000 mg/kg i.p. (in 0.2 ml saline) respectively. Eighteen hours after endotoxin administration, blood sampleε were collected for determination of serum chemistry values of indicators of tissue damage.

Results:

Uridine produced a dose-dependent protection of tissues against damage from endotoxin administration. ALT, AST, and SDH are specific indicators of liver damage; CPK is an indicator of damage to muscle; LDH is releaεed from both liver and muεcle. The moεt effective uridine dose in mice in this experiment was 2000 mg/kg.

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Table 1: Uridine attenuateε endotoxin-induced tissue damage

ALT AST LDH CPK SDH

Basal (No LPS) 198±124 137126 708±177 9061211 49±2

Control (LPS) 37681482 41761459 84061850 11628+2398 11701157

Uridine 500 25681678 30901871 598811225 883211089 8341192

Uridine 1000 13381401* 12061314* 31011860* 443111529* 404195*

Uridine 2000 6051236* 6201174* 19901642* 453112139* 125145*

Uridine 4000 11201970* 7441457* 344112378* 868016746* 135175*

* = Different from Control (LPS i.p.), P < .02

ALT = Alanine Aminotransferaεe AST = Aεpartate A inotranεferase LDH = Lactate Dehydrogenase CPK = Creatine PhosphokJnase SDH = Sorbitol Dehydrogenase

Example 3: Oral triacetyluridine improves survival of mice treated with a lethal dose of Salmonella Typhimurium endotoxin

Purpose:

Sepsiε εyndrome cauεed by gram-negative bacteria iε mediated primarily through endotoxin, a lipopolyεaccharide constituent of the bacterial wall. The purpose of this experiment was to determine the effect of an orally-

administered uridine prodrug (Triacetyluridine; TAU) on survival of mice treated with a lethal dose of purified Salmonella Typhimurium endotoxin (LPS).

Methods:

Twenty female Balb/C mice (eight weeks old) were divided into two groupε of ten animalε each. All mice received 100 microgramε of Salmonella Typhimurium endotoxin by intraperitoneal injection in 0.2 ml of saline. One group of mice received triacetyluridine (6000 mg/kg in a vehicle of 1:1 corn oil/water containing 2.5 % Tween 80) by oral intubation. Survival was monitored for one week.

Results:

All ten of the animals which received endotoxin alone died within 48 hours. Nine of the ten mice that received oral TAU survived for the seven day observation period and appeared to have recovered completely.

Example 4: Oral triacetyluridine reduces tissue damage caused by endotoxin

Purpose:

Bacterial endotoxin causes damage to the liver and other organs which can be assessed and quantified by determining serum levels of enzymeε and other markerε of tiεsue integrity and function. The purpoεe of thiε study was to determine the dose-response characteristicε of orally-adminiεtered triacetyl uridine (TAU) in attenuating tiεsue damage due to endotoxin.

_£2-

Methods :

Female Balb/C mice (eight weeks old) were divided into groups of six animals each. One group of animals remained untreated to provide basal values for serum chemistry indices of tisεue damage. Mice in the other four groups received 100 micrograms of Salmonella Typhimurium endotoxin by i.p. injection, in a volume 0.2 ml εaline. Three groupε of endotoxin-treated mice alεo received TAU 24, 6 , and 2 hourε before endotoxin in doεeε of 2000, 4000, and 6000 mg/kg by oral intubation in a volume of 0.4 ml; an additional group received a single dose of 6000 mg/kg of TAU two hours before endotoxin. The TAU waε formulated aε a suspenεion in 1 % carboxymethylcelluloεe in water. The remaining group (Controlε) received the carboxymethylcellulose vehicle by oral intubation.

Results:

Oral TAU administration reduced the levels of εeru chemiεtry indicatorε of tiεεue damage. 1'he beneficial effect on prevention of endotoxin-induced organ damage waε doεe dependent. A εingle doεe of 6000 mg/kg TAU 2 hourε before endotoxin provided approximately the same degreee of protection aε three εuch doεeε 24, 6, and 2 hourε before endotoxin.

