In vitro evaluation of platinum,titanium and ruthenium metal complexes in cisplatin-sensitive and-resistant rat ovarian tumors

Summary The antitumor activity of eight new metal complexes (three platinum, one titanium, four ruthenium derivatives) was investigated in a cisplatin (DDP)-sensitive (O-342) and a DDP-resistant (O-342/DDP) ovarian tumor line using the bilayer soft-agar assay. A continuous exposure set up at logarithmically spaced concentrations was used to test the drugs; to uncover possible pharmacokinetic features, a short-term exposure was additionally included for selected compounds. DDP served as the reference drug. The following compounds were investigated: 18-crown-6-tetracarboxybis-diammineplatinum(II) (CTDP),cis-aminotrismethylenephosphonato-diammineplatinum(II) (ADP),cis-diamminecyclohexano-aminotrismethylenephosphonato-platinum(II) (DAP), diethoxybis(1-phenylbutane-1,3-dionato)titanium(IV) (DBT, budotitane),trans-imidazolium-bisimidazoletetrachlororuthenate(III) (ICR),trans-indazolium-tetrachlorobisindazoleruthenate(III) (IndCR),cis-triazolium-tetrachlorobistriazoleruthenate(III) (TCR) andtrans-pyrazolium-tetrachlorobispyrazoleruthenate(III) (PCR). Of the new metal complexes, CTDP was the most active compound in O-342, resulting in a percentage of control plating efficiency (±SE) of 1±1, 12±8 and 40±21 following continuous exposure to 10, 1 and 0.1 m, respectively, and was thus comparable to DDP at equimolar concentrations. In the resistant line, 10 m CTDP reduced colony growth to 18%±8%, whereas an equimolar concentration of DDP effected a reduction to 26%±9%. During short-term exposure, CTDP was inferior to DDP, which may be ascribed to the stability of the bis-dicarboxylate platinum ring system. The titanium compound DBT, in contrast, showed promising effects at its highest concentration (100 m) during short-term exposure in both lines; at this concentration the activity in O-342/DDP was higher than that in O-342 (7%±7% vs 34%±17% of control plating efficiency at 100 m). All ruthenium complexes showed higher activity in the resistant line O-342/DDP than in the sensitive counterpart. ICR was the most active compound. Following continuous exposure of O-342/DDP cells to 10 m ICR, colony growth was reduced to 18%±4% that of controls. Further studies should concentrate on CTDP and ICR for the following reasons: the activity of CTDP was equal to that of DDP at equimolar concentrations during continuous exposure; considering that the in vivo toxicity of DDP was 3-fold that of CTDP, an increase in the therapeutic index of CTDP would be expected. ICR showed the best effect of all ruthenium complexes; it was superior to DDP in the resistant line.     

Enhancement of the cytotoxicity of cisplatin by the cholecystokinin antagonist MK-329 in a human pancreatic cancer cell line

Cholecystokinin (CCK) is an important trophic hormone for the pancreas, and CCK receptors are present on pancreatic carcinoma cells. We sought to determine whether either CCK itself or an antogonist of CCK could modulate the sensitivity of the human pancreatic cell line MIA-PaCa2 to cisplatin (DDP). The IC₅₀ for a 1-h exposure to DDP was 35.3±3.2(SD) M. Exposure to CCK8 octapeptide at physiologic and supra-physiologic concentrations did not alter the sensitivity of MIA-PaCa2 cells to DDP. The CCK receptor antagonist MK-329 was directly cytotoxic to the MIA-PaCa2 cells on a constant exposure schedule with an IC₅₀ of 9.5±1.4 (SD) M. MK-329 enhanced the sensitivity of MIA-PaCa2 cells to DDP by a factor of 3.5, and the interaction between DDP and MK-329 was shown to be synergistic by median-effect analysis. At a level of 50% cell kill, the combination index was 0.58±0.10. The ability of MK-329 to sensitize cells to DDP was schedule-dependent and required prolonged exposure to the antagonist following a 1-h exposure to DDP. MK-329 had no effect on the uptake of a radiolabeled analog of cisplatin ([³H]-dichloro(ethylenediamine)platinum) and did not affect intracellular glutathione content. MK-329 had no effect on the cell-cycle-phase distribution of MIA-PaCa2 cells and did not alter the DDP-induced cell-cycle perturbations. The cytotoxic effect of MK-329 and its ability to interact synergistically with DDP could not be reversed by CCK. We conclude that MK-329 is directly cytotoxic to pancreatic carcinoma cells and can enhance their sensitivity to DDP by a mechanism not related to its ability to antagonize the CCK receptor.Abbreviations DDP cisplatin - DEP cis-dichloro(ethylenediamine)-platinum(II) - CCK cholecystokinin - IC₅₀ concentration producing 50% cell kill in a clonogenic assay - CI combination index - CI₅₀ combination index at 50% cell kill - GSH glutathione - MBB monobromobimane - PBS phosphate-buffered saline This work was supported by the Foundation for Surgical Education, UCSD Department of Surgery, and grant CA23100 from the National Institutes of Health. This work was conducted in part by the Clayton Foundation for Research — California Division. Dr. S.B. Howell is a Clayton Foundation investigator     

