Introduction
Zidovudine (AZT) is a widely used anti HIV drug, which exhibits a vastly demonstrated clinical efficacy. Unfortunately, this drug presents several adverse effects that are consequence of its suboptimal pharmacotherapeutic properties. To improve these properties, several strategies have been applied, among which the design and synthesis of prodrugs has been pursued. Considering that these prodrugs require adequate stability, both in aqueous and physiological conditions, the need to evaluate and rationalize this behavior is critical. In this context, the general objective of this work is to study the chemical and enzymatic stability of three new prodrugs of AZT with essential aminoacids that were previously obtained in our research group,1 namely AZT-l-Leu (l-leucine), AZT-l-Pha (l-phanylalanine) and AZT-d-Pha.
Zidovudine (AZT) is a widely used anti HIV drug, which exhibits a vastly demonstrated clinical efficacy. Unfortunately, this drug presents several adverse effects that are consequence of its suboptimal pharmacotherapeutic properties. To improve these properties, several strategies have been applied, among which the design and synthesis of prodrugs has been pursued. Considering that these prodrugs require adequate stability, both in aqueous and physiological conditions, the need to evaluate and rationalize this behavior is critical. In this context, the general objective of this work is to study the chemical and enzymatic stability of three new prodrugs of AZT with essential aminoacids that were previously obtained in our research group,1 namely AZT-l-Leu (l-leucine), AZT-l-Pha (l-phanylalanine) and AZT-d-Pha.
Materials and Methods
The chemical stabilities of AZT-l-Leu, AZT-l-Pha and AZT-d-Pha (1 x10-4M) were determined at pH 2, 5, 7.4 and rat plasma. Sampling was performed during 50 minutes, after which quantitation analyses were performed by HPLC-UV. Prior to analyses, samples were subjected to a preparation procedure involving solid phase extraction, sample concentration and a reconstitution step (100 μl). This procedure was adequately developed and validated in our laboratory according to FDA guidelines (results not presented in this work). The stability of the different prodrugs was evaluated from the corresponding concentration vs. time profiles.
l-Leu, AZT-l-Pha and AZT-d-Pha (1 x10-4M) were determined at pH 2, 5, 7.4 and rat plasma. Sampling was performed during 50 minutes, after which quantitation analyses were performed by HPLC-UV. Prior to analyses, samples were subjected to a preparation procedure involving solid phase extraction, sample concentration and a reconstitution step (100 μl). This procedure was adequately developed and validated in our laboratory according to FDA guidelines (results not presented in this work). The stability of the different prodrugs was evaluated from the corresponding concentration vs. time profiles.Results and Discussion
The chemical stability of the three prodrugs is highly dependent on the pH of the medium, with a high stability found at pH 2 and 5, while a higher hydrolysis rate was found at pH 7,4 (Table 1). Taking into account these prodrugs pKa reported values, the hydrolysis ratio showed to be independent of the degree of ionization of the aminoacid sidechain. Regarding the plasma stability of these three prodrugs, all of them are efficiently hydrolyzed by plasma esterases, with almost all the prodrugs having regenerated AZT in an extent of 90% at 10 minutes of incubation in this medium. Although no quantitative determination of plasma half-life time was possible due to this high rate of hydrolysis, qualitative observation of the corresponding area vs time plots (Figure 1) showed that the enzymatic stability of the three compounds follows the following trend: AZT-d-Pha < AZT-l-Pha < AZT-l-Leu. This observation may confer some hints for to establish a structure-esterase affinity relationship in future works.
d-Pha < AZT-l-Pha < AZT-l-Leu. This observation may confer some hints for to establish a structure-esterase affinity relationship in future works.Conclusion
The three studied prodrugs exhibited a wide stability ratio under physiologic and pseudo-physiologic conditions. We also concluded that the linker between the AZT moiety and the aminoacid template should be redesigned in order to obtain AZT prodrugs with higher resistance to esterase mediated hydrolysis.
References
1- Moroni GN, Bogdanov PM, Briñón MC. "Synthesis and in vitro antibacterial activity of novel 5′-O-analog derivatives of zidovudine as potential prodrugs". Nucleosides, Nucleotides and Nucleic Acids. 2002;21(3):231-241. *Quevedo, M.A. E-mail: alfredoq@fcq.unc.edu.ar
*Quevedo, M.A. E-mail: alfredoq@fcq.unc.edu.ar|
Table 1: Stability data of AZT-d-Pha, AZT-l-Pha and AZT-l-Leu. Prodrugs AZT-d-Pha, AZT-l-Pha and AZT-l-Leu. Prodrugs |
pH 2 |
pH 5 |
pH 7,4 |
Plama |
|
AZT-l-Leu l-Leu |
Stable |
Stable |
Unstable (t1/2: 16.70 min.) t1/2: 16.70 min.) |
Unstable |
|
AZT-d-Pha d-Pha |
Stable |
Stable |
Unstable (t1/2: 10.53 min.) t1/2: 10.53 min.) |
Unstable |
|
AZT-l-Pha l-Pha |
Stable |
Stable |
Unstable (t1/2: 16.70 min.) t1/2: 16.70 min.) |
Unstable |
