Grace K. Poon

Phase I and pharmacokinetic study of D-limonene in patients with advanced cancer

Purpose: d-Limonene is a natural monoterpene with pronounced chemotherapeutic activity and minimal toxicity in preclinical studies. A phase I clinical trial to assess toxicity, the maximum tolerated dose (MTD) and pharmacokinetics in patients with advanced cancer was followed by a limited phase II evaluation in breast cancer. Methods: A group of 32 patients with refractory solid tumors completed 99 courses of d-limonene 0.5 to 12 g/m2 per day administered orally in 21-day cycles. Pharmacokinetics were analyzed by liquid chromatography-mass spectrometry. Ten additional breast cancer patients received 15 cycles of d-limonene at 8 g/m2 per day. Intratumoral monoterpene levels were measured in two patients. Results: The MTD was 8 g/m2 per day; nausea, vomiting and diarrhea were dose limiting. One partial response in a breast cancer patient on 8 g/m2 per day was maintained for 11 months; three patients with colorectal carcinoma had prolonged stable disease. There were no responses in the phase II study. Peak plasma concentration (Cmax) for d-limonene ranged from 10.8 ± 6.7 to 20.5 ± 11.2 μM. Predominant circulating metabolites were perillic acid (Cmax 20.7 ± 13.2 to 71 ± 29.3 μM ), dihydroperillic acid (Cmax 16.6 ± 7.9 to 28.1 ± 3.1 μM ), limonene-1,2-diol (Cmax 10.1 ± 8 to 20.7 ± 8.6 μM ), uroterpenol (Cmax 14.3 ± 1.5 to 45.1 ± 1.8 μM ), and an isomer of perillic acid. Both isomers of perillic acid, and cis and trans isomers of dihydroperillic acid were in urine hydrolysates. Intratumoral levels of d-limonene and uroterpenol exceeded the corresponding plasma levels. Other metabolites were trace constituents in tissue. Conclusions:d-Limonene is well tolerated in cancer patients at doses which may have clinical activity. The favorable toxicity profile supports further clinical evaluation.

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

Abstract 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)hydroxoplatinum (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 enzyatic reaction. Thus, the biotransformation profile following oral administration of JM216 shows a variety of Pt(IV) and Pt(II) metabolites in plasma that differ significantly from other systemically applied platinum drugs.

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.

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.

Electrospray ionization mass spectrometry for analysis of low-molecular-weight anticancer drugs and their analogues.

In this study, several anticancer drugs and their analogues consisting of organic and organometallic compounds were analyzed by electrospray ionization mass spectrometry (ESI/MS) using a quadrupole mass spectrometer. Protonated molecular ions [M+H]+ were observed for all of the compounds studied, and in the case of the two steroid sulfates, deprotonated molecular ions [M-H]− were obtained. Tandem mass spectrometry was performed on these quasimolecular ions, and the product ions formed provided useful fragmentation patterns that were characteristic for the compounds. This study provides evidence that ESI/MS is a sensitive technique for structure confirmation and identification of small organic and organometallic molecules.

Effects of 4-Hydroxyandrost-4-Ene-3,17-Dione and Its Metabolites on 5α-Reductase Activity and the Androgen Receptor

The steroidal aromatase inhibitor, 4-hydroxyandrost-4-ene-3,17-dione (4OHA) and its metabolites, 4-hydroxytestosterone (4OHT), 3 beta,17-dihydroxy-5 alpha-androstan-4-one (metabolite A) and 3 alpha, 17-dihydroxy-5 beta-androstan-4-one (metabolite B) were evaluated as inhibitors of the human prostatic 5 alpha-reductase enzyme and for binding to the rat prostatic androgen receptor. 4OHA and 4OHT were weak inhibitors of 5 alpha-reductase with IC50 values of 15-29 microM. Metabolites A and B had no significant inhibitory activity. 4OHA and metabolites A and B bound weakly to the androgen receptor. The binding affinities (RBA) relative to mibolerone (RBA = 100) were 0.085, 0.485 and 0.016, respectively. However, 4OHT (RBA = 75) was a more potent binder than the endogenous androgen 5 alpha-dihydrotestosterone (RBA = 66). The ability of these metabolites, in particular 4OHT, to bind to the androgen receptor may explain the in vivo androgenic activity of 4OHA.

The Clinical Development of the Oral Platinum Anticancer Agent JM216

The platinum antitumour agents cisplatin and carboplatin have brought significant benefits to the treatment of a wide variety of malignancies1. However, drug resistance remains a problem and toxicity can be significant. Where the aim of treatment is symptom palliation, which is more often than not the case, it is important to consider the side effects and convenience of treatment. A collaborative programme of research into new platinum anticancer agents conducted by The Institute of Cancer Research and Johnson Matthey Technology has focused on the twin objectives of developing agents which lack cross-resistance with cis- and carboplatin and agents which can be given by the oral route. A key lead was the identification of the ammine/amine platinum (IV) dicarboxylates2 which not only exhibit good oral bioavailability but also show activity in certain cisplatin resistant human ovarian cancer cell lines in vitro.3 The specific aims of the oral development programme were to identify an agent with good oral bioavailability, low emetogenicity, no significant specific organ toxicity, such as nephrotoxicity, ototoxicity, or neurotoxicity, and dose limiting myelosuppression. These selection criteria were used to identify bis-acetato-ammine-dichloro-cyclohexylamine-platinum (IV), or JM216 for clinical development4,5 (Fig. 1). Of these, one of the most stringent was emesis, which was tested in a ferret model in comparison with a number of other agents, especially cisplatin. Preclinical studies of neurotoxicity were conducted in parallel with the clinical trials and while confirming the lack of neurotoxicity compared with cisplatin and ormaplatin they did not play a part in the selection process.6 The phase I studies with JM216 demonstrated that the preclinical objectives had been amply fulfilled7 and phase II studies are currently underway to evaluate its antitumour efficacy.