Background The poly(ADP-ribose) polymerase (PARP) inhibitor olaparib potentiated radiation and temozolomide (TMZ) chemotherapy in preclinical glioblastoma models but brain penetration was poor. Clinically, PARP inhibitors exacerbate the hematological side effects of TMZ. The OPARATIC trial was conducted to measure penetration of recurrent glioblastoma by olaparib and assess the safety and tolerability of its combination with TMZ. Methods Preclinical pharmacokinetic studies evaluated olaparib tissue distribution in rats and tumor-bearing mice. Adult patients with recurrent glioblastoma received various doses and schedules of olaparib and low-dose TMZ in a 3 + 3 design. Suitable patients received olaparib prior to neurosurgical resection; olaparib concentrations in plasma, tumor core and tumor margin specimens were measured by mass spectrometry. A dose expansion cohort tested tolerability and efficacy of the recommended phase II dose (RP2D). Radiosensitizing effects of olaparib were measured by clonogenic survival in glioblastoma cell lines. Results Olaparib was a substrate for multidrug resistance protein 1 and showed no brain penetration in rats but was detected in orthotopic glioblastoma xenografts. Clinically, olaparib was detected in 71/71 tumor core specimens (27 patients; median, 496 nM) and 21/21 tumor margin specimens (9 patients; median, 512.3 nM). Olaparib exacerbated TMZ-related hematological toxicity, necessitating intermittent dosing. RP2D was olaparib 150 mg (3 days/week) with TMZ 75 mg/m2 daily for 42 days. Fourteen (36%) of 39 evaluable patients were progression free at 6 months. Olaparib radiosensitized 6 glioblastoma cell lines at clinically relevant concentrations of 100 and 500 nM. Conclusion Olaparib reliably penetrates recurrent glioblastoma at radiosensitizing concentrations, supporting further clinical development and highlighting the need for better preclinical models.

1. In vitro studies were conducted to evaluate potential inhibitory and inductive effects of the poly(ADP-ribose) polymerase (PARP) inhibitor, olaparib, on cytochrome P450 (CYP) enzymes. Inhibitory effects were determined in human liver microsomes (HLM); inductive effects were evaluated in cultured human hepatocytes. 2. Olaparib did not inhibit CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2D6 or CYP2E1 and caused slight inhibition of CYP2C9, CYP2C19 and CYP3A4/5 in HLM up to a concentration of 100 μM. However, olaparib (17-500 μM) inhibited CYP3A4/5 with an IC50 of 119 μM. In time-dependent CYP inhibition assays, olaparib (10 μM) had no effect against CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1 and a minor effect against CYP3A4/5. In a further study, olaparib (2-200 μM) functioned as a time-dependent inhibitor of CYP3A4/5 (KI, 72.2 μM and Kinact, 0.0675 min-1). Assessment of the CYP induction potential of olaparib (0.061-44 μM) showed minor concentration-related increases in CYP1A2 and more marked increases in CYP2B6 and CYP3A4 mRNA, compared with positive control activity; however, no significant change in CYP3A4/5 enzyme activity was observed. 3. Clinically significant drug-drug interactions due to olaparib inhibition or induction of hepatic or intestinal CYP3A4/5 cannot be excluded. It is recommended that olaparib is given with caution with narrow therapeutic range or sensitive CYP3A substrates, and that prescribers are aware that olaparib may reduce exposure to substrates of CYP2B6.

