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.

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.

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.

Upregulation of p27 and its inhibition of CDK2/cyclin E activity following DNA damage by a novel platinum agent are dependent on the expression of p21.

The cisplatin analogue 1R,2R-diaminocyclohexane(trans-diacetato)(dichloro)platinum(IV) (DAP) is a DNA-damaging agent that will be entering clinical trials for its potent cytotoxic effects against cisplatin-resistant tumour cells. This cytotoxicity may reside in its ability to selectively activate G1-phase checkpoint response by inhibiting CDKs via the p53/p21 pathway. We have now evaluated the role of another CDK inhibitor p27 as a contributor to DAP-mediated inhibition of G1-phase CDK2 activity. Our studies in ovarian A2780 tumour cells demonstrate that p27 levels induced by DAP are comparable to or greater than those seen for p21. The induction of p27 is not through a transcriptional mechanism, but rather is due to a four-fold increase in protein stabilisation through a mechanism dependent on p21. Moreover, DAP-induced p21 promoted the selective increase of p27 in the CDK2 complex, but not in CDK4 complex, and this selective increase contributed to inhibition of the CDK2 kinase activity. The inhibited complex contained either p27 or p21, but not both, with the relative levels of cyclin E associated with p27 and p21 indicating that about 25% of the inhibition of CDK2 activity was due to p27 and 75% due to p21. This study provides the first evidence that p27 upregulation is directly attributable to activation of the p53/p21 pathway by a DNA-damaging agent, and promulgates p53/p21/p27 axis as a significant component of checkpoint response.

Phase I clinical and pharmacological study of intraperitoneal cis-bis-neodecanoato( trans- R, R-1, 2-diaminocyclohexane)-platinum II entrapped in multilamellar liposome vesicles.

PURPOSE: To perform a phase I study of intraperitoneal cis-bis-neodecanoato ( trans- R, R-1, 2-diaminocyclohexane)-platinum II entrapped in multilamellar vesicles (L-NDDP) for peritoneal carcinomatosis or sarcomatosis. METHODS: Eligible patients had normal renal, hematologic, and liver functions. Laparoscopy was performed on the first two courses for evaluation, adhesiolysis, and chemotherapy administration. Afterwards, chemotherapy was administered through a peritoneal catheter. Up to six courses were allowed. Peritoneal imaging with technetium-labeled sulfur colloid was used to determine adequate distribution prior to each course. Volunteering patients underwent pharmacokinetics studies during the second course. RESULTS: Fifteen of 16 registered patients, seven women and eight men (median age 53 years (range 26-76) and median performance status of 1) were assessable. Diagnoses were: malignant mesothelioma (six patients), signet ring cell (three), colon adenocarcinoma, pseudomyxoma peritonei, gastrointestinal stromal tumor (two each), and ovarian carcinoma (one). Median number of courses was two (range, one to six) Dose-limiting toxicity symptoms were fatigue and abdominal pain. Hematologic toxicities were minimal. Peri-operative complications included one colonic perforation requiring primary closure, a peritoneal catheter malfunction, a port site hematoma, and an ascites leak requiring re-suture. Five patients survived at least 3 years. Pharmacokinetics studies indicated a rapid but low absorption of drug into the systemic circulation, with a prolonged retention of platinum in the plasma compartment. Peritoneal L-NDDP exposure was 17 to 49-times greater than in the plasma compartment. CONCLUSIONS: Peritoneal cavity exposure to L-NDDP is prolonged, and systemic absorption is limited, yielding a high peritoneal/plasmatic ratio. The recommended dose for phase II studies is 400 mg/m2 every 28 days.

Bimodal effects of 1R,2R-diaminocyclohexane(trans-diacetato)(dichloro)platinum(IV) on cell cycle checkpoints.

