Recruitment of trimeric proliferating cell nuclear antigen by G1-phase cyclin-dependent kinases following DNA damage with platinum-based antitumour agents.

BACKGROUND: In cycling tumour cells, the binary cyclin-dependent kinase Cdk4/cyclin D or Cdk2/cyclin E complex is inhibited by p21 following DNA damage to induce G1 cell-cycle arrest. However, it is not known whether other proteins are also recruited within Cdk complexes, or their role, and this was investigated. METHODS: Ovarian A2780 tumour cells were exposed to the platinum-based antitumour agent 1R,2R-diaminocyclohexane(trans-diacetato)(dichloro)platinum(IV) (DAP), which preferentially induces G1 arrest in a p21-dependent manner. The Cdk complexes were analysed by gel filtration chromatography, immunoblot and mass spectrometry. RESULTS: The active forms of Cdk4 and Cdk2 complexes in control tumour cells have a molecular size of ~140 kDa, which increased to ~290 kDa when inhibited following G1 checkpoint activation by DAP. Proteomic analysis identified Cdk, cyclin, p21 and proliferating cell nuclear antigen (PCNA) in the inhibited complex, and biochemical studies provided unequivocal evidence that the increase in ~150 kDa of the inhibited complex is consistent with p21-dependent recruitment of PCNA as a trimer, likely bound to three molecules of p21. Although p21 alone was sufficient to inhibit the Cdk complex, PCNA was critical for stabilising p21. CONCLUSION: G1 Cdk complexes inhibited by p21 also recruit PCNA, which inhibits degradation and, thereby, prolongs activity of p21 within the complex.

Carboplatin dosage: prospective evaluation of a simple formula based on renal function.

A dosage formula has been derived from a retrospective analysis of carboplatin pharmacokinetics in 18 patients with pretreatment glomerular filtration rates (GFR) in the range of 33 to 136 mL/min. Carboplatin plasma clearance was linearly related to GFR (r = 0.85, P less than .00001) and rearrangements of the equation describing the correlation gave the dosage formula dose (mg) = target area under the free carboplatin plasma concentration versus time curve (AUC) x (1.2 x GFR + 20). In a prospective clinical and pharmacokinetic study the formula was used to determine the dose required to treat 31 patients (GFR range, 33 to 135 mL/min) with 40 courses of carboplatin. The target AUC was escalated from 3 to 8 mg carboplatin/mL/min. Over this AUC range the formula accurately predicted the observed AUC (observed/predicted ratio 1.24 +/- 0.11, r = 0.886) and using these additional data, the formula was refined. Dose (mg) = target AUC x (GFR + 25) is now the recommended formula. AUC values of 4 to 6 and 6 to 8 mg/mL. min gave rise to manageable hematological toxicity in previously treated and untreated patients, respectively, and hence target AUC values of 5 and 7 mg/mL min are recommended for single-agent carboplatin in these patient groups. Pharmacokinetic modeling demonstrated that the formula was reasonably accurate regardless of whether a one- or two-compartment model most accurately described carboplatin pharmacokinetics, assuming that body size did not influence nonrenal clearance. The validity of this assumption was demonstrated in 13 patients where no correlation between surface area and nonrenal clearance was found (r = .31, P = .30). Therefore, the formula provides a simple and consistent method of determining carboplatin dose in adults. Since the measure of carboplatin exposure in the formula is AUC, and not toxicity, it will not be influenced by previous or concurrent myelosuppressive therapy or supportive measures. The formula is therefore applicable to combination and high-dose studies as well as conventional single-agent therapy, although the target AUC for carboplatin will need to be redefined for combination chemotherapy.

Axial ligands and alicyclic ring size modulate the activity and biochemical pharmacology of ammine/cycloalkylamine-platinum(IV) complexes in tumor cells resistant to cis-diamminedichloroplatinum(II) or trans-1R,2R-1S,2S-diaminocyclohexanetetrachloroplatinum(IV).

