Antitumor activity of halogen analogs of phosphoramide,isophosphoramide, and triphosphoramide mustards,the cytotoxic metabolites of cyclophosphamide,ifosfamide, and trofosfamide

A series of halogen analogs of phosphoramide mustard, isophosphoramide mustard, and triphosphoramide mustard, the cytotoxic metabolites of the antitumor drugs cyclophosphamide, ifosfamide, and trofosfamide, respectively, was evaluated in vitro against human tumor cell lines and in vivo against experimental tumors to investigate the effect of replacement of chlorine with bromine or fluorine on the antitumor activity of the parent phosphoramide mustards. In the experimental tumors L1210 leukemia, B16 melanoma, mammary adenocarcinoma 16/C, and ovarian sarcoma M5076, the antitumor activity of the analogs was observed to be generally comparable with that of the parent mustards when chlorine was replaced by bromine but uniformly lower when chlorine was replaced by fluorine. Furthermore, the monobromo analog of isophosphoramide mustard displayed equal or somewhat greater activity in comparison with cyclophosphamide when evaluated against subcutaneously implanted L1210 leukemia with intraperitoneal drug treatment and against mammary adenocarcinoma 16/C.This work was financially supported by NIH, NCI grant PO1 CA34200     

Pharmacokinetics and tissue distribution of idarubicin and its active metabolite idarubicinol in the rabbit

 A three-compartment model was fitted to idarubicin data in a NONMEM pooled-data approach. Clearance (CL) of 221.7 ml/min was relatively high, and drug distribution was rapid (CL_D=248.3 ml/min) and extensive [steady-state volume of distribution (V_ss) 24 l]. The area under the concentration-time curve (AUC) of idarubicinol was 8 times that of idarubicin. Concentrations of idarubicin (idarubicinol) measured in the myocardium at 24 h after i.v. administration of idarubicin were 20 (5) times those determined in plasma. Tissue concentrations of idarubicinol were up to 400 times those of idarubicin, indicating that the active metabolite contributes significantly to the overall drug action. Received: 16 June 1996 / Accepted: 4 November 1996     

Pharmacokinetics of the multidrug-resistance-converting drug dexniguldipine and its pyridine metabolite M-1 in the plasma,tumor, and renal tissue of tumor-bearing Wag / Rij rats

The pharmacokinetics of oral dexniguldipine, a new multidrug-resistance-modifying agent under clinical evaluation, and its pyridine metabolite M-1 were determined in plasma, tumor, and renal tissue in Wag/Rij rats bearing a multidrug-resistant CC531 colon adenocarcinoma tumor under the renal capsule. The pharmacokinetics were studied in four experiments. After a single administration of dexniguldipine (30 mg/kg), tumors and kidneys were collected after 5 (experiment 1), 24 (experiment 2), and 48 h (experiment 3). In the fourth experiment, dexniguldipine was given once daily for 3 consecutive days at a dose of 30 mg/kg. In all experiments, plasma samples were collected at regular intervals. The concentrations of dexniguldipine and M-1 could be determined in plasma in most of the rats at up to 32 h after drug administration. The area under the curve (AUC) of dexniguldipine and M-1 varied by a factor of 2–6 in the four experiments. High tumor-tissue concentrations of dexniguldipine were observed. The concentrations were highest in the multiple-dose experiment (2014 ± 1005 ng/g tissue). High degrees of correlation (>0.8) were established between the concen-trations of dexniguldipine measured in plasma and tumor as well as renal tissue. Overall, tumor-tissue concentrations of M-1 comprised one-third of the dexniguldipine concentrations measured. Received: 7 January 1997 / Accepted: 1 June 1997     

Comparative preclinical toxicology and pharmacology of isophosphoramide mustard, the active metabolite of ifosfamide

