U.S. patent application number 13/375463 was filed with the patent office on 2012-06-21 for treatment of portal hypertension and restoration of liver function using l-ornithine phenylacetate.
This patent application is currently assigned to UCL Business PLC. Invention is credited to Keith Anderson, Rajiv Jalan.
Application Number | 20120157526 13/375463 |
Document ID | / |
Family ID | 43309444 |
Filed Date | 2012-06-21 |
United States Patent
Application |
20120157526 |
Kind Code |
A1 |
Jalan; Rajiv ; et
al. |
June 21, 2012 |
TREATMENT OF PORTAL HYPERTENSION AND RESTORATION OF LIVER FUNCTION
USING L-ORNITHINE PHENYLACETATE
Abstract
Disclosed herein are methods of treating and/or preventing
portal hypertension and/or restoring liver function using
L-ornithine phenylacetate.
Inventors: |
Jalan; Rajiv; (Chislehurst,
GB) ; Anderson; Keith; (San Diego, CA) |
Assignee: |
UCL Business PLC
London
GB
|
Family ID: |
43309444 |
Appl. No.: |
13/375463 |
Filed: |
June 8, 2010 |
PCT Filed: |
June 8, 2010 |
PCT NO: |
PCT/US10/37838 |
371 Date: |
February 17, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61185158 |
Jun 8, 2009 |
|
|
|
61240748 |
Sep 9, 2009 |
|
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61296377 |
Jan 19, 2010 |
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Current U.S.
Class: |
514/555 ;
514/564 |
Current CPC
Class: |
A61K 31/195 20130101;
A61P 29/00 20180101; A61K 31/192 20130101; A61K 31/19 20130101 |
Class at
Publication: |
514/555 ;
514/564 |
International
Class: |
A61K 31/205 20060101
A61K031/205; A61P 29/00 20060101 A61P029/00; A61P 25/00 20060101
A61P025/00; A61P 7/04 20060101 A61P007/04; A61K 31/198 20060101
A61K031/198; A61P 1/16 20060101 A61P001/16; A61P 9/12 20060101
A61P009/12 |
Claims
1. A method of treating a condition in a subject, comprising
administering to the subject L-ornithine in combination with at
least one of phenylacetate and phenylbutyrate, thereby relieving
the condition, wherein the condition is portal hypertension,
variceal bleeding, or ascites.
2. The method of claim 1, wherein the subject is suffering from
liver disease.
3. The method of claim 2, wherein the liver disease is a chronic
liver disease or an acute liver failure.
4. The method of claim 3, wherein the chronic liver disease is
cirrhosis.
5. (canceled)
6. The method of claim 1, wherein the treatment of the condition is
achieved by reducing the level of proinflammatory cytokines or
increasing endothelial nitric oxide synthase activity in the
subject.
7. (canceled)
8. The method of claim 1, wherein said L-ornithine and
phenylacetate is administered as L-ornithine phenyl acetate.
9. The method of claim 1, wherein separate physiologically
acceptable salts of said L-ornithine and at least one of
phenylacetate and phenylbutyrate are administered to the
subject.
10. The method of claim 1, wherein said L-ornithine is present
administered as a free monomeric amino acid or physiologically
acceptable salt thereof.
11. The method of claim 1, wherein at least one of phenylacetate
and phenylbutyrate is administered as a sodium phenylacetate or
sodium phenylbutyrate.
12. The method of claim 1, wherein said administration is oral,
intravenous, intraperitoneal, intragastric, or intravascular
administration.
13. The method of claim 12, wherein said administration is
intravenous administration.
14. The method of claim 12, wherein said administration is oral
administration.
15. The method of claim 1, wherein the dose of the L-ornithine and
the phenylacetate or phenylbutyrate administered is between 20 g
and 40 g.
16. A method of delaying or reducing the likelihood of onset of
portal hypertension in a subject, comprising administering to the
subject L-ornithine in combination with at least one of
phenylacetate and phenylbutyrate, thereby delaying or reducing the
likelihood of onset of portal hypertension.
17. The method of claim 16, wherein the subject is suffering from
liver disease.
18. The method of claim 17, wherein the liver disease is a chronic
liver disease or an acute liver failure.
19. The method of claim 18, wherein the chronic liver disease is
cirrhosis.
20. (canceled)
21. The method of claim 16, wherein the delay or reduction in the
likelihood of onset of portal hypertension is achieved by reducing
the level of proinflammatory cytokines or increasing endothelial
nitric oxide synthase activity in the subject.
22. (canceled)
23. The method of claim 16, wherein said L-ornithine and
phenylacetate is administered as L-ornithine phenyl acetate.
24. The method of claim 16, wherein separate physiologically
acceptable salts of said L-ornithine and at least one of
phenylacetate and phenylbutyrate are administered to the
subject.
25. The method of claim 16, wherein said L-ornithine is
administered as a free monomeric amino acid or physiologically
acceptable salt thereof.
26. The method of claim 16, wherein at least one of phenylacetate
and phenylbutyrate is administered as a sodium phenylacetate or
sodium phenylbutyrate.
27. The method of claim 16, wherein said administration is oral,
intravenous, intraperitoneal, intragastric, or intravascular
administration.
28. (canceled)
29. (canceled)
30. The method of claim 16, wherein the dose of the L-ornithine and
the phenylacetate or phenylbutyrate administered is between 20 g
and 40 g.
31. A method of restoring liver function in a subject having poor
liver function, comprising administering to the subject L-ornithine
in combination with at least one of phenylacetate and
phenylbutyrate to said subject and thereby improving liver
function.
32. The method of claim 31, wherein the subject is suffering from
portal hypertension.
33. The method of claim 32, wherein improving liver function
reduces the portal hypertension.
34. The method of claim 31, wherein improving liver function
comprises increasing liver perfusion.
35. The method of claim 31, wherein said restoration of liver
function is achieved by reducing the level of proinflammatory
cytokines or increasing endothelial nitric oxide synthase activity
in the subject.
36. (canceled)
37. The method of claim 31, wherein said L-ornithine and
phenylacetate is administered as L-ornithine phenyl acetate.
38. The method of claim 31, wherein separate physiologically
acceptable salts of said L-ornithine and at least one of
phenylacetate and phenylbutyrate are administered to the
subject.
39. The method of claim 31, wherein said L-ornithine is present
administered as a free monomeric amino acid or physiologically
acceptable salt thereof.
40. The method of claim 31, wherein at least one of phenylacetate
and phenylbutyrate is administered as a sodium phenylacetate or
sodium phenylbutyrate.
41. The method of claim 31, wherein said administration is oral,
intravenous, intraperitoneal, intragastric, or intravascular
administration.
42. (canceled)
43. (canceled)
44. The method of claim 31, wherein the dose of the L-ornithine and
the phenylacetate or phenylbutyrate administered is between 20 g
and 40 g.
45.-66. (canceled)
67. A method of protecting against brain injury in a patient with
acute liver failure or acute liver decompensation, the method
comprising: identifying a patient as suffering from acute liver
failure or acute liver decompensation; and administering to the
patient L-ornithine in combination with at least one of
phenylacetate and phenylbutyrate, thereby reducing the likelihood
that the patient will develop brain injury.
68. The method of claim 67, wherein said administering occurs
immediately after said identifying.
69. The method of claim 67, further comprising treating the acute
liver failure or acute liver decompensation.
70. The method of claim 69, wherein said treating comprises liver
transplantation.
71. The method of claim 67, further comprising treating a
complication caused by the acute liver failure or acute liver
decompensation.
72. The method of claim 71, wherein said complication comprises
variceal bleeding.
73. The method of claim 67, wherein the dose of the L-ornithine and
the phenylacetate or phenylbutyrate administered is between 20 g
and 40 g.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefits of U.S. Provisional
Patent Applications 61/185,158 filed Jun. 8, 2009, 61/240,748 filed
Sep. 9, 2009 and 61/296,377 filed Jan. 19, 2010. The contents of
each of these related applications are hereby incorporated by
reference in their entirety.
BACKGROUND
[0002] 1. Field
[0003] The present application relates to the fields of
pharmaceutical chemistry, biochemistry and medicine. One aspect
relates to the treatment and/or prevention of portal hypertension
using L-ornithine in combination with at least one of phenylacetate
and phenylbutyrate. Another aspect relates to restoration of liver
function using L-ornithine in combination with at least one of
phenylacetate and phenylbutyrate.
[0004] 2. Description of the Related Art
[0005] Portal hypertension is an increase in the blood pressure
within the portal vein and its tributaries. It is a condition that
can develop in patients with liver disease such as cirrhosis and
hepatic fibrosis. Portal hypertension may also be caused by
scarring of the liver, thrombosis, or clotting in the portal
vein.
[0006] Various prevention, treatment and management strategies for
portal hypertension are currently available depending upon the
severity of the symptoms. There is a need for additional therapies
for treat the above conditions.
SUMMARY
[0007] Some embodiments disclose methods of treating portal
hypertension in a subject, comprising administering to the subject
L-ornithine in combination with at least one of phenylacetate and
phenylbutyrate, thereby reducing portal hypertension.
[0008] Some embodiments disclose methods of delaying or reducing
the likelihood of onset of portal hypertension in a subject,
comprising administering to the subject L-ornithine in combination
with at least one of phenylacetate and phenylbutyrate, thereby
delaying the onset of portal hypertension.
[0009] Some embodiments disclose methods of restoring liver
function in a subject having poor liver function, comprising
administering to the subject L-ornithine in combination with at
least one of phenylacetate and phenylbutyrate, thereby improving
liver function.
[0010] Some embodiments disclose methods of treating variceal
bleeding in a subject suffering from portal hypertension,
comprising administering to the subject L-ornithine in combination
with at least one of phenylacetate and phenylbutyrate, thereby
reducing the variceal bleeding.
[0011] Some embodiments disclose methods of treating ascites in a
subject suffering from portal hypertension, comprising
administering to the subject L-ornithine in combination with at
least one of phenylacetate and phenylbutyrate, thereby reducing
ascites.
[0012] Some embodiments disclose methods of protecting against
brain injury in a patient with acute liver failure, the method
comprising identifying a patient as suffering from acute liver
failure and administering to the patient L-ornithine in combination
with at least one of phenylacetate and phenylbutyrate, thereby
reducing the likelihood that the patient will develop brain injury.
In some embodiments, the administering occurs immediately after
said identifying. Some embodiments further comprise treating the
acute liver failure. In some embodiments, the treating comprises
liver transplantation. Some embodiments further comprise treating a
complication caused by the acute liver failure. In some
embodiments, the complication comprises variceal bleeding.
[0013] In some embodiments, the subject is suffering from portal
hypertension. In some embodiments, the subject with portal
hypertension also suffers from liver disease, such as cirrhosis. In
some embodiments, the subject is suffering from liver disease. In
some embodiments, the subject with liver disease also suffers from
portal hypertension. In some embodiments, the liver disease is a
chronic liver disease (for example, cirrhosis) or an acute liver
failure. In some embodiments, the treatment of portal hypertension
is achieved by reducing the level of proinflammatory cytokines in
the subject. In some embodiments, the treatment of portal
hypertension is achieved by increasing endothelial nitric oxide
synthase activity. In some embodiments, L-ornithine and
phenylacetate is administered as L-orthine phenyl acetate. In some
embodiments, separate physiologically acceptable salts of
L-ornithine and at least one of phenylacetate and phenylbutyrate
are administered to the subject. In some embodiments, L-ornithine
is present administered as a free monomeric amino acid or
physiologically acceptable salt thereof. In some embodiments, at
least one of phenylacetate and phenylbutyrate is administered as a
sodium phenylacetate or sodium phenylbutyrate. In some embodiments,
administration is oral, intravenous, intraperitoneal, intragastric,
or intravascular administration. In some embodiments, improving
liver function reduces the portal hypertension. In some
embodiments, improving liver function comprises increasing liver
perfusion. In some embodiments, the dose of the L-ornithine and the
phenylacetate or phenylbutyrate administered is between 20 g and 40
g.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIGS. 1A-D. Plasma and TNF-.alpha. expression: Shows that
compared with sham-operated controls, there was A) a significant
increase in plasma TNF-.alpha. in bile duct ligation (BDL) rats
(*p<0.05), which was markedly augmented by administration of
lipopolysaccharide (LPS) (.sup.##p<0.01), and ameliorated by
administration of L-ornithine, phenylacetate (OP) treatment; B) a
significant increase in brain TNF-.alpha. in BDL rats (*p<0.05),
which was markedly augmented by administration of LPS
(.sup.#p<0.05), and ameliorated by administration of OP
treatment (.sup.$p<0.05); C) a near significant increase in
plasma IL-6 in BDL rats was augmented by administration of LPS
(.sup.##p<0.01); and D) although there were similar trends in
IL-6 brain concentrations with BDL and treatment intervention by OP
failed to reach significance.
