WO2011044638A1 - Procédé de traitement de maladie de foie - Google Patents

Procédé de traitement de maladie de foie Download PDF

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Publication number
WO2011044638A1
WO2011044638A1 PCT/AU2010/001372 AU2010001372W WO2011044638A1 WO 2011044638 A1 WO2011044638 A1 WO 2011044638A1 AU 2010001372 W AU2010001372 W AU 2010001372W WO 2011044638 A1 WO2011044638 A1 WO 2011044638A1
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Prior art keywords
diltiazem
calcium channel
flavonolignan
channel blocker
liver
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PCT/AU2010/001372
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English (en)
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Howard John Smith
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Howard J. Smith & Associates Pty Ltd
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Priority claimed from AU2009905039A external-priority patent/AU2009905039A0/en
Application filed by Howard J. Smith & Associates Pty Ltd filed Critical Howard J. Smith & Associates Pty Ltd
Priority to CN2010800469847A priority Critical patent/CN102573864A/zh
Publication of WO2011044638A1 publication Critical patent/WO2011044638A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/554—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one sulfur as ring hetero atoms, e.g. clothiapine, diltiazem
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics

Definitions

  • the present invention relates to a method of treatment of liver disease and its complications including cirrhosis of the liver and portal hypertension.
  • the invention relates to a method of treatment of non-malignant liver disease such as viral or toxic hepatitis, and fatty liver, where direct or indirect damage to the liver results, and to compositions for use in treatment of liver disease.
  • Non-malignant liver diseases include toxic hepatitis such as alcoholic hepatitis, which is the most common form of toxicity, hepatitis caused by infection such as viral hepatitis, fatty liver, and less commonly, the immunological liver diseases characterised by chronic inflammation.
  • toxic hepatitis such as alcoholic hepatitis, which is the most common form of toxicity
  • hepatitis caused by infection such as viral hepatitis, fatty liver, and less commonly, the immunological liver diseases characterised by chronic inflammation.
  • silymarin The side effects of silymarin are most severe in diseased patients such as those with viral hepatitis C and patients with non-alcoholic fatty liver disease in which case the levels of accumulated flavanolignans have been reported as four or five times or more than that of healthy subjects (Schrieber et al., Drug Metab. Dispos. 2008 Sept. 36(9)1909- 16).
  • the side effects of silymarin include upset stomach, nausea, mild headaches, diarrhoea, vomiting and joint pain.
  • Silymarin has also been reported to reduce the effectiveness of oral contraceptives and in less common cases the side effects can include severe stomach pain, sweating and muscle weakness.
  • calcium blocking agents verapamil, diltiazem, and amlodipine (Mason et al, 1999), and also nitrendipine (Thurman and colleagues, 1998).
  • these agents acted directly on liver cells to block calcium entry as they do in excitable tissues such as the myocardium and in arteries (Liang and Thurman, 1992), however, it was soon realised that the liver does not have voltage-gated calcium channels, which are the target of these drugs in excitable tissues. Therefore, if these drugs were acting on the liver, they needed to act in a different way. Thus, it was found that many calcium blockers were also powerful antioxidants (Heo et al., 1997).
  • calcium channel blockers may dilate the hepatic artery to increase delivery of oxygenated blood to the liver (McLean. 1998).
  • drugs such as verapamil, diltiazem (Liang and Thurman, 1992; Romero and colleagues, 1994) and the other calcium blockers occurs in isolated cells, and are therefore independent of drug-induced alteration in blood flow.
  • the effects on hepatic arterial blood flow of calcium blockers administered orally in low doses have not been confirmed.
  • Diltiazem has also been shown to have no effect on microvascular blood flow within the liver (Marteau and colleagues, 1988).
  • a method of treatment for patients suffering from liver disease comprising the administration of (i) an oral slow-release formulation of a calcium channel blocker with antioxidant effects and (ii) at least one flavonolignan.
  • calcium channel blocker is diltiazem and is administered at a dose in the range of from 10 to 70 mg/day.
  • a method of treatment of a patient suffering from liver disease comprising the administration of (i) an oral slow- release formulation of a calcium channel blocker with antioxidant effects and (ii) an antioxidant effective amount of flavonolignan.
  • the flavonolignan is generally administered in an amount of from 1 to 5000mg per day.
  • thiamine is also administered and typical doses used may be in the range of from 1 to 200mg per day and preferably in the range of from 20 to 200mg thiamine per day.
  • the calcium channel blocker and flavonolignin may be present in the same unit dosage or may be administered separately.
  • the diltiazem is in a slow release formulation which in one embodiment also includes the flavonolignin and in another embodiment does not include the flavonolignin which is administered separately either in a slow release or non slow release formulation.
  • the invention further provides a pharmaceutical composition for treatment or prevention of liver disease comprising (i) a calcium channel blocker with antioxidant effects and (ii) at least one flavonolignan.
  • the pharmaceutical composition is typically a slow-release composition, providing slow-release of each of the calcium channel blockers and flavonolignan.
  • the slow release composition may comprises a unit dose of from 1 to 5000 mg of at least one flavanolignan (preferably 5 to 3000 mg such as 10 to 2000 mg or 50 to 1000 mg) and diltiazem in an amount of from 10 to 70 mg such as 10 to 50mg.
  • silymarin as a hydrophilic antioxidant complements the antioxidant effect of diltiazem to protect both cell membranes and mitochondrial membranes of cells exposed to reactive oxygen species (ROS).
  • ROS reactive oxygen species
  • Diltiazem as a calcium blocker inhibits calcium entry into mitochondria during conditions of calcium overload because it inhibits the calcium-gated calcium channel in the mitochondrial membrane which is activated when the intracellular calcium concentration rises. This action allows damaged or tired cells to continue to produce energy (ATP). This effect has a positive effect on liver cell function under conditions that resemble disease conditions.
