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
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
  • A61K38/4813—Exopeptidases (3.4.11. to 3.4.19)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• the present invention relates to the use of an enzyme having carboxypeptidase G activity, and in particular to its use in combating toxicity caused by Pemetrexed and related antifolate compounds.
• Natural folates are used by cells in the folate pathway to synthesise DNA, RNA and in protein synthesis, and are therefore essential dietary requirements (Jolivet et ⁇ /,1983; Pinedo et al, 1976; Goldman 1975).
• DHFR dihydrofolate reductase
• TS thymidylate synthase
• GARFT glycinamide ribonucleotide formyltransferase
• SHMT serine hydroxymethyltransferase
• tetrahydrofolate supplies reducing equivalents for the conversion of the methylene group of MTHF to the methyl group of thymidylate (dTMP).
• dTMP methyl group of thymidylate
• DHF dihydrofolate
• DHFR which utilizes NADPH as the reductant.
• DHFR also catalyzes the conversion of folic acid to DHF.
• GARFT catalyzes the third in the series of ten reactions required for de novo purine biosynthesis, the conversion of glycinamide ribonucleotide to formylglycinamide ribonucleotide utilizing 10-formylTHF as the formyl donor.
• GARFT occurs in mammals as a trifunctional protein which catalyzes the second and the fifth steps on this pathway in addition to the third.
• GARFT activity resides in the carboxy-terminal portion of this tri-functional protein.
• De novo purine biosynthesis leads to the formation of inosine monophosphate, the precursor of the formation of inosine monophosphate, the precursor of ATP and GTP necessary for RNA formation and of dATP and dGTP necessary for DNA formation.
• Inhibition of DHFR leads to a deficiency of dTMP because DHF cannot be recycled for use in the TS reaction. This in turn leads to deficient DNA synthesis, DNA breakdown and cell death.
• Direct inhibition of TS likewise leads to a deficiency of dTMP and cell death.
• Direct inhibition of GARFT leads to depletion of purine nucleotides, which also leads to cell death, but the degree of cell kill is generally less than that produced by an equally growth-inhibitory concentration of a TS inhibitor (Kisliuk et al, 2003).
• Methotrexate a synthetic folate analogue
• MTX Methotrexate
• RA rheumatoid arthritis
• MS multiple sclerosis
• psoriasis psoriasis
• Non-Hodgkin's Lymphoma NHL
• ALL acute lymphoblastic leukemia
• soft tissue tumours such as osteosarcoma
• MTX san still be toxic to healthy cells in a dose and time dependent manner through two Drinciple mechanisms. The first is common to all antifolates. This mechanism is -hrough the inhibition of DNA synthesis and cellular metabolism, which is the jnderlying mechanism that is responsible for MTX' s cytotoxic anti-cancer action. The risk of significant toxicity to healthy cells correlates with increasing doses of VUX and the time of exposure. MTX therapy is associated with a spectrum of oxicities, with myelo suppression, mucositis, acute hepatitis and nephrotoxicity being the most frequent and serious complications (Bleyer 1978). Additional toxicities seen with high dose therapy are acute desquamative dermatitis, B- lymphocyte dysfunction, and neurological effects. Similar common toxicities are also caused by other antifolate drugs, although they have generally been administered at lower doses than MTX.
• the second mechanism is MTX induced renal tubular obstruction and consequent renal dysfunction (MTX nephrotoxicity).
• MTX is metabolised by liver aldehyde oxidase to 7-hydroxy-MTX.
• the aqueous solubility of 7-hydroxy-MTX is 3 to 5 times lower than that of the parent compound and, under certain conditions, is known to precipitate in the renal tubules which is thought to be a principal mechanism in the pathogenesis of the MTX nephrotoxicity (Kintzel 2001, Condit 1969). Normal kidney function will accommodate removal of a particular load in a given time, thereafter accumulation and damage will ensue.
• Renal toxicity has been recorded with other antifolate compounds, but this may not be due to an analogous 7-hydroxylation of the compound. 7-OH-MTX toxicity occurs at high MTX doses, while the typical administered doses of the further antifolates described herein would only be considered to be 'intermediate' doses for MTX.
• Leucovorin calcium is the calcium salt of 5-formyl tetrahydrofolic acid (also known as folinic acid/folinate), the DHFR metabolite of folic acid and an essential coenzyme for nucleic acid synthesis and is not inhibited by MTX (Immunex Corporation 2001). As a result, leucovorin calcium is able to rescue MTX inhibited cells. However, at high MTX concentrations, leucovorin calcium may fail to prevent systemic toxicities (Goldman, 1975; Pinedo et al, 1976). Reversal of MTX by leucovorin calcium is competitive, with relatively higher concentrations required as the MTX concentration increases. When concentrations of MTX reach 100 ⁇ M, even ten-fold higher leucovorin calcium concentrations (1,000 ⁇ M) are unable to protect bone marrow cells from toxicity (Pinedo et al, 1976).
• MTX remains the most widely used antifolate anticancer agent in clinical use to this date. Because of the relative safety and utility of MTX, considerable effort has been invested in attempting to design more therapeutically selective ant ⁇ folates or antifolates with a wider tumor spectrum. Initially, the design was based on the burgeoning knowledge of fo late-dependent pathways and the determinants of the mechanism of action of MTX. These determinants include transport, the tight- binding inhibition of its target, DHFR, and metabolism of MTX to poly- ⁇ - glutamate (GIu n ) metabolites.
• GIu n poly- ⁇ - glutamate
• edatrexate has shown no improvement over MTX, with similar or slightly worse toxicity (primarily mucositis).
• G-CSF granulocyte-colony stimulating factor
• NSCLC non-small-cell lung cancer
• Lometrexol a folate analogue that specifically inhibits GARFT
• this agent requires polyglutamation for its effect on GARFT. It is transported into the cell via both the RFC and FR systems.
• Lometrexol has no effect on DHFR, TS, or AICARFT, and thus its effectiveness is purely related to decreasing purine synthesis.
• Early phase I trials of lometrexol were confounded by cumulative delayed myelosuppression that prevented repetitive administration. As with Pemetrexed, further preclinical studies suggested that coadministration of folic acid might favourably modulate lometrexol toxicity without eliminating potential antitumor activity.
• Lometrexol is currently being evaluated in phase I combination studies with each of the following agents: temozolomide, doxorubicin, carboplatin, gemcitabine, and paclitaxel as well as in single-agent phase II studies in soft-tissue sarcoma, melanoma, breast cancer, NSCLC, and head and neck cancer (reviewed by Purcell & Ettinger, 2003).
• Pemetrexed a potent polyglutamatable classic antifolate TS inhibitor formerly called LY23154 and now manufactured as Alimta ® (Eli Lilly).
• Pemetrexed has been reviewed by Calvert (2004) and Norman (2001).
