|dc.description.abstract||The oral route of drug administration has for years remained the mainstay drug delivery route because of its ease of use and good patient compliance. Orally administered drugs need to pass through a number of barriers before entering the systemic blood circulation. The biological membranes of the gastro-intestinal tract are lipophilic in nature and contain various proteins responsible for active or facilitated transport of polar and large molecules. Apart from active and facilitated transport passive diffusion is one of the major absorption processes for most drugs. Water soluble drugs have a greater difficulty in crossing these membranes due to their hydrophilic nature compared to their lipophilic counterparts. For hydrophilic molecules, in order to passively cross the lipophilic membranes, they need to be rendered lipophilic. One way to address this problem is through linkage of the hydrophilic drugs to lipophilic moieties.
Human African trypanosomiasis (HAT), or sleeping sickness, is a vector-borne parasitic disease caused by protozoa of the species Trypanosoma brucei. HAT is responsible for 40 000 to 50 000 deaths each year. The disease covers 15% of Africa's population with 0.5 to 0.8% of that population contracting the disease each year. There are currently only 4 drugs (suramin, pentamidine, melarsoprol and eflornithine) approved for the treatment of HAT. The latest of these drugs, eflornithine, was approved 20 years ago.
Eflornithine (DFMO) is a selective irreversible inhibitor of ornithine decarboxylase (ODC), an enzyme responsible for polyamine synthesis in humans and trypanosoma. Eflornithine is the second line treatment for late stage T. b. gambiense infections or melarsoprol relapse patients. The drug is very hydrophilic and is primarily administered intravenously which contributes to it being expensive and labour intensive. Eflornithine can however be given orally but is not favoured due to a low oral bioavailability of 54%. Consequently the drug needs high doses to achieve the minimum effective concentration of 50 µM in the brain. This is explained by the hydrophilic nature of the drug limiting its oral absorption as well as transport over the blood-brain barrier.
The object of this study was to synthesise lipophilic amides of DFMO, determine their physicochemical properties, evaluate their intrinsic activity and assess their oral absorption in an attempt to improve the bioavailability of this drug.
Seven o-amides were synthesised by means of acylation whereby lipophilic moieties were attached to the o-amino group of eflornithine through amide bond formation. The structures of the products were confirmed by nuclear magnetic resonance spectroscopy (NMR) and
mass spectroscopy (MS).
The aqueous solubility of DFMO (control) and all its derivatives were determined experimentally in phosphate buffer (pH 7.4) at 37°C. All the derivatives except 2-amino-2(difluoromethyl)-5-acetamidopentanoic acid demonstrated a decrease in water solubility ranging from 28 to 19 mg/ml compared to that of DFMO (34.96 mg/ml), which corresponds to an increase in log D in the range of 4.6 to 9.47 mg/ml. 2-amino-2-(difiuoromethyl)-5-(2phenylacetamido)pentanoic acid (Sw = 11.13 mg/ml, log D = -0.07) was the most lipophilic and was therefore expected to be the most absorbed. The biggest increases in lipophilicity were observed with aryl-containing derivatives.
The in vivo oral absorption tests conducted at the University of Göteborg, Sweden, were done on Sprague-Dawley rats after oral administration of the compounds. Blood samples were drawn and analysed with HPLC. Results for the compounds tested showed no metabolism into efiornlthine, possible due to the stable amide bond. The in vivo results do not represent the concentration of the synthesised compound but that of eflornithine in the blood stream. Thus no conclusive evidence was attained to confirm oral absorption.
T. b. brucei was used to determine the intrinsic activity of the synthesised compounds in vitro and was expressed as IC50 values. 2-amino-2-(difluoromethyl)-5-propanamidopentanoic acid and 2-amino-2-(difluoromethyl)-5-[(4-methoxyphenyl)formamido]pentanoic acid showed a moderate increase in activity of 32.05 and 35.45 µM respectively, compared to that of eflornithine (36.22 µM).
No correlation was found between physicochemical properties, oral absorption and intrinsic activity. The study does, however, prove that derivatisation can influence the lipophilicity. Only 2-amino-2-(difluoromethy/)-5-propanamidopentanoic acid and 2-amino-2(difluoromethyl)-5-[(4-methoxyphenyl)formamido]pentanoic acid showed an increased lipophilicity and intrinsic activity.||