Drug – bio-affecting and body treating compositions – Plant material or plant extract of undetermined constitution...
Reexamination Certificate
2000-08-16
2002-12-17
Tate, Christopher R. (Department: 1651)
Drug, bio-affecting and body treating compositions
Plant material or plant extract of undetermined constitution...
C424S070510, C514S022000, C514S023000, C514S783000, C514S893000
Reexamination Certificate
active
06495170
ABSTRACT:
DESCRIPTION
FIELD OF THE INVENTION
This invention relates to a method of increasing the presence of glutathione in cells including treating or preventing of impaired liver function by administering to a mammal in the need thereof a dietetic or pharmaceutical composition and to a corresponding dietetic or pharmaceutical composition.
BACKGROUND OF THE INVENTION
The liver is the major organ involved in metabolism of protein, carbohydrates, and fats, but is also the major organ for detoxification. Potential toxic compounds are converted into inactive metabolites by phase I-metabolising enzymes and excreted. Alternatively, metabolites are further conjugated by phase II-metabolising enzymes and excreted after all.
Many drugs and toxins can be detoxified by conjugation with glutathione. When the levels of these drugs or toxins, however, exceed the liver concentration of reduced glutathione; such components become acutely hepatotoxic. Striking is that in several kinds of liver disorders, glutathione levels are decreased, for example: in hepatitis infection, where the grade of activity of the liver disease is correlated with reduction of GSH in acute liver toxification, e.g. acetaminophen intoxication, and in alcoholics with liver failure.
Both hepatitis C and the exposure to liver toxins can lead to hepatocarcinoma, possibly via the same mechanism of incompetence of the liver to respond to toxins, either endo-toxins or exo-toxins.
Reduced glutathione (GSH) is the substrate of glutathione peroxidase (GPX) and as such contributes to the antioxidant defence mechanism as well.
Glutathione reductase enzymes reduce GSSG back to GSH, predominantly in the presence of NADPH, which is provided by e.g. the oxidative pentose phosphate pathway. The capacity of the glutathione system to cope with H
2
O
2
in liver depends on the activity of GPX and giutathione reductase, the rate of NADPH supply, and the GSH content. Except for its effects on hydrogen peroxide, GSH can also react with e.g. OH
o
, HOCl, peroxynitrite, RO
o
, and RO
2
o
in vitro. Upon reacting with free radicals, thiyl radicals (Gaf) are produced, which generate superoxide. Moreover, superoxide can also inactivate GPX in the absence of GSH. Hence, superoxide dismutase (SOD) or antioxidants can co-operate with GSH to remove free radicals in vivo.
Many xenobiotics supplied to living organisms are metabolised by conjugation with GSH. This process is catalysed by glutathione S-transferase enzymes (GST).
GSH is synthesised in two steps, catalysed by two different enzymes. During the first step, &ggr;-glutamylcysteine synthetase (GCS) catalyses the formation of L-&ggr;-glutamyl-L-cysteine from glutamate and cysteine. The second step incorporates glycine under influence of glutathione synthetase, yielding GSH.
One example of a drug that can deplete the concentration of reduced glutathione in the liver is the commonly used sedative acetaminophen (paracetamol). Acetaminophen has caused severe hepatic necrosis when ingested in large amounts, e.g. in suicide attempts or accidentally by children. Acetaminophen hepatotoxicity is mediated by a toxic reactive metabolite formed from the parent compound by the cytochrome P450 mixed-function oxidase system of the hepatocyte. The metabolite is then detoxified by conjugation with glutathione. If excessive amounts are formed, the glutathione reserves of the liver are depleted, and the quinonimine reacts with constituents of the liver cells, leading to necrosis. The hepatic injury may be potentiated by the use of alcohol or other drugs and also starvation and cachexia, conditions in which liver glutathione are lower, may potentiate the effect.
Milk thistle, or
Silybum marianum
(L.) Gaertn. (Compositae) is in Western countries well-known for its hepatoprotective effects. Its key components are flavanolignans, collectively known as silymarin. A composition for the protection, treatment and repair of liver tissue containing Milk thistle is for instance described in WO 99/43336.