  • silymarin does not appear to have a direct effect on mitochondria other than contributing to the preservation of membranes on account of its antioxidant properties, the energy-protective effect of diltiazem on the mitochondria will produce an additional beneficial effect by enabling the cells to continue to metabolize the silymarin.
  • Preservation of the metabolism of silymarin which is enabled by the direct mitochondrial effect of diltiazem, has two important clinical sequelae. First, it will help reduce the side effects of silymarin that may occur under conditions of liver disease if the metabolic capacity of the liver cells is reduced.
  • sustained metabolism of silymarin by the liver cells under disease conditions will help maintain the liver-selective delivery of silymarin because in addition to being administered in low dose, and in a slow-release formulation, a key feature of liver-selective drug delivery is that the drug must have a short half-life produced by first-pass clearance by the liver
  • a method of treatment of a subject suffering from liver disease comprising the administration of (i) an oral slow-release formulation of a calcium channel blocker with antioxidant effects and (ii) at least one flavonolignan.
  • the invention also provides the use of (i) a calcium channel blocker with antioxidant effects and (ii) at least one flavonolignan, which is also an antioxidant, in preparation of a medicament for treatment of liver disease by co administration thereof in one or more compositions for providing slow-release of calcium channel blocker.
  • the invention further provides a pharmaceutical composition for treatment or prevention of liver disease comprising (i) a calcium channel blocker with antioxidant effects and (ii) at least one flavonolignan which is also an antioxidant.
  • the pharmaceutical composition is typically a slow-release composition that releases the active agents (lipophilic calcium channel blocker with antioxidant effects plus hydrophilic flavonolignan antioxidant, over a period of several hours, preferably 8 - 12 or more hours.
  • the liver-selective slow-release formulation is required because many of the drugs suitable for this treatment are vaso-active or have systemic effects that may be avoided if they are concentrated preferentially within the liver.
  • the low dose, slow-release composition will preferably provide a delivery rate of calcium channel blocker sufficient to provide a clinical effective blood level in the portal vein and less than required to provide a clinically effective level in the peripheral circulation to thereby provide a delivery rate having a selective effect on the liver.
  • Thiamine may be administered in addition to the calcium channel blocker and flavonolignan separately or concurrently.
  • Thiamine which is well known as a B vitamin acting within mitochondria as a mitochondrial protective agent, that is, as a cofactor of pyruvate dehydrogenase, is also a hydrophilic antioxidant. It is used in patients with nutritional deficiency and in some patients with viral hepatitis.
  • the addition of thiamine to the calcium antagonist with antioxidant properties also exploits both the facilitator effect of the vitamin on energy production within mitochondria, and its own antioxidant effect within the cell.
  • the liver is perfused at low pressure with venous blood. Therefore, more than any other organ in the body, it is at risk of hypoxia when cell swelling of any cause inhibits blood flow. Hypoxia of the liver is a feature of almost every form of non-malignant liver disease, and accounts for 50% or more of the pathology and impairment of function.
  • oxygen free radicals are produced by the inflammatory processes outside the cells.
  • hypoxia free radicals are produced inside the cells (as levels of NADPH rise). Both areas of free radical production are in the aqueous phase.
  • flavanolignins tend to accumulate in diseased liver cells their effectiveness as antioxidants is restored in the presence of the calcium chanel blocker.
  • the metabolism of flavonolignins in diseased cells is also enhanced thereby reducing the risk of side effects of flavanolignins in subjects with liver disease.
  • the invention uses at least one antioxidant selected from flavonolignans.
  • Silibinin also known as silybin, is the major active constituent of silymarin, the mixture of flavonolignans extracted from blessed milk thistle (Silybum marianum).
  • flavonolignan includes silymarin and its active constituent silibinin whether in the form of the purified compound (believed to be 3,5,7-trihydroxy-2-(3-(3-hydroxy-4-methoxyphenyl)-2-(hydroxymethyl)-2,3- dihydrobenzo[b][1 ,4]dioxin-6-yl)chroman-4-one or pharmaceutically acceptable salts such as silibinin dihydrogen disuccinate disodium (trade name Legalon SIL a solution for injection), inclusion complexes such as ⁇ -cyclodextrin inclusion complexes and glycosides of silybin, which show better water solubility and even stronger hepatoprotective effect.
  • the invention allows the side effects associated with sylmarin to be reduced or generally eliminated allowing sustained use of the combination therapy without adverse effects over an extended period.
  • the composition will be administered at least once daily for at least ten consecutive days, preferably at least 20 and more preferably, at least 30 consecutive days.
  • the composition contains thiamine in a unit dosage form containing from 1 to 200mg thiamine, preferably 20 to 200mg thiamine.
  • Patients which suffer from liver disease may have thiamine deficiency which may be treated by administration of thiamine in an amount of from 1 to 5 mg per day.
  • the use of relatively high doses of 20 to 200mg by contrast is well above the level required to treat vitamin deficiency.
  • This dosage in the composition of the invention will provide a significant therapeutic effect believed to result from the antioxidant effects of the thiamine at relatively high dosage.
  • the preferred calcium channel blockers are lipophilic to ensure that they penetrate deep with cells and within membranes so that their therapeutic effect is located where free radicals are acting.
  • drugs acting within the cytosol or cellular milieu need to be more hydrophilic.
  • Most calcium blocking drugs including verapamil, diltiazem, amlodipine, and nitrendipine have antioxidant properties as a component of a membrane stabilizing effect but the shorter half-life of diltiazem and verapamil makes these agents more suitable for formulation as a liver-selective membrane-stabilizing agent.
  • the preferred agent is diltiazem administered as a slow-release formulation at doses of less than 70mg per day such as 10 to 70 mg per day or 20 to 70 mg per day and preferably less than 50 mg per day such as 10 to 50 mg per day. These doses are much lower that the doses of the drug used in the treatment of angina and hypertension. Diltiazem may be in the form of the hydrochloride salt or other pharmaceutically acceptable salt.