1R,2R-Diaminocyclohexane(trans-diacetato)(dichloro)-platinum(IV) (DACH-acetato-Pt) is a novel platinum-based agent that is highly effective against cisplatin-resistant ovarian tumor cells. To probe its cellular mechanism, the effects of DACH-acetato-Pt (0-6.4 microM) on cell cycle checkpoints were examined using the ovarian cancer A2780 cell line as the model system. We found that DACH-acetato-Pt at > or =0.2 microM dramatically inhibited cell growth and induced cell death. At concentrations < or =0.6 microM (low effective concentrations), DACH-acetato-Pt specifically induced G(1) phase arrest by selectively inhibiting cyclin-dependent kinase 4 (Cdk4) and Cdk2 activities. The Cdc2 activity, which regulates G(2)-M phase progression, was unaffected by the drug at these concentrations. At concentrations >0.6 microM (high effective concentrations), DACH-acetato-Pt first transiently inhibited S-phase progression and then blocked cell cycle progression at both G(1) and G(2) phases. These cell cycle effects were associated with sequential inhibitions of Cdk2/cyclin A activity, Cdk4 and Cdk2 activities, and Cdc2 kinase activity. Following the cell cycle effects, both the low and high effective concentrations of DACH-acetato-Pt induced cell death through apoptosis. These results indicate that DACH-acetato-Pt activates multiple cell cycle checkpoints in a bimodal manner and suggest that the cell cycle effects demonstrated in these studies may be linked to its ability to induce apoptosis.

Synthesis, characterization and cytotoxicity of new platinum(IV) axial carboxylate complexes: crystal structure of potential antitumor agent [PtIV(trans-1R,2R-diaminocyclohexane)trans(acetate)2Cl2].

A series of new platinum(IV) complexes of the type [PtIV(DACH)trans(L)2Cl2] (where DACH = trans-1R,2R-diaminocyclohexane, and L = acetate, propionate, butyrate, valerate, hexanoate, or heptanoate) bearing the carboxylate groups in the axial positions have been synthesized and characterized by elemental analysis, IR, and 195Pt NMR spectroscopy. The crystal structure of the analogue [PtIV(DACH)trans(acetate)2Cl2] was determined by single crystal X-ray diffraction method. There were two crystallographically independent molecules, both of which lie on crystallographic two-fold axes. The bond lengths and bond angles of both the molecules were the same within the experimental error. The compound crystallizes in the monoclinic space group C2, with a = 11.180(2) A, b = 14.736(3) A, c = 10.644(2) A, beta = 112.38(3) degrees, Z = 4 and R = 0.0336, based upon a total of 1648 collected reflections. In this complex, the platinum had a slightly distorted octahedron geometry owing to the presence of a geometrically strained five-member ring. The two adjacent corners of the platinum plane were occupied by the two amino nitrogens of DACH, whereas the other two equatorial positions were occupied by two chloride ions. The remaining two axial positions were occupied by the oxygens of acetate ligands. The DACH ring was in a chair configuration. An intricate network of intermolecular hydrogen bonds held the crystal lattice together. These analogues were evaluated in vitro and demonstrated cytotoxic activity against the human ovarian 2008 tumor cell line (IC50 = 0.001-0.06 microM). Structure-activity study revealed that activity was highest for the analogue where L = butyrate.

EGF receptor and p21WAF1 expression are reciprocally altered as ME-180 cervical carcinoma cells progress from high to low cisplatin sensitivity.

Cell cycle regulators and signal transduction pathways can influence apoptotic sensitivity of tumor cells, and we previously described an association between EGFr overexpression, reduced DNA repair activity, and increased apoptotic sensitivity of ME-180 cervical carcinoma cells toward cis-diammedichloroplatinum (cDDP; K. Nishikawa, et al., Cancer Res., 52: 4758-4765, 1992). In the present study, the characteristics of ME-180 cells selected for high or low apoptotic sensitivity to cDDP (or camptothecin) were examined and compared to determine whether signal transduction components and cell cycle regulation were distinct in these isogenic drug response variant populations. As ME-180 cells progressed from high to low cDDP sensitivity [IC50 approximately 80 ng/ml in cDDP sensitive (PT-S) to approximately 2000 ng/ml in cDDP-resistant (Pt-R) cells], there was a significant decrease in EGFr expression that paralleled the relative reduction in cDDP apoptotic responsiveness (approximately 30-fold). cDDP-resistant cells had the slowest rate of growth and more effectively reduced DNA adduct levels following cDDP exposure than parental cells. Cellular levels of the cell cycle inhibitor p21WAF1 inversely correlated with cDDP responsiveness with high levels of p21WAF1 expressed in drug-resistant Pt-R cells in the absence of elevated p53. cDDP stimulated a 2-fold increase in p53 levels in both drug-sensitive and drug-resistant cells but caused a delayed reduction in p21WAF1 levels, suggesting p53-independent regulation of p21WAF1 in ME-180 cells. Activation of EGFr in Pt-R cells stimulated cell cycle progression (2-fold), reduced p21WAF1 levels (>2-fold), and increased sensitivity to cDDP (3-fold), suggesting that receptor signaling enhanced the efficacy of cDDP to induce cell death by relieving cell cycle restriction. These results demonstrate that the transition of ME-180 cells from a drug-sensitive to drug-resistant phenotype correlates with reciprocal changes in EGFr and p21WAF1 expression and provides additional evidence that the pathways controlled by these proteins may contribute to some forms of drug resistance.

