AN ABSTRACT OF THE THESIS OF
Beom-Jin Lee for the degree of Doctor of Philosophy in
Pharmacy (Pharmaceutics) presented on December 8,
1992.
Title: Development and Evaluation of an Oral Controlled
Release and a Transdermal Delivery System for Melatonin in
Human Sublects.
Abstract approved:
Redacted for privacy
James W. Ayres,
Melatonin (MT) concentration in non-biological fluids
was determined using a HPLC system with UV detection.
Retention time and sensitivity (0.01 M acetate/methanol
(50:50, v/v%), mobile phase) were about 4.5 minutes and 10
ng/ml, respectively, at a flow rate of 1.1 ml/min.
MT was very pure as received from the company. MT
solubility increased linearly as 2-hydroxypropy1-0cyclodextrin (2-HPCD) concentration (<30 w/v%)
increased,
but increased exponentially as propylene glycol (PG)
concentration (<60 v/v%) increased. Solubilization
capacity of 2-HPCD was decteased and less efficient as PG
concentration increased in the presence of 2-HPCD. MT was
unstable in acidic solution.
Sugar spheres loaded with MT were coated with
variable amounts of Aquacoat* (aqueous ethylcellulose
suspension) to obtain a desired controlled release of MT
over 8 hours. Twenty percent Aquacoat' coating of 8-10
mesh beads containing MT showed a controlled release of
drug over 8 hours in dissolution profiles and in vivo
human studies. An oral preparation containing 0.1 mg
immediate release MT (uncoated beads) and 0.4 mg
controlled release MT (20% coated beads) produced the
plasma MT concentration versus time profile which was
similar in shape to the computer simulated profile.
However, maximal plasma MT concentration was three times
greater than predicted. Urinary excretion rate of 6sulphatoxymelatonin (6-STMT) was closely related with
plasma MT concentration, as expected.
Flux (Ag/hr/cm2) of MT through excised hairless mouse
skin using vertical Franz' diffusion cells was in the
order, PG 40%/2-HPCD 30% > CD 30% > PG 40% > PG 20% >
buffer. Flux of MT through the ethylene vinyl acetate
membrane was 5-20 times less than through excised hairless
mouse skin and no MT through the microporous polyethylene
membrane was diffused over 12 hours. PG and 2-HPCD were
ineffective for the diffusion of MT through synthetic
membranes. In human pilot'studies using the Hill Top
Chamber' containing MT in 40% PG solution as a transdermal
delivery device (TDD), MT could be delivered
transdermally. Urinary excretion rate of 6-STMT was also
related with plasma MT concentration, as expected.
DEVELOPMENT AND EVALUATION OF AN ORAL CONTROLLED RELEASE
AND
A TRANSDERMAL DELIVERY SYSTEM FOR MELATONIN
IN HUMAN SUBJECTS
by
Beom-Jin Lee
A THESIS
Submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Completed December 8, 1992
Commencement June 1993
APPROVED:
Redacted for privacy
of Major
Professor of Pharmacy in
Redacted for privacy
Dean of College of Pharmacy
Redacted for privacy
Dean of Gra
te Schoo
Date thesis is presented
December 8, 1992
Typed by
Beom-Jin Lee
ACKNOWLEDGEMENT
Science is not simply a collection of facts; it is a
discipline of thinking about rational solutions to
problems after establishing the basic facts derived from
observations. It is hypothesizing from what is known to
what might be, and then attempting to test hypotheses.
The love for logical thinking must come first; the
facts can later fall into place.
Rosalyn S. Yalow
Mount Sinai School of Medicine
Chronicle of Higher Education, June, 1988.
I would like to dedicate this thesis in bereavement to my
father for his love, encouragement and support until he
died July, 24, 1992.
My deepest gratitude goes to my wife, Mina Lee. Her
understanding, patience and love make this truly
meaningful. I would like to give my earnest love and
blessing for my two daughters, Rita and Tina for their
healthy growing and understanding.
I would like to thank Dr. James W. Ayres for his guidance
and valuable advice. I greatly appreciate Dr. Keith A.
Parrott for his support, assistance, advice and
friendship. Special thanks must go to Dr. Robert L. Sack
of School of Medicine, Oregon Health Science University
for his financial support and guiding in vivo human
studies.
I would like to thank my committee members for their
advice and comments. I also appreciate my fellow graduate
students for their help and friendship. I would like to
thank all staff in College of Pharmacy for their helpful
guidance and assistance. Special thanks go to all advisers
in the Office of International Education for their
assistance and guidance to adjust myself to new
surroundings.
TABLE OF CONTENTS
1
INTRODUCTION
CHAPTER I:
DETERMINATION OF MELATONIN BY
REVERSED-PHASE HPLC WITH UV
DETECTION
3
ABSTRACT
3
INTRODUCTION
4
MATERIALS AND METHODS
5
RESULTS AND DISCUSSION
7
CONCLUSIONS
15
REFERENCES
16
CHAPTER II:
PREFORMULATION EVALUATION OF
PHYSICOCHEMICAL PROPERTIES OF
MELATONIN
19
ABSTRACT
19
INTRODUCTION
21
MATERIALS AND METHODS
24
RESULTS AND DISCUSSION
28
CONCLUSIONS
52
REFERENCES
53
CHAPTER III:
DEVELOPMENT AND CHARACTERIZATION
OF AN ORAL CONTROLLED RELEASE
DELIVERY SYSTEM FOR MELATONIN
56
ABSTRACT
56
INTRODUCTION
57
MATERIALS AND METHODS
60
RESULTS AND DISCUSSION
69
CONCLUSIONS
83
REFERENCES
84
CHAPTER IV:
EVALUATION AND PHARMACOKINETICS
OF AN ORAL CONTROLLED RELEASE
DELIVERY SYSTEM FOR MELATONIN IN
HUMAN SUBJECTS
87
ABSTRACT
87
INTRODUCTION
89
MATERIALS AND METHODS
92
RESULTS AND DISCUSSION
99
CONCLUSIONS
118
REFERENCES
119
CHAPTER V:
IN VITRO DIFFUSION STUDIES OF
MELATONIN THROUGH HAIRLESS MOUSE
SKIN AND SYNTHETIC MEMBRANES
122
ABSTRACT
122
INTRODUCTION
124
MATERIALS AND METHODS
127
RESULTS AND DISCUSSION
132
CONCLUSIONS
154
REFERENCES
156
CHAPTER VI:
PRELIMINARY EVALUATION OF
TRANSDERMAL DELIVERY DEVICE FOR
MELATONIN IN HUMAN SUBJECTS
160
ABSTRACT
160
INTRODUCTION
162
MATERIALS AND METHODS
164
RESULTS AND DISCUSSION
167
CONCLUSIONS
175
REFERENCES
176
BIBLIOGRAPHY
178
APPENDICES
189
LIST OF FIGURES
Figure
Page
1.1
A typical chromatogram of a mixture of MT
(peak 1) and MP (peak 2) as an internal
standard.
10
1.2
Standard calibration curve for known MT
concentration (0.04 to 0.5 mg/m1) versus
peak area ratio of MT and MP (internal
standard).
11
11.1
The chemical structure of melatonin (top)
and 2-hydroxypropyl-fl-cyclodextrin
25
(bottom).
11.2
FT-IR spectrum of MT.
29
11.3
Thermogram of MT by DSC.
31
11.4
Construction for determining phase
transition temperature of MT. A and B
indicate lower and upper limit
temperature and phase transition
temperature is indicated by asterisk
32
(*lc).
11.5
MT solubility as a function of PG
concentration (v/v%) at 25 °C in 0.05 M
phosphate buffer (pH 6.1)/PG solutions.
Arrow indicates the turning point of MT
solubility.
11.6
Log solubility of MT as a function of PG
concentrations (v/v %) at 25 °C in 0.05 M
phosphate buffer (pH 6.1)/PG solutions.
38
11.7
MT solubility as a function of 2-HPCD
concentration at 25 °C in 0.05 M
phosphate buffer (pH 6.1).
39
11.8
MT solubility in PG with varying
concentrations of 2-HPCD solutions at 25
°C in 0.05 M phosphate buffer (pH 6.1).
41
11.9
Comparison of observed (open circle) and
theoretical (solid line) MT solubility in
20 % (v/v) PG with varying concentrations
of 2-HPCD at 25 °C in 0.05 M phosphate
buffer (pH 6.1).
42
37
11.10
Comparison of observed (open circle) and
theoretical (solid line) MT solubility in
40% (v/v) PG with varying concentrations
of 2-HPCD at 25 °C in 0.05 M phosphate
buffer (pH 6.1).
43
11.11
Effect of concentrations of PG on the
stability constant of MT (1/M) and slope
calculated from the linear portion of MT
solubility (mM) versus 2-HPCD
concentration (mM) curves.
45
11.12
Comparison of MT solubility (mM) in
various vehicles.
47
111.1
Cross section of STREA-1 SPRAY COATER
fully assembled.
62
111.2
Dissolution profiles of MT from 18-20
mesh beads coated with Aquacoat'. Coating
amounts: open circle, 5%; solid circle,
10%; and trangle, 20%. Each data point
represents the mean ± standard deviation
except where the standard deviation is
too small to show.
74
111.3
Dissolution profiles of MT from 8-10 mesh
beads coated with Aquacoat% Coating
amounts: open circle, 5%; solid circle,
10%; and trangle, 20%. Each data point
represents the mean ± standard deviation
except where the standard deviation is
too small to show.
75
Time for 50% MT dissolution as a function
of coating amounts (theoretical weight
gain) in 18-20 and 8-10 mesh coated
beads. Each data point represents the
mean ± standard deviation except where
the standard deviation is too small to
77
show.
111.5
Dissolution profiles of MT from 8-10 mesh
coated beads with 20% coating as a
function of stirring speed. Each data
point represents the mean ± standard
deviation except where the standard
deviation is too small to show.
78
111.6
Time for 50% MT dissolution as a function
of stirring speed. Each data point
represents the mean ± standard deviation
except where the standard deviation is
too small to show.
79
111.7
Dissolution profiles of MT from 8-10 mesh
beads with 20 % coating after 6 months'
storage at room temperature. Each data
point represents the mean ± standard
deviation except where the standard
deviation is too small to show.
82
IV.1
Computer simulated plasma MT
concentrations from different dosage
forms in human subjects. An 0.5 mg
immediate release MT in curve 1 and an
oral controlled release delivery system
containing 0.1 mg immediate release MT
from uncoated beads and 0.4 mg controlled
release MT from 20% Aquacoat. coated
beads over 8 hours (total dose = 0.5 mg)
in curve 2.
100
IV.2
Mean plasma MT concentration versus time
profiles after administration of
controlled release delivery system to six
human subjects at two different doses.
Values are expressed as mean ± standard
deviation (n=4 for low dose, n=2 for high
102
dose).
IV.3
Comparison of the profiles of observed
plasma MT concentration (mean ± standard
deviation, n=4) and computer simulated
plasma MT concentration at low dose.
103
IV.4
Estimated MT input rate (µg /hr) by
deconvolution analysis resulting from an
oral controlled release delivery system
at two different doses of MT.
105
IV.5
Estimated cumulative amount input (Mg) of
MT by deconvolution analysis resulting
from an oral controlled release delivery
system at two different doses of MT.
106
IV.6
Urinary excretion rate (ng/hr) of 6-STMT
determined at the midpoints of each urine
collection interval after administration
of the oral controlled release delivery
system to six human subjects. Values are
expressed as mean ± standard deviation
(n=4 for low dose, n=2 for high dose).
110
IV.7
Comparison of the profiles of plasma MT
concentration and urinary excretion rate
of 6-STMT determined at the midpoints of
each urine collection interval at the low
dose of MT (0.5 mg) in six human
subjects. Values are expressed as mean ±
standard deviation (n=4).
111
IV.8
Comparison of the profiles of plasma MT
concentration and urinary excretion rate
of 6-STMT determined at the midpoints of
each urine collection interval at the
high dose of MT (1.0 mg) in six human
subjects. Values are expressed as mean ±
standard deviation (n=2).
112
IV.9
Cumulative amount of urinary
for 6 hours at two different
Values are expressed as mean
deviation (n=4 for low dose,
114
6-STMT (mg)
doses of MT.
± standard
n=2 for high
dose).
IV.10
Relationship between plasma MT
concentrations and urinary excretion
rates of 6-STMT determined at the
midpoints of each urine collection
interval in six human subjects.
115
IV.11
Computer-simulated plasma MT
concentration versus time profiles at
various dosing of MT. Total doses and
percentage of immediate release portion
are as follows: Curve 1, 0.8 mg, 10%;
Curve 2, 0.5 mg, 20%; Curve 3, 0.4 mg,
12.5%; Curve 4, 0.4 mg, 10%; Curve 5, 0.2
mg, 10%. Open circles indicate observed
plasma MT concentrations at low dose MT
studied (0.5 mg, 20%). Kel=0.9 hrl, Ka=1.74
hr4, Vd=35.2 1, and F=0.3 for immediate
release MT and F=0.1 for controlled
release MT.
116
Scheme of a vertical Franz. diffusion
129
V.1
cell.
V.2
Profiles of cumulative amount (4g) of MT
released from vehicles saturated with MT
through excised hairless mouse skin.
Values are expressed as mean ± standard
deviation (n=6).
134
V.3
Effects of vehicles on the flux
(Ag/hr/cm2) of MT through excised
hairless mouse skin. Vehicle 1-5, and 2-5
are statistically different (p<0.05) by
Tukey method of multiple comparison
analysis. Values are expressed as mean ±
standard deviation (n=6).
136
V.4
Correlation between flux (Ag/hr/cm2) of
MT from vehicles through excised hairless
mouse skin and MT solubility (mM).
Vehicles are indicated by numbers. 1,
buffer, 2, PG 20% (v/v), 3, PG 40% (v/v),
4, 2-HPCD 30% (w/v), and 5, PG 40%
(v/v)/2-HPCD 30% (w/v). Values are
expressed as mean ± standard deviation
139
(n=6).
V.5
Effects of vehicles on the lag time (hrs)
of MT through excised hairless mouse
skin. Values are expressed as mean ±
standard deviation (n=6).
140
V.6
Effects of hydration time (minutes)
between excision of hairless mouse skin
and start of diffusion on the flux
(Ag/hr/cm2) of MT from vehicles.
142
V.7
Profiles of cumulative amount (Ag) of MT
released from vehicles through ethylene
vinyl acetate membrane. Values are
expressed as mean ± standard deviation
143
(n=4).
V.8
Effects of vehicles on the flux
(Ag/hr/cm2) of MT through ethylene vinyl
acetate membrane. Vehicle 1-2, 1-3, 1-4,
2-3, and 2-5 are statistically different
(p<0.05) by Tukey method of multiple
comparison analysis due to high precision
of data. Values are expressed as mean ±
standard deviation (n=4).
145
V.9
Correlation between flux (Ag/hr/cm2) of
MT from vehicles through ethylene vinyl
acetate membrane and MT solubility (mM).
Vehicles are indicated by numbers. 1,
buffer, 2, PG 40% (v/v), 3, 2-HPCD 30%
(w/v), and 4, PG 40% (v/v)/2-HPCD 30%
(w/v). Values are expressed as mean ±
standard deviation (n=4).
147
Comparison of flux (Ag/hr/cm2) of MT
through excised hairless mouse skin and
ethylene vinyl acetate membrane. Bar
values of microporous polyethylene
membrane are not shown due to
undetectable flux. Values are expressed
as mean ± standard deviation.
150
VI.1
Plasma MT concentration versus time
profiles after application of TDD to four
human subjects over 8 hours.
169
VI.2
Profiles of urinary excretion rate of 6STMT (ng/hr) determined at the midpoints
of each urine collection interval after
application of TDD to four human
subjects. TDS was applied at 11:00 AM
(t=0) in the morning. Asterisks show the
time of TDD removal.
171
VI.3
Cumulative amount of urinary 6-STMT for 6
hours (11:00-17:00) after application of
172
V.10
TDD.
VI.4
Correlation between plasma MT
concentrations and urinary excretion rate
of 6-STMT determined at the midpoints of
each urine collection interval after
application of TDD to four human
subjects.
173
LIST OF TABLES
Page
Table
1.1
Optimization of HPLC analytical conditions
8
for MT.
1.2
Melatonin analysis: Peak processing
parameters for the Shimadzu Chromatopac CR3A integrator.
1.3
Slope, intercept (I), and coefficient of
determination (R2) calculated from
standard calibration curves of MT.
12
1.4
Precision and accuracy of standard
calibration curves (n=7) for assay of MT
concentration in water.
14
II.1
Melting point (mp), heat of fusion, and
purity of MT as received from the company
by DSC analysis.
30
11.2
Group contribution method for calculating
solubility parameter (6) of MT.
34
11.3
Solubility of MT in varying concetrations
of PG and/or 2-HPCD solutions.
36
11.4
Percentage of intact MT as a function of
48
9
pH at 70 °C.
11.5
Observed degradation rate constant (k,,,
day') and half-life (t1,2, days) of MT as a
function of PG and 2-HPCD at 70°C in NaClHC1 buffer (pH 1.4).
49
111.1
Typical processing conditions for coating
of MT-loaded beads (STREA-1, Aeromatic).
63
111.2
Formulation for the application of MT to
nonpareil sugar spheres.
64
111.3
Coating formulations for Aquacoat. coating
of MT-loaded beads.
66
111.4
Amount of MT (mg per gram of bead) assayed
70
in beads.
111.5
Estimated coating amounts based on
approximate weight gain of beads (batch
size: 90 g of MT-loaded beads).
71
111.6
Amount of MT (mg per gram of bead) in
coated beads after storage at room
temperature (24±2 °C).
80
IV.1
Selec ed pharmacokinetic parameters of MT
for c mputer simulation in human subjects.
93
IV.2
Vital statistics of six human subjects.
94
IV.3
Sampl ng schemes for blood and urine
collection in six human subjects.
96
IV.4
Nonco partmental pharmacokinetic
param ters for MT following administration
of the oral controlled release delivery
syste to six human subjects.
107
V.1
MT so ubility in various vehicles
133
V.2
Flux Ag/hr/cm2), permeability coefficient
(cm /hi), and lag time of MT from vehicles
throu h excised hairless mouse skin (n=6).
135
V.3
Flux Ag/hr/cm2), and permeability
coeff cient (cm/hr) of MT from vehicles
throu h synthetic membranes (n=4).
146
V.4
Mass alance studies of MT from a vehicle
of PG 40% (v/v) with 2-HPCD 30% (w/v)
throu h synthetic membranes (n=4).
148
V.6
Volum
conta
with
studi
micro
11)
changes (ml) of donor phase
ning MT in a vehicle of PG 40% (v/v)
-HPCD 30% (w/v) during diffusion
s through ethylene vinyl acetate and
orous polyethylene membranes.
152
VI.l
Vital statistics of four male subjects.
165
VI.2
Plasm MT concentration (pg/ml) after
appli ation of TDD containing MT in
propy ene glycol 40% (v/v) to four human
subjects.
168
LIST OF APPENDIX FIGURES
Page
Figure
A.1
Profiles of plasma MT concentration and
urinary excretion rate of 6-STMT versus
time for subject 1. Total 0.5 mg of MT
doses (0.1 mg immediate release and 0.4
mg controlled released from 20%
Aquacoat.-coated beads) were
administered.
190
A.2
Profiles of plasma MT concentration and
urinary excretion rate of 6-STMT versus
time for subject 2. Total 0.5 mg of MT
doses (0.1 mg immediate release and 0.4
mg controlled released from 20%
Aquacoat.-coated beads) were
administered.
191
A.3
Profiles of plasma MT concentration and
urinary excretion rate of 6-STMT versus
time for subject 3. Total 0.5 mg of MT
doses (0.1 mg immediate release and 0.4
mg controlled released from 20%
Aquacoat.-coated beads) were
administered.
192
A.4
Profiles of plasma MT concentration and
urinary excretion rate of 6-STMT versus
time for subject 4. Total 0.5 mg of MT
doses (0.1 mg immediate release and 0.4
mg controlled released from 20%
Aquacoat.-coated beads) were
administered.
193
A.5
Profiles of plasma MT concentration and
urinary excretion rate of 6-STMT versus
time for subject 5. Total 1 mg of MT
doses (0.2 mg immediate release and 0.8
mg controlled released from 20%
Aquacoat.-coated beads) were
administered.
194
A.6
Profiles of plasma MT concentration and
urinary excretion rate of 6-STMT versus
time for subject 6. Total 1 mg of MT
doses (0.2 mg immediate release and 0.8
mg controlled released from 20%
Aquacoat-coated beads) were
administered.
195
LIST OF APPENDIX TABLES
Page
Table
B.1
Preparation of buffer solutions
197
C.1
Dissolution data: The percentage of MT
released from Aquacoat - coated beads at
various coating amounts (n=3).
199
C.2
Dissolution data: The percentage of MT
released from 8-10 mesh Aquacoat- coated
beads with 20% coating as a function of
stirring speed.
200
C.3
Dissolution data: The percentage of MT
released from 8-10 mesh Aquacoat- coated
beads with 20% coating after 6 months'
storage at room temperature.
201
D.1
Plasma MT concentration (pg /mi) versus
time data after administration of the oral
controlled release delivery system to
human subjects.
203
D.2
Urinary excretion rate of 6-STMT (ng/hr)
versus time data after administration of
the oral controlled release delivery
system to human subjects.
204
E.1
Mean cumulative amount (Ag) of MT and flux
(Ag/hr/cm2) from vehicles through excised
hairless mouse skin.
206
E.2
Mean cumulative amount (Ag) of MT and flux
(Ag/hr/cm2) from vehicles through ethylene
vinyl acetate membrane.
207
E.3
Comparison of ethylene vinyl acetate and
microporous polyethylene membranes
208
DEVELOPMENT AND EVALUATION OF AN ORAL CONTROLLED RELEASE
AND
A TRANSDERMAL DELIVERY SYSTEM IN HUMAN SUBJECTS
INTRODUCTION
Melatonin (N- Acetyl -5- Methoxytryptamine) is a
neurohormone secreted by the pineal gland in a circadian
rhythm. Currently, no commercial dosage forms of melatonin
(MT) that mimic endogenous MT circadian rhythm are
available. Dosage forms which mimic the physiologically
produced endogenous MT plasma concentration versus time
profile will be valuable to fully evaluate the clinical
potential of MT in the treatment of disordered sleep
syndrome, jet-lag, seasonal affective disease, shift work
syndrome, and other circadian disorders. Dosage forms must
deliver MT over 8-10 hours in a controlled fashion so that
endogenous circadian rhythms are mimicked. The overall
goal of this research is to develop and evaluate an oral
controlled release dosage form and a transdermal dosage
form of MT for use in human subjects.
Chapter I deals with development of a HPLC analysis
for MT determination in non-biological fluids. The HPLC
procedure was fine tuned to achieve optimal peak
resolution and retention time.
In Chapter II, the preformulation evaluation of MT
2
and determination of MT physicochemical properties is
described. Thermal behavior of MT by DSC, and solubility
and stability of MT in varying concentrations of propylene
glycol (PG) and 2-hydroxypropyl cyclodextrin (2-HPCD) are
also discussed.
