Metabolism, protein binding and in vivo activity of the oral platinum drug JM216 and its biotransformation products.

This study evaluates the metabolism of the oral platinum drug JM216 [bis(acetato) amminedichloro (cyclohexylamine)platinum (IV)] following oral administration to Balb C- mice. JM216 was detectable 1 h post administration in mice but not in patients. Also, a late eluting metabolite observed in patients was not detected in mice. JM118 [amminedichloro(cyclohexylamine) platinum (II)], the platinum II species which is the major metabolite in patients was rapidly converted following i.v. administration to a compound having the same retention time as JM383 [bis(acetato)ammine(cyclohexylamine)dihydroxo platinum(IV)] indicating that the levels of JM383 following JM216 administration have probably been overestimated. The metabolite D observed in patients for which a structure has not been assigned, was also detected in mice. However, it did not originate from any of the identified biotransformation products. The protein binding evaluated in plasma, and buffer with physiological levels of albumin and globulin showed that only Platinum (II) species have significant binding and that Jm118 showed the same affinity to albumin and globulin (t 1/2 of 4.2 and 4.8 h) while cisplatin bound more readily to albumin (t 1/2 3.4 h) than globulin IV (t 1/2 8.2 h). JM216 itself failed to bind to either of the proteins tested indicating extensive reduction in patients, animals or plasma incubation medium. JM118 and JM518 [bis(acetato)amminechloro(cyclohexylamine) hydroxoplatinum (IV)] were significantly more active than the platinum IV complexes JM216 and JM383 when given i.p. to ADJ/PC6 plasmacytoma bearing mice (ED90 of 1.0 and 0.4 versus 5.7 and 4.2 mg/kg, TI (therapeutic index) of 14 and 37 versus 5.3 and 4.2). When given orally, JM216 was the most potent drug (ED90 of 5.8 versus 11,12 and 42 mg/kg and TI of 57 versus 12 12 and 16) for JM118 and JM383. There data indicates that JM216 biotransformation products are potent but that the levels of JM383 determined in our analytical conditions could have been overestimated.

Novel trans platinum complexes: comparative in vitro and in vivo activity against platinum-sensitive and resistant murine tumours.

Two pairs of cis/trans platinum complexes, JM118 (cis-ammine(cyclohexylamine) dichloro platinum(II)) and its trans counterpart, JM334 and JM149 (cis-ammine(cyclohexylamine) dichloro-dihydroxo platinum(IV)) and its trans counterpart JM335 have been evaluated (both in vitro and in vivo) against two murine tumour models of historical importance in the discovery of novel platinum drugs; the ADJ/PC6 plasmacytoma and the L1210 leukaemia and sublines selected for resistance to platinum drugs. In vitro, results showed that the trans complexes induced comparable growth inhibitory properties to those observed for cisplatin and their respective cis isomers. Moreover, retention of activity was observed in a series of 5 acquired platinum drug (cisplatin, carboplatin, iproplatin, tetraplatin and JM149)-resistant L1210 sublines whereas at least partial cross-resistance was observed to the cis isomer JM149 in the acquired carboplatin and iproplatin-resistant lines (in addition to being 11-fold resistant in the line selected for resistance to JM149 itself). In vivo, JM355 showed activity against both the ADJ/PC6 and L1210 models of acquired cisplatin resistance. Furthermore, JM355 was active against an ADJ/PC6 subline possessing resistance to iproplatin and a L1210 subline possessing resistance to its cis isomer JM149. Interestingly, the trans platinum(II) counterpart of JM335(JM334) was inactive in vivo. These data indicate that the trans platinum(IV) complex JM335 possess several in vitro growth inhibitory- and in vivo antitumour properties which are distinct from those observed for cisplatin (or its cis isomer). Thus, JM335 contravenes the original structure-activity rules determined for platinum-containing compounds and, because of its level of activity against cisplatin-resistant tumours, establishes the complex as of interest in the search for new platinum drugs active against cisplatin-resistant disease.

Biotransformation of the platinum drug JM216 following oral administration to cancer patients.

