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.

Neutron activation of NDDP, a liposomal platinum antitumor agent.

UNLABELLED: Cis-bis-neodecanoato-trans-R,R-1,2-diamminocyclohexane platinum (II) [NDDP] is a liposome-entrapped platinum compound currently, in phase II clinical trials, that has been shown to undergo intraliposomal activation. The objective of this study was to determine the feasibility of activating NDDP and using the induced radioactivity to monitor NDDP distribution and penetration. METHODS: Neutron activation analysis (NAA) was done on NDDP using the nuclear reactor at Texas A & M University, College Station, Texas. After a 3-hour irradiation, the NDDP samples were analyzed using an HPGE (high purity germanium) detector to determine the activation of the radioisotopic platinum. This was followed by HPLC-UV analysis to determine the stability of NDDP after exposure to the reactor's core. RESULTS: Platinum radioisotopes were produced along with potassium-40 and sodium-24. Irradiation did not result in any significant degradation of NDDP. CONCLUSIONS: (1) Irradiating fully synthesized NDDP is feasible for diagnostic use if a purification step is taken after the irradiation, and (2) radiation exposure is lessened by irradiating NDDP after synthesis rather than starting with high-specific-activity isotopes.

Intraliposomal chemical activation patterns of liposomal cis-bis-neodecanoato-trans-R,R-1,2-diaminocyclohexane platinum (II) (L-NDDP)-a potential antitumour agent.

L-NDDP is a liposome-entrapped platinum compound currently in phase 2 clinical trials that has been shown to undergo intraliposomal activation. The degradation/activation kinetics of liposome entrapped cis-bis-neodecanoato-trans-R,R-1,2-diamminocyclohexane platinum (II) [L-NDDP] at different conditions of pH, and temperature is presented. Liposomes were reconstituted in a solution of 0.9% sodium chloride (NaCl) in water (pH 5) at room temperature (formulation conditions currently used in the ongoing clinical trials). In the temperature experiments, L-NDDP 0.9% sodium chloride liposomes were incubated in a water-bath at 40, 60, and 80 degrees C. In the pH experiments, these solutions were compared to water, phosphate with and without chloride ion present, phosphate buffer without chloride ion at pH 3.1, 5.0, and 7.4, and glycine buffer with and without chloride ion. In 0.9% sodium chloride at room temperature, the chemical degradation/activation of liposome-bound NDDP was biphasic, with most of the degradation (approximately 45% conversion) occurring during the first hour after formation of the liposome suspension. NDDP degradation was pH dependent: when using pH 3 phosphate buffer as a reconstituting solution, liposome-bound NDDP degraded rapidly, whereas in pH 7.4 phosphate buffer it was stable for > 72 h. NDDP degradation was also temperature-dependent, the 50% point decreasing from 12 h at 25 degrees C to 9.5 h at 40 degrees C, 3.8 h at 60 degrees C, and 0.3 h at 80 degrees C when using 0.9% NaCl in water as a reconstituting solution. Using glycine buffer solution with and without NaCl at room temperature, no NDDP degradation over a 72 h period was observed at 25 degrees C; however, at 40 degrees C, only 68% NDDP remained intact at 72 h. Atomic absorption spectrophotometry (AAS) analysis of the eluting fractions after injection of L-NDDP samples reconstituted in chloride-containing and non chloride-containing solutions clearly indicated that the formation of DACH-Pt-Cl2 was only observed when chloride-containing solutions were used and was first detected at 3 h when using 0.9% NaCl in water as a reconstituting solution. These results indicate that pH and temperature, and not the presence of chloride ion, are the main factors leading to the activation of NDDP. Since 45% of NDDP is already degraded at 1 h in the same conditions, it is concluded that (1) the first active intermediates of L-NDDP formed within the liposomes are the DACH-Pt chloro-aquo and diaquo intermediates, and (2) the in vivo, antitumour activity of L-NDDP is most likely mediated by direct intracellular delivery of the active species.

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.

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.

Evaluation of toxicosis of liposome-encapsulated cis-bis-neodecanoato-trans-R,R-1,2-diaminocyclohexane platinum (II) in clinically normal cats.

