WO2001016358A2

WO2001016358A2 - Method of screening for triacyglycerol hydrolase inhibitors

Info

Publication number: WO2001016358A2
Application number: PCT/EP2000/008262
Authority: WO
Inventors: Catherine Sylvia Borg-Capra; Richard Jiri Lehner; Dennis Edward Vance
Original assignee: Glaxo Group Limited; Governors Of The University Of Alberta
Priority date: 1999-08-28
Filing date: 2000-08-24
Publication date: 2001-03-08
Also published as: US6861233B1; GB9920334D0; JP2003535572A; EP1206569A2; WO2001016358A3; AU7412800A

Abstract

The invention relates to methods of identifying therapeutic agents which inhibit triacyglycerol hydrolase (TGH) activity, defined as TGH inhibitors, which are useful in the treatment of conditions resulting from elevated circulating levels of TG, VLDL/LDL-cholesterol and ApoB-100. Also claimed are therapeutic agents which are TGH inhibitors, identifiable by such methods and their use in combating diseases associated with elevated circulating levels of TG, VLDL/LDL-cholesterol and ApoB-100.

Description

Method Of Screening For Triacyglycerol Hydrolase Inhibitors

The present invention relates to the use of triacyglycerol hydrolase (TGH), it's use in methods of screening for agents which inhibit TGH, and to agents having TGH inhibition characteristics for use in combating diseases associated with elevated lipid levels.

There is convincing evidence that hyperlipidemia is a major risk factor for coronary heart disease (CHD) [1-2]. Several studies of lipid-lowering drugs have demonstrated a reduction in coronary endpoints accompanied with a beneficial effect on the progression of atherosclerosis [3-5].

Lipids are transported in the blood plasma and from different tissues in the body in the form of lipoproteins. Very-low-density lipoprotein (VLDL) is the principal vehicle for the transport of endogenous triglycerides (TG), and, ultimately, through its metabolic product, low-density lipoprotein (LDL), of cholesterol as well. VLDL is synthesized in the liver. Although many dysiipidaemia are characterized by excessive rate of production and secretion of hepatic VLDL [6- 7], little is known of the molecular mechanisms involved in the origin and transfer of lipid, particularly TG to the developing VLDL particle. However, it seems likely that TG synthesized de novo in the endoplasmic reticulum from fatty acids are not immediately transferred to nascent VLDL [8]. Instead TG, that are destined for incorporation into VLDL are stored temporarily within the cell cytosol [9]. In vitro and in vivo evidence supports the concept that this storage pool is the source of much of the TG which appear in VLDL (70%) [9-11]. TG from storage droplets are mobilized by lipolysis, and the fatty acid re-esterified before incorporation in the VLDL [11-12]. The rate at which this process operates may determine the effective availability of TG at the site of VLDL assembly and therefore may represent an important regulatory step for VLDL secretion. The nature of the lipases involved in the cycle of lipolysis/re-esterification is currently unknown as is their exact location within the cell. A candidate lipolytic enzyme, lysosomal acid lipase was considered. However, since chloroquine did not affect the bulk of intracellular hydrolysis of TG, it appeared that other lipases might be involved in the mobilization of TG for VLDL synthesis and secretion [12]. Also, the lipolysis/re-esterification cycle was resistant to insulin, suggesting that it is not a hormone-sensitive lipase similar to that, which occurs in adipose tissue [12].

A microsomal TG hydrolase purified from porcine liver has been described [13]. The enzyme is located in the endoplasmic reticulum and mitochondria- associated membranes, organelles where de novo TG synthesis and assembly take place. The triacylglycerol hydrolase (TGH) has been shown to be associated with lipid droplets. TGH is expressed in rat liver toward the end of the suckling period that coincides with the ontology of lipoprotein secretion. TGH is present exclusively to liver cells surrounding the capillary vessels, an area that it is likely to be active in lipoprotein production and secretion. In addition, the enzyme is absent from liver-derived HepG2 and McArdIeRH7777 hepatoma cells which are known to have impaired VLDL assembly and secretion [14]. Taking these results together, it has been suggested that TGH may participate in the mobilization of TG for assembly into VLDL.

It has now been found by the inventors that TGH modulates circulating TG levels in a mammalian subject, which provides the use of TGH as a target for screening for the identification of compounds for the treatment of diseases which are ameliorated by lowering TG levels, such as pancreatitis. The inventors have additionally found that TGH modulates circulating VLDULDL-cholesterol and apolipoprotein B-100 (ApoB-100) levels in a mammalian subject. Thus, TGH is also a target for screening for the identification of compounds useful in the treatment of diseases which are ameliorated by lowering VLDULDL-cholesterol and ApoB-100 levels, such as mixed dislipidemia.

