An exploratory, prospective, open-label clinical trial was carried out at the “Hermanos Ameijeiras” Hospital, Dermatology Service, Havana, Cuba. The protocol was approved by the Ethics Committee of this hospital and by the Cuban Regulatory Authority, the State Center for the Control of Drugs, Equipment & Medical Devices (CECMED, reference number: 999/16.016.09.B). The trial was in compliance with the Helsinki Declaration and its amendments. All patients prior to study enrollment provided their written informed consent.
Subjects
Twelve patients, both genders, 18 to 75 years-old, with clinically and histologically proven mycosis fungoides, at stages IB to III, were recruited for the study. Other eligibility criteria included a measurable disease, a life expectancy of at least 24 weeks, Karnofsky’s index ≥ 60%, with more than 1 month of previous disease specific treatments or more than 3 months in case of steroid use. Patients also had adequate hematological, hepatic, and renal function. Exclusion criteria were other uncompensated chronic diseases or neoplasias, pregnancy or nursing and severe psychiatric dysfunction.
HeberPAG formulation
A stabilized formulation containing 3.5 x106 IU of a synergistic combination of human recombinant, produced in E. coli, IFNs alpha-2b and gamma (HeberPAG®, Heber Biotec, Havana, Cuba), was used for all cases. Each vial also contains 18.87 mg sodium phosphates, 13 mg Dextran-40, 0.24 mg kalium phosphate, 16.59 mg sodium chloride, 0.18 mg kalium chloride, 5.0 mg manitol, 5.0 mg saccharose, and 5.5 mg human albumin. This lyophilized powder formulation was reconstituted with 2 mL bacteriostatic water for injection.
Study design
The study was designed to calculate pharmacokinetic and pharmacodynamic parameters after a first single high dose of HeberPAG®. Each patient received, intramuscularly, in the gluteus region, a single 23 x106 IU HeberPAG® dose, chosen to obtain detectable serum levels of both IFNs. Antipyretic medication was given orally at the same time as the HeberPAG® injection and every 4 hours thereafter, up to 12 hours or more if needed in order to mitigate the expected IFN-dependent flu-like syndrome. Patients were hospitalized during the first 96 hours after the injection under strict medical supervision. After this period the study continued to evaluate efficacy and safety of this product in the same group of patients. Then, they received 11 x106 IU twice a week during one year.
Laboratory evaluations
Blood samples for serum IFN alpha-2b and IFN gamma concentration determinations were collected by venipuncture before and 1, 3, 6, 12, 24, 48, 72, and 96 hours after injection. Pharmacodynamics was assessed by serum neopterin and β2M concentrations at the same times and by the induction of 2’-5’ OAS1 mRNA expression before and at 6, 12, 24, 48, 72 and 96 hours. Routine hematological and biochemical determinations were taken as safety variables, every 24 hours during the first 96 hours. These included hemoglobin, hematocrit, leukocytes and platelets counts, transaminases, bilirubin, creatinine and urea. Patients were regularly checked for vital signs and symptoms during the whole hospitalization.
Vacutainers were used to collect blood samples to determine serum concentrations and biochemistry (8.5 mL Z Serum Sep, Greiner bio-one) and for hematology analysis (4 mL K3E K3EDTA, Greiner bio-one). Blood samples for total RNA purification were collected in PAXgene Blood RNA Tubes (2.5 mL, QIAGEN, US).
Serum IFNs, neopterin and β2M levels were measured using commercially available kits according to the manufacturer’s instructions using sera stored at −80°C until be tested. IFN alpha-2b and IFN gamma were quantified in serum with high sensitivity enzyme immunoassay (EIA) kits specific for IFN alpha (Catalogue: BMS216CE, Bender MedSystem, GMBH) or IFN gamma (Catalogue: BMS228CE, Bender MedSystem, GMBH), respectively. Neopterin was determined by a commercial EIA kit (HENNING test, BRAHMS Diagnostica GmbH, Berlin, Germany) as well as serum β2M (Quantikine® IVD®, R&D System, Inc, Minneapolis).
Quantification of OAS1 mRNA levels was performed using the Real-Time Polimerase Chain Reaction (qPCR) method. Total RNAs were obtained by PAXgene purification protocol (PreAnalytiX/Qiagen, US). RNA quality was checked in a Spectrophotometer Nanodrop 1000 (ThermoScientific, US), reporting a 260/280 nm OD relation between 1.7 and 2.2. Agarose electrophoresis allowed to visualize 28S rRNA and 18S rRNA bands in a proportion higher than 1.5. Complementary DNA (cDNA) was used as template in qPCR experiments; the synthesis was carried out using Superscript II RT kit and protocol (Invitrogen, US) from total RNA samples at each time point. qPCR experiments were performed in 20 μL using ABsolute QPCR SYBR Green Mixes (ThermoScientific, ABgene, UK) and 0.3 μM of primers for amplification of OAS1 target gene (F: 5’ AGCCTCATCCGCCTAGTCAA 3’; R: 5’ CTCGCTCCCAAGCATAGACC 3’) and reference genes GAPDH (F: 5’ CCATGGGTGGAATCATATTGGA 3’; R: 5’ TCAACGGATTTGGTCGTATTGG 3’) and HMBS (F: 5’ GGAATGTTACGAGCAGTGATGC 3’; R: 5’ CCTGACTGGAGGAGTCTGGAGT 3’). All of them were designed on the basis of the GenBank database information using primer3 software [6]. All experiments were in triplicates, rendering amplifications curves between cycles 15 and 30 in RT™Cycler equipment (Capitalbio, China) with a standard program of 15 min at 95°C for enzyme activation followed by 40 cycles of 15 s at 95°C, 30s at 60°C and 30s at 72°C. Capitalbio software reports of Ct values, as the 2nd derivative maximum, and values of fluorescence per cycle, which were used for efficiency calculation by LinReg software (version 11.3, 2009), were used for calculations of relative OAS1 gene expression, at each time point respect to time 0 h, using REST-MCS (version 2, 2006) software [7], after the normalization with reference genes.
Hematological counts and blood chemistry were done according to usual clinical laboratory procedures, using advanced automated analyzers.
Data analysis
The drug disposition data analysis was performed per patient by a non-compartmental method with a combined linear/log - linear trapezoidal rule approach. The linear trapezoidal rule was used up to peak level and the logarithmic trapezoidal rule thereafter. The first-order rate constant associated with the curve terminal (log linear) portion (λ) and terminal half-life (t1/2) were estimated by linear regression of the included terminal data points. Time-to-peak values (Tmax) were determined directly from the experimental data as the time of maximum observed level (Cmax) considering the entire curve. Area under the serum concentration-time curve from 0 to 96 hours (AUC96) was calculated using the linear/log linear trapezoidal rule. Mean residence time (MRT) was also calculated using the moments of the drug disposition curve. Parameters that were extrapolated to infinity, such as AUC (area under disposition curve) and AUMC (area under first moment of the disposition curve) were computed based on the last predicted value from the linear regression performed to estimate λ and t1/2. Some similar kinetic parameters were estimated for the pharmacodynamic markers, corrected for baseline values, neopterin and β2M in order to describe the kinetic behavior of the IFN-induced immunological response: Rmax (maximum response), T(Rmax) (time to reach maximum response), λ effect (effect dissipation constant), t1/2 effect (effect half-life), AUEC (area under the effect curve), MET (mean effect time) [8]. The WinNonlin professional software (Version 2.1, Pharsight Inc., 1997, NC, USA) was used for all these purposes. A descriptive statistic was done using SPSS for Windows version 15.0.