WO2009098451A2 - Biomarkers of aminopeptidase inhibition - Google Patents

Abstract

This invention relates to biomarkers for aminopeptidase inhibition. These biomarkers may be useful, for example, in assessing the extent of aminopeptidase inhibition in vivo or in monitoring cancer treatments using aminopeptidase inhibitors, such as bestatin or CHR-2797.

Description

Biomarkers of Aminopeptidase Inhibition

This invention relates to biomarkers of arαinopeptidase inhibition. These biomarkers may be useful, for example, in determining the efficacy of aminopeptidase inhibitors in therapeutic applications.

Aminopeptidases catalyse the sequential removal of amino acids from the amino terminus of peptide substrates (Taylor A. FASEB J 1993/7:290-8) . Known functions of these enzymes include activation and inactivation of biologically active peptides, removal of the N- terminal methionine of newly synthesised proteins, trimming of antigens for MHC class 1 presentation and determination of protein stability (Albiston AL et al . Protein Pept Lett 2004; 11:491-500; Sato Y. Biol Pharm Bull 2004; 27:772-6; Saveanu L et al Nat Immunol 2005; 6:689-97; Varshavsky A. Proc Natl Acad Sci USA 1996; 93 : 12142-9) .

The prototypical aminopeptidase inhibitor, bestatin, is a potent inhibitor of a number of members of the M (metallopeptidase) family of aminopeptidases (Rawlings ND et al Nucleic Acids Res 2006; 34 : D270-2; Scornik OA et al Curr Drug Metab 2001; 2 : 67-85) and exerts antiproliferative effects across a range of human tumour cell lines (Sakuraya M et al. J Int Med Res 2000; 28 : 214-21) . Furthermore, it has demonstrated clinical efficacy in non-lymphocytic leukemia and squamous cell lung carcinoma (Ota K, ϋzuka Y et al . Biotherapy 1992;4:205-14; Ichinose Y et al J Natl Cancer Inst 2003; 95: 605-10) . The aminopeptidases may, therefore, be considered validated anticancer drug targets .

CHR-2797 is a hydroxamate-containing dipeptidic ester that is converted inside cells to a pharmacologically active acid, CHR-79888, which is a potent inhibitor of a number of intracellular aminopeptidases, including puromycin-sensitive aminopeptidase, leukotriene A₄ hydrolase and leucine aminopeptidase (Needham LA et al (2008) Cancer Research In Press) . The compound induces apoptosis in a number of haematopoietic tumour cell lines and its anti-proliferative effects are selective for transformed over non-transformed cells. Across a wide range of tumour cell lines originating from diverse tumour types, it is between 300-1000 times more potent than bestatin. Furthermore, there is significant clinical interest in the compound which is in phase II clinical trials in both myeloid leukemia and non small cell lung cancer (Davies F et al Blood 2007; 110; Abst 443; Protheroe A et al J Clin Oncol 2007; 25 (18S) :Abst 3537) .

Although aminopeptidase inhibition by both bestatin and CHR-2797 has been shown to exert anti-proliferative effects in tumour cells, the mechanism by which inhibition of these enzymes leads to cell death is not fully elucidated.

In the early clinical development of anti-cancer agents, clinical trials are typically designed to evaluate the safety, tolerability, and pharmacokinetics, as well as to identify a suitable dose and schedule for further clinical evaluation. Increasingly, there is a need to evaluate the pharmacological effects of novel agents in early clinical trials, particularly in cases where dosing to maximum tolerated doses may not be appropriate.

The present inventors have identified biomarkers that correlate with the inhibition of aminopeptidases . These may be useful, for example, in evaluating the pharmacological effects of aminopeptidase inhibitors, such as bestatin and CHR-2797, in vivo, for example in clinical trials.

One aspect of the invention provides a method for assessing aminopeptidase inhibition in an individual comprising:

(a) administering an aminopeptidase inhibitor to the individual; and,

(b) measuring the level or amount of at least one biomarker selected from the biomarkers of Tables 1 and 2 in a sample obtained from the individual . The amount of the at least one biomarker present in the sample may be indicative of the amount or extent of aminopeptidase inhibition in the individual after administration of the aminopeptidase inhibitor.

A change in the amount of the at least one biomarker following administration of the aminopeptidase inhibitor may be indicative of the amount or extent of aminopeptidase inhibition in the individual. For example, the amount of increase in the level of a biomarker from Table 1 or the amount of decrease in the level of a biomarker from Table 2 may be indicative of the amount or extent of aminopeptidase inhibition in the individual.

In some embodiments, the level or amount of at least one biomarker selected from the biomarkers of Tables 3 and 5 may be measured in a sample obtained from the individual, an increase in the level of the biomarker being indicative of aminopeptidase inhibition in the individual.

In some embodiments, the level or amount of at least one biomarker selected from the biomarkers of Tables 4 and 6 may be measured in a sample obtained from the individual, a decrease in the level of the biomarker being indicative of aminopeptidase inhibition in the individual .

The methods described herein may be useful, for example, in assessing the effect of an aminopeptidase inhibitor on the individual.

Aminopeptidase inhibition may be assessed in an individual having a cancer condition. Aminopeptidase inhibition is known to be beneficial in the treatment of cancer conditions. The biomarkers described herein may therefore be useful in determining, assessing or predicting the response of a cancer condition to treatment with an aminopeptidase inhibitor.

The methods described herein may be useful, for example, in assessing the responsiveness of a cancer condition in an individual to treatment with an aminopeptidase inhibitor; monitoring the efficacy and/or progress of treatment of a cancer condition with an aminopeptidase inhibitor; or determining whether or not a cancer condition is responding to treatment with an aminopeptidase inhibitor. For example, a method for assessing the responsiveness of a cancer condition in an individual to treatment with an aminopeptidase inhibitor may comprise:

(a) administering the aminopeptidase inhibitor to the individual; and,

(b) measuring the amount of at least one biomarker selected from the biomarkers of Tables 1 and Table 2 in a sample obtained from the individual .

A change in the amount of the at least one biomarker following administration of the aminopeptidase inhibitor is indicative that the cancer condition is responsive to treatment with the aminopeptidase inhibitor i.e. the aminopeptidase inhibitor is efficacious in treating the cancer condition in the individual.

For example, an increase in the level or amount of a biomarker of Table 1 may be indicative of aminopeptidase inhibition in the individual and a decrease in the level of a biomarker of Table 2 may be indicative that the cancer condition is responsive to treatment with the aminopeptidase inhibitor.

In some embodiments, the level or amount of at least one biomarker selected from the biomarkers of Tables 3 and 5 may be measured in a sample obtained from the individual, an increase in the level of the biomarker being indicative that the cancer condition is responsive to treatment with the aminopeptidase inhibitor.

In some embodiments, the level or amount of at least one biomarker selected from the biomarkers of Tables 4 and 6 may be measured in a sample obtained from the individual, a decrease in the level of the biomarker being indicative that the cancer condition is responsive to treatment with the aminopeptidase inhibitor. The amount of change in level of the at least one biomarker may be indicative of the amount or extent of responsiveness of the cancer condition in the individual. For example, the amount of increase in the level of a biomarker from Table 1 or the amount of decrease in the level of a biomarker from Table 2 may be indicative of the amount or extent of responsiveness of the cancer condition in the individual.

The amount of at least 1, at least 2, at least 3, at least 4, at least

5 at least 10, at least 20 or at least 30 biomarkers shown in Table 1, more preferably Table 3 or 5, and/or Table 2, more preferably Table 4 or 6, may be determined in the sample.

A change in the amount of the at least one biomarker may be determined relative to the amount in a sample obtained from the individual before administration. For example, the amount of the at least one biomarker may be measured in a sample obtained from the individual before administration of the aminopeptidase inhibitor and compared to the amount measured in the sample obtained following administration. A method may thus comprise: (a) measuring the amount of at least one biomarker selected from the biomarkers of Tables 1 and 2 in a first sample obtained from the individual;

(b) administering the aminopeptidase inhibitor; and,

(c) measuring the amount of the at least one biomarker in a second sample obtained from the individual, wherein a difference between the amounts of the at least one biomarker in the first and second samples is indicative that the cancer condition is responsive to treatment with the aminopeptidase inhibitor.

In some embodiments, the at least one biomarker may be selected from the biomarkers of Table 3 or 5 and/or the biomarkers of Table 4 or 6.

The extent of the difference between the amounts of the at least one biomarker in the first and second samples may be indicative of the extent of responsiveness of the cancer condition to treatment with the aminopeptidase inhibitor.