OBJECTIVE: To determine adverse effects of single and multiple doses of liposome-encapsulated cis-bis-neodecanoato-trans-R,R-1,2-diaminocyclohexane platinum (II) (L-NDDP) administered IV to healthy adult cats. ANIMALS: 10 healthy adult cats. PROCEDURE: 8 cats were given a single dose of L-NDDP (at rates of 75, 100, 150, or 200 mg/m2), and 2 cats were given liposomal lipid (1,500 mg/m2). Six of the 10 cats were given doses of L-NDDP at the maximum tolerated dosage (100 mg/m2) or a lower dosage (75 mg of L-NDDP/m2) at 21-day intervals, for a total of 4 treatments. Hematologic and serum biochemical analyses, urinalyses, and physical examinations were used to monitor effects of L-NDDP. RESULTS: All cats had transient pyrexia, lethargy, vomiting (1 to 3 times/24 h), inappetence, and an acute species-specific infusion reaction that was prevented by administration of atropine-diphenhydramine. Dose-limiting toxicosis was evident as a 10-day course of lethargy, intermittent vomiting, and diarrhea. In cats given multiple doses, dose-related thrombocytopenia, cumulative myelosuppression, transient increased hepatic transaminase activity, and mild to moderate hepatic hydropic degeneration and proximal renal tubular lipidosis in excess of lipidosis expected for this species were detected. Bone marrow hypoplasia was detected in some cats that received higher doses (cumulative dosages of 300 or 400 mg of L-NDDP/m2). CONCLUSION: Cats can safely be given L-NDDP at potentially therapeutic dosages without inducing renal or pulmonary toxicoses. CLINICAL RELEVANCE: Because L-NDDP has better tumoricidal activity than cisplatin (in vivo and in vitro) and is not cross resistant, it may be similarly or more efficacious than cisplatin in humans and dogs.

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.

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.

Intraliposomal conversion of lipophilic cis-bis-carboxylato-trans-R,R-1,2-diaminocyclohexane-platinum (II) complexes into cis-bis-dichloro-trans-R,R-1,2-diaminocyclohexane-platinum (II).

Cis-bis-neodecanoato-trans-R,R-1,2-diaminocyclohexane platinum (II) (NDDP) is a lipophilic platinum complex (Pt complex) developed in a liposomal carrier. Prior studies have suggested that NDDP is a prodrug that exerts its biological activity through activation within the liposome bilayers containing dimyristoyl phosphatidylglycerol (DMPG) before in vivo administration. In order to understand the kinetics of the intraliposomal degradation/activation of different liposomal Pt complexes, we studied the effects of their structure, lipid composition, content of acidic phospholipids and size, and the effects of pH, temperature and the presence of residual chloroform on their stability, in vitro cytotoxicity, and in vivo antitumor activity. The following factors were found to enhance the intraliposomal degradation/activation of Pt complexes: (1) the size and spatial configuration of the Pt complex, (2) an acidic pH, (3) a high temperature, (4) the presence and amount of acidic phospholipids, and (5) the presence of residual chloroform. Liposome size did not affect the intraliposomal stability of different Pt complexes. Good inverse relationships between the extent of drug degradation and in vitro cytotoxicity and between the extent of drug degradation and in vivo antitumor potency were observed, thus confirming that the biological activity of these complexes is exerted through the intraliposomal formation of certain active intermediate(s). The only active intermediate that could be identified was cis-bis-dichloro-trans-R,R-1,2-diaminocyclohexane platinum(II) whose structure was confirmed by 1H, 13C, and 195Pt nuclear magnetic resonance (NMR) spectroscopy.

In vivo antitumor activity of cis-bis-neodecanoato-trans-R,R-1, 2-diaminocyclohexane platinum(II) formulated in long-circulating liposomes.

A lipophilic cisplatin derivative, cis-bis-neodecanoato-trans- R,R-1,2-diaminocyclohexane platinum (II) (NDDP), was formulated in liposomes composed of phosphatidylcholine (PC) and cholesterol (Chol) additionally containing monosialoganglioside (Gm1) or polyethyleneglycol conjugated to phosphatidylethanolamine (PEG-PE). These NDDP-containing long-circulating liposomes were examined for in vivo antitumor activity using the mouse RIF-1 solid tumor as a target residing outside the reticuloendothelial system (RES). Biodistribution studies, using C3H/HeJ mice and 111In-labelled DTPA-SA as a lipid marker, showed that the activity of GM1 and PEG-PE in prolonging the circulation times of liposomes was preserved in the presence of 3.0 mol% of NDDP in the liposome membranes. The high levels of liposomes remaining in the blood for PC/Chol/GM1 and PC/Chol/PEG3000-PE liposomes were associated with high levels of platinum in the blood as determined by atomic absorption spectrophotometry. These NDDP-containing long-circulating liposomes showed approximately a three-fold increase in tumor accumulation as compared to the conventional PC/Chol liposomes. In vitro cytotoxicity studies using RIF-1 tumor cells showed that the presence of PEG-PE, but not Gm1, significantly enhanced the cytotoxicity of liposomal NDDP. RIF-1 tumor-bearing C3H/HeJ mice were treated twice with 25 mg/kp NDDP in various liposomal formulations on days 12 and 16 after tumor cell inoculation. A significant reduction in the tumor growth rate was observed when NDDP was formulated in PC/Chol/PEG3000-PE liposomes which support both efficient tumor accumulation and enhanced cytotoxicity of liposomal NDDP. On the other hand, NDDP formulated in PC/Chol/GM1 liposomes, which display only a high tumor accumulation, had no effect on the tumor growth rate. Furthermore, NDDP formulated in dimyristoylphosphatidylglycerol (DMPG)-containing liposomes, exhibiting in vitro cytotoxicity comparable to NDDP formulated in PC/Chol/PEG3000-PE liposomes, but showing poor tumor accumulation, was also not effective. These results indicate a potential effectiveness of NDDP formulated in PEG-PE-containing liposomes for therapy of tumors in non-RES organs.

