A Phase I clinical study of cisplatin-incorporated polymeric micelles (NC-6004) in patients with solid tumours.

BACKGROUND: On the basis of preclinical studies of NC-6004, a cisplatin-incorporated micellar formulation, we hypothesised that NC-6004 could show lower toxicity than cisplatin and show greater anti-tumour activity in phase I study. METHODS: A total of 17 patients were recruited in a range of advanced solid tumour types. NC-6004 was administered intravenously (i.v.) every 3 weeks. The dose escalation started at 10 mg m(-2) and was increased up to 120 mg m(-2) according to the accelerated titration method and modified Fibonacci method. RESULTS: One dose-limiting toxicity (DLT) occurred in a patient who was given 90 mg m(-2) of NC-6004, otherwise any significant cisplatin-related toxicity was not observed or generally mild toxicity was observed. Despite the implementation of post-hydration and pre-medication regimen, renal impairment and hypersensitivity reactions still developed at 120 mg m(-2), which led to the conclusion that the maximum tolerated dose was 120 mg m(-2), and the recommended dose was 90 mg m(-2), although DLT was not defined as per protocol. Stable disease was observed in seven patients. The maximum concentration and area under the concentration-time curve of ultrafilterable platinum at 120 mg m(-2) NC-6004 were 34-fold smaller and 8.5-fold larger, respectively, than those for cisplatin. CONCLUSION: The delayed and sustained release of cisplatin after i.v. administration contributes to the low toxicity of NC-6004.

Influence of pharmacogenetics on response and toxicity in breast cancer patients treated with doxorubicin and cyclophosphamide.

BACKGROUND: Doxorubicin and cyclophosphamide (AC) therapy is an effective treatment for early-stage breast cancer. Doxorubicin is a substrate for ABCB1 and SLC22A16 transporters. Cyclophosphamide is a prodrug that requires oxidation to 4-hydroxycyclophosphamide, which yields a cytotoxic alkylating agent. The initial oxidation is catalysed by cytochrome P450 enzymes including CYP2B6, CYP2C9, CYP2C19 and CYP3A5. Polymorphic variants of the genes coding for these enzymes and transporters have been identified, which may influence the systemic pharmacology of the two drugs. It is not known whether this genetic variation has an impact on the efficacy or toxicity of AC therapy. METHODS: Germ line DNA samples from 230 patients with breast cancer on AC therapy were genotyped for the following SNPs: ABCB1 C1236T, G2677T/A and C3435T, SLC22A16 A146G, T312C, T755C and T1226C, CYP2B6*2, *8, *9, *3, *4 and *5, CYP2C9*2 and *3, CYP3A5*3 and CYP2C19*2. Clinical data on survival, toxicity, demographics and pathology were collated. RESULTS: A lower incidence of dose delay, indicative of less toxicity, was seen in carriers of the SLC22A16 A146G, T312C, T755C variants. In contrast, a higher incidence of dose delay was seen in carriers of the SLC22A16 1226C, CYP2B6*2 and CYP2B6*5 alleles. The ABCB1 2677A, CYP2B6*2, CYP 2B6*8, CYP 2B6*9, CYP 2B6*4 alleles were associated with a worse outcome. CONCLUSION: Variant alleles in the ABCB1, SLC22A16 and CYP2B6 genes are associated with response to AC therapy in the treatment of breast cancer.

Topotecan in combination with carboplatin: phase I trial evaluation of two treatment schedules.

