EP0952834A2 - Isopentylpyrophosphate isomere (ipi) und/oder prenyltransferase hemmern - Google Patents

Isopentylpyrophosphate isomere (ipi) und/oder prenyltransferase hemmern

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Publication number
EP0952834A2
EP0952834A2 EP97921954A EP97921954A EP0952834A2 EP 0952834 A2 EP0952834 A2 EP 0952834A2 EP 97921954 A EP97921954 A EP 97921954A EP 97921954 A EP97921954 A EP 97921954A EP 0952834 A2 EP0952834 A2 EP 0952834A2
Authority
EP
European Patent Office
Prior art keywords
ipi
prenyl transferase
inhibitor
bisphosphonate
drug
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97921954A
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English (en)
French (fr)
Inventor
Richard John Brown
Donald Jeremy Watts
Robert Graham Goodwin Russel
Michael John University of Sheffield ROGERS
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University of Sheffield
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University of Sheffield
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Publication date
Priority claimed from GBGB9610174.6A external-priority patent/GB9610174D0/en
Priority claimed from GBGB9708329.9A external-priority patent/GB9708329D0/en
Application filed by University of Sheffield filed Critical University of Sheffield
Publication of EP0952834A2 publication Critical patent/EP0952834A2/de
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/662—Phosphorus acids or esters thereof having P—C bonds, e.g. foscarnet, trichlorfon
    • A61K31/663—Compounds having two or more phosphorus acid groups or esters thereof, e.g. clodronic acid, pamidronic acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/533—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving isomerase

Definitions

  • the present invention relates to prenyl transferase (farnesyl pyrophosphate synthase) and isopentenyl pyrophosphate isomerase (IPI) inhibitors, and to the use of such inhibitors in various forms of therapy and prophylaxis.
  • the present invention relates to the field of bone metabolism (e.g. bone resorption), and in particular to the use of prenyl transferase and IPI inhibitors in the treatment of various diseases and disorders of bone metabolism and to the use of prenyl transferase in the screening, isolation, synthesis and evaluation of osteoactive drugs.
  • Lipids occur in the blood mainly as cholesterol and triglycerides, with smaller amounts of phospholipids, fatty acids, and fatty acid esters.
  • cholesterol and triglycerides are complexed with proteins (known as apolipoproteins) and transported in the form of lipoprotein particles.
  • the lipoprotein particle surface is composed largely of phospholipid, free cholesterol and protein, and the core contains mostly triglyceride and cholesterol esters.
  • Lipoprotein particles are divided into four major groups on the basis of density, composition and electrophoretic mobility. Chylomicrons are formed in the intestine and are large triglyceride-rich particles derived from dietary fat. Very low density lipoproteins (hereinafter VLDLs) are composed largely of endogenous triglycerides and are synthesised in the liver. Low density lipoproteins (hereinafter LDLs) are rich in cholesterol and are mainly end products of VLDL catabolism. High density lipoproteins (HDLs) contain about 50% protein, are produced in the liver and intestine and act as acceptors of lipids, especially free cholesterol.
  • VLDLs Very low density lipoproteins
  • LDLs Low density lipoproteins
  • HDLs High density lipoproteins
  • Hyperlipidaemia (or hyperlipoproteinaemia) embraces a group of disorders associated with elevated levels of lipids in the blood. Hyperlipidaemias may be inherited or secondary to dietary factors, excessive alcohol intake or primary disease states (such as hypothyroidism and diabetes mellitus) , and can also arise as a side effect of various drug treatments (e.g. the administration of beta-blockers, corticosteroids, oestrogens, oral contraceptives or thiazide diuretics). Other factors affecting the lipid composition of the blood include physical activity, age and sex.
  • Type I hyperchylomicronaemia characterised by the presence of chylomicrons and by normal or only slightly increased concentrations of very low- density lipoproteins;
  • Type Ila hyper- ⁇ -lipoproteinaemia characterised by an elevation in the concentration of low-density lipoproteins
  • Type lib characterised by an elevation in the concentration of low-density lipoproteins and of very low-density lipoproteins
  • Type III (floating ⁇ " or “broad fl” pattern) characterised by the presence of very low-density lipoproteins having an abnormally high cholesterol content and an abnormal electrophoretic mobility;
  • Type IV hyperpre- ⁇ -lipoproteinaemia characterised by an elevation in the concentration of very low- density lipoproteins , but no increase in the concentration of low-density lipoproteins, and by the absence of chylomicrons ;
  • Typ e v hyperpre- ⁇ -lipoproteinaemia and chylomicronaemia characterised by an elevation in the concentration of very low-density lipoproteins and the presence of chylomicrons.
  • Primary hyperlipidaemias may also be classified according to the genetic and metabolic disorder resulting in the following categories:
  • familial hypercholesterolaemia which is usually heterozygous but very rarely may be homozygous, characterised by a type Ila pattern (occasionally a type lib pattern is present) ;
  • familial combined hyper lipidaemia characterised by elevated cholesterol only, elevated triglyceride only, or elevated cholesterol and triglyceride - type Ila, type IV, or type lib patterns may be found;
  • familial dysbetalipoproteinaemia familial dysbetalipoproteinaemia (remnant hyperlipoproteinaemia or broad- ⁇ disease) showing the type III pattern;
  • hypolipidaemic agents can find application in the treatment and prophylaxis of a wide range of cardiovascular disorders associated with hyperlipidaemia.
  • bile acid sequestrants colestipol hydrochloride and cholestyramine
  • niacin probucol
  • gemfibrozil neomycin sulphate
  • statins are the "statins" (Hoeg and Brewer, 1987, JAMA, Vol.258 (24), pages 3532-3536). These compounds act at the beginning cf the cholesterol biosynthetic pathway (see Figure 1) by competitively inhibiting 3 -hydroxy- 3 -methylglut aryl coenzyme A reductase (herinafter HMGCoAR, the rate determining enzyme for cholesterol synthesis). All of the statins presently in use are HMG-CoA analogues, and include mevastatin (compactin), lovastatin (mevinolin), prevastatin (eptastatin) and simvastatin (synvinolin) .
