U.S. patent application number 10/601863 was filed with the patent office on 2004-02-19 for pyrimidine nucleotide precursors for treatment of systemic inflammation and inflammatory hepatitis.
This patent application is currently assigned to Pro-Neuron Inc.. Invention is credited to Bamat, Michael K., Hiltbrand, Bradley M., von Borstel, Reid W..
Application Number | 20040033981 10/601863 |
Document ID | / |
Family ID | 27537437 |
Filed Date | 2004-02-19 |
United States Patent
Application |
20040033981 |
Kind Code |
A1 |
von Borstel, Reid W. ; et
al. |
February 19, 2004 |
Pyrimidine nucleotide precursors for treatment of systemic
inflammation and inflammatory hepatitis
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 W.;
(Potomac, MD) ; Bamat, Michael K.; (Potomac,
MD) ; Hiltbrand, Bradley M.; (Columbia, MD) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
1100 N GLEBE ROAD
8TH FLOOR
ARLINGTON
VA
22201-4714
US
|
Assignee: |
Pro-Neuron Inc.
|
Family ID: |
27537437 |
Appl. No.: |
10/601863 |
Filed: |
June 24, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10601863 |
Jun 24, 2003 |
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08266897 |
Jul 1, 1994 |
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08266897 |
Jul 1, 1994 |
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08158799 |
Dec 1, 1993 |
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08158799 |
Dec 1, 1993 |
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07987730 |
Dec 8, 1992 |
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07987730 |
Dec 8, 1992 |
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07438493 |
Jun 26, 1990 |
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07438493 |
Jun 26, 1990 |
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07115929 |
Oct 28, 1987 |
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Current U.S.
Class: |
514/50 ;
514/269 |
Current CPC
Class: |
A61K 31/7072 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 31/505 20130101;
A61K 2300/00 20130101; A61K 31/7088 20130101; A61K 31/505 20130101;
C07H 19/06 20130101; A61K 45/06 20130101; A61K 31/515 20130101;
A61K 31/515 20130101; A61K 31/00 20130101; A61K 31/70 20130101;
A61K 31/7064 20130101; A61K 31/515 20130101; A61K 31/715 20130101;
A61K 31/513 20130101; A61K 31/7068 20130101; A61K 31/715 20130101;
A61K 31/70 20130101 |
Class at
Publication: |
514/50 ;
514/269 |
International
Class: |
A61K 031/7072; A61K
031/513 |
Claims
What is claimed is:
1. A method for treating or preventing tissue 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 is 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 further comprising administering an
inhibitor of uridine phosphorylase.
6. A method for treating or preventing sepsis comprising
administering to an animal a therapeutically effective amount of an
inhibitor of uridine phosphorylase.
7. A method for reducing toxicity of a therapeutic cytokine or
inflammatory stimulus 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.
8. A method as in claim 7 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.
9. A method as in claim 8 wherein said acyl derivative of uridine
is triacetyluridine.
10. A method as in claim 7 wherein said cytokine or said stimulus
is selected from the group consisting of interleukin 1,
interleukin-2, interleukin 6, tumor necrosis factor, endotoxin,
fungal polysaccharides, and double-stranded RNA.
11. A method as in claim 7 further comprising the step of
administering an inhibitor of uridine phosphorylase.
12. A method for reducing toxicity of a therapeutic cytokine or
inflammatory stimulus 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 stimulus.
13. A method as in claim 12 wherein said cytokine or said stimulus
is selected from the group consisting of interleukin 1,
interleukin-2, interleukin 6, tumor necrosis factor, endotoxin,
fungal polysaccharides, and double-stranded RNA.
14. 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
a pyrimidine nucleotide precursor prior to, during, or after
administration of said cytokine or said stimulus.
15. A method as in claim 14 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.
16. A method as in claim 15 wherein said acyl derivative of uridine
is triacetyluridine.
17. A method as in claim 14 wherein said cytokine or said stimulus
is selected from the group consisting of interleukin 1,
interleukin-2, interleukin 6, tumor necrosis factor, endotoxin,
fungal polysaccharides, and double-stranded RNA.
18. A method as in claim 14 further comprising the step of
administering an inhibitor of uridine phosphorylase.
19. 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 phosphorylase prior to, during, or after
administering said cytokine or said stimulus.
20. A method as in claim 19 wherein said cytokine or said stimulus
is selected from the group consisting of interleukin 1,
interleukin-2, interleukin 6, tumor necrosis factor, endotoxin,
fungal polysaccharides, and double-stranded RNA.
21. 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.
22. A method as in claim 21 wherein said inflammatory hepatitis is
due to viral infection.
23. A method as in claim 21 wherein said inflammatory hepatitis is
due to autoimmune processes.
24. A method as in claim 21 wherein said inflammatory hepatitis is
due to alcohol consumption.
25. A method as in claim 21 wherein said acyl derivative of uridine
is triacetyluridine.
26. A method as in claim 21 including the further step of
administering an inhibitor of uridine phosphorylase.
27. A method for treating or preventing inflammatory hepatitis
comprising administering to an animal a therapeutically effective
amount of an inhibitor of uridine phosphorylase.
28. A method for treating or preventing inflammatory hepatitis
comprising administering to an animal a therapeutically effective
amount of uridine or cytidine.
29. A method as in claim 28 wherein from 2 to 40 grams of uridine
or cytidine are administered per day.
30. A method for treating or preventing hepatic damage in an animal
receiving parenteral nutrition comprising administering
intravenously to said animal a therapeutically effective amount of
a pyrimidine nucleotide precursor.
31. A method as in claim 30 wherein said hepatic damage is due to
said animal receiving parenteral nutrition.
32. A method as in claim 30 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.
33. A method as in claim 30 wherein from 2 to 40 grams of said
pyrimidine nucleotide precursor are administered per day.
34. A method as in claim 30 including the further step of
administering an inhibitor of uridine phosphorylase.
35. A method for treating or preventing hepatic damage in an animal
receiving total parenteral nutrition comprising administering to
said animal an inhibitor of uridine phosphorylase.
36. A method for treating or preventing hepatic damage in an animal
receiving a liver transplant comprising administering to said
animal a therapeutically effective amount of a pyrimidine
nucleotide precursor.
37. A method as in claim 36 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.
38. A method as in claim 36 wherein from 2 to 40 grams of said
pyrimidine nucleotide precursor are administered per day.
39. A method as in claim 36 including the further step of
administering an inhibitor of uridine phosphorylase.
40. A method for treating or preventing hepatic damage in an animal
receiving a liver transplant comprising administering to said
animal an inhibitor of uridine phosphorylase.
41. A composition comprising: a) an acyl derivative of a pyrimidine
nucleotide precursor and; b) an inhibitor of uridine
phosphorylase
42. A composition comprising: a) an acyl derivative of a pyrimidine
nucleotide precursor and; b) a purine nucleotide precursor.
43. A composition as in claim 42 where said pyrimidine nucleotide
precursor is uridine, cytidine, or orotate.
44. A composition as in claim 42 where said purine nucleotide
precursor is inosine, adenosine, or an acyl derivative of inosine
or adenosine.
45. A composition comprising a parenteral nutrition formula and 2
to 40 grams of a pyrimidine nucleotide precursor per daily
portion
46. A composition as in claim 45 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.
47. A method of providing nutrition to a mammal receiving nutrition
intravenously comprising administering to said mammal the
composition of claim 45.
48. A composition comprising a) glucose, and b) a pyrimidine
nucleotide precursor.
49. A composition as in claim 48 wherein said composition is an
aqueous solution containing 1 to 10% glucose.
50. A composition as in claim 48 wherein said composition is an
aqueous solution containing 5% glucose.
51. A composition as in claim 48 wherein said pyrimidine nucleotide
precursor is uridine or cytidine.
52. A method of treating a mammal during or after liver
transplantation comprising administering the composition of claim
48.
53. A method for reducing the effects of ethanol intoxication
comprising administering to a mammal in need of such treatment
uridine, cytidine, orotic acid, or an acyl derivative of uridine,
cytidine, or orotic acid, or a pharmaceutically acceptable salt
thereof.
54. A method of treating ethanol intoxication comprising
administering to an intoxicated mammal uridine, cytidine, orotic
acid, or an acyl derivative of uridine, cytidine, or orotic acid,
or a pharmaceutically acceptable salt thereof.
55. A method as in claim 54 wherein said administering step
comprises administering triacetyluridine.
56. A method as in claim 54 wherein said administering step
comprises administering uridine or cytidine.