Background Isophosphoramide mustard (IPM) is the cytotoxic alkylating metabolite of Ifosfamide (IFOS). IPM is being readied for a phase I clinical trial. In the present preclinical study, IPM was evaluated for usage in multidose intravenous (IV) infusion protocols.Methods Mice and dogs received IV IPM daily for 3 days. Single-day dosing—oral and IV—to mice, rats, and monkeys is also reviewed for comparison. Complete toxicology studies were completed in the mice and dogs. For mice, dogs and monkeys, IV pharmacokinetic studies were conducted and compared.Results For mice, the LD₁₀ for the 3-day IV schedule for IPM was calculated to be 119 mg/kg (with 95% confidence limits of 87–134 mg/kg) (combined sexes), and for adult male dogs the maximum tolerated dose (MTD) was 5 mg/kg. Pharmacokinetic studies in mice, dogs and monkeys were compared and projected to human dosing. For dogs that received 10 mg/kg of IPM, T_1/2 was 0.99 h, and clearance was constant (1.01 l/h/kg). IPM was detected from 0 h to 1.5 h after the 5 mg/kg dose and from 0 h to 2 h after the 10 mg/kg dose; none was detected after 2 h. The IV MTD in dogs was 5 mg/kg per day for 3 days. Renal tubular necrosis and bone marrow failure were the causes of death. Transient liver, renal and bone marrow toxicity and gastrointestinal dysfunction were seen at low doses (<5 mg/kg) in dogs. In mice (receiving 100 mg/kg IV) plasma concentrations disappeared in less than 1 h (T_1/2 2 min), with a clearance of 8.44 l/h/kg. For monkeys, the mean T_1/2 was 4.2 h. Median clearance was 1.65 l/h/kg and no IPM was detected 4 h after dosing. No potential IPM metabolites could be detected in any of the studies. In vitro, plasma protein bound 90% of IPM within 5 min of incubation.Conclusions Predictions for human pharmacokinetic parameters and dosing are made from allometric analysis using the above three species. Data predicted an acceptable starting dose of 30 mg/m² with a clearance of 39.5 l/h, and a T_1/2 of 1 h 45 min for a 70-kg patient.     

Pharmacokinetics of a HER2 tyrosine kinase inhibitor CP-724,714 in patients with advanced malignant HER2 positive solid tumors: correlations with clinical characteristics and safety

Purpose CP-724,714 is an orally available, small molecule, potent HER-2 tyrosine kinase inhibitor under development for the treatment of advanced HER2-overexpressing cancers. In this study, the influence of baseline clinical characteristics and pathophysiological variables on the pharmacokinetics (PK) of CP-724,714, and the correlation between PK exposure and safety were examined in patients treated in the First-in-Human trial. PK and safety were also simulated for a Phase 2 trial at the recommended Phase 2 dose (RP2D) to assess if the simulated PK exposures of CP-724,714 covered the preclinically predicted efficacious concentrations, and if the predicted incidence of hepatic toxicities (≥CTC grade 3) was acceptable. Methods Patients (n = 30) with advanced malignant HER2 positive solid tumors were enrolled in this open label dose-escalation study, and treated with daily oral dosing of CP-724,714 in 21-day cycles at the following dose levels: 250 mg QD, 250 mg BID, 400 mg BID, and 250 mg TID. PK parameter values were estimated using noncompartmental techniques. PK exposure parameters were correlated with the baseline pathophysiological variables, clinical characteristics, and safety. The simulations of PK exposures and the incidence of ≥grade 3 liver toxicity at the recommended Phase 2 dose were performed by nonparametric bootstrap (n = 1,000). Results C _max and AUC increased in an approximate dose proportional manner. The terminal t _1/2 was approximately 4.5 h, and was constant across the dose range from 250 to 400 mg. There was some accumulation with BID and TID dosing with a mean AUC accumulation ratio ∼1.2–1.5, consistent with the t _1/2. Inter-patient variability in PK parameters was 31–65%, resulting in a considerable overlap of systemic exposure parameters (C _max and AUC) at higher doses (i.e., 250 mg TID and 400 mg BID), as expected for the narrow dose range. Significant correlations were observed for body size and oral clearance (CL/F) (r = 0.574, P = 0.001) and oral steady-state volume of distribution (V _dss/F) (r = 0.669, P = 0.0001). The most frequently encountered toxicities were elevated ALT and AST, hyperbilirubinemia, rash, asthenia, and nausea/vomiting (N/V). The steady-state AUC0-24 h was significantly correlated with the elevation of total bilirubin (r = 0.670, P = 0.001), ALT (r = 0.548, P = 0.002), and AST (r = 0.461, P = 0.010). The simulation of the Phase 2 trial at 250 mg BID predicted that the 95% confidence interval of the simulated mean concentrations of CP-724,714 were above the preclinically predicted efficacious concentrations throughout the majority of the dosing interval. The probability for ≥33% incidence of grade 3 or greater elevations of liver function test (LFT) was low (1.1%). Conclusions CP-724,714 demonstrates linear single-dose and multiple-dose PK. Both CL/F and V _dss/F correlate with body size. Elevations of ALT, AST, and total bilirubin positively correlate with the steady-state AUC0-24 h. The Phase 2 trial simulation suggests that CP-724,714 will be well tolerated and that PK exposures will exceed the preclinically predicted efficacious level at the recommended Phase 2 dose (250 mg BID), supporting further evaluation of CP-724,714 in the Phase 2 trial.     

