WO2011030104A1

WO2011030104A1 - Phenylacetate and/or phenylbutyrate (not ornithine) for reducing portal blood pressure

Info

Publication number: WO2011030104A1
Application number: PCT/GB2010/001705
Authority: WO
Inventors: Rajiv Jalan; Rajeshwar P. Mookerjee
Original assignee: Rajiv Jalan; Mookerjee Rajeshwar P
Priority date: 2009-09-09
Filing date: 2010-09-09
Publication date: 2011-03-17

Abstract

The present invention provides a method of reducing portal blood pressure comprising administering to a subject in need thereof a formulation comprising phenylacetate and/or phenylbutyrate, wherein the formulation does not comprise ornithine.

Description

PHENYLACETATE AND/OR PHENYLBUTYRATE (NOT ORNITHINE) FOR REDUCING PORTAL BLOOD PRESSURE

Field of the invention

The present invention relates to the treatment of portal hypertension.

Background to the invention

Statistics from the NIH for the period 1976-80 suggest that deaths from liver cirrhosis in the US were greater than 26,000. If this data is extrapolated to incorporate the increasing burden of viral and alcoholic liver disease currently in The West and also in the under-developed world, the number exceeds millions of cases per year world-wide. This figure is likely to continue to increase with the recognition of the new entity of Nonalcoholic fatty liver disease (in association with diabetes and the metabolic syndrome) which is increasingly recognized as a chronic liver disease with risk of progression.

Cirrhosis is associated with severe morbidity and mortality largely from portal hypertension. Increased blood pressure in the portal blood vessels may result from either increased volume of blood flowing through the vessels or increased resistance to the blood flow through the liver. In Western countries, the most common cause of portal hypertension is increased resistance to blood flow caused by extensive scarring of the liver in cirrhosis, which is most often due to chronic excessive alcohol intake.

Portal hypertension leads to the development of new veins (called collateral vessels) that directly connect the portal blood vessels to the general circulation, bypassing the liver. Because of this bypass, substances (such as toxins) that are normally removed from the blood by the liver can pass into the general circulation. Collateral vessels develop in particular at the lower end of the esophagus and at the upper part of the stomach. Here, the vessels can become variceal. These engorged variceal vessels are fragile and prone to bleeding, sometimes seriously and occasionally with fatal results.

Current treatment to lower portal pressure to decrease risk from variceal bleeding is limited to about 40% efficacy, in part due to tolerability of agents such as beta- blockers. Moreover, there is a suggestion that such agents decrease liver perfusion which may further compromise liver function, as hepatic blood flow is already low in cirrhosis, despite systemic vasodilatation. Summary of the invention

The present invention relates to methods for the reduction of portal blood pressure. In accordance with the present invention, this is achieved by administering phenylacetate and/or phenylbutyrate to a patient in need thereof. Accordingly, the invention provides a method of reducing portal blood pressure comprising administering to a subject in need thereof a formulation comprising phenylacetate and/or

phenylbutyrate, wherein the formulation does not comprise ornithine. Similarly, the invention provides a formulation comprising phenylacetate and/or phenylbutyrate which does not comprise ornithine for use in the reduction of portal pressure.

The formulation may consist essentially of phenylacetate and/or phenylbutyrate.

The formulation may consist essentially of phenylacetate or consist essentially of phenylbutyrate.

The subject to be treated in accordance with the present invention may have increased portal blood pressure. For example, the subject may have portal hypertension. The subject to be treated may have cirrhosis.

Brief description of the drawings

Figure 1 reports the portal pressure in bile duct ligated (BDL) and sham treated rats. One group of BDL rats were treated with vehicle (BDL) and another group was treated with phenylacetate. Portal pressure was significantly reduced (P < 0.01 , n = 12) in the phenylacetate treated rats compared to the BDL animals treated with saline.

Figure 2 shows that the expression of eNOS was significantly elevated in BDL rats compared to sham (* p<0.0l). Phenylacetate treatment had no alteration in eNOS expression compared to BDL alone.

