Kenneth R. Harrap

The comparative pharmacokinetics of carboplatin and cisplatin in mice and rats

The plasma, urinary and biliary clearances of cisplatin and its non-nephrotoxic analogue, Carboplatin (cis-diammine-1,1-cyclobutane dicarboxylate platinum II, CBDCA, JM8) have been determined in mice and rats following intravenous administration of the compounds. The plasma concentration-time curves were biphasic during the time period studied (0-60 min), with t1/2 alpha of 2-3 min for both platinum complexes and t1/2 beta of 10-15 min for cisplatin and 25-26 min for Carboplatin. The kinetic rate constants, k12 and k21, were similar for both Carboplatin and cisplatin, indicating that there was no appreciable net accumulation of the compounds in the peripheral tissues. Immediately after administration, Carboplatin became reversibly bound to plasma proteins in vivo to the extent of about 20%. Appreciable irreversible binding appeared after the first 60 min and increased steadily, so that by 4 hr only 34% of the compound was present in the plasma as the free drug. In comparison, binding of cisplatin to plasma was exclusively irreversible and, after the first 10 min, free drug disappeared rapidly, such that by 60 min free platinum was not detectable. The plasma clearance of free cisplatin (26.1 ml/min/kg) was significantly greater than that of either Carboplatin (10.3 ml/min/kg) or insulin (10.1 ml/min/kg). The main route of excretion of the two platinum complexes was via the urine, with 80-90% of Carboplatin and 43-48% of cisplatin being excreted within 4 hr. In the rat, the Carboplatin excreted in the urine was predominantly as the unchanged compound. The renal clearance of cisplatin (12.3 ml/min/kg) was significantly greater than that of either Carboplatin (9.3 ml/min/kg) or insulin (9.6 ml/min/kg), suggesting that cisplatin was excreted by an active renal secretory mechanism whilst Carboplatin was eliminated by glomerular filtration alone. Biliary excretion of the two compounds was only 0.4-1.2% of the administered dose in 6 hr, with biliary clearance of cisplatin (0.27 ml/min/kg) being fivefold greater than that of Carboplatin (0.053 ml/min/kg). The results indicate that the major pharmacokinetic differences between Carboplatin and cisplatin relate to their renal handling and their reactivity with macromolecules. These differences may well underline the substantial lack of Carboplatin nephrotoxicity in comparison with cisplatin.

Flameless atomic absorption spectrophotometric determination of platinum in tissues solubilized in hyamine hydroxide

Processing biological samples by solubilization in Hyamine hydroxide (methylbenzethonium hydroxide) as an alternative to wet ashing with concentrated nitric acid for atomic absorption determination of platinum has been explored. The concentrations of platinum in tissues removed from rats 2 h after they had received the antitumor drug Carboplatin (60 mg/kg, iv) were comparable using either of the procedures, indicating the utility of tissue solubilization. Limits of detection were also comparable between the Hyamine hydroxide (0.25 microgram platinum/g) and nitric acid (0.15 microgram/g) procedures. The solubilization method, however, has advantages over wet digestion because of its simplicity and greater safety.

ANTITUMOR, TOXIC AND BIOCHEMICAL PROPERTIES OF CISPLATIN AND EIGHT OTHER PLATINUM COMPLEXES

Publisher Summary The discovery of the bactericidal activities of platinum complexes and their subsequent demonstration of the antitumor properties of this class of compounds stimulated the clinical development and utilization of cisplatin (cis-dichlorodiammine platinum II) in man. Although this material exhibits activity against several human malignancies, its therapeutic efficacy is compromised by the occurrence of severe dose-limiting toxic side effects. Predominant among these are nephrotoxicity, severe nausea and vomiting, myelotoxicity, and ototoxicity. The need for a less toxic antitumor platinum derivative is unequivocal. The growth inhibitory properties of the eight compounds have been compared against several transplantable rodent tumors and against a human epidermoid carcinoma of the bronchus (P246) grown in immune-deprived mice. The potential nephrotoxicities of these complexes have been assessed in the rat by following changes in blood urea levels: The histopathology of kidneys and other normal tissues has also been examined. In view of the similarities that exist between the mechanisms of action of platinum complexes and alkylating agents, the effects of the chosen platinum compounds on nuclear protein phosphorylation in the nuclei of tumor, kidney, and liver tissues.

