Biopharmaceutics and pharmacokinetics of the macrolide antibiotic Josamycin

The investigations detailed herein have been conducted to address various aspects of the biopharmaceutics and pharmacokinetics of josamycin which to-date, have received little or no attention in the literature. Areas of investigation have included the selective determination of josamycin in serum and urine samples, the stability of josamycin in stored biological samples, intrinsic dissolution rates, solubility, acid and alkali stability and bioavailability and pharmacokinetics after dosing with a solution, powder and tablets.

High performance liquid chromatography (HPLC) was used as the main analytical tool throughout these studies and proved to be highly versatile for the determination of josamycin in a number of different media. HPLC analysis afforded simple yet accurate determination of josamycin in samples from dissolution, solubility, tablet content and stability studies. Furthermore, the specificity afforded by HPLC was particularly useful for the separation of josamycin from degradation products formed in acid and alkali media.

Since metabolites of josamycin are microbiologically active, microbiological assays do not determine the concentration solely of josamycin. An analytical method 
capable of the selective determination of josamycin in serum and urine samples is therefore required for the procurement of reliable bioavailability and pharmacokinetic 
data. HPLC affords this selectivity and a method for the selective determination of josamycin in serum and urine was successfully developed. The assay was simple yet precise, accurate and sensitive. Furthermore, it was well suited to the determination of josamycin in a large number of biological samples. Its success was largely due to the use of a solid phase extraction step using C₁₈ extraction columns, with a highly specific wash sequence followed by a phase separation step after elution from the extraction column. Chromatography was performed on a C₁₈ reversed-phase analytical column with UV detection of josamycin and internal standard at 231 nm and at 204 nm 
respectively using a programmable multi-wavelength detector. Only slight modification of the assay described should enable the selective determination of the metabolites of josamycin. This assay, therefore, lays the groundwork for future investigations into the pharmacokinetics of these metabolites.

The re-usability of extraction columns was assessed in an attempt to reduce the cost of sample analysis. It was found that extraction columns could be used twice for the extraction of serum samples and up to four times for the extraction of urine samples. The difference between the re-usability of extraction columns for serum and urine samples was ascribed to various differences in the composition of the sample matrix. The stability of josamycin in stored serum and urine samples was also assessed. Josamycin was found to be stable for up to 72 hrs at +4°C and up to 84 days at -15°C in ex-vivo and in-vitro serum samples. However, josamycin was less stable in urine samples and a significant change in concentration was detected during the storage periods studied.

Intrinsic dissolution rate-pH and solubility-pH profiles were determined over the pH range of the gastro-intestinal tract to assess the possible role of dissolution in the absorption of josamycin. Furthermore, intrinsic dissolution rales at each pH were determined at 50, 100, 200 and 300 rpm, and then at infinite rotation speed (G∞) by an extrapolation procedure. G∞ was determined in order to compare dissolution rates in different dissolution media without the influence of a diffusion layer. The intrinsic 
dissolution rate and solubility were found to be highly pH dependent. G∞ decreased >8500 fold from 5.15 mg.cm⁻².s⁻¹ at pH 1.2 to 5.815 x 10⁻⁴ mg.cm⁻².s⁻¹ at pH 7.5. The solubility of josamycin decreased from 212 mg.ml⁻¹ at pH 5.45 to 0.18 mg.ml⁻¹ at about 9.0. These profiles suggest that josamycin may be subject to dissolution and solubility limited absorption from the intestinal tract, particularly at higher intestinal pH's. This hypothesis is supported by the reduced bioavailability of josamycin from a powder compared to a solution. Determination of intrinsic dissolution rates at infinite rotation speed (when the diffusion layer is theoretically equal to zero) proved to be a useful means by which the dissolution characteristics of pure drug in different dissolution media could be compared.

Dissolution profiles of josamycin powder and two tablet preparations used in bioavailability studies were determined at various pH's over the gastro-intestinal pH-range. The effect of formulation on dissolution from the tablet preparations was assessed by comparison of the powder with tablet dissolution profiles at each pH. Dissolution profiles of Josamycin 200mg tablets from pH 1.2 to 5.0 showed rapid dissolution and were similar to powder dissolution profiles. However, above pH 5.0, dissolution of the powder was retarded by reduced intrinsic dissolution rate and solubility. Dissolution from the tablet preparation above pH 5.0 was further reduced by formulation factors. Dissolution of these tablets will therefore be rapid in acidic gastric fluids. However, if intact tablets or granules pass into the less acidic environment of the duodenum, dissolution will be significantly reduced and absorption is likely to be dissolution rate limited.

Comparison of josamycin powder with Josacine 500mg tablet profiles showed that dissolution from these tablets was severely limited by formulation factors over the entire pH range, particularly at higher pH's. Absorption from these tablets is therefore likely to be dissolution rate limited whether dissolution occurs in gastric or intestinal fluids.