Long-term pharmacokinetic behavior of platinum after cisplatin administration

Purpose: The platinum concentration in plasma was studied in 19 patients treated by 3 or 4 successive courses of chemotherapy including cisplatin for head and neck cancers.Methods: Cisplatin was given i.v. daily at 25 mg/m² by 1-h infusions for 4 days every 3 weeks. Total and ultrafiltrable platinum were measured in plasma using an inductively coupled plasma mass spectrometry (ICPMS) technique.Results: A progressive accumulation of total platinum in plasma was observed with consecutive infusions. The mean (±SD) total plasma platinum level detected at the end of cisplatin infusion was 1134±234, 1407±268, and 1618±282 g/l at the end of the first, second, and third courses, respectively. The minimal platinum concentration measured before the second and third courses also increased to 221±59 and 309±76 g/l, respectively. The steady state was not reached before the third course. However, differences in the evolution of platinum plasma levels were found among the 19 patients. In 14 patients the pharmacokinetics of platinum was characterized by low initial levels, a progressive accumulation, and a long terminal half-life with a very late steady state. In 5 patients, the pharmacokinetic behavior of platinum was different: platinum levels were directly high, without progressive accumulation, the steady state being reached as early as the first course. Significant levels of ultrafiltrable platinum were found throughout the treatment, even during the intervals between courses with this very sensitive analytical method. A close equilibrium between ultrafiltrable and total platinum (ratio, 6%) persisted for as long as 3 weeks after cisplatin administration.Discussion: These results underline the importance of individual differences in platinum metabolism. The relationship between total and ultrafiltrable platinum are discussed.     

Blood clearance of three radioactively labelled platinum complexes: cis-dichlorodiammine platinum II,cis, trans-dichlorodihydroxy-bis-(isopropylamine) platinum IV,and cis-dichloro-bis-cyclopropylamine platinum II,in patients with malignant disease

Summary The blood clearances of three platinum compounds, cis-dichlorodiammine platinum II (DDP), cis, trans-dichlorodihydroxy-bis-(isopropylamine) platinum IV (CHIP), and cis-dichloro-bis-cyclopropylamine platinum II (CP), were determined in nine patients with malignant disease. The complexes were prepared using radioactive platinum (¹⁹¹Pt and ¹⁹³Pt). A 10-Ci dose of each complex, containing the equivalent of 1–2 mg elemental platinum, was injected IV into groups of three patients. Serial blood and urine samples were collected over 72 h.No obvious difference was found between the three complexes for blood clearance, median t_1/2 being 16.8 (range 11.2–23.5) min and median t_1/2 89 (range 63.7–127) h. The urinary excretion was greatest for CHIP, 60% of injected dose as against 42.6% for CP and 38.8% for DDP.Differences in renal excretion of DDP analogues could indicate potentially less nephrotoxic agents. The use of radioactive Pt will allow in vivo dynamic imaging of the distribution of platinum compounds in areas of interest.     

Tumor-tissue and plasma concentrations of platinum during chemotherapy of non-small-cell lung cancer patients

Summary Tumor-tissue and plasma concentrations of platinum were studied prospectively in two groups of eight patients who were suffering from advanced non-small-cell lung cancer. Treatments including two different schedules of cisplatin administration (25 vs 100 mg/m² on day 1) were compared. At 30 min after the beginning of the cisplatin infusion, blood samples and bronchoscopically obtained biopsy specimens were taken for determinations of platinum concentrations by means of flameless atomic absorption spectrophotometry. The procedure did not induce any complication. Total plasma platinum concentrations at 30 min were significantly lower (P<0.01) in patients receiving 25 mg/m² (0.49±0.23 g Pt/ml) than in those receiving 100 mg/m² (1.44±0.62 g Pt/ml), whereas no significant difference was observed in tumor-tissue platinum concentrations (22.49±53.89 ng Pt/mg in patients receiving 25 mg/m² vs 51.13±65.52 ng Pt/mg in those receiving 100 mg/m²). There was a weak correlation between simultaneous plasma and tumor-tissue platinum concentrations at 30 min. Tumor-tissue platinum concentrations seem to be poorly influenced by the cisplatin dose. This finding suggests a great interindividual variability of platinum tumor-diffusion properties in non-small-cell lung cancer.Supported in part by a grant from the Ligue Nationale Française contre le Cancer     

