Abstract
Purpose: The aim of this thesis was to investigate the stability of penethamate (PNT), an ester prodrug of benzylpenicillin (BP), used intramuscularly (i.m.) in the treatment of bovine mastitis. The specific objectives were to understand the chemical stability of PNT in aqueous and oily vehicles; to develop some understanding of the effect of absorption rate from the i.m. site on milk levels of BP; to formulate a stable ready-to-use (RTU) product and to test this product in a pilot study in the target species (cow).
Methods: A reversed phase HPLC assay was developed to study the degradation kinetics of PNT in aqueous vehicles. Several formulation approaches such as cosolvents, cyclodextrins, common ion and oily vehicles to enhance the
chemical stability of PNT were assessed. A simulation model was constructed for the prediction of absorption rate from i.m. injection site and concentration of BP, the hydrolytic product of PNT, in milk. A pilot animal study in cows was conducted to compare milk levels of BP after i.m. injection of a stable oily formulation of PNT with a marketed aqueous reconstituted suspension formulation.
Results: A reversed phase HPLC assay for simultaneous determination of PNT and its hydrolytic product BP using an isocratic system with photo diode array detection was developed and validated. The assay was linear over the concentration range 1-100 μg ml-1 for both analytes (r > 0.99) with satisfactory inter-day and intraday
precision (RSD ≤ l %) and accuracy (98-99%) for PNT.
The degradation of PNT in aqueous solutions followed pseudo-first-order kinetics over the pH range 2 - 9.3 with a V- shaped pH-rate profile with a sigmoid portion in the pH range 7.5 - 9.3 corresponding to the pKa (8.4) of PNT. The
minimum degradation rate of PNT was at pH 4.5 (half-life (t1/2) = 44 h at 30 °C). The impact of buffer concentration and ionic strength on PNT stability was small but at pH 6 the type of buffer salt had some influence with solutions in acetate being about twice as stable as those in phosphate. The Arrhenius activation energies determined at pH 3.01, 6.01 and 8.04 were 62.1, 74.1 and 98.8 kJ mol-1 respectively.
In aqueous solutions, propylene glycol (PG) increased the t1/2 of PNT from 1.8 days to 4.3 days at 30 °C, whereas hydroxypropyl- ß-cyclodextrin (HP-ß-CD) and ß-CD did not influence the t1/2 and iodide (I-) resulted in a decrease in t½ from 1.8 days to 1.1 days. The estimated shelf-life (t90) of PNT in solution of about 0.3 days (5% PG) and 0.7 days (60% PG) increased to 15 days (5% PG) and 11 days (60% PG) in a 50% PNT suspension. With increasing concentration of HP-ß-CD, the solubility of PNT increased linearly and resulted in a decrease in the estimated t90 of aqueous suspensions. The decrease in solubility of PNT due to the common ion effect resulted in an increase in the t90 (26 days) in aqueous suspension.
PNT stability (% drug remaining) in oily suspensions after 105 days was in the order LP (light liquid paraffin) (96.2%) > MIG (miglyol 812) (95.4%) > EO (ethyl oleate) (94.1%) > SO (sunflower oil) (86.4%). PNT degradation was rapid in oily solutions of LP, MIG, EO and SO and less than 10 % remained after 7-15 days.
The simulation model provided insights into the absorption rate of PNT from the i.m. injection site. Sensitivity analysis suggested that absorption rate constants (ka), clearance from plasma to milk (PA) and volume of distribution (Vd) are critical parameters for predicting concentrations of BP in milk. The developed oily suspension formulation of PNT in EO with 0.15 % Polysorbate 80 showed good physical and chemical stability. A pilot animal study in cow suggested that the oily formulation achieves concentrations of BP in milk similar to those obtained from a marketed aqueous suspension formulation of PNT. The AUC0-48 and t1/2el of BP in milk after i.m. administration of the marketed aqueous suspension formulation of PNT were 3.56 ± 0.17 mg.h L-1 and 4.9 ± 0.3 h respectively, while the corresponding data for Formulation B were 4.9 ± 1.4 mg.h L-1 and 4.6 ± 1.2 h respectively.
Conclusion: The chemical stability of PNT was greatly enhanced in oily suspensions with around 95% PNT remaining for over 3 months under intermediate storage conditions (30 °C). PNT can be formulated as a physically and chemically stable ready-to-use suspension. This suspension probably gives milk levels of BP comparable with the existing marketed aqueous suspension formulation and a larger animal study is warranted to test this.