Development of an oral controlled release dosage form
containing MT is presented in Chapter III. Coated MT
pellets with the desired controlled release rate were
developed using fluid-bed coating technology. Coating
technique, dissolution, and stability of the coated
pellets is also described.
Chapter IV
deals with evaluation of an oral
controlled release delivery system for MT in human
subjects. This chapter describes computer simulation for
in vivo studies, and the plasma MT and urinary 6sulphatoxymelatonin (a major renally excreated metabolite
of MT) concentrations achieved in human subjects.
Chapter V deals with in vitro diffusion studies of MT
through excised hairless mouse skin and two synthetic
membranes. The diffusional behavior of MT through two
synthetic membranes, ethylene vinyl acetate and
microporous polyethylene membrane, and excised hairless
mouse skin is described.
In Chapter VI, the preliminary evaluation of a
transdermal delivery system in human subjects is
presented.
3
CHAPTER I
DETERMINATION OF MELATONIN BY REVERSED-PHASE HPLC
WITH UV DETECTION
ABSTRACT
An analytical procedure for determination of
melatonin (MT) concentration in non-biological fluids
using reversed-phase radial compression column (C18, 4A),
with UV detection (229 nm) is described. Optimal elution
conditions were determined by studying several variables:
pH, buffer concentration, and ratio of mobile phase
components. Mobile phase consisting of 50% (v/v) methanol
(HPLC grade) in 0.01 M acetate buffer (pH 4.7) at a flow
rate of 1.1 ml/min provided good resolution of MT and
methylparaben (internal standard) peaks with a retention
time of less than 7 minutes. Because this method has
insufficient sensitivity (10 ng/ml), it may not be used
for analysis of endogenous MT in plasma but is suitable
for determination of MT concentration in aqueous solution.
The method is simple, reliable, accurate, and precise.
4
INTRODUCTION
It is well known that several indole compounds, which
exert neurochemically important physiological actions,
exist in the pineal gland. Among the pineal indoles,
melatonin (MT; N- acetyl -5- methoxytryptamine) is implicated
in the physiology of circadian rhythms (1) and
reproductive development (2).
Quantitative measurements of MT have mainly depended
on bioassay (3), gas chromatography-mass spectroscopy (47), gas chromatography (8), radioimmunoassay (9-12), and
high performance liquid chromatography (13-20). A very
sensitive detection system is necessary because of the
extremely low circulating MT concentrations in blood (10
to 100 pg/ml). All these methods have particular merits
and demerits in respect to sensitivity, selectivity,
specificity, and convenience (21).
The purpose of the present study was to develop a
simple and rapid technique for the determination of MT in
non-biological fluids by HPLC with UV detection.
5
MATERIALS AND METHODS
MT was purchased from Regis Chemical Co. (Morton
Grove, IL). Sodium acetate (reagent grade) was from EM
Industrial, Inc. (Gibbstown, NJ). Glacial acetic acid
(reagent grade) was from J.T. Baker, Inc. (Phillisburg,
NJ). Methanol (HPLC grade) was purchased from Mallinckrodt
Specialty Chemicals Co.
(Paris, KY). Methylparaben (p-
hydroxybenzoic acid methyl ester) was from Sigma Chemical
Co.
(St. Louis, MO). Water was deionized before use.
HPLC Analysis
MT concentration in solution was determined using a
HPLC system consisting of a delivery pump (M-600A, Waters
Associates, Milford, MA), an automatic sample injector
(WISP 712B, Water Associates, Milford, MA), a reversed-
phase C18, 4 gm) radial compression column, a Model 441
absorbance detector with 229 nm light source (Waters
Associates, Milford, MA), and a C-R3A Chromatopac
Integrator (Shimadzu Corp, Kyoto, Japan). Mobile phase
consisted of 50% (v/v) methanol in 0.01 M sodium acetate
buffer (pH 4.7). The flow rate to deliver mobile phase was
1.1 ml/min. Mobile phase was filtered using 0.45 gm nylon
filter (Cole-Parmer Instrument Co. Chicago, IL) under
vacuum. Mobile phase was then further degassed by
sonication under vacuum for 1 hour. Methyparaben (MP) was
6
prepared as an aqueous solution (16.7 µg /ml) and used as
an internal standard. MT standard solutions ranging in
concentration from 0.04 to 0.5 µg /ml were used for the
standard calibration curve. MT and internal standard
solutions (100 Al and 25 Al, respectively) were mixed and
40 Al was injected in duplicate for HPLC analysis. The
standard calibration curve was constructed by plotting the
ratio of MT peak area/internal standard peak area versus
known MT concentrations (µg /m1). There were no detectable
peaks from vehicles or solvents.
7
RESULTS AND DISCUSSION
Composition of mobile phase, pH, and concentration of
acetate buffer were changed to optimize retention time and
resolution of MT and MP peaks. The volume ratio of acetate
buffer and methanol had a significant effect on retention
time of MT and MP (see Table I.1). Excellent resolution
and short retention times were obtained at 50 (v/v)% of
methanol in 0.01 M sodium acetate buffer (pH 4.70).
Peak processing and recording parameters as shown in
Table 1.2 provided the optimal condition for measurement
of MT and MP peak areas. The area normalization method was
used for the construction of standard calibration curve.
Under the conditions described, MT and internal
standard were clearly separated and eluted within 7
minutes (Figure I.1). Mean retention time for MT and MP
was 4.5 and 6.1 minutes, respectively. Figure 1.2 shows a
standard calibration curve for MT. Injections were made in
duplicate. Slope, intercept, and coefficient of
determination were calculated using linear regression
analysis. Mean slope, intercept, and coefficient of
determination were 2.001, 0.0071, and 0.9998, respectively
(see Table 1.3) for seven standard curves produced over a
one year interval. Slope and linearity were very
reproducible, and reliable. The intercept was never
significantly different from zero but did have a
8
Table 1.1 Optimization of HPLC analytical conditions for
MT.
Acetate Buffer
Conc.
(M)
ratio'
pH
retention time (min)
MT
MP
0.04
5.40
65:35
14
19
0.04
4.70
65:35
13
17
0.04
4.25
65:35
14
18
0.04
4.70
70:30
No peakb
No peakb
0.01
4.70
60:40
10
14
0.01
4.70
50:50
4.6
6.1
'Volume ratio of acetate buffer and methanol.
bNo peaks were found within 25 minutes.
Table 1.2 Melatonin analysis: peak processing parameters for the Shimadzu
Chromatopac C-R3A integrator.
Peak Processing
Calculation
Recording
Attenuation
Width (sec)
5
Drift (AV/min)
5000
(mV/full scale)
Slope (4V/min)
3469
Speed (mm/min)
Min. Area (count)
1000
Stop Time (min)
9
3
Area Normalization Method
5
to
10
START
CC?
--4755s
Peak 1
6. 13
Peak 2
CHROMATOPAC
C-R3A
SAMPLE MO
0
REPORT NO
3564
P<M0
1
2
3
FILE
METHOD
TIME
AREA
2.667
4.558
6.13
145954
25949
83176
MK
E
IDNO
COMC
0
41
MAME
57.2193
18.1729
32.6078
TOTAL ---33101
Figure 1.1
A typical chromatogram of a mixture of MT
(peak 1) and MP (peak 2) as an internal
standard.
13
1.2
1.0
2.004 X 0.007
= 0.999
y ==
R2
0.8
0.6
0.4
0.2
0.0
Figure 1.2
0.4
0.5
0.2
0.3
MT Concentration (µg /ml))
0.1
0.6
Standard calibration curve for known MT
concentration (0.04 to 0.5 µg /ml) versus
peak area ratio of MT and MP (internal
standard).
Table 1.3 Slope, intercept (I), and coefficient of determination (R2) calculated
from standard calibration curves of MT.
0.0455
0.0833
0.1154
0.1667
0.5
Slope
I
R2
0.0833
0.1595
0.2156
0.3135
0.9740
1.9613
-0.0081
0.9999
0.0860
0.1576
0.2182
0.3055
0.9627
1.9327
-0.0061
0.9998
0.0754
0.1608
0.2090
0.3188
0.9683
1.9575
-0.0102
0.9998
0.0861
0.1641
0.2201
0.3291
0.9962
2.0037
-0.0059
0.9999
0.0844
0.1755
0.2372
0.3321
1.0467
2.1078
-0.0088
0.9997
0.0769
0.1569
0.2195
0.3195
0.9664
1.9491
-0.0072
0.9999
0.0921
0.1755
0.2444
0.3344
1.0467
2.0961
-0.0032
0.9997
Mean
0.0835
0.1643
0.2234
0.3218
0.9974
2.0012
0.0071
0.9998
S.Dh
0.0057
0.0080
0.0126
0.0105
0.0373
0.0722
0.0023
0.0001
CV (%)'
6.8747
4.8841
5.6427
3.2709
3.7554
3.6083
32.224
0.0090
Concentration
(µg /m1)
P.A.R.a
aPeak area ratio of MT and methylparaben (internal standard).
bStandard deviation.
`Coefficient of variation, CV (%) =[ S.D./Mean] x 100
13
coefficient of variation (C.V.) of 32 %. This was a minor
variability in real value as the range was only from
-
0.0102 to -0.0032. The precision and accuracy of the HPLC
is shown in Table 1.4. Mean percent of theory and C.V.
were 100.15 % and 1.82 %, respectively.
The lower limit of detection for this procedure is
approximately 10 ng/ml. Consequently this assay is not
appropriate for measurement of plasma MT where MT
concentration is in the 10-100 pg/ml range. However, it
may be possible to increase the sensitivity by
concentrating plasma samples or increasing injection
volume and/or changing detectors. Sagara et al. could
detect plasma MT concentration of 19.3 pg/ml using HPLC
with electrochemical detection (18). Even though the
method described here has insufficient sensitivity to
measure plasma MT, it is suitable for the determination of
MT in non-biological solutions.
Table 1.4 Precision and accuracy of standard calibration curves (n=7) for
assay of MT concentration in water.
% Theory'
C.V.
(%)b
Actual (µg /m1)
Observed
0.04546
0.04524 ± 0.0016c
99.52 ± 3.59
3.61
0.08333
0.08560 ± 0.0013
102.72 ± 1.53
1.49
0.11539
0.11515 ± 0.0023
99.80 ± 1.96
1.96
0.16667
0.16440 ± 0.0032
98.64 ± 1.90
1.92
0.5
0.50045 ± 0.0006
100.09 ± 0.13
0.13
100.15 ± 1.53
1.82± 1.24
(µg /m1)
Overall Mean
3% theory = Observed 4- Actual x 100
bCoefficient of variation, C.V. (%)= S.D. + % Theory x 100
`Mean ± standard deviation, n=7
15
CONCLUSIONS
Reversed-phase radial pak compression with UV
detection could detect 10 ng/ml of aqueous MT solution.
The method was reliable, accurate, and precise. Even
though this method is not appropriate for measurement of
plasma MT where endogenous MT concentration is in the 10200 pg/ml range because of an insufficient detection
limit, the method is suitable for the determination of MT
in non-biological fluids which is very useful in drug
dosage form development.
16
REFERENCES
1.
Waldhauser, F. and Dietzel, M. Daily and annual
rhythms in human melatonin secretion: Role in
puberty. Ann., New York Acad. Sci., 453, 205-214
(1985) .
2.
Tamarkin, L., Baird, C.J., and Almeida, O.F.X.
Melatonin: A coordinating signal for mammalian
reproduction? Science, 227, Feb., 15, 714-720 (1985).
3.
Ralph, C.I. and Lynch, H.J. A quantitative melatonin
bioassay. Gen. Comp. Endocrinol., 15, 334-338 (1970).
4.
Cattabeni, F., Koslow, S.H., and Costa, E. Gas
chromatographic-mass spectrometric assay of four
indole alkylamines of rat pineal. Science, 178, 166168 (1972).
5.
Wilson, B.W., Snedden, W., Silman R.E., Smith, I.,
and Mullen, P. A gas chromatography-mass spectrometry
method for the quantitative analysis of melatonin in
plasma and cerebrospinal fluid. Anal Biochem., 81,
283-291 (1977).
6.
Lewy, A.J., and Markey, S.P. Analysis of melatonin in
human plasma by gas chromatography negative chemical
ionization mass spectrometry.
Science, 201(25), 741743 (1978).
7.
Lee, C.R. and Esnaud, H. Determination of melatonin
in blood plasma using capillary gas chromatography
and electron impact medium resolution mass
spectrometry. Biomed. Environ. Mass Spectrom., 15,
249-252 (1988).
8.
Greiner, A.C. and Chan, S.C. Melatonin content of the
human pineal gland. Science, 199, Jan., 6, 83-84
(1978) .
9.
Rollag, M.D. and Niswender, G.D. Radioimmunoassay of
serum concentration of melatonin in sheep exposed to
different lightening regimens. Endocrinol., 98, 482489 (1976).
10
Wetterberg, L.,
Eriksson, 0., Friberg, Y., and
Vangbo, B. A simplified radioimmunoassay for
melatonin and its application to biological fluids.
Preliminary observations on the half-life of plasma
melatonin in man. Clinica Chimica Acta, 86, 169-177
(1978) .
17
11.
Fraser, S., Cowen, P., Franklin, M., Franey, C., and
Arendt, J. Direct radioimmunoassay for Melatonin in
plasma,
29(2), 396-397 (1983).
12.
Vakkuri, 0., Leppaluoto
J., and Vuolteenaho, O.
Development and validation of a melatonin
radioimmunoassay using radioiodinateded melatonin as
tracer, Acta Endocrinologica, 106, 152-157 (1984).
13.
Anderson, G.M., Young J.G., Cohen, D.J., and Young,
S.N. Determination of indoles in human and rat
pineal. J. Chromatogr., Biomed. Appl, 228, 155-163
,
(1982).
14.
Yamada, K., Sakamoto, Y., and Satoh, T. A simple
method for the measurement of pineal melatonin with a
high performance liquid chromatography-UV detection.
Res. Comm. Chem. Pathol. & Pharmacol. 46(2), 283-287
(1984).
15.
Wakabayashi, H., Shimada, K., and Aizawa, Y.
Determination of serotonin and melatonin in rat
pineal gland by high-performance liquid
chromatography with ultraviolet and fluorometric dual
detection. Chem. Pharm. Bull., 33(9), 3875-3880
(1985).
16.
Malcolm H. Mills and Maurice G. King, Melatonin
determination in human urine by high-performance
liquid chromatography with fluorescence detection, J.
Chromatography, 377, 350-355 (1986).
17.
Mills, M.H. and Finlay, D.C. Determination of
melatonin and monoamines in rat pineal using
reversed-phase ion-interaction chromatography with
fluorescence detection. J. Chromatogr., 564, 93-102
(1991).
18.
Sagara, Y., Okatani, Y., Yamanaka, S., and Kiriyama,
T. Determination of plasma 5-hydroxytryptophan, 5hydroxytryptamine, 5-hydroxyindoleacetic acid,
tryptophan and melatonin by high performance liquid
chromatography with electrochemical detection, J.
Chromatography, 431, 170-176 (1988).
19.
Raynaud, F. and Pevet, P. Determination of 5methoxyindoles in pineal gland and plasma samples by
high-performance liquid chromatography with
electrochemical detection. J. Chromatogr., 564, 103113 (1991).
18
20.
Hernandez, G., Abreu, P., Alonso, R., and Calzadilla,
C.H. Determination of pineal melatonin by highperformance liquid chromatography with
electrochemical detection: Application for rhythm
studies and tissue explants. J. Pineal Res., 8, 11-19
(1990).
21.
Lynch, H. Assay methodology, The Pineal, edited by
Relkin, R. p129-150 (1983).
19
CHAPTER II
PREFORMULATION EVALUATION OF
PHYSICOCHEMICAL PROPERTIES OF MELATONIN
ABSTRACT
The physicochemical properties of melatonin (MT) were
characterized. Melting point and heat of fusion obtained
were 116.9 ± 0.24 °C and 7386 ± 87 cal/mol, respectively,
as determined by differential scanning calorimetry (DSC).
HPLC, IR and DSC analysis indicate that MT as received is
identifiable and very pure. Effects of propylene glycol
(PG) and 2-hydroxypropy1-0-cyclodextrin (2-HPCD) on the
solubility and stability of MT were investigated.
Solubility of MT increased linearly as 2-HPCD (0-30 w/v%)
increased but increased exponentially as PG (0-60 v/v%)
increased. Solubility of MT in varying concentrations of
2-HPCD solution mixed with PG (20 or 40 v/v%) increased
linearly, but was less than the sum of its solubility in
2-HPCD and PG individually. As PG increased in the
presence of 2-HPCD, solubilization capacity of 2-HPCD was
decreased and solubilization was less efficient although
apparent MT solubility was highest in the mixture of 40%
(v/v) PG and 30% (w/v) 2-HPCD. PG may have competitively
displaced MT molecules from the 2-HPCD cavity. Stability
of MT was dependent on pH. MT was unstable in HC1-NaC1
20
buffer (pH 1.4), but was relatively stable in acetate
buffer (pH 4.7), phosphate buffer (pH 6.8), and carbonate
buffer (pH 10) at 70 °C. In HC1-NaC1 buffer, the presence
of PG made MT more unstable as compared to its stability
in water. However, MT stability was unchanged compared to
aqueous, nonbuffered solution in the presence of low
concentrations of 2-HPCD (<0.3 w/v%). Information from
these studies will aid in the ongoing development of an
oral controlled release and a transdermal dosage form of
MT.
21
INTRODUCTION
Melatonin (MT; N- acetyl -5- methoxytryptamine) is an
indole amide neurohormone (1). It is primarily secreted by
the pineal gland in a circadian rhythm (2,3). Exogenous MT
has been shown to be effective in resetting circadian
rhythms in humans (4-6) and may be useful in the treatment
of disordered sleep syndrome (7,8), jet lag (9), shift
work syndrome (10), and seasonal affective diseases (11)
in humans. Before a MT delivery system which mimics the
endogenous MT circadian rhythm can be developed, the
physicochemical properties of MT must be evaluated.
Unfortunately, little information on the physicochemical
properties of MT is available.
MT has a relatively low solubility in water (12).
Various approaches may be employed to improve MT
solubility. Cosolvents or complexing agents are commonly
used to overcome solubility and stability problems of
drugs in the pharmaceutical industry. Propylene glycol
(PG) is among the most useful cosolvents for drugs because
of its ability to increase aqueous solubility. It is
inexpensive, stable, nontoxic, and used widely in
commercial preparations (13). The solubility parameter of
PG is 14.8
(cal/cm3)112, which is similar to MT (11.6
(cal/cm3)1/2), based on the group contribution theory of
22
Hildebrand (14). Thus, one might expect that the presence
of PG would increase MT solubility in water.
Another approach to increase drug solubility is to
use cyclodextrins (CDs) which complex with lipophilic
drugs. CDs are oligomers of glucose which are produced
from enzymatically modified starches. The outer surface of
a CD molecule is hydrophilic, but the internal cavity is
apolar and lipophilic. Inclusion complexes are formed when
the hydrophobic portion of CD interacts with the
hydrophobic portion of a drug molecule by noncovalent
physical forces (15-16). The highly water soluble 2-
hydroxypropy1-0-cyclodextrin (2-HPCD) is a commercially
useful complexing agent used to encapsulate numerous
compounds at the molecular level. 2-HPCD is utilized by
formulators to increase aqueous solubility and chemical
stability (17-19). Although many drugs can be complexed,
there are some limitations such that CD complexation
either is not possible or yields no advantages. However,
MT's molecular structure provides conditions for which CD
complexation may be applied (20).
Purposes of the present research were to: a)
characterize the thermal behavior and purity of MT using
DSC, and b) to identify MT as received from the
manufacture using IR and HPLC, and c) to determine the
aqueous solubility and stability of MT in the presence of
PG and/or 2-HPCD. Since no data that establish formulation
23
parameters for MT delivery in humans are available,
preformulation studies will aid in the ongoing development
of an oral controlled release and a transdermal dosage
form of MT.
24
MATERIALS AND METHODS
Materials
Melatonin (MT) was purchased from Regis Chemical Co.
(Morton Grove, IL, USA).
2- Hydroxypropyl-13- cyclodextrin
(2-HPCD) was provided courtesy of American Maize-Product
Company (Hammond, ID, USA).
The average molecular weight,
average degree of molar substitution and range of molar
substitution are 1542, 6.5 moles of hydroxypropyl per mole
of 0-cyclodextrin, and 5-8, respectively. Figure II.1
shows the structure of MT and 2-HPCD, respectively. All
other chemicals used were of reagent grade. Deionized
water was used throughout the study.
HPLC and IR Analysis of MT
All lots of MT received from the company were tested
for identity using HPLC and Fourier transform infrared
(FT-IR) spectroscopy.
IR spectra were obtained using a Nicolet Model DXB
FTIR-spectrometer. Samples were prepared in potassium
bromide using a Wig-L-Bug mixer, filled into the
macrosample cup of the Collector Cell, and leveled with a
spatula. IR spectrum of MT was compared with a reference
spectrum provided by the Regis company.
HPLC retention time was also checked. Details of the
HPLC analysis are described in Chapter I.
25
CH 0
NHCOCH3
CH2OR
O
"HO
ROCH2
OH
0
HO
OH
OH
R = -CH2-CH(OH)-CH3
Figure 11.1
The chemical structure of melatonin
(top) and 2-hydroxypropY1-0cyclodextrin (bottom).
26
DSC Analysis of MT
A Perkin-Elmer differential scanning calorimetry
(DSC) was used to characterize thermal properties of MT,
including melting point, heat of fusion, and purity of MT.
The DSC apparatus consists of a Thermal Analysis Data
Station (TADS) system, Interface, System 4 Microprocessor
Controller, DSC-4 Analyzer with sampling provision, and
recorder. Dried nitrogen gas continuously purged the DSC
sample holder. Temperature was scanned from 50 to 150 °C
at a rate of 10 °C/min. A pure indium sample with melting
point 156.6 °C and transition energy 6.80 cal/gram was
used for temperature calibration. The phase transition
temperature determined for indium
was within ± 0.2 °C.
Solubility Studies
Solubility studies were carried out according to the
method of Higuchi and Connors (21). Excess amounts of MT
were added to 0.05 M phosphate buffer (pH 6.1) containing
various concentrations of PG and 2-HPCD. The resulting
mixtures were sonicated and vortexed, and then placed in a
25.0 ± 0.1 °C constant-temperature water bath. Parafilm
was used to cover the top to prevent evaporation.
Equilibrium solubility was reached within 24 hours.
Samples were centrifuged at 10,000 g for 15 minutes and
the clear supernatant diluted with phosphate buffer to
place the concentration in the correct range for assay.
27
The concentration of MT was determined by HPLC (Chapter
Stability Studies
Stability of MT was investigated under conditions of
varying pH and varying concentrations of PG and 2-HPCD.
HC1-NaC1 Buffer (pH 1.4), acetate buffer (pH 4.7),
phosphate buffer (pH 7.4), and carbonate buffer (pH 10)
were used to study the effect of pH on stability of MT.