Heat enhances the cytotoxicity ofcis-diamminedichloroplatinum(II) and its analoguescis-1,1-cyclobutane-dicarboxylato(2R)-2-methyl-1,4-butanediammineplatinum(II) andcis-diammine(glycolato)platinum in vitro

cis-1,1-Cyclobutanedicarboxylato(2R)-2-methyl-1,4-butanediammineplatinum(II) (NK121) andcis-diammine(glycolato)platinum (254-S), analogues ofcis-diamminedichloroplatinum (II) (CDDP) with reduced nephrotoxicity, are under clinical phase trial in Japan. Since CDDP has been shown to be more cytotoxic under conditions of an elevated temperature, we tested the cytotoxicity and cellular uptake of these analogues at 37° and 43°C using EMT6/KU cells in vitro. The cytotoxicity of CDDP was enhanced at 43°C, and that of NK121 and 254-S was also enhanced, in a dose- and time-dependent manner. The 90% cytotoxic concentration (IC₉₀) of each drug was reduced 2.9-fold for CDDP, 2.5-fold for NK121, and 2.2-fold for 254-S. Cytotoxicity was maximal when the two modalities were used simultaneously for all three drugs. The intracellular platinum concentration was assayed using flameless atomic absorption spectrophotometry. When exposed to IC₉₀ drug concentration at 43°C for 2 h simultaneously, the intracellular platinum concentration increased to 0.095±0.007 g/10⁷ cells (a 1.9-fold increase) for CDDP, to 0.198±0.012 g/10⁷ cells (a 1.3-fold increase) for NK121, and to 0.090±0.014 g/10⁷ cells (a 1.3-fold increase) for 254-S; respectively, as compared with the level measured after drug exposure at 37°C (P<0.05 for all drugs). The elevation in platinum concentration may be one of mechanism related to a synegistic effect of the two treatment modalities. The concomitant use of CDDP analogues and heat shows potential for possible clinical application.     

Cytotoxicity and cellular accumulation of a new cis-diammineplatinum (II) complex containing procaine in murine L1210 cells sensitive and resistant to cis-diamminedichloroplatinum (II)

 The emergence of drug resistance during tumor chemotherapy is one of the main problems associated with cancer treatment, particularly with cisplatin (cis-DDP). In the hope of overcoming this problem, various cis-DDP-derived compounds have been synthesized, and their pharmacological activity was compared with that of cis-DDP. In this paper we report on studies on the cytotoxic activity induced by cis-diamminechloro-[2-(diethylamino)ethyl-4-aminobenzoate, N ⁴]-chlorideplatinum(II) monohydrochloride monohydrate (DPR), a new complex of platinum containing procaine. All experiments were carried out on murine leukemic cells, which were either sensitive (L1210) or resistant (L1210/DDP) to cis-DDP. A tetrazolium dye (MTT) assay conducted 5 days after a 2-h exposure of cells to both drugs was utilized to determine the resistance factor (RF) of L1210/DDP cells as compared with the sensitive wild-type cells. Drug accumulation and efflux, together with the amount of platinum bound to DNA, were also investigated. The activity of DPR on sensitive cells was not significantly different from that of cis-DDP. Conversely, DPR was 4.3 times more effective than cis-DDP on resistant cells. A decreased drug accumulation is one of the mechanisms of resistance to cis-DDP of L1210/DDP cells. However, DPR accumulation was not significantly different in sensitive and resistant L1210 cells. Under culture conditions that yielded similar intracellular platinum concentrations, treatment with DPR produced significantly greater DNA platination than did treatment with cis-DDP in both cell lines. No difference in efflux was observed between L1210 and L1210/DDP cells exposed to either cis-DDP or DPR. Our results show that in parental cells, DPR is as potent as cis-DDP on a molar basis, and it is also minimally cross-resistant with cis-DDP in L1210/DDP cells. A direct implication of our results is that DPR could be useful in those human tumors showing a mechanism of resistance similar to that of L1210/ DDP cells. Received: 11 March 1994/Accepted: 15 July 1994     

Lack of effect of cisplatin on i. v. L-PAM plasma pharmacokinetics in ovarian cancer patients

Summary Melphalan (L-PAM) pharmacokinetics were investigated in nine ovarian cancer patients before and after cisplatin (DDP) treatment. When L-PAM was given 24 h before DDP, the elimination half-life (t_1/2), plasma clearance (Clp), and volume of distribution (Vd) of L-PAM were, respectively: 46.4±6.7 min, 20.5±3.7 l/m², and 306.8±34.4 ml/min per square meter. When L-PAM was inoculated 24 h after DDP, t_1/2, Clp, and Vd were 47.5±6.3 min, 20.4±2.8 l/m², and 322.0±54.1 ml/min per square meter. Thus, DDP pretreatment does not significantly affect L-PAM pharmacokinetics. Regression analysis showed a significant correlation between the L-PAM elimination rate constant () and renal function assessed by creatinine clearance. One patient who received this sequence of treatment for six courses showed a threefold decrease of L-PAM clp after the last treatment. The reported high myelotoxicity of the combination of DDP and L-PAM when DDP was given 24 h before L-PAM cannot be attributed to DDP-induced changes in L-PAM kinetics but might to some extent be related to a loss of renal function consequent to many courses of treatment.This work was supported by a Grant from the Swiss League against Cancer     

Bioavailability and pharmacokinetics of the cardioprotecting flavonoid 7-monohydroxyethylrutoside in mice

