Phase I Study Of The Poly(ADP-Ribose) Polymerase Inhibitor, AG014699, In Combination With Temozolomide in Patients with Advanced Solid Tumors

Trial design and patient recruitment

The study was performed in two parts in accordance with the Declaration of Helsinki (2000). The protocol was approved by a multi-center research ethics committee, as well as by Cancer Research UK and local site institutional review boards. All patients gave written informed consent prior to participation and undergoing any study-related procedures. Patients were recruited over an eighteen month period between 2003-2005.

Inclusion criteria included histological/cytological proof of malignancy, WHO performance status of 0-1, age ≥18 years and adequate bone marrow, liver and renal function. Patients were excluded if they had prior treatment with TMZ, nitrosoureas, dacarbazine or mitomycin C, symptomatic brain metastases, primary brain tumours, other current malignancy at a second site, or other significant co-morbidity.

AG014699 was given as a 30 minute daily IV infusion followed by oral TMZ for 5 consecutive days repeated every 4 weeks. Disease response was assessed according to RECIST criteria every 2 cycles (23). Toxicity was graded according to National Cancer Institute Common Toxicity Criteria for Adverse Events (CTCAE) version 3.0 (2006). Dose limiting toxicity (DLT) was defined as a drug-related event occurring in the first 4-week cycle of treatment, as follows: neutropenia grade 4 lasting ≥5 days; fever associated (≥38.5 °C) with ≥ grade 3 neutropenia; thrombocytopenia - platelets ≤25 × 10⁹/l; anaemia ≥grade 3; grade 3 or 4 non-haematological toxicity; drug-related death. A standard three patient cohort dose escalation design was used, but with 2 separate dose escalations, part 1 and part 2.

Part 1 was open to patients of all tumour types and aimed to establish the PID of AG014699 in peripheral blood lymphocytes (PBLs). Patients received a single IV dose of AG014699 one week before starting combination therapy (day -7) to investigate the toxicity, PK and PD profile of AG014699 alone. The TMZ dose was fixed at 100mg/m²/day for this part of the study to allow for the possibility that PARP inhibition might increase the myelotoxicity of TMZ. The PID was defined as maximal achievable (at least 50%) reduction in PARP activity 24h after this first dose of AG014699 with no increase in the degree of PARP inhibition over the preceding AG014699 dose level.

Once the PID had been identified, patients received this dose of AG014699 and the dose of TMZ was escalated until the MTD of the combination was established or the TMZ dose reached a maximum of 200 mg/m² (the standard single-agent dose for this schedule). In part 2 of the study, participation was confined to patients with chemonaive melanoma with tumor deposits that were amenable to pre- and post-treatment biopsy.

PARP activity assay

PARP enzyme inhibition was assessed in PBLs at baseline, end of infusion, 4-6 and 24 hours after dosing on days -7, 1 and 4 of cycle 1 during part 1 of the study to establish the PID. An additional sample on day 8 (3 days after last dose of AG014699) was taken at the PID to explore the duration of inhibition. Paired tumour biopsies were obtained from all patients in part 2 of the study to examine target tissue PARP inhibition; biopsies being taken at baseline and 4 or 24 hours after treatment with AG014699. All samples were analysed using a previously validated and published activity assay (24) employing quantitative immunological detection of PAR formation ex vivo.

AG014699 and temozolomide pharmacokinetics

Plasma samples for pharmacokinetic analysis were collected from all patients on Day -7 and on Day 1 and 4 of cycle 1 (combination treatment) pre-infusion, 15 min, end of infusion (T0), 15 min, 30 min and before TMZ dose, 60 min, 2 h,4 h, 6 h, 8 to 12 h and 24 h post-infusion. Validated and published methods were used for the measurement of temozolomide plasma concentrations [17], plasma samples were also analysed for AG014447 concentrations (free base of AG014699) by high-performance liquid chromatography (HPLC) and tandem mass spectrometry analysis. Temozolomide and AG014699 pharmacokinetics were characterized by non-compartmental methods using WinNonlin version 3.1.

Pharmacogenomic analysis for metabolic phenotype of CYP2D6

A single 5 ml blood sample was collected at baseline in EDTA and frozen immediately at -20°C for pharmacogenomic analysis. TaqMan allelic discrimination assays were developed and validated for six of these alleles (CYP2D6 *3, *4, *6, *7, *8 and *10).

DNA strand break assessment

The method used for the alkaline Comet assay was a modified version of that described originally by Olive to detect DNA strand breaks (25). An increase in Comet tail size demonstrates an increased percentage of fragmented more mobile DNA within the cell indicating the degree of DNA strand breaks.

Slides were stained with SYBR gold and the percentage DNA in the tail and olive moment determined using KOMET5 software (Kinetic Imaging Ltd). Fifty cells from two slides were counted for each sample and the mean percentage tail DNA and Olive Tail moment was calculated.

Patient demographics and treatment

A total of 32 patients (21 male, 11 female; mean age 52) were recruited and received at least one dose of AG014699: 17 patients in part 1 and 15 in part 2 of the study (table 1). All patients were evaluable for toxicity. Twenty-nine patients received 2 cycles of treatment and were evaluable for tumour response. Dose levels and the number of cycles delivered are described in table 2.

In part 1, the dose of AG014699 was escalated through 5 dose levels. No DLT was observed and the PID was established as 12 mg/m²/day. In part 2 of the study it proved possible to administer 200 mg/m² (the licensed dose) of temozolomide with the PARP inhibitory dose of AG014699 without DLT, so the trial had reached its primary objective. One further dose level, increasing the dose of AG014699 by 50% to 18 mg/m²/day in combination with 200 mg/m²/day temozolomide was explored to establish whether more profound tumour PARP inhibition could be achieved. In view of the toxicities described below at this dose, AG014699 12 mg/m²/day (the PID) with TMZ 200 mg/m²/day x5 every 28 days is recommended for future studies.