The cDNA encoding the rat hepatic TGH has been cloned and expressed [15]. McArdle RH7777 rat hepatoma cell lines stably expressing the rat liver TGH displayed a higher utilization of intracellular triacylglycerol pools for secretion, and a higher secretion of ApoB-100 in the medium than the non transfected cell lines. These results strengthen the finding of the active role of TGH in the mobilization of stored TG, which can be used for lipoprotein assembly. Thus, according to a first aspect, the present invention provides a method for identifying compounds which will be useful in the treatment of conditions resulting from elevated circulating levels of: i) TG; ii) TG, VLDULDL-cholesterol and ApoB-100; or iii) VLDULDL-cholesterol and ApoB-100; comprising the step of determining whether the compound inhibits TGH activity.

As a preferred aspect, the treatment is of conditions resulting from elevated circulating levels of VLDULDL-cholesterol and ApoB-100.

As a further preferred aspect, the method comprises detecting or assaying the extent or result of enzymatic activity or lipolysis of TGH on a control substrate, in the presence of and absence of said TGH inhibitor.

Methods of detection of enzyme activity according to the present invention comprise any suitable methods known in the art. Thus, a control substrate, as defined herein, may comprise a labelled compound, (i.e. one which is radioactive or fluorescent) and/or one which is photo-activable. An example of a suitable control substrate is 4-methylumbeHiferyl butyrate.

The present invention demonstrates that an agent which inhibits TGH enzymatic activity (lipolysis) decreases to an equally strong extent the circulating levels of TG, VLDULDL-cholesterol and apolipoprotein B-100 in a mammalian subject. The effect of TGH inhibition on TG is by no means certain from the teaching of the prior art. In fact, TGH is only one of a number of lipases which may have been involved in the process, and there is no teaching that inhibition of TGH alone would exhibit a sufficient therapeutic effect. The further observation of the effect of TGH inhibition on VLDULDL-cholesterol and apolipoprotein B-100 is surprising in that this effect is not linked to the role of TGH in decreasing circulating TG. Thus, the additional finding represents a further effective method of treating specific diseases associated with elevated lipid levels, which is neither taught nor suggested by the prior art. As used herein, the 'methods for identification' include any screen or assay whereby the action of an agent capable of modulating, affecting, influencing or interfering with the enzymatic activity of TGH is investigated, and includes inhibition assays in which a single agent or compound is investigated as well as assays in which more than one compound, such as an array of compounds, or a library of compounds is tested. In the case of testing more than one agent, these tests may be either simultaneous or sequential. The methods of detection and assay include any quantitative, qualitative or semiquantitative assessment of whether there is any inhibition of enzymatic activity of TGH on a substrate in the presence of the agent being tested, compared with that in the absence of said agent.

In one aspect, the method of the invention comprises an inhibition assay whereby the difference in enzymatic activity of TGH on a fluorogenic control substrate in the presence of a test TGH inhibitor, with that in the absence of said test inhibitor is compared.

In another aspect, the present invention comprises a functional in vivo assay whereby the extent of hypolipidemic activity is determined when the test TGH inhibitor is administered to a test mammalian subject, for example, a hamster.

Viewed from a further aspect, the present invention provides a therapeutic agent (a TGH inhibitor'), identified or identifiable by the aforementioned methods according to the present invention, and its use in combating diseases associated with elevated circulating levels of lipids.

In a further alternative or yet further aspect, there is provided a method for the treatment of conditions resulting from elevated circulating levels of: i) TG; ii) TG, VLDULDL-cholesterol and ApoB-100; or iii) VLDULDL-cholesterol and ApoB-100; comprising administration of a compound identified by the aforementioned method for identification of suitable compounds. The invention provides, as a further aspect, the use of a compound which inhibits the action of TGH, or a physiologically acceptable salt, solvate or derivative thereof, in the preparation of a medicament for the treatment of conditions resulting from elevated circulating levels of: i) TG; ii) TG, VLDULDL-cholesterol and ApoB-100; or iii) VLDULDL-cholesterol and ApoB-100.

The use, above, in the preparation of a medicament of conditions resulting from elevated circulating levels of VLDULDL-cholesterol and ApoB-100 is preferred.

In an alternative or further aspect, there is provided a method for the treatment of a mammal, including man, in particular in the treatment of conditions resulting from elevated circulating levels of: i) TG; ii) TG, VLDULDL-cholesterol and ApoB-100; or iii) VLDULDL-cholesterol and ApoB-100; comprising administration of an effective amount of a compound which inhibits the action of TGH, or a physiologically acceptable salt, solvate or derivative thereof.