Abstract 1. In vitro assessments were conducted to examine interactions between olaparib (a potent oral inhibitor of poly[ADP-ribose] polymerase) and drug transporters. 2. Olaparib showed inhibition of the hepatic drug uptake transporters OATP1B1 (IC50 values of 20.3 μM and 27.1 μM) and OCT1 (IC50 37.9 μM), but limited inhibition of OATP1B3 (25% at 100 μM); inhibition of the renal uptake transporters OCT2 (IC50 19.9 μM) and OAT3 (IC50 18.4 μM), but limited inhibition of OAT1 (13.5% at 100 μM); inhibition of the renal efflux transporters MATE1 and MATE2K (IC50s 5.50 μM and 47.1 μM, respectively); inhibition of the efflux transporter MDR1 (IC50 76.0 μM), but limited inhibition of BCRP (47% at 100 μM) and no inhibition of MRP2. At clinically relevant exposures, olaparib has the potential to cause pharmacokinetic interactions via inhibition of OCT1, OCT2, OATP1B1, OAT3, MATE1 and MATE2K in the liver and kidney, as well as MDR1 in the liver and GI tract. Olaparib was found to be a substrate of MDR1 but not of several other transporters. 3. Our assessments indicate that olaparib is a substrate of MDR1 and may cause clinically meaningful inhibition of MDR1, OCT1, OCT2, OATP1B1, OAT3, MATE1 and MATE2K.

Drug delivery in glioblastoma (GBM) is challenging; poor activity may result from low biological efficacy and/or poor brain penetration. While the blood-brain barrier (BBB) is compromised in contrast-enhancing regions, little is known about BBB integrity and drug penetration in non-enhancing regions of GBM including tumour margins. Adverse clinical outcomes might reflect poor drug delivery to viable, invasive tumour cells in these regions. We evaluated tumour core and margin concentrations of olaparib (Lynparza), a small molecule inhibitor of poly(ADP-ribose) polymerase (PARP). Preclinical studies indicate that >90% PARP inhibition is achieved at 10-100 nM olaparib. Pre-clinically, BBB penetration was assessed in MDCKII cells expressing MDR1 and by autoradiography of rats and mice treated with [14C]-olaparib. Clinically, seventeen patients with recurrent resectable GBM underwent tumour resection after four days of olaparib (tablet) dosing (100 – 400 mg daily). In three patients, additional biopsies were taken from tumour margin regions identified by 5-aminolaevulinic acid guided resection. Olaparib levels in snap frozen specimens were measured by LC-MS. Olaparib was a substrate for MDR1 and efflux was blocked by ketoconazole. Radioactivity was not detected in the central nervous systems of healthy rats or mice after single dose [14C]-olaparib, indicating no BBB penetration. Despite this, olaparib was detected in 49/50 tumour core specimens from 17 patients and 9/9 tumour margin specimens from 3 patients. Olaparib concentrations in tumour core specimens ranged from 138 to 1233 nM (mean 584 nM), similar to those observed in previous breast cancer studies. Tumour margin olaparib concentrations ranged from 308 to 1090 nM (mean 681 nM). Within individual patients, margin:core ratios ranged from 0.5 to 3.8 (mean 1.8). Olaparib is excluded from the CNS under normal conditions but reliably penetrates recurrent GBM at concentrations expected to effectively inhibit PARP. Preliminary results demonstrate similar penetration of tumour margins.

2025 Background: Drug delivery is a major problem in the treatment of glioblastoma (GBM). Tumour pharmacokinetics (PK) of small molecule targeted agents in GBM are not well understood, and poor activity may result from lack of biological efficacy or adverse PK. Olaparib, a small molecule inhibitor of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), has potential to overcome treatment resistance of GBM. Despite radiological responses in brain metastases, GBM penetration by olaparib has not been studied. Methods: Preclinically, blood-brain barrier penetration was assessed by directional transport of [14C]-olaparib across MDCKII cells expressing MDR1 and autoradiography of rats and mice treated with [14C]-olaparib. Clinically, 8 patients with recurrent GBM underwent dynamic contrast enhanced (DCE) MRI at baseline followed by tumour resection after 4 days of oral olaparib (tablet: 100 mg QD, n=5; 200 mg BID, n=3). Olaparib levels were measured in tumour and plasma by LC-MS. Results: Olaparib was a substrate for MDR1 and efflux was blocked by the MDR1 inhibitor ketoconazole. Radioactivity was not detected in the central nervous systems (CNS) of rats or mice after single dose [14C]-olaparib, but significant levels were measured in subcutaneous HCT-116 tumour xenografts up to 96 hrs. Olaparib was detected in 24/24 resected GBM specimens from 8 patients (Table) at concentrations similar to those in previous breast cancer studies in which PARP inhibition and tumour responses were observed. Pre-treatment DCE-MRI showed increased vascular permeability in tumours, and tumour cellularity parameters correlated with olaparib levels. Conclusions: Olaparib is excluded from the CNS under normal conditions but reliably penetrates recurrent GBM at therapeutic levels. Small molecule PK in GBM are poorly predicted by standard pre-clinical models. Clinical trial information: NCT01390571.mtgabstract Subject Daily olaparib dose (mg) Olaparib concentration (day 4) Tumor section (ng/g) Mean tumor (ng/g) Plasma (ng/ml) 1 400 137 238 5290 210 367 2 400 71.2 94.7 3840 72.9 140 3 400 431 282 1330 133 281 4 100 177 112 876 105 55.0 5 100 324 364 773 354 413 6 100 494 413 617 345 401 7 100 53.1 59.8 1290 65.8 60.6 8 100 203 175 1460 156 166

