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Journal of Controlled Release 75 (2001) 307–315

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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


eliminate the need for frequent administration. Bio-
Controlled release dosage forms have been investi- degradable microspheres were shown to improve the
bioavailability of peptides by protecting them from
*Corresponding author. Tel.: 11-859-257-1831; fax: 11-859- physical degradation and proteolysis in body fluids.
323-0242. Poly( D,L-lactide) (PLA) and poly( D,L-lactide-co-gly-
E-mail address: ppdelu1@pop.uky.edu (P.P. DeLuca). 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- 2. Materials and methods


degradable microspheres [1–8].
Leuprolide, the peptide drug used in this study, is 2.1. Materials
a potent agonist of luteinizing hormone-releasing
hormone (LHRH) currently used for the treatment of Leuprolide, [Des-Gly 10 , D-Leu 6 , Pro 9 ]-LHRH
prostatic cancer, endometriosis and precocious pu- ethylamide, was obtained from Bachem (Torrance,
berty [9,10]. Utilizing an in-water drying method CA, USA). Poly( D,L-lactide) (m.w. 11 000 Da,
based on the w / o / w emulsion technique, 1-, 3- and Resomer  R202H, PLA) was supplied by Boehringer
4-month release leuprolide microcapsules, Lupron  , Ingelheim (Ingelheim, Germany). Polyvinyl alcohol
have been developed [11–16]. In the in-water-drying (m.w. 30 000–70 000; PVA) was supplied by Sigma
method leuprolide acetate was dissolved in aqueous (St Louis, MO, USA). Dialysis tubes (Tube-O-
gelatin or water, dispersed into a polymer solution Dialyzer  ) were purchased from Research Products
which was then emulsified into an external aqueous International Co. (Mount Prospect, IL) and
phase to form droplets. The microcapsules solidified Spectrapor  CE dialysis membranes (MWCO 7000–
after solvent removal and were freeze-dried. Several 300 000 Da) were supplied by Spectrum Medical
publications have described in vivo pharmacological Industries (Houston, TX). All other chemicals were
and pharmacokinetic evaluations of these microcap- obtained commercially as analytical grade reagents.
sules [12–16].
In most literature reports of in vitro release, the 2.2. Preparation of microspheres
microspheres were incubated in a certain volume of
release medium, generally phosphate buffered saline Leuprolide PLA microspheres were prepared by
(PBS) with a preservative, using glass or plastic test dispersing the homogeneous solution of polymer and
tubes at 378C with optional agitation [17]. In these drug into a PVA solution followed by solvent
methods, the pH of the release medium consistently extraction / evaporation / dilution as previously de-
dropped due to the formation and accumulation of scribed [21]. A solution of leuprolide in methanol
acidic polymer degradation products [18,19]. Con- was added into a 34% (w / w) solution of polymer in
tinuous removal of the acidic polymer fragments and methylene chloride to form the clear solution. The
released drug by dialysis might be desirable to resulting solution was then slowly injected into
prevent the pH drop of the release medium. A 0.35% aqueous PVA solution while mixed with a
dialysis in vitro release method for an LHRH Silverson  L4R mixer (Silverson Machines, MA,
antagonist, orntide, loaded PLGA microspheres has USA) at 7000 rpm. The solvents were removed by
been developed by using a small dialysis unit and 0.1 stirring and continuous phase replacement at 38–
M acetate buffer, pH 4.0 [20]. The in vitro release 408C for 1 h. The solidified microspheres were
profile obtained with the dialysis method permitted a recovered by filtration and dried under vacuum at
better correlation with in vivo release. However, only room temperature for 48 h.
a little information is available on in vitro degra-
dation kinetics and correlation between in vitro and 2.3. Particle characterization
in vivo release of long-term release peptide loaded
PLA microspheres. 2.3.1. Particle size distribution
The goal of this study was to prepare and char- Particles were sized by laser diffractometry using
acterize a 4-month formulation of leuprolide micro- a Malvern 2600 laser sizer (Malvern 2600c Particle
spheres using a dispersion method and to study in Sizer, Malvern, UK). The average particle size was
vitro drug release and in vivo efficacy. Also, an expressed as the volume mean diameter Vmd in
attempt to develop a modified dialysis method for microns.
testing in vitro release from biodegradable micro-
spheres, which would exhibit a good in vitro–in vivo 2.3.2. Surface morphology
correlation, was undertaken. The surface morphology was examined by scan-
B.H. Woo et al. / Journal of Controlled Release 75 (2001) 307 – 315 309

