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 13.5 mg/kg AST-001 to Sprague-Dawley rats and once daily 1 h intravenous infusions of 0.5, 1.5, and 4.5 mg/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 7 days of repeated dosing. In rats, the half-life of AST-001 was dose independent and ranged from 4.89 to 5.75 h. In cynomolgus monkeys, the half-life of AST-001 was from 1.66 to 5.56 h 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 5 mg/m2 in the first-in-human dose escalation study.

Aldophosphamide-thiazolidine (NSC-613060) an oxazaphosphorine cytostatic that crosses the blood brain barrier.

The pharmacologically active metabolite of cyclophosphamide is aldophosphamide. With cysteine, aldophosphamide forms stable aldophosphamide-thiazolidine which under physiological pH and temperature conditions hydrolyzes to aldophosphamide and cysteine. Aldophosphamide-thiazolidine was synthesized and tested for its ability as a cytostatic. The LD50 after a single intraperitoneal injection in mice was determined to be 2162 mg/kg, but after intravenous bolus administration of 500 mg/kg or in chronic toxicity tests with daily intraperitoneal injections, neurological side effects were observed. Antitumor activity was determined in therapy experiments in CD2F1 mice bearing subcutaneously transplanted P388 mouse leukemia cells. Administration of 100 mg/kg (less than 5% LD50) days 1-5 after tumor transplantation yielded an ILS of 100%. Organ distribution studies showed that aldophosphamide-thiazolidine is evenly distributed in all tissues examined, including brain tissue. The possibilities to increase the antitumor activity of aldophosphamide-thiazolidine by modulating the alkylating function are discussed.

pH-Sensitive PEGylated liposomes for delivery of an acidic dinitrobenzamide mustard prodrug: Pathways of internalization, cellular trafficking and cytotoxicity to cancer cells.

This paper aims to develop and evaluate a pH-sensitive PEGylated liposomal (pPSL) system for tumor-targeted intracellular delivery of SN25860, a weakly acidic, poorly water-soluble dinitrobenzamide mustard prodrug which is activated by the E. coli nitroreductase nfB. pPSL and non pH-sensitive liposomes (nPSL), as reference, were formulated by thin-film hydration; an active drug loading method was developed with the aid of solubilizers. Cytotoxicity was evaluated in an nfsB-transfected EMT6 mouse mammary carcinoma cell line. Cellular uptake of liposomes was evaluated by both high performance liquid chromatography and flow cytometry. Intracellular trafficking was visualised by confocal microscopy. High drug loading (7.0±0.2% w/w) was achieved after systematic optimization of drug loading conditions. pPSL-SN25860 demonstrated a 21 and 24- fold increase in antiproliferative potency compared to nPSL-SN25860 and free drug, respectively. Cells treated with pPSL had a 1.6-2.5- fold increase in intracellular drug concentration compared to nPSL. This trend was consistent with flow cytometry results. Cells treated with chlorpromazine demonstrated reduced uptake of both nPSL (40%) and pPSL (46%), indicating clathrin-mediated endocytosis was the major pathway. Confocal microscopy showed that pPSL had not only undergone faster and greater endocytosis than nPSL but was also homogeneously distributed in the cytosol and nuclei suggesting endosome escape, in contrast to nPSL.

Different Efficiency of Liposomal Forms with Hydrophilic and Hydrophobic Antitumor Agents in Relation to Solid Transplants of Mouse Tumor and Its Metastases in the Liver.

Experiments were performed on the model of transplanted mouse tumor with high incidence of liver metastases. Hydrophilic drug cycloplatam (injected intravenously in liposomes) was more potent than "free cycloplatam" (injected intravenously or intraperitoneally in physiological saline) in inhibiting the growth of natural and experimental metastases in the liver. By contrast, liposomal cycloplatam had lower efficiency than free cycloplatam in suppressing the growth of solid tumor. Liposomal and free cortifen (hydrophobic hormonal cytostatic) produced nearly the same effects on solid tumor growth. Our results suggest that liposomal forms of hydrophobic compounds producing nonselective effect on tumor cells (e.g., actinomycin D or Cosmegen), should not have advantages over free forms.

Pharmacokinetic and pharmacodynamic profile of bendamustine and its metabolites.