This study evaluates the metabolic profile of JM216 [bis(acetato)ammine-dichloro(cyclohexylamine) platinum(IV)], the first orally administrable platinum complex, in plasma ultrafiltrates of 12 patients (n = 2-4 time points per patient) following different doses of drug (120, 200, 340, 420, 560 mg/m2). The biotransformation profile was evaluated by high-performance liquid chromatography (HPLC) followed by atomic absorption spectrophotometry (AA). The AA profiles were compared with those previously identified by HPLC on line with mass spectrometry (HPLC-MS) in plasma incubated with JM216. A total of six platinum peaks (Rt = 5.5, 7.2, 10.6, 12.4, 15.6, and 21.6 min, respectively) were observed in patients' plasma ultrafiltrate samples, of which only four appeared during the first 6 h post-treatment. Four of these coeluted with those observed and identified previously in plasma incubation medium. No parent JM216 was detected. The major metabolite seen in patients was the Pt II complex JM118 [cis-amminedichloro-(cyclohexylamine)platinum(II)] and was observed in all the patients. Interestingly, the second metabolite was shown to coelute with the Pt IV species JM383 [bis-acetatoammine(cyclohexylamine)dihydroxoplatinum (IV)]. Both JM118 and JM383 were identified by HPLC-MS in a clinical sample. Peak C, which was a minor product (less than 5% of the free platinum), coeluted with JM559 [bis-acetatoammine-chloro(cyclohexylalamine)hydroxoplatin um(IV)]. The cytotoxicity profile of all three metabolites in a panel of cisplatin-sensitive and -resistant human ovarian carcinoma cell lines was very close to that of the parent drug. In addition, the concentrations of JM118 reached in patients' plasma ultrafiltrate were comparable with the cytotoxic levels of the compound determined in the ovarian carcinoma panel of cell lines. Two metabolites were seen in patients but not in the in vitro incubation medium, suggesting the involvement of a possible enzymatic reaction. Thus, the biotransformation profile following oral administration of JM216 shows a variety of Pt(IV) and Pt(Il) metabolites in plasma that differ significantly from other systemically applied platinum drugs.

Determination of metabolites of a novel platinum anticancer drug JM216 in human plasma ultrafiltrates.

The present study describes the application of on-line liquid chromatography-electrospray ionisation in conjunction with a high resolution magnetic sector mass spectrometer to identify metabolites of a platinum(IV) anticancer drug JM216 [bis(acetato)amminedichloro(cyclohexylamine)platinum(IV)] in human plasma. Four metabolites were identified following incubation of JM216 in human plasma: JM118 [amminedichlorocyclohexylamineplatinum(II)], a platinum(II) complex; JM383 [bis(acetato)amminedihydroxo(cyclohexylamine)platinum(IV)]; JM518 [bis(acetato)amminechloro(cyclohexylamine)hydroxoplatinum (IV)] and its isomer JM559. The platinum complexes mass spectra were dominated by the natriated [M + Na]+ ion. Elemental compositions of these natriated ions were confirmed by accurate mass measurement on a magnetic sector mass spectrometer in the course of LC/MS analysis. This study demonstrates the capability of direct LC-ESI/MS with accurate mass measurement for analysis of platinum complexes in biological samples. Our results suggest that LC-ESI/MS is a powerful technique for structure elucidation of novel metabolites, and could make valuable contributions to drug metabolism research.

DNA-binding properties of novel cis- and trans platinum-based anticancer agents in 2 human ovarian carcinoma cell lines.

We have investigated the comparative initial DNA binding properties of 7 platinum-based anticancer drugs: 5 cis-oriented compounds, cisplatin, tetraplatin (Ormaplatin), JM118 [cis ammine dichloro (cyclohexylamine) platinum (II)], JM216 [bisacetato cis ammine dichloro (cyclohexylamine) platinum (IV)] and JM149 [cis ammine dichloro (cyclohexylamine) trans dihydroxo platinum (IV)], and 2 trans-oriented compounds, transplatin and JM335 [trans ammine dichloro (cyclohexylamine) dihydroxo platinum (IV)] in SKOV-3 and CHI human ovarian carcinoma cells. Unlike transplatin, the trans complex JM335 was comparably cytotoxic to its cis isomer JM149 and cisplatin. No significant correlation was observed between levels of total platinum bound to DNA after exposure to the 7 drugs and cytotoxicity in either cell line. Using a competitive enzyme-linked immunoabsorbent assay, DNA extracted from CH1 cells exposed to the 5 cis platinum drugs was recognized by the monoclonal antibody ICR4 (raised against DNA platinated by cisplatin) in the order JM118 > cisplatin > JM216 > tetraplatin > JM149; a strong positive correlation which just attained statistical significance was observed between recognition by ICR4 and cytotoxicity. In contrast, DNA extracted from CH1 cells exposed to the trans platinum drugs transplatin and JM335 was no more immunoreactive than control DNA. Using alkaline elution, interstrand cross-link levels after exposure to drug did not correlate with cytotoxicity in either cell line. The 5 cis drugs formed interstrand cross-links in both cell lines, whereas transplatin formed very low levels in SKOV-3 and undetectable levels in CH1. JM335 was efficient at forming interstrand cross-links in SKOV-3 but, notably, none were observed in CH1. In contrast, in the CH1 cells, single-strand breaks were observed with JM335 (but not with any other drug). The novel trans complex JM335 was unique, among the platinum drugs studied, in its ability to form both DNA interstrand cross-links and single strand breaks (DNA lesion formation being cell line dependent), a property which may account for its cytotoxicity.