[0015] FIGS. 2A-B. Brain iNOS and NFkB expression: Shows that
compared with sham-operated controls, there was A) a significant
increase in brain iNOS protein expression in BDL rats
(***p<0.01), which was ameliorated by administration of OP
(.sup.$p<0.01); and B) a significant increase in NFkB in BDL
rats (***p<0.05), which was also ameliorated by administration
of OP (.sup.$$$p<0.01). These were associated with significant
reduction in arterial and brain TNF.alpha., IL1b and IL-6 in the OP
treated animals.
[0016] FIGS. 3A-B. Plasma and brain TNF-.alpha. levels: Shows that
compared to sham controls, there was A) a significant elevation in
plasma TNF-.alpha. level in BDL rats, which was reversed by
administration of OP; and B) a significant elevation in plasma
TNF-.alpha. level in BDL rats, which was also reversed by
administration of OP.
[0017] FIGS. 4A-C. eNOS activity and protein expression, and iNOS
protein expression: Shows that compared to sham controls, there was
A) a significant decrease in eNOS activity in BDL rats, which was
reversed by administration of OP; B) an increase in eNOS protein
expression in BDL rats; and C) a significant increase in iNOS
protein expression in BDL rats.
[0018] FIGS. 5A-C. Plasma ADMA and cerebral caveolin-1 protein
expression: Shows that compared to sham control, there was A) a
significant increase in plasma ADMA in BDL rat plasma, which was
non-significantly reduced after administration of OP; B) a
significant increase in plasma ADMA in BDL brain homogenates, which
was significantly reduced by administration of OP; and C) a
significant increase in cerebral caveolin-1 protein expression in
BDL rat, which was reversed by administration of OP.
[0019] FIGS. 6A-C. Cerebral DDAH-1 and DDAH-2 protein expression
and activity: Shows that compared to sham control, there was A) a
significant decrease in cerebral DDAH-1 protein expression in BDL
rats, which was reversed by administration of OP; B) a significant
increase in cerebral DDAH-2 protein expression in BDL rats, which
was reversed by administration of OP; and C) a significant increase
in DDAH activity in BDL rat brain, which was reversed by
administration of OP.
[0020] FIGS. 7A-F. eNOS activity and protein expression, protein
expression of DDAH-1, NF.kappa.-B, hepatic caveolin-1, and portal
pressure: Show that compared to sham control, there was A) a
significant decrease in eNOS activity in BDL rats, which was
reversed by administration of OP; B) a significant increase in eNOS
protein expression in BDL rats, which was also reversed by
administration of OP; C) a significant increase in DDAH-1 protein
expression in BDL rats, which was reversed by administration of OP;
D) a significant increase in NF.kappa.-B protein expression in BDL
rats and the increase was significantly reduced by administration
of OP; E) a significant increase in caveolin-1 protein expression
in BDL rats and the increase was significantly reduced by
administration of OP; and F) a significant increase in portal
pressure in BDL rats and the administration of OP resulted in a 30%
reduction of portal pressure.
DETAILED DESCRIPTION
Definitions
[0021] As used herein, a "subject" refers to an animal that is the
object of treatment, observation or experiment. "Animal" includes
cold- and warm-blooded vertebrates and invertebrates such as fish,
shellfish, reptiles and, in particular, mammals. "Mammal" includes,
without limitation, mice; rats; rabbits; guinea pigs; dogs; cats;
sheep; goats; cows; horses; primates, such as monkeys, chimpanzees,
and apes, and, in particular, humans.
[0022] As used herein, a "patient" refers to a subject that is
being treated by a medical professional, such as a Medical Doctor
(i.e. Doctor of Allopathic medicine or Doctor of Osteopathic
medicine) or a Doctor of Veterinary Medicine, to attempt to cure,
or at least ameliorate the effects of, a particular disease or
disorder or to prevent the disease or disorder from occurring in
the first place.
[0023] As used herein, "administration" or "administering" refers
to a method of giving a dosage of a pharmaceutically active
ingredient to a vertebrate.
[0024] As used herein, a "dosage" refers to an amount of
therapeutic agent administered to a patient.
[0025] As used herein, a "daily dosage" refers to the total amount
of therapeutic agent administered to a patient in a day.
[0026] As used herein, the term "therapeutic agent" means a
substance that is effective in the treatment of a disease or
condition.
[0027] As used herein, "therapeutically effective amount" or
"pharmaceutically effective amount" is meant an amount of
therapeutic agent, which has a therapeutic effect. The dosages of a
pharmaceutically active ingredient which are useful in treatment
are therapeutically effective amounts. Thus, as used herein, a
therapeutically effective amount means those amounts of therapeutic
agent which produce the desired therapeutic effect as judged by
clinical trial results and/or model animal studies.
[0028] As used herein, a "therapeutic effect" relieves, to some
extent, one or more of the symptoms of a disease or disorder. For
example, a therapeutic effect may be observed by a reduction of the
subjective discomfort that is communicated by a subject (e.g.,
reduced discomfort noted in self-administered patient
questionnaire).
Abbreviations
[0029] BDL=bile duct ligation;
[0030] OP=ornithine, phenylacetate;
[0031] LPS=lipopolysaccharide.
[0032] iNOS=inducible nitric oxide synthase
[0033] eNOS=endothelial nitric oxide synthase
Portal Hypertension
[0034] Portal hypertension is an increase in the pressure within
the portal vein (the vein that carries the blood from the digestive
organs to the liver). The main symptoms and complications of portal
hypertension include, but are not limited to, gastrointestinal
bleeding, for example, black, tarry stools or blood in the stools,
or vomiting of blood due to the spontaneous rupture and hemorrhage
from varices; ascites, for example, an accumulation of fluid in the
abdomen; encephalopathy, for example, confusion and forgetfulness
caused by poor liver function and the diversion of blood flow away
from the liver; and reduced levels of platelets or decreased white
blood cell count.
[0035] Portal hypertension can be a symptom or a result of an
underlying condition (e.g., liver disorder), and therefore a
subject may have portal hypertension that is associated with a one
or more conditions. In some embodiments, the portal hypertension is
associated with a liver disease.
[0036] Non-limiting examples of liver disease include intrahepatic
cholestasis (alagille syndrome, biliary liver cirrhosis), fatty
liver (alcoholic fatty liver, reye syndrome), hepatic vein
thrombosis, hepatolentricular degeneration, hepatomegaly, liver
abscess (amebic liver abscess), liver cirrhosis (alcoholic, biliary
and experimental), alcoholic liver diseases (fatty liver,
hepatitis, cirrhosis), parasitic (hepatic echinococcosis,
fascioliasis, amebic liver abscess), jaundice (hemolytic,
hepatocellular, and cholestatic), cholestasis, portal hypertension,
liver enlargement, ascites, hepatitis (alcoholic hepatitis, animal
hepatitis, chronic hepatitis (autoimmune, hepatitis B, hepatitis C,
hepatitis D, drug induced), toxic hepatitis, viral human hepatitis
(hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E),
Wilson's disease, granulomatous hepatitis, secondary biliary
cirrhosis, hepatic encephalopathy, varices, primary biliary
cirrhosis, primary sclerosing cholangitis, hepatocellular adenoma,
hemangiomas, bile stones, liver failure (hepatic encephalopathy,
acute liver failure), and liver neoplasms (angiomyolipoma,
calcified liver metastases, cystic liver metastases, epithelial
tumors, fibrolamellar hepatocarcinoma, focal nodular hyperplasia,
hepatic adenoma, hepatobiliary cystadenoma, hepatoblastoma,
hepatocellular carcinoma, hepatoma, liver cancer, liver
hemangioendothelioma, mesenchymal hamartoma, mesenchymal tumors of
liver, nodular regenerative hyperplasia, benign liver tumors
(Hepatic cysts [Simple cysts, Polycystic liver disease,
Hepatobiliary cystadenoma, Choledochal cyst], Mesenchymal tumors
[Mesenchymal hamartoma, Infantile hemangioendothelioma, Hemangioma,
Peliosis hepatis, Lipomas, Inflammatory pseudotumor,
Miscellaneous], Epithelial tumors [Bile duct epithelium (Bile duct
hamartoma, Bile duct adenoma), Hepatocyte (Adenoma, Focal nodular
hyperplasia, Nodular regenerative hyperplasia)], malignant liver
tumors [hepatocellular, hepatoblastoma, hepatocellular carcinoma,
cholangiocellular, cholangiocarcinoma, cystadenocarcinoma, tumors
of blood vessels, angiosarcoma, Karposi's sarcoma,
hemangioendothelioma, other tumors, embryonal sarcoma,
fibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, carcinosarcoma,
teratoma, carcinoid, squamous carcinoma, primary lymphoma]),
peliosis hepatis, erythrohepatic porphyria, hepatic porphyria
(acute intermittent porphyria, porphyria cutanea tarda), Zellweger
syndrome).
[0037] In some embodiments, the liver disorder is hepatitis,
cirrhosis, cholestasis or liver failure. In some embodiments, a
subject suffering from a liver disease has hepatic
encephalopathy.
[0038] In some embodiments, the portal hypertension is associated
with a chronic liver disease. In some embodiments, the chronic
liver disease is cirrhosis. In cirrhosis, the scar tissue blocks
the flow of blood through the liver, which consequently results in
portal hypertension. Increased pressure in the portal vein causes
large veins (varices) to develop across the esophagus and stomach
to bypass the blockage. The pressure in the varices increases and
may rupture. In some embodiments, reducing portal hypertension
reduces the likelihood of onset of hepatic encephalopathy.
[0039] Portal hypertension may also be caused by thrombosis or
clotting in the portal vein. Portal hypertension in humans and
laboratory animals can be associated with a hyperkinetic
circulation, vasodilation in the splanchnic territory and a
hypersplenism. The hypersplenism can lead to an important
pancytopenia.
Treatment of Portal Hypertension
[0040] In some embodiments, L-ornithine is co-administered with
phenylacetate or phenylbutyrate to a subject to treat and/or
prevent portal hypertension. In some embodiments, L-ornithine is
co-administered with phenylacetate or phenylbutyrate to a subject
to delay or reduce the likelihood of the onset of portal
hypertension. In some embodiments, the treatment results in
restoration of liver function (e.g., increasing liver perfusion)
and thereby improves portal hypertension. In some embodiments,
partial liver function is restored. In some embodiments, entire
liver function is restored. Restoration of liver function (e.g.,
increasing liver perfusion) may be indicated by one or more of the
following measurements: the alanine transaminase (ALT) test,
aspartate aminotransferase (AST) test, alpha glutathione
S-transferase (GST) test, albumin (Alb) test, prothrombin time
test, and composite scores (for example, child-pugh score' MELD
score). Additionally, liver hemodynamics can be measured by
detecting liver blood flow and/or portal pressure.