A change in the level or amount of the at least one biomarker may be determined relative to a predetermined standard level. A standard level may be determined by measuring the level or amount of the biomarker in an individual or a population of individuals who have not been subjected to treatment with an aminopeptidase inhibitor. For example, the standard level may be calculated from the mean level of the biomarker in individuals within the population. Standard levels may be recorded or stored for subsequent use in determining changes in biomarker levels as described herein.

A change in the level or amount of the at least one biomarker may be determined over time following administration of the aminopeptidase inhibitor. For example, a method may comprise;

(a) administering the aminopeptidase inhibitor to the individual; and,

(b) measuring the level or amount of at least one biomarker selected from the biomarkers of Tables 1 and 2 in samples obtained from the individual at two or more time points following said administration.

A change in the level or amount of the at least one biomarker over the two or more time points is indicative that the cancer condition is responsive to treatment with the aminopeptidase inhibitor.

A cancer condition which is responsive to treatment with an aminopeptidase inhibitor may display a positive clinical effect (e.g. inhibition of the progress of the cancer condition, a reduction in the rate of progress, a halt in the rate of progress, amelioration of the cancer condition or one or more symptoms thereof, and cure of the cancer condition) in response to treatment with the aminopeptidase inhibitor. For example, the size or number of tumours in the individual may be reduced or stabilized or the rate of tumour growth may be slowed. Cancer cells which are responsive to aminopeptidase inhibition may display reduced proliferation, invasiveness or metastasis and/or increased cell death following treatment with an aminopeptidase inhibitor. The methods described herein may be useful in predicting whether or not an individual will or is likely to display a positive clinical response to the treatment. In particular, the methods may be used at an early stage in the treatment when the clinical response of the individual cannot be readily determined by standard medical evaluation methods.

An aminopeptidase inhibitor is a compound which inhibits, reduces or represses the activity of one or more aminopeptidases (EC 3.4.11) . For example, an aminopeptidase inhibitor may inhibit one or more of aminopeptidase N (CD13; EC 3.4.11.2; GenelD: 290; Nucleic acid Genbank entry NM_001150.2 GI: 157266299; Amino acid Genbank entry NP_001141.2 GI: 157266300); leucine aminopeptidase 3 (LAP3 GenelD: 51056; Nucleic acid Genbank entry NM_015907.2 GI: 41393560, Amino acid Genbank entry NP_056991.2 GI : 41393561) , aminopeptidase puromycin sensitive (NPEPPS; Gene ID 9520; Nucleic acid Genbank entry NM__006310.3 GI : 158937235; Amino acid Genbank entry NP_006301.3 GI : 158937236) and leukotriene A4 hydrolase (LTA4H; Gene ID: 4048; Nucleic acid Genbank entry

NM_000895.1 GI: 4505028; Amino acid Genbank entry; NP_000886.1 GI:4505029) .

Compounds which inhibit reduce or repress the activity of an aminopeptidase, for example an aminopeptidase set out above, may be identified using techniques which are well-known in the art. For example, the ability of a compound to inhibit the cleavage of a peptide substrate by an aminopeptidase, such as the peptide Leu-Gly- GIy by LAP3, the fluorogenic peptide substrate AIa-AMC by NPEPPS, or the fluorogenic peptide substrate Arg-AMC by LTA4H, may be determined (Needham LA et al (2008) Cancer Research In Press) .

Examples of suitable aminopeptidase inhibitors include bestatin (CAS 65391-42-6; N- (3R-amino-2S-hydroxy—oxo-4-phenylbutyl) -L-leucine) ; betstatin analogues, such as AHPA-VaI ( (2S, 3R) -3-amino-2-hydroxy-4- phenylbutanoyl-L-valine) , lapstatin (3-amino-2-hydroxy-4- methylpentanoylvaline) , Z4212 (N- [(2 S₅ 3R) -3-Amino-2-hydroxy-4- (4-3. methylsulphonyl-phenyl) -1-oxobuty I] -I- aminocyclopentanecarboxylic) and Z1796 ( (2S) -N- [ (2S, 3R) -3-Amino-2-hydroxy-4- ( 4-methylsulphonyl- phenyl) -1- oxobutyl] -L-leucine) ; puromycin (CAS 58-58-2; (S) 3'-[[2- Amino-3- (4-methoxyphenyl) -1-oxopropyl] amino] -3 ' -deoxy-N,N- dimethyladenosine, 3 ' - (a-Amino-p-methoxy-hydocinnamamido) -3 ' -dexoy-N,N- dimethyladenosine) ; actinonin (CAS 13434-13-4; 3-((l-((2- [Hydroxymethyl] -1-pyrrolidinyl) carbonyl) -2- methylpropyl) carbamoyl) octanohydroxamic acid); and CHR-2797 (CAS 240489-34-3; 2S- [2R- (S-Hydroxy-hydroxycarbamoyl-methyl) -4- methylpentanoylamino] -2-phenylethanoic acid cyclopentyl ester; WO 9946241) .

Other suitable aminopeptidase inhibitors are described in WO9946241.

In any of the methods described herein, the level or amount of at least one biomarker in Table 1 may be determined. Preferably, the level or amount of at least one biomarker in at least one biomarker in Table 3 and/or Table 5 may be determined. An increase in the level or amount of the at least one biomarker from Table 1, Table 3 and/or

Table 5 following administration of the aminopeptidase inhibitor is indicative that the cancer condition responds to treatment with the aminopeptidase inhibitor i.e. the aminopeptidase inhibitor is efficacious in treating the cancer condition in the individual.

In any of the methods described herein, the level or amount of at least one biomarker in Table 2 may be determined. Preferably, the level or amount of at least one biomarker in Table 4 and/or Table 6 may be determined. A decrease in the level or amount of the at least one biomarker from Table 2, Table 4 and/or Table β, following administration of the aminopeptidase inhibitor is indicative that the cancer condition is responsive to treatment with the aminopeptidase inhibitor i.e. the aminopeptidase inhibitor is efficacious in treating the cancer condition in the individual . In some preferred embodiments, the at least one biomarker may be a secreted polypeptide set out in Table 5 or 6. The level or amount of secreted biomarker polypeptide may be conveniently determined in samples of biological fluid, such as blood, using immunological techniques, as described below.

In some especially preferred embodiments of the methods described herein, the level or amount of a biomarker selected from stanniocalcin 2 and/or CCL5 may be determined. An increase in the level or amount of stanniocalcin 2 and/or CCL5 following administration of the aminopeptidase inhibitor is indicative that the cancer condition responds to treatment with the aminopeptidase inhibitor

Stanniocalcin 2 (STC2; GenelD: 8614) may have the amino acid sequence of NCBI database entry NP_003705.1 GI: 4507267 or may be an allelic variant of this sequence and may be encoded by the nucleic acid sequence of NCBI database entry NM_003714.2 GI: 61676085 or an allelic variant of this sequence.

CCL5 (GenelD: 6352) may have the amino acid sequence of NCBI database entry NP_002976.2 GI: 22538814 or may be an allelic variant of this sequence and may be encoded by the nucleic acid sequence of NCBI database entry NM_002985.2 GI: 22538813 or an allelic variant of this sequence .

An allelic variant is a variant of a reference nucleotide or amino acid sequence which occurs naturally in one or more individuals in a population (e.g. humans) . An allelic variant of a reference sequence may comprise a sequence having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the reference sequence .

Amino acid identity is generally defined with reference to the algorithm GAP (GCG Wisconsin Package™, Accelrys, San Diego CA) . GAP uses the Needleman & Wunsch algorithm (J. MoI. Biol. (48) : 444-453

(1970) ) to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, the default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST or TBLASTN (which use the method of Altschul et al. (1990) J. MoI. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448) , or the Smith-Waterman algorithm (Smith and Waterman (1981) J. MoI Biol. 147: 195-197), generally employing default parameters.

An allelic variant of a reference amino acid sequence of a biomarker may differ from a reference sequence by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20 20-30 or 30-50 amino acids. An allelic variant of a reference nucleotide sequence may differ from a reference nucleotide sequence of a biomarker by insertion, addition, substitution or deletion of 1 nucleotide, 2, 3, 4, 5-10, 10-20 20-30 or 30-50 nucleotides.

A biomarker which is measured as described herein, such as Stanniocalcin 2 or CCL5, may be glycosylated.

The type of sample in which the level or amount of the at least one biomarker is measured will depend on a variety of factors such as the particular biomarker (s) , where and when the particular biomarker is synthesized, where the biomarker may be stored in the tissues, and into what biological tissue or fluid it may be released or otherwise accumulate .

