Phase I study of DFP-11207, a novel oral fluoropyrimidine with reasonable AUC and low Cmax and improved tolerability, in patients with solid tumors

Summary 5-fluorouracil (5-FU) and 5-FU derivatives, such as capecitabine, UFT, and S-1, are the mainstay of chemotherapy treatment for gastrointestinal cancers, and other solid tumors. Compared with other cytotoxic chemotherapies, these drugs generally have a favorable safety profile, but hematologic and gastrointestinal toxicities remain common. DFP-11207 is a novel oral cytotoxic agent that combines a 5-FU pro-drug with a reversible DPD inhibitor and a potent inhibitor of OPRT, resulting in enhanced pharmacological activity of 5-FU with decreased gastrointestinal and myelosuppressive toxicities. In this Phase I study (NCT02171221), DFP-11207 was administered orally daily, in doses escalating from 40 mg/m2/day to 400 mg/m2/day in patients with esophageal, colorectal, gastric, pancreatic or gallbladder cancer (n = 23). It was determined that DFP-11207 at the dose of 330 mg/m2/day administered every 12 hours was well-tolerated with mild myelosuppressive and gastrointestinal toxicities. The pharmacokinetic analysis determined that the 5-FU levels were in the therapeutic range at this dose. In addition, fasted or fed states had no influence on the 5-FU levels (patients serving as their own controls). Among 21 efficacy evaluable patients, 7 patients had stable disease (33.3%), of which two had prolonged stable disease of >6 months duration. DFP-11207 can be explored as monotherapy or easily substitute 5-FU, capecitabine, or S-1 in combination regimens.

Introduction 5-Fluorouracil (5-FU), an antimetabolite, was introduced by Heidelberger et al. [1] in 1957, and has since been widely used as a single agent or in combination with other drugs [2][3][4] mainly in localized or metastatic gastrointestinal cancers and breast cancer. Clinical response and toxicity of 5-FU are remarkably influenced by its dosing schedule and a prolonged exposure by continuous infusion of 5-FU has been found to increase tumor response rates [5][6][7][8][9][10][11][12].
Interindividual and intraindividual variations in plasma 5-FU concentrations are mainly caused by differing levels of dihydropyrimidine dehydrogenase (DPD), the primary catabolic enzyme of 5-FU [43][44][45]. Deficiency of DPD is associated with severe hematologic and gastrointestinal toxicity after 5-FU administration [46]. Inhibition or inactivation of DPD has emerged as a potential strategy to reduce the pharmacokinetic variability and improve the efficacy of 5-FU [12,47].
To minimize the 5-FU-induced toxicities without compromising its antitumor activity, Delta-Fly Pharma, Inc. has developed DFP-11207, a novel cytotoxic agent that combines a 5-FU pro-drug (1-ethoxymethyl-5-fluorouracil; EM-FU) [26] with a reversible DPD inhibitor CDHP [48] and a potent inhibitor of orotate phosphoribosyl transferase (citrazinic acid; CTA). This combination results in enhanced pharmacological activity of 5-FU, with lower C max and AUC values but longer T max and T 1/2 values of 5-FU, respectively than S-1, which suggests that DFP-11207 may be superior in preventing the 5-FU-induced severe hematological and gastrointestinal toxicities. CTA, that is mainly retained in gastrointestinal tract cells, protects the gastrointestinal tract from injury by inhibiting 5-FU phosphorylation. DFP-11207's self-controlled toxicity profile may allow this molecule to improve the tolerability and efficacy of 5-FUbased treatment for cancer patients as a monotherapy or in combination therapy [49].

Study design
This Phase I, open-label, single arm, single-center, dose escalation, safety, tolerability, and pharmacokinetic (PK) study of DFP-11207 in patients with advanced solid tumors (NCT02171221) was sponsored by Delta-Fly Pharma, Inc. (Tokushima, Japan).
The primary objective of this study was to determine the maximum tolerated dose (MTD), the recommended Phase II dose (RP2D) and the dose-limiting toxicity (DLT) of DFP-11207 in patients with advanced solid tumors, and to assess pharmacokinetic (PK) profiles of DFP-11207 under fed and fasted conditions. Secondary objectives were to perform PK analysis of DFP-11207 and to assess the antitumor activity of DFP-11207 in patients with advanced solid tumors.

Eligibility
Eligible patients were males or females of at least 18 years of age, with solid tumors refractory by standard therapies or for which conventional chemotherapy was not reliably effective or no effective therapy was available. Patients must have had adequate bone marrow function as defined by absolute neutrophil count of ≥1.5 × 10 9 /L and platelets of ≥100 × 10 9 /L. Adequate liver and kidney function were required. Patient who had current malignancies of another type, patients after extensive prior radiotherapy, or prior bone marrow/stem cell transplantation, and patients with clinically evident CNS metastases or leptomeningeal disease were not eligible for the study. Prior exposure to chemotherapy, immunotherapy, radiotherapy or any other investigational therapy within 4 weeks was not permitted. Patients with cardiac dysfunction and known bleeding disorder were excluded.

