A novel polymer drug conjugate for the sustained release of ganciclovir

Ocular manifestation of cytomegalovirus (CMV retinitis) remains the most common AIDS-related ocular complication which occurs in 40% of HIV patients and causes loss of vision or blindness [1]. Intravitreal injection of ganciclovir (an inhibitor of the viral DNA polymerase [2]) is the current gold standard to treat CMV retinitis. Since there is no sustained release drug delivery system currently available [3], patients suffer from side effects of the frequent intravitreal injections such as retinal detachment and hemorrhage [4, 5]. This study aims to explore a ganciclovir-loaded polymer drug conjugate for the long-term sustained release for the treatment of CMV retinitis. Considering the high drug loading, is necessary to be able to reduce the number of intravitreal injections, a PAA-ValG conjugate comprising polyacrylic acid (PAA) as polymer backbone, L-valine as linker and ganciclovir as drug, were chosen. The conjugate was synthesized by EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) coupling of valganciclovir (L-valine ester prodrug of ganciclovir) to polyacrylic acid in aqueous buffer solution. Multiple conjugates of different molecular weights were synthesized in different stoichiometric ratios of reactants. HPLC analysis was used to quantify ganciclovir, as well as valganciclovir and impurities or degradation products from the extracted (free drug) or hydrolyzed (assay) reaction products. It was shown that the 10 batches of higher stoichiometric ratios (1, 1.5 or 2 mol coupling reagent per mol COOH from PAA) contained 42.0% ganciclovir (w/w) on average, which were higher than all other studies in literature reported [6-12]. An effect of the chosen PAA (1.2, 8, 15 and 30 kDa) as well as the stoichiometric ratio was not observed, hence the standard deviation for the drug loading (assay) over all 10 samples was only 1.1%. This constant conjugation of 26.6% of all COOH groups from PAA was expected to be caused by steric hindrance. The content of free drug was < 0.5% of the total drug and unknown ingredients were < 0.2% for all mentioned samples, which showed that the purification process worked. Analysis of the samples after 4 months of storage showed no significant release of drug (free drug) or increase in degradation products, which showed the high storage stability of the conjugates. The in vitro release of ganciclovir from PAA-ValG was performed in a release medium which has all relevant properties of vitreous humor, with the exception of biological ingredients. A long-term sustained release over a period of more than 1 year was observed, which was much longer than all other ganciclovir-releasing systems reported [6-12]. The release rates were noticed to have decreased slightly over time, from 8.2% per month in the 1st month, to 2.2% per month in the 12th month on average. Ex vivo release experiments in pig-vitreous showed a similar release in the solid state and approximately 8 times faster release in the soluble state of PAA-ValG, in comparison to the in vitro release testing. Similar behavior of polymer drug conjugates in biological fluids was also confirmed by D’Souza et. al., since release enhancing enzymes like esterase can enter polymer drug conjugates in the soluble state, but not in the solid state [13]. Since PAA-ValG was observed to change from the solid to the soluble state during release, depending on the molecular weight, concentration and amount released, the in vivo release of PAA-ValG is expected to be faster, but also more constant than the observed in vitro release profile indicated. In the best case, one injection of around 11 mg PAA-ValG (depending on the exact drug content), which is equivalent to 4.5 mg ganciclovir will be sufficient for 6 to 8 months of treatment, as it was in the later discontinued ganciclovir-releasing implant Vitrasert [3, 14], but with less side effects, due to injectability. Considering the high drug loading, purity, stability and the long-term sustained release, as well as the predicted properties from literature, PAA-ValG seems to be an excellent candidate for in vivo studies, with the goal to control cytomegalovirus retinitis with fewer intravitreal injections as per the current treatment. To complete the work, the release mechanism of ganciclovir from PAA-ValG in a simulated physiological ocular environment was studied. Acylated valganciclovir (Ac-ValG) was synthesized to study the ester hydrolysis without the effects of the polymer, since its chemical structure next to relevant ester bond is similar. The reaction constants for the pseudo-first-order release rate of Ac-ValG were determined and found to be pH dependent and increasing linearly with the hydroxide ion concentration in the release medium. Furthermore the penetration of hydrolyzing agent into the polymer (degree of hydration) was studied by tablet swelling experiments, as previously performed by Pitt and Shah [15] and found to be increasing with time and pH. Also the decrease in the microclimate pH during release, as it is known from other biodegradable polymers [16] was studied via titration and found to be stronger at high pH values, and the beginning of the release. The combination of reaction constant, microclimate pH, concentration of cleavable drug, and the degree of hydration leads to a formula which describes the release rate accurately.Doctor of Philosoph