In some embodiments, the sample may be blood, a blood component such as serum or plasma, cerebrospinal fluid (CSF) , saliva, or urine; preferably blood, serum, plasma, or CSF; most preferably blood, serum, or plasma. Preferably, the level or amount of a secreted biomarker, such as stanniocalcin 2 and CCL5, may be measured in blood samples, including whole blood, plasma or serum, obtained from the individual. Immunoassay formats may be particularly suitable for testing such samples. In other embodiments, the biological sample may be a tissue sample and in particular a biopsy from a cancerous tissue e.g. a tumour biopsy. Where more than one biomarker is analyzed, the analysis can be conducted on the same or different biological samples obtained from the patient. The sample may be freshly obtained from the individual or may be stored prior to testing. For example, the sample may be frozen or refrigerated prior to testing.

Other aspects of the invention relate to the design and optimisation of treatment regimens for aminopeptidase inhibitors, in particular, regimens for the treatment of cancer conditions. For example, methods described herein may be useful in selecting an appropriate treatment or treatment regimen for an individual.

A method for determining the responsiveness of an individual with a cancer condition to a regimen of treatment with an aminopeptidase inhibitor may comprise:

(a) subjecting the individual to a regimen of treatment with the aminopeptidase inhibitor; and (b) measuring in the individual the level or amount of at least one biomarker from Tables 1 and 2.

A change in the level or amount of the at least one biomarker, for example an increase in the level of a biomarker of Table 1 or a decrease in the level of a biomarker of Table 2, is indicative that the individual is responsive to the regimen. The regimen is therefore suitable for treatment of the individual.

Preferably, the level or amount of at least one biomarker from Tables 3 and/or 5 or the level or amount of at least one biomarker from Tables 4 and/or 6 is determined.

As described above, the amount of at least 1, at least 2, at least 3, at least 4, at least 5 at least 10, at least 20 or at least 30 biomarkers shown in Tables 1 and 2 in the sample may be determined. The level or amount of the at least one biomarker may be measured in samples obtained at one or more time points during or after the treatment. The amount of change in the level or amount of biomarker may be indicative of the level of responsiveness of the cancer condition.

A treatment regimen is a predetermined scheme or program which defines the parameters of the treatment to which the individual is to be subjected. For example, the regimen may set out the dosage, the mode of administration and the timetable or schedule of administration of the aminopeptidase inhibitor with which the individual is to be treated.

An appropriate regimen of treatment with the aminopeptidase inhibitor can vary from patient to patient. Determining the appropriate dosage, mode and schedule of administration will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments. For example, the initial dosage level and schedule will depend on a variety of factors including, but not limited to, the activity of the particular aminopeptidase inhibitor, the chosen route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the individual. The parameters of the regimen may be optimised for an individual using the methods described below.

The initial treatment regimen will ultimately be at the discretion of the physician, although generally the dosage and other parameters will be selected in order to achieve aminopeptidase inhibition as assessed using the methods described herein, without causing substantial harmful or deleterious side-effects.

In the absence of any change in the level or amount of the at least one biomarker, the regimen may be altered, for example by increasing the dosage, frequency of administration and/or duration of treatment, and the responsiveness of the individual to the altered regimen determined. This may be repeated until a change in the level or amount of the at least one biomarker is observed. A treatment regimen which changes the level or amount of the at least one biomarker may be identified.

In some embodiments, a treatment regimen which produces a change in the level or amount of the at least one biomarker may be altered, for example, by increasing the dosage, frequency of administration and/or duration of treatment, and the responsiveness of the individual to the altered regimen determined. This may be repeated until no further change in the level or amount of the at least one biomarker is observed. A treatment regimen which produces a maximal change in the level or amount of the at least one biomarker may be identified.

In some embodiments, following identification of a treatment regimen which changes the level or amount of the at least one biomarker or produces a maximal change in the level or amount of the at least one biomarker, the safety, tolerability and/or pharmacokinetic effects of the regimen may be assessed in one or more individuals.

In some embodiments, the clinical effect of the aminopeptidase inhibitor on the cancer condition in the individual may be assessed during and/or after the treatment regimen. The clinical effect of the aminopeptidase inhibitor may be correlated with the change in the level or amount of the at least one biomarker (i.e. the relationship between the clinical effect and the change in the level or amount of the at least one biomarker may be determined) . The correlation may be expressed numerically, for example as an algorithm or graphically, for example as a response curve. This allows the clinical effect of a treatment regimen on an individual to be predicted from the change in the level or amount of the at least one biomarker which is observed in the individual during and/or after the treatment regimen. The progress of aminopeptidase inhibitor treatment may be monitored in an individual using the methods described herein, for example to ensure that the pharmacological effect is sustained in the individual throughout the duration of the treatment. A method for monitoring the treatment of a cancer condition in individual with an aminopeptidase inhibitor may comprise:

(a) subjecting the individual to a regimen of treatment with the aminopeptidase inhibitor; and

(b) monitoring in the individual the level or amount of at least one biomarker selected from the biomarkers of Tables 1 and 2 during said treatment.

Preferably, the level or amount of at least one biomarker from Tables 3 and/or 5 or the level or amount of at least one biomarker from Tables 4 and/or 6 is monitored.

The level or amount of at least one biomarker may be monitored by periodically obtaining samples from the individual and measuring the level or amount of biomarker in the samples obtained.

A change in the level or amount of the at least one biomarker in response to the regimen which is sustained over its duration, for example, because biomarker levels remain above or below a predetermined value or within a predetermined range of values throughout the treatment, is indicative that the regimen is effective for treating the cancer condition in the individual.

Preferably, the change in the level or amount of the at least one biomarker in response to the regimen is a maximal change i.e. no further changes in the level or amount of the at least one biomarker occur when the dosage of aminopeptidase inhibitor is increased further.

A change in the level or amount of the at least one biomarker in response to the regimen which is not sustained over its duration, for example, because biomarker levels move beyond a predetermined value or outside a predetermined range of values, is indicative that the regimen is ineffective, partially effective or not fully effective in treating the cancer condition in the individual .

A regimen which is found to be not fully effective may be altered, for example by altering the dosage or schedule, to restore the change in the level or amount of the at least one biomarker; for example, by restoring biomarker levels to above or below a predetermined value or within a predetermined range of values. In some embodiments, the dosage or frequency of administration of the aminopeptidase inhibitor may be increased if the level or amount of at least one biomarker from Table 1 falls below a predetermined value or decreased if the level of the at least one biomarker from Table 1 rises above a predetermined value. The dosage or frequency of administration of the aminopeptidase inhibitor may be decreased if the level or amount of at least one biomarker from Table 2 falls below a predetermined value or increased if the level of the at least one biomarker from Table 2 rises above a predetermined value.

A regimen which is found to be produce toxicological effects in the individual may be altered, for example by reducing the dosage, to reduce or abolish the toxicological effects.

The predetermined values for the level or amount of the biomarker in the sample may, for example, be the amount of the biomarker which is higher (Table 1 biomarkers) or lower (Table 2 biomarkers) statistically than the amount which is determined to be present in a biological sample obtained from the patient without treatment with the aminopeptidase inhibitor. The predetermined value depends upon the particular biomarker.

The methods described herein may be useful in optimising a treatment regimen for an individual. A method for optimising a regimen of cancer treatment with an aminopeptidase inhibitor for an individual may comprise: (a) subjecting the individual to an initial regimen of aminopeptidase inhibitor treatment;

(b) monitoring the level of at least one biomarker selected from the biomarkers of Tables 1 and 2 in the individual, wherein a change in the level or amount of the at least one biomarker in response to the regimen which is sustained over its duration is indicative that the regimen is optimised for the treatment of cancer in the individual .

Preferably, the level or amount of at least one biomarker from Tables 3 and/or 5 or the level or amount of at least one biomarker from Tables 4 and/or 6 is monitored.

In some embodiments, a change in the level of the at least one biomarker beyond a pre-determined value which is sustained over the duration of the treatment is indicative that the treatment is optimised for the treatment of cancer in the individual.