  • diltiazem and flavonolignans such as silymarin protected the cells against oxidative stress compared to either drug alone.
  • Cell viability was statistically enhanced.
  • Diltiazem, flavonolignan and the combination drug treatment were all associated with enhanced ATP levels in both cell types with the combination drug treatment showing a much greater protective effect.
  • Bax and Bax mRNA levels were statistically reduced by drug treatment, again with the combination showing a much greater beneficial effect than either treatment alone.
  • the combination of diltiazem and flavonolignan provided an additional hepatoprotective effect than either drug alone.
  • the protective nature extends to reducing levels of Bax a pro-apoptotic protein.
  • Diltiazem is preferably administered at a daily dose which is less than half (more preferably less than one third) of the dose prescribed for treatment of cardiovascular disease.
  • the dose prescribed for treatment of cardiovascular disease is provided in medical texts relating to prescription drugs. We have referred, in particular, to the Australian Edition of MIMS Annual (2001 ) which lists the average optimum dose of diltiazem as 180 to 240 mg per day.
  • the preferred calcium channel blocker will be a membrane-stabilizing agent (or combination of agents) which has effects across several of the principal components of the membrane destructive process.
  • the preferred calcium channel blocker will act as an intracellular and intramembranous antioxidant, limit calcium entry into the mitochondria, inhibit phospholipase activity, and to facilitate or maintain energy production by the cells.
  • Mitochondria in all cells have a calcium channel which is calcium-gated.
  • the channel is activated as intracellular levels of calcium rise so that calcium is "shunted" into the mitochondria.
  • This channel is different from the voltage-gated channel of excitable tissues (muscle and arteries), but is inhibited by almost all calcium blockers (calcium antagonists).
  • Patients with liver disease frequently have low normal blood pressure, and there is a need to ensure that treatment does not lower blood pressure to levels that may contribute to fatigue and impairment of liver function.
  • Liver-selective drug delivery of a short-acting calcium blocker is one solution to the risk of hypotension and fatigue, but diltiazem as a benzothiazine, and in contrast to verapamil and the dihydropyridines, results in very little blood- pressure lowering effect in normotensive patients and is therefore preferred over other calcium antagonists.
  • Subcellular studies have shown that protective effect of diltiazem appears to occur at concentrations of diltiazem that are much higher than the those used in whole cell studies or than plasma concentrations of the drug observed after oral administration.
  • the lipophilic nature of the drug means that membranes absorb it achieving a concentration gradient of 100:1 or more between the membrane and plasma. This also explains why the mitochondrial calcium blocking effect can take 2 hours or more to develop after the drug is applied to cell cultures.
  • Diltiazem which is cis-(+)-3-(acetyloxy)-5-[2-(dimethylamino)ethyl]-2,3-dihydro- 2-(4-methoxyphenyl)-1 ,5-benzothiazepin-4(5H)-one, referred to herein as "diltiazem", is a benzothiazine derivative possessing calcium antagonist activity. Diltiazem has heretofore been used clinically to block the influx of calcium ions in smooth and cardiac muscle and thus exert potent cardiovascular effect.
  • Diltiazem has been shown to be useful in alleviating symptoms of chronic heart disease, particularly angina pectoris and myocardial ischemia and hypertension, while displaying a low incidence of side effects.
  • diltiazem is available as diltiazem hydrochloride in tablet form in strengths of 30, 60, 90 and 120 mg. and in capsule form in strengths of 60, 90, 120, 180, 240 and 300 mg.
  • Diltiazem is also available in injectable form at a concentration of 5 mg/ml.
  • Diltiazem therapy for treatment of cardiovascular disease typically starts with 30 mg. administered 4 times daily.
  • the dosage is gradually increased to 180 to 360 mg/day, given in divided doses three or four times daily, at one- to two- day intervals until an optimum response is obtained.
  • Diltiazem is extensively metabolized by the liver. According to professional use information issued by Marion Merrell Dow Inc., diltiazem in CARDIZEM.RTM. brand tablets is absorbed to about 80% and is subject to an extensive first-pass effect, giving an absolute bioavailability, compared to intravenous administration, of about 40%.
  • Single oral doses of 30 to 120 mg. of CARDIZEM.RTM. diltiazem tablets result in peak plasma levels two to three hours after administration. Detectable plasma levels occur within 30 to 60 minutes after administration indicating that CARDIZEM.RTM.
  • diltiazem tablets are readily absorbed. The plasma elimination half-life following single or multiple administration is approximately 3.5 hours. Therapeutic blood levels of CARDIZEM.RTM. diltiazem tablets appear to be in the range of 50 to 200 ng./ml. In contrast with prior art formulation of calcium channel blockers the first pass clearance of drugs such as diltiazem, which is viewed as an impediment to effective treatment of cardiovascular disease, becomes a virtue in treating liver disease as it allows the clinical effect of the drug to be confined to the liver.
  • the calcium channel blocker be present in a low dose and as a slow-release formulation to provide a clinical effective blood level in the portal vein and a dose less than required to provide a clinically effective level in the peripheral circulation.
  • the method and composition of the invention thus provide a delivery rate having a selective effect on the liver.
  • This method of hepatic protection using diltiazem in combination with flavonolignans and optionally also thiamine applies to any disease state of the liver in which the cell membranes have been damaged either primarily or secondarily by oxidising agents or oxidising processes.
  • the use of a liver- selective formulation of these membrane-stabilizing agents is complementary to the treatment of the primary disease.
  • the direct mitochondrial effect of diltiazem which enables preservation of liver function under disease conditions also has a clinically important effect on the metabolic clearance of silymarin.
  • Second, sustained metabolism of silymarin by the liver cells under disease conditions will help maintain the liver-selective delivery of silymarin because in addition to being administered in low dose, and in a slow-release formulation, a key feature of liver-selective drug delivery is that the drug must have a short half-life produced by first-pass clearance by the liver
  • liver-protective agents may be co-prescribed or co- formulated with therapeutic agents used in the primary management of the disease, such as ribavirin or other orally-administered antiviral agent in the management of hepatitis C.