Cytotoxicity and pharmacokinetics of cladribine metabolite, 2-chloroadenine in patients with leukemia

The nucleoside analog 2-chlorodeoxyadenosine (Cladribine, CdA) is used in the treatment of patients with several hematological malignancies. After administration of CdA, the major catabolite measured in plasma and urine is 2-chloroadenine (CAde). This study was performed to determine the pharmacokinetics after oral and intravenous (iv) infusion of CdA in patients treated for chronic lymphocytic leukemia and to evaluate the toxicity of CAde to leukemia cells in vitro. CdA and CAde were also determined in plasma from 31 patients and in urine from 16 patients with reversed-phase high-performance liquid chromatographic. The toxicity of CdA and CAde was also determined in leukemic cells from 7 patients by fluorometric microculture cyotoxicity assay. Five times more CAde was quantified after oral treatment compared with an iv infusion of CdA. After iv infusion, the half-life was the same for CdA and CAde, but after oral administration the half-life was doubled for CAde. Excreted amount of CAde in urine constituted about 1.1% after iv infusion and 4.7% after oral CdA treatment. In vitro exposure of leukemia cells to CAde showed that it was eight times less toxic as compared to CdA. We conclude that CAde has a lower cytotoxic effect than CdA but may contribute significantly to the cytotoxicity after oral administration.     

Pharmacokinetics and distribution of liposomal busulfan in the rat: a new formulation for intravenous administration

The plasma pharmacokinetics and tissue distribution of busulfan (Bu) were investigated after intravenous injection of free Bu (D-Bu) and freshly prepared liposomal Bu (L-Bu). Liposomal Bu was prepared using l-α-phosphatidylcholine, 1,2-dioleolyl-sn-glycero-3-phosphate, and cholesterol. The liposomes formed were unilamellar vesicles measuring 220 ± 14 nm in diameter and containing a Bu concentration of 0.31 ± 0.03 mg/ml. The half-life of Bu in the present formulation was determined to be 8.7 ± 2.7 days at 4 °C. The liposomes in the new formulation were stable for 20 days at 4 °C. After the intravenous administration of L-Bu or D-Bu (dissolved in a mixture of DMSO, ethanol, and propylene glycol) to the rats a higher bone marrow exposure to Bu as expressed in AUC marrow/AUC blood was achieved using L-Bu as compared with D-Bu (1.59 and 0.83, respectively). A higher distribution volume was observed for L-Bu as compared with D-Bu (1.39 versus 0.67 l/kg, respectively). The elimination half-lives were significantly longer in both blood and marrow after the administration of L-Bu as compared with D-Bu (2.52 and 3.08 versus 1.53 and 1.75 h, respectively). The new liposomal Bu showed linear pharmacokinetics within the range of 0.5–3.5 mg/kg, which is comparable with that obtained for D-Bu. A slight difference was observed in systemic exposure to L-Bu as compared with D-Bu as expressed in AUC (9.93 and 11.82 μg h ml⁻¹, respectively). The distribution study using ¹⁴C-labeled Bu showed that the radioactivity was significantly higher over 18 h in the bone marrow (3-fold) and spleen (2-fold; P < 0.01) in a comparison of L-Bu with D-Bu. However in the brain, lungs, and heart the distribution of radioactivity after the administration of L-Bu was significantly lower (P < 0.05) than that␣obtained using D-Bu. On the basis of the present study, the new formulation of liposomal Bu seems to be a promising preparation for clinical trails, since it appears to target bone marrow and spleen with no accumulation in the liver or other organs known for Bu toxicity. Received: 19 February 1998 / Accepted: 2 June 1998     

Pharmacokinetics and whole-body distribution of the new chemotherapeutic agent β-D-glucosylisophosphoramide mustard and its effects on the incorporation of [methyl-3H]-thymidine in various tissues of the rat