[0041] In some embodiments, the co-administration of L-ornithine
and phenylacetate or phenylbutyrate results in modulating of
endothelial nitric oxide synthase (eNOS) activity, and thereby
treat or ameliorate one or more symptoms associated with decreased
eNOS activity. In some embodiments, the decreased eNOS activity is
associated with an increase in endogenous nitric oxide synthase
inhibitors, including Caveolin-1 and asymmetric-dimethylarginine
(ADMA). In some embodiments, the decreased eNOS activity is
associated with an increase in NF.kappa.B. In some embodiments, the
decreased eNOS activity is associated with an increase in ammonia.
In some embodiments, the decreased eNOS activity is associated with
a liver disease, including chronic liver disease (for example,
cirrhosis) and acute liver failure. In some embodiments, the
co-administration is used to treat hepatic inflammation. In some
embodiments, the co-administration is used to improve the function
of organ systems that have deranged nitric oxide signaling in liver
disease (for example, cirrhosis).
[0042] In some embodiments, the co-administration is useful to
reduce pro-inflammatory cytokines, which further promotes its
ability to treat or reduce the likelihood of portal hypertension.
In some embodiments, portal hypertension is prevented in patients
with existing chronic liver disease such as cirrhosis by the
administration of the combination. Thus, in some embodiments, the
combination is administered to a patient having chronic liver
disease also having a portal hypertension. In some embodiments, the
co-administration is used to restore partial or entire liver
function.
[0043] While not being bound by any particular theory, in some
embodiments, the co-administration prevents or relieves the
condition of portal hypertension through effects on inflammatory
pathways. In some embodiments, decreasing the level of inflammatory
cytokines and/or iNOS (inducible nitric oxide synthase) results in
the restoration of partial or complete liver function and treatment
of portal hypertension.
[0044] The L-ornithine and phenylacetate or phenylbutyrate may be
administered separately or in a single dosage form. In one
embodiment, the combination is administered as the L-ornithine
phenylacetate salt or as a solution of the L-ornithine
phenylacetate salt.
[0045] Different forms of composition of L-ornithine in combination
with at least one of phenylacetate (or phenyl acetate salts) and
phenylbutyrate are described in U.S. Patent Publication No.
US2008/0119554 and U.S. patent application Ser. No. 12/753763 filed
Apr. 2, 2010, which are hereby incorporated by reference in their
entireties. In some embodiments, L-ornithine and phenylacetate is
present and/or administered as L-orthine phenyl acetate or
physiologically acceptable salt thereof. In some embodiments,
L-ornithine is present and/or administered as a free monomeric
amino acid or physiologically acceptable salt thereof In some
embodiments, at least one of phenylacetate and phenylbutyrate is
present and/or administered as a sodium phenylacetate or sodium
phenylbutyrate. In some embodiments, a physiologically acceptable
salt of L-ornithine and a physiologically acceptable salt of at
least one of phenylacetate and phenylbutyrate are administered to
the subject.
Protection against Brain Injury
[0046] In other embodiments, L-ornithine is co-administered with
phenylacetate or phenylbutyrate to a subject with acute liver
failure or acute liver decompensation in subjects with chronic
liver disease to protect against brain injury. In some embodiments,
the combination is administered prophylactically to a subject at
risk of acute liver failure (e.g., a subject with a Tylenol
overdose who has not yet manifested acute liver failure) or having
chronic liver disease without acute liver decompensation. While not
being bound by any particular theory, in some embodiments, early
administration of L-ornithine with phenylacetate or phenylbutyrate
to a patient with acute liver failure or acute liver decompensation
can prevent brain injury from developing through its action on
suppressing inflammatory pathways as described herein. Accordingly,
in some embodiments, L-ornithine is co-administered with
phenylacetate or phenylbutyrate prior to or immediately upon
diagnoses with acute liver failure or acute liver decompensation,
regardless of the further course of treatment, which may include
liver transplantation. In some embodiments, such early
administration prevents variceal bleeding, onset of encephalopathy,
onset of raised intracranial pressure, onset of coma, need for
intubation and ICU treatment, and mitigates or reverses
hyperammonemia, and thereby protects against brain injury caused by
such complications.
Pharmaceutical Compositions
[0047] In another aspect, the present disclosure relates to a
pharmaceutical composition comprising a physiologically acceptable
surface active agents, carriers, diluents, excipients, smoothing
agents, suspension agents, film forming substances, and coating
assistants, or a combination thereof; and a compound disclosed
herein. Acceptable carriers or diluents for therapeutic use are
well known in the pharmaceutical art, and are described, for
example, in Remington's Pharmaceutical Sciences, 18th Ed., Mack
Publishing Co., Easton, Pa. (1990), which is incorporated herein by
reference in its entirety. Preservatives, stabilizers, dyes,
sweeteners, fragrances, flavoring agents, and the like may be
provided in the pharmaceutical composition. For example, sodium
benzoate, ascorbic acid and esters of p-hydroxybenzoic acid may be
added as preservatives. In addition, antioxidants and suspending
agents may be used. In various embodiments, alcohols, esters,
sulfated aliphatic alcohols, and the like may be used as surface
active agents; sucrose, glucose, lactose, starch, crystallized
cellulose, mannitol, light anhydrous silicate, magnesium aluminate,
magnesium methasilicate aluminate, synthetic aluminum silicate,
calcium carbonate, sodium acid carbonate, calcium hydrogen
phosphate, calcium carboxymethyl cellulose, and the like may be
used as excipients; magnesium stearate, talc, hardened oil and the
like may be used as smoothing agents; coconut oil, olive oil,
sesame oil, peanut oil, soya may be used as suspension agents or
lubricants; cellulose acetate phthalate as a derivative of a
carbohydrate such as cellulose or sugar, or
methylacetate-methacrylate copolymer as a derivative of polyvinyl
may be used as suspension agents; and plasticizers such as ester
phthalates and the like may be used as suspension agents.
[0048] The ornithine and the phenylacetate and/or phenylbutyrate
can be formulated for administration with a pharmaceutically
acceptable carrier or diluent. The ornithine and the phenylacetate
and/or phenylbutyrate can be formulated as a medicament with a
standard pharmaceutically acceptable carrier(s) and/or excipient(s)
as is routine in the pharmaceutical art. The exact nature of the
formulation will depend upon several factors including the desired
route of administration. Typically, ornithine and the phenylacetate
and/or phenybutyrate are formulated for oral, intravenous,
intragastric, intravascular or intraperitoneal administration.
[0049] The term "pharmaceutical composition" refers to a mixture of
a compound or compounds disclosed herein with other chemical
components, such as diluents or carriers. The pharmaceutical
composition facilitates administration of the compound(s) to an
organism. Multiple techniques of administering a compound exist in
the art including, but not limited to, oral, injection, aerosol,
parenteral, and topical administration. Pharmaceutical compositions
can also be obtained by reacting compound(s) with inorganic or
organic acids such as hydrochloric acid, hydrobromic acid, sulfuric
acid, nitric acid, phosphoric acid, methanesulfonic acid,
ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the
like.
[0050] The term "carrier" defines a chemical compound that
facilitates the incorporation of a compound into cells or tissues.
For example dimethyl sulfoxide (DMSO) is a commonly utilized
carrier as it facilitates the uptake of many organic compounds into
the cells or tissues of an organism.
[0051] The term "diluent" defines a chemical compound diluted in
water that will dissolve the compound of interest as well as
stabilize the biologically active form of the compound. Salts
dissolved in buffered solutions are utilized as diluents in the
art. One commonly used buffered solution is phosphate buffered
saline because it mimics the salt conditions of human blood. Since
buffer salts can control the pH of a solution at low
concentrations, a buffered diluent rarely modifies the biological
activity of a compound.
[0052] The term "physiologically acceptable" defines a carrier or
diluent that does not abrogate the biological activity and
properties of the compound.
[0053] The pharmaceutical compositions described herein can be
administered to a human patient per se, or in pharmaceutical
compositions where they are mixed with other active ingredients, as
in combination therapy, or suitable carriers or excipient(s).
Techniques for formulation and administration of the compound or
combination of compounds disclosed herein may be found in
"Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton,
Pa., 18th edition, 1990.
[0054] Some embodiments provide the compound(s) or combination of
compounds disclosed herein in tablets, film coated tablets,
capsules, caplets, pills, gel caps, pellets, beads, or dragee
dosage forms. Preferably, the formulations disclosed herein can
provide favorable drug processing qualities, including, for
example, but not limited to, rapid tablet press speeds, reduced
compression force, reduced ejection forces, blend uniformity,
content uniformity, uniform dispersal of color, accelerated
disintegration time, rapid dissolution, low friability (preferable
for downstream processing such as packaging, shipping,
pick-and-pack, etc.) and dosage form physical characteristics
(e.g., weight, hardness, thickness, friability) with little
variation.
[0055] The compound(s) or combination of compounds disclosed herein
can be formulated readily, for example, by combining the drug
substance with any suitable pharmaceutically acceptable
excipient(s) for example, but not limited to, binders, diluents,
disintegrants, lubricants, fillers, carriers, coatings, glidants,
flavours, color additives, and the like, as set forth below. Such
compositions can be prepared for storage and for subsequent
processing.
Excipients
[0056] Acceptable excipients for therapeutic use are well known in
the pharmaceutical art, and are described, for example, in Handbook
of Pharmaceutical Excipients, 5th edition (Raymond C Rowe, Paul J
Sheskey and Sian C Owen, eds. 2005), and Remington: The Science and
Practice of Pharmacy, 21st edition (Lippincott Williams &
Wilkins, 2005), each of which is hereby incorporated in its
entirety. The term "carrier" material or "excipient" herein can
mean any substance, not itself a therapeutic agent, used as a
carrier and/or diluent and/or adjuvant, or vehicle for delivery of
a therapeutic agent to a subject or added to a pharmaceutical
composition to improve its handling or storage properties or to
permit or facilitate formation of a dose unit of the composition
into a discrete article such as a capsule, tablet, film coated
tablet, caplet, gel cap, pill, pellet, bead, and the like suitable
for oral administration. Excipients can include, by way of
illustration and not limitation, diluents, disintegrants, binding
agents, wetting agents, polymers, lubricants, glidants, coatings,
sweetens, solubilizing agens substances added to mask or counteract
a disagreeable taste or odor, flavors, colorants, fragrances, and
substances added to improve appearance of the composition.
[0057] The compositions and formulations can include any other
agents that provide improved transfer, delivery, tolerance, and the
like. These compositions and formulations can include, for example,
powders, pastes, jellies, waxes, oils, lipids, lipid (cationic or
anionic) containing vesicles (such as Lipofectin.TM.), DNA
conjugates, anhydrous absorption pastes, oil-in-water and
water-in-oil emulsions, emulsions carbowax (polyethylene glycols of
various molecular weights), semi-solid gels, and semi-solid
mixtures containing carbowax.
[0058] Any of the foregoing mixtures can be appropriate in
treatments and therapies in accordance with the disclosure herein,
provided that the active ingredient in the formulation is not
inactivated by the formulation and the formulation is
physiologically compatible and tolerable with the route of
administration. See also Baldrick P. "Pharmaceutical excipient
development: the need for preclinical guidance." Regul. Toxicol.
Pharmacol. 32(2):210-8 (2000), Charman W N "Lipids, lipophilic
drugs, and oral drug delivery-some emerging concepts." J. Pharm.
Sci. 89(8):967-78 (2000), and the citations therein for additional
information related to formulations, excipients and carriers well
known to pharmaceutical chemists.
[0059] In some embodiments, one or more, or any combination of the
listed excipients can be specifically included or excluded from the
formulations and/or methods disclosed herein. As will be
appreciated by those of skill in the art, the amounts of excipients
will be determined by drug dosage and dosage form size.
