Stability of vincristine sulfate, doxorubicin hydrochloride and etoposide phosphate admixtures in polyisoprene elastomeric pump supporting transition of the EPOCH regimen to outpatient care.

BACKGROUND: The EPOCH regimen, consisting of vincristine sulfate, doxorubicin hydrochloride, and etoposide phosphate, is typically administered by continuous infusion over four days to oncology inpatients. If the EPOCH regimen was available to be administered through portable elastomeric pumps, chemotherapy could be transitioned to an outpatient setting, reducing inpatient bed days and overall healthcare costs. However, a lack of stability data for the admixtures in the elastomeric infusion devices currently prevents the transition of the regime to an outpatient setting. The purpose of this study is to determine the physical and chemical stability of the admixture in polyisoprene elastomeric pumps under different storage conditions to support the transition of the EPOCH regime to an outpatient setting. METHODS: The physico-chemical stability of three admixtures at a range of clinically relevant concentrations compounded in polyisoprene elastomeric infusors was determined when refrigerated at 2-6℃ over a 14-day period followed by 35℃ up to 7 days in the dark, and under standardized fluorescent light to simulate scenarios in clinical practice. RESULTS: All tested admixtures were compatible and the drugs were stable in the elastomeric infusors for up to 14 days when stored at 2-6℃ followed by 7 days at 35℃ in the dark, with nominal losses of <5%. The major degradant of etoposide phosphate was its active form etoposide. There was no degradation (<1% loss) found when the admixture was exposed to a standardized fluorescent light dose of 80 klux-h (25℃) for 10 h. The temperature and light conditions the infusors were exposed to during the stability study were more severe than the conditions determine during clinical administration. CONCLUSION: The extended stability of the three infusional admixtures compounded in elastomeric infusion pumps demonstrated herein permits advance preparation and storage of these drugs, reducing pharmacy compounding resources. The demonstrated stability at 35℃ and under light exposure, conditions more severe than those experienced during clinical practice, support continuous infusions for up to seven days from the elastomeric infusors without a loss of potency. The proven stability of the EPOCH regimens in the tested elastomeric infusion device supports the transition of treatment to an outpatient setting which will reduce inpatient bed days and overall healthcare costs.

Phase I/II study of the hypoxia-activated prodrug PR104 in refractory/relapsed acute myeloid leukemia and acute lymphoblastic leukemia.

We previously demonstrated vast expansion of hypoxic areas in the leukemic microenvironment and provided a rationale for using hypoxia-activated prodrugs. PR104 is a phosphate ester that is rapidly hydrolyzed in vivo to the corresponding alcohol PR-104A and further reduced to the amine and hydroxyl-amine nitrogen mustards that induce DNA cross-linking in hypoxic cells under low oxygen concentrations. In this phase I/II study, patients with relapsed/refractory acute myeloid leukemia (n=40) after 1 or 2 prior treatments or acute lymphoblastic leukemia (n=10) after any number of prior treatments received PR104; dose ranged from 1.1 to 4 g/m(2). The most common treatment-related grade 3/4 adverse events were myelosuppression (anemia 62%, neutropenia 50%, thrombocytopenia 46%), febrile neutropenia (40%), infection (24%), and enterocolitis (14%). Ten of 31 patients with acute myeloid leukemia (32%) and 2 of 10 patients with acute lymphoblastic leukemia (20%) who received 3 g/m(2) or 4 g/m(2) had a response (complete response, n=1; complete response without platelet recovery, n=5; morphological leukemia-free state, n=6). The extent of hypoxia was evaluated by the hypoxia tracer pimonidazole administered prior to a bone marrow biopsy and by immunohistochemical assessments of hypoxia-inducible factor alpha and carbonic anhydrase IX. A high fraction of leukemic cells expressed these markers, and PR104 administration resulted in measurable decrease of the proportions of hypoxic cells. These findings indicate that hypoxia is a prevalent feature of the leukemic microenvironment and that targeting hypoxia with hypoxia-activated prodrugs warrants further evaluation in acute leukemia. The trial is registered at clinicaltrials.gov identifier: 01037556.

Zinc finger nuclease knock-out of NADPH:cytochrome P450 oxidoreductase (POR) in human tumor cell lines demonstrates that hypoxia-activated prodrugs differ in POR dependence.

