WO1999038507A1

WO1999038507A1 - Reversal and prevention of clinically significant aspects of aging through oral use of hepatic artery vasodilator and other agents which increase hepatic oxygenation

Info

Publication number: WO1999038507A1
Application number: PCT/AU1999/000065
Authority: WO
Inventors: David George Le Couteur; Allan Joseph Mclean
Original assignee: David George Le Couteur; Allan Joseph Mclean
Priority date: 1998-01-30
Filing date: 1999-02-01
Publication date: 1999-08-05

Abstract

This invention relates to methods and compositions for preventing senescence induced-hypoxia in the liver. The use of vasodilators to increase blood flow and hence oxygen supply to the liver is contemplated. Anti-oxidants administered either alone or co-administered with vasodilators, with or without food, are also contemplated for maintaining normal levels of oxygen supply to the liver to prevent senescence of the organ.

Description

1

REVERSAL AND PREVENTION OF CLINICALLY SIGNIFICANT ASPECTS OF

AGING THROUGH ORAL USE OF HEPATIC ARTERY VASODILATOR AND

OTHER AGENTS WHICH INCREASE HEPATIC OXYGENATION

The present invention relates to methods for the prevention or treatment of liver senescence and age-related diseases.

The liver is a large gland situated in the upper part of the abdomen on the right side. It's domed upper surface fits closely against the inferior surface of the right diaphragm. It has a double blood supply consisting of one component from the hepatic artery (oxygenated arterial blood) and another from the portal vein (deoxygenated venous blood) carrying substances absorbed from the stomach, small intestine and large intestine as well as hormones and autocoids derived from spleen, pancreas and gut. It comprises thousands of minute lobules (lobuli hepatis), the functional units of the liver. Its manifold functions include the storage and filtration of blood, the secretion of bile, the excretion of bilirubin and other substances formed elsewhere in the body, and numerous metabolic functions. One important example is blood sugar regulation, achieved through the synthesis of sugar , and dynamic conversion and retrieval within glycogen stores, as well as the regulation of insulin action locally on the liver and the regulation of it's release into the general circulation after secretion from the pancreas. The liver also has important synthetic activities where the products are used in other specialised cells. One useful example is cholesterol and sterol synthesis. Due to these supportive roles for whole body metabolism and maintenance, the liver is essential to life.

Oxygen is also essential for life. The major biological role of oxygen is the production of energy in a form that is available for cellular processes, primarily the aerobic metabolism of carbohydrates and fatty acids to adenosine triphosphate. The central chemical equation for human and mammalian biology is cellular respiration:

Cn.H2n.On + nθ2 — > nCOi + H.H2O + energy .

In aged animals and humans the capacity to utilise oxygen falls markedly regardless of the presence of good health, indicating a general deficit in oxygen utilisation.

Thus, it is not surprising that the role of oxygen in energy generation has been implicated in the aging process by at least one influential theory of aging, the mitochondrial theory of aging. This theory states that the primary deficit of aging is impaired mitochondrial use of oxygen (mitochondrial respiration). It is believed that this functional deficit is initiated by oxygen-derived free radical damage, leading to reduced energy availability. A corollary of the mitochondrial theory of aging is that cellular levels of oxygen are normal, or even increased secondary to reduced oxygen consumption by mitochondria.

In steps leading up to the present invention, the inventors deduced that ageing is related to an intracellular deficit of oxygen (hypoxia), initially and principally in the liver. It was recognised that there is a consistent reduction in categories of liver metabolism which are directly oxygen-dependent, whilst oxygen-independent metabolism was preserved. On the basis of this pivotal observation and other evidence, it was concluded that aging begins in the liver, associated with the development of an oxygen diffusion deficit, most likely due to a diffusion barrier at the hepatocyte surface membrane in contact with the sinusoid. Restricted delivery of oxygen to oxygen-dependent systems develops, resulting in reduced function generally in oxygen-sensitive enzyme systems as exemplified by mitochondrial respiration, oxidative detoxification in the endoplasmic reticulum within the cytoplasm, changes in carbohydrate regulation through changes in glucose oxidase function, and changes in purine and pyrimidine metabolism by way of alteration in xanthine oxidase function. That is, the decline in oxygen use probably results from decreased access to sites of oxygen utilisation, rather than from an incapacity to use oxygen.

There are no therapies available to improve age-related changes in liver function. Prior art treatments of liver diseases have not been proposed or used for preventing or treating changes resulting from old age. For example, choline has been administered as an adjunct to the dietary treatment of fatty acid infiltration and early cirrhosis of the liver. Methionine has a lipotropic action similar to choline. It has also been used as an adjunct in the treatment of liver diseases in patients unable to take an adequate diet, though there is evidence that in cases of severe liver damage, large doses of methionine may aggravate the toxaemia. Litrison is a composition of methionine, choline, vitamins of the B complex and Vitamin E. It has been used for the treatment of hepatic parenchymal degenerative changes and to maintain the function of the liver. Neurogem is a composition of high potency essential Vitamin B-complex and Vitamin C which has been used for supplementary or maintenance therapy. Finally, Ripason is a protein-free total extract from livers of healthy animals. It has been used to treat chronic hepatitis, cirrhosis, medicarnentous liver damage and liver parenchyma disorders.

None of these agents are designed to reverse the relative hypoxia, or lack of oxygen, which appears to contribute to age-related changes in liver function. Accordingly, one object of the present invention is to overcome, or at least alleviate, one of more of the difficulties or deficiencies related to the prior art.

In a first aspect of the present invention, there is provided a method for the treatment of hypoxia in the senescent liver.

In a preferred embodiment, the invention relates to a method for the treatment of senescence induced-hypoxia of the liver, which method includes administering orally to a subject in need thereof a vasodilating agent at a dose less than the oral dose required to lower peripheral blood pressure thereby to selectively increase hepatic arterial inflow and alleviate the hypoxia.

The hepatic artery supplies highly oxygenated blood to the liver. If the flow of such blood in an intact liver, or the flow of a physiological solution with red cells in an isolated liver is increased, the oxygen uptake is increased. Pharmacological agents that act to relax or vasodilate the hepatic artery can be used to improve the supply of oxygenated blood to the liver. In dogs, this increase in hepatic artery flow applies from the lowest doses until the vasodilator agents begin to penetrate into the general circulation and the arterial blood pressure in the general circulation begins to fall, at which point the blood flow in the hepatic artery falls. Thus, the doses of arterial vasodilator agents to be used to increase local flow in the hepatic artery after oral administration must be at a dose significantly less than the antihypertensive dose or any dose, producing other actions on the heart or general circulation. At doses generally used in treatment of cardiovascular conditions, the vasodilators on reduces blood pressure in the peripheral circulation, and the ability of such agents to selectively increasing hepatic arterial blood flow is therefore lost.

Oral administration of some agents in accordance with the present invention are possible for increasing hepatic arterial flow selectively, even when such agents have either never been administered by this route, or their use by this route has been abandoned. Glyceryl-trinitrate exemplifies this class of compound, as its use by the oral route has been abandoned due to difficulty in showing any systemic effect of the drug despite high oral dose administration.

The intravenous, intramuscular, sublingual, subcutaneous or transdermal dosing with this vasodilator produce effects on the general circulation, including reduction of blood pressure. Other agents given by these routes cannot produce specific and local increases in hepatic artery flow required due to the fact that blood pressure is reduced, even at very low doses.

In one embodiment of the present invention, a low dose of glyceryl trinitrate e.g., approximately 0.5 mg to 10mg per dose, more preferably approximately 0.5 mg to 2.5 mg/dose is administered. Lower doses will be used for the oldest ages (0.5mg- 2.5mg/day) while higher doses (2.5-30 mg/day) will be used in those individuals who are younger up to and including those in the age range 40-50 years old.

Accordingly, in a second aspect, the invention provides a method for the treatment of senescence induced-hypoxia of the liver, which method includes administering orally to a subject in need thereof 0.5-30 mg/day of glyceryl trinitrate thereby to selectively increase hepatic arterial inflow and alleviate the hypoxia without lowering peripheral blood pressure.

