Synthesis and transdermal permeation of lamivudine derivatives
Van Heerden, Jacobus
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The skin is the organ of the body that comes in contact with the outer environment. This means that it has certain functions like protecting the body from excessive water loss and inhibiting the entry of potentially harmful chemicals. This is called the barrier function of the skin and is enforced by the outermost layer of the skin, the stratum corneum (SC). The transdermal route of delivery has several advantages over the common oral route which include improving patient compliance by decreasing the amount of medication, circumventing the first pass metabolism in the liver, eliminating certain unwanted side-effects which are associated with oral administration (nausea, diarrhoea) and to have better control over the input kinetics of the drug. The SC is a very lipophilic membrane, inhibiting the penetration of hydrophilic molecules. This means that in order for molecules to permeate through the skin, they must have specific physicochemical properties like a specific aqueous solubility, octanol-water partition coefficient (log P), melting point and molecular mass. Since the epidemic started in 1981, more than 25 million people have died of AIDS worldwide. Statistics at the end of 2007 showed that 33 million people are living with HIV and that 2.7 million people become infected with it each year. There are a total of 16 drugs that have been approved by the U.S. Food and Drug Administration (FDA) for the chemotherapy of AIDS whereof seven are nucleoside reverse transcriptase inhibitors (NRTI), making them the most important class of compounds in the treatment of HIV. Lamivudine (4-amino-1-[2- (hydroxymethyl)-1 ,3-oxathiolan-5-yl]-1 H-pyrimidin-2-one, 3TC) is in this class and is used in combination with zidovudine (AZT) against HIV-1 and HIV-2 because of their synergism. The most common adverse effects of lamivudine are usually associated with the gastro-intestinal system and include vomiting, diarrhoea, abdominal cramps and pain, and nausea. The objective of this study was to determine the in vitro transdermal permeation through the human SC of lamivudine and the synthesised methoxypoly(ethylene glycol) (MPEG) carbonates and carbamates thereof, with and without the use of Pheroid™ as delivery system and to establish a relationship, if any, with selected physicochemical properties. Three. N4-methoxypoly(ethylene glycol) carbamates and three 6'-O -metoxypoly(ethylene glycol) carbonates were synthesised by alkoxy carbamylation and carbonylation of lamivudine with acylating agents containing the corresponding sho,rt-chain methoxypoly(ethylene glycols). The structures of the products were confirmed by nuclear magnetic resonance spectroscopy (NMR) and liquid chromatography mass spectroscopy (LC-MS). The aqueous solubility of only lamivudine (188.02 mg/ml) and 2',3'-dideoxy-3'-thiacytidin-N4-yl-methoxy(ethylene glycol) carbamate (8 .53mg/ml) could be determined experimentally. The relationship between the aqueous solubility of lamivudine and the other derivatives with their flux could not be determined because these derivatives were oils infinitely miscible with PBS. Theoretical equations using the physicochemical properties like log P were employed to calculate the aqueous solubility. The derivatives became more soluble in aqueous medium as the chain length increased because of the increasing number of intra-chain oxygen atoms forming bonds with surrounding water molecules. However, as the length increased so did the number of ethylene units which ultimately also increased lipid solubility. The experimental octanol-PBS partition coefficient of lamivudine (-0.83) was lower than that of its derivatives (ranging from -0.16 to -0.63). This meant that the synthesised derivatives were more lipophilic than lamivudine. As the chain length increased the partition coefficient value decreased. This correlates with the increasing aqueous solubility as the chain length increased. In vitro penetration was measured through excised female human abdominal skin in Franz diffusion cells. The steady-state flux (Jss) of lamivudine in PBS (4.23 µmol/cm2/h) was higher than that of lamivudine in Pheroid™ (0.20 µmol/cm2/h) . In both PBS and Pheroid™ the median flux of lamivudine was higher than that of the derivatives (0.04 to 2.07 µmol/cm2/h in PBS and 0.002 to 0.11 µmol/cm2/h in Pheroid™) except for the 6'-0-methoxy(ethylene glycol) carbonate (n = 1) which had a higher flux value (0.23 µmol/cm2/h) in Pheroid™. Of all the derivatives of lamivudine, the N4-methoxy(ethylene glycol) carbamate (n = 1) (2.07 µmol/cm2/h) and the N4- methoxytri(ethylene glycol) carbamate (n = 3) (1 .32 µmol/cm2/h) (in PBS) presented the highest flux. Two methods for measuring transdermal flux were used: in Method 1 donor solutions were prepared in PBS while Method 2 used the Pheroid™ delivery system for donor solutions. A t-test yielded a p-value of 0.0002 indicating that there was a highly statistically significant difference between the mean of the overall flux of all the synthesised derivatives of Method 1 and that of Method 2, showing that the flux in PBS was significantly higher than that in Pheroid™. The results of the ANOVA for Method 2 indicated that there were no statistically significant differences between the mean flux values of the different compounds (p-value of 0.26). Thus in Pheroid™ there were no statistically significant differences between the mean flux of any compound and the parent drug. In the case of Method 1, the ANOVA indicated that a significant difference between the means of the flux of all the compounds existed (p-value of < 0.0001 ). A Dunnett test showed that except for 2',3'-dideoxy-3'-thiacytidin-N4-yl-(methoxy(ethylene glycol)) carbamate, the mean flux values in PBS of all the other compounds were statistically significantly lower than the mean flux of the parent drug. Although a small data set of only six compounds was used in this study no relationship between the aqueous solubility, partition coefficient (log P) and transdermal flux of any of the compounds was found . Neither derivatisation of lamivudine nor the use of the Pheroid™ delivery system improved the transdermal flux of this drug. This shows that a clinically useful transdermal administration of the derivatives synthesised in this study will not be feasible.
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