Compounds of the invention which inhibit TGH activity are of use in the treatment of disease associated with elevated lipid levels. Diseases which result from elevated levels of circulating TG (i.e. hyperthglyceridemia, hyperbetalipoproteinemia) include pancreatitis and obesity. Diseases in which elevated levels of TG, VLDULDL-cholesterol and ApoB-100 are implicated (i.e. mixed dyslipidemia, hypercholesterolemia, hyperbetalipoproteinemia) include non-insulin dependent diabetes mellitus (NIDDM), coronary arterial disease, peripheral vascular disease and cerebro- vascular disease. Diseases in which elevated levels of VLDULDL-cholesterol and ApoB-100 are implicated (i.e. hypercholesterolemia, hyperbetalipoproteinemia) include non-insulin dependent diabetes mellitus (NIDDM), coronary arterial disease, peripheral vascular disease and cerebro-vascular disease. Further, TGH is involved in the cycle of lipolysis/re-esterification of TG, early steps in the assembly of TG in VLDL. TGH is also present in the intestine [13] and is expected to have similar function. Therefore, TGH in the intestine might participate in the assembly of TG into chylomicrons and as a consequence modulate lipid absorption. Inhibition of TGH specifically in the intestine or in concert with hepatic TGH inhibition by compounds of the further aspects of the present invention may decrease the absorption of dietary lipid. Yet further, it is well recognized that lipids and associated lipoproteins and apolipoproteins play a significant role in the formation and progression of atherosclerosis disease. Numerous angiographic trials have shown that reducing cholesterol levels in patients with coronary heart disease can significantly slow progression and in some cases actually cause regression, of atherosclerosis in these patients [5, 16]. Therefore, TGH inhibitors of the further aspects of the present invention, by reducing TG, VLDULDL-cholesterol and Apo-B100, may represent an effective treatment of atherosclerosis.

It will be appreciated that reference to treatment is intended to include prophylaxis in patients deemed to be at risk of suffering a clinical event as a result of elevated lipids levels (primary prevention), also in patients deemed to be at risk of suffering a second or further clinical event as a result of elevated lipids levels (secondary prevention), as well as the alleviation of established symptoms. TGH inhibitors according to the invention may be administered as the raw chemical but the active ingredient is preferably presented as a pharmaceutical formulation.

Accordingly, the invention also provides a pharmaceutical composition which comprises at least one TGH inhibitor, or a physiologically acceptable salt, solvate or derivative thereof, together with one or more pharmaceutically acceptable derivatives and formulated for administration by any convenient route.

Such compositions are preferably in a form adapted for use in medicine, in particular human medicine, and can conveniently be formulated in a conventional manner using one or more pharmaceutically acceptable carriers or excipients. Thus, TGH inhibitors according to the present invention may be formulated for oral, buccal, parenteral, transdermal, topical (including ophthalmic and nasal), depot or rectal administration or in a form suitable for administration by inhalation or insufflation (either through the mouth or nose), by methods well known in the art.

For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g. lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrants (e.g. potato starch or sodium starch glycollate); or wetting agents (e.g. sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g. sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g. lecithin or acacia); non-aqueous vehicles (e.g. almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g. methyl or propyl- p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavouring, colouring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to give controlled release of the active compound.

For buccal administration the composition may take the form of tablets or lozenges formulated in conventional manner.

For transdermal administration the compounds according to the invention may be formulated as creams, gels, ointments or lotions or as a transdermal patch. Such compositions may for example be formulated with an aqueous or oily base with the addition of suitable thickening, gelling, emulsifying, stabilising, dispersing, suspending, and/or colouring agents.

The compounds of the invention may be formulated for parenteral administration by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form e.g. in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile pyrogen-free water, before use.

The compounds of the invention may be formulated for topical administration in the form of ointments, creams, gels, lotions, pessaries, aerosols or drops (e.g. eye, ear or nose drops). Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Ointments for administration to the eye may be manufactured in a sterile manner using sterilised components.

Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilising agents, dispersing agents, suspending agents, thickening agents, or colouring agents. Drops may be formulated with an aqueous or non aqueous base also comprising one or more dispersing agents, stabilising agents, solubilising agents or suspending agents. They may also contain a preservative.

The compounds of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, e.g. containing conventional suppository bases such as cocoa butter or other glycerides.

The compounds of the invention may also be formulated as depot preparations. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds of the invention may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

For intranasal administration, the compounds of the invention may be formulated as solutions for administration via a suitable metered or unit dose device or alternatively as a powder mix with a suitable carrier for administration using a suitable delivery device.

The compositions may contain from 0.1% upwards, e.g. 0.1 - 99% of the active material, depending on the method of administration. A proposed dose of the compounds of the invention is 0.25mg/kg to about 125mg/kg bodyweight per day e.g. 20mg/kg to 100mg/kg per day. It will be appreciated that it may be necessary to make routine variations to the dosage, depending on the age and condition of the patient and the precise dosage will be ultimately at the discretion of the attendant physician or veterinarian. The dosage will also depend on the route of administration and the particular compound selected.

TGH inhibitors according to the invention may, if desired, be administered with one or more therapeutic agents and formulated for administration by any convenient route in a conventional manner. Appropriate doses will be readily appreciated by those skilled in the art. For example, TGH inhibitors according to the invention may be administered in combination with other lipid lowering drugs acting through cholesterol depletion or by reducing VLDL production, for instance inhibition of enzymes involved in cholesterol biosynthesis such as an HMGCo-A reductase inhibitor, or a microsomal triglyceride transfer protein

(MTP) inhibitor and/or a bile acid sequestrant or bile acid transporter inhibitor.