Table 2 : TAU attenuates endotoxin-induced tissue damage

ALT AST LDH SDH

Basal (No LPS) 130146 148132 5631132 9821329 4115

Control (LPS) 36791703 47981927 699811064 2263+247 11281174

TAU 2000 26321915 315111085 541911561 1053180 7931294

TAU 4000 14631382* 19401456* 38781672* 28111395* 3451106*

TAU 6000 4911199* 4841161* 14481334* 18201612* 172195*

TAU 6000 365191* 403161* 12211181* 11831151* 104118*

(εingle doεe)

* = Different from Control (LPS i.p. + vehicle p.o.), P < .02

ALT = Alanine Aminotranεferase AST - Aspartate A inotransferase LDH = Lactate Dehydrogenase CPK = Creatine Phosphokinaεe SDH = Sorbitol Dehydrogenase

Example 5: Uridine reduces tissue damage in mice treated with carrageenan as a potentiator of endotoxin toxicity

Carrageenan is a polysaccharide derived from seaweed which modifies the activity of macrophages, which are principal cellular mediators of systemic inflammatory responεe to endotoxin. Macrophageε release inflammatory peptides and other compounds in responεe to endotoxin. Carrageenan

pretreatment sensitizeε macrophages εo that much leεε endotoxin than normal is required to elicit a serious syεtemic inflammatory reεponse. Furthermore, a somewhat different spectrum of inflammatory mediators is involved in the toxic effects of the combination of carrageenan plus endotoxin compared to endotoxin alone (Franks et al.. Infection and Immunity. 59: 2609-2614 [1991]). The purpose of this experiment was to determine the effect of uridine on tisεue damage induced by a combination of carrageenan and endotoxin.

Methods:

Female Balb/C mice (eight weeks old) were divided into five groups of six animals each. One group of animals remained untreated to provide baεal valueε for serum chemistry indices of tisεue damage. Mice in the other four groupε received 2 mg of lambda carrageenan in 0.2 ml saline by i.p. injection; three of these groups also received, one hour later, 2 micrograms of Salmonella Typhimurium endotoxin, alεo by i.p. injection in a volume 0.2 ml saline. Two of the groups that received both carrageenan and endotoxin also received uridine (2000 mg/kg i.p. in 0.2 ml saline); one group was treated with uridine 30 minuteε after adminiεtration of endotoxin, and the other received 3 uridine pretreatmentε, 24, 6, and 2 hourε before endotoxin adminiεtration, at 2000 mg/kg/doεe i.p. Eighteen hours after endotoxin adminiεtration, blood samples were collected for determination of serum chemistry valueε of indicatorε of tissue damage.

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Results :

The combination of carrageenan with a low dose of endotoxin (2 mg) resulted in εignificant tiεεue damage aε evaluated by serum chemistry indices. Treatment with uridine either before or after administration of endotoxin resulted in significant attenuation of tisεue damage due to the carrageenan-endotoxin combination. Data are εhown below.

Table 3: Uridine attenuates endotoxin-induced tissue damage in carrageenan-sensitized mice

ALT ______ LDH CPK SDH

Basal (No LPS) 223177 141135 7001145 7471278 3311

Control (LPS) 19371235 20721149 73601354 11612+1513 107117

Uridine 8171202* 9891139* 43851454* 548511638* 80112*

Uridine 7701141* 891179* 44161283* 50331565* 11719

(postreatment)

* = Different from Control, P<.05

ALT = Alanine Aminotranεferaεe AST = Aεpartate Aminotranεferase LDH = Lactate Dehydrogenase CPK = Creatine Phosphokinaεe SDH = Sorbitol Dehydrogenaεe

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Example 6: Uridine improves survival in Zymosan-treated mice

Purpose:

Zymosan iε a yeaεt component, primarily polysaccharide, which induces systemic inflammation and activation of complement. In fungal infections in general (including but not limited to yeaεt infectionε), εuch polyεaccharideε participate in the induction of a sepsis response. Zymosan administration to rodents iε conεidered to be a suitable model for multiple organ failure syndrome (Goriε et al. (1986) Arch. Surα. 121:897-901; Steinberg et al. (1989) Arch. Surσ. 124:1390-1395). Mortality at minimum lethal doεes of zymosan is due in part to gut damage leading to translocation of bacteria and bacterial toxins from the gut into the bloodstream (Deitch et al., (1992) J. Trauma 32:141-147).