Purpose The pharmacokinetics and bioavailability of monoHER, a promising protector against doxorubicin-induced cardiotoxicity, were determined after different routes of administration.Methods Mice were treated with 500 mg.kg^–1 monoHER intraperitoneally (i.p.), subcutaneously (s.c.) or intravenously (i.v.) or with 1000 mg.kg^–1 orally. Heart tissue and plasma were collected 24 h after administration. In addition liver and kidney tissues were collected after s.c. administration. The levels of monoHER were measured by HPLC with electrochemical detection.Results After i.v. administration the AUC_{0–120 min} values of monoHER in plasma and heart tissue were 20.5±5.3 mol.min.ml^–1 and 4.9±1.3 mol.min.g^–1 wet tissue, respectively. After i.p. administration, a mean peak plasma concentration of about 130 M monoHER was maintained from 5 to 15 min after administration. The AUC_{0–120 min} values of monoHER were 6.1±1.1 mol.min.ml^–1 and 1.6±0.4 mol.min.g^–1 wet tissue in plasma and heart tissue, respectively. After s.c. administration, monoHER levels in plasma reached a maximum (about 230 M) between 10 and 20 min after administration. The AUC_{0–120 min} values of monoHER in plasma, heart, liver and kidney tissues were 8.0±0.6 mol.min.ml^–1, 2.0±0.1, 22.4±2.0 and 20.5±5.7 mol.min.g^–1, respectively. The i.p. and s.c. bioavailabilities were about 30% and 40%, respectively. After oral administration, monoHER could not be detected in plasma, indicating that monoHER had a very poor oral bioavailability.Conclusions MonoHER was amply taken up by the drug elimination organs liver and kidney and less by the target organ heart. Under cardioprotective conditions (500 mg/kg, i.p.), the C_max was 131 M and the AUC was 6.3 M.min. These values will be considered endpoints for the clinical phase I study of monoHER.     

Cisplatin efflux, binding and intracellular pH in the HTB56 human lung adenocarcinoma cell line and the E-8/0.7 cisplatin-resistant variant

Purpose: Many cell lines resistant to cisplatin (DDP) have reduced DDP accumulation. We postulated that reduced accumulation of DDP in resistant cells might be due to decreased intracellular DDP binding, leading to increased passive efflux. Methods: The total cellular ([T-DDP]), intracellular ultrafiltrable ([F-DDP]) and precipitable cellular bound ([B-DDP]) DDP concentrations were all compared in the HTB56 human lung adenocarcinoma cell line and its E-8/0.7 variant that has acquired DDP resistance. Cells were exposed to 509 μM DDP for 20 min. Ultrafiltration with a 500 molecular weight cut-off separated cellular free from bound cisplatin. Fragmentation by sonication and microcentrifugal spinning precipitated cellular bound cisplatin. Flow cytometry was used to measure the intracellular pH (pH_i) of the HTB56 cell line, the E-8/0.7 cell line, as well as of the OV2008 cell line and its C13 resistant variant. The DNA-bound DDP and protein-bound DDP ([P-DDP]) were also compared when equal [T-DDP] was achieved for both sensitive and resistant cells by exposing them for 1 h to two pairs of DDP concentrations, i.e. 509 vs 911 μM DDP, and 111 vs 666 μM DDP, respectively. Platinum was assayed by flameless atomic absorption spectrophotometry. Results: At time 0 (end of cisplatin exposure), [T-DDP] and [B-DDP] were significantly higher in the sensitive HTB56 parent cell line (P < 0.02 and P < 0.001, respectively), whereas [F-DDP] did not differ significantly (P = 0.62). Two distinct phases of T-DDP efflux were observed. In the first 10 s after DDP exposure, the rate constant for resistant cells (K_R1) was 0.17 s⁻¹, whereas that for sensitive cells (K_S1) was 0.14 s⁻¹. From 10 s to 50 s, however, K_R2 and K_S2 became 0.005 s⁻¹ and 0.004 s, respectively. [T-DDP] remained lower in resistant cells than in sensitive cells at 10, 30 and 50 s (all P < 0.0001). For 1 h drug exposure to 509 vs 911 μM cisplatin concentrations designed to give comparable [T-DDP] in the sensitive and resistant cell lines, only [DNA-bound DDP] was found to be significantly higher in sensitive cells (P = 0.002), whereas both [F-DDP] and [P-DDP] did not differ significantly (P = 0.18, P = 0.75, respectively). On the other hand, there were no significant differences found in [F-DDP], [P-DDP] and [DNA-bound DDP] between the two cell lines when 111 vs 666 μM DDP was used. Flow cytometry data indicated that the pH_i was significantly higher in the E-8/0.7 (P < 0.0186) and C13 (P < 0.0169) resistant variants than in the sensitive parent cell lines. Conclusions: DDP binds more slowly in resistant than in sensitive lung cancer cells, despite comparable amounts of free drug. Early efflux is higher in the resistant variant. Differences between the lines with respect to DNA binding may be DDP concentration-dependent. We speculate that the reduced early binding and increased early efflux in the resistant line may be related to the higher pH in this line. A higher pH is supposed to favor production of neutral hydroxyl metabolites rather than charged aquated metabolites, and these neutral metabolites would be expected to react less readily with intracellular molecules and to efflux more readily across cell membranes. Since we have previously documented a threefold increase in glucose utilization and lactate production in the DDP-resistant variants of the human HTB56 lung cancer cell lines, and this increased lactate production would have been expected to reduce the intracellular pH instead of raising it, it is possible that our alkaline-resistant cells have a higher Na⁺/H⁺ exchanger activity which would protect them from intracellular acidification. Received: 4 September 1998 / Accepted: 3 December 1998     