A platinum(II) and 3 platinum(IV) ammine/cycloalkylamine homologous series were evaluated for cytotoxicity and biochemical pharmacology in murine leukemia L1210/0, cis-diamminedichloroplatinum(II)- resistant L1210/DDP, and diaminocyclohexaneplatinum-resistant L1210/1,2-diaminocyclohexane (DACH) cells. Within each series, which contained 4 homologues with differing alicyclic (cycloalkyl) ring size (cyclopropane, cyclobutane, cyclopentane, or cyclohexane), cytotoxicity increased with increasing ring size. This appeared to be due to an increase in partition coefficient, and the resulting increase in drug accumulation and intracellular DNA adducts in ascending each of the series. There were exceptions to this generalization, predominantly in L1210/DACH cells, where the biochemical pharmacology was not entirely consistent with the cytotoxic response and suggested that other factors may be at play. The relationship between structure and ability to circumvent cis-diamminedichloroplatinum(II) and/or trans-1R,2R-1S,2S- diaminocyclohexanetetrachloroplatinum(IV) resistance was complex. Ascending the platinum(II) series caused resistance factors to decrease in L1210/DDP cells but increase in L1210/DACH cells. An increase in resistance factors was also observed in ascending the axial chloroplatinum(IV) series in the L1210/DACH line. In contrast, ascending the axial chloroplatinum(IV) series in the L1210/DDP line and axial acetatoplatinum(IV) and axial hydroxoplatinum(IV) series in both cell lines resulted in increases in resistance factors for the first stepwise increase in the cycloalkylamine ring size, but resistance factors then decreased progressively with further increases in ring size. Reduction of the platinum(IV) analogues to the platinum(II) congener appears to be necessary for binding to DNA. The similarity in biological actions between the platinum(II) and axial chloroplatinum(IV) series is likely due to the rapid reduction of tetravalent members to platinum(II) forms. The axial acetatoplatinum(IV) and axial hydroxoplatinum(IV) complexes were reduced more slowly, which may explain their lower potency, but not the ability of the higher member to circumvent both cis-diamminedichloroplatinum(II) and trans-1R,2R-1S,2S- diaminocyclohexanetetrachloroplatinum(IV) resistances. Explanation for this will require additional studies. The results have demonstrated high dependencies on ring size of the carrier amine ligand, valence state of platinum, and the nature of the axial ligand for modulation of potency, cross-resistance property, and biochemical pharmacology of ammine/cycloalkylamine complexes.

Biochemical pharmacology of homologous alicyclic mixed amine platinum(II) complexes in sensitive and resistant tumor cell lines.

Three homologous alicyclic mixed amine cis-(NH3)(R-NH2)Cl2Pt(II) complexes, in which R = C3H5, C6H11, and C8H15 (complexes abbreviated C3, C6, and C8, respectively), were evaluated with reference compounds cisplatin and tetraplatin for antitumor activities and biochemical pharmacology in wild-type (murine leukemia L1210/0 and human ovarian A2780) and corresponding variant cell lines resistant to cisplatin (L1210/DDP and 2780CP) and tetraplatin (L1210/DACH and 2780TP). Cytotoxicities, measured by either a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or a clonogenic assay, were maximal for the C6 complex, which was up to 12-, 40-, and 6-fold more potent than C3 against wild-type, cisplatin-resistant, and tetraplatin-resistant models, respectively, and up to 2-fold more potent than C8 against these cell lines. In general, cross-resistance to mixed amine analogues was partial in cisplatin- and tetraplatin-resistant cells and decreased (in L1210/DDP and 2780CP) or increased (in L1210/DACH and 2780TP) with increase in the alicyclic ring size. The increase in ring size resulted in a corresponding increase in partition coefficient, which correlated directly with intracellular accumulations of mixed amine analogues in all cell lines. However, the intracellular DNA-platinum adducts, and not cellular platinum content, was the pharmacological entity that corresponded closely to potencies of the molecules. DNA adduct formation was disproportionate to the level of cellular drug accumulation. For instance, complex C8, which accumulated to the greatest extent in any given cell line, produced adduct levels that were similar to or lower than those produced by C6. A partial explanation for this observation was the demonstrated reduced rate of binding of C8 to DNA. This study has highlighted the significance of alicyclic ring size in modulating the potency, cross-resistance profile, and biochemical pharmacology of mixed amine platinum(II) complexes in sensitive and cisplatin- or tetraplatin-resistant tumor cells.

Independent pathways of p53 induction by cisplatin and X-rays in a cisplatin-resistant ovarian tumor cell line.

The p53 tumor suppressor gene is critical in regulating cell proliferation following DNA damage, and disruption of p53 protein function by mutation has been implicated as a factor responsible for resistance of tumor cells to chemotherapeutic agents. Our studies were initiated by asking whether the translational product of the p53 gene is associated with cisplatin resistance in the 2780CP human ovarian tumor model. We have demonstrated by single-strand conformation polymorphism analysis and sequencing that p53 in parental cisplatin-sensitive A2780 cells was wild type. In 2780CP cells, however, a mutation was found in exon 5 at codon 172 (Val to Phe). Interestingly, exposure to X-rays resulted in p53 induction in both A2780 and 2780CP tumor models. The p53 increases by the ionizing radiation were accompanied by concomitant increases in levels of the p53-regulated p21Waf1/Cip1 protein and led to arrest of cells in G1 phase of the cell cycle. A yeast functional assay confirmed that p53 in A2780 was wild type, but, more importantly, it provided evidence that the p53 mutation in 2780CP cells was temperature sensitive and heterozygous. These experiments demonstrate that sensitive and resistant cells have normal p53 functions, despite the presence of p53 mutation in the 2780CP model. In parallel investigations using the Western technique, exposure of A2780 cells to clinically relevant concentrations of cisplatin (1-20 microM) resulted in time- and dose-dependent increases in p53, together with coordinate increases in p21Waf1/Cip1. In contrast, cisplatin did not induce these proteins in 2780CP cells to any significant degree. The results indicate that a defect exists in the signal transduction pathway for p53 induction following cisplatin-induced DNA damage in 2780CP cells, and this may represent a significant mechanism of cisplatin resistance. Furthermore, induction of p53 in 2780CP cells by X-rays, but not cisplatin, strongly suggests that independent pathways are involved in p53 regulation for the two DNA-damaging agents.

