Aminopeptidase

The HL-60 cell growth media has more stanniocalcin 2 (STC2) protein when tosedostat (CHR-2797) is used. After two hours of treatment with Tosedostat (60 nM), SLC7A11 expression rose. U-937 and HuT 78 cell lines are blocked by tosedostat treatment, which also causes U-937 cells but not HuT 78 cells to express more of the incremental response (AADR) gene [1]. The mean MCA production of untreated control cells was lowered to 77.8% by tosedostat (0.01 μM; the IC50 for 24 hours was 10 nM and >10 μM, respectively); likewise, MCA production was decreased to 51.3% at 1 μM and 51.3% at 5 μM, and to 38.5% at μM and 35.3% at 10 μM [2].

In animal cancer models that are pregnant, tosedostat (CHR-2797) is active and exhibited a dose-response relationship in both models; the effect of tosedostat is less pronounced when the tumor burden is at its highest prior to treatment [1].

In Vitro Enzyme Assays [2]
LAP[2]
LAP activity was determined by measuring the hydrolysis of the tripeptide, LGG, which was detected by the derivatization reagent OPA in the presence of βmercaptoethanol. The enzyme was resuspended in H2O, then buffer exchanged on a PD-10 colum into 50mM HEPES, pH 8.0. Protein concentration was determined by BCA assay, and the concentration was adjusted to 10µg/ml. LGG was diluted to 0.5mM in 50mM HEPES. The assay was carried out in 96-well assay plates. Wells contained diluted inhibitor/vehicle (5µl), 10µg/ml LAP (5µl) and 40µl of 0.5mM LGG. Samples were assayed in triplicate. The plate was shaken briefly, and incubated for 90min at 37o C. The reaction was terminated by addition of 200µl of OPA/β-Mercaptoethanol per well. The plate was read on the Victor Wallac3 plate reader: excitation, 355nm and emission, 460nm, and IC50 values calculated.

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PuSA[2]
PuSA activity was determined using the fluorogenic substrate Ala-AMC. Incubations contained diluted inhibitor/vehicle (20µl), substrate (125µM Ala-AMC in 0.125M TrisHCl buffer, pH 7.5; 40µl) and enzyme (40µl). After incubation for 2h at 37o C, the reaction was stopped by addition of 100µl 3% (v/v) acetic acid. Fluorescence was measured using a SLT Fluostar fluorimeter.

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LTA4 Hydrolase[2]
LTA4 hydrolase was assayed using the fluorogenic substrate Arg-AMC. Incubations contained diluted inhibitor/vehicle (10µl), substrate (285µM Arg-AMC in PBS-0.01% (v/v) Brij-35; 70µl) and enzyme (20µl). After incubation for 1h at 37o C, the reaction was stopped by addition of 100µl 3% (v/v) acetic acid. Fluorescence was measured using a SLT Fluostar fluorimeter.

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PILSAP[2]
2 PILSAP activity was determined using the colorimetric substrate l-leucine pnitroanilide according to the manufacturer’s instructions.

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Aminopeptidase N Aminopeptidase N was assayed using the fluorogenic substrate, Ala-AMC. Incubations contained diluted inhibitor/vehicle (20µl), substrate (40µl; final concentration, 60µM) and enzyme (40µl, 1:8000 dilution) and were incubated for 60min at 37o C prior to addition of 100µl 3% (v/v) acetic acid, to stop the reaction. Fluorescence was measured using a SLT Fluostar fluorimeter.

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Aminopeptidase B[2]
Aminopeptidase B was assayed using the fluorescent substrate, Arg-AMC. Incubations contained diluted inhibitor/vehicle (20µl), substrate (40µl; final concentration 200µM) and enzyme (40µl) and were incubated for 120min at 37o C prior to addition of 100µl 3% (v/v) acetic acid, to stop the reaction. Fluorescence was measured using a SLT Fluostar fluorimeter.

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MetAP-2[2]
MetAP-2 was assayed using Met-Ala-Ser as substrate, and released Met was detected by OPA in the presence of reducing agents to generate a fluorescent product. Incubations contained substrate Met-Ala-Ser-OH (1.67mM) in assay buffer (83mM sodium phosphate buffer pH 7.2, 83mM NaCl, 0.266mM CoCl2; 60µl), diluted inhibitor/vehicle (20µl) and enzyme (20µl). They were incubated for 90min at 37o C prior to addition of 150µl stop reagent (0.2mM OPA/0.2mM β-mercaptoethanol in 50mM borax pH 9.5). Fluorescence was measured using a SLT Fluostar fluorimeter.

Cell proliferation, survival and cell cycle assays[1]
Inhibition of proliferation was measured using a WST-1 assay as per the manufacturer's instructions. Cell death was measured by flow cytometry using the AnnexinV:FITC Apoptosis Detection Kit I on a FACSCaliburTM. Cell cycle status was measured by fixing cells on ice in 70% ethanol, re-suspending in PBS and treated for 30 minutes at 37°C with 100 μg/ml RNase, then stained with 50 μg/ml Propidium Iodide (PI) and analysed by flow cytometry.