Intraperitoneal administration of silymarin (200 mg/kg) in rats increased the total glutathione content and improved the reduced glutathione/oxidised glutathione ratio in the liver, intestine, and stomach, while levels of kidney, lung, and spleen were not affected (Valenzuela et al., Selectivity of silymarin on the increase of gutathione content in different tissues of the rat, Planta Medica 55, 420-2 (1989)). The main component of silymarin, silybin, exerted inhibitory effects on superoxide and hydrogen peroxide production in human neutrophils and increased the activity of both superoxide dismutase and glutathione peroxidase in human erythrocytes. Silymarin protects against acetaminophen-induced GSH depletion and cytotoxicity in hepatoblastoma Hep G2 cells in vitro. Silymarin and silybin protect in vivo against hepatic glutathione depletion induced by ethyl alcohol and paracetamol in rats. It was shown that silybin reduces the GSH depletion induced by acetaminophen, but does not affect GSH depletion by buthionine sulfoximide in isolated rat hepatocytes. This suggests that it enhances GSH oxidation or conjugation, without affecting glutamylcysteine synthetase or GSH synthesis (Garrido et al.,
Pharmacology an Toxicology
69, 9-12 (1991)).
Furthermore, silybin was found to be a potent, non-toxic, inhibitor of glutathione-S-transferase (Bartholomeus et al.,
Xenobiotica
24, 17-24, (1994)). This observation leads to the conclusion that, although silymarin might be effective as a hepatoprotective agent, secondary detoxification by conjugation to GSH, as is catalysed by GSH-transferase, is inhibited.
Silymarin treatment (Legalon, 420 mg, 6 months) in patients with chronic alcoholic liver disease in a double blind placebo controlled trial increased the serum level of free —SH groups and the activity of GSH peroxidase. Other clinical trials confirm the hepatoprotective effect of standardised Silybum extracts.
RO 102689 (Intr. Medicamente Biofarm) discloses the extraction of silymarin from armurariu fruit. Said extract shall be useful in the treatment of active chronic hepatitis, cirrhosis of the liver and for the protection of liver cells during administration of hepatoxic agents.
Picrorhiza kurrooa
Royle (Scrophulariaceae) has been used in Ayurveda mainly for the treatment of liver disorders. Iridoid glucosides have been regarded as its active constituents; however, other compounds like acetophenones might also play a role.
Several iridoid glucosides have been isolated from
P. kurrooa
, most of them conjugates of catalpol with either benzoyl- or cinnamoyl-derived side chains.
Picroliv is a standardised fraction of
Picrorhiza kurrooa
containing the iridoid glycosides picroside I and kutkoside in a ratio of 1:1.5 to 1.2 (50-70%) and a mixture of cucurbitacin glycosides (4-5%) (U.S. Pat. No. 5,145,955). Picroside II has also been isolated from
Picrorhiza kurrooa
and 50 mg/kg p.o. in mice treated with CCl4 showed a hepatoprotective activity (DE-A 2203884).
Picroliv has been tested in a number of liver damage models in vitro and in vivo. These studies have demonstrated its antihepatotoxic, hepatoregenerative, choleretic, and hypolipidemic activities (reviewed by Dhawan,
Medicinal Chemistry Research
5, 595-605, (1995)). Hepatoregenerative effects were associated with an increased recovery of the liver anti-oxidant system after partial hepatectomy or carbon tetrachloride-induced liver damage of Picroliv-treated rats. Furthermore, Picroliv significantly reversed paracetamol-induced biochemical changes in several liver cell markers after oral administration to rats (6 and 12 mg/kg) for 7 days. Injection of carbon tetrachloride in rats induced a drastic impairment of the hepatic mixed-function oxidase system, as indicated by several drug-metabolising enzymes such as glutathione-S-transferase and reduced glutathione. Administration of Picroliv (6 mg/kg) for 7 days significantly prevented liver damage (Rastogi et al.,
Drug Development Research
41, 44-47, (1997)). Picroliv, given to rats during the last 15 days (12 mg/kg/day p.o.) of a 45
Hageman Robert Johan Joseph
Smit Hobbe Friso
van Norren Klaske
Bacon & Thomas
Flood Michele C.
N. V. Nutricia
Tate Christopher R.
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