In a third aspect, the invention provides a composition suitable for the treatment of senescence induced-hypoxia of the liver, which composition includes an oral daily dosage of a vasodilating agent that is less than the oral daily dosage required to lower peripheral blood pressure and that increases the hepatic arterial inflow and alleviates the hypoxia, and a pharmaceutically acceptable carrier or diluent therefor.

Other vasodilating agents may be used. These may include any of a variety of agents known to dilate the hepatic artery. Preferred vasodilators include: agents acting on the nerve supply of the artery, agents acting to deplete transmitter stores or to prevent release of nerve transmitter, agents acting to block receptors for neurotransmitter on nerve or arterial smooth muscle, agents acting to block receptor maction of circulating agents causing arterial contraction, and agents acting to prevent arterial contraction or promoting arterial relaxation. The vasodilators used in accordance with the method of the invention may produce vasodilation at these sites of action.

One class of vasodilators that are especially preferred are the adrenergic neurone blockers which interfere with transmission in the nerve. Several nerve types may be acted upon to produce vasodilation, depending on the pharmacological category of the agent. The vasodilators in this class include debrisoquine which is available under the trade name DECLINAX.

Further classes of vasodilators act on pharmacological receptors on the nerve membranes. These include presynaptic receptor blockers and vasodilators which reduce the amount of chemical messenger in the synaptic vesicles which provide the point of contact with the smooth muscle. An example of the former type is clonidine (CATAPRES) and examples of the latter type include guanethidine (ISMELIN) and reserpine available under the trade name SERPASIL.

One specific class of vasodilators act on catecholamine transmitters and receptor sites including alpha receptor blockers such as prazosin (MINIPRESS, MIPRAZ, PRAZIG,PRESSIN), doxazocin (CARDURAN), labetalol (TRANDATE), phenoxybenzamine (DIBENYLINE,) phentolamine (REGITINE), betahistine (SERC), ergotamine (CAFERGOT) and sumatripton available under the trade name IMMIGPAN. There are several other receptor types present on the smooth muscle cell which mediate contractions. Vasodilation results when actuation of these receptors is interfered with. Angiotensin II receptors mediate such contractions, and agents which block these processes indirectly or directly are vasodilators. ACE inhibitors and Angiotensin II receptor antagonists are two categories which are known and have commercially marketed representatives. Angiotensin II receptor antagonists include ibesartan (KARVEA, AVAPRO). The ACE inhibitors include quinapril (ACCUPRIL, ASIG) captopril (ACENORM, CAPACE, DBL CAPTOPRIL, ENZACF, SBA CAPTOPRIL WL CAPTOPRIL), enalapril (AMPRACE, RENITEC), perindopril (COVERSYL), trandolapril (GOPTEN, ODRIK), cilazapril (INHIBACE) fosinopril (MONOPRIL), lisinopril (PRINIVIL, ZESTRIL) and ramipril (RAMACE, TRITACE).

There are further major classes of vasodilators which act directly in the smooth muscle membrane by way of a variety of non-receptor mechanisms exemplified in the example provided by agents acting via calcium channel blockade. They include hydrallazine (ALPHAPRESS), verapamil (ANPEC), diltiazem (CARDIZEM), felodipine (FELDOURER), minoxidil (LONITEN), amlodipine (NORVASC), nicorandil (IKOREL), dipyridamole (PERSANTIN), multiple actives (PROFLO), aiprostadil (PROSTIN VR), hydroxyethyl rutosides & tartrazine (VAREMOID), and nimodipine (NIMOTOP).

There are other membrane-based mechanisms whereby the production of a vasodilator is activated, or the breakdown of such a class of vasodilator is inhibited. Nitrigic mechanisms exemplify this category of mechanism . Glyceryl trinitrate (ANGININE, IMDUR) and isosorbide mononitrate (DURIDE) activate this system, while sildenafil (VIAGRA) and oxpentifyiline (TRENTAL) inhibit the phosphodiesterase enzyme system which terminates the action of the mediator of vasodilatation. Another example is adenosine (ADENOSCAN).

There are other nerve processes other than adrenergic which modify contraction. Known examples of these are the purinergic and neuropeptide Y transmitter and receptor systems. Vasodilators which act on these nerve processes may be used in accordance with the invention. Thus, there is a range of receptor types which may be targeted to provide the vasodilator effect. These include alpha-1- adrenergic (including 1A, 1 B and IC), alpha-2 -adrenergic (including (2A, 2B and 2C), Neuropeptide Y (including Y1and Y2) and purinergic (including P2x1 , P2x2, P2x3, P2x4, P2x5, P2x6, P2x7 blockers).

The following example illustrates the compositions which can be used in accordance with the invention. A preferred composition is that originally used in the NITRONG sustained release tablet [previously produced by Wharton Laboratories, Division of US Ethicals Inc, Long Is NY ref Winsor & Berger 1975, American Heart Journal 90:611-626].

In general, the exact dosage used will depend upon the type of the drug used, and the dosage at which systemic vasodilation occurs.

Examples of vasodilators and the daily dosages that can be used in accordance with the invention are provided below.

Vasodilator Daily Dose

Debrisoquine 5 to 20 mg clonidine 10 to 50 mg doxazosin 0.5 to 10 mg prazosin 0.3 to 1 mg 8

labetalol 10 to 400 mg irbesartan 2.5 to 50 mg nifedipine 0.5 to 10 mg hydrallazine 2.5 to 35 mg verapamil 2.5 to 30 mg perindopril 0.2 to 2 mg cilazapril 0.5 to 2 mg trandolapril 0.05 to 0.5 mg

1 isinopril 0.5 to 8 mg irbesartan 2.5 to 60 mg amiodipine 0.05 to 2.5 mg quinapril hydrochloride 0.2 to 20 mg captopril 0.2 to 20 mg enalapril maleate 0.05 to 5 mg fosinopril 0.05 to 15 mg ramipril 0.02 to 2 mg sildenafil 0.02 to 25 mg

The composition suitable for the treatment and prevention of age-related changes in the liver and like indications may be in the form of a unit daily dosage comprising an amount of vasodilating agent less than required to produce a significant effect on the heart or peripheral circulation. The oral dosage per unit of a preferred vasodilator such as glyceryl trinitrate is approximately 0.25mg-3.5mg , preferably 0.5 mg to 2.5 mg in a pharmaceutically acceptable diluent or carrier therefor.

The pharmaceutically acceptable diluent or carrier may be of any suitable type. The pharmaceutically acceptable diluent or carrier may be a pharmaceutical organic or inorganic carrier material suitable for enteral or oral administration. The composition is formulated so as to allow suitable oral administration to the patient. The oral route is used as the active ingredient is able to reach the liver directly, that is through the portal vein.

Oral administration by the use of tablets, capsules, powders or in liquid form such as suspensions, solutions, emulsions or syrups is particularly advantageous. When formed into tablets, conventional excipients (e.g. sodium citrate, lactose, microcrystalline cellulose, starch, etc.), lubricating agents (e.g. anhydrous silicic acid, hydroyzed castor oil, magnesium stearate, sodium lauryl sulfate, tale, etc-) and binding agents (e.g. starch paste glucose, lactose, gum acacia, gelatin, mannitol, magnesium trisilicate, talc, etc.) can be used.

When administered as liquids, conventional liquid carriers can be employed. In the case of solid preparations, each unit dosage form of the active ingredient can contain from about 5 to about 95% of the same by weight of the entire composition with the remainder comprising conventional pharmaceutical carriers. When the therapeutic agent is used as aqueous solution, i.e., injection, the solution may contain about 0.05 to about 0.5% of the same by weight of the entire solution. Preferably the composition may be of the sustained release type, for example to allow for a once-daily administration. A suitable slow release formulation may be achieved for example when the active ingredient is bound to a suitable polymer. A once daily composition is able to supply sufficient quantity of active ingredient to the patient .

The senescence-related changes in liver function can also be treated by provision of inhaled oxygen. However this method has limitations due to the inherent toxicity of continued exposure to high doses of oxygen. Exposure to oxygen supplementation does increase the concentrations of oxygen in the hepatic artery without change in the oxygen concentration in portal vein blood. Thus, this approach can be used as an adjunct to the method of increasing local oxygen 10

supply to the liver and hepatic cells by increasing flow specifically and selectively in the hepatic artery. Supplementation of inhaled oxygen for periods of time which do not result in oxygen toxicity to the subject under therapy is preferred.

