COMPOUNDS

Abstract: Polymethacrylate polymers and their use in therapy as cholesterol lowering agents.

Description: COMPOUNDS The present invention relates to novel anion exchange polymers, processes for their preparation, pharmaceutical compositions containing them and their use in the lowering of plasma cholesterol levels in humans. Coronary Heart Disease (CHD) is one of the most serious health problems of contemporary society. Worldwide epidemiological studies have shown that the incidence of CHD is related to a number of independent risk factors, in particular, for example, high concentrations of serum cholesterol (hypercholesterolaemia) . Such adverse factors lead to atherosclerosis, and ultimately, in severe cases, intermittent claudication, cerebrovascular insufficiency, thrombosis and cardiac arrest. It is known that ion exchange polymers can be used as sequestering agents to bind bile acids and salts in the intestinal tract, forming complexes which are then excreted in the faeces. This sequestering leads to a decrease in the amount of bile acids returning to the liver via enterohepatic circulation. The synthesis of replacement bile acids from hepatic cholesterol depletes hepatic cholesterol, regulates hepatic LDL receptors and consequently reduces plasma cholesterol levels. Such sequestering polymers have been recognised as useful for the treatment of hypercholesterolaemia and it is now proven that reducing serum cholesterol with bile acid sequestrants has a beneficial effect on protecting against the occurrence of coronary heart disease. SUBSTITUTE SHEET One particular agent which is currently used to lower serum cholesterol levels in humans by binding bile acids in the intestinal tract is cholestyramine. Cholestyramine is a cross-linked anion exchange polystyrene polymer bearing an ionisable trimethylammonium group bound to the polymer backbone. However, the use of this agent is associated with a number of undesirable side-effects, for example, it is unpalatable and must be taken in high doses and causes, in some cases, bloating, constipation and other gut side- effects. Furthermore, its ability to bind bile acids is inefficient with respect to the amounts of resin which it is necessary to use (up to 36 g per person per day) . In addition, other polymers have been disclosed in the art as sequestering agents, in particular US 3787474 discloses the use of polymers derived from acrylic monomers of structure RCH=CHR 1 A in which R is methyl or ethyl, R 1 is hydrogen or methyl and A is for example, C0 2 (CH ) 2 N(R 3 ) R 4 X in which R 3 is methyl or ethyl, and R 4 is hydrogen, methyl or ethyl and X is Cl~, Br~, I~ or CH 3 SO 3 - cross-linked with methyl bisacrylamide or ethylene glycol bis methacrylate; US 4393145 discloses further polymers derived from acrylic monomers cross-linked through divinyl benzene (10 to 12%) , and SE 7906129 discloses acrylic polymers cross-linked by 10-12% of a divinyl cross-linking monomer. However, despite these disclosures, no such acrylic polymers are available for human use and there remains a need for effective bile acid sequestering agents which do not have the disadvantages associated with agents currently in use. The present invention therefore provides in a first aspect, cross-linked polymers of structure (I) SUBSTITUTE SHEET in which a, b, c and d indicate the relative molar percentages of the units present in the polymer, (a) and (b) together being from about 25 to about 99.5 molar percent, and (c) being from about 0.5 to about 8 molar percent; X is a cross-linking unit; X 1 is a comonomer unit; R and R' are the same or different and are each hydrogen or C 1 _ alkyl; R 1 and R 2 are the same or different and are each C 1 _ 4 alkyl, and R 3 is C 1 _ 20 alkyl or C- L _ 20 aralkyl; or R 1 is C _ 4 alkyl and R 2 and R 3 together with the nitrogen atom to which they are attached form a saturated ring, optionally containing one or more further heteroatoms; or R 1 to R 3 together with the nitrogen atom to which they are attached form an unsaturated ring, optionally containing one or more further heteroatoms; SUBSTITUTE SHEET R 1 ' and R 2 ' are the same or