Figure 3 shows that DDAH-1 protein expression was significantly higher in sham animals compared to BDL (* p<0.01). Following phenylacetate treatment in BDL rats, DDAH-1 expression increased significantly and reverts to sham levels ($ p<0.01).

Figure 4 shows that caveolin-1 protein expression was significantly higher in BDL animals compared to sham (* p<0.05). Following phenylacetate treatment Caveolin-1 protein expression decreased significantly ($ p<0.05).

Figure 5 shows that mean arterial pressure was significantly decreased in BDL rats (* p<0.05) compared to sham. Phenylacetate treatment in BDL rats shows no change in MAP.

* p<0.01 $ p<0.0l Figure 6 shows that portal pressure was significantly increased in BDL rats (* p<0.0001) compared to sham. Phenylacetate treatment given to BDL rats showed significant drop in portal pressure ($ p<0.05) (p=0.02). Detailed description of the invention

The present invention derives from the inventors' finding that administration of phenylacetate to bile duct ligated (BDL) rats leads to a decrease in portal pressure. The BDL rat is an animal model in which increased portal pressure is seen. Increased portal pressure in the BDL rat is similar to portal hypertension in the human, such as portal hypertension associated with liver cirrhosis. The present invention therefore relates to methods for reducing portal pressure, for example in portal hypertension, by

administering phenylacetate or a related molecule such as phenylbutyrate to a subject in need thereof. Therapeutic agent

The present invention utilises phenylacetate or a similar agent such as

phenylbutyrate in the treatement or prophylaxis of portal hypertension. The

phenylacetate or other agent used in the present invention may be used in a pure crystalline form. Isolated forms of the phenylbutyrate or other agent are typically used. Any active form of the phenylacetate or other agent may be used to prevent or treat portal hypertension or otherwise to decrease portal pressure.

At least one of phenylacetate and phenylbutyrate may be used. Phenylacetate and/or phenylbutyrate and/or a related molecule may be in physiologically acceptable salt form, such as an alkali metal or alkaline earth metal salt. The salt may be sodium phenylacetate or sodium phenylbutyrate. The salt form of phenylacetate and

phenylbutyrate or a related molecule may be in free form. Therefore the agent such as phenylacetate, phenylbutyrate, phenylacetate salt or phenylbutyrate salt is typically not chemically bound, or covalently linked to any other agent.

Pharmaceutical formulations

The phenylacetate and/or phenylbutyrate and/or other related molecule are typically provided in a formulation for administration. Such a formulation may comprise or consist essentially of phenylacetate and/or phenylbutyrate and/or another related molecule.

The phenylacetate and/or phenylbutyrate and/or other related molecule are typically formulated for administration with a pharmaceutically acceptable carrier or diluent. The phenylacetate and/or phenylbutyrate and/or other related molecule may thus be formulated as a medicament with a standard pharmaceutically acceptable carrier(s) and/or excipient(s) as is routine in the pharmaceutical art. The exact nature of the formulation will depend upon several factors including the desired route of

administration. Typically, the phenylacetate and/or phenylbutyrate and/or other related molecule are formulated for oral, intravenous, intragastric, intravascular or intraperitoneal administration.

The pharmaceutical carrier or diluent may be, for example, an isotonic solution such as physiological saline. Solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, gum arabic, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs;

sweeteners; wetting agents, such as lecithin, polysorbates, lauryl sulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film-coating processes.

Liquid dispersions for oral administration may be syrups, emulsions or suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.

Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with at least one of phenylacetate, phenylbutyrate and other related molecule, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.

The formulation or medicament may comprise or consist essentially of at least one of phenylacetate and phenylbutyrate. The formulation or medicament may comprise or consist essentially of phenylacetate and/or phenylbutyrate and a pharmaceutically acceptable carrier.

The formulation or medicament may comprise substantially no amino acids. For example, the formulation or medicament preferably does not comprise ornithine or comprises substantially no ornithine.

