Formulation of Hyaluronidase Enzyme Sensitive Topical Nanomicrobicides for HIV Virus Transmission Prevention

Title from PDF of title page, viewed August 14, 2017Dissertation advisor: Bi-Botti C. YouanVitaIncludes bibliographical references (pages 159-187)Thesis (Ph.D.)--School of Pharmacy and Department of Chemistry, University of Missouri--Kansas City, 2015The objective of this dissertation is to design and optimize a nanoformulations (nanoparticle and nanofiber) delivery system loaded with anti-HIV topical microbicides for HIV prevention in women. In chapters 1 and 2, the overview of the problem, research objectives as well as the literature review of the technical and scientific background of this dissertation are introduced. In chapter 3, a study was designed to test the hypothesis that a triggered release of a topical anti-HIV microbicide (tenofovir: TFV) from hyaluronic acid based nanoparticles (HA NPs) can be achieved under the influence of hyaluronidase (HAase) enzyme. The Fractional Factorial Experimental Design (FFED) was employed to examine the formulation variables such as: molar concentrations of adipic acid dihydrazide (X₁) and 1-Ethyl-3-[3 dimethylaminopropyl]carbodiimide hydrochloride (X₂), volume of acetone (X₃) and reaction time (X₄), and their influence on the responses such as Y₁; particle mean diameter: PMD (nm), Y₂; polydispersity index: PDI and Y₃; zeta (ζ) potential: (mV). The cross-linking efficiency of NPs was characterized by Fourier Transform Infra-Red (FT-IR), and ¹³C-nuclear magnetic iv resonance (NMR) analyses. When formulated with X₁; 2.49 mM, X₂; 9.96 mM, X₃; 60 mL, X₄; 6 h, the HA-NPs exhibited a spherical shape with PMD, PDI, ζ potential, and drug loading of 70.6 ± 4.1 nm, 0.07 ± 0.02, -38.2 ± 2.8 mV, and 26.1 ± 1.2% w/w, respectively, (n = 3). Unlike for HA based gel, HAase notably triggered the drug release and HA degradation from the NPs after 24 h (~90% w/w and 65% w/w, respectively); whereas, in its absence, these values were ~39% w/w and 26% w/w, respectively. The NPs were non-cytotoxic to human vaginal VK2/E6E7 and End1/E6E7 cells and had no effect on Lactobacillus viability. These data suggested the possibility of using HA-NPs as a delivery system for intravaginal delivery of topical microbicides for the prevention of HIV transmission. In chapter 4, a study was designed to test the hypothesis that a stimuli-sensitive, safe and mucoadhesive thiolated hyaluronic acid (HA) based nanofibers (NFs) loaded with a topical vaginal microbicide (TFV) can be used for the prevention of HIV virus vaginal transmission in women. To test this, a novel thiolated sulfhydryl (-SH) group modified HA (HA-SH) was synthesized to fabricate the TFV loaded HA-SH-NFs (mean diameter ~75 nm) using electrospinning method. Sulfhydryl (-SH) group modified HA (HA-SH) were characterized for their size distribution, surface morphology, surface chemistry, crystallinity, mucoadhesion property, and in vitro drug release profile using size exclusion chromatography, powder X-ray diffraction, FT-IR, and ¹H-NMR analyses. Mucin interaction and ellipsometer measurements confirmed that mucoadhesion of HA-SH-NFs was increased compared to that of native HA polymer based on an increase in the size (~4 fold), thickness (~3 fold) and adsorbed mucin amount (~2 fold) after 3 h incubation of HA-SH-NFs with mucin. A triggered drug release (~87% w/w) from NFs (drug loading ~17% w/w) occurred after 1 h in the presence of seminal hyaluronidase enzyme. It was observed that in the absence of HAase, the drug release from NFs followed the Peppas kinetic model whereas, in the presence of HAase, NFs followed Weibull model. The HA-SH-NFs were non-cytotoxic to vaginal VK2/E6E7 and End1/E6E7 v cells and L. crispatus bacteria for 48 h. The results suggested that TFV loaded HA-SH-NFs templates developed in this study have the potential of vaginal delivery of topical microbicides for the prevention of HIV transmission. In chapter 5, in vivo evaluations of the developed HA-NPs and HA-SH-NFs were performed in female C57BL/6 mice. The histological analysis on the mice genital tract and other organs did not show any signs of damage upon once-daily administration of HA-NPs or HA-SH-NFs up to 7 days. Following 24 h exposure, HA-NPs or HA-SH-NFs did not show any significant immune (CD45) cell infiltration in mice vaginal tissues. The cytokines ((IL-1α, IL 1β, IL-6, IP-10, IL-7, MKC, TNF-α) levels (pg/mL) in cervicovaginal lavage and cervicovaginal tissues were not significantly changed compared to control mice data analyzed after 24 h. The cytokine results confirmed the non-immunogenicity of developed nanoformulations. The in vitro anti-HIV activity of HA-NPs and HA-SH-NFs was analyzed at the MOI of 10,000, 5,000, and 1,000 using a luciferase assay. The pseudotyped HIV virus particles were generated using lipofectamine plasmid transfection method. The size distribution, mean diameter (~128 nm), and titer (~3.07×10¹⁰) of pseudotyped virus particles was analyzed using nanoparticle tracking analysis measurements. The in vitro anti-HIV activity data showed that the TFV loaded HA-NPs and HA-SH-NFs were able to inhibit the pseudotyped HIV virus replication. Moreover, the results also confirmed that the structural integrity and anti-HIV activity of TFV was preserved after the nanofabrication processes. The in vivo results illustrated that these nanoformulations (HA-NPs and HA-SH-NFs) are promising delivery systems and offered a safe delivery of anti-HIV microbicide candidates. Overall, the data presented here highlight the applicability and potential of TFV loaded HA-NPs and HA-SH-NFs templates for the topical vaginal delivery of anti-HIV/AIDS microbicide candidates.Introduction -- Overview and pandemic of HIV infections -- Development of hyaluronidase sensitive tenofovir loaded hyaluronic acid based nanoparticles -- Fabrication of hyaluronidase sensitive tenofovir loaded hyaluronic acid based mucoadhesive nanofibers -- Preclinical safety and in vitro HIV efficacy evaluaitons of hyaluronidase sensitive tenofovir loaded hyaluronic acid based nanoformulations -- Summary, conclusion and future directions -- Appendi