different and are each C 1-4 alkyl, and R 3 is C 1 _ 20 alkyl or C 1 _ 20 aralk y 1 or R 1 ' is C 1 _ 4 alkyl and R 2 ' and R 3 ' together with the nitrogen atom to which they are attached form a saturated ring, optionally containing one or more further heteroatoms; or R 1 ' to R 3 ' together with the nitrogen atom to which they are attached form an unsaturated ring, optionally containing one or more further heteroatoms; n and n' are the same or different and are each 1 to 20; p is a number indicating the degree of polymerisation of the polymer; and Y~ is a physiologically acceptable counter ion. Suitably, (a) plus (b) is from about 25 to about 99.5 molar percent; preferably from about 60 to about 99.5 molar percent. Suitably, (c) is from about 0.5 to about 8 molar percent; preferably from about 0.5 to about 5.0 molar percent. Suitably, X is a cross-linking unit i.e. a unit which provides a random distribution of cross-links between chains of polymers. SUBSTITUTE SHEET Preferred such units include, for example, divinylbenzene, alkylene glycol bis methacrylates of structure (i) in which m is 2 to 6, z is 1 to 4 and (c) comprises from about 0.5 to about 8 molar percent of said polymer; and trismethacrylates of structure (ii) Suitably, z is l to 4, preferably z is 1. Suitably, m is 2 to 6, preferably m is 2. Suitably X 1 is a comonomer unit. Preferably X 1 is styrene, an alkyl alkylate of structure (ii) or an alkylstyrene of structure (iii) SUBSTITUTE SHEET in which R and d are as described for structure (I) and R 4 is C 1 _ 0 alkyl. In such comonomers groups R is preferably methyl and R 4 is preferably C 6 _ 12 alkyl. Suitably R 1 to R 3 or R 1 ' to R 3 ' together with the nitrogen atom to which they are attached form an unsaturated ring optionally containing one or more further heteroatoms. Suitable examples of such rings unsaturated 5 or 6 membered rings such as imidazolyl and pyridyl. More suitably, R 1 or R 1 is C 1 _ 4 alkyl and R 2 and R 3 or R 2 ' and R 3 together with the nitrogen atom to which they are attached form a saturated ring optionally containing one or more further heteroatoms. Suitable examples of saturated rings include, for example, morpholino, piperidino and piperazino rings, and in addition, bicyclic rings i.e. those in which the R 1 or R J group forms a bridge between the two nitrogen atoms of a saturated ring e.g. diazabicyclo [2.2.2] octane rings of structure Θ -N N. Preferably, R 1 and R 2 or R 1 and R 2 ' are the same or different and are each C 1 _ alkyl; more preferably R 1 and R 2 or R 1' and R are the same and are each C 1-4 alkyl, in particular methyl; and R 3 or R 3 ' is C 1 _ 20 alkyl or C 1 _ 0 aralkyl, preferably C 1 _ 0 alkyl, most preferably ~ l -" .2 ---~- r ~ ' - P rt i cu l ar C 12 alkyl. SUBSTITUTE SHEET Suitably, n is 1 to 20; preferably n is 8 to 20; most preferably n is 8 to 12. Suitably, n' is 1 to 20; preferably n' is 8 to 20; most preferably n' is 8 to 12. p is a number indicating the degree of polymerisation of the polymer. Owing to the three dimensional cross- linkage, precise figures cannot be given for p, but in any case will be greater than 1,000. Suitably Y~ is a physiologically acceptable counter ion such as a bicarbonate, carbonate, formate, acetate, sulphonate, propionate, malonate, succinate, maleate, tartrate, citrate, maleate, fumarate, ascorbate, sulphate, phosphate, halide or glucuronate; or the anion of an a ino acid such as aspartic or glutamic acid. Preferably Y~ is a sulphate, phosphate or halide ion; more preferably a halide ion, in particular a chloride ion. It is to be noted that C 1-4 alkyl and C 1 _ 20 alkyl groups as herein defined include both straight and branched alkyl groups. The polymers of the present invention are also characterised by their total exchange capacity i.e. the theoretical maximum capacity of the resin if each counter ion were to be exchanged with bile acid. In this specification the total exchange capacity is defined in terms of the number of milliequivalentε of counter ion per gram of dry weight of polymer. Suitable total exchange capacities are in the range of, for example where the counter ion Y ~ is chlorine, SUBSTITUTE