A pharmaceutical formulation in accordance with the present invention may further comprise one or more additional therapeutic agents. For example, the formulation may comprise phenylacetate and/or phenylbutyrate and/or a related molecule as described here and also one or more additional therapeutic agents. Preferably the additional therapeutic agent(s) are agents which will assist in the treatment or prophylaxis of the individual to be treated. For example, one or more agents that are effective at treating portal hypertension may be administered as part of a formulation as described herein. One or more agents that are effective at treating an underlying liver condition or symptom thereof in the patient may be administered as part of a formulation as described herein.

Treatment

The present invention provides methods for the reduction of portal blood pressure, for example in a subject with portal hypertension. Accordingly, the invention provides a method of reducing portal blood pressure comprising administering to a subject in need thereof phenylacetate and/or phenylbutyrate and/or a related molecule. Similarly, phenylacetate and/or phenylbutyrate and/or a related molecule may be provided for use in a method of reducing portal blood pressure. Also provided is the use of phenylacetate and/or phenylbutyrate and/or a related molecule in the manufacture of a medicament for the reduction of portal blood pressure.

The phenylacetate and/or phenylbutyrate and/or a related molecule as described herein is thus administered to a subject in order to reduce portal pressure in the subject. Phenylacetate and/or phenylbutyrate and/or a related molecule as described herein can thus be administered to improve the condition of a subject, for example a subject suffering from portal hypertension. The phenylacetate and/or phenylbutyrate and/or a related molecule as described herein may be administered to alleviate the symptoms of a subject, for example the symptoms associated with portal hypertension. The

phenylacetate and/or phenylbutyrate and/or a related molecule as described herein may be administered to combat or delay the onset of portal hypertension or any symptom associated therewith, such as varices. The invention can therefore prevent the medical consequences of portal hypertension. Use of phenylacetate and/or phenylbutyrate and/or a related molecule as described herein may thus extend the life of a patient with liver disease.

The subject is treated with phenylacetate and/or phenylbutyrate and/or a related molecule as described herein. As described above, the phenylacetate and/or

phenylbutyrate and/or a related molecule may be administered alone or in the form of a pharmaceutical formulation. The formulation may comprise one or more of

phenylacetate and/or phenylbutyrate and/or a related molecule and may additionally comprise one or more additional therapeutic or prophylactic agents. The formulation preferably does not comprise ornithine. The present methods of treatment preferably do not comprise the administration of ornithine to the subject being treated. The subject being treated is preferably not administered ornithine during the course of treatment in accordance with the present invention.

Two or more agents selected from phenylacetate, phenylbutyrate and a related molecule as described herein may be used in combination to treat a subject. For example, a combination of phenylacetate and phenylbutyrate may be used. The two or more agents may be administered together, in a single formulation, at the same time, in two or more separate formulations, or separately or sequentially as part of a combined administration regimen.

An agent or formulation of the invention may be administered by any suitable route. Preferably it is administered by oral, intravenous, intragastric, intraperitoneal or intravasular routes.

The agent or formulation is administered in a therapeutically effective amount. A suitable dose of phenylacetate and/or phenylbutyrate and/or a related molecule can be determined according to various parameters such as the age, weight and condition of the subject to be treated; the type and severity of the liver disease; the route of administration; and the required regimen. A typical dose of phenylacetate or phenylbutyrate or a related molecule is from 0.02 to 1.25, for example from 0.1 to 0.5, g per kg of body weight.

Consequently, a dosage of phenylacetate or phenylbutyrate or a related molecule may be from 1 g to 50 g such as from 5 g to 30 g. A physician will be able to determine the required dosage of phenylacetate and/or phenylbutyrate for any particular subject.

The present invention is broadly applicable to therapeutic methods and is relevant to the development of prophylactic and/or therapeutic treatments. It is to be appreciated that all references herein to treatment include curative, palliative and prophylactic treatment.

Prophylaxis or therapy includes but is not limited to administering a suitable dose of phenyl acetate and/or phenylbutyrate and/or a related molecule in order to cause a reduction in portal pressure, or in order to prevent or reduce an increase in portal pressure. For example, prophylaxis or therapy may result in the reduction of portal pressure in a subject with increased portal pressure such as a subject with portal hypertension.

