Background
PR-104 is a phosphate ester dinitrobenzamide mustard pre-prodrug that is rapidly hydrolyzed systemically to PR-104A, a bioreductive prodrug. PR-104A is in turn activated via reduction by NADPH:cytochrome P450 oxidoreductase and other one-electron reductases in hypoxic cells, and by aldo-keto reductase 1C3 (AKR1C3) independently of oxygen, to the corresponding hydroxylamine (PR-104H) and amine (PR-104M) metabolites. Subsequently, these reactive nitrogen mustards crosslink DNA and cause cytotoxicity in cells [
1‐
5]. Hypoxia is a common feature of many solid tumours and is associated with poor prognosis and resistance to conventional radiotherapy and chemotherapy [
5‐
8]. In addition, AKR1C3 has been shown to be up-regulated in several types of human cancer [
3,
9‐
11]. Targeting ACK1C3 and hypoxia in tumours is therefore a novel and promising approach to cancer therapy.
PR-104 is known to have preclinical antitumour activity in human tumour xenograft models as mono-therapy and in combination with radiotherapy and chemotherapy [
1,
2,
12]. In rats and dogs its dose-limiting toxicity (DLT) was myelosuppression [
13]. A previous phase I study of PR-104 given as a 1-hour intravenous infusion once every three weeks identified fatigue, neutropenic sepsis and infection with normal neutrophil counts as the DLTs and a maximum tolerated dose (MTD) of 1100 mg/m
2 3-weekly [
13,
14]. The current phase I study of weekly PR-104 was undertaken to explore whether its therapeutic index is related to the schedule of administration. If so, then weekly administration may provide higher dose-intensity and greater tumour activation of PR-104, as well as being a schedule potentially suitable for combination with fractionated radiotherapy or with weekly chemotherapy. For this reason, we carried out the phase I study we now report whose primary objective was to determine the MTD of intravenous PR-104 administered weekly in subjects with solid tumours. Secondary objectives included characterising the pharmacokinetics of PR-104, PR-104A and its major metabolites [
15,
16] and their relationship to toxicity, and assessing evidence of anti-tumour activity.
Methods
Patient selection
Eligibility criteria for entry into this study included: age 18 years or more; histologically confirmed malignancy for which no effective therapy existed; measurable or evaluable disease; ECOG Performance Status of 0 or 1; ability to provide written informed consent; no or stable dose of systemic steroid for at least two weeks; adequate bone marrow function (absolute neutrophil count ≥ 1.5 × 109/L, platelet count ≥ 100 × 109/L, haemoglobin level > 90 g/L not maintained by red blood cell transfusion, prothrombin and activated partial thromboplastin times < 1.1 × upper limit of normal (ULN)); adequate liver function (serum bilirubin within normal limits, ALT and AST < 2.5 × ULN), and serum creatinine < 1.5 × ULN. Exclusion criteria included: licensed or investigational anti-cancer therapy (including radiotherapy but excluding androgen deprivation therapy) within four weeks; nitrosoureas or mitomycin C within 6 weeks; prior radiotherapy to more than 25% of bone marrow; prior high-dose chemotherapy; prior receipt of more than three chemotherapy regimens; pregnancy, breast feeding or plans for becoming pregnant during the study; unwillingness to use effective contraception during the study and for 30 days following the last dose of study medication; other medical disorder or laboratory finding that in the opinion of the investigator compromised subject safety; less than four week since major surgery, or; HIV, Hepatitis B surface Antigen or Hepatitis C positivity with abnormal liver function tests.
Study design
This was a phase I, two-centre, open label, uncontrolled, serial cohort, dose-escalation study evaluating weekly intravenous administration of PR-104. Patients were enrolled in serial cohorts of three patients each using a conventional phase I study design to establish the MTD. Recruitment to dose-levels was expanded if DLT was observed.
Drug administration
A lyophilized cake of 400 mg of PR-104 was reconstituted in two mL of water for injection, further diluted in 250 mL of 5% dextrose in water and administered as an intravenous infusion over one hour on days 1, 8 and 15 of a 28 day treatment cycle. Prophylactic anti-emetics were administered to all study patients. Other anticancer treatments (except androgen deprivation therapy), prophylactic haematopoietic growth factors and radiotherapy were not permitted during the study.
Definition of DLT and MTD
Toxicity was assessed according to the National Cancer Institute Common Toxicity Criteria for Adverse Events (version 3.0). DLT was assessed during the first four weeks following the start of PR-104 administration and was defined as any one of the following: grade four thrombocytopenia (platelets < 25 × 109/L) of any duration; other grade four haematological toxicity that lasted for five days or more (haemoglobin < 65 g/L, neutrophils < 0.5 × 109/L); non-haematological toxicity ≥ grade three despite appropriate treatment; neutropenic fever; grade two or higher neurotoxicity lasting one week or more; any toxicity of grade two or higher that has not resolved within two weeks of the end of cycle one (except grade two alopecia). The MTD was defined as a dose level at which one or fewer subjects in six exhibited DLT and for which the next highest dose level demonstrated two or more of six subjects with DLT.