In vitro stability,plasma protein binding and blood cell partitioning of 14C-carboplatin

Summary Radiochemically pure ¹⁴C-labeled carboplatin, cis-diammine [1,1-cyclobutane (1-¹⁴C) dicarboxylato (2-)-0,0'] platinum (II), was added to fresh human, dog and rat plasma, at concentrations ranging from 1 to 100 g ¹⁴C-carboplatin/ml. After 10 min incubation at ambient temperature, the plasma was ultrafiltered in Amicon Centrifree micropartition units to generate protein-free plasma ultrafiltrate (PU). Total radioactivity was determined by liquid scintillation counting. A mean (±SD) of 102%±2.0%, 99.5%±1.9%, and 99.0%±1.0% of the ¹⁴C-carboplatin added to fresh human, dog and rat plasma respectibely was recovered in the PU. ¹⁴C-carboplatin was incubated at 37°C with fresh plasma (60g/ml) and urine (200 g/ml) from humans and dogs for 120 h, and samples were removed at appropriate times for analysis of carboplatin, 1,1-cyclobutane dicarboxylic acid and cyclobutane carboxylic acid. The latter were separated by HPLC on a C-18 column with a mobile phase of H₂O/CH₃CN/0.3 M tetrabutylammonium phosphate (880:50:20 v/v/v), and the column eluants at the retention time of each compound were collected and counted for total radioactivity. Carboplatin degraded in each of the matrices with a corresponding release of 1,1-cyclobutane dicarboxylic acid. ¹⁴C-carboplatin (50 g/ml) was incubated at 37°C with fresh human, dog and rat blood and the distribution of radioactivity into the cellular fraction was determined. Radioactivity did not distribute into the blood cells of humans or dogs, but after 5 h, 44% of the radioactivity in rat blood was associated with the cellular fraction. These results show that carboplatin, at physiological concentrations, does not bind instantaneously and reversibly to the plasma proteins of rat, dog or human, and that the molecule slowly degrades in plasma and urine in vitro with the release of 1,1-cyclobutane dicarboxylic acid. The remaining diammine platinum (II) portion of the molecule therefore accounts for the essentially irreversible protein binding of the platinum from carboplatin.     

Preclinical toxicology and tissue platinum distribution of novel oral antitumour platinum complexes: ammine/amine platinum(IV) dicarboxylates

The preclinical toxicology and tissue platinum distribution of a series of six orally given antitumour platinum complexes [ammine/amine platinum(IV) dicarboxylates] with structural variations of their alicyclic amine (c-C₅, c-C₆ or c-C₇), axial dicarboxylate (CH₃, C₃H₇ or NHC₂H₅) or leaving substituents (Cl₂ or OCOOCO) was studied in the mouse. Platinum tissue levels measured at 48 h after a single oral dose at 0.5 of the MTD were highest in the liver (6.0–19 m/g) and second highest in the kidney (2.8–12 g/g), and these levels were up to 5 times higher than those reported with equi-toxic doses of i.v. cisplatin and i.v. carboplatin. Platinum levels in the lung, heart, spleen, skin, skeletal muscle and brain were all<-3.1 g/g at this dose level. Liver platinum levels measured at 2 h, 2 days, 6 days and 10 days after a single oral dose at the MTD ranged widely (from 15 to 109 g platinum/g), were related to the number of carbon atoms in the axial dicarboxylate and alicyclic amine groups (r=0.9389) and showed a diversity of time-course profiles. Elevations of plasma ALT activity were recorded with single oral doses of JM225 and JM256 at the MTD. Accumulation of platinum in the liver with repeated oral dosing weekly for 4 consecutive weeks at 0.5 of the MTD occurred with JM269 (3.3-fold increase,P<0.05) and JM 225 (2.4-fold increase,P<0.05), and elevated plasma ALT activity (44±33 IU/I) was recorded with repeated oral doses of JM269. JM216 was selected from this series of analogues for further study on the basis of the elevated plasma ALT activity (JM225, JM256 and JM269), liver platinum accumulation (JM269 and JM225), poor activity against human ovarian carcinoma xenografts (JM291) or severe emetogenesis (JM221) of other examples. Following a single oral dose of JM216 at the MTD, transient reductions in the WBC (nadir, 1.6×10⁹/ 1,2 days, 74% reduction), platelet count (nadir, 613×10⁹/l, 10 days, 33% reduction) and bone marrow cellularity (nadir, 0.5×10⁷ nucleated cells/femur, 4 days, 75% reduction) were found, and these had recovered by 21 days after treatment. Jejunal mucosal disaccharidase activity following single MTDs indicated that small-intestinal mucosal damage was less severe for oral JM216 (nadir maltase activity, 68%±16% of control, NS) than for i.v. cisplatin (nadir maltase activity, 35%±6.0% of control,P<0.01) and i.v. carboplatin (nadir maltase activity, 38%±6.4% of control,P<0.01). In conclusion, the liver is the major tissue platinum deport for orally delivered ammine/amine platinum(IV) dicarboxylates and is a site of toxicity for examples of this class. Oral JM216 causes dose-limiting leucopenia but produces less gastrointestinal toxicity than either i.v. cisplatin or i.v. carboplatin at the MTD in the mouse.Abbreviations ALP alkaline phosphatase - ALT alanine aminotransferase; carboplatin,cis-diamminecyclobutanedicarboxylatoplatinum(II); cisplatin,cis-diamminedichloroplatinum(II) - EDTA ethylenediaminetetraacetic acid - MTD maximally tolerable dose - NS not significant Institute of Oncology, The Prince of Wales Hospital, High Street, Randwick, Sydney NSW 2031, Australia     