Lubricants
[0060] In some embodiments, lubricants are employed in the
manufacture of certain dosage forms. For example, a lubricant will
often be employed when producing tablets. In some embodiments, a
lubricant can be added just before the tableting step, and can be
mixed with the formulation for a minimum period of time to obtain
good dispersal. In some embodiments, one or more lubricants can be
used. Examples of suitable lubricants include, but are not limited
to, magnesium stearate, calcium stearate, zinc stearate, stearic
acid, talc, glyceryl behenate, polyethylene glycol, polyethylene
oxide polymers (for example, available under the registered
trademarks of Carbowax.RTM. for polyethylene glycol and Polyox.RTM.
for polyethylene oxide from Dow Chemical Company, Midland, Mich.),
sodium lauryl sulfate, magnesium lauryl sulfate, sodium oleate,
sodium stearyl fumarate, DL-leucine, colloidal silica, and others
as known in the art. Typical lubricants are magnesium stearate,
calcium stearate, zinc stearate and mixtures of magnesium stearate
with sodium lauryl sulfate.
Color Additives
[0061] In some embodiments, color additives also can be included.
The colorants can be used in amounts sufficient to distinguish
dosage form strengths. Preferably, color additives approved for use
in drugs (21 CFR 74, which is incorporated herein by reference in
its entirety) are added to the commercial formulations to
differentiate tablet strengths. The use of other pharmaceutically
acceptable colorants and combinations thereof are encompassed by
the current disclosure.
Binders
[0062] Binders can be used, for example, to impart cohesive
qualities to a formulation, and thus ensure that the resulting
dosage form remains intact after compaction. Suitable binder
materials include, but are not limited to, microcrystalline
cellulose, gelatin, sugars (including, for example, sucrose,
glucose, dextrose and maltodextrin), polyethylene glycol, waxes,
natural and synthetic gums, polyvinylpyrrolidone, pregelatinized
starch, povidone, cellulosic polymers (including, for example,
hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose
(HPMC), methyl cellulose, hydroxyethyl cellulose, and the like),
hydroxypropyl cellulose (HPC), and the like. Accordingly, in some
embodiments, the formulations disclosed herein can include at least
one binder to enhance the compressibility of the major
excipient(s). In some embodiments, the binder(s) is(are) sprayed on
from solution, e.g. wet granulation, to increase binding
activity.
Disintegrants
[0063] In some embodiments, disintegrants are used, for example, to
facilitate tablet disintegration after administration, and are
generally starches, clays, celluloses, algins, gums, or crosslinked
polymers. Suitable disintegrants include, but are not limited to,
crosslinked polyvinylpyrrolidone (PVP-XL), sodium starch glycolate,
alginic acid, methacrylic acid DYB, microcrystalline cellulose,
crospovidone, polacriline potassium, sodium starch glycolate,
starch, pregelatinized starch, croscarmellose sodium, and the like.
If desired, the pharmaceutical formulation can also contain minor
amounts of nontoxic auxiliary substances such as wetting or
emulsifying agents, pH buffering agents and the like, for example,
sodium acetate, sorbitan monolaurate, triethanolamine sodium
acetate, triethanolamine oleate, sodium lauryl sulfate, dioctyl
sodium sulfosuccinate, polyoxyethylene sorbitan fatty acid esters,
etc. and the like.
Coatings
[0064] In some embodiments, the formulations can include a coating,
for example, a film coating. Where film coatings are involved,
coating preparations can include, for example, a film-forming
polymer, a plasticizer, or the like. Also, the coatings can include
pigments and/or opacifiers. Non-limiting examples of film-forming
polymers include hydroxypropyl methylcellulose, hydroxypropyl
cellulose, methylcellulose, polyvinyl pyrrolidine, and starches.
Non-limiting examples of plasticizers include polyethylene glycol,
tributyl citrate, dibutyl sebecate, castor oil, and acetylated
monoglyceride. Furthermore, non-limiting examples of pigments and
opacifiers include iron oxides of various colors, lake dyes of many
colors, titanium dioxide, and the like.
Diluents
[0065] In some embodiments, diluents are used, and are generally
selected from one or more of the compounds sucrose, fructose,
glucose, galactose, lactose, maltose, invert sugar, calcium
carbonate, lactose, starch, microcrystalline cellulose, lactose
monohydrate, calcium hydrogen phosphate, anhydrous calcium hydrogen
phosphate, a pharmaceutically acceptable polyol such as xylitol,
sorbitol, maltitol, mannitol, isomalt and glycerol, polydextrose,
starch, or the like, or any mixture thereof.
Surfactants
[0066] In some embodiments, surfactants are used. The use of
surfactants as wetting agents in oral drug forms is described in
the literature, for example in H. Sucker, P. Fuchs, P. Speiser,
Pharmazeutische Technologie, 2nd edition, Thieme 1989, page 260. It
is known from other papers, such as published in Advanced Drug
Delivery Reviews (1997), 23, pages 163-183, that it is also
possible to use surfactants, inter alia, to improve the permeation
and bioavailability of pharmaceutical active compounds. Examples of
surfactants include, but are not limited to, anionic surfactants,
non-ionic surfactants, zwitterionic surfactants and a mixture
thereof. Preferably, the surfactants is selected from the group
consisting of poly(oxyethylene) sorbitan fatty acid ester,
poly(oxyethylene) stearate, poly(oxyethylene) alkyl ether,
polyglycolated glyceride, poly(oxyethylene) caster oil, sorbitan
fatty acid ester, poloxamer, fatty acid salt, bile salt, alkyl
sulfate, lecithin, mixed micelle of bile salt and lecithin, glucose
ester vitamin E TPGS (D-.alpha.-tocopheryl polyethylene glycol 1000
succinate), sodium lauryl sulfate, and the like, and a mixture
thereof.
Glidants
[0067] In some embodiments, glidants are used. Examples of glidants
which may be used include, but are not limited to, colloidal
silicon dioxide, magnesium trisilicate, powdered cellulose, starch,
talc and calcium phosphate, or the like, and mixtures thereof.
[0068] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, topical, or intestinal administration;
parenteral delivery, including intramuscular, subcutaneous,
intravenous, intramedullary injections, as well as intrathecal,
direct intraventricular, intraperitoneal, intranasal, or
intraocular injections. The compound or combination of compounds
disclosed herein can also be administered in sustained or
controlled release dosage forms, including depot injections,
osmotic pumps, pills, transdermal (including electrotransport)
patches, and the like, for prolonged and/or timed, pulsed
administration at a predetermined rate.
[0069] The pharmaceutical compositions of the present disclosure
may be manufactured in a manner that is itself known, e.g., by
means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping
or tabletting processes.
[0070] Pharmaceutical compositions for use in accordance with the
present disclosure thus may be formulated in conventional manner
using one or more physiologically acceptable carriers comprising
excipients and auxiliaries which facilitate processing of the
active compounds into preparations which can be used
pharmaceutically. Proper formulation is dependent upon the route of
administration chosen. Any of the well-known techniques, carriers,
and excipients may be used as suitable and as understood in the
art; e.g., in Remington's Pharmaceutical Sciences, above.
[0071] Injectables can be prepared in conventional forms, either as
liquid solutions or suspensions, solid forms suitable for solution
or suspension in liquid prior to injection, or as emulsions.
Suitable excipients are, for example, water, saline, dextrose,
mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine
hydrochloride, and the like. In addition, if desired, the
injectable pharmaceutical compositions may contain minor amounts of
nontoxic auxiliary substances, such as wetting agents, pH buffering
agents, and the like. Physiologically compatible buffers include,
but are not limited to, Hanks's solution, Ringer's solution, or
physiological saline buffer. If desired, absorption enhancing
preparations (for example, liposomes), may be utilized.
[0072] For transmucosal administration, penetrants appropriate to
the barrier to be permeated may be used in the formulation.
[0073] Pharmaceutical formulations for parenteral administration,
e.g., by bolus injection or continuous infusion, include aqueous
solutions of the active compounds in water-soluble form.
Additionally, suspensions of the active compounds may be prepared
as appropriate oily injection suspensions. Suitable lipophilic
solvents or vehicles include fatty oils such as sesame oil, or
other organic oils such as soybean, grapefruit or almond oils, or
synthetic fatty acid esters, such as ethyl oleate or triglycerides,
or liposomes. Aqueous injection suspensions may contain substances
which increase the viscosity of the suspension, such as sodium
carboxymethyl cellulose, sorbitol, or dextran. Optionally, the
suspension may also contain suitable stabilizers or agents that
increase the solubility of the compounds to allow for the
preparation of highly concentrated solutions. Formulations for
injection may be presented in unit dosage form, e.g., in ampoules
or in multi-dose containers, with an added preservative. The
compositions may take such forms as suspensions, solutions or
emulsions in oily or aqueous vehicles, and may contain formulatory
agents such as suspending, stabilizing and/or dispersing agents.
Alternatively, the active ingredient may be in powder form for
constitution with a suitable vehicle, e.g., sterile pyrogen-free
water, before use.
[0074] For oral administration, the compound(s) or combination of
compounds disclosed herein can be formulated readily by combining
the active compound with pharmaceutically acceptable carriers well
known in the art. Such carriers enable the compound or combination
of compounds disclosed herein to be formulated as tablets, film
coated tablets, pills, dragees, capsules, liquids, gels, get caps,
pellets, beads, syrups, slurries, suspensions and the like, for
oral ingestion by a patient to be treated. Pharmaceutical
preparations for oral use can be obtained by combining the active
compound with solid excipient, optionally grinding a resulting
mixture, and processing the mixture of granules, after adding
suitable auxiliaries, if desired, to obtain tablets or dragee
cores. Suitable excipients are, in particular, fillers such as
sugars, including lactose, sucrose, mannitol, or sorbitol;
cellulose preparations such as, for example, maize starch, wheat
starch, rice starch, potato starch, gelatin, gum tragacanth, methyl
cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If
desired, disintegrating agents may be added, such as the
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate. Dragee cores are provided with
suitable coatings. For this purpose, concentrated sugar solutions
may be used, which may optionally contain gum arabic, talc,
polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or
titanium dioxide, lacquer solutions, and suitable organic solvents
or solvent mixtures. Dyestuffs or pigments may be added to the
tablets or dragee coatings for identification or to characterize
different combinations of active compound doses. For this purpose,
concentrated sugar solutions may be used, which may optionally
contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments may be added to the tablets or dragee coatings for
identification or to characterize different combinations of active
compound doses. In addition, stabilizers can be added. All
formulations for oral administration should be in dosages suitable
for such administration. In some embodiments, formulations of the
compound(s) or combination of compounds disclosed herein with an
acceptable immediate release dissolution profile and a robust,
scalable method of manufacture are disclosed.
[0075] Pharmaceutical preparations which can be used orally 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 can contain the active ingredients in
admixture with filler 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 may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for such administration.
[0076] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0077] For administration by inhalation, the compound or
combination of compounds disclosed herein is conveniently delivered
in the form of an aerosol spray presentation from pressurized packs
or a nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of, e.g., gelatin for use in an inhaler or insufflator
may be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0078] Further disclosed herein are various pharmaceutical
compositions well known in the pharmaceutical art for uses that
include intraocular, intranasal, and intraauricular delivery.
Suitable penetrants for these uses are generally known in the art.
Pharmaceutical compositions for intraocular delivery include
aqueous ophthalmic solutions of the active compounds in
water-soluble form, such as eyedrops, or in gellan gum (Shedden et
al., Clin. Ther., 23(3):440-50 (2001)) or hydrogels (Mayer et al.,
Ophthalmologica, 210(2):101-3 (1996)); ophthalmic ointments;
ophthalmic suspensions, such as microparticulates, drug-containing
small polymeric particles that are suspended in a liquid carrier
medium (Joshi, A., J. Ocul. Pharmacol., 10(1):29-45 (1994)),
lipid-soluble formulations (Alm et al., Prog. Clin. Biol. Res.,
312:447-58 (1989)), and microspheres (Mordenti, Toxicol. Sci.,
52(1):101-6 (1999)); and ocular inserts. All of the above-mentioned
references are incorporated herein by reference in their
entireties. Such suitable pharmaceutical formulations are most
often and preferably formulated to be sterile, isotonic and
buffered for stability and comfort. Pharmaceutical compositions for
intranasal delviery may also include drops and sprays often
prepared to simulate in many respects nasal secretions to ensure
maintenance of normal ciliary action. As disclosed in Remington's
Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa.