A phase I and pharmacology study of an oral platinum complex, JM216: dose-dependent pharmacokinetics with single-dose administration

JM216 [bis-acetato-ammine-dichloro-cyclohexylamine-platinum (IV)] is an oral platinum complex with in vivo activity against murine and human tumor models and a lack of nephro- and neurotoxicity in rodents. During a phase I study of a single-dose schedule, JM216 was given in dry-filled hard gelatin capsules by mouth without hydration or diuresis. In all, 37 patients were given a total of 88 courses at doses ranging from 60 to 700 mg/m2. The study was stopped before the MTD was reached because of nonlinear pharmacokinetics. Myelosuppression was manifest by leucopenia or thrombocytopenia and showed marked variability at 420–700 mg/m2. Vomiting was mild and controllable by antiemetics in approximately 50% of courses. The onset of vomiting was delayed to 4 h after during ingestion. There was no nephro-, oto- or neurotoxicity. A partial response was recorded in a patient with recurrent ovarian cancer, and significant falls in plasma tumour markers (CA125) were seen in two further cases. Plasma pharmacokinetics were linear and showed moderate interpatient variability at dose levels of ≤120 mg/m2. At dose levels of ≥200 mg/m2, Cmax and AUC increased less than proportionally to dose. This was associated with greater interpatient pharmacokinetic variability and reduced urinary platinum recovery. A significant sigmoidal relationship existed between ultrafilterable plasma AUC and the percentage of reduction in platelet count (r2=0.78). Nonlinear absorption was a limitation to this single-dose schedule of oral NM216; however, little nonhaematological toxicity was seen at doses associated with myelosuppression and antitumour activity. Clinical studies of divided dose schedules using doses within the range of pharmacokinetic linearity (≤120 mg/m2) are now being investigated.

cis-Diamminedichloroplatinum(II)-induced cell death through apoptosis in sensitive and resistant human ovarian carcinoma cell lines

We have studied the effects of the chemotherapeutic drug cis-diamminedichloroplatinum(II) (cisplatin) on three human ovarian carcinoma cell lines one sensitive to the drug (CHI), one with acquired resistance (CHlcisR) and one with intrinsic resistance (SKOV-3). Previous work has shown that the 50% inhibitory concentrations (IC50 values) after a 2-h exposure to the drug are: CHI, 2.5 (µM; CHlcisR, 7.5 µM; and SKOV-3, 33 µM. Despite the variation in sensitivity, the amount of Pt bound to DNA and the rate of removal of Pt was similar for the three lines. There were significant differences in the rates of formation of DNA cross-links but these were not large enough to account for the high resistance of the SKOV-3 line. We have reported that in the L1210 murine leukaemia cell line there are two mechanisms of cisplatin-induced cell death — one of which involves apoptosis. In this paper, we report on an investigation into whether sensitivity to apoptosis played a role in the resistance of these ovarian lines towards cisplatin. After a 2-h incubation with the drug, cells from the three lines showed evidence of death through apoptosis. The cells detached from the culture dish in a time- and dose-dependent fashion. These cells morphologically were quite distinctive from the attached cells and showed changes in their chromatin structure indicative of apoptosis. Their DNA had not been degraded into oligonucleosomal fragments (200 bp and multiples thereof) but had been cut into larger fragments (30 kilobase pairs, kbp) of a size associated with chromatin domains (chromatin loops). At equitoxic doses of drug, the quantity of cells undergoing apoptosis was similar for the three cell lines. The most prominent effect on cell-cycle kinetics was a slowdown in S-phase transit during which the cells underwent apoptosis. Cells that successfully completed the S phase subsequently suffered a temporary G2 block. We propose that the sensitivity of these cell lines to cisplatin was governed by their ability to handle damage caused by platination of the DNA and that the major mechanism of cisplatin-induced cell death in all three cell lines was the induction of apoptosis.

Phase I trial of ZD1694, a new folate-based thymidylate synthase inhibitor, in patients with solid tumors.