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.

Overexpression of edg-2/vzg-1 induces apoptosis and anoikis in ovarian cancer cells in a lysophosphatidic acid-independent manner.

Lysophosphatidic acid (LPA) is one of the major growth factors in ascites from ovarian cancer patients and appears to play an important role in proliferation, survival, and invasion of ovarian cancer cells. Recently, several groups have shown that Edg-2, which belongs to the G-protein coupled receptor family, is a functional LPA receptor. Northern blot analysis showed that most ovarian cancer cell lines express Edg-2. Edg-2 expression was especially high in the cisplatin-resistant and slowly proliferating 2780cp cell line and was almost absent from the cisplatin-sensitive and rapidly proliferating A2780 cell line. We thus assessed whether Edg-2 could contribute to changes in cell viability, cell proliferation, or cisplatin resistance. Stable overexpression of Edg-2 in A2780 cells induced an exogenous LPA-independent decrease in proliferation but did not alter cisplatin sensitivity. The LPA-independent decrease in growth rate induced by overexpression of Edg-2 could be explained, at least in part, by Edg-2-induced apoptosis rather than by effects on cell cycle progression. In agreement with the results in stably transfected A2780 cells, transient expression of Edg-2 in Jurkat T cells also induced apoptosis. When cells were separated from the extracellular matrix, they underwent a specialized form of apoptosis called anoikis, which is particularly important in survival of cells in the circulation during metastasis. A2780 cells engineered to overexpress Edg-2 were particularly sensitive to anoikis. These observations suggest that Edg-2 may be a negative regulator for ovarian epithelial cell growth and metastasis.

Role of p53 in the ability of 1,2-diaminocyclohexane-diacetato-dichloro-Pt(IV) to circumvent cisplatin resistance.

Several reports indicate that the mechanism of resistance to cisplatin is multifactorial. However, DNA damage tolerance appears to be the more significant mechanism. It is clear that resistance in general is a major clinical concern, and a number of approaches have been taken to circumvent this clinical impediment. One approach is through analog development, and we have identified 1,2-diaminocyclohexane-diacetatodichloro-platinum(IV) as an analog with activity in cisplatin resistance. The activity is greatest against ovarian tumor cell lines where the latent, non-inducible wild-type p53 function can be reactivated by the analog. This functional activation of p53 also corresponds to a reduced threshold for tolerance to DNA damage induced by the analog. Interestingly, cell lines with mutant or null p53 are cross-resistant to the analog. The data indicate that cisplatin resistance due to an increase in DNA damage tolerance can arise through a loss of p53 function, and that functional activation of latent wild-type p53 by the analog facilitates cell death and circumvents this resistance mechanism.

Preparation, characterization, and antitumor activity of new cisplatin analogs with homopiperazines: crystal structure of [PtII(1-methylhomopiperazine)(methylmalonato)].2H2O.