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Cell Cycle Analysis[2]
Cytokinetic investigations into the effects of CHR-2797 or CHR-5346 were analysed on U-937 and HuT 78 tumour cell lines using a bivariate BrdUrd-PI staining FACS protocol, based on the method of Dolbeare et al (Proc Natl Acad Sci USA 1983;80:5573-77). This method also identified the presence of sub-G1 apoptotic DNA profiles. For annexin analysis, the presence of externalised phosphatidylserine was detected using the Annexin V-FITC staining kit-1. Samples were analysed by flow cytometry on a FACS Canto using a 488nm excitation laser, with a 530nm/30nm bandpass filter for FITC, and a 670nmLP filter for PI fluorescence emission.

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CHR-2797 is a novel metalloenzyme inhibitor that is converted into a pharmacologically active acid product (CHR-79888) inside cells. CHR-79888 is a potent inhibitor of a number of intracellular aminopeptidases, including leucine aminopeptidase. CHR-2797 exerts antiproliferative effects against a range of tumor cell lines in vitro and in vivo and shows selectivity for transformed over nontransformed cells. Its antiproliferative effects are at least 300 times more potent than the prototypical aminopeptidase inhibitor, bestatin. However, the mechanism by which inhibition of these enzymes leads to proliferative changes is not understood. Gene expression microarrays were used to profile changes in mRNA expression levels in the human promyelocytic leukemia cell line HL-60 treated with CHR-2797. This analysis showed that CHR-2797 treatment induced a transcriptional response indicative of amino acid depletion, the amino acid deprivation response, which involves up-regulation of amino acid synthetic genes, transporters, and tRNA synthetases. These changes were confirmed in other leukemic cell lines sensitive to the antiproliferative effects of CHR-2797. Furthermore, CHR-2797 treatment inhibited phosphorylation of mTOR substrates and reduced protein synthesis in HL-60 cells, both also indicative of amino acid depletion. Treatment with CHR-2797 led to an increase in the concentration of intracellular small peptides, the substrates of aminopeptidases. It is suggested that aminopeptidase inhibitors, such as CHR-2797 and bestatin, deplete sensitive tumor cells of amino acids by blocking protein recycling, and this generates an antiproliferative effect. CHR-2797 is orally bioavailable and currently undergoing phase II clinical investigation in the treatment of myeloid leukemia.[2]

Rat mammary (HOSP.1P) carcinoma model: lung colonisation [2]
Female rats (CBH/cbi) were randomised into groups (n=8-10/group) to receive CHR2797 (3, 10 or 30mg/kg/day) or vehicle. HOSP.1P cells (passage 6/7, 30,000 in 0.4 ml) were injected i.v. into all rats under halothane anaesthesia. Rats were dosed daily p.o. commencing 2 days after tumour cell inoculation and continuing until day 32. Lungs were removed from all rats 24 hours after the final dose. After tumour dissection and fixation in Methacarn, their number, mean weights and total tumour burden were determined.[2]

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Rat chondrosarcoma (HSN LV10) model: liver colonisation [2]
Male (CBH/cbi) rats were randomised into groups (n=9-10/group) to receive CHR2797 (10 or 100mg/kg/day) or vehicle. Animals were injected with HSN LV10 cells (3,000 tumour cells in 0.2ml; passage 17, propagated in vitro) via the mesenteric vein under halothane anaesthesia, by exteriorising a small portion of the small intestine. The wound was closed using a silk suture for the musculature and a Michel clip for the skin. Animals were dosed daily p.o. commencing 2 days after tumour cell inoculation and continuing until day 20. Blood was taken from all drug-treated rats 24 hours after the last dose, for estimation of drug and CHR-79888 concentrations. Tumours in the liver were dissected and ten tumours (where possible), of representative sizes, from each drug-treated rat were weighed and snap-frozen in liquid nitrogen for analyte estimation by LC/MS (see Figure 1D). The remaining tumours were fixed in Methacarn and their number, mean weight and total tumour burden determined, including those removed for analysis. [2]

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Human tumour xenograft model: MDA-MB-435 breast carcinoma [2]
Nude mice (MF1 (nu/nu); Harlan, Bicester, UK) were inoculated sub-cutaneously into the mammary fat pad with MDA-MB 435 cells (106 cells/mouse) on day 0. The tumours were allowed to grow to an average volume of 350µl/animal. On day 23, the mice were divided into two treatment groups based on a composite of tumour volume and the rate of tumour growth. Animals were dosed with either vehicle or CHR-2797 (100 mg/kg/day p.o.) from day 23 until day 37. The primary tumour was resected on day 28 and the study ended on day 61. Lungs were dissected out at the end of the study and fixed in Bouin’s solution. Under a dissecting microscope, the diameter of all tumours was measured using callipers. Lung tumour numbers were also counted for each animal and the total tumour burden assessed.