Toxicity

No toxicity of any kind attributable to AG014699 alone was observed. The combination with temozolomide was well tolerated with no toxic deaths. Myelosuppression, the dose limiting toxicity predicted by pre-clinical models, was observed at the maximum dose of AG014699 evaluated (18 mg/m²/day given with TMZ 200 mg/m²/day): one patient suffered grade 4 thrombocytopenia and neutropenia, with grade 3 neutropaenic fever but the patient made a full recovery by day 29. Three other patients in the cohort had cycle 2 delayed by 1, 8 and 14 days respectively due to grade 3 neutropenia with slow recovery of the white cell count. These 3 patients continued on treatment with a dose reduction, receiving at least 2 further cycles without toxicity. Three patients with tumour responses on part 1 of the trial had the dose of AG014699 increased without subsequent toxicity.

Pharmacokinetics

Pharmacokinetic of AG014447 (the free base of AG014699) pharmacokinetics are detailed in table 3. The data are summarized below giving mean values for the treated population (CV%). The drug demonstrated linear pharmaocokinetics with the Cmax at the end of the infusion and a mean terminal half-life of 9.5 (50.4) hours. The mean volume of distribution was 212 (65.9) L, indicating extensive distribution into tissues, and the mean percentage of dose recovered over 24 hours in the urine after a single dose was 11%, indicating that the kidneys were not the major elimination route. Further analysis showed that AUC_0-24h normalized for actual dose and AUC_0-24h normalized for mg/m² dose did not correlate to BSA and inter-subjectsubject variability was equivalent (54% in both cases) suggesting that AG014699 can be given either as a fixed dose or based upon surface area. There is no evidence that TMZ has an effect on AG-014447 pharmacokinetics either after a single dose or multiple doses. Temozolomide PKs were similar to those previously reported, suggesting that they were not affected by the co-administration of AG014699 (data not shown).

AG014447 was detectable in all tumour biopsy homogenates (range 5-110 ng/g tumour protein) in samples taken 5 hours (24 hr in 3 patients) after the administration of the drug. Concentrations varied up to 20-fold between patients treated at the same dose level and there was no apparent correlation with the degree of PARP inhibition.

Pharmacodynamics

PARP inhibition in PBLs was seen at all AG014699 dose levels studied with profound inhibition (>90%) at the end of infusion. At the lower dose levels there was recovery of enzyme activity over 24 hours; however, at the doses above 8 mg/m² no recovery was observed over the 24 hours (figure 2 a and b), indicating that PARP was inhibited throughout the period TMZ exposure induces DNA strand breaks (26). Analysis of PARP inhibition on cycle 1 day 8 in patients dosed with 12 mg/m²/day demonstrated that enzyme activity had recovered in PBLs to ∼50% of baseline 72 hours after the last dose of AG014699.

Based on surrogate tissue enzyme inhibition 12 mg/m² was established as the PID for Part 2 of the study. In part 2 paired tumour biopsies were taken in all patients and PARP inhibition of > 50% was observed in all biopsies, and a trend towards AG014699 dose-dependency was observed (figure 2c), small numbers making formal statistically comparison not feasible. Although 2 doses of AG014699 were investigated in part 2 (12 and 18 mg/m²) such profound and consistent inhibition was observed in PBMC no correlation has been possible between the degree of inhibition in PBMC and tumour.

Comet analysis in PBLs showed evidence of DNA damage in all patients treated with AG014699 and TMZ but not AG014699 alone. The duration of DNA damage was dependent on the dose of AG014699, at lower doses (1-8 mg/m²) tail size was smaller at 24 h than at 4 h on Day 1 (mean decrease 8%) indicating repair of some strand breaks, whereas at PARP inhibitory doses (12-18 mg/m²) Comet tail size was maintained or increased at the later time point (mean increase 3%, p=0.03). Additionally there was evidence that DNA damage was retained during the treatment week with an increase, compared to baseline, in percentage DNA in the Comet tail prior to treatment on Day 4, significantly more damage remaining at 12 mg/m² than for lower doses (t-test, p=0.002, figure 2d).

Pharmacogenomics

The genotype of CYP2D6 was estimated in 26/32 patients. In 22 patients, the homozygous wild type for both of the CYP2D6 alleles or a heterozygous genotype containing at least one wild type allele was observed. In the remaining four patients (# 3, #5, #11 and #25), mutations in the CYP2D6 G1846A allele, designated as CYP2D6 *4, were observed. These patients were homozygous for the *4, *4 genotype and were predicted to be associated with poor metabolism. Three of these 4 patients were in the group who benefited from the combination, receiving 8, 8 and 16 cycles, including 2 patients with melanoma who had confirmed partial and complete responses respectively. The 4^th patient with the 4* genotype died after his test dose of a disease-related acute complication (bronchial obstruction). However, PK analysis showed that the exposure measured by AUC_0-24hr were similar between patients with predicted extensive or poor metabolism..

Response

Clinical benefit was observed for a number of patients in parts 1 and 2 of the study. There was one documented complete and one partial response in patients with metastatic melanoma (both patients had received no prior chemotherapy for melanoma) and a further partial response in a patient with a desmoid tumour (previously treated with extensive surgery and imatinib). Seven further patients had prolonged disease stabilisation (≥ 6 months), 4 with melanoma and one each with prostate cancer, pancreatic cancer and leiomyosarcoma.