  • statins work by increasing the cataboiism of LDL as well as decreasing its synthesis.
  • LDL particles contain apolipoprotein B (apoB) , and are removed from circulation by means of high affinity LDL receptors which recognize apoB. These receptors are upregulated by the cholesterol depletion induced by the statins.
  • apoB apolipoprotein B
  • LDL receptors which recognize apoB.
  • HMGCoAR inhibitors as hypolipidaemic agents is associated with several problems.
  • Adverse reactions include gastro-intestinal disturbances, headache, rash and pruritus. Liver damage can occur in hepatocompromized subjects, as well as myositis with increased values for creatine phosphokinase.
  • severe muscle effects including myolysis, rhabdomyoiysis, myositis and myolitis
  • the inhibitors may also interfere with DNA replication and there are reports of carcinogenicity and teratogenicity.
  • Nonresponders can amount to 20% of the target patient population, not including those patients lacking functional LDL receptors (such as those homozygous for familial hypercholesterolaemia) which also fail to respond.
  • statins Some workers have ascribed certain side effects to the fact that the metabolic block induced by the statins effectively shuts down the isoprenoid biosynthetic pathways which lead from farnesyl pyrophosphate to a large number of different products (including ubiquinone and dolichol) . Since many of these products are essential for cell viability, it has been suggested that at least some of the side effects associated with the statins might arise from the block imposed on the isoprenoid pathways rather than from that on cholesterol biosynthesis per se.
  • statins particularly those associated with the alleviation of cardiovascular disease events associated with hyperlipidaemia
  • mevalonate pathway products other than cholesterol particularly the isoprenoids
  • eff ective inhibition of the cholesterol biosynthetic pathway depends to a significant extent on negative feedback arising from suppression of other isoprenoid pathways .
  • the squalene synthase inhibitors may not be as effective as the statins in alleviating cardiovascular disease attendant on hyper lipidaemia , and may be less effective in lowering cholesterol levels .
  • hypolipidaemics which exhibit reduced side effects and increased efficacy and breadth of application .
  • Bone destruction can result from various cancers and also from rheumatoid arthritis .
  • Metabolic bone disorders commonly involve excessive bone resorption and include
  • Paget ' ⁇ disease hypercalcaemia (both tumour-induced and non-tumour induced), bone metastases and osteoporosis.
  • Paget's disease (a focal increase in bone turnover) is fairly common, and in some countries affects up to 5% of the population over 50 years of age.
  • the disease may be caused by a slow virus infection, and leads to bone pain, deformities and fractures.
  • Bone metastases can induce bone destruction either through local invasion or via the secretion of bone- resorbing agents into the blood stream.
  • Hypercalcaemia can result either from an increase in the flow of calcium from bone or the intestine to the blood, or from an increase in tubular reabsorption of calcium in the kidney. It can induce a wide variety of physiological disturbances and can be life threatening.
  • Osteoporosis is characterized by a reduction in the quantity of bone leading to atrophy of skeletal tissue.
  • osteoporosis is another common symptom.
  • Primary osteoporosis is the result of an identifiable disease process or agent, while primary osteoporosis (which constitutes about 90% of all cases) includes postmenopausal osteoporosis, age-associated osteoporosis (affecting a majority of individuals over the age of 70) and idiopathic osteoporosis affecting middle-aged and younger men and women.
  • Bone remodeling occurs throughout life, renewing the skeleton and maintaining the strength of bone.
  • This remodelling involves the erosion and filling of discrete sites on the surface of bones by an organized group of cells called “basic multicellular units” or "BMUs".
  • BMUs basic multicellular units
  • 3MUs primarily consist of osteoclasts, osteoblasts, and their cellular precursors.
  • bone is resorbed at the site of an "activated" BMU by an osteoclast, forming a resorption cavity. This cavity is then filled with bone by osteoblasts.
  • compositions and methods are described in the medical literature for the treatment of the above- described diseases and conditions, and most attempt to either slow the loss of bone or produce a net gain in bone mass.
  • oestrogen has been used as a means both to prevent and to treat osteoporosis in postmenopausal women.
  • the use of oestrogen has been associated with certain side effects, such as uterine bleeding.
  • Other treatments are based on the administration of parathyroid hormone.
  • the hormone calcitonin has also been used to treat Paget's disease (and to a lesser extent tumour bone disease) , and can be effective in decreasing bone turnover.
  • Paget's disease and to a lesser extent tumour bone disease
  • the incidence of relapse is high, and side effects on the vascular system limit the therapeutic usefulness of calcitonin.
  • One of the most successful class of drugs f or the treatment of the above diseases has proved to be the bisphosphonates .
  • inorganic pyrophosphate and polyphosphate have high affinity for bone mineral and are able to inhibit the precipitation and dissolution of calcium phosphate crystals in vitro and to inhibit bone mineralization in vivo .
  • These activities are thought to arise from direct physicochemical effects (such as adsorption to hydroxyapatite , inhibition of dissolution of hydroxyapatite and crystal growth inhibition) .
  • Pyrophosphates have therefore found application as bone imaging agents for diagnostic purposes ( usually in the form of ' ' " technetium derivatives ) and as antitartar agents for use in toothpastes .
  • inorganic pyrophosphates are rapidly hydrolysed following administration (particularly oral administration) due to the presence of the relatively labile phosphorus-oxygen bond P-O-P .
  • This severely limits their pharmaceutical utility and has prompted a search for PPi analogues which exhibit similar physicochemical activities while resisting enzymatic hydrolysis in vivo .