  • the invention thus also provides a method of treatment or prevention of liver disease by co-administering or co-formulating together with other active agents such as are used to treat portal hypertension or viral hepatitis, the liver protective therapy detailed in this invention that includes a liver-selective formulation of a calcium channel blocker and antioxidant such as diltiazem with at least one flavonolignan and optionally also a further antioxidant agent such as thiamine which is relatively hydrophilic compared with the calcium channel blocker.
  • a liver-selective formulation of a calcium channel blocker and antioxidant such as diltiazem with at least one flavonolignan and optionally also a further antioxidant agent such as thiamine which is relatively hydrophilic compared with the calcium channel blocker.
  • the two components of the composition may be co-formulated or may be administered in a separate composition.
  • the agents may be administered by the same route (particularly oral administration) or may be administered by different routes.
  • one active agent may be administered intravenously or parenterally and the calcium channel blocker administered orally.
  • thiamine may, when used, be in the form of the salt from such a hydrochloride salt or other pharmaceutically acceptable derivative.
  • Patients suffering from toxic hepatitis have an impaired ability to absorb thiamine despite having low thiamine concentration in the blood that are often difficult to measure. The production of energy in the mitochondria is thus severely compromised in cells of a diseased liver.
  • At least one agent selected from agents used to treat portal hypertension or an antiviral agent is co- formulated with calcium channel blocker, flavonolignan and optionally thiamine.
  • the invention provides a method of treatment or prevention of toxic hepatitis (for example alcoholic hepatitis, but also including fatty liver) comprising co-administration of the liver-selective formulation of a calcium blocker and thiamine. It is particularly advantageous if thiamine, which is hydrophilic, is co-formulated with the calcium channel blocker component. Accordingly in the preferred embodiment we provide a composition for treatment of toxic hepatitis comprising a liver selective formulation of a calcium channel blocker such as diltiazem and a flavonolignan particularly silymarin, silibinin and pharmaceutically acceptable antioxidant derivatives thereof.
  • a calcium channel blocker such as diltiazem and a flavonolignan particularly silymarin, silibinin and pharmaceutically acceptable antioxidant derivatives thereof.
  • the preferred calcium channel blocker is diltiazem in an amount of from 20 to 70 mg/day and more preferably 25 to 50 mg/day.
  • the daily dosage of flavonolignan such as silibinin or silimarin is in the range of from 1 to 5000mg/day and preferably from 10 to 2000mg/day.
  • the ratio of the daily dosage of diltiazem:flavonolignan is in the range of from 50:1 to 1 :120 and more preferably from 2:1 to 1 :120.
  • thiamine When used it is preferably present in an amount to provide delivery of from 1 to 200 mg per day and more preferably 20 to 200 mg per day.
  • vitamin thiamine adds a complementary mitochondrial effect in the mitochondria where as a co-factor of pyruvate dehydrogenase, that facilitates energy production.
  • This therapy may be used to protect the liver and maintain hepatic function when the liver has been damaged by any form of chronic non- malignant disease in which the cell membranes have been damaged either by primary or secondary processes such that the liver cells swell sufficient to impede portal venous blood flow and cause hypoxia of the liver.
  • Diseases in which the treatment of the present invention may have application include all forms of viral hepatitis (including hepatitis B and C), alcoholic liver disease, cirrhosis of the liver, toxic hepatitis, autoimmune hepatitis, fatty liver, damage to the liver sustained during chemotherapy or radiotherapy, and ageing of the liver.
  • liver-selective drug delivery requires that a drug with a short half- life be administered as low dose and as a slow-release formulation. This is very different from an enteric coating and provides a release over a period of preferably at least 8 hours and up to 24 hours as the drug descends through the small intestine. After crossing the gastrointestinal wall, the drug reaches the relatively small volume of the portal venous system and is carried to the liver. Here a significant portion is removed from the circulation by metabolism with the remainder passing into the much larger volume systemic circulation. In this way, a stable concentration gradient is achieved where the concentration of the drug is up to 5 or more times higher in the liver and portal circulation than in the systemic circulation. The achieved concentration gradient may be higher in cirrhosis or other conditions with sluggish portal circulation, however, this effect may be offset by the development of significant collateral vessels between the portal and systemic circulations.
  • liver-selective delivery also reduces the total daily dose of a drug required to 20 - 25% of the full systemic dose that would normally be required if the drug was being administered to achieve a systemic effect.
  • diltiazem where the doses required for systemic effects used in the treatment of angina pectoris and hypertension are generally in the range of 120 - 360 mg/day, a dose of 50mg or less per day of diltiazem will retain a therapeutic effect in the liver but not the rest of the body.
  • a membrane-stabilizing agent with these properties and including diltiazem formulated for liver-selective delivery with or without thiamine can be used to protect the liver in:
  • Viral hepatitis including Hepatitis B, and C, and other forms.
  • NASH Non-alcoholic Steatohepatitis
  • liver-selective membrane-stabilizing agent with both antioxidant and mitochondrial effects is complementary to the primary treatment.
  • liver-selective membrane-stabilizing agent such as diltiazem or related molecules that can minimise or contain symptoms becomes an affordable alternative.
  • a liver-selective membrane-stabilizing agent with or without added thiamine can therefore be prescribed as monotherapy, be co-prescribed with interferon and ribavirin or with other appropriate antiviral therapy, or be co- formulated with ribavirin or other orally-administered component of the antiviral therapy so that both or all drugs are administered together as a liver-selective formulation.
  • thiamine (Vitamin B1 ) has been used in management of alcoholic liver disease because patients with this disease frequently have a nutritional deficiency of the vitamin.
  • Thiamine acts as a co-enzyme for several reactions that cleave carbon-carbon bonds including key metabolic processes within the hepatic mitochondria (Wilson, 1998).
