Delta-9 THC can be detected and quantified in the semen of men who are chronic users of inhaled cannabis.

PURPOSE: The purpose of this proof-of-concept study was to determine whether delta-9-tetrahydrocannabinol (THC) and THC metabolites (11-OH THC and THC-COOH) can be detected in semen. METHODS: Twelve healthy men aged 18-45 years who identified as chronic and heavy users of inhaled cannabis were recruited. THC and THC metabolite levels were measured in serum, urine, and semen of the participants. Semen analyses were performed. Serum reproductive hormones were measured. RESULTS: The median age and BMI of participants were 27.0 years and 24.7 kg/m2, respectively. Over half the participants were daily users of cannabis for over 5 years. Serum reproductive hormones were generally within normal ranges, except prolactin, which was elevated in 6 of 12 participants (mean 13.9 ng/mL). The median sperm concentration, motility, and morphology were 75.5 million/mL, 69.5%, and 5.5%, respectively. Urinary THC-COOH was detected in all 12 participants, and at least one serum THC metabolite was present in 10 of 12 participants. Two semen samples had insufficient volume to be analyzed. THC was above the reporting level of 0.50 ng/mL in the semen of two of the remaining participants. Seminal THC was moderately correlated with serum levels of THC (r = 0.66), serum 11-OH THC (r = 0.57), and serum THC-COOH (r = 0.67). Seminal delta-9 THC was not correlated with urinary cannabinoid levels or semen analysis parameters. CONCLUSION: This is the first study to identify and quantify THC in human semen, demonstrating that THC can cross the blood-testis barrier in certain individuals. Seminal THC was found to be moderately correlated with serum THC and THC metabolites.

Stabilities of neutral and basic esters of bendamustine in plasma compared to the parent compound: kinetic investigations by HPLC.

Esters of the cytostatic bendamustine (1), previously demonstrated to be much more potent than the parent compound as antiproliferative agents in vitro, were investigated for stability in buffer and plasma, as well as against porcine liver esterase in the presence of different amounts of albumin using a validated RP-HPLC method with fluorescence detection. The hydrolysis of the nitrogen mustard moiety was retarded (for 1: approximately 130 vs. 11 min) in the presence of plasma proteins. For the derivatives, both cleavage of ester and nitrogen mustard moieties were analyzed. Enzymatic hydrolysis was very fast in the case of 2-pyrrolidino-, 2-piperidino- and 2-(4-methylpiperazino)-ethyl esters, whereas methyl, ethyl, morpholinoethyl and branched 2-pyrrolidinoethyl esters were considerably more stable (half-lives between 41 and 116 min, compared to <5 min). Inhibition by physostigmine indicated unspecific cholinesterases to be involved in the rapid ester cleavage. Due to lower protein content and higher enzymatic activity in murine compared to human plasma, reduced stability of all investigated esters in mouse plasma (t½<2 min) has to be taken into account with respect to the design of animal studies.

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.

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.

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).

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.

PR-104 plus sorafenib in patients with advanced hepatocellular carcinoma.

PURPOSE: PR-104 is activated by reductases under hypoxia or by aldo-keto reductase 1C3 (AKR1C3) to form cytotoxic nitrogen mustards. Hepatocellular carcinoma (HCC) displays extensive hypoxia and expresses AKR1C3. This study evaluated the safety and efficacy of PR-104 plus sorafenib in HCC. METHODS: Patients with advanced-stage HCC, Child-Pugh A cirrhosis, and adequate organ function, were assigned to dose escalating cohorts of monthly PR-104 in combination with twice daily sorafenib. The plasma pharmacokinetics (PK) of PR-104 and its metabolites were evaluated. RESULTS: Fourteen (11 men, 3 women) HCC patients: median age 60 years, ECOG 0-1, received PR-104 at two dose levels plus sorafenib. Six patients were treated at starting cohort of 770 mg/m(2). In view of one DLT of febrile neutropenia and prolonged thrombocytopenia, a lower PR-104 dose cohort (550 mg/m(2)) was added and accrued 8 patients. One patient had a partial response and three had stable disease of ≥8 weeks in the 770 mg/m(2) cohort. Three patients at the 550 mg/m(2) had stable disease. There were no differences in PK of PR-104 or its metabolites with or without sorafenib, but the PR-104A AUC was twofold higher (P < 0.003) than in previous phase I studies at equivalent dose. CONCLUSIONS: PR-104 plus sorafenib was poorly tolerated in patients with advanced HCC, possibly because of compromised clearance of PR-104A in this patient population. Thrombocytopenia mainly and neutropenia were the most clinically significant toxicities and led to discontinuation of the study. PR-104 plus sorafenib is unlikely to be suitable for development in this setting.

