First-in-Human Study of CH5132799, an Oral Class I PI3K Inhibitor, Studying Toxicity, Pharmacokinetics, and Pharmacodynamics, in Patients with Metastatic Cancer

The intracellular phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway regulates cellular functions such as cell survival, proliferation, growth, apoptosis, protein synthesis, and glucose metabolism (1–6). Of the three classes of PI3K (I to III), class IA is implicated most in cancer (5). Class IA PI3K heterodimers comprise a p85-regulatory and a p110-catalytic subunit with several isoforms (5). Mutation or amplification of PIK3CA, the gene encoding the catalytic subunit of PI3K (p110α), promotes oncogenic activation of the PI3K pathway and occurs frequently in human cancers such as ovarian cancer and breast cancer (1, 6–12). Phosphatase and tensin homolog (PTEN), a key negative regulator of AKT, can be inactivated via mutations, downregulation, or loss of protein expression, and is associated with tumorigenesis in prostate, gastric, and other cancers (1, 13–19). Moreover, it is likely that PI3K pathway activation is associated with resistance to both chemotherapy (20–22) and targeted agents (23–26). Selective inhibition of the PI3K–AKT–mTOR pathway in cancer represents a promising therapeutic approach and has been the focus of significant research efforts, including the clinical development of novel agents targeting this pathway (1, 19, 27–37).

CH5132799 (Chugai Pharmaceutical Co Ltd.) is an oral PI3K inhibitor with specific and potent activity against class I PI3Ks, especially demonstrated in wild-type and mutant PI3Kα isoforms, and PI3Kγ, at nanomolar concentrations (38). CH5132799 has no inhibitory activity against class III PI3K, or mTOR (38). In vitro experiments demonstrated a strong antiproliferative effect of CH5132799 on human cancer cell lines with alterations in the PI3K pathway (38, 39). In vivo, CH5132799 demonstrated significant antitumor activity in human tumor xenograft models with PIK3CA mutations, with good correlation between CH5132799 exposure and inhibition of PI3K signaling (39).

The primary objective of this first-in-human, phase I, dose-escalation study was to determine the MTD of CH5132799 using a continuous oral schedule in patients with advanced solid tumors. Secondary objectives included the characterization of CH5132799 PK, and the PD profile of PI3K inhibition in tumor and in surrogate tissues such as peripheral blood samples, and by [¹⁸F] fluorodeoxyglucose positron emission tomography (FDG-PET).

Patients had been diagnosed with advanced solid tumors that were not amenable or were refractory to standard therapy. Patients of ages ≥18 years with an Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 2 and adequate bone marrow, renal, hepatic, and cardiac function (see Supplementary data for detailed inclusion and exclusion criteria) and a life expectancy of ≥12 weeks were enrolled.

This study was approved by an independent ethics committee (The Royal Marsden Research Ethics Committee, London, United Kingdom) and conducted in accordance with the Declaration of Helsinki and Good Clinical Practice (GCP). Written informed consent was obtained from all patients before carrying out any study-related procedures.

This open-label dose-escalation study was conducted at four centers. Before the first treatment cycle, CH5132799 was administered as a single oral dose followed by a 5- to 7-day washout (run-in period). A classic “3+3” design was used for dose escalation with once daily to the early patient cohorts, and then twice daily to others, continuously in 4-week cycles. Dose escalation was determined by the nature and grade/severity of toxicities.

The primary objective was to determine the MTD of CH5132799 based on DLTs observed during the run-in period and first 4-week cycle. The MTD was defined as the highest dose level at which no more than 1 of 6 patients experienced a DLT. A starting dose of 2 mg was chosen on the basis of the highest nonsevere toxic dose in a non-rodent species and the severely toxic dose (10% lethal dose) in rat, which means 7.8-fold and 30-fold safety margins were applied to the two metabolically/kinetically relevant animal species, respectively.

Medical history and demographic data were collected at baseline. Physical examination, monitoring of vital signs, and other safety assessments were performed throughout the study. All toxicities were documented using Common Terminology Criteria for Adverse Events (CTCAE) V4.03 (40). DLTs were defined as grade ≥3, nonhematologic toxicity despite adequate treatment; grade 4, neutropenia lasting ≥7 days; and febrile neutropenia, grade 4 thrombocytopenia lasting ≥7 days or requiring a platelet transfusion.