The initial regimen, e.g. the initial dosage, frequency of administration and duration of treatment of the aminopeptidase inhibitor may be determined by a medical practitioner. If the initial regimen of the aminopeptidase inhibitor treatment is insufficient to cause a sustained change in the level or amount of the biomarker, the regimen may be altered or adjusted until the level of the biomarker in the individual changes. In some embodiments, if the initial regimen of the aminopeptidase inhibitor treatment is insufficient to change the level or amount of the biomarker beyond a predetermined value, the regimen may be altered or adjusted until the level of the biomarker in the individual is beyond the pre-determined value. A method may thus comprise the further steps;

(c) altering the regimen of aminopeptidase inhibitor treatment and subjecting the individual to the altered regimen;

(d) monitoring the level of at least one biomarker selected from the biomarkers of Tables 1 and 2 in the individual, and (e) repeating steps c) and d) until a change in the level of the one or more biomarkers is observed, wherein a change in the level or amount of the at least one biomarker in response to the regimen which is sustained over its duration is indicative that the regimen is optimised for the treatment of cancer in the individual.

In some embodiments, step (e) may comprise repeating steps c) and d) until the level or amount of the one or more biomarkers changes beyond a predetermined value, wherein a change in the level of the at least one biomarker beyond the predetermined value is indicative that the regimen is optimised for the treatment of cancer in the individual.

The regimen may be altered by increasing one or more of the dosage, frequency of administration, and/or duration of the aminopeptidase inhibitor treatment. The skilled person is readily able to alter or modify the treatment regimen and monitor resultant biomarker levels as described herein.

In some embodiments, if a regimen of the aminopeptidase inhibitor treatment changes the level or amount of the biomarker, the regimen may be altered or adjusted until the maximum change in the level or amount of the biomarker in the individual is observed. A method may thus comprise the further steps;

(c) subjecting the individual to an increased dose regimen; (d) monitoring the level of at least one biomarker selected from the biomarkers of Tables 1 and 2 in the individual, and

(e) repeating steps c) and d) until no further change in the level of the one or more biomarkers is observed, wherein no further change in the level or amount of the at least one biomarker in response to the increased dose regimen is indicative that the regimen is optimised for the treatment of cancer in the individual .

The dose regimen may be progressively increased until the maximal effect on the level or amount of the at least one biomarker is observed. The toxicological effect of the increased dose regimen may be assessed in the individual. Toxicological effects may be assessed by a medical practitioner and determined to be acceptable or unacceptable in accordance with standard criteria. A regimen which is found to cause unacceptable toxicological effects in the individual may be altered, for example by reducing the dosage, to reduce or abolish the toxicological effects.

The level or amount of biomarker in a sample may be measured at the polypeptide level. Many suitable methods are available in the art for determining the amount of a biomarker peptide, polypeptide or protein in a sample including, for example, Western blot analysis, immunohistochemistry (Angele S et al (2000) Clin. Cancer Res. 6, 3536- 3544) and immunoassay (Butch AW et al (2004) Clinical Chemistry 50, 2303-2308) .

For example, the level or amount of a biomarker in a sample may be determined by contacting the sample with a specific binding member, such as an antibody, directed against the biomarker peptide or protein, and the amount of binding of the specific binding member to the sample determined. The presence or amount of binding of the specific binding member is indicative of the level or amount of the biomarker within the sample.

Numerous techniques and formats for determining the presence or amount of a target polypeptide using an antibody or other specific binding member are known in the art. Any appropriate immunoassay method can be used, including radioimmunoassays, sandwich enzyme-linked immunoassays, competitive binding assays, homogeneous assays, and heterogeneous assays. The assay can be carried out in various formats including, e.g., in a microtiter plate format, using automated immunoassay analyzers known in the art. Antibodies to protein biomarkers which are suitable for use in immunoassays may be produced using standard techniques or obtained from commercial sources (for example, Abnova Corp, TW; Bethyl Laboratories Inc, USA; Abgent Inc, USA; Abeam Ltd, UK) .

Alternatively, the amount of biortiarker peptide or protein can be determined using other techniques such as magnetic resonance spectroscopy, HPLC, or mass spectrometry.

The level of biomarker peptide or polypeptide may be measured as described above in a sample of biological fluid obtained from the individual, such as a sample of blood or a blood fraction.

Preferred biomarker peptides or polypeptides for detection as described above may include secreted biomarker peptides or polypeptides, for example a biomarker peptide or polypeptide set out in Tables 5 and 6.

In other embodiments, the level or amount of biomarker in the sample may be determined at the nucleic acid level. The amount of the biomarker may, for example, be measured by measuring the level or amount of biomarker nucleic acid in the sample. Any convenient technique may be employed and numerous suitable techniques are available in the art (see, for example, Molecular Cloning: a Laboratory Manual: 3rd edition, Sambrook & Russell (2001) Cold Spring Harbor Laboratory Press NY; Current Protocols in Molecular Biology, Ausubel et al . eds . John Wiley & Sons (1992); DNA Cloning, The

Practical Approach Series (1995), series eds. D. Rickwood and B. D. Hames, IRL Press, Oxford, UK and PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif) . For example, the amount of transcribed biomarker nucleic acid, such as mRNA or cDNA, in one or more cells within the sample (e.g. cancer cells) may be measured. This may be performed using standard techniques such as Northern blotting, RT-PCR or microarray techniques.

In some preferred embodiments, the level of biomarker nucleic acid is measured in a tissue biopsy, for example a tumour biopsy, obtained from the individual. A cancer condition which may be treated with an aminopeptidase inhibitor as described herein may be any type of solid or non-solid cancer or malignant lymphoma and especially leukaemias, such as acute or chronic myeloid leukaemia and acute or chronic lymphocytic leukaemia, multiple myeloma, myelodysplastic syndromes, sarcomas, skin cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, non small cell lung cancer, small cell lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, oesophageal cancer, pancreatic cancer, renal cancer, stomach cancer and cerebral cancer.

A individual suitable for assessment as described herein may include a eukaryote, an animal, a vertebrate animal, a mammal, a rodent (e.g. a guinea pig, a hamster, a rat, a mouse), a murine (e.g. a mouse), a canine (e.g. a dog), a feline (e.g. a cat), an equine (e.g. a horse), a primate, such as a simian (e.g. a monkey or ape), a monkey (e.g. marmoset, baboon), an ape (e.g. gorilla, chimpanzee, gibbon), or a human .

Treatment with an aminopeptidase inhibitor as described herein may comprise administering the aminopeptidase inhibitor to the individual in a therapeutically effective dosage. Treatment may be in accordance with a predetermined treatment regimen, for example a predetermined dosage, frequency of administration and duration of treatment, as described above. While it is possible for an aminopeptidase inhibitor to be administered alone, it is preferable to present it as a pharmaceutical composition (e.g., formulation) comprising at least one aminopeptidase inhibitor as defined above, together with one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilisers, preservatives, lubricants, or other materials well known to those skilled in the art and optionally other therapeutic or prophylactic agents.

Pharmaceutical compositions comprising an aminopeptidase inhibitor described herein admixed or formulated together with one or more pharmaceutically acceptable carriers, excipients, buffers, adjuvants, stabilisers, or other materials, as described herein, may be used in the methods described herein.

The term "pharmaceutically acceptable" as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical

Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well-known in the art of pharmacy. Such methods include the step of bringing the active compound into association with a carrier which may constitute one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

Formulations may be in the form of liquids, solutions, suspensions, emulsions, elixirs, syrups, tablets, lozenges, granules, powders, capsules, cachets, pills, ampoules, suppositories, pessaries, ointments, gels, pastes, creams, sprays, mists, foams/ lotions, oils, boluses, electuaries, aerosols, patches or implants. The compound (s) or pharmaceutical composition comprising the compound (s) may be administered to a subject by any convenient route of administration, whether systemically/ peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion) or parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot, for example, subcutaneously or intramuscularly.

In some embodiments, the compound (s) or pharmaceutical composition comprising the compound(s) may be administered by mechanical methods, for example by means of a patch or implant, such as a cutaneous or sub-cutaneous patch.

Formulations suitable for oral administration (e.g., by ingestion) may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous or nonaqueous liquid; or as an oil-in-water liquid emulsion or a water-in- oil liquid emulsion; as a bolus; as an electuary; or as a paste.

Formulations suitable for parenteral administration (e.g. by injection, including cutaneous, subcutaneous, intramuscular, intravenous and intradermal) , include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions which may contain anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs . Examples of suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the active compound in the solution is from about 1 ng/ml to about 10 μg/ml, for example, from about 10 ng/ml to about 1 μg/ml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.

Suitable pharmaceutical formulations of aminopeptidase inhibitors, such as bestatin and CHR-2797, are well-known in the art (Ota K, Uzuka Y et al. Biotherapy 1992; 4 : 205-14; Ichinose Y et al J Natl Cancer Inst 2003; 95: 605-10; Needham LA et al (2008) Cancer Research In Press; Davies F et al Blood 2007; 110; Abst 443; Protheroe A et al J Clin Oncol 2007;25(18S) :Abst 3537) .

