Journal of Controlled Release 75 (2001) 307–315

www.elsevier.com / locate / jconrel

Preparation, characterization and in vivo evaluation of 120-day

poly( D,L-lactide) leuprolide microspheres
Byung H. Woo a , Janusz W. Kostanski b , Sisay Gebrekidan c , Bhas A. Dani a ,
d a,
B.C. Thanoo , Patrick P. DeLuca *
a
Faculty of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 800 Rose Street, Lexington, KY 40536, USA
b
Bristol-Myers Squibb Pharmaceutical Research Institute, One Squibb Drive, PO Box 191, New Brunswick, NJ 08903, USA
c
Wyeth-Ayerst Laboratories, 2 Esterbrook Lane, Cherry Hill, NJ 08002, USA
d
Oakwood Laboratories, 7670 First Place - Suite A, Oakwood, OH 44146, USA

Received 12 February 2001; accepted 21 May 2001

Abstract

A 120-day poly( D,L-lactide) (PLA) microsphere delivery system for a luteinizing hormone-releasing hormone (LHRH)
analogue, leuprolide, was prepared and evaluated. Leuprolide microspheres were prepared with PLA (m.w. 11 000 Da) by a
dispersion / solvent extraction-evaporation method and characterized for drug load by HPLC, particle size by laser
diffractometry and surface morphology by scanning electron microscopy. In vitro peptide release and polymer degradation
were studied using a modified dialysis method. Serum peptide and testosterone levels were analyzed after subcutaneous
administration using a rat model. Spherical microspheres with a mean diameter of 52 mm containing 13.4% peptide released
10% of the peptide within 24 h, followed by a linear release for 150 days. Serum leuprolide levels increased immediately
after administration of the microspheres to 45.6 ng / ml, but then fell to 4.3 ng / ml at 15 days and |2.0 ng / ml at 30 days
where they remained for 120 days. The testosterone levels increased initially to 15 ng / ml and then decreased to below 0.5
ng / ml by day 4 where they remained for 120 days. In conclusion, a 120-day microsphere formulation of leuprolide was
developed with excellent controlled peptide release characteristics and in vivo efficacy.  2001 Elsevier Science B.V. All
rights reserved.

Keywords: Leuprolide; Luteinizing hormone-releasing hormone (LHRH) analogue; Poly( D,L-lactide); Microspheres; Testosterone suppres-
sion

1. Introduction gated to improve the efficacy of peptide drugs and

Controlled release dosage forms have been investi-
*Corresponding author. Tel.: 11-859-257-1831; fax: 11-859-
323-0242.
E-mail address: ppdelu1@pop.uky.edu (P.P. DeLuca).
eliminate the need for frequent administration. Bio-
degradable microspheres were shown to improve the
bioavailability of peptides by protecting them from
physical degradation and proteolysis in body fluids.
Poly( D,L-lactide) (PLA) and poly( D,L-lactide-co-gly-
colide) (PLGA) are the most widely used and well-