(1990), which is incorporated herein by reference in its entirety,
and well-known to those skilled in the art, suitable formulations
are most often and preferably isotonic, slightly buffered to
maintain a pH of 5.5 to 6.5, and most often and preferably include
antimicrobial preservatives and appropriate drug stabilizers.
Pharmaceutical formulations for intraauricular delivery include
suspensions and ointments for topical application in the ear.
Common solvents for such aural formulations include glycerin and
water.
[0079] The compound(s) or combination of compounds disclosed herein
may also be formulated in rectal compositions such as suppositories
or retention enemas, e.g., containing conventional suppository
bases such as cocoa butter or other glycerides.
[0080] In addition to the formulations described previously, the
compound or combination of compounds disclosed herein may also be
formulated as a depot preparation. Such long acting formulations
may be administered by implantation (for example subcutaneously or
intramuscularly) or by intramuscular injection. Thus, for example,
the compound or combination of compounds disclosed herein may be
formulated with suitable polymeric or hydrophobic materials (for
example as an emulsion in an acceptable oil) or ion exchange
resins, or as sparingly soluble derivatives, for example, as a
sparingly soluble salt.
[0081] For hydrophobic compounds, a suitable pharmaceutical carrier
may be a cosolvent system comprising benzyl alcohol, a nonpolar
surfactant, a water-miscible organic polymer, and an aqueous phase.
A common cosolvent system used is the VPD co-solvent system, which
is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar
surfactant Polysorbate 80.TM., and 65% w/v polyethylene glycol 300,
made up to volume in absolute ethanol. Naturally, the proportions
of a co-solvent system may be varied considerably without
destroying its solubility and toxicity characteristics.
Furthermore, the identity of the co-solvent components may be
varied: for example, other low-toxicity nonpolar surfactants may be
used instead of POLYSORBATE 80.TM.; the fraction size of
polyethylene glycol may be varied; other biocompatible polymers may
replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other
sugars or polysaccharides may substitute for dextrose.
[0082] Alternatively, other delivery systems for hydrophobic
pharmaceutical compounds may be employed. Liposomes and emulsions
are well known examples of delivery vehicles or carriers for
hydrophobic drugs. Certain organic solvents such as
dimethylsulfoxide also may be employed, although usually at the
cost of greater toxicity. Additionally, the compounds may be
delivered using a sustained-release system, such as semipermeable
matrices of solid hydrophobic polymers containing the therapeutic
agent. Various sustained-release materials have been established
and are well known by those skilled in the art. Sustained-release
capsules may, depending on their chemical nature, release the
compounds for a few weeks up to over 100 days. Depending on the
chemical nature and the biological stability of the therapeutic
reagent, additional strategies for protein stabilization may be
employed.
[0083] Agents intended to be administered intracellularly may be
administered using techniques well known to those of ordinary skill
in the art. For example, such agents may be encapsulated into
liposomes. All molecules present in an aqueous solution at the time
of liposome formation are incorporated into the aqueous interior.
The liposomal contents are both protected from the external
micro-environment and, because liposomes fuse with cell membranes,
are efficiently delivered into the cell cytoplasm. The liposome may
be coated with a tissue-specific antibody. The liposomes will be
targeted to and taken up selectively by the desired organ.
Alternatively, small hydrophobic organic molecules may be directly
administered intracellularly.
[0084] Additional therapeutic or diagnostic agents may be
incorporated into the pharmaceutical compositions. Alternatively or
additionally, pharmaceutical compositions may be combined with
other compositions that contain other therapeutic or diagnostic
agents.
Methods of Administration
[0085] The compound(s) or combination of compounds disclosed herein
or pharmaceutical compositions may be administered to the patient
by any suitable means. Non-limiting examples of methods of
administration include, among others, (a) administration though
oral pathways, which includes administration in capsule, tablet,
granule, spray, syrup, or other such forms; (b) administration
through non-oral pathways such as rectal, vaginal, intraurethral,
intraocular, intranasal, or intraauricular, which includes
administration as an aqueous suspension, an oily preparation or the
like or as a drip, spray, suppository, salve, ointment or the like;
(c) administration via injection, subcutaneously,
intraperitoneally, intravenously, intramuscularly, intradermally,
intraorbitally, intracapsularly, intraspinally, intrasternally, or
the like, including infusion pump delivery; (d) administration
locally such as by injection directly in the renal or cardiac area,
e.g., by depot implantation; as well as (e) administration
topically; as deemed appropriate by those of skill in the art for
bringing the compound or combination of compounds disclosed herein
into contact with living tissue.
[0086] Pharmaceutical compositions suitable for administration
include compositions where the compound(s) or combination of
compounds disclosed herein is contained in an amount effective to
achieve its intended purpose. The therapeutically effective amount
of the compound or combination of compounds disclosed herein
required as a dose will depend on the route of administration, the
type of animal, including human, being treated, and the physical
characteristics of the specific animal under consideration. The
dose can be tailored to achieve a desired effect, but will depend
on such factors as weight, diet, concurrent medication and other
factors which those skilled in the medical arts will recognize.
More specifically, a therapeutically effective amount means an
amount of compound effective to prevent, alleviate or ameliorate
symptoms of disease or prolong the survival of the subject being
treated. Determination of a therapeutically effective amount is
well within the capability of those skilled in the art, especially
in light of the detailed disclosure provided herein.
[0087] As will be readily apparent to one skilled in the art, the
useful in vivo dosage to be administered and the particular mode of
administration will vary depending upon the age, weight and
mammalian species treated, and the specific use for which the
compound or combination of compounds disclosed herein are employed.
The determination of effective dosage levels, that is the dosage
levels necessary to achieve the desired result, can be accomplished
by one skilled in the art using routine pharmacological methods.
Typically, human clinical applications of products are commenced at
lower dosage levels, with dosage level being increased until the
desired effect is achieved. Alternatively, acceptable in vitro
studies can be used to establish useful doses and routes of
administration of the compositions identified by the present
methods using established pharmacological methods.
[0088] As used herein, a "dosage" refers to the combined amount of
the active ingredients (e.g., L-ornithine and phenylacetate or
phenylbutyrate).
[0089] In non-human animal studies, applications of potential
products are commenced at higher dosage levels, with dosage being
decreased until the desired effect is no longer achieved or adverse
side effects disappear. The dosage may range broadly, depending
upon the desired effects and the therapeutic indication. Typically,
dosages may be between about 0.1 mg/kg and 4000 mg/kg body weight,
preferably between about 80 mg/kg and 1600 mg/kg body weight.
Alternatively dosages may be based and calculated upon the surface
area of the patient, as understood by those of skill in the
art.
[0090] Depending on the severity and responsiveness of the
condition to be treated, dosing can also be a single administration
of a slow release composition, with course of treatment lasting
from several days to several weeks or until cure is effected or
diminution of the disease state is achieved. The amount of a
composition to be administered will, of course, be dependent on
many factors including the subject being treated, the severity of
the affliction, the manner of administration, the judgment of the
prescribing physician. The compound or combination of compounds
disclosed herein may be administered orally or via injection at a
dose from 0.1 mg/kg to 4000 mg/kg of the patient's body weight per
day. The dose range for adult humans is generally from 1 g to 100
g/day. Tablets or other forms of presentation provided in discrete
units may conveniently contain an amount of the compound or
combination of compounds disclosed herein which is effective at
such dosage or as a multiple of the same, for instance, units
containing 1 g to 60 g (for example, from about 5 g to 20 g, from
about 10 g to 50 g, from about 20 g to 40 g, or from about 25 g to
35 g). The precise amount of compound administered to a patient
will be the responsibility of the attendant physician. However, the
dose employed will depend on a number of factors, including the age
and sex of the patient, the precise disorder being treated, and its
severity. Also, the route of administration may vary depending on
the condition and its severity. A typical dose of ornithine, or of
phenylacetate or phenylbutyrate is from 0.02 g to 1.25 g per kg of
body weight, for example from 0.1 g to 0.5 g per kg of body weight,
depending on such parameters. In some embodiments, a dosage of
ornithine, or of phenylacetate or phenylbutyrate can be from 1 g to
100 g, for example, from 10 g to 80 g, from 15 g to 60 g, from 20 g
to 40 g, or from 25 g to 35 g. In some embodiments, the ornithine
and phenylacetate/phenylbutyrate can be administered in a weight
ratio from 10:1 to 1:10, for example, from 5:1 to 1:5, from 4:1 to
1:4, from 3:1 to 1:3, from 2:1 to 1:2, or about 1:1. A physician
will be able to determine the required dosage of ornithine and of
phenylacetate or phenylbutyrate for any particular subject.
[0091] The exact formulation, route of administration and dosage
for the pharmaceutical compositions of the compound or combination
of compounds disclosed herein can be chosen by the individual
physician in view of the patient's condition. (See, e.g., Fingl et
al. 1975, in "The Pharmacological Basis of Therapeutics," which is
hereby incorporated herein by reference, with particular reference
to Ch. 1). Typically, the dose range of the composition
administered to the patient can be from about 0.1 to about 4000
mg/kg of the patient's body weight. The dosage may be a single one
or a series of two or more given in the course of one or more days,
as is needed by the patient. In instances where human dosages for
compounds have been established for at least some condition, the
present disclosure will use those same dosages, or dosages that are
between about 0.1% and about 5000%, more preferably between about
25% and about 1000% of the established human dosage. Where no human
dosage is established, as will be the case for newly-discovered
pharmaceutical compounds, a suitable human dosage can be inferred
from ED.sub.50 or ID.sub.50 values, or other appropriate values
derived from in vitro or in vivo studies, as qualified by toxicity
studies and efficacy studies in animals.
[0092] It should be noted that the attending physician would know
how to and when to terminate, interrupt, or adjust administration
due to toxicity or organ dysfunctions. Conversely, the attending
physician would also know to adjust treatment to higher levels if
the clinical response were not adequate (precluding toxicity). The
magnitude of an administrated dose in the management of the
disorder of interest will vary with the severity of the condition
to be treated and to the route of administration. The severity of
the condition may, for example, be evaluated, in part, by standard
prognostic evaluation methods. Further, the dose and perhaps dose
frequency, will also vary according to the age, body weight, and
response of the individual patient. A program comparable to that
discussed above may be used in veterinary medicine.
[0093] Although the exact dosage will be determined on a
drug-by-drug basis, in most cases, some generalizations regarding
the dosage can be made. In cases of administration of a
pharmaceutically acceptable salt, dosages may be calculated as the
free base. In some embodiments, the composition is administered 1
to 4 times per day. Alternatively the compositions of the compound
or combination of compounds disclosed herein may be administered by
continuous intravenous infusion, preferably at a dose of each
active ingredient up to 100 g per day. As will be understood by
those of skill in the art, in certain situations it may be
necessary to administer the compound disclosed herein in amounts
that exceed, or even far exceed, the above-stated, preferred dosage
range in order to effectively and aggressively treat particularly
aggressive diseases or infections. In some embodiments, the
compound or combination of compounds disclosed herein will be
administered for a period of continuous therapy, for example for a
week or more, or for months or years.
[0094] In some embodiments, the dosing regimen of the compound(s)
or combination of compounds disclosed herein is administered for a
period of time, which time period can be, for example, from at
least about 1 week to at least about 4 weeks, from at least about 4
weeks to at least about 8 weeks, from at least about 4 weeks to at
least about 12 weeks, from at least about 4 weeks to at least about
16 weeks, or longer. The dosing regimen of the compound(s) or
combination of compounds disclosed herein can be administered three
times a day, twice a day, daily, every other day, three times a
week, every other week, three times per month, once monthly,
substantially continuously or continuously.