Known concentrations of melatonin were stored in an
incubator at 70 ± 2 °C in the dark over 9 months. The
degradation rate of MT with varying concentrations of PG
and 2-HPCD was determined only in the acidic NaCl-HC1
buffer (pH 1.4) because MT was stable at other pH values.
Concentrations of intact MT were determined by HPLC
(Chapter I). Degradation rates were calculated by fitting
data to a first order kinetic degradation model.
28
RESULTS AND DISCUSSION
HPLC and IR Analysis of MT
MT as received from the manufacture was identified by
HPLC and IR spectroscopy before use. A typical HPLC
chromatogram of MT in water for identification of MT is
shown in Figure I.1 (Chapter I). Only MT and internal
standard peaks were identified. The IR spectrum of MT was
compared with that of a reference standard. Figure 11.2
shows an IR spectrum of MT as received. IR spectrum of MT
is the same as that of the reference standard. These
results suggest that MT as received from company is
identifiable and suitable for further study.
DSC Analysis of MT
DSC evaluation was also undertaken to determine the
purity of MT as received from the supplier to decide
whether or not this product was suitable for evaluation in
solubility and stability study, as well as for a proposed
in vivo administration to people as an investigational new
drug. DSC is commonly used to determine melting point,
heat of fusion, and purity of compounds. DSC data obtained
are summarized in Table 11.1. A sharp endothermic peak of
fusion was found for MT on a DSC trace (Figure 11.3). The
transition temperature (marked as **) of MT is shown in
Figure 11.4. The transition temperature is that
XTRANSMITTANCE
10. 731
25. 121
31. 510
27. 800
44. 288
O
D
.
,.
.00
.
icxxxxxxxxxerr
8
10,410;;;;;
0..0.60 OOOOO .1,
f
4P!'PPPrrt,Prt
grATAISMIa
30
Table 11.1
Melting point (mp), heat of fusion, and
purity of MT as received from the company
by DSC analysis.
mp (°C)
Heat of Fusion
Purity (%)
(cal/mol)
116.8 ± 0.25
7249 ± 217
99.97 ± 0.05
Note: MT samples accurately weighed using microbalance
were heated in the calorimeter cell from 50 to 150 °C.
Heating rate was 10°C/min. Two lots of MT samples were
used. Values are expressed as mean ± standard deviation
(total number of replication = 13).
31
10.00
MT (LOT: P89-114-1. A 1)
3. 30 mg
WTI
SCAN RATE,
10.00 deg/min
A
O
5.004
_J
UX
0.00
50.00
70.00
40. 00
90. Ce
100.00
110.D3
12100
130.00
TFMPFRATURE <C)
Figure 11.3
Thermogram of MT by DSC.
140.00
150.00
16400
DSC
32
10.00
MT CLOT' P89-114-1. A2-2)
YTS
3.22 ns
SCAN RATE,
10. DC degIn i n
PEAK FROM. 115.01
TO. 122.51
117. 37
CAL /CRAM, 31. 87
ONSET.
5. 00 +
A
1
0.00
105.00
110.00
115.00
1
00
125. 00
TEMPERATURE CC)
Figure 11.4
Construction for determining phase
transition temperature of MT. A and B
indicate lower and upper limit
temperature. Phase transition
temperature is indicated by asterisk
(**)
DSC
33
temperature at which melting of sample is first detected,
and is independent of variation in sample size. Heat of
fusion of MT was determined by comparison of peak area of
MT to the indium calibrant. Peak sharpness and peak width
of the melting peak reflects purity and crystalline
perfection of samples. The purity of MT was at least
99.9%. Thus, the commercially obtained MT is sufficiently
pure to be used for in vivo human studies.
Solubility Studies
MT has a relatively low solubility in water (12).
Information on MT solubility is useful for dosage form
design, especially transdermal delivery systems. Group
contribution theory based on the thermodynamic partition
of each structural moiety is often useful to calculate
solubility parameters, which express the cohesion forces
between like molecules (14). The calculated solubility
parameter (6) of MT was 11.6 (cal/cm3) 1/2 (Table 11.2).
PG was selected as a solubilizer for MT because it
has a solubility parameter (6=14.8 (cal/cm3) In, which is
j
similar to MT. PG is a common solvent in pharmaceutical
formulations. In addition, 2-HPCD was selected as a
complexing agent to increase solubility of MT. 2-HPCD is
widely used and commercially available.
MT solubility, as a function of concentration of PG
or 2-HPCD, and in mixtures of PG and 2-HPCD is summarized
34
Table 11.2
Group contribution method for calculating
solubility parameter (6) of MT.
Atoms or
Number
AU; (KJ/mol)*
AV;
(CM3/M01) a
Group
CH3
2
4.71 x
2
=
9.42
33.5 x
2
= 67.0
CH2
2
4.94 x
2
=
9.88
16.1 x
2
= 32.2
CH
4
4.11 x
4
= 16.44
13.5 x
4
= 54.0
0
1
3.35
3.8
C=0
1
17.40
10.8
NH
2
8.40 x
2
C
4
4.31 x
4=
17.24
ring closure
2
1.05 x
2
=
2.10
16.0 x
2
= 32.0
conjugation
4
1.67 x
4
=
6.68
-2.2 x
4
= -8.8
= 16.80
EAU; = 100.11
6 = (rdiudalv, )
4.5 x
2
=
9.0
-5.5 x 4= -22.0
EAV, = 178
un
= 11.6 (cal/cm3)1/2
'AU; and AV; are the additive atomic and group contribution
for the energy of vaporization and molar volume,
respectively (14).
35
in Table 11.3.
MT solubility increased as the
concentration of PG increased. The solubility of MT in PG
solution increased slowly until reaching 40% PG (indicated
by an arrow) and then steeply increased (Figure 11.5).
According to a linear combination model (13), mixed
solvents can be treated as a linear combination of their
components. That is, there is a linear relationship
between log solubility of the solute in a mixed solvent
and volume fraction of solvent. The log MT solubility as a
function of volume fraction of PG did have a linear
tendency although deviation from linearity at low and high
volume fraction was observed (Figure 11.6). This curvature
may result from nonideality of solvent or solute.
Nonideality refers to water-PG interaction or interaction
between MT and either itself or one of the mixed solvent
components.
The solubility of MT as a function of 2-HPCD
concentration is shown in Figure 11.7. Solubility of MT
increased linearly as the concentration of 2-HPCD
increased (R2=0.993). According to the definition provided
by Higuchi and Connors (21), the two types of solubility
profiles are A and B.
Curve AL indicates a linear
relationship between CD concentration and drug
concentration. Curve Ap and AN types indicate positive and
negative deviations from linearity. On the other hand,
B-type relationship indicates the formation of complexes
36
Table 11.3
Solubility of MT in varying concentrations
of PG and/or 2-HPCD solutions.
Solubilizer
PG (v/v%)
2-HPCD (w/v%)
0
--
10
20
40
60
AM. OM
-------
0.05
0.15
0.50
1.50
5.00
10.00
20.00
30.00
Solubility (mM)a
6.489
7.400
12.125
41.272
280.392
5.921
6.113
7.416
10.276
23.000
35.814
57.981
78.135
Mixtures of solubilizers
20
20
20
20
20
20
20
20
0.05
0.15
0.50
1.50
5.00
10.00
20.00
30.00
11.864
10.211
12.590
17.219
23.747
31.070
54.807
70.055
40
40
40
40
40
40
40
40
0.05
0.15
0.50
1.50
5.00
10.00
20.00
30.00
36.130
38.839
40.735
42.075
49.647
58.269
75.568
87.852
a n=3, mM = mmole/1
37
300
250
200
150
100
50
10
20
30
40
50
60
PG (v/v%)
Figure 11.5
MT solubility as a function of PG
concentration (v/v%) at 25 °C in 0.05
M phosphate buffer (pH 6.1)/PG
solutions. Arrow indicates the
turning point of MT solubility.
38
10
Figure 11.6
20
30
40
PG (v/v %)
50
60
Log MT solubility as a function of PG
concentrations (v/v %) at 25 °C in
0.05 M phosphate buffer (pH 6.1)/PG
solutions.
39
40
120
80
160
2 HPCD (mM)
.
0
5
10
15
20
25
.
i
30
2-HPCD (w/v %)
Figure 11.7
MT solubility (mM) as a function of
2-HPCD concentration at 25 °C in 0.05
M phosphate buffer (pH 6.1).
40
with limited solubility. Because the solubility profile of
MT in 2-HPCD solution looks an AL-type curve, MT likely
forms soluble complexes with 2-HPCD. MT solubility in the
mixtures of PG and 2-HPCD also increased linearly as 2HPCD increased (Figure 11.8). In the mixtures of PG and 2HPCD, observed MT solubility must be equivalent to
theoretical MT solubility (sum of MT solubility in PG and
2-HPCD individually) if PG and 2-HPCD behave independently
as solubilizers. Figure 11.9 and 10 show that observed MT
solubility is less than theoretical MT solubility. These
results suggest that interaction between PG, 2-HPCD,
and/or MT may be present. MT solubility in the mixtures of
PG and 2-HPCD was not additive.
The initial linear portion of a solubility curve
which involves complexation can be useful to examine the
efficiency of complexation since the slope reflects the
solubility increase as a function of 2-HPCD concentration.
The steeper the slope, the more efficient the
complexation. If the slope equals 1.0, then 1.0 mole of 2HPCD is complexing 1.0 mole of MT, i.e., the process is
100% efficient. If the slope is greater than one, higher
order complexation is occurring. Figure 11.8 provides that
the initial slope obtained in buffer, PG 20%, and PG 40%
as a function of 2-HPCD concentration are 0.38, 0.31, and
0.26, respectively. Complexation efficiency between MT and
2-HPCD was not strong. However, the slope is useful to
41
100
o 0 % PG
20 % PG
80
v 40 % PG
60
20
0
40
80
120
2-HPCD (mM)
160
1..111t1111111111/111 111111.1111
0
5
10
15
20
25
30
2-HPCD (w/v %)
Figure 11.8
MT solubility (mM) in PG with varying
concentrations of 2-HPCD at 25 °C in
0.05 M phosphate buffer (pH 6.1).
42
100
80
E
..'N
--6
,-E,
o
40
01
E-4
20
0
40
80
120
2-HPCD (mM)
0
5
10
15
20
160
25
30
2-HPCD (w/v %)
Figure 11.9
Comparison of observed (open circle)
and theoretical (solid line) MT
solubility (mM) in 20 % (v/v) PG with
varying concentrations of 2-HPCD at
25 °C in 0.05 M phosphate buffer (pH
6.1).
43
120
100
80
60
40
20
40
0
80
120
160
2-HPCD (mM)
0
5
10
15
20
25
30
2-HPCD (w/v %)
Figure 11.10
Comparison of observed (open circle)
and theoretical (solid line) MT
solubility in 40 % (v/v) PG with
varying concentrations of 2-HPCD at
25 °C in 0.05 M phosphate buffer (pH
6.1).
44
calculate the stability constant (or complex formation) on
the basis of a 1:1 stoichiometric ratio between drug and
complexing agent. Although more than one drug molecule is
complexed with 2-HPCD, an apparent stability constant,
calculated with the assumption of only 1:1 stoichiometry
is often used to describe the system because an AL type
diagram would still be observed, and a series of complexes
with more than one drugs might not be distinguishable from
that for 1:1 complex alone (21). The equation used is as
follows.
Stability constant
(K1:1)
=
slope
So (1-slope)
where S, refers to the solubility of drug in water without
PG and 2-HPCD. The stability constant (1/M) and the slope
of the solubility isotherm are given in Figure II.11. The
stability constant and the slope of the solubility
isotherm were decreased without deviating from linearity
as the concentration of PG was increased. Combination of
2-HPCD with PG reduced the solubilization capacity of 2-
HPCD so that solubilization of MT was less efficient. In
the mixtures of PG and 2-HPCD, PG may have competitively
displaced MT molecules from 2-HPCD complexed cavity.
Because 2-HPCD complexes MT in the inner ring closure by
noncovalent bonding or hydrophobic properties, PG may
influence surrounding forces such as a hydrogen bridge
between 2-HPCD and MT by changing water structure and
45
80
60
71
0
C.)
40
erS
20
0
40
20
PG (v/v%)
Figure 11.11
Effect of concentrations of PG on the
stability constant of MT (1/M) and
slope calculated from the linear
portion of MT solubility versus 2HPCD concentration (mM) curves.
46
entropy inside the hydrophobic ring (22). The overall MT
solubility in various vehicles is compared in Figure
11.12. Apparent MT solubility was the highest in the
mixture of PG (40 v/v %) and 2-HPCD (30 w/v %) although
efficiency of MT solubilization in 2-HPCD decreased as PG
increased as mentioned previously.
Stability Studies
An amide linkage has lower reactivity than many other
leaving groups since cleavage is usually in the order,
acid halide > anhydrides > esters > amides. The amide
linkage of MT is relatively insensitive to degradation
compared with these other bond linkages (23). However, an
amide linkage is sensitive to acid catalyzed hydrolysis in
low pH solution (24). The percentage of intact MT as a
function of pH at 70 °C is given in the Table 11.4. MT is
degraded by first order kinetics and was unstable in
strong acidic solution (HC1-NaC1 buffer, pH 1.4) but
relatively stable in other pH values of 4-10.
The stability of MT in the presence of PG and/or 2-
HPCD was also investigated in acidic NaCl-HC1 buffer (pH
1.4)
(Table 11.5). The use of mixed solvents to achieve
greater aqueous solubility may result in altering the
stability of drugs (25). Degradation of MT was accelerated
in the presence of PG. MT in 10% PG solution was degraded
57 times more quickly than in aqueous solution without PG.
47
Water
/
PG 20%
41.4
PG 40%
78.1
2-HPCD 30%
70.1
PG 20%a-11PCD 30%
I 87.9
PG 40%:2-HPC1) 30%
1.1.1.1.1.1.1.1.
0
10
20
30
40
50
60
70
80
MT Solubility (mM)
Figure 11.12
Comparison of MT solubility in
various vehicles.
90
48
Table 11.4
Percentage of intact MT as a function of pH
at 70
°C
pH
Day
kobs
t1f2
1.4
4.7
7.4
10
0
100
100
100
100
0.25
96.5
0.5
86.9
1
81.3
2
71.3
3
61.7
4
56.5
5
51.1
6
47.0
8
41.6
14
12.8
27
2.26
97.3
95.2
94.0
60
0
175
0
73.9
67.2
58.2
283
0
62.5
63.2
37.1
(day')'
1.11x10'1
1.67x103
1.68x10-3
3.46x10-3
(day) b
6.2
415
412
200
'Observed degradation rate constant of MT at each pH was
determined by fitting data to a first order kinetic model.
"Half-life of MT, t12=0.692/kob,
Table 11.5
Observed degradation rate constant (1(0,0 days-') and half-life (t1,2,
days) of MT as a function of 2-HPCD and PG at 70 °C in NaCl-HC1
buffer (pH 1.4).
PG (v/v
Water
2-HPCD (w/v
%)
10
20
40
0.005
0.03
0.1
%)
0.3
1(06,
(days')'
0.112
6.336
1.217
0.638
0.106
0.111
0.107
0.146
tw
(days)"
6.19
0.11
0.62
1.09
6.54
6.24
6.48
4.75
'Degradation rates were determined by fitting data to the first order kinetic
model.
"Half -life,
t112 = 0.692/k
50
Acid catalyzed degradation of MT slowed in the higher
concentration of PG. Because MT is a neutral drug, there
would be a competition between water and PG in acid
catalyzed degradation of MT. The system may contain both
hydronium ions and glycolated proton in the reaction
mixture. A decrease in dielectric constant, i.e., increase
in PG concentration, resulted in an increased reaction
rate as compared to MT in water only. In other words,
degradation rate may be dependent on water content and
solvent polarity (dielectric constant) (23, 25). In 10%
PG, a glycolated proton would be a stronger acid than the
hydronium ions, thus achieving a greater catalytic effect.
However, as PG concentrations increased, the "water"
reaction significantly decreased since water content
decreased. Thus, overall acid catalyzed hydrolysis may be
slowly decreased as water content decreases.
On the other hand, the 2-HPCD at the lower
concentrations (< 0.3%) did not change the degradation
rate constant compared to the only aqueous solution. Acid
catalyzed degradation of MT is less sensitive in 2-HPCD
solutions than PG solutions. In 2-HPCD solutions, the
moiety of MT structure which is sensitive to acid
catalyzed degradation may be protected by 2-HPCD. The
degradation mechanism of MT and complexation phenomena
between MT and 2-HPCD are still under consideration.
Degradation mechanism of MT can be studied by comparing
51
melting point of MT using DSC, HPLC retention time, IR,
and NMR spectrum of reaction mixture with MT standard.
Complexation phenomena between MT and 2-HPCD can also be
studied using computer modeling and spectroscopic changes
using circular dichroism, IR, NMR, and UV (19,26,27).
52
CONCLUSIONS
MT as received from a supplier was identified by HPLC
and IR. MT was highly pure, at least 99.9%. MT was
endothermally melted as identified by a DSC tracing. PG
increased MT solubility exponentially over 60%
concentration. MT solubility increased linearly as 2-HPCD
increased. MT solubility in the mixtures of PG and 2-HPCD
increased linearly but was less than the sum of its
solubility in 2-HPCD and PG individually. As PG
concentrations increased in the presence of 2-HPCD,
solubilization capacity of 2-HPCD was decreased and
solubilization was less efficient although apparent MT
solubility was the highest in the mixture of 40% PG and
30% (w/v) 2-HPCD. In the mixtures of PG and 2-HPCD, PG may
have competitively displaced MT molecules from the 2-HPCD
cavity.
MT stability was pH dependent. MT was unstable in
strong acidic solution (NaCl-HC1 buffer, pH 1.4) but
relatively stable at pH values of 4-10. MT was more
unstable in the presence of PG than in the aqueous
solution only.
MT stability was unchanged in the presence
of low concentrations of 2-HPCD (< 0.3 w/v%).
53
REFERENCES
1.
Lerner, A.B., Case, J.D., Heinzelman, R.V. Structure
of melatonin. J. Am. Chem. Soc., 81, 6084-6085
(1959).
2.
Waldhauser, F., Dietzel, M. Daily and annual rhythms
in human melatonin secretion: Role in puberty. Ann.,
New York Acad. Sci., 453, 205-214 (1985).
3.
Pelham, R.W., Vaughan, G.M., Sandock, K.L., and
Vaughan, M.K. Twenty-four hour cycles of
melatonin-like substances in the plasma of human
males. J. Clin. Endocrinol. Metab., 37, 341-344
(1973).
4.
Arendt, J., Aldhous, M., and Wright, J.
Synchronization of a disturbed sleep-wake cycle
in a blind man by melatonin treatment. The
Lancet, April 2, 773 (1988).
5.
Petterborg, L.J., Thalen, B.E., Kjellman, B.F., and
Wetterberg, L. Effects of melatonin replacement on
serum hormone rhythm in a patient lacking endogenous
emlatonin. Brain Res. Bull., 27(2), 181-185 (1991)
.
6.
Sack, R.L., Lewy, A.J., Blood, M.L., Stevenson, J,
and Keith, L.D. Melatonin administration to blind
people: Phase advances and entrainment. J. Biol.
Rhythm, 6(3), 249-262 (1991).
7.
Cramer, H., Rudolph, J., Consbruch, U., and Kendel,
K. On the effects of melatonin on sleep and behavior
in man.
Adv. in Biochem. Psychopharmacol., 11, 187191 (1974).
8.
Dahlitz, M., Alvarez, B., Vignau, J., English, J.,
Arendt, J., and Parkes, J.D. Delayed sleep phase
syndrome response to melatonin. Lancet, 337(8750),
1121-1124 (1991).
9.
Petrie, K., J.V. Conaglen, L. Thompson, K.
Chamberlain (1989) Effects of melatonin on jet lag
after long haul flight.
Br. Med. J., 298, 705-707.
10.
Waldhauser, F., Vierhapper, H., and Oirich, K.
Abnormal circadian melatonin secretion in night-shift
workers. The New Eng. J. Med., 7, 441-446 (1986).
54
11.
Rosenthal, N.E., Sack, D.A., Gillin, J.C., Lewy,
A.J., Goodwin, F.K., Davenport, Y., and Mueller,
P.S., et. al., Seasonal affective disorder.
A
description of the syndrome and preliminary findings
with light therapy. Arch. Gen, Psychiatry, 41, 71-80
(1984) .
12.
Lewy, A.J. Biochemistry and regulation of mammalian
melatonin production. The Pineal Gland, edited by
Richard Relkin, Elsevier Science Publishing Co., Inc.
(1983) .
13.
Yalkowsky, S.H., and Rubino, J.T. Solubilization
by cosolvents I: Organic solutes in propylene
glycol-water mixtures. J. Pharm. Sci., 74(4),
416-421 (1985).
14.
Barton, A.F.M. Handbook of solubility parameters and
other cohesion parameters. CRC Handbook, CRC Press,
Inc. (1983).
15.
Duchene, D. and Wouessidjewe, D. Physicochemical
characteristics and pharmaceutical uses of
cyclodextrin derivatives, Part I. Pharm. Tech.,
14(6), 26-34
(1990).
16.
Yoshida, A., Arima, H., Uekama, K., and Pitha, J.
Pharmaceutical evaluation of hydroxyalkyl ehters of
0-cyclodextrin. Int. J. Pharm., 46, 217-222 (1988).
17.
Muller, B.W. and Brauns, V. Solubilization of drugs
by modified 0-cyclodextrins. Int. J. Pharm., 26, 7788 (1985).
18.
Loftsson, T., Bjornsdottir, S., Palsdottir, G., and
Bodor, N. The effects of 2-hydroxypropy1-0cyclodextrin on the solubility and stability of
chlorambucil and melphalan in aqueous solution. Int.
J. Pharm. 57:63-72 (1989).
19.
Backensfeld, T., Muller, B.W., Wiese, M., and Seydel,
J.K. Effects of cyclodextrin derivatives on
indomethacin stability in aqueous solution. Pharm.
Res., 7(5), 484-490 (1990).
20.
Szejtli, J. Cyclodextrins in drug formulations:
PartII. Pharm. Tech., August, 24-38 (1991).
21.
Higuchi, T. and Connor, K. Phase solubility
techniques. Adv. Anal. Chem. Instr., 4. 117-212
(1965) .
55
22.
Muller, B.W. and Albers, E. Effects of hydrotropic
substances on the complexation of sparingly soluble
drugs with cyclodextrin derivatives and the influence
of cyclodextrin complexation on the pharmacokinetics
of the drugs. J. Pharm. Sci., 80(6), 599-604 (1991).
23.
Connors, K.A., Amidon, G.L., and Kennon, L. Chemical
stability of pharmaceuticals, A handbook for
pharmacists. John Wiley & Sons, 33-79 (1979).
24.
Dilbeck, G.A., Field, L., Gallo, A.A., and Gargiulo,
R.J. Biologically oriented organic sulfur chemistry.
