Evaluation of the safety, immunogenicity, and pharmacokinetic profile of a new, highly purified, heat-treated equine rabies immunoglobulin, administered either alone or in association with a purified, Vero-cell rabies vaccine.

A clinical evaluation of a new, purified, heat-treated equine rabies immunoglobulin (PHT-Erig), F(ab')2 preparation, was carried out in Thailand and in the Philippines-two countries where rabies is endemic. An initial prospective, randomised, controlled trial (Study 1), compared the safety and pharmacokinetics (serum concentrations of rabies antibodies) after administration either of PHT-Erig or of a commercially-available, equine rabies immune globulin (Erig PMC). A second trial (Study 2) simulated post-exposure rabies prophylaxis by using a reference cell culture vaccine, the purified Vero-cell rabies vaccine (PVRV), administered in association with either Erig PMC or PHT-Erig. In Study 1, 27 healthy, Thai adults received a 40 IU kg(-1) dose of either Erig PMC (n = 12) or PHT-Erig (n = 15) via the intramuscular (i.m.) route; half of the dose was injected into the deltoid area and the other half into the buttocks. Serum for rabies antibody determination and F(ab')2 concentration was collected at hours (H) 0, 6 and 12, and on day (D) 2, 3, 4, 6, 8, 10, 12 and 15. Both products were safe, with no serious adverse events, and in particular, no anaphylactic reactions or serum sickness was reported. A statistical comparison of the pharmacokinetic parameters did not demonstrate bioequivalence of the two products. Nonetheless, the relative bioavailability of 93% and the similar absorption rates suggest the pharmacokinetic profiles of Erig and PHT-Erig are similar. The antibody level in either group were low throughout the 15-day study period. The geometric mean titer (GMT) values ranged from group 0.027-0.117 IU ml(-1) in the Erig group and from 0.029 to 0.072 IU ml(-1) in the PHT-Erig. There was no significant difference between the evolution of GMT values for the two groups. In Study 2, 71 healthy volunteers received 40 IU kg(-1) via the intramuscular route of either Erig PMC (n = 36) or PHT-Erig (n = 35) on D0, in association with five doses of PVRV on D0, D3, D7, D14 and D28. The safety evaluation was performed during the 28-day follow-up and serum samples for anti-rabies antibody titration were collected on D0 (before injection) D3, D7, D14 and D28. No serious reactions were reported in either group. In particular, no immediate (anaphylactic type) or delayed (serum sickness) allergic reactions were observed. Over the 28-day follow-up period, GMT profiles of the two groups were statistically equivalent. On D14, 100% of the subjects had protective antibody titers (anti-rabies antibodies > or = 0.5 IU ml(-1), which is the WHO-recommended level of seroconversion), and Erig PMC and PHT-Erig were indistinguishable according to the clinical definition chosen. On D28, the GMT values were 33.2 IU ml(-1) (95% CI, 23.8-46.1 IU ml(-1)) in the Erig PMC/PVRV group and 31.4 IU ml(-1) (95% confidence interval, CI, 23.4-42.2 IU ml(-1)) in the PHT-Erig/PVRV group, showing evidence of adequate vaccine-induced antibody responses in both groups. The increased purity, the heat-treatment step introduced in the manufacturing process of PHT-Erig, and the good clinical results substantiate the use of this new generation, purified equine F(ab')2 preparation in the post-exposure prophylaxis of rabies.

Preclinical assessment of immunoreactivity of a new purified equine F(ab')2 against European viper venom.

The immunological and pharmacokinetic properties of a new, further purified, pasteurized preparation of equine F(ab')2 (VIPERFAV) against Vipera aspis, Vipera berus, and Vipera ammodytes venom were compared with the current equine F(ab')2 preparation (IPSER Europe). Affinity constants of the V. aspis-specific F(ab')2 were determined using biosensor technology and found to be in the range of 10(8) M-1 for the four antigenic fractions of V. aspis toxins and for both F(ab')2 preparations. The improvement of 51% in the specific activity (LD50 mg-1) of the new F(ab')2 was in close agreement with the 1.8-fold increase in the immunoreactive fraction of the new preparation. In vivo investigations of venom immunocomplexation by F(ab')2 in rabbits confirmed the ability of F(ab')2 to neutralize and redistribute toxin venom. Infusion of a stoichiometric molar ratio (i.e., 1 mg kg-1) of the new antivenom induced a 2.3-fold elevation of the plasma venom concentration with a Tmax observed 8 h after F(ab')2 administration and a decline in the terminal half-life from 31.92 +/- 4.49 h to 16.73 +/- 4.34 h, in contrast, for the venom alone. The area under the curve was 1.4-fold greater in the VIPERFAV group than in the IPSER Europe group during the post-F(ab')2 infusion period. Increasing the F(ab')2 dose to 3 mg kg-1 increased by 27% the percent of venom bound to F(ab')2. Finally, the greater the venom distribution, the smaller and less pronounced the plasma redistribution. These results demonstrate that the purification and pasteurization steps involved in the preparation of the new F(ab')2 have no deleterious influence on F(ab')2 affinity but, on the contrary, improve the protective efficacy. Alteration of viper venom kinetics by specific F(ab')2 antivenom was also shown to be dependent on the interval between of F(ab')2 administration and venom bite and on the specific F(ab')2 dose administered.