PURPOSE: Bendamustine is a unique alkylating agent indicated for the treatment of chronic lymphocytic leukemia and rituximab-refractory, indolent B cell non-Hodgkin's lymphoma. Despite the extensive experience with bendamustine, its pharmacokinetic profile has only recently been described. This overview summarizes the pharmacokinetics, pharmacokinetic/pharmacodynamic relationships, and drug-drug interactions of bendamustine in adult and pediatric patients with hematologic malignancies. METHODS: A literature search and data on file (including a human mass balance study, pharmacokinetic population analyses in adult and pediatric patients, and modeling analyses) were evaluated for inclusion. RESULTS: Bendamustine concentrations peak at end of intravenous infusion (~1 h). Subsequent elimination is triphasic, with the intermediate t 1/2 (~40 min) as the effective t 1/2 since the final phase represents <1 % of the area under the curve. Bendamustine is rapidly hydrolyzed to monohydroxy-bendamustine and dihydroxy-bendamustine, which have little or no activity. Cytochrome P450 (CYP) 1A2 oxidation yields the active metabolites γ-hydroxybendamustine and N-desmethyl-bendamustine, at low concentrations, which contribute minimally to cytotoxicity. Minor involvement of CYP1A2 in bendamustine elimination suggests a low likelihood of drug-drug interactions with CYP1A2 inhibitors. Systemic exposure to bendamustine 120 mg/m(2) is comparable between adult and pediatric patients; age, race, and sex have been shown to have no significant effect on systemic exposure in either population. The effect of hepatic/renal impairment on bendamustine pharmacokinetics remains to be elucidated. Higher bendamustine concentrations may be associated with increased probability of nausea or infection. No clear exposure-efficacy response relationship has been observed. CONCLUSIONS: Altogether, the findings support dosing based on body surface area for most patient populations.

Obinutuzumab plus fludarabine/cyclophosphamide or bendamustine in the initial therapy of CLL patients: the phase 1b GALTON trial.

Obinutuzumab is a type 2, glycoengineered, anti-CD20 antibody recently approved with chlorambucil for the initial therapy of chronic lymphocytic leukemia (CLL). In this nonrandomized, parallel-cohort, phase 1b, multicenter study, we explored the safety and preliminary efficacy of obinutuzumab-bendamustine (G-B) or obinutuzumab fludarabine cyclophosphamide (G-FC) for the therapy of previously untreated fit patients with CLL. Patients received up to 6 cycles of G-B (n = 20) or G-FC (n = 21). The primary end point was safety, with infusion-related reactions (88%, grade 3-4 20%) being the most common adverse event and grade 3-4 neutropenia in 55% on G-B and 48% on G-FC. Mean cycles completed were 5.7 for G-B and 5.1 for G-FC, with 2 and 7 early discontinuations, respectively. The objective response rate (ORR) for G-B was 90% (18/20) with 20% complete response (CR) and 25% CR with incomplete marrow recovery (CRi). The ORR for G-FC was 62% (13/21), with 10% CR and 14% CRi, including 4 patients not evaluable. With a median follow-up of 23.5 months in the G-B cohort and 20.7 months in the G-FC cohort, no patient has relapsed or died. We conclude that obinutuzumab with either B or FC shows manageable toxicity and has promising activity. This study was registered at www.clinicaltrials.gov as #NCT01300247.

Population pharmacokinetics and pharmacokinetics/pharmacodynamics of bendamustine in pediatric patients with relapsed/refractory acute leukemia.

