Nitrogen Mustard Prodrug Research Articles

Hydrogen peroxide responsive theranostics for cancer-selective activation of DNA alkylators and real-time fluorescence monitoring in living cells

Triple negative breast cancer (TNBC) is a notoriously difficult disease to treat, and many of the existing TNBC chemotherapeutics lack tumor selectivity and the capability for simultaneously visualizing and monitoring their own activity in the biological context. However, TNBC cells have been known to generate high levels of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2). To this end, three novel small molecule theranostics 1a, 1c, and 2 consisting of both H2O2-responsive nitrogen mustard prodrug and profluorophore character have been designed, synthesized, and evaluated as targeted cancer therapeutics and bioimaging agents. The three theranostics comprise of boronate esters that deactivate nitrogen mustard functional groups and fluorophores but allow their selective activation through H2O2-specific oxidative deboronation for the release of the active drug and fluorophore. The three theranostics demonstrated H2O2-inducible DNA-alkylating capability and fluorescence turn-on properties in addition to selective anticancer activity. They are particularly effective in killing TNBC MDA-MB-468 cells with high H2O2 level while safe to normal epithelial MCF-10A cell. The conjugated boron-masked fluorophores in 1c and 2 are highly responsive towards H2O2, which enabled tracking of the theranostics in living cellular mitochondria and nucleus organelles. The three theranostics 1a, 1c, and 2 are capable of both selective release of the active drug to take effect in H2O2-rich cancer sites and simultaneously monitoring its activity. This single molecule system is of utmost importance to understand the function, efficacy, and mechanism of the H2O2-activated prodrugs and theranostics within the living recipient.

Characterization of AST-001 non-clinical pharmacokinetics: A novel selective AKR1C3-activated prodrug in mice, rats, and cynomolgus monkeys.

AST-001 is a chemically synthesized inactive nitrogen mustard prodrug that is selectively cleaved to a cytotoxic aziridine (AST-2660) via aldo-keto reductase family 1 member C3 (AKR1C3). The purpose of this study was to investigate the pharmacokinetics and tissue distribution of the prodrug, AST-001, and its active metabolite, AST-2660, in mice, rats, and monkeys. After single and once daily intravenous bolus doses of 1.5, 4.5, and 13.5mg/kg AST-001 to Sprague-Dawley rats and once daily 1 h intravenous infusions of 0.5, 1.5, and 4.5mg/kg AST-001 to cynomolgus monkeys, AST-001 exhibited dose-dependent pharmacokinetics and reached peak plasma levels at the end of the infusion. No significant accumulation and gender differences were observed after 7days of repeated dosing. In rats, the half-life of AST-001 was dose independent and ranged from 4.89 to 5.75h. In cynomolgus monkeys, the half-life of AST-001 was from 1.66 to 5.56h and increased with dose. In tissue distribution studies conducted in Sprague-Dawley rats and in liver cancer PDX models in female athymic nude mice implanted with LI6643 or LI6280 HepG2-GFP tumor fragments, AST-001 was extensively distributed to selected tissues. Following a single intravenous dose, AST-001 was not excreted primarily as the prodrug, AST-001 or the metabolite AST-2660 in the urine, feces, and bile. A comprehensive analysis of the preclinical data and inter-species allometric scaling were used to estimate the pharmacokinetic parameters of AST-001 in humans and led to the recommendation of a starting dose of 5mg/m2 in the first-in-human dose escalation study.

Safety, pharmacokinetics, and clinical activity of OBI-3424, an AKR1C3-activated prodrug, in patients with advanced or metastatic solid tumors: A phase 1 dose-escalation study.

