Regular Abstract Submission
Nongnuj TANPHAICHITR
Professor Emeritus
Ottawa Hospital Research Institute
Ottawa, Ontario, Canada
Background: Human cathelicidin, LL-37, its truncated peptides (GI-20, GF-17), and 17BIPHE2 (engineered peptide based on the GF-17 sequence) are known for their microbicidal activities. We have demonstrated that these peptides have spermicidal activity on both human and mouse sperm. Their contraceptive activity in vivo, as well as their microbicidal activity on Neisseria gonorrhoeae, has been shown. Female mice transcervically injected with any of these peptides together with motile sperm fail to become pregnant. However, these small peptides (2.1-4.5 kDa) rapidly diffuse out from the vagina or uterus following administration in the tissue lumen, making it impossible to test for their contraceptive efficacy by natural mating of the administered females with fertile males. Formulation of the peptide for vaginal delivery is needed, and we have selected 17BIPHE2 in this development, since it is more resistant to body fluid proteases due to possession of D-amino acids. The hydrogel approach (using 2% hydroxyethylcellulose (HEC), known as a universal hydrogel) is chosen because of its simplicity.
Aims: Develop 17BIPHE2 formulation in 2% HEC and test for its contraceptive efficacy in vivo via natural mating, as well as safety of 17BIPHE2-HEC application to the female reproductive tract (FRT) tissues.
Methods: 17BIPHE2 (various concentrations) formulated into 2% HEC made in bicarbonate/CO2 salt buffer, pH 7.4 was first determined for the spermicidal activity on Percoll-gradient-centrifuged sperm (100% motile), layered on top of it. 17BIPHE2 (86.4 µM)-HEC completely immobilized sperm and therefore used to determine its contraceptive efficacy. Females were tracked for their estrous cycle stages and transcervically injected with 17BIPHE2-HEC or HEC (negative control) one hour before the start of the estrus dark phase. They were then co-caged (1:1) with fertile males for two days, and subsequently assessed for pregnancy by pup delivery. The in vivo spermicidal activity of 17BIPHE2-HEC was also evaluated by videomicroscopy of uterine sperm recovered from 17BIPHE2-HEC-administered females right after the mating. Moreover, toxicity of 17BIPHE2-HEC was histologically determined on FRT tissues in females 24 h after transcervical injection of 17BIPHE2-HEC. Reversibility of the contraceptive effect of 17BIPHE2-HEC was also assessed. After 3 weeks of resting, the unpregnant 17BIPHE2-HEC females were co-caged with fertile males and monitored for pregnancy.
Results: Fifteen out of 16 females injected with 17BIPHE2-HEC failed to become pregnant, whereas pregnancy occurred in 70% of 14 females injected with HEC. However, the only pregnancy in 17BIPHE2-HEC females ended in a stillbirth. As expected, uterine sperm collected from the 17BIPHE2-HEC administered females were immotile in contrast to the robust hyperactivated motility of uterine sperm from HEC injected females. The vagina, cervix and uterus of 17BIPHE2-HEC injected females were histologically normal like HEC-injected or PBS-injected females. Among 15 unpregnant 17BIPHE2-HEC females, 14 became pregnant (93%) in the reversibility experiment.
Conclusions: 17BIPHE2-HEC had a high contraceptive efficacy but with >90% reversibility. Together with its lack of toxicity to the FRT tissues, it is a promising candidate to be developed into an MPT (multipurpose prevention technology) agent. Women intravaginally administered with 17BIPHE2-HEC will have empowerment to protect themselves against both unplanned pregnancies and sexually transmitted infections.
Funded by Canadian Institutes of Health Research