OBJECTIVE: The pharmacokinetic (PK) profile of bendamustine has been characterized in adults with indolent non-Hodgkin lymphoma (NHL), but remains to be elucidated in pediatric patients with hematologic malignancies. This analysis used data from a nonrandomized pediatric study in patients with relapsed/refractory acute lymphocytic leukemia or acute myeloid leukemia. METHODS: Bendamustine 90 or 120 mg/m(2) (60-minute infusion) was administered on days 1 and 2 of 21 day cycles. The population PK base model was adjusted for body surface area (BSA), and the appropriateness of the final model was evaluated by visual predictive check. A covariate analysis explored PK variability. Bayesian PK parameter estimates and concentration-time profiles for each patient were generated. Bendamustine PK in pediatric patients was compared with that of adults with indolent NHL. PK/pharmacodynamic analyses were conducted for fatigue, nausea, vomiting, and infection. RESULTS: Thirty-eight patients (median age: 7 years; range: 1-19 years) receiving bendamustine 120 mg/m(2) and an additional five patients receiving bendamustine 90 mg/m(2) (median age: 12 years; range: 8-14 years) were included in the population PK analysis. Peak plasma concentrations of bendamustine (Cmax) occurred at the end of infusion (about 1 h). Decline from peak showed a rapid distribution phase (t½α = 0.308 h) and a slower elimination phase (t½ß = 1.47 h). Model-predicted mean Cmax and area under the curve values from time 0-24 h were 6806 ng/mL and 8240 ng*h/mL, respectively. When dosed based upon BSA, it appeared that age, body weight, race, mild renal (n = 3) or hepatic (n = 2) dysfunction, cancer type, and cytochrome P450 1A2 inhibitors (n = 17) or inducers (n = 3) did not affect systemic exposure, which was comparable between pediatric and adult patients. Infection was the only adverse event associated with bendamustine Cmax. However, due to the small sample size for some subgroups, the observed trends should be interpreted with caution. CONCLUSIONS: At the recommended dose (120 mg/m(2)), bendamustine systemic exposure was similar across the pediatric population and comparable to adults. The similarity in exposure despite the large range of BSA across pediatric and adult populations confirms the appropriateness of BSA-based dosing, which was utilized to attain systemic exposures in pediatric patients reflective of the therapeutic range in adults. Probability of occurrence of infection increased with higher bendamustine Cmax.

Phase I study of bendamustine with concurrent whole brain radiation therapy in patients with brain metastases from solid tumors.

A phase I study was conducted to evaluate the dose-limiting toxicities (DLTs) and to determine the maximum tolerated dose (MTD)/recommended phase II dose of bendamustine with concurrent whole brain radiation (WBR) in patients with brain metastases (BM) from solid tumors. Four doses of intravenous weekly bendamustine were administered with 3 weeks of WBR at three dose levels (60, 80, and 100 mg/m(2)) according to a standard 3 + 3 phase I design. A total of 12 patients with solid tumor BM were enrolled in the study (six with non-small cell lung cancer, four with melanoma, one with breast cancer, and one with neuroendocrine carcinoma). The first two dose levels had three patients each, and the third dose level had six total patients. Plasma pharmacokinetic studies of bendamustine demonstrated no significant differences from pharmacokinetic characteristics of bendamustine in other studies. No DLTs were noted at any dose levels, and no grade 4 toxicities occurred. The MTD of weekly bendamustine with concurrent WBR was 100 mg/m(2). The majority of trial patients died from progressive systemic disease rather than their brain disease. The combination of weekly bendamustine with concurrent WBR was acceptably tolerated. The efficacy of this combination may be evaluated in a phase II trial with stratification by histologies.

Phase 1b study of otlertuzumab (TRU-016), an anti-CD37 monospecific ADAPTIR™ therapeutic protein, in combination with rituximab and bendamustine in relapsed indolent lymphoma patients.

PURPOSE: CD37 is cell surface tetraspanin present on normal and malignant B cells. Otlertuzumab (TRU-016) is a novel humanized anti-CD37 protein therapeutic that triggers direct caspase independent apoptosis of malignant B cells and induces antibody-dependent cell-mediated cytotoxicity. This study evaluated the safety, pharmacokinetics, and efficacy of otlertuzumab administered in combination with rituximab and bendamustine to patients with relapsed, indolent B-cell non-Hodgkin Lymphoma (NHL). METHODS: Patients with relapsed or refractory NHL received otlertuzumab (10 or 20 mg/kg) intravenously (IV) on days 1 and 15, bendamustine (90 mg/m(2)) on days 1 and 2, and rituximab (375 mg/m(2)) on day 1 for up to six 28 day cycles. Responses were determined using standard criteria. RESULTS: Twelve patients were treated with 6 patients at each dose level; median age was 57 years (range, 51-79), and median number of prior regimens was 3 (range, 1-4). All patients had relapsed after prior rituximab including 7 refractory to their most recent previous treatment. In the 10 and 20 mg/kg dose cohorts, the mean half-life was 8 and 10 days following the first dose, and 12 or 14 days following 12 doses of otlertuzumab, respectively. Overall response rate was 83% (10/12) with 4 CRs (32%). The most frequent adverse events were neutropenia, nausea, fatigue, leukopenia, and insomnia; most were grade 1 or 2. CONCLUSIONS: Otlertuzumab in combination with rituximab and bendamustine was well tolerated and induced responses in the majority of patients with relapsed indolent B-NHL. NCI Clinical Trials Network registration: NCT01317901.