Evaluation of Antihyperlipidemic Activity of Ethanolic Extract of Lathyrus Sativus Linn. on Rats.

Introduction Hyperlipidemia is a major contributor to atherosclerosis and its accompanying conditions, including coronary heart disease, ischemic cerebrovascular disease, and peripheral vascular disease. The majority of morbidity and mortality among middle-aged people\u27s mortality and morbidity and older adults still results from atherosclerosis-related events, although their incidence has decreased in the United States. As a result of the obesity epidemic and the ageing of the American population, both the incidence and the absolute number of annual events will rise over the coming ten years.. Objectives Evaluation of Antihyperlipidemic Activity of Ethanolic Extract of Lathyrus sativus Linn. on rats.       Methods: Screening of antihyperlipidemic was done by using Lathyrus sativus Linn.. Leaves part extracts used for antihyperlipidemic activity leaves (200mg/kg). Hyperlipidemia was administered in experimental animals using triton X-100 administered intraperitoneally at a dose of (100 mg/kg). In order to collect blood for triton-induced hemolysis, retroorbital punctures were performed every 24 hours. hyperlipidemia and 28 days for high fat diet induced hyperlipidemia. Antihyperlipidemic activity of Lathyrus sativus Linn. plant extracts were evaluated by estimation of lipid profiles and standard drug Atorvastatin for 28 days. The biochemical parameters such as The levels of Total Cholesterol, Triglycerides, Very Low Density Lipoprotein (VLDL), High Density Lipoprotein (HDL), and Low Density Lipoprotein (LDL) are assessed. Results: Estimation of lipid profile shows that Lathyrus sativus Linn. leaves extract (200mg/kg), Lathyrus sativus Linn. leaves extract (200mg/kg) shows the less significant antihyperlipidemic activity. It decreases TC, TG, LDL, VLDL and increases HDL levels. Lathyrus sativus Linn. leaves extract 200 mg/kg shows significant antihyperlipidemic activity as standard drug Atorvastatin. Conclusions: This finding tends to reveal that the  hyperlipidemic effects of Lathyrus sativus Linn. are similar to the effect of standard drug Atorvastatin. This plant can get in consideration for the searching new drug to treat hyperlipidemic from plant

Formulation Development of Dual-Compartment Topical Inserts Combining Tenofovir Alafenamide and Elvitegravir for Flexible On-Demand HIV Prevention