  • treatment with thiamine may improve drug-induced mitochondrial damage in patients with hepatitis B and C (Kontorinis and Dieterich, 2003).
  • Other workers have shown that thiamine deficiency is common in patients with cirrhosis caused by both alcoholic liver disease and Hepatitis C, but not in Hepatitis C without cirrhosis (Levy and colleagues, 2002.).
  • thiamine has been shown to improve liver function in patients with Hepatitis B (Wallace and Weeks, 2001 ). These workers have suggested that the vitamin may have a protective effect on mitochondrial function in patients with viral hepatitis. Another key component of the protective affect of thiamine it its antioxidant properties, which appear to be active in the cytosol on account of its hydrophilic profile.
  • thiamine vitamin
  • Co-prescription or co-formulation of a liver- selective membrane-stabilizing agent and thiamine may be helpful in these patients.
  • the complementary energy-facilitating role of thiamine and its antioxidant effect together with the membrane stabilizing agent provide a significant advantage.
  • the immediate treatment is correction of the underlying causative liver disease, and then reduction of the vascular resistance to portal venous flow, or immediate reduction of portal venous flow with propranolol or other non-selective beta-adrenergic antagonist. Therefore the composition with both antioxidant and mitochondrial calcium inhibition is complementary to this primary treatment.
  • the composition may be co-prescribed with propranolol.
  • attack of the cell membranes of the liver cell by oxidising agents and free radicals from both within and without the cell is the principle pathological mechanism of the disease.
  • Radiotherapy also generates free radicals. While this may be a key mechanism targeted at organs with cancer when the liver is irradiated either necessarily or intentionally, the normal physiology with low pressure perfusion by portal venous blood makes the organ particularly at risk of hypoxia and deterioration of function. Therefore the coadministration of the calcium channel blocker and flavonolignan is complementary to drug or toxin withdrawal, management of the immune disease, treatment with systemic chemotherapy, or after radiotherapy. In the ageing liver, there is a reduction of the oxidising properties of the liver such that the liver is less able to protect itself from naturally occurring oxidising effects in the diet.
  • the dosage forms according to the present invention may be controlled-release dosage forms.
  • the mechanism of release of these dosage forms can be controlled by diffusion and or erosion.
  • the formulation comprises polymer-coated multiparticulates, polymer-coated tablets or minitablets, or hydrophilic matrix tablets.
  • a slow-release formulation of a membrane-stabilizing drug designed to act as a hepatoprotective agent may be designed to release the drug over a period of about 6 to about 24 hours following administration, thereby permitting once-a-day administration and providing a sustained exposure of the drug to the liver.
  • formulations releasing the drug over extended periods of time may have more than one timed-release component to affect time coverage.
  • the present invention relates to the finding that a protective effect of low-doses of membrane-stabilizing agents (including diltiazem at doses of less than 50 mg/day) administered as a slow-release formulation, is mediated by a direct protective effect on the liver itself rather than on the vasculature, that the drug acts as a lipophilic antioxidant to penetrate cells and protect both cell membranes and mitochondria from the damaging effects of phospholipid oxygenation, and that it acts as a mitochondrial calcium antagonist.
  • membrane-stabilizing agents including diltiazem at doses of less than 50 mg/day
  • the slow-release composition may be of a type previously reported for treatment of cardiovascular disease with the exception that the dose of diltiazem is reduced to within the range of from 20 to 70 mg per day. As discussed above the slow- release composition will preferably also contain thiamine in an amount of from 1 to 5 mg per day. The composition may be designed for once daily administration, twice daily administration or more often but once daily administration is particularly preferred.
  • U.S. Pat. Nos. 4,721 ,619, 4,891 ,230, 4,917,899 and 5,219,621 disclose diltiazem formulations that purport to require administration once every twelve hours (i.e., twice a day).
  • U.S. Pat. Nos. 4,894,240 and 5,002,776 disclose diltiazem formulations that purport to require administration once every 24 hours (i.e., once a day).
  • the formulations disclosed require a multi-layer membrane that coats the central core and an organic acid in the active core and/or in the multi-layer membrane.
  • Suitable organic acids disclosed in these patents are adipic acid, ascorbic acid, citric acid, fumaric acid, malic acid, succinic acid and tartaric acid.
  • CD diltiazem capsule is a sustained release diltiazem capsule containing 120, 180, 240 or 300 mg. diltiazem hydrochloride with a suggested dosage of one capsule a day.
  • CD diltiazem capsule include fumaric acid, an organic acid, and a multi-layer membrane that coats the central core. According to the aforementioned patents, the pellets must be dried for a number of hours during and after the coating process.
  • the slow-release composition comprises a core containing diltiazem and one or more polymeric coatings.
  • the core may and preferably will contain the thiamine component.
  • the core may be formed on a seed of, for example, an inert material such as a sugar sphere.
  • Pharmaceutically acceptable binders such as hydroxypropyl cellulose may be used in the core.
  • the one or more coatings may comprise a polymer which is permeable to diltiazem and water and a polymer which is relatively less permeable to diltiazem and water.
  • a diltiazem permeable polymer is the cationic polymer synthesized from acrylic and methacrylic acid ester with a low content of quaternary ammonium groups, known as EUDRAGIT RL (manufactured by Rohm Pharma GmbH) ethylcellulose, cellulose acetate, cellulose propionate (lower, medium or higher molecular weight), cellulose acetate propionate, cellulose acetate butyrate, cellulose acetate phthalate, cellulose triacetate, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octt
  • Substances that can be used that are less permeable to diltiazem and water include a cationic polymer known as EUDRAGIT RS (manufactured by Rohm Pharma GmbH. EUDRAGIT RS is less permeable than EUDRAGIT RL because EUDRAGIT RS has fewer ammonium groups) ethylcellulose, cellulose acetate, cellulose propionate (lower, medium or higher molecular weight), cellulose acetate propionate, cellulose acetate butyrate, cellulose acetate phthalate, cellulose triacetate, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(is
  • the coating layer includes a lubricant and a wetting agent.