  • Bisphosphonates which are characterized by phosphorus- carbon (P-C-P) bonds, are stable analogues of naturally occurring inorganic pyrophosphates which to a great extent overcome the limitations associated with inorganic pyrophosphates. Bisphosphonates are resistant to chemical and enzymatic hydrolysis but retain the therapeutic activity of PPi.
  • bisphosphonates exhibit properties which extend beyond those attributable to purely physicochemical phenomena.
  • bisphosphonates have been found to be inhibitors of osteoclast-mediated bone resorption in organ cultures of bone and in animal models.
  • Bisphosphonates therefore have broader clinical utility than PPi, and have found widespread application in four main clinical areas: (a) as bone imaging agents for diagnostic purposes, usually in the form of ' ' " technetium derivatives, (b) as anti- resorptive agents to combat bone loss associated with Paget 's disease, hypercalcaemia associated with malignancy and bone metastases or osteoporosis, (c) as calcification inhibitors in patients with ectopic calcification and ossification, and (d) as a ⁇ titartar agents for use in toothpastes.
  • Bisphosphonates are now among the most important therapeutic agents for the treatment of pathological disorders of bone metabolism, including osteoporosis . Moreover, since some bisphosphonates appear to have anti- inflammatory as well as anti-resorptive effects in vivo , they may also have utility in the treatment of inflammation and rheumatoid arthritis .
  • bisphosphonates may affect the differentiation and recruitment of osteoclast precursors or alter the capacity of mature osteoclasts to resorb bone by altering the permeability of the osteoclast membrane to small ions .
  • Another hypothesis is that they act by affecting lysosomal enzyme production or cell metabolism or through toxic effects on osteoclasts .
  • a further suggestion is that bisphosphonates affect other cells in the bone microenvironment that regulate the activity of the osteoclasts that are involved in the antiresorptive mechanism.
  • the bisphosphonates represent a important class of drug which has opened up new approaches in the therapy of bone diseases .
  • the bisphosphonates are characterized by poor intestinal absorption and the ideal bisphosphonate would show substantial and consistent intestinal absorption, consistent and reversible effects on bone turnover, low toxicity and (for appropriate treatment regimens) shortened residence time in bone.
  • Present bisphosphonates fulfil these ideals only to a limited degree.
  • prenyl transferase catalyzes the condensation of isopentenyl pyrophosphate with an allylic pyrophosphate at a point in the sterol biosynthetic pathway just prior to the branchpoint into the sterol and isoprenoid pathways (see Figures 1 and 3).
  • Isopentenyl pyrophosphate isomerase catalyses interconversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate ( Figure 3 ⁇ via a carbocation intermediate.
  • IPI and/or prenyl transferase inhibitors for use in various forms of therapy and prophylaxis.
  • the IPI and/or prenyl transferase inhibitors of the present invention find particular utility in the modulation of lipid metabolism, cell proliferation, isoprenoid-related cellular apoptosis and cellular signal transduction.
  • the IPI and/or prenyl transferase inhibitors are also useful as fungicides or herbicides.
  • the inhibitors may have dual inhibitory activity, and inhibit both IPI and prenyl transferase. They may also inhibit other isoprenoid synthetic enzymes.
  • the present invention also provides a new class of antiresorptive (osteoactive) drugs based on the presence of IPI and/or prenyl transferase inhibitory activity, and provides methods for rapidly and efficiently screening a large number of potential osteoactive drugs (e.g. improved osteoactive bisphosphonates) and for the identification and synthesis of compounds having improved antiresorptive and/or anti-inflammatory activity.
  • osteoactive drugs e.g. improved osteoactive bisphosphonates
  • Prenyl transferase inhibitors, IPI inhibitors and inhibitors of IPI and/or prenyl transferase lipid metabolism, cell proliferation, apoptosis and signal transduction
  • Prenyl transferase catalyzes the condensation of isopentenyl pyrophosphate with an allylic pyrophosphate
  • the prenyl transferase inhibitors of the present invention are inhibitors of this enzyme. It is specific for isopentenyl pyrophosphate but can use either the 5-carbon dimethylallyl pyrophosphate (farnesyl diphosphate synthase I) or the 10 carbon geranyl pyrophosphate
  • the site of action of the IPI and prenyl transferase inhibitors of the present invention is shown in Figure 1 , along with those of the known HMGCoA reductase and squalene synthase inhibitors. It can be seen that the IPI and/or prenyl transferase inhibitors of the present invention act at a point in the cholesterol biosynthetic pathway before the synthesis of farnesyl pyrophosphate. In contrast, the known squalene synthase inhibitors act after the synthesis of farnesyl pyrophosphate.
  • the IPI and/or prenyl transferase inhibitors of the invention are upstream inhibitors, in that they inhibit both the cholesterol biosynthetic pathway and the various isoprenoid pathways.
  • the IPI and/or prenyl transferase inhibitors of the invention act at the step immediately preceding the branch point into the various cholesterol/isoprenoid pathways. This has important consequences with respect to the nature of the perturbations induced in the pools of mevaionate metabolites, as explained below.
  • HMGCoA reductase mediates the rate-determining step of cholesterol biosynthesis and is the most elaborately regulated enzyme of the cholesterol biosynthetic pathway. It is subject to control by both competitive and allosteric mechanisms, by phosphorylation and dephosphorylation as well as by long-term regulation. Cholesterol and other metabolites are feedback regulators of the enzyme.
  • inhibition of HMGCoA reductase profoundly perturbs the sizes of a large number of different metabolite pools, and some of the side effects associated with the use of the statins arise from these perturbations.
  • the reduction of cholesterol levels relieves negative feedback on the HMGCoA reductase and results in a large increase in the effective concentration of the enzyme. This blunts the effect of the statins and limits the extent of cholesterol reductions attainable.