ZD4054 is an oral specific endothelin-A receptor antagonist in development for the treatment of hormone-resistant prostate cancer. Both renal and metabolic processes contribute to its overall clearance. Two preclinical in vitro studies investigated the metabolism of ZD4054 using human liver microsomes, individual cytochrome P450 (CYP) isozymes, and flavin-containing monooxygenase isoforms. Two Phase I open-label crossover volunteer studies subsequently investigated in vivo drug interactions between ZD4054 and the CYP450 inducer rifampicin or CYP3A4 inhibitor itraconazole. The most abundant metabolite produced in in vitro incubations accounted for 12.8% of radioactivity after ZD4054 was incubated with CYP3A4. No significant flavin-containing monooxygenase metabolism of ZD4054 was observed. In the in vivo studies, rifampicin co-administration reduced the area under the concentration–time curve and maximum plasma concentration of ZD4054 by 68% and 29%, respectively, whilst co-administration with itraconazole was associated with an increase in ZD4054 area under the curve of approximately 28%. While co-administration of CYP450 inducers might be associated with reduced efficacy of ZD4054, dose reduction is unlikely to be required with concomitant administration of CYP3A4 inhibitors.

The in vitro metabolism of M445,526 (ZD6,126 phenol) was investigated by incubating [(14)C]-M445,526 at a concentration of 10 microg ml(-1) with human hepatic microsomes (4 mg ml(-1)) or human hepatocytes (2 x 10(6) cells ml(-1)) for up to 180 min. Following incubation with microsomes and hepatocytes, up to 78% and 40% of [(14)C]-M445,526 was metabolized after 180 and 120 min, respectively. High-performance liquid chromatography (HPLC) with radiochemical detection confirmed extensive metabolism of [(14)C]-M445,526 by microsomes and hepatocytes. Mass spectrometry and (1)H-NMR spectroscopy enabled structural identification of up to eight metabolites. Human liver microsomes formed one major (O-desmethyl) and three minor (a further O-desmethyl and two different hydroxylated) phase I metabolites. Human hepatocytes produced one major metabolite, a sulphate conjugate of the major O-desmethyl metabolite formed by microsomes. Four minor metabolites were also formed, primarily by O-demethylation with subsequent glucuronidation. Taken collectively, [(14)C]-M445,526 underwent extensive in vitro metabolism by human liver fractions. These data were confirmed by subsequent human in vivo studies.