Improved antitumor activity of cis-Bis-neodecanoato-trans-R,R-1,2- diaminocyclohexaneplatinum (II) entrapped in long-circulating liposomes.

Cis-bis-neodecanoato-trans-R, R-1,2-diaminocyclohexaneplatinum (II) (NDDP) has previously been formulated in conventional liposomes and shown to be nonnephrotoxic in humans, not cross-resistant with cisplatin in different in vitro and in vivo systems, and more active than cisplatin against murine models of experimental liver metastasis. The activity was attributed to the avid uptake of liposomes by the liver. To extend to the treatment of solid tumors outside the liver, NDDP was formulated, in this study, in long-circulating liposomes composed of egg phosphatidylcholine (PC), cholesterol (Chol) and polyethyleneglycol conjugated to phosphatidylethanolamine (PEG-PE). In vitro, PC/Chol/NDDP liposomes were barely toxic to murine melanoma cells B16-F0 (IC50 = 195.6 microM) as evaluated by tetrazolium dye colorimetric assay. Inclusion of PEG3000-PE into PC/Chol/NDDP liposomes significantly enhanced their cytotoxicity to a level that was comparable to that of DMPC/DMPG/NDDP liposomes (7.3 vs. 7.9 microM). Biodistribution study indicated that PC/Chol/PEG3000-PE/NDDP liposomes could preferentially localize in s.c. melanoma in C57BL/6 mice. PC/Chol/PEG3000-PE/NDDP liposomes were much more efficient in inhibiting tumor growth in vivo than free cisplatin, free NDDP or NDDP formulated in liposomes of other lipid composition. Compared with cisplatin, the in vivo toxicities of PC/Chol/PEG3000-PE/NDDP liposomes were also significantly reduced. Furthermore, if the tumor was treated with local hyperthermia after injection of PC/Chol/PEG3000-PE/NDDP liposomes, the tumor uptake of liposomes increased by 60%. The tumor inhibitory effect of PC/Chol/PEG3000-PE/NDDP was also significantly improved when combined with local hyperthermia.

Tetravalent platinum complexes with ammine/amine carrier ligand configuration: circumvention of platinum resistance in vivo.

A platinum(II) and three platinum(IV) ammine/cycloalkylamine homologous series, the latter possessing either chloro, acetato or hydroxo axial ligands, were evaluated for efficacies in mice bearing tumor cells sensitive (leukemia L.1210/0 and reticulosarcoma M5076) or resistant to cisplatin (L1210/DDP) and tetraplatin (L1210/DACH). Within each series, which contained four homologs, potency increased (optimal dose decreased) as alicyclic ring size increased incrementally from cyclopropane to cyclohexane. All analogs were active at maximally tolerated doses against L1210/0 (%T/C = 125-426), with good associated therapeutic ratios of 2 to > 8 that, like the therapeutic index, provided indications of the drug's safety margin. Most complexes had activities that were similar to cisplatin (%T/C = 239) and tetraplatin (%T/C = 310). Antitumor activities were seen for all four platinum(II) complexes against L1210/DDP cells (%T/C = 133-167). In the three platinum(IV) series, on the other hand, only cyclopentane (C5) and cyclohexane (C6) analogs met or exceeded the minimum criterion for activity. These activities were similar to that seen with the positive control agent tetraplatin (%T/C = 133), but higher than that of cisplatin (%T/C = 94). Long-term survivors, which were frequently observed with these complexes in the L1210/0 model, were also seen in the L1210/DDP model, but to a lesser extent. Against L1210/DACH cells, which were sensitive to cisplatin (%T/C = 155), but resistant to tetraplatin (%T/C = 113), the C5 and C6 congeners in the platinum(IV) series were effective with %T/C in the range 148-189, while corresponding members in the platinum(II) series were only marginally active. In the solid M5076 model, complexes C5 in platinum(II) and in the acetato- and hydroxoplatinum(IV) series, and C6 from the hydroxo-platinum(IV) series, were as effective or more effective than cisplatin, which itself gave a tumor growth delay of 27.5 days. In summary, the results indicate that alicyclic ring size and, in the platinum(IV) series, axial ligand, are important modulators of efficacies of ammine/cycloalkylamine platinum congeners in both sensitive and platinum-resistant models. However, the cyclopentylamine or cyclohexylamine carrier ligand with acetato or hydroxo axial ligands in the platinum(IV) configuration are optimal combinations for circumventing both cisplatin and tetraplatin resistances.