Similar to other low molecular weight protein drugs, alpha or gamma interferons (IFNs) have a relatively short serum half-life. Consequently, if vascular retention is considered to be desired for enhanced efficacy, strategies that can improve a drug’s pharmacokinetic (PK) and pharmacodynamic (PD) properties might improve its therapeutic benefits. Novel injectable drug delivery systems have been developed in attempts to improve PK/PD properties of therapeutic peptides and proteins. This can be achieved either by modification of the drug molecule itself (e.g. pegylation) or through a change in formulation (e.g. controlled-release formulations, liposomal preparations) [1].

Another alternative is to potentiate the pharmacodynamics of the therapeutic drug by the combination of two active principles that can act synergistically. This approach could have the same potential advantages of novel delivery mechanisms that include an increased or prolonged pharmacological activity without additional toxicity, less frequent injections, and a better patient’s compliance and quality of life.

IFNs have been widely used in the treatment of human solid and hematological malignancies. However, despite antitumor activity of IFNs is well-known at present, no major advances have been achieved in the last decade. A hopeful option could be the combination of IFN alpha-2b and IFN gamma, two molecules with recognized synergistic antiproliferative effects on several tumor cell lines [2].

Takaoka et al. demonstrated in mouse embryonic fibroblasts that IFN gamma response is substantially augmented through autocrine IFN alpha/beta. Additionally, they observed that cross-recruitment and phosphorylation of one of the IFN alpha/beta receptor subunits (IFNAR1) occurred in response to IFN gamma [3]. Similarly, other authors showed that IFNGR1-IFNAR2 (ligand binding domains of both receptor systems) are associated in the presence of IFN alpha and IFN gamma [4]. The physical interaction between both IFN receptor complexes may be the first step for the triggering of intracellular signals that promote the synergism between both IFNs. In the clinics, the peri- and intralesional administration of a new pharmaceutical stabilized formulation containing IFNs alpha-2b and gamma (HeberPAG®), was safe and effective for the treatment of elder patients with advanced, recurrent or resistant to previous treatments basal and squamous cell skin carcinomas [5].

The main objective of this study was to characterize the PK/PD of HeberPAG® in patients with mycosis fungoides. Classical IFN-inducible biological markers, neopterin, β2-microglobulin (β₂M), and 2’-5’ oligoadenylate synthetase (2’-5’ OAS1), were used as indicators of their pharmacodynamic action. A secondary objective was the registration of adverse events.

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.

Twelve patients, 7 females and 5 males, with a disease stage IIA and III, were recruited. They were between 33 and 74 years-old (mean: 53.3 yrs), with a mean corporal surface of 1.79 m² (range: 1.46 – 2.28). Three clinical variants of mycosis fungoides were represented; 6 patients had plaques, 2 had erythroderma, and the rest had atypia of the epidermis. Only five patients had received prior systemic antitumor therapy, four of them IFN alpha and three methotrexate. Other previous treatments (cyclosporine, steroid cream, radiotherapy) were received by a single patient each one. Initial mean LDH was 216 U/L (range: 69 – 355). Most of the patients complied with the evaluations as previewed, except for patient No.8 whose serum samples were not available at 72 and 96 hours.

Pharmacokinetic analysis

Except for two patients, who had 7.1 and 114 pg/mL of IFN alpha-2b, all had undetectable or very low endogenous pre-dose serum IFN concentrations (median = 0 pg/ml). The average concentration profiles obtained for both IFNs are showed in Figure 1. IFN alpha-2b concentrations started to increase notably from the first hour post-injection. At 3 hours, more than half of the patients reached 100 pg/mL. Maximum values (> 200 pg/mL) were obtained at 6–12 hours in most of the cases (Figure 1a). Patient No. 12, who had the higher baseline value, reached 440 pg/mL at 12 hours. Increments in IFN gamma levels were more discreet with a peak at 12 hours post-injection, where 5 patients reached 15 pg/mL or more (Figure 1b). A pronounced drop of IFN concentrations was detected since 24 hours. At 48–96 hours after the injection, these values had returned near to the initial values, so the AUC₉₆ obtained covered more than 95% of the AUC extrapolated to infinite.