0168-3659 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved.
PII: S0168-3659( 01 )00403-5
308 B.H. Woo et al. / Journal of Controlled Release 75 (2001) 307 – 315
characterized materials for the preparation of bio-
degradable microspheres [1–8].
Leuprolide, the peptide drug used in this study, is
a potent agonist of luteinizing hormone-releasing
hormone (LHRH) currently used for the treatment of
prostatic cancer, endometriosis and precocious pu-
berty [9,10]. Utilizing an in-water drying method
based on the w / o / w emulsion technique, 1-, 3- and
4-month release leuprolide microcapsules, Lupron  ,
have been developed [11–16]. In the in-water-drying
method leuprolide acetate was dissolved in aqueous
gelatin or water, dispersed into a polymer solution
which was then emulsified into an external aqueous
phase to form droplets. The microcapsules solidified
after solvent removal and were freeze-dried. Several
publications have described in vivo pharmacological
and pharmacokinetic evaluations of these microcap-
sules [12–16].
In most literature reports of in vitro release, the
microspheres were incubated in a certain volume of
release medium, generally phosphate buffered saline
(PBS) with a preservative, using glass or plastic test
tubes at 378C with optional agitation [17]. In these
methods, the pH of the release medium consistently
dropped due to the formation and accumulation of
acidic polymer degradation products [18,19]. Con-
tinuous removal of the acidic polymer fragments and
released drug by dialysis might be desirable to
prevent the pH drop of the release medium. A
dialysis in vitro release method for an LHRH
antagonist, orntide, loaded PLGA microspheres has
been developed by using a small dialysis unit and 0.1
M acetate buffer, pH 4.0 [20]. The in vitro release
profile obtained with the dialysis method permitted a
better correlation with in vivo release. However, only
a little information is available on in vitro degra-
dation kinetics and correlation between in vitro and
in vivo release of long-term release peptide loaded
PLA microspheres.
The goal of this study was to prepare and char-
acterize a 4-month formulation of leuprolide micro-
spheres using a dispersion method and to study in
vitro drug release and in vivo efficacy. Also, an
attempt to develop a modified dialysis method for
testing in vitro release from biodegradable micro-
spheres, which would exhibit a good in vitro–in vivo
correlation, was undertaken.
2. Materials and methods
2.1. Materials
Leuprolide, [Des-Gly 10 , D-Leu 6 , Pro 9 ]-LHRH
ethylamide, was obtained from Bachem (Torrance,
CA, USA). Poly( D,L-lactide) (m.w. 11 000 Da,
Resomer  R202H, PLA) was supplied by Boehringer
Ingelheim (Ingelheim, Germany). Polyvinyl alcohol
(m.w. 30 000–70 000; PVA) was supplied by Sigma
(St Louis, MO, USA). Dialysis tubes (Tube-O-
Dialyzer  ) were purchased from Research Products
International Co. (Mount Prospect, IL) and
Spectrapor  CE dialysis membranes (MWCO 7000–
300 000 Da) were supplied by Spectrum Medical
Industries (Houston, TX). All other chemicals were
obtained commercially as analytical grade reagents.
2.2. Preparation of microspheres
Leuprolide PLA microspheres were prepared by
dispersing the homogeneous solution of polymer and
drug into a PVA solution followed by solvent
extraction / evaporation / dilution as previously de-
scribed [21]. A solution of leuprolide in methanol
was added into a 34% (w / w) solution of polymer in
methylene chloride to form the clear solution. The
resulting solution was then slowly injected into
0.35% aqueous PVA solution while mixed with a
Silverson  L4R mixer (Silverson Machines, MA,
USA) at 7000 rpm. The solvents were removed by
stirring and continuous phase replacement at 38–
408C for 1 h. The solidified microspheres were
recovered by filtration and dried under vacuum at
room temperature for 48 h.
2.3. Particle characterization
2.3.1. Particle size distribution
Particles were sized by laser diffractometry using
a Malvern 2600 laser sizer (Malvern 2600c Particle
Sizer, Malvern, UK). The average particle size was
expressed as the volume mean diameter Vmd in
microns.
2.3.2. Surface morphology
The surface morphology was examined by scan-