Simultaneous determination of bendamustine and its active metabolite, gamma-hydroxy-bendamustine in human plasma and urine using HPLC-fluorescence detector: application to a pharmacokinetic study in Chinese cancer patients.

A simple, sensitive and cost-effective assay based on reversed phase high performance liquid chromatography (RP-HPLC) with isocratic mode for simultaneous determination of bendamustine (BM) and its active metabolite, gamma-hydroxy-bendamustine (γ-OH-BM) in human plasma and urine was developed and validated. Sample preparation involved protein precipitation by 10% perchloric acid-methanol solution. The peaks were recorded by using fluorescence detector (excitation wavelength 328 nm and emission wavelength 420 nm). The calibration curves were linear over concentration ranges of 8.192-10,000 ng mL(-1) and 5-1,000 ng mL(-1) for BM in human plasma and urine as well as 10-1,000 ng mL(-1) and 5-1,000 ng mL(-1) for γ-OH-BM in human plasma and urine, respectively. Intra- and inter-run precisions of BM and γ-OH-BM were less than 15% and the bias were within ± 15% for both plasma and urine. This validated method was successfully applied to a pharmacokinetic study enrolling 10 Chinese patients with indolent B-cell non-Hodgkin lymphoma and chronic lymphocytic leukemia administered a single intravenous infusion of 100 mg m(2) bendamustine hydrochloride.

An evaluation of the potential for drug-drug interactions between bendamustine and rituximab in indolent non-Hodgkin lymphoma and mantle cell lymphoma.

PURPOSE: Bendamustine plus rituximab has been reported to be effective in treating lymphoid malignancies. This analysis investigated the potential for drug-drug interactions between the drugs in patients with indolent non-Hodgkin lymphoma or mantle cell lymphoma. METHODS: Data were derived from a bendamustine-rituximab combination therapy study, a bendamustine monotherapy study, and published literature on rituximab monotherapy and combination therapy. Analysis of the potential for rituximab to affect bendamustine systemic exposure included comparing bendamustine concentration-time profile following monotherapy to that following combination therapy and comparing model-predicted Bayesian bendamustine clearance in the presence and absence of rituximab. Analysis of the potential for bendamustine to affect rituximab systemic exposure included plotting observed minimum, median, and maximum serum rituximab concentrations at the end of rituximab infusion (EOI) and 24 h and 7 days post-infusion in patients receiving combination therapy versus concentrations reported in literature following rituximab monotherapy. RESULTS: The established population pharmacokinetic model following bendamustine monotherapy was evaluated to determine its applicability to combination therapy for the purpose of confirming lack of pharmacokinetic interaction. The model adequately described the bendamustine concentration-time profile following monotherapy and combination therapy in adults. There was no statistically significant difference in estimated bendamustine clearance either alone or in combination. Also, rituximab concentrations from EOI to 24 h and 7 days demonstrated a pattern of decline similar to that seen in rituximab studies without bendamustine, suggesting that bendamustine does not affect the rituximab clearance rate. CONCLUSIONS: Neither bendamustine nor rituximab appears to affect systemic exposure of the other drug when coadministered.

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.

Determination of tissue distribution of potent antitumor agent ureidomustin (BO-1055) by HPLC and its pharmacokinetic application in rats.

Ureidomustin hydrochloride (BO-1055) was designed as a water-soluble nitrogen-mustard, which exhibited potent anticancer activity and was selected as a candidate for preclinical studies. However, up to date, there is rarely an easy and economic method to quantize ureidomustin in the biological samples. The aim of this study is to develop a simple yet valid quantization method to tackle this challenge. Here we present a combined high-performance liquid chromatography with photodiode array (HPLC-PDA) method in quantizing the ureidomustin in the plasma and various organs of Sprague-Dawley rats. The method was validated in terms of precision, accuracy, and extraction recovery. Furthermore, the established method was applied to study pharmacokinetics of ureidomustin in the rat's plasma and verified via a liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Calibration curves of the plasma and organ samples were falling at the range between 0.5-50µg/mL and 0.1-50µg/mL (r(2)≥0.999 and CV≤±15%), respectively. The limits of detection (LOD) were 0.1µg/mL for plasma samples and 0.05µg/mL for organ samples, while the detection limits of quantification (LOQ) were 0.5µg/mL for plasma samples and 0.1µg/mL for organ samples. The average recovery of ureidomustin was about 83%. These results demonstrated a linear pharmacokinetic pattern at dosages of 10 and 30mg/kg. The pharmacokinetic data revealed that ureidomustin was best fitted to a two-compartment model with a rapid distribution phase and a slow elimination phase. Besides, after a short intravenous administration time at the dose of 10mg/kg, ureidomustin was found to be quickly distributed to all organs in rats, accumulated mainly in the kidney, and only a limited amount was detected in the brain.