Immunoreactivity of a new generation of horse F(ab')2 preparations against European viper venoms and the tetanus toxin.

The immunoreactivity of the current and the new more purified, pasteurized preparations of horse F(ab')2 against the tetanus toxin and Vipera aspis venom was investigated with a biosensor based on technology using the optical phenomenon of surface plasmon resonance. Immunoreactivity data were compared with seroneutralization titres to investigate immunoreactivity-immunoprotection efficacy relationships. The association-dissociation rate and affinity constants of the current and the new tetanus toxin-specific F(ab')2 preparations were similar, at about 10(4) M-1 sec-1, 10(-4) sec-1 and 10(8) M-1, respectively. Similar values were found using a solid immunoradiometric assay. To assess the immunoreactivity of V. aspis venom-specific horse F(ab')2, the mol. wt and percentage of the antigenic fractions of V. aspis venom were determined. Western blotting of electrophoresis gels showed four antigenic fractions of V. aspis venom (mol. wts 17,500, 28,500, 32,000 and 60,000), which represented 6, 3.4, 17.7 and 5% of total venom, respectively. Association and dissociation rate constants were in the same range as those of the tetanus toxin-F(ab')2 interactions for each of the four antigenic fractions. Seroneutralization of both tetanus toxin and V. aspis by the corresponding specific F(ab')2 showed that the LD50 mg-1 protein was 1.76-fold and 1.51-fold higher with the new than with the current preparations, respectively. These improvements in efficacy were in close agreement with the higher immunoreactive fraction ratios, which were 2-fold and 1.8-fold higher with the new preparations. These results demonstrate that the removal of non-IgGT immunoglobulins and the pasteurization treatment have no overall influence on F(ab')2 affinity but improve the specific activity of these new antitoxin horse F(ab')2.

Immunoreactivity and pharmacokinetics of horse anti-scorpion venom F(ab')2-scorpion venom interactions.

The immunoreactivity and pharmacokinetics of a new horse F(ab')2 scorpion antivenom and its effect on Buthus occitanus mardochei venom plasma disposition in the rabbit were studied. The scorpion venom-specific F(ab')2 affinity constant determined by immunoradiometric assay was 1.6 +/- 0.6 10(8) M-1. One group received a F(ab')2 bolus dose of 9.57 mg.kg-1 i.v. bolus or i.m.. The plasma F(ab')2 concentration followed a biexponential decline after i.v. administration with distribution and elimination half-lives of 2.54 +/- 0.36 and 49.52 +/- 3.07 hr, respectively. The total volume of distribution (Vdss or Vd beta) was between 230 and 255 ml.kg-1. Total body clearance was 3.56 +/- 0.34 ml.kg-1.hr-1. After intramuscular administration, Tmax was 48 hr and the absolute bioavailability was 36%. Two other groups of rabbits received i.v.60 micrograms.kg-1 B. occitanus mardochei venom either alone (control group) or followed by 3 mg.kg-1 scorpion venom-specific F(ab')2 administered by intravenous infusion 1.75 hr later. In the rabbits treated with horse F(ab')2 antivenom the venom concentration profile was initially identical to that observed in the control group which received venom alone before F(ab')2 administration. Subsequent infusion of antivenom induced a 1.5-fold elevation of the plasma venom concentration with a Tmax 0.5 hr after F(ab')2 administration. The AUC was 10-fold higher in the F(ab')2-treated group than in the control group in the post-F(ab')2 infusion period. Twelve hours after F(ab')2 administration the venom disposition declined with a terminal half-life equal to that of F(ab')2 (49.49 +/- 7.53 hr). These data show the ability of F(ab')2 to alter venom pharmacokinetics and demonstrate that the scorpion toxins adopt the F(ab')2 elimination properties.