Tuesday 30 August 2016

Calorimetric Evaluation of Amoxicillin Stability in Aqueous Solutions



The thermal properties of amoxicillin suspension in aqueous solutions were analyzed by differential scanning calorimetry under various solvent conditions to identify changes in thermodynamic parameters closely related to drug absorption and pharmacokinetic behavior. Analysis of thermal profiles of amoxicillin in solid form and in aqueous suspension showed several phase transitions, not reported previously, which correspond to different transformations in drug integrity induced by temperature. The low temperature endothermic transition in temperature range 20-50°C is described as associated with decomposition of crystal-like structure of amoxicillin in liquid suspension, which cooperatively dissembles with increase of temperature. The low temperature endothermic transition is following exothermic transition in temperature range 60-125°C with a pH dependent temperature maximum occurring in 80-114°C range. While the maximum temperature for the endothermic transition is practically independent of the solution pH, the exothermic transition displays strong pH dependence, decreasing its Tmax as the pH of solution decreased from pH 7 to pH 2.0. During heating of the amoxicillin suspension the exothermic transition demonstrates a complex character with several maximums occurring on the calorimetric thermogram. 

The physical and chemical changes in the amoxicillin are associated with the degradation of amoxicillin, as evident by the yellowing of the solution as it is heated. The temperature induced changes observed for the amoxicillin aqueous suspension contrast those reported for the solid form of amoxicillin where only endothermic thermal transition in temperature range 55-125°C are observed. The latter is associated with dehydration loss crystallinity of the solid form. Based on comprehensive calorimetric analysis, this manuscript describes the thermal properties as well as transformations between the different states of amoxicillin during heating.Amoxicillin trihydrate is a beta lactam antibiotic that has been used worldwide to treat a broad spectrum of bacterial infections including the upper respiratory tract, cystitis, peritonitis, gonorrhea, intra-abdominal sepsis, skin and soft tissue infections. The antibiotic formulations are available in varioussolid and liquid dosages consisting of numbers of excipients to improve its effectiveness. Reconstitution of amoxicillin powder for the injection of high doses (250mg, 500mg and 1g) is considered when the oral route is unsuitable or urgent treatment of severe infection is required. Unfortunately, due to the wide variability in the quality of drugs in solid form, their dissolution and pharmacokinetic profiles may behave unpredictably under conditions which differ from those tested by the manufacture. Most of the drugs developed in the pharmaceutical industry have poor solubility or are practically insoluble in water. 

The low aqueous solubility may result in new chemical entities which are usually associated with poor parental and oral bioavailability. Upon reconstitution in aqueous solution some fraction of drug is solubilized while another one remains in highly hydrated insoluble forms at concentrations above the drug’s solubility point. Although, the description of the thermal behavior of amoxicillin solid polymorphs has already been given in the literature, their thermodynamic characteristics in aqueous suspension are not well clarified. For the category of poor soluble drugs that require reconstitution from solid form to the injectable liquid, determination of physicochemical properties, thermodynamic stability and inter-conversion conditions are essential for optimization of drug absorption and pharmacokinetics. It has been noted that differences in solubility may affect antibacterial activity more than pharmacokinetics. 

Therefore it is important for pharmaceutical practice to know stability and physical properties of drug molecules and their interactions forming the heterogeneous mixture of drug suspension to predict its pharmacokinetic and pharmacodynamic behavior. The presence of insoluble forms may impact drug permeability at the site of absorption. The nature of the physical forms of amoxicillin, which belongs to the category of poorly soluble drugs, plays a key role in affecting its dissolution from a solid dispersion when given orally in high doses or parentally. The aim of this study, therefore, was to investigate specific changes in the structural arrangement of amoxicillin in aqueous suspension at different temperatures and to characterize the physical and chemical states of the drugAmoxicillin trihydrate (Teva Pharmaceuticals, USA) was provided as a 500mg encapsulated dry powder by the Medical Unit of NASA Ames Research Center (Moffett Field, CA). The molecular mass of amoxicillin trihydrate MW = 365.4 g/mol has been used in this analysis. All reagents used in this study were of analytical grade (Sigma-Aldrich, Germany). Gly-HCl buffer was used for preparation of samples with pH 2 and pH 3. Buffers with pH 4 and pH 5 were prepared from sodium acetate. Buffers at pH=7 and pH=8 were prepared from sodium phosphate. The pH of buffers and solutions of amoxicillin trihydrate were measured using a pH meter (IQ Scientific Instruments, San Diego, USA), calibrated with buffer standards with error ± 0.02 pH units. Amoxicillin powder was reconstituted in select buffer solutions (pH 2-8) at ambient temperature and shaken for an hour to homogenize the amoxicillin preparations. The preparation was considered as homogeneous following visual evaluation. Reconstituted samples can be stored at 4°C temperature over period of 7 days without any detectable degradation or changes in solubility as assayed by UV-spectroscopy. For solubility testing of amoxicillin at different pH values, the suspensions were filtered through Milipore filter with pore size 0.45mm and its amount of soluble fraction was determined spectrophotometrically using the molar extinction coefficient of amoxicillin at 254 nm (ɛAM = 1050 M−1 cm−1). Lambda 35 UV- double beam recording spectrophotometer (PerkinElmer Inc.) with a 1 mm length quartz cuvette were used for all absorbance measurements. 

The concentration of amoxicillin was corrected for light scattering when measured spectrophotometrically. Its value before and after filtration enables a precise measurement of the soluble and insoluble fraction under each given solvent conditions. Calorimetric experiments were performed using a Microcal VP-DSC microcalorimeter with a heating rate of 1 deg C°/min and external pressure 32 atm. The amoxicillin concentration in calorimetric experiments varied in the range of 0.625mg/ml - 14 mg/ml depending on conditions of each experiment. Deconvolution analysis of the excess heat capacity function obtained in the calorimetric experiments was not performed due to irreversible, non-equilibrium nature of the transitions. The amoxicillin in solid powder form was analyzed using conventional Q100 DSC (TA Instrument, USA). Approximately 2 to 5 mg of each sample was heated in an open aluminum pan from 30 to 250°C at a scanning rate of 10°C/min under a stream of nitrogen gas. An optical microscope Axioskop 2 (Carl Zeiss Microscopy, LLC, USA) with a digital camera attached to a PC utilizing software was used to record the images of amoxicillin samples spreading onto glass slabs. 

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