 β-D-Glucosylisophosphoramide mustard (β-DGlc-IPM) is a new, potential chemotherapeutic agent currently under investigation. Its pharmacokinetics in plasma and elimination of the parent drug and its metabolites via urine, bile, and exhaled air were studied in female Sprague-Dawley rats after bolus injection of 315 mg/kg. Typically, the drug’s disposition from plasma follows a linear two-compartment model with half-lives (t _1/2) of 1.8 (t _1/2α) and 32 min (t _1/2β). The rate of clearance is 0.0046 (range 0.0030–0.0071) l min^-1 kg^-1, and the steady-state volume of distribution (V _ss) is 0.18 (0.08–0.042) l/kg (mean±interindividual standard deviation). In human plasma, 28.1±2.6% (mean±SD) of the drug (concentration range 0.5–5 mg/ml) is bound to plasma proteins (predominantly to albumin). Biliary excretion of the parent drug accounts for 2.9±1.7% of the dose; its elimination in the form of ¹⁴CO₂ via exhaled air is less than 1%. Within 24 h, 63.5±4.9% of the ¹⁴C-labeled drug is excreted unchanged in the urine, whereas 17.5±5.1% is excreted in the urine as metabolites. In addition, β-D-Glc-[¹⁴C]-IPM was given as a bolus injection to female Sprague-Dawley rats at dose levels of 315 and 56.2 mg/kg. The distribution of radioactivity into tissue was examined qualitatively by whole-body autoradiography (WBA). Parallel experiments were carried out using the high dose of the L-derivative. After dosing with the D-compound, the highest levels of radioactivity were found in the liver, kidneys, thymus, thyroid gland, and central nervous system, including the brain. A similar distribution pattern was observed for the L-compound, except in the brain, which contained negligible levels of radioactivity. The distribution of the D-compound (high dose) was also investigated in male Copenhagen rats bearing a Dunning prostate tumor. The results were similar to those obtained in healthy Sprague-Dawley rats. Additionally, radioactivity was found in the tumor at 1 h after dosing with the drug and remained there even after 24 h. The effects of β-D-Glc-IPM on the incorporation of [methyl-³H]-thymidine into the DNA of the liver, kidneys, thymus, spleen, esophagus, and bone marrow of the rat were examined following tissue excision and liquid scintillation counting at 2, 8, and 24 h after administration of the drug. β-D-Glc-IPM showed no effect on the incorporation of [methyl-³H]-thymidine in the liver and an insignificant reduction in kidney DNA (maximal reduction: −27.3%). However, after 8 h there was a marked reduction in the incorporation rate in the thymus (−83.7%), spleen (−74.6%), and esophagus (−87.2%), with a tendency toward recovery within 24 h. In bone marrow cells a reduction of −75.5% (8 h) and −73.3% (24 h) was observed. Received: 15 March 1995/Accepted: 6 October 1995     

Inhibitory Effect of Ellagic Acid on N-2-Fluorenylacetamide-induced Liver Carcinogenesis in Male ACI/N Rats

The effect of ellagic acid (EA) on the hepatocarcinogenesis induced by N-2-fluorenylacetamide (FAA) was investigated in male ACI/N rats. Rats were fed diet containing 200 ppm FAA and 400 ppm EA for 16 weeks, and diet containing 400 ppm EA alone was fed to the animals for one week before FAA exposure and one week after the carcinogen treatment. Animals were killed at intervals up to 20 weeks after cessation of the carcinogen. Liver altered foci and neoplasms were quantified using γ-glutamyl transpeptidase reaction as well as conventional staining for identification. Exposure to FAA alone induced a substantial number of altered foci and at the end of experiment (week 36), the incidence of hepatocellular neoplasms was 100%. In the group receiving EA together with FAA, the number of altered foci was decreased at all time points and at termination, the final incidence of hepatocellular neoplasms (30%) was also reduced. Thus, EA inhibited the hepatocarcinogenesis induced by FAA when it was administered concurrently with the carcinogen.     