3030 Background: OBI-3424 is a novel nitrogen mustard prodrug that can be selectively converted in the presence of the AKR1C3 enzyme into the bis-alkylating agent OBI-2660, which forms intra- and inter-strand DNA crosslinks resulting in cell death. This selective mode of activation distinguishes OBI-3424 from traditional alkylating agents, and potentially improves the safety profile and antitumor activity against chemo- and radio-resistant tumors. We conducted a first-in-human, phase 1, dose-escalation study of OBI-3424 monotherapy in patients with advanced solid tumors (NCT03592264). Methods: OBI-3424 was administered intravenously at doses of 1, 2, 4, 6, 8, or 12 mg/m2 (Days 1 and 8 every 21 days, Schedule A) or 8, 10, 12, or 14 mg/m2 (Day 1 every 21 days, Schedule B). A “3+3” design was used for dose escalation. Patients received study treatment until disease progression, unacceptable toxicity, or up to 2 years of treatment. Results: Overall, 39 adult patients were treated. In patients receiving Schedule A, the maximum tolerated dose (MTD) of OBI-3424 was determined to be 8 mg/m2. Dose limiting toxicities (DLTs) were reported at the 12 mg/m2 dose level, including thrombocytopenia (Grade 3-4, 5 of 6 patients) and anemia (Grade 3-4, 5 of 6 patients). Platelet count nadirs were noted on Day 15 or Day 22. These cytopenias led to dose modification and resulted in protocol amendment (Schedule B). In Schedule B, the MTD was not reached at the maximum dose tested (14 mg/m2). Grade 3 or higher anemia was noted in 3 of 6 patients treated at 14 mg/m2; the recommended phase 2 dose (RP2D) is 12 mg/m2 (Day 1 every 21 days). Treatment-related AEs (TRAEs) were noted in 82% (32/39) of patients. The most common TRAEs were anemia (64%), thrombocytopenia (51%), nausea (26%), and fatigue (21%). No patient had a fatal TRAE, 49% had ≥Grade 3 TRAEs, including 3 patients who had serious TRAEs. OBI-3424 showed linear pharmacokinetics from 1 to 14 mg/m2, with minimal accumulation after repeated dosing. A retrospective validated automated immunohistochemistry assessment indicated that 27% of patients had high AKR1C3 staining (H-score ≥135). Best confirmed response was stable disease in 21 patients (54%). Conclusions: We completed the dose-escalation portion of the study. The RP2D was determined to be 12 mg/m2 once every 3 weeks. OBI-3424 was well tolerated. Dose-dependent, non-cumulative thrombocytopenia and anemia were dose limiting. We are currently enrolling patients with pancreatic cancer and other tumor types with overexpression of AKR1C3 (H-score ≥135) in the expansion phase of the study. Clinical trial information: NCT03592264.

Tissue Pharmacokinetic Properties and Bystander Potential of Hypoxia-Activated Prodrug CP-506 by Agent-Based Modelling.

Hypoxia-activated prodrugs are bioactivated in oxygen-deficient tumour regions and represent a novel strategy to exploit this pharmacological sanctuary for therapeutic gain. The approach relies on the selective metabolism of the prodrug under pathological hypoxia to generate active metabolites with the potential to diffuse throughout the tumour microenvironment and potentiate cell killing by means of a “bystander effect”. In the present study, we investigate the pharmacological properties of the nitrogen mustard prodrug CP-506 in tumour tissues using in silico spatially-resolved pharmacokinetic/pharmacodynamic (SR-PK/PD) modelling. The approach employs a number of experimental model systems to define parameters for the cellular uptake, metabolism and diffusion of both the prodrug and its metabolites. The model predicts rapid uptake of CP-506 to high intracellular concentrations with its long plasma half-life driving tissue diffusion to a penetration depth of 190 µm, deep within hypoxic activating regions. While bioreductive metabolism is restricted to regions of severe pathological hypoxia (<1 µM O2), its active metabolites show substantial bystander potential with release from the cell of origin into the extracellular space. Model predictions of bystander efficiency were validated using spheroid co-cultures, where the clonogenic killing of metabolically defective “target” cells increased with the proportion of metabolically competent “activator” cells. Our simulations predict a striking bystander efficiency at tissue-like densities with the bis-chloro-mustard amine metabolite (CP-506M-Cl2) identified as a major diffusible metabolite. Overall, this study shows that CP-506 has favourable pharmacological properties in tumour tissue and supports its ongoing development for use in the treatment of patients with advanced solid malignancies.

Assessment of Phenylboronic Acid Nitrogen Mustards as Potent and Selective Drug Candidates for Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) has limited treatment options and the worst prognosis among all types of breast cancer. We describe two prodrugs, namely, CWB-20145 (1) and its methyl analogue FAN-NM-CH3 (2) that reduced the size of TNBC-derived tumors. The DNA cross-linking of nitrogen mustard prodrugs 1 and 2 was superior to that of chlorambucil and melphalan once activated in the presence of H2O2. The cellular toxicity of 1 and 2 was demonstrated in seven human cancer cell lines. The TNBC cell line MDA-MB-468 was particularly sensitive toward 1 and 2. Compound 2 was 10 times more cytotoxic than chlorambucil and 16 times more active than melphalan. An evaluation of the gene expression demonstrated an upregulation of the tumor suppressor genes p53 and p21 supporting a transcriptional mechanism of a reduced tumor growth. Pharmacokinetic studies with 1 showed a rapid conversion of the prodrug. The introduction of a methyl group generated 2 with an increased half-life. An in vivo toxicity study in mice demonstrated that both prodrugs were less toxic than chlorambucil. Compounds 1 and 2 reduced tumor growth with an inhibition rate of more than 90% in athymic nude mice xenografted with MDA-MB-468 cells. Together, the in vivo investigations demonstrated that treatment with 1 and 2 suppressed tumor growth without affecting normal tissues in mice. These phenylboronic acid nitrogen mustard prodrugs represent promising drug candidates for the treatment of TNBC. However, the mechanisms underlying their superior in vivo activity and selectivity as well as the correlation between H2O2 level and in vivo efficacy are not yet fully understood.