Measurement of in vivo glucose transport from blood to tissue of experimentally-induced glioma in rat brain

Summary In experimentally-induced F98 glioma of rat brain, regional blood flow and glucose transfer were assessed by means of double tracer autoradiography to measure Michaelis-Menten constants for the determination of unidirectional glucose transport across the blood-tumor and blood-brain barrier. In brain regions opposite the tumor hemisphere, the maximal glucose transport rate constant, T_m, ranged from 1.41 ± 0.12 to 3.22 ± 0.29 mol/g/min and the half saturation transport constant of glucose, K_t, varied from 2.78 ± 0.83 to 5.6 ± 1.94 mol/ml (estimate ± standard error of the estimate) yielding a normoglycemic unidirectional glucose inward transport which ranged from 1.24 ± 0.24 to 1.97 ± 0.13 mol/g/min (mean ± standard deviation). In the tumor periphery, the T_m and the Kt values were 3.64 ± 0.56 mol/g/ml and 7.32 ± 2.12 mol/min, and in the tumor center, 1.77 ± 0.25 mol/ml and 2.76 ± 1.13 mol/min, respectively. The unidirectional glucose influx of tumor periphery and center in normoglycemia was 1.98 ± 0.22 and 1.34 ± 0.16 mol/g/min, respectively. Despite comparable unidirectional glucose influxes, however, glucose metabolism of tumor tissue located in the periphery (0.83 ± 0.12 mol/g/min) and the center (0.41 ± 0.10 mol/g/min) of the tumor mass exceeded that of normal gray matter by about 68% and 100% which indicates uncoupling between glucose transport and phosphorylation in experimentally-induced F98 glioma of rat brain.     

Extensive and saturable accumulation of paclitaxel by the human platelet

Little is known about the cellular distribution of paclitaxel in humans. In the present study we examined the distribution of [³H]-paclitaxel in human blood. When 1 M paclitaxel was incubated with fresh blood at 37°C, the platelet/plasma concentration ratio was 240±17 (mean±SEM), whereas the red blood cell (RBC)/plasma concentration ratio was only 0.59±0.05. In kinetics experiments using platelet-rich plasma, we observed that the platelet accumulation of paclitaxel was highly temperature- and concentration-dependent. Scatchard analysis of the 37°C uptake data demonstrated a dissociation constant (K _app) of 0.80±0.10 M and a maximal binding capacity of 672±102 pmol/10⁹ platelets. It is proposed that the platelet accumulation of paclitaxel reflects binding to microtubules and may serve as a useful model for binding to less accessible cellular sites.     

Cisplatin disposition in children and adolescents with cancer

Summary The disposition of cisplatin was evaluated in 28 children and adolescents with cancer, as part of a phase II clinical trial. Patients received either 30 mg/m² (11) or 90 mg/m² (17) of cisplatin as a 6-h IV infusion. Serum samples and divided urine collections were obtained over 48 h following completion of the cisplatin infusion, and were assayed in duplicate for total platinum by atomic absorption spectrophotometry. Serum samples obtained up to 4 h after three cisplatin infusions were assayed for parent (free) cisplatin following ultrafiltration. The mean (±SE) elimination half-life of free cisplatin in serum was 1.3 (±0.4) h. Serial serum concentrations of total platinum following 90 mg/m² dosages were adequately described by a biexponential equation. The mean (±SE) serum T_1/2 of total platinum was 0.42 (±0.10) h and the mean (±SE) T_1/2 was 44.43 (±8.24) h. The intercompartment distribution rate constants of a two-compartment kinetic model indicate extensive tissue accumulation of total platinum, with a rate of transport into tissue compartments (K₁₂) that is about six times the rate of transport out of tissues (K₂₁). The mean (±SE) renal clearance of total platinum from 0–3 h was 37.36 (±11.96) ml/min/m² and 35.8 (±13.6) ml/min/m² for the 30 mg/m² and 90 mg/m² groups, respectively. This value decreased to 3.25 (±0.94) and 2.16 (±0.4) ml/min/m² for the two groups by the 6–12 h interval, and remained between 1 and 3 ml/min/m² for the duration of the observation period. The ratio of total plantinum clearance to creatinine clearance decreased significantly(P<0.05) beginning 3 h post-infusion. The change in renal clearance of total platinum is apparently a function of two independent first-order processes for renal clearance of parent drug and cisplatin metabolites.     