Treatment
In the Phase I Study, patients received DFP-11207 capsules orally, daily in 28-day cycles. Patients in the 40 to 250 mg/ m 2 /day cohorts received once-daily DFP-11207 dosing. For patient compliance, the DFP-11207 dosing schedule was changed at doses of 330 mg/m 2 /day (1 patient) or 440 mg/m 2 / day (4 patients) to every 12 hours on all treatment days except on Cycle 1, Days 1 and 29, when DFP-11207 was administered as a single dose for PK sampling purposes and later for the subsequent 5 patients in the 330 mg/m 2 /day cohort, DFP-11207 dosing schedule was changed to every 12 hours on all treatment days. Subsequently, a Food Effect Study was added, and 6 patients were treated with DFP-11207 at the dose of 600 mg/day administered as 300 mg every 12 hours.

Criteria for evaluation
Safety Safety data including laboratory parameters, vital signs, and adverse events were collected for all patients. All patients who received any amount of DFP-11207 were included in the safety analysis. Safety parameters evaluated include adverse events, vital signs, and clinical laboratory results. Adverse events were classified according to the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) Version 4.0.
Efficacy Although response was not the primary endpoint of this study, patients with measurable disease were assessed using RECIST version 1.1, where possible after every 2 cycles. For patients with less than 2 cycles of study therapy, if there was clear evidence of clinical progression then they were considered eligible for the efficacy evaluation.
Plasma pharmacokinetics, food effect study Following oral administration of DFP-11207, blood samples were collected during Cycle 1 (fed or fasted study days) on Day 1 at 0 hour (pre-dose), and 4, 10, 24 and 48 hours after the study drug administration, Day 8 at 2 hours after the morning DFP-11207 administration, Day 14 at 2, 10 and 24 hours after DFP-11207 administration, Day 16 at 0 hour (pre-dose), and 4, 10 and 24 hours after DFP-11207 administration, Day 18 at pre-dose, Day 23 at 2 hours after the morning DFP-11207 administration, Day 29 at 2, 10 and 24 houras after DFP-11207 administration, and pre-dose on Cycle 2 Day 1.
For both the Phase I Study and the Food Effect Study, whole blood (5 mL) was collected in chilled heparin collection tubes to harvest plasma. High performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used to determine plasma and urine concentrations of DFP-11207 metabolites 5-FU, EM-FU, CDHP, and CTA.
Urine pharmacokinetics, phase I study Urine samples (8 mL) were also collected and pooled at pre-dose (−12 to 0 hour) and after the start of DFP-11207 treatment at: 0 to 12 hours, 12 to 24 hours, 24 to 36 hours, and 36 to 48 hours. DFP-11207 metabolites were then measured.

Statistical approach
The primary endpoint of this study was to assess the toxicity of DFP-11207 by determining the dose level at which DLTs were observed. AEs were arranged by decreasing frequency of AEs. Laboratory data was graded according to NCI CTCAE (Version 4.0) and tabulated based on maximum grade. AEs were coded using the Medical Dictionary for Drug Regulatory Activities (MedDRA ® ) Version 17.0.
Secondary endpoints of this study were to examine the efficacy and PKs of DFP-11207. For efficacy analysis, overall response was assessed using RECIST version 1.1. The objective antitumor response rate [complete response (CR) or partial response (PR)] was calculated as the proportion of patients with responsive disease (CR + PR) and 95% confidence interval for response was calculated for the median time. Duration of response and time to tumor progression were evaluated using Life Table methods. Life Table estimates were calculated using Kaplan-Meier methodology.
Plasma concentration and time data of DFP-11207 metabolites (5-FU, EM-FU, CDHP and CTA) in the Food Effect Study were determined using non-compartmental methods (WinNonLin ® ). PK parameters to be calculated included AUC extrapolated to infinity (AUC inf ), peak concentration (C max ), time at C max (T max ) and elimination half-life (T 1/2 ). Statistical analysis of PK parameters was to be performed to compare each fed condition to the fasted condition by using analysis of variance which were compared by using the Wilcoxon matched-pairs test.

Baseline disease characteristics
The majority of patients (19 patients; 82.6%) had histopathology diagnosis of adenocarcinoma not otherwise specified (NOS); 4 patients (17.4%) had carcinoma NOS as the predominant histopathology with primary tumors sites of esophagus or rectum (both 6 patients each), large intestine (4 patients), pancreas or stomach (3 patients each) and gallbladder and extrahepatic bile duct (1 patient) (see Tables 1, 2 and 3).