19. Synthesis and properties of 2-amino-5-mercapto-5methylhexanoic acid, a bishomologue of penicillamine.
Use of boron trifluoride etherate for catalyzing
Markownikoff addition of a thiol to an olefin. J.
Org. Chem., 43(24), 4593-4596 (1978).
25.
Marcus, A.D. and Taraszka, A.J. A kinetic study of
the specific hydrogen ion catalyzed solvolysis of
chloramphenicol in water-propylene glycol system. J.
Am. Pharm. Asso., Scientific edition, 48 (2), 77-84
(1959).
26.
Puglisi, G., Santagati, N.A., Pignatello, R.,
Ventura, C., Bottino, F.A., Mangiafico, S., and
Mazzone, G. Inclusion complexation of 4biphenylacetic acid with 0-cyclodextrin. Drug Dev.
Ind. Pharm., 16(3), 395-413 (1990).
27.
Xiang, T. and Anderson, B.D. Inclusion complexes of
purine necleosides with cyclodextrins. II.
Investigation of inclusion complex geometry and
cavity microenvironment. Int. J. Pharm., 59, 45-55
(1990).
56
CHAPTER III
DEVELOPMENT AND CHARACTERIZATION OF AN ORAL CONTROLLED
RELEASE DELIVERY SYSTEM FOR MELATONIN
ABSTRACT
Sugar spheres loaded with melatonin (MT) were coated
with Aquacoat' (aqueous polymeric ethylcellulose
suspension) to control the release rate of MT. Dissolution
was evaluated using the USP basket method. With 18-20 mesh
beads, Tm(time to release 50% of drug) for 5%, 10% and
20% coatings was 10 min, 35 min and 60 min, respectively.
Most drug was released within 2 hours so that prolonged
release was not achieved. However, a zero order release
pattern over 8 hours was obtained with 20% coating on 8-10
mesh beads. T50% for 5%, 10% and 20% coatings was about 1,
2, and 4 hours, respectively. MT in 20% coated beads was
quite stable during storage at room temperature with less
than 5% MT degraded during 6 months' storage. Dissolution
profiles from 8-10 mesh beads with a 20% coating were
unchanged after 6 months, which implies that coated films
are relatively stable at room temperature. An effective
drug delivery system has been developed which mimics the
endogenous circadian MT pattern in vivo can be provided as
a result of prospective application of pharmacokinetics
and pharmaceutical coating technology.
57
INTRODUCTION
Melatonin (MT, N- acetyl -5- methoxytryptamine) is an
indole amide neurohormone (1). It is primarily secreted by
the pineal gland in a circadian rhythm (2) which is quite
stable in humans. MT concentrations increase in the
evening about 10 pm, which lasts for approximately 8 hours
above the arithematic mean levels of MT (30 pg/ml). It is
desirable to develop controlled release dosage forms which
deliver MT over 8 hours so that the normal endogenous
circadian fashion of MT may be re-established in selected
individuals whose MT production differs from the normal
subjects. Currently, MT's clinical usefulness may be
limited because a proper dosage form that mimics the
physiological, endogenous plasma MT concentration versus
time profile is not available.
Since exogenous MT has been used as a circadian
rhythm synchronizer in humans (3,4), appropriate dosage
forms for MT have clinical potential to treat a variety of
circadian rhythm disorders including sleep disorders
(5,6), jet lag (7), shift work syndrome (8), seasonal
depression (9), and may help blind and elderly people with
desynchronized rhythms (3,10).
Application of aqueous polymeric film coatings to
drug loaded nonpareils (sugar spheres, NF) has become an
increasingly popular method of developing controlled
58
release dosage forms (11-14). Aqueous polymeric
ethylcellulose suspension (Aquacoat.) has replaced the
conventional organic solvent-based coating methods because
of the potential toxicity and high costs associated with
organic solvents (15,16). Aquacoat' is an ethylcellulose
dispersion stabilized by sodium lauryl sulfate and cetyl
alcohol (17). Plasticizers must be added to these
polymeric suspensions prior to their use. Plasticizers
provide flexibility for the coating polymer by lowering
its glass transition temperature, promoting film
coalescence, and making the polymer more susceptible to
deformation. Diethyl phthalate, dibutyl sebacate and
triethyl citrate are commonly used as plasticizers
(11,17,18).
The fluid bed process is increasingly popular for
coating of drug-loaded particles such as granules, beads,
and sugar spheres to control drug release rates (19,20).
Fluid-bed coating utilizes three basic approaches to spray
coating materials: top, tangential, or bottom spray
patterns (Wurster). The fluidized bed process using
bottom-spraying with a Wurster air-suspension column
provides the best conditions for coalescence of small
polymeric particles, coating efficiency, homogenous drug
disposition, and reproducible release characteristics
(12,13,19,21).
Drug release through polymeric films occurs not only
59
via diffusion, but also through pores in the polymeric
networks formed during coating process (22,23). Drug
release rate from a polymer coated system is sensitive to
various parameters such as coating thickness, coating
morphology, amount of additives and plasticizers,
physicochemical properties of drugs, and polymeric
components. Therefore, optimizing processing and
formulation parameters is required to ensure desired
release rates of drugs (21, 24-26).
The purpose of this study was to develop an oral
controlled release delivery system that releases MT over
an 8 hour period. The effect of sugar pellet size, coating
amount, and stirring rate of the dissolution basket on the
release rate of MT were characterized. The chemical
stability and release rate of MT from coated beads during
storage was also determined.
60
MATERIALS AND METHODS
Materials
MT was purchased from Regis Chemical Co.(Morton
Grove, IL). Core sugar spheres (USP/NF) as a substrate for
MT loading were from Paulaur Co. (Robbinsville, NJ).
Polyvinylpyrrolidone (PVP, average molecular weight
40,000, Lot # 05907CW) and hydroxypropylcellulose (HPC,
average molecular weight 300,000, Lot # 80253BP) were from
Aldrich Chemical Co. (Milwaukee, WI).
Polymeric
ethylcellulose suspension (Aquacoat': Type ECD-30, Lot #
J0211) was from FMC Corp.
(Philadelphia, Pennsylvania,
USA). Sebatic acid dibutyl ester (DBS, grade II, lot #
40H0279) was from Sigma Chem. Co.
(St. Louis, MO), and
triethyl citrate (TEC) was from Aldrich Chemical Co.
(Milwaukee, WI, USA) as plasticizers, respectively. 95%
ethanol (USP grade) was from Georgia-pacific Co.
Equipment
The apparatus for MT-loading onto sugar spheres and
applying the polymeric film coating consists of a
laboratory scale spray coater having a Wurster column
(2"x7", STREA -1, Aeromatic Inc., Columbia, MD) inside
clear plexiglass column mounted on a fluid-bed dryer (LabLine/P.R.L. Hi-Speed Fluid Bed Dryer, Lab-Line Instruments
Inc., Melrose Park, IL). A peristaltic pump is used to
61
deliver solutions to the spray nozzle. The entire
equipment is located in a ventilating hood. A cross
section of the STREA-1 SPRAY COATER is shown in Figure
111.1. Typical processing conditions for MT loading and
coating of MT-loaded beads is given in Table 111.1.
Preparation of MT-loaded Bead
The formulation for the production of MT loaded beads
is listed in Table 111.2. Polyvinylpyrrolidone (PVP) and
hydroxypropylcellulose (HPC) were used as binders
dissolved in 95% ethanol. MT was then dissolved in the
binder solution. 300 grams of core sugar spheres retained
on a either 18-20 or a 8-10 mesh screen were prewarmed in
a fluid-bed coating chamber at 40°C for about 15-30
minutes. The solution containing MT and binders was then
applied to the sugar spheres to produce MT-loaded beads
using a 0.8 mm nozzle with continuous fluidizing air
supply. The solution was delivered at 4 ml/min using a
peristaltic pump. After the solution was applied, MT beads
were further dried for 30 min in the coating chamber using
fluidizing air at 40°C.
Coating Process
Aqueous polymeric coating suspension was prepared to
provide rate controlling membranes. The coating
formulation for Aquacoatscoating of MT-loaded beads is
62
plexiglass column
Wurster colonn
pins with win.
nut screws
mesh base screen
tubing to
air compressor
fluid
mounting
Figure 111.1
Cross section of STREA-1 SPRAY COATER fully
assembled
63
Table III.1
Typical processing conditions for coating
of MT-loaded beads (STREA-1, Aeromatic)
Wurster insert
bottom spray
Nozzle size
0.8 mm
Inlet temperature'
50 °C
Atomization air
12-15 psi
Fluidization air blower
50-70% of full capacity
Flow rateb
1 ml/min and 4 ml/min
'40 °C was used for the preparation of MT-loaded beads.
biOnly 4 ml/min was used for the preparation of MT-loaded
beads. Peristaltic pump was manually switched between
"on" or "off" as necessary to control clumping of beads
during the coating process.
64
Table 111.2
Formulation for application of MT to
nonpareil sugar spheres.
MT
1.2 g
Sugar spheres
300 g
PVV"
0.24 g
HP&"
0.12 g
95% ethanol
200 ml
alpolyvinylpyrrolidone, Average Mw 40,000
bliydroxypropylcellulose, Average Mw 300,000
"Total binders, 30% (w/w) based on MT content.
65
listed in Table 111.3. Plasticizers (TEC and DBS) were
combined and added at 30% (15% each, based on the solid
content of Aquacoat.) to Aquacoat. suspension. Aquacoat'
suspension was then diluted with deionized water
(w /w,l:l). The coating solutions were stirred for at least
2 hours to ensure that plasticizers were well mixed with
the polymeric suspension prior to coating, and were
continuously stirred throughout the coating process.
Coating solutions were applied to 90 g of MT-loaded beads
to achieve the desired coatings with continuous air supply
at a rate of 1 ml/min for 10 minutes and then 4 ml/min
using a peristaltic pump. 31.4g, 62.7 g, or 125.4 g of
coating solution produces a 5%, 10% or 20% theoretical
coating based on solids content of Aquacoat'. If all
coating solids attach to the MT beads, some solids are
lost to the walls of the coating chamber which means all
beads will contain less than the theoretical amount of
coating, which has been retained as 5%, 10% or 20% coating
for convenience. The final coated beads were dried in the
chambers for 30 minutes and then further air dried in a
hood.
Assay for MT Content from Beads
About 2 g of MT-loaded or coated beads were ground
into a fine powder using a mortar and pestle. About 100 mg
of powder was weighed exactly and transferred to 1 ml of
66
Table 111.3
Coating formulations for Aquacoat. coating
of MT-loaded beads.
Aquacoat'
100 g
DBSb-
4.5 g
4.5 g
Water
100 g
`Aqueous ethylcellulose suspension containing 30 %
solids. Total solids per 100 g Aquacoat' = 30 grams.
tibutyl sebacate
`Triethyl citrate
"Total amount of plasticizers added was 30% based on
solids content of Aquacoat'.
67
95 % ethanol. The suspension was agitated and centrifuged
three times at 10,000 g for 10 min. The supernatant was
collected and diluted with deionized water so that the
expected MT concentration was within the range of the
standard calibration curve. MT concentration was
determined by HPLC.
In vitro Dissolution
In vitro dissolution of each formulation was
performed in triplicate using the USP dissolution
apparatus I (Basket method) at 37 ± 0.5 °C. The stirring
rate was 50 rpm except when the effect of the stirring
rate on the release rate of MT was evaluated. Dissolution
media for the first 2 hours was 900 ml of enzyme free
simulated gastric fluid (pH 1.4 ± 0.1) followed by enzyme
free simulated intestinal fluid (pH 7.4 ± 0.1).
Dissolution samples were collected at 0.25, 0.5, 0.75,
1.0, 1.5 and 2 hr (in gastric fluid), and 3, 4,
5,
6,
8,
12 and 24 hr (in intestinal fluid) with replacement of
equal volume with temperature equilibrated media. The
effect of sugar bead size, stirring rate of dissolution
basket, and theoretical coating amounts on the percentage
of MT released were studied. MT concentrations in
dissolution samples were determined by HPLC. The
percentage of MT released is the amount of MT released
divided by amount of MT loaded, multiplied by 100.
68
Stability of MT from Coated Beads
Uncoated and Aguacoate coated MT beads were placed in
polyethylene containers and stored at room temperature (24
± 2 °C)
in a laboratory drawer (dark) to evaluate their
stability. Samples were evaluated in duplicate at 0, 6 and
9 months. MT contents were determined according to the
procedure described previously in Chapter I. Dissolution
of MT from 20% coated beads (8-10 mesh) after 6 months
storage was tested to determine whether the dissolution
profile was the same as that of the initial coated beads.
69
RESULTS AND DISCUSSION
Evaluation of Manufacturing Process
The amount of MT assayed from beads is summarized in
Table 111.4. Because some MT is lost to the coating
chamber wall, supply tube, Wurster column, and through
exhaust from the chamber during processing, the content of
MT assayed was about 80% of total MT applied. As coating
amounts increased, the content of MT per gram of bead
decreased because MT-loaded beads gains more weight by the
coating process.
It is desirable to describe coating amount by
determining the coating thickness. Coating amount are
described as a function of weight gain (solids content) as
a matter of convenience. Table 111.5 shows estimated
coating amounts based on the weight gain of coating
materials onto beads (see Table V.5 footnote for method of
estimation). Weight gains of coated beads recovered was
less than expected because a certain amount of coating
materials may be lost initially to the coating chamber
wall, supply tube, Wurster column, and through exhaust
from the chamber. At the higher coating amounts, a smaller
fraction of coating material is lost. Fraction of coating
solids lost was higher at lower coatings than at higher
coatings. Estimated coating amounts using weight gain
revealed 22-88% of theoretical coat being deposited. This
70
Table 111.4
Amount of MT (mg per gram of bead)
assayed in beads
Size
Theoretical
% coating
Amount
(mg/g bead)
0
3.26±0.11
8-10
5
2.71±0.07
mesh
10
2.70±0.09
20
2.49±0.10
0
3.50±0.10
5
3.50±0.05
10
3.40±0.03
20
3.05±0.12
18-20
mesh
Table 111.5
Estimated coating amounts based on approximate weight gain of
beads (batch size: 90 g of MT-loaded beads).
Size (mesh)
Coating (%)'
bwt
1
lit 2
dWt
3
8-10
5
31.4
5.9
88.7
es,
fio
g__
(Estimated (%)
8--
10
62.7
11.7
95.1
0.56
4.36
18-20
20
5
125.4
23.4
105.5
0.33
13.4
31.4
5.9
94.5
0.23
3.85
10
62.7
11.7
99.8
0.16
8.40
20
125.4
23.4
110.7
0.11
17.6
'Theoretical coatings.
bTotal amount (g) of coating solution applied to 90 g MT-loaded beads.
`Total solids content (g) of coating solution applied. Aguacoat. contains 30%
solids. Total amounts of plasticizers added was 30% based on solids content of
Aquacoat%
dTotal weight of coated beads recovered after coating.
`Fraction of coating solids lost = 1- [Wt3-90)/Wt2
Estimated coating (%) = a x (1-e)
gNot available because weight of coated beads recovered was less than weight of
batch size (90 g).
Note: Less than complete recovery of coated beads occurrs which results in all
estimated % coating's being slightly underestimated since calculation assumed 90
gm of beads which is about 3-5 gm more than was actually recovered.
72
large deviation implies that estimating coating amounts
using weight gain is not accurate, especially at the low
coating level because weight loss for these small batches
is not accurately predictable during the coating process.
A direct measure of coating materials to determine coating
amount is desirable. Coating thickness measured by
scanning electron microscopy is an useful parameter to
describe coating thickness, but was not studied in the
current works.
On the other hand, fraction of coating materials
lost was less for the 18-20 mesh beads than for the 8-10
mesh beads. Coating efficiency based on weight gain was
greater for the 18-20 mesh beads. Since 18-20 mesh beads
have larger numbers of beads per unit batch size (90 g),
18-20 mesh beads have a larger surface area which results
in greater coating efficiency. In addition, it was noted
that 8-10 mesh beads readily crack and break in the
coating chamber compared to 18-20 mesh beads when
fluidized in the air. As a result, 18-20 mesh beads are
more efficiently coated than 8-10 mesh beads. However,
even though coating efficiency by weight gain is greater
for the 18-20 mesh beads, coat thickness is less because
of the greater surface area per unit weight, when compared
to 8-10 mesh beads. These results are supported by
dissolution analysis of coated beads showing the release
73
rate of MT was faster for the 18-20 mesh beads, which is
consistent with a thinner coat and larger surface area.
In vitro Dissolution
Release rates of drugs are affected by many
parameters. Factors affecting release rate of drug in the
development of controlled release delivery system by
fluidized-bed methods were reviewed in the literatures
(21,24-26). Several processing and formulation parameters
must be controlled to ensure the reproducibility of drug
release rates. In this study, the effects of stirring rate
of dissolution basket, size of core sugar spheres, and
coating amounts were evaluated on the dissolution profile
of MT from coated beads.
Dissolution profiles of MT from 18-20 mesh beads
coated with Aquacoate are shown in Figure 111.2. Most MT
was released in simulated gastric fluid, and the desired
controlled release was not achieved. A possible
interpretation of MT release from the coated beads is that
the coating may be incomplete and porous so that the drug
is readily released. The amounts of coating up to 20%
theoretical coating was not enough to produce the desired
controlled release. On ther other hand, dissolution
profiles of MT from 8-10 mesh beads coated with Aguacoat.
are shown in Figure 111.3.
A desired controlled release
pattern was observed over 8 hours at 20% coating. At 20%
74
100
7
80
100
80
60
60
40
40
20
.
20
0.0
0.5
1.0
1.5
2.0
12
15
18
21
24
1,,I,,I.,1,,t IiI,,t,It
3
6
9
Time (hrs)
Figure 111.2
Dissolution profiles of MT from 18-20 mesh
beads coated with Aguacoat% Coating
Amounts: Open circle, 5%; solid circle,
10%, and triangle, 20%. Each data point
represents the mean ± standard deviation
except where the standard deviation is too
small to show.
75
100
80
60
40
20
3
6
9
12
15
18
21
24
Time (hrs)
Figure 111.3
Dissolution profiles of MT from 8-10 mesh
beads coated with Aguacoat% Coating
amounts: Open circle, 5%; solid circle,
10%, and triangle, 20%. Each data point
represents the mean ± standard deviation
except where the standard deviation is too
small to show.
76
coating of 8-10 mesh beads, drug release is predominantly
governed by diffusion through the film barrier.
T50% (time to release 50% of drug release) of MT from
18-20 mesh and 8-10 mesh coated beads is compared in
Figure 111.4. As coating amounts are increased,
T50% of MT
is increased, as expected. T50% was about 4 hours for the
20 % coating of 8-10 mesh beads which provided the
controlled release over 8 hours.
Dissolution profiles of MT from 20% coated beads (810 mesh) as a function of stirring speed are compared in
Figure 111.5. No statistically significant change of MT
release rate was observed in 20% coated beads (8-10 mesh)
as stirring speed increased from 50 to 100 rpm. T50% was
essentially unchanged as a function of stirring rate
(Figure 111.6).
Stability of MT from Beads
The amount of MT determined per gram of beads and the
percentage of intact MT from coated beads stored at room
temperature for up to 9 months is given in Table 111.6. MT
was degraded relatively slowly during storage. Degradation
of MT may be related to transfer of heat and water through
the coated membranes (27,28). However, dissolution of MT
from the 20% Aguacoat° coated beads (8-10 mesh) after 6
months' storage produced a release of MT from stored beads
which was similar to the originally coated beads drug
77
5
o 8-10 mesh
18-20 mesh
s.
3
0
to
Ei
2
1
0
0
5
10
15
20
Coating Levels (%)
Figure 111.4
Time for 50% MT dissolution as a function
of coating amounts (theoretical weight
gain) in 18-20 and 8-10 mesh coated beads.
Each data point represents the mean ±
standard deviation except where the
standard deviation is too small to show.
25
78
100
80
60
Stirring Speed
50 rpm
77 rpm
40
v 100 rpm
20
It
0
0
3
1
It 'It lilt
6
9
12
I
It
15
I
I
18
I
11 til
21
24
Time
Figure 111.5
Dissolution profiles of MT from 8-10 mesh
coated beads with 20% coating as a function
of stirring speed. Each data point
represents the mean ± standard deviation
except where the standard deviation is too
small to show.
79
5
4
1
50
Figure 111.6
I
77
Stirring Rate (rpm)
1
100
Time for 50% MT dissolution as a function
of stirring speed. Each data point
represents the mean ± standard deviation
except where the standard deviation is too
small to show.
Amount of MT (mg per gram of bead) in coated beads
after storage at room temperature (24 ± 2 °C)
Table 111.6
size
Time (month)
Coating
(%)
0
6
9
0
3.26±0.11 2.91±0.07 (10.6)*
2.84±0.05 (12.9)
8-10
5
2.71±0.07 2.55±0.03
2.32±0.04 (14.1)
mesh
10
2.70±0.09 2.65±0.08 (4.41)
2.43±0.04
20
2.49±0.10 2.35±0.03
(5.48)
2.11±0.06 (15.3)
0
3.50+0.10 2.98±0.07
(14.8)
2.92±0.04
5
3.50+0.05 2.94±0.01 (16.1)
2.58±0.05 (26.4)
10
3.40+0.03 2.76±0.03
2.68±0.05
20
3.05±0.12 2.61±0.20 (14.4)
18-20
mesh
(5.90)
(18.8)
(9.76)
(16.5)
(21.3)
2.51±0.03 (17.7)
'Number in parenthesis indicates the percentage of MT degraded based on
initial amount of MT.
81
release (Figure 111.7). These data suggest that, although
MT in coated beads was slowly degraded, the wall coat
integrity was unchanged under the storage conditions.
82
0 T = 0
T = 6 naontias
100
80
60
40
20
0
0
3
6
9
12
15
18
21
24
Time (hrs)
Figure 111.7
Dissolution profiles of MT from 8-10 mesh
beads with 20 % coating after 6 months'
storage at room temperature. Each data
point represents the mean ± standard
deviation except where the standard
deviation is too small to show.
83
CONCLUSIONS
Based on weight gain, estimated coating amounts were
less than theoretical due to loss of coating materials
during the coating process. Larger beads (8-10 mesh) were
coated less efficiently than smaller beads (18-20 mesh).
The smaller beads gain more weight as coating materials
are sprayed but have a thinner coat because of larger
total surface area per unit batch.
Desired controlled release over 8 hours was not
observed with the 18-20 mesh coated beads at 5,
10 or 20%
coatings. However, 8-10 mesh beads overlaid with small
quantities of MT coated with 20% Aquacoat' showed the
desired controlled release over 8 hours. Stirring speed of
the dissolution basket at three different settings (50, 77
and 100 rpm) did not affect the release rate of MT from
the 20% coated beads (8-10 mesh). MT from coated beads was
degraded relatively slowly at room temperature but the
dissolution profile of MT from 20% coated beads (8-10
mesh) was unchanged after 6 months storage.
84
REFERENCES
1.
Lerner, A.B., Case, J.D., Heinzelman, R.V. Structure
of melatonin. J. Am. Chem. Soc., 81, 6084-6085
(1959).