  • the IPI and/or prenyl transferase inhibitors of the present invention do not act directly on HMGCoA reductase and so disrupt a smaller range of metabolite pools than the statins. They therefore have important benefits as hypolipidaemics. in particular, they exhibit reduced side effects, greater activity as hypolipidaemics and can be used in a broader class of patients.
  • the IPI and/or prenyl transferase inhibitors of the invention can be used as hypolipidaemic agents, and in particular as hypocholesterolaemic agents. They find application in lowering serum cholesterol levels, in the treatment or prophylaxis of hypercholesterolaemia, hyperlipidaemia, hyperlipoproteinaemia, nephrotic hyperlipidaemia or atherosclerosis, in increasing HDL cholesterol levels while lowering LDL cholesterol and serum triglyceride levels, in the treatment or prophylaxis of cardiovascular disease (e.g. atherosclerosis and other arterial lesions) and for the prevention of restenosis after coronary angioplasty.
  • cardiovascular disease e.g. atherosclerosis and other arterial lesions
  • IP! and/or prenyl transferase inhibitors of the invention may be administered to any or all of the following classes of patients: (a) hepatocompromized individuals; (b) individuals with a prior history of liver dysfunction;
  • mammals e.g. humans
  • IPI and/or prenyl transferase inhibitors of the invention may be used in the treatments of any cf the following types of hyperlipoproteinaemias:
  • Type I hyperchylomicronaemia characterised by the presence of chylomicrons and by normal or only slightly increased concentrations of very low- density lipoproteins;
  • Type Ila hyper- ⁇ -lipoproteinaemia characterised by an elevation in the concentration of low-density lipoproteins
  • Type lib characterised by an elevation in the concentration of low-density lipoproteins and of very low-density lipoproteins
  • Type III ('floating ⁇ ' or 'broad ⁇ pattern) characterised by the presence of very low-density lipoproteins having an abnormally high cholesterol content and an abnormal electrophoretic mobility;
  • Type IV hyperpre- ⁇ -lipoproteinaemial characterised by an elevation in the concentration of very low- density lipoproteins, but no increase in the concentration of low-density lipoproteins, and by the absence of chylomicrons
  • Type V 'hyperpre- ⁇ -lipoproteinaemia and chylomicronaemia' characterised by an elevation in the concentration of very low-density lipoproteins and the presence of chylomicrons.
  • familial hypercholesterolaemia which is usually heterozygous but very rarely may be homozygous, characterised by a type Ila pattern (occasionally a type lib pattern is present) ;
  • familial dysbetalipoproteinaemia familial dysbetalipoproteinaemia (remnant hyperiipoproteinaemia or broad- ⁇ disease) showing the type III pattern; and (e) lipoprotein lipase deficiency or apolipoprotein C-ll deficiency showing a type I of type V pattern.
  • the IPI and/or prenyl transferase inhibitors of the invention also find application in the modulation of cell proliferation and in particular in the treatment of prophylaxis of disorders involving cell proliferation (for example various cancers, including cancers and cancer metastases in bone) .
  • IPI and/or prenyl transferase inhibitors block the synthesis of these products, and are therefore also useful in the treatment of diseases characterised by cell proliferation.
  • the prenyl transferase and/or IPI inhibitors of the invention also potentiate the effects of other drugs and treatments affecting cellular growth, such as radiotherapy, surgery and drugs used in cancer chemotherapy, immunotherapy or immunosuppression.
  • the IPI and/or prenyl transferase inhibitors of the present invention may be administered as adjuncts to radiotherapy, chemotherapy, immunotherapy or surgery in the treatment of cancer, particularly of ras-related cancers.
  • ras- related cancers include lung, bladder, colon and brain cancers.
  • Protein-prenyl transferase acts at a point downstream of farnesyl pyrophosphate (see Figure 1 ) and the inhibitors described in EP0537008 do not block or reduce the flow of farnesyl pyrophosphate into the various prenylation pathways. In this respect they differ fundamentally from the IPI and/or prenyl transferase inhibitors of the present invention.
  • the anti-cancer activity of the IPI and/or prenyl transferase inhibitors of the invention arise from a block of protein prenylation (for example, prenylation of CaaX box containing proteins such as the G proteins, members of the ras family and other oncoproteins) by reducing or eliminating input into the isoprenoid pathways shown in Figure 1 .
  • protein prenylation for example, prenylation of CaaX box containing proteins such as the G proteins, members of the ras family and other oncoproteins
  • doiichol pools blocks the synthesis of glycoproteins and changes membrane fluidity, which are important for cell growth.
  • the IPI and/or prenyl transferase inhibitors of the present invention reduce metabolic pools of prenyls (e.g. farnesyl pyrophosphate) which are involved in the post-translational modification of CaaX box containing proteins (including proteins in the r_as family and G-proteins).
  • prenyls e.g. farnesyl pyrophosphate
  • the inhibitors of the present invention reduce metabolic pools of farnesyl pyrophosphate, which is the donor of the farnesyl group to CaaX box containing proteins such as the ras p21 oncoprotein.
  • the activity of CaaX containing proteins such as the ras oncoprotein and G proteins can be blocked by preventing their post-translational modification by administration of the inhibitors of the invention.
  • the prenyl transferase and/or IPI inhibitors of the invention can be used to prevent prenylation
  • the inhibitors of the present invention find application in the treatment and/or prophylaxis of tumours.
  • the prenyl transferase and/or IPI inhibitors of the invention are used to prevent or reduce the farnesylation of ras in the modulation of cell proliferation, isoprenoid-related cellular apoptosis or cellular signal transduction.
  • the inhibitors of the present invention also find application in the modulation of isoprenoid-related cellular apoptosis, for example in the treatment or proyphlaxis of various autoimmune diseases (such as rheumatoid arthritis) and chronic inflammatory diseases.