Modulation of cytotoxicity and cellular pharmacology of 1,2-diaminocyclohexane platinum (IV) complexes mediated by axial and equatorial ligands.

Isomers (R,R-, S,S-, and cis-) of 1,2-diaminocyclohexane (DACH) platinum(IV) complexes with selected axial and equatorial ligands were synthesized and evaluated for in vitro antitumor activity, cellular uptake, and total DNA-Pt adducts. L1210 cells, sensitive to cis-diamminedichloroplatinum(II) (CDDP) and tetraplatin (L1210/0), 160-fold resistant to CDDP [L1210/diamminedichloroplatinum (DDP)], or 70-fold resistant to tetraplatin (L1210/DACH), were used in conjunction with compounds having the general structure DACH-Pt(IV)-X2Y2, where X and Y are axial and equatorial ligands and X2Y2 are specifically Cl2Cl2,Ac2Cl2, (TFA)2Cl2, (OH)2Cl2, and Cl2CBDCA (Cl, chloro; Ac, acetato; TFA, trifluoroacetato; OH, hydroxo; CBDCA, 1,1-cyclobutanedicarboxylato). Comparison of cytotoxicities between isomers of Cl2Cl2,Ac2Cl2, or Cl2CBDCA indicated that R,R-isomers were the most effective against all three cell lines. The relatively lower activity of the S,S- and cis-isomers was cell line dependent: against L1210/DACH, both isomers of Cl2Cl2 were only 2- to 3-fold less effective, and this contrasted with 7- and 26-fold lower cytotoxicities, respectively, against L1210/DDP. Cross-resistance factors in the L1210/DDP and L1210/DACH lines depended on both isomeric form and the nature of axial or equatorial ligand. The L1210/DDP cells were 6- to 9-fold cross-resistant to the R,R-isomer, 8- to 15-fold to S,S-isomer, and 13- to 38-fold to cis-isomer. The L1210/DACH line was only 4- to 7-fold cross-resistant to the three isomers of Ac2Cl2 but cross-resistance to the isomers was 47- to 79-fold for Cl2Cl2 and 22- to 56-fold for Cl2CBDCA complexes. Compared with CDDP, accumulation (2 h at 100 microM drug concentration) of Ac2Cl2 in the three L1210 cell lines was 26-50%, while uptake of Cl2Cl2 and (TFA)2Cl2 was 100-170% and 320-570%, respectively. The greatest DNA binding was seen with Cl2Cl2 in all cell lines, followed by (TFA)2Cl2, CDDP, and Ac2Cl2. DNA binding correlated directly with potency (1/concentration producing 50% inhibition) in the L1210/0 model (r = 0.973, P < 0.016) but not in the L1210/DDP and L1210/DACH models. Accumulation and DNA-binding studies indicated that binding efficiency to DNA was: Cl2Cl2 > Ac2Cl2 > CDDP > (TFA)2Cl2. In a nonreducing environment, the Pt(IV) complexes (20 microM) did not react with salmon sperm DNA. Reduced glutathione (100 microM), as a reducing agent, rendered full binding capacity to Cl2Cl2; binding was 25-30% of the expected maximum for (TFA)2Cl2, while Ac2Cl2 remained inert.(ABSTRACT TRUNCATED AT 400 WORDS)

Enhanced sensitivity of human colon tumor cell lines in vitro in response to thermochemoimmunotherapy.

We have investigated the cytotoxic responses in vitro of three human colon tumor cell lines with epithelial-like morphology, DLD-1, HCT-15, and HT-29, to thermochemoimmunotherapy with hyperthermia (42 degrees C for 2 h), carboplatin, and recombinant human tumor necrosis factor (TNF). Dose ranges of carboplatin and recombinant human TNF were administered essentially simultaneously and were followed 1 h later by hyperthermia. A two-tiered approach was used to evaluate cytotoxicity. In the first tier, a 5-day microcytotoxicity assay using vital dye staining was done; the effect on surviving fraction of simultaneously varying carboplatin and recombinant human TNF doses was evaluated by response surface methodology. From this analysis doses were selected for use in the second-tier clonogenic survival assays. A similar treatment protocol was used in clonogenic assays. Both assays revealed significant interline treatment response heterogeneity. Only the HCT-15 cells were sensitive to TNF alone; carboplatin activity against all three tumor cell lines was enhanced by TNF. Hyperthermia had minimal effect as a sole agent but enhanced the effects of carboplatin and TNF in DLD-1 and HCT-15 cells. Triple modality treatment resulted in 3-4-log decreased survival and could reduce cytotoxic resistance expressed against single- or dual-modality treatments by some of these cells.