2.
Waldhauser, F. Dietzel, M. Daily and annual rhythms
in human melatonin secretion: Role in puberty. Ann.,
New York Acad. Sci., 453, 205-214 (1985).
3.
Arendt, J., Aldhous, M., and Wright, J.
Synchronization of a disturbed sleep-wake cycle
in a blind man by melatonin treatment. The
Lancet, April 2, 773 (1988).
4.
Petterborg, L.J., Thalen, B.E., Kjellman, B.F., and
Wetterberg, L. Effects of melatonin replacement on
serum hormone rhythm in a patient lacking endogenous
melatonin. Brain Res. Bull., 27(2), 181-185 (1991).
5.
Cramer, H., Rudolph, J., Consbruch, U., and Kendel,
K. On the effects of melatonin on sleep and behavior
in man. Adv. in Biochem. Psychopharmacol., 11, 187191 (1974).
6.
Dahlitz, M., Alvarez, B., Vignau, J., English, J.,
Arendt, J., and Parkes, J.D. Delayed sleep phase
syndrome response to melatonin. Lancet, 337(8750),
1121-1124 (1991).
7.
Petrie, K., Conaglen, J.V., Thompson, L.,
Chamberlain, K. Effects of melatonin on jet lag after
Br. Med. J., 298, 705-707 (1989).
long haul flight.
8.
Waldhauser, F., Vierhapper, H., and Oirich, K.
Abnormal circadian melatonin secretion in night-shift
workers. The New Eng. J. Med., 7, 441-446 (1986).
9.
Rosenthal, N.E., Sack, D.A., Gillin, J.C., Lewy,
A.J., Goodwin, F.K., Davenport, Y., Mueller, P.S.,
et. al., Seasonal affective disorder. A description
of the syndrome and preliminary findings with light
therapy. Arch. Gen, Psychiatry, 41, 71-80 (1984).
10.
Sack, R.L., Lewy, A.J., Blood, M.L., Stevenson, J.,
and Keith, L.D. Melatonin administration to blind
people: Phase advances and entrainment. J. Biol.
Rhythm, 6(3), 249-262 (1991).
85
11.
Ghebre-sellassie, I., Iyer, U., Kubert, D., and
Fawzi, M.B. Characterization of a new waterbased coating for modified release preparations,
Pharm. Tech., Sep., 96-106 (1988).
12.
Mehta, A.M., and Jones, D.M. Coated pellets
under the microscope. Pharm. Tech., 52-60
(1985) .
13.
Jackson, I.M., Robert, S., Timmins, P., and Sen, H.
Comparison of laboratory-scale processing techniques
in the production of coated pellets. Pharm. Tech.,
Feb., 50-56 (1990).
14.
Rekhi, G.S., Mendes, R.W., Porter, S.C., and
Jambhekar, S.S. Aqueous polymeric dispersions
for controlled drug delivery- Wurster process.
Pharm. Tech., March, 112-125 (1989).
15.
Bodmeier, R., and Paeratakul, 0. Drug release
from laminated polymeric film prepared from
aqueous latexes. J. Pharm. Sci., 79(1), 32-36
(1990).
16.
Appel, L.E., and Zentner, G.M. Use of modified
ethylcellulose lattices for microporous coating
of osmotic tablets. Pharm. Res., 8(5), 600-604
(1991) .
17.
FMC Aquacoat. manual, aqueous polymeric dispersion
18.
Lyer, U., Hong, W.-H., Das, N. and GhebreSellassie, I. Comparative evaluation of three
organic solvent and dispersion-based
ethylcellulose coating formulations. Pharm.
Tech., 14(9) 68-86 (1990).
19.
Mehta, A.M., Valazza, M.J., and Abele, S.E.
Evaluation of fluid-bed processes for enteric
coating systems. Pharm. Tech., April, 46-56
(1986).
20.
Mehta, A.M. Scale-up considerations in the
fluid-bed process for controlled release
products, Pharm. Tech., Feb., 46-52 (1988).
21.
Variables that
Hossain, M. and Ayres, J.W.
influence coat integrity in a laboratory spray
coater. Pharm. Tech., Oct., 72-82 (1990).
86
22.
Higuchi, T. Mechanisms of sustained-action medicine:
Theoretical analysis of rate of release of solid
drugs dispersed in solid matrices. J. Pharm. Sci.,
50, 874-875 (1961).
23.
Zhang, G., Schwartz, J.B., and Schnaare, R.L.
Bead coating. I. Change in release kinetics (and
mechanism) due to coating levels. Pharm. Res.,
8(3), 331-335 (1991).
24.
Abdou, H.M. Dissolution, bioavailability and
bioequivalence. Mack Publishing Company, Easton, PA.
(1989).
25.
Lippold, B.H., Sutter, B.K., and Lippold, B.C.
Parameters controlling drug release from pellets
coated with aqueous ethyl cellulose dispersion.
Int. J. Pharm., 54, 15-25 (1989).
26.
Li, S.P., Mehta, G.N., Buehler, J.D., Grim, W.M., and
Harwood, R.J. The effect of film-coating additives on
the in vitro dissolution release rate of ethyl
cellulose-coated theophylline granules. Pharm.
March, 20-24 (1990).
27.
Banker, G.S., Core, A.Y., and Swarbrick, J. Water
vapor transmission properties of free polymer films.
J. Pharm. Pharmacol., 18, 457-466 (1966).
28.
Porter, S.C. The practical significance of the
permeability and mechanical properties of polymer
film used for the coating of pharmaceutical solid
dosage forms. Int. J. Pharm. & Prod. Mfr., 3(1), 2125 (1982).
87
CHAPTER IV
EVALUATION AND PHARMACOKINETICS OF AN ORAL CONTROLLED
RELEASE DELIVERY SYSTEM FOR MELATONIN IN HUMAN'SUBJECTS
ABSTRACT
An oral preparation containing melatonin (MT)loaded uncoated pellets for immediate release and coated
pellets for controlled release over 8 hours was
evaluated in six human subjects. The desired drug
release pattern was determined based on a computer
simulation program (MAXSIM.) using population average
pharmacokinetics of MT. When total 0.5 mg MT as low dose
(immediate release portion of MT, 0.1 mg) was
administered to four subjects, average peak plasma MT
concentration was reached at about 600 pg/ml and
maintained at about 100 pg/ml over 8 hours. Maximal
plasma MT concentrations were three times greater than
predicted MT concentrations by computer simulation. When
total 1 mg MT as high dose (immediate release portion of
MT, 0.2 mg) was administered to two subjects, average
peak plasma MT concentrations were doubled and terminal
elimination phase was parallell with low dose. Plasma MT
concentration versus time profiles were similar in shape
to MAXSIM. computer-simulated profiles. However, maximal
plasma MT concentrations produced were three times
88
higher than MT concentrations predicted by computer
simulation. Therefore, the results indicate that proper
dosing of immediate and controlled release MT are
required to produce physiological MT concentration
profiles. Input drug rate and cumulative amount of MT
absorbed were estimated by deconvolution analysis of
plasma MT concentrations. Deconvolution and
pharmacokinetic analysis revealed that less than 20% MT
doses administered were absorbed until 8 hour. A low
bioavailability may result from extensive first pass
metabolism and remaining amounts of MT from controlled
beads. A good correlation between plasma MT
concentration and urinary excretion rate of 6sulphatoxymelatonin (6-STMT), a major metabolite of MT
was observed. As plasma MT concentration increased,
urinary excretion rate of 6-STMT increased
concomittantly. Linear relationship between urinary
excretion rate of 6-STMT and plasma MT among subjects
was statistically significant (r = 0.75, F18 2.28,
p<0.001). If relationship between urinary 6-STMT and
plasma MT is recognized, urinary 6-STMT may be used as
an index of circadian rhythms of MT in humans.
89
INTRODUCTION
MT (N- acetyl -5- methoxytryptamine) is an indole
amide neurohormone secreted by the pineal gland in a
circadian fashion (1,2). MT concentration is very low
during the daytime (< 10 pg/ml). MT concentrations start
to rise in the late evening and are maintained at 25-120
pg/ml during the night (over 8 hours) until MT levels
return to the daytime baseline (2,3). This circadian
rhythms of MT may be largely affected by endogenous and
exogenous factors, called zeitgebers (3-5). The full
potential significance of introducing exogenous MT at
physiological and pharmacological concentrations is
still unknown (6,7). Exogenous MT may have clinical
potential as a circadian synchronizer to treat or
entrain circadian rhythm disorders including sleep
disorders (8-10), jet lag (11), shift work syndrome, and
seasonal affective diseases (12,13) in human subjects.
The pharmacokinetics of MT are controversial. MT is
extensively metabolized by a first pass effect and then
eliminated in the urine as a sulphate conjugate (14,15).
The half-life of MT reported is to be about 40 min
(16,17). Physicochemical properties of MT, disease
state, and nutritional state may affect pharmacokinetic
behavior of MT (17-21). Le Bars et. al., suggested that
sustained release dosage forms which possess a longer
90
biologically effective half life may be of interest to
evaluate clinical potential of MT by offsetting rapid
brain turnover or mimicking sustained nocturnal
secretion of melatonin (22).
The usual physiological nocturnal secretion over 8
hours coupled with the short half-life of MT prompted us
to develop a controlled release delivery system for MT
which mimics endogenous plasma MT concentration versus
time profiles. Although Strassman and colleagues have
used intravenous infusion to produce endogenous plasma
MT concentration profiles (23), lack of clinically
useful dosage forms may hinder evaluating the full
clinical potential of MT. Polymer coating technology has
been coupled with pharmacokinetic simulation to produce
an oral controlled release dosage form (24). Computer
simulation program aids to determine the dosing regime
of drug and predict plasma concentration versus time
profiles.
The purpose of this study was to determine whether
or not an oral controlled release drug delivery system
for MT produced plasma MT concentration versus time
profiles similar to endogenously produced MT profiles
in human subjects. An oral controlled release delivery
system consists of MT-loaded uncoated beads for
immediate release and MT-loaded coated beads with
aqueous ethylcellulose suspension (Aquacoat') for
91
controlled release in gelatin capsules. Deconvolution
and pharmacokinetics analysis were performed to analyze
plasma MT concentrations after oral administration of
the new dosage form to huam subjects. Correlation
between plasma MT concentrations and urinary excretion
rate of 6-STMT, a major metabolite of MT, was also
evaluated.
92
MATERIALS AND METHODS
Computer Simulations
Plasma MT concentrations after administration of an
oral controlled drug delivery system containing 0.1 mg
MT for immediate release and 0.4 mg MT for controlled
release over 8 hours were estimated by computer
simulation (MAXSIM', version 3.01, Uppsala, Sweden).
Pharmacokinetic parameters of MT for MAXSIM. computer
simulation were selected from the literature (Table
IV.1). Simulations were performed assuming an one
compartment open model.
In Vivo Study Protocol
Six human volunteers (5 males and 1 female)
participated in the study after giving informed consent
approved by Oregon Health Science University,
Instruction Review Board (Table IV.2). All subjects were
admitted to the Oregon Health Science University
Clinical Research Center at 10 o'clock, A.M. on the day
of the study. Subjects fasted at least 2 hours before
the study began. Gelatin capsules containing 0.1 mg MTloaded uncoated beads for immediate release and 0.4 mg
MT-loaded beads which were coated with 20% Aquacoat.
(aqueous polymeric suspension, courtesy of FMC Corp.,
Philadelphia, USA) for controlled release over 8 hours
Table IV.1
Selected pharmacokinetic parameters of MT for computer
simulation in human subjects.
Ka
(hr-1)
K,
(hr-1)
0.90
V,
13
( 1 )
Vd
( 1 )
F
35.2
References
Iguchi et al (17)
1.74
Waldhauser et al (18)
0.1
Lane et al (15)
94
Table IV.2
Subject No.
Vital statistics of six human subjects
Sex
Age
Weight
Height
(yrs)
(Kg)
(cm)
1
M
49
82
182.9
2
M
32
102
185.4
3
M
30
73
177.8
4
F
30
49
165.0
5
M
30
59
169.5
6
M
45
65
180.3
36 ± 9
71.7 ±18.7
176.7-1-8.1
Mean t S.D.
95
were administered to six subjects. Methods to produce
20% coated beads and amount of MT loaded per gram of
uncoated and coated beads are described previously in
Chapter III. Each capsule contained 0.1 mg of immediate
release MT on uncoated beads and 0.4 mg of controlled
release MT on 20% ethylcellulose coated beads designated
as low dose (0.5 mg of total dose of MT). Thus, 31 mg of
uncoated beads and 161 mg of 20% coated beads (total
weight: 192 mg) provides 0.1 mg of immediate release MT
and 0.4 mg of controlled release MT, respectively. Two
subjects were also given two capsules of this
formulation designated as high dose (1.0 mg total dose
of MT)
.
Blood samples were collected through an indwelling
intravenous catheter at 0, 0.5, 1, 1.5, 2, 3, 4, 6, and
8 hours and kept in heparinized test tube in the freezer
until analysis. Urine was collected every two hours for
determination of 6-STMT, a major metabolite of MT. Blood
and urine samples were collected before administration
of the oral controlled delivery system to provide
baseline values with regard to the daytime concentration
of MT and 6-STMT. Sampling schemes for blood and urine
in the six human subjects are provided in Table IV.3.
Plasma MT concentrations were determined by high
sensitivity GC/MS (25). Urinary 6-STMT concentration
were determined by radioimmunoassay (26).
96
Table IV.3
Sampling scheme for blood and urine
collection in six human subjects.
Time (hrs)
Clock
Blood
Urine
0
10:30
Yes
Yes
0.5
11:00
Yes
No
1
11:30
Yes
No
1.5
Noon
Yes
No
2
12:30
Yes
Yes
3
13:30
Yes
No
4
14:30
Yes
Yes
6
16:30
Yes
Yes
8
18:30
Yes
Yes
97
Deconvolution and Pharmacokinetic Evaluation
Input rates (µg /hr) and cumulative amounts of MT
absorbed were estimated using an interactive
deconvolution program, PCDCON (version 1.0, courtesy of
Dr. Gillespie, College of Pharmacy, University of
Texas). Plasma MT data following an intravenous bolus of
MT by Iguchi et. al., were used for impulse response
specification of MT (17). Plasma MT concentrations
obtained after administration of the oral controlled
release delivery system was used for the input response
specification of MT.
Noncompartmental pharmacokinetic parameters (27)
for MT including AUC (area under the drug concentration-
time curve), AUMC (area under the moment curve) and MRT
(mean residence time, AUC/AUMC) were obtained RSTRIP II
(Micromath Scientific Software, Salt Lake City, UT).
This program employs a least square minimization
procedure based on a modification of Powell's algorithm.
"Estimated" bioavailability (FEsT) of the oral controlled
release delivery system containing MT was estimated from
AUC in this study (AUC
)
divided by AUC from a
different study (AUCIV) followed by intravenous injection
of MT by Iguchi et. al. (17). "Relative" bioavailability
(Fm) of the oral controlled release delivery system was
estimated from AUC in this study (AUCmm) divided by AUC
from a different study (AUCro) followed by oral
98
administration of immediate release MT capsule by
Waldhauser et. al. (18). The equations used are as
follows.
AUCDDS
FEST =
)
DDDS
F REL =
where Dims, Div, and
k
AUCIV )
DIV
AUCDDS )
AUC po )
DDDS
PO
Dm indicate MT doses for the new
oral controlled release in the study, and intravenous
injection, and oral immediate release administration in
a different study, respectively.
Statistical Analysis
Regression analysis was used to calculate slope,
correlation coefficient (r), and coefficient of
determination (r2). The aptness of the linear models was
evaluated by comparing F-test for lack of fit (28). The
estimators of the linear regression parameters are
obtained according to the methods of least squares
minimization.
99
RESULTS AND DISCUSSION
Computer Simulation
Before administering the oral controlled release
delivery system to human subjects, plasma MT
concentrations were predicted from dissolution data of
dosage form and population pharmacokinetic parameters of
MT using computer simulation (MAXSIM.). An ideal dosage
form would deliver MT to mimic the endogenous circadian
pattern of MT. Computer-simulated plasma MT
concentration versus time profile in human subjects for
the new dosage form is shown in Figure IV.1. A
conventional (immediate release) MT capsule (curve 1)
does not produce the desired sustained drug
concentration because MT has a short half life of MT
(16,17,19). Curve 2 shows that a mixture of uncoated
beads containing 0.1 mg MT for immediate release and
beads coated with 20% Aquacoate containing 0.4 mg MT for
controlled release are predicted to produce a plasma MT
concentration versus time profile which mimics the
profile known to be produced by endogenous release of MT
during the night.
Deconvolution and Pharmacokinetic Evaluation
The study was conducted during the day. Because
normal daytime concentration of MT is below 10-30 pg/ml,
100
900
IMelatonin Simulation
I
800
700
01
600
;
2500
w
400
E
.-42
300
200
100
Figure IV.1
Computer-simulated plasma MT concentrations
from different dosage forms in human
subjects. An 0.5 mg immediate release MT in
curve 1 and an oral controlled release
delivery system containing 0.1 mg immediate
release MT from uncoated beads and 0.4 mg
controlled release MT from 20% Aquacoat.-
coated beads, over 8 hours (total dose =
0.5 mg) in curve 2.
101
the contribution of endogenous MT to exogenously
produced MT concentration is negligible. Observed mean
plasma MT concentration versus time profile after
administration of the oral controlled release delivery
system to human subjects is shown in Figure IV.2.
When low dose of MT was administered to four subjects,
average peak plasma MT concentration was reached at
about 600 pg/ml and maintained around 100 pg /mi over 8
hours. When high dose of MT was administered to two
subjects, average peak plasma MT concentrations were
doubled, as expected and terminal slope was parallel
with low dose. The shape of the plasma MT concentration
versus time profile was similar for the two different
doses. Peak plasma MT reached about 600 pg/ml as a
result of immediate release of MT and extended MT
concentrations over 8 hours result from the coated
beads. As predicted by computer simulation previously in
Figure IV.1, Figure IV.3 shows that the delivery system
produces a plasma MT concentration versus time profile
which is in shape similar to that produced at night as a
result of endogenous MT release (2). However, maximal
plasma MT concentrations produced were three times
higher than MT concentrations predicted by computer
simulation. These differences primarily resulted from
the fraction of bioavailability (F=0.1) selected for
computer simulation and intrinsic metabolic function
102
0 Low Dose (0.5 mg)
High Dose (1 mg)
I,I,1 t III
4
3
1
5
6
t
7
1
8
,
1
(1
10
9
postdose (hrs)
12
14
16
18
20
Time of the Day
Figure IV.2
Mean plasma MT concentration versus time
profiles after administration of controlled
release delivery system to six human
subjects at two different doses. Values are
expressed as mean ± standard deviation (n=4
for low dose, n=2 for high dose).
103
1000
0 Low Dose (0.5 mg)
A 800
MAXSI1L Simulation
tv
04
0 600
0
0
.4..$
,as
i
400
ei
0
rn
200
ccs
rai
2
1
12
Figure IV.3
3
4 5
6
7 8
postdose (hrs)
14
16
18
Time of the Day
10
9
20
12
11
22
Comparison of the profiles of observed
plasma MT concentrations (mean ± standard
deviation, n=4) and computer-simulated
plasma MT concentration at low dose.
104
among subjects. Therefore, proper dosing regime based on
the observed plasma MT concentration is required to
produce physiological MT concentration profiles.
The rate and extent of in vivo drug release was
obtained by deconvolving the plasma concentration versus
time profile. Estimated MT input rate after
administration of the delivery system is shown in Figure
IV.4. The profile of estimated input rate was coincident
with that of observed plasma MT concentrations versus
time. The initial sharp increase of input rate results
from the immediate release MT. The slow declining curve
indicates input of MT from the controlled release beads.
As expected, the high dose of MT provided a larger input
rate than the lower dose. Estimated cumulative amount of
MT input after oral administration of the controlled
delivery system is also shown in Figure IV.5. The input
slope was steeper during the first 2 hours, followed by
slow cumulative input until 8 hour studied. The
cumulative amounts input at the low and high dose were
about 0.10 mg and 0.15 mg, respectively. This suggests
that only 15-20% of administered dose of MT is absorbed.
Noncompartmental pharmacokinetic parameters
obtained using RSTRIP II program are summarized in Table
IV.4. Ratio of area under the curve (AUC) and area under
the moment curve (AUMC) gives mean residence time (MRT).
MRT has been defined as the mean time for intact drug to
105
150 n-
120
Low Dose (0.5 mg)
High Dose (1 mg)
90
60
30
0
0
2
4
6
8
Time (hrs)
Figure IV.4
Estimated MT input rate (µg /hr) by
deconvolution analysis resulting from an
oral controlled release delivery system at
two different doses of MT.
106
200
Low Dose (0.5 mg)
- High Dose (1 mg)
eo
150
a.
2 100
-3
a'
50
0'
2
4
6
8
rime (h s)
Figure IV.5
Estimated cumulative amount input (Mg) of
MT by deconvolution analysis resulting from
an oral controlled release delivery system
at two different doses of MT.
Noncompartmental pharmacokinetic parameters for MT following
Table IV.4
administration of the oral controlled release delivery system to human
subjects.
Low Dose (0.5 mg)
Values
1
Slope
(hr-1)`
2
3
Mean ± S.D.'
4
0.505 0.089 0.278 0.162
C.V.°
( %)
High Dose (1 mg)
5
Mean ± S.D.
6
0.259 ± 0.18
70.3
0.345 0.179
c.v.b
(%)
0.262 ± 0.117
44.8
AUCd
3729
2127
2063
1538
2364 ± 947
40.1
4742
3820
4281 ± 652
15.2
AUMC°
13205 5202
4985
4503
6974 ± 4164
59.7
10966
19812
15389 ± 6255
40.6
MRTt
3.54
2.42
2.93
2.83 ± 0.53'
18.7
2.32
5.18
3.75 ± 2.02
53.9
C,"
774.8 744.4 628.2 456.7
651.0 ± 144.1
22.1
1666.2
1178 ± 690.4
58.6
Tm.,°
1.77
0.72
1.21
0.58
1.07 ± 0.54
50.4
0.72
0.45
0.59 ± 0.19
32.6
F.,,i
0.30
0.17
0.16
0.13
0.19 ± 0.07
39.6
0.19
0.15
0.17 ± 0.03
16.6
Fd
1.28
0.73
0.71
0.53
0.82 ± 0.32
39.9
0.82
0.66
0.74 ± 0.11
15.3
2.44
689.8
'Standard deviation.
°Coefficient of variation, C.V.(%)= S.D./mean *100
`Apparent slope of terminal phase by least square regression.
'Area under the curve (pg-hr/ml).
`Area under the moment curve (pg- hr2 /ml).
'Mean residence time (hr), MRT=AUC/AUMC
tobserved maxsimum plasma MT concentration (pg/ml).
'Time to reach maxsimum plasma MT concentration (hr).
°Absolute bioavailability based on the I.V. data of MT in different subjects (17), AUC,= 247.6 pghr/ml, Dose=10 pg.