  • the inhibitors of the present invention also find application in the modulation of cellular signal transduction, for example in the treatment or prophylaxis of graft (e.g. allograft) rejection.
  • Protein prenylation increases protein hydrophobicity and promotes protein- membrane interactions.
  • Prenytated ras protein localizes to the inner cell membrane and appears to function as a mediator through which various peptide growth factors and cytokines signal to stimulate intracellular events.
  • Binding of these substances to their respective receptors on target cells activates ras and triggers inter alia cell proliferation, differentiation, and T-cell activation.
  • the inhibitors of the invention block or reduce ras prenylation, and ras has now been discovered to play an important role in chronic allograft rejection (O'Donnell et al. , 1995,
  • the prenyl transferase inhibitors of the invention ameliorate chronic rejection.
  • the IPI and/or prenyl transferase inhibitors of the present invention can also be used to block sterol synthesis in plants and fungi . They therefore find utility as fungicides and/or herbicides.
  • IPI and/or prenyl transferase inhibitors of the invention are thought to act as stable analogues of the carbocation intermediate shown in Figure 2, thereby blocking the synthesis of farnesyl pyrophosphate.
  • prenyl transferase inhibitors of the present invention preferably comprise structural and/or functional analogues of the carbocation:
  • analogues may be bisphosphonates (either geminal or non geminal bisphosphonates), and may be structural and/or functional analogues of the carbocation:
  • R is an alkyl side chain, for example having > 5 (for example about 10) carbon atoms.
  • the analogues may also be sulphonates, carboxylates, phosphonocarboxylates, phosphonosulphonates, phosphonophosphates, or pyrophosphates.
  • Preferred inhibitors constitute a subclass of bisphosphonates which inhibit either or both of IPI and prenyl transferase (the latter hereinafter referred to as prenyl transferase inhibitory bisphosphonates or PIBs and the former as IPI inhibitory bisphosphonates or IPIBs), or analogues or derivatives thereof.
  • the IPIB/PIB is preferably selected from the large number of known osteoactive bisphosphonates (see e.g. Fleisch, H., 1993, Bisphosphonates in bone disease, ISBN 3-9520459-0-X, the contents of which are incorporated herein by reference).
  • the IPI and/or prenyl transferase inhibitor of the invention may also be an analogue or derivative of the osteoactive IPIB or PIB, for example a derivative having a group for promoting cellular uptake (e.g. a lipophilic group or a dipeptide group).
  • a derivative having a group for promoting cellular uptake e.g. a lipophilic group or a dipeptide group.
  • the IPI and/or prenyl transferase inhibitors have a positively charged nitrogen atom.
  • Particularly preferred are PIBs or IPIBs having a positively charged nitrogen atom.
  • Examples of such bisphosphonates are alendronate, pamidronate and ibandronate, and analogues or derivatives thereof including nitrogen ring-containing (heterocyclic) compounds.
  • the IPI prenyl transferase inhibitors may be modified in a variety of ways to modify their pharmacokinetics and in vivo localization.
  • the IPl/prenyl transferase inhibitors may be modified or derivatized to render them more lipophilic using techniques known to those skilled in the art.
  • the inhibitors may be modified by the attachment of a lipophilic group or of a dipeptide group permitting uptake via the cellular plasma membrane peptide carrier system (see e.g. work by Breuer et al at the School of Pharmacy at the Hebrew University of Jerusalem).
  • inhibitors of the invention may be prodrugs having enhanced bioavailablity.
  • the prenyl transferase/IPI inhibitors When used to treat cancers, the prenyl transferase/IPI inhibitors may be modified to target them to the tumour site.
  • the IPIBs/PIBs accumulate in bone, and so are particularly preferred in the treatment of cancers and cancer metastases in bone.
  • the invention also relates to the use of an IPI and/or prenyl transferase inhibitor for the manufacture of a medicament for the regulation of bone metabolism, for example in the treatment of Paget's disease, hypercalcaemia (both tumour-induced and non- tumour induced), bone metastases or osteoporosis in both men and women (e.g. associated with secondary causes such as administration of glucocorticoids), wherein the inhibitor is not a geminal bisphosphonate.
  • the invention permits the identification of improved phosphonate drugs with reduced affinity for bone.
  • the invention relates to an IPI and/or prenyl transferase inhibitor for use in therapy or prophylaxis, e.g. in the manufacture of a medicament for the regulation of bone metabolism, for example in the treatment of Paget's disease, hypercalcaemia (both tumour-induced and non-tumour induced), bone metastases or osteoporosis, wherein the inhibitor is not a geminal bisphosphonate.
  • bisphosphonate is used herein in a broad sense to cover not only bisphosphonate sensu stricto, but also bisphosphonate analogues.
  • Bisphosphonate analogues are those ligands which can compete with osteoactive bisphosphonate for binding to the IPI and/or prenyl transferase of the invention, or which can compete ijn vitro (for example, on an affinity column) with osteoactive bisphosphonate (in either the free state or in the form of a derivative linked to an affinity column) for binding to the IPI and/or prenyl transferase of the invention.
  • Bisphosphonate analogues include pyridoxal phosphate, 0- phosphorylethanolami ⁇ e, o-phosphorylcholine, phosphatidyl ethanolamine and phospholipid bisphosphonate analogues.
  • Osteoactive drugs are those which act as antiresorptive and/or anti-inflammatory drugs in vivo.
  • the prenyl transferase and/or IPI inhibitor for use in the therapies listed above is preferably a structural and/or functional analogue of the carbocation:
  • analogues may be bisphosphonates (either geminal or non geminal bisphosphonates), and may be structural and/or functional analogues of the carbocation:
  • R is an alkyl side chain, for example having >5 (for example about 10) carbon atoms.
  • the inhibitors may also be sulphonates, carboxylates, phosphonocarboxylates, phosphonosulphonates, phosphonophosphates, or pyrophosphates.