Increased therapeutic gain of combined cis-diamminedichloroplatinum (II) and whole body hyperthermia therapy by optimal heat/drug scheduling.

To maximize therapeutic gain, the timing sequence of whole body hyperthermia (WBH) and cis-diamminedichloroplatinum (II) (DDP) was examined. Normal tissue injury as well as growth of a s.c. transplanted fibrosarcoma were measured in F344 rats treated with variable schedules of WBH and DDP. Simultaneous application of DDP (2 mg/kg i.v.) with WBH (120 min at 41.5 degrees C) resulted in severe renal injury, body weight loss, and mortality; while sequential use of the modalities caused minimal to no toxicity. DDP or WBH alone produced only minimal tumor growth delay, whereas supraadditive antitumor effects occurred with all tested schedules of DDP combined with WBH, regardless of sequence or interval between the two modalities. We designated the ratio of antitumor effect to nephrotoxicity as specific therapeutic efficacy (STE). DDP given simultaneously with WBH produced the lowest STE (0.6-1.2), which was less than or equal to either DDP (STE = 1.2) or WBH (STE = 1.5) alone. On the other hand, schedules of DDP prior to and after WBH resulted in a STE of 1.8-3.0, a supraadditive effect. These results indicate that an optimal scheduling of DDP with WBH significantly improves therapeutic gain by reducing normal tissue injury while maintaining enhanced antitumor activity.

Pharmacokinetics of cis-diammine-1,1-cyclobutane dicarboxylate platinum(II) in patients with normal and impaired renal function.

cis-Diammine-1,1-cyclobutane dicarboxylate platinum(II) (CBDCA, JM8) is a nonnephrotoxic analogue of cisplatin currently undergoing clinical evaluation. Pharmacokinetic studies have been performed in patients receiving CBDCA (20 to 520 mg/sq m) as a 1-hr infusion without hydration or diuresis. Following the end of the infusion, plasma levels of total platinum and ultrafilterable (Mr less than 50,000) platinum (free platinum) decayed biphasically with first-order kinetics (total platinum t alpha 1/2 = 98 min; t beta 1/2 range, 399 to greater than 1440 min; free platinum t alpha 1/2 = 87 min; t beta 1/2 = 354 min). During the first four hr, binding of platinum to plasma protein was limited (24%), with most of the free platinum in the form of unchanged CBDCA (94%). However, by 24 hr, the majority of platinum was protein bound (87%). The major route of elimination was renal, 65% of the platinum administered being excreted in the urine within 24 hr, with 32% of the dose excreted as unchanged CBDCA. No evidence was found from studies on the renal clearance of free platinum to indicate renal tubular secretion (mean free platinum renal clearance, 69 ml/min). However, the plasma clearance of free platinum did correlate positively with glomerular filtration rates (p = 0.005). None of the pharmacokinetic parameters determined were dose dependent. In vitro studies with plasma and urine demonstrated that, in contrast to cisplatin, CBDCA is a stable complex [t 1/2 - 37 degrees; plasma, 30 hr, and urine (range), 20 to 460 hr]. The differences in the pharmacokinetics of cisplatin and CBDCA may explain why the latter complex is not nephrotoxic.

Protective effect of ICRF-187 against normal tissue injury induced by adriamycin in combination with whole body hyperthermia.

The use of [(+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)]propane (ICRF-187) as a protective agent against normal tissue toxicity caused by combined Adriamycin (ADR) and whole body hyperthermia (WBH; 2 h at 41.5 degrees C) was assessed in a rat model. The effect of ICRF-187 on the antitumor response induced by the combination of ADR and WBH was also investigated in order to assess alterations in the therapeutic index of this combined therapeutic modality treatment. ICRF-187 significantly reduced ADR-mediated body weight loss, renal toxicity, and cardiomyopathy under both normothermic and hyperthermic conditions as shown by morphological and functional assays. ADR-induced neuropathy (seen only in normothermic rats) was also ameliorated by ICRF-187. Although this study did not show a pronounced effect of ICRF-187 on ADR-induced acute myelosuppression, ADR-mediated chronic anemia, leukocytosis, and thrombocytosis were reduced by ICRF-187 in both normothermic and WBH-treated rats. The effect of ICRF-187 on antitumor response was evaluated with a tumor growth delay assay using an in vivo transplantable fibrosarcoma. ICRF-187 caused no significant change in tumor growth delay induced by either ADR alone or ADR combined with WBH. Indeed, the only complete tumor regression following treatment resulted from the combination of ICRF-187 plus ADR plus WBH. Thus, ICRF-187 significantly increases the therapeutic index of the combined modality treatment of ADR and WBH by selectively reducing normal tissue toxicity without interfering with antitumor efficacy.