The invention will now be described with reference to the following non-limiting examples in which:

Figure 1 shows the deduced amino acid sequence of the Human TGH from the cDNA sequence

Figure 2 shows a comparison between the N-terminal residues of purified human TGH, porcine TGH and cloned human TGH; Figure 3 shows the dose-dependent inhibition of TGH enzymatic activity with a test TGH inhibitor;

Figure 4 shows the enzymatic activity of LPL in the presence of and absence of test TGH inhibitor; and Figure 5 shows the decrease in various lipid levels resulting from administration of test TGH inhibitor to normal fed hamsters.

Biological Experimental Methods

Materials

Test TGH inhibitor, 4,4,4-trifluoro-2-[2-(3-methylphenyl)hydrazono]-1-(2- thienyl)butane-1 ,3-dione was obtained from Maybridge. The high Q and Hydroxyapatite cartridges were from Bio-Rad S.A. [9,10-³H(N)Trioleoylglycerol was purchased from Dupont-NEN. Enzymatic assay kits for cholesterol and triglycerides were obtained from Bio Merieux. The polyclonal antibody against the porcine TGH was provided by Dr Lehner and Dr Vance from the University of Alberta, Edmonton. All other reagents including the fluorogenic substrate 4- methylumbelliferyl butyrate and lipoprotein lipase were purchased from Sigma.

1. Cloning of the Human TGH

Two 40 nucleotides long oligos, P-TGHI

(δ'GCATCTGGGGATTCTTCAGCACAGGGGATGAACACAGCCGS') and P- TGHII (5OAGCAAAGTTGGCCCAGTATTTCATCACCATTTTGCTGAG3'), corresponding to highly conserved sites between mouse, rat and pig TGH cDNAs (15), were used to amplify a 1 kb fragment using PCR (in 50 ml 1XPCR buffer: 1 mg human liver Igt11 cDNA library, 0.4 mM of each primer, 0.25 mM dNTPs, 2 mM MgCI2, 2.5U Tag polymerase; 5 min hot-start 95°C, 1 min 95°C, 1 min 52°C, 1 min 72°C, 30 cycles, 5 min 72oC). This fragment was sequenced and compared to existing data base using BLAST search. It was identified as human carboxylesterase I (hCEI).

hTGH protein was purified from human liver. Upon amino acid sequencing of the first 20 residues, it was found to be the same as hCEI's first 20 amino acids. From these 2 lines of evidence, it is confirmed that hTGH is the same as hCEI. Two 22 nucleotides long oligos, hCE5Εor(5y\ACTGTCGCCCTTCACGATGTG3') and hCE3'Rev (5TCACAGCTCTATGTGTTCTGTCTGG3'), corresponding to the 5' and 3' of hCEI respectively, were used to amplify the 1.7 kb complete cDNA using PCR (in 50 ml 1XPCR buffer: 1 mg phage cDNA library, 0.4 mM of each primer, 0.25 mM dNTPs, 2 mM MgCI2, 2.5U Tag polymerase; 5 min hot-start 95oC, 1 min 95°C, 1 min 54°C, 1 min 72°C, 40 cycles, 5 min 72°C). The cDNA was ligated to pCR2.1TOPO (Invitrogen) to obtain cloning sites, sequenced to ensure fidelity, before finally cloned into mammalian expression vector pCI (Promega) between Xho I and Xba I.

2. Purification Of Human Liver TGH

Human liver TGH was purified according to Lehner and Verger's protocol described for the porcine liver TGH [13]. Briefly, the enzyme from microsomal membranes of liver tissues was solubilised by the zwitterionic detergent 3-[(3- cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and was purified to apparent homogeneity by sequential chromatography on Q-sepharose and hydroxyapatite.

3. Effect of test agents on human liver TGH.

The enzymatic activity of TGH was evaluated with the fluorogenic substrate 4- methylumbelliferyl butyrate (4-MU-butyrate). Briefly, 10 μl of a solution of 25mM of 4-MU-butyrate in tetrahydrofuran was injected in 2 ml of a buffer containing Tris (20 mM), pH 8.0, EDTA (1 mM) and taurodeoxycholate (300 μM). TGH activity was assayed at 1.16 nM in a final volume of 100 μl. The compound was dissolved in DMSO to be tested at various concentrations (from 1 nM to 2 μM) and was incubated with the enzyme 15-min prior to the addition of 20 μl of the substrate, which gives the starting point of the enzymatic reaction. The reaction mixture comprised of 60 μl of buffer (Tris 20 mM, pH 8, EDTA 1 mM), 10 μl of the compound at various dilutions or 10 μl of corresponding DMSO concentrations (for the 100 % TGH activity), 10 μl of TGH and 20 μl of substrate. The basal level of fluorescence of the substrate was evaluated in a reaction mixture of 70 μl of buffer, 10 μl of DMSO (at the appropriate dilution) and 20 μl of substrate, and was subtracted from each other data . Rates of lipolysis were determined from continuous increase in fluorescence intensity at 460 nm (Excitation: 355 nm).

