Date of Award

2014

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Pharmaceutical Sciences

First Advisor

Keykavous Parang

Abstract

2′,3′-Dideoxynucleoside (ddNs) analogs are the most widely used anti-HIV drugs in the market. Even though these drugs display very potent activities, they have a number of limitations when are used as therapeutic agents. The primary problem associated with ddNs is significant toxicity, such as neuropathy and bone marrow suppression. Due to their hydrophilic nature, they have poor cellular uptake. Some of these nucleoside analogs develop resistance over a period of time. Furthermore, these analogs themselves are not pharmacologically active, and undergo three-step phosphorylation in order to become active. Of these steps, the first step is usually a slow and rate-limiting process.

Herein, we report the synthesis and evaluation of 2′,3′-dideoxynucleoside conjugates with fatty acids with varying chain length and fatty acyl poly-L-arginine derivatives. The hypothesis of this project was that conjugating the nucleosides with fatty acids or fatty acyl poly-L-arginine derivatives will generate multifunctional agents with enhanced anti-HIV activity and cellular uptake, and less drug resistance development when compared to that of the parent nucleoside alone. Furthermore, the compounds will also have longer duration of action through sustained intracellular release of the parent nucleosides. The fatty acid conjugates of nucleosides could have higher uptake into the cells because of their enhanced lipophilicity. This dissertation will be revealed in two manuscripts.

In the first Manuscript, the synthesis and anti-HIV activities of 5′-O-dicarboxylic fatty acyl monoester derivatives of 3'-azido-3'-deoxythymidine (zidovudine, AZT), 2',3'-didehydro-2',3'-dideoxythymidine (stavudine, d4T), and 3'-fluoro-3'- deoxythymidine (alovudine, FLT) are discussed. The compounds were synthesized to improve the lipophilicity and potentially the cellular delivery of parent polar 2′,3′-dideoxynucleoside (ddN) analogs. Three different fatty acids with varying chain length of suberic acid (octanedioic acid), sebacic acid (decanedioic acid), and dodecanedioic acid were used for the conjugation with the nucleosides. Among all the compounds, 5′-O-suberate derivative of AZT (1, EC50 = 0.10 nM) exhibited the best anti-HIV profile when compared to other fatty acyl derivatives. The compound showed 80-fold higher anti-HIV activity than AZT without any significant toxicity (TC50  500 nM). This work was published in Tetrahedron Letters (2014, http://dx.doi.org/10.1016/j.tetlet.2014.02.001).

In the second Manuscript, synthesis, anti-HIV activity, and preformulation tests of poly-L-arginine cell-penetrating peptides (CPPs) conjugated with fatty acyl derivatives of anti-HIV nucleosides, FLT, lamivudine (3TC) and emtricitabine (FTC) are discussed. All conjugates exhibited less anti-HIV activity when compared with the parent nucleoside analogues. For example, poly-L-arginine-fatty acyl derivative of 3-fluoro-3-deoxythymidine, FLT-CO-(CH2)12-CO-(Arg)7, exhibited EC50 values of 2.9 μM and 3.1 μM against X4 and R5 cell-free virus, respectively, while the FLT had EC50 values of 0.2 μM and 0.1 μM, suggesting the limited uptake or intracellular hydrolysis to the parent analogue. Further preformulation studies were done on the FLT-conjugate by determining the lipid solubility (partition coefficient) and solution state degradation. The compound was found to be stable in acidic and alkaline conditions. The partition coefficient (Log P) of FLT conjugate was found to be -0.34. The derivative was also evaluated in dissolution studies using four different hydrogels with and without a thermogelling polymer. Gel formulations of the compound were manufactured using non-ionic (HPC-SL) and anionic (Carbopol) polymers with and without the inclusion of a thermo-reversible gelling (Pluronic F-127) polymer. The compound was found to be unstable in the manufactured formulations as shown by HPLC profiles. This work has been submitted to a peer-reviewed journal.

In summary, the studies provided more insights in designing new generation of anti-HIV nucleoside conjugates with fatty acids and poly-arginine CPPs. Overall, we have established a strategy for generation more potent nucleoside conjugates as shown in Manuscript I. Furthermore, the data indicated that addition of positively-charged poly-arginine to the nucleoside analogs is not beneficial in generating more potent anti-HIV conjugates as demonstrated in Manuscript II, possibly because of the presence of the positively charged viral glycoprotein gp120. Further optimization of conjugated CPP-fatty acid-nucleoside conjugates is required to generate compounds with improved anti-HIV activity and optimized stability and formulation performance.

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