Methods:

Female Balb/C mice (eight weeks old) were divided into groupε of five animalε each:

1. Zymoεan 15 mg

2. Zymoεan 15 mg + Uridine

3. Zymoεan 20 mg

4. Zymoεan 20 mg + Uridine

5. Baεal

Zymoεan A was εuεpended in mineral oil at a concentration of 50 mg/ml and adminiεtered by intraperitoneal injection. Uridine (2000 mg/kg) waε adminiεtered by intraperitoneal

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injection in a volume of 0.2 ml two hours before administration of Zymosan.

18 hours after administration of Zymosan, blood samples were collected from both groups of mice that received 20 mg Zymosan and from a basal (untreated) group for subsequent measurement of serum chemistry indices of tissue damage.

Results:

A. Survival at 48 hours:

Group Survival

Zymosan 15 mg/kg 0/5

Zymosan 15 mg/kg + Uridine 5/5

Zymosan 20 mg/kg 0/5

Zymosan 20 mg/kg + Uridine 3/5

B. Survival at 14 days (complete recovery)

Zymosan 15 mg/kg 0/5

Zymoεan 15 mg/kg + Uridine 4/5

Uridine εignificantly improved εurvival time and incidence of long-term εurvivorε among mice treated with Zymosan.

C.Serum chemistry indiceε of tiεsue damage

Table 4: Uridine attenuates Zymosan-induced tissue damage

ALT LDH CPK SDH

Basal 50122 93141 8991198 5321731 52125

Zymosan 3971140 392+97 19741392 210711172 81115

Zymosan + Uridine 1201126 2731131 1419+244 7541370 58122

ALT = Alanine Aminotransferaεe AST = Aεpartate Aminotranεferaεe LDH - Lactate Dehydrogenase CPK = Creatine Phosphokinaεe SDH = Sorbitol Dehydrogenaεe

Example 7: Comparison of effects of uridine versus arginine on survival of endotoxin-treated mice

Purpose:

The amino acid arginine is reported to have beneficial effects in sepεiε εyndrome (Leon et al. J. Parenteral and Enteral Nutrition. 1991, 15:503-508). The purpoεe of thiε εtudy waε to compare the efficacy of uridine with that of arginine, an agent which supports liver function in sepsis syndrome and which is clinical uεe for thiε purpose.

Methods:

Female Balb/C mice weighing 25 grams were divided into five groups of five or six animals each. Mice in the

remaining five groups received 125 micrograms of Salmonella

Typhimurium endotoxin (LPS) by i.p. injection in a volume 0.2 ml saline. Two hours prior to endotoxin administration, the five groupε of mice received injections of:

1) Saline (Controlε)

2) Uridine 2000 mg/kg

3) Arginine 25 mg/kg

4) Arginine 250 mg/kg

5) Arginine 1250 mg/kg

All drugε were administered i.p. in 0.2 ml saline. The numbers of surviving mice in each group were determined 16, 20, and 24 hours.

Results:

Only one of the Control animals was alive 16 hours after LPS; in contrast, the majority of the animals treated with uridine or arginine were alive at this point. However, by 24 hours after administration of endotoxin, the only surviving animals were in the group treated with uridine. All three doses of arginine did improve survival time (but did not produce any long-term survivors), and the lowest dose (25 mg/kg) was more effective than the highest dose (1250 mg/kg). Uridine was clearly more effective than arginine in promoting survival of endotoxin-treated animals.

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Table 5: Effect of uridine vs arginine on survival after LPS administration

e after LPS (hr.

Groups

1. Control

2. Uridine

3. Arg 25

4. Arg 250

5. Arg 1250

Example 8: Orotic acid improves survival of mice treated with Salmonella Typhimurium endotoxin

Purpose:

Sepsis syndrome caused by gram-negative bacteria is mediated primarily through endotoxin, a lipopolysaccharide conεtituent of the bacterial wall. The purpose of this experiment was to determine the effect of orotate on survival of mice treated with a lethal dose of purified Salmonella Typhimurium endotoxin.