A series of new platinum(II) and (IV) complexes with homopiperazine have been synthesized and characterized by elemental analysis, infrared, and 195Pt nuclear magnetic resonance spectroscopic techniques. The complexes are of two types: [PtIILX] (where L = homopiperazine (hpip), 1-methylhomopiperazine (mhpip), or 1,4-dimethylhomopiperazine (dmhpip), and X = 1,1-cyclobutanedicarboxylato (CBDCA), or methylmalonato ligand) and [PtIV(L-)trans-(Y)2Cl2] (where Y = hydroxo, acetato, or chloro ligand). Among the complexes synthesized, the crystal structure of [PtII(mhpip)(methylmalonato)].2H2O was determined by the single crystal X-ray diffraction method. The crystallographic parameters were orthorhombic, P2(1)2(1)2(1) (no. 19), a = 7.2014(14), b = 7.3348(15), c = 26.971(5) A, and Z = 4. The structure refinements converged to R1 = 0.0641 and wR2 = 0.1847. In this complex, platinum has a slightly distorted square planar geometry with the two adjacent corners being occupied by two nitrogens of the mhpip ligand, whereas the remaining cis positions are coordinated with two oxygen atoms of the methylmalonato group. The mhpip ligand is in a boat conformation and forms five and six membered chelating rings with platinum. The intricate network of intermolecular hydrogen bonds holds the crystal lattice together. Some of these synthesized cisplatin analogs have good in vitro cytotoxic activity against the cisplatin-sensitive human ovarian A2780 (IC50 = 0.083-17.8 microM) and the isogenic cisplatin-resistant 2780CP (IC50 = 20.1-118.1 microM) cell lines.

Synthesis, characterization, and antitumor activity of new platinum(IV) trans-carboxylate complexes: crystal structure of [Pt(cis-1,4-DACH)trans-(acetate)2Cl2].

A series of novel platinum(IV) cisplatin analogues of the type [Pt(cis-1,4-DACH)trans-(L)2Cl2] (where cis-1,4-DACH = cis-1,4-diaminocyclohexane and L = acetate, propionate, butyrate, valerate, hexanoate, heptanoate, octanoate, nonanoate, or decanoate) was synthesized and characterized by elemental analysis, IR, 13C-NMR, and 195Pt-NMR spectroscopy. The structure of [Pt(cis-1,4-DACH)trans-(acetate)2Cl2] (1) was determined by X-ray crystallography. The crystals were monoclinic, space group P2(1)/n (no. 14) with a = 10.193(2), b = 10.687(2), c = 14.265(3) A, beta = 99.67(3) degrees, Z = 4. The total reflections collected were 2556. The structure refinement converged to R1 = 0.0539 and wR2 = 0.1531. In this complex, platinum has distorted octahedral geometry, and cis-1,4-DACH is in a unique twist-boat configuration. cis-1,4-DACH forms a seven-member chelating ring with platinum, leading to considerable strain in bidentate DACH binding. The strain is evidenced by a large 126.5(9) degrees C-N-Pt angle. The N-Pt-N angle is expanded to 97.4(5) degrees owing to geometric constraints of the cis-1,4-DACH geometry. Three lower homologs of the cis-1,4-DACH-Pt(IV) series were tested in the murine L1210/0 leukemia model for antitumor activity. The results indicate that activity decreases in ascending the homologous series, and that the activity of two of the complexes is substantially better than that of cisplatin with respect to increase in life span and cures.

Cytotoxicity and tolerance to DNA adducts of alicyclic mixed amine platinum(II) homologs in tumor models sensitive and resistant to cisplatin or tetraplatin.

Structure-cytotoxicity relationships for six alicyclic cis-(NH3)(R-NH2)Cl2Pt(II) complexes, where R=C3H5, C4H7, C5H9, C6H11, C7H13 and C8H15 (complexes abbreviated C3, C4, C5, C6, C7 and C8, respectively), were evaluated against four sensitive (L1210/0, A2780, FSaIIC and Colon 26), two cisplatin-resistant (L1210/DDP and 2780CP) and two tetraplatin-resistant (L1210/DACH and 2780TP) murine and human tumor cell lines. The studies demonstrated that in general the structure of C6 was optimal within the homologous series for cytotoxic potency against these tumor models. Biochemical pharmacologic studies indicated that the greater sensitivity of cells to C6 could be correlated with their low tolerance to DNA damage induced by this homolog. These results provide evidence for the alicyclic ring size as a structural determinant of DNA damage tolerance and anti-tumor activity in sensitive and 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.

Physiologic and pathologic drug resistance in ovarian carcinoma--a hypothesis based on a clonal progression model.