Tumor response was assessed according to Response Evaluation Criteria In Solid Tumors (RECIST; version 1.1) with imaging at baseline and every two cycles (41).

Plasma PK samples were collected on cycle 0, day 1, followed by cycle 1, days 1, 8, 15, and 22. Plasma concentrations of CH5132799 were measured by a validated LC-MS/MS assay method (Chugai Pharmaceutical Co. Ltd.; data on file) and PK parameters calculated by noncompartmental analysis with first-order oral absorption (WinNonlin Version 5.3 and Phoenix WinNonlin Version 6.1; Pharsight Corporation). The CH5132799-related inhibition of AKT phosphorylation (pAKT) was studied in platelet-rich plasma (PRP). Blood for PRP samples was collected at 0 (predose), 1, 2, 6, 24, 48, and 72 hours postdose on day 1 of the run-in period (cycle 0, day 1) and at 0 (predose) on cycle 1, day 15. Blood samples were collected into BD Vacutainer sodium citrate coagulation tubes and centrifuged at 200 ×g at 4°C for 15 minutes. The isolated PRP layer was incubated with PhosSTOP (Roche) to stabilize the phosphorylation signals and then lysed using the Cell Lysis Buffer (Cell Signaling Technology) containing phenylmethane sulfonyl fluoride (Sigma-Aldrich) and then snap-frozen on dry ice. PD analysis with PRP was performed by The Institute of Cancer Research. All PRP samples were analyzed for the levels of phosphorylated and total forms of PD biomarker AKT by a Meso Scale Discovery electrochemiluminescent (ECL) assay. The assay was modified into a GCP-compliant quantitative assay for AKT by inclusion of a standard curve of recombinant active AKT protein on every plate. The quality, accuracy, and precision of the assays were monitored using quality control (QC) samples created by spiking three known quantities of recombinant AKT into 10% human plasma.

Biopsies were performed at screening and at cycle 1, day 15. Flash frozen tumor biopsies were homogenized using a micro tissue grinder in the Cell Lysis Buffer (w/v) containing PhosSTOP. The tumor lysates were centrifuged to remove debris and the protein concentration measured using the BCA assay (Pierce). Tumor biopsy samples were analyzed for the levels of phosphorylated and total forms of PD biomarker AKT by a quantitative ICR-modified GCP compliant Meso Scale Discovery ECL assay. FDG-PET imaging was performed within 28 days before the first dosing of CH5132799, cycle 1, day 8, and cycle 3, day 1. Lesions with the highest degree of FDG uptake were selected for quantitative analysis and a circular/spherical region of interest drawn. A SUV_max was measured for each selected lesion and the delta change in SUV_max was calculated. Tumor biopsies were taken after FDG-PET scanning to avoid interference of biopsy on FDG uptake.

PIK3CA/KRAS/BRAF mutations were studied with the OncoCarta panel v1.0 and detected by mass ARRAY System.

Descriptive statistics were used for the analysis of PK, PD, safety, and tumor response data.

Thirty-eight patients received at least one dose of CH5132799 (Table 1), of whom 23 received CH5132799 once daily (2–96 mg/day). The decision to dose twice daily was made as there had been no DLTs and no significant increase in drug concentrations at doses above 56 mg once daily along with PD measures, i.e., recovery of p-AKT after 12 hours. Subsequently, 15 further patients then received CH5132799 twice daily (96–144 mg/day). Overall, patients received a median of 2 cycles (range, 0–6), with a median duration of 52 days of treatment (range, 1–164 days). Archival tumor samples were available in 30 patients. PIK3CA mutations were found in 10% (3 of 30) of the samples studied (Table 1).