The present inventors have been able to reproduce the functional state described in the aged liver by exposure of young healthy livers to H₂O₂ via the portal vein under conditions of experimental liver perfusion. The results show that propranolol metabolism (cytochrome P450 dependent) is altered in parallel with a reduction in O₂ uptake by the livers, while the metabolism of morphine (through glucuronidation) is unaltered.

Thus, in a fourth aspect, the invention relates to prophylactic measures aimed at preventing development of a diffusional block or a diffusional barrier at the hepatocyte surface membrane.

In a preferred embodiment, there is provided a method for the prevention of oxidation of hepatic cell membranes, which method includes the steps of administering to a subject in need thereof a prophylactically effective amount of an anti-oxidant, said anti-oxidant being substantially absorbed by the portal vascular- system.

Preferably, the anti-oxidant is provided in the form of a composition that can be taken by itself, with solid or liquid food or drinks, or as a food-additive.

Thus, in a fifth aspect, the invention relates to a composition suitable for the prevention of oxidisation of hepatic cell membranes, which composition includes a prophylactically effective amount of an anti-oxidant, said anti-oxidant being substantially absorbed by the portal vascular system, and a pharmaceutically acceptable diluent, or carrier therefor, in a form suitable for oral administration.

The anti-oxidant is preferably water-soluble, most preferably includes but is not limited to vitamin C, N-acetylcysteine, cysteine glutathione or urate. 11

Preferably, the antioxidant is delivered at maximum concentration at the time of exposure to environmentally sourced xenobiotics with oxidative potential (i.e. food intake together with contaminants, additives and reactive "normal constituents", or food constituent exposure, food additive exposure, food processing byproduct exposure and food-induced mammalian metabolic byproduct exposure). Mammalian oxidative metabolism also generates oxidative free radicals and other prooxidative products, reinforcing the need for postprandial antioxidant. The metabolism of resident microbial flora of the bowel also generate oxidatively active products which would require sustained presence of antioxidant, indicating the need for a strategy based on antioxidant coinciding with feeding and the postprandial period, as well as a sustained release formulation to sustain a level of antioxidant during the fasting interval. Therefore, preferred embodiments of the invention include the administration of the anti-oxidant at or around the times of exposure to: food-induced bacterial metabolic byproducts, and maximum bacterial metabolic byproducts.

Following ingestion of food, the supply of arterial blood to a normal liver declines markedly. This reduction could contribute to oxygen deprivation of the liver and affect its function. When this co-exists with diffusional blocks at the hepatic cell membranes described above, the liver could be very susceptible to accelerated "ageing".

Thus, in a sixth aspect, the invention relates to a method for the prevention of oxidation of hepatic cell membranes and resultant lack of oxygen, which method includes the step of maintaining blood flow in the hepatic artery to maintain delivery of oxygen thereby to prevent reduction in oxygen supply to the liver.

In a preferred embodiment, the blood flow in the hepatic artery is maintained by administering to a subject a hepatic artery vasodilator together with a meal thereby to delivery 10 mg of glyceryl trinitrate in rapid release form in the first hour, followed by 250 μg per hour from a slow release form over 5 hours. 12

Other preferred embodiments of the invention include:

a method for the prevention of oxidation of hepatic cell membranes including the step of introducing water soluble anti-oxidant agents into the portal vein and maintaining active anti-oxidant concentrations to block oxidant activity of food constituents, food additives including preservatives and colourants, food processing byproduct, food contaminants, byproducts of food storage, increase in liver blood flow from food, metabolic by-products of mammalian cell metabolism and metabolic by-products of resident bacterial flora in the gut,

a method for the prevention of oxidation of hepatic cell membranes including the step of administering to a subject a formulation of an anti-oxidant delivered into the portal vein at a dose of 100mg of vitamin C per hour for 5 hours,

a method for the prevention of oxidation of hepatic cell membranes including the step of administering to a subject a formulation of an anti-oxidant agent during increased hepatic arterial blood flow, and wherein the anti-oxidant agent is delivered into the portal vein at a dose of 100mg of vitamin C per hour for

8 hours,

a method for the prevention of oxidation of hepatic cell membranes as a result of bacterial metabolism, including the step of administering a slow release formulation which yields a water soluble product and delivers 600mg of vitamin C to the portal vein over a 12 hour period at a constant rate of 50 mg per hour,

a method for reversing metabolic effects of oxidation of hepatic cell membranes and resultant lack of oxygen by maintaining the blood flow in the hepatic artery with maintained delivery of oxygen,

a method for reversing the metabolic effects of oxidation of hepatic cell membranes and resultant oxygen by maintaining blood flow in the hepatic artery, including the step of administering to a subject a formulation of hepatic artery 13

vasodilator with a meal thereby to deliver 1.0 mg of glyceryl trinitrate in rapid release form in the first hour, followed by 250 microgram per hour from a slow release form over 5 hours,

a method for reversing metabolic effects of oxidation of hepatic cell membranes and resultant lack of oxygen, including the step of increasing the blood flow in the hepatic artery by administering to a subject a formulation of hepatic artery vasodilator at the time of retiring to bed thereby to deliver 500 microgram glyceryl trinitrate released per hour from a slow release form over an 8 hour period,

- a method for reversing metabolic effects of oxidation of hepatic cell membranes and resultant lack of oxygen by maintaining blood flow in the hepatic artery, including the step of administering to a subject a formulation of hepatic artery vasodilator together with a meal thereby to target 1.Omg vasodilator in rapid release form in the first hour, followed by 250 microgram per hour from a slow release form over 2 hours, and administering a formulation of vitamin C thereby to deliver into the portal vein a dose of 100mg of vitamin C per hour for 3 hours to prevent oxidation of hepatic cell membranes,

a method for reversing metabolic effects of oxidation of hepatic cell membranes and resultant lack of oxygen, including the step of administering to a subject at the time of retiring to bed 500 microgram glyceryl trinitrate released per hour from a slow release form over an 8 hour period, and administering a formulation of an anti-oxidant agent thereby to deliver into the portal vein a dose of 100mg of vitamin C per hour for 8 hours to prevent further oxidation of hepatic cell membranes,

- a method for reversing metabolic effects of oxidation of hepatic cell membranes and resultant lack of oxygen by maintaining the blood flow in the hepatic artery including the step of administering a subject a formulation of hepatic artery vasodilator with a meal to target 1.0 mg of vasodilator in rapid release form 14

in the first hour during food absorption, followed with 250 microgram per hour from a slow release form over 2 hours, administering to a subject a formulation of antioxidant agent which is water soluble to deliver into the portal vein a dose of 100mg of vitamin C per hour for 5 hours, and administering a slow release formulation thereby to deliver 600mg of vitamin C to the portal vein over a 12 hour period at a constant rate of 50 mg per hour, to prevent oxidation of hepatic cell membranes,

a method for reversing metabolic effects of oxidation of hepatic cell membranes and resultant lack of oxygen, including the step of administering to a subject 500 microgram hepatic artery vasodilator in rapid release form in the first hour during food absorption, followed by 250 microgram per hour from a slow release form over 5 hours, administering a formulation of an anti-oxidant agent thereby to deliver into the portal vein a dose of 10Omg of vitamin C per hour for 5 hours to prevent further oxidation of hepatic cell membranes at the time of metabolic formation of oxidative products, and administering a slow release formulation to deliver 600mg of vitamin C to the portal vein over a 12 hour period at a constant rate of 50 mg per hour to prevent oxidation of the hepatic cell membranes by bacterial metabolic products,

a method for reversing metabolic effects of oxidation of hepatic cell membranes and resultant lack of oxygen including the step of administering to a subject a formulation of hepatic artery vasodilator at the time of retiring to bed 500 microgram glyceryl trinitrate released per hour from a slow release form over an 8 hour period, administering a formulation of an anti-oxidant agent thereby to deliver into the portal vein a dose of 100mg of vitamin C per hour for 8 hours to prevent further oxidation of hepatic cell membranes at the time of maximum metabolic formation of oxidative products, and administering a slow release formulation thereby to deliver 600mg of vitamin C to the portal vein over a 12 hour period at a constant rate of 50 mg per hour to prevent oxidation hepatic cell membranes by bacterial metabolic products. 15

In an especially preferred embodiment, vitamin C at a concentration of 50micM/L and observing a daily limit of 3.3 gm to prevent renal stone formation is used. Preferably, the preventive formulation includes about 500mg ascorbic acid in readily absorbed form which is released over about 5 hours commencing at the time of ingestion together with 600 mg vitamin C in a sustained release formulation released over a 12 hour period at a rate of 50 mg per hour. Three tablets are given daily with each of the three daily main meals. In the event that only 2 meals are eaten each day, then 2 tablets are taken with the major meal in the evening. Similar strategies can apply to other anti-oxidant agents contemplated for use in accordance with the invention.