PURPOSE To perform a phase I clinical and pharmacologic study of ZD1694 (Tomudex, Alderley Park, United Kingdom), a new folate-based thymidylate synthase (TS) inhibitor, in patients with advanced malignancy. PATIENTS AND METHODS From February 1991 to January 1993, 61 patients with a range of solid tumor received 161 courses of ZD1694 given as a single 15-minute intravenous infusion every 3 weeks, at escalating doses from 0.1 to 3.5 mg/m2. Pharmacokinetic (PK) analysis was performed with the first two courses of treatment. There were 33 men and 28 women with a median age of 53 years (range, 21 to 73). Fifty-five patients (90%) had previously received chemotherapy. RESULTS Reversible liver toxicity and dose-related gastrointestinal (GI) and bone marrow toxicity occurred at > or = 1.6 mg/m2. Liver function usually returned to normal with repeated treatment, but GI and bone marrow toxicities generally became more severe. No renal toxicity was observed. The maximum-tolerated dose (MTD) was 3.5 mg/m2, at which, in addition to antiproliferative toxicities, four of six patients (67%) developed severe malaise that consisted of anorexia, nausea, and asthenia, with rapidly decreasing performance status that limited re-treatment. Abnormal liver function was also seen in four patients (67%). At 3.0 mg/m2, grades III and IV diarrhea were seen in six of 23 patients (26%) and grade IV myelosuppression in two others. Liver toxicity was self-limiting and not associated with severe malaise. Two patients had a partial response to treatment. PK analysis showed that plasma elimination was triexponential, with pronounced variability in the mean terminal half-life (t1/2gamma) for a given dose ranging from 8.2 to 105 hours. There was a linear relationship between dose and both the area under the concentration-time curve (AUC) and maximum concentration (Cmax), but no clear association between these parameters and response or toxicity. CONCLUSION The dose of ZD1694 recommended for phase II trials is 3.0 mg/m2.

The clinical pharmacokinetics of the novel antifolate N10-propargyl-5,8-dideazafolic acid (CB 3717)

SummaryThe pharmacokinetics of the new antifolate CB 3717 were studied in 20 patients during its phase-I clinical evaluation. The drug was administered at doses of 100–550 mg/m2 in 1-h and 12-h infusions, resulting in peak plasma concentrations of CB 3717 of 40–200 μM. There was a linear relationship between the dose and both CB 3717 AUC and peak plasma levels. Following a 1-h infusion, drug levels in the plasma decayed biphasically (t1/2α=49±9 min, t1/2β=739±209 min). 27%±2% of the dose was excreted in urine in the 24-h period after treatment, suggesting that the major route of elimination was via the bile. Furthermore, the parent compound CB 3717 and its desglutamyl metabolite, CB 3751, were found in a faecal collection although the metabolite was not detected in plasma or urine samples. Plasma protein binding of CB 3717 was extensive (97.6%±0.1%). Significant quantities of CB 3717 penetrated into ascitic fluid but not into cerebrospinal fluid.Residual drug was detected in postmortem kidney tissue from a patient who died of progressive disease 8 days after treatment with 330 mg/m2 CB 3717. Thus, dose-limiting renal toxicity (maximum tolerated dose 600 mg/m2) may be due to drug precipitation in the renal tubules. Elevation of liver enzymes, in particular transaminases, occurred frequently as a toxic manifestation of CB 3717 therapy. In 11 patients studied after their first treatment there was a positive correlation between the rise in serum alanine transaminase and peak drug levels (r=0.69, P=0.02)These pharmacokinetic studies have shown that, by analogy with experimental systems, cytotoxic plasma levels of CB 3717 are archieved in man. In addition, they have been valuable in interpreting toxicities observed during phase-I clinical studies.