DLTs were observed in 5 of 38 evaluable patients who received CH5132799 twice daily and completed the first cycle (Table 2). Of 7 patients at 48 mg twice daily, 1 patient with hepatocellular carcinoma experienced grade 3 elevated transaminases. Although the patient had liver metastases with elevated LFTs (grade 2) at baseline, a causal link to CH5132799 could not be excluded because clear evidence of disease progression in liver metastasis was not observed. At the next dose level, 2 of 3 patients receiving 72 mg twice daily had DLTs. One had grade 3 fatigue that was resolved following cessation of CH5132799 and she restarted CH5132799 with reduced dose when fatigue had been resolved and she did not experience the event later on, whereas the other developed grade 3 posterior reversible encephalopathy syndrome (PRES) that was presented with seizures. The diagnosis was made on characteristic MRI findings and the symptoms resolved on cessation of CH5132799 as well as instigation of supportive care without restarting CH5132799. Two of 5 patients receiving the intermediate dose of 56 mg twice daily had grade 3 diarrhea, the other grade 3 diarrhea with grade 3 stomatitis, which resolved following cessation of CH5132799 and instigation of supportive treatment; in both cases, study treatment was then restarted at a reduced dose without further DLTs. Therefore, the MTD and recommended phase II dose (RP2D) of CH5132799 administered orally was established at 48 mg twice daily.

The most common AEs were diarrhea, nausea, stomatitis, fatigue, and rash, which occurred in 34%, 32%, 29%, 29%, and 24% of patients, respectively (Table 3). Diarrhea was predominantly grade 1 or 2, with no grade 4 diarrhea reported. Four patients experienced grade 3 diarrhea (96 mg once daily, n = 2; 56 mg twice daily, n = 2). One patient experienced grade 3 nausea (96 mg once daily). No grade ≥3 gastrointestinal AEs occurred in the cohort who received CH5132799 at the R2PD (48 mg twice daily). Hyperglycemia was predicted because of the mechanism of action of CH5132799. Five patients experienced grade ≥3 hyperglycemia in the twice-daily dosing schedule, suggesting a dose-dependent effect (48 mg twice daily, n = 1; 56 mg twice daily, n = 2; 72 mg twice daily; n = 2 including one grade 4). No episodes of diabetic ketoacidosis occurred and hyperglycemia was controlled with oral metformin as required. No drug-related deaths were reported.

Following oral administration of a single dose of CH5132799, plasma concentrations peaked with a T_max at 2.60 hours and t_1/2 of 10.2 hours at the MTD (Fig. 1A). Dose proportionality of AUC was shown in the dose range of 2 to 56 mg (Fig. 2B). Steady state was reached by cycle 1, day 8 at the latest with little subsequent accumulation; there was no further increase in AUC at higher doses. At the MTD of 48 mg twice daily, mean C_max and AUC_last were 172 ng/mL and 1,270 ng·h/mL after a single dose, respectively. Mean C_max and AUC_0–24h were 175 ng/mL and 1,550 ng·h/mL at steady state, respectively (Table 4).

Significant inhibition of PI3K signaling was observed in PRP, with reduction of AKT phosphorylation at doses of 32 mg and above (Fig. 2A). The temporal relationship between reduction in phosphorylation of AKT and dosing administration with CH5132799 suggested target engagement for less than 24 hours (Fig. 2B). This observation contributed to the decision to increase dosing frequency to twice daily.

Pre- and posttreatment tumor biopsies were optional and the minimum number to be conducted during the study was not based on statistical assumptions. Tumor biopsy samples were collected before and after CH5132799 from 3 patients (one patient each on 56 mg once daily, 96 mg once daily, and 56 mg twice daily). There was a more than 50% reduction in normalized p-AKT levels in 2 of the 3 patients (Fig. 2C).

A decrease in FDG avidity between baseline and cycle 1, day 8 was observed in 74% of patients (17 of 23) who underwent serial PET imaging (Fig. 2D). It was interesting that 5 of 7 patients at the RP2D who had pre-and post-(cycle1, day 8) treatment PET scans showed a reduction in maximum standardized uptake value (SUV_max); 2 had a reduction in SUV_max of 55% and 44%.

There was no correlation between the PD changes in the limited number of biopsies, PET responses, or PRP inhibition.