• Phase I/II studies on Pemetrexed have been reviewed by Hanauske et al (2004), and selected phase II and III clinical trials of Pemetrexed are outlined in Table 3 of Purcell & Ettinger (2003).
• Clinical trials involving Pemetrexed have been summarised by the US Food and Drug Administration (Hazarika et al (2004) and Cohen et al (2005)). The entire disclosure of each of these references in relation to Pemetrexed is incorporated herein by reference.
• Pemetrexed is polyglutamated inside the cell and shows high affinity for folylpolyglutamate synthetase (FPGS).
• the polyglutamate derivatives are also potent inhibitors of DHFR and GARFT and show less potent inhibition of amino imidazole carboxamide ribonucleotide formyltransferase (AICARFT). Therefore, Pemetrexed has been referred to as a "multitargeted" antifolate.
• Preclinical studies have shown that Pemetrexed inhibition of GARFT and AICARFT, in addition to DHFR and TS, is important in that cells need both thymidine and hypoxanthine to overcome the antitumour effect.
• Pemetrexed TS inhibition is 30 to 200 times greater than GARFT or AICARFT inhibition and seven times greater than DHFR inhibition.
• Toxicities of Pemetrexed in several early trials included significant myelo suppression, mucositis, and diarrhoea.
• plasma levels of homocysteine and methylmalonic acid were studied as sensitive surrogate markers for folic acid and vitamin Bi 2 status, respectively.
• Low levels of homocysteine and methylmalonic acid were found to be strongly correlated with the development of serious " drug-related toxicities, suggesting that toxicity was related to related to relative folic acid or vitamin Bi 2 deficiency in some cancer patients.
• Pemetrexed is active in many solid tumors, it has been studied most extensively in mesothelioma and NSCLC. Pemetrexed is also under investigation for breast, gastrointestinal, head and neck urothelial, and cervical cancer.
• AAGl 13-161 is a recently developed analogue of Pemetrexed and is a dual inhibitor of DHFR and TS. It was considered that replacing the 4-oxo moiety of Pemetrexed with a methyl group to form AAGl 13-161 would enhance DHFR binding by enabling a hydrophobic interaction of the 4-methyl group with Phe31 and Leu22 of human DHFR.
• X-Ray crystallographic studies of AAGl 13-161 bound to P. carinii DHFR showed it to be bound in the proposed 2,4-diamino mode and that the 4-methyl group of AAGl 13-161 does occupy the hydrophobic environment predicted. In fact, for L.
• AAGl 13-161 is 10,000 times more inhibitory than Pemetrexed, and for human DHFR it is 8 time more inhibitory. With regard to TS, AAGl 13-161 is 55 times more inhibitory than Pemetrexed for E. coli TS and 10 times more inhibitory than Pemetrexed for human TS. AAGl 13- 161 has IC50 values for CCRF-CEM human leukaemia and FaDu head and neck squamous ceU. carcinoma cell lines of 12.5 nM and 7.0 nM, respectively.
• AAGl 13-161 is a dual inhibitor of DHFR and TS.
• hypoxanthine alone at 50 ⁇ M caused no reversal of inhibition with AAGl 13-161.
• AAGl 13-161 is an excellent substrate for human FPGS. Its K m value is below the limit of detection of the assay (reviewed by Kisliuk, 2003).
• one major drawback to the clinical use of the newer antifolate drugs is an unacceptable level of toxicity.
• the ability to degrade these antifolate drugs rapidly in vivo would have two major clinical advantages. Firstly, it would minimize toxicity caused by the antifolate drugs. It also allows a higher dose of the antifolate compounds to be administered, potentially leading to a greater clinical effect. The lower toxicity and higher efficacy may be sufficient to realise the clinical promise of a number of drugs that have not progressed through clinical trials. Furthermore, as toxicity associated with antifolate drugs is often duration- related rather than dose-related, the ability to rapidly remove excess free drug at a given time point may be therapeutically very useful.
• Carboxypeptidase G 2 (CPG 2 ) is an enzyme from Pseudomonas sp. strain RS-16 (now reclassified as Variovorax paradoxus) and is a zinc-dependent dimeric protein of 83,000 - 84,000 Dalton (Kalghati & Bertino, 1981; Chabner et al, 1972; McCullough et al, 1971; Minton et al, 1983; and Sherwood et al, 1985).
• the International Nonproprietary Name (INN) of recombinant carboxypeptidase G 2 is glucarpidase, and it is commercially available as VoraxazeTM (Protherics).
• Glucarpidase cleaves methotrexate (MTX) into its inactive metabolites, 4-deoxy-4-amino-N 10 - methylpteroic acid (DAMPA) and glutamate, and thus may provide an alternative route of MTX elimination particularly in patients who develop renal dysfunction due to MTX nephrotoxicity (Adamson et al, 1991; Mohty et al, 2000; von Poblozki et al, 2000; Widemann et al, 2000).
• DAMPA 4-deoxy-4-amino-N 10 - methylpteroic acid
• Para-aminobenzoyl glutamate is a substrate of glucarpidase, as are a number of mustard prodrugs based on p-aminobenzoyl glutamate (Springer et al (1995); Do well et al (1996)).
• glucarpidase a number of mustard prodrugs based on p-aminobenzoyl glutamate
• any folate compounds other than folic acid, MTX, 5 -methyl THF, and 5-formyl THF were substrates for glucarpidase. It was not known, and could not predicted, whether any of the new generation of antifolate drugs are substrates for glucarpidase cleavage.
• Pemetrexed (Alimta ® ) is a substrate for glucarpidase and has a K m of 25.4 ⁇ M and a k cat of 1808 s "1 as measured by spectrophotometric assay. All of the known substrates for glucarpidase have N (e.g. an amino or substituted amino group) in the para position to the benzene ring marked by R 4 in Formula I, see below. Pemetrexed, by contrast, has a carbon at this position which is not an isosteric or functional replacement for the nitrogen.
• N e.g. an amino or substituted amino group
• Antifolate compounds are useful in treating a range of medical conditions, particularly cancers, and being able to combat toxicity associated with these compounds will significantly increase their therapeutic value.