Methods:

Twenty female Balb/C mice (eight weeks old) were divided into two groups of ten animals each. One group of mice received four treatmentε with lysine orotate (200 mg/kg/dose; 9 AM and 2 PM on each of two consecutive days). Lyεine

O 94/13687

orotate is a water-soluble salt of orotic acid; lysine alone does not improve survival of endotoxin-treated mice. Control animals received 0.2 ml of sterile water on the same treatment schedule. All mice received 100 icrograms of Salmonella Typhimurium endotoxin (LPS) by intraperitoneal injection in 0.2 ml of saline immediately after the laεt dose of lysine orotate. Survival was monitored for one week.

Results:

All of the mice in the Control group died within 48 hours. Nine of the ten mice treated with Lysine Orotate survived the full 72 hour observation period and were still alive and appeared to recover completely one week after LPS administration.

Table 6: Orotate improves survival of endotoxin-treated mice

Survival after endotoxin treatment

Time (hr after LPS) 24 26 28 22. 4-8 J72

Control 6/10 4/10 3/10 2/10 0/10 0/10

LOR 10/10 10/10 10/10 10/10 9/10 9/10

Example 9: Orotic acid protects tissues against endotoxin damage

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Purpose:

The purpose of this study was to demonstrate the protective effect σf orotic acid in prevention of inflammatory tissue damage caused by endotoxin.

Methods:

Female Balb/C mice (eight weeks old) were divided into three groups of six animals each. One group of animals remained untreated to provide basal values for serum chemistry indices of tisεue damage. Mice in the remaining two groupε received 100 microgramε of Salmonella Typhimurium endotoxin (LPS) by i.p. injection in a volume 0.2 ml saline. Two hours prior to endotoxin administration, mice in one group received lyεine orotate in a dose corresponding to 100 mg/kg of free orotic acid. Eighteen hours after endotoxin administration, blood samples were collected for determination of serum chemistry content of indicatorε of tiεεue damage.

Results:

Orotate protected tiεεueε against damage from endotoxin administration.

A

Table 7: Orotate attenuates endotoxin-induced tissue damage

ALT AST LDH CPK SDH

Basal (No LPS) 132114 165121 681+552 1258+233 42+1

Control (LPS) 2827+413 28601506 683311167 68201365 6801142

Orotate + LPS 252199* 415177* 16411274* 10401283* 8917*

* = Different from Control (LPS i.p.), P < .02

ALT = Alanine Aminotransferase AST = Aεpartate Aminotransferase LDH = Lactate Dehydrogenase CPK = Creatine Phosphokinaεe SDH - Sorbitol Dehydrogenase

Example 10: Uridine and triacetyluridine attenuate hepatic damage caused by Concanavalin A

Purpose:

Interleukin-2 (IL-2) is used clinically for treatment of several varieties of cancer. Hepatic toxicity in reεponse to IL-2, is not uncommon in patients receiving therapeutic doses of IL-2 for cancer treatment (Viens et al., J. Immunother. 1992 11:218-24). In an experimental model of autoimmune hepatitiε induced by administration of Concanavalin A (Con A) to mice, hepatic damage is reported to be related to elevated production of endogenous IL-2 (Tiegs et al., J. Clin. Invest.

1992 90:196-203). The purpose of this study was to demonstrate the utility of the compounds and methods of the invention in attenuating hepatic damage initiated by administration of Con A.

Methods:

Female Balb/C mice (eight weeks old) were divided into four groups of five animals each. One group of animalε remained untreated to provide baεal valueε for εerum chemistry indices of tissue damage. Mice in the remaining three groups received 10 mg/kg Concanavalin A by intravenous (tail vein) injection in a volume of- 0.2 ml saline. Two hours prior to receiving Con A, one of these groups of mice received uridine (2000 mg/kg i.p. in 0.2 ml saline) and another group received triacetyluridine (6000 mg/kg orally, in 0.6 ml of a 1:1 corn oil/water e ulεion containing 2.5 % Tween 80); the remaining Con A-treated group (Control) received 0.2 ml saline i.p. two hours prior to Con A. Twenty hours after administration of Con A, blood εampleε were collected from all mice for determination of serum levels of various indiceε of tissue damage or metabolic dysfunction.

Results:

Con A administration reεulted in significant damage to the liver, as asεeεεed by εerum levelε of the enzymeε ALT, AST, and SDH. Con A did not εignficantly elevate levels of creatine phosphokinaεe (CK), an enzyme found primarily in muεcle; tiεεue damage due to Con A in thiε model is more specifically localized in the liver than is damage due to

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endotoxin. Uridine and TAU both reduced the liver damage produced by Con A administration, as shown in Table 8 below.