Prophylaxis or therapy may result in the maintenance of a particular level of portal pressure in a patient where portal pressure has been increasing or in which portal pressure is expected to increase. Prophylaxis or therapy may result in an increase in portal pressure in an individual being reduced or slowed compared to the increase that would have been seen, or would have been expected, in the absence of such treatment.

Prophylaxis or therapy may have similar effects in relation to any of the symptoms or consequences of portal hypertension described herein. That is, treatment in accordance with the present invention may lead to a lessening in the severity of such symptoms or consequences, maintenance of an existing level of such symptoms or consequences or a slowing or reduction in the worsening of such symptoms or consequences.

Administration may be via any suitable route. Typically, the phenylacetate and/or phenylbutyrate and/or other related molecule are administered orally, intravenously, intragastrically, intravascularly or intraperitoneally.

Patients to be Treated

The present invention relates to the reduction of portal pressure in individuals in need thereof. An individual to be treated in accordance with the present invention may therefore have portal hypertension or may be at increased riak of portal hypertension. For example, the subject may have cirrhosis. Portal hypertension may be defined as increased blood pressure in the portal vein and its tributaries. The portal vein is the large vein that brings blood from the intestine to the liver. Portal hypertension may be defined as clinically significant when there is a portal pressure gradient (the difference in pressure between measurements of a catheter wedged in the hepatic veins or the portal vein and the free pressure readings in the hepatic vein or inferior vena cava) of > 10 mm Hg or greater.

Methods for diagnosing portal hypertension are well known in the art and in particular to clinicians and veterinarians in the field. Preferably, the subject will have been diagnosed as having portal hypertension, for example by a medical or veterinarian professional. The subject may display one or more symptoms associated with portal hypertension.

For example, portal hypertension can lead to accumulation of ascitic fluid. This can lead to the patient's abdomen swelling. The patient may also have an enlarged spleen. An ultrasound scan may be used to examine blood flow in the portal vein and nearby blood vessels and to detect fluid in the abdomen.

Collateral vessels may be visible on the skin over the abdominal wall or around the rectum. Esophageal and gastric varices bleed easily and sometimes massively. An ultrasound or computed tomography (CT) scan can be used to look for and examine collateral vessels.

Because most people with portal hypertension also have severe liver dysfunction, they may have symptoms of liver failure, such as a tendency to bleed.

Portal pressure may be measured directly. For example, a catheter may be inserted through an incision in the neck and threaded through blood vessels into the liver or spleen to directly measure pressure in the portal blood vessels.

The individual to be treated may have been diagnosed as suffering from portal hypertension, for example by any of these methods. The individual to be treated may have been diagnosed as being at risk of portal hypertension. For example, the individual may have been diagnosed with one or more symptoms that are associated with portal hypertension. For example, portal hypertension is commonly found in patients with liver cirrhosis. The individual to be treated may have liver cirrhosis. Portal hypertension can also result from advanced liver disease including alcoholic hepatitis, idiopathic non- cirrhotic portal hypertension, congenital hepatic fibrosis, partial nodular transformation, Budd-Chiari syndrome, portal vein thrombosis, right heart failure or schistosomiasis infection. The individual to be treated may have any one or more of these conditions.

The subject to be treated may be any individual that is susceptible to increased portal pressure such as portal hypertension. The subject may be male or female. Women may be more susceptible to the adverse effects of alcohol than men. Women can develop alcoholic chronic liver disease in a shorter time frame and from smaller amounts of alcohol than men.

The subject to be treated may be a human. The subject to be treated may be a non-human animal. The subject to be treated may be a farm animal for example, a cow or bull, sheep, pig, ox, goat or horse or may be a domestic animal such as a dog or cat. The subject may or may not be an animal model for liver disease. The animal may be any age, but will often be a mature adult subject.

Example

Phenylacetate treatment reduces portal pressure

These experiments utilised an established animal model of cirrhosis, the bile duct Iigated (BDL) rat.