57. A method of reducing inflammatory liver injury in an animal in
need of such treatment comprising administering to said animal a
therapeutically effective amount of an acyl derivative of uridine,
cytidine or orotic acid, or a pharmaceutically acceptable salt
thereof.
Description
[0001] This application is a continuation-in-part application of
copending U.S. application Ser. No. 158,799, filed Dec. 1, 1993,
which in turn is a continuation-in-part application of copending
U.S. application Ser. No. 987,730, filed Dec. 8, 1992, which in
turn is a continuation-in-part application of U.S. application Ser.
No. 438,493, filed June. 26, 1990, which in turn is a
continuation-in-part application of U.S. application Ser. No.
115,929 filed Oct. 28, 1987. All of these applications are hereby
incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention relates generally to pyrimidine 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
[0003] 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.
[0004] 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.
[0005] 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 bacteremia, 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.
[0006] 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.
[0007] 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.
[0008] Endotoxin toxicity is in part mediated by endogenous
cytokines and other bioactive molecules released from macrophages,
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., 1987, 139:4225-4231; Echtenacher et al., J. 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.
[0009] 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, Hepatology, 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.
Histochem. 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. Surg., 1991,
126:219-224)
[0010] 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 endo toxin or TNF, in some cases by several orders of
magnitude.
[0011] 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, 1979, 76:5939-5943).
[0012] Similarly, endotoxin hypersensitivity in mice deliberately
treated with the rodent hepatotoxin TCDD was partially reversed by
administration of uridine (Rosenthal et al., Toxicology, 1989
56:239-251).
[0013] However, in contrast to these situations wherein uridine
partially reversed 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-normal
resistance to endotoxin.
[0014] Uridine, cytidine, and orotate have been tested for effects
on liver function in hepatic disorders and in experimental models,
with mixed results. Shafer and 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-1,2-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.
[0015] Hata et al. (U.S. Pat. Nos. 4,027,017 and 4,058,601)
disclose that uridinediphosphate and uridinediphosphoglucuronic
acid reduce blood alcohol content and inhibit accumulation of
neutral lipids in the liver after administration of ethanol.
[0016] 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).
[0017] 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 (U.S. patent applications Ser. No. 438,493,
115,929, and 903,107, hereby incorporated by reference).
OBJECTS OF THE INVENTION
[0018] 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.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] It is a further object of the invention to provide a family
of compounds to treat or prevent inflammatory hepatitis.
[0023] It is a further object of the invention to provide compounds
which can be administered orally or parenterally.
SUMMARY OF THE INVENTION
[0024] These and other objects of the invention are achieved by
precursors of pyrimidine 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.
[0025] 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.
[0026] 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.
[0027] 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.
[0028] One embodiment of the invention involves the use of the
compounds and compositions of the invention in treatment and
prevention of inflammatory hepatitis.
[0029] Compounds of the Invention
[0030] The compounds useful in enhancing resistance to inflammatory
stimuli or inflammatory mediators have the following
structures:
[0031] 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.
[0032] (1) Uridine or an acyl derivative of uridine having the
formula: 1
[0033] wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are the same
or different and each is hydrogen or an acyl radical of a
metabolite, or a pharmaceutically acceptable salt thereof.
[0034] (2) Cytidine or an acyl derivative of cytidine having the
formula: 2
[0035] wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are the same
or different and each is hydrogen or an acyl radical of a
metabolite or a pharmaceutically acceptable salt thereof.
[0036] (3) An acyl derivative of uridine having the formula: 3
[0037] wherein R.sub.1, R.sub.2, and R.sub.3 are the same, or
different, and each is hydrogen or an acyl radical of
[0038] a. an unbranched fatty acid with 5 to 22 carbon atoms,
[0039] 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,
[0040] c. a dicarboxylic acid having 3-22 carbon atoms,
[0041] 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.
[0042] (4) An acyl derivative of cytidine having the formula: 4
[0043] wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are the same,
or different, and each is hydrogen or an acyl radical of
[0044] a. an unbranched fatty acid with 5 to 22 carbon atoms,
[0045] 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,
[0046] c. a dicarboxylic acid having 3-22 carbon atoms,
[0047] 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.
[0048] (5) An acyl derivative of uridine having the formula: 5
[0049] wherein at least one of R.sub.1, R.sub.2, or R.sub.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.
[0050] (6) An acyl derivative of cytidine having the formula: 6
[0051] wherein at least one of R.sub.1, R.sub.2, R.sub.3 or R.sub.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.
[0052] (7) Orotic acid or salts thereof: 7
[0053] 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.
[0054] 8) Alcohol-substituted orotate derivatives: 8
[0055] wherein R.sub.1 is a radical of an alcohol containing 1 to
20 carbon atoms joined to orotate via an ester linkage.
[0056] Also encompassed by the invention are the pharmaceutically
acceptable salts of the above-noted compounds.
[0057] Advantageous compounds of the invention are short-chain (2
to 6 carbon atoms) fatty acid esters of uridine or cytidine.
Particularly advantageous compounds are triacetyluridine,
triacetylcytidine or salts of orotic acid.
[0058] Inhibitors of Uridine Phosphorylase
[0059] 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.
[0060] 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-1-[(1-hydroxy-2-ethoxy)methyl] barbiturate,
5-benzyloxybenzylacetyl-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,
benzyloxybenzylacyclouridine, aminomethyl-benzylacyclouridine,
aminomethylbenzyloxybenzylacyclouridine,
hydroxymethylbenzylacyclouridine- , and
hydroxymethyl-benzyloxybenzylacyclouridine. See also WO 89/09603
and WO 91/16315, hereby incorporated by reference.
DETAILED DESCRIPTION OF THE INVENTION
[0061] 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.
[0062] 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.
[0063] A. Definitions
[0064] 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.
[0065] 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 as intermediary metabolites.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] 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.
[0070] The term "dicarboxylic acids" as used herein means fatty
acids with a second carboxylic acid substituent.
[0071] The term "therapeutically effective amount" as used herein
refers to that amount which provides therapeutic effects for a
given condition and administration regimen.
[0072] 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.
[0073] 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.
[0074] 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.
[0075] B. Compounds of the Invention
[0076] 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.
[0077] 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:
[0078] 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.
[0079] (1) An acyl derivative of uridine having the formula: 9
[0080] wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.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.
[0081] (2) An acyl derivative of cytidine having the formula:
10
[0082] wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.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.
[0083] The compounds of the invention useful in enhancing
resistance to endotoxin include:
[0084] (3) An acyl derivative of uridine having the formula: 11
[0085] wherein R.sub.1, R.sub.2, and R.sub.3 are the same, or
different, and each is hydrogen or an acyl radical of
[0086] a. an unbranched fatty acid with 5 to 22 carbon atoms,
[0087] 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,
[0088] c. a dicarboxylic acid having 3-22 carbon atoms,
[0089] 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.
[0090] (4) An acyl derivatives of cytidine having the formula:
12
[0091] wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are the same,
or different, and each is hydrogen or an acyl radical of
[0092] a. an unbranched fatty acid with 5 to 22 carbon atoms,
[0093] b. an amino acid selected from the group consisting of
glycine, the L forms of phenylalanine, alanine, valine, leucine,
isoleucine, tyrosine, praline, hydroxyproline, serine, threonine,
cystine, cysteine, aspartic acid, glutamic acid, arginine, lysine,
histidine carnitine and ornithine,
[0094] c. a dicarboxylic acid having 3-22 carbon atoms,
[0095] 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.
[0096] (5) An acyl derivative of uridine having the formula: 13
[0097] wherein at least one of R.sub.1, R.sub.2, or R.sub.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.
[0098] (6) An acyl derivative of cytidine having the formula:
14
[0099] wherein at least one of R.sub.1, R.sub.2, R.sub.3 or R.sub.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.
[0100] (7) Orotic acid or salts thereof: 15
[0101] 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.
[0102] 8) Alcohol-substituted orotate derivatives: 16
[0103] wherein R.sub.1 is a radical of an alcohol containing 1 to
20 carbon atoms joined to orotate via an ester linkage.
[0104] Also encompassed by the invention are the pharmaceutically
acceptable salts of the above-noted compounds.
[0105] Advantageous compounds of the invention are short-chain (2
to 6 carbon atoms) fatty acid esters of uridine or cytidine.
Particularly advantageous compounds are triacetyluridine or
triacetylcytidine.