 B.H. Woo et al. / Journal of Controlled Release 75 (2001) 307 – 315
ning electron microscopy (Hitachi Model S800,
Japan) after palladium / gold coating of the micro-
sphere sample on an aluminum stub.
2.3.3. Peptide content
A 10-mg amount of the microspheres was dis-
solved in 2.0 ml methylene chloride. The peptide
was extracted from the polymer solution by addition
of 10 ml of 0.1 M acetate buffer (pH 4.0) followed
by agitation for 1 h. The peptide was assayed in the
aqueous phase by HPLC method: Bondclone 10 C 18
column (15033.9 mm; Phenomenex, Torrance, CA,
USA); mobile phase: 27% (v / v) acetonitrile, 0.1%
trifluoroacetic acid; flow rate 1.0 ml / min; UV de-
tection at 215 nm.
2.4. In vitro release study
In vitro release was carried out in 0.1 M PBS at
378C using a modified dialysis method [20], which
allowed control of the pH of the release medium and
complete sample recovery. Dialysis membranes with
various MWCOs and dialysis tubing with a large
surface area were used to optimize the release
method by assessing the permeability of the peptide
through the membranes and the recovery of the
peptide. A 50-mg amount of microspheres was
suspended in 6 ml 0.1 M phosphate buffered saline
(pH 7.4, PBS) containing 0.02% sodium azide in a
7-ml Tube-O-Dialyzer  tube (1.5 cm i.d.35.5 cm
length). The dialysis tubes were capped with a
dialysis membrane-windowed screw cap (molecular
weight cut off 300 000), placed in a 4-l dialysis tank
filled with the same release medium and incubated at
378C with continuous agitation. At each time point,
remaining microspheres were recovered by filtration
through a 0.8-mm filter. The amount of leuprolide
remaining in the microspheres was determined by
HPLC as described previously.
2.5. In vitro polymer degradation
2.5.1. Mass loss and hydration
At each time point, recovered wet mass was
weighed accurately (wet weight, Ww ), dried for 48 h
under vacuum at room temperature and reweighed
309
(dry weight, Wd ). The mass remaining (MR) and the
degree of hydration (DH) were calculated as follows:
MR (%) 5 (Wd /W0 ) ? 100 (1)
DH 5 (Ww 2 Wd ) /Wd (2)
2.5.2. Molecular weight ( Mw ) of polymer
The molecular weight distribution of PLA was
determined by gel permeation chromatography
(GPC) as described previously [22]. A Waters M-45
solvent delivery system with a Waters 990 Photo-
diode Array Detector was used. Two Ultrastyragel
columns connected in series (7.83300 mm each, one
4 ˚ 3 ˚
with 10 -A pores and one with 10 -A pores) were
used. Samples, 1 mg / ml, were eluted with tetrahy-
drofuran at 1 ml / min. The weight average molecular
weight (Mw ) of each sample was calculated using
monodisperse polystyrene standards, molecular
weight 1000–50 000.
2.6. In vivo study
Male Sprague–Dawley rats (n56) weighing |300
g were used to evaluate in vivo performance of
leuprolide microspheres. The microspheres were
injected subcutaneously at the back of the neck (12
mg leuprolide / kg) after reconstitution in a suitable
vehicle (1% carboxymethylcellulose and 2% man-
nitol, w / v). Blood samples were collected from the
tail vein at specific time points. The samples were
centrifuged in Microtainer  tubes (Becton Dickin-
son, Franklin Lakes, NJ) and serum was collected.
Serum samples were frozen and stored at 2208C
until analysis.
Serum leuprolide levels in rats were assessed
using a radioimmunoassay (RIA) method as de-
scribed previously [23,24]. The lower detection limit
of the assay was 8 pg / ml. The intra- and interassay
coefficients of variation were 6 and 9%, respectively.
Serum testosterone levels were assayed using
ActiveE Testosterone RIA DSL-4000 kits (Diagnos-
tic Systems, Webster, TX). The lower limit of
detection for this assay was 0.08 ng / ml and the
intra- and interassay coefficients of variation were 10
and 9%, respectively. The cross-reactivity of the
testosterone antiserum was less than 6%.
310 B.H. Woo et al. / Journal of Controlled Release 75 (2001) 307 – 315
3. Results and discussion
3.1. Microsphere characterization
Leuprolide microspheres were spherical with a
relatively non-porous surface (Fig. 1, A). The aver-
age particle size was 51.7 mm, which is suitable for
intramuscular or subcutaneous injections. The target