Treatment may be effected in one dose, continuously or intermittently (e.g. in divided doses at appropriate intervals) throughout the course of treatment. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. The most effective means and dosage of administration may vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated and methods of determining the most effective means and dosage of administration in any particular case are described herein.

In general, a suitable dose of the aminopeptidase inhibitor is in the range of about 100 μg to about 100 nag per kilogram body weight of the subject per day. Where the active compound is a salt, an ester, prodrug, or the like, the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.

Other aspects of the invention provide kits for use in performing methods described above, e.g. for assessing aminopeptidase inhibition in an individual .

A kit may comprise reagents for determining the amount of biomarker nucleic acid in a sample, such a tumour biopsy. For example, the kit may comprise one or more of a microarray comprising one or more biomarker nucleic acids immobilised on a solid support; one or more primers for use in a PCR or RT-PCR reaction and one or more probes for use in a hybridisation reaction. A kit may, for example, comprise suitable reagents and materials for performing RT-PCR, microarray analysis, or Northern blotting as described herein.

A kit may comprise reagents for determining the amount of biomarker protein or peptide in a sample, such a blood sample. For example, a kit may comprise one or more antibodies or other ligands for a biomarker protein and/or one or more detection reagents, including secondary antibodies, labels, and enzymatic substrates. A kit may, for example, comprise suitable reagents and materials for performing immunoassays, such as enzyme linked immunosorbent assays (ELISAs) , immunoblotting, e.g. Western blots, or in situ hybridization, as described herein.

A kit may also include suitable means for detection, buffers, calibration and any other reagent for use in a protein peptide or nucleic acid assay. A kit may comprise one or more controls, standards or references for use in a test. The kit may comprise software for use in a method described herein.

Reagents may be sealed in a suitable container that protects its contents from the external environment.

A kit typically also comprises instructions for using the kit components in a method described herein.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

All documents mentioned in this specification are incorporated herein by reference in their entirety. The sequences set out in the database entries mentioned in this specification are also incorporated by reference. Unless stated otherwise, the sequence of a database entry refers to the sequence version in the database entry which is current at the filing date.

"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example ^λλA and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures and tables described below.

Figure 1 shows a dose response to CHR-2797 of the expression of SLC7A11 (Top) and TRIB3 (Bottom) in HL-60 cells treated with 0.06-6μM CHR-2797 over a 72hr time course.

Figure 2 shows the expression of SLC7A11, TRIB3 and ASNS in a number of human haematopoetic tumour cell lines treated with 6μM CHR-2797 for 6hr.

Figure 3 shows Western blot results which show increased stanniocalcin 2 secretion following treatment of HL-60 cells with 6 μM of CHR-2797 over 48 hours.

Figure 4 shows Western blot results which show a dose response to CHR- 2797 of stanniocalcin 2 secretion following treatment of HL-60 cells with 0.06-6 μM of CHR-2797 for 48 hours. Figure 5 shows Western blot results which show increased CCL5 secretion following treatment of H23 cells with 6 μM of CHR-2797 over 72 hours.

Figure 6 shows Western blot results which show a dose response to CHR- 2797 of CCL5 secretion following treatment of H23 cells with 0.06-6 μM of CHR-2797 for 72 hours.

Figure 7 shows by quantitative ELISA, data showing increased CCL5 secretion following treatment of H23 cells with 6 μM of CHR-2797 over 72 hours.

Figure 8 shows, by quantitative ELISA, data showing a response to CHR- 2797 of CCL5 secretion following treatment of H23 cells with 0.06-6 μM of CHR-2797 for 72 hours.

Table 1 shows the genes whose expression is increased by ≥ than 2 fold upon treatment of the human promyelocytic leukaemia cell line, HL-60, or the human lung adenocarcinoma cell line, H23, with 6 μM of CHR-2797 compared to vehicle control.

Table 2 shows the genes whose expression is decreased by ≥ than 2 fold upon treatment of the human promyelocytic leukaemia cell line, HL-60, or the human lung adenocarcinoma cell line, H23, with 6 μM of CHR-2797 compared to vehicle control.

Table 3 shows a subset of genes from Table 1 whose expression, by quantitative PCR, is increased by > than 2 fold upon treatment of the human promyelocytic leukaemia cell line, HL-60, or the human lung adenocarcinoma cell line, H23, with 6 μM of CHR-2797 compared to vehicle control and whose expression is also increased with 200 μM of the prototypical aminopeptidase inhibitor, bestatin compared to vehicle control.

Table 4 shows a subset of the genes from Table 2 whose expression, by quantitative PCR, is decreased by > than 2 fold upon treatment of the human promyelocytic leukaemia cell line, HL- 60, or the human lung adenocarcinoma cell line, H23, with 6 μM of CHR-2797 compared to vehicle control and whose expression is also decreased with 200 μM of the prototypical aminopeptidase inhibitor, bestatin compared to vehicle control.

Table 5 shows a subset of genes from Table 1 whose expression is increased by ≥ than 2 fold upon treatment of the human promyelocytic leukaemia cell line, HL-60, or the human lung adenocarcinoma cell line, H23, with 6 μM of CHR-2797 compared to vehicle control, and whose protein products are secreted.

Table 6 shows a subset of genes from Table 2 whose expression is decreased by ≥ than 2 fold upon treatment of the human promyelocytic leukaemia cell line, HL-60, or the human lung adenocarcinoma cell line, H23, with 6 μM of CHR-2797 compared to vehicle control, and whose protein products are secreted.

Materials and Methods Compounds

All reagents were from Sigma (Poole, UK) unless otherwise stated and were of the highest chemical grade . All compounds were prepared as 1OmM stocks in dimethyl sulfoxide (DMSO) (except bestatin, 10OmM) .

Cell Culture

HL-60 and H23 cell lines were obtained from the American Type Culture Collection (LGC Promochem, Teddington, UK) . Cells were grown in RPMI 1640 media supplemented with 2mM glutamine, 10% foetal bovine serum (FBS; HyClone, Logan, UT), lOOU/ml penicillin and lOOμg/ml streptomycin (H23 cells were also supplemented with 10% RPMI 1640 vitamins solution) . Cell cultures were maintained in logarithmic growth phase in a humidified incubator at 37⁰C in 5% CO2.

Microarray Studies The human promyelocytic leukemia cell line, HL-60, and the human lung adenocarcinoma cell line, H23, were treated with 6μM CHR-2797, equivalent to 20Ox the IC50 for its inhibition of proliferation in either cell line (3OnM) (Needham LA et al (2008) Cancer Research In Press), for 6 and 24hr. Control cells were treated with vehicle (0.06% DMSO) for 6hr. Total RNA was extracted from vehicle or compound- treated cells using commercially available kits (Qiagen, Crawley, UK) . Independent triplicate total RNA samples were pooled, and biotin- labelled cRNA was synthesised and fragmented before hybridisation to Affymetrix Human Genome U133 Plus 2.0 arrays (Almac Diagnostics Ltd., Craigavon, UK) using the manufacturer's standard protocols.

RNA extraction and gene expression microarray studies Total RNA was extracted from vehicle or compound-treated cells using commercially available kits (Qiagen, Crawley, UK) . Biotin-labelled cRNA was synthesised and fragmented before hybridisation to Affymetrix Human Genome U133 Plus 2.0 arrays (Almac Diagnostics Ltd., Craigavon, UK) using the manufacturer's standard protocols.

Real time quantitative PCR

Gene expression changes identified by microarray analysis were validated by real time quantitative PCR. cDNA was synthesised from lOOOng total RNA using Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA) and anchored oligo-dT primers (Abgene, Epsom, UK) . For all genes (except TRIB3, for which the TaqMan® Gene Expression Assay was used; Applied Biosystems, Foster City, CA) , SYBR Green quantitative PCR was performed using the 7300 Real Time PCR

System (Applied Biosystems) . Thermal cycling conditions were 95⁰C for lOmin followed by 40 cycles of 95°C for 15s and 60°C for lmin. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was amplified as the endogenous control gene from all samples and reactions were performed in triplicate. Data were analysed using the 7300 Sequence Detection software (Applied Biosystems) .