[0106] Inhibitors of Uridine Phosphorylase
[0107] 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-1-[(1-hydroxy-2-ethoxy)methyl] barbiturate,
5-benzyloxybenzylacetyl-1-[(1-hydroxy-2-ethoxy)methyl] barbiturate,
and 5-methoxybenzylacetylacyclobarbiturate,
2,2'-anhydro-5-ethyluridine, 5-ethyl-2-deoxyuridine and
acyclouridine compounds, particularly 5-benzyl substituted
acyclouridine congeners including but not limited to
benzylacyclouridine, benzyloxybenzylacyclouridine,
aminomethyl-benzylacyclouridine,
aminomethylbenzyloxybenzylacyclouridine,
hydroxymethyl-benzylacyclouridine, and
hydroxymethyl-benzyloxybenzylacycl- ouridine. See also WO 89/09603
and WO 91/16315, hereby incorporated by reference.
[0108] Compositions of the Invention
[0109] 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.
[0110] 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).
[0111] 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-1-[(1-hydroxy-2-ethoxy)methyl] barbiturate,
5-benzyloxybenzylacetyl-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,
benzyloxybenzylacyclouridine, aminomethyl-benzylacyclouridine,
aminomethylbenzyloxybenzylacyclouridine,
hydroxymethyl-benzylacyclouridine, and
hydroxymethyl-benzyloxybenzylacycl- ouridine. See also U.S. Pat.
No. 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.
[0112] 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; purine nucleotide precursors
including inosine, adenosine, or acyl derivatives thereof; cyclic
AMP or acyl derivatives thereof.
[0113] In another embodiment of the invention, the composition
comprises at least one compound of the invention and an
antibacterial, antifungal, or antiviral compound.
[0114] Therapeutic Uses of the Compounds and Compositions of the
Invention
[0115] 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.
[0116] The compounds and compositions of the invention are useful
in treating mammals including 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.
[0117] 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.
[0118] The invention is furthermore embodied in the oral or
systemic administration of a pharmaceutical compound or composition
containing pyrimidine nucleotide precursors and/or agents which
inhibit uridine catabolism, for the purpose of enhancing resistance
to endotoxin, other inflammatory stimuli, or their mediators.
[0119] SIRS, Sepsis and Septic Shock
[0120] 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.
[0121] The compounds and compositions of the invention are
administered to patients with symptoms of sepsis, e.g. fever,
neutropenia, hypotension, etc., or prophylactically to patients at
risk for sepsis, e.g. surgical patients, patients with serious
burns or wounds, or patients with urinary tract catheters.
[0122] The compounds 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.
[0123] 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 nasogastric 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 and compositions 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.
[0124] Following an episode of shock, trauma or sepsis, patients
often enter into a persistent state of hypermetabolism 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 patients.
As demonstrated in the Examples, the compounds, compositions and
methods of the invention are effective in reducing tissue damage
and improving survival in animals subjected to endotoxin or other
inducers of sepsis and organ failure. The compounds, compositions,
and methods of the invention are useful in the treatment of
patients at risk for hypermetabolic organ failure.
[0125] A serious consequence of sepsis is a propensity toward
coagulation disorders, especially disseminated intravascular
coagulation (DIC). In DIC, both blood coagulation and fibrinolysis
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 is
the primary site for synthesis 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).
[0126] Reduction of Toxicity of Therapeutic Cytokines
[0127] Many of the biological effects of endotoxin and other
inflammatory stimuli are mediated by the release of endogenous
bioactive molecules (mediators) from target cells, particularly
macrophages and Kupffer cells (sessile macrophages in the liver).
Evidence for this is that macrophages in the C3H/HEJ strain of mice
are genetically non-responsive to endotoxin (in terms of releasing
cytokines upon exposure to endotoxin), and endotoxin is relatively
nontoxic in this strain. These mice are however sensitive to
bioactive peptides normally released from macrophages, e.g. tumor
necrosis factor (TNF), and toxicity of LPS is restored by
transplantation of normal macrophages. TNF is generally held to be
a primary mediator of endotoxin toxicity, but interleukin-1 (IL-1)
and other agents also participate in the expression of endotoxin
toxicity and sepsis.
[0128] Compounds, compositions, and methods of the invention are
thus useful in modifying biological effects of inflammatory
cytokines, whether produced endogenously (especially from
macrophages), or introduced into the body from exogenous sources
(e.g. polypeptides produced by recombinant DNA and fermentation
technology).
[0129] Various inflammatory cytokines and even endotoxin itself
have potential therapeutic applications. Tumor necrosis factor, as
suggested by its name, can destroy tumors and synergizes with
interferon-alpha in inhibiting viral infections. Thus, TNF, and
even bacterial endotoxin itself (which elicits the release of
endogenous TNF), have been administered to patients for the
treatment of cancer. Classes of inflammatory cytokines with both
therapeutic activity and toxicity which limits, their clinical use
include TNF, interleukins and interferons. The compounds,
compositions and methods of the invention are useful in preventing
or treating toxicity which occurs during therapeutic administration
of such cytokines as well as inflammatory stimuli.
[0130] When endotoxin is administered to cancer patients by
intravenous infusion, hepatic toxicity limits the dose of endotoxin
which can be administered (Engelhardt R et al., Cancer. Res. 1991
51:2524-30). In non-hepatic cancers, protection of the liver from
endotoxin permits administration of higher doses of endotoxin in
order to maximize its antitumor 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 Chemother. Pharmacol. 1987, 20:223-229). Inflammatory
stimuli of yeast or fungal origin, such as 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-stranded RNA, such polyinosine-polycytidine,
also has therapeutic activity as an inflammatory stimulus for
treatment of cancer or infections.
[0131] The inflammatory peptide, Interleukin-1 (IL-1), which
mediates some actions of endotoxin, similarly has important
therapeutic potential (e.g. in restoring hematopoiesis after damage
caused by cancer chemotherapy), but its use is limited by toxic
side effects which may be attenuated by utilization of the
compounds, compositions, and methods of the invention.
[0132] Interleukin-2 (IL-2) is used 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 response to vaccines. Hepatic toxicity in response
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 hepatitis
induced by administration of concanavalin 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); as demonstrated
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 side effects when administered in
conjunction with IL-2; furthermore, the compounds, compositions,
and methods of the inventions are useful in treating autoimmune
hepatitis.
[0133] Interleukin 6, which has therapeutic potential in improving
blood platelet production, induces hepatic TNF receptors, thus
increasing tissue sensitivity to TNF. The compounds, compositions,
and methods of the invention are thus useful for use in combination
with IL-6 or similar agents which affect tissue senstivity to, or
production of, TNF (Van Bladel et al., Cytokine, 1991
3:149-54).
[0134] The combination of a particular therapeutic cytokine and a
pyrimidine nucleotide precursor and/or a uridine phosphorylase
inhibitor is used for treatment of the disorders for which the
particular therapeutic cytokine is known to be effective. For
example, interleukin 2 is used for treatment of renal cancer, colon
cancer, melanoma, lymphoma, leukemia and other neoplastic
conditions. TNF has antitumor efficacy against a variety of cancer
types, but its use in therapy has heretofore been limited by its
toxicity, (Kimura et al., Cancer. Chemother. Pharmacol. 1987;
20:223-9). Endotoxin has shown significant antitumor efficacy
(Engelhardt R et al., Cancer. Res. 1991 51:2524-30).
[0135] For prevention or treatment of toxicity due to
administration of therapeutic cytokines, approximately 0.5 to 40
grams of a pyrimidine nucleotide precursor is administered daily
for one to several days, depending on the duration of the cytokine
treatment. The pyrimidine nucleotide precursors are administered
before, during, or after administration of the therapeutic
cytokine. The therapeutic cytokines are administered in the
particular doses and regimens already established for experimental
and clinical treatment of various forms of cancer, except that
increased doses of cytokines may be tolerated when the pyrimidine
nucleotide precursors of the invention are administered as would be
determined in simple dose-escalation studies for each cytokine or
inflammatory stimulus.
[0136] Inflammatory Hepatitis: Liver Disorders Involving Endotoxin
or Mediators
[0137] 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 syndrome, or alcohol) which either increase hepatic
sensitivity to endotoxin or inhibit endotoxin clearance, is in part
mediated by bacterial endotoxin (normally present 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, Hepatology 1989
10:887-91). 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).
[0138] As is demonstrated in the Examples below, the compounds,
compositions, and methods of the invention significantly reduce
hepatic damage induced by endotoxin and other inflammatory stimuli
and mediators. The compounds, compositions, 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 other inflammatory stimuli or mediators are
implicated in their etiology (whether or not systemic
sepsis-syndrome is present). Conditions in which damage to the
liver by endotoxin or its mediators (e.g. TNF) are implicated
include but are not limited to the following disease states:
[0139] A. Reye's Syndrome
[0140] Reye's syndrome is characterized by rapid hepatic failure
and is most 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).