• a first aspect of the invention provides a method of combating toxicity caused by an antifolate compound of Formula I 5
• R 1 represents NH 2 , OH or CH 3 ;
• R 2 represents NHo or Ci -4 alkyl;
• the group B represents a structural fragment of Formula Ha, lib, Hc, Hd or He,
• Hd lie in which groups the dashed lines indicate the point of ring fusion with the pyrimidinyl ring and the wavy lines indicate the point of attachment of the bicyclic heterocycle to the rest of the molecule;
• R 7a to R 7e independently represent H or C M alkyl;
• a 1 represents C(R 8a ) or N;
• a 2 represents CH or N;
• a 3 represents C(H)R Sb , NR Sc or S;
• a 4 and A 5 independently represent CH 2 , NH, O or S;
• R 8a to R 8c independently represent H or Ci -4 alkyl, or R 8c represents C(O)R 8d ; R 8d represents H or Ci -4 alkyl;
• R 3 represents H, Ci . 6 alkyl, C3-6 alkenyl or C 3- 6 alkynyl;
• R 4 represents H or one or two substituents selected from halo, C M alkyl and Ci -4 alkoxy, or R 4 , together with R 3 , when R 4 is attached at a position that is ortho- to the position to which the moiety C(O)NR 5 is attached, represents Ci -2 77-alkylene;
• R 3 represents H or C 1-4 alkyl, or R 5 , together with R 4 , when R 4 is attached at a position that is ortho- to the position to which the moiety C(O)NR 3 is attached, represents Ci -2 w-alkylene;
• R 6 represents -CH 2 C(R 9a )(R 9b )-D;
• R 9a and R 9b independently represent H or C M alkyl, or R 9a and R 9b together represent ⁇ C(H)R 10 ;
• R 10 represents H or Ci -4 alkyl
• R 11 represents H or C(O)R 12 ;
• R 12 represents H or phenyl substituted by C(O)OH and optionally substituted by one or two further substituents selected from halo, Ci -4 alkyl and Ci -4 alkoxy;
• alkyl, alkenyl and alkynyl groups, as well as the alkyl part of alkoxy groups, may be substituted by one or more halo atoms;
• halo when used herein, includes fluoro, chloro, bromo and iodo.
• Pharmaceutically acceptable salts of compounds of Formula I include both acid addition salts and metal (e.g. alkali metal, such as sodium or potassium) salts.
• Solvates that may be mentioned include hydrates.
• R 1 represents NH 2 or, particularly, CH 3 or OH
• R 2 represents methyl or, particularly, NH 2 ;
• the group B represents a structural fragment of Formula Ha, Hc or lid;
• a 1 and A 2 both represent CH or, particularly, both represent N;
• R 7a , R 7c and R 7d independently represent H or, when A 1 and A 2 both represent CH,
• R 7a may also represent methyl
• a 3 represents S or, particularly, CH 2 ;
• a 4 represents NH
• R 3 represents 3-prop-l-ynyl or, particularly, H, methyl or ethyl
• 'R 4 represents methyl or, particularly, H
• R 5 represents H;
• R 11 represents phenyl ⁇ z-t/z ⁇ -substituted by C(O)OH.
• R represents NH 2 ; the group B represents a structural fragment of Formula Hd;
• a J represents NH
• R 7d represents H
• R 3 represents H
• D represents C(O)OH
• R 9a and R 9b both represent H.
• the compound of Formula I is one of those listed at (1) to (5) below.
• R 3 H (10-deazaaminopterin);
• R 3 CH 3 ( 10-methyl- 10-deazaaminopterin) ;
• R 3 CH 2 CH 3 (10-ethyl-l O-deazaaminopterin, Edatrexate);
• R 3 CH 2 C ⁇ CH (10-propargyl- 1 O-deazaaminopterin).
• the compound of Formula I is Edatrexate, AAGl 13-161 or, most preferably, Pemetrexed.
• Pemetrexed may exhibit tautomerism, in particular with respect to the 0/OH groups at position R 1 , and Pemetrexed it is often depicted in its alternative tautomeric form, as shown in Formula Ia.
• an enzyme that has carboxypeptidase G activity we include the meaning of an enzyme that hydrolyses the C-terminal L-glutamic acid residue from folic acid, folate analogues, and sub-fragments of folic acid eg, p-aminobenzoyl glutamate.
• the enzyme that has carboxypeptidase G activity is glucarpidase (recombinant carboxypeptidase G 2 (CPG 2 )), EC number 3.4.22.12.
• CPG 2 carboxypeptidase G 2
• the sequence of the gene encoding glucarpidase and the glucarpidase amino acid sequence can be found in GenBank Accession Nos. M12599 and AAA62842 and in Minton et al ⁇ Gene 31(1-3), 31-38 (1984)), Minton and Clarke (J MoI. Appl. Genet. 3(1), 26-35 (1985)); and Chambers et al (Appl. Microbiol. Biotechnol. 29, 572-578 (1998)) and the amino acid sequence is listed in Figure 1.
• the enzyme that has carboxypeptidase G activity may be a derivative of glucarpidase that has carboxypeptidase G activity.
• a “derivative" of glucarpidase we include a fragment, variant, modification or fusion of glucarpidase, or combinations thereof, which has carboxypeptidase G activity.
• the derivatives may be made using protein chemistry techniques for example using partial proteolysis (either exolytically or endolytically), or by de novo synthesis.
• the derivatives may be made by recombinant DNA technology. Suitable techniques for cloning, manipulation, modification and expression of nucleic acids, and purification of expressed proteins, are well known in the art and are described for example in Sambrook et al (2001) ''Molecular Cloning, a Laboratory Manual", 3 rd edition, Sambrook et al (eds), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, incorporated herein by reference.
• fragment of glucarpidase we mean any portion of the full length enzyme that has carboxypeptidase G activity. Typically, the fragment has at least 30% of the carboxypeptidase G activity of glucarpidase. It is more preferred if the fragment has at least 50%, preferably at least 70% and more preferably at least 90% of the activity of CPG 2 . Most preferably, the fragment has 100% or more of the carboxypeptidase G activity of glucarpidase.
• carboxypeptidase G activity of a derivative of glucarpidase can readily be determined by a person of skill in the art using the enzyme assay described on page 448 of Sherwood et al (1985). The entire disclosure of Sherwood et al (1985) is incorporated herein b)' reference.
• a "variant" of glucarpidase refers to glucarpidase that has been altered by an amino acid insertion, deletion and/or substitution, either conservative or non-conservative, at one or more positions.
• B) 7 "conservative substitutions" is intended combinations such as GIy, Ala; VaI, lie, Leu; Asp, GIu; Asn, GIn; Ser, Thr; Lys, Arg; and Phe, Tyr. Such modifications may be made using the methods of protein engineering and site- directed mutagenesis, as described in Sambrook et al 2001, supra.
• one or more residues of one or both of the active site of the enzyme may beneficially alter the specificity or activity of the enzyme.
• the crystal structure of glucarpidase was published by Rowsell et al (1997) and identifies the active sites of the enzyme. In other embodiments, it may be advantageous not to modify residues in the active sites. Sequence variants, typically outside the active sites, may protect the enzyme from in vivo metabolism or decrease antigenicity. Additionally, it may be advantageous to add one or more Cys residues to allow disulphide bonds to be formed.