  • a lubricant is talc and the wetting agent is sodium lauryl sulfate.
  • Suitable alternatives for sodium lauryl sulfate may include agents such as acacia, benzalkonium chloride, cetomacrogol emulsifying wax, cetostearyl alcohol, cetyl alcohol, cholesterol, diethanolamine, docusate sodium, sodium stearate, emulsifying wax, glyceryl monostearate, hydroxypropyl cellulose, lanolin alcohols, lecithin, mineral oil, monoethanolamine, poloxamer, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, propylene glycol alginate, sorbitan esters, stearyl alcohol and triethanolamine, or a mixture of any two or more of the foregoing.
  • agents such as acacia, benzalkonium chloride, cetomacrogol emulsifying wax, cetostearyl alcohol, cetyl alcohol, cholesterol, diethanolamine, do
  • talc Suitable alternatives for talc that may be included in the coating are calcium stearate, colloidal silicon dioxide, glycerine, magnesium stearate, mineral oil, polyethylene glycol, and zinc stearate, aluminium stearate or a mixture of any two or more of the foregoing.
  • a plasticizing agent is preferably included in the coating to improve the elasticity and the stability of the polymer film and to prevent changes in the polymer permeability over prolonged storage. Such changes could affect the drug release rate.
  • Suitable conventional plasticizing agents include acetylated monoglycerides, acetyltributylcitrate, acetyltriethyl citrate, castor oil, citric acid esters, dibutyl phthalate, dibutylsebacate, diethyloxalate, diethyl malate, diethylfumarate, diethylphthalate, diethylsuccinate, diethylmalonate, diethyltartarate, dimethylphthalate, glycerine, glycerol, glyceryl triacetate, glyceryltributyrate, mineral oil and lanolin alcohols, petrolatum and lanolin alcohols, phthalic acid esters, polyethylene glycols, propylene glycol, rape oil
  • the core or coating may comprise an organic acid such as adipic acid, ascorbic acid, citric acid, fumaric acid, malic acid, succinic acid, tartaric acid and fumaric acid.
  • an organic acid such as adipic acid, ascorbic acid, citric acid, fumaric acid, malic acid, succinic acid, tartaric acid and fumaric acid.
  • An alternative method of controlled release is to use membrane coating about a core comprising the active agents which membrane has at least one pore therein.
  • Such an arrangement is described by Chen et al in US 6,866,866 for providing slow-release of metformin for a once a day dose.
  • Chen et al also describe a slow-release formulation for diltiazem in US Patent 6,524,620 which may be utilised with the low dose composition of the invention for treatment of liver disease.
  • silymarin has a complex composition
  • other workers have shown that it is possible to formulate this herbal agent in erodable matrix based on systems based on glyceryl monostearate and polyethylene glycol 6000 or poloxamer 188 (Cheng et al 2007.) It follows that silymarin and other herbal antioxidants can be co-formulated with diltiazem using many of the standard techniques for preparation of slow-release formulation.
  • the claims of this invention relate to the concept of co-formulation a herbal antioxidant with low-dose diltiazem in a slow- release formulation rather than to the specific chemical nature of the formulation used. Comparative Example
  • Isolated rat liver mitochondria were incubated with low concentrations of Ca 2+ to partly uncouple oxidative phosphorylation, and oxygen consumption and acceptor control ratios measured.
  • concentration of diltiazem in the extra mitochondrial medium was measured using LC-mass spectrometry.
  • Rat hepatocytes isolated by collagenase digestion and cultured on coverslips and in multiwell plates were incubated with H 2 O 2 (to generate reactive oxygen species) and cell viability was assessed by measuring lactate dehydrogenase release using a Cobas Fara autoanalyser.
  • the measured concentrations of diltiazem were 142 ⁇ 14 (5), 275 ⁇ 10 (6) and 553 ⁇ 38 (3) ⁇ at 205, 410 and 790 ⁇ added diltiazem, respectively.
  • Incubation of isolated rat hepatocytes with 0.5 mM H 2 0 2 for 24 h induced 87% cell death.
  • diltiazem, but not low concentrations of silymarin substantially reduced H 2 0 2 -induced hepatocyte death.
  • diltiazem at low concentrations, is effective in protecting hepatocytes from damage induced by reactive oxygen species. This may, in part, be due to the actions of diltiazem in modulating Ca 2+ movement and preserving the capacity of mitochondria to synthesise ATP. Under the conditions employed, diltiazem was considerably more effective than low concentrations of silymarin. These results suggest that diltiazem could play a valuable role in protecting hepatocytes from damage induced by reactive oxygen species and other agents but that Silymarin has a relatively small effect.
  • This Example compares the response of hepatocytes under oxidative stress to the actions of diltiazem, silymarin and a combination of diltiazem and silymarin.
  • DCFH 2 -DA dichlorofluorescein diacetate
  • DMEM Dulbecco's modified Eagle's medium
  • MTT cell proliferation
  • ATPase kits were purchased from Sigma (St. Louis, MO).
  • Human hepatoma cell lines Chang and PLC/PRF/5 cells were purchased from American Type Culture Collection (ATCC, Rockville, MD).
  • Chang and PLC/PRF/5 hepatoma cells were maintained in 25 cm 2 culture flasks supplemented with DMEM containing 10% FBS (DF-10), penicillin and streptomycin (50 units/ml) at 37 C in a humidified atmosphere of 95% air and 5% CO2 for at least 24 hours.
  • Cells were plated onto culture plates (100 mm 2 ) and exposed to DF-10 media and drug treatment for 24 hours for Western blot and RT-PCR studies.
  • For DCF and MTT assays cells were subcultured onto 96-well black plates (Corning, NY, USA) and allowed to attach for 8 hours prior to any drug treatment. Following plate confluence, cells were exposed to DF-10 media containing drugs for 24 hours. Control cells were incubated without any drug.
