BIOPHARMACEUTICS AND PHARMACOKINETICS OF THE MACROLIDE ANTIBIOTIC ...

231 Pages·2013·25.38 MB·English

BIOPHARMACEUTICS AND PHARMACOKINETICS OF THE
MACROLIDE ANTIBIOTIC - JOSAMYCIN
by
Michael F. Skinner
A Thesis Submitted to
Rhodes University
in Fulfilment of the
Requirements for the Degree
of
DOCTOR OF PHILOSOPHY
December 1991
School of Pharmaceutical Sciences
Rhodes University
Graharnstown
TO MY PARENTS
ABSTRACT
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 C extraction columns, with a highly
II
specific wash sequence followed by a phase separation step after elution from the
extraction c·olumn. Chromatography was performed on a C reversed-phase analytical
II
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 difHirences in the composition of the sample
11
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. Gao 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·'.s·' at pH 1.2 to 5.815 x 10·' mg.cm·'.s·' at pH 7.5.
The solubility of josamycin decreased from 212 mg.ml·' at pH 5.45 to 0.18 mg.ml·'
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 200 mg 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 500 mg 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.
111
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 (k,) was 54.11 M·'.hr·' whilst the rate constant for catalysis by
hydroxide ion (k,) was 60.35 M-'.hr·'. Catalysis due to water was insignificant and
the water catalysed rate constant was found to be 3.370 x 10-' br-'. 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 bipbasic
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 witb 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 bitberto not
been reported and tbe bioavailability of josamycin witbout the influence of formulation
factors has not been establisbed. Bioavailability of josamycin from a solution and
powder were tberefore conducted. Furth'*more, tbe bioavailability of two tablet
preparations was assessed. A multiple dose study was also conducted and parameters
obtained from pbarmacokinetic modelling of both single dose solution and multiple
dose solution profiles compared.
IV
The results showed that josamycin IS inherently rapidly absorhed from a
buffered solution. After a 1 gram dose, a mean Cmu of 1.64 I'g/ml was reached
within a mean 1m .. of 0.39 hrs after a lag time of about 10 mins. Absorption from
emu
powder, however, is slower and a mean of 0.755 ",g/m1 was attained with a mean
tm .. = 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 (Ci = 6.50 hr",
k21 = 1.46 hr"). The mean volume of distribution was 161.7 I 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 t'h of 1.66 hrs with a total clearance of
4.71 l.kg·'.hr" . The pharmacokinetics of josamycin however, changed significantly
after multiple dosing. Both distribution coefficients decreased (Ci = 2.99 hr",
k21 = 0.41 hr") as did the volume of distribution (Vd = 34.3 I). This suggested that
saturation of the peripheral compartment occurred. Furthermore, a decrease in
clearance (CI = 0.63 l.kg" .hr') with a corresponding increase in elimination half-life
(t'h = 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.
v
ACKNOWLEDGEMENTS
I would like to express my sincere thanks to the following people:-
My supervisor, Professor I. Kanfer for his guidance and assistance throughout the
course of my studies. Also, for providing the excellent laboratory facilities, financial
support and the opportunity to attend the 3rd International Conference on Drug
Absorption in Edinburgh.
All my colleagues in the laboratory for their congenial company, support and
numerous forms of assistance. Special thanks to Chris Stubbs and Neil Sparrow for
their invaluable assistance during the early days. Also Rod Walker, Sue Terespolsky
and Roz Dowse for their help with operating the main frame computer, Rod for his
help with proof reading and Dr. A. Kench for her invaluable help during
bioavailability trials.
Andy Soper and Owen Campbell for maintaining and repairing equipment, to Sagaran
Abboo for his help in the laboratory and during bioavailability trials and also Leon
Purdon for his various forms of assistance. Special thanks also go to Carol Hubbard
for her patience and help in printing.
Professor H. Parolis and staff of the School of Pharmaceutical Sciences for the use
of departmental facilities.
The Council for Scientific and Industrial Research and the Foundation for
Pharmaceutical Education for financial assistance.