[0141] B. Alcoholic Liver Damage
[0142] Excessive consumption of ethanol, in addition to problems
associated with impaired mental and physical control associated
with ethanol intoxication, is a significant cause of liver injury
in humans. Endotoxin and TNF contribute to hepatic problems
associated with exposure to alcohol. (Nolan JP, Hepatology 1989
10:887-91; Arai M, Nakano S, Okuno F, et al. Hepatology 1989;
9:846-851; McClain C J and Cohen D A, Hepatology 1989;
9:349-351).
[0143] C. Fulminant Hepatitis
[0144] 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 Hypotheses, 1988
27:193-6)
[0145] D. Viral Hepatitis
[0146] Endotoxin contributes to hepatocyte damage occurring during
viral hepatitis. Viral hepatitis reduces the LD.sub.50 of endotoxin
in animal models, and exclusion of endogenous endotoxin from
experimental animals (by colectomy or by using axenic rodents)
reduces the hepatic damage caused by a viral challenge. (Gut et
al., J. Infect. Disease., 1984, 149:621). In some cases 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, compositions, and
methods of the invention are useful for treating hepatic damage
related to viral infection. Example 14 demonstrates that the
compounds and methods of the invention improve survival in an
animal model of viral hepatitis.
[0147] Immunopathology contributes to liver injury in viral
hepatitis in humans. Hepatitis B and C viruses do not necessarily
directly injure cells. There is substantial evidence that immune
responses to infected cells contributes significantly to liver
injury. Activated cytotoxic T lymphocytes attack antigen-bearing
infected cells, but also release cytokines like interferon-gamma
which then recruit and activate inflammatory leukocytes in the
liver and enhance hepatic sensitivity to macrophage activators like
endotoxin (Ando et al., J. Exp Med. 178:1541-1554, 1993). In
Examples 10 and 12, beneficial effects of compounds, compositions,
and methods of the invention are presented in experimental models
mimicking the key features of T Cell-mediated hepatic inflammatory
injury with and without secondary exacerbation caused by endotoxin.
These Examples support the utility of the compounds, compositions,
and methods of the invention in viral hepatitis, as well as in
autoimmune hepatitis and cell-mediated liver graft rejection.
[0148] E. Parasitic Infections
[0149] Hepatic damage and morbidity which occurs during malaria
infection is mediated in part by TNF (Clark et al., Am. J. Pathol.
1987, 129:192-9).
[0150] F. Hepatic Damage During Total Parenteral Nutrition
[0151] Hepatic complications are common in patients receiving total
parental nutrition (TPN) and who have no underlying liver disease;
exacerbation of pre-existing liver injury also occurs during TPN.
Pappo et al. (J. Surg. Res., 1991, 51:106-12) reported that
endotoxin (LPS) derived from the overgrowth of intestinal
gram-negative bacteria is responsible for TPN-associated hepatic
steatosis, 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 is in fact mediated in part by endotoxin or
TNF. Therefore, inclusion of effective amounts of compounds of the
invention in TPN solutions is useful for reducing TPN-induced liver
damage, as well as for treating underlying inflammatory liver
injury. Compounds of the invention, especially uridine, cytidine,
orotic acid, or water soluble salts and esters thereof, are either
included in a TPN formulation or are administered separately but
concurrently with TPN infusion. A typical TPN formula contains the
basic nutrients needed to fulfill nutritional requirements in a
form that is acceptable for intravenous administration. Thus,
macromolecular dietary constituents like proteins or starches are
provided in partially or fully digested form, e.g. as amino acids
or sugars. A typical TPN formulation contains not only amino acids
and sugars, but also other required nutrients like vitamins,
minerals, and fats. Preferred doses of compounds of the invention
to be used in conjunction with, or as constituents of, TPN formulas
are in the range of 1 to 40 grams per day (usually in the range of
2 to 20 grams per day) either as a bolus injection or as a
sustained infusion.
[0152] In the context of this embodiment of the invention, a
patient need not be receiving all of his or her nutrient
requirements by the parenteral route in order to obtain benefit
from the compounds, compositions, and methods of the invention.
However, this embodiment of the invention is particularly
advantageous where patients are receiving 50% or more of their
nutrient requirements by intravenous infusion.
[0153] Lead Poisoning
[0154] 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. Sure. Res., 1985, 17:140-9).
[0155] H. Partial Hepatectomy
[0156] Following partial hepatectomy (e.g. for removal of cancerous
tissue), morbidity and mortality from hepatic failure is not
uncommon. Liver tissue undergoing regeneration after partial
hepatectomy in animals is hypersensitive to the deleterious effects
of endotoxin and mediators (Shirai et al., Acta Pathol. Jpn., 1987,
37:1127-1134).
[0157] I. Postanesthetic Hepatitis
[0158] 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., Anesth. Analg., 1972, 51:264-270); the compounds
of the invention are thus useful for administration to patients
(prophylactically, therapeutically, or both) undergoing inhalation
anesthesia for preventing and treating hepatitis. Trauma itself may
contribute to postanesthetic hepatitis. Trauma furthermore often
induces translocation of bacteria and endotoxin from the gut into
other tissues 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 precursors (before, during, or after surgery)
significantly improves their resistance to endotoxin poisoning.
[0159] J. Cholestatic Hepatitis
[0160] 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).
[0161] K. Liver Transplantation
[0162] In patients receiving liver transplants, the presence of
high levels of endotoxin or inflammatory mediators preoperatively
and at the end of the anhepatic period is associated with graft
failure and a high mortality. Patients with primary nonfunction of
their transplants typically have severe endotoxemia. Endotoxemia is
implicated as 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 doses ranging from about 1 to
about 40 grams per day, though typically 2 to 20 grams,
advantageously divided into one to about four doses or else
administered as a continuous or intermittent enteral or parenteral
infusion. A compound of the invention is also optionally
incorporated into an enteral or parenteral nutrition formulation
prior to administration. Patients often receive intravenous
isotonic (5%) glucose for several days after liver transplantation
as an alternative to more complete parenteral or enteral nutrition.
A compound of the invention, especially uridine or cytidine, is
advantageously formulated in an aqueous solution of 1 to 10%
glucose. In a preferred embodiment, 1 to 40 grams per day,
advantageously 2 to 20 grams, of a pyrimidine nucleotide precursor
are administered per day. A secondary benefit of pyrimidine
nucleotide precursors in liver disease or in recovery from a
transplant is improved peripheral glucose utilization.
[0163] The donor liver can also be perfused with a solution
containing a compound of the invention, advantageously uridine,
cytidine, orotic acid or salts or acyl derivatives thereof, prior
to or during implantation in the recipient. A pyrimidine nucleotide
precursor, especially uridine, is included in a liver perfusion
solution (also containing appropriate ions and other metabolites
like glucose) at concentrations ranging from 10 micromolar to 10
millimolar.
[0164] Endotoxins and inflammatory mediators are also involved in
other hepatic disorders; the diversity of the specific examples
discussed 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.
[0165] For treatment of inflammatory hepatitis, 0.5 to 40 grams
(advantageously 3 to 30 grams) of a pyrimidine nucleotide precursor
are administered daily, advantageously divided into one to about
four doses. The duration of the treatment regimen depends on
improvement of clinical symptoms; acute inflammatory liver
disorders will typically require a shorter course of treatment than
chronic degenerative conditions.
[0166] Other Disorders
[0167] As is demonstrated in Examples 2, 4-6, and 9, the compounds
of the invention protect tissues other than liver, e.g. muscle, as
indicated by serum creatine phosphokinase (CPK) levels in animals
treated with endotoxin or the fungal inflammatory agent zymosan.
Serum CPK activity is elevated as a consequence of damage to
skeletal or heart muscle.
[0168] Cachexia, a syndrome of weight loss, tissue wasting and
misutilization of nutrients is a common complication in patients
with cancer. TNF and other inflammatory cytokines are 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.
[0169] Clearance of ethanol from the circulation is a process that
is largely dependent on energy metabolism and redox balance in the
liver, in addition to levels of the enzyme alcohol dehydrogenase.
Example 13 demonstrates that compounds of the invention improve
recovery from severe ethanol intoxication. The compounds and
compositions of the invention are useful in reducing the severity
of both the mental and physical impairment due to alcohol
intoxication as well as longer-term health consequences of chronic
alcohol ingestion such as liver injury. Compounds of the invention
(e.g. triacetyluridine, uridine or cytidine) are administered
orally before, during, or after ingestion of ethanol in doses of
0.5 to 40 grams per day, advantageously 1 to 20 grams.