Hypoxia, a ubiquitous feature of tumors, can be exploited by hypoxia-activated prodrugs (HAP) that are substrates for one-electron reduction in the absence of oxygen. NADPH:cytochrome P450 oxidoreductase (POR) is considered one of the major enzymes responsible, based on studies using purified enzyme or forced overexpression in cell lines. To examine the role of POR in HAP activation at endogenous levels of expression, POR knock-outs were generated in HCT116 and SiHa cells by targeted mutation of exon 8 using zinc finger nucleases. Absolute quantitation by proteotypic peptide mass spectrometry of DNA sequence-confirmed multiallelic mutants demonstrated expression of proteins with residual one-electron reductase activity in some clones and identified two (Hko2 from HCT116 and S2ko1 from SiHa) that were functionally null by multiple criteria. Sensitivities of the clones to 11 HAP (six nitroaromatics, three benzotriazine N-oxides, and two quinones) were compared with wild-type and POR-overexpressing cells. All except the quinones were potentiated by POR overexpression. Knocking out POR had a marked effect on antiproliferative activity of the 5-nitroquinoline SN24349 in both genetic backgrounds after anoxic exposure but little or no effect on activity of most other HAP, including the clinical stage 2-nitroimidazole mustard TH-302, dinitrobenzamide mustard PR-104A, and benzotriazine N-oxide SN30000. Clonogenic cell killing and reductive metabolism of PR-104A and SN30000 under anoxia also showed little change in the POR knock-outs. Thus, although POR expression is a potential biomarker of sensitivity to some HAP, identification of other one-electron reductases responsible for HAP activation is needed for their rational clinical development.

Novel nitroimidazole alkylsulfonamides as hypoxic cell radiosensitisers.

A novel class of nitroimidazole alkylsulfonamides have been prepared and evaluated as hypoxia-selective cytotoxins and radiosensitisers. The sulfonamide side chain markedly influences the physicochemical properties of the analogues: lowering aqueous solubility and raising the electron affinity of the nitroimidazole group. The addition of hydroxyl or basic amine groups increased aqueous solubility, with charged amine groups contributing to increased electron affinity. The analogues covered the range of electron affinity for effective radiosensitisation with one-electron reduction potentials ranging from -503 to -342mV. Cytotoxicity under normoxia or anoxia against a panel of human tumour cell lines was determined using a proliferation assay. 2-Nitroimidazole sulfonamides displayed significant hypoxia-selective cytotoxicity (6 to 64-fold), while 4- and 5-nitroimidazole analogues did not display hypoxia-selective cytotoxicity. All analogues sensitised anoxic HCT-116 human colorectal cells to radiation at non-toxic concentrations. 2-Nitroimidazole analogues provided modest sensitisation due to the relatively low concentrations used while several 5-nitroimidazole analogues provided equivalent sensitisation to misonidazole and etanidazole at similar molar concentrations.

PR-104 a bioreductive pre-prodrug combined with gemcitabine or docetaxel in a phase Ib study of patients with advanced solid tumours.