  • the active intracellular 2',7'-dichlorodihydrofluorescein (DCFH) form of DCFH 2 -DA was prepared as described by Tollefson et al. 14 . Briefly, the diacetate moiety of DCFH 2 -DA was cleaved by incubating 100 ⁇ _ stock DCFH 2 -DA in the presence of 200 ⁇ _ methanol and 100 ⁇ _ 2N NaOH at room temperature in the dark. After 2 hours the resulting DCFH solution was diluted with Ca/Mg-PBS to achieve a final probe concentration of 20 ⁇ with the pH adjusted to 7.4 immediately before use. Oxidative stress induced by H 2 0 2
  • Chang and PLC cells were cultured in a 96- well culture plates (Costar# 3603, Corning Incorporated) at a cell density of 25,000 cells per well. After overnight incubation in DMEM-10% FBS, cultures were washed twice with PBS and then incubated with 10 ⁇ H 2 DCFDA in 95% air/5% C0 2 for 30 min at 37°C. Extracellular H 2 DCFDA was removed by washing the culture twice with warm PBS. Cellular oxidative stress was induced by incubating cells with 400 ⁇ H 2 0 2 in PBS containing Ca ++ and Mg ++ for 20 min at 37°C in the dark. Negative controls were performed using the same conditions but without H 2 0 2 .
  • the MTT assay was used to assess cell growth and viability. Living cells reduce MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) to purple formazan by functional mitochondria. Assay conditions were as outlined by the manufacturer (Sigma, St. Louis, MO). Briefly, Chang and PLC cells were cultured in 96-well plates at a concentration of 3x10 4 cells/well. Following exposure to diltiazem and/or silymarin for 24 hours and H 2 O 2 (400 ⁇ , 20 minute) treatment, 10 ⁇ of the MTT solution (10% of the culture volume) was added to each well.
  • a bioluminescent assay was employed for the determination of total cellular ATP levels. Procedural details were followed as outlined by the manufacturer (Sigma, St. Louis, MO).
  • 4X 250 mmol/L Tris- HCI, pH 6.8, 8 % SDS, 20 % glycerol, 0.2 % bromophenol blue and 5 % ⁇ - mercaptoethanol
  • Nonspecific antibody binding was blocked by pre-incubation of the membranes in dry skim milk in 1X Tris-buffered-saline-Tween (TBS-T, 2.42 g Tris base, 29.25 g NaCI, 0.5 ml Tween 20 per liter) for 1 hour at room temperature.
  • TBS-T Tris-buffered-saline-Tween
  • 2X TBS-T Tris base, 29.25 g NaCI, 0.5 ml Tween 20 per liter
  • Membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibody (goat anti- rabbit antibody for and Bax and anti-mouse antibody for ⁇ -Actin (1 :1000 dilution).
  • the antigen-antibody complexes were detected by enhanced chemiluminescence (ECL system, Amersham).
  • ECL system enhanced chemiluminescence
  • the optical density (OD) values of each target protein band were determined by using NIH Imaging software. Protein loading variation was corrected by normalization of ⁇ -Actin.
  • TRIzol LS reagent was employed to extract total RNA of the Chang cells as described in the manufacturer's manual.
  • the first-strand cDNA was synthesized by an Advantage RT-for-PCR kit. Briefly, 1 g of total RNA was dissolved in diethylpyrocarbonate-treated doubled-distilled water (ddH20) to achieve a final volume of 12.5 ⁇ . Subsequently, 1 ⁇ oligo(dT) primer, 4 ⁇ of 5 ⁇ reaction buffer, 1 ⁇ of 10 mmol/l dNTP mix, 0.5 ⁇ recombinant RNase inhibitor, and 1 ⁇ Moloney murine leukemia virus reverse transcriptase were added and incubated for 1 h at 42°C.
  • PCR was performed by using the Advantage PCR kit, polymerase mix, and oligonucleotides synthesized by GIBCO-BRL (Burlington, ON).
  • Gene-specific PCR primers for Bax were purchased from Sigma (St. Louis, MO).
  • the oligonuleotide primers were 5'-GTT TCA TCC AGG ATC GAG CAG-3' (sense), 5'-CAT CTT CTT CCA GAT GGT GA-3' (antisense).
  • Product length was 487bp.
  • the specific rat GAPDH amplimers were from Clontech (5507-3) with an expected GAPDH size of 986 base pairs.
  • PCR amplification was carried out in 30 cycles of denaturation at 94 ° C for 45 seconds, annealing at 53 ° C (Bax) and 60°C (GAPDH) for 45 seconds, elongation at 72 ° C for 120 seconds with an additional 7-minute final extension at 72 ° C using an Eppendorf MasterCycler (Eppendorf, Westbury, NY).
  • the PCR product was analyzed using a 1 .2% agarose gel. Identity of PCR products was confirmed by sequencing at the DNA- sequencing facility of the Manitoba Institute of Cell Biology (Winnipeg, Canada). Statistical Analyses
  • Data are presented as mean ⁇ SEM. The n value refers to the number of replicates performed for each study. Data were analyzed using one-way ANOVA with Student-Newman-Keuls post hoc test taking p ⁇ 0.05 as the level for significance (statistical significance set at p ⁇ 0.05).
  • Figure 2 Cell growth as assessed by the MTT assay for PLC ( Figure 2a) Chang ( Figure 2b) cells following diltiazem and/or silymarin. Cells were cultured in 96- well plates.
  • Figure 7. RT-PCR image of Bax and loading control GAPDH in control and drug treated Chang cells.
  • FIG. 1 shows DCF fluorescence from H 2 0 2 induced oxidative stress treated with diltiazem (2.5 and 10 ⁇ ), silymarin (10 and 1000 ⁇ g L) or the combination diltiazem (2.5 ⁇ or 10 ⁇ ) and silymarin (1000 ⁇ g L) for PLC cells (top) or diltiazem (2.5 ⁇ ) and silymarin (1000 ⁇ g L) or diltiazem (10 ⁇ ) and silymarin (10 ⁇ g L) for Chang cells (bottom).