Although macrolide antibiotics are known to be susceptible to acid degradation, there is a dearth of information on the stability of josamycin. This prompted a comprehensive investigation into the stability of josamycin from pH 1.0 to 12.0, together with the effect of ionic strength and buffer concentration on the rate of degradation in acid. Further studies were conducted utilising simulated gastric and intestinal fluids to assess the extent of degradation after an oral dose.

The pH-rate profile for josamycin over the pH range 1 - 12 was comprehensively characterised and the equation describing the profile determined. Results indicated that josamycin is subject to specific acid catalysis but catalysis in alkali media appeared to be more complex. The rate constant for catalysis by hydronium ion (k₁) was 54.11 M⁻¹.hr⁻¹ whilst the rate constant for catalysis by hydroxide ion (k₃) was 60.35 M⁻¹ .hr⁻¹. Catalysis due to water was insignificant and the water catalysed rate constant was found to be 3.370 x 10⁻⁵ hr⁻¹. The pH of 
maximum stability was determined as pH 6.5 whilst degradation at pH 1.0 and 12.0 is about five orders of magnitude greater than at pH 6.5. The degradation of 
josamycin in acid is subject to a significant primary salt effect, however, no secondary salt effect was evident. Consideration of the ionic strength of the reaction medium 
is therefore essential when undertaking stability studies of josamycin in aqueous media. Concentration vs. time profiles for josamycin in acidic media were biphasic which indicated that the degradation reaction did not follow a simple pathway whereby josamycin degrades directly to products. Further investigations demonstrated that josamycin undergoes a reversible isomerisation step, with subsequent degradation of josamycin and possibly its isomer, by cleavage of the mycarose moiety to the desmycarose compounds.

Studies to determine the stability of josamycin in simulated gastric fluids demonstrated that acid degradation could be appreciable after oral administration. However, extensive degradation in-vivo will only occur at the most acidic gastric pH's of about pH 1.0 to 2.0. Josamycin was, however, found to be significantly more stable than erythromycin, suggesting that problems with acid degradation in-vivo would be less for josamycin than the extensively used macrolide antibiotic, erythromycin.

The administration of josamycin as a solution and powder have hitherto not been reported and the bioavailability of josamycin without the influence of formulation factors has not been established. Bioavailability of josamycin from a solution and powder were therefore conducted. Furthermore, tbe bioavailability of two tablet preparations was assessed. A multiple dose study was also conducted and parameters obtained from pharmacokinetic modelling of both single dose solution and multiple dose solution profiles compared.

The results showed that josamycin is inherently rapidly absorbed from a buffered solution. After a 1 gram dose, a mean Cmax of 1.64 μg/ml was reached within a mean tmax of 0.39 hrs after a lag time of about 10 mins. Absorption from 
powder, however, is slower and a mean Cmax of 0.755 μg/ml was attained with a mean tmax = 0.50 hrs. Furthermore, the bioavailability of the powder was only ±50% that of the solution. It was therefore concluded that absorption from the powder was limited by the dissolution rate and solubility. The bioavailability from two different tablet formulations was still lower than from the powder, with absorption hindered not only by the intrinsic dissolution rate and solubility, but also by formulation factors. 
Furthermore, absorption from the tablets was highly erratic. It was concluded that intact tablets or granules passed into the duodenum, after which dissolution from the 
tablets or granules was retarded and pH-dependent. Investigations into the in-vitro dissolution characteristics of the tablet preparations and comparison with powder dissolution profiles over the entire gastro-intestinal pH range rather than at a limited number of pH's provided invaluable information for the explanation of the poor profiles obtained after tablet administration.

Modelling of single dose solution data showed that the pharmacokinetics of josamycin can be adequately described by a two body-compartment model with rapid and extensive distribution into a peripheral compartment (∝ = 6.50 hr⁻¹, k₂₁ = 1.46 hr⁻¹). The mean volume of distribution was 161.7 l which suggests that josamycin distributes to "deep" tissue and is consistent with the considerable accumulation of josamycin observed in various organs. Josamycin was 51% bioavailable in this study and had a t½ of 1.66 hrs with a total clearance of 4.71 l.kg⁻¹.hr⁻¹. The pharmacokinetics of josamycin however, changed significantly after multiple dosing. Both distribution coefficients decreased (∝ = 2.99 hr⁻¹, k₂₁ = 0.41 hr⁻¹ ) as did the volume of distribution (Vd = 34.3 l). This suggested that saturation of the peripheral compartment occurred. Furthermore, a decrease in clearance (Cl = 0.63 l.kg⁻¹.hr⁻¹) with a corresponding increase in elimination half-life (t½ = 3.61) were also observed. This suggested possible saturation of metabolic enzyme pathways. These changes in pharmacokinetic parameters were presumably 
responsible for the non-linear kinetics observed during the multiple dose studies.