load was 16.5% and the actual peptide content was
determined to be 13.4%. Leuprolide encapsulation
efficiency was 81.2%. The major difference between
the microspheres prepared in this study and the
microcapsules of 4-month Lupron  depot is the drug
distribution in the polymer matrix. Lupron  mi-
crocapsules are prepared using a water-in-oil-in-
water (w / o / w) emulsion technique and the particles
contain discrete internal pockets of drug, hence
called microcapsules. The microspheres in this study
are prepared from a clear homogeneous solution of
polymer and drug, and the drug is believed to be
molecularly distributed in the PLA matrix.
3.2. In vitro release
Table 1 indicates that membranes with higher
MWCOs showed higher rates of peptide permeabili-
ty, faster equilibrium between the dialysis cells and
the media and good peptide recovery. The dialysis
tubing with a large surface area showed complete
equilibrium after 48 h; however, it showed lower
peptide recovery compared to the dialysis cells due
to higher adsorption of the peptide on the large
surface area. Based on these results, a dialysis
membrane (MWCO 300 000) was selected for the in
vitro release study. The dialysis method used in this
study showed several advantages over the conven-
tionally used test tube methods. This method elimi-
nated: (a) the undesired loss of microspheres during
sample preparation and handling; (b) pH changes of
the release medium; and (c) shear stresses due to the
centrifugation for sample recovery. Use of higher
buffer capacity and frequent replacement of the
release medium in the conventional tube method
could also prevent a pH change. However, centrifu-
gation is necessary to remove the supernatant with-
out loss of microspheres for the conventional tube
method. This will cause the packing of microspheres
at the bottom of the tube that could affect the
physical characteristics of the microspheres.
The in vitro release study of leuprolide micro-
spheres is shown in Fig. 2. In the initial 15 days,
29% peptide released. Thereafter release was nearly
linear through day 150 with |0.5% released per day
during this period. Release in the first 24 h is |10%,
probably due to the fast diffusion of the surface-
associated peptide. The initial 24-h release is illus-
trated in the small panel. A 5% initial burst was
observed in 30 min, followed by a decrease to |2%
in the next 5 h. Then a linear release ensued
thereafter. The initial decrease in release in the first
few hours has been a typical observation with
leuprolide microspheres and the only reason which
can be offered is that adsorption of released peptide
occurs to the hydrated polymer surface in the early
phase of release. Once the binding sites are saturated
then the diffusional release proceeds.
3.3. In vitro polymer degradation
Both the peptide loaded and blank PLA micro-
spheres showed slow mass loss in the first 50 days
followed by linear profiles for 150 days (Fig. 3). The
initial mass loss within 24 h was only 2.0%, confirm-
ing that the initial release was caused by diffusion of
the peptide located on the surface of the micro-
spheres. Blank microspheres showed lower mass loss
than drug loaded microspheres. The peptide loaded
and blank microspheres were slowly hydrated within
50 days (Fig. 4). Hydration rate increased between
50 and 90 days, with the peptide loaded micro-
spheres showing a higher degree of hydration. The
drug loading of PLA microspheres led to higher
uptake of water into the polymer matrices and a
faster mass loss. The molecular weight of residual
polymers from both drug loaded and blank micro-
spheres gradually decreased from 11 000 to 7000 Da
after 90 days (Table 2). There was no significant
difference in polymer degradation rate between drug
loaded and blank microspheres. Blank and drug
loaded microspheres showed relatively smooth sur-
faces with spherical shape at the beginning of the
degradation study (Fig. 1). The drug loaded micro-
spheres possessed very porous interior structures
while the blank microspheres showed an extremely
shrunken shape after 35 days. After 70 days, the drug
 B.H. Woo et al. / Journal of Controlled Release 75 (2001) 307 – 315 311