Pre-exposure prophylaxis (PrEP) has emerged as a prominent approach for the prevention of HIV infections. While the latest advances have resulted in effective oral and injectable product options, there are still gaps in on-demand, event-driven, topical products for HIV prevention that are safe and effective. Here we describe the formulation development of a dual-compartment topical insert containing tenofovir alafenamide fumarate (TAF) and elvitegravir (EVG) that may be administered when needed, vaginally or rectally, pre- or post-coitus, for flexible HIV prophylaxis. Specifically, we describe the lab-scale formulation development, preclinical mucosal safety and pharmacokinetics (PK) testing in rabbits, long-term stability, and scale-up clinical manufacturing of the lead TAF/EVG (20 mg/16 mg) inserts, which are currently in clinical stages of development. As designed, the inserts are small, discreet and portable, offering a number of promising attributes, such as simple and robust direct-compression manufacturing, fast initial disintegration/dissolution, and suitable mechanical strengths showing low hardness (\u3c8 kg), friability (\u3c1 %), and moisture content (\u3c1 %). The inserts initiated disintegration quickly (~ ≤ 15 min) providing full in vitro release (\u3e90 %) of TAF and EVG within 60 min of dissolution. The lead insert was selected from formulation prototypes that met the evaluation criteria for manufacturability and characterization, together with a dose-ranging PK study in non-human primates. Successful technology transfer for clinical development of the lead TAF/EVG (20 mg/16 mg) insert was confirmed under cGMP conditions. Based on the 12 months (lab-scale) and 24 months (clinical batch) stability data, the TAF/EVG inserts are projected to have a long shelf life of over 2 years, if stored at or below 30 °C/65 % RH. Overall, these newly designed topical inserts have formulation properties that enable stable storage and fast release of the antiretroviral payload from a small, portable and discreet dosage form. They are safe and effective when applied vaginally or rectally, before or after coitus, providing the basis for a new method of flexible on-demand HIV prevention for cisgender and transgender women and men. The TAF/EVG inserts are currently the most clinically advanced on-demand topical product, as attested by their completed and ongoing clinical trials

Development and Preclinical Investigation of Physically Cross-Linked and pH-Sensitive Polymeric Gels as Potential Vaginal Contraceptives

This study explored the development of cross-linked gels to potentially provide a physical barrier to vaginal sperm transport for contraception. Two types of gels were formulated, a physically cross-linked iota-carrageenan (Ci) phenylboronic acid functionalized hydroxylpropylmethyacrylate copolymer (PBA)-based (Ci-PBA) gel, designed to block vaginal sperm transport. The second gel was pH-shifting cross-linked Ci-polyvinyl alcohol-boric acid (Ci-PVA-BA) gel, designed to modulate its properties in forming a viscoelastic, weakly cross-linked transient network (due to Ci gelling properties) on vaginal application (at acidic pH of ~3.5-4.5) to a more elastic, densely cross-linked (due to borate-diol cross-linking) gel network at basic pH of 7-8 of seminal fluid, thereby acting as a physical barrier to motile sperm. The gels were characterized for dynamic rheology, physicochemical properties, and impact on sperm functionality (motility, viability, penetration). The rheology data confirmed that the Ci-PBA gel was formed by ionic interactions whereas Ci-PVA-BA gel was chemically cross-linked and became more elastic at basic pH. Based on the screening data, lead gels were selected for in vitro sperm functionality testing. The in vitro results confirmed that the Ci-PBA and Ci-PVA-BA gels created a barrier at the sperm-gel interface, providing sperm blocking properties. For preclinical proof-of-concept, the Ci-PBA gels were applied vaginally and tested for contraceptive efficacy in rabbits, demonstrating only partial efficacy (40-60%). Overall, the in vitro and in vivo results support the development and further optimization of cross-linked gels using commercially available materials as vaginal contraceptives

The exciting potential of nanotherapy in brain-tumor targeted drug delivery approaches

Delivering therapeutics to the central nervous system (CNS) and brain-tumor has been a major challenge. The current standard treatment approaches for the brain-tumor comprise of surgical resection followed by immunotherapy, radiotherapy, and chemotherapy. However, the current treatments are limited in providing significant benefits to the patients and despite recent technological advancements; brain-tumor is still challenging to treat. Brain-tumor therapy is limited by the lack of effective and targeted strategies to deliver chemotherapeutic agents across the blood-brain barrier (BBB). The BBB is the main obstacle that must be overcome to allow compounds to reach their targets in the brain. Recent advances have boosted the nanotherapeutic approaches in providing an attractive strategy in improving the drug delivery across the BBB and into the CNS. Compared to conventional formulations, nanoformulations offer significant advantages in CNS drug delivery approaches. Considering the above facts, in this review, the physiological/anatomical features of the brain-tumor and the BBB are briefly discussed. The drug transport mechanisms at the BBB are outlined. The approaches to deliver chemotherapeutic drugs across the CNS into the brain-tumor using nanocarriers are summarized. In addition, the challenges that need to be addressed in nanotherapeutic approaches for their enhanced clinical application in brain-tumor therapy are discussed