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.

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.

Bendamustine-associated hemolytic anemia.

OBJECTIVE: To report a case of probable bendamustine-related hemolytic anemia. CASE SUMMARY: A 64-year-old white female had recently received treatment with bendamustine for stage III follicular lymphoma. After her fourth cycle, she was admitted to an outside facility with severe right upper quadrant pain across her back and findings consistent with obstructive jaundice. She was found to have pancytopenia and elevations in total bilirubin, alkaline phosphatase, and transaminase levels. A bone marrow biopsy showed no evidence of lymphoma and presence of megakaryocytes on 2 occasions. Upon transfer to West Virginia University Hospitals, her haptoglobin was found to be undetectable, total bilirubin 10.3 mg/dL (unconjugated bilirubin 4.9 mg/dL), reticulocyte count 21.4% (reticulocyte index > or = 2%), alkaline phosphatase 1125 U/L, and lactate dehydrogenase 421 U/L. The peripheral smear showed evidence of spherocytes and very rare schistocytes. Based on these findings, the woman was diagnosed with hemolytic anemia secondary to bendamustine exposure. She was started on prednisone 1 mg/kg (60 mg) daily and, soon after, her platelets and hemoglobin stabilized. DISCUSSION: Drug-induced hemolytic anemia is an acquired or extrinsic process that results in antibody-mediated red blood cell destruction. The patient was not taking any medications commonly associated with hemolytic anemia; however, her laboratory test results were consistent with hemolytic anemia. Based on bendamustine's structural similarity to fludarabine and fludarabine's association with causing hemolytic anemia, we considered exposure to bendamustine to be the most likely contributory factor for her diagnosis. According to the Naranjo probability scale, a probable likelihood was reflected in bendamustine causing the hemolytic anemia. CONCLUSIONS: Continued monitoring of postmarketing data is necessary to correlate this occurrence of hemolytic anemia with bendamustine therapy.

Rapid and sensitive ultra-high-pressure liquid chromatography-tandem mass spectrometry analysis of the novel anticancer agent PR-104 and its major metabolites in human plasma: Application to a pharmacokinetic study.

PR-104 is a dinitrobenzamide mustard currently in clinical trial as a hypoxia-activated prodrug. It is converted systemically to the corresponding alcohol, PR-104A, which is activated by nitroreduction to the hydroxylamine (PR-104H) and amine (PR-104M). PR-104A is also metabolised to the O-glucuronide (PR-104G), and by oxidative debromoethylation to the semi-mustard PR-104S. We now report a validated ultra-high-pressure liquid chromatography and tandem mass spectrometry (UHPLC-MS/MS) method for the determination of these metabolites in human plasma. Plasma proteins were precipitated with acidified methanol and the supernatant diluted into water. Aliquots were analysed by UHPLC-MS/MS using a Zorbax Eclipse XDB-C18 Rapid Resolution HT (50mmx2.1mm, 1.8microm) column and gradient of acetonitrile and 0.01% formic acid with a 6min run time. The method had a linear range of 0.1-50microM for PR-104, PR-104A and PR-104G, 0.05-5microM for PR-104H, 0.025-2.5microM for PR-104M and 0.01-1microM for PR-104S. The intra-day and inter-day precision and accuracy were within 14%. The extraction recovery of all analytes was over 87%. The validated method was illustrated by using it to study the pharmacokinetics of PR-104 and its metabolites in a human patient.