[0170] Veterinary Applications
[0171] In horses and other large animals, there is a common
syndrome known as laminitis, 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 tissue damage due to
endotoxin, are useful in treating or preventing laminitis and other
effects of endotoxin poisoning in animals.
[0172] Administration and Formulation of Compounds and Compositions
of the Invention
[0173] The compounds and compositions of the invention are
administered orally, by parenteral injection, intravenously, or by
other means, depending on the condition being treated and the
status of the patient.
[0174] The compounds and compositions of the invention are
administered chronically, intermittently, or acutely as needed. In
the case of an event which involves endotoxin toxicity or systemic
inflammatory syndrome, the compounds and compositions are
administered prior to, during, or after such event.
[0175] The pharmacologically active compounds optionally are
combined with suitable pharmaceutically acceptable carriers
comprising excipients and auxiliaries which facilitate processing
of the active compounds. 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).
[0176] For parenteral administration by injection or intravenous
infusion, the active compounds are suspended or dissolved in
aqueous medium such as sterile water or saline solution. Injectable
solutions or suspensions optionally contain a surfactant agent such
as polyoxyethylenesorbitan esters, sorbitan esters, polyoxyethylene
ethers, or solubilizing agents like propylene glycol or ethanol.
The solution typically contains 1 to 25% of the active compounds.
In one embodiment of the invention, the aqueous medium is a
solution of 1 to 10% glucose in water or isotonic saline. In some
circumstances concurrent intravenous adminisration of glucose and a
compound of the invention, especially uridine, is advantageous.
Uridine (and acyl derivatives of uridine) improve glucose
peripheral glucose utilization, and insulin (which is generally
released from the pancreas in response to glucose or other
carbohydrates or some amino acids) enhances nucleoside uptake and
utilization by cells.
[0177] For use in conjunction with parenteral nutrition, compounds
of the invention are dissolved or suspended in parenteral nutrition
products, either during manufacture of such products or shortly
prior to their administration to patients. The concentration of
pyridimine nucleotide precursor is adjusted in the parenteral
nutrition formulation so that 1 to 40 grams, generally 2 to 20
grams, are delivered per day during infusion of the parenteral
nutrition product. A typically parenteral nutrition formula
contains and delivers nutritionally adequate portions of amino
acids, carbohydrates, fats, vitamins, and minerals in sterile
compositions suitable for intreavenous adminstration.
[0178] Suitable excipients include fillers such as sugars, for
example lactose, sucrose, mannitol or sorbitol, cellulose
preparations and/or calcium phosphates, for example tricalcium
phosphate or calcium hydrogen phosphate, as well as binders such as
starch paste, using, for example, maize starch, wheat starch, rice
starch or potato starch, gelatin, tragacanth, methyl cellu-lose,
hydroxypropylmethyl cellulose, sodium carboxymethyl cellulose
and/or polyvinyl pyrrolidone.
[0179] Auxiliaries include flow-regulating agents and lubricants,
for example, silica, 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
sugar solutions 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 as
acetylcellulose phthalate or hydroxypropylmethylcell- ulose
phthalate are used. Dyestuffs or pigments are optionally added to
the tablets or dragee coatings, for example, for identification or
in order to characterize different compound doses.
[0180] 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(s) with solid excipients, optionally grinding the
resulting mixture and processing the mixture of granules, after
adding suitable auxiliaries, if desired or necessary, to obtain
tablets or dragee cores.
[0181] Other pharmaceutical preparations which are useful for oral
delivery include push-fit capsules made of gelatin, as well as
soft-sealed capsules made of gelatin and a plasticizer such as
glycerol or sorbitol. The push-fit capsules contain the active
compound(s) in the form of granules which optionally are mixed with
fillers 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, suspensions, or emulsions. In particular, a liquid form
suitable for administration via an enteral catheter, e.g. a
nasogastric tube, is advantageous, particularly for bedridden or
unconscious patients.
[0182] Pharmaceutical preparations which are used rectally include,
for example, suppositories which consist of a combination of active
compounds with a suppository base. Suitable suppository bases are,
for example, natural or synthetic triglycerides, paraffin
hydrocarbons, polyethylene glycols or higher alkanols. In addition,
gelatin rectal capsules which consist of a combination of the
active compounds with a base are useful. Base materials include,
for example, liquid triglycerides, polyethylene glycols, or
paraffin hydrocarbons.
[0183] Suitable formulations for parenteral administration include
aqueous solutions of the active compounds in water soluble form,
for example, water soluble salts. In addition, suspensions of the
active compounds as appropriate in oily injection suspensions 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 suspensions optionally include substances which increase
the viscosity of the suspension which include, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension
optionally contains stabilizers.
[0184] Synthesis of the Compounds of the Invention
[0185] Acylated derivatives of pyrimidine nucleosides are
synthesized 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 is
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 nucleosides or congeners with
coupling reagents like dicyclohexylcarbodiimide (DCC).
[0186] 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 groups, these groups are blocked with protecting groups,
e.g., t-butyldimethylsilyl ethers or t-BOC groups, respectively,
before preparation of the anhydride. For example, lactic acid is
converted to 2-t-butyldimethylsiloxypropionic acid with
t-butyldimethylchlorosilane, followed by hydrolysis of the
resulting silyl ester with aqueous base. The anhydride is formed by
reacting the protected acid with DCC. With amino acids, 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.
[0187] 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 methods using DCC in a
suitable solvent, particularly a mixture of (i) methylene chloride
and (ii) dimethylacetamide or dimethylformamide.
[0188] Carbyloxycarbonyl derivatives of non-methylated pyrimidine
nucleosides are prepared by reacting the nucleoside with the
appropriate carbylchloroformate in a solvent such as pyridine or
pyridine plus dimethylformamide under anhydrous conditions. The
solvent is removed under vacuum, and the residue is purified by
column chromatography.
[0189] It will be obvious to the person skilled in the art that
other methods of synthesis can be used to prepare the compounds of
the invention.
[0190] 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 skilled in the art are within the spirit and scope
of this invention.
EXAMPLES
Example 1
Triacetyluridine and Uridine Improve Survival in Mice Treated with
Killed E. Coli
[0191] Purpose:
[0192] Sepsis syndrome 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 purpose 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
[0193] Methods:
[0194] 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% Tween 80) by oral intubation. Survival was
monitored for one week.
[0195] A. n=6 E. Coli (Control)
[0196] B. n=6 E. Coli (Control)+Urd i.p.
[0197] C. n=6 E. Coli (Control)+TAU p.o.
[0198] Results:
[0199] Animals in the Control group appeared to be in shock and
were hypothermic 18 hours after administration of the E. Coli
powder. Animals in the treated groups were active and maintaining
body temperature, although their coats were scruffy throughout the
first 48 hours of the observation period. Animals surviving 48
hours recovered completely. All of the mice treated only with E.
Coli died within 48 hours. All mice treated with either uridine or
triacetyluridine survived administration of killed E. Coli.
Example 2
Dose-Response Study of Uridine in Protection of Tissues from
Endotoxin Damage
[0200] Purpose:
[0201] The purpose of this study was to determine the dose-response
characteristics for uridine in prevention of inflammatory tissue
damage caused by endotoxin (LPS).
[0202] Methods:
[0203] 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 indices 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 hours prior to endotoxin administration, the
five groups 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 samples were collected
for determination of serum chemistry values of indicators of
tissue-damage.
[0204] Results:
[0205] 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 released from both liver and muscle. The most
effective uridine dose in mice in this experiment was 2000
mg/kg.
1TABLE 1 Uridine attenuates endotoxin-induced tissue damage ALT AST
LDH CPK SDH Basal (No LPS) 198 .+-. 124 137 .+-. 26 708 .+-. 177
906 .+-. 211 49 .+-. 2 Control (LPS) 3768 .+-. 482 4176 .+-. 459
8406 .+-. 850 11628 .+-. 2398 1170 .+-. 157 Uridine 500 2568 .+-.
678 3090 .+-. 871 5988 .+-. 1225 8832 .+-. 1089 834 .+-. 192
Uridine 1000 1338 .+-. 401* 1206 .+-. 314* 3101 .+-. 860* 4431 .+-.