Coupling of a Novel TIMP3 Peptide to Carboxypeptidase G2 for Pro-Drug Activation at the Tumour Site.

Broad-spectrum cytotoxic drugs have been used in cancer therapy for decades. However, their lack of specificity to cancer cells often results in serious side-effects, limiting efficacy. For this reason, antibodies have been used to attempt to specifically target cytotoxic drugs to tumours. One such approach is antibody-directed enzyme prodrug therapy (ADEPT) which uses a tumour-directed monoclonal antibody, coupled to an enzyme, to convert a systemically administered non-toxic prodrug into a toxic one only at the tumour site. Among the main drawbacks of ADEPT is the immunogenicity of the antibody-enzyme complex, which is exacerbated by slow clearance due to size, hence limiting repeated administration. Additionally, the mono-specificity of the antibody could potentially result in drug resistance with repeated administration. We have identified a novel short peptide sequence, p700, derived from a human tissue inhibitor of metalloproteinases-3 (TIMP-3), which binds to and inhibits a number of tyrosine kinase growth factor receptors (VEGFRs1-3, FGFRs 1-4 and PDGFRα) which are known to be upregulated in many tumours and tumour vasculature. In this report, we fused p700 to His-tagged, codon-optimised, carboxypeptidase G2 (CPG2). CPG2 is a bacterial enzyme used in ADEPT, which activates potent nitrogen-mustard pro-drugs by removal of an inhibitory glutamic acid residue. Recombinant CPG2-p700 was highly expressed in Escherichia coli and successfully purified by nickel affinity chromatography. Biolayer interferometry showed that CPG2-p700 had a 100-fold increase in binding affinity for VEGFR2 compared with CPG2 alone and retained its catalytic activity, as determined by methotrexate cleavage. In the presence of CPG2-p700, the ZD2676P pro-drug showed significant cytotoxicity for 4T1 cells compared with prodrug alone or CPG2 alone. p700 is, therefore, a potentially useful alternative to monoclonal antibodies for enzyme pro-drug therapy and could equally be used for effective delivery of other cytotoxic drugs to tumour tissue.

Reduced Toxicity of Liposomal Nitrogen Mustard Prodrug Formulation Activated by an Intracellular ROS Feedback Mechanism in Hematological Neoplasm Models.

Nitrogen mustard (NM) is among the earliest drugs used to treat malignant tumors and it kills tumor cells by cross-linking DNA. Unfortunately, because of the short half-life and unfavorable selectivity, NM causes significant damage to normal tissues. As NM can increase the levels of reactive oxygen species (ROS) in tumor cells, a ROS-activated nitrogen mustard prodrug (NM-Pro) was synthesized and mixed with NM at a specific ratio to obtain an "NM-ROS-NM-Pro-NM" positive feedback system, which ultimately achieves a specific lethal effect on hematological neoplasms. The further encapsulation of NM/NM-Pro in liposomes allows the sustained release of the drug and prolongs the residence time in vivo. Here, we prepared stable liposomes with a uniform particle size of 170.6 ± 2.2 nm. The optimal ratio of NM to NM-Pro in this study was 2:1. The active drug NM in the NM/NM-Pro system continuously stimulated ROS production by the cells, which in turn further activated the NM-Pro to continuously generate NM. The positive feedback pathway between the NM and NM-Pro resulted in the specific death of tumor cells. Furthermore, the K562 hematological neoplasm model was utilized to evaluate the therapeutic effect of NM/NM-Pro liposomes in vivo. After encapsulation in liposomes, the targeting of tumor cells was increased approximately two times compared with that of normal cells, and NM/NM-Pro liposomes exhibited reduced toxicity, without an increase in drug activity compared to the NM/NM-Pro combination. The NM/NM-Pro delivery system exerts a positive feedback effect on ROS production in tumor cells and displays good potential for the specific killing of hematoma cells.