Reference is now made to the drawings which accompany the application wherein:

Figure 1 shows the effects of a vasodilator on hepatic artery flow in the rat.

Figure 2 shows the mean hepatic artery flow during administration of nitroglycerine in the dog.

Figure 3 shows the vasodilating effect of PPADS tetrodotoxin, benextramine and guanethidine on the hepatic artery.

The present invention will now be more fully described with reference to the accompanying examples. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

EXAMPLE 1

AIMS

To determine whether the metabolic deficit in the liver associated with ageing 16

could be reproduced by a standardised pro-oxidant/free radical exposure.

The parameters to be assessed included oxygen uptake, a measure of oxygen sensitive metabolism propranolol metabolic clearance, and a measure of oxygen insensitive metabolism- morphine metabolic clearance.

MATERIALS & METHOD

Male Wistar rats (334±82 g, n=5) were obtained from the John Curtin School of Medical Research. The study was approved by the Australian National University Animal Experimentation Ethics Committee. Fed rats were anaesthetised with 50 mg/kg i.p. pentobarbital sodium. The livers were perfused at 20 ml/min in situ via the portal vein and in a single pass mode. The perfusate was Krebs Henseleit buffer, containing 1 % bovine serum albumin (Sigma Chemical Co, Sydney, Australia), 4 mg/l propranolol (Sigma Chemical Co, Sydney, Australia), 4 mg/l morphine tartrate (David Bull Laboratories, Mulgrave, Victoria) and equilibrated with 95% O₂ - 5% CO₂ (Linde Gas, Sydney, Australia). Oxygen delivery to the livers was kept at approximately 15 mol/min because this is below the oxygen threshold identified for propranolol clearance. Viability was assessed by macroscopic appearance, portal venous pressure, oxygen consumption and assays of outflow samples for bilirubin, alanine transaminase and glutamyl transpeptidase.

After allowing the liver to equilibrate for 15 minutes, inflow and outflow samples were collected for measurement of PO₂, propranolol and morphine. Oxidative stress was then induced by perfusing the livers for ten minutes with buffer containing 10 mM hydrogen peroxide. Samples were collected ten minutes after this treatment had been completed to allow hydrogen peroxide to be completely flushed from the liver.

Oxygen concentrations were measured with an ABL Automatic Blood Gas System (Melbourne, Australia). Propranolol concentrations were measured by HPLC 17

(Waters 715 Ultra Wisp, Sydney, Australia). Samples were extracted with a C18 Bond-Elut column, separated with a 10 m Bondapak HPLC column using acetonitrile:water:triethylamine (33:69:1 , pH 3.5) as mobile phase, and measured with a fluorescence detector (Schoeffel Instruments Corp, Sydney, Australia). Free morphine concentrations by were determined by GCMS (Micromass BG Trio 2000, United Kingdom) using pentafluoropropionyl derivatives and the multiple ion detector focussed at m/z 414, and m/z 417 for the internal standard

The hepatic extraction ratio (E) was calculated from the inflow (Cin) and outflow (Cout) concentrations by the formula:

E = (Cin - Cout) / Cin.

The intrinsic clearance (Cli) of propranolol and morphine was determined from the extraction ratio using the parallel-tube model according to the relationship15:

Cli = {-Q.ln(1 - E)} / fu.

where Q was the flow rate, and fu was the unbound fraction (propranolol 0.07 and morphine 0.65). Statistical significance was determined using the two-tailed, paired t test.

Results

Towards the end of the perfusion period with buffer containing hydrogen peroxide, the livers became swollen and then returned to normal after reperfusion with the control buffer. Ten minutes after treatment with hydrogen peroxide, oxygen consumption decreased by 14±9% (P = 0.03). There was an increase in alanine transaminase and portal venous pressure (Table I) but bilirubin and -glutamyl transpeptidase remained negligible.

Propranolol extraction decreased from 0.992±0.013 to 0.927±0.038 and morphine extraction from 0.666±0.046 to 0.514±0.038. The percentage decrease 18

in the intrinsic clearance of propranolol was greater than that of morphine (57±14% vs 34±7 % P < 0.005, Table I).

Table 1: The effect of oxidative injury induced by perfusion for 10 min with 10 mM hydrogen peroxide on parameters of liver viability and drug metabolism

Parameter Initial Perfusion After treatment Percentage with hydrogen Change peroxide oxygen delivery (μmol/min) 14.8 zt 1.1 15.3 ±0.8 oxygen uptake (μmol/min/g) 0.75 ±0.12 0.65 ±0.12 14±9

ALT(U/1) 8±3 54 ±16

Propranolol extraction 0.992 ±0.013 0.927 ± 0.038 clearance (ml/min/g) 1.36 ±0.28 1.27 ± 0.28 intrinsic clearance-venous 128896 ±127188 359 ± 270 93 ±10 equilibrium model

(ml/min/g) intrinsic clearance-parallel 144 ±74 54 ±17 57 ±14 tube model (ml/min/g)

Morphine extraction 0.666 ± 0.046 0.514 ±0.038 clearance (ml/min/g) 0.92 ± 0.22 0.71 ±0.16 intrinsic clearance-venous 4.3 ±1.4 2.3 ±0.6 47 ±9^* equilibrium model

(ml/min/g) intrinsic clearance-parallel 2.3 ±0.6 1.5 ±0.4 34±7⁺ tube model (ml/min/g)

^*significantly less than propranolol (P<0.0005) Significantly less than propranolol (P<0.005)

COMMENT ON RESULTS 19

The data establish that the two cardinal features of the metabolic deficit in the aged liver are reproduced by exposure to doses of hydrogen peroxide which are known from prior art to produce effects on the surface membranes of a variety of cells in the body (for example see van der ZEE and others, Biochemica et Biophysica Acta (1985): 818,38-44. "Peroxide-induced membrane damage in human erythrocytes"). Anti-oxidants will block the lipid peroxidation inherent in this process, as well as the oxidation of sulphydryl groups.

Increase in arterial blood inflow will also minimise these reactions through inhibition of the formation of methaemoglobin, and the supply of oxygen to leukocytes. Supplementary processes which minimise the effects of hydrogen peroxide are derived from the activation of oxygen-dependent enzymes within the hepatocytes.

EXAMPLE II

AIMS

To determine whether the oxygen status of the liver could be improved by inhaled oxygen supplementation.

MATERIALS & METHOD

Aged male Wistar rats (600±82 g, n=12) were obtained from the John Curtin School of Medical Research. The study was approved by the Australian National University Animal Experimentation Ethics Committee. Fed rats were allocated to the oxygen supplementation group or sham supplementation group. Animals were placed in standard housing cages with or without pure oxygen being directed into the cage at a rate of 2 litres per minute.

Animals were injected with 50 mg/kg i.p. pentobarbital sodium and observed until they were anaesthetised. 20

Blood samples were then taken from the aorta and portal vein with the order of sampling randomised within each group.

Oxygen concentrations were measured with an ABL Automatic Blood Gas System (Melbourne, Australia). Statistical significance was determined using the two- tailed, paired t test.

RESULTS

The pAO2 in the aorta (surrogate for the hepatic artery) was 109+/- 15mmHg in the rats without oxygen supplementation and 425 +/- 35mmHg in the supplemented rats. The pAO2 in the portal vein of unsupplemented rats was 42+/- 10 mmHg and 80+/- 12mmHg in the supplemented rats.