4. Effect of TGH inhibitors on the lipoprotein lipase activity: In order to evaluate the specificity of TGH inhibitors toward TGH, test compound was tested on the enzymatic activity of lipoprotein lipase (LPL) from bovine milk. For this purpose radiolabelled trioleoylglycerol (250 μM, specific activity 1 mCi/mmole) was emulsified in mixture of 10% gum arabic by sonication. Long- chain triacylglycerol hydrolysis was assayed in a final volume of 200 μl containing LPL (1.5 μg/ml), Tris (50 mM), pH 8.0; MgCI_2, CaCI₂ (1 mM), and 150mM NaCl with 1 mg/ml BSA as a fatty acid acceptor for 30 min at 37°C. The reaction was terminated with the addition of 3.25 ml of methanol/chloroform/heptane (3.85:3.42:2.73 by volume); 0.3 mi of 150 mM NaCl, lipid carriers (100 μg of unlabelled oleic acid), and 50 μl of 1 N NaOH. The mixture was vortexed and centrifuged. One ml of the upper phase (containing the fatty acids that have been hydrolyzed) was mixed with 10 ml of Cytoscint and counted. Test compound is incubated with the enzyme 15 minutes prior to the addition of the substrate.

5. Hypolipidemic activity of the TGH inhibitor in hamster:

Ten normal fed hamsters were randomly allocated into 2 groups. Animals from one group were orally gavaged with a test TGH inhibitor in DMSO/ Labrafil (10/90%), 25 mg/kg, twice a day for 3 days while the animals from the other group were gavaged with the solvent (DMSO/labrafil) twice a day for 3 days. The animals were sacrificed 4 hours following the last administration and the plasma lipids and lipoproteins were analyzed.

Total cholesterol (TC) and triglyceride (TG) levels in plasma were determined enzymatically with reagents from Bio Merieux. VLDULDL lipoprotein fractions were separated from the HDL lipoproteins by gradient centrifugation (d=1.063). Cholesterol in the VLDULDL as well as in the HDL fraction was also determined using the Bio Merieux kit. Apolipoprotein B-100 (Apo-B100) in the VLDULDL fraction was visualized by using SDS-PAGE under reducing conditions using resolving gels containing a 5% to 12% gradient. Biological Results

1. Cloning of the human TGH

The Human TGH cDNA was isolated by PCR using specific primers designed on the rat TGH cDNA [15] and human liver cDNA library as a template. As illustrated in Figure 1 , the cDNA encodes a human carboxylesterase previously identified as human carboxylesterase EST-1 (Accession number P23141). Since the identification of human TGH as the human carboxylesterase, a paper was uncovered that mentioned the purification and cloning of a human enzyme with Acyl coenzyme A:cholesterol acyltransferase activity identical to the human carboxylesterase EST-1 [17].The putative dual function in TG hydrolysis and cholesterol esterification is relevant to the function of TGH in the liver and intestine. Indeed it implies that the enzyme impairs the assembly of the VLDL particles by acting at both levels: lipolysis and re-esterification

2. Purification Of Human Liver TGH

The purified protein migrated in SDS-polyacrylamide gel electrophoresis as a single band of an apparent molecular weight of 62 kDa which is comparable to the porcine TGH (60 kDa). As shown by Figure 2, the amino acid sequence of 28 N-terminal residues shared a high degree of homology with the porcine TGH and was identical to the human TGH. Finally, the polyclonal antibody raised against the porcine TGH which has been shown to be specific to the enzyme [13] cross-reacted with the purified human protein as well as the rat TGH [13]. These data present strong evidence that the purified protein is the human TGH.

3. Effect of test agents on human liver TGH

As shown by Figure 3, pre-incubation of human liver TGH with test inhibitor, 4,4,4-trifluoro-2-[2-(3-methylphenyl)hydrazono]-1-(2-thienyl)butane-1 ,3-dione, resulted in a dose-dependent inhibition of the enzymatic activity. The concentration of 4,4,4-trifluoro-2-[2-(3-methylphenyl)hydrazono]-1-(2- thienyl)butane-1 ,3-dione which resulted in 50% inhibition of TGH was evaluated to be 4 nM.

4. Effect of TGH inhibitors on the lipoprotein lipase activity: 4,4,4-Trifluoro-2-[2-(3-methylphenyl)hydrazono]-1-(2-thienyl)butane-1 ,3-dione was tested at 5 μM and was incubated with the enzyme 15-min prior to the addition of the substrate. As shown by Figure 4, 4,4,4-trifluoro-2-[2-(3- methylphenyl)hydrazono]-1-(2-thienyl)butane-1 ,3-dione at 5 μM did not affect the enzymatic activity of LPL.

5. Hypolipidemic activity of the TGH inhibitors in hamster: As shown in Figure 5, the oral administration of 4,4,4-trifluoro-2-[2-(3- methylphenyl)hydrazono]-1-(2-thienyl)butane-1 ,3-dione resulted in significant reduction in the plasma TG concentration (-55 % from the solvent treated animals) as well as in the VLDULDL cholesterol (-40 % from the solvent treated animals) while HDL cholesterol level was not significantly affected. Apo-B100 was also reduced to the same extent as Apo-B100 containing particles (-39 % decrease compared to control animals).