  • the IPI and/or prenyl transferase inhibitor may comprise a positively charged nitrogen atom.
  • the invention also contemplates the use of IPI and/or prenyl transferase for binding a bisphosphonate, for example in an in vitro assay.
  • the IPI and/or prenyl transferase is one which, in vivo, mediates the physiological (e.g. antiresorptive) effects of osteoactive bisphosphonate. It may also be a Dictvostelium spp., for example Dictvostelium discoideum, prenyl transferase.
  • the invention also embraces a method for screening for osteoactive drugs (e.g. bisphosphonates) comprising the steps of: (a) contacting a putative drug (e.g. a bisphosphonate) with the IPI and/or prenyl transferase of the invention, and (b) determining whether interaction (e.g. binding) between the putative drug and IPI and/or prenyl transferase occurs, wherein interaction is indicative of an osteoactive drug (e.g. an osteoactive bisphosphonate).
  • the relative affinity (interaction) with IPI and prenyl transferase may also be determined in the methods of the invention.
  • the presence or degree of interaction between the putative drug and IPI and/or prenyl transferase is measured or determined by an enzyme inhibition assay.
  • enzyme inhibition assays may be conducted according to any of the standard IPI and/or prenyl transferase assay protocols known to those skilled in the art.
  • the method described above is useful for screening large numbers of synthetic drugs for therapeutic activity. It may also be used to classify potential osteoactive or anti-arthritic bisphosphonates into different groups according to their mode of action (or their cellular targets) . Preferably, the method is employed in high throughput screening of drug candidates. Compounds identified by the method of the invention can be further modified or used directly as therapeutic compounds, for example in the treatment of osteoporosis.
  • a method for evaluating the therapeutic activity of a putative drug comprising the steps of: (a) contacting the drug with IPI and/or prenyl transferase, and (b) measuring the binding affinity of the putative drug for the prenyl transferase and/or IPI, or the relative binding affinities for prenyl transferase and IPI.
  • a putative drug e.g. a bisphosphonate
  • the method described above is useful e.g. for ranking the therapeutic activity of potential drugs (e.g. potential bisphosphonate drugs).
  • the invention in another aspect relates to a method for synthesising a therapeutically active (e.g. antiresorptive or antiarthritic) drug (e.g. a bisphosphonate) comprising the steps of: (a) providing a three- dimensional model comprising a catalytic site of the prenyl transferase of the invention (e.g. by computer analysis of its amino- acid sequence or by X-ray crystallography of the prenyl transferase or fragment thereof), and (b) modelling the therapeutically active drug with reference to the three-dimensional model generated in step (a).
  • a therapeutically active drug e.g. antiresorptive or antiarthritic
  • a bisphosphonate e.g. a bisphosphonate
  • the three- dimensional model is generated by computer analysis of the amino- acid sequence of all or a portion of the IPI prenyl transferase (for example a catalytic site thereof or a bisphosphonate binding domain thereof). Examples of such models are known to those skilled in the art.
  • the three-dimensional model could be generated by X- ray crystallography of prenyl transferase/IPI (or fragments/derivatives thereof), or by NMR techniques. These techniques could also be applied to the IPI/prenyl transferase-drug (e.g. IPI/prenyl transferase-bisphosphonate) complex, the results of which could also be used as the basis for the rational design of therapeutic agents.
  • IPI/prenyl transferase-drug e.g. IPI/prenyl transferase-bisphosphonate
  • the invention also covers therapeutically active drugs (e.g. a bisphosphonate) which have been screened, evaluated or synthesised by the methods of the invention.
  • therapeutically active drugs e.g. a bisphosphonate
  • the invention also contemplates an antibody (e.g. a monoclonal antibody) which binds to the IPI and/or prenyl transferase of the invention.
  • an antibody e.g. a monoclonal antibody which binds to the IPI and/or prenyl transferase of the invention.
  • the antibodies of the invention may advantageously bind specifically to the IPI and/or prenyl transferase of the invention (e.g. specifically to both) .
  • Antibodies specific for the catalytic site of IPI and/or prenyl transferase may act as bisphosphonate mimetics.
  • osteoactive drug targets e.g. bisphosphonate targets
  • degree of occupancy of drug targets in patients undergoing therapy may be exploited in imaging techniques, for example to assess the extent to which osteoactive drug targets (e.g. bisphosphonate targets) are available for drug action, or to determine the degree of occupancy of drug targets in patients undergoing therapy.
  • the invention also contemplates antibody derivatives, including antibody fragments (e.g. Fab fragments), chimaeric antibodies
  • antibody derivatives including humanized antibodies and antibody derivatives (such as fusion derivatives comprising an antibody-derived variable region and a non-immunoglobulin peptide having for example enzyme or conjugative activity).
  • test kits comprising the IPI and/or prenyl transferase of the invention, for example for use in the screening or evaluation methods of the invention.
  • the IPI and/or prenyl transferase may be bound to a solid support and/or
  • the kit further may comprise a labelled (e.g. radiolabelled or f luorescently labelled) bisphosphonate and/or
  • the kit may further comprise the antibody of the invention.
  • kits per se.
  • the invention contemplates the IPI prenyl transferase of the invention when bound to a solid support.
  • Also contemplated by the invention is a mimetic or
  • the mimetic for example consisting essentially of the catalytic or bisphosphonate binding site of IPI or prenyl transferase .
  • the invention also contemplates various therapeutic applications for the antibody , mimetic or antagonist of the invention .
  • the invention relates to a process for producing an osteoactive drug comprising the step of providing an analogue of the carbocation:
  • the invention relates to a process for selectively inhibiting an enzyme involved in sterol/isopren ⁇ id biosynthesis comprising the step of providing an analogue of the carbocation :
  • analogue has a positively charged nitrcger. atom in an alkvl chain thereof.