Effect of carboplatin combined with whole body hyperthermia on normal tissue and tumor in rats.

The antitumor activity and normal tissue toxicity of cis-diammine-1,1-cyclobutane dicarboxylate platinum (II) (carboplatin) in combination with whole body hyperthermia (WBH) (41.5 degrees C, 120 min.) were examined in an F344 rat model. Carboplatin data were compared with those of cis-diamminedichloroplatinum (II) (cisplatin). At 37 degrees C, carboplatin showed minimal activity against a rat fibrosarcoma, but when combined with WBH, the antitumor effect-of the drug was greatly enhanced. The major carboplatin-induced acute toxicity at both normothermic and hyperthermic temperatures was marked hypocellularity of the bone marrow. A significant decrease in peripheral blood platelet counts was caused by the maximum tolerated doses (MTD) of carboplatin alone and with WBH. While the lethal dose of carboplatin alone caused only minimal renal damage, mild acute tubular necrosis was observed at the MTD of carboplatin with WBH, although no significant increase in blood urea nitrogen occurred. Therapeutic ratios of the combined chemotherapy and WBH modalities were calculated by comparing tumor growth response at the MTD of drug alone and drug combined with WBH. The combination of the nephrotoxic cisplatin with WBH resulted in a therapeutic ratio of only 0.8, whereas when carboplatin was combined with WBH, a value of 3.0 was obtained, representing a 3- to 4-fold increase over cisplatin in the therapeutic ratio. These data indicate that the less nephrotoxic carboplatin in combination with WBH improves therapeutic gain and may provide a more promising clinical combination for cancer treatment than cisplatin combined with WBH.

Effect of adriamycin combined with whole body hyperthermia on tumor and normal tissues.

Thermal enhancement of Adriamycin-mediated antitumor activity and normal tissue toxicities by whole body hyperthermia were compared using a F344 rat model. Antitumor activity was studied using a tumor growth delay assay. Acute normal tissue toxicities (i.e., leukopenia and thrombocytopenia) and late normal tissue toxicities (i.e., myocardial and kidney injury) were evaluated by functional/physiological assays and by morphological techniques. Whole body hyperthermia (120 min at 41.5 degrees C) enhanced both Adriamycin-mediated antitumor activity and toxic side effects. The thermal enhancement ratio calculated for antitumor activity was 1.6. Thermal enhancement ratios estimated for "acute" hematological changes were 1.3, whereas those estimated for "late" damage (based on morphological cardiac and renal lesions) varied between 2.4 and 4.3. Thus, while whole body hyperthermia enhances Adriamycin-mediated antitumor effect, normal tissue toxicity is also increased, and the potential therapeutic gain of the combined modality treatment is eroded.

o-(beta-Hydroxyethyl)-rutoside-mediated protection of renal injury associated with cis-diamminedichloroplatinum(II)/hyperthermia treatment.

A bioflavonoid, o-(beta-hydroxyethyl)-rutoside, has been investigated for its potential to increase the therapeutic index of the combined treatment modalities of whole body hypothermia (WBH) (41.5 degrees C) and chemotherapy (cisplatin) in studies utilizing a transplantable fibrosarcoma solid tumor model in Fischer rats. When whole body WBH was induced 45 min after cisplatin administration, a significantly increased tumor growth delay was noted beyond that achieved by either treatment modality alone. The combination of WBH and cisplatin treatments, however, produced an unacceptable increase in renal injury. o-(beta-Hydroxyethyl)-rutoside administration was found to effectively block the renal injury without interfering with the antitumor efficacy of the combined regimen. Potential explanations for the ability of o-(beta-hydroxyethyl)-rutoside to affect the increase in WBH-cisplatin therapeutic regimen are discussed.

Cellular pharmacology of liposomal cis-bis-neodecanoato-trans-R,R-1,2-diaminocyclohexaneplatinum (II) in mouse resident peritoneal macrophages, Kupffer cells, and hepatocytes.