Fig. 1. Scanning electron microscopy of: (a) leuprolide microsphere; (b) leuprolide microsphere incubated in PBS at 378C for 35 days; (c)
PLA blank microspheres; and (d) PLA blank microspheres incubated in PBS at 378C for 35 days.
312 B.H. Woo et al. / Journal of Controlled Release 75 (2001) 307 – 315

Table 1
Equilibrium of leuprolide through dialysis membranes and peptide
recovery
MWCO Dialysis Equilibrium Peptide
(Da) unit (%) recovery
(%)
8000 Dialysis cell 66.662.1 88.861.2
14 000 Dialysis cell 67.363.4 88.360.5
50 000 Dialysis cell 96.461.8 92.360.2
300 000 Dialysis cell 97.361.8 91.960.6
300 000 Dialysis tubing 102.562.2 77.062.4

loaded microspheres showed a deformed cake-like

shape compared to the lumpy shape of blank micro-
spheres. This suggests that the presence of peptide
affects the morphological changes in the polymer
matrix during in vitro release. The overall in vitro
release of the peptide correlated well with the
polymer degradation profile.
Fig. 3. Mass loss of peptide loaded and blank microspheres in
PBS at 378C (n53).
3.4. In vivo leuprolide level

Fig. 5A shows serum leuprolide levels in rats after release from the microspheres. After the initial
the subcutaneous administration of the microspheres. elevation, the levels fell to 4.3 ng / ml at day 15
Serum leuprolide levels increased immediately after followed by a plateau level around 2 ng / ml or less
administration to 45.6 ng / ml due to the initial burst over 120 days. A second dose of the microspheres at

Fig. 2. In vitro release of leuprolide loaded PLA microspheres in Fig. 4. Hydration of peptide loaded and blank microspheres in
PBS at 378C for 150 days (large panel) and initial release within PBS at 378C (n53).
24 h (small panel).
 B.H. Woo et al. / Journal of Controlled Release 75 (2001) 307 – 315
Fig. 5. In vivo serum leuprolide (A) and testosterone concen-
trations (B) after single administration of PLA microspheres
(dose: 12 mg / rat, n56, small panel) and a second dose (large
panel). Arrows in B indicate challenges of 100 mg / kg leuprolide.
123 days resulted in a serum peptide level similar to
the first dose.
3.5. In vivo testosterone suppression
The small panel of Fig. 5B shows testosterone
levels in rats after a single subcutaneous administra-
tion of the peptide-containing PLA microspheres.
313
Fig. 6. Comparison of in vitro leuprolide release profile with in
vivo release profile, the latter plotted as cumulative area under
serum peptide curve normalized as percent of the total area (large
panel) and in vitro–in vivo correlation plot (small panel).
The testosterone levels were suppressed to 0.5 ng / ml
at day 4 and the levels remained near this level for
50 days before falling below 0.5 ng / ml through day
120. The challenges of 100 mg of aqueous leup-
rolide / kg at 40, 64, 89, 105 and 120 days did not
elevate the testosterone levels, which suggests that
the pituitary LHRH receptors were occupied by the
analogue and down-regulated. Leuprolide challenges
at 135 and 160 days caused an elevation of the
testosterone level suggesting that the microspheres
were only effective for 120 days. The large panel of
Fig. 5B shows that following the initial elevation of
testosterone, the levels were suppressed below 0.5
ng / ml through 123 days and a second dose did not
cause a similar initial elevation of testosterone.
Fig. 6 compares the in vitro and in vivo release
profiles plotted as cumulative area under serum
leuprolide normalized as percent of the total area
between days 0 and 123 (total area under the curve
(AUC) 0 – 123d 5447 ng / day per ml). The overall in
vitro release rate was slower than the estimated in
vivo release. However, the in vitro release of leup-
rolide obtained with the modified dialysis method
314 B.H. Woo et al. / Journal of Controlled Release 75 (2001) 307 – 315

Table 2
Molecular weight changes of the residual polymer of leuprolide loaded and blank PLA microspheres
Time Drug loaded Blank
(day) Mw a Mn b PD c Mw Mn PD
0 10 100 6780 1.49 11 000 6830 1.61
7 9800 5850 1.68 10 700 6420 1.67
14 9710 5350 1.81 9830 5950 1.65
24 8870 5510 1.61 8780 5210 1.69
35 7910 4920 1.61 8530 6450 1.32
50 7830 5050 1.55 8540 5120 1.67
70 7340 4720 1.56 7610 5070 1.50
90 6910 4480 1.54 7060 4640 1.52
a
Weight average of molecular weight with three-significant figures.
b
Number average of molecular weight.
c
Polydispersity (PD 5 Mw /Mn ).

correlated rather well with the estimated in vivo
release (r50.995) as shown in Fig. 6, small panel.
4. Conclusions
A controlled release 4-month formulation of leup-
rolide microspheres was fabricated from PLA. A
dialysis method for measurement of in vitro release
was developed and successfully applied. In vitro
release of leuprolide correlated well with polymer
degradation as well as with in vivo release. The
leuprolide PLA microspheres are potentially useful
as a 120-day formulation for testosterone suppres-
sion.
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