  • the carbocation preferably is :
  • R is an alkyl side chain, for example having >5 ( for example about 10 or about 15 ) carbon atoms .
  • analogues are bisphosphonates having a formula selected from:
  • the enzyme to be selectively inhibited is preferably selected from the enzymes squalene synthase , protein prenyl transferase, cis-prenyl transferase and trans- prenyl transferase (geranylgeranylpyrophosphate synthase) .
  • the selectivity of inhibition need not be absolute, and the compounds of the invention may inhibit two or more of the above-listed enzymes.
  • the spectrum of inhibitory activity over the enzymes listed above i.e. the relative or absolute inhibitory specificity
  • greater inhibitory selectivity can be achieved by providing a long (e.g. about C; s or longer) carbon side chain.
  • the invention therefore also contemplates a method of varying the inhibitory profile of an inhibitor of enzyme(s) involved in sterol/isoprenoid biosynthesis by varying the length of the carbon side chain shown in the general formula below:
  • Such inhibitors preferably have the formula:
  • Drugs based on or derived from such inhibitors would allow specific inhibition of prenylation of proteins that are modified by a geranylgeranyl group without inhibiting sterol biosynthesis or protein farnesylation .
  • the drug or inhibitor may not be a bisphosphonate .
  • the drug or inhibitor may be a bisphosphonate (for example a geminal or non geminal bisphosphonate) , sulphonate , carboxylate , phosphonocarb ⁇ xylate , phosphonosulphonate , ph ⁇ sphoncphosphate or pyrophosphate .
  • the drug or inhibitor may be further modified to facilitate cellular uptake , for example by the attachment of a liDCDhiiic group or by the attachment of a dipeptide group permitting uptake via the cellular plasma membrane peptide carrier system.
  • the invention in another aspect relates to a process for increasing the therapeutic efficacy of an osteoactive -4V- bisphosphonate comprising the step of introducing a positively charged nitrogen atom into an alkyl chain thereof.
  • the IPI and/or prenyl transferase inhibitors of the invention can be administered in a variety of forms, including oral and parental routes.
  • Parental administration can be intravenous, intramuscular, subcutaneous, intrasynovial, transdermal, intraocular, sublingual, buccal, topically or rectal.
  • Other routes include nasal inhalation e.g. via a nebulizer, atomizer or aerosol.
  • Tne IPI and/or prenyl transferase inhibitors may be formulated in an inert diluent or with an assimilable edible carrier. It may be enclosed in hard or soft shell capsules or compressed into tablets.
  • the active compound may be incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups or wafers.
  • Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 10 and about 1000 mg (e.g. between 50 and 300 mg) of active compound.
  • the tablets, troches, pills, capsules and the like may also contain a binder (for example corn starch or gelatin) , excipients such as dicalcium phosphate, disintegrating agents, lubricants and sweetening cr flavouring agents.
  • a binder for example corn starch or gelatin
  • excipients such as dicalcium phosphate, disintegrating agents, lubricants and sweetening cr flavouring agents.
  • the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier.
  • Unit dosage forms are pharmaceutically pure.
  • the inhibitors may be incorporated into sustained-release preparations and formulations.
  • the active compound may also be administered carenterally or intraperitoneally.
  • Solutions or suspensions of the active compounds or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant.
  • Dispersion can also be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and oils.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the dosage of the inhibitors is determined by the physician and varies with the form of administration, the active compound selected and the patient under treatment.
  • the therapeutic dosage will generally be from about 0.1 to about 100 mg/kg/dy and may be administered in several different unit dosage form. Higher dosages may be required for oral administration.
  • Example 1 Inhibition of farnesyl diphosphate synthase (prenyl transferase) and IPI by bisphosphonates
  • Clodronate, alendronate, ibandronate, YM 1 75 and risedronate were provided by Procter and Gamble Pharmaceuticals, Cincinnati, OH.
  • the bisphosphonates were dissolved in PBS, the pH adjusted to 7.4 with 1 N NaOH, then filter-sterilised by using a 0.2 ⁇ m filter.
  • Mevastatin also known as compactin
  • the pH of the solution was adjusted to approximately pH8 using 1 N HC1 , then filter-sterilised .
  • a stock solution of 10mM mevaionic acid lactone was prepared by dissolving the solid in dry ethanol , while farnesylpyrophosphate ( FPP) and geranylgeranylpyrophosphate (GGPP) , purchased from Sigma, were dried to remove solvent then resuspended in culture medium immediately before use.
  • FPP farnesylpyrophosphate
  • GGPP geranylgeranylpyrophosphate
  • Methionine and mevalonolactone was from Amersham , Aylesbury , U K . All other chemicals were from Sigma Chemical Co, Poole, U K , unless stated otherwise.
  • J774.2 cells were obtained from the European Collection of Animal Cell Cultures (Porton Down , UK) . Cultures were grown at 37° C in Dulbecco's Modified Eagle Medium (GIBCO, Paisley , UK) containing 10% heat inactivated foetal calf serum , 100 U/ml penicillin , 100 ⁇ g/ml streptomycin and 1 mM glutamine in 5% CCL atmosphere.
  • Dulbecco's Modified Eagle Medium GIBCO, Paisley , UK
  • Apoptosis in J774 macrophages was identified on the basis of characteristic changes in nuclear morphology i . e. condensation of chromarin and fragmentation of nuclei into apoptoric bodies , that distinguish apoptoric cells from cells undergoing necrotic cell death .
  • J774 cells were seeded into 12 well plates ( Costar) at a density of 10 u per well . After 24h , the medium was replaced with fresh medium containing either 1 -1 OO ⁇ M mevastatin , 100 ⁇ M alendronate or 15 ⁇ M mevastatin , with or without 0.5 ⁇ M cycloheximide, 200 ⁇ M FPP, 200 ⁇ M GGPP or 200 ⁇ M mevaionic acid lactone . After 48h, both adherent and non-adherent cells were collected , fixed with 4% (v/v) formaldehyde than cytospun onto slides and visualised as described by Rogers et al.