Metabolic studies of an orally active platinum anticancer drug by liquid chromatography-electrospray ionization mass spectrometry.

Bis(acetato)amminedichloro(cyclohexylamine) platinum(IV) (JM216) is a new orally administered platinum complex with antitumor properties, and is currently undergoing phase II clinical trials. When JM216 was incubated with human plasma ultrafiltrate, 93% of the platinum species were protein-bound and 7% were unbound. The unbound platinum complexes in the ultrafiltrates of human plasma were analysed using a liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method. Apart from the parent drug, four metabolites were identified and characterised. These include JM118 [amminedichloro(cyclohexylamine) platinum(II)], JM383 [bis(acetato)ammine(cyclohexylamine)dihydroxo platinum(IV)] and the two isomers JM559 and JM518 [bis(acetato)amminechloro(cyclohexylamine) hydroxo platinum(IV)]. Their elemental compositions were determined by accurate mass measurement during the LC analysis, to confirm their identities. Quantitation of these metabolites by off-line LC atomic absorption spectroscopy demonstrated that JM118 is the major metabolite in plasma from patients receiving JM216 treatment.

Synthesis and in vitro and in vivo antitumor activity of a series of trans platinum antitumor complexes.

The synthesis of a series of platinum complexes of trans coordination geometry [centered around the general formula, trans-ammine(amine)dichlorodihydroxoplatinum(IV) plus corresponding tetrachloroplatinum(IV) or Pt(II) counterparts] is described as part of a drug discovery program to identify more effective platinum-based anticancer drugs, particularly targeted toward the circumvention of resistance to cisplatin. Complexes have been evaluated for antitumor activity using in vitro and in vivo tumor models. In vitro against a panel of cisplatin-sensitive and -resistant human tumor cell lines (predominantly ovarian), many of the trans platinum complexes studied (e.g., 1, R = cyclohexyl) exhibited comparable potency to cisplatin and also overcame acquired cisplatin resistance, where resistance was due mainly to either reduced drug uptake or enhanced platinum-DNA adduct removal. Moreover, 14 trans complexes showed significant in vivo antitumor activity against the subcutaneous murine ADJ/PC6 plasmacytoma model; all were platinum(IV) complexes, 13/14 possessing axial hydroxo ligands the other possessing axial ethylcarbamato ligands. Where tested, all of their respective platinum(II) or tetrachloroplatinum(IV) counterparts were inactive. Notably, three dihydroxoPt(IV) complexes (18, 29, 34) (R = c-hexyl, c-heptyl, and 1-adamantyl) retained some efficacy against a cisplatin-resistant variant of the ADJ/PC6. Compounds 18 (trans-[PtCl2(OH)2NH3-(RNH2)]) R = c-C6H11, 22, R = Me3C, 27, R = n-C6H13, 28, R = PhCH2, and 36 (trans-[PtBr2(OH)2NH3(c-C6H11NH2)]) also produced evidence of antitumor activity (> 5 days growth delay) against subcutaneously grown advanced stage human ovarian carcinoma xenografts. These data demonstrate that a series of trans-ammine(amine)dichlorodihydroxoplatinum(IV) complexes are active in vivo against both murine and human subcutaneous tumor models and represent potential leads to a new generation of platinum-based anticancer drug.

Bis(acetato)amminedichloro(cyclohexylamine)platinum(IV), an orally active anticancer drug.

The structure of the anticancer drug bis(acetato)-amminedichloro(cyclohexylamine)platinum(IV), [PtCl2-(C2H3O2)2(C6H13N)(NH3)], is reported. The acetato groups are axial to the square plane composed of the chlorine and amine substituents. The cyclohexane ring may sterically hinder one of the acetato groups for metabolic attack. The amine groups are hydrogen bonded to the carbonyl O atoms of the acetato groups.

A novel trans-platinum coordination complex possessing in vitro and in vivo antitumor activity.