ning electron microscopy (Hitachi Model S800, (dry weight, Wd ). The mass remaining (MR) and the
Japan) after palladium / gold coating of the micro- degree of hydration (DH) were calculated as follows:
sphere sample on an aluminum stub.
MR (%) 5 (Wd /W0 ) ? 100 (1)
2.3.3. Peptide content
DH 5 (Ww 2 Wd ) /Wd (2)
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 2.5.2. Molecular weight ( Mw ) of polymer
of 10 ml of 0.1 M acetate buffer (pH 4.0) followed The molecular weight distribution of PLA was
by agitation for 1 h. The peptide was assayed in the determined by gel permeation chromatography
aqueous phase by HPLC method: Bondclone 10 C 18 (GPC) as described previously [22]. A Waters M-45
column (15033.9 mm; Phenomenex, Torrance, CA, solvent delivery system with a Waters 990 Photo-
USA); mobile phase: 27% (v / v) acetonitrile, 0.1% diode Array Detector was used. Two Ultrastyragel
trifluoroacetic acid; flow rate 1.0 ml / min; UV de- columns connected in series (7.83300 mm each, one
4 ˚ 3 ˚
tection at 215 nm. 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
2.4. In vitro release study weight (Mw ) of each sample was calculated using
monodisperse polystyrene standards, molecular
In vitro release was carried out in 0.1 M PBS at weight 1000–50 000.
378C using a modified dialysis method [20], which
allowed control of the pH of the release medium and
2.6. In vivo study
complete sample recovery. Dialysis membranes with
various MWCOs and dialysis tubing with a large
Male Sprague–Dawley rats (n56) weighing |300
surface area were used to optimize the release
g were used to evaluate in vivo performance of
method by assessing the permeability of the peptide
leuprolide microspheres. The microspheres were
through the membranes and the recovery of the
injected subcutaneously at the back of the neck (12
peptide. A 50-mg amount of microspheres was
mg leuprolide / kg) after reconstitution in a suitable
suspended in 6 ml 0.1 M phosphate buffered saline
vehicle (1% carboxymethylcellulose and 2% man-
(pH 7.4, PBS) containing 0.02% sodium azide in a
nitol, w / v). Blood samples were collected from the
7-ml Tube-O-Dialyzer  tube (1.5 cm i.d.35.5 cm
tail vein at specific time points. The samples were
length). The dialysis tubes were capped with a
centrifuged in Microtainer  tubes (Becton Dickin-
dialysis membrane-windowed screw cap (molecular
son, Franklin Lakes, NJ) and serum was collected.
weight cut off 300 000), placed in a 4-l dialysis tank
Serum samples were frozen and stored at 2208C
filled with the same release medium and incubated at
until analysis.
378C with continuous agitation. At each time point,
Serum leuprolide levels in rats were assessed
remaining microspheres were recovered by filtration
using a radioimmunoassay (RIA) method as de-
through a 0.8-mm filter. The amount of leuprolide
scribed previously [23,24]. The lower detection limit
remaining in the microspheres was determined by
of the assay was 8 pg / ml. The intra- and interassay
HPLC as described previously.
coefficients of variation were 6 and 9%, respectively.
Serum testosterone levels were assayed using
2.5. In vitro polymer degradation ActiveE Testosterone RIA DSL-4000 kits (Diagnos-
tic Systems, Webster, TX). The lower limit of
2.5.1. Mass loss and hydration detection for this assay was 0.08 ng / ml and the
At each time point, recovered wet mass was intra- and interassay coefficients of variation were 10
weighed accurately (wet weight, Ww ), dried for 48 h and 9%, respectively. The cross-reactivity of the
under vacuum at room temperature and reweighed testosterone antiserum was less than 6%.
310 B.H. Woo et al. / Journal of Controlled Release 75 (2001) 307 – 315