'Relative bioavailability based on the oral data of MT in different subjects (18), AUC po= 464480 pghr/ml, Dose=80 mg (18)
jMRT for I.V. data was 0.71 hr when data of Iguchi et al was analyzied by RSTRIP II (17).
108
transit through the body (27). The apparent terminal
slopes were prolonged as would be expected for a
controlled release preparations. MRT calculated from the
intravenous data of Iguchi et al (17) from a different
study was 0.71 hours. The new oral dosage form produced
a MRT of 5 times longer compared to the intravenous
injection of MT. Observed MRT for the new oral dosage
form ranges from 2.4 to 5 hours. "Estimated"
bioavailability (FasT) obtained was about 19% and 18% for
low and high MT dose, respectively. These results were
very consistent with fraction of the cumulative amount
input of MT estimated by deconvolution method in Figure
IV.5. The "relative" bioavailability compared to oral
immediate release MT from a different study by
Waldhauser et. al., was about 82% and 74% for low and
high dose MT, respectively.
A low bioavailability may result from extensive
first pass metabolism and remaining amounts of MT from
controlled beads. The main metabolite of MT excreted in
urine is 6-STMT (14,15). Extensive first pass metabolism
may be involved in the apparent low fraction of dose
absorbed. This may suggest that plasma MT profiles are
greatly influenced by the liver function. Iguchi et.
al., found that patients with liver cirrhosis have a
prolonged metabolic clearance of MT, maybe due to a
decrease in liver flow, lowered activity of 60-
109
hydroxylase, or competition with bilirubin in the
intrahepatic transport system (17).
It is interesting to correlate urinary excretion
rate of 6-STMT with plasma MT because MT is extensively
metabolized by the liver and excreted as 6-STMT. Urinary
excretion rate of 6-STMT was plotted as a function of
both postdose and time of the day (Figure IV.6). Urinary
excretion rates of 6-STMT during the daytime in subjects
not receiving MT was very low (<200 ng/hr). When the
oral controlled release delivery system of MT was
administered, the urinary excretion rate of 6-STMT
greatly increased compared to the control urine. The
terminal slopes of urinary excretion rate profiles of 6STMT were parallel for the two different doses.
A correlation between plasma MT and urinary 6-STMT
has been reported elsewhere (29,30). If relationship
between plasma MT concentration and urinary excretion
rate of 6-STMT is well recognized, urinary 6-STMT can be
used as an non-invasive method to assess circadian
rhythms of MT in humans. The plasma MT concentration and
urinary excretion rate profiles of 6-STMT as a function
of time at low dose and high dose MT are compared in
Figure IV.7 and Figure IV.8, respectively. The profiles
of plasma MT and urinary excretion rates of 6-STMT are
parallel at each dose. As expected, as plasma MT
concentration increased, urinary excretion rate of 6-
110
High Dose (1mg)
O Low Dose (0.5mg)
Control Urine
#74%
to
50000
E-4
C/3
to
10000
s..4
0
Q)
Qi
C4
.9
100
U
cr3
t,
10
c.
0
2
1
4
3
5
8
6
Postdose (hrs)
12
Figure IV.6
14
16
Time of the Day
18
10
9
20
Urinary excretion rate (ng/hr) of 6-STMT
determined at the midpoints of each urine
collection interval after administration of
the oral controlled release delivery system
to six human subjects. Values are expressed
as mean ± standard deviation (n=4 for low
dose, n=2 for high dose).
111
50000 '-0
cs
1000
30000
tO
04
a0
10000 47
7000
100
/5000
700
70'
0
10
500
Low Dose (0.5 tag)
rn
czi
tIlltItIt I tItli I
a,
0
2
1
3
4
1.111
5
6
7
8
300
10
9
Postdose (hrs)
12
14
16
18
20
Time of the Day
Figure IV.7
Comparison of the profiles of plasma MT
concentration and urinary excretion rate of
6-STMT determined at the midpoints of each
urine collection interval at the low dose
of MT (0.5 mg) in human subjects. Values
are expressed as mean ± standard deviation
(n=4).
112
50000
II
30000
0 1000
V)
c0
.51
0
al
al
10000
at
4.3
1:4
100
500
al
High Dose (1 mg)
5
M
rn
O
10
o
ti
1
.1
I
,
1
,
1
.1
,
a
0
II
0
1
.1.
300
4-4
0
2
1
3
4
5
6
8
7
10
9
Postdose (hrs)
12
Figure IV.8
16
14
Time of the Day
18
20
Comparison of the profiles of plasma MT
concentration and urinary excretion rate of
6-STMT determined at the midpoints of each
urine collection interval at the high dose
of MT (1.0 mg) in human subjects. Values
are expressed as mean ± standard deviation
(n=2).
113
STMT also increased concomitantly. Urinary 6-STMT
appears to reflect behavior of plasma MT concentration.
Cumulative amounts of urinary 6-STMT for 6 hours were
compared at two different doses of MT (Figure IV.9). As
MT dose was doubled, urinary 6-STMT was also twice
increased. The difference was statistically significant.
Figure IV.10 shows the relationship between urinary
excretion rate of 6-STMT and plasma MT concentration in
six human subjects. The linear relationship between
urinary excretion rate of 6-STMT and plasma MT
concentration was highly significant (F 1, 18 = 2.28,
r2=0.56, p <0.001). Furthermore, the linear relationship
was much greater when only past peak plasma MT
concentrations were correlated (r=0.838).
Although observed plasma MT concentration versus
time profiles were similar in shape to the computer
predicted MT concentration curves, maximal plasma MT
concentrations produced were three times higher than MT
concentrations predicted by computer simulation at the
low dose MT assuming 10% bioavailability. Therefore,
computer simulation curves with appropriate dosing
adjustments are regenerated in Figure IV.11. Total dose
of MT administered and ratio of immediate release MT and
controlled release MT were varied. Fraction of drug
absorbed (F) was obtained based on the observed plasma
MT concentration using pharmacokinetic analysis. In
114
0.3
0.2
0.1
0.0
Figure IV.9
Low Dose
High Dose
Cumulative amount of urinary 6-STMT (mg)
for 6 hours at two different doses of MT.
Values are expressed as mean ± standard
deviation (n=4 for low dose, n=2 for high
dose).
115
70000
R = .747
40
p 60000
EEco
50000
0
40000
0 Subject 1
30000
Subject 2
0
20000
7 Subject 3
Subject 4
Subject 5
Subject 6
10000
400
800
1200
1600
Plasma Melatonin (pg/m1)
Figure IV.10
Relationship between plasma MT
concentrations and urinary excretion rates
of 6-STMT determined at the midpoints of
each urine collection interval in six human
subjects.
116
O
O
500
----=
E
---...
a,
0.
g 400
7-,
s_'''
4.,
4.C
2 300
0
1
-
(..)
C
oc
'
'''"
200
4J
5
E'C'
tra
cr:
0
100
50
T
0
Figure IV.11
2.0
,
6.0
4.0
Time (hrs)
B.0
Computer-simulated plasma MT concentration
versus time profiles at various dosing of
MT. Total doses and percentage of immediate
release portion are as follows: Curve 1,
0.8 mg, 10%; Curve 2, 0.5 mg, 20%; Curve 3,
0.4 mg, 12.5%; Curve 4, 0.4 mg, 10%; Curve
5, 0.2 mg, 10%. Open circles indicate
observed plasma MT concentrations at low
dose MT studied (0.5 mg, 20%). Ke1 =0.9 hr 1,
K,=1.74 hr-I, Vd=35.2 1, and F =0.3 for
immediate release MT and F=0.1 for
controlled release MT.
117
other words, the simulated curve 2 approximated observed
plasma MT concentrations (open circle) in the study at
low dose MT when F=0.3 for immediate release MT and
F=0.1 for controlled release MT for computer simulation
were utilized. Five curves at various doses of MT were
thereafter simulated. This simulation suggests that a
total 0.2 mg MT dose (curve 5) containing 10% immediate
release is desirable to approximate a nighttime
endogenous plasma MT concentration versus time profile.
Inherent is the assumption that F is larger for
immediate release MT than controlled release MT due to a
decreased first pass effect when the drug is rapidly
absorbed. This assumption certainly needs confirmation
prior to acceptance. Another evaluation of an oral
controlled release delivery system for MT in both young
and elderly people is under investigation.
118
CONCLUSIONS
An oral preparation designed to release 0.1 mg MT
immediately from uncoated beads, and 0.4 mg MT from
coated beads in a controlled release fashion over 8
hours produced about 600 pg/ml average peak plasma MT
concentration, and then maintained MT concentrations at
about 100 pg/ml. Plasma MT concentration versus time
profile was very similar in shape to the computersimulated profile. However, maximal plasma MT
concentrations were about three times greater than
predicted. The input rate and cumulative amount of MT
were estimated using plasma MT concentrations and a
deconvolution program. Deconvolution and pharmacokinetic
analysis revealed that less than 20% of the MT dose
administered was absorbed. A low bioavailability
observed may result from extensive first pass metabolism
and incomplete absorption from coated beads. Urinary
excretion rate of 6- suiphatoxymelatonin (6-STMT) profile
was closely related with plasma MT concentration.
Urinary 6-STMT may be used as non-invasive method to
assess pineal gland activity of MT in humans.
119
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variance and experimental design, 2nd edition.
Richard D. Irwin, Inc., Homewood, IL (1985).
29. Brown, G. M., Bar-Or, A., Grossi, D., Kashur, S.,
Johannson, E., and Yie, S.M. Urinary 6sulphatoxymelatonin, an index of pineal function in
the rat. J. Pineal Res., 10, 141-147 (1991).
30. Nowak, R., Mcmillen, I.C., Redman, J., and Short,
R.V. The correlation between serum and salivary
melatonin concentration and urinary 6hydroxymelatonin sulphate excretion rate: Two noninvasive techniques for monitoring human circadian
rhythmicity. Clin. Endocrinol., 27, 445-452 (1987).
122
CHAPTER V
IN VITRO DIFFUSION STUDIES OF MELATONIN
THROUGH HAIRLESS MOUSE SKIN AND SYNTHETIC MEMBRANES
ABSTRACT
The effects of vehicles on the diffusion rate of
melatonin (MT) through excised hairless mouse skin and two
synthetic membranes (ethylene vinyl acetate and
microporous polyethylene) using vertical Franz. diffusion
cell was evaluated. Flux (gg/hr/cm2) of MT were dependent
on the type of membrane and the formulation vehicles. Flux
of MT was greatest in excised hairless mouse skin followed
by ethylene vinyl acetate membrane and microporous
polyethylene membrane. Flux of MT from vehicle
formulations through excised hairless mouse skin in
increasing order was: 40% (v/v) of propylene glycol
(PG)/30% (w/v) of 2-hydroxypropy1-0-cyclodextrin (2-HPCD)
> 30% 2-HPCD > 40% PG > 20% PG > buffer. Flux of MT
through excised hairless mouse skin was proportional to MT
solubility in vehicles and the relationship was
statistically significant (F13= 10.87, p < 0.05). A lag
time of MT from PG 40% vehicle was the shortest through
excised hairless mouse skin. Flux of MT was slightly
changed as hydration time (<1.5 hours) prior to start of a
diffusion experiment increased but no correlation was
123
observed. Flux of MT from four vehicles through ethylene
vinyl acetate membrane was 5-20 times lower than those
through excised hairless mouse skin. Flux of MT through
ethylene vinyl acetate membrane in increasing order was:
buffer > PG 40% > 2-HPCD 30 %> PG 40%/2-HPCD 30%. Unlike
the excised hairless mouse skin, flux of MT through
ethylene vinyl acetate membrane decreased as MT solubility
increased. These contradictory results need to be
explained by other mechanisms rather than solubility only.
No MT was detected over 12 hours after starting diffusion
through microporous polyethylene membrane. PG and 2-HPCD
appears to be ineffective for the penetration of MT
through synthetic membranes. Interestingly, for the
microporous polyethylene membrane, volume of donor phase
increased to 2.5 times the initial applied volume (1.5
m1). This indicates that water flows from receptor to
donor phase. Flux of MT through two synthetic membranes
were so low or negligible that it could not be used as a
rate controlled membrane. On the other hand, flux of MT
through the excised hairless mouse skin needs to be
optimized.
124
INTRODUCTION
Melatonin (MT) is a neurohormone secreted by the
pineal gland in a circadian rhythm (1). Exogenous MT may
be effective in resetting circadian rhythm disorders of MT
(2,3) such as sleep disorder (4,5), jet-lag (6), shift
work syndrome (7), and seasonal affective disease (8) in
humans. However, useful MT treatment may depend on dosage
forms that mimic the endogenous circadian rhythms of MT.
No appropriate dosage forms are currently available.
Transdermal delivery is one of promising routes to
deliver drug molecules for a desired length of time (911). Factors related to skin penetration by drugs includes
the physicochemical properties of drugs, partition
coefficient, hydration of skin, age, and presence or
absence of penetration enhancers (9,13-18).
Long lag times, small permeability coefficient, and
low solubility may be a physical obstacle to transdermal
delivery (12). Solubility is a common and measurable index
which is related to the diffusional behavior of drugs.
Development of a transdermal delivery system for MT may be
limited because MT has a relatively low solubility (<1.5
mg/ml) in water (19). The poor permeability of most drugs
through the skin has led to use of a variety of
transdermal penetration enhancers which may increase the
thermodynamic activity of the drug and change the barrier
125
function of the stratum corneum so as to improve drug
permeability (16-18).
Propylene glycol (PG) has been widely used as a
cosolvent or penetration enhancer in transdermal delivery
systems (16-18,20). 2- hydroxypropy1- 3- cyclodextrin (2-
HPCD) is also widely used in the pharmaceutical industry
because of its ability to improve the solubility,
stability, and bioavailability of many drugs (21-23). It
has been reported that 2-HPCD improved the topical and
transdermal absorption of 4-biphenyl acetic acid (24) and
17 /3- estradiol (25). Pfister et. al., suggested 2-HPCD as a
penetration enhancer compatible with silicone medical
adhesives (17). However, 2-HPCD has not been extensively
studied in a transdermal delivery system. Therefore it is
of interest to incorporate these additives into vehicles
and to determine if penetration of MT through skin and
model membranes is enhanced.
It is desirable to conduct in vitro transdermal
delivery experiments with human skin. However,
alternatives to human skin may be considered because human
skin is, for many good reasons, not readily available. Of
course there is no animal skin that completely mimics the
penetration characteristics of human skin (9,11). The
majority of in vitro experiments reported have been
conducted using hairless mouse skin due to its
similarities to human skin, and ready availability.
126
Inconsistent results have been reported with both animal
and human skin (26,27). Many polymeric membranes have also
been used to control or evaluate drug diffusion and
develop transdermal delivery devices (11,28). Diffusion of
drugs through rate controlling membranes is dependent on
membrane type employed, thickness, pore size, and membrane
composition (17,28).
Partition and diffusion of MT from PG solution
through excised hairless mouse skin was previously studied
in our laboratory (29). The purpose of this research was
to extensively evaluate diffusional behavior of MT through
excised hairless mouse skin and two synthetic membranes
(ethylene vinyl acetate and microporous polyethylene).
Donor vehicles for these studies included buffer, PG
and/or 2-HPCD.
127
MATERIALS AND METHODS
Materials
Melatonin (MT) was purchased from Regis Chemical Co
(Morton Grove, IL). Ten week old, male hairless mice
(HRS/J strain) were used in the study. Propylene glycol
(PG) was obtained from J.T. Baker, Inc., (Phillisburg,
NJ). 2-hydroxypropy1-0-cyclodextrin (2-HPCD; molecular
weight, 1542; average degree of molar substitution, 6.5
moles of hydroxypropyl/mole of 0-cyclodextrin; range of
molar substitution, 5-8) was provided courtesy of American
Maize-Product company (Hammond, ID). Ethylene vinyl
acetate (3M CoTranTlm controlled caliper film #9702) and
microporous polyethylene (3M CoTranTm microporous film
#9710) were provided courtesy of 3M company (St. Paul.
MN). All chemicals used were of reagent grade. Deionized
water was used throughout the study.
Equilibrium Solubility of MT
Excess amounts of MT were added to 0.05 M phosphate
buffer (pH 6.1) containing various concentrations of PG
and 2-HPCD. The resulting mixtures were placed in a 25±0.1
°C constant temperature water bath. Parafilm was used to
cover the top to prevent evaporation. Equilibrium
solubility was reached within 24 hours. Samples were
centrifuged at 10,000 g for 15 minutes and the clear
128
supernatant was collected and diluted with phosphate
buffer. HPLC analysis of MT concentration was carried out
as described in Chapter I.
Skin Sources and Preparation
The mice were housed individually at the College of
Pharmacy, Oregon State University. The mice were
sacrificed by exposure to CO2. Full thickness abdominal
skin samples were excised and kept in 0.9 % physiological
NaC1 equilibrated at 30 °C prior to mounting on the
diffusion cell.
Diffusion Studies
Skin and synthetic membrane samples were mounted
between the donor and receptor phase in vertical Franz'
diffusion cells as shown in Figure V.1. The crosssectional area and diameter for diffusion was 3.14 cm2 and
2 cm, respectively. The temperature was maintained at 30
°C (approximate skin temperature) with a circulating water
bath during the entire experiment. Normal saline solution
was used as receptor media. One and a half mililiter (1.5
ml) of vehicle solution containing saturated MT was
applied to donor chamber using a pipette. The saturated MT
concentration in vehicles was determined based on
solubility studies. Stirring of the media within the
receptor chamber was achieved by externally driven
129
Open to Air
Saline Solution
Injection Port
Cell Cap (Donor)
. Thin Finite Dose
Isotonic Saline
Solution Chamber
Skin
Water Out
Water Jacket
Cell Body (Receptor)
Water In
Figure V.1
Stirring Bar
Scheme of a vertical Franz. diffusion cell.
130
magnetic stirring bars. 150 ul of samples from the
receptor chamber were collected using a pipette attached
with teflon tube through the sampling port of the
diffusion cell at 0.5, 1, 1.5, 2, 2.5, 3.5, 4.5, 6, 9, and
12 hours. The receptor phase was replenished with an equal
volume of saline solution after each sample were
collected. Sink conditions were maintained in the
receptor. Analysis of each subsequent sample was corrected
for all previous samples (150 Al) that had been removed
for analysis.
until analysis.
The samples were stored in the freezer
Samples were centrifuged at 7000 g for 10
min to remove any insoluble residue before HPLC analysis.
MT concentration was determined by HPLC as described in
Chapter I.
Data Analysis
The cumulative amount of MT (Ag) permeating through
excised hairless mouse skin or other membranes during each
sampling interval was calculated based on the MT
concentration and volume in the receptor phase. The flux
(µg /hr /cm2) and lag time (hr) of MT release from each
vehicle were calculated from the slope and the intercept
of the plot of cumulative amount of MT (Mg) in the
receptor phase at steady state against time using linear
regression analysis (29). Diffusional area was 3.14 cm2.
Permeability coefficient (P) was obtained by dividing
131
the flux (J) by corresponding solubility of MT (C)
in
vehicles based on the following equation (12).
J =KDC/h=PC
The amount of drug diffused through a unit area of
membrane per unit time (J)
is the product of the diffusion
coefficient (D), skin-vehicle partition coefficient (K),
and the drug solubility in the vehicle or delivery system
(C) divided by the thickness of the skin (h). The above
equation is commonly expressed as product of permeability
coefficient (P) and drug solubility (C) if there is an
excess supply of drug in the delivery system since D, K
and h are experimentally entangled.
All values are expressed as mean ± standard deviation
of at least three determinations. Statistical
significances in flux among vehicles were evaluated using
Tukey's method of multiple comparison (29). The aptness of
the linear models was evaluated by comparing F-test for
lack of fit.
132
RESULTS AND DISCUSSION
While transdermal delivery has several potential
advantages by delivering drug over a period of desired
time, and avoiding first pass metabolism, it requires an
understanding of both functions of skin and drugs (10,12).
Drugs with reasonable potency, oil/water partitioning
coefficient, and solubility can be a good candidate for
transdermal delivery (10,12,16). Parameters which affect
transdermal delivery of drugs must be investigated to
optimize flux of drug, and to finally fabricate
transdermal delivery device.
In this study, vehicles which affect MT solubility
were utilized to increase MT flux through the membranes.
MT solubility obtained in various vehicles is shown in
Table V.I. MT solubility in PG and/or 2-HPCD solution
markedly increased compared with buffer solution. The
profiles of cumulative amount of MT released vs time
through excised hairless mouse skin is shown in Figure
V.2. Flux, permeability coefficient (P), and lag time of
MT calculated are also given in Table V.2. Flux of MT
through excised hairless mouse skin was increased as MT
solubility increased. Flux of MT from PG 40% with 2-HPCD
30% was the largest and statistically different (p<0.05)
compared to buffer and PG 20% (Figure V.3). There was no
statistically significant difference for MT flux between
133
Table V.1 MT solubility in various vehicles
Vehicles
Solubility' (mg/ml)
Buffer
1.507
PG 20% (v/v)
2.817
PG 40% (v/v)
9.587
2-HPCD 30% (w/v)
18.151
PG 40% (v/v)/2-HPCD 30% (w/v)
20.408
"n=3, Mw=232.3
134
o
12
Buffer
PG 20%
v PG 40%
2-HPCD 30%
PG 40%/2-HPCD 30%
9
6
3
3
6
9
12
Time (hrs)
Figure V.2
Profiles of cumulative amount (Ag) of MT
released from vehicles saturated with MT
through excised hairless mouse skin. Values
are expressed as mean ± standard deviation
(n=6).
Table V.2 Flux (µg /hr /cm2), permeability coefficient (cm/hr), and lag time (hr) of
MT from vehicles through excised hairless mouse skin (n=6).
Flux
Solubility
P
Lag time
(µg /hr /cm2)'
(mg/ml)
(cm/hrx10-5)b
(hrs)'
Buffer
0.198±0.012
1.507
7.029
1.867±0.781
PG 20% (v/v)
0.128±0.015
2.817
1.335
2.341±0.496
PG 40% (v/v)
0.236±0.026
9.587
1.300
1.028±0.113
2-HPCD 30% (w/v)
0.274±0.060
18.151
1.510
2.361±1.147
PG 40% (v/v)/2HPCD 30% (w/v)
0.404±0.050
20.408
1.980
2.361±1.147
Vehicles
3Slope of terminal steady state of cumulative amount of MT (Ag) versus time curve
fitted by linear regression divided into diffusional area of Franz cell (3.14 cm2).
hPermeability coefficient (P)=flux (µg /hr /cm2) + MT solubility (µg /ml).
`Intercept of terminal steady state of cumulative amount of MT released versus time
curve fitted by linear regression.
136
PG 40%/
2 -HPCD 30%
2-HPCD 30%
PG 40%
3
2
PG 20%
Buffer
0.0
0.1
0.2
Flux (,ug/hr/cm
Figure V.3
0.4
0.3
0.5
)
Effects of vehicles on the flux (gg/hr/cm2)
of MT through excised hairless mouse skin.