BACKGROUND: The purpose of this phase Ib clinical trial was to determine the maximum tolerated dose (MTD) of PR-104 a bioreductive pre-prodrug given in combination with gemcitabine or docetaxel in patients with advanced solid tumours. METHODS: PR-104 was administered as a one-hour intravenous infusion combined with docetaxel 60 to 75 mg/m2 on day one given with or without granulocyte colony stimulating factor (G-CSF) on day two or administrated with gemcitabine 800 mg/m2 on days one and eight, of a 21-day treatment cycle. Patients were assigned to one of ten PR-104 dose-levels ranging from 140 to 1100 mg/m2 and to one of four combination groups. Pharmacokinetic studies were scheduled for cycle one day one and 18F fluoromisonidazole (FMISO) positron emission tomography hypoxia imaging at baseline and after two treatment cycles. RESULTS: Forty two patients (23 females and 19 males) were enrolled with ages ranging from 27 to 85 years and a wide range of advanced solid tumours. The MTD of PR-104 was 140 mg/m2 when combined with gemcitabine, 200 mg/m2 when combined with docetaxel 60 mg/m2, 770 mg/m2 when combined with docetaxel 60 mg/m2 plus G-CSF and ≥770 mg/m2 when combined with docetaxel 75 mg/m2 plus G-CSF. Dose-limiting toxicity (DLT) across all four combination settings included thrombocytopenia, neutropenic fever and fatigue. Other common grade three or four toxicities included neutropenia, anaemia and leukopenia. Four patients had partial tumour response. Eleven of 17 patients undergoing FMISO scans showed tumour hypoxia at baseline. Plasma pharmacokinetics of PR-104, its metabolites (alcohol PR-104A, glucuronide PR-104G, hydroxylamine PR-104H, amine PR-104M and semi-mustard PR-104S1), docetaxel and gemcitabine were similar to that of their single agents. CONCLUSIONS: Combination of PR-104 with docetaxel or gemcitabine caused dose-limiting and severe myelotoxicity, but prophylactic G-CSF allowed PR-104 dose escalation with docetaxel. Dose-limiting thrombocytopenia prohibited further evaluation of the PR104-gemcitabine combination. A recommended dose was identified for phase II trials of PR-104 of 770 mg/m2 combined with docetaxel 60 to 75 mg/m2 both given on day one of a 21-day treatment cycle supported by prophylactic G-CSF (NCT00459836).

Glucuronidation of anticancer prodrug PR-104A: species differences, identification of human UDP-glucuronosyltransferases, and implications for therapy.

PR-104, the phosphate ester of a dinitrobenzamide mustard [PR-104A; 2-((2-bromoethyl)-2-{[(2-hydroxyethyl) amino] carbonyl}-4,6-dinitroanilino)ethyl methanesulfonate], is currently in clinical trial as a hypoxia- and aldo-keto reductase 1C3 (AKR1C3)-activated prodrug for cancer therapy. Here, we investigate species (human, dog, rat, mouse) differences in metabolism to the corresponding O-glucuronide, PR-104G, and identify the human UDP-glucuronosyltransferase (UGT) isoforms responsible. After intravenous PR-104, plasma area under the concentration-time curve ratios (PR-104G/PR-104A) decreased in the order of dog (2.3) > human (1.3) > mouse (0.03) > rat (0.005). The kinetics of uridine 5'-diphosphoglucuronic acid-dependent glucuronidation by liver microsomes in vitro fitted the single-enzyme Michaelis-Menten equation with similar K(m) (∼150 µM) but differing V(max) (472, 88, 37, and 14 nmol/h/mg for dog, human, rat, and mouse, respectively), suggesting that facile glucuronidation is responsible for the anomalously rapid clearance of PR-104A in dogs. In vitro-in vivo extrapolation of PR-104A glucuronidation kinetics is consistent with this also being a major clearance pathway in humans. Recombinant UGT screening identified UGT2B7 as the only commercially available human isoform able to conjugate PR-104A, and UGT2B7 protein concentrations were highly correlated (r = 0.93) with PR-104A glucuronidation by liver microsomes from 24 individuals. The active hydroxylamine metabolite of PR-104A, PR-104H, was also glucuronidated by UGT2B7, although with slightly lower specificity and much lower rates. UGT2B7 mRNA expression was highly variable in human tumor databases. Glucuronidation of PR-104A greatly suppressed nitroreduction by AKR1C3 and NADPH-supplemented anoxic human liver S9 (9000g postmitochondrial supernatant). In conclusion, PR-104A is glucuronidated by UGT2B7 with high specificity and seems to make a major contribution to clearance of PR-104A in humans, but it also has the potential to confer resistance in some human tumors.

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

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 with advanced solid tumours 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 tumours (2 pts) or other solid tumours (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 tumour 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.

Initial testing of the hypoxia-activated prodrug PR-104 by the pediatric preclinical testing program.