Pharmacokinetics and excretion of 14C-bendamustine in patients with relapsed or refractory malignancy.

BACKGROUND: Bendamustine is an alkylating agent with clinical activity against a variety of hematologic malignancies and solid tumors. To assess the roles of renal and hepatic drug elimination pathways in the excretion and metabolism of bendamustine, a mass balance study was performed in patients with relapsed or refractory malignancies. METHODS: A single 60-minute intravenous dose of 120 mg/m(2), 80-95 µCi (14)C-bendamustine hydrochloride was administered to six patients, followed by collection of blood, urine, and fecal samples at specified time points up to day 8 or until the radioactivity of the 24-hour urine and fecal collections was below 1% of the administered dose (whichever was longer). Total radioactivity (TRA) was measured in all samples, and concentrations of unchanged bendamustine and its metabolites γ-hydroxy-bendamustine (M3), N-desmethyl-bendamustine (M4), and dihydroxy bendamustine (HP2) were determined in plasma and urine, using validated liquid chromatography-tandem mass spectrometry methods. RESULTS: The mean recovery of TRA in excreta was 76% of the radiochemical dose. Approximately half of the administered dose was recovered in urine and a quarter in feces. Less than 5% of the administered dose was recovered in urine as unchanged bendamustine. Bendamustine clearance from plasma was rapid, with a half-life of ~40 minutes. Plasma concentrations of M3, M4, and HP2 were very low relative to bendamustine concentrations. Plasma levels of TRA were higher and more sustained as compared with plasma concentrations of bendamustine, M3, M4, and HP2, suggesting the presence of one or more longer-lived (14)C-bendamustine-derived compounds. Fatigue (50%) and vomiting (50%) were the most frequent treatment-related adverse events. A grade 3/4 absolute lymphocyte count decrease occurred in all patients at some point during the study. CONCLUSION: Bendamustine is extensively metabolized, with subsequent excretion in both urine and feces. Accumulation of bendamustine is not anticipated in cancer patients with renal or hepatic impairment, because of the dose administration schedule and short half-life.

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).

Pharmacokinetic evaluation and therapeutic activity of bendamustine in B-cell lymphoid malignancies.

INTRODUCTION: Bendamustine , a cytotoxic agent comprising structural features of both an alkylating drug and a purine nucleoside analog, was approved by the US FDA for treatment of chronic lymphocytic leukemia (CLL) and indolent B-cell non-Hodgkin's lymphoma (NHL). Additionally, in Europe the drug has been also approved for treatment of multiple myeloma (MM) and in Asia, especially in Japan for refractory/relapsed NHL and mantle cell lymphoma. AREAS COVERED: The authors present the chemical structure, mechanism of action, pharmacokinetic properties and clinical application of bendamustine in hematological malignancies. Publications in English related to the above, up to June 2012, have been surveyed in the MEDLINE database. Conference proceedings reports from the last 5 years are also included. Additional relevant publications have been obtained by reviewing the references from the chosen articles. EXPERT OPINION: The availability of bendamustine provides an effective treatment option for patients with lymphoid malignancies. Several recent clinical trials have documented the activity of bendamustine in CLL, NHL and MM, both as a single agent and in combination with other cytotoxic drugs. However, doses, schedules and also the role of bendamustine in treatment of patients with hematological malignancies should be further investigated to establish its real place in the management of these diseases.

Metabolite profiling of bendamustine in urine of cancer patients after administration of [14C]bendamustine.