Mechanistic aspects of the cytotoxic activity of glufosfamide, a new tumour therapeutic agent

Beta-D-glucosyl-ifosfamide mustard (D 19575, glc-IPM, INN = glufosfamide) is a new agent for cancer chemotherapy. Its mode of action, which is only partly understood, was investigated at the DNA level. In the breast carcinoma cell line MCF7 glufosfamide inhibited both the synthesis of DNA and protein in a dose-dependent manner, as shown by the decreased incorporation of [3H-methyl]-thymidine into DNA and [14C]-methionine into protein of these cells. Treatment of MCF7 cells with 50 microM glufosfamide was sufficient to trigger poly(ADP-ribose) polymerase (PARP) activation, as revealed by immunofluorescence analysis. Both CHO-9 cells, which are O6-methylguanine-DNA methyltransferase (MGMT)-deficient, and an isogenic derivative, which has a high level of MGMT, showed the same cytotoxic response to beta-D-glc-IPM, indicating that the O6 position of guanine is not the critical target for cytotoxicity. By contrast, a sharp decrease in survival of cross-link repair deficient CL-V5 B cells was observed already at concentrations of 0.1 mM beta-D-glc-IPM, whereas the wild-type V79 cells showed a 90% reduction in survival only after treatment with 0.5 mM of this compound. The therapeutically inactive beta-L-enantiomer of glufosfamide also showed genotoxic effects in the same assays but at much higher doses. This was probably due to small amounts of ifosfamide mustard formed under the conditions of incubation. The results indicate that the DNA crosslinks are the most critical cytotoxic lesions induced by beta-D-glc-IPM.     

Pharmacokinetics of oral fenretinide in neuroblastoma patients: indications for optimal dose and dosing schedule also with respect to the active metabolite 4-oxo-fenretinide

PURPOSE: Pharmacokinetic data on fenretinide (4-HPR) are scant, thus limiting the rational use of the drug. We investigated the pharmacokinetics of 4-HPR and its active metabolite 4-oxo-fenretinide (4-oxo-4-HPR). EXPERIMENTAL DESIGN: Pharmacokinetics were assessed in 18 children (3 for each dose) with neuroblastoma who received oral 4-HPR once daily for 28 days at the doses of 100, 300, 400, 600, 1,700 and 4,000 mg/m(2)/day. 4-HPR and 4-oxo-4-HPR were determined by HPLC in plasma collected up to 48 h after the first and 28th administration. RESULTS: After single administration, 4-HPR mean C (max) ranged from 0.9 to 6.6 muM and these concentrations roughly doubled at steady state (range 1.6-14.5 muM). 4-HPR mean t (1/2) was 22 h. 4-HPR pharmacokinetics were linear in the dose range 100-1,700 mg/m(2); less than dose-proportional increase in exposure was found at 4,000 mg/m(2). At steady state, pharmacologically relevant plasma concentrations (range 0.7-10 muM and 0.4-5 muM for 4-HPR and 4-oxo-4-HPR, respectively) were maintained during the 24 h dosing interval in the dose range 300-4,000 mg/m(2). CONCLUSIONS: 4-HPR pharmacokinetics supports once-daily dosing. Steady state concentrations of 4-HPR and 4-oxo-4-HPR in children with neuroblastoma are in line with those found to have in vitro growth inhibitory effects in neuroblastoma cells.     

Pharmacokinetics and tissue distribution of Kendine 91, a novel histone deacetylase inhibitor, in mice

Purpose  The present investigation was undertaken to characterize the pharmacokinetics and oral bioavailability of Kendine 91 in mice and to compare it with other HDAC (histone deacetylases) inhibitors. Methods  After administration of a single intravenous dose (10 mg/kg) or a single oral dose (50 mg/kg) blood and tissues samples were collected and analysed by HPLC/MS/MS. Results  Elimination half-life was higher than that of SAHA (5.87 vs. 0.38 h after intravenous (IV) administration and 10.29 versus 0.75 h after oral administration). Absolute oral bioavailability was found to be 18%. Total body clearance (7.72 l/h/kg) was greater than the hepatic blood flow of 5.4 l/h/kg in mice and larger than glomerular filtration rate in mice (0.84 l/h/kg). Tissue levels and distribution volume indicate a high capacity of Kendine 91 to distribute into tissues. Conclusions  This preliminary pharmacokinetic evaluation prompts us to believe that it is worth pursuing further development of Kendine 91 as an anticancer drug.     