[0095] Some embodiments provide a method to use an effective amount
of the compound(s) or combination of compounds disclosed herein in
the treatment of portal hypertension and/or restoration of liver
function in a patient comprising administering to the patient a
dosage of the compound(s) or combination of compounds disclosed
herein containing an amount of about 1 g to about 100 g of drug per
dose of the compound or combination of compounds disclosed herein,
orally, three times per month, once monthly, once weekly, once
every three days, once every two days, once per day, twice per day,
or three times per day substantially continuously or continuously,
for the desired duration of treatment.
[0096] Some embodiments provide a method to use an effective amount
of the compound(s) or combination of compounds disclosed herein in
the treatment of portal hypertension and/or restoration of liver
function in a patient comprising administering to the patient a
dosage of the compound or combination of compounds disclosed herein
containing an amount of from 0.1 mg to about 4000 mg of drug per
kilogram of body weight per dose of the compound or combination of
compounds disclosed herein, orally, three times per month, once
monthly, once weekly, once every three days, once every two days,
once per day, twice per day, or three times per day substantially
continuously or continuously, for the desired duration of
treatment.
[0097] Dosage amount and interval may be adjusted individually to
provide plasma levels of the active moiety which are sufficient to
maintain the modulating effects, or minimal effective concentration
(MEC). The MEC will vary for each compound but can be estimated
from in vitro data. Dosages necessary to achieve the MEC will
depend on individual characteristics and route of administration.
However, HPLC assays or bioassays can be used to determine plasma
concentrations.
[0098] Dosage intervals can also be determined using MEC value. In
some embodiments, compositions can be administered using a regimen
which maintains plasma levels above the MEC for 10-90% of the time,
for example, between 15-30%, 20-45%, 25-50%, 30-55%, 35-60%,
40-65%, 45-70%, 50-75%, 55-80%, 60-90%, 65-75%, 70-80%, 75-85%,
15-90%, 20-90%, 25-90%, 30-90%, 35-90%, 40-90%, 45-90%, 50-90%,
55-90%, 60-90%, 65-90%, 70-90%, 75-90%, or 80-90%. In some
embodiments, compositions can be administered using a regimen which
maintains plasma levels above the MEC for 20-90% of the time. In
some embodiments, compositions can be administered using a regimen
which maintains plasma levels above the MEC for 30-90% of the time,
between 40-90% and most typically between 50-90%.
[0099] In cases of local administration or selective uptake, the
effective local concentration of the drug may not be related to
plasma concentration.
[0100] The amount of composition administered may be dependent on
the subject being treated, on the subject's weight, the severity of
the affliction, the manner of administration and the judgment of
the prescribing physician.
[0101] The compound(s) or combination of compounds disclosed herein
can be evaluated for efficacy and toxicity using known methods. For
example, the toxicology of the compound or combination of compounds
disclosed herein may be established by determining in vitro
toxicity towards a cell line, such as a mammalian, and preferably
human, cell line. The results of such studies are often predictive
of toxicity in animals, such as mammals, or more specifically,
humans. Alternatively, the toxicity of the compound or combination
of compounds disclosed herein in an animal model, such as mice,
rats, rabbits, or monkeys, may be determined using known methods.
The efficacy of the compound or combination of compounds disclosed
herein may be established using several recognized methods, such as
in vitro methods, animal models, or human clinical trials.
Recognized in vitro models exist for nearly every class of
condition, including but not limited to cancer, cardiovascular
disease, and various immune dysfunction. Similarly, acceptable
animal models may be used to establish efficacy of chemicals to
treat such conditions. When selecting a model to determine
efficacy, the skilled artisan can be guided by the state of the art
to choose an appropriate model, dose, and route of administration,
and regime. Of course, human clinical trials can also be used to
determine the efficacy of a compound in humans.
[0102] The compositions may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms
containing the active ingredient. The pack may for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration. The pack or dispenser may also be accompanied with
a notice associated with the container in form prescribed by a
governmental agency regulating the manufacture, use, or sale of
pharmaceuticals, which notice is reflective of approval by the
agency of the form of the drug for human or veterinary
administration. Such notice, for example, may be the labeling
approved by the U.S. Food and Drug Administration for prescription
drugs, or the approved product insert. Compositions comprising the
compound or combination of compounds disclosed herein formulated in
a compatible pharmaceutical carrier may also be prepared, placed in
an appropriate container, and labeled for treatment of an indicated
condition.
[0103] An effective amount of the compound(s) or combination of
compounds disclosed herein may be determined by one of ordinary
skill in the art. It will be understood that the specific dose
level and frequency of dosage for any particular subject may be
varied and will depend upon a variety of factors including the
activity of the specific compound employed, the metabolic stability
and length of action of that compound, the species, age, body
weight, general health, sex and diet of the subject, the mode and
time of administration, rate of excretion, drug combination, and
severity of the particular condition. Preferred subjects for
treatment include animals, most preferably mammalian species such
as humans, and domestic animals such as dogs, cats and the like,
subject to portal hypertension.
[0104] Pharmaceutical compositions comprising the compound(s) or
combination of compounds disclosed herein capable of treating
portal hypertension in an amount effective therefore, and a
pharmaceutically acceptable vehicle or diluent are also disclosed.
The compositions of the present disclosure may contain other
therapeutic agents as described below, and may be formulated, for
example, by employing conventional solid or liquid vehicles or
diluents, as well as pharmaceutical additives of a type appropriate
to the mode of desired administration (for example, excipients,
binders, preservatives, stabilizers, flavors, etc.) according to
techniques such as those well known in the art of pharmaceutical
formulation or called for by accepted pharmaceutical practice.
[0105] The compound(s) or combination of compounds disclosed herein
may be administered by any suitable means, for example, orally,
such as in the form of tablets, capsules, granules or powders;
sublingually; buccally; parenterally, such as by subcutaneous,
intravenous, intramuscular, or intrasternal injection or infusion
techniques (e.g., as sterile injectable aqueous or non-aqueous
solutions or suspensions); nasally such as by inhalation spray;
topically, such as in the form of a cream or ointment; or rectally
such as in the form of suppositories; in dosage unit formulations
containing non-toxic, pharmaceutically acceptable vehicles or
diluents.
[0106] The compound(s) or combination of compounds disclosed
herein, for example, may be administered in a form suitable for
immediate release or extended release. Immediate release or
extended release may be achieved by the use of suitable
pharmaceutical compositions comprising the compound(s) or
combination of compounds, or, particularly in the case of extended
release, by the use of devices such as subcutaneous implants or
osmotic pumps.
[0107] The compound(s) or combination of compounds disclosed herein
may also be administered liposomally. For example, the active
substance can be utilized in a composition such as tablet, capsule,
solution or suspension containing the compound or combination of
compounds disclosed herein or in topical form for wound healing
(0.01 to 5% by weight the compound or combination of compounds
disclosed herein, 1 to 5 treatments per day).
[0108] The compound(s) or combination of compounds disclosed herein
may be compounded in a conventional manner with a physiologically
acceptable vehicle or carrier, excipient, binder, preservative,
stabilizer, flavor, etc., or with a topical carrier.
[0109] The compound(s) or combination of compounds disclosed herein
can also be formulated in compositions such as sterile solutions or
suspensions for parenteral administration. The compound or
combination of compounds disclosed herein may be compounded with a
physiologically acceptable vehicle, carrier, excipient, binder,
preservative, stabilizer, etc., in a unit dosage form as called for
by accepted pharmaceutical practice. The amount of active substance
in these compositions or preparations is preferably such that a
suitable dosage in the range indicated is obtained.
[0110] Exemplary compositions for oral administration include
suspensions which may contain, for example, microcrystalline
cellulose for imparting bulk, alginic acid or sodium alginate as a
suspending agent, methylcellulose as a viscosity enhancer, and
sweeteners or flavoring agents such as those known in the art; and
immediate release tablets which may contain, for example,
microcrystalline cellulose, dicalcium phosphate, starch, magnesium
stearate and/or lactose and/or other excipients, binders,
extenders, disintegrants, diluents and lubricants such as those
known in the art. Molded tablets, compressed tablets or
freeze-dried tablets are exemplary forms which may be used.
Exemplary compositions include those formulating the compound or
combination of compounds disclosed herein with fast dissolving
diluents such as mannitol, lactose, sucrose and/or cyclodextrins.
Also included in such formulations may be high molecular weight
excipients such as celluloses (avicel) or polyethylene glycols
(PEG). Such formulations may also include an excipient to aid
mucosal adhesion such as hydroxy propyl cellulose (HPC), hydroxy
propyl methyl cellulose (HPMC), sodium carboxy methyl cellulose
(SCMC), maleic anhydride copolymer (e.g., Gantrez), and agents to
control release such as polyacrylic copolymer (e.g., Carbopol 934).
Lubricants, glidants, flavors, coloring agents and stabilizers may
also be added for ease of fabrication and use.
[0111] Exemplary compositions for nasal aerosol or inhalation
administration include solutions in saline which may contain, for
example, benzyl alcohol or other suitable preservatives, absorption
promoters to enhance bioavailability, and/or other solubilizing or
dispersing agents such as those known in the art.
[0112] Exemplary compositions for parenteral administration include
injectable solutions or suspensions which may contain, for example,
suitable non-toxic, parenterally acceptable diluents or solvents,
such as mannitol, 1,3-butanediol, water, Ringer's solution, an
isotonic sodium chloride solution, or other suitable dispersing or
wetting and suspending agents, including synthetic mono- or
diglycerides, and fatty acids, including oleic acid.
[0113] Exemplary compositions for rectal administration include
suppositories which may contain, for example, a suitable
non-irritating excipient, such as cocoa butter, synthetic glyceride
esters or polyethylene glycols, which are solid at ordinary
temperatures, but liquify and/or dissolve in the rectal cavity to
release the drug.
[0114] Exemplary compositions for topical administration include a
topical carrier such as Plastibase (mineral oil gelled with
polyethylene). For example, the compound or combination of
compounds disclosed herein may be administered topically to treat
peripheral vascular diseases and as such may be formulated as a
cream or ointment.
[0115] The compound(s) or combination of compounds disclosed herein
may be employed alone or in combination with other suitable
therapeutic agents useful in the treatment of portal hypertension
or restoration of liver function. For example, the compound or
combination of compounds disclosed herein can be administered in
combination with vasopressin analogues such as terlipressin,
ornipressin and vasopressin; somatostatin and its analogues such as
octreotide; non-selective beta blockers such as propranolol and
nadolol; vasodilating beta blockers such as carvedilol; nitrates
such as isosorbide mono-nitrate and glycerine tr-nitrate; and
statins such as Atorvastatin, Fluvastatin, Lovastatin and
Simvastatin
[0116] The above other therapeutic agents, when employed in
combination with the compound or combination of compounds disclosed
herein, may be used, for example, in those amounts indicated in the
Physicians' Desk Reference (PDR) or as otherwise determined by one
of ordinary skill in the art.
EXAMPLES
[0117] Embodiments of the present application are disclosed in
further detail in the following examples, which are not in any way
intended to limit the scope of the present disclosure.
Example 1
[0118] In this example, experiments were conducted on the
clinically relevant, bile duct-ligated (BDL) rat model of
cirrhosis, which exhibits clinical characteristics of low-grade
systemic and brain proinflammatory state indicated by elevated
cytokines such as tumour necrosis factor-alpha (TNF-.alpha.)) along
with low-grade brain oedema. In BDL rats, administration of
bacterial Lipopolysaccharide (LPS) leads to a clinical situation
that mimics clinical ACLF.