Bendamustine is an alkylating agent consisting of a mechlorethamine derivative, a benzimidazole group, and a butyric acid substituent. A human mass balance study showed that bendamustine is extensively metabolized and subsequently excreted in urine. However, limited information is available on the metabolite profile of bendamustine in human urine. The objective of this study was to elucidate the metabolic pathways of bendamustine in humans by identification of its metabolites excreted in urine. Human urine samples were collected up to 168 h after an intravenous infusion of 120 mg/m(2) (80-95 µCi) [(14)C]bendamustine. Metabolites of [(14)C]bendamustine were identified using liquid chromatography (high-resolution)-tandem mass spectrometry with off-line radioactivity detection. Bendamustine and a total of 25 bendamustine-related compounds were detected. Observed metabolic conversions at the benzimidazole and butyric acid moiety were N-demethylation and γ-hydroxylation. In addition, various other combinations of these conversions with modifications at the mechlorethamine moiety were observed, including hydrolysis (the primary metabolic pathway), cysteine conjugation, and subsequent biotransformation to mercapturic acid and thiol derivatives, N-dealkylation, oxidation, and conjugation with phosphate, creatinine, and uric acid. Bendamustine-derived products containing phosphate, creatinine, and uric acid conjugates were also detected in control urine incubated with bendamustine. Metabolites that were excreted up to 168 h after the infusion included products of dihydrolysis and cysteine conjugation of bendamustine and γ-hydroxybendamustine. The range of metabolic reactions is generally consistent with those reported for rat urine and bile, suggesting that the overall processes involved in metabolic elimination are qualitatively the same in rats and humans.

Development and validation of LC-MS/MS assays for the quantification of bendamustine and its metabolites in human plasma and urine.

A sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assay is described for the quantification of the anti-cancer agent bendamustine and its phase I metabolites γ-hydroxy-bendamustine (M3) and N-des-methylbendamustine (M4) and for its product of two-fold hydrolysis, dihydroxy-bendamustine (HP2), in human plasma and urine. Like most alkylating nitrogen mustards, bendamustine is prone to chemical hydrolysis in aqueous solution. To minimize degradation of bendamustine, urine samples were stabilized by a 100-fold dilution with human plasma and then processed identically to plasma samples. Sample aliquots of 200 µL were mixed with an internal standard solution and acidified before separation of the analytes from the biomatrix with solid phase extraction. Dried and reconstituted extracts were injected on a Synergi Hydro RP column for the analysis of bendamustine, M3 and M4 or a Synergi Polar RP column for the analysis of HP2. Gradient elution was applied using 5mM ammonium formate with 0.1% formic acid in water and methanol as mobile phases. Analytes were ionized using an electrospray ionisation source in positive mode and detected with a triple quadrupole mass spectrometer. The quantifiable range for bendamustine, M3 and M4 was 0.5-500 ng/mL in plasma and 0.5-50 µg/mL in urine, and that for HP2 was 1-500 ng/mL in plasma and 0.1-50 µg/mL in urine. The assays were accurate and precise, with inter-assay and intra-assay accuracies within ± 20% of nominal and CV values below 20% at the lower limit of quantification and within ± 15% of nominal and below 15% at the other concentration levels tested. These methods were successfully applied to evaluate the pharmacokinetic profile of bendamustine and its metabolites in cancer patients treated with bendamustine.

Preclinical pharmacokinetic and toxicology assessment of red blood cells prepared with S-303 pathogen inactivation treatment.

BACKGROUND: A system has been developed to inactivate a wide spectrum of blood-borne pathogens in red blood cells (RBCs) before transfusion. The system utilizes S-303 to target nucleic acids of pathogens and white blood cells. The safety of pathogen inactivated RBC was assessed using S-303-treated RBCs (S-303 RBCs) and S-300, the primary degradation product of S-303. STUDY DESIGN AND METHODS: As part of a preclinical safety evaluation program, intravenous toxicity, safety pharmacology, toxicokinetic, and pharmacokinetic studies were conducted in rats and dogs with S-303 RBCs and S-300. RESULTS: Single and repeated transfusions of S-303 RBCs were well tolerated in rats and dogs at S-303 concentrations up to five times higher than that used to prepare RBCs for clinical use. For S-300, the doses ranged from the lowest level representative of a clinical exposure from transfusion of 1 unit (0.052 mg/kg/day) to up to the amount of S-300 that would result from exposure to more than 1900 units of RBCs (100 mg/kg/day). There were no related effects of S-303 RBCs or S-300 on mortality, clinical status, body weight, or clinical laboratory assessments and no evidence of organ toxicity. S-300 did not accumulate in the plasma of rats and dogs after repeated transfusions. For all the studies, plasma S-303 was consistently below the limit of quantitation. CONCLUSION: The level of residual S-303 and S-300 in the treated blood component is well below that at which no adverse effects were observed. These results support further clinical development of S-303 RBCs for prevention of transfusion-transmitted infections.

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.