BDL rats may be generated by methods known in the art. For example, male Sprague-Dawley rats (200-250g) may be used for this procedure. Following

anaesthetisation, a mid-line laparotamy may be performed, the bile duct exposed, triply Iigated with 4.0 silk suture, and severed between the second and third ligature. The wound is then closed in layers with absorbable suture, and the animal allowed to recover in a quiet room before being returned to the animal storage facility.

Molecular analysis was performed by Western blot. Extracts containing equal amounts of protein were denatured and separated on 4-12% NuPAGE Bis-Tris Gels and blotted on to PVDF membranes (Invitrogen, UK). The membranes were then being incubated with using different goat anti DDAH 1 (1 :1000) mouse anti-eNOS (1 :500; Transduction Laboratories/Pharmingen, San Jose, CA), and anti-caveolin-1 antibodies and later with respective HRP-conjugated secondary antibodies. The bands were visualized using an enhanced ECL detection kit and quantified by densitometry. Loading accuracy was evaluated via membrane rehybridization with antibodies against mouse and rabbit anti-a tubulin (1.1000; upstate and Cell Signaling Technology, respectively).

Figure 1 shows the results of experiments in which BDL rats were treated with either phenylacetate or a vehicle (control). As shown in Figure 1, bile duct ligation led to a significant increase in portal pressure when compared to sham animals. This confirms that the BDL rat model is a suitable experimental model of portal hypertension.

Administration of phenylacetate to BDL rats led to a significant decrease of about 30% in portal pressure (P < 0.01 , n = 12) compared to animals treated with BDL animals treated with vehicle alone.

Figures 2 to 6 show that treatment with phenylacetate significantly reduced the actions of several inhibitors of nitric oxide synthase, namely caveolin-1 and asymmetric dimethylarginine (ADMA), and thus resulted in a lowering of portal pressure. ADMA was in turn reduced by a significant increase in its metabolizing enzyme DDAH-1.

In particular, expression of eNOS was significantly elevated in BDL rats compared to sham (* p<0.01). Phenylacetate treatment had no alteration in eNOS expression compared to BDL alone (Figure 2). DDAH-1 protein expression was significantly higher in sham animals compared to BDL (* p<0.01). Following phenylacetate treatment in BDL rats, DDAH-1 expression increased significantly and reverts to sham levels ($ p<0.01 ) (Figure 3). Caveolin-1 protein expression was significantly higher in BDL animals compared to sham (* p<0.05). Following phenylacetate treatment Caveolin-1 protein expression decreased significantly ($ p<0.05) (Figure 4). Mean arterial pressure was significantly decreased in BDL rats (* p<0.05) compared to sham. Phenylacetate treatment in BDL rats shows no change in MAP.

* p<0.01 $ p<0.01 (Figure 5). Portal pressure was significantly increased in BDL rats (* p<0.0001) compared to sham. Phenylacetate treatment given to BDL rats showed significant drop in portal pressure ($ p<0.05) (p=0.02) (Figure 6).

It is important to note that the action on portal pressure was without any systemic haemodynamic compromise on mean arterial pressure suggesting that the action on eNOS is a local effect within the liver, the desired end-point for treatments targeting intrahepatic resistance lowering to reduce portal pressure.

Claims

1. A method of reducing portal blood pressure comprising administering to a subject in need thereof a formulation comprising phenylacetate and/or phenylbutyrate, wherein the formulation does not comprise ornithine.

2. A formulation comprising phenylacetate and/or phenylbutyrate which does not comprise ornithine for use in the reduction of portal pressure.

3. A method or formulation according to claim 1 or 2 wherein said formulation consists essentially of phenylacetate and/or phenylbutyrate.

4. A method or formulation according to claim 1 or 2 wherein said formulation

(a) consists essentially of phenylacetate; or

(b) consists essentially of phenylbutyrate.

5. A method or formulation according to any one of the preceding claims wherein said subject to be treated has portal hypertension.

6. A method or formulation according to any one of the preceding claims wherein said subject to be treated has liver cirrhosis.