• the variant of glucarpidase has at least 70% sequence identity with SEQ ID No: 1. It is more preferred if variant glucarpidase has at least 80%, preferably at least 85% and more preferably at least 90% sequence identity with SEQ ID No: 1. Most preferably, the variant glucarpidase has 91 or 92 or 93 or 94 or 95 or 96 or 97 or 98 or 99% or more sequence identity with SEQ ID No: 1.
• the percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally.
• the alignment may alternatively be carried out using the Clustal W program (Thompson et al, (1994) Nucleic Acids Res 22, 4673-80). The parameters used may be as follows:
• Fast pairwise alignment parameters K-tuple(word) size; I 5 window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent.
• the variant of glucarpidase, or a fragment of the variant retains at least 30% of the carboxypeptidase G activity of glucarpidase. It is more preferred if variant glucarpidase has at least 50%, preferably at least 70% and more preferably at least 90% of the activity of glucarpidase. Most preferably, the variant of glucarpidase has 100% or more of the carboxypeptidase G activity of glucarpidase.
• the variant of glucarpidase has a substitution at one or more of the Asn residues at positions 222, 264 and 272 which are N-glycosylation sites.
• Asn 222 is substituted with GIn;
• Asn 264 is substituted with Thr or Ser, most preferably Ser;
• Asn 272 is substituted with GIn, independently or in combination.
• the most preferred combination of substitutions has GIn at positions 222 and 272 and Ser at residue 264.
• This QSQ motif results in a high catalytic activity and a low K 1n for MTX (US 2004/0014187).
• a “modification" of glucarpidase refers to glucarpidase in which one or more of the amino acid residues has been chemically modified. Such modifications include forming salts with acids or bases, especially physiologically acceptable organic or inorganic acids and bases, forming an ester or amide of a terminal carboxyl group, attaching amino acid protecting groups such as N-t-butoxycarbonyl and glycosylation. Such modifications may protect the enzyme from in vivo metabolism or decrease antigenicity.
• the glucarpidase may be present as single copies or as multiples, for example tandem repeats.
• the invention also includes a fusion of glucarpidase, or a fragment or variant thereof which has carboxypeptidase G activity, to another compound.
• the fusion retains at least 30% of the activity of glucarpidase. It is more preferred if the fusion has at least 50%, preferably at least 70% and more preferably at least 90% of the activity of glucarpidase. Most preferably, the fusion has 100% or more of the carboxypeptidase G activity of glucarpidase.
• the invention may be used to alleviate symptoms of toxicity caused by the antifolate compound of Formula I in an individual (ie palliative use), or may be used to reduce the severity of the toxicity in an individual, or may be used to treat toxicity in an individual or may be used prophylactically to prevent toxicity in an individual.
• combining toxicity we include the meaning of treating, reducing or preventing toxicity caused by the antifolate compound or alleviating the symptoms of it.
• the enzyme that has carboxypeptidase G activity typically acts to combat toxicity caused by the antifolate compound of Formula I by rapidly lowering plasma levels of the drug, thereby reducing the duration of exposure of normal tissues to the drug and preventing longer-term uptake.
• Whether or not a particular patient is one who is expected to benefit from treatment may be determined by the physician.
• preventing toxicity we include the meaning of treating a patient at risk of toxicity, for example due to high levels and/or delayed elimination of the antifolate compound of Formula I. Any patient who has been administered the antifolate compound may be considered to be at risk of toxicity caused by it.
• the individual at risk of toxicity may be one who has been administered the antifolate compound and who has not been tested for the presence of a clinical marker of toxicity caused by the antifolate compound.
• the individual at risk of toxicity may be one who has been administered the antifolate compound and has one or more clinical markers or indications of toxicity caused by it.
• Pemetrexed is not metabolised in the body to an appreciable extent and it is primarily excreted in the urine (EH Lilly, 2004).
• 70-90% of the Pemetrexed is recovered unchanged in the urine in the 24 hours following administration, with an elimination half life of 3.5 hours.
• Creatinine clearance can be used as a marker for renal function, and a creatinine clearance of ⁇ 45 rnL/min, can be considered to be a clinical marker or indication of Pemetrexed toxicity.
• the method may comprise the prior step of determining whether the individual who has been administered the antifolate compound of Formula I has a clinical marker of toxicity caused by the antifolate compound.
• the clinical marker of toxicity caused by the antifolate compound of Formula I may be a level of the compound, such as a plasma level, greater than a predetermined level at a given time after administration of the compound.
• the predetermined plasma level of the antifolate compound indicating toxicity may be 0.1 or 0.2 or 0.3 or 0.4 or 0.5 or 0.6 or 0.7 or 0.8 or 0.9 ⁇ mole per litre, or 1 or 2 or 3 or 4 or 5 ⁇ mole or more per litre at 24 hours after administration of the antifolate compound, or at 48, or 72 or 96 or 120 hours, or more, after administration of the antifolate compound.
• the method may comprise the prior step of determining the level of the antifolate compound in the individual at a given time after administration of the compound to the individual, such as at 24 or 48, or 72 or 96 or 120 hours, or more, after administration of the antifolate compound.
• the invention includes administering an enzyme that has carboxypeptidase G activity to an individual who has been administered an antifolate compound of Formula I as defined above, whether or not the individual has any symptoms of toxicity caused by the compound.
• the invention can be considered to be an in vivo method of cleaving an antifolate compound of Formula I as defined above.
• the individual at risk of toxicity may be one who has been administered the antifolate compound and has one or more clinical symptoms of toxicity caused by it.
• the method may comprise the prior step of determining whether the individual who has been administered the antifolate compound has a clinical symptom of toxicity caused by the antifolate compound.
• Symptoms of toxicity for various of the antifolate compounds of Formula I as defined above are well known.
• toxicities for Pemetrexed include, mucositis, myelosuppression, thrombocytopaenia, neutropaenia, neutropenic sepsis, septicaemia, fatigue, neurotoxicity, anaemia, parasthesia, dyspnea, nausea and diarrhoea, although somewhat reduced by supplementation with folic acid and vitamin Bn, skin rash, fatigue and stomatitis (Martin et al, 2003; Hanauske ef al, 2004); and toxicities for Edatrexate include mucositis, myelo suppression and leukopaenia (Purcell & Ettinger., 2003); and toxicities for Lometrexol include thrombocytopaenia and mucositis (Purcell & Ettinger, 2003).
• the individual is typically administered the enzyme that has carboxypeptidase G activity between about 24 and 48 hours after being administered the antifolate compound.
• the individual may be administered the enzyme between about 12 and 24 hours or between about 48 and 72 hours, or between about 72 and 96 hours, or between about 96 and 120 hours, or more, after being administered the antifolate compound.