Table 8: Uridine and Triacetyluridine attenuate liver damage caused by Concanavalin A

______ __S ______ __£__ SDH

Basal (No Con A) 144118 217127 790190 23921370 5112

Con A 26521847 276511030 433511385 25721486 1114131

Con A + Uridine 2891115* 3941114* 9731202* 19961317 163168*

Con A + TAU 5751286* 6131221 13801270 19511435 283+143

* - Different from Control (LPS i.p.), P < .02

ALT = Alanine Aminotransferase AST = Aspartate Aminotransferase LDH = Lactate Dehydrogenase CPK = Creatine Phosphokinase SDH = Sorbitol Dehydrogenaεe

Liver damage in the Con A model uεed in thiε study is related to elevated IL-2 levels, and is mediated through T lymphocyteε. Therefore, the compoundε and methodε of the invention are uεeful in reducing side effects due to therapeutic administration of IL-2; furthermore, the compounds and methods of the inventions are useful in treating autoimmune hepatitis.

Example 11: Uridine attenuateε sepsis induced alterations in blood coagulation

Purpose:

Disseminated Intravascular Coagulation (DIC) is a serious conεequence of εepεis, in which both blood coagulation and fibrinolysiε are activated, εo that blood clotting factorε are rapidly conεumed. DIC can reεult in hemorrhage or thrombuε formation. The liver iε the primary εite for εyntheεiε of clotting factorε and for clearing micro-aggregates of thrombin from the circulation. Thiε purpose of this experiment was to determine the effect of pyrimidine nucleotide precursorε on coagulation diεorderε induced by sepsis. Partial thro boplastin time was used aε an index of the εtatuε of the blood coagulation εyεtem.

Methods:

Thirty female Balb/C mice (eight weeks old) were divided into three groups of ten animals each. One group of mice remained untreated, and was uεed to determine baεal valueε for partial thromboplastin time. Two groups of mice received received 30 mg/kg killed E. Coli (εtrain 0111:B4); Two hourε before E. Coli adminiεtration, one group received uridine (2000 mg/kg) by intraperitoneal injection. 20 hourε after E. Coli adminiεtration, plaεma samples were collected from all thirty mice for determination of partial thromboplastin time (PTT). 0.27 ml of blood was collected via the retro-orbital plexus into a tube containing 0.03 ml of 3.5% sodium citrate, pH 4. Plasma was separated by

centrifugation, and 100 microliters of plasma was transferred to a clean 1.5 ml Eppendorf tube for determination of PTT with a commercial kit.

Results:

Administration of killed E. Coli resulted in a prolongation of the normal partial thromboplastin time. Uridine attenuated the sepεis-induced change in coagulation time, aε εhown in Table 9.

Table 9: Uridine attenuateε sepsis-induced alterations in partial thromboplastin time

Partial Thromboplastin Time

Group PTT (seconds)

Baεal (Normal) 32.311.3

E. Coli 69.815.4

E. Coli + Uridine 51.212.1 *

* = different from control (E.Coli alone) value, P < .05

Example 12: Combined liver injury due to T cells and endotoxin

Several important forms of viral hepatitis as well as autoimmune hepatitis are initiated by cytotoxic T cells which attack hepatocytes bearing appropriate viral or other antigens. Since endotoxin participateε in liver damage

initiated by a number of other agents like carbon tetrachloride, choline deficiency, ethanol, or choleεtaεis, εtudies were conducted to determine whether liver injury caused by T cells induces hepatic hypersenεitivity to endotoxin. Following thiε experiment, the effect of TAU on combined liver injury due to both T lymphocyteε and endotoxin was inveεtigated.

Example 12A: Concanavalin A potentiates endotoxin-induced tissue damage

Groups (n=6) of female Balb/C mice, age eight weeks, received Concanavalin A (2.5 mg/kg i.v.), endotoxin (Salmonella Typhimurium, 0.5 mg/kg), or a combination of Con A and endotoxin. The Con A was administered twenty four hourε before endotoxin. Blood εampleε were taken 18 hours after injection of endotoxin (or its vehicle in the groups of mice that did not receive endotoxin) . The "Basal" group of mice received vehicle only (saline) instead of Con A or endotoxin.