From these data it is possible to assume incomplete (50%) saturation of Hb in the portal vein of unsupplemented rats and 100% saturation of Hb in the supplemented rats. Assuming that the ratio of hepatic artery flow to portal venous flow is 20%, it is possible to calculate that the sinusoidal blood in non- supplemented rats will not be fully saturated and that the pAO2 will not exceed 50 mmHg. Similarly it is possible to calculate that the pAO2 of supplemented rats will be of the order of 150mmHg.

COMMENT ON RESULTS

The data establish that supplementation with oxygen has the potential to increase the diffusion of oxygen by a factor no more than three fold in the liver due to a limitation of pO2 increment in the sinusoid as a result of desaturation of portal blood.

The degree of oxygen supplementation used in rats is not sustainable in man as recent studies by Froomes and coworkers have shown (Froomes personal communication-data in press Gastroenterology 1999). In summary, Froomes showed that normal subjects have a mean pAO2=95+/-10 (SD) mmHg on room air 21

which increases to pAO2=272+/-29(SD) mmHg when pure oxygen was delivered via a plastic face mask at 12LJmin.

By the same principles applied above, it is possible to estimate that the increase in sinusoidal pO2 will be limited to a level of some 75mmHg, representing an increase in diffusion capacity of less than 50% .

These data mandate an examination of the capacity to increase delivery of flow to hepatocytes by increase in hepatic artery flow.

EXAMPLE III

AIMS

To determine whether the oxygen status of the liver could be improved by increase in perfusion via the hepatic artery. The experimental strategy adopted was to use an isolated perfused liver system with the novel features of perfusing both artery and vein (isolated perfused livers are always perfused by the portal vein alone unless stated to the contrary) and the perfusion conditions were to mimic physiological conditions in terms of flow rates, pressures and partial pressures of oxygen.

MATERIALS & METHOD

Materials

Propranolol, bovine serum albumin (BSA) and taurocholic acid were obtained from Sigma Chemical Co (Sydney, Australia), phenobarbitone sodium from David Craig and Co (Sydney, Australia), pentobarbitone sodium from Boehringer Ingelheim Pty Ltd (Sydney, Australia), O2/CO2 and N2/CO2 gases from Linde Gas (Canberra, Australia) and acetonitrile and triethylamine from Ajax Chemicals (Sydney, Australia). 22

Induction of cirrhosis

Male Wistar rats (80-100 g) were obtained from the John Curtin School of Medical Research, Australian National University (Canberra, Australia).

Rats were treated with sodium phenobarbitone for 2 weeks then with corn oil orally for 10 weeks before study. The study was approved by the Australian National University Animal Experimentation Ethics Committee.

Bivascular liver perfusion

After anaesthesia with pentobarbitone sodium (I.P. 60 mg/kg), laparotomy incision was made. The bile duct was cannulated with a 5 cm length of polyethylene tubing (I.D. 0.28 mm, O.D. 0.61 mm), the portal vein was cannulated with a 18G intravenous cannula and the thoracic inferior vena cava with a 16G intravenous catheter and perfusion commenced. The hepatic artery was cannulated via the abdominal aorta with an 18G intravenous cannula and branches of the hepatic artery were ligated. The liver was perfused with Krebs Henseleit buffer containing 20% out-of-date human erythrocytes (Red Cross Blood Bank, Canberra, Australia), 1% w/v BSA, 0.1% w/v glucose, 30M taurocholic acid and 2 g/ml propranolol. The livers were perfused in situ in a 37°C cabinet in a single pass mode with a total flow rate of 1-1.3 ml/min/g over a period of 60 min. The portal vein and hepatic artery were perfused using separate circuits allowing the flow rates and PO2 to be adjusted separately. The flow rates were determined by timed collections of each circuit performed before and after each perfusion.

Viability was assessed by macroscopic appearance, portal venous pressure (OHMEDA P23XL transducer and MacLab), bile production, oxygen consumption (AVL Automatic Blood Gas System) and assays of outflow samples for bilirubin, alanine transaminase and glutamyl transpeptidase. Liver tissue was sampled after perfusion had been completed for microscopic examination, using hematoxylin- and-eosin and Masson tri-chrome stains, and assessed by an independent 23

blinded pathologist. Cirrhosis was confirmed by the presence of bridging fibrosis and nodular regeneration.

Experimental design

Prior to surgery, the experimental flow rate was estimated as 1 ml/min/g of liver assuming that liver was 3% of body weight (Lautt and Greenway, details? 1987). The portal vein was perfused at a constant flow rate of 9-12 ml/min. To replicate physiological partial pressures the PO2 of the portal vein was adjusted to 40 mmHg and the hepatic artery PO2 to 100 mmHg using mixtures of 95% 02 / 5% CO2 and 95% N2 / 5% CO2. Propranolol was added in equal concentrations (2 mg/ml) to the hepatic arterial and portal venous perfusates.

The per usions were performed in three 20 minutes phases in randomised order. The hepatic artery flow rate was adjusted at each phase to low (1-3 ml/min), medium (2-5 ml/min) and high (5-7 ml/min) flow rates. Samples for assay of propranolol concentration were collected and viability parameters measured after a 10 and 20 minutes interval at each hepatic artery flow rate.

Sample analysis

Propranolol concentrations were measured by HPLC (Waters 715 Ultra Wisp, Sydney, Australia) according to the method of Harrison et al (Harrison et al., 1985). Samples were extracted with a C18 Bond-Elut column, separated with a 10 m Bondapak HPLC column using acetonitrile:water:triethylamine (33:69:1 , pH 3.5) as mobile phase, and measured with a fluorescence detector (Schoeffel Instruments Corp, Sydney, Australia).

Data analysis

The hepatic extraction ratio (E) was calculated from the inflow (Cin) and outflow (Cout) concentrations by the formula: 24

E = (Cin - Cout) / Cin. (1)

The clearance of propranolol was calculated from E by the formula:

CI=E (QPV + QHA) (2)

where QPV and QHA are the flow rates of the portal vein and hepatic artery, respectively.

The intrinsic clearance (Cli) of propranolol, which is a model-dependent measure of total metabolising enzyme activity, was determined from the extraction ratio using the parallel tube model according to the relationship:

Cli = {-(QPV + QHA).ln(1 - E)} / fu. (3)

where fu is the unbound fraction of propranolol.

Statistical analyses

Data are presented as mean standard deviation. Statistical significance was determined using linear regression analysis, ANOVA and the two-tailed t test and differences were considered significant when P < 0.05. Forward stepwise progression was performed using Sigmastat version 2.0 (SPSS Inc, Chicago, lllinios).

RESULTS

Influence of hepatic artery flow rate on propranolol metabolism

There were no sequential changes in macroscopic appearance, portal venous resistance or enzyme release during perfusions. Other parameters changed according to the changes in hepatic artery flow. 25

As expected, there were positive linear relationships between propranolol clearance and hepatic artery flow rate in the perfused livers of control (slope 1.2, P < 0.001 )(Figure 1 ). The slope approximated unity consistent with flow-limited clearance.

There were no significant relationships between intrinsic clearance and either hepatic artery flow rate or oxygen consumption in the livers of control rats when analysed using linear regression or stepwise regression.

There also was no relationship between oxygen consumption and the calculated intrinsic clearance .

The effect of blood flow on oxygen consumption

There was a positive relationship between hepatic artery flow and oxygen consumption (slope 2.0, P < 0.001), (Fig IIIA).

COMMENT ON RESULTS

The major finding in this study is that oxygen uptake and propranolol clearance have a position, linear relationship. The degree of change observed in both oxygen consumption and propranolol clearance was greater than 200% of baseline across the physiological range of flow range. The capacity of arterial flow change to alter oxygen consumption is four-fold over the likely contribution of oxygen supplementation (see section above).

EXAMPLE IV

AIMS

The aim of this study was to determine the flow responses of hepatic and mesenteric artery and systemic circulatory responses to dosing into the gut with a vasodilator (glyceryl trinitrate ) whose action is likely to be confined to the vascular 26

compartment of the gut due to extensive extraction by vascular and hepatic clearance.