Evidence for a role of chloroethylaziridine in the cytotoxicity of cyclophosphamide

A number of investigators have observed that the use of 4-hydroperoxycyclophosphamide (4-HC) in multiwell plate cytotoxicity assays can be associated with toxicity to cells in wells that contain no drug. Previous reports have implicated diffusion of 4-HC decomposition products, and acrolein in particular, as the active species. Purpose: The purpose of this study was to elucidate the species responsible for the airborne cytotoxicity of 4-HC, and to devise ways to minimize such effects in chemosensitivity assays. Methods: To this end, analogues of 4-HC were synthesized to identify the contributions of individual cyclophosphamide metabolites to cytotoxicity. The analogues were then tested for activity against three human breast tumor cell lines (including a line resistant to 4-HC), and one non-small-cell lung carcinoma line. Cytotoxicity was evaluated by assays that quantitate cellular metabolism and nucleic acid content. Results: Didechloro-4-hydroperoxycyclophosphamide, a compound that generates acrolein and a nontoxic analogue of phosphoramide mustard, gave no cross-well toxicity. In contrast, a significant neighboring well effect was observed with phenylketophosphamide, a compound that generates phosphoramide mustard but not acrolein. Addition of authentic chloroethylaziridine reproduced the airborne toxicity patterns generated by 4-HC and phenylketophosphamide. Increasing the buffering capacity of the growth medium and sealing the microtiter plates prevented airborne cytotoxicity. Conclusions: Since it is unlikely that phosphoramide mustard is volatile, these findings implicate chloroethylaziridine rather than acrolein as the volatile metabolite of 4-HC that is responsible for airborne cytotoxicity. The fact that chloroethylaziridine is generated in amounts sufficient to volatilize, diffuse across wells and cause cytotoxicity indicates that it is an important component in the overall cytotoxicity of 4-HC in vitro. Furthermore, these findings suggest that chloroethylaziridine may also contribute to the toxicity of cyclophosphamide in vivo. Received: 9 July 1999 / Accepted: 19 November 1999     

Pharmacokinetics of the hypoxic cell cytotoxic agent tirapazamine and its major bioreductive metabolites in mice and humans: retrospective analysis of a pharmacokinetically guided dose-escalation strategy in a phase I trial

 Tirapazamine (3-amino-1,2,4-benzotriazine-1,4-di-N-oxide; SR 259075) is a selective hypoxic cell cytotoxic agent that is bioreductively activated in tumours to a reactive-drug free radical. Preclinically the agent has been shown to possess additive and synergistic anti-tumour activity in combination with radiotherapy and chemotherapy regimens. In the present study the pharmacokinetics and metabolism of tirapazamine were investigated in mice and patients as part of pre-clinical and phase I investigations. The objectives of this work were twofold; firstly, to evaluate retrospectively the utility of a pharmacokinetically guided dose-escalation (PGDE) strategy for tirapazamine, and secondly, to investigate if pharmacologically relevant plasma concentrations could be achieved at tolerable doses. Pharmacokinetic studies for PGDE were conducted in mice at four dose levels ranging from one-tenth of the LD₁₀ to the LD₅₀. The AUC at the LD₁₀ (2932 μg ml^-1min) was used to determine a target AUC value of 1173 μg ml^-1min (equivalent to 40% of the mouse LD₁₀ AUC) for clinical studies. A phase I study to investigate the tolerance of a single i.v. infusion of tirapazamine (once every 3 weeks) was initiated with close pharmacokinetic monitoring. The starting dose (36 mg/m²) was based on toxicity data obtained in the mouse, rat and dog. Doses were escalated by increases in the volume and duration of infusion. A retrospective analysis of the pharmacokinetic and toxicity data was then made to determine the utility of a PGDE approach. The drug exhibited a steep dose-lethality relationship in mice (LD₁₀ 294 mg/m², LD₅₀ 303 mg/m²). The major gross toxicities were body-weight loss (15–20%), pilo-erection and hypoactivity at all dose levels. Sporadic ptosis and conjunctivitis were observed at doses of >300 mg/m². The plasma elimination of tirapazamine fitted a monoexponential open model, with rapid elimination from the plasma (t _1/2=36±0.65 min) occuring at the LD₁₀ dose of 294 mg/m². A 10.3-fold increase in dose resulted in a 25.0-fold increase in AUC. Clinically, doses were escalated over the range of 36–450 mg/m². Ototoxicity (tinnitus and reversible hearing loss) was dose-limiting at 450 mg/m² and the MTD was 390 mg/m² for this schedule. Pharmacokinetic analyses in patients revealed that the elimination of tirapazamine in patients was generally bi-phasic, with low inter-patient variability being found in clearance. A 12.5-fold increase in dose resulted in a 19.0-fold increase in AUC. There was good quantitative agreement in metabolite formation between mice and humans with respect to the two- and four-electron bioreductive metabolites. AUC values recorded for tirapazamine at the MTD of 390 mg/m² (range 1035–1611 μg ml^-1min) were similar to the target AUC in mice. Importantly, these levels are consistent with the levels required for radiation-dose enhancement and effective combination with cisplatin in mice. Given (a) the similarities in plasma pharmacokinetics and metabolism observed at the target AUC/MTD in mice, rats, dogs and humans, (b) the similar degree of plasma protein binding seen between species and (c) the relatively low inter-patient variability noted in drug clearance, a successful PGDE approach should have been feasible. The results also indicate that potentially therapeutic levels of tirapazamine are achievable in patients at tolerable doses. Received: 27 May 1996 / Accepted: 30 September 1996     