[0119] It has been shown that naive rats exposed to ammonia
followed by intravenous LPS, developed an inflammatory response,
cerebral vasodilation and intracranial hypertension, which did not
occur in animals administered LPS alone, indicating the important
role of ammonia in `priming` the brain to the deleterious effect of
LPS. There is an exaggeration of both systemic and brain
inflammatory response which results in worsening of the cytotoxic
brain oedema resulting in a decline in consciousness to
pre-coma/coma stages.
Animal Models
[0120] Thirty-four male Sprague-Dawley rats, body weight 200-250 g
were used. All rats were housed in the unit and given free access
to standard rodent chow and water, with a light/dark cycle of 12
hours, at a temperature of 19-23.degree. C. and humidity of
approximately 50%.
[0121] Bile duct-ligation (BDL): Rats underwent bile duct-ligation
to induce biliary cirrhosis under anesthesia--intravenous (IV)
diazepam (1 mg/kg), followed by a 150 .mu.l/kg of intramuscular
Hypnorm.RTM. (Janssen Pharmaceutica, Belgium).
[0122] Sham-operated (sham): Rats underwent sham-operation under
anesthesia. BDL rats were administered a high protein/ammoniagenic
diet for 7 days prior to inclusion in the study. The diet consisted
of a liquid rodent feed (Bioserve, Frenchtown, N.J. 08825, USA) and
a tailor-made mixture mimicking the amino-acid composition of
haemoglobin molecule (4 g/Kg/day Nutricia, Cuijk, The Netherlands)
mixed with commercially available gelatin to prevent sedimentation.
This regimen produces chronic hyperammonaemia.
Study Design
[0123] The effect of treating superimposed inflammation on the
background of hyperammonemia indicative of ACLF was investigated by
administering OP. Four weeks after surgery, BDL rats were
randomized to receive 3 days of successive intraperitoneal (IP)
injections of OP (0.3 g/kg), the mouse chimeric anti-TNF-.alpha.
monoclonal antibody, or saline (placebo). Three hours before
termination, all BDL rats were administered IP-1 mg/kg LPS (Sigma.
Poole, UK). As controls, sham-operated rats only received IP
saline. Study groups were 1) sham-operated (n=6), 2) BDL+saline
(n=6), 3) BDL+LPS (n=6), and 4) BDL+OP+LPS (n=6).
[0124] A further study was conducted in order to determine the
ammonia-lowering effect of OP on brain inflammatory responses in
just BDL (non-LPS treated) rats. Four weeks after surgery, BDL rats
were randomised to receive 3 days of successive intraperitoneal
(IP) injections of OP (0.3 g/kg) and/or saline (placebo). As
controls, sham-operated rats only received IP saline. Study groups
were 1) sham-operated (n=6), 2) BDL+saline (n=6) and 3) BDL+OP
(n=6). As per protocol, the rats were allowed free access to food
and water for the period of 3 hours post-intervention in a
temperature controlled environment and were then sacrificed by
exsanguination under anesthesia--IP Hypnorm.RTM. (200 .mu.L/kg), 20
minutes after IP diazepam (1 mg/kg). Blood was withdrawn from the
descending aorta and immediately put into ice cold heparin/EDTA
containing tubes (until full exsanguination), centrifuged at
3,120.times.g and 4.degree. C., and the plasma collected and stored
at -80.degree. C. until assayed.
Plasma and Cortical Brain Cytokines
[0125] Plasma and cortical brain samples were snap frozen
(-80.degree. C.) and stored. Prior to analysis, 100 .mu.g of
cerebral cortex was homogenised and deproteinised (using a glass
tube Teflon pestle homogeniser) in 300 .mu.l of ice-cold cell lysis
buffer solution. After centrifugation at 12,000.times.g for 10
minutes at 4.degree. C., the supernatants were collected for
processing. Following protein concentration quantification of
equilibrated brain protein samples and plasma supernatants (50
.mu.l) were analysed for cytokine levels (pg/ml) by flow cytometry
using the Becton Dickinson (BD.TM. biosciences) rat inflammation
cytometric bead array (CBA) kit as described by the manufacturer's
instructions. These included the proinflammatory
cytokines--TNF-.alpha. and interleukin-6 (IL-6). Samples were
analysed by measuring the fluorescence produced by the CBA beads on
an FACS Canto.TM. II flow cytometry system (BD.TM. Sciences) and
the data analysed with BD.TM. CTA software.
Western Blot Analysis
[0126] Snap frozen (-80.degree. C.) and stored 100 .mu.g cortical
brain samples were homogenised and deproteinised (using a glass
tube Teflon pestle homogenizer) in 300 .mu.l of ice-cold cell lysis
buffer solution. After centrifugation at 12,000 g for 10 minutes at
4.degree. C., the supernatants were collected for processing.
Following quantification of sample protein concentration (of
equilibrated 50 .mu.l frontal cortical brain tissue), western-blot
was performed on prepared samples for protein separation and
transfer using a NuPAGE.RTM. pre-cast gel system (Invitrogen Ltd,
UK). Specific protein bands were detected using an iNOS mouse
primary (BD Biosciences, UK) and secondary goat polyclonal antibody
to mouse IgG, HRP conjugated (Hycult biotechnology, Netherlands),
and p65 NFkB-rabbit primary (cell signalling, UK) with a secondary
goat polyclonal antibody to rabbit IgG, HRP conjugated (Hycult
biotechnology, Netherlands). Antibody to alpha-tubulin
(.alpha.-tubulin; Santa Cruz Biotechnology, Inc. USA), a ubiquitous
cellular cytoskeletal protein, was measured to establish accurate
differences in total protein expression between sample tissues;
requiring a secondary goat polyclonal antibody to mouse IgG, HRP
conjugated (Hycult biotechnology, Netherlands) for detection. All
antibodies were used at a dilution of 1:1000. Protein bands were
visualized using Amersham ECL.TM. advance western blotting
detection reagents and Hyperfilm.TM. (GE Healthcare, UK).
Densitometry measurements where made using Image-J software
(freeware; rsbweb.nih.gov/ij).
Statistics
[0127] Data are expressed as mean.+-.SEM. Significance of
difference was tested with Newman-Keuls multiple comparison test or
two-way ANOVA; p<0.05 was taken to be statistically significant.
Paired t test or Wilcoxon Signed Rank test was used for comparison
of two groups as appropriate. Kaplan-Meier survival analyses were
performed for the time to pre-coma/coma in the different treatment
groups and the log-rank test was used for statistical analysis of
the data comparing the survival curves. Software used included
Microsoft Excel 2003 (Microsoft Corp., Redmond, Wash.) and GraphPad
Prism 4.0 (GraphPad Software, Inc., San Diego, Calif.).
Results
[0128] All rats continued to gain weight following surgery. From
the final weight taken immediately prior to termination, BDL rats
(mean.+-.SEM; 342 g.+-.42) were not significantly different to
sham-operated controls (mean.+-.SEM; 380 g.+-.38). The systemic
haemodynamics in the BDL animals were well maintained as previously
shown.
Plasma and Cortical Brain Cytokines
[0129] Throughout all study groups, the mean frontal cortical brain
tissue cytokine levels were in the order of 10 fold higher compared
to their respective circulating plasma levels (FIGS. 1A-D and Table
1). Multiple comparison group analysis revealed the following:
[0130] Plasma cytokines: When compared to sham-operated rats, bile
duct-ligation was associated with a significant increase in the
plasma levels of the proinflammatory cytokines TNF-.alpha.
(p<0.05) (FIG. 1A). In BDL rats, LPS challenge significantly
increased plasma TNF-.alpha. and IL-6 when compared to
sham-operated controls (p<0.001, respectively) and
saline-treated BDL controls (p<0.01, respectively) (FIGS. 1B-C).
When compared to BDL+LPS rats, there was a significant reduction in
plasma TNF-.alpha. (p<0.01) and trend towards reduction in IL-6
following administration of OP (FIGS. 1B-D).
[0131] Cortical brain cytokines: When compared to sham-operated
rats, bile duct-ligation was associated with a significant increase
in the brain levels of TNF-.alpha. (p>0.05, Table 1), which was
augmented by LPS (p<0.001). In LPS treated BDL rats,
administration of OP ameliorated the elevated cortical TNF-.alpha.
brain tissue levels (p<0.05). There was a similar trend with
brain IL-6 levels following bile duct-ligation and treatment
intervention.
Plasma and Cortical Brain Cytokines
[0132] Plasma cytokines: When compared to sham-operated rats
(114.8.+-.40.4 pg/ml), BDL was associated with a significant
increase in the plasma levels of TNF-.alpha. (902.2.+-.176.1 pg/ml;
p<0.001), which were significantly reduced by pre-treatment with
OP (270.5.+-.59.4 pg/ml; p<0.001).
[0133] Cortical brain cytokines: When compared to sham-operated
rats (34.2.+-.4.6 pg/ml), BDL was associated with a significant
increase in the brain levels of TNF-.alpha. (178.7.+-.62.9 pg/ml;
p<0.05), which were significantly reduced by pre-treatment with
OP (54.7.+-.15.3 pg/ml; p<0.05).
iNOS Expression
[0134] When compared to sham-operated rats (1.46.+-.0.17; FIG. 2A),
there was a significant rise in brain iNOS with BDL (2.87.+-.0.14;
p<0.001), which was reduced following pre-treatment with OP
(2.32.+-.0.17; p<0.05).
NF.kappa.B Expression
TABLE-US-00001 [0135] TABLE 1 Cytokine levels (pmol/L) Sham BDL BDL
+ LPS BDL + OP + LPS Plasma 90 .+-. 1859 .+-. 417* 4143 .+-. 528***
1919 .+-. 828.sup.$$ TNF-a 25 Plasma 153 .+-. 1470 .+-. 422 4135
.+-. 560***/.sup.## 3730 .+-. 832 IL-6 52 Brain 45 .+-. 237 .+-.
79* 402 .+-. 57*** 165 .+-. 12.sup.$ TNF-a 13 Brain 26 .+-. 144
.+-. 99 285 .+-. 104 126 .+-. 84 IL-6 5 Data are expressed as mean
.+-. standard error of mean (SEM) Symbols represent;- *p < 0.05
and ***p < 0.001 compared to sham-operated control rats; .sup.$p
< 0.05 and .sup.$$p < 0.01 compared to BDL + LPS rats and
.sup.##p < 0.01 compared to BDL rats. Abbreviations: sham,
sham-operated. BDL, bile duct-ligation. LPS,
lipopolysaccharide.
[0136] When compared to sham-operated rats (2.65.+-.0.17; FIG. 2B),
there was a significant rise in brain NF.kappa.B with BDL
(4.25.+-.0.13; p<0.001), which was markedly reduced following
pre-treatment with OP (2.52.+-.0.19; p<0.001).
[0137] In this example, BDL rats for 4-weeks prior to the study
represents chronic liver disease with hyperammonaemia and a
proinflammatory state indicated by elevated arterial and brain
cytokines. Additionally, the administration of LPS to this model is
reflective of a second hit, and in this context represents an
infective episode with evidence of exaggeration of the inflammatory
response manifested by an increase in TNF-.alpha. and IL-6. The
cirrhotic brain exhibits classical `cytotoxic oedema` even in the
LPS treated group. Ammonia and inflammation work simultaneously
(and in synergy) to produce brain oedema and coma.
[0138] It was observed that OP treatment was associated with a
reduction in plasma and brain proinflammatory cytokines as
well.
Modulation of Brain eNOS Activity
Example 2
[0139] Asymmetric dimethylarginine (ADMA) is an endogenous
inhibitor of eNOS, the levels of which are increased in liver
failure. A study was conducted to determine whether administration
of combinations of L-ornithine and phenylacetate impacts upon the
NO pathway. This example addresses the questions: (a) is eNOS
activity reduced in cirrhotic brains? (b) is ADMA level increased
and di-hydro diamino hydrolase (DDAH, enzyme that breaks down ADMA)
decreased in cirrhotic brains: (c) are other regulators of eNOS
activity altered in cirrhotic brains, and whether these are
restored by L-ornithine in combination with phenylacetate (OP)?