1529* 404 .+-. 95* Uridine 2000 605 .+-. 236* 620 .+-. 174* 1990
.+-. 642* 4531 .+-. 2139* 125 .+-. 45* Uridine 4000 1120 .+-. 970*
744 .+-. 457* 3441 .+-. 2378* 8680 .+-. 6746* 135 .+-. 75* *=
Different from Control (LPS i.p.), P < .02 ALT = Alanine
Aminotransferase AST = Aspartate Aminotransferase LDH = Lactate
Dehydrogenase CPK = Creatine Phosphokinase SDH = Sorbitol
Dehydrogenase
Example 3
Oral Triacetyluridine Improves Survival of Mice Treated with a
Lethal Dose of Salmonella Typhimurium Endotoxin
[0206] Purpose:
[0207] Sepsis syndrome caused by gram-negative bacteria is mediated
primarily through endotoxin, a lipopolysaccharide 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).
[0208] Methods:
[0209] Twenty-female Balb/C mice (eight weeks old) were divided
into two groups of ten animals each. All mice received 100
micrograms 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.
[0210] Results:
[0211] 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
[0212] Purpose:
[0213] Bacterial endotoxin causes damage to the liver and other
organs which can be assessed and quantified by determining serum
levels of enzymes and other markers of tissue integrity and
function. The purpose of this study was to determine the
dose-response characteristics of orally-administered triacetyl
uridine (TAU) in attenuating tissue damage due to endotoxin.
[0214] Methods:
[0215] Female Balb/C mice (eight weeks old) were divided into
groups of five animals each. One group of animals remained
untreated to provide basal values for serum chemistry indices of
tissue damage. Mice in the other four groups received 100
micrograms of Salmonella Typhimurium endotoxin by i.p. injection,
in a volume 0.2 ml saline. Three groups of endotoxin-treated mice
also received TAU 2 hours before endotoxin in doses of 2000, 4000,
and 6000 mg/kg by oral intubation in a volume of 0.4 ml. The TAU
was formulated as a suspension in 1% carboxymethylcellulose in
water. The remaining group (Controls) received the
carboxymethylcellulose vehicle by oral intubation.
[0216] Results:
[0217] Oral TAU administration reduced the levels of serum
chemistry indicators of tissue damage. The beneficial effect on
prevention of endotoxin-induced organ damage was dose
dependent.
2TABLE 2 TAU attenuates endotoxin-induced tissue damage ALT AST LDH
SDH Basal (No LPS) 130 .+-. 46 148 .+-. 32 563 .+-. 132 41 .+-. 5
Control (LPS) 3679 .+-. 703 4798 .+-. 927 6998 .+-. 1064 1128 .+-.
174 TAU 2000 2632 .+-. 915 3151 .+-. 1085 5419 .+-. 1561 793 .+-.
294 TAU 4000 1463 .+-. 382* 1940 .+-. 456* 3878 .+-. 672* 345 .+-.
106* TAU 6000 365 .+-. 91* 403 .+-. 61* 1221 .+-. 181* 104 .+-. 18*
*Different from Control (LPS i.p. + vehicle p.o.), P < .02 ALT =
Alanine Aminotransferase AST = Aspartate Aminotransferase LDH =
Lactate Dehydrogenase CPK = Creatine Phosphokinase SDH = Sorbitol
Dehydrogenase
Example 5
Uridine Reduces Tissue Damage in Mice Treated with Carrageenan as a
Potentiator of Endotoxin Toxicity
[0218] Carrageenan is a polysaccharide derived from seaweed which
modifies the activity of macrophages, which are principal cellular
mediators of systemic inflammatory response to endotoxin.
Macrophages release inflammatory peptides and other compounds in
response to endotoxin. Carrageenan pretreatment sensitizes
macrophages so that much less endotoxin than normal is required to
elicit a serious systemic inflammatory response. 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 tissue damage induced by a
combination of carrageenan and endotoxin.
[0219] Methods:
[0220] Female Balb/C mice (eight weeks old) were divided into five
groups of six animals each. One group of animals remained untreated
to provide basal values for serum chemistry indices of tissue
damage. Mice in the other four groups 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, also 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 minutes after administration
of endotoxin, and the other received 3 uridine pretreatments, 24,
6, and 2 hours before endotoxin administration, at 2000 mg/kg/dose
i.p. Eighteen hours after endotoxin administration, blood samples
were collected for determination of serum chemistry values of
indicators of tissue damage.
[0221] Results:
[0222] The combination of carrageenan with a low dose of endotoxin
(2 mg) resulted in significant tissue damage as evaluated by serum
chemistry indices. Treatment with uridine either before or after
administration of endotoxin resulted in significant attenuation of
tissue damage due to the carrageenan-endotoxin combination. Data
are shown below.
3TABLE 3 Uridine attenuates endotoxin-induced tissue damage in
carrageenan-sensitized mice ALT AST LDH CPK SDH Basal (No LPS) 223
.+-. 77 141 .+-. 35 700 .+-. 145 747 .+-. 278 33 .+-. 1 Control
(LPS) 1937 .+-. 235 2072 .+-. 149 7360 .+-. 354 11612 .+-. 1513 107
.+-. 17 Uridine 817 .+-. 202* 989 .+-. 139* 4385 .+-. 454* 5485
.+-. 1638* 80 .+-. 12* Uridine 770 .+-. 141* 891 .+-. 79* 4416 .+-.
283* 5033 .+-. 565* 117 .+-. 9 (postreatment) *= Different from
Control, P < .05 ALT = Alanine Aminotransferase AST = Aspartate
Aminotransferase LDH = Lactate Dehydrogenase CPK = Creatine
Phosphokinase SDH = Sorbitol Dehydrogenase
Example 6
Uridine Improves Survival in Zymosan-Treated Mice
[0223] Purpose:
[0224] Zymosan is a yeast component, primarily polysaccharide,
which induces systemic inflammation and activation of complement.
In fungal infections in general (including but not limited to yeast
infections), such polysaccharides participate in the induction of a
sepsis response. Zymosan administration to rodents is considered to
be a suitable model for multiple organ failure syndrome (Goris et
al. (1986) Arch. Surg. 121:897-901; Steinberg et al. (1989) Arch.
Surg. 124:1390-1395). Mortality at minimum lethal doses 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).
[0225] Methods:
[0226] Female Balb/C mice (eight weeks old) were divided into
groups of five animals each:
[0227] 1. Zymosan 15 mg
[0228] 2. Zymosan 15 mg+Uridine
[0229] 3. Zymosan 20 mg
[0230] 4. Zymosan 20 mg+Uridine
[0231] 5. Basal
[0232] Zymosan A was suspended in mineral oil at a concentration of
50 mg/ml and administered by intraperitoneal injection. Uridine
(2000 mg/kg) was administered by intraperitoneal injection in a
volume of 0.2 ml two hours before administration of Zymosan.
[0233] 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.
[0234] Results:
4 Group Survival A. Survival at 48 hours: 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 Zymosan 15 mg/kg + Uridine 4/5
[0235] Uridine significantly improved survival time and incidence
of long-term survivors among mice treated with Zymosan.
[0236] C. Serum Chemistry Indices of Tissue Damage
5TABLE 4 Uridine attenuates Zymosan-induced tissue damage ALT AST
LDH CPK SDH Basal 50 .+-. 22 93 .+-. 41 899 .+-. 198 532 .+-. 731
52 .+-. 25 Zymosan 397 .+-. 140 392 .+-. 97 1974 .+-. 392 2107 .+-.
1172 81 .+-. 15 Zymosan + 120 .+-. 126 273 .+-. 131 1419 .+-. 244
754 .+-. 370 58 .+-. Uridine 22 ALT = Alanine Aminotransferase AST
= Aspartate Aminotransferase LDH = Lactate Dehydrogenase CPK =
Creatine Phosphokinase SDH = Sorbitol Dehydrogenase
Example 7
Comparison of Effects of Uridine Versus Arginine on Survival of
Endotoxin-Treated Mice
[0237] Purpose:
[0238] The amino acid arginine is reported to have beneficial
effects in sepsis syndrome (Leon et al. J. Parenteral and Enteral
Nutrition, 1991, 15:503-508). The purpose of this study was to
compare the efficacy of uridine with that of arginine, an agent
which supports liver function in sepsis syndrome and which is
clinical use for this purpose.
[0239] Methods:
[0240] 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 groups of mice received
injections of:
[0241] 1) Saline (Controls)
[0242] 2) Uridine 2000 mg/kg
[0243] 3) Arginine 25 mg/kg
[0244] 4) Arginine 250 mg/kg
[0245] 5) Arginine 1250 mg/kg
[0246] All drugs were administered i.p. in 0.2 ml saline. The
numbers of surviving mice in each group were determined 16, 20, and
24 hours.