SHEET from about 1.5 to about 5.0 meq Cl~ per gram of resin. Preferred within this range are polymers having a total exchange capacity of between 2 and 3 meq Cl~/gram of resin. It is to be noted that the term 'bile acid' when used herein shall be taken to include bile acids, bile salts and conjugates thereof. The polymers of the present invention can be prepared by processes analogous to those known in the art. The present invention therefore provides, in a further aspect, a process for preparing the polymers of structure (I) which comprises reaction of a polymer of structure (II) in which a, b, c, d, p, R, R' , X, X 1 , n and n' are as described for structure (I) , and Z is a group displaceable by an amine, with a compound of structure R 1 R 2 R 3 N, in which R 1 to R 3 are as described for structure (I) ; and a compound of structure R 1 R 2 R 3 'N in which R 1 ' to R- are as described for structure (I) Suitable groups Z displaceable by an amine will be apparent to those skilled in the art and include for example halogen, such as bromine. SUBSTITUTESHEET The reaction between a polymer of structure (II) and a compound of structure R 1 R 2 R 3 N and a compound of structure R 1, R 2 'R 3 'N can be carried out in a suitable solvent at elevated temperature. Suitable solvents include for example, a C j ^al a ol such as ethanol, N-methyl- pyrrolidone, dimethylformamide, tetrahydrof ran, nitro- ethane or sulpholane. Preferably the reaction is carried out in methanol at a temperature of about 40° for a period of up to 24 hours or until the reaction is complete. The intermediate polymers of structure (II) can be prepared from readily available materials by methods known to those skilled in the art. For example polymers of structure (II) in which X is a cross-link of structure (i) in which 2 is 1 and is 2, and Z is bromine and R is methyl can be prepared by reaction of the appropriate bromo alkyl methacrylate, ethylene glycol bis methacrylate, and, optionally, for example, a C 1 _ 20 alkyl alkacrylate (if a comonomer unit X 1 is desired in the final polymer) in an aqueous suspension comprising polyvinyl alcohol in the presence of an initiator at elevated temperature. Suitable initiators will be apparent to those skilled in the art and include, for example azobisisobutyronitrile benzoyl peroxide and WAKO V601 (Trade name - Me0 2 C(CH 3 )CN=NC(CH 3 ) 2 C0 2 Me) . The starting monomers can be prepared by methods apparent to those skilled in the art. For example, chloro or bromo alkyl methacrylates can be prepared by reaction of the corresponding chloro- or bromoalkanol and methacrylic anhydride in the presence of 4-dimethylaminopyridine (DMAP) in a suitable solvent such as pyridine, or by reaction of the corresponding chloro or bromo alkanol with methacryloyl chloride in the presence of a base in a suitable solvent - SUBSTITUTE SHEET suitable combinations of bases and solvents include, for example sodium bicarbonate in petroleum spirit as a solvent and pyridine as a base in toluene as solvent (cf. method described in Polymer (1987) , _l__, 325-331, and Br.Polymer J. (1984) 16, 39-45) . The polymers of structure (I) have been found to bind bile acids both in in vitro and in in vivo models. As indicated earlier it is recognised that removal of bile acids from the intestinal tract in this way lowers serum cholesterol levels and also has a beneficial effect on protecting against atherosclerosis and its dependent clinical conditions. The present invention therefore provides in a further aspect, polymers of structure (I) for use in therapy, in particular for the lowering of serum cholesterol levels in mammals, including humans. In addition the polymers of structure (I) are expected to be of use in protecting against atherosclerosis and its sequelae, and for example, in the treatment of pruritus and diarrhoea. When used in therapy polymers of structure (I) are in general administered in a pharmaceutical composition. in a still further aspect of the present invention there is therefore provided a pharmaceutical composition comprising a polymer of structure (I) in association with a pharmaceutically acceptable