Table 1 shows the results of the pharmacokinetic parameters calculated from the above commented profiles. A high patient -dependant variability is observed, mainly with IFN gamma (see SD). Additionally, an elevated distribution volume and fast blood clearance of both IFNs was obtained (data not shown).

Parameter	IFN alpha-2b	IFN gamma
Cmax (pg/mL)	263 ± 129	9.3 ± 7.0
Tmax (h)	12.0 ± 6.0	6.0 ± 0.8
λ (h-1)	0.16 ± 0.06	0.07 ± 0.05
t1/2 (h)	4.9 ± 1.4	13.4 ± 27.1
AUC (pg.h/mL)	4483 ± 4485	87.5 ± 89.9
MRT (h)	13.9 ± 7.9	13.5 ± 8.2

Data are reported as mean ± standard deviation, except for Tmax expressed as median ± quartile range.

Pharmacodynamic analysis

The time courses for the induction of neopterin and β₂M are showed in Figure 2. Both variables were strongly stimulated by simultaneous administration of both IFNs with highest inductions around 24–48 hours after intramuscular administration. HeberPAG® induced an average increment of serum neopterin at 48 hours about approximately six times, until 9.6 pg/mL compared to pre-dose values (Figure 2a). Individually, five patients surpassed 10 pg/mL at this time. Figure 2b shows that mean serum β₂M peaked around the double from baseline at 24–48 hours. For both variables the values remained clearly upper baseline levels until 96 hours. Notably, neopterin remained three times superior to pre-dose value at the end of the sampling period.

A pharmacokinetic-like analytical procedure was carried out with both variables (Table 2). Since patients could have pre-treatment levels of these biological markers, a more real interpretation of kinetics has to be showed as fold increases over baseline. For neopterin Rmax was 8.0 ng/mL, with a t_1/2 effect = 40 hours and a MET around 80 hours. For β₂M, T(Rmax) was 2.7 μg/mL, and the effect dissipation phase occurred similarly slow compared to neopterin. A high intra-patient variability was again evidenced.

Parameter	Neopterin	β2-microglobulin
Rmax	8.0 ± 4.2 ng/mL	2.7 ± 1.4 μg/mL
T(Rmax)	48 ± 24 h	24 ± 24 h
λ effect	0.02 ± 0.01 h-1	0.02 ± 0.01 h-1
t1/2 effect	40.3 ± 12.9 h	37.6 ± 15.9 h
AUEC	661 ± 322 ng.h/mL	195 ± 117 μg.h/mL
MET	80.6 ± 17.4 h	74.8 ± 21.9 h

Data are reported as mean ± standard deviation, except for T(Rmax) expressed as median ± quartile range.

Since the individual induction of the enzyme 2’-5’ OAS1 was measured through the relative amount of its mRNA expression using the Real-Time PCR method, an important variability could be expected. Therefore, parameters could not be rigorously calculated from the experimental data and the analysis was essentially qualitative. Figure 3 shows individual relative OAS1 mRNA levels at each time point with respect to time zero, normalized with GAPDH/HMBS levels. A single intramuscular dose of 23 x10⁶ IU of HeberPAG® resulted in high increases of this variable with regard to their pre-dose values in all patients, in a factor between 8 and 122 times in ten patients, primarily within the first 24 hours after dosing. The expression levels return to initials by 96 hours in most of the patients.

Treatment with unmodified IFNs for several malignancies and chronic viral affections requires frequent injections (e.g., daily or three times weekly) over the course of therapy due to the molecule’s short circulating half-life in humans. Increase of doses and prolonged therapy could favor a better clinical response but it could also lead to magnify adverse events. Besides, patient’s compliance under long-term dosing regimens is difficult to preserve. The development of more slowly cleared IFNs has allowed to reduce dosing frequency and to enhance response rates in patients with chronic hepatitis C [9]. However, modified high molecular IFNs could have more difficulties to penetrate the tumor niches bearing a reduction in their antitumor effects. A significant reduction of in vitro biological activity has been demonstrated for pegylated IFNs due to non optimal interaction with IFN receptor [10].