[0247] Results:
[0248] 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.
6TABLE 5 Effect of uridine vs arginine on survival after LPS
administration Time after LPS (hr) Groups 16 20 24 1. Control 1/6
0/6 0/6 2. Uridine 5/5 5/5 5/5 3. Arg 25 5/5 3/5 0/5 4. Arg 250 4/5
2/5 0/5 5. Arg 1250 4/6 1/6 0/6
Example 8
Orotic Acid Improves Survival of Mice Treated with Salmonella
Typhimurium Endotoxin
[0249] Purpose:
[0250] sepsis syndrome caused by gram-negative bacteria is mediated
primarily through endotoxin, a lipopolysaccharide constituent 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.
[0251] Methods:
[0252] Twenty female Balb/C mice (eight weeks old) were divided
into two groups of ten animals each. One group of mice received
four treatments with lysine orotate (200 mg/kg/dose; 9 AM and 2 PM
on each of two consecutive days). Lysine 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
micrograms of Salmonella Typhimurium endotoxin (LPS) by
intraperitoneal injection in 0.2 ml of saline immediately after the
last dose of lysine orotate. Survival was monitored for one
week.
[0253] Results:
[0254] 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.
7TABLE 6 Orotate improves survival of endotoxin-treated mice
Survival after endotoxin treatment Time (hr after LPS) 24 26 28 32
48 72 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
[0255] Purpose:
[0256] The purpose of this study was to demonstrate the protective
effect of orotic acid in prevention of inflammatory tissue damage
caused by endotoxin.
[0257] Methods:
[0258] 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 tissue
damage. Mice in the remaining two groups received 100 micrograms 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 lysine 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 indicators of tissue damage.
[0259] Results:
[0260] Orotate protected tissues against damage from endotoxin
administration.
8TABLE 7 Orotate attenuates endotoxin-induced tissue dama ALT AST
LDH CPK SDH Basal (No LPS) 132 .+-. 14 165 .+-. 21 681 .+-. 552
1258 .+-. 233 42 .+-. 1 Control (LPS) 2827 .+-. 413 2860 .+-. 506
6833 .+-. 1167 6820 .+-. 365 680 .+-. 142 Orotate + LPS 252 .+-.
99* 415 .+-. 77* 1641 .+-. 274* 1040 .+-. 283* 89 .+-. 7* *=
Different from Control (LPS i.p.), P < .02 ALT = Alanine
Aminotransferase AST = Aspartate Aminotransferase LDH = Lactate
Dehydrogenase CPK = Creatine Phosphokinase SDH = Sorbitol
Dehydrogenase
Example 10
Uridine and Triacetyluridine Attenuate Hepatic Damage Caused by
Concanavalin A
[0261] Purpose:
[0262] Interleukin-2 (IL-2) is used clinically for treatment of
several varieties of cancer. Hepatic toxicity in response 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 hepatitis 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.
[0263] Methods:
[0264] Female Balb/C mice (eight weeks old) were divided into four
groups of five animals each. One group of animals remained
untreated to provide basal values for serum 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 emulsion 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 samples were collected from all mice for determination
of serum levels of various indices of tissue damage or metabolic
dysfunction.
[0265] Results:
[0266] Con A administration resulted in significant damage to the
liver, as assessed by serum levels of the enzymes ALT, AST, and
SDH. Con A did not signficantly elevate levels of creatine
phosphokinase (CK), an enzyme found primarily in muscle; tissue
damage due to Con A in this model is more specifically localized in
the liver than is damage due to endotoxin. Uridine and TAU both
reduced the liver damage produced by Con A administration, as shown
in Table 8 below.
9TABLE 8 Uridine and Triacetyluridine attenuate liver damage caused
by Concanavalin A ALT AST LDH CPK SDH Basal (No Con A) 144 .+-. 18
217 .+-. 27 790 .+-. 90 2392 .+-. 370 51 .+-. 2 Con A 2652 .+-. 847
2765 .+-. 1030 4335 .+-. 1385 2572 .+-. 486 1114 .+-. 318 Con A +
Uridine 289 .+-. 115* 394 .+-. 114* 973 .+-. 202* 1996 .+-. 317 163
.+-. 68* Con A + TAU 575 .+-. 286* 613 .+-. 221 1380 .+-. 270 1951
.+-. 435 283 .+-. 143* *= Different from Control (LPS i.p.), P <
.02 ALT = Alanine Aminotransferase AST = Aspartate Aminotransferase
LDH = Lactate Dehydrogenase CPK = Creatine Phosphokinase SDH =
Sorbitol Dehydrogenase
[0267] Liver damage in the Con A model used in this study is
related to elevated IL-2 levels, and is mediated through T
lymphocytes. Therefore, the compounds and methods of the invention
are useful 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 Attenuates Sepsis Induced Alterations in Blood
Coagulation
[0268] Purpose:
[0269] Disseminated Intravascular Coagulation (DIC) is a serious
consequence of sepsis, in which both blood coagulation and
fibrinolysis are activated, so that blood clotting factors are
rapidly consumed. DIC can result in hemorrhage or thrombus
formation. The liver is the primary site for synthesis of clotting
factors and for clearing micro-aggregates of thrombin from the
circulation. This purpose of this experiment was to determine the
effect of pyrimidine nucleotide precursors on coagulation disorders
induced by sepsis. Partial thromboplastin time was used as an index
of the status of the blood coagulation system.
[0270] Methods:
[0271] 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 used to determine basal values for partial
thromboplastin time. Two groups of mice received received 30 mg/kg
killed E. Coli (strain 0111:B4); Two hours before E. Coli
administration, one group received uridine (2000 mg/kg) by
intraperitoneal injection. 20 hours after E. Coli administration,
plasma 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.
[0272] Results:
[0273] Administration of killed E. Coli resulted in a prolongation
of the normal partial thromboplastin time. Uridine attenuated the
sepsis-induced change in coagulation time, as shown in Table 9.
[0274] Table 9: Uridine Attenuates Sepsis-Induced Alterations in
Partial Thromboplastin Time
10 Partial Thromboplastin Time Group PTT (seconds) Basal (Normal)
32.3 .+-. 1.3 E. Coli 69.8 .+-. 5.4 E. Coli + Uridine 51.2 .+-.
2.1* * = different from control (E. Coli alone) value, P <
.05
[0275] *=different from control (E. Coli alone) value,
P<0.05
Example 12
Combined Liver Injury Due to T Cells and Endotoxin
[0276] 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 participates in liver damage initiated by a number
of other agents like carbon tetrachloride, choline deficiency,
ethanol, or cholestasis, studies were conducted to determine
whether liver injury caused by T cells induces hepatic
hypersensitivity to endotoxin. Following this experiment, the
effect of TAU on combined liver injury due to both T lymphocytes
and endotoxin was investigated.
Example 12A
Concanavalin A Potentiates Endotoxin-Induced Tissue Damage
[0277] 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 hours before endotoxin.
Blood samples 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.
11TABLE 1 Concanavalin A potentiates endotoxin-induced tissue
damage ALT AST LDH CPK SDH Basal 87 .+-. 15 110 .+-. 9 656 .+-. 41
413 .+-. 87 39 .+-. 2 Con A 2.5 mg/kg 117 .+-. 19 170 .+-. 16 915
.+-. 46 419 .+-. 132 42 .+-. 4 LPS 0.5 mg/kg 119 .+-. 23 256 .+-.
22 881 .+-. 10 426 .+-. 82 41 .+-. 3 Con A + LPS 1130 .+-. 494 2119
.+-. 910 4370 .+-. 1303 1525 .+-. 450 471 .+-. 267 ALT = Alanine
Aminotransferase AST = Aspartate Aminotransferase LDH = Lactate
Dehydrogenase CPK = Creatine Phosphokinase SDH = Sorbitol
Dehydrogenase
[0278] Endotoxin or Con A alone at the doses used in this
experiment produced minimal damage to liver and muscle as
determined by serum enzyme levels (ALT, AST, LDH and SDH are
markers for liver damage; CPK is an indicator for muscle damage).
However, in mice treated with the combination of Con A and
endotoxin, significantly greater damage was observed. The toxicity
of Con A in this model is believed to be specifically related to T
Lymphocyte-mediated liver damage (Tiegs et al., J. Clin. Invest.
90:196-203, 1992). Therefore, these results 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 insults including carbon tetrachloride, choline
deficiency, D-galactosamine, and viral infections.