Vehicle 1-5, and 2-5 are statistically
different (p < 0.05) by Tukey method of
multiple comparison analysis. Values are
expressed as mean ± standard deviation
(n=6).
137
PG 40%, 2-HPCD 30%, and PG 40% with 2-HPCD 30%. Although
MT solubility in PG 40% with 2-HPCD 30% increased about 13
times compared to buffer, flux was increased twice only.
Flux of MT may be more compensated by the decreasing
partitioning coefficient of MT compared to the increasing
MT solubility. Is the flux of MT (0.40 Ag/hr/cm2) in PG
40% with 2-HPCD 30% enough to reach normal endogenous
plasma MT concentration? Transdermal delivery rate can be
treated as intravenous infusion assuming no metabolism and
degradation are occurred in the skin. Infusion rate can be
calculated by the product of MT concentration desired, and
volume of distribution and elimination constant. When 100
pg/ml of plasma MT concentration desired, 34.2 liter of
volume of distribution, and 0.9 hr'' of elimination
constant are used (34), delivery rate is 3.17 pg /hr. Since
diffusional area is 3.14 cm2, flux expected is 1.01
Ag/hr/cm2. Therefore, if hairless mouse skin may simulate
human skin, flux of MT through the excised hairless mouse
skin is not enough to reach normal plasma MT concentration
by transdermal route. More detailed study is required to
develop a transdermal delivery device.
In contrast, permeability coefficient (P) of MT
through excised hairless mouse skin was the greatest in
buffer followed by PG 40% with 2-HPCD 30%, 2-HPCD, PG 40%,
and PG 20% (Table V.2). The permeability coefficient (P)
is uniquely dependent on the chemical structure of the
138
drug, the physicochemical nature of the medium of
application, and physicochemical nature of the membrane
(12). Figure V.4 shows the correlation between flux and
solubility of MT among vehicles. The linear relationship
between flux of MT and MT solubility was significant
(F0=10.8, p<0.05, r=0.89). PG as a cosolvent, acts on the
skin to enhance permeation of the drug (18). 2-HPCD
appeared to be more useful to enhance permeation of MT.
The enhancing effects of 2-HPCD on MT flux rate may result
from the enhancement of MT solubility by complexation.
Direct membrane disruption due to extraction of lipids
from the skin may contribute to penetration enhancement.
Altering effects of the lipid barrier of the absorption
site by cyclodextrin derivatives have been studied for the
nasal delivery of insulin (32).
Extrapolation of the linear portion of the cumulative
amount of MT vs time curves (Figure V.2) to axis gives an
apparent diffusional lag time. The lag time due to
membrane (stratum corneum) resistance is directly
proportional the thickness of the membrane and inversely
proportional to the membrane diffusion coefficient (9,12).
Lag time is a measure of the time it takes for the
permeant's concentration gradient to become stabilized
across the membrane. A lag time for MT permeation through
excised hairless mouse skin was observed (Figure V.5).
Although the lag time of MT was the shortest in PG 40%, no
139
0.5
0
0.4
5
N
E
0
-...
2a
0.3
biz,
g
0.2
0.1
0.0
0
20
40
60
80
100
MT Solubility (mM)
Figure V.4
Correlation between flux (Ag/hr/cm2) of MT
from vehicles through excised hairless
mouse skin and MT solubility (mM). Vehicles
are indicated by numbers. 1, buffer, 2, PG
20% (v/v), 3, PG 40% (v/v), 4, 2-HPCD 30%
(w/v), and 5, PG 40% (v/v)/2-HPCD 30%
(w/v). Values are expressed as mean ±
standard deviation (n=6).
140
PG 40%/
2-HPCD 30%
2-HPCD 30%
PG 40%
PG 20%
Buffer
0
I
I
1
2
_1
I
I
3
4
I
5
Lag Time (hrs)
Figure V.5
Effects of vehicles on the lag time (hrs)
of MT through excised hairless mouse skin.
Values are expressed as mean ± standard
deviation (n=6).
141
statistically significant difference among vehicles was
observed.
It is known that prolonged skin hydration may
mechanically disrupt mouse skin so that increased
permeability of drugs is expected as hydration time
increases (15,27). Figure V.6 shows flux of MT as a
function of hydrating time (interval between excision of
skin and start of experiment). Flux of MT was changed in a
small number of replication as hydrating time (<1.5 hours)
of skin increased but it was difficult to explain the
correlation because of the short time interval and
variation of data. Merwe et. al., reported that evaluation
of drug diffusion through skin within 24 hr skin
collection and temperature less than 37 °C is desirable in
order to avoid deterioration of the skin (15). In this
study, excised hairless mouse skin was hydrated for less
than 1.5 hours at 30 °C.
Selecting polymer films for use as rate controlling
membranes or device matrices for a transdermal delivery
system requires consideration of many parameters (11,17).
Two commercially available 3M CoTranTM films, ethylene
vinyl acetate and polyethylene films were evaluated as
potential components of a transdermal delivery device
because no information on diffusion of MT through these
membranes have been available. Figure V.7 shows the
cumulative amount of MT released versus time profiles
142
o Buffer
0.5
PG 20%
o PG 40%
v 2-HPCD 30%
0.4
E
C.)
0.3
V
V
0
an
0.2
Q
0
0.1
II..1,,,,,...(1....1
0.0
0
20
40
60
80
100
Hydration Time (min)
Figure V.6
Effects of hydration time (minutes)
between
excision of hairless mouse skin and start
of diffusion on the flux (mg/hr/cm2) of MT
from vehicles.
143
o
Buffer
PG 40%
v 2-HPCD 30%
PG 40%/2-HPCD 30%
3
6
9
12
Time (hrs)
Figure V.7
Profiles of cumulative amount (Ag) of MT
released from vehicles through ethylene
vinyl acetate membrane. Values are
expressed as mean ± standard deviation
(n=4).
144
through the ethylene vinyl acetate membrane. Flux rates of
MT in decreasing order are: buffer >PG 40% >2-HPCD 30%>PG
40%/2-HPCD 30%. Flux of MT from a vehicle of PG 40% with
2-HPCD 30% was lowest and statistically different from PG
40% and buffer (p<0.05)
(Figure V.8). The flux,
permeability coefficient (P), and lag time through the
ethylene vinyl acetate membrane are given in Table V.3.
Interestingly, flux of MT decreased as MT solubility
increased and was inversely proportional to MT solubility
(Figure V.9). The negative linear relationship between
flux rate of MT and MT solubility was significant
(F12 =62.3, p<0.05, r=0.98). It is not clear why the
relationship between solubility and flux of MT through
excised hairless mouse skin and ethylene vinyl acetate
membrane are inconsistent. However, these results suggest
that permeability of MT through ethylene vinyl acetate
membrane is dependent on factors other than MT solubility
only. Further, no MT diffusion occurred through the
microporous polyethylene membrane no matter which vehicle
was used. This finding was verified by mass balance
studies of MT from a vehicle of PG 40% with 2-HPCD 30%
saturated with MT and applied to both synthetic membranes
(Table V.4). PG and 2-HPCD appears to be ineffective for
the penetration of MT through synthetic membranes.
Although flux of MT through the ethylene vinyl acetate
membrane was higher than the microporous polyethylene,
145
PG 40%/
2-HPCD 30%
4
2-HPCD 30%
PG 40%
Buffer
1
t
0.00
0.01
0.02
Flux
Figure V.8
0.03
0.04
0.05
(ii,g/hr/cm2)
Effects of vehicles on the flux (Ag/hr/cm2)
of MT through ethylene vinyl acetate
membrane. Vehicle 1-2, 1-3, 1-4, 2-3, and
2-5 are statistically different (p<0.05) by
Tukey method of multiple comparison
analysis due to high precision of data.
Values are expressed as mean ± standard
deviation (n=4).
Table V.3 Flux (Ag/hr/cm2), and permeability coefficient (cm/hr) of MT from
vehicles through ethylene vinyl acetate membrane (n=4).
Vehicles
Flux
(Ag/hr/cm2)4
Solubility
P
(mg/ml)
(cm/hrx10-5) b
Buffer
0.039±0.001
1.507
2.588
PG 40% (v/v)
0.034±0.002
2.817
0.355
2-HPCD 30% (w/v)
0.022±0.001
9.587
0.121
PG 40% (v/v)/2-
0.019±0.003
18.151
0.093
HPCD 30% (w/v)
'Slope of terminal steady state of cumulative amount of MT (Ag) versus time curve
fitted by linear regression divided into diffusional area of Franz cell (3.14cm2).
bPermeability coefficient (P, cm/hr)=flux rate (Ag/hr/cm2) /MT solubility (µg /ml).
Note) Calculation of lag time is insignificant because intercept of terminal steady
state of cumulative amount of MT released versus time curve fitted by linear
regression is essentially zero.
147
0.05
0.04
(,1
c.)
I.
0.03
=
an
3x
0.02
=
0.01
0.00
0
20
40
60
80
100
MT Solubility (mM)
Figure V.9
Correlation between flux (Ag/hr/cm2) of MT
from vehicles through ethylene vinyl
acetate membrane and MT solubility (mM).
Vehicles are indicated by numbers. 1,
buffer, 2, PG 40% (v/v), 3, 2-HPCD 30%
(w/v), and 4, PG 40% (v/v)/2-HPCD 30%
(w/v). Values are expressed as mean ±
standard deviation (n=4).
148
Table V.4 Mass balance studies of MT from a vehicle of PG
40% (v/v) with 2-HPCD 30% (w/v) through
synthetic membranes (n=4).
Synthetic membranes
Ethylene
Microporous
vinyl acetate
Polyethylene
22.17
22.17
Donor (mg)
21.98±0.45
22.07±0.39
Membranes (4g)
NDb
ND
Receptor(Ag)
1.08 ± 0.37
ND
Applied (mg)
Recovered'
`MT assayed at 12 hours after MT diffusion.
bNot detectable
149
fluxes of MT was so low that normal plasma MT
concentration could not be achieved when these membranes
were used as rate controlled membranes.
Flux (Ag/hr/cm2) of MT from four vehicles through the
membranes are compared in Figure V.10. Fluxes of MT were
more dependent on the type of membrane selected than on MT
solubility. Flux of MT was greatest in excised hairless
mouse skin, followed by the ethylene vinyl acetate
membrane and microporous polyethylene membrane. Flux of MT
through the ethylene vinyl acetate membrane were 5-20
times lower than those through excised hairless mouse
skin, while no MT diffused through the microporous
polyethylene membrane over 12 hours. Flux of drug through
synthetic membranes is dependent on structural difference,
interaction of vehicle with membranes, and physicochemical
properties of drug (10). Effects of PG and 2-HPCD on
disruption of lipid barriers and partitioning of vehicles
may be least important for the synthetic membranes. The
possible lipid extraction effect of 2-HPCD (32,33) may not
be considered in the synthetic membranes compared to the
excised hairless mouse skin. The content of vinyl acetate
increases flux of drug (10). In addition, since the
ethylene vinyl acetate membrane contains 9 % of vinyl
acetate in polyethylene as a copolymer, ethylene vinyl
acetate membrane had higher flux of MT than the
microporous polyethylene membrane.
150
1=1
Hairless Mouse Skin
Ethylene Vinyl Acetate Membrane
PG 40%/
2-HPCD 30%
1
2-HPCD 30%
I
1
PG 40%
7)
H
Buffer
0.0
H
I
0.1
0.2
Flux
Figure V.10
t
I
0.3
0.4
0.5
(p,g/hr/cm2)
Comparison of flux (Ag/hr/cm2) of MT
through excised hairless mouse skin and
ethylene vinyl acetate membrane. Bar values
of microporous polyethylene membrane are
not shown due to undetectable flux.
151
If solvent transport across membranes is involved in
drug permeability (31), volume changes of donor phase can
affect MT flux rate through the membranes. Table V.5 shows
that volumes of donor phase applied to the microporous
polyethylene membrane are markedly increased. With the
ethylene vinyl acetate membrane, no significant volume
changes occurred. This result indicates that water flows
from receptor to the donor phase for the microporous
polyethylene membrane. It was not clear why the liquid
traveled into the donor phase through the microporous
polyethylene membrane but this water flux apparently
prevents the drug from migrating to the receptor phase.
Structural differences between membranes clearly influence
the flux of MT and water. Since microporous polyethylene
membrane have pores, liquid may travel through the pores
unlike to the ethylene vinyl acetate membrane with
continuous film. One might think that osmotic pressure
difference between donor and receptor phase are involved.
Additional diffusion studies in which the donor and
receptor phases are isotonic will be necessary to
determine whether or not MT is able to across the
microporous polyethylene membrane.
Flux of MT in the vehicles selected through the
excised hairless mouse skin was lower than expected to
reach the normal plasma MT concentration. However,
transdermal delivery of MT may be possible if flux of MT
152
Table V.5 Volume changes (ml) of donor phase containing MT
in a vehicle of PG 40% (v/v) with 2-HPCD 30%
(w/v) during diffusion studies through ethylene
vinyl acetate and microporous polyethylene
membranes.
Membranes*
Surfaceb
Applied`
Recoveredd
(ml)
(ml)
Ml
1.5
1.45
-3.33
M1
1.5
1.40
-0.07
A Vol (%)`
M1
NG
1.5
1.35
-10.0
M1
NG
1.5
1.40
-0.07
M2
1.5
3.80
153.3
M2
1.5
3.60
140.0
M2
NG
1.5
3.80
153.3
M2
NG
1.5
3.65
143.3
*Synthetic membranes, Ml, ethylene vinyl acetate, M2,
microporous polyethylene.
bMembrane surface exposed to donor phase, G, glassy, NG,
non-glassy.
`Total volume (ml) of vehicle solution containing MT
applied to donor phase.
dTotal volume (ml) of vehicle solution recovered from
donor phase at 12 hours after diffusion.
`Volume changes, A Vol (%)=(recovered-1.5)
1.5 x 100.
153
was optimized by selecting approprite enhancers and
vehicle compositions. Flux of MT through ethylene vinyl
acetate membrane was so low that it could not be used as
rate controlled membrane. Microporous polyethylene
membrane may be used as backing materials because no MT
was penetrated through the membrane.
154
CONCLUSIONS
PG and 2-HPCD enhanced penetration of MT through
excised hairless mouse skin. Flux of MT from the 40% PG
with 2-HPCD 30% was greatest and statistically different
compared to buffer and PG 20%. (p<0.05).
In contrast, flux of MT through the ethylene vinyl
acetate membrane was 5-20 times lower than through the
excised hairless mouse skin and MT did not diffuse through
microporous polyethylene membranes over 12 hrs. No
markedly enhancing effect of vehicles on flux of MT was
observed for the synthetic membranes.
As MT solubility in various vehicles increased, flux
of MT linearly increased through excised hairless mouse
skin. However, flux of MT through the ethylene vinyl
acetate membrane was inversely proportional to MT
solubility, which is quite unexpected. Flux of MT is
considered as a function of product of solubility and
partitioning coefficient of MT rather than individually.
However, flux of MT obtained appeared to be too low
to reach normal plasma MT concentration in this study.
Fluxes of MT were highly dependent on the type of
membranes. Flux of MT through the membranes may result
from structural difference, interaction of vehicle with
membranes, and physicochemical properties of drug. The
lack of flux of MT through the microporous membrane was
155
associated content of vinyl acetate, and volume expansion
of donor phase due to flux of water from the receptor
phase. Selecting vehicles and model membranes is important
in developing a transdermal delivery device. Information
reported here-in will be used to prepare a transdermal
delivery device.
156
REFERENCES
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Waldhauser, F., Dietzel, M. Daily and annual rhythms
in human melatonin secretion: Role in puberty. Ann.,
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2.
Petterborg, L.J., Thalen, B.E., Kjellman, B.F., and
Wetterberg, L. Effects of melatonin replacement on
serum hormone rhythm in a patient lacking endogenous
emlatonin. Brain Res. Bull., 27(2), 181-185 (1991).
3.
Sack, R.L., A.J. Lewy, D.L. Blood, M.L., Stevenson,
J., and Keith, L.D. Melatonin administration to blind
people: Phase advances and entrainment. J. Biol.
Rhythm, 6(3), 249-262 (1991)
.
4.
Cramer, H., Rudolph, J., Consbruch, U., and Kendel,
K. On the effects of melatonin on sleep and behavior
in man. Adv. in Biochem. Psychopharmacol., 11, 187191 (1974).
5.
Dahlitz, M., Alvarez, B., Vignau, J., English, J.,
Arendt, J., and Parkes, J.D. Delayed sleep phase
syndrome response to melatonin. Lancet, 337(8750),
1121-1124 (1991).
6.
Petrie, K., Conaglen, J.V. Thompson, L., and
Chamberlain, K. Effects of melatonin on jet lag after
long haul flight. Br. Med. J., 298, 705-707 (1989).
7.
Waldhauser, F., Vierhapper, H., and Oirich, K.
Abnormal circadian melatonin secretion in night-shift
workers. The New Eng. J. Med., 7, 441-446 (1986).
8.
Rosenthal, N.E., Sack, D.A., Gillin, J.C., Lewy,
A.J., Goodwin, F.K., Davenport, Y., and Mueller,
P.S., et. al.,
Seasonal affective disorder.
A
description of the syndrome and preliminary findings
with light therapy. Arch. Gen, Psychiatry, 41, 71-80
(1984).
9.
10.
Kydonieus, A.F. and Berner, B. Transdermal delivery
of drugs. Vol. I, II, & III. CRC Press, Inc., Boca
Raton, FL (1987).
Guy, R.H. and Hadgraft, J. Transdermal drug
delivery: A perspective.
J. Controlled Release
4, 237-251(1987).
157
11.
Hadgraft, J. and Guy, R.H. Transdermal drug
delivery. Developmental issues and research
initiatives. Marcel Dekker, Inc., NY (1989).
12.
Flynn, G.L. and Stewart, B. Percutaneous drug
penetration: Choosing candidates for transdermal
development. Drug Dev. Res., 13, 169-185 (1988).
13.
Higo, N., Hinz, R.S., Lau, D.T.W., Benet, L.Z., and
Guy, R.H. Cutaneous metabolism of nitroglycerin in
vitro. II. Effects of skin condition and penetration
enhancement. Pharm. Res., 9(3), 303-306 (1992).
14.
Dick, I.P. and Scott, R.C. The influence of different
strains and age on in vitro rat skin permeability to
water and mannitol. Pharm. Res., 9(7), 884-887
(1992) .
15.
Merwe, E.V.D., Ackermann, C., and Wyk, C.J.V. Factors
affecting the permeability of urea and water through
nude mouse skin in vitro. I. Temperature and time of
hydration.
16.
Pfister, W.R. and Hsieh, D.S.T. Permeation
enhancers compatible with transdermal drug
delivery systems.
Part I: Selection and
formulation considerations. Pharm Tech. Sep.,
132-140 (1990).
17.
Pfister, W.R. and Hsieh, D.S.T. Permeation
enhancers compatible with transdermal drug
delivery systems.
Part II: System design
considerations. Pharm Tech. Oct., 54-60 (1990).
18.
Rolf, D. Chemical and physical methods of
enhancing transdermal drug delivery. Pharm.
Tech. Sep. 130-140 (1988).
19.
Lewy, A.J. Biochemistry and regulation of mammalian
melatonin production. The Pineal Gland, edited by
Richard Relkin, Elsevier Science Publishing Co., Inc.
(1983) .
20.
Yalkowsky, S.H. and Rubino, J.T. Solubilization
by cosolvents I: Organic solutes in propylene
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416-421 (1985).
21.
Duchene, D. and Wouessidjewe, D. Physicochemical
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22.
Duchene, D. and Wouessidjewe, D. Pharmaceutical uses
of cyclodextrins and derivatives. Drug Dev. & Ind.
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23.
Szejtli, J. Cyclodextrins in drug formulations: Part
II. Pharm. Tech., August, 24-38 (1991).
24.
Arima, H., Adachi, H., Irie, T., Uekama, K., and
Pitha, J. Enhancement of the antiinfalmmatory effect
of ethyl 4-biphenyl acetate in ointment by gcyclodextrin derivatives: Increased absorption and
localized activation of the prodrugs in rats. Pharm
Res., 7(11), 1152-1156 (1990).
25.
Loftsson, T. and Border, N. Effects of 2hydroxypropy1-0-cyclodextrin on the aqueous
solubility of drugs and transdermal delivery of 170estradiol. Acta Pharm Nordica 1, 185-194 (1989).
26.
Rigg, P.C. and Barry, B.W. Shed snake skin and
hairless mouse skin as model membranes for human
skin during permeation studies. J. Invest.
Dermatol., 94, 234-240 (1990).
27.
Bond, J.R. and Barry, B.W. Hairless mouse skin is
limited as a model for assesing the effects of
penetration enhancers in human skin. J. Invest.
Dermatol., 90, 810-813 (1988).
28.
Hsieh, D.S.T. Multiple lamination for transdermal
patches. Controlled release systems: Fabrication
technology. Volume I, edited by Dean Hsieh, CRC
Press, Inc., Boca Raton, FL (1988).
29.
Lee, Y. Development of a transdermal delivery system
for melatonin. M.S. Thesis, College of Pharmacy,
Oregon State University (1989).
30.
Mallo, C., Zaidan, R., Galy, G., Vermeulen, E., Brun,
J., Chazot, G., and Claustrat, B. Pharmacokinetics of
melatonin in man after intravenous infusion and bolus
injection. Eur. J. Clin. Pharmacol., 38, 297-301
(1990) .
31.
Neter, J., Wasserman, W., and Kutner, M.H. Applied
linear statistical models: Regression, analysis of
variance and experimental design, 2nd edition.
Richard D. Irwin, Inc., Homewood, IL (1985).
159
32.
Aungst, B.J., Blake, J.A., and Hussain, M.A.
Contribution of drug solubilization, partitioning,
barrier disruption, and solvent permeation to the
enhancement of skin permeation of various compounds
with fatty acids and amines. Pharm. Res., 7(7), 712718 (1990).
33.
Shao, Z., Krishnamoorthy, R., and Mitra, A.K.
Cyclodextrins as nasal absorption promoters of
insulin: Mechanistic evauation. Pharm. Res., 9(9),
1157-1163 (1992).
160
CHAPTER VI
PRELIMINARY EVALUATION OF TRANSDERMAL DELIVERY DEVICE FOR
MELATONIN IN HUMAN SUBJECTS
ABSTRACT
The Hill Top Chamber' with Webril' pad as an
occlusive transdermal delivery device (TDD) was applied to
four human subjects to investigate the penetration
behavior of melatonin (MT) through human skin. Saturated
MT solution in 40% propylene glycol was loaded in Webril'
pad of TDD. A total TDD surface area of 3.80 cm2 was
applied to the forearm of each subject. Plasma MT
concentrations increased and above the baseline in
approximately 2-4 hours although steady state was not
achieved in the 8 hour study period. Urinary excretion
rate of 6-STMT also increased as plasma MT concentration
increased. Urinary excretion rate of 6-STMT following TDD
application was higher than control excretion. Cumulative
amounts of urinary 6-STMT over 6 hours when TDD was
applied were three times greater than control excretion.