The in vitro and in vivo interaction of liposomal cis-bis-neodecanoato-trans-R,R-1,2-diaminocyclohexaneplatinum++ + (II) (L-NDDP) with mouse resident peritoneal macrophages (RPM), Kupffer cells (KC), and hepatocytes was studied. The peak in vitro uptake of L-NDDP by RPM was 12.5 ng elemental platinum/100 micrograms cell protein and constituted 0.2% of the platinum available for phagocytosis. The subsequent release of platinum by RPM was rapid initially, with a 20-fold increase over the first 4 h, followed by a plateau; ultrafilterable (free) platinum constituted 50% of the total platinum released at 24 h. The retained intracellular platinum in RPM at 24 h was close to 50% of that initially present. The peak in vitro uptake of L-NDDP by KC was 11.3 ng platinum/100 micrograms cell protein and amounted to 0.2% of the platinum available for phagocytosis. The release of platinum by KC was detectable only after 4 h of incubation and increased 3-fold over the next 14 h. The ultrafilterable platinum released by KC at 18 h was 40% of the total platinum released. The retained intracellular platinum in KC at 18 h was 33% of that initially present. The peak in vitro uptake of L-NDDP by hepatocytes was almost 50 ng platinum/100 micrograms cell protein and constituted 0.8% of the platinum available for intake. Following the i.v. injection of L-NDDP, hepatocytes contained up to 6-fold higher platinum concentrations than KC. This observation was supported by transmission electron microscopy showing a higher concentration of multilamellar vesicles within hepatocytes than in KC, 5 min after i.v. injection of L-NDDP. These findings suggest that L-NDDP becomes available to the liver following i.v. injection, that both macrophages and hepatocytes play a role in the metabolism of L-NDDP, and that Kupffer cells could mediate a sustained release of platinum in the liver following the interaction with L-NDDP, indicating the potential of L-NDDP for the treatment of tumors in the liver.

Increased therapeutic efficacy induced by tumor necrosis factor alpha combined with platinum complexes and whole-body hyperthermia in rats.

This study examined the effect of a trimodality therapy of the combination of recombinant human tumor necrosis factor alpha (TNF), whole-body hypertheria (WBH), and cis-diamminedichloroplatinum(II) (CDDP) or cis-diammine-1,1-cyclobutane dicarboxylate platinum(II) (CBDCA) on a fibrosarcoma and normal tissue in F344 rats. TNF (1 x 10(5) units/kg) increased the antitumor effect of both CDDP (1.5 mg/kg) + WBH (2 h at 41.5 degrees C) and CBDCA (30 mg/kg) + WBH. Tumor growth delay, which was 1.9 days for CDDP + WBH and 2.7 days for CBDCA + WBH (P less than 0.01 compared to control), was significantly increased to 2.9 days with TNF + CDDP + WBH and 5.4 days with TNF + CBDCA + WBH (P less than 0.05). WBH, TNF, CDDP or CBDCA alone, TNF + CDDP, TNF + CBDCA, or TNF + WBH had no significant effect on tumor growth. In contrast, administration of TNF did not enhance the CDDP- or CBDCA-mediated dose limiting normal tissue toxicity. CDDP + WBH-mediated acute renal injury and CBDCA + WBH-mediated acute myelosuppression, as determined by blood urea nitrogen and peripheral blood cell counts, respectively, were not increased with the addition of TNF to either dual modality therapy. Histopathologically, addition of TNF produced no significant alterations in the kidney and the bone marrow as compared to CDDP + WBH or CBDCA + WBH. These data show that TNF enhanced the platinum + WBH-mediated antitumor effect without increasing normal tissue toxicity, suggesting that TNF may increase the therapeutic efficacy of CDDP or CBDCA combined with WBH.

Heterozygous p53(V172F) mutation in cisplatin-resistant human tumor cells promotes MDM4 recruitment and decreases stability and transactivity of p53.

Cisplatin is an important antitumor agent, but its clinical utility is often limited by multifactorial mechanism of resistance. Loss of tumor suppressor p53 function is a major mechanism that is affected by either mutation in the DNA-binding domain or dysregulation by overexpression of p53 inhibitors MDM2 and MDM4, which destabilize p53 by increasing its proteosomal degradation. In the present study, cisplatin-resistant 2780CP/Cl-16 ovarian tumor cells expressed a heterozygous, temperature-sensitive p53(V172F) mutation, which reduced p53 half-life by two- to threefold compared with homozygous wild-type (wt) p53 in parental A2780 cells. Although reduced p53 stability in 2780CP/Cl-16 cells was associated with moderate cellular overexpression of MDM2 or MDM4 (<1.5-fold), their binding to p53 was substantially enhanced (five- to eightfold). The analogous cisplatin-resistant 2780CP/Cl-24 cells, which express loss of p53 heterozygosity, retained the p53(V172F) mutation and high p53-MDM4 binding, but demonstrated lower p53-bound MDM2 that was associated with reduced p53 ubiquitination and enhanced p53 stability. The inference that p53 was unstable as a heteromeric p53(wt)/p53(V172F) complex was confirmed in 2780CP/Cl-24 cells transfected with wt p53 or multimer-inhibiting p53(L344P) mutant, and further supported by normalization of p53 stability in both resistant cell lines grown at the permissive temperature of 32.5 °C. Surprisingly, in 2780CP/Cl-16 and 2780CP/Cl-24 models, cisplatin-induced transactivity of p53 was attenuated at 37 °C, and this correlated with cisplatin resistance. However, downregulation of MDM2 or MDM4 by small interfering RNA in either resistant cell line induced p53 and restored p21 transactivation at 37 °C, as did cisplatin-induced DNA damage at 32.5 °C that coincided with reduced p53-MDM4 binding and cisplatin resistance. These results demonstrate that cisplatin-mediated p53(V172F) mutation regulates p53 stability at the normothermic temperature, but it is the increased recruitment of MDM4 by the homomeric or heteromeric mutant p53(V172F) complex that inhibits p53-dependent transactivation. This represents a novel cellular mechanism of p53 inhibition, and, thereby, induction of cisplatin resistance.