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Human tumour xenograft model: MDA-MB-468 breast carcinoma [2]
Nude mice (MF1 (nu/nu) were inoculated sub-cutaneously into the mammary fat pad with MDA-MB-468 cells (2x106 cells/mouse) on day 0. Either on day 22 (Supplementary Figure S4, bottom) or day 39 (Figure S4, top), they were randomised into groups (n ≥ 14 per group) based on a composite of tumour mass and % change in tumour mass/unit time. From days 22-49 or days 40-69, mice were treated i.p., u.i.d. with either CHR-2797 (100mg/kg) or vehicle. Every 3 or 4 days, tumour volume was assessed using callipers. Mice were culled on day 50 or 70.

[1]. The combination of HDAC and aminopeptidase inhibitors is highly synergistic in myeloma and leads to disruption of the NFκB signalling pathway. Oncotarget. 2015 Jul 10;6(19):17314-27.

[2]. CHR-2797: an antiproliferative aminopeptidase inhibitor that leads to amino acid deprivation in human leukemic cells. Cancer Res. 2008 Aug 15;68(16):6669-79.

[3]. Aminopeptidase inhibition by the novel agent CHR-2797 (tosedostat) for the therapy of acute myeloid leukemia. Leuk Res. 2011 May;35(5):677-81.

[4]. The combination of HDAC and aminopeptidase inhibitors is highly synergistic in myeloma and leads to disruption of the NFκB signalling pathway. Oncotarget. 2015 Jul 10;6(19):17314-27.

(2S)-2-[[(2R)-2-[(1S)-1-hydroxy-2-(hydroxyamino)-2-oxoethyl]-4-methyl-1-oxopentyl]amino]-2-phenylacetic acid cyclopentyl ester is a secondary carboxamide, a hydroxamic acid and a carboxylic ester.
Tosedostat has been used in trials studying the treatment and supportive care of AML, Leukemia, Pancreas Cancer, Multiple Myeloma, and Pancreatic Cancer, among others. Tosedostat is an inhibitor of the M1 family of aminopeptidases, in particular PuSA, and LTA4 hydrolase. It has demonstrated anti-tumour activity in a number of models of cancer, both as a single agent and in synergy with cytotoxic agents such as carboplatin and paclitaxel. It entered the clinical trial in patients with haematological malignancies.
Tosedostat is a proprietary orally bioavailable inhibitor of the M1 family of aminopeptidases with potential antineoplastic activity. Aminopeptidase inhibitor CHR-2797 is converted intracellularly into a poorly membrane-permeable active metabolite (CHR-79888) which inhibits the M1 family of aminopeptidases, particularly puromycin-sensitive aminopeptidase (PuSA), and leukotriene A4 (LTA4) hydrolase; inhibition of these aminopeptidases in tumor cells may result in amino acid deprivation, inhibition of protein synthesis due to a decrease in the intracellular free amino acid pool, an increase in the level of the proapoptotic protein Noxa, and cell death. Noxa is a member of the BH3 (Bcl-2 homology 3)-only subgroup of the proapoptotic Bcl-2 (B-cell CLL/lymphoma 2) protein family.

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Mechanism of Action
Tosedostat is anti-proliferative agent which induces apoptosis in leukemic cell lines in vitro. The mechanism underlying these anti-cancer actions is unclear, particularly since normal cells are much less sensitive to the agents than transformed cells. It exerts potent anti-proliferative, pro-apoptotic and anti-angiogenic effects in vitro and shows selectivity for transformed over non-transformed cells. It inhibits a number of M1 aminopeptidase enzyme family members in vitro (eg puromycin-sensitive aminopeptidase (PSA), leukotriene A4 hydrolase (LTA4H)).

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Pharmacodynamics
Tosedostat has pleiotropic effects against a range of human tumor cell lines originating from diverse tumor types in vitro and in vivo.

C21H30N2O6

406.4727

406.21

C, 62.05; H, 7.44; N, 6.89; O, 23.62

238750-77-1

15547703

White to off-white solid powder

1.2±0.1 g/cm3

1.566

2.23

4

6

10

29

555

3

O(C([C@]([H])(C1C([H])=C([H])C([H])=C([H])C=1[H])N([H])C([C@@]([H])([C@@]([H])(C(N([H])O[H])=O)O[H])C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])=O)=O)C1([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H]

FWFGIHPGRQZWIW-SQNIBIBYSA-N

InChI=1S/C21H30N2O6/c1-13(2)12-16(18(24)20(26)23-28)19(25)22-17(14-8-4-3-5-9-14)21(27)29-15-10-6-7-11-15/h3-5,8-9,13,15-18,24,28H,6-7,10-12H2,1-2H3,(H,22,25)(H,23,26)/t16-,17+,18+/m1/s1

(S)-cyclopentyl 2-((R)-2-((S)-1-hydroxy-2-(hydroxyamino)-2-oxoethyl)-4-methylpentanamido)-2-phenylacetate

CHR2797; CHR-2797; CHR 2797; KZK563J2UW; C21H30N2O6; Tosedostat (CHR2797); Tosedostat.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~25 mg/mL (~61.51 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.15 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.15 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.15 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)