Table 1: Concanavalin A potentiates endotoxin-induced tissue damage

ALT AST LDH CPK SDH

Basal 87115 11019 656141 413187 3912

Con A 2.5 mg/kg 117119 170116 915+46 4191132 4214

LPS 0.5 mg/kg 119123 256122 881+10 426182 4113

Con A + LPS 11301494 21191910 437011303 15251450 4711267

( ?

ALT = Alanine Aminotransferase AST = Aspartate Aminotransferase LDH = Lactate Dehydrogenase CPK = Creatine Phosphokinase SDH = Sorbitol Dehydrogenase

Endotoxin or Con A alone at the doεeε uεed in this experiment produced minimal damage to liver and muεcle aε determined by εerum enzyme levels (ALT, AST, LDH and SDH are markers for liver damage; CPK is an indicator for muεcle damage). However, in mice treated with the combination of Con A and endotoxin, εignificantly greater damage waε obεerved. The toxicity of Con A in thiε model iε believed to be εpecifically related to T Lymphocyte-mediated liver damage (Tiegε et al. , J. Clin. Invest . 90:196-203, 1992). Therefore, theεe reεults support the view that enterally-derived endotoxin participates in liver damage attributed to cytotoxic T lymphocytes (i.e. in viral and autoimmune hepatitis), as has been demonstrated for liver damage initiated by other primary insultε including carbon tetrachloride, choline deficiency, D-galactoεamine, and viral infectionε.

Example 12B: TAU attenuateε combined liver injury due to CTL's and endotoxin

Experimental hepatitis initiated by intravenous administration of concanavalin A (Con A) is mediated by activation of cytoxic T lymphocytes. Liver injury in this model results in a marked increase in sensitivity to toxic effects of bacterial endotoxin. Sequential administration of

Con A and endotoxin resultε in greater-than-additive hepatic

injury (εee Example 12 A). Hepatocyte injury in viral and autoimmune hepatitiε involveε εimilar mechaniεmε, with damage initiated by T cellε and exacerbated by enterally-derived endotoxin and other inflammatory proceεεeε.

TAU protectε the liver of experimental animalε from damage initiated by either endotoxin or Con A. In thiε experiment, TAU was tested for hepatoprotective effects in mice subjected to combined liver injury caused by sequential administration of both Con A and endotoxin.

Methods:

Female Balb/C mice (eight weeks old) were divided into three groups of seven animals each. One group of animals remained untreated to provide basal valueε for εerum chemiεtry indiceε of tiεεue damage. Mice in the remaining two groupε received 2 mg/kg Concancavalin A by intravenouε (tail vein) injection in a volume of 0.2 ml εaline, followed 24 hourε later by Salmonella Typhimurium endotoxin (10 microgramε i.p.). One of theεe groupε of mice received TAU (6000 mg/kg orally, in 0.6 ml of 0.5% methylcelluloεe two hourε before Con A and again 2 hourε before endotoxin; the remaining Con A/endotoxin-treated group (Control) received vehicle (methylcelluloεe) alone. Eighteen hours after administration of endotoxin, blood sampleε were collected from all mice for determination of serum levels of various indiceε of tiεεue damage or metabolic dysfunction.

7

Results :

Sequential administration of Con A and endotoxin resulted in significant liver injury, aε assessed by serum chemistry indices of liver damage. TAU, administered orally, markedly attenuates thiε combined liver injury.

Oral TAU attenuates liver damage caused by Concanavalin A + LPS

ALT AST LDH CPK SDH

Basal 118133 162114 522180 15211235 5613

Con A/LPS 22951309 34081389' 56961560 468411569 700169

Con A/LPS 285167* 451187* 13411236* 20981465* 122119* + TAU

* - Different from Control (LPS i.p.), P < .02

ALT = Alanine Aminotransferaεe AST = Aεpartate Aminotranεferaεe LDH = Lactate Dehydrogenase CPK - Creatine Phosphokinaεe SDH = Sorbitol Dehydrogenaεe

The foregoing iε intended as illustrative of the present invention but not limiting. Numerous variationε and modifications may be effected without departing from the true spirit and scope of the invention.

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