A phase I evaluation of the quinazoline antifolate thymidylate synthase inhibitor, N10-propargyl-5,8-dideazafolic acid, CB3717.

CB3717 is a quinazoline antifolate whose cytotoxic activity is mediated by inhibition of thymidylate synthase (TS). A phase I clinical trial commenced in September 1981 and 99 patients have received 296 treatments. Doses were dissolved in 0.15 mol/L NaHCO3 (pH 9.0) at a concentration of 4 mg/mL infused over one hour or in a total volume of 1 L infused over 12 hours. Doses were repeated every 3 weeks. The starting dose of 140 mg/m2 was escalated to 600 mg/m2. Renal toxicity, detected by a decrease in the 51Cr EDTA clearance, was dose-related and occurred in seven of ten patients receiving greater than 450 mg/m2. Reversible hepatic toxicity often associated with malaise occurred in 223 of 288 assessable courses (77%). Fifty-nine courses (20%) were associated with increases in alanine transaminase (ALT) levels to greater than 2.5 times the upper limit of the normal laboratory range. Increases in alkaline phosphatase levels also occurred, but were less marked. The severity and prevalence of these elevations were unaffected by the duration of the infusion. A self-limiting rash appeared in 12 patients and a radiation recall reaction was seen in two. Leukopenia developed in 17 patients (WBC less than 3 X 10(9)/L), and thrombocytopenia occurred in six patients (platelets less than 100 X 10(9)/L). The mean leucocyte nadir occurred on day 10 and was followed by recovery at 11 to 19 days. Neither the incidence nor the severity of any of these latter toxicities was dose related. The maximum tolerated dose was in the region of 600 mg/m2 with renal toxicity being dose limiting, although the inter-patient variation did not allow a precise definition. Seventy-six patients were evaluable for response. Responses occurred at doses greater than or equal to 200 mg/m2 and were ovary, one complete response (CR), one partial response (PR), seven minor responses (MR) in 30 cases; breast, two PRs and one MR in eight cases; adenocarcinoma of the lung, one MR in 5 cases; mesothelioma, one PR in five cases; and colon, two MRs in four cases. CB3717 has activity in heavily pretreated patients. The recommended phase II dose for good-risk patients is 400 mg/m2 using the one-hour infusion schedule of administration.

Carboplatin dosage: prospective evaluation of a simple formula based on renal function.

A dosage formula has been derived from a retrospective analysis of carboplatin pharmacokinetics in 18 patients with pretreatment glomerular filtration rates (GFR) in the range of 33 to 136 mL/min. Carboplatin plasma clearance was linearly related to GFR (r = 0.85, P less than .00001) and rearrangements of the equation describing the correlation gave the dosage formula dose (mg) = target area under the free carboplatin plasma concentration versus time curve (AUC) x (1.2 x GFR + 20). In a prospective clinical and pharmacokinetic study the formula was used to determine the dose required to treat 31 patients (GFR range, 33 to 135 mL/min) with 40 courses of carboplatin. The target AUC was escalated from 3 to 8 mg carboplatin/mL/min. Over this AUC range the formula accurately predicted the observed AUC (observed/predicted ratio 1.24 +/- 0.11, r = 0.886) and using these additional data, the formula was refined. Dose (mg) = target AUC x (GFR + 25) is now the recommended formula. AUC values of 4 to 6 and 6 to 8 mg/mL. min gave rise to manageable hematological toxicity in previously treated and untreated patients, respectively, and hence target AUC values of 5 and 7 mg/mL min are recommended for single-agent carboplatin in these patient groups. Pharmacokinetic modeling demonstrated that the formula was reasonably accurate regardless of whether a one- or two-compartment model most accurately described carboplatin pharmacokinetics, assuming that body size did not influence nonrenal clearance. The validity of this assumption was demonstrated in 13 patients where no correlation between surface area and nonrenal clearance was found (r = .31, P = .30). Therefore, the formula provides a simple and consistent method of determining carboplatin dose in adults. Since the measure of carboplatin exposure in the formula is AUC, and not toxicity, it will not be influenced by previous or concurrent myelosuppressive therapy or supportive measures. The formula is therefore applicable to combination and high-dose studies as well as conventional single-agent therapy, although the target AUC for carboplatin will need to be redefined for combination chemotherapy.