BACKGROUND: PR-104 is rapidly hydrolyzed to PR-104A in vivo, which is activated by reduction to the corresponding 5-hydroxylamine (PR-104H) and amine (PR-104M) to produce DNA interstrand cross-links. PR-104 activation can occur via hypoxia-dependent reductases and also independently of hypoxia by aldo-keto reductase (AKR) 1C3. PROCEDURES: PR-104A was tested against the PPTP in vitro panel (10 nM to 100 µM), and PR-104 in vivo using a weekly × 6 schedule at its maximum tolerated dose (MTD) of 550 mg/kg. Subsequently PR-104 was tested at 270 and 110 mg/kg. Pharmacokinetics for PR-104 and its metabolites were determined, as were levels of AKR1C3 RNA and protein in xenografts. RESULTS: In vitro, the leukemia models were most sensitive to PR-104A. In vivo, PR-104 induced objective responses at its MTD in 21/34 solid tumor models and maintained complete responses against 7/7 acute lymphoblastic leukemia (ALL) models. At 270 mg/kg and lower dose levels, PR-104 did not induce solid tumor regressions, suggesting a steep dose-response relationship. Pharmacokinetic analysis suggests higher systemic exposures to PR-104A and its metabolites in mice compared to those achievable in patients. Levels of AKR1C3 protein did not correlate with tumor responsiveness. CONCLUSIONS: As monotherapy, PR-104 demonstrated a high level of activity against both solid tumor and ALL models at its MTD, but the activity was almost completely lost at half the MTD dose for solid tumors. Pharmacokinetic data at the PR-104 MTD from human trials suggest that PR-104 metabolites may not reach the plasma exposures in children that were associated with high-level preclinical activity.

Metabolism and excretion of the novel bioreductive prodrug PR-104 in mice, rats, dogs, and humans.

PR-104 is the phosphate ester of a 3,5-dinitrobenzamide nitrogen mustard (PR-104A) that is reduced to active hydroxylamine and amine metabolites by reductases in tumors. In this study, we evaluate the excretion of [(3)H]PR-104 in mice and determine its metabolite profile in mice, rats, dogs, and humans after a single intravenous dose. Total radioactivity was rapidly and quantitatively excreted in mice, with cumulative excretion of 46% in urine and 50% in feces. The major urinary metabolites in mice were products from oxidative N-dealkylation and/or glutathione conjugation of the nitrogen mustard moiety, including subsequent mercapturic acid pathway metabolites. A similar metabolite profile was seen in mouse bile, mouse plasma, and rat urine and plasma. Dogs and humans also showed extensive thiol conjugation but little evidence of N-dealkylation. Humans, like rodents, showed appreciable reduced metabolites in plasma, but concentrations of the cytotoxic amine metabolite (PR-104M) were higher in mice than humans. The most conspicuous difference in metabolite profile was the much more extensive O-beta-glucuronidation of PR-104A in dogs and humans than in rodents. The structure of the O-beta-glucuronide (PR-104G) was confirmed by independent synthesis. Its urinary excretion was responsible for 13 +/- 2% of total dose in humans but only 0.8 +/- 0.1% in mice. Based on these metabolite profiles, biotransformation of PR-104 in rodents is markedly different from that in humans, suggesting that rodents may not be appropriate for modeling human biotransformation and toxicology of PR-104.

Hypoxic selectivity and solubility--investigating the properties of A-ring substituted nitro seco-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-ones (nitroCBIs) as hypoxia-activated prodrugs for antitumor therapy.

Nitro seco-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-ones (nitroCBIs) are a new class of prodrugs for antitumor therapy that undergo hypoxia-selective metabolism to form potent DNA minor groove alkylating agents. Although hindered by poor aqueous solubility, several examples have shown activity against hypoxic tumor cells in vivo. Here we investigate structural properties that influence hypoxic selectivity in vitro, and show that for high hypoxic selectivity nitroCBIs should combine an electron-withdrawing group of H-bond donor capacity on the A-ring, with a basic substituent on the minor groove-binding side chain. Substitution on the A-ring is compatible with the introduction of functionality that can improve water solubility.

The SIRT2-mediated deacetylation of AKR1C1 is required for suppressing its pro-metastasis function in Non-Small Cell Lung Cancer.