  • DCF dichlorofluorescein
  • silymarin to diltizem further enhanced the ATP levels than did diltiazem alone (p ⁇ 0.001 ) or silymarin alone (p ⁇ 0.01 ). There was no statistical enhancement on ATP levels when the diltiazem dose was increased (2.5 ⁇ to 10 ⁇ ) and silymarin (1000 M g/L).
  • FIG. 7 shows a representative scan of RT-PCR Bax mRNA. It is apparent that diltiazem suppresses Bax mRNA significantly more than the use of silymarin alone.
  • Intracellular free radical levels were assessed using DCF fluorescence following exposure of cells to an oxidative stress of 400 ⁇ H2O2 for 20 min. Diltiazem significantly reduced the DCF fluorescence signal following 5 and 10 ⁇ as did Silymarin at 1000 pg/L. The combination of diltiazem and silymarin further protected the cells against oxidative stress (p ⁇ 0.001 ) compared to either drug alone. Cell viability was assessed using the MTT assay which showed that diltiazem and silymarin statistically enhanced cell growth (p ⁇ 0.01 ) with the combination also providing a further protective effect. The nature of the protective effect was assessed by investigating levels of ATP, Bax and Bax mRNA.
  • Diltiazem, silymarin and the combination drug treatment were all associated with enhanced ATP levels in both cell types with the combination drug treatment showing a much greater protective effect (p ⁇ 0.001 ).
  • Bax and Bax mRNA levels were statistically reduced by drug treatment, again with the combination showing a much greater beneficial effect than either treatment alone (p ⁇ 0.001 ).
  • low dose diltiazem and silymarin (1 pg/ml) provides a hepatoprotective effect against free radical damage due to oxidative stress.
  • the combination of dilitiazem and silymarin provided an additional hepatoprotective effect compared with either drug alone.
  • the protective nature extends to reducing levels of Bax a pro-apoptotic protein.
  • rat liver cell line the H4-IIE cell line.
  • This is a well established model liver cell.
  • the cell line exhibits all major characteristics of normal differentiated rat hepatocytes.
  • the cell line has been employed in Medical Biochemistry research on liver function for the last 20 years and its properties compared favourably with those of normal freshly-isolated rat hepatocytes.
  • Oxidative damage is induced using an established method which employs hydrogen peroxide. Liver cells in culture in plastic multiwall plates are incubated with 0.5 mM hydrogen peroxide for 1 hour, then for 12 hours in normal medium under standard incubation conditions. This regime induces oxidative damage to target cells. Cell damage is assessed by measuring exclusion of the dye Trypan blue and release of the cytoplasmic enzyme, lactate dehydrogenase.
  • the cells are incubated with a 5 ⁇ concentration of silymarin for 4 - 10 hours under normal cell culture incubation conditions.
  • the experiments are conducted with normal untreated liver cells and cells previously treated with hydrogen peroxide to induce oxidative damage.
  • the metabolism of silymarin through the liver conjugation and oxidative drug metabolising enzyme pathways is measured using liquid chromatography in order to separate the different metabolites and mass spectrometry in order to identify and quantitate the metabolites.
  • This effect may be allied to the protection and enhancement of the mitochondrial function enabling preservation of energy production. It may be that this effect of diltiazem on the mitochondria enable liver cells to more effectively metabolise silymarin than would otherwise be the case when they are exposed to oxygen free radicals, or are mildly damaged.

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Abstract

L'invention concerne un procédé de traitement d'un sujet atteint d'une maladie de foie, lequel procédé comprend l'administration (i) d'une préparation orale à libération lente d'un inhibiteur calcique comportant des effets antioxydants et (ii) d'au moins une flavonolignane.
PCT/AU2010/001372 2009-10-15 2010-10-15 Procédé de traitement de maladie de foie WO2011044638A1 (fr)

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Cited By (3)

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CN102942551A (zh) * 2012-12-04 2013-02-27 贵阳医学院 含有3-硝基丙酰基的黄烷醇和含其的药物组合物及其在制药中的应用
US10702568B2 (en) 2015-03-19 2020-07-07 Cydex Pharmaceuticals, Inc. Compositions containing silymarin and sulfoalkyl ether cyclodextrin and methods of using the same
US11786539B2 (en) 2015-09-02 2023-10-17 Eloxx Pharmaceuticals Ltd. Aminoglycoside derivatives and uses thereof in treating genetic disorders

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WO2005102353A1 (fr) * 2004-04-22 2005-11-03 Howard J Smith & Associates Pty Ltd Traitement de soutien d'une maladie du foie

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US20050203612A1 (en) * 2000-12-22 2005-09-15 Avantec Vascular Corporation Devices delivering therapeutic agents and methods regarding the same
CN100584333C (zh) * 2004-04-22 2010-01-27 霍华德·J·史密斯及同仁控股有限公司 治疗肝病的药物组合物

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WO2005102353A1 (fr) * 2004-04-22 2005-11-03 Howard J Smith & Associates Pty Ltd Traitement de soutien d'une maladie du foie

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102942551A (zh) * 2012-12-04 2013-02-27 贵阳医学院 含有3-硝基丙酰基的黄烷醇和含其的药物组合物及其在制药中的应用
US10702568B2 (en) 2015-03-19 2020-07-07 Cydex Pharmaceuticals, Inc. Compositions containing silymarin and sulfoalkyl ether cyclodextrin and methods of using the same
US11382944B2 (en) 2015-03-19 2022-07-12 Cydex Pharmaceuticals, Inc. Compositions containing silymarin and sulfoalkyl ether cyclodextrin and methods of using the same
US11786539B2 (en) 2015-09-02 2023-10-17 Eloxx Pharmaceuticals Ltd. Aminoglycoside derivatives and uses thereof in treating genetic disorders

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