Pentetration of carboplatin and cisplatin into rat peritoneal tumor nodules after intraperitoneal chemotherapy

Summary Platinum distribution was studied in rat peritoneal tumors after i.p. treatment with equimolar doses of carboplatin and cisplatin. Low platinum concentrations (4 ppm) were detected in the periphery of the tumor after carboplatin treatment, whereas no platinum was detected 0.5 mm in from the periphery. In contrast, after cisplatin treatment, high platinum concentrations (29 ppm) were measured in the periphery of the tumor and moderate concentrations (14 ppm) were measured in the center. Only following increased carboplatin doses were low platinum concentrations detectable in the tumor. The total platinum concentration in the tumors was determined after equimolar administration of both drugs. In all, 7 times more platinum was detected after cisplatin treatment than after carboplatin treatment, and 10 times more carboplatin than cisplatin had to be injected to obtain comparable platinum concentrations in the tumors. When single cells were incubated with equimolar concentrations of carboplatin and cisplatin, 6–7 times more platinum was found in cells treated with cisplatin. However, pharmacokinetic studies favored i.p. administration of carboplatin because the clearance of this compound from the peritoneal cavity, expressed ast _1/2, was lower than that of cisplatin (239 vs 78 min), resulting in an AUC in the peritoneal cavity for both total and ultrafiltered drug that was almost 3 times higher for carboplatin than cisplatin. The AUC for ultrafiltered carboplatin in plasma was 2-fold that for cisplatin (2,801±210 vs 1,334±431 M m). The present study demonstrated that in spite of the pharmacological advantages of carboplatin, its capacity to penetrate into peritoneal tumors and tumor cells is far lower than that of cisplatin.This work was supported by grant NKI 86-5 from the Dutch Cancer Society     

Pharmacokinetics and toxicity of the antitumour agentN-[2-(dimethylamino)ethyl]acridine-4-carboxamide after i.v. administration in the mouse

Summary The pharmacokinetics, tissue distribution and toxicity of the antitumour agentN-[2-(dimethylamino)ethyl]acridine-4-carboxamide(AC) were studied after i.v. administration to mice. Over the dose range of 9–121 mol/kg (3–40 mg/kg), AC displayed linear kinetics with the following model-independent parameters: clearance (C), 21.0±1.9 l h^–1 kg^–1; steady-state volume of distribution (V_ss), 11.8±1.4 l/kg; and mean residence time (MRT), 0.56±0.02 h. The plasma concentration-time profiles for AC fitted a two-compartment model with the following parameters:C _c, 19.4±2.3 l h^–1 kg^–1; V_c, 7.08±1.06 l/kg;t _1/2 13.1±3.5 min; andt _1/2Z, 1.60±0.65 h. AC displayed moderately high binding in healthy mouse plasma, giving a free fraction of 15.9%–25.3% over the drug concentration range of 1–561 M. After the i.v. administration of 30 mol/kg [³H]-AC, high radioactivity concentrations were observed in all tissues (especially the brain and kidney), showing a hight _1/2c value (37–59 h). At 2 min (first blood collection), the AC concentration as measured by high-performance liquid chromatography (HPLC) comprised 61% of the plasma radioactivity concentration (expressed as AC equivalents/l). By 48 h, 73% of the dose had been eliminated, with 26% and 47% of the delivered drug being excreted by the urinary and faecal routes, respectively; <1% of the total dose was excreted as unchanged AC in the urine. At least five distinct radiochemical peaks were distinguishable by HPLC analysis of plasma extracts, with some similar peaks appearing in urine. The 121-mol/kg dose was well tolerated by mice, with sedation being the only obvious side effect and no significant alterations in blood biochemistry or haematological parameters being recorded. After receiving a dose of 152 mol/kg, all mice experienced clonic seizures for 2 min (with one death occuring) followed by a period of sedation that lasted for up to 2h. No leucopenia occurred, but some mild anaemia was noted. There was no significant change in blood biochemistry. A further 20% increase in the i.v. dose (to 182 mol/kg) resulted in mortality, with death occurring within 2 min of AC administration.Supported by the Auckland Medical Research Foundation and the Cancer Society of New Zealand     

Effect of homoharringtonine on the viability of murine leukemia P388 cells resistant to either adriamycin,vincristine, or 1-β-D-arabinofuranosylcytosine