Aldo-keto reductase family 1 member C1 (AKR1C1) promotes malignancy of Non-Small Cell Lung Cancer (NSCLC) by activating Signal Transducer and Activator of Transcription 3 (STAT3) pathway. However, how the pro-metastatic functions of AKR1C1 are switched on/off remains unknown. Methods: Immunoprecipitation and LC-MS/MS analyses were performed to identify the acetylation on AKR1C1 protein, and the functional analyses (in vitro and in vivo) were performed to depict the contribution of acetylation to the pro-metastatic effects of AKR1C1. Results: Here we report that acetylated AKR1C1 on two lysine residues K185 & K201 is critical to its pro-metastatic role. The acetylation modification has no impact on the canonical enzymatic activity of AKR1C1, while it is required for the interaction between AKR1C1 to STAT3, which triggers the downstream transduction events, ultimately mobilizing cells. Importantly, the deacetylase Sirtuin 2 (SIRT2) is capable of deacetylating AKR1C1, inhibiting the transactivation of STAT3 target genes, thus suppressing the migration of cells. Conclusion: Acetylation on Lysines 185 and 201 of AKR1C1 dictates its pro-metastatic potential both in vitro and in vivo, and the reverting of acetylation by Sirtuin 2 provides potential therapeutic targets for treatment against metastatic NSCLC patients with high AKR1C1 expression.

Hypoxia-selective radiosensitisation by SN38023, a bioreductive prodrug of DNA-dependent protein kinase inhibitor IC87361.

DNA-dependent protein kinase (DNA-PK) plays a key role in repair of radiation-induced DNA double strand breaks (DSB) by non-homologous end-joining. DNA-PK inhibitors (DNA-PKi) are therefore efficient radiosensitisers, but normal tissue radiosensitisation represents a risk for their use in radiation oncology. Here we describe a novel prodrug, SN38023, that is metabolised to a potent DNA-PKi (IC87361) selectively in radioresistant hypoxic cells. DNA-PK inhibitory potency of SN38023 was 24-fold lower than IC87361 in cell-free assays, consistent with molecular modelling studies suggesting that SN38023 is unable to occupy one of the predicted DNA-PK binding modes of IC87361. One-electron reduction of the prodrug by radiolysis of anoxic formate solutions, and by metabolic reduction in anoxic HCT116/POR cells that overexpress cytochrome P450 oxidoreductase (POR), generated IC87361 efficiently as assessed by LC-MS. SN38023 inhibited radiation-induced Ser2056 autophosphorylation of DNA-PK catalytic subunit and radiosensitised HCT116/POR and UT-SCC-54C cells selectively under anoxia. SN38023 was an effective radiosensitiser in anoxic HCT116 spheroids, demonstrating potential for penetration into hypoxic tumour tissue, but in spheroid co-cultures of high-POR and POR-null cells it showed no evidence of bystander effects resulting from local diffusion of IC87361. Pharmacokinetics of IC87361 and SN38023 at maximum achievable doses in NIH-III mice demonstrated sub-optimal exposure of UT-SCC-54C tumour xenografts and did not provide significant tumour radiosensitisation. In conclusion, SN38023 has potential for exploiting hypoxia for selective delivery of a potent DNA-PKi to the most radioresistant subpopulation of cells in tumours. However, prodrugs providing improved systemic pharmacokinetics and that release DNA-PKi that elicit bystander effects are needed to maximise therapeutic utility.

Mechanism of action and preclinical antitumor activity of the novel hypoxia-activated DNA cross-linking agent PR-104.

PURPOSE: Hypoxia is a characteristic of solid tumors and a potentially important therapeutic target. Here, we characterize the mechanism of action and preclinical antitumor activity of a novel hypoxia-activated prodrug, the 3,5-dinitrobenzamide nitrogen mustard PR-104, which has recently entered clinical trials. EXPERIMENTAL DESIGN: Cytotoxicity in vitro was evaluated using 10 human tumor cell lines. SiHa cells were used to characterize metabolism under hypoxia, by liquid chromatography-mass spectrometry, and DNA damage by comet assay and gammaH2AX formation. Antitumor activity was evaluated in multiple xenograft models (PR-104 +/- radiation or chemotherapy) by clonogenic assay 18 h after treatment or by tumor growth delay. RESULTS: The phosphate ester "pre-prodrug" PR-104 was well tolerated in mice and converted rapidly to the corresponding prodrug PR-104A. The cytotoxicity of PR-104A was increased 10- to 100-fold by hypoxia in vitro. Reduction to the major intracellular metabolite, hydroxylamine PR-104H, resulted in DNA cross-linking selectively under hypoxia. Reaction of PR-104H with chloride ion gave lipophilic cytotoxic metabolites potentially able to provide bystander effects. In tumor excision assays, PR-104 provided greater killing of hypoxic (radioresistant) and aerobic cells in xenografts (HT29, SiHa, and H460) than tirapazamine or conventional mustards at equivalent host toxicity. PR-104 showed single-agent activity in six of eight xenograft models and greater than additive antitumor activity in combination with drugs likely to spare hypoxic cells (gemcitabine with Panc-01 pancreatic tumors and docetaxel with 22RV1 prostate tumors). CONCLUSIONS: PR-104 is a novel hypoxia-activated DNA cross-linking agent with marked activity against human tumor xenografts, both as monotherapy and combined with radiotherapy and chemotherapy.