carrier. The compositions of the present invention can be prepared by techniques well known to those skilled in the art of pharmacy. SUBSTITUTE SHEET The polymers are preferably administered as formulations in admixture with one or more conventional pharmaceutical excipients which are physically and chemically compatible with the polymer, which are non- toxic, are without deleterious side-effects but which confer appropriate properties on the dosage form. In general, for liquid formulations, aqueous pharmaceutically acceptable carriers such as water itself or aqueous dilute ethanol, propylene glycol, polyethylene glycol or glycerol or sorbitol solutions are preferred. Such formulations can also include preservatives and flavouring and sweetening agents such as sucrose, fructose, invert sugar, cocoa, citric acid, ascorbic acid, fruit juices etc. In general, digestible oil or fat based carriers should be avoided or minimised as they contribute to the condition sought to be alleviated by use of the polymers. They are also subject to absorption by the polymers during prolonged contact, thus reducing the capacity of the polymer to absorb bile acids after administration. The polymers can also be prepared as 'concentrates' , for dilution prior to administration, and as formulations suitable for direct oral administration. They can be administered orally ad libitum, on a relatively continuous basis for example by dispersing the polymer in water, drinks or food, for example in a granule presentation suitable for admixture with water or other drink to provide a palatable drinking suspension. Preferably, the polymers are administered in tablet form or in gelatin capsules containing solid particulate polymer or a non-aqueous suspension of solid polymer SUBSTITUTE SHEET containing a suitable suspending agent. Suitable excipients for such formulations will be apparent to those skilled in the art and include, for example, for tablets and capsules, lactose, microcrystalline cellulose, magnesium, stearate, povidone, sodium starch, glycollate and starches; and for suspensions in capsules, polyethylene glycol, propylene glycol and colloidal silicone dioxide. If desired these dosage forms in addition optionally comprise suitable flavouring agents. Alternatively, a chewable tablet or granule presentation incorporating suitable flavouring and similar agents may be used. Preferably the polymer is administered in unit dosage form, each dosage unit containing preferably from 0.5 g to 1.5 g of polymer. The daily dosage regimen for an adult patient may be, for example, a total daily oral dose of between 1 and 10 g, preferably 1-5 g, the compound being administered 1 to 4 times a day. Suitably the compound is administered for a period of continuous therapy of one month or more sufficient to achieve the required reduction in serum cholesterol levels. in addition the polymers of the present invention can be co-administered (together or sequentially) with further active ingredients such as HMGCoA reductase inhibitors and other hypocholesterolaemic agents, and other drugs for the treatment of cardiovascular diseases. The following data and examples indicate the properties and preparation of the polymers of the present invention. Temperatures are recorded in degrees celsius. The exchange capacity of the ammonium substituted polymers SUBSTITUTE SHEET was determined by elemental analysis and/or potentiometric titration of chloride ion. Figures quoted are expressed as milli equivalents of exchangeable chloride ion per gram of dry resin weight; and the percent cross-linking values given are based on the ratios of the starting monomers used in the polymerisation stage. SUBSTITUTE SHEET EXAMPLE 1 (a) A suspension of 11-bromoundecanol (lOOg) and 4-dimethylaminopyridine (DMAP) (lg) in methacrylic 5 anhydride (60 ml) and pyridine (37ml) was stirred for 48 hours at room temperature. Water (400ml) was added and the aqueous phase brought to pH 3 using dilute hydrochloric acid. The aqueous phase was extracted with hexane (3 x 300ml) , water (200ml) , saturated sodium hydrogen 0 carbonate solution (2 x 400ml) and finally water (200ml) . After drying (MgS0 4 ) , the solution was concentrated in vacuo to give a clear oil (108g) . This was subjected to column chromatography on silica gel with hexane as eluent, to give 11-bromoundecyl methacrylate as a clear oil 5 (70.8g; 56% yield) . (b) 11-Bromoundecyl methacrylate (49.5g), ethylene glycol bismethacrylate (0.5g) and azobisisobutyronitrile (AIBN) (0.5g) were mixed to give a suspension and added to 0 a solution of poly(vinyl alcohol) (M.W. 125,000) (l.Og) in distilled water (500ml) . The mixture was then stirred at 80°C under an atmosphere of nitrogen at such a rate as to maintain the monomers in suspension. After 7 hours the stirring was stopped and the mixture poured into distilled 5 water. The resin formed was washed by decantation with cold and hot water, filtered, and washed with acetone and diethyl ether. Drying under reduced pressure gave an approximately 1.6 molar % cross-linked 11-bromoundecyl methacrylate co-polymer containing 3.1 meq Br/g (44.9g). 0 (c) The above 1.6 molar % cross-linked 11-bromo- undecyl methacrylate co-polymer (lOg) was suspended in dimethylformamide (lOOml) , N,N-dimethyldodecylamine (3.3g) added, and the reaction mixture heated at 70°C for 24 ; SUBSTITUTE SHEET hours. The polymer was filtered and washed with dimethyl- formamide, methanol, diethyl ether and dried under vacuum to give cross-linked ll-bromoundecyl methacrylate [ll-N,N- dimethyl-N-dodecylammonioundecyl methacrylate]chloride co-polymer containing 2.2 meq Br/g (12.46g). (d) The above co-polymer (lOg) was suspended in dimethylformamide (100ml) , 33% trimethylamine in ethanol (75ml) added, and the reaction mixture heated at 70°C for 26 hours. Additional 33% trimethylamine in ethanol (50ml) was added at 6h. After cooling the mixture was passed through a 53 micron sieve and the retained beads were washed with methanol. Anion exchange was accomplished by stirring the polymer in aqueous 2N HClr ethanol (250ml:250ml) and then allowing to stand overnight. The polymer was then filtered and washed with aqueous 2N HCl, water, methanol and diethyl ether and finally dried under vacuum to give cross-linked [11-N,N- dimethyl-N-dodecylammonioundecyl methacrylate]chloride- [ll-N,N,N-trimethylammonioundecyl methacrylate]chloride co-polymer beads (9.65g), (exchange capacity = 2.3 meq Cl " /g) . EXAMPLE 2 (a) Methacrylic anhydride (336ml) was added to a solution of 8-bromooctanol (508.9g) and pyridine (186ml) in hexane (1.5/) with cooling to 10°C in an ice-bath. After stirring at 10°C for 1 hour, 4-dimethylaminopyridine (5g) was added and the reaction was stirred for 72 hours at room temperature. The reaction mixture was washed with 2N HCl (1/) and then the aqueous phase was washed with hexane (500ml) . The combined organic extracts were washed with 2N HCl (2 x 500ml) , water, saturated sodium SUBSTITUTE SHEET hydrogen carbonate solution (2 x 500ml) , water ( 11) , dried (MgS0 ) and concentrated in vacuo to give a clear oil. This oil was subjected to column chromatography on silica gel with a hexane:dichloromethane gradient as eluent, to give 8-bromooctyl methacrylate as a clear oil (230.2g, 37%) . (b) 8-Bromooctyl methacrylate (24.75g), ll-bromo¬ undecyl methacrylate (24.75g) and ethylene glycol bismethacrylate (0.5g) were copolymerised as in Example 1(b) to give, after washing, an approximately 1.5 molar % cross-linked 8-bromooctyl methacrylate ll-bromoundecyl methacrylate co-polymer containing 3.3 meq Br/g (45.5g). (c) The above cross-linked 8-bromooctyl methacrylate ll-bromoundecyl methacrylate co-polymer (20g) was suspended in dimethylformamide (180ml) , 33% trimethyl¬ amine in ethanol (80ml) added, and the reaction mixture heated at 70°C for 30 hours. Additional 33% trimethyl- amine in ethanol (100ml) was added at 24h. Anion exchange and washing as in Example 1(d) gave [8-N,N,N-trimethyl- ammonioocty1 methacrylate]chloride[11-N,N,N-trimethy1- ammonioundecyl methacrylate]chloride co-polymer beads (20.3g), exchange capacity = 3.16 meq Cl~/G) . SUBSTITUTE SHEET Example A A chewable tablet composition can be prepared from the following : mg/tablet Compound of structure (I) : 1250 Silicon dioxide 15 Microcrystalline cellulose 280 