Evaluation of CI-973, a platinum analogue, in refractory or relapsed acute leukemia.

The purpose of the study was to define the maximally tolerated dose (MTD), major toxicities, and possible antitumor activity of CI-973 a new platinum analogue, in patients with refractory or relapsed acute leukemia. CI-973 was given as a 5-day continuous infusion every 3 to 4 weeks to patients with refractory or relapsed acute leukemia, at doses ranging from 150 mg/m2 to 1350 mg/m2 per course. Thirty-six patients were treated including 18 patients with acute myelogenous leukemia (AML), four with acute lymphocytic leukemia (ALL) and 14 with chronic myelogenous leukemia in blastic phase (CML-BP). Severe gastrointestinal and renal side-effects were the dose-limiting toxicities occurring in four of five patients treated with CI-973 1200 to 1350 mg/m2 per course. At the MTD of 1000 mg/m2 per course, three of 13 patients treated (23%) had moderate to severe nausea and vomiting, three (23%) had moderate diarrhea and one had moderate mucositis. Among 21 patients treated at > or = 1000 mg/m2 (15 AML, 6 CML-BP) no objective complete or partial responses were observed. Twelve of 18 patients (66%) with evaluable marrows on day 14 showed significant suppression of marrow blasts percentage and marrow leukemic infiltrate percentage. Tests for measurement of DNA adduct formation in leukemic cells in vivo after CI-973 therapy, and in vitro following exposure of leukemic cells to CI-973 were developed. This study defined the MTD of CI-973 to be 1000 mg/m2 by continuous infusion over 5 days every 3 to 4 weeks in patients with refractory or relapsed acute leukemia. Gastrointestinal and renal side-effects were dose-limiting. No objective responses were noted in this heavily resistant population. Correlations between CI-973-induced DNA adduct formation and individual patient response to CI-973 will help to define its role in leukemia subsets.

Expression of p53 in cisplatin-resistant ovarian cancer cell lines: modulation with the novel platinum analogue (1R, 2R-diaminocyclohexane)(trans-diacetato)(dichloro)-platinum(IV).

The compound (1R,2R-diaminocyclohexane)(transdiacetato)(dichloro)platinum(IV) (DACH-acetato-Pt) is a novel platinum-based antitumor agent with clinical potential against cisplatin-resistant disease that is under development in our laboratory. In view of the central role of the wild-type p53 tumor suppressor gene in drug-induced apoptosis, we evaluated the cytotoxicity of cisplatin and DACH-acetato-Pt in a panel of cisplatin-resistant ovarian tumor models with differing p53 status. Cisplatin was relatively more effective against mutant or null p53 cell lines (continuous drug exposure IC50, 1.2-3.3 microM) than it was against those harboring wild-type p53 (IC50, 2.8-9.9 microM). In contrast, DACH-acetato-Pt was considerably more active in wild-type p53 models (IC50, 0.17-1.5 microM) than it was in mutant or null models (IC50, 2.7-11.3 microM). Inactivation of wild-type p53 function in OVCA-429 cells by the human papillomavirus type 16 (HPV 16) E6 plasmid increased resistance to DACH-acetato-Pt by 3-5-fold, which confirmed the drug's dependence on wild-type p53 for its high cytotoxic potency. Differences between the two platinum agents were also evident in cell cycle studies: cisplatin arrested both wild-type and mutant p53 cells in G2-M, whereas DACH-acetato-Pt arrested wild-type p53 cells in G1 and mutant p53 cells in G2-M. The G1 arrest by DACH-acetato-Pt was abrogated in HPV 16 E6 transfectant clones of OVCA-429 cells. In agreement with effects on cell cycle progression, a 2-h pulse exposure to low concentrations (< or =25 microM) of DACH-acetato-Pt induced marked increases in p53 and p21Waf1/Cip1 expression in OVCA-429 cells. Cisplatin, in direct contrast, had no effect on expression of p53 or p21Waf1/Cip1 until the drug concentration was increased to 125 microM. In HPV 16 E6 transfectants of OVCA-429 cells, induction of p53 by the two agents was severely attenuated, and corresponding increases in p21Waf1/Cip1 were abrogated. This suggests that p21Waf1/Cip1 increases were p53 dependent. Collectively, the results demonstrate that DACH-acetato-Pt is very distinct from cisplatin. In particular, the greater activity of DACH-acetato-Pt in cisplatin-resistant wild-type p53 ovarian tumor models can be ascribed to its ability to more efficiently induce p53 protein and activate p53 functions.