Tailored Design of an ROS-Responsive Drug Release Platform for Enhanced Tumor Therapy via "Sequential Induced Activation Processes".

The reactive oxygen species (ROS)-responsive intelligent drug delivery system has developed rapidly in recent years. However, because of the low concentration of ROS in most types of tumor cells, it is not possible to rapidly and effectively stimulate the drug delivery system to release the active drug. Here, we introduced "sequential induced activation processes" for efficient tumor therapy by designing a new ROS-responsive drug release platform. β-Lapachone, a positively charged nitrogen mustard (NM) prodrug, and two diblock molecules (mPEG-AcMH and PAsp-AcMH) are self-assembled to form prodrug primary micelles, which are further aggregated into nanoparticles that facilitate drug codelivery. When administered by intravenous injection, the nanoparticles reach the tumor site and enter the tumor cells by endocytosis. The β-lapachone released in the tumor cells induces a large amount of H2O2, and the ROS-responsive NM prodrug is activated to form activated NM, quinone methide, and boric acid under the induction of H2O2. The activated NM leads to tumor cell apoptosis.

Nitrogen Mustards as Anticancer Chemotherapies: Historic Perspective, Current Developments and Future Trends.

Nitrogen mustards, a family of DNA alkylating agents, marked the start of cancer pharmacotherapy. While traditionally characterized by their dose-limiting toxic effects, nitrogen mustards have been the subject of intense research efforts, which have led to safer and more effective agents. Even though the alkylating prodrug mustards were first developed decades ago, active research on ways to improve their selectivity and cytotoxic efficacy is a currently active topic of research. This review addresses the historical development of the nitrogen mustards, outlining their mechanism of action, and discussing the improvements on their therapeutic profile made through rational structure modifications. A special emphasis is made on discussing the nitrogen mustard prodrug category, with Cyclophosphamide (CPA) serving as the main highlight. Selected insights on the latest developments on nitrogen mustards are then provided, limiting such information to agents that preserve the original nitrogen mustard mechanism as their primary mode of action. Additionally, future trends that might follow in the quest to optimize these invaluable chemotherapeutic medications are succinctly suggested.

Massively Parallel Protein Design to Develop Enzymes for Next‐Generation Chemotherapy

Most chemotherapy targets dividing cells, rather than just tumor cells. This causes the failure of chemotherapy, as dosing limited by side-effects leads to drug resistance and relapse. One strategy to overcome this, referred to as Directed Enzyme Prodrug Therapy (DEPT), involves administration of an inactive prodrug that is activated by a foreign enzyme at the tumor site. Enzyme targeting can be achieved using mechanisms that include tumor-directed antibodies or viruses. Clinical trials have demonstrated the effectiveness of DEPT, but were hampered by the complex dosing schemes needed to ensure that the enzyme-antibody complex had cleared circulation and was entirely localized the tumor before the prodrug administered. Otherwise, the circulating enzyme would activate the circulating prodrug, resulting in systemic toxicity. We are employing a massively-parallel computationally-guided design approach using Rosetta, validated with a novel selection system and deep sequencing, to overcome this shortcoming in a model DEPT system, in which the bacterial enzyme, carboxypeptidase G2 (CPG2), is used to activate nitrogen mustard prodrugs. We have designed a “pro-enzyme” form of CPG2 that is activated by tumor-overexpressed proteases at the tumor site through removal of the designed “pro-domain.” The pro-enzyme will be dormant in circulation, thereby avoiding activation of the prodrug by circulating enzyme. Proteolytic processing at the tumor site by tumor overexpressed proteases removes the inhibitory “pro-domain,” restoring the activity exclusively in the tumor microenvironment. In addition to the design approach, the structural and biochemical characterization of our designed enzyme, in comparison to the native form, will be discussed. In addition, data showing the pro-enzyme's effectiveness in cancer cell culture will be shown. Support or Funding Information BJY is supported by fellowships from the Canadian Institutes of Health Research (CIHR) and the Mistletoe Research Foundation. Summary of the general experimental strategy for the design of a stimulus-responsive enzyme. The designed “pro-domain” is shown in green. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evaluating the abilities of diverse nitroaromatic prodrug metabolites to exit a model Gram negative vector for bacterial-directed enzyme-prodrug therapy