Conventional models for successful chemotherapy suggest that early-stage ovarian cancer should be more responsive to cytotoxic drugs than advanced disease, that multiple drugs will prove more efficacious than single agents and that more intensive chemotherapy will prove superior to conventional doses. The purpose of numerous clinical studies has been to test these predictions and the results often fall short of the expectations. In trials to date, adjuvant chemotherapy for high-risk, early-stage ovarian cancer has provided only a modest, equivocal increase in disease-free survival. Combination chemotherapy produces higher response rates but this has not consistently resulted in prolonged survival. Dose intensification with high-dose chemotherapy increases rate of response further, but most responses are of short duration. The objective of this review is to integrate clinical and molecular biological observations into a novel model of drug resistance. We hypothesize that drug sensitivity is an acquired characteristic of neoplastic cells, and that there are two broad cellular forms of resistance to cytotoxic drugs. 'Physiological drug resistance' is a cellular state when drug sensitivity of cancer cells is similar to that of the corresponding normal tissue. This form of drug resistance can precede the acquisition of drug sensitivity and may be predominant in the early stages of neoplastic progression. A distinct, 'pathological drug resistance' can reappear later during tumor progression or during chemotherapy as a result of increased detoxification, upregulation of repair pathways or defective apoptosis. Tumors are formed of heterogeneous cell populations and in terms of drug sensitivity three distinct cell types may be present: drug-sensitive, physiologically drug-resistant and pathologically drug-resistant. Clinical response to chemotherapy is determined by the relative contribution of these different cell populations to the total cellular mass. Depending on the predominant type of surviving population, distinct patterns of clinical failure may develop that require different treatment strategies for optimal management. For chemosensitive cells that survived insufficient chemotherapy, consolidation with further, perhaps, high-dose chemotherapy is a rational option. For pathologically drug-resistant cells, pharmacological manipulation of drug resistance, and signaling pathways in combination with chemotherapy could be exploited. For physiologically drug-resistant cells, non-chemotherapy-based, 'chemopreventive' strategies to arrest tumor progression may prove beneficial.

A phase II and pharmacokinetic study of enloplatin in patients with platinum refractory advanced ovarian carcinoma.

This was a study of enloplatin in 18 evaluable patients with platinum refractory ovarian cancer. They received an i.v. infusion of enloplatin over 1.5 h without prehydration every 21 days. One patient had a partial response (6%; 95% CI 0-26%) lasting 2.8 months. The median survival was 9.4 months (95%; CI 5.1-19.7%). Neutropenia was the dose-limiting toxicity. Nephrotoxicity was manageable. Enloplatin is the major form of the free drug in plasma. However, 13.5 h after initiation of treatment, 85% of the drug in plasma is protein bound. Elimination of the drug is mainly renal. Enloplatin pharmacokinetics is similar to that of carboplatin. Thus, the plasma pharmacokinetics of enloplatin is dictated by the cyclobutanedicarboxylato (CBDCA) ligand and not the novel amino ligand.

Phase I clinical and pharmacological study of liposome-entrapped NDDP administered intrapleurally in patients with malignant pleural effusions.