Immunohistochemical study of keratin in proliferative bladder epithelium induced by freezing, cyclophosphamide or N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide in the rat

The distribution of intracellular keratin proteins was examined in a variety of urinary bladder lesions of the rat using the immunoperoxidase staining technique. In ethanol-fixed sections, the normal epithelium was strongly positive for keratin staining. Focal regenerative hyperplasia of the bladder epithelium induced by freezing exhibited relatively weak staining. However, diffuse regenerative hyperplasia induced by a single intraperitoneal injection of cyclophosphamide (CP) showed an intensely positive reaction throughout the epithelium. Of the sections fixed with Bouin's solution, the staining reaction was drastically reduced in the normal bladder and the staining was totally negative in the regenerative hyperplasia caused by freezing. Simple hyperplasia induced by a 4-week feeding of N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) as 0.2% of the diet exhibited strong reactivity, and nodular and papillarly hyperplasia induced by a 10-week feeding of FANFT was also positive for keratin throughout the lesions. In contrast to the preneoplastic lesions, FANFT-induced transitional cell carcinoma showed differential staining within the tumors. These results suggest that different keratin expression is involved in the proliferative bladder lesions induced mechanically by freezing and chemically by CP or by the carcinogen FANFT.     

In vivo microdialysis to characterize drug transport in brain tumors: analysis of methotrexate uptake in rat glioma-2 (RG-2)-Bearing rats

 Brain microdialysis was applied to sample free methotrexate (MTX) concentrations in brain extracellular fluid of normal and RG-2 glioma-bearing rats. All animals received 50 mg/kg of MTX intraarterially following which serial blood and interstitial fluid samples were collected for 3 h and measured for MTX by an HPLC assay. Retrodialysis was used to estimate the in vivo recovery of MTX from brain. A linear two-compartment model was fitted to the plasma MTX concentration-time data in both the normal and RG-2 groups. The mean total body clearance and volume of distribution at steady state of MTX varied from 0.90±0.3 to 0.24±0.02 l h^-1 kg^-1 (P<0.05) and from 0.58±0.24 to 0.21±0.16 l kg^-1 (P<0.05) in control and tumor rats, respectively. The significant reductions in clearance and volume of distribution at steady-state were attributed in part to a cachectic state in the RG-2 animals in which total body water was reduced. The mean MTX area under the interstitial fluid concentration-time curve (AUC) was 171.6±69.14 μg min ml^-1(control) and 583.5±296.7 μg min ml^-1 (brain tumor-bearing rats). The significantly higher AUC values obtained with RG-2 rats compared with control rats may have resulted from high plasma MTX concentrations and a more permeable blood–tumor barrier (BTB). A hybrid physiologically based pharmacokinetic model was used to characterize the mechanisms responsible for the high MTX brain tumor concentrations. In conclusion, a microdialysis technique was successfully utilized to examine the extracellular uptake of MTX in brain. This technique can be a powerful tool to evaluate intracerebral drug kinetics and the delivery of drugs to brain tumors. Received: 11 July 1995/Accepted: 1 February 1996     

A phase I study of SAR405838, a novel human double minute 2 (HDM2) antagonist,in patients with solid tumours