Starting dose and dose escalation scheme
The starting dose was 135 mg/m2 based on results of toxicology studies in rats and dogs, of which the rat was the most sensitive pre-clinical species to PR-104 (MTD 1328 and 2678 mg/m2 in rats and dogs, respectively by weekly dosing for 4 consecutive weeks). Five dose-levels were explored: 135, 270, 540, 675 and 900 mg/m2. Dose-levels were escalated by 100% or less according to the toxicity at preceding dose-levels after review of safety data by the study safety committee.
Patient evaluation and follow-up
After gaining informed consent, baseline evaluations included a history and physical examination, assessment of performance status, complete blood count (CBC), blood chemistry profile, coagulation studies (INR and APPT), urinalysis, pregnancy test and serum tumour markers. Vital signs and electrocardiogram were taken before, during and after the administration of the first dose of PR-104. With each cycle, weekly assessments included performance status, symptom-directed physical examination, laboratory investigations (CBC, coagulation studies, serum chemistry and urinalysis), intercurrent adverse events and concomitant medication use. Extent of disease was determined by computed tomography or magnetic resonance scans prior to enrolment and repeated once every two treatment cycles. Efficacy was assessed using Response Evaluation Criteria in Solid Tumours (RECIST) criteria version 1.0 [
17]. Where possible, complete blood counts data was collected after the end of the study participation until death or commencement of other cancer therapy.
Pharmacokinetic analyses
To evaluate the pharmacokinetics of PR-104, PR-104A and their major metabolites, blood samples were collected in EDTA vacutainer tubes pre-dose, 45 minutes into the infusion, immediately following the completion of the infusion and at 5, 10, 20, 30, 45, 60, 120, 240 minutes and 24 (±8) hours post-infusion of the first dose. Blood samples were centrifuged for five minutes to prepare plasma. Plasma was then immediately deproteinised by addition of nine volumes of methanol:ammonium acetate: acetic acid (1000:3.5:0.2 v/w/v) and stored at -70°C. Extracts were assayed by validated ultra high-performance liquid chromatography methods [
18] using triple quadrupole mass spectrometric detection with tetradeuterated internal standards [
19]. Non-compartmental pharmacokinetic analyses using WinNonLin (v4.0.1), and actual infusion times and doses, was used to derive the maximum plasma concentration (C
max), area under the plasma concentration time curve extrapolated to infinity (AUC
(0-inf)), clearance (Cl), volume of distribution at steady state (V
ss) and elimination half-life (t
1/2).
Statistics
Data were analysed using descriptive statistics including the median, range and proportion, and mean and standard deviation for normally distributed data. Relationships between dose-levels, pharmacokinetic parameters, percentage change in platelet and neutrophil counts and creatinine clearance were assessed by nonparametric correlation analysis. A P < 0.05 was regarded as being statistically significant.
Discussion
We describe a phase I trial evaluating weekly administration of PR-104, a novel nitrogen mustard pro-drug activated by hypoxia and AKR1C3. Tumour hypoxia is common in solid tumours and associated with poor prognosis and resistance to radiotherapy and chemotherapy [
6‐
8]. AKR1C3 has been shown to be up-regulated in some human cancers [
9‐
11]. On this basis, targeting tumour hypoxia and AKR1C3 is a promising approach to cancer therapy.
In the current study, we determined the MTD of weekly PR-104 to be 675 mg/m2 given on days 1, 8 and 15 every 28 days. This MTD was established by the occurrence of protocol-defined DLTs in one of six (17%) of patients at the 540 mg/m2 dose-level, and in two of four (50%) of patients at the 900 mg/m2 dose-level. DLTs included two episodes of grade four thrombocytopenia, and a single episode of grade four neutropenia lasting greater than five days. In contrast, there was no DLT at the MTD dose-level in a total of seven patients given a total of 12 treatment cycles (median 2, per patient) and 33 infusions (median, 5 per patient), although all seven experienced moderate to severe (grade 2 to 4) haematological toxicity. Thus, this study met its primary objective of determining the DLT and MTD of weekly PR-104.