Gene-directed enzyme-prodrug therapy (GDEPT) employs tumour-tropic vectors including viruses and bacteria to deliver a genetically-encoded prodrug-converting enzyme to the tumour environment, thereby sensitising the tumour to the prodrug. Nitroreductases, able to activate a range of promising nitroaromatic prodrugs to genotoxic metabolites, are of great interest for GDEPT. The bystander effect (cell-to-cell transfer of activated prodrug metabolites) has been quantified for some nitroaromatic prodrugs in mixed multilayer human cell cultures, however while these provide a good model for viral DEPT (VDEPT) they do not inform on the ability of these prodrug metabolites to exit bacterial vectors (relevant to bacterial-DEPT (BDEPT)). To investigate this we grew two Escherichia coli strains in co-culture; an activator strain expressing the nitroreductase E. coli NfsA and a recipient strain containing an SOS-GFP DNA damage responsive gene construct. In this system, induction of GFP by reduced prodrug metabolites can only occur following their transfer from the activator to the recipient cells. We used this to investigate five clinically relevant prodrugs: metronidazole, CB1954, nitro-CBI-DEI, and two dinitrobenzamide mustard prodrug analogues, PR-104A and SN27686. Consistent with the bystander efficiencies previously measured in human cell multilayers, reduced metronidazole exhibited little bacterial cell-to-cell transfer, whereas nitro-CBI-DEI was passed very efficiently from activator to recipient cells post-reduction. However, in contrast with observations in human cell multilayers, the nitrogen mustard prodrug metabolites were not effectively passed between the two bacterial strains, whereas reduced CB1954 was transferred efficiently. Using nitroreductase enzymes that exhibit different biases for the 2- versus 4-nitro substituents of CB1954, we further showed that the 2-nitro reduction products exhibit substantially higher levels of bacterial cell-to-cell transfer than the 4-nitro reduction products, consistent with their relative bystander efficiencies in human cell culture. Overall, our data suggest that prodrugs may differ in their suitability for VDEPT versus BDEPT applications and emphasise the importance of evaluating an enzyme-prodrug partnership in an appropriate context for the intended vector.

Drug-DNA adducts as biomarkers for metabolic activation of the nitro-aromatic nitrogen mustard prodrug PR-104A

PR-104A is a clinical-stage nitrogen mustard prodrug that is activated for DNA alkylation by reduction of a nitro group to the corresponding hydroxylamine (PR-104H) or amine (PR-104M). Metabolic reduction is catalysed by flavoreductases such as cytochrome P450 oxidoreductase (POR) under hypoxia, or by aldo-ketoreductase 1C3 (AKR1C3) independently of hypoxia. The unstable reduced metabolites are challenging to measure in biological samples, and biomarkers of the metabolic activation of PR-104A have not been used in the clinical evaluation of PR-104 to date. Here, we employ a selected reaction monitoring mass spectrometry assay for DNA crosslinks to assess the capacity of human cancer cells to bioactivate PR-104A. We also test whether the more abundant DNA monoadducts could be used for the same purpose. DNA monoadducts and crosslinks from PR-104A itself, and from its reduced metabolites, accumulated over 4 h in AKR1C3-expressing TF1 erythroleukaemia cells under hypoxia, whereas intracellular concentrations of unstable PR-104H and PR-104M reached steady state within 1 h. We then varied rates of PR-104A reduction by manipulating hypoxia or reductase expression in a panel of cell lines, in which AKR1C3 and POR were quantified by targeted proteomics. Hypoxia or reductase overexpression induced large increases in PR-104A sensitivity (inhibition of proliferation), DNA damage response (γH2AX formation), steady-state concentrations of PR-104H/M and formation of reduced drug-DNA adducts but not DNA adducts retaining the dinitro groups of PR-104A. The fold-change in the sum of PR-104H and PR-104M correlated with the fold-change in reduced crosslinks or monoadducts (R2 = 0.87 for both), demonstrating their potential for assessing the capacity of cancer cells to bioactivate PR-104A.

Critical Considerations in Anticancer Drug Development and Dosing Strategies: The Past, Present, and Future.

Critical Considerations in Anticancer Drug Development and Dosing Strategies: The Past, Present, and Future.