• the individual may be administered the enzyme that has carboxypeptidase G activity about 6 hours, or about 12 hours, or about 18 hours, or about 24 hours, or about 30 hours, or about 36 hours, or about 42 hours, or about 48 hours, or about 54 hours, or about 60 hours, or about 72 hours, or about 84 hours, or about 96 hours, or about 108 hours, or about 120 hours, or more, after being administered the antifolate compound.
• the enzyme that has carboxypeptidase G activity is preferably administered as soon as possible once the error is noticed in order to combat toxicity caused by it.
• the individual has a clinical marker of toxicity caused by the antifolate compound, or a clinical symptom of toxicity caused by the antifolate compound, it may also be preferable to administer the enzyme as soon as possible.
• the invention thus includes administering to an individual in need thereof, as described herein, a high dose of an antifolate compound of Formula I, such as 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or more times the above doses, and subsequently administering an enzyme that has carboxypeptidase G activity to the individual.
• a high dose of an antifolate compound of Formula I such as 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or more times the above doses
• the invention thus includes administering a dose of Pemetrexed equivalent to about 1000 mg/m 2 or 1.5 or 2 or 2.5 or 3 or 3.5 or 4 or 5 or 6 or 7 or 8 or 9 or 10 g/m 2 or more on a 3 -weekly schedule, and subsequently administering an enzyme that has carboxypeptidase G activity to the individual.
• the invention thus includes administering a dose of Edatrexate equivalent to about 5 or 6 or 7 or 8 or 9 or 10 or 15 or 20 or 25 g/m 2 or more, and subsequently administering an enzyme that has carboxypeptidase G activity to the individual.
• the enzyme that has carboxypeptidase G activity or a formulation thereof may be administered by any conventional method including parenteral (eg subcutaneous or intramuscular) injection.
• the treatment may consist of a single dose or a plurality of doses over a period of time.
• the enzyme that has carboxypeptidase G activity or a formulation thereof is administered intravenously.
• the enzyme that has carboxypeptidase G activity or a formulation thereof may be administered intrathecally, typically when the antifolate compound of formula I has been administered intrathecal! ⁇ '.
• glucarpidase Studies in rhesus monkej's indicate that the half life of glucarpidase in plasma following intravenous administration is between 52 and 58 minutes. Following intrathecal administration to rhesus monkeys, glucarpidase's half life in cerebrospinal fluid has been estimated at between 3.3 and 3.9 hours.
• the enzyme that has carboxypeptidase G activity Whilst it is possible for the enzyme that has carboxypeptidase G activity to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers.
• the carrier(s) must be "acceptable” in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof.
• the carriers will be water or saline which will be sterile and pyrogen free.
• the enzyme that has carboxypeptidase G activity is stored as a freeze-dried powder ready to be made up as a solution for injection as required.
• the contents of a vial of freeze dried enzyme are reconstituted with sterile normal saline (0.9% w/v), immediately before use.
• the formulation of the enzyme that has carboxypeptidase G activity also contains lactose as an inactive ingredient, except for patients with hypersensitivity to lactose.
• the enzyme that has carboxypeptidase G activity is administered to the individual at a dose of about 50 Units per kg body weight (1 unit corresponds to the enzyme activity that cleaves 1 micromole of MTX per minute at 37°C) intravenously over 5 minutes.
• the enzyme can be administered at lower doses of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10,- 15, 20, 25, 30, 35, 40 or 45 Units per kg/body weight. It is also appreciated that the enzyme can be administered at higher doses of about 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or 200 or higher Units per kg/body weight.
• the frequency, timing and dosage of administration of the enzyme that has carboxypeptidase G activity may be determined by the physician, using knowledge of the properties of the enzyme, the levels of the antifolate enzyme in the patient, and the degree of any symptoms of toxicity in the patient.
• proteins and peptides may be delivered using an injectable sustained-release drug delivery system. These are designed specifically to reduce the frequency of injections.
• An example of such a system is Nutropin Depot which encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that, once injected, release rhGH slowly over a sustained period.
• ReGeI injectable system An alternative method of protein and peptide delivery is the ReGeI injectable system that is thermo-sensitive. Below body temperature, ReGeI is an injectable liquid while at body temperature it immediately forms a gel reservoir that slowly erodes and dissolves into known, safe, biodegradable polymers. The active drug is delivered over time as the biopolymers dissolve.
• the individual may be administered a polynucleotide that encodes the enzyme that has carboxypeptidase G activity, leading to in vivo expression of the enzyme.
• Suitable vectors and methods are well known to a person of skill in the art. For example, Schepelmann et al (2005) describe systemic gene-directed enzyme prodrug therapy using a targeted adenovirus to express carboxypeptidase G2.
• the individual to be treated may be any individual who would benefit from such treatment.
• the individual to be treated is a human.
• the methods of the invention may be used to treat mammals, such as the cows, horses, pigs, sheep, cats and dogs.
• the methods have uses in both human and veterinary medicine.
• the method of combating toxicity caused by an antifolate compound of Formula I as defined above further comprises administering a folate pathway rescue agent to the individual.
• a folate pathway rescue agent we include the meaning of an agent that can rescue the folate pathway which is blocked by the antifolate compound.
• the most commonly used folate pathway rescue agent is leucovorin, the calcium salt of 5- formyl tetrahydrofoHc acid.
• Alternative rescue agents may include other salts of 5- formyl tetrahydrofolic acid, thymidine and folic acid.
• the antifolate compound is an inhibitor of DHFR or of GARFT 5
• the folate pathway rescue agent is leucovorin
• the folate pathway rescue agent is leucovorin
• the folate pathway rescue agent is leucovorin
• the folate pathway rescue agent is thymidine.
• Edatrexate is known to be an inhibitor of DHFR
• Lometrexol is known to be a GARFT inhibitor
• Pemetrexed is a multitargeted antifolate that is known to inhibit DHFR, GARFT and TS (Table 2, Purcell & Ettinger, 2003).
• the appropriate folate pathway rescue agent can readily be determined.
• the individual is administered the enzyme that has carboxypeptidase G activity prior to the folate pathway rescue agent.
• the individual may be administered the folate pathwaj' rescue agent prior to the enzyme that has carboxypeptidase G activity.
• the individual may be administered the folate pathway rescue agent and the enzyme that has carboxypeptidase G activity substantially simultaneously.
• the antifolate compounds may be useful in treating a range of cancers, and being able to combat toxicity associated with these compounds increases their therapeutic value.
• the invention includes a method of treating cancer comprising administering an antifolate compound of Formula I as defined above to the individual, and subsequently administering to the individual an enzyme that has carboxypeptidase G activity.
• the subsequent administration of the enzyme that has carboxypeptidase G activity is to combat toxicity caused by the antifolate compound as described above.
• the invention includes a method of treating cancer comprising administering an antifolate compound of Formula I as defined above to the individual and combating toxicity caused by the antifolate compound by administering an enzyme that has carboxypeptidase G activity.