METHODS:

Greyhounds were used in this study. All dogs were present in the animal house for <1 week prior to surgery, and all were deemed clinically sound. Dogs were given 15 minutes of exercise prior to arriving at the theatre. On arrival, they were clipped on the abdomen, forelimbs and hindquarters, and anaesthesia was induced with sodium pentobarbitone (Nembutal for Injection ) given intravenously . Subjects were intubated and connected to a respirator. Table heating was used to maintain body temperature. An initial infusion of 1 litre of Hartmann's solution was given throughout the surgical procedure, with bicarbonate being administered as required according to blood gas estimation. The abdomen was opened, and the gastro-duodenal branch of the common hepatic artery was located and ligated. Electromagnetic flow probes were placed on the common hepatic artery and the anterior mesenteric artery. A branch of the splenic vein was exposed and a catheter introduced and advanced into the portal vein. A catheter was also placed in the left hepatic vein using a purse string technique. An indwelling catheter was placed in a branch of the mesenteric vein, in close proximity to another catheter placed in the lurnen of the jejunum. The abdomen was then closed and a catheter introduced into the femoral artery. And a Statham Strainguage Arterial Pressure Transducer was connected to measure arterial blood pressure. The dogs' circulation and temperature were allowed to stabilise prior to the commencement of the experimental stage. At the end of the study, the dogs were euthanised with sodium pentobarbitone.

RESULTS:

After 90 minutes stabilisation, the first dose of glyceryl trinitrate (0.5 mg; 0.002 mg/kg) was given into the jejunal lumen. Time was allowed for any changes in blood flow before the next dose was given. Effects on flow reached a plateau by 27

20-30 minutes, when the next dose was given. Cumulative doses were given: 0.5, 1.5, 3.5, 7.5 mg/kg.

Five animals were studied. In all dogs, there were good flow responses confined entirely to the hepatic artery (ie there was no response in the mesenteric artery) with no change in systemic blood pressure. This pattern was maintained until 3.5 mg cumulative dosing, then systemic blood pressure fell and hepatic artery flow began to decline as shown in Figure 2.

A further 3 dogs were studied with portal vein and femoral vein administration alternating. When nitroglycerin was given into the portal vein the blood flow through the hepatic artery increased and oxygen uptake was increased. By contrast when nitroglycerin was given systemically, hepatic blood flow decreased, oxygen uptake decreased and systemic blood pressure was reduced.

28

COMMENT ON RESULTS

It can be concluded that glyceryl trinitrate represents an example of a drug whose action can be largely confined to the presystemic/ splanchnic circulation. An increase in hepatic artery circulation alone can be recorded without increase in mesenteric artery flow. This action is maintained until very high cumulative doses (> 3..5 mg) were given.

This local increase in hepatic artery flow was associated with increase in oxygen uptake.

In contrast, administration of glyceryl trinitrate into the general circulation caused a decrease in mean arterial pressure, and hepatic artery flow from the lowest doses.

EXAMPLE V

AIM

To study the range of physiological and pharmacological mechanisms controlling the tone of the hepatic artery using novel techniques applied to a new preparation of small muscular artery segments of the hepatic arterial bed.

METHODS

Male Wistar rats aged between 4 and 5 weeks postnatal (80-100 g) were obtained from the John Curtin School of Medical Research, Australian National University (Canberra, Australia).

The study was approved by the Australian National University Animal Experimentation Ethics Committee.

Animals were anaesthetised with ether anaesthetic and killed by cervical dislocation. For all experiments the hepatic artery proper was dissected from the 29

point where it separated from the coeliac artery to the point where its branches enter the liver parenchyma.

After dissection, the hepatic arterial tree was immobilised by pinning the adjacent mesentery in a 1 ml bath whose base was covered in a thin layer of silicone (Sylgard, Dow Coming Corporation, Midland U.S.A.). Preparations were superfused with Krebs' gassed with 5% CO₂195% O₂, at 34°C in the tissue bath. Hyoscine hydrochloride (10-6, M) and capsaicin (10-6 M) were added to the Krebs' solution at all times to prevent the effects of cholinergic and sensory nerves respectively. Preparations were allowed to equilibrate for 45 min prior to transmural stimulation (10 Hz, 10 s, 6OV, 0.1 msec pulse duration) every 20 min via platinum electrodes placed 5 mm apart on opposite sides of the preparation. Preliminary experiments were performed to determine the minimal stimulation parameters, with regard to frequency and duration, that would produce a response of reasonable size that could be blocked by tetrodotoxin. The artery was visualised using video microscopy and the vessel diameter was continually monitored (DIAMTRAK). Data were collected and measured on a MacLab Chart Recorder (AD Instruments U.S.A.). All experiments were performed on second or third order branches of the hepatic artery within the mesentery (mean resting diameter, 78.52 + 2.23 urn, n = 113). These diameters would be larger than the actual vessel diameter in vivo due to the pinning of the surrounding presented, however vessels were not occluded and blood movement was always observed during nerve stimulation.

Control experiments were performed to determine the time period over which consistent responses could be achieved. The magnitude of the vasoconstrictor response to nerve stimulation was expressed as a percentage of the resting vessel diameter. This was done in order to standardise nerve-mediated responses in vessels of different resting diameter. Prior to the addition of any drug, the average of two to three nerve-mediated responses in control Krebs' was calculated. For each drug or specific drug concentration, at least two responses were averaged once a consistent response appeared. This meant that drugs were 30

perfused for at least 20 min, the time between sequential nerve stimuli. The response in the drug solution was expressed as a percentage of the control response. Experimental values are given as the mean + s.e. mean of results from at least four preparations, where each preparation was obtained from different animals. All results were obtained with the appropriate drug present in, solution except for the irreversible c-adrenergic blocker benextramine, whose effect was determined after a washout period of 20 min to avoid any non-specific actions. Statistical significance was tested using a paired two tailed Students t-test and a P value of <0,05 was taken as significant. Concentration response curves were constructed using Axograph (Axon Instruments) and a Hills equation to fit the curves. The half maximal inhibitory concentration was calculated directly from the curves.

The following drugs were used: tetrodotoxin, hyoscine (scopolamine) hydrochloride, guanethidine sulphate (GE), benextramine tetrachloride, prazosin hydrochloride U.S.A. alpha,beta-mATP, pyridoxal phosphate-6-azophenyl 2'-4'- disulphonic acid tetrasodium (PPADS). All drugs were made up as at least 100 x stocks in water except for capsaicin (100% ethanol), prazosin (20% vv methanol) and 5-methyl-urapidil and WB41 01 (0.1 M hydrochloric acid). Dilutions of all stocks were made in Krebs' for final concentrations. Diluents were tested at appropriate concentrations. Appropriate precautions were taken for light-sensitive drugs, including illuminating the preparations with only long wavelength light (>610 nm).

Reverse transcription PCR techniques with appropriate primers for alpha-1 and alpha-2-adrenergic receptors, neuropeptide Y(Y1&Y2), purinergic receptors .

RESULTS

Figure 3 shows the effects of the cumulative consecutive application of (a) benextramine (BNX 10^"3M) and alpha,beta-mATP (MATP, 3 x 10^'6M) or (b) BNX and PPAADS (10^"5M) on the nerve-mediated contractile response of arteries in the 31

rat hepatic mesentery. Tetrodotexin (TTX, 10-5 M) and/or quanethidine (GE, 5 x 10^"6 M) abolished the small residual contraction. Columns represent the means + s.e. mean of at least 4 preparations. Results are expressed as % of the contractile response in control Krebs solution. Control response in (a) was 20.78 + 2.64% of resting vessel diameter, n=7 and in (b) was 17.92 +/- 1.68%, n=8. The symbol "^*" in Fig. 3(a) and 3(b) indicates a significant difference from the control (P>0.05).

Studies of the responses to nerve stimulation in the presence of alpha-1- adrenergic blockers showed 82.2% inhibition of contraction in the presence of 10^"8M prazosin.

Evidence for a variety of receptor types was obtained as detailed in Table 2.