Synthesis and Reactions of a New Class of Orally Active Pt(IV) Antitumor Complexes

The advent of cisplatin was a breakthrough in the chemotherapy of certain cancers. Its success, in spite of adverse effects such as nephrotoxicity, nausea and vomiting, ototoxicity and myelosuppression, attests to its efficacy1. Still, the cost of treatment, in terms of patient quality of life, underscores the need for an efficacious drug with milder side effects. Carboplatin is an example of an agent specifically developed to reduce side effects while retaining the antitumor activity of cisplatin2. Its tremendous success, following its introduction in Europe and the US, attests to the importance of addressing patient quality of life. Although, oral chemotherapeutic agents are not presently a significant factor in cancer treatment, a properly designed agent could offer significant advantages in terms of a patients’ comfort and convenience, and anticipates the possibility of outpatient chemotherapy. At Johnson Matthey, in conjunction with the Institute of Cancer Research and Bristol-Myers Squibb, a portion of our platinum antitumor drug discovery program is devoted to the design and development of an orally active platinum antitumor drug. This paper describes the synthesis, reactions, and a few of the biological properties of a new class of antitumor agents that possess many characteristics required of an orally active antitumor agent.

Genomic Imbalances Associated with Acquired Resistance to Platinum Analogues

During the past several years, a panel of human tumor cell lines (predominantly ovarian) with acquired resistance to cisplatin, the orally bioavailable analogue JM216, and the structurally hindered analogue AMD473, has been established and characterized for underlying mechanisms of resistance. We have examined these resistant cell lines for gains and losses of DNA associated with the acquisition of resistance using the molecular cytogenetic technique of comparative genomic hybridization. Our comparison of three analogues has shown the most frequently observed changes to include amplification of 4q (5/7) and 6q (5/7), followed by amplification of 5q (3/7). We have defined four minimal common overrepresented regions, two each on 4q and 6q, which are potential loci of genes associated with platinum analogue resistance. Additional consistent abnormalities appear to be associated with cell lines sharing specific resistance mechanisms. For example, amplification of 12q was observed in the CH1 lines made respectively resistant to JM216 and AMD473 in which increased DNA repair appears to be a major mechanism of resistance for both agents. Hence, these comparative genomic hybridization studies have identified distinct chromosomal aberrations which may correlate with defined mechanisms of resistance and contain hitherto unrecognized genes that may provide targets for future therapeutic intervention.

The effect of the nucleoside transport inhibitor dipyridamole on the incorporation of [3H]thymidine in the rat

Dipyridamole is a non-specific inhibitor of nucleoside transport into mammalian cells. It is currently undergoing clinical evaluation in combination with various antimetabolites in an attempt to enhance the activity of these anticancer drugs by blocking the salvage of extracellular nucleosides, an important determinant of their cytotoxicity. In the present study, the effect of i.v. infusions of dipyridamole on [3H]thymidine incorporation into DNA has been examined in the anaesthetized rat. The tissues studied were bone marrow, gastrointestinal tract epithelium and the ascitic form of the Walker carcinosarcoma. Dipyridamole at 10 mg/kg, given over 3 hr, led to plasma levels of less than 5 microM and did not reduce [3H]thymidine incorporation into any of the tissues studied. At 40 mg/kg dipyridamole (plasma levels 10-15 microM) [3H]thymidine incorporation into the DNA of bone marrow and gastrointestinal tract epithelium was reduced to 20-30% of control values. Increasing the dose to 100 mg/kg did not lead to a further suppression of incorporation. Measurement of [3H]thymidine plasma pharmacokinetics and the intracellular distribution of tritium suggested that the inhibition of [3H]thymidine incorporation was due to reduced cellular uptake. In contrast to the effects on normal tissues, even at a lethal dose (200 mg/kg) dipyridamole did not significantly inhibit [3H]thymidine incorporation into Walker tumour cells. The levels of dipyridamole found in the ascitic fluid, at 100 mg/kg approximately half those in plasma, argue against a pharmacokinetic basis for this difference. Dipyridamole was found to bind extensively (97%) to rat plasma proteins, which may explain the discrepancy between the concentrations of dipyridamole required to inhibit nucleoside incorporation in vitro, in serum-free media, and those needed in vivo. From a comparison of the plasma levels of dipyridamole which cause an inhibition of [3H]thymidine incorporation in the rat with those which can be achieved safely in patients, it is concluded that dipyridamole is unlikely to markedly reduce nucleoside salvage in man.