BACKGROUND: Topotecan and cisplatin combinations have shown schedule-dependent toxicity, which may in part be due to cisplatin nephrotoxicity. As carboplatin is less nephrotoxic and increasingly replacing cisplatin in clinical practice, the aim of this study was to define the optimal sequence and dose for topotecan in combination with carboplatin. PATIENTS AND METHODS: Two parallel phase I trials, with pharmacokinetic studies, were conducted administering carboplatin on day 1 with topotecan on days 1-5 (schedule A) or days 8-12 (schedule B). repeated every 3 weeks. RESULTS: Twenty-one patients were treated over two dose levels, carboplatin AUC 4 [glomerular filtration rate (GFR) calculated from 51Cr-EDTA clearance] with topotecan 0.5 or 0.75 mg/m2. At the first dose level, six patients were evaluable for each schedule. With schedule A, from 34 cycles, there were two dose reductions and 10 treatment delays due to myelosuppression. With schedule B from 25 cycles, there was one reduction and 10 delays. At dose level 2, both patients in schedule A had dose-limiting neutropenia. In contrast, there was no dose-limiting toxicity with schedule B in six patients, although the majority of cycles were delayed. CONCLUSION: The combination of topotecan and carboplatin using these 3-weekly schedules lead to significant myelotoxicity with attendant dose reductions and delays; the optimal scheduling of these agents remains to be defined.

Pharmacological study of paclitaxel duration of infusion combined with GFR-based carboplatin in the treatment of ovarian cancer.

PURPOSE: To determine the effect on systemic pharmacology and clinical toxicity of dose and mode of administration of paclitaxel combined with carboplatin in the treatment of ovarian cancer. PATIENTS AND METHODS: A total of 18 patients were treated with a dose of carboplatin determined by GFR, to attain a target AUC of 6 or 7 mg/ml x min. The paclitaxel dose was 175 or 200 mg/m2 administered over approximately 1 or 3 h. The duration of infusion was randomized, crossing over to the alternative treatment for the second course. Blood samples were analysed for carboplatin, paclitaxel and for the excipients of the paclitaxel formulation, ethanol and Cremophor. RESULTS: Overall the three-weekly schedule of administration of the combination of carboplatin and paclitaxel was well tolerated. There were no clinical differences in the toxicities observed between courses where a 1-h infusion was used compared with those with a 3-h infusion. The target AUC of carboplatin was achieved (mean +/- SD 114 +/- 20% of target). Analysis of paclitaxel pharmacokinetics did not show a difference in the AUC or time above a pharmacological threshold for the two infusion durations. The peak concentration of paclitaxel obtained at the end of the infusion (9.1 vs 4.5 microg/ml), and the plasma ethanol concentration (40.0 vs 20.5 mg/dl) were higher following the shorter duration infusion. Peak concentrations of Cremophor were not different. CONCLUSION: The combination of paclitaxel at a dose of 175 mg/m2 and carboplatin at a target AUC of 6-7 mg/ml min can safely be administered every 3 weeks. Also, a 1-h infusion of paclitaxel has no acute clinical disadvantage over a 3-h infusion and these durations of administration are pharmacologically equivalent.

Reversal of drug resistance in human tumor xenografts by 2'-deoxy-5-azacytidine-induced demethylation of the hMLH1 gene promoter.

Loss of DNA mismatch repair because of hypermethylation of the hMLH1 gene promoter occurs at a high frequency in a number of human tumors. A role for loss of mismatch repair (MMR) in resistance to a number of clinically important anticancer drugs has been shown. We have investigated whether the demethylating agent 2'-deoxy-5-azacytidine (DAC) can be used in vivo to sensitize MMR-deficient, drug-resistant ovarian (A2780/cp70) and colon (SW48) tumor xenografts that are MLH1 negative because of gene promoter hypermethylation. Treatment of tumor-bearing mice with the demethylating agent DAC at a nontoxic dose induces MLH1 expression. Re-expression of MLH1 is associated with a decrease in hMLH1 gene promoter methylation. DAC treatment alone has no effect on the growth rate of the tumors. However, DAC treatment sensitizes the xenografts to cisplatin, carboplatin, temozolomide, and epirubicin. Sensitization is comparable with that obtained by reintroduction of the hMLH1 gene by chromosome 3 transfer. Consistent with loss of MMR having no effect on sensitivity in vitro to Taxol, DAC treatment has no effect on the Taxol sensitivity of the xenografts. DAC treatment does not sensitize xenografts of HCT116, which lacks MMR because of hMLH1 mutation. Because there is emerging data on the role of loss of MMR in clinical drug resistance, DAC could have a role in increasing the efficacy of chemotherapy for patients whose tumors lack MLH1 expression because of hMLH1 promoter methylation.