Table 2: Expression of mRNA for o^ -adrenergic, α₂-adrenergic, neuropeptide Y and purinergic receptors in arteries of the hepatic mesentery

Receptor subtype mRNA expression level αι -adrenergic

0C1A + + + αιe + αiD +

0.2-adrenergic α_2A +

0C2B + + α₂c +

Neuropeptide Y

Y-i + + +

Y₂ 0

Purinergic

P2 x 1 + + +

P≥ x 2 + 32

P2x3 +

P_2x + + +

P2x5 + + P₂χ6 0

P2x7 + +

Crosses represent detection of PCR product which correspond to the size of the desired cDNA band for each set of subtype specific primers. The number of crosses reflects the amount of amplified product generated after 35 cycles. [0]=never detected; [+]weakly detected; [++]=strongly detected; [+++]=very strongly detected

COMMENT ON RESULTS

The results establish that diverse nerve and receptor blocking mechanisms apply to the control of the hepatic artery bed.

The major nerve type involved is adrenergic neurones involving the neurotransmitters noradrenaline, ATP and neuropeptide Y.

A variety of receptor types modulate the activity of the system including presynaptic purinergic P2x and alpha-2 adrenrgic receptors as well as post- synaptic alpha-1 -adrenergic, P2x purinergic and neuropeptide Y1 receptors.

A variety of agents acting on the nerve mechanisms or receptors can be considered for therapeutic modification of this arterial system.

Example VI

The major principle of preventative vasodilator therapy is that it is most desirably given before meals with the strategy of maintaining (not increasing) hepatic artery during responses to food. Accordingly, delivery in man of glyceryl trinitrate using a standard unit formulation targets 1.0mg in rapid release form in the first hour, followed by 250 microgram per hour from a slow release form over the next 2 33

hours. Such formulations is taken 3 times a day before meals. Similarly, prazosin formulated as a standard unit formulation is taken with meals containing 125 microgram in a rapid release form, together with 50microgm per hour from a sustained release over the next 2 hours.

Example VII

The second major principle of preventative anti-oxidant therapy involves cellular antioxidant mechanisms that in turn involve agents and mechanisms such as glutathione and glutathione transferase respectively. Many such processes are heavily energy dependent with respect to their own synthesis, transfer to protective cellular sites (particularly glutathione), and for exerting their action. For these reasons, they are sensitive to oxygen deprivation.

The supply of oxygen is heavily dependent on the hepatic artery. Indeed the viability of the liver in man and dog and other animal species is absolutely dependent on the hepatic artery supply remaining intact. When there is an intake of food, the supply of arterial blood to the normal liver declines markedly (Dauzat and others, Eur. J Appl Physiol 1994;68:373; Numata and others, J Clin Ultrasound 1998;26:137). The degree of the decline is 53+/-3%.

Based on these and a model of the complete period of time of reduction in flow, a period slightly in excess of 2 hours, provision of oxygen is made for an intervention over a period of 2.5-3.0h to abolish this reduction in flow and oxygen supply during feeding and the postpriandial phase.

These reductions in hepatic artery flow as recorded in normals do not occur in cirrhotic subjects. Accordingly these considerations of maintaining blood flow with vasodilators following feeding is not appropriate to patients with cirrhosis.

It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or 34

evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

It will be understood that the term "comprises" or its grammatical variants as used herein is equivalent to the term "includes" and is not to be taken as excluding the presence of other elements or features.

DISCUSSION

The liver protects the rest of the body from ingested toxins that may predispose to age-related diseases and aging itself based on the Oxygen Diffusion Barrier reported here. Toxins from the gut, whether swallowed, ingested in the diet or produced by gut bacteria are delivered to the liver by the portal vein. The liver acts as a gatekeeper against chemical exposures which will potentially cause toxic change in bodily cells generally. The compounds types acting to produce degenerative diseases include MPTP(1-methyl-4-phenyl-1 , 2,3,6- tetrahydrophyrine) acting locally on cells of the substantial nigra to produce Parkinsonism, streptozotocin acting locally on pancreas to produce diabetes, and compounds acting generally on mitochondria to inhibit or uncouple oxidative phosphorylation such as cyanide, sodium azide and dinitrophenol producing syndromes of optic atrophy, deafness, ataxia of gait, seizures, myoclonus and Parkinsonism-like syndromes (Wallace, Science 1992;256: 628-632).

In addition, the liver is also the main organ for the detoxification of circulating toxins such as oxygen derived free radicals, cholesterol, neurotoxins and compounds which act as proxidants such as polycyclical hydrocarbons from foodstuffs and cigarette smoke. Therefore, any age-related changes in the detoxification system within the liver will have profound effects on the rest of the body, predisposing to age-related diseases that have a direct toxic etiology (eg Parkinsons disease and others above) and/or are related to the effects of free radicals (degenerative vascular disease and malignancies). Immune cells have been shown to be prone to oxidation from minor change in pro-oxidant chemical 35

exposure (Poulsen and others, Nature 1998;395:231-232 ), accordingly a mechanism for secondary change in cells and cell membranes of diverse cells outside the liver.

The liver is involved in diverse synthetic activities. Accordingly, specialised cells such as nerve and sensory cells will be susceptible to deficiency in cell maintenance if these synthetic activities of the aged liver are compromised. Thus, a mechanism for indirect consequences not involving toxic chemicals and byproducts appears to contribute to changes in metabolism of the ageing or senescent liver.

As shown hereinabove and in accordance with the present invention, the changes in liver metabolism when established may be reversed or alleviated by treatment processes involving oral delivery of pharmaceutical formulations which increase local oxygen supply to hepatic cells through selective increase in hepatic artery flow, either alone or in combination with defined supplementation of inhaled oxygen. This restoration of liver function in middle-aged and elderly subjects will restrict the exposure of the body to toxic materials from the environment , gut, pancreas and liver itself which lead to the development of degenerative diseases associated with ageing including Parkinson's disease, diabetes, atherosclerotic and degenerative vascular disease, and the development of cancers.

These newly defined changes in liver metabolism may be prevented by oral delivery of pharmaceutical formulations which enhance cellular antioxidant systems and deliver defined doses of antioxidants over critically defined time periods.

Claims

36THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method for the treatment of senescence induced-hypoxia of the liver, which method includes administering orally to a subject in need thereof a vasodilating agent at a dose less than the oral dose required to lower peripheral blood pressure thereby to selectively increase hepatic arterial inflow and alleviate the hypoxia.

2. A method according to claim 1 , wherein the daily dose of vasodilating agent is such that the vasodilating agent is essentially confined to the splanchnic circulation, and the systemic circulation is essentially free of said vasodilator.

3. A method according to claim 1 or 2, wherein the vasodilator is selected from the group consisting of glyceryl trinitrate, nifedipine, felodipine, veraparmil, nitroglycerine, debrisoquine, clonidine, doxazosin, prazosin, labetalol, irbesartan, quinapril, hydrallazine, minoxidil, amlodipine, sildenafil, alpha-1 adrenergic blocker, alpha-2 adrenergic blocker, neuropeptide Y and purinergic blocker.

4. A method according to any one of claims 1 to 3, wherein the vasodilating agent is administered in an amount of about 0.02 to 60 mg per day.

5. A method according to any one of claims 1 to 4, wherein the vasodilating agent is administered in the form of a sustained release formulation once daily.

6. A method for the treatment of senescence induced-hypoxia of the liver, which method includes administering orally to a subject in need thereof 0.5-30 mg/day of glyceryl trinitrate, thereby to selectively increase hepatic arterial inflow and alleviate the hypoxia without lowering peripheral blood pressure.

7. A composition suitable for the treatment of senescence induced-hypoxia of the liver, which composition includes an oral daily dosage of a vasodilating agent that is less than the oral daily dosage required to lower peripheral blood pressure and that increases the hepatic arterial inflow and alleviates the hypoxia, and a 37

pharmaceutically acceptable carrier or diluent therefor.

8. A composition in accordance with claim 7, wherein the composition is in the form of a tablet, capsule, powder, suspension, emulsion or syrup

9. A composition in accordance with claim 7 or 8, wherein the composition is in unit dosage solid form and wherein the vasodilating agent is present in an amount of from about 5 to about 95% by weight and the remainder includes a conventional pharmaceutical carrier.