• the invention includes a method of treating cancer, the method comprising administering Pemetrexed to the individual, and combating toxicity caused by Pemetrexed by administering an enzyme that has carboxypeptidase G activity.
• the cancer to be treated by administration of Pemetrexed may be leukaemia, mesothelioma, NSCLC 5 lung, breast, colon, pancreas, kidney, bladder, gastrointestinal cancer, head and neck cancer, urothelial cancer or cervical carcinoma (Martin et at (2003); Thodtmarm et al (2003); Ettinger (2002); Calvert (2004)).
• the invention includes a method of treating cancer, the method comprising administering AAGl 13-161 to the individual, and combating toxicity caused by AAGl 13- 161 by administering an enzyme that has carboxypeptidase G activity.
• the cancer to be treated by administration of AAGl 13-161 may be leukaemia, mesothelioma. NSCLC, breast, colon, pancreas, kidney, bladder, gastrointestinal cancer, head and neck cancer, urothelial cancer or cervical carcinoma.
• the invention includes a method of treating cancer, the method comprising administering Edatrexate to the individual, and combating toxicity caused by Edatrexate by administering an enzyme that has carboxypeptidase G activity.
• the cancer to be treated by administration of Edatrexate may be breast, lung, head and neck squamous cell carcinoma, NSCLC, non-Hodgkin's lymphoma, germ cell tumour, pleural mesothelioma or malignant fibrous histiocytoma (Kuriakose et al (2002); Dreicer et al (1997); Pisters et al (1996); and Meyers et al (1998-9)).
• the invention includes a method of treating cancer, the method comprising administering Lometrexol to the individual, and combating toxicity caused by Lometrexol by administering an enzyme that has carboxypeptidase G activity.
• the cancer to be treated by administration of Lometrexol may be soft tissue sarcoma, NSCLC, breast, head and neck cancer or melanoma.
• the method of treating cancer may also comprise administering an additional anticancer agent to the individual.
• suitable cancer chemotherapeutic agents may include: alkylating agents including nitrogen mustards such as mechlorethamine (HN 2 ), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin) and chlorambucil; etliylenimines and methyknelamines such as hexamethylmelamine, thiotepa; alky ⁇ sulphonates such as busulphan; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU) and streptozocin (streptozotocin); and triazenes such as decarbazine (DTIC; dimethyltriazenoimidazole-carboxamide); Antimetabolites including pyrimidine analogues such as fluorouracil (5-fluorouracil; 5-FU), fi
• Natural Products including vinca alkaloids such as vinblastine (VLB) and vincristine; - epipodophyllotoxins such as etoposide and teniposide; antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin C); enzymes such as L- asparaginase; and biological response modifiers such as interferon alphenomes.
• VLB vinblastine
• - epipodophyllotoxins such as etoposide and teniposide
• antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin C)
• enzymes such as L- aspara
• Miscellaneous agents including platinum coordination complexes such as cisplatin (cis- DDP) and carboplatin; anthracenedione such as mitoxantrone and anthracycline; substituted urea such as hydroxyurea; methyl hydrazine derivative such as procarbazine (N-methyll ⁇ .ydrazine, MTH); and adrenocortical suppressant such as mitotane ip,p'- DDD) and aminoglutethimide; taxol and analogues/derivatives; and hormone agonists/antagonists such as flutamide and tamoxifen.
• platinum coordination complexes such as cisplatin (cis- DDP) and carboplatin
• anthracenedione such as mitoxantrone and anthracycline
• substituted urea such as hydroxyurea
• methyl hydrazine derivative such as procarbazine (N-methyll ⁇ .y
• preferred anticancer agents include cisplatin, carboplatin, oxaliplatin, vinorelbine, doxorubicin, epirubicin, cyclophosphamide, paclitaxel, irinotecan, gemcitabine and docetaxel.
• a second aspect of the invention provides the use of an enzyme that has carboxypeptidase G activity in the preparation of a medicament for combating toxicity caused by an antifolate compound of Formula I as defined above in the first aspect of the invention.
• antifolate compound of Formula I in this and subsequent aspects of the invention are as described above with respect to the first aspect of the invention.
• Compounds of Formula I that can be mentioned include Pemetrexed, AAGl 13-161, Edatrexate and Lometrexol. Pernetrexed is most preferred.
• the invention includes use of an enzyme that has carboxypeptidase G activity for combating toxicity in an individual who has one of more clinical signs, sjinptoms or markers of toxicity caused by said compound, as described above.
• the invention includes use of the enzyme that has carboxypeptidase G activity in the preparation of a medicament for combating toxicity caused by an antifolate compound of Formula I as defined above in an individual who is administered a folate pathway rescue agent.
• the individual may be administered the folate pathway rescue agent prior to the medicament, or the individual may be administered the folate pathway rescue agent after the medicament, or the individual may be administered the folate pathway rescue agent and the medicament substantially simultaneously.
• a third aspect of the invention provides the use of a folate pathway rescue agent in the preparation of a medicament for combating toxicity caused by an antifolate compound of Formula I as defined above in an individual who is administered an enzyme that has carboxypeptidase G activity.
• the individual may be administered the enzyme prior to the medicament, or the individual may be administered the enzyme after the medicament, or the individual may be administered the enzyme and the medicament substantially simultaneously.
• a fourth aspect of the invention provides the use of an enzyme that has carboxypeptidase G activity and a folate pathway rescue agent in the preparation of a medicament for combating toxicity caused by an antifolate compound of Formula I as defined above.
• the invention includes the use as defined above in the second, third and fourth aspects of the invention for combating toxicity caused by an antifolate compound of Formula I in an individual who is being treated for a cancer by administration of the antifolate compound, as detailed above.
• a fifth aspect of the invention provides the use of an antifolate compound of Formula I as implanted above in the preparation of a medicament for combating cancer in an individual who is subsequently administered an enzyme that has carboxypeptidase G activity.
• the type of cancer that can be combated by any specific antifolate compound of Formula I is known to the person skilled in the art and can be determined by the physician.
• the individual is also adrninistered a folate pathway rescue agent.
• the enzyme that has carboxypeptidase G activity may be adrninistered before, after, or substantially simultaneously with the folate pathway rescue agent.
• the invention includes the use of Pemetrexed in the preparation of a medicament for treating cancer in an individual who is adrninistered an enzyme that has carboxypeptidase G activity.
• the invention also includes the use of an enzyme that has carboxypeptidase G activity in the preparation of a medicament for combating toxicity caused by Pemetrexed in an individual who has been administered the Pemetrexed to treat cancer.
• Cancers that can be treated by administration of Pemetrexed are known in the art and include those listed above.
• cancers that can be treated by administration of a medicament containing AAGl 13-161, Edatrexate, Lometrexol and other antifolate compounds of Formula I are known in the art, and include those listed above.