There were no RECIST partial or complete responses, but 1 patient with PIK3CA H1047R-mutated clear cell ovarian cancer had a GCIG CA125 response having been treated at 48 mg twice daily and remained on study for 6 cycles (Fig. 3A). A second patient with heavily pretreated triple-negative breast cancer bearing no PIK3CA or AKT mutations who was treated at the 72 mg twice daily dose level had marked symptomatic and objective improvement in her cutaneous skin lesions and remained on treatment for 6 cycles (Fig. 3B). Disease stabilization was seen in 8 patients up to week 16 (approximately 4 cycles), including 2 patients with PIK3CA mutation.

We report the first-in-human study of an oral PI3K class I inhibitor CH5132799. No DLTs occurred at the once daily schedule, but the twice-daily schedule was selected on the basis of the duration of target inhibition observed in platelet-rich plasma and the lack of dose-dependent incremental elevation in concentrations of drug above 56 mg once daily. The twice-daily schedule of CH5132799 was well tolerated and showed evidence of clinical and PD activity with a RP2D of 48 mg twice daily.

The most frequently observed toxicities were gastrointestinal, including diarrhea, nausea, and stomatitis. These have been described previously in studies of PI3K, AKT, and m-TOR inhibitors (28, 30–38). Reversible grade 3 LFT elevation was observed in 1 of 7 patients treated at the MTD of 48 mg twice daily. This patient with hepatocellular carcinoma had liver metastases and had previously undergone hepatic resection; therefore, the event was possibly caused by disease progression and/or overload of a liver with insufficient metabolic capability. No other cases of drug-related LFT abnormalities were noted, although they have been described in trials of other PI3K inhibitors (30–32, 34, 42). Of note, we observed one case of PRES at a dose above the MTD. This has not been reported previously in the trials of other drugs in this class; nevertheless, it raised the possibility that CH5132799 crosses the blood–brain barrier, which may be of future relevance in the treatment of patients with intracerebral malignancies. Mood alterations have been described as AEs in with other PI3 kinase inhibitors (30, 42), however, was not seen in this trial. Although skin-related toxicities have been frequently observed in the trials of other PI3K inhibitors (28–30, 32, 33, 35–37, 42–44), CH5132799 was associated with only mild-to-moderate skin toxicities at a lower frequency. On-target toxicity such as hyperglycemia was observed with CH5132799, as with other PI3K, AKT, and m-TOR inhibitors (28–30, 35–37, 43, 44) and was not associated with ketoacidosis. The hyperglycemia was well controlled by metformin. The favorable toxicity profile of CH5132799 may make it suitable to combine with other targeted agents such as epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) or MAPK/ERK kinase (MEK) inhibitors.

The PK data show that it was possible to attain CH5132799 concentrations in patients that had been efficacious in xenograft models (39). The AUC increased proportionally with dose from 2 to 56 mg. The PD data indicated target modulation using phosphorylation of AKT at Sr473 as a proof-of-mechanism biomarker. Levels of p-AKT were reduced in PRP consistently at dose levels 8 to 96 mg. The maximal inhibition was approximately 50% compared with baseline and this was achieved at dose levels of 32 mg. p-AKT levels in PRP frequently recovered by 24 hours following the treatment. The duration of PD biomarker changes in normal tissue and the fact that there was no significant further reduction in p-AKT levels after the 32 mg cohort, along with the fact that the AUC of CH5132799 did not rise significantly above 56 mg, led us to explore a twice-weekly schedule, despite not reaching an conventional MTD at 96 mg on the once-daily dosing cohorts. At the recommended phase II dose of 48 mg twice daily, levels of p-AKT were reduced in PRP and in a limited number of posttreatment tumor biopsies. There was a reduction in levels of p-AKT (Ser473) in 2 of 3 post-treatment tumor biopsies at the higher dose levels, but there was considerable variation in reduction between patients (Fig. 2C). The degree of p-AKT reduction in the tumor from patients was less than what was seen in xenograft models (39), however, this could reflect different platforms to assess AKT phosphorylation and other factors such as intratumoral heterogeneity.

Further evidence of tumor target modulation was observed with FDG-PET scans with all patients at recommended dose. All 5 patients scanned at the RP2D had a reduction in SUV_max and two of them achieved a PET response by PERCIST criteria (45). Although the number of patients is too small to draw definitive conclusions, these PD data are highly encouraging. There was no correlation between the PD changes in the limited number of biopsies and PET responses or PRP inhibition. The FDG PET was done pretreatment and on day 8 of treatment, whereas tumor biopsies were performed pretreatment and day 15 of treatment, while the detailed p-AKT studies in PRP were done on day 1 of treatment. The small number of biopsies and the disparities in the days when tests were conducted could have led to the lack of correlation between p-AKT levels in tumor, PRP, and changes in SUV_max on PET.