As part of a drug discovery program to discover more effective platinum-based anticancer drugs, a series of platinum complexes of trans coordination geometry centered on trans-ammine(cyclohexylaminedichlorodihydroxo)platinum(IV) (JM335) has been evaluated in vitro against a panel of cisplatin-sensitive and cisplatin-resistant human tumor cell lines (predominantly ovarian). In vitro, against 5 human ovarian carcinoma cell lines, JM335 was comparably cytotoxic to cisplatin itself and over 50-fold more potent than transplatin (mean 50% inhibitory concentrations: JM335, 3.1 microM; cisplatin, 4.1 microM; transplatin, 162 microM). With the use of seven pairs of human tumor cell lines (parent and subline with acquired resistance to cisplatin and encompassing all of the known major mechanisms of resistance to cisplatin) JM335 exhibited a different cross-resistance pattern to that of its cis isomer (JM149). JM335 showed non-cross-resistance in six of the seven resistant lines, cross-resistance in the A2780cisR line possibly being associated with high levels of glutathione. Preliminary intracellular DNA binding studies showed that in contrast to transplatin, JM335 was efficient at forming DNA-DNA interstrand cross-links. In vivo, JM335 produced growth delays in excess of 15 days against 4 of 6 human ovarian carcinoma xenografts and was unique among the complexes studied in retaining some efficacy against a cisplatin-resistant subline of the murine ADJ/PC6 plasmacytoma. JM335 is the first trans-platinum complex to demonstrate marked antitumor efficacy against both murine and human s.c. tumor models and represents a significant structural lead to complexes capable of circumventing cross-resistance to cisplatin.

Circumvention of acquired tetraplatin resistance in a human ovarian carcinoma cell line by a novel trans platinum complex, JM335 [trans ammine (cyclohexylamine) dichloro dihydroxo platinum (IV)].

Acquired resistance to tetraplatin [d,1-trans-1,2-diaminocy-clohexane tetrachloroplatinum (IV)] has been generated in vitro in the human ovarian carcinoma cell line PXN94; the derived line, PXN94tetR, was 24-fold resistant to tetraplatin. Intracellular tetraplatin accumulation was reduced in PXN94tetR compared with PXN94 by an average of 1.3-fold across the concentration range 1-100 microM (2 hr exposure). There was no significant difference in glutathione levels between the 2 cell lines. PXN94tetR was 1.6-fold more resistant to cadmium chloride than PXN94, suggesting that metallothionein levels may be elevated. However, no significant difference was observed between PXN94 and PXN94tetR in the levels of total platinum bound to DNA or DNA interstrand cross-links immediately after tetraplatin exposure (10-100 microM x 2 hr). There was also no significant difference between the 2 cell lines in the rate of removal of total platinum or interstrand cross-links from DNA following 2 hr exposure to 25 microM tetraplatin. Hence the major mechanism of acquired tetraplatin resistance in PXN94tetR appears to be increased tolerance of platinum-DNA adducts. PXN94tetR was partially cross-resistant to the bifunctional alkylating agents melphalan, chlorambucil and mitomycin C. Partial cross-resistance was also observed to Adriamycin, bleomycin, etoposide, 5-fluorouracil and vinblastine; however, no elevation in P-glycoprotein levels was apparent in PXN94tetR. No cross-resistance was observed to taxotere. PXN94tetR was partially cross-resistant to cisplatin, carboplatin and several novel cis platinum complexes. In contrast, resistance was completely circumvented by the novel trans platinum complex JM335 [trans ammine (cyclohexylamine) dichloro dihydroxo platinum (IV)].

Evaluation of novel ammine/amine platinum (IV) dicarboxylates in L1210 murine leukaemia cells sensitive and resistant to cisplatin, tetraplatin or carboplatin.

Seventeen alkylamine ammine dicarboxylatodichloroplatinum(IV) complexes of general structure c,t,c-[PtCl2(OCOR1)2NH3(RNH2)], where R = aliphatic or alicyclic and R1 = aliphatic or aromatic, have been evaluated against L1210 cell lines with acquired resistance to cisplatin (10-fold), tetraplatin (34-fold) or carboplatin (14-fold) using an in vitro growth-delay assay. All of these compounds overcame cisplatin, tetraplatin and carboplatin resistance. Potency increased as the number of carbon atoms in the axial aliphatic ligands (R1) increased, for example comparing JM216 (R = cyclohexyl, R1 = CH3, IC50 = 1.2 microM) with JM274 (R = cyclohexyl, R1 = n-C4H9, IC50 = 0.05 microM) against the parent sensitive line (L1210/S). The most active compounds were those possessing aromatic ligands at R1, regardless of whether R = aliphatic or alicyclic, for example JM244 (R = n-C3H7, R1 = C6H5, IC50 = 0.028 microM) and JM2644 (R = c-C6H11, R1 = C6H5, IC50 = 0.031 microM) against L1210/S. For an alicyclic alkylamine series in which R is varied from c-C3H7 to C-C7H13, with R1 = n-C3H7 for each compound, cytotoxic potency was maximised at c-C6H11 (JM221, IC50 = 0.06 microM against L1210/S). Preliminary biochemical studies, at equitoxic doses, comparing JM221 (0.1 microM) with cisplatin (0.6 microM) identified five times more platinum associated with JM221 treated cells and 1.5 times more platinum bound to the DNA of JM221-treated cells. The lipophilic properties of some of these platinum(IV) dicarboxylates may contribute to both the potency and circumvention of resistance by these compounds.