Tablet compositions

The following compositions A and B can be prepared by wet granulation of ingredients (a) to (c) and (a) to (d) with a solution of povidone, followed by addition of the magnesium stearate and compression.

Composition A mg/tablet mg/tablet

(a) Active ingredient 250 250

(b) Lactose B.P. 210 26 ( (cc)) SSooddiiuumm SSttaarrcchh GGllvyccoollllaattee 2 200 12

(d) Povidone B.P. 15 9

(e) Magnesium Stearate 5 _3

500 300

Composition B mg/tablet mg/tablet

(a) Active ingredient 250 250

(b) Lactose 150 150

(c) Avicel PH 101 60 26 (d) Sodium Starch Glycollate 20 12 (e) Povidone B.P. 15 9

(f) Magnesium Stearate 5 _3

500 300

Composition C mg/tablet

Active ingredient 100

Lactose 200

Starch 50

Povidone 5

Magnesium Stearate _4 359

The following compositions D and E can be prepared by direct compression of the admixed ingredients. The lactose used in composition E is of the direct compression type.

Composition D mg/tablet

Active ingredient 250

Magnesium Stearate 4

Pregelatinised Starch NF15 146 400

Composition E mg/tablet

Active ingredient 250

Magnesium Stearate 5

Lactose 145

Avicel 100 500

Composition F (Controlled release composition) mg/tablet

(a) Active ingredient 500 (b) Hydroxypropylmethylcellulose 112 (Methocel K4M Premium)

(c) Lactose B.P. 53

(d) Povidone B.P.C. 28 (e) Magnesium Stearate _7

700

The composition can be prepared by wet granulation of ingredients (a) to (c) with a solution of povidone, followed by addition of the magnesium stearate and compression.

Composition G (Enteric-coated tablet)

Enteric-coated tablets of Composition C can be prepared by coating the tablets with 25mg/tablet of an enteric polymer such as cellulose acetate phthalate, polyvinylacetate phthalate, hydroxypropylmethyl- cellulose phthalate, or anionic polymers of methacrylic acid and methacrylic acid methyl ester (Eudragit L). Except for Eudragit L, these polymers should also include 10% (by weight of the quantity of polymer used) of a plasticizer to prevent membrane cracking during application or on storage. Suitable plasticizers include diethyl phthalate, tributyl citrate and triacetin.

Composition H (Enteric-coated controlled release tablet)

Enteric-coated tablets of Composition F can be prepared by coating the tablets with 50mg/tablet of an enteric polymer such as cellulose acetate phthalate, polyvinylacetate phthalate, hydroxypropylmethyl- cellulose phthalate, or anionic polymers of methacrylic acid and methacrylic acid methyl ester (Eudragit L). Except for Eudragit L, these polymers should also include 10% (by weight of the quantity of polymer used) of a plasticizer to prevent membrane cracking during application or on storage. Suitable plasticizers include diethyl phthalate, tributyl citrate and triacetin.

(ii) Capsule compositions Composition A Capsules can be prepared by admixing the ingredients of Composition D above and filling two-part hard gelatin capsules with the resulting mixture. Composition B (infra) may be prepared in a similar manner.

Composition B mg/capsule

(a) Active ingredient 250

(b) Lactose B.P. 143

(c) Sodium Starch Glycollate 25

(d) Magnesium Stearate _2

420

Composition C mg/capsule

(a) Active ingredient 250

(b) Macrogol 4000 BP 350

600

Capsules can be prepared by melting the Macrogol 4000 BP, dispersing the active ingredient in the melt and filling two-part hard gelatin capsules therewith.

Composition D mg/capsule Active ingredient 250 Lecithin 100

Arachis Oil 100

450

Capsules can be prepared by dispersing the active ingredient in the lecithin and arachis oil and filling soft, elastic gelatin capsules with the dispersion.

Composition E (Controlled release capsule) mg/capsule (a) Active ingredient 250 (b) Microcrystalline Cellulose 125 (c) Lactose BP 125

(d) Ethyl Cellulose _13

513

The controlled release capsule composition can be prepared by extruding mixed ingredients (a) to (c) using an extruder, then spheronising and drying the extrudate. The dried pellets are coated with a release controlling membrane (d) and filled into two-part, hard gelatin capsules.

Composition F (Enteric capsule) mg/capsule

(a) Active ingredient 250

(b) Microcrystalline Cellulose 125

(c) Lactose BP 125 (d) Cellulose Acetate Phthalate 50

(e) Diethyl Phthalate _5

555 The enteric capsule composition can be prepared by extruding mixed ingredients (a) to (c) using an extruder, then spheronising and drying the extrudate. The dried pellets are coated with an enteric membrane (d) containing a plasticizer (e) and filled into two-part, hard gelatin capsules.

Composition G (Enteric-coated controlled release capsule)

Enteric capsules of Composition E can be prepared by coating the controlled-release pellets with 50mg/capsule of an enteric polymer such as cellulose acetate phthalate, polyvinylacetate phthalate, hydroxypropylmethylcellulose phthalate, or anionic polymers of methacrylic acid and methacrylic acid methyl ester (Eudragit L). Except for Eudragit L, these polymers should also include 10% (by weight of the quantity of polymer used) of a plasticizer to prevent membrane cracking during application or on storage.