The 8 study participants (21%) showing a best response of stable disease by RECIST included 2 participants who had clear clinical responses. In reports of other PI3K, AKT, and mTOR inhibitors as single agent in patients with solid tumors, disease stabilization was observed, but only a few cases of radiologic response have been reported (28–32, 34–36, 43). Therefore, there was clinical evidence as well as PD activity for CH5132799. These results indicated that CH5132799 was comparable with other PI3K, AKT, and m-TOR inhibitors in terms of single-agent activity.

Three patients were found to have PIK3CA mutations. Although there were no objective radiologic responses, it was interesting that 1 of these patients (with clear cell ovarian cancer) had a GCIG-defined partial response in her CA125 tumor marker. Another patient with triple-negative breast cancer had a visible response in cutaneous metastases. This patient with breast cancer did not have a PIK3CA mutation, although triple-negative breast cancer was known frequently to diseases associated with other perturbations in the PI3K pathway (46). Preclinical models suggest that PIK3CA mutations predict for sensitivity to PI3K inhibitors as either single agents (47, 48) or in combination (49). This was also seen with preclinical studies of CH5132799 (39). However, the clinical correlation between PIK3CA mutations and PI3K inhibitors to this has not been fully realized (50) in the clinic, and it is possible that other activating mutations (51) or intratumoral heterogeneity (52) may play a role.

We conclude that CH5132799 at RP2D of 48 mg twice daily is well tolerated and attains drug concentrations that show evidence of achieving target engagement in both normal tissues and tumor. CH5132799 has clinical, as well as PD activity, with evidence of proof of mechanism from a clinical response in 1 patient with PIK3CA-mutant ovarian cancer. The favorable toxicity profile and activity observed with single-agent CH5132799 suggests that it is suitable for combination with other targeted therapies and for exploration in subsets of patients with cancers harboring predefined mutations.

U. Banerji reports receiving a commercial research grant from Chugai Pharma Europe Ltd. No potential conflicts of interest were disclosed by the other authors.

Conception and design: S. Blagden, K. Noguchi, R. Shiokawa, M. Inatani, J. Prince, J. Spicer, U. Banerji

Development of methodology: S. Blagden, A. Olmin, S. Decordova, K. Noguchi, R. Shiokawa, M. Inatani, J. Prince, J. Spicer, U. Banerji

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Blagden, A. Olmin, D. Josephs, C. Stavraka, A. Zivi, D.J. Pinato, A. Anthoney, K. Swales, L. Pope, K. Noguchi, R. Shiokawa, M. Inatani, C. Twelves, J. Spicer, U. Banerji

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A. Olmin, D.J. Pinato, S. Decordova, K. Swales, L. Pope, K. Noguchi, R. Shiokawa, M. Inatani, C. Twelves, J. Spicer, U. Banerji

Writing, review, and/or revision of the manuscript: S. Blagden, A. Olmin, C. Stavraka, A. Zivi, D.J. Pinato, A. Anthoney, K. Swales, R. Riisnaes, L. Pope, K. Noguchi, R. Shiokawa, M. Inatani, J. Prince, K. Jones, C. Twelves, J. Spicer, U. Banerji

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C. Stavraka, K. Swales, R. Riisnaes, K. Noguchi, R. Shiokawa, M. Inatani

Study supervision: S. Blagden, C. Stavraka, J. Prince, C. Twelves, U. Banerji

The authors thank all the patients who took part in this study, together with their families and caretakers.

This study was funded by Chugai Pharmaceutical Co. Ltd. Infrastructure; support was provided by a Cancer Research UK Joint Phase I Clinical Core Grant (grant number C51/A6883) to The Institute of Cancer Research and The Royal Marsden. The recruiting sites acknowledge infrastructural funding from the Experimental Cancer Medicine Centres and NIHR Biomedical Research Centre initiatives.

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