Summary Cultured murine leukemia P388 cell populations were derived from P388 cells resistant to vincristine (P388/VCR), adriamycin (P388/ADR), and 1--D-arabinofuranosylcytosine (P388/ARA-C) that were developed in vivo and to the parental drug-sensitive cells (P388/O) that were passaged in vivo. The doubling times of the cultured cell populations (mean±SD) between cell densities of 5×10⁴ and 1×10⁶ cells/ml were 14.2±2 h (P388/O), 16.5±1.9 h (P388/VCR), 16.9±1.2 h (P388/ADR), and 15.0±1.4 h (P388/ARA-C). Exponentially proliferating cultured cell populations were exposed to selected homoharringtonine (HHT) concentrations for 24 h and the surviving cell fractions were determined by colony formation in semisolid medium. The results, based on differential sensitivity of the cell populations to HHT, indicated that cultured P388/VCR cells were cross-resistant to 0.018–1.8 g/ml HHT, P388/ADR cells were cross-resistant to 0.058–1.8 g/ml HHT, and P388/ARA-C cells were collaterally sensitive to 0.09–0.36 g/ml HHT. The results with the cultured P388/VCR, P388/ADR, P388/ARA-C, and P388/O cell populations were confirmed in animal experiments. CD2F₁ mice bearing intraperitoneal (i.p.) implants of 1×10⁶ P388/VCR, P388/ADR, P388/ARA-C, or P388/O leukemia cells were given HHT i.p. qd on days 1–9 postimplantation. Optimal treatment (LD₁₀) produced in vivo cell kills of 2 to 3 log₁₀ units in P388/O and about 7 log₁₀ units in P388/ARA-C, whereas P388/VCR and P388/ADR cells actually increased by 1–2 log₁₀ units during treatment. The results of this study indicate that cross-resistance (P388/VCR and P388/ADR) or collateral sensitivity to HHT (P388/ARA-C) is a function of the cellular properties of the target tumor cell populations that is independent of host factors.This work was supported by Contract NO1-CM-97309 with the Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, Bethesda, Md 20892     

BCNU-sensitivity in parental cells and clones from four freshly resected near-diploid human gliomas: An astrocytoma,an anaplastic astrocytoma and two glioblastomas multiforme

Summary We compared the BCNU sensitivity of 4 freshly resected tumors (astrocytoma WM, and malignant gliomas MK, MB, and AM) and their clones to their karyology. The majority of primary cells in all 4 tumors had near-diploid chromosome numbers (2n±) and all were resistant to concentrations of BCNU exceeding 10g/ ml. Followingin vitro cultivation, the cells from tumors WM and MB retained their 2n ± modal chromosome number with little change in the complexity of the karyotype. In contrast, tumors MK and AM demonstrated a more unstable genome. The modal chromosome number of MK shifted from 45 to 86 and that of tumor AM from 45 to 90. Karyotyping demonstrated additional ploidy changes and new marker chromosomes in both tumors. The colony forming assay (CFA) performed on thein vitro cultivated cells demonstrated little change in the sensitivity to BCNU in tumors WM and MB, while tumors MK and AM exhibited greater than a one log cell kill at 10.0g/ml and 15.0g/ml BCNU, respectively. The modal chromosome number and BCNU sensitivity followed a similar pattern in the 30 clones that were isolated; 21 clones with near-diploid and pseudodiploid chromosome numbers were all resistant to BCNU doses at or greater than 10/ml. In contrast, 9 clones isolated from the 3 malignant gliomas with 3n ± and 4n ± modal chromosome numbers were sensitive to this concentration of BCNU. The karyotypes of the hyperdiploid clones were more complex; they contained 5 or more ploidy changes and/or had marker chromosomes. These studies confirm the association of diploidy and BCNU-resistance in freshly resected malignant gliomas.     

The effect of cimetidine on cyclophosphamide metabolism in rabbits

Summary Six female rabbits were given 20 mg/kg cyclophosphamide (containing 100 Ci [³H-chloroethyl]-cyclophosphamide) alone or 1 h following 100 mg/kg cimetidine. Serial plasma and urine specimens were collected and levels of cyclophosphamide and its metabolites (4-hydroxycyclophosphamide, 4-ketocyclophosphamide, phosphoramide mustard, and carboxyphosphamide) were measured. 4-Ketocyclophosphamide was the major metabolite present in rabbit plasma and urine, with lesser amounts of 4-hydroxycyclophosphamide, carboxyphosphamide, and phosphoramide mustard also being identified. Cimetidine pretreatment resulted in prolongation of cyclophosphamide's half-life from 24.3±7.3 to 33.5±9.5 min (mean ± SD;P=0.036) but did not significantly alter the AUC_{0–8 h} for the latter drug. Cimetidine pretreatment resulted in a significantly greater AUC_{0–8 h} for 4-hydroxycyclophosphamide (189.4±77 vs 364.6±126.7 mol min/l^–1;P=0.016) as compared with control values. A higher AUC_{0–8 h} value for phosphoramide mustard (53.7±69.2 vs 95.7±34.7 mol min/l^–1) was also observed after cimetidine dosing but the difference was not significant (P=0.21). Kinetics of 4-ketocyclophosphamide and carboxyphosphamide were not significantly affected by cimetidine treatment. Cimetidine was added to hepatic microsomes isolated from phenobarbital-treated rabbits; it did not inhibit cyclophosphamide's metabolism in vitro, suggesting that its in vivo effect may be mediated through mechanisms other than cytochrome P-450 inhibition. Cimetidine pretreatment increases exposure to cyclophosphamide and its major activated metabolite, 4-hydroxycyclophosphamide. Potentiation rather than inhibition of cyclophosphamide's pharmacodynamic effect is to be predicted when cimetidine is given concomitantly with the former. Alterations in hepatic blood flow or mechanisms other than microsomal inhibition by cimetidine may explain this potentiation.Supported in part by the Department of Veteran Affairs and grant CA-49186 from the National Institutes of Health (NIH)Department of Clinical Pharmacology, Sun Yat-sen University of Medical Sciences, Guangzhou, People's Republic of China