Yamanouchi Pharmaceutical Company for their generous donation of Josamycin tablets
and josamycin powder, and Pharmuka for their generous donation of Josacine tablets.
My parents for their continued support, encouragement and assistance throughout my
studies.
VI
CONTENTS
Abstract
Acknowledgements
V
List of Figures xiv
List of Tables xxiv
CHAPTER 1
INTRODUCTION 1
1.1 ISOLATION. STRUCTURE AND PHYSICO-CHEMICAL PROPERTIES
OF JOSAMYCIN 1
1.2 PHARMACOLOGY 3
1.2.1 Mode of Action 3
1.2.2 Spectrum of Activity 4
1.2.3 Drug Interactions 5
1.2.4 Tolerance and Side Effects 5
1.3 PHARMACOKINETICS 6
1.3.1 Absorption 7
1.3.2 Distribution 9
1.3.3 Metabolism 10
1.3.4 Excretion 11
1.3.5 Pharmacokinetic Modelling 12
vii
CHAPTER TWO
QUANTITATIVE DETERMINATION OF JOSAMYCIN 13
2.1 ANALYTICAL METHODS FOR USE WITH RAW MATERIAL,
INTRINSIC DISSOLUTION RATE STUDIES AND DOSAGE FORM
ANALYSIS. 13
2.1.1 HPLC Apparatus 13
2.1.2 Chemicals 14
2.1.2 Method Development 14
2.1.3.1 Principles of Reversed-Phase Chromatography 14
2.1.3.2 Choice of Column 15
2.1.3.3 Internal Standard 16
2.1.3.4 Choice of Mobile Phase 16
2.1.3.5 UV Detection of Josamycin 18
2.1.4 Chromatographic Conditions 18
2.1.5 Intrinsic Dissolution Rate Studies 20
2.1.5.1 Preparation of Standards 20
2.1.5.2 Sample Preparation 20
2.1.5.3 Precision and Accuracy 21
2.1.6 Tablet Content Analysis 21
2.1.6.1 Preparation of Standards 21
2.1.6.2 Sample Preparation 22
2.1.6.3 Results and Discussion 22
2.1.7 Discussion 24
2.2 ANALYTICAL METHODS FOR DEGRADATION, SOLUBILITY, AND
TABLET AND POWDER DISSOLUTION STUDIES USING HPLC 25
2.2.1 HPLC Apparatus 25
2.2.2 Method Development 26
2.2.2.1 Choice of Column 26
2.2.2.2 Mobile Phase 26
Vll1
2.2.3 Chromatographic Conditions 29
2.2.4 Assessment of Peak Purity 29
2.2.4.1 UV Diode Array Detection 29
2.2.4.2 Ratio Plot 31
2.2.4.3 Assessment of losamycin Peak Purity after
Alkali Degradation. 32
2.2.5 Degradation Studies 32
2.2.5.1 Preparation of Standards and Controls 32
2.2.5.2 Sample Preparation 33
2.2.5.3 Precision and Accuracy 34
2.2.6 Solubility Studies 34
2.2.6.1 Preparation of Standards 34
2.2.6.2 Sample Preparation 34
2.2.6.3 Precision and Accuracy 35
2.2.7 Powder and Tablet Dissolution Studies - pH 1.2 to 4.0 35
2.2.7.1 Preparation of Standards and Controls 35
2.2.7.2 Sample Preparation 36
2.2.8 Discussion 36
2.3 TABLET AND POWDER DISSOLUTION STUDIES USING
SPECTROPHOTOMETRIC METHODS 38
2.3.1 Method Development 38
2.3.2 Analysis of Samples from Dissolution Studies at
pH 5.0 to 9.0 39
2.3.2.1 Preparation of Standards and Controls 39
2.3.2.2 Sample Preparation 40
2.3.2.3 Precision and Accuracy 40
2.3.3 Analysis of Samples from Mass Balance Analysis Studies 41
2.3.3.1 Preparation of Standards 41
2.3.3.2 Sample Preparation 41
2.3.4 Discussion 41