Urinary excretion rate of 6-STMT was correlated with
plasma MT concentration among subjects (coefficient of
determination = 0.77). These data suggest that urinary
excretion rate of 6-STMT can be used as an index of MT
circadian rhythm in human subjects. An intersubject
161
variability in both plasma MT concentration and urinary
excretion rate of 6-STMT was observed. However, it was
evident that MT could be delivered transdermally in human
subjects.
162
INTRODUCTION
Melatonin (MT; N-Acetyl -5-Methoxytryptamine) is an
indole amide neurohormone secreted primarily from the
pineal gland (1). The circadian pattern of endogenous
production may be described by low levels during the day
time (< 30 pg/ml) and higher constant values (50-200
pg/ml) during the night (2). Although exogenously
administered MT is known to entrain (reset) circadian
rhythms in animals and humans (3,4), MT's potential
clinical values may be limited because no dosage forms are
currently available which provide sustained release of MT
so as to mimic the normal physiological plasma MT
concentrations vs time profile. Since MT has a short halflife, about 40 minutes (12), a sustained release
preparation is necessary to maintain the plasma MT
concentration in the physiological range of 50-200 pg/ml
over 8-10 hours (2). Exogenously administered MT in a
sustained release dosage form could play a role in
treating disruptions of circadian rhythm such as delayed
sleep syndrome (5,6), jet lag (7), shift work syndrome
(8), and winter depression (9).
Because of the need for MT to be delivered in a
sustained manner over 8-10 hours, transdermal delivery may
be a viable route for MT administration. MT may be a good
candidate for transdermal delivery. It has low molecular
163
weight (Mw 232.3) and is organic soluble (11). Lee et.
al., studied the diffusion of MT across hairless mouse
skin in the in vitro system (12). A vehicle of phosphate
buffer (pH 6.1) with 40% (v/v) propylene glycol as a
cosolvent gave the maximum flux of MT compared to other
buffer/propylene glycol mixtures.
The purpose of this preliminary study was to
characterize the plasma MT concentration and urinary
excretion rate of its metabolite, 6-sulphatoxymelatonin
(6-STMT), after administration of TDD containing MT to
human subjects.
164
MATERIALS AND METHODS
Subjects
Four healthy male subjects who had received no
medication in the previous 48 hours were admitted to the
Oregon Health Science University Clinical Research Center
on the study day at approximately 9 A.M. Subjects were
allowed food and fluids freely throughout the study.
Characterization of the four subjects are shown in Table
VI.1.
Study Protocol
The study was conducted in late August. The Hill Top
Chambers with Webril pad. (occlusive device containing 0.95
cm2 absorbent pads; Hill Top Research, Inc., Cincinnati,
OH, USA) was used as a transdermal delivery device (TDD).
One hundred milligrams of MT (Regis Chemical Co., Morton
Grove, IL) were dissolved in 10 ml of pH 6.1 phosphate
buffer containing 40% propylene glycol (J.T. Baker Inc.,
Phillipsburg, NJ). Thereafter, 200 Al MT solution was
loaded on each Webril pad. which was in the Hilltop
Chambers.. Four Chambers with Webril pads were applied to
the hairless surface of an each subject's forearm and
covered with a tape adhesive. TDD was applied at 11 AM.
Total surface area
and total MT contents are 3.80 cm2 and
8 mg, respectively. An indwelling catheter was set in the
165
Table VI.1
Subjects
Vital statistics of four male subjects.
Age
Weight
Height
(yr)
(Kg)
(cm)
1
30
59
170
Asian
2
45
65
180
Caucasian
3
48
82
183
Caucasian
4
33
73
173
Asian
Mean±S.D.
39 ± 9
70 ± 10
177 ± 6
Race
166
opposite forearm for blood collection. Blood and urine
samples were collected before TDD application.
Approximately 5 ml of blood was withdrawn through the
indwelling catheter at 1,
2,
4,
6, and 8 hours after
application of TDD. Heparin solution was used to prevent
clotting within the catheter. The TDD was removed from
subject 1 and 2. The skin area was gently washed with
water after the TDD was removed. After a two week washout
period, daytime control urines were collected from subject
1 and 2.
Analytical Methods
Plasma MT concentrations were determined by a highly
sensitive and specific GC/MS assay (13).
Urinary 6-STMT
concentrations were determined by radioimmunoassay (14).
167
RESULTS AND DISCUSSION
Preliminary diffusion studies using excised abdominal
hairless mouse skin showed that MT in 40 % (v/v) propylene
glycol had a flux rate of 0.24 pg/hr/cm2. Input rate of MT
from the TDD is 0.912 gg/hr over 3.80 cm2 of total skin
surface area applied. Transdermal delivery rate of MT can
be treated as intravenous infusion assuming that no
metabolism and degradation of MT are occurred in the skin.
The expected MT plasma concentration by transdermal route
will be about 30 pg/ml at steady state when 35.2 liter of
volume of distribution of MT and 0.9 hr-' of elimination
are used (12).
Plasma MT concentrations for each subject are shown
in Table VI.2 and Figure VI.1. Plasma MT concentration
increased above the baseline in approximately 2-4 hours
although steady state was not achieved in the eight hour
study period. The in vivo flux rate of MT was not
determined directly but it appeared to be greater than the
in vitro flux of MT through excised hairless mouse skin.
The baseline MT concentration in the four subjects at
11:00 AM before TDD application ranged from 9.7 to 24.3
pg/ml (see Table VI.2). The variation in baseline MT
concentration among subjects may be related to
interindividual differences of circadian rhythm (15), age
(16), sleeping cycles (6) or light sensitivity (17).
Plasma MT concentration (pg/ml) after administration of TDD
Table VI.2
containing MT in propylene glycol 40% (v/v) to four human subjects.
Subjects
Time
(hrs)
Clock Time
On
11:00
1
12:00
2
13:00
24.3
4
15:00
24.3
6
17:00
75.3b
8
19:00
#1
24.3
#2
#3
23.0
mean ± SD
#4
9.7
8.3
110.3
30.3
9.7
19.4
20.9±8.7
19.4
48.6
30.8±15.6
43.01'
58.3
20.0
24.3
58.9±16.2
170.1
'TDD was applied at 11:00 AM.
bTDD was removed and then skin was gently washed with water.
`Subject 3 and 4 only.
19.0±8.1
97.2±103.1`
169
100
10
Subject 1
1
100
10 r
Subject 2
1
100 r
10
Subject 3
10
Subject 4
1
0
2
4
6
8
Post TDD (hrs)
Figure VI.1
Plasma MT concentration versus time
profiles after application of TDD to four
human subjects over 8 hours. Asterisks show
the time of TDD removal.
170
MT is extensively metabolized to 6-STMT (18). The
correlation between plasma MT concentration and urinary
excretion rate of 6-STMT was reported (19). If 6-STMT is
closely related to plasma MT concentration, urinary
excretion rate of 6-STMT may be used as a non-invasive
method to study circadian rhythms of MT. Figure VI.2 shows
urinary excretion rate of 6-STMT determined at the
midpoint of urine collection interval in four subjects. As
plasma MT concentration increased, urinary excretion rate
of 6-STMT also increased during TDD application.
Cumulative amount of urinary 6-STMT over 6 hours in
subject 1 and 2 is compared in Figure VI.3. The cumulative
amount of urinary 6-STMT when TDD was applied was three
times greater than control excretion. This result
suggested that MT could be delivered by the transdermal
route. Correlation between plasma MT concentration and
urinary excretion rate of 6-STMT is shown in Figure VI.4.
Although the correlation was not strong due to large
intersubject variation, the linear relationship was
statistically significant (F1,0 = 45, p < 0.0001).
Significant intersubject variations in plasma MT
concentrations and urinary excretion rates of 6-STMT were
also observed in this small study group.
In conclusion, MT could be delivered transdermally in
human subjects. Further development and testing of the TDD
are necessary. This includes proper vehicle composition to
171
1000
100
Subject 1
10
1000
100
Subject 2
10
1000
100
Subject 3
10
1000
100
Subject 4
10
0
2
4
6
8
Post TDD (hrs)
Figure VI.2
Profiles of urinary excretion rate of 6
STMT (ng/hr) determined at the midpoints of
each urine collection interval after
application of TDD to four human subjects.
TDD was applied at 11:00 AM (t=0) in the
morning. Asterisks show the time of TDD
removal.
172
5,000
c.
4.
4,000
3,000
E
2,000
1,000
E
U
0
Subject 1
Figure VI.3
Subject 2
Cumulative amount of urinary 6-STMT (ng)
for 6 hours (11:00-17:00) after application
of TDD.
173
2000
-731)
r = 0.915
E-4
E-0
1500
\cs
0
rsa)
1000
0
o
0
4-a
Subject 1
Subject 2
Subject 3
Subject 4
ct.)
c.)
500
."4
0
7
I
I
I
50
I
1
I
1
I
100
I
t
150
I
200
Plasma MT Concentration (pg/ml)
Figure VI.4
Correlation between plasma MT
concentrations and urinary excretion rate
of 6-STMT determined at the midpoints of
each urine collection interval after
application of TDD to four human subjects.
174
provide maximum flux of MT through human skin, and
fabrication of transdermal delivery system for clinical
application.
175
CONCLUSIONS
Plasma MT concentration increased after
administration of TDD to human subjects. Urinary excretion
rate of 6-STMT also increased as plasma MT concentration
increased. Cumulative amount of urinary 6-STMT over 6
hours when TDD was applied was three times greater than
control urine. Urinary 6-STMT was related with plasma MT
concentration among subjects. A significant intersubject
variation in plasma MT concentration and urinary 6-STMT
was also observed in a small study group. A linear
correlation between urinary 6-STMT and plasma MT suggest
that urinary 6-STMT can be used as an index of MT
circadian rhythm in humans.
176
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189
APPENDIX A
PROFILES OF PLASMA MT CONCENTRATION AND URINARY EXCRETION
RATE OF 6-STMT VERSUS TIME FOR INDIVIDUAL SUBJECTS AFTER
ADMINISTRATION OF ORAL CONTROLLED RELEASE DELIVERY SYSTEM
190
=
1000
-61:1
cn
=
tvi
10000
oo
40
a)
=
tt4
=
0
0
U
0
10
ccS
cC
0
100
2
4
6
8
Time (hrs)
Figure A.1
Profiles of plasma MT concentration and
urinary excretion rate of 6-STMT versus
time for subject 1. Total 0.5 mg of MT
doses (0.1 mg immediate release and 0.4 mg
controlled released from 20% Aguacoaecoated beads) were administered.
191
.."..
$
.1:4
to
=
1000
E--4
E--4
con
15
4.m
0
0
100
1 0000
1 00
t`l
P4
=
2.
te
10
I-
0
.
w
cci
.z.44
1
2
1
,
4
100
1
6
8
Time (hrs)
Figure A.2
Profiles of plasma MT concentration and
urinary excretion rate of 6-STMT versus
time for subject 2. Total 0.5 mg of MT
doses (0.1 mg immediate release and 0.4 mg
controlled released from 20% Aguacoat.coated beads) were administered.
192
1..
.-=
to
=
I 1000
E-4
ii)
E-4
c.)
=
10000 ±
o
t41
o
100
a)
-44
1'4
0
=
a4
(.)
- 1000 .40,
=
o
F-4
o
I0
10
x
W
m
E
w
>,
m
=
5.:
o
2
I
I
4
6
100
8
Time (hrs)
Figure A.3
Profiles of plasma MT concentration and
urinary excretion rate of 6-STMT versus
time for subject 3. Total 0.5 mg of MT
doses (0.1 mg immediate release and 0.4 mg
controlled released from 20% Aguacoaecoated beads) were administered.
193
1000
,-,
El
100DP`4-4 D
0
0
100
P4
=
0
O
1000
10
0
X
44
.a
100
2
4
6
D
8
Time (hrs)
Figure A.4
Profiles of plasma MT concentration and
urinary excretion rate of 6-STMT versus
time for subject 4. Total 0.5 mg of MT
doses (0.1 mg immediate release and 0.4 mg
controlled released from 20% Aguacoat.coated beads) were administered.
194
81)
1000
C/)
=
2
4-
0
100
0
0
/10000 a
=
0
C.)
E.4
:244
0
1.4
10
iC
cd
100
2
4
6
8
Time (hrs)
Figure A.5
Profiles of plasma MT concentration and
urinary excretion rate of 6-STMT versus
time for subject 5. Total 1 mg of MT doses
(0.2 mg immediate release and 0.8 mg
controlled released from 20% Aguacoat.-
coated beads) were administered.
195
1 1000
ao
0
ti
100
a'
0
=
0
10
*-41
0
100
2
4
6
8
Time (hrs)
Figure A.6
Profiles of plasma MT concentration and
urinary excretion rate of 6-STMT versus
time for subject 6. Total 1 mg of MT doses
(0.2 mg immediate release and 0.8 mg
controlled released from 20% Aguacoat.-
coated beads) were administered.
196
APPENDIX B
PREPARATION OF BUFFER SOLUTION
197
Table B.1 Preparation of buffer solutions
1.
0.01 M acetate buffer (pH 4.70) for HPLC analysis
Weigh 0.8203 g of sodium acetate (Mw, 82.03) and
dissolve in about 950 ml of deionized water. Add 0.1 M of
glacial acetic acid until pH reaches 4.70. Volume of 0.1 M
acetic acid consumed is about 10 to 20 ml. Finally, adjust
volume to 1000 ml by water.
2.
NaCl-HC1 buffer (pH 1.4 ± 0.1) as simulated gastric
fluid
Dissolve 6.0 g of Nadi in 2900 ml of deionized water.
Add 7.4% of diluted HC1 and adjust pH 1.4. Volume of 7.4%
of diluted HC1 consumed is about 40 to 55 ml. Finally,
adjust volume to 3000 ml by water.
3.
Phosphate buffer (pH 7.4 ± 0.1) as simulated
intestinal fluid.
Dissolve 20.4 g of potassium phosphate monobasic
(KH2PO4) in about 2800 ml of deionized water. Mix and
adjust resulting solution with 1 N NaOH and adjust pH
7.4±0.1. Volume of 1N NaOH consumed is about 100 to 110
ml. Dilute and adjust with deionized water to 3000 ml.
198
APPENDIX C
DISSOLUTION DATA:
PERCENTAGE OF MT RELEASED FROM COATED BEADS
Table C.1 Dissolution data: The percentage of MT released from
Aquacoat'- coated beads at various coating amounts (n=3).
Coating (%)
18-20 mesh
8-10 mesh
Size
5
10
20
5
10
20
0.25
17.6
12.5
9.5
54.7
29.9
22.8
0.5
29.5
19.8
13.1
75.3
48.2
35.4
1
49.9
30.9
19.0
85.2
67.5
50.6
1.5
63.9
40.0
25.2
89.7
76.9
61.9
2
73.4
47.9
29.8
90.0
80.0
69.6
4
86.5
75.6
52.7
92.0
86.8
85.1
6
90.2
86.9
66.3
87.3
89.5
9
92.9
94.6
80.4
87.3
91.6
12
91.4
97.1
88.9
90.0
91.1
24
91.2
98.9
101.5
90.2
91.9
Time (hrs)
200
Table C.2 Dissolution data: The percentage of MT released
from 8-10 mesh Aguacoat.-coated beads with 20%
coating as a function of stirring speed.
rpm
50
77
100
Time (hrs)
9.5
8.7
9.4
0.5
13.1
13.2
12.0
1.0
19.1
18.7
18.7
1.5
25.2
23.3
23.1
2.0
29.8
31.3
27.9
3.0
42.9
43.6
40.7
4.0
52.7
53.4
51.0
6.0
66.3
68.4
67.2
9.0
80.4
83.7
82.3
12.0
88.9
93.1
93.1
24.0
101.5
104.5
101.1
0.25
201
Table C.3 Dissolution data: The percentage of MT released
from 8-10 mesh Aguacoat.-coated beads with 20%
coating after 6 months' storage at room
temperature.
Time (hrs)
Original
0.25
9.5
6 months
9.3
0.5
13.1
13.0
1.0
19.1
18.8
1.5
25.2
24.2
2.0
29.8
29.2
3.0
42.9
39.5
4.0
52.7
48.5
6.0
66.3
63.4
9.0
80.4
78.9
12.0
88.9
86.9
24.0
101.5
101.7
MT content loaded for dissolution (mg): 7.47 mg at t=0;
7.06 mg at t=6 months
202
APPENDIX D
PLASMA MT CONCENTRATION AND URINARY EXCRETION RATE OF
6-STMT VERSUS TIME DATA FOR INDIVIDUAL SUBJECTS AFTER
ADMINISTRATION OF ORAL CONTROLLED RELEASE DELIVERY SYSTEM.
Table D.1 Plasma MT concentration (pg/ml) versus time data after administration of
the oral controlled release delivery system to human subjects.
High Dose
Low Dose
Time
(hrs)
#3
#2
#1b
#4
Mean
S.D.°
C.V.d
#5
#6
Mean
S.D.
C.V.
(%)
(%)
0'
2
3
16
5
6.4
6.5
101.9
16
12
14
2.8
20.2
0.5
480
700
490
450
530
114.6
21.6
1575
690
1133
625.8
55.3
1.0
510
750
440
440
535
147.1
27.5
1600
575
1088
724.8
66.6
1.5
660
435
800
341
559
209.1
37.4
1400
590
995
572.8
57.6
2.0
1075
550
570
300
624
324.9
52.1
975
600
788
265.2
33.7
3.0
610
201
295
168
318
201.8
63.4
590
425
508
116.7
23.0
4.0
490
146
141
148
231
172.6
74.7
375
315
345
42.4
12.3
6.0
142
135
88
65
107
37.3
34.7
205
220
213
10.6
5.0
8.0
105
120
66
86
94
23.4
24.8
170
170
--
°Start of experiment, clock time: 10:30 A.M.
bSubject number (see also Table IV.2).
`Standard deviation.
dCoefficient of variation.
204
Table D.2 Urinary excretion rate of 6-STMT (ng/hr) versus
time data after administration of the oral
controlled delivery system.
Low Dose
Time'
(hrs)
#1b
#2
#3
Mean
#4
S.D.`
C.V.d
(%)
0
339
572
1104
201
554
397
71.8
1
20551
35954
37824
31981
31578
7744
24.5
3
29575
26129
12615
18367
21671
7643
35.2
5
11840
13370
9734
7488
10608
2558
24.1
7
8552
*m.
6352
7452
1556
20.8
High Dose
Time
(hrs)'
#51,
#6
Mean
S.D.`
C.V.d
(%)
0
469
441
455
20.8
4.4
1
45562
51773
48667
4392
9.0
3
34918
5
11242
12491
62.2
7
12240
34918
28907
20075
'Midpoints of each urine collection interval after start
of experiment at 10:30 A.M.
bSubject number (see also Table IV.2).
`Standard deviation.
d Coefficient of variation.
205
APPENDIX E
CUMULATIVE AMOUNT OF MT RELEASED FROM VEHICLES THROUGH
EXCISED HAIRLESS MOUSE SKIN AND ETHYLENE VINYL ACETATE
MEMBRANE
Table E.1 Mean cumulative amount (Ag) of MT and flux (µg /hr/cm2) from vehicles
through excised hairless mouse skin.
Time (hrs)
0.5
1.0
1.5
2.0
2.5
3.5
4.5
6.0
8.0
10.0
12.0
Flux'
PG 40%
2-HPCD 30%
(v/v)
(w/v)
PG 40%(v/v)/2HPCD 30%(w/v)
0.34±0.33
0.38±0.37
0.50±0.45
0.71±0.24
0.89±0.29
1.32±0.33
1.72±0.29
2.48±0.24
3.67±0.19
4.98±0.20
6.36±0.31
0.91±1.10
1.06±1.16
1.30±1.20
1.67±1.06
1.87±0.96
2.58±1.01
3.18±0.80
4.46±0.74
5.87±1.03
7.78±0.47
8.93±0.23
0.21±0.26
0.38±0.30
0.48±0.38
0.73±0.51
0.99±0.66
1.50±0.99
2.83±0.96
3.93±1.49
4.94±2.53
6.76±3.39
8.70±4.15
0.54±0.40
0.65±0.32
0.96±0.48
1.22±0.32
1.53±0.23
2.28±0.46
3.03±0.72
4.67±1.17
7.27±1.40
9.62±2.68
12.60±3.22
0.198±0.012
0.236±0.026
0.274±0.060
0.404±0.050
1.867±0.781
1.028±0.113
2.361±1.147
2.361±1.147
Buffer
(µg /hr /cm2)
Lag Timeb
(hrs)
'Slope of terminal steady state of cumulative amount of MT released versus time
curve fitted by linear regression divided into diffusional area of Franz. cell
(3.14 cm2).
bIntercept of terminal steady state of cumulative amount of MT released versus time
curve fitted by linear regression.
Table E.2 Mean cumulative amount (Ag) of MT and flux (Ag/hr/cm2) from vehicles
through ethylene vinyl acetate membrane (n=4).
Time (hrs)
0.5
1.0
1.5
2.0
2.5
3.5
4.5
6.0
8.0
10.0
12.0
Flux'
(Ag/hr/cm2)
PG 40%(v/v)/2HPCD 30%(w/v)
PG 40%
2-HPCD 30%
(v/v)
(w/v)
0.17+0.34
0.18±0.36
0.31±0.25
0.36±0.33
0.28±0.25
0.48±0.15
0.54±0.10
0.82±0.25
1.12±0.19
1.34±0.18
1.57±0.20
0.14±0.24
0.14±0.25
0.36±0.33
0.45±0.43
0.47±0.43
0.65±0.31
0.70±0.36
0.87±0.31
1.14±0.29
1.30±0.32
1.54±0.37
0
0
0
0.06±0.11
0.34±0.07
0.44±0.04
0.61±0.03
0.72±0.07
0.85±0.04
0.29±0.57
0.29±0.57
0.37±0.55
0.38±0.56
0.43±0.51
0.57±0.46
0.63±0.45
0.73±0.45
0.84±0.44
0.95±0.44
1.10±0.39
0.039±0.001
0.034±0.002
0.022±0.001
0.019±0.003
Buffer
0
0
'Slope of terminal steady state of cumulative amount of MT released versus time
curve fitted by linear regression divided into diffusional area of Franz cell (3.14
cm2).
Table E.3 Comparison of ethylene vinyl acetate and microporous polyethylene
membranes.
Properties
Ethylene Vinyl
Acetate'
Caliper
Vinyl Acetate Content
Tensile, g/25 mm
Elongation (%)
Gurly Porosity
Void Volume
Bubble Point
Heat Shrinkage (65 °C)
Pore Deformation
Safety
Heat Sealing
Storage
0.051 mm
9.0 %
MD 2000
CD 900
MD 300
CD 200
Microporous
Polyethyleneb
0.051 mm
0 %
24 sec/50cc (air)
78 %
0.18A
4 %
115 °C
Yes
Yes
Temp.=10-27 °C
Humidity 40-60%
Yes
Yes
Temp.=10-27
Humidity 40-60%
'3M CoTranTM controlled caliper film #9702, homogenous membranes.
b3M CoTranTM microporous membranes.