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.

Next-Generation Hypoxic Cell Radiosensitizers: Nitroimidazole Alkylsulfonamides.

Innovations in the field of radiotherapy such as stereotactic body radiotherapy, along with the advent of radio-immuno-oncology, herald new opportunities for classical oxygen-mimetic radiosensitizers. The role of hypoxic tumor cells in resistance to radiotherapy and in suppression of immune response continues to endorse tumor hypoxia as a bona fide, yet largely untapped, drug target. Only nimorazole is used clinically as a radiosensitizer, and there is a dearth of new radiosensitizers in development. Here we present a survey of novel nitroimidazole alkylsulfonamides and document their cytotoxicity and ability to radiosensitize anoxic tumor cells in vitro. We use a phosphate prodrug approach to increase aqueous solubility and to improve tumor drug delivery. A 2-nitroimidazole and a 5-nitroimidazole analogue demonstrated marked tumor radiosensitization in either ex vivo assays of surviving clonogens or tumor regrowth delay.

Analysis of the hypoxia-activated dinitrobenzamide mustard phosphate pre-prodrug PR-104 and its alcohol metabolite PR-104A in plasma and tissues by liquid chromatography-mass spectrometry.

PR-104 is a dinitrobenzamide mustard pre-prodrug that is activated by reduction to a cytotoxic hydroxylamine metabolite in hypoxic tumour cells; it has recently commenced Phase I clinical trial. Here, we report two validated methods for the determination of PR-104 and its alcohol hydrolysis product, PR-104A in plasma and tissues across species. A high pH LC/MS/MS method was optimised for rapid and sensitive analysis of both analytes in rat, dog and human plasma. This assay was linear over the concentration range 0.005-2.5 microg/ml for PR-104 and 0.05-25 microg/ml for PR-104A (0.005-2.5 microg/ml for rat). A second method, using a low pH LC separation, was designed to provide higher chromatographic resolution, facilitating identification of metabolites. Both methods were successfully applied to the plasma pharmacokinetics of PR-104 and PR-104A in rats. In addition, cytotoxic reduced metabolites of PR-104A were identified in human tumour xenografts by the higher chromatographic resolution method.

Reductive Metabolism Influences the Toxicity and Pharmacokinetics of the Hypoxia-Targeted Benzotriazine Di-Oxide Anticancer Agent SN30000 in Mice.