AKR1C3 is a biomarker of sensitivity to PR-104 in preclinical models of T-cell acute lymphoblastic leukemia

AbstractPR-104, a phosphate ester of the nitrogen mustard prodrug PR-104A, has shown evidence of efficacy in adult leukemia clinical trials. Originally designed to target hypoxic cells, PR-104A is independently activated by aldo-keto-reductase 1C3 (AKR1C3). The aim of this study was to test whether AKR1C3 is a predictive biomarker of in vivo PR-104 sensitivity. In a panel of 7 patient-derived pediatric acute lymphoblastic leukemia (ALL) xenografts, PR-104 showed significantly greater efficacy against T-lineage ALL (T-ALL) than B-cell-precursor ALL (BCP-ALL) xenografts. Single-agent PR-104 was more efficacious against T-ALL xenografts compared with a combination regimen of vincristine, dexamethasone, and l-asparaginase. Expression of AKR1C3 was significantly higher in T-ALL xenografts compared with BCP-ALL, and correlated with PR-104/PR-104A sensitivity in vivo and in vitro. Overexpression of AKR1C3 in a resistant BCP-ALL xenograft resulted in dramatic sensitization to PR-104 in vivo. Testing leukemic blasts from 11 patients confirmed that T-ALL cells were more sensitive than BCP-ALL to PR-104A in vitro, and that sensitivity correlated with AKR1C3 expression. Collectively, these results indicate that PR-104 shows promise as a novel therapy for relapsed/refractory T-ALL, and that AKR1C3 expression could be used as a biomarker to select patients most likely to benefit from such treatment in prospective clinical trials.

Dual targeting of hypoxia and homologous recombination repair dysfunction in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive malignancy with poor clinical outcome and few validated drug targets. Two prevalent features of TNBC, tumor hypoxia and derangement of homologous recombination (HR) repair, are potentially exploitable for therapy. This study investigated whether hypoxia-activated prodrugs (HAP) of DNA-damaging cytotoxins may inhibit growth of TNBC by simultaneously addressing these two targets. We measured in vitro activity of HAP of DNA breakers (tirapazamine, SN30000) and alkylators (TH-302, PR-104, SN30548) in TNBC cell lines and isogenic models, and related this to measures of HR repair and expression of prodrug-activating enzymes. Antitumor activity of HAP was examined in isogenic BRCA2-knockout xenograft models and compared with platinum chemotherapy. All five HAP selectively inhibited growth of TNBC cell lines under hypoxia. Sensitivity to HAP was not strongly associated with BRCA1 genotype. However, HAP sensitivity was enhanced by suppression of HR (assessed by radiation-induced RAD51 focus formation) when BRCA1 and PALB2 were knocked down in a common (MDA-MB-231) background. Furthermore, knockout of BRCA2 markedly sensitized DLD-1 cells to the clinical nitrogen mustard prodrugs TH-302 and PR-104 and significantly augmented sterilization of clonogens by these agents in xenografts, both as monotherapy and in combination with radiotherapy, but had less effect on activity of the benzotriazine di-N-oxide SN30000. PR-104 monotherapy was more effective than cisplatin at inhibiting growth of BRCA2-knockout tumors at equitoxic doses. This study demonstrates the potential for HAP of nitrogen mustards to simultaneously exploit hypoxia and HR defects in tumors, with translational implications for TNBC and other HR-deficient malignancies.

Aromatic nitrogen mustard-based prodrugs: activity, selectivity, and the mechanism of DNA cross-linking.

Three novel H2O2-activated aromatic nitrogen mustard prodrugs (6-8) are reported. These compounds contain a DNA alkylating agent connected to a H2O2-responsive trigger by different electron-withdrawing linkers so that they are inactive towards DNA but can be triggered by H2O2 to release active species. The activity and selectivity of these compounds towards DNA were investigated by measuring DNA interstrand cross-link (ICL) formation in the presence or absence of H2O2. An electron-withdrawing linker unit, such as a quaternary ammonia salt (6), a carboxyamide (7), and a carbonate group (8), is sufficient to deactivate the aromatic nitrogen mustard resulting in less than 1.5 % cross-linking formation. However, H2O2 can restore the activity of the effectors by converting a withdrawing group to a donating group, therefore increasing the cross-linking efficiency (>20 %). The stability and reaction sites of the ICL products were determined, which revealed that alkylation induced by 7 and 8 not only occurred at the purine sites but also at the pyrimidine site. For the first time, we isolated and characterized the monomer adducts formed between the canonical nucleosides and the aromatic nitrogen mustard (15) which supported that nitrogen mustards reacted with dG, dA, and dC. The activation mechanism was studied by NMR spectroscopic analysis. An in vitro cytotoxicity assay demonstrated that compound 7 with a carboxyamide linker dramatically inhibited the growth of various cancer cells with a GI50 of less than 1 μM, whereas compound 6 with a charged linker did not show any obvious toxicity in all cell lines tested. These data indicated that a neutral carboxyamide linker is preferable for developing nitrogen mustard prodrugs. Our results showed that 7 is a potent anticancer prodrug that can serve as a model compound for further development. We believe these novel aromatic nitrogen mustards will inspire further and effective applications.