  • the adherent and non-adherent cells were collected and centrifuged at 1000g for 5 min .
  • the cells were then lysed in 0.5ml RIPA buffer ( PBS , 0.1% (w/v) sodium dodecyl sulfate, 0.5% (w/v) sodium deoxycholate, 10 ⁇ g/ml phenylmethylsulfonyl fluoride) .
  • the protein concentration of the lysates was determined using the BCA protein assay ( Pierce) .
  • Equal quantities of protein ( usually 50 ⁇ g ) of each lysate were then electrophoresed on 12% polyacrylamide-SDS gels under reducing conditions . After electrophoresis , the gels were dried then visualised after exposed to a high sensitivity phosphoimaging screen ( BioRad ) for 3 days .
  • Lysates for immunoprecipitation of Rab6 were precleared by using 1 ⁇ g of rabbit IgG and 20 ⁇ l protein A agarose slurry , followed by addition of 2 ⁇ g polyclonal rabbit a ⁇ ti-Rab6 (santa Cruz) for 2h , then 50 ⁇ l protein A-agarose and incubation overnight. Ras was immunoprecipitated by overnight incubation , at 4° , of 1 ml lysate with 30 ⁇ l pan-Ras antibody . Y13-259 conjugated to agarose beads (Oncogene Science) .
  • Immunoprecipitates were washed 5 times with 1 ml RIPA buffer, then bound proteins were removed by boiling for 4 minutes in 30 ⁇ l Laemmli sample buffer. Finally, samples were electrophoresed on 12.5% polyacrylamide-SDS gels under reducing conditions and detected as described above.
  • Mevastatin appeared to be more potent at inducing apoptosis than alendronate or risedronate, since concentrations of approximately 10 ⁇ M mevastatin , 30 ⁇ M alendronate or 3 ⁇ M risedronate caused 50% loss of total cell viability after 48h ( Fig .4) .
  • Mevastatin-i ⁇ duced loss of cell viability could be prevented by co-incubating J774 cells with 15 ⁇ M mevastatin and 0.5 ⁇ M cycloheximide during the 48h culture period ( Fig .5) .
  • Analysis of the proportion of morphologically apoptoric cells also demonstrated that cycloheximide prevented apoptosis (not shown) .
  • Co- incubation with 200 ⁇ M FPP , GGPP or especially mevaionic acid lactone also prevented (at least partially) mevastatin-induced loss of cell viability and apoptosis ( Fig .6) .
  • Bisphosphonates inhibit post-translational prenylation of proteins
  • J774 cells metabolically-labelled with mevalonolactone for 24h contained radiolabelled proteins that could be separated by electrophoresis on 12% polyacrylamide gel into proteins of molecular weight 21-26kDa (mostly geranygeranylated GTP-binding proteins , but also farnesylated Ras proteins) , 60-70kDa (farnesylated lamin B and prelamin A) , 17kDa and 46kDa.
  • a broad band at the migrating front of the gels (which did not stain with Coomassie blue and was not affected by prior treatment of cell
  • Bisphosphonates inhibit farnesylation of Ras and geranylgeranylation of Rab6
  • cycloheximide and actinomycin D prevent Ras transcription and hence prevent accumulation of non-prenylated , cytoplasmic Ras .
  • Mevastatin is an inhibitor of HMG-CoA reductase and thus prevents synthesis of mevalonate.
  • mevastatin-induced apoptosis could be prevented by addition of mevaionic acid lactone or the mevalonate-derived compounds FPP and GGPP.
  • addition of either FPP or GGPP but not mevaionic acid lactone, partially prevented alendronate-induced apoptosis .
  • alendronate appears to inhibit enzymes later in the mevalonate pathway than HMG-CoA reductase.
  • Rho and Rac proteins play an important role in regulating cytoskeletal organisation and cell morphology, including membrane ruffling, and whose function is essential for bone resorption by osteoclasts .
  • Loss of function of Rab proteins could affect intracellular membrane trafficking and vesicular transport from the Golgi , and could therefore prevent the insertion of proton pumps into the osteoclast ruffled border.
  • Induction of osteoclast apoptosis could result from effects on lamins or on Ras signalling , as discussed above. It is most li kely that inhibition of bone resorption in vivo is actually a consequence of lack of prenylation of a multitude of osteoclast proteins .
  • the extent to which prenylation is inhibited in osteoclasts may determine whether the cells simply lose the ability to function normally (i .e . fail to resorb bone) or whether this leads to induction of apoptoric cell death .
  • bisphosphonates affect osteoclasts and other cells by interfering with cellular metabolism .

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GBGB9610174.6A GB9610174D0 (en) 1996-05-15 1996-05-15 Prenyl transferase inhibitors
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US6984400B2 (en) * 1998-07-14 2006-01-10 Yissum Research Development Company Of The Hebrew University Of Jerusalem Method of treating restenosis using bisphosphonate nanoparticles
US6423519B1 (en) * 1998-07-15 2002-07-23 Gpc Biotech Inc. Compositions and methods for inhibiting fungal growth
WO2000038660A2 (de) * 1998-12-23 2000-07-06 Jomaa Pharmaka Gmbh Verwendung von bisphosphonaten zur prophylaxe und zur behandlung von infektiösen prozessen
WO2000052198A1 (en) * 1999-03-05 2000-09-08 Merck & Co., Inc. Methods for identifying compounds useful for inhibiting farnesyl diphosphate synthase
ES2315411T3 (es) * 2001-07-20 2009-04-01 Bioagency Ag Compuestos organofosforados para la activacion de celulas t gamma/delta.
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