The subject of this study was the determination of the major urinary and biliary metabolites of [(14)C]-ZD6126 following i.v. administration to female and male bile duct cannulated rats at 10 mg/kg and 20 mg/kg, respectively, and male bile duct cannulated dogs at 6 mg/kg by HPLC-NMR spectroscopy. ZD6126 is a phosphorylated pro-drug, which is rapidly hydrolysed to the active metabolite, ZD6126 phenol. The results presented here demonstrate that [(14)C]-ZD6126 phenol is subsequently metabolised extensively by male dogs and both, male and female rats. Recovery of the dose in bile and urine was determined utilising the radiolabel, revealing biliary excretion as the major route of excretion (93%) in dog, with the majority of the radioactivity recovered in both biofluids in the first 6 h. In the rat, greater than 92% recovery was obtained within the first 24 h. The major route of excretion was via the bile 51-93% within the first 12 h. The administered phosphorylated pro-drug was not observed in any of the excreta samples. Metabolite profiles of bile and urine samples were determined by high performance liquid chromatography with radiochemical detection (HPLC-RAD), which revealed a number of radiolabelled components in each of the biofluids. The individual metabolites were subsequently identified by HPLC-NMR spectroscopy and HPLC-MS. In the male dog, the major component in urine and bile was the [(14)C]-ZD6126 phenol glucuronide, which accounted for 3% and 77% of the dose, respectively. [(14)C]-ZD6126 phenol was observed in urine at 1% of dose, but was not observed in bile. A sulphate conjugate of demethylated [(14)C]-ZD6126 phenol was identified in bile by HPLC-NMR and confirmed by HPLC-MS. In the rat, the bile contained two major radiolabelled components. One was identified as the [(14)C]-ZD6126 phenol glucuronide, the other as a glucuronide conjugate of demethylated [(14)C]-ZD6126 phenol. However, a marked difference in the proportions of these two components was observed between male and female rats, either due to a sex difference in metabolism or a difference in dose level. The glucuronide conjugate of the demethylated [(14)C]-ZD6126 phenol was present at higher concentration in the bile of male rats (4-34%), while the phenol glucuronide was present at higher concentration in the bile of female rats (8-70%) over a 0-6 h collection period. A third component was only observed in the bile samples (0-6 h and 6-12 h) of male rats. This was identified as being the same sulphate conjugate of demethylated [(14)C]-ZD6126 phenol as the one observed in dog bile. The rat urines contained two main metabolites in greatly varying concentrations, namely the demethylated [(14)C]-ZD6126 phenol glucuronide and the glucuronide of [(14)C]-ZD6126 phenol. Again, the differences in relative amounts between male and female rats were observed, the major metabolite in the urines from male rats being the demethylated [(14)C]-ZD6126 phenol (0-17% in 0-24 h), whilst the phenol glucuronide, accounting for 0.5-50% of the dose over 0-24 h, was the major metabolite in females. Methanolic extracts of the pooled biofluid samples were submitted for HPLC-NMR for the quick identification of the major metabolites. Following a single injection of the equivalent of 6-28 ml of the biofluids directly onto the HPLC-column with minimal sample preparation, the metabolites could be largely successfully isolated. Despite severe column overloading, the major metabolites of [(14)C]-ZD6126 could be positively identified, and the results are presented in this paper.

The in vitro metabolism of [(14)C]-gefitinib (1-3 microM) was investigated using human liver microsomes and a range of expressed human cytochrome P450 enzymes, with particular focus on the formation of O-desmethyl-gefitinib (M523595), the major metabolite observed in human plasma. High-performance liquid chromatography with ultraviolet light, radiochemical and mass spectral analysis, together with the availability of authentic standards, enabled quantification and structural identification of metabolites. On incubation with pooled human liver microsomes, [(14)C]-gefitinib underwent rapid and extensive metabolism to a number of metabolites, although M523595 was only a minor microsomal product. Formation of most metabolites was markedly decreased by ketoconazole, but M523595 production was inhibited only by quinidine. Gefitinib was metabolized extensively by expressed CYP3A4, producing a similar range of metabolites to liver microsomes, but M523595 was not formed. CYP1A2, 2C9 and 2C19 produced no measurable metabolism of gefitinib, while CYP3A5 produced a range of metabolites similar to CYP3A4, but to a much lower degree. In contrast, CYP2D6 catalysed rapid and extensive metabolism of gefitinib to M523595. While formation of M523595 was CYP2D6 mediated, the overall metabolism of gefitinib was dependent primarily on CYP3A4, and this was not obviously diminished in liver microsomes from CYP2D6 poor metabolizers.