3. Results and discussion at the bottom of the tube that could affect the
physical characteristics of the microspheres.
The in vitro release study of leuprolide micro-
3.1. Microsphere characterization
spheres is shown in Fig. 2. In the initial 15 days,
29% peptide released. Thereafter release was nearly
Leuprolide microspheres were spherical with a
linear through day 150 with |0.5% released per day
relatively non-porous surface (Fig. 1, A). The aver-
during this period. Release in the first 24 h is |10%,
age particle size was 51.7 mm, which is suitable for
probably due to the fast diffusion of the surface-
intramuscular or subcutaneous injections. The target
associated peptide. The initial 24-h release is illus-
load was 16.5% and the actual peptide content was
trated in the small panel. A 5% initial burst was
determined to be 13.4%. Leuprolide encapsulation
observed in 30 min, followed by a decrease to |2%
efficiency was 81.2%. The major difference between
in the next 5 h. Then a linear release ensued
the microspheres prepared in this study and the
thereafter. The initial decrease in release in the first
microcapsules of 4-month Lupron  depot is the drug
few hours has been a typical observation with
distribution in the polymer matrix. Lupron  mi-
leuprolide microspheres and the only reason which
crocapsules are prepared using a water-in-oil-in-
can be offered is that adsorption of released peptide
water (w / o / w) emulsion technique and the particles
occurs to the hydrated polymer surface in the early
contain discrete internal pockets of drug, hence
phase of release. Once the binding sites are saturated
called microcapsules. The microspheres in this study
then the diffusional release proceeds.
are prepared from a clear homogeneous solution of
polymer and drug, and the drug is believed to be
3.3. In vitro polymer degradation
molecularly distributed in the PLA matrix.
Both the peptide loaded and blank PLA micro-
3.2. In vitro release spheres showed slow mass loss in the first 50 days
followed by linear profiles for 150 days (Fig. 3). The
Table 1 indicates that membranes with higher initial mass loss within 24 h was only 2.0%, confirm-
MWCOs showed higher rates of peptide permeabili- ing that the initial release was caused by diffusion of
ty, faster equilibrium between the dialysis cells and the peptide located on the surface of the micro-
the media and good peptide recovery. The dialysis spheres. Blank microspheres showed lower mass loss
tubing with a large surface area showed complete than drug loaded microspheres. The peptide loaded
equilibrium after 48 h; however, it showed lower and blank microspheres were slowly hydrated within
peptide recovery compared to the dialysis cells due 50 days (Fig. 4). Hydration rate increased between
to higher adsorption of the peptide on the large 50 and 90 days, with the peptide loaded micro-
surface area. Based on these results, a dialysis spheres showing a higher degree of hydration. The
membrane (MWCO 300 000) was selected for the in drug loading of PLA microspheres led to higher
vitro release study. The dialysis method used in this uptake of water into the polymer matrices and a
study showed several advantages over the conven- faster mass loss. The molecular weight of residual
tionally used test tube methods. This method elimi- polymers from both drug loaded and blank micro-
nated: (a) the undesired loss of microspheres during spheres gradually decreased from 11 000 to 7000 Da
sample preparation and handling; (b) pH changes of after 90 days (Table 2). There was no significant
the release medium; and (c) shear stresses due to the difference in polymer degradation rate between drug
centrifugation for sample recovery. Use of higher loaded and blank microspheres. Blank and drug
buffer capacity and frequent replacement of the loaded microspheres showed relatively smooth sur-
release medium in the conventional tube method faces with spherical shape at the beginning of the
could also prevent a pH change. However, centrifu- degradation study (Fig. 1). The drug loaded micro-
gation is necessary to remove the supernatant with- spheres possessed very porous interior structures
out loss of microspheres for the conventional tube while the blank microspheres showed an extremely
method. This will cause the packing of microspheres 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 – 315 313

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
Fig. 5. In vivo serum leuprolide (A) and testosterone concen-
the pituitary LHRH receptors were occupied by the
trations (B) after single administration of PLA microspheres
(dose: 12 mg / rat, n56, small panel) and a second dose (large analogue and down-regulated. Leuprolide challenges
panel). Arrows in B indicate challenges of 100 mg / kg leuprolide. 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
123 days resulted in a serum peptide level similar to ng / ml through 123 days and a second dose did not
the first dose. 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
3.5. In vivo testosterone suppression between days 0 and 123 (total area under the curve
(AUC) 0 – 123d 5447 ng / day per ml). The overall in
The small panel of Fig. 5B shows testosterone vitro release rate was slower than the estimated in
levels in rats after a single subcutaneous administra- vivo release. However, the in vitro release of leup-
tion of the peptide-containing PLA microspheres. 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 ).

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