Discussion
The patient experience in this Phase I dose escalation study of DFP-11207 indicates successful implementation of a twopronged strategy to control the toxicity of orally administered 5-FU while maintaining effective circulating levels of 5-FU. CTA is mainly retained in the gastrointestinal tract cells where it inhibits 5-FU phosphorylation, thus protecting the gastrointestinal tract from injury as the 5-FU is absorbed into the circulation. Secondly, CDHP reversibly inhibits DPD to delay the enzymatic degradation of 5-FU in the circulation while associated with the degradation of 5-FU prodrug, resulting in a prolonged systemic 5-FU exposure profile with lower C max and similar AUC compared to S-1. Owing to the concomitant presence of these DFP-11207 components, treatment at the DFP-11207 dose of 330 mg/m 2 /day administered orally every 12 hours produced a steady state circulating 5-FU level of 5.27-23.5 ng/mL (C max < 25 ng/mL) and was well tolerated without any significant myelosuppression or gastrointestinal toxicity in advanced solid tumor patients, while patients treated at 440 mg/m 2 /day had steady state circulating 5-FU levels of 23-45.3 ng/mL (C max 49.7-54.7 ng/mL) associated with severe myelosuppression and moderate mucosal inflammation, fatigue and palmar-plantar erythrodysesthesia.
The food-effect study demonstrates maintenance of bioavailability when DFP-11207 is administered as a twicedaily 300 mg flat-dose with or without food (mean steady state levels of 27.6 and 16.7 ng/mL, mean AUC of 809 and 535 ng· h/mL, respectively) and no significant myelosuppressive or gastrointestinal adverse events. These results are in contrast to S-1 for which the maximum tolerated dose of 40 mg/m 2 / day has an associated 5-FU C max of 128 ng/mL and 5-FU AUC of 724 ng·h/mL [24] and S-1 dosing at 30 mg/m 2 BID has an associated 5-FU C max of approximately 150 ng/mL and 5-FU AUC of approximately 800 ng·h/mL [50,51]. S-1 doses above these MTDs were associated with Grade 3 or 4 gastrointestinal and myelosuppressive toxicities.
The PK characteristics of DFP-11207 continuous dosing indicate 5-FU concentration levels and 5-FU AUCs conducive to an anti-tumor effect and minimal toxicity, supported by preliminary evidence of anti-tumor activity suggest promise for future clinical trials of DFP-11207 in monotherapy or combination with standard chemotherapeutic drugs, specifically as a substitution for 5-FU, capecitabine or S-1 within standard 5-FU or oral 5-FU derivative treatment regimens for the treatment of a variety of 5-FU-responsive cancer indications.    1 Overall Response based on patients with either a Complete Response (CR) or Partial Response (PR) 2 Number of Patients used as denominator to calculate percentages 3 Clopper-Pearson method used for the calculation of the 95% confidence interval 4 Includes patients with no post-baseline tumor assessments and/or symptomatic deterioration and/or death due to any cause Invest New Drugs (2020) 38:1763-1773 Funding information The work was supported by Delta-Fly Pharma, Inc. and by Prof. Jaffer Ajani.

Compliance with ethical standards
Conflict of interest Jaffer Ajani declares receiving grants/research support from Delta-Fly Pharma, Inc., ProLynx, Bristol-Myers Squibb, Merck, Astellas, Roche, Zymeworks, Eli Lilly, Taiho Pharmaceutical, and honoraria or consultation fees from Bristol-Myers Squibb, Merck, Astellas, Eli Lilly, AstraZeneca, Daiichi Sankyo, Roche. Milind Javle declares receiving honoraria or consultation fees from Rafael Pharmaceuticals, Incyte, Pieris Pharmaceuticals, Merck, Novartis, Seattle Genetics, BeiGene, QED Therapeutics, Bayer. Cathy Eng declares that she has no conflict of interest. David Fogelman declares receiving grants / research support from Delta-Fly Pharma, Inc. Jackie Smith declares that she has no conflict of interest. Barry Anderson declares that he has no conflict of interest. Chun Zhang is an employee of Delta-Fly Pharma, Inc. Kenzo Iizuka is an executive officer of Delta-Fly Pharma, Inc.
Ethical approval This study was conducted in accordance with International Conference on Harmonization Good Clinical Practice (ICH/GCP), the protocol, all applicable regulatory requirements, and guiding principles of the Declaration of Helsinki and its later amendments.
Informed consent Informed consent was obtained from all individual participants included in the study.
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