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.

Synthesis and characterization of some new phase II metabolites of the alkylator bendamustine and their identification in human bile, urine, and plasma from patients with cholangiocarcinoma.

The alkylating agent bendamustine is currently in phase III clinical trials for the treatment of hematological malignancies and breast, lung, and gastrointestinal tumors. Renal elimination mainly as the parent compound is thought to be the primary route of excretion. Because polar biliary conjugates were expected metabolites of bendamustine, three cysteine S-conjugates were synthesized, purified by quantitative high-performance liquid chromatography (HPLC), and characterized by NMR spectroscopy and mass spectrometry (MS). HPLC assays with MS, as well as fluorescence detection of bile, urine, and plasma after single-dose intravenous infusion of 140 mg/m(2) bendamustine in five subjects with cholangiocarcinoma, indicated the existence of these phase II metabolites, which were identified as cysteine S-conjugates by comparison with the previously characterized synthetic reference standards. The sum of the three cysteine S-conjugates of bendamustine was determined in human bile and urine to be 95.8 and 26.0%, respectively, expressed as mean percentage of the sum of the parent compound and identified metabolites. The percentage of administered dose recovered in urine as cysteine S-conjugates ranged from 0.9 to 4.1%, whereas the total percentage of the administered dose excreted in urine as the parent drug and seven metabolites ranged from 3.8 to 16.3%. The identification of cysteine S-conjugates provide evidence that a major route of bendamustine metabolism in humans involves conjugation with glutathione. Results indicate the importance of phase II conjugation in the elimination of bendamustine, besides phase I metabolism and hydrolytic degradation, and require further investigation.

Selective uptake of the anticancer drug bendamustine by liver and kidney tissues following its intravenous administration to mice.

Distribution of 14C-bendamustine following intravenous (i.v.) administration to mice was examined by whole body autoradiographic (WBAR) and quantitative techniques. The WBAR study showed that 14C-bendamustine-derived radioactivity was distributed extremely unevenly at each time interval investigated. After 5 min of administration the highest density of radioactive material was found in the liver and in the kidney. At all time intervals investigated the brain remained free of the label. In a detailed quantitative distribution study it was found that 14C-bendamustine-derived radioactivity was also unevenly distributed throughout the mouse tissues. At 5 min postdosing the level of 14C was by one order higher in the liver and in the kidney in comparison to the lungs, heart, spleen, and muscle. The results of both WBAR and quantitative tissue distribution studies suggest that bendamustine was selectively taken up from the blood by liver and kidney tissues. Because of this pharmacokinetic property, dose modification should be taken into consideration when administering the drug to patients suffering from hepatobiliary and kidney disorders.

Determination of tallimustine in human plasma by high-performance liquid chromatography.

A sensitive and reproducible HPLC method for the determination of tallimustine (I) in human plasma has been developed and validated. Compound I was extracted from plasma by solid-phase extraction using a C18 cartridge from which the test compound was eluted with a methanol-formic acid mixture. The methanol solution was evaporated to dryness and the residue dissolved in a 0.2 M formic acid in methanol-water (1:1, v/v) mixture, then injected onto the HPLC column. The chromatographic separation was performed isocratically by a reversed-phase column filled with ODS, using a 50 mM KH2PO4-acetonitrile mixture as the mobile phase. The flow-rate was 1 ml/min. The eluate was monitored at 314 nm. No peak interfering with that of I was observed when blank human plasma was assayed. Linearity was established in the concentration range 0.5-85.5 nanograms of I per millilitre of plasma. Four calibration curves in plasma, prepared and run on four different days, showed correlation coefficients higher than 0.99 and good reproducibility of the slope (C.V. = 4.5%). The intra-day precision, evaluated at three concentrations (in the low, mid and high range of the standard curve) and expressed as C.V. ranged from 0.9 to 14.4%. The inter-day precision evaluated at the same concentrations was better than 10.2%. The inter-day accuracy evaluated in the same samples and expressed as the ratio of found/added amount of I, ranged from 86.2 to 108.5%. The limit of quantitation was 0.5 ng/ml plasma. The HPLC method described here was successfully employed for the determination of I in some plasma samples obtained during a phase I clinical trial with the test compound.