SDS-PAGE and immunoblotting

Proteins in cell culture medium were resolved by SDS-PAGE (Invitrogen, Paisley, UK) and transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat milk in tris-buffered saline/0.1% Tween (TBS-T) for lhr, incubated with primary antibody overnight at 4°C, washed in TBS-T and incubated with horseradish-peroxidase-labeled secondary antibodies (Cell Signalling Technology, Danvers, MA) for lhr at room temperature. Detection was achieved using an enhanced chemiluminescence reagent (Amersham, Little Chalfont, UK) .

Protein Secretion Studies- HL-60 cells

Culture medium from HL-60 cells treated with 0.06% DMSO vehicle or 0.06-6μM CHR-2797 over a 48hr time course was harvested. Following SDS-PAGE, Western blots were probed with antibodies to stanniocalcin 2 (R&D Systems, Abingdon, UK) . Recombinant stanniocalcin 2 (0.5ng) was obtained from Abeam (Cambridge, UK) .

Protein Secretion Studies- H23 cells Culture medium from H23 cells treated with 0.06% DMSO vehicle or 0.06- 6μM CHR-2797 over a 72hr time course was harvested. Following SDS- PAGE, Western blots were probed with antibodies to CCL5 (Abeam) . CCL5 secretion was also quantitated by ELISA using the Quantikine kit for human CCL5 (RANTES) (R&D Systems) .

Results

Gene expression profiling in HL-60 and H23 cells treated with CHR-2797 The human promyelocytic leukemia cell line, HL-60, and the human lung adenocarcinoma cell line, H23, were treated with 6μM CHR-2797, equivalent to 20Ox the IC50 for its inhibition of proliferation in either cell line (3OnM) (Needham LA et al (2008) Cancer Research In Press ), for 6 and 24hr. Control cells were treated with vehicle (0.06% DMSO) for 6hr. Independent triplicate total RNA samples were pooled, and gene expression changes were analysed using Affymetrix Whole Genome U133 Plus 2.0 microarrays.

Genes whose expression was observed to change by ≥ than 2 fold upon treatment of the human promyelocytic leukaemia cell line, HL-60, or the human lung adenocarcinoma cell line H23 with 6 μM of CHR-2797 compared to vehicle control are shown in Tables 1 and 2. Genes whose expression is increased are shown in Table 1. Genes whose expression is decreased are shown in Table 2.

The human promyelocytic leukemia cell line, HL-60, and the human lung adenocarcinoma cell line, H23, were treated with 200μM of the prototypical aminopeptidase inhibitor, bestatin, equivalent to 1Ox the IC50 for its inhibition of proliferation in either cell line (20μM) (Needham LA et al (2008) Cancer Research In Press ) , for 6 and 24hr. Control cells were treated with vehicle (0.06% DMSO) for 6hr. Gene expression changes for a subset of the genes in Tables 1 and 2 were analysed using quantitative PCR.

Genes whose expression was observed to change by ≥ than 2 fold upon treatment of the human promyelocytic leukaemia cell line, HL-60, or the human lung adenocarcinoma cell line H23 with 200 μM of bestatin compared to vehicle control are shown in Tables 3 and 4. Genes whose expression is increased are shown in Table 3. Genes whose expression is decreased are shown in Table 4.

Preferred subsets of the genes in Tables 1 and 2 are shown in Tables 5 and 6. The protein products of these genes are either known to be, or predicted to be, secreted (according to the annotation in the Secreted Protein Database, SPD; http://spd.cbi.pku.edu.cn/) . Genes whose protein products are secreted and whose expression is increased are shown in Table 5. Genes whose protein products are secreted and whose expression is decreased are shown in Table 6.

Gene expression dose response in CHR-2797 treated cells

Since cells were treated with 20Ox the IC₅₀ for CHR-2797 in the microarray study, gene expression induced by lower concentrations of the compound was also examined. Figure 1 shows a dose-response and time course of CHR-2797 treatment for two representative genes, SLC7A11 (Top) and TRIB3 (Bottom) . Increases in gene expression were detectable at a dose 10Ox lower (0.06μM; equivalent to 2x the IC₅₀ for inhibition of proliferation) than that used in the microarray study, and as early as 2hr post-treatment. Cell line selectivity of the transcriptional response to CHR-2797 treatment

To investigate the cell line selectivity of the transcriptional response to CHR-2797, three further leukemic cell lines were treated with the compound; U-937 (histiocytic lymphoma) , MOLT-4 (acute lymphoblastic leukemia) and HuT 78 (T-cell lymphoma) . The IC50s for the inhibition of proliferation by CHR-2797 in these cell lines are 1OnM, 145OnM and >10μM respectively (Needham LA et al (2008) Cancer Research In Press) . CHR-2797 treatment increased expression of the same genes in the two additional cell lines most sensitive to the anti-proliferative effects of the compound, but not in HuT 78 cells which are insensitive to the anti-proliferative effects (Figure 2) .

Secretion of Stanniocalcin 2 in CHR-2797 treated cells

Treatment of HL-60 cells with CHR-2797 led to an increase in the secretion of Stanniocalcin 2 (STC2) protein into the growth medium. Increased Stanniocalcin 2 secretion was observed by Western Blot following treatment of HL-60 cells with 6μM CHR-2797 over 48 hours (Figure 3) . A dose response to CHR-2797 of Stanniocalcin 2 secretion following treatment of HL-60 cells with 0.06-6 μM of CHR-2797 for 48 hours was also demonstrated (Figure 4) .

Secretion of CCL5 in CHR-2797 treated cells Treatment of H23 cells with CHR-2797 led to an increase in the secretion of CCL5 protein into the growth medium. Increased CCL5 secretion was observed by both Western Blot (Figure 5) and quantitative ELISA (Figure 7) following treatment of H23 cells with 6μM CHR-2797 over 72 hours. A dose response to CHR-2797 of CCL5 secretion was demonstrated by both Western Blot (Figure 6) and quantitative ELISA (Figure 8) following treatment of H23 cells with 0.06-6 μM of CHR-2797 for 72 hours. Sequences

1 mcaerlgqfm tlalvlatfd pargtdatnp pegpqdrssq qkgrlslqnt aeiqhclvna

61 gdvgcgvfec fennsceirg lhgicmtflh nagkfdaqgk sfikdalkck ahalrhrfgc 121 isrkcpaire mvsqlqrecy lkhdlcaaaq entrvivemi hfkdlllhep yvdlvnlllt

181 cgeevkeait hsvqvqceqn wgslcsilsf ctsaiqkppt apperqpqvd rtklsrahhg

241 eaghhlpeps sretgrgakg ergskshpna hargrvgglg aqgpsgssew edeqseysdi

301 rr

STC2 amino acid sequence (SEQ ID NO:1)

1 tttctccttc cctccacggg ccgggtgaga aagtagccgg gggctatccc gacccggcgg

61 ttcttgggga gggggccgaa caagaaaagg gaggagatgg agataacttc cccggattta

121 gcttttttgt ctttgttttt gttctcacca cttccatcgg atgactggag agtaaaaggg

181 aacccggagc ggggtggcga gcagcgcttt gagaaaatgc aggagtgtgt ttggagacgc 241 gtaaagttgc ctttcaagct ctggcctccg ggcacgcgat gctccgcggc gggctgactc

301 agggctgcct tgggcctccc tgccaccctc ctggaaatga tgcaagtcct gactgtcacc

361 tggatccctg cagcccagcc tggaatgcgt ctggattagg ggaaagacga gaaacgacac

421 tccaggtgtt gcacggccca ccaaagcggg aagatagggc agttgctcag accaaatact

481 gtatctagtg cttctgctcc tatcttcaat cgtggggttc tttttaatgc aaagtgtcac 541 aaggccagga attcccatgt gtgctcagtt ggcccacagc atcattgtgc ctaggaaact

601 gcttcaattt atcaagtcct ctgggctggg aatctcactg aattccaaac ggcggaaaga

661 ggaaactttc ccaacccgat gtgggtgtga cgcgagccag gggccccagg gacactgtcc

721 cagagcacac cgtccccctt taacagcaac tggagcttgg attcgctctt atattgtaca

781 gtcctttcga ccattgccct ggagcacccg cacacgcgca cgcatctccg gccgcgctca 841 cacacactca tacacacgca cgcaaacgcg tggccgccgc caggtcggca actttgtccg

901 gcgctcccag cggcgctcgg cttcctcctg tagtagttga gcgcaggccc cgcctcccgg

961 ccgtgttgtc aaaagggccg gggtctcgga ttggtccagc cgccgggaca acacctgctc

1021 gactccttca ttcaagtgac accagagctt ccagggatat ttgaggcacc atccctgcca

1081 ttgccgggca ctcgcggcgc tgctaacggc ctggtcacat gctctccgga gagctacggg 1141 agggcgctgg gtaacctcta tccgagccgc ggccgcgagg aggagggaaa aggcgagcaa