Example 12B
TAU Attenuates Combined Liver Injury Due to CTL's and Endotoxin
[0279] 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 results
in greater-than-additive hepatic injury (see Example 12A).
Hepatocyte injury in viral and autoimmune hepatitis involves
similar mechanisms, with damage initiated by T cells and
exacerbated by enterally-derived endotoxin and other inflammatory
processes.
[0280] TAU protects the liver of experimental animals from damage
initiated by either endotoxin or Con A. In this experiment, TAU was
tested for hepatoprotective effects in mice subjected to combined
liver injury caused by sequential administration of both Con A and
endotoxin.
[0281] Methods:
[0282] 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 values for serum chemistry indices of
tissue damage. Mice in the remaining two groups received 2 mg/kg
Concancavalin A by intravenous (tail vein) injection in a volume of
0.2 ml saline, followed 24 hours later by Salmonella Typhimurium
endotoxin (10 micrograms i.p.). One of these groups of mice
received TAU (6000 mg/kg orally, in 0.6 ml of 0.5% methylcellulose
two hours before Con A and again 2 hours before endotoxin; the
remaining Con A/endotoxin-treated group (Control) received vehicle
(methylcellulose) alone. Eighteen hours after administration of
endotoxin, blood samples were collected from all mice for
determination of serum levels of various indices of tissue damage
or metabolic dysfunction.
[0283] Results:
[0284] Sequential administration of Con A and endotoxin resulted in
significant liver injury, as assessed by serum chemistry indices of
liver damage. TAU, administered orally, markedly attenuates this
combined liver injury.
12 Oral TAU attenuates liver damage caused by Concanavalin A + LPS
ALT AST LDH CPK SDH Basal 118 .+-. 33 162 .+-. 14 522 .+-. 80 1521
.+-. 235 56 .+-. 3 Con A/LPS 2295 .+-. 309 3408 .+-. 389 5696 .+-.
560 4684 .+-. 1569 700 .+-. 69 Con A/LPS + TAU 285 .+-. 67* 451
.+-. 87* 1341 .+-. 236* 2098 .+-. 465* 122 .+-. 19* *= Different
from Control (LPS i.p.), P < .02 ALT = Alanine Aminotransferase
AST = Aspartate Aminotransferase LDH = Lactate Dehydrogenase CPK =
Creatine Phosphokinase SDH = Sorbitol Dehydrogenase
Example 13
Oral Triacetyluridine Improves Recovery from Ethanol
Intoxication
[0285] Ethanol intoxication results in depression of activity in
the central nervous system. Recovery is dependent upon clearance of
ethanol from the system. Ethanol clearance from the circulation
occurs primarily in the liver, regulated by both the enzyme alcohol
dehydrogenase and the redox balance and metabolic state of the
liver.
[0286] In these experiments, ethanol-intoxicated mice were treated
with triacetyluridine (TAU) in order to determine whether metabolic
support through provision of uridine to the liver and other tissues
affected recovery from ethanol intoxication.
[0287] Experiment 1: Fasted Mice
[0288] Methods:
[0289] Female Balb/C mice weighing an average of 22 grams were
fasted for 24 hours. 9 mice received oral TAU 2000 mg/kg p.o., and
8 received vehicle (0.75% Hydroxypropylmethylcellulose in
water).
[0290] One hour later, all animals received 5.7 ml/kg ethanol (0.5
ml of 25% aqueous solution p.o.).
[0291] One hour after EtOH, mice received an additional dose of TAU
or vehicle. All mice were basically comatose at this time.
[0292] Behavior was monitored at hourly intervals, beginning 3
hours after ethanol administration. The scale for behavioral
assessment is as follows:
[0293] Behavioral Recovery After Ethanol Intoxication
[0294] Deep coma: Unresponsive to stimuli. Slow respiration
[0295] Prostrate: Mice laying flat but not moving. Eyelid reflex to
touching with a probe. Rapid respiration.
[0296] Righting reflex: When placed on its back, animal attempts to
turn over within 5 seconds. Category includes all "mobile" animals
and some "prostrate".
[0297] Mobile: Animal is capable of walking.
13 TAU accelerates recovery from ethanol intoxication in fasted
mice Control n = 8 mice Time Dead Deep coma Prostrate Righting
Reflex Mobile 3 hr 1 8 0 0 0 4 hr 1 5 3 0 0 5 hr 2 3 3 3 0 6 hr 2 2
2 4 2 7 hr 2 1 1 4 4 8 hr 2 1 1 5 4
[0298]
14 TAU n = 9 mice Time Dead Deep coma Prostrate Righting Reflex
Mobile 3 hr 0 5 4 4 0 4 hr 0 4 5 4 0 5 hr 0 2 7 6 0 6 hr 0 1 1 7 7
7 hr 0 1 0 8 8 8 hr 0 0 1 8 8 Mice = Female Balb/C, 22 grams,
fasted 24 hours Ethanol dose = 5.7 ml/kg p.o. at time = 0 hr (0.7
ml of 25% EtOH) TAU (2 g/kg) or vehicle (control group) were
administered one hour before and one hour after ethanol
[0299] Experiment 2: Non-Fasted Mice
[0300] Female Balb/C mice weighing an average of 22 grams were
al;lowed free access to food up to the time of the experiment. 10
mice received oral TAU 2000 mg/kg p.o., and 10 received vehicle
(0.75% HPMC).
[0301] One hour later, all animals received 8 ml/kg ethanol (0.7 ml
of 25% aqueous solution p.o.).
[0302] One hour after EtOH, mice received an additional dose of TAU
or vehicle.
[0303] Behavior was monitored at intervals of 2, 3, 4, and 6 hours
after ethanol administration. The scale for behavioral assessment
was the same as in the test on fasted mice above.
15 TAU accelerates recovery from ethanol intoxication in non-fasted
mice Control n = 10 mice Time Dead Deep coma Prostrate Righting
Reflex Mobile 2 hr 0 9 1 0 0 3 hr 0 7 3 2 0 4 hr 1 1 6 5 2 6 hr 1 0
1 8 8
[0304]
16 TAU n = 10 mice Time Dead Deep coma Prostrate Righting Reflex
Mobile 2 hr 0 5 5 2 0 3 hr 0 1 5 5 3 4 hr 0 0 3 8 7 6 hr 0 0 0 10
10 Mice = Female Balb/C, 22 grams, fed ad libitum Ethanol dose = 8
ml/kg p.o. at time = 0 hr (0.7 ml of 25% EtOH) TAU (2 g/kg) or
vehicle (control group) were administered one hour before and one
hour after ethanol
[0305] TAU clearly improved behavioral recovery in mice subjected
to severe ethanol intoxication. Non-fasted mice were given a higher
dose of ethanol than fasted animals (8 ml/kg versus 5.7 ml/kg), but
nevertheless recovered somewhat faster. This observation highlights
the importance of energy metabolism in recovery from ethanol
intoxication. TAU accelerates recovery from ethanol intoxication in
both fed and fasted animals.
Example 14
Triacetyluridine Reduces Mortality in Viral Hepatitis in Mice
[0306] Frog virus 3 (FV3) induces a rapidly fatal hepatitis in
mice, which is mediated in part by secondary damage due to
endogenous endotoxin (Gut et al., J. Infect. Disease., 1984,
149:621).
[0307] Triacetyluridine (TAU) was tested in this model to
demonstrate that this agent and other compounds of the invention
have useful therapeutic effects in viral hepatitis.
[0308] Methods:
[0309] Lyophilized FV3 was reconstituted in phosphate-buffered
saline to a density of 1.times.10.sup.a plaque-forming units (PFU)
per ml.
[0310] Female Balb/C mice weighing 25 grams received FV3 virus at
doses corresponding to the approximated LD.sub.50 by
intraperitoneal or intravenous (tail vein) injection. TAU (3000
mg/kg) or vehicle (0.75 hydroxypropylmethylcellulose) was
administered orally one hour before FV3 and on subsequent
afternoons and mornings. Animals were observed for three days; the
animals that did not survive this observation period all died
approximately 24-30 hours after virus administration.
[0311] Intraperitoneal Administration of FV3
[0312] Virus: FV3 4.times.10.sup.7 PFU/mouse i.p.
17 Survival Control 6/10 TAU 10/10
[0313] Intravenous Administration of FV3
[0314] Virus: FV3 2.times.10.sup.7 PFU/mouse i.v.
18 Survival Control 5/10 TAU 10/10
[0315] The foregoing is intended as illustrative of the present
invention but not limiting. Numerous variations and modifications
may be effected without departing from the true spirit and scope of
the invention.
* * * * *