The in vitro metabolism of gefitinib was investigated by incubating [14C]-gefitinib, as well as M537194, M387783 and M523595 (the main metabolites of gefitinib observed in man), at a concentration of 100 microM with human liver microsomes (4 mg ml(-1)) for 120 min. These relatively high substrate and microsomal protein concentrations were used in an effort to generate sufficient quantities of metabolites for identification. HPLC with ultraviolet light, radiochemical and mass spectral analysis, together with the availability of authentic standards, enabled quantification and structural identification of a large number of metabolites. Although 16 metabolites were identified, metabolism was restricted to three regions of the molecule. The major pathway involved morpholine ring-opening and step-wise removal of the morpholine ring and propoxy side chain. O-demethylation of the quinazoline methoxy group was a quantitatively less important pathway, in contrast to the clinical situation, where O-desmethyl gefitinib (M523595) is the predominant plasma metabolite. The third metabolic route, oxidative defluorination, was only a minor route of metabolism. Some metabolites were formed by a combination of these processes, but no metabolism was observed in other parts of the molecule. Incubation of gefitinib produced ten identified metabolites, but the use of the three main in vivo metabolites as additional substrates enabled a more comprehensive metabolic pathway to be constructed and this has been valuable in supporting the more limited data available from the human in vivo study.

1. D0870, an azole antifungal agent, produced dose-related increases in total cytochrome P450 and aldrin epoxidase when administered as 14 daily oral doses (0, 0.5, 2.5 and 12.5 mg/kg/day) to the male rat. Administered as single doses, D0870 increased pentobarbitone-sleeping time in a dose-related manner. 2. In human hepatic microsomal incubations, D0870 produced pronounced inhibition of CYP2C9 (tolbutamide hydroxylase) and, to a lesser degree, CYP3A4 (testosterone 6beta-hydroxylase), but had more limited effects on CYP1A2, 2C19 and 2D6 activity. In comparison with ketoconazole, itraconazole and fluconazole, D0870 was the most potent inhibitor of CYP2C9 activity. It is predicted that D0870 may inhibit the in vivo clearance of CYP2C9 substrates by approximately 58%, thereby increasing their steady-state concentrations by 2.4 times, which would be of clinical significance for some compounds. 3. During incubation of [14C]-D0870 with cultured human hepatocytes for up to 72 h, two discrete metabolites (A and B) were formed. Formation of metabolite A was abolished by both quinidine and ketoconazole and is probably CYP3A4-mediated, whereas generation of metabolite B did not appear to be dependent on cytochrome P450. 4. D0870 has potential to produce both induction and inhibition of cytochrome P450 enzymes in man.

1. Casodex, a non-steroidal antiandrogen, was eliminated primarily in faeces by rat, mouse, rabbit and dog. Rat, mouse and rabbit eliminated 20-30% of a single oral dose (8-25 mg/kg) in urine; only 3-4% was excreted in urine by dog (2.5 mg/kg). Oral absorption was about 80% in rat, mouse, rabbit and dog. 2. Most of the dose was recovered in 48 h from rat, mouse and rabbit. In rat, < 1% of the dose was exhaled as 14CO2 and < 1% remained in the carcass after 7 days. Recovery from dog was incomplete in 4 days but consistent with the long plasma elimination half-life of 7-7.5 days. Casodex was eliminated from rat plasma with a half-life of 17-21 h. 3. Examination of urine indicated extensive metabolism of Casodex and showed a marked species difference. In rat, mouse and dog, Casodex was cleaved at the amide to yield a carboxylic acid and an aromatic amine which subsequently underwent ring hydroxylation with sulphate conjugation. In rabbit, the major urinary metabolite was Casodex glucuronide, conjugated on the tertiary hydroxyl. 4. The major component in faeces of all species was unchanged Casodex; some hydroxy-Casodex was also observed in rat faeces. Analysis of rat and dog bile indicated that Casodex and hydroxy-Casodex were eliminated in bile primarily as glucuronide conjugates.