10. A composition according to any one of claims 7 to 9, in the form of a sustained release composition.

11. A method for the prevention of oxidation of hepatic cell membranes, which method includes the step of administering to a subject in need thereof a prophylactically effective amount of an anti-oxidant, said anti-oxidant being substantially absorbed by the portal vascular system.

12. A method according to claim 11 , wherein anti-oxidant is administered at the time of food constituent exposure.

13. A method according to claim 11 , wherein anti-oxidant is administered at the time of food additive exposure.

14. A method according to claim 11 , wherein anti-oxidant is administered at the time of food processing byproduct exposure.

15. A method according to claim 11 , wherein anti-oxidant is administered at the time of food storage byproduct exposure.

16. A method according to claim 11 , wherein anti-oxidant is administered at the time of food-induced mammalian metabolic byproduct exposure. 38

17. A method according to claim 11 , wherein anti-oxidant is administered at the time of mammalian metabolic byproduct exposure.

18. A method according to claim 11 , wherein anti-oxidant is administered at the time of food-induced bacterial metabolic byproduct exposure.

19. A method according to claim 11 , wherein anti-oxidant is administered during food intake, post-prandially and/or during fasting.

20. A method according to claim 19, wherein anti-oxidant administered during fasting is in the form of a sustained release formulation.

21. A method according to any one of claims 11 to 20, wherein the anti-oxidant is selected from the group consisting of vitamin C, N-acetyl cysteine, cysteine, glutathione and urate.

22. A method according to claim 21 , wherein the anti-oxidant includes 3 unit doses each including 500 mg ascorbic acid in readily absorbed form which is released over about 5 hours commencing at the time of ingestion together with 600 mg vitamin C in a sustained release formulation released over a 12 hour period at a rate of 50 mg per hour.

23. A composition suitable for the prevention of oxidation of hepatic cell membranes, which composition includes a prophylactically effective amount of an anti-oxidant, said anti-oxidant being substantially absorbed by the portal vascular system, and a pharmaceutically acceptable diluent, or carrier therefor, in a form suitable for oral administration.

24. A composition according to claim 23, wherein the anti-oxidant is selected from the group consisting of vitamin C, N-acetyl cysteine, cysteine, glutathione and urate.

25. A method for the prevention of oxidation of hepatic cell membranes and 39

resultant lack of oxygen, which method includes the step of maintaining blood flow in the hepatic artery to maintain delivery of oxygen thereby to prevent reduction in oxygen supply to the liver.

26. A method according to claim 25, wherein the blood flow in the hepatic artery is maintained by administering to a subject a hepatic artery vasodilator together with a meal thereby to deliver 1.0 mg of glyceryl trinitrate in rapid release form in the first hour, followed by 250 ╬╝g per hour from a slow release form over 5 hours.

27. A method for the prevention of oxidation of hepatic cell membranes including the step of delivering a water soluble anti-oxidant agent into the portal vein and maintaining active anti-oxidant concentration to block oxidant activity of one or more of food constituents, food additives including preservatives and colourants, food processing byproduct, food contaminants, byproducts of food storage, increase in liver blood flow from food, metabolic by-products of mammalian cell metabolism or metabolic by-products of resident bacterial flora in the gut.

28. A method according to claim 27, wherein the anti-oxidant is vitamin C delivered into the portal vein at a dose of 100mg per hour for 5 hours.

29. A method according to claim 27, wherein the anti-oxidant is vitamin C delivered into the portal vein at a dose of 100mg per hour for 8 hours.

30. A method according to claim 27, wherein the anti-oxidant is vitamin C delivered into the portal vein at a dose of 600mg over a 12 hour period at a constant rate of 50 mg per hour, thereby to prevent oxidation of hepatic cell membranes as a result of bacterial metabolism

31. A method for reversing metabolic effects of oxidation of hepatic cell membranes and resultant lack of oxygen by maintaining blood flow in the hepatic 40

artery with maintained delivery of oxygen.

32. A method according to claim 31 , wherein blood flow in the hepatic artery is maintained by administering to a subject a formulation of glyceryl trinitrate to deliver 1.0 mg in rapid release form in the first hour, followed by 250 microgram per hour from a slow release form over 5 hours.

33. A method according to claim 31 , wherein blood flow in the hepatic artery is maintained by administering to a subject a formulation of glyceryl trinitrate to deliver 500 microgram per hour from a slow release form over an 8 hour period.

34. A method according to claim 32 or 33, wherein the vasodilator is administered with a meal or prior to bed time.

35. A method for reversing metabolic effects of oxidation of hepatic cell membranes and resultant lack of oxygen by maintaining blood flow in the hepatic artery, including the step of administering to a subject a formulation of hepatic artery vasodilator and a formulation of anti-oxidant.

36. A method according to claim 35, wherein the formulation of hepatic artery vasodilator and formulation of anti-oxidant are co-administered.

37. A method according to claim 35, wherein administration of the formulation of hepatic artery vasodilator is followed by administration of the formulation of antioxidant.

38. A method according to any one of claims 35 to 37, wherein the formulation of hepatic artery vasodilator is administered prior to bed time or with a meal.

39. A method according to any one of claims 35 to 38, wherein the formulation of hepatic artery vasodilator delivers 1.Omg glyceryl trinitrate in rapid release form in the first hour, followed by 250 microgram per hour from a slow release form over 2 hours, and the formulation of anti-oxidant is vitamin C administered to 41

deliver into the portal vein a dose of 100mg per hour for 3 hours to prevent oxidation of hepatic cell membranes.

40. A method according to any one of claims 35 to 38, wherein the formulation of hepatic artery vasodilator is administered to deliver 500 microgram glyceryl trinitrate released per hour from a slow release form over an 8 hour period, and the formulation of an anti-oxidant agent is vitamin C administered to deliver into the portal vein a dose of 100mg per hour for 8 hours to prevent further oxidation of hepatic cell membranes.

41. A method according to any one of claims 35 to 38, wherein the formulation of hepatic artery vasodilator is administered to deliver 1.0 mg of vasodilator in rapid release form in the first hour during food absorption, followed with 250 microgram per hour from a slow release form over 2 hours, and the formulation of anti-oxidant agent is vitamin C administered to deliver into the portal vein a dose of 100mg per hour for 5 hours, followed by administration of a slow release formulation to deliver 600mg of vitamin C to the portal vein over a 12 hour period at a constant rate of 50 mg per hour, to prevent further oxidation of hepatic cell membranes.

42. A method according to any one of claims 35 to 38, wherein the formulation of hepatic artery vasodilator is administered to deliver 500 microgram vasodilator in rapid release form, followed by 250 microgram per hour from a slow release form over 5 hours, and the formulation of anti-oxidant agent is vitamin C administered to deliver into the portal vein a dose of 100mg per hour for 5 hours followed by administration of a slow release formulation to deliver 600mg of vitamin C to the portal vein over a 12 hour period at a constant rate of 50 mg per hour to prevent further oxidation of the hepatic cell membranes.

43. A method according to any one of claims 35 to 38, wherein the formulation of vasodilator is administered to deliver 500 microgram glyceryl trinitrate released per hour from a slow release form over an 8 hour period, the formulation of anti- 42

oxidant agent is vitamin C administered to deliver into the portal vein a dose of 100mg per hour for 8 hours, followed by administration of a slow release formulation to deliver 600mg of vitamin C to the portal vein over a 12 hour period at a constant rate of 50 mg per hour to prevent further oxidation hepatic cell membranes.

44. A method according to claim 42, wherein 500 microgram of hepatic artery vasodilator in rapid release form is administered in the first hour during food absorption, and 100 g of vitamin C per hour for 5 hours is delivered to prevent oxidation of hepatic cell membranes at the time of metabolic formation of oxidative products.

45. A method according to claim 43, wherein 100 mg of vitamin C is delivered per hour for 8 hours to prevent oxidation of hepatic cell membranes at the time of maximum metabolic formation of oxidative products.

46. A method according to any one of claims 42 to 45, wherein the 600 mg of vitamin C delivered to the portal vein over a 12 hour period at a constant rate of 50 mg per hour is administered to prevent oxidation of the hepatic cell membranes by bacterial metabolic products.