[0140] Sprague-Dawley rats were studied 4-weeks after bile duct
ligation (BDL) (n=16) or sham operation (n=8) and randomized to
treatment with placebo or OP (0.6 gm/kg) i.p. Arterial blood,
frontal brain tissue and urine were collected at the time of
sacrifice. Ammonia and amino-acids were measured in the plasma
using Cobas-MiraS and HPLC respectively. Brain water was measured
using the dry weight technique. Urinary phenylacetyglutamine and
plasma and brain ADMA were measured using LCMS. eNOS activity was
measured using radiolabelled .sup.1H Arginine and protein
expression for eNOS, DDAH-1 and Caveolin measured using Western
Blotting.
[0141] Treatment of BDL rats with OP resulted in normalization of
arterial ammonia (p<0.001), brain water (p<0.001) and
increased urinary phenylacetylglutamine (p<0.01). eNOS activity
was significantly lower but eNOS protein expression was greater in
BDL animals compared with sham operated controls which was restored
towards sham values in the OP treated animals. Brain ADMA levels
were significantly higher in BDL compared with sham and brain
DDAH-1 was significantly lower which was restored on treatment with
OP. Brain Caveolin was significantly lower in BDL animals, which
was increased towards sham values in the OP treated animals.
[0142] This example showed that the brain nitric oxide pathway is
adversely affected by hyperammonemia, which can be restored by
treatment with OP. These results demonstrated that OP may be used
for the treatment of organ systems in cirrhosis that are known to
have deranged NO signalling.
Example 3
[0143] Sham operated Sprague-Dawley rats (n=10) and BDL rats (n=10)
were compared four weeks after BDL surgery, and in an additional
BDL group (n=6), after administration of 3 g/kg i.p. OP twice a day
for 5 days. Ammonia and amino-acids were measured in the plasma
using Cobas-Integra and HPLC, respectively. Brain water was
measured using the dry weight technique. TNF.alpha. was measured by
FACS bead array. Urinary phenylacetylglutamine and plasma and brain
ADMA were measured using LC-MS/MS-respectively. eNOS and DDAH
activity was measured radiometrically. Protein expression for eNOS,
DDAH-1&2 and caveolin-1 were measured by western blotting.
[0144] As shown below, plasma (FIG. 3A) and brain (FIG. 3B)
TNF-.alpha. levels were significantly elevated in BDL rats compared
to sham (p<0.01, for both). OP treatment reverts these changes
near to sham and are significant when compared to BDL alone
(p<0.05; p<0.01, respectively).
[0145] As shown in FIG. 4, eNOS activity (FIG. 4A) was
significantly decreased in BDL rat brain compared to sham despite
increased eNOS protein expression (FIG. 4B). Following treatment
with OP, eNOS activity reverted to sham levels, with similar
normalization of eNOS protein expression. iNOS protein was also
significantly elevated in BDL rat (FIG. 4C). OP treatment
significantly down regulated iNOS protein as compared to BDL alone
(FIG. 4C).
[0146] As shown in FIG. 5, plasma ADMA (FIG. 5A) was significantly
elevated in BDL rat plasma (p<0.01) and brain homogenates (FIG.
5B) (p<0.05) compared with sham. Following OP treatment, there
was a non-significant reduction in plasma ADMA but a significant
reduction in brain ADMA concentration as compared to BDL. Moreover,
cerebral caveolin-1 protein expression (FIG. 5C) was increased
significantly in BDL rat compared to sham (p<0.01). Treatment
with OP reverted caveolin-1 protein to sham levels (p<0.05).
[0147] As shown in FIG. 6, cerebral DDAH-1 (FIG. 6A) protein
expression was decreased significantly (p<0.05) in BDL rat.
Conversely, cerebral DDAH-2 (FIG. 6B) protein expression was
significantly elevated (p<0.05) compared to sham. Following OP
administration, DDAH-1 increased significantly (p<0.05) and
DDAH-2 protein expression decreased significantly (p<0.05)
compared to BDL. Moreover, DDAH activity (FIG. 6C) was
significantly elevated in BDL rat brain compared to sham. OP
treatment significantly (p<0.01) decreased DDAH activity as
compared to BDL alone (p<0.01).
Modulation of Liver eNOS Activity
Example 4
[0148] In cirrhosis, portal hypertension is associated with hepatic
inflammation which contributes to reduced intrahepatic endothelial
nitric oxide synthase (eNOS) activity, which is associated with the
increased endogenous NOS inhibitors, Caveolin-1 and
asymmetric-dimethylarginine (ADMA). This example is to determine
whether treatment with L-ornithine phenylacetate combinations (OP)
reduces NF.kappa.B and increases intrahepatic NO availability
through modulation of these inflammatory dependent inhibitors of
endothelial NOS and thereby reduces portal pressure.
[0149] Sprague-Dawley rats were studied 4-weeks after BDL surgery
(n=16) or sham operation (n=8) and randomized to treatment with
placebo or OP (0.6 gm/kg) i.p. for 5 days prior to study. Ra ts
underwent direct portal pressure measurement under anaesthesia at
the time of sacrifice and plasma and liver tissue was harvested for
subsequent analysis. Plasma ammonia and biochemistry were measured
using a Cobas-MiraS analyser, and eNOS activity was determined
radiometrically. Protein expression for eNOS, DDAH-1, Caveolin-1,
and NF.kappa.B were measured using Western Blotting.
[0150] Treatment with OP resulted in a reduction in hyperammonaemia
(p<0.001) towards sham values and an increase in hepatic eNOS
activity towards sham levels. This was associated with a
significant reduction in portal pressure compared with placebo
treated group (11.+-.0.4 vs. 14.+-.0.7 mmHg, p=0.01). Moreover, OP
treatment significantly reduced the expression of Caveolin-1
(p<0.05) and increased expression of
dimethylarginine-dimethylamainohydrolase-1 (p<0.05) [DDAH 1 is
responsible for metabolism of ADMA], whilst also significantly
lowering hepatic phosphorylated NF.kappa.B expression, compared
with placebo treated animals.
[0151] This example showed that treatment of hyperammonemia with OP
reduces the severity of portal hypertension in a clinically
relevant model of cirrhosis through restoration of the hepatic eNOS
activity by modulating NF.kappa.B and the expression of eNOS
regulators, DDAH1-ADMA and Caveolin-1.
Example 5
[0152] Four weeks after BDL and sham surgery in Sprague-Dawley
rats, BDL rats were given i.p. OP 3 g/kg twice a day or vehicle
alone (n=14/group), and treated for 5 days. After the 5th treatment
day, all rats underwent direct portal pressure measurement under
anaesthesia prior to sacrifice. Plasma and liver tissue was
harvested for subsequent analysis. Plasma ammonia and biochemistry
were measured using a Cobas-Integra analyzer. Plasma TNF.alpha. was
measured by FACS bead array. eNOS activity was determined
radiometrically. Protein expression for eNOS, DDAH-1, caveolin-1,
and NF.kappa.B were measured using standard Western Blotting
techniques.
[0153] Effect of OP on arterial ammonia and plasma biochemistry of
BDL rats is indicated in Table 2. Statistical significance was
calculated using student t' test Mann Witney comparisons test.
*-P<0.01 versus sham; **-P<0.001 versus sham;
.dagger.-P<0.01 versus BDL; .sctn.-P<0.05 versus BDL; NS-no
significance.
TABLE-US-00002 TABLE 2 Parameters Sham BDL BDL + OP Ammonia
(.mu.mol/L) 55.86 .+-. 4.05 222.3 .+-. 24.01** 127.1 .+-.
47.8.dagger. ALT (U/L) 26.88 .+-. 2.61 159.8 .+-. 10.81** 108.3
.+-. 5.66.dagger. Bilirubin (.mu.mol/L) 32.52 .+-. 2.43 304.9 .+-.
25.97** 267.7 .+-. 14.5NS Creatinine (.mu.mol/L) 23.07 .+-. 0.94
40.88 .+-. 2.37* 32.38 .+-. 1.44.sctn. TNF-.alpha. (pg/ml) 121.9
.+-. 46.4 822.9 .+-. 203.1* 270.5 .+-. 59.4.sctn.
[0154] As shown in FIGS. 7A and 7B, eNOS activity was significantly
decreased in BDL animals compared to sham (p<0.05) despite
increased eNOS protein expression (p<0.01). Following treatment
with OP, eNOS activity reverted to sham levels, with similar
normalisation of eNOS protein expression (p<0.05). As shown in
FIG. 7C, DDAH-1 protein expression was significantly higher in sham
animals compared to BDL (p<0.01). Following OP administration to
BDL rats, DDAH-1 expression increases significantly and reverts to
sham levels (p<0.05). As shown in FIG. 7D, protein expression of
NFkB was significantly elevated in BDL rats compared to sham
(p<0.01). OP treatment produces a marked reduction in NF.kappa.B
expression compared to BDL (p=0.05). As shown in FIG. 7E, the
expression of caveolin-1 was significantly elevated in BDL rats
compared to sham (p<0.01). Following intervention with OP,
Caveolin-1 expression decreased significantly (p<0.05) compared
to BDL alone. As shown in FIG. 7C, portal pressure was
significantly increased in BDL rats compared to sham (p<0.0001).
OP treatment results in a 30% reduction of portal pressure as
compared to BDL (P<0.01).
Example 6
[0155] This example is to determine whether treatment with
L-ornithine phenylacetate combinations (OP) improves liver function
(e.g., perfusion).
[0156] Sprague-Dawley rats are studied 4-weeks after bile duct
ligation (BDL) and randomized to treatment with placebo or OP. The
liver function of the BDL rats is measured by a variety of methods.
For example, liver injury is detected by the alanine transaminase
(ALT) test, aspartate aminotransferase (AST) test, and/or alpha
glutathione S-transferase (GST) test. Liver function is measured by
albumin (Alb) test; prothrombin time test; and/or composite scores,
such as child-pugh score' MELD score. Liver hemodynamics is
measured by detecting liver blood flow and/or portal pressure.
[0157] The administration of OP is effective in improving and/or
restoring liver function, e.g., increasing liver perfusion, in the
BDL rats. OP treatment is also effective in reducing portal
pressure in the BDL rats.
Example 7
[0158] This example is to determine whether treatment with
L-ornithine phenylacetate combinations (OP) decreases variceal
bleeding in BDL rats with portal hypertension.
[0159] Sprague-Dawley rats are studied 4-weeks after bile duct
ligation (BDL) and randomized to treatment with placebo or OP.
Portal pressure of the BDL rats is measured. The BDL rats are also
detected for variceal bleeding and the extent of variceal bleeding
of the BDL rats is measured.
[0160] The administration of OP is effective in reducing portal
pressure and decreasing variceal bleeding in the BDL rats.
Example 8
[0161] This example is to determine whether treatment with
L-ornithine phenylacetate combinations (OP) decreases ascites in
BDL rats with portal hypertension.
[0162] Sprague-Dawley rats are studied 4-weeks after bile duct
ligation (BDL) and randomized to treatment with placebo or OP.
Portal pressure of the BDL rats is measured. The BDL rats are also
detected for ascites and the extent of ascites of the BDL rats is
measured.
[0163] The administration of OP is effective in reducing portal
pressure and ascites in the BDL rats.
[0164] Although the present disclosure has been described with
reference to embodiments and examples, it should be understood that
numerous and various modifications can be made without departing
from the spirit of the present disclosure. Accordingly, the present
disclosure is limited only by the following claims.
[0165] All references cited herein, including patents, patent
applications, papers, text books, and the like, and the references
cited herein, to the extent that they are not already, are hereby
incorporated by reference in their entirety. In the event that one
or more of the incorporated literature and similar materials differ
from or contradict this application, including but not limited to
defined terms, term usage, described techniques, or the like, this
application controls.
* * * * *