Suitable plasticizers include diethyl phthalate, tributyl citrate and triacetin.

(iii) Intravenous injection composition

Active ingredient 0.200g Sterile, pyrogen-free phosphate buffer (pH 9.0) to 10 ml

The active ingredient is dissolved in most of the phosphate buffer at 35-40°C, then made up to volume and filtered through a sterile micropore filter into sterile 10 ml glass vials (Type 1) which are sealed with sterile closures and overseals.

(iv) Intramuscular injection composition

Active ingredient 0.20 g

Benzyl Alcohol 0.10 g

Glycofurol 75 1.45 g

Water for Injection q.s. to 3.00 ml

The active ingredient is dissolved in the glycofurol. The benzyl alcohol is then added and dissolved, and water added to 3 ml. The mixture is then filtered through a sterile micropore filter and sealed in sterile 3 ml glass vials (Type 1).

(v) Syrup composition

Active ingredient 0.25g

Sorbitol Solution 1.50g

Glycerol 1.00g

Sodium Benzoate 0.005g

Flavour 0.0125ml Purified Water q.s. to 5.0ml

The sodium benzoate is dissolved in a portion of the purified water and the sorbitol solution added. The active ingredient is added and dissolved. The resulting solution is mixed with the glycerol and then made up to the required volume with the purified water.

(vi) Suppository composition

mg/suppository Active ingredient 250 Hard Fat, BP (Witepsol H1δ - Dynamit NoBel) 1770

2020

One-fifth of the Witepsol H1δ is melted in a steam-jacketed pan at 4δ°C maximum. The active ingredient is sifted through a 200lm sieve and added to the molten base with mixing, using a Silverson fitted with a cutting head, until a smooth dispersion is achieved. Maintaining the mixture at 4δ°C, the remaining Witepsol H1δ is added to the suspension which is stirred to ensure a homogenous mix. The entire suspension is then passed through a 2δ0lm stainless steel screen and, with continuous stirring, allowed to cool to 40°C. At a temperature of 38-40°C, 2.02g aliquots of the mixture are filled into suitable plastic moulds and the suppositories allowed to cool to room temperature.

(vii) Pessary composition mg/pessary

Active ingredient (63lm) 250

Anhydrous Dextrose 380

Potato Starch 363

Magnesium Stearate _7

1000

The above ingredients are mixed directly and pessaries prepared by compression of the resulting mixture.

(viii) Transdermal composition

Active ingredient 200mg Alcohol USP 0.1ml Hydroxyethyl cellulose

The active ingredient and alcohol USP are gelled with hydroxyethyl cellulose and

2 packed in a transdermal device with a surface area of 10 cm .

References: I] LaRosa JC, Hunninghake D, Grundy SM, Wilson PW, Clarkson TB, Hay JW. The cholesterol facts. A summary of the evidence relating dietary habits, serum cholesterol, and coronary heart disease. A joint statement by the American Heart Association and the National Heart, Lung, and Blood Institute. Circulation

5 1990;81 :1721-1733.

2] Neaton JD, Wentworth DF,. Serum cholesterol, blood pressure, cigarette smoking and death from coronary heart disease. Overall findings and differences by age for 316,099 white men. Arch Intern Med 1992;152:δ6-63.

3] Blankenhom DH, Azen SP, Kramsch DM Mack WJ. Cashin-Hemphill L. Hodis 0 HN. DeBoer LW. Mahrer PR. Masteller MJ. Vailas LI. et al. Coronary changes with lovastatin therapy: the monitored atherosclerosis regression study (MARS).

Ann Intern Med 1993;119:969-976.

4] Effect of Simvastatin on coronary atheroma: the Multicentre Anti-Atheroma

Study (MAAS). Lancet 1994;344:633-638. δ δ] Blankenhom DH, Nessim A, Johnson RL, Sanmarco ME, Azen SP, Cashin-

HemphillL. Beneficial effects of combined colestipolniacin therapy on coronary atherosclerosis and coronary venous bypass grafts. J Am Med Assoc

1987;2δ7:3233-3240.

6] Howard BV . Lipoprotein metabolism in diabetes mellitus . J Lipid Res 0 1987;28:613-628

7] Laws A. Free fatty acids, insulin resistance and lipoprotein metabolism. Curr.

Opin. Lipidol 1996;7:172-177.

8] Gibbons GF. Bartlett SM. Sparks CE. Sparks JD. Extracellular fatty acids are not utilized directly for the synthesis of very-low-density lipoprotein in primary δ cultures of rat hepatocytes. Biochemical Journal 1992 ; 287:749763.

9] Francone OL. Kalopissis AD. Griffaton G. Contribution of cytoplasmic storage triacylglycerol to VLDL-triacylglycerol in isolated rat hepatocytes. Biochimica et

Biophysica Acta. 1989; 1002:28-36.