3-(3-Morpholinopropyl)-7,8-dihydro-6H-indeno[5,6-e][1,2,4]triazine 1,4-dioxide (SN30- 000), an analog of the well-studied bioreductive prodrug tirapazamine (TPZ), has improved activity against hypoxic cells in tumor xenografts. However, little is known about its biotransformation in normal tissues. Here, we evaluate implications of biotransformation of SN30000 for its toxicokinetics in NIH-III mice. The metabolite profile demonstrated reduction to the 1-N-oxide (M14), oxidation of the morpholine side-chain (predominantly to the alkanoic acid M18) and chromophore, and subsequent glucuronidation. Plasma pharmacokinetics of SN30000 and its reduced metabolites was unaffected by the presence of HT29 tumor xenografts, indicating extensive reduction in normal tissues. This bioreductive metabolism, as modeled by hepatic S9 preparations, was strongly inhibited by oxygen indicating that it proceeds via the one-electron (radical) intermediate previously implicated in induction of DNA double strand breaks and cytotoxicity by SN30000. Plasma pharmacokinetics of SN30000 and M14 (but not M18) corresponded closely to the timing of reversible acute clinical signs (reduced mobility) and marked hypothermia (rectal temperature drop of ∼8°C at nadir following the maximum tolerated dose). Similar acute toxicity was elicited by dosing with TPZ or M14, although M14 did not induce the kidney and lung histopathology caused by SN30000. M14 also lacked antiproliferative potency in hypoxic cell cultures. In addition M14 showed much slower redox cycling than SN30000 in oxic cultures. Thus a non-bioreductive mechanism, mediated through M14, appears to be responsible for the acute toxicity of SN30000 while late toxicities are consistent with DNA damage resulting from its one-electron reduction. A two-compartment pharmacokinetic model, in which clearance of SN30000 is determined by temperature-dependent bioreductive metabolism to M14, was shown to describe the non-linear PK of SN30000 in mice. This study demonstrates the importance of non-tumor bioreductive metabolism in the toxicology and pharmacokinetics of benzotriazine di-oxides designed to target tumor hypoxia.

Improved HPLC method for the simultaneous determination of tramadol and O-desmethyltramadol in human plasma.

This paper describes an HPLC method for the determination of tramadol and its major active metabolite, O-desmethyltramadol (ODT), in human plasma. Sample preparation involved liquid-liquid extraction with diethyl ether-dichloromethane-butanol (5:3:2, v/v/v) and back extraction with sulphuric acid. Tramadol, ODT and the internal standard, sotalol, were separated by reversed phase HPLC using 35% acetonitrile and an aqueous solution containing 20 mM sodium phosphate buffer, 30 mM sodium dodecyl sulphate and 15 mM tetraethylammonium bromide pH 3.9. Detection was by fluorescence with excitation and emission wavelengths of 275 and 300 nm, respectively. The method was linear for tramadol (3-768 ng/ml) and ODT (1.5-384 ng/ml) with mean recoveries of 87.2% and 89.8%, respectively. Intra- and inter-day precisions were 10.34% and 8.43% for tramadol and 9.43% and 8.75% for ODT at the respective limits of quantitation (3 and 1.5 ng/ml). Accuracy for tramadol ranged from 96.2% to 105.3%. The method was applied to a pharmacokinetic study of tramadol in human volunteers.

Pre-clinical activity of PR-104 as monotherapy and in combination with sorafenib in hepatocellular carcinoma.

PR-104 is a clinical stage bioreductive prodrug that is converted in vivo to its cognate alcohol, PR-104A. This dinitrobenzamide mustard is reduced to activated DNA cross-linking metabolites (hydroxylamine PR-104H and amine PR-104M) under hypoxia by one-electron reductases and independently of hypoxia by the 2-electron reductase aldo-keto reductase 1C3 (AKR1C3). High expression of AKR1C3, along with extensive hypoxia, suggested the potential of PR-104 for treatment of hepatocellular carcinoma (HCC). However, a phase IB trial with sorafenib demonstrated significant toxicity that was ascribed in part to reduced PR-104A clearance, likely reflecting compromised glucuronidation in patients with advanced HCC. Here, we evaluate the activity of PR-104 in HCC xenografts (HepG2, PLC/PRF/5, SNU-398, Hep3B) in mice, which do not significantly glucuronidate PR-104A. Cell line differences in sensitivity to PR-104A in vitro under aerobic conditions could be accounted for by differences in both expression of AKR1C3 (high in HepG2 and PLC/PRF/5) and sensitivity to the major active metabolite PR-104H, to which PLC/PRF/5 was relatively resistant, while hypoxic selectivity of PR-104A cytotoxicity and reductive metabolism was greatest in the low-AKR1C3 SNU-398 and Hep3B lines. Expression of AKR1C3 in HepG2 and PLC/PRF/5 xenografts was in the range seen in 21 human HCC specimens. PR-104 monotherapy elicited significant reductions in growth of Hep3B and HepG2 xenografts, and the combination with sorafenib was significantly active in all 4 xenograft models. The results suggest that better-tolerated analogs of PR-104, without a glucuronidation liability, may have the potential to exploit AKR1C3 and/or hypoxia in HCC in humans.