• the invention thus includes the use of an enzyme that has carboxypeptidase G activity in the preparation of a medicament for complementing the therapy of a cancer that is being treated by administration of an antifolate compound of Formula L
• a sixth aspect of the invention provides an in vitro method of cleaving a terminal L-glutamate moiety from a compound of Formula I as defined above, the method comprising contacting the compound with an enzyme that has carboxypeptidase G activity.
• a seventh aspect of the invention provides a method of determining the rate and/or extent of cleavage of a compound of Formula I as defined above by an enzyme that has carboxypeptidase G activity, the method comprising: providing a compound of Formula I, contacting the compound of Formula I with an enzyme that has carboxypeptidase G activity under conditions such that cleavage of the compound can occur, and monitoring the rate and/or extent of cleavage of the compound of Formula I over time.
• the providing step comprises providing a known amount or concentration of the compound of Formula I.
• the monitoring step comprises monitoring the amount and/or concentration of the compound of Formula I over time. Additionally or alternatively, the monitoring step comprises monitoring the amount and/or concentration of one or more break-down products of the compound of Formula I over time.
• the method of determining the rate and/or extent of cleavage can be performed in vitro, or can be performed in vivo.
• the method can be used either in vivo or in vitro to monitor the level of the compound of Formula I that remains uncleaved after treatment with the enzyme that has carboxypeptidase G activity.
• the method can be used to monitor the effectiveness of the enzyme that has carboxypeptidase G activity in combating toxicity associated with the compound of Formula I.
• the method may further comprise determining whether an additional dose of the enzyme that has carboxypeptidase G activity is required in order to reduce the amount of the compound of Formula I to a predetermined level, typically a level which does not cause toxicity.
• the amount of enzyme to be administered in the additional dose may also be determined.
• the method may therefore also comprise contacting the compound of Formula I with an additional dose of the enzyme that has carboxypeptidase G activity under conditions such that cleavage of the compound of Formula I can occur.
• An eighth aspect of the invention provides a therapeutic system (or it may be termed a "kit of parts") consisting of or comprising an antifolate compound of Formula I as defined above and an enzyme that has carboxypeptidase G activity.
• the therapeutic system may also contain a folate pathway rescue agent.
• the therapeutic system contains a preferred compound of Formula I as defined above in the first aspect of the invention, most preferably Pemetrexed. Still preferably, the therapeutic system contains glucarpidase, or a derivative thereof that has carboxypeptidase G activity, as defined above in the first aspect of the invention. Preferred folate pathway rescue agents are also as defined in the first aspect of the invention.
• the therapeutic system or kit of parts may suitably contain both the compound of Formula I and the enzyme that has carboxypeptidase G activity, and optionally the folate pathway rescue agent, packaged and presented in a suitable formulation either for storage or for use.
• the glucarpidase may be a freeze-dried powder ready to be reconstituted as a solution for injection, or may already be reconstituted as a solution for injection.
• Pemetrexed may be a freeze-dried powder ready to be reconstituted as a solution for injection, or may already be reconstituted as a solution for injection.
• the compound of Formula I and the enzyme are for separate administration in a particular treatment regime, thus they are packaged or formulated separately.
• the enzyme and the folate pathway rescue agent may be administered together, and thus may be formulated for co- administration.
• a preferred therapeutic system or kit as defined above comprises Pemetrexed and Glucarpidase, and optionally but preferably, folic acid and/or vitamin Bi 2 .
• a ninth aspect of the invention provides a method of cleaving a compound comprising a structural fragment of Formula IV 5
• R 3 to R 6 are as hereinbefore died
• the method comprising contacting the compound comprising the structural fragment of Formula IV with an enzyme that has carboxypeptidase G activity.
• the method may be performed in vitro. In an alternative embodiment, the method may be performed in vivo.
• the compound comprising the structural fragment of Formula IV is an antifolate compound.
• the invention provides a method of combating toxicity caused by the antifolate compound comprising the structural fragment of Formula IV, the method comprising administering to the individual an enzyme that has carboxypeptidase G activity.
• the invention includes the use of an enzyme that has carboxypeptidase G activity in the preparation of a medicament for combating toxicity caused by an antifolate compound comprising a structural fragment of Formula IV.
• Figure 1 is the amino acid sequence of glucarpidase (SEQ ID No: 1).
• Figure 2 shows the chemical structure of five substrates of CPG 2 . Pemetrexed, AAGl 13-161, Edatrexate, Lometrexol and Methotrexate (prior art).
• Figure 3 is a graph showing spectral differences in Pemetrexed absorbance pre- and post- glucarpidase addition.
• Figure 4 is a graph of a Michaelis-Menten plot for MTX (prior art substrate).
• Figure 5 is a graph of a Michaelis-Menten plot of Pemetrexed (experiment 1).
• Figure 6 is a graph of a Michaelis-Menten plot of Pemetrexed (experiment 2).
• Example 1 Determination of Kinetic Property of Cleavage of Pemetrexed (Alimta ® ) by Glucarpidase fVoraxazeTM)
• glucarpidase may have clinical utility as intervention therapy for Pemetrexed toxicity and have a long term role in planned use with Pemetrexed in several malignant conditions.
• a 1000 Unit vial of lyophilised glucarpidase (Protherics, batch CAMR 004/1991) was resuspended in ImI of 5OmM Tris-HCl pH 7.4 containing 0.2 mM Zn 2 ", then diluted 1/20 followed by 1/50 to give a working solution 1/1000 of the original concentration, i.e. 1 U/ml. lO ⁇ l of the working solution, 0.01 Units, was used per assay.
• the carboxypeptidase activity is determined by measuring the MTX hydrolysis speed at 37 0 C.
• the reactive mixture 1000 ⁇ l contains Tris/HCl (pH 7.4), MTX and ZnAc 2 .2H 2 ⁇ . .
• the reaction is initiated by adding the solution containing the enzyme to be assayed and the absorbance at 320 nm is monitored on the Hitachi U- 2010 spectrophotometer.
• a unit of carboxypeptidase activity is defined as the quantity of enzyme needed to catalyse the hydrolysis of a micromole of MTX in 1 minute, in 1 ml of the reactive mixture at 37°C.
• CA carboxypeptidase activity
• ⁇ A the absorbance variation measured in the period of time selected for measuring the activity
• DiI the dilution of the sample to be assayed
• ⁇ time the duration of the period selected for measuring the activity (min);
• V react the volume of the reactive mixture
• V sam the volume of the sample used for measuring
• ⁇ M 320 the variation of the molar extinction coefficient (in M "1 . cm “1 ).