The determination of cyclophosphamide and its metabolites in blood plasma as stable trifluoroacetyl derivatives by electron capture chemical ionization gas chromatography/mass spectrometry.

A method is described for the determination of the antitumour drug cyclophosphamide and six stable metabolites in plasma of cancer patients, namely dechloroethyl-cyclophosphamide, 4-keto-cyclophosphamide, carboxy-phosphamide, alcophosphamide, nor-nitrogen mustard and the N-chloroethyl-1,3-oxazolidine-2-one, as methyl and/or trifluoroacetyl derivatives by single ion monitoring gas chromatography/mass spectrometry, mostly in the electron capture chemical ionization mode. The isolation of most metabolites was performed by solid-phase C-18 extraction in weakly acidic medium. The phosphoramide mustard isolated under these conditions decomposes readily to the nor-nitrogen mustard during derivatization. The original nor-nitrogen mustard and the chloroethyl-1,3-oxazolidine-2-one were isolated by liquid extraction with ethyl acetate in alkaline medium. Recoveries of 75-99% were measured using spiked blank plasma samples. Quantitation of metabolites in patient plasma samples was performed using two sets of calibration curves for the concentration ranges of 1-100 ng and 0.1-10 micrograms of metabolite per millilitre of original plasma.

Uptake and retention of estramustine and the presence of estramustine binding protein in malignant brain tumours in humans.

Estraumustine phosphate (EMP), a cytotoxic drug used in the treatment of prostatic carcinoma, has been shown to exert cytotoxic effects on glioma cells in vitro. The drug uptake is assumed to depend on a specific estramustine binding protein (EMBP). One of the main difficulties in achieving cytotoxic effect in malignant brain tumours is believed to be due to the poor penetration of cytotoxic drugs into tumour tissue. In patients with malignant supratentorial brain tumours we have analysed the uptake of EMP metabolites in tumour tissue after oral administration and demonstrated EMBP in the same tissue specimens. Sixteen patients were given 280 mg EMP orally 14 h prior to surgery. Specimens from brain tumour tissue, cystic fluid, and serum were collected during surgery. Using gas chromatography the metabolites of EMP, estramustine (EaM) and estromustine (EoM), were quantified, EMBP was demonstrated by immunohistochemistry. The mean concentrations of EaM and EoM, expressed in ng g-1, were 60.3 and 38.4 in tumour tissue and 3.5 and 56.3 in serum, respectively. An accumulation of EaM in tumour tissue was found with a mean concentration gradient of 16.1 versus serum, while the gradient for EoM was 0.76. EMBP was demonstrated with a high degree of staining in all but one tumour. The high concentrations of EaM and EoM found in malignant brain tumour tissue correspond to potentially cytotoxic levels. The present results as well as the earlier in vitro demonstrated cytotoxic effects on glioma cells strengthen the possibility of a therapeutic effect of EMP in the treatment of malignant brain tumours.

Identification of prodrug, active drug, and metabolites in an ADEPT clinical study.

Antibody-directed enzyme prodrug therapy (ADEPT) involves two phases. The first is an antibody-enzyme conjugate that localizes to tumor. The second phase is a prodrug that is administered when the enzyme-conjugate has cleared from blood and other nontumor tissues. In the pilot-scale clinical trial, the prodrug has been measured--in the plasma of patients, by liquid chromatography (HPLC) and by liquid chromatography-mass spectrometry (LC-MS). Active drug has been detected and metabolites identified. An indirect measurement of enzyme-conjugate in the plasma of patients has also been developed.