1201 aaaggaagag tgggaggagg aggggaagcg gcgaaggagg aagaggagga ggaggaagag

1261 gggagcacaa aggatccagg tctcccgacg ggaggttaat accaagaacc atgtgtgccg

1321 agcggctggg ccagttcatg accctggctt tggtgttggc cacctttgac ccggcgcggg

1381 ggaccgacgc caccaaccca cccgagggtc cccaagacag gagctcccag cagaaaggcc 1441 gcctgtccct gcagaataca gcggagatcc agcactgttt ggtcaacgct ggcgatgtgg

1501 ggtgtggcgt gtttgaatgt ttcgagaaca actcttgtga gattcggggc ttacatggga

1561 tttgcatgac ttttctgcac aacgctggaa aatttgatgc ccagggcaag tcattcatca

1621 aagacgcctt gaaatgtaag gcccacgctc tgcggcacag gttcggctgc ataagccgga

1681 agtgcccggc catcagggaa atggtgtccc agttgcagcg ggaatgctac ctcaagcacg 1741 acctgtgcgc ggctgcccag gagaacaccc gggtgatagt ggagatgatc catttcaagg

1801 acttgctgct gcacgaaccc tacgtggacc tcgtgaactt gctgctgacc tgtggggagg

1861 aggtgaagga ggccatcacc cacagcgtgc aggttcagtg tgagcagaac tggggaagcc

1921 tgtgctccat cttgagcttc tgcacctcgg ccatccagaa gcctcccacg gcgccccccg 1981 agcgccagcc ccaggtggac agaaccaagc tctccagggc ccaccacggg gaagcaggac 2041 atcacctccc agagcccagc agtagggaga ctggccgagg tgccaagggt gagcgaggta 2101 gcaagagcca cccaaacgcc catgcccgag gcagagtcgg gggccttggg gctcagggac 2161 cttccggaag cagcgagtgg gaagacgaac agtctgagta ttctgatatc cggaggtgaa 2221 atgaaaggcc tggccacgaa atctttcctc cacgccgtcc attttcttat ctatggacat 2281 tccaaaacat ttaccattag agagggggga tgtcacacgc aggattctgt ggggactgtg 2341 gacttcatcg aggtgtgtgt tcgcggaacg gacaggtgag atggagaccc ctggggccgt 2401 ggggtctcag gggtgcctgg tgaattctgc acttacacgt actcaaggga gcgcgcccgc 2461 gttatcctcg tacctttgtc ttctttccat ctgtggagtc agtgggtgtc ggccgctctg 2521 ttgtggggga ggtgaaccag ggaggggcag ggcaaggcag ggcccccaga gctgggccac 2581 acagtgggtg ctgggcctcg ccccgaagct tctggtgcag cagcctctgg tgctgtctcc 2641 gcggaagtca gggcggctgg attccaggac aggagtgaat gtaaaaataa atatcgctta 2701 gaatgcagga gaagggtgga gaggaggcag gggccgaggg ggtgcttggt gccaaactga 2761 aattcagttt cttgtgtggg gccttgcggt tcagagctct tggcgagggt ggagggagga 2821 gtgtcatttc tatgtgtaat ttctgagcca ttgtactgtc tgggctgggg gggacactgt 2881 ccaagggagt ggcccctatg agtttatatt ttaaccactg cttcaaatct cgatttcact 2941 ttttttattt atccagttat atctacatat ctgtcatcta aataaatggc tttcaaacaa 3001 agcaactggg tcattaaaac cagctcaaag ggggtttaaa aaaaaaaaac cagcccatcc 3061 tttgaggctg atttttcttt tttttaagtt ctattttaaa agctatcaaa cagcgacata 3121 gccatacatc tgactgcctg acatggactc ctgcccactt gggggaaacc ttatacccag 3181 aggaaaatac acacctgggg agtacatttg acaaatttcc cttaggattt cgttatctca 3241 ccttgaccct cagccaagat tggtaaagct gcgtcctggc gattccagga gacccagctg 3301 gaaacctggc ttctccatgt gaggggatgg gaaaggaaag aagagaatga agactactta 3361 gtaattccoa tcaggaaatg ctgacctttt acataaaatc aaggagactg ctgaaaatct 3421 ctaagggaca ggattttcca gatcctaatt ggaaatttag caataaggag aggagtccaa 3481 ggggacaaat aaaggcagag agaagagaca gaactaaaaa tacgaggaaa ggagagtgag 3541 gattttcatt aaaagtctca gcagtgggtt tcttgggtta tttaaaacat cacctaaata 3601 ggccttttct tcctaattgg ccatcaaatt aaagcctatc ctttctcaag caggagctgg 3661 tattgtaggg agtggccggg tattctgggc tgggctcttc tggagtaggg ggtcagcaaa 3721 cattgtctgc aaagggccag atactgaatc cagtactttc agtttggcga gccgtgaggt 3781 ctctgtcgaa actactcaac tctgccgtcc tagcacaaaa gcagccatag acaacacaca 3841 aacgagaggg cttggctccc ttccaggaag atttatttaa caggctccca gctgaatttc 3901 actcacagga cacagtttac tgatctctgt tctagtgagt gggtcaaaaa gcatatgcat 3961 ccttatccgt caactcatca gctcttcctc aaggcaacct gaggccagac accaagaaac 4021 caagcgtatc tgctctaaaa tgacttgttc ctggggaatg ccttcaacca aaacacagct 4081 agtatttcta tgccccaaat ccaatcccag tctttcatga tccatgccgg cggttgggtg 4141 gggaggggaa tcattggttg ggggaaggga ggaaacccca cctccagccc ccgccaccgg 4201 gctccctggg cacccagcaa gatctggggc tgcagagaac agaagagctg gtgcacttaa 4261 tccagctctg cccttggggg gaggaggacc tgtgtgtcag gctctgccat gggaacgagt 4321 gtaaaccgtg gctgtctcct gcagtgagcc accgcggcag gcacgttgac tgttttactg 4381 acatcactca aaagctaaag caataacatt ctcctgcgtt gctgagtcag ctgttcattt 4441 gtccgccagc tcctggactg gatgtgtgaa aggcatcaca tttccatttt cctccgtgta 4501 aatgttttat gtgttcgcct actgatccca ttcgttgctt ctattgtaaa tatttgtcat 4561 ttgtatttat tatctctgtg ttttccccct aaggcataaa atggtttact gtgttcattt 4621 gaacccattt actgatctct gttgtatatt tttcatgcca ctgctttgtt ttctcctcag

4681 aagtcgggta gatagcattt ctatcccatc cctcacgtta ttggaagcat gcaacagtat

4741 ttattgctca gggtcttctg cttaaaactg aggaaggtcc acattcctgc aagcattgat

4801 tgagacattt gcacaatcta aaatgtaagc aaagtagtca ttaaaaatac accctctact 4861 tgggctttat actgcataca aatttactca tgagccttcc tttgaggaag gatgtggatc

4921 tccaaataaa gatttagtgt ttattttgag ctctgcatct taacaagatg atctgaacac

4981 ctctcctttg tatcaataaa tagccctgtt attctgaagt gagaggacca agtatagtaa

5041 aatgctgaca tctaaaacta aataaataga aaacaccagg ccagaactat agtcatactc

5101 acacaaaggg agaaatttaa actcgaacca agcaaaaggc ttcacggaaa tagcatggaa 5161 aaacaatgct tccagtggcc acttcctaag gaggaacaac cccgtctgat ctcagaattg

5221 gcaccacgtg agcttgctaa gtgataatat ctgtttctac tacggattta ggcaacagga

5281 cctgtacatt gtcacattgc attatttttc ttcaagcgtt aataaaagtt ttaaataaat

5341 ggcaaaaaaa aaaaaaaaaa a

STC2 nucleotide sequence (SBQ ID NO: 2]

1 mkvsaaalav iliatalcap asaspyssdt tpccfayiar plprattikey fytsgkcsnp 61 awfvtrkπr qvcanpekkw vreyinslein s CCL5 amino acid sequence (SEQ ID NO: 3)