A novel fluorometric assay for aldo-keto reductase 1C3 predicts metabolic activation of the nitrogen mustard prodrug PR-104A in human leukaemia cells

Aldo-keto reductase 1C3 (AKR1C3, EC 1.1.1.188) metabolises steroid hormones, prostaglandins and xenobiotics, and activates the dinitrobenzamide mustard prodrug PR-104A by reducing it to hydroxylamine PR-104H. Here, we describe a functional assay for AKR1C3 in cells using the fluorogenic probe coumberone (a substrate for all AKR1C isoforms) in conjunction with a specific inhibitor of AKR1C3, the morpholylurea SN34037. We use this assay to evaluate AKR1C3 activity and PR-104A sensitivity in human leukaemia cells. SN34037-sensitive reduction of coumberone to fluorescent coumberol correlated with AKR1C3 protein expression by immunoblotting in a panel of seven diverse human leukaemia cell lines, and with SN34037-sensitive reduction of PR-104A to PR-104H. SN34037 inhibited aerobic cytotoxicity of PR-104A in high-AKR1C3 TF1 erythroleukaemia cells, but not in low-AKR1C3 Nalm6 pre-B cell acute lymphocytic leukaemia (B-ALL) cells, although variation in PR-104H sensitivity confounded the relationship between AKR1C3 activity and PR-104A sensitivity across the cell line panel. AKR1C3 mRNA expression showed wide variation between leukaemia patients, with consistently higher levels in T-ALL than B-ALL. In short term cultures from patient-derived paediatric ALL xenografts, PR-104A was more potent in T-ALL than B-ALL lines, and PR-104A cytotoxicity was significantly inhibited by SN34037 in T-ALL but not B-ALL. Overall, the results demonstrate that SN34037-sensitive coumberone reduction provides a rapid and specific assay for AKR1C3 activity in cells, with potential utility for identifying PR-104A-responsive leukaemias. However, variations in PR-104H sensitivity indicate the need for additional biomarkers for patient stratification.

Activity of the Hypoxia-Activated Prodrug, TH-302, in Preclinical Human Acute Myeloid Leukemia Models

Acute myeloid leukemia (AML) is an aggressive hematologic neoplasm. Recent evidence has shown the bone marrow microenvironment in patients with AML to be intrinsically hypoxic. Adaptive cellular responses by leukemia cells to survive under low oxygenation also confer chemoresistance. We therefore asked whether therapeutic exploitation of marrow hypoxia via the hypoxia-activated nitrogen mustard prodrug, TH-302, could effectively inhibit AML growth. We assessed the effects of hypoxia and TH-302 on human AML cells, primary samples, and systemic xenograft models. We observed that human AML cells and primary AML colonies cultured under chronic hypoxia (1% O2, 72 hours) exhibited reduced sensitivity to cytarabine-induced apoptosis as compared with normoxic controls. TH-302 treatment resulted in dose- and hypoxia-dependent apoptosis and cell death in diverse AML cells. TH-302 preferentially decreased proliferation, reduced HIF-1α expression, induced cell-cycle arrest, and enhanced double-stranded DNA breaks in hypoxic AML cells. Hypoxia-induced reactive oxygen species by AML cells were also diminished. In systemic human AML xenografts (HEL, HL60), TH-302 [50 mg/kg intraperitoneally (i.p.) 5 times per week] inhibited disease progression and prolonged overall survival. TH-302 treatment reduced the number of hypoxic cells within leukemic bone marrows and was not associated with hematologic toxicities in nonleukemic or leukemic mice. Later initiation of TH-302 treatment in advanced AML disease was as effective as earlier TH-302 treatment in xenograft models. Our results establish the preclinical activity of TH-302 in AML and provide the rationale for further clinical studies of this and other hypoxia-activated agents for leukemia therapy.