Therefore sustained full IFN-receptor interactions with more potent antiproliferative activity are desired in the treatment of cancer. This is possible to obtain combining IFN-alpha and IFN gamma that synergize for their biological activities. HeberPAG® is a new formulation containing a mixture of recombinant IFN alpha-2b and IFN gamma at synergistic proportions. This formulation was created to improve antiproliferative and other biological effects of conventional IFNs with an adequate tolerability leading to administer fewer doses similar to others currently available therapy. This is the first PK/PD study in humans with this variety of IFN formulation.

The pharmacodynamic variables measured in this trial to characterize HeberPAG® formulation are well-known IFN-induced genes, classical surrogate markers of IFN biological actions. Neopterin is a sensitive marker of T helper 1-cell immune response, because it is primarily produced by monocytes/macrophages after activation by IFNs and augments the production of tumor necrosis factor in peripheral blood mononuclear cells [11]. Beta2-microglobulin plays an important role in the tumor growth control and metastases [12]. Progression of the cell cycle is mediated by 2’- 5’OAS levels stimulated by IFN [13]. Additionally, antiviral effects subsequent to IFNs addition are initiated by synthesis of 2’- 5’oligoadenylates that activate an endoribonuclease to cleave double-stranded viral RNA [14].

The most remarkable result was the six-fold increase of serum neopterin concentrations respect to basal value. This high increment induced by HeberPAG® has not been described before in the literature with any subtype or variant of IFN, even for pegylated forms [15–20]. The induction by pegylated IFN-alpha could only approximately tripled the neopterin basal values as maximum 48 hours after injection as reported [17, 19, 20]. In the case of PEG-IFN beta although half-life was greatly extended by pegylation, the neopterin response was not affected [18]. On the other hand, two times higher levels than baseline were recorded for serum β₂M 24–48 hours after injection, superior to those increments detected by other authors, which were around 60% with natural or pegylated IFN-alpha [19–21]. For both pharmacodynamic markers their more slow return to initial levels has not been observed with conventional IFN in the reports above-cited. This last result could lead to space the dosage interval for the IFN mixture formulation until twice or once a week. Recent data obtained with the same markers but in healthy male volunteers [article in preparation] emphasize that possibility. Efficacy trials evaluating these frequencies of administration in several oncologic pathologies are under development.

After the single intramuscular injection, 2’- 5’-OAS1 mRNA levels were extensively increased which was also recently found in a group of healthy male volunteers who received 24.5 × 10⁶ IU of a similar formulation. Although mRNA induction does not ensure the presence of active protein, it has been reported that 2’-5’ OAS enzyme activity in the serum of IFN-treated patients appears to increase since the first 6 hours and maintains elevated levels for as long as 4 to 8 months after the initiation of daily IFN treatment [22].

At molecular level this beneficial pharmacodynamic effects could be explained by synergistic effects in the expression and activation of several genes regulated by both IFNs [23].

Concerning pharmacokinetics, no interferences by simultaneous administered IFNs were observed in their typical similar serum profiles. Parameters as Tmax and t_1/2 were within the reported ranges for these conventional IFNs after systemic administration either in patients or healthy volunteers even considering the expected high variability [24–27]. For IFN alpha-2b Cmax was also very similar to a previous report ours in healthy male volunteers [21].

Flu-like symptoms and other clinical and laboratory adverse events associated with HeberPAG® have been previously reported for recombinant IFN treatment [28]. Fever began 2 to 4 hours after intramuscular administration and peaked at 6 to 12 hours coincident with maximum IFN serum levels. However, the mechanisms of fever induction appear to be different between IFNs. IFN-alpha has been shown to be intrinsically pyrogenic and the body temperature rise is related to the interaction of IFN alpha to hypothalamic μ-opioid receptors [29]. Meanwhile the administration of IFN gamma stimulates the release of other lymphokines such as interleukin-1 [30], an endogenous pyrogen [31].

The co-administration of IFN alpha-2b and IFN gamma with potent synergistic actions will allow us to obtain a more favorable pharmacodynamics introducing new promissory perspectives in the use of IFNs to treat several malignancies. Efficacy trials can be carried out to ratify the obtained results.