Kramzer, Lindsay
(2015)
PREFORMULATION AND BIOLOGICAL EVALUATIONS FOR THE INTRAVAGINAL DELIVERY OF GRIFFITHSIN FOR HIV PREVENTION.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
HIV/AIDS persists as a global health concern, with the overwhelming majority of infections caused by sexual transmission. The lack of effective prevention modalities for women has prompted research of novel approaches to prevent disease spread. One such approach includes vaginal microbicides. The work presented within this dissertation project contributes significantly to the vaginal microbicide field, focusing on preformulation assessments, stability in the biological environment, and drug targeting of the biopharmaceutical microbicide candidate Griffithsin (GRFT).
We hypothesized that GRFT will undergo physical and chemical instabilities that will affect successful formulation and vaginal delivery of this microbicide candidate. We further hypothesized that GRFT will undergo binding interactions within cervicovaginal secretions that will negatively impacting GRFT-gp120 binding and that GRFT will not be able to inhibit HIV binding to the DC-SIGN receptor and transfer to CD4 cells because GRFT will not permeate cervical tissue.
Identification of degradation pathways for GRFT was conducted by performing preformulation experiments under selected conditions of temperature, light, shear, ionic strength, and oxidation. Analytical methods included HPLC, CD, UV-spectroscopy, ELISA, and SDS-PAGE. GRFT chemical modifications, including intact mass analyses and peptide sequencing, were evaluated in the presence of hydrogen peroxide and human cervicovaginal secretions. The effects of human cervicovaginal secretions on GRFT-gp120 binding was also assessed with ELISA. Tissue permeability and localization of GRFT was evaluated using excised human ectocervical tissue.
Major findings from this dissertation indicate that: (1) GRFT is prone to oxidation, by both hydrogen peroxide exposure and human cervicovaginal secretion exposure; (2) Methionine at position 78 in the amino acid sequence of GRFT is oxidized; (3) GRFT-gp120 binding is inhibited in human cervicovaginal secretions containing normal microflora, but not in secretions with BV; and (4) GRFT does not permeate deep into or through human cervical tissue, but does adheres to the superficial epithelial tissue.
Overall, this dissertation has created more knowledge about a drug candidate in the microbicide field and will guide further development of GRFT. Further, the methodologies implemented throughout this dissertation can be used or adapted as part of a strategy to preclinically evaluate other vaginal microbicide candidates.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
1 May 2015 |
| Date Type: |
Publication |
| Defense Date: |
23 March 2015 |
| Approval Date: |
1 May 2015 |
| Submission Date: |
1 May 2015 |
| Access Restriction: |
1 year -- Restrict access to University of Pittsburgh for a period of 1 year. |
| Number of Pages: |
192 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Pharmacy > Pharmaceutical Sciences |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Griffithsin, GRFT, microbicide, HIV prevention |
| Date Deposited: |
01 May 2015 17:52 |
| Last Modified: |
19 Dec 2016 14:42 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/25102 |
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