10] Gibbons GF. Wiggins D. Intracellular triacylglycerol lipase: its role in the 0 assembly of hepatic very-low-density lipoprotein (VLDL). Advances in Enzyme

Regulation. , 199δ ; 36:179-198

I I] Yang LY. Kuksis A. Myher JJ. Steiner G. Contribution of de novo fatty acid synthesis to very low density lipoprotein triacylglycerols: evidence from mass isotopomer distribution analysis of fatty acids synthesized from [2H6]ethanol. δ Journal of Lipid Research. 1996; 37:262-74. 12] Wiggins D. Gibbons GF. The lipolysis/esterification cycle of hepatic triacylglycerol. Its role in the secretion of very-low-density lipoprotein and its response to hormones and sulphonylureas. Biochemical Journal. 1992; 284:467- 62. δ 13] Lehner R. Verger R. Purification and characterization of a porcine liver microsomal triacylglycerol hydrolase. Biochemistry. 1997 ; 36:1861-1868. 14] Lehner R. Cui Z. Vance DE. Subcellullar localization, developmental expression and characterization of a liver triacylglycerol hydrolase. Biochemical Journal. 1999 ;338:761-768. 0 16] Lehner R and Vance DE. Cloning and expression of a cDNA encoding a hepatic microsomal lipase that mobilizes stored triacylglycerol. Biochemical Journal. (October) 1999;343:1-1 OR

16] Buchwald H. Vargo RL. Matts JP, Long JM, Fitch LL, Campbell GS, Pearce MB, Yellin AE, Edmiston WA, Smink RD Jr et al. Effect of partial ilial bypass on δ mortality and morbidity from coronary artery disease in patients with hypercholesterolemia- Report of the Program on surgical Control of the Hyperlipidemia (POSCH). New Engl J Med. 1990 ;323 :946. 17] Becker A.Bόttcher A. Lackner KJ. Fehringer P. Notka F. Aslanidis C. Schmitz G. Purification, cloning, and purification of a Huamn enzyme with Acyl coenzymz 0 A:cholesterol acyltransferase activity, which is identical to liver carboxylesterase. Arterioscler Thromb. 1994; 14:1346-13δδ.

Claims

1. A method for identifying compounds which will be useful in the treatment of conditions resulting from elevated circulating levels of: i) TG; ii) TG, VLDULDL-cholesterol and ApoB-100; or iii) VLDULDL-cholesterol and ApoB-100; comprising the step of determining whether the compound inhibits TGH activity. 0

2. A method according to claim 1 where the TGH is human TGH.

3. A method according to either claim 1 or 2 where the conditions result from elevated circulating levels of VLDULDL-cholesterol and ApoB-100. 5

4. A method according to any one of claims 1 to 3 which comprises an inhibition assay whereby the difference in enzymatic activity of TGH on a fluorogenic control substrate in the presence of a test TGH inhibitor, with that in the absence of said test inhibitor is compared. 0

5. A compound identified by a method according to any one of claims 1 to 4.

6. A pharmaceutical composition comprising a compound according to claim δ, or a physiologically acceptable salt, solvate or derivative thereof, together δ with one or more pharmaceutically acceptable carriers and optionally, one or more physiologically active agents.

7. Use of a compound according to claim 5, or a physiologically acceptable salt or solvate thereof in medical therapy. 0

8. The use of a compound according to claim 5, or a physiologically acceptable salt, solvate or derivative thereof, in the preparation of a medicament for the treatment of conditions resulting from elevated circulating levels of: i) TG; 5 ii) TG, VLDULDL-cholesterol and ApoB-100; or iii) VLDULDL-cholesterol and ApoB-100.

9. A method for the treatment of a mammal, including human, of conditions resulting from elevated circulating levels of: 5 i) TG; ii) TG, VLDULDL-cholesterol and ApoB-100; or iii) VLDULDL-cholesterol and ApoB-100; comprising administration of a compound according to claim δ.

0 10. The use of a compound which inhibits the action of TGH, or a physiologically acceptable salt, solvate or derivative thereof, in the preparation of a medicament for the treatment of conditions resulting from elevated circulating levels of: i) TG; δ ii) TG, VLDULDL-cholesterol and ApoB-100; or iii) VLDULDL-cholesterol and ApoB-100.

11.The use according to claim 10 where the TGH is human TGH.

0 12. The use according to either claim 10 or 11 where conditions resulting from elevated circulating levels of VLDULDL-cholesterol and ApoB-100.

13. The use according to claim 12 where the conditions include hypercholesterolemia, hyperbetalipoproteinemia, non-insulin dependent δ diabetes mellitus (NIDDM), coronary arterial disease, peripheral vascular disease and cerebro-vascular disease.

14. A method for the treatment of a mammal, including human, of conditions resulting from elevated circulating levels of: 0 i) TG; ii) TG, VLDULDL-cholesterol and ApoB-100; or iii) VLDULDL-cholesterol and ApoB-100; comprising administration of a compound which inhibits the action of TGH.

δ 16. The method according to claim 14 where the TGH is human TGH.