1 gctgcagagg attcctgcag aggatcaaga cagcacgtgg acctcgcaca gcctctccca

61 caggtaccat ςaaggtctcc gcggcagccc tcgctgtcat cctcattgct actgccctct

121 gcgctcctgc atctgcctcc ccatattcct cggacaccac accctgctgc tttgcctaca

181 ttgoccgccc actgccccgt gcccacatca aggagtattt ctacaccagt ggcaagtgct

241 ccaacccagc agtcgtcttt gtcacccgaa agaaccgcca agtgtgtgcc aacccagaga 301 agaaatgggt tcgggagtac atcaactctt tggagatgag ctaggatgga gagtccttga

361 acctgaactt acacaaattt gcctgtttct gcttgctctt gtcctagctt gggaggcttc

421 ccctcactat cctaccccac ccgctccttg aagggcccag attctaccac acagcagcag

481 ttacaaaaac cttccccagg ctggacgtgg tggctcacgc ctgtaatccc agcactttgg

541 gaggccaagg tgggtggatc acttgaggtc aggagttcga gaccagcctg gccaacatga 601 tgaaacccca tctctactaa aaatacaaaa aattagccgg gcgtggtagc gggcgcctgt

661 agtcccagct actcgggagg ctgaggcagg agaatggcgt gaacccggga ggcggagctt

721 gcagtgagcc gagatcgcgc cactgcactc cagcctgggc gaoagagcga gactccgtct

781 caaaaaaaaa aaaaaaaaaa aaaatacaaa aattagccgg gcgtggtggc ccacgoctgt

841 aatcccagct actcgggagg ctaaggcagg aaaattgttt gaacccagga ggtggaggct 901 gcagtgagct gagattgtgc cacttcactc σagcctgggt gacaaagtga gactccgtca

961 caacaacaac aacaaaaagc ttccccaact aaagcctaga agagcttctg aggcgctgct

1021 ttgtcaaaag gaagtctcta ggttctgagc tctggctttg ccttggcttt gccagggctc

1081 tgtgaccagg aaggaagtca gcatgcctct agaggcaagg aggggaggaa cactgcactc

1141 ttaagcttcc gccgtctcaa cccctcacag gagcttactg gcaaacatga aaaatcggct 1201 taccattaaa gttctcaatg caaccataaa aaaaaaa

CCL5 nucleotide sequence (SEQ ID NO: 4)

1. Fold Change (microarray)

2. Fold Change (Q-PCR) Table 3

1. Fold Change (microarray)

2. Fold Change (Q-PCR)

Table 4

Claims

Claims :

1. A method for assessing aminopeptidase inhibition in an individual following:

(a) administering an aminopeptidase inhibitor to the individual; and,

(b) measuring the level or amount of at least one biomarker selected from the biomarkers of Tables 1 and 2 in a sample obtained from the individual.

2. A method according to claim 1 wherein the amount of the at least one biomarker present in the sample may be indicative of the amount or extent of aminopeptidase inhibition in the individual following administration of the aminopeptidase inhibitor.

3. A method for assessing the responsiveness of a cancer condition in an individual to treatment with an aminopeptidase inhibitor comprising: (a) administering the aminopeptidase inhibitor to the individual; and,

(b) measuring the amount of at least one biomarker selected from the biomarkers of Tables 1 and 2 in a sample obtained from the individual .

4. A method according to claim 3 wherein a change in the level or amount of the at least one biomarker after the administration is indicative that the cancer condition is responsive to treatment with the aminopeptidase inhibitor.

5. A method according to claim 4 wherein the level or amount of the at least one biomarker is measured in a first sample obtained from the individual before said administration and in a second sample obtained from the individual after said administration, a difference between the level or amount of the at least one biomarker in the first and second samples being indicative that the cancer condition is responsive to treatment with the aminopeptidase inhibitor.

6. A method according to claim 5 wherein the level or amount of the at least one biomarker selected from the biomarkers of Tables 1 and 2 is measured in samples obtained from the individual at two or more time points following said administration, wherein a change in the level or amount of the at least one biomarker in the samples obtained at the two or more time points is indicative that the cancer condition is responsive to treatment with the aminopeptidase inhibitor.

7. A method according to any one of claims 4 to 6 wherein the level or amount of at least one biomarker selected from the biomarkers of Table 1 is measured and an increase in the level or amount of the at least one biomarker after the administration is indicative that the cancer condition is responsive to treatment with the aminopeptidase inhibitor.

8. A method according to claim 7 wherein the level or amount of at least one biomarker selected from the biomarkers of Table 3 or Table 5 is measured and an increase in the level or amount of the at least one biomarker after the administration is indicative that the cancer condition is responsive to treatment with the aminopeptidase inhibitor.

9. A method according to claim 8 wherein the level or amount of stanniocalcin 2 and/or CCL5 is measured and an increase in the level or amount of stanniocalcin 2 and/or CCL5 after the administration is indicative that the cancer condition is responsive to treatment with the aminopeptidase inhibitor.

10. A method according to any one of claims 4 to 6 wherein the level or amount of at least one biomarker selected from the biomarkers of Table 2 is measured and a decrease in the level or amount of the at least one biomarker after the administration is indicative that the cancer condition is responsive to treatment with the aminopeptidase inhibitor.

11. A method according to claim 10 wherein the level or amount of at least one biomarker selected from the biomarkers of Table 4 or Table 6 is measured and a decrease in the level or amount of the at least one biomarker after the administration is indicative that the cancer condition is responsive to treatment with the aminopeptidase inhibitor.

12. A method according to any one of the preceding claims wherein the aminopeptidase inhibitor is bestatin.

13. A method according to any one of claims 1 to 11 wherein the aminopeptidase inhibitor is CHR-2797.

14. A method according to any one of the preceding claims wherein the sample is a blood, plasma or serum sample.

15. A method according to any one of claims 1 to 13 wherein the sample is a tumour biopsy.

16. A method according to any one of the preceding claims wherein the level or amount of the at least one biomarker is measured by measuring the level or amount of biomarker polypeptide in the sample.

17. A method according to claim 16 wherein the level of biomarker polypeptide in the sample is measured using an antibody which binds to the biomarker polypeptide .

18. A method according to claim 17 wherein the antibody is contacted with the sample and the binding of biomarker polypeptide in the sample to the antibody determined.

19. A method according to claim 17 or claim 18 wherein the antibody is immobilised.

20. A method according to any one of claims 1 to 15 wherein the level or amount of the at least one biomarker is measured by the level or amount of biomarker nucleic acid in the sample

21. A method according to claim 20 wherein the level of biomarker nucleic acid in the sample is measured using a probe nucleic acid which hybridises to the biomarker nucleic acid.

22. A method according to claim 20 or claim 21 wherein the probe nucleic acid is contacted with the sample and the binding of biomarker nucleic acid in the sample to the probe nucleic acid determined.

23. A method according to claim 21 or claim 22 wherein the probe nucleic acid is immobilised.

24. A method according to claim 23 wherein the probe nucleic acid is comprised in a microarray.

25. A method according to claim 21 or claim 22 wherein the probe nucleic acid is an oligonucleotide primer and binding is determined by amplification of the biomarker nucleic acid.

26. A method for monitoring the treatment of a cancer condition in individual with an aminopeptidase inhibitor comprising:

(a) subjecting the individual to a regimen of treatment with the aminopeptidase inhibitor; and (b) monitoring in the individual the level or amount of at least one biomarker selected from the biomarkers of Tables 1 and 2 during said treatment, wherein a change in the level or amount of the at least one biomarker which is sustained during the treatment is indicative that the regimen is effective for treating the cancer condition in the individual .

27. A method according to claim 26 wherein, in the absence of sustained changes in the level or amount of the at least one biomarker during the treatment, the method further comprises;

(c) altering the regimen of aminopeptidase inhibitor treatment and subjecting the individual to the altered regimen;

(d) monitoring the level of at least one biomarker selected from the biomarkers of Tables 1 and 2 in the individual, and

(e) repeating steps c) and d) until a sustained change in the level of the one or more biomarkers is observed. wherein a change in the level or amount of the at least one biomarker which is sustained during the treatment is indicative that the altered regimen is effective for treating the cancer condition in the individual .

28. A method according to claim 26 or claim 27 wherein, in the presence of sustained changes in the level or amount of the at least one biomarker during the treatment, the method further comprises;

(c) altering the regimen of aminopeptidase inhibitor treatment and subjecting the individual to the altered regimen,-

(d) monitoring the level of at least one biomarker selected from the biomarkers of Tables 1 and 2 in the individual, and (e) repeating steps c) and d) until a maximal change in the level of the one or more biomarkers is observed. wherein a maximal change in the level or amount of the at least one biomarker which is sustained during the treatment is indicative that the altered regimen is effective for treating the cancer condition in the individual.