Evaluation of dihydropyrimidine dehydrogenase activity in South-west Asian, Kenyan and Ghanaian populations.

AIMS: Dihydropyrimidine dehydrogenase (DPD) reduces endogenous pyrimidines and therapeutic analogues such as the anticancer agent 5-fluorouracil (5FU). Among Caucasian populations DPD activity is highly variable and subject to polymorphic regulation. To evaluate interethnic influence, DPD activity was assessed in South-west Asian, Kenyan and Ghanaian populations. METHODS: DPD activity was determined in peripheral mononuclear cells using[14C]-5-fluorouracil and h.p.l.c. analysis. RESULTS: A high degree of variation in DPD activity was observed within each population (range CV = 34-48%). Median DPD activity also varied between these populations. South-west Asian and Kenyan subjects exhibited almost identical median values (192 and 193.5 pmol min(-1) mg(-1), respectively), which were similar to Caucasians (median 215 pmol min(-1) mg(-1). A significantly lower median DPD activity (119 pmol min(-1) mg(-1)) was observed in the Ghanaian population. CONCLUSIONS: The similarity in DPD activity between Caucasian, Kenyan and South-west Asian populations suggests that the incidence of 5FU-related toxicity may be comparable in these groups. The pharmacokinetic implications of lower activity amongst Ghanaians needs to be evaluated.

The encapsulation of bleomycin within chitosan based polymeric vesicles does not alter its biodistribution.

Polymeric vesicles have recently been developed from an amphiphilic chitosan derivative--palmitoyl glycol chitosan. Their potential as a drug delivery system was evaluated using the anti-cancer compound bleomycin as a model drug. Palmitoyl glycol chitosan (GCP41) was synthesised by conjugation of palmitoyl groups to glycol chitosan. Bleomycin-containing vesicles (669 nm diameter) were prepared from a mixture of GCP41 and cholesterol by remote loading. The vesicles were imaged by freeze-fracture electron microscopy and their in-vitro stability tested. Incubation of the larger vesicles with plasma in-vitro led to a reduction of mean size by 49%, a reaction not seen with control sorbitan monostearate niosomes (215 nm in size). They also showed a higher initial drug release (1 h), but GCP41 and sorbitan monostearate vesicles retained 62% and 63% of the encapsulated drug after 24h, respectively. The biodistribution of smaller vesicles (290 nm) prepared by extrusion through a 200-nm filter was also studied in male Balb/c mice. Encapsulation of bleomycin into polymeric vesicles did not significantly alter the pharmacokinetics of biodistribution of bleomycin in male Balb/c mice although plasma and kidney levels were slightly increased. It is concluded that the extruded GCP41 vesicles break down in plasma in-vivo and hence are unlikely to offer any therapeutic advantage over the free drug.

Phase I and pharmacokinetic study of DACA (XR5000): a novel inhibitor of topoisomerase I and II. CRC Phase I/II Committee.

DACA, also known as XR5000, is an acridine derivative active against both topoisomerase I and II. In this phase I study, DACA was given as a 3-h intravenous infusion on 3 successive days, repeated every 3 weeks. A total of 41 patients were treated at 11 dose levels between 9 mg m(-2) d(-1) and the maximum tolerated dose of 800 mg m(-2) day(-1). The commonest, and dose-limiting, toxicity was pain in the infusion arm. One patient given DACA through a central venous catheter experienced chest pain with transient electrocardiogram changes, but no evidence of myocardial infarction. At the highest dose levels, several patients also experienced flushing, pain and paraesthesia around the mouth, eyes and nose and a feeling of agitation. Other side-effects, such as nausea and vomiting, myelosuppression, stomatitis and alopecia, were uncommon. There was one minor response but no objective responses. DACA pharmacokinetics were linear and did not differ between days 1 and 3. The pattern of toxicity seen with DACA is unusual and appears related to the mode of delivery. It is possible that higher doses of DACA could be administered using a different schedule of administration.

Phase I/pharmacokinetic study of the topoisomerase I inhibitor GG211 administered as a 21-day continuous infusion.