Homologous recombination repair-dependent cytotoxicity of the benzotriazine di- N-oxide CEN-209: Comparison with other hypoxia-activated prodrugs

CEN-209 (SN30000) is a second-generation benzotriazine di- N-oxide currently in advanced preclinical development as a hypoxia-activated prodrug (HAP). Herein we describe the DNA repair-, hypoxia- and one-electron reductase-dependence of CEN-209 cytotoxicity. We deployed mutant CHO cell lines to generate DNA repair profiles for CEN-209, and compared the profiles with those for other HAPs. Hypoxic selectivity of CEN-209 was significantly greater than PR-104A and the nitro-chloromethylbenzindoline (nCBI/SN29428) and comparable to tirapazamine and TH-302. CEN-209 was selective for homologous recombination (HR) repair-deficient cells ( Rad51d −/−), but less so than nitrogen mustard prodrugs TH-302 and PR-104A. Further, DNA repair profiles for CEN-209 differed under oxic and hypoxic conditions, with oxic cytotoxicity more dependent on HR. This feature was conserved across all three members of the benzotriazine di- N-oxide class examined (tirapazamine, CEN-209 and CEN-309/SN29751). Enhancing one-electron reduction of CEN-209 by forced expression of a soluble form of NADPH:cytochrome P450 oxidoreductase (sPOR) increased CEN-209 cytotoxicity more markedly under oxic than hypoxic conditions. Comparison of oxygen consumption, H 2O 2 production and metabolism of CEN-209 to the corresponding 1-oxide and nor-oxide reduced metabolites suggested that enhanced oxic cytotoxicity in cells with high one-electron reductase activity is due to futile redox cycling. This study supports the hypothesis that both oxic and hypoxic cell killing by CEN-209 is mechanistically analogous to tirapazamine and is dependent on oxidative DNA damage repaired via multiple pathways. However, HAPs that generate DNA interstrand cross-links, such as TH-302 and PR-104, may be more suitable than benzotriazine di- N-oxides for exploiting reported HR repair defects in hypoxic tumour cells.

Abstract C21: A phase I trial of PR-104, a pre-prodrug of the bioreductive prodrug PR-104A, given weekly to solid tumor patients.

Abstract Background: The phosphate ester PR-104 is rapidly converted in vivo to the alcohol PR-104A, a nitrogen mustard prodrug that is metabolised to hydroxylamine (PR-104H) and amine (PR-104M) DNA crosslinking agents by one-electron reductases in hypoxic cells and by aldo-keto reductase 1C3 independently of oxygen. In a previous phase I study using a q 3 week schedule of PR-104, the maximum tolerated dose (MTD) was 1100 mg/m2 and fatigue, neutropenic fever and infection were dose-limiting. The primary objective of the current study was to determine the dose-limiting toxicity (DLT) and MTD of weekly PR-104. Methods, patients and treatment: Patients with advanced solid tumors received PR-104 as a 1-hour intravenous infusion on days 1, 8 and 15 every 28 days with assessment of pharmacokinetics on cycle 1 day 1. Twenty-six patients (pts) were enrolled (16 male/10 female; median age 58 yrs, range 30 to 70 yrs) who had received a median of two prior chemotherapy regimens (range, 0 to 3) for melanoma (8 pts), colorectal or anal cancer (3 pts), NSCLC (3 pts), sarcoma (3 pts), glioblastoma (2 pts), salivary gland tumors (2 pts) or other solid tumors (5 pts). PR-104 was administered at 135 mg/m2 (3 pts), 270 mg/m2 (6 pts), 540 mg/m2 (6 pts), 675 mg/m2 (7 pts) and 900 mg/m2 (4 pts) for a median of two treatment cycles (range, 1 to 7 cycles) and five infusions (range, 1 to 18) per patient. Results: Dose-limiting toxicities (DLTs) during cycle one included grade four thrombocytopenia at 540 mg/m2 (1 of 6 pts) and grade four thrombocytopenia and neutropenia at 900 mg/m2 (2 of 4 pts). At an intermediate dose of 675 mg/m2, there were no DLTs among a total of seven patients given 12 treatment cycles but all experienced moderate to severe (grade 2 to 4) haematological toxicity. Thrombocytopenia was delayed in its onset and nadir, and its recovery was protracted and incomplete in many patients. There were no complete or partial tumor responses. PR-104-induced thrombocytopenia and neutropenia correlated with plasma AUC of PR-104, PR-104A and an oxidative semi-mustard metabolite (PR-104S1), but no more strongly than with PR-104 dose-level. There was no significant correlation between plasma AUC for the reduced metabolites and myelotoxicity. Conclusions: Thrombocytopenia, and to a lesser extent neutropenia, was the DLT of weekly PR-104. The MTD was 675 mg/m2/week. PR-104 given weekly may be a suitable protocol for further clinical evaluation as a short course of treatment with fractionated radiotherapy or haematopoietic stem cell support, as its duration of dosing is restricted by delayed-onset and protracted thrombocytopenia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C21.