cis-Bis-neodecanoato-trans-R,R-1,2-diaminocyclohexane platinum(II) (NDDP) is a lipophilic non-cross-resistant platinum compound formulated in large multilamellar liposomes (1-3 micrometer). The maximum tolerated dose (MTD) of liposomal-entrapped NDDP (L-NDDP) administered i.v. in humans is 300 mg/m2, and myelosuppresion is the dose-limiting toxicity. L-NDDP administered i.p. is absorbed slowly from the peritoneal cavity of rats. Recently, i.p. cisplatin has been shown to be superior to i.v. cisplatin in improving the survival of patients with ovarian carcinoma and minimal residual disease. We conducted a Phase I study to determine the MTD, side effects, kinetics of absorption into the systemic circulation, and preliminary antitumor activity of L-NDDP administered intrapleurally in patients with free-flowing malignant pleural effusions. Twenty-one patients were treated with escalating doses of L-NDDP by intrapleural administration over 30 min every 21 days. Fourteen patients had adenocarcinoma of the lung, 5 patients had malignant pleural mesothelioma (MPM), and 2 patients had ovarian carcinoma. The dose-limiting toxicity of L-NDDP was chest pain secondary to chemical pleuritis, which was severe in three of four patients treated at 550 mg/m2. The MTD was 450 mg/m2. At this dose, the only toxicity observed was grade 1-2 nausea and vomiting presenting 6-8 h after drug administration. Neither myelosuppression nor nephrotoxicity was observed. Loculation of residual pleural fluid with continued decrease over a period of weeks to months was observed in seven patients; in one of these patients (MPM), the pleural effusion disappeared without evidence of recurrence for 19 + months, and in six patients (three adenocarcinoma of the lung, two MPM, and one ovarian carcinoma), the pleural effusion was reduced by >50% for 5+, 10+, 18+, 8, 5+, and 2+ months. Plasma pharmacokinetic studies showed that the absorption of L-NDDP from the pleural cavity was rapid during the first 2 h, with levels becoming steady (bioavailable or free platinum) or increasing slowly (total plasma platinum) between 6 and 24 h after administration. Urinary excretion was negligible (1-3%). We conclude that: (a) the MTD of intrapleural L-NDDP is 50% higher than the MTD after i.v. administration; (b) intrapleural L-NDDP causes mild nausea and vomiting and no myelosuppression at the MTD; and (c) the absorption of L-NDDP into the systemic circulation is much slower than that of the parent compound cisplatin. Because of the favorable depot effect, lack of systemic toxicity, and control of the pleural effusion in three of five patients with MPM, a disease similar to ovarian carcinoma in that it tends to remain confined to a body cavity, a Phase II study of intrapleural L-NDDP administered in patients with MPM is in progress.

Chemical and biological studies on a series of novel (trans-(1R,2R)-, trans-(1S,2S)-, and cis-1,2-diaminocyclohexane)platinum(IV) carboxylate complexes.

A series of novel platinum(IV) complexes of the type DACH-PtIV-trans-(Y)2-cis-X (where DACH = trans-(1R,2R)-, trans-(1S,2S)-, or cis-1,2-diaminocyclohexane; X = diacetate, oxalate, malonate, methylmalonate, cyclobutanecarboxylate (CBCA), or 1,1-cyclobutanedicarboxylate (CB-DCA); and Y = acetate or trifluoroacetate) has been synthesized and characterized by elemental analysis, IR, and 195Pt-NMR spectroscopy. The compounds have been tested against cisplatin-sensitive L1210/0 leukemia, cisplatin-resistant L1210/DDP leukemia, and M5076 reticulosarcoma cell lines in vivo. Most of these analogs displayed reasonable activity against L1210/0 cells (%T/C = 135 to > 700). There were no gross differences in activity between analogs containing isomers of DACH. Selected compounds were evaluated against L1210/DDP tumor models in which they demonstrated reduced but significant activity compared with activity in the L1210/0 model. Interestingly, complex 20, PtIV(trans-1R,2R-DACH)-trans-(acetate)2-methylmalonate, was highly active against M5076, although it had no activity against the L1210 lines. The results demonstrate that specific combinations of axial and equatorial carboxylate ligands, together with the DACH carrier ligand, can favorably modulate the antitumor properties of platinum complexes and enhance circumvention of cisplatin resistance.

Synthesis, characterization, and antitumor activity of a series of novel cisplatin analogs with cis-1,4-diaminocyclohexane as nonleaving amine group.

A series of novel platinum(II) complexes of the type PtII(cis-1,4-DACH)X (where cis-1,4-DACH = cis-1,4-diaminocyclohexane and X = dichloro, sulfato, oxalato, malonato, methylmalonato, tartronato, or 1,1-cyclobutanedicarboxylato [CBDCA) ligand and novel platinum(IV) complexes of the types PtIV(cis-1,4-DACH)trans(Y)2Cl2 (where Y = chloro, hydroxo, acetato, propionato, or butyrato ligand) and PtIV(cis-1,4-DACH)trans(Cl)2(X) (where X = oxalato or CBDCA ligand) were synthesized and characterized by elemental analysis, infrared, and 195Pt NMR spectroscopic techniques. Platinum complexes had good in vitro cytotoxic activity against murine leukemia L1210/0 (IC50 = 0.0032-1.60 microM) and human ovarian A2780 (IC50 = 0.045-0.430 microM) cell lines.