BACKGROUND: Preclinical results support a prolonged schedule of administration for topoisomerase I inhibitors, and we have previously demonstrated the safety and activity of the novel water-soluble topoisomerase I inhibitor GG211 when given as a 72-hour continuous infusion to cancer patients. PATIENTS AND METHODS: In a three-center international phase I trial, 38 patients received GG211 doses from 0.3 to 0.5 mg/m2/day by continuous intravenous infusions for seven, 14, and 21 days. Patients' median performance status was 1; nearly half had colorectal cancer, and 35 patients had prior chemotherapy. RESULTS: The first patient cohort received 0.3 mg/m2/day for seven days with no significant toxicities. Subsequent cohorts received continuous infusions for 14 and 21 days at this dose level with only mild myelosuppression noted. Dose-escalation on the 21-day schedule was then performed. No dose-limiting toxicity occurred at the 0.4 mg/m2/day dose level. Thrombocytopenia was dose-limiting with 0.5 mg/m2/day dosing but was not cumulative. Other grade 3 4 toxicities included neutropenia, nausea, vomiting, diarrhea, and fatigue. Partial responses occurred with 21-day infusion in two patients with breast and ovarian cancer at the 0.3 and 0.4 mg/m2/day dose levels, respectively. Mean GG211 lactone Css ranged from 0.17 to 0.64 ng/ml. CONCLUSION: The maximum tolerated dose of GG211 administered as a 21-day continuous infusion is 0.4 mg/m2/day with antitumor activity noted at tolerable doses.

Dihydropyrimidine dehydrogenase pharmacogenetics in Caucasian subjects.

AIMS: Dihydropyrimidine dehydrogenase (DPD) catalyses the reduction of pyrimidines, including the anticancer agent 5-fluorouracil (5FU). Impaired 5FU degradation, through low DPD activity, has led to severe, life-threatening or fatal toxicity after administration of 5FU. Complete DPD deficiency is associated with the inherited metabolic disease thymine uraciluria. Several mutations in the gene encoding DPD have recently been identified, but the phenotype-genotype concordance of these alterations in the general population has not been reported. METHODS: Mononuclear cells were isolated from whole blood and DPD activity was determined after ex vivo incubation with 14C-5FU followed by h.p.1.c. analysis of 5FU metabolites. Analysis of mutations in the DPD gene at an exon splice site, codons 534, 543, and 732, and a deletion at base 1897 (deltaC1897) were performed in 30 subjects with the lowest and 30 subjects with the highest enzyme activity using PCR-RFLP. RESULTS: DPD activity was measured in 226 Caucasian subjects and was highly variable (range 19.1-401.4 pmol min(-1)mg(-1) protein). Mutations were frequently observed at codons 543 (allele frequency 28%), 732 (allele frequency 5.8%), and 534 (allele frequency 0.8%), but were not associated with low DPD activity. There were no splice site or deltaC1897 mutations found in this population. CONCLUSIONS: The five mutations analysed in this study are insufficient for identification of patients at risk for 5FU toxicity or thymine uraciluria. Both the splice site mutation and deltaC1897 are relatively rare in the general Caucasian population. Therefore, identification of further molecular alterations is required to facilitate the use of DPD analysis in genetic diagnosis and cancer therapeutics.

Liver dihydropyrimidine dehydrogenase activity in human, cynomolgus monkey, rhesus monkey, dog, rat and mouse.

Interspecies differences in dihydropyrimidine dehydrogenase (DPD), the initial and rate-limiting enzyme in pyrimidine degradation, were assessed in cytosol from livers isolated from human, monkey, dog, rat, and mouse. Hepatic DPD activity was measured by an HPLC assay with on-line radioactivity detection, using 14C-5-fluorouracil as a substrate. Activity was highly variable within each species and significant interspecies differences in liver DPD activity were observed. The order of activity was mouse > rat > human > dog > or = cynomolgus monkey > rhesus monkey. These data suggest that careful selection must be made when choosing in vivo models of human DPD for the preclinical development of novel fluoropyrimidine anticancer agents and DPD inhibitors.

Characterization of dihydropyrimidine dehydrogenase in human colorectal tumours.

Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme for degradation of 5-fluorouracil (5-FU). DPD activity is highly variable in liver and peripheral mononuclear cells (PMNCs) and it has not been well studied in human tumours. Characterization of DPD in colorectal cancer is of clinical interest through its role in the regulation of 5-FU, the main chemotherapeutic agent used in this disease. Therefore, DPD activity was analysed in colorectal tumour and adjacent normal tissue from 63 patients, including three liver metastasis. DPD activity was highly variable in all tissues studied (coefficient of variation 43-61%) and was higher in normal tissue than in tumour. The tumour-normal activity ratio ranged from 0.19 to 3.32 (median 0.76). PMNC DPD activity was available for 57 patients and was correlated with tumour activity (r(s) = 0.29, P < 0.001). A higher correlation was observed between PMNCs and tumour samples that were both obtained in the morning (r(s) = 0.49), consistent with circadian variation in DPD activity. Normal tissue DPD activity was not correlated with either tumour (r(s) = 0.11) or PMNC activity (r(s) = -0.06). This study provides the first analysis of DPD activity in colorectal cancer and illustrates the large degree of variation in tumour activity. The tumour-normal activity ratio results suggest that elevated tumour DPD can play a role in 5-FU resistance through increased inactivation in tumour cells, but is an uncommon event in colorectal tumours. The results support the use of PMNCs for monitoring tumour DPD activity, particularly when circadian variation is taken into account. As a large degree of the variation in tumour DPD activity is not explained by PMNC activity, more accurate alternatives are needed before DPD activity can be used for targeting 5-FU therapy.

Dihydropyrimidine dehydrogenase pharmacogenetics in patients with colorectal cancer.

Individuals with a deficiency in the enzyme dihydropyrimidine dehydrogenase (DPD) may experience severe life-threatening toxicity when treated with 5-fluorouracil (5-FU). As routine measurement of enzyme activity is not practical in many clinical centres, we have investigated the use of DNA mutation analysis to identify cancer patients with low enzyme levels. We have identified two new mutations at codons 534 and 543 in the DPD cDNA of a patient with low enzyme activity and screened the DNA from 75 colorectal cancer patients for these mutations and the previously reported splice site mutation (Vreken et al, 1996; Wei et al, 1996). In all cases, DPD enzyme activity was also measured. The splice site mutation was detected in a patient (1 out of 72) with low enzyme activity whereas mutations at codons 534 (2 out of 75) and 543 (11 out of 23) were not associated with low enzyme activity. These studies highlight the need to combine DPD genotype and phenotype analysis to identify mutations that result in reduced enzyme activity.

Autoregulation of 5-fluorouracil metabolism.

5-Fluorouracil (5-FU) is a commonly used anticancer agent for the treatment of gastrointestinal, head and neck, and breast tumours. This study determined the influence of 5-FU on dihydropyrimidine dehydrogenase (DPD) activity, the enzyme responsible for its in vivo degradation. DPD activity was measured in mononuclear cells obtained prior to and after the administration of 5-FU in 20 patients with colorectal cancer. Following the results from the human studies, DPD activity was measured in Sprague-Dawley rat liver up to 72 h after administration of 5-FU 200 mg/kg as a single injection. Total liver P450 content and the production of testosterone metabolites (indicative of CYP3A activity) were also analysed to determine the specificity of 5-FU-associated alteration in rat liver metabolism. Human mononuclear cell DPD activity decreased by a median of 38.7% following the administration of 5-FU (P = 0.001). 5-FU-induced alterations in rat liver DPD were also observed, with the lowest activity occurring 48 h after injection (50% of control activity; P = 0.009). Rat liver DPD activity returned to near control values by 72 h postinjection. Rat liver total P450 content and CYP3A activity were not significantly different in 5-FU treated or control tissues. Thus, 5-FU demonstrates autoregulation of its metabolism through inhibition of DPD activity. Although this inhibition appears to be specific for DPD, the mechanism for enzyme inhibition is not clear. These findings may aid in the design of 5-FU treatment regimens and provide the basis for further studies into the regulation of DPD.