• the variation of the molar extinction coefficient ( ⁇ M 320 ) of the methotrexate is — 8200 M “1 . cm “1 , which means that, for a lcm optical path, the total hydrolysis of a IM methotrexate solution is accompanied by a decrease in 8200 of the absorbance at 320 am,
• V r ⁇ act lOOO ⁇ l
• V sam 50 ⁇ l
• CA carboxypeptidase activity (in U.ml "1 );
• PC protein concentration (mg/ml) determined by UV Spectrophotometry. Assay Procedure
• the samples are diluted in the dilution buffer at 40°C just before the assay.
• the activity (U/ml) of a sample at a given dilution is obtained by the average of two replicates. These two tests are carried out using two independent dilutions.
• the second dilution of the sample is also prepared just before the assay.
• the freeze-dried product activity test is carried out on 10 pools of 2 freeze-dried vials of CPG2 each one resuspended in 1 ml of water to obtain a carboxypeptidase G2 activity of close to 1000 U/ml. Further dilutions in the dilution buffer (+ lactose) of 200Ox, 250Ox and 300Ox can be used for the measurements.
• a negative control (without enzyme) is tested.
• the A j2 ° is measured and is between 0.80 and 1.00 and does not vary by more than 0.01 units in 1 minute.
• the carboxypeptidase activity is measured by adding 50 ⁇ l of the diluted sample to be assayed to the sample cell, mixing, and measuring the A 320 once every second for 40 seconds.
• the carboxypeptidase activity is defined as:
• the rate of the turnover for a fixed, known amount of Glucarpidase was measured over a range of nine different substrate concentrations: 60 ⁇ M, 40 ⁇ M, 20 ⁇ M, 16 ⁇ M, 12 ⁇ M, lO ⁇ M, 8 ⁇ M 5 6 ⁇ M and 4 ⁇ M.
• the rate of cleavage at each different substrate concentration was measured in triplicate.
• the substrate concentrations were chosen to cover a range that provided data at both saturation and substrate limiting levels in order to allow accurate curve fitting. Rate measurements were made by evaluation of the change in absorbance by Pemetrexed at 250nm using a thermostatted Hitachi U2010 Spectrophotometer s/n 121-01222 with UV solutions vl .2 software.
• Pemetrexed was not soluble in aqueous buffer and was therefore dissolved in DMSO at 5.97mg/ml to give a stock solution of 1OmM, 1-1 O ⁇ l of which was used to prepare cuvettes containing 10-60 ⁇ M Pemetrexed; for preparation of cells containing ⁇ 10 ⁇ M, a further 1/10 dilution in DMSO was carried out to give a ImM stock solution.
• the volume of DMSO in the cuvette was kept constant at lO ⁇ l by addition of red DMSO as required. Previous studies have demonstrated that the presence of 1 %v/v DMSO is not inhibitory to glucarpidase.
• Figure 4 shows the action of methotrexate with glucarpidase over a concentrated range and fitted to demonstrate Michaelis Menten kinetics.
• a Km (affinity constant) for methotrexate can be calculated, and hy dividing the calculated Vmax by the known enzyme concentration (starting concentration of glucarpidase was 2.15mg/vial) the kcat was calculated.
• FIGS 5 and 6 show equivalent data for two studies on Pemetrexed. The results from each experiment are shown below, and are summarised in Table I.
• Vmax (limiting maximal rate): 0.067 ⁇ 0.004 (standard error value)
• AAGl 13-161 is a dual inhibitor of DHFR and TS. AAGl 13-161 is contacted with glucarpidase in vitro and is found to be a substrate of glucarpidase.
• Deglutamylation of AAGl 13-161 by glucarpidase is measured spectrophotometrically.
• a stock solution of 1OmM AAGl 13-161 in 10OmM Tris- HCl pH7.3 is prepared and used to prepare a series of dilutions from 0-100 ⁇ M in 10OmM Tris-HCl pH 7.3. 990 ⁇ l of each dilution is placed in a quartz cuvette and pre- warmed to 37°C. lO ⁇ l of enzyme solution is added to initiate the reaction and the progress of the reaction is followed by measuring the rate of change in absorbance at a suitable wavelength.
• Edatrexate is a DHFR inhibitor (Ciba Geigy). Edatrexate is contacted with glucarpidase in vitro and is found to be a substrate of glucarpidase.
• Deglutamylation of Edatrexate by glucarpidase is measured spectrophotometrically.
• a stock solution of 1OmM Edatrexate in 10OmM Tris-HCl pH7.3 is prepared and used to prepare a series of dilutions from 0-100 ⁇ M in 10OmM Tris-HCl pH 7.3. 990 ⁇ l of each dilution is placed in a quartz cuvette and pre-warmed to 37 0 C. 10 ⁇ l of enzyme solution is added to initiate the reaction and the progress of the reaction is followed by measuring the rate of change in absorbance at a suitable wavelength.
• the change in extinction coefficient of Edatrexate following complete conversion of a lOO ⁇ M solution by an excess of glucarpidase is measured in order to calculate the change in extinction coefficient due to the complete conversion of lOOnmol of Edatrexate. With this data, measured rates are converted to values of ⁇ mol/min. Measurement of the rate of reaction at a range of substrate concentrations, from 1- lOO ⁇ M allows the K m and k cat for the enzyme to be determined using Enzfitter computer software.
• Lometrexol is a GARFT inhibitor (Tularik, originated Lilly). Lometrexol is contacted with glucarpidase in vitro and is found to be a substrate of glucarpidase.
• Deglutamylation of Lometrexol by glucarpidase is measured spectrophotometrically.
• a stock solution of 1OmM Lometrexol in 10OmM Tris- HCl pH7.3 is prepared and used to prepare a series of dilutions from 0-100 ⁇ M in 10OmM Tris-HCl pH 7.3. 990 ⁇ l of each dilution is placed in a quartz cuvette and pre-warmed to 37 0 C. lO ⁇ l of enzyme solution is added to initiate the reaction and the progress of the reaction is followed by measuring the rate of change in absorbance at a suitable wavelength.
• the change in extinction coefficient of Lometrexol following complete conversion of a lOO ⁇ M solution by an excess of glucarpidase is measured in order to calculate the change in extinction coefficient due to the complete conversion of lOOnmol of Lometrexol. With this data, measured rates are converted to values of ⁇ mol/min. Measurement of the rate of reaction at a range of substrate concentrations, from l-100 ⁇ M allows the K m and k cat for the enzyme to be determined using Enzfitter computer software.
• a patient who has been administered Pemetrexed and has toxic plasma levels of Pemetrexed is administered a dose of 50 Units per kg body weight of gluearpidase by intravenous injection over a period of about 5 minutes.
• the patient's plasma concentration of Pemetrexed is reduced to non-toxic levels.
• Kisliuk RL (2003) “Deaza analogs of folic acid as antitumor agents.” Current Pharmaceutical Design 9(31): 2615-2625.

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