Thrombocytopenia was the most significant clinical toxicity associated with weekly PR-104 in this phase I study. It was the major DLT and necessitated platelet transfusions for very low counts and/or bleeding in some patients but specific details of blood product support were not collected prospectively. The onset and nadir of thrombocytopenia were delayed relative to the commencement of PR-104 treatment by a median of six and ten weeks, respectively. Thereafter, thrombocytopenia persisted without recovery to baseline or to normal levels in most patients in whom it was feasible to undertake longer term monitoring of platelet counts. Persisting thrombocytopenia limited the delivery of weekly treatment with PR-104 beyond two treatment cycles at dose-levels of 540 mg/m2 or more. The extent and significance of protracted thrombocytopenia may have been underestimated by this and previous studies of PR-104 because of their use of definitions for DLTs confined to adverse-events occurring within the first four weeks of treatment, their lack of prospectively defined plans for longer term monitoring of haematological toxicity and difficulties doing so in study populations with short survival. In contrast, neutropenia was earlier in onset and more reversible than thrombocytopenia but also common and dose-limiting. Other toxicities included anaemia, fatigue and gastrointestinal symptoms but were less clinically significant and not dose-limiting.
One hypothesis for the mechanism underlying the myelotoxicity of PR-104 is that the circulating reduced metabolites (PR-104H and PR-104M), which are activated nitrogen mustards, might be responsible. However, the lack of correlation between systemic exposure to these metabolites and either neutropenia or thrombocytopenia (Table
8) argues against this hypothesis. This in turn suggests that myelotoxicity might result from activation of PR-104A locally in the bone marrow, as a result of expression of AKR1C3 in myeloid progenitor cells [
22] and/or hypoxia in bone marrow stem cell niches as reported in mice [
23].
The findings of this study suggest that treatment protocols for giving PR-104 at weekly intervals at or about a dose of 675 mg/m
2 may be feasible for a short course of treatment only as the total duration of treatment would be restricted by toxicity. Continued treatment at this dose-level beyond six to eight weeks may not be feasible because of delayed onset thrombocytopenia that would then persist, necessitating the deferral, reduction or omission of subsequent PR-104 dosing. Single doses of PR-104 of 675 mg/m
2 were shown by pharmacokinetic studies carried out on day one of cycle one in the current study to achieve systemic exposures to PR-104A ranging between 10.9 to 25.9 uM.hr. These PR-104A AUC values were close to but lower than those (> 30 μM.hr) achieved in a previous phase I study of q 3 weekly dosing of PR-104 [
14], and those associated with preclinical activity of PR-104 in human tumour xenograft models (> 30 μM.hr) [
1,
2,
12]. Repeated dosing of PR-104 at 675 mg/m
2 was shown to achieve cumulative exposures to PR-104A exceeding these threshold levels required for experimental activity, since a target AUC of 30 μM.hr or more was exceeded after two or three doses (Table
7). In addition, reductions in tumour dimensions of > 20% seen in two patients at > 540 mg/m
2, and significant myelosuppression likely related to the main pharmacological action of PR-104, provides further supporting evidence for therapeutically active exposures having been achieved on the phase I trial of weekly treatment. Thus, treatment protocols for giving PR-104 weekly at or about a dose of 675 mg/m
2 over 4 to 6 weeks may be suitable for future study to explore its clinical feasibility and activity as a single agent or in combination therapy.
Short courses of weekly PR-104 may be suitable for use concurrently with fractionated radiotherapy or as high-dose chemotherapy prior to bone marrow reconstitution. Many conventional fractionated radiotherapy regimens are limited by tumour hypoxia and given over a short period of up to six weeks, to which it may be feasible to add weekly PR-104. The delayed-onset myelosuppresion of PR-104, combined with its minimal non-haematological toxicity, may allow its delivery at or beyond the current MTD prior to haematological stem cell support via infusions of autologous bone marrow or peripheral blood stem cells. It will be necessary however to establish the feasibility of treatment protocols based on weekly PR-104 combined with fractionated radiotherapy and/or bone support in further early phase clinical studies with careful longer term monitoring of haematological toxicity and provision of blood product support as required. An exploratory study of PR-104 in relapsed and treatment-refractory acute leukaemia is currently underway (ClinicalTrials.gov Identifier: NCT01037556;
http://clinicaltrials.gov/ct2/show/NCT01358227?term=pr104&rank=3) that is supported by evidence of preclinical activity in this disease setting [
24].
Competing interests
WRW is a stock holder and advisor to Proacta, Inc. TJM is an employee of Proacta, Inc. The authors have no other competing interests to declare.
Authors' contributions
MJM contributed to the study design, patient recruitment, clinical study procedures, data interpretation and preparation of the final manuscript. YG and WRW contributed to the pharmacokinetic study procedures, data interpretation and preparation of the final manuscript. AH and KA contributed to patient recruitment and clinical study procedures. TJM contributed to the study design, data interpretation and preparation of the final manuscript. MBJ contributed to the study design, patient recruitment, clinical study procedures, data interpretation and preparation of the final manuscript. All authors read and approved the final manuscript.