Sorbitol 445 Lactose 450 Sweetener 5 Peppermint 30 Magnesium Stearate 25 2500mg Example B A food additive composition, for example, a sachet for reconstitution or mixing with food, is prepared by incorporating into a powder formulation compound of structure (I) (250 mg) , sodium carboxymethylcellulose (50 mg) , sucrose (2400 mg) and flavours (50 mg) . SUBSTITUTE SHEET DATA In vitro Dissociation assay The following assay provides a measure of affinity of the polymers of the invention for the bile acid, glycocholate (GC) based on the amount of GC bound at a subsaturating concentration of 5mM (t=0) , and an estimate of the rate at which this bile acid dissociates into a large volume of buffer. The results are obtained as initial amounts of GC bound (t=0) and amounts remaining bound after 2 minutes in buffer (t=2min) ; from these figures the % dissociation i.e. the proportion of bound GC dissociated from the polymer after 2 minutes can be obtained. The lower the % dissociation the more efficient the polymer can be expected to be in extracting bile acids in vivo. Method Test compound (150 mg) is equilibrated with 5mM sodium glycocholate (30 ml) in Krebs' buffer. The compound is separated by centrifugation and the total bound determined by subtraction of the amount in the supernatant from the total bile acid used. Dissociation is measured by resuspending the compound in Krebs' buffer, shaking and sampling the mixture through a filter at several time points up to 20 minutes. Radioactivity and hence bile acid dissociated is determined in the filtrate. SUBSTITUTE SHEET

Claims: Claims 1. A polymer of structure (I) in which a, b, c and d indicate the relative molar percentages of the units present in the polymer, (a) and (b) together being from about 25 to about 99.5 molar percent, and (c) being from about 0.5 to about 8 molar percent; X is a cross-linking unit; X I is a comonomer unit; R and R' are the same or different and are each hydrogen or C 1 _ 4 alkyl; R 1 and R 2 are the same or different and are each C 1 _ alkyl, and R 3 is C 1 _ 2 o a l k yl or C 1 _ 20 a ralkyl; or R 1 is C 1 _ 4 alkyl and R 2 and R 3 together with the nitrogen atom to which they are attached form a saturated ring, optionally containing one or more further heteroatoms; SUBSTITUTE SHEET or R 1 to R 3 together with the nitrogen atom to which they are attached form an unsaturated ring, optionally containing one or more further heteroatoms; R 1 ' and R 2 ' are the same or different and are each C 1 _ 4 alkyl, and R 3 ' is C 1 _ 20 alkyl or C 1 _ 20 aralkyl; or R 1 ' is C 1 _ 4 alkyl and R 2 ' and R 3 ' together with the nitrogen atom to which they are attached form a saturated ring, optionally containing one or more further heteroatoms; or R 1 ' to R 3 together with the nitrogen atom to which they are attached form an unsaturated ring, optionally containing one or more further heteroatoms; n and n' are the same or different and are each l to 20; p is a number indicating the degree of polymerisation of the polymer; and γ ~ is a physiologically acceptable counter ion. 2. A polymer of structure (I) as claimed in claim 1 for use in therapy. 3. A polymer of structure (I) as claimed in claim 1 for use in the lowering of serum cholesterol levels. 4. A pharmaceutical composition comprising a polymer of structure (I) as claimed in claim 1, in association with a pharmaceutically acceptable carrier. SUBSTITUTE SHEET 5. A process for preparing a polymer as claimed in claim 1 which comprises reaction of a polymer of structure (II) in which a, b, c, d, p, R, R', X, X 1 , n and n' are as described for structure (I) , and Z is a group displaceable by an amine, with a compound of structure R 1 R R 3 N, in which R 1 to R 3 are as described for structure (I) ; and a compound of structure R 1 R 2 R 3 N in which R 1 ' to R- are as described for structure (I) . SUBSTITUTE SHEET

Inventors: BENSON GREGORY MARTIN, HICKEY DEIRDRE MARY BERNADETTE, BENSON, GREGORY, MARTIN, HICKEY, DEIRDRE, MARY, BERNADETTE
Applicants: SMITH KLINE FRENCH LAB, SMITH KLINE & FRENCH LABORATORIES LIMITED
Application year: 1991
Main classification: C08F8
IPC codes: A61K31, A61P3, C08F8