PurposeIn tumours with wild-type TP53, the tumour-suppressive function of p53 is frequently inhibited by HDM2. This phase I, dose-escalating study investigated the maximum tolerated dose (MTD), safety, pharmacokinetics and pharmacodynamics of SAR405838, an HDM2 inhibitor, in patients with advanced solid tumours (NCT01636479).MethodsIn dose escalation, patients with any locally advanced/metastatic solid tumour with TP53 mutation prevalence below 40%, or documented as TP53 wild-type, were eligible. In the MTD expansion cohort, only patients with de-differentiated liposarcoma were included. Primary end-points were MTD and efficacy in the MTD expansion cohort. Secondary end-points included safety, pharmacokinetics and pharmacodynamics biomarkers.ResultsSeventy-four patients were treated with SAR405838 (50–800 mg once daily [QD], 800–1800 mg weekly and 1800 mg twice weekly). Two patients treated with SAR405838 400 mg QD had thrombocytopaenia as a dose-limiting toxicity (DLT). The MTD for the QD schedule of SAR405838 was 300 mg QD. No DLTs were observed with the weekly schedule; one patient had a DLT of nausea with the 1800 mg twice-weekly dose. Treatment with SAR405838 was associated with increased plasma MIC-1, reflecting p53 pathway activation. In the de-differentiated liposarcoma MTD cohort, 89% of the patients had HDM2 amplification at baseline and no TP53 mutations were observed; best response was stable disease in 56% and progression-free rate at 3 months was 32%.ConclusionSAR405838 had an acceptable safety profile with limited activity in patients with advanced solid tumours. The MTD of SAR405838 was 300 mg QD; MTD was not reached with the weekly schedule.     

Pharmacokinetics of amifostine and its metabolites in the plasma and ascites of a cancer patient

 The pharmacokinetics of amifostine, a protector against chemotherapy and radiation-induced toxicities, was investigated in the plasma and ascites of a cancer patient. A high-performance liquid chromatography (HPLC) procedure with electrochemical detection was used to measure amifostine, its active metabolite, WR 1065, and the disulfides (symmetrical plus mixed disulfides). Both amifostine and WR 1065 were rapidly cleared from the plasma (95% and 50% of the peak concentration within 1 h, respectively). The disulfides, which were rapidly formed from WR 1065, were cleared much more slowly (final half-life 13.6 h). Multiple dosing resulted in a tendency toward increasing peak levels of WR 1065 and decreasing peak levels of the disulfides. Only 1% of the delivered dose appeared in the ascites. Therefore, it is not plausible that the presence of ascites or other third spaces would have an impact on the pharmacokinetics of amifostine. Received: 21 January 1996 / Accepted: 24 June 1996     

Effect of filgrastim on the pharmacokinetics of MX2 hydrochloride in patients with advanced malignant disease

Purpose: To investigate the effect of granulocyte colony-stimulating factor (G-CSF) on the pharmacokinetics and pharmacodynamics of the new morpholino anthracycline drug MX2. Methods: A total of 25 patients with advanced malignant disease participated in a dose-escalation study in the first cycle of treatment given i.v. at doses of 50–80 mg/m² (74–152 mg) with concomitant filgrastim (G-CSF, 5 μg/kg) given daily beginning at 24 h after the dose of MX2. Results: The mean fast distribution half-life (1.5 ± 1.0 min) and the mean plasma clearance (2.18 ± 0.95 l/min) were significantly lower than the respective mean values found in a previous study in which 27 patients had received MX2 (16.8–107.5 mg) alone (3.3 ± 2.2 min and 2.98 ± 1.68 l/min, respectively; P < 0.05). There was no correlation between plasma clearance and the delivered dose for the combined MX2-alone and MX2-filgrastim groups, indicating that the lower clearance observed in the G-CSF group was probably not due to the higher dose. Elimination half-lives of the metabolites M1 and M4 were significantly greater in the filgrastim group (19.8 ± 14.7 and 11.8 ± 5.0 h for M1 and 14.8 ± 4.1 and 12.3 ± 6.3 h for M2, respectively). Unlike the MX2-alone group, there was no relationship in the MX2- filgrastim group between the relative nadir neutrophil count and the dose or between the duration of grade IV neutropenia and the dose of MX2. Conclusions: This study shows that filgrastim decreased the plasma clearance of MX2 by approximately 25%, possibly by inhibition of metabolism. Received: 1 June 1997 / Accepted: 17 September 1997