IN VITRO STUDIES

2. IN VITRO STUDIES

2.1. Characterization Studies The morphology and chemical homogeneity of active and pla-

cebo microspheres were investigated using scanning electron microscopy (SEM), mercury intrusion porosimetry, infrared spectroscopy (IR), and static secondary ion mass spectrome- try (SSIMS). The changes that take place during in vitro dissolution testing were studied using these techniques and in addition nuclear magnetic resonance spectroscopy (NMR) and gel permeation chromatography (GPC) were used.

Freshly manufactured microspheres were all similar in that they comprised spherical or near-spherical particles in the size range 25–125 mm ( Fig. 1 ).

In Vitro=In Vivo Release from Injectable Microspheres 545

Figure 1 SEM image showing freshly manufactured active micro- spheres (

SEM images at high magnification (Fig. 2) showed the presence of a discontinuous surface coating of rod-like crys- tals, determined by IR and SSIMS to be mannitol (containing residual drug in the case of the active microspheres). Further- more, the active microspheres, but not the placebo, exhibited voids that were confined to the surface layer of the particles. These were distributed randomly, in patches or in bands,

Figure 2 SEM images of placebo (left) and active (right) micro- sphere sections (

546 Clark et al.

and were shown by mercury intrusion porosimetry to be non- porous.

Cross-sections of active microspheres, embedded in epoxy resin, were examined, in a north–south and east–west direc- tion at 10 mm intervals to determine the distribution of drug throughout the polymer matrix (Fig. 3). The data, expressed as peak area for each absorption band examined, show a homogeneous distribution of drug associated with polymer throughout the sample.

Further SEM analysis was performed on cross-sections of the agglomerated mass evident following 7 days exposure to dissolution medium in a preliminary in vitro release test. In this test, 10 mg of microspheres were placed in a vial, 10 mL of pre-heated isotonic phosphate-buffered saline (PBS, pH 7.4) was added, and the vial sealed and incubated at 37

C without agitation. At pre-determined time-points (typically 1,4,7,11,14,18,21 and 28 days), 5 mL samples of supernatant liquid were withdrawn and the concentration of dissolved drug measured by UV spectroscopy, and 5 mL of pre-heated replacement buffer were added to the dissolution vial to maintain constant volume. Sink conditions may be

Figure 3 IR imaging of a sectioned active microsphere, showing chemical homogeneity for both north–south (left hand group, 1–20 mm) and east–west (right hand group, 22–41 mm) traverses, as evidenced by constant active =PLGA ratio.

In Vitro=In Vivo Release from Injectable Microspheres 547

Figure 4 SEM images ( active (right) microspheres after 7 days of the preliminary in vitro release test (isotonic PBS, pH 7.4, 37 C).

assumed as the solubility of the active substance is greater than 100 mg =mL in the dissolution medium.

Low magnification images (Fig. 4) reveal extensive agglo- meration of microspheres; the image for the active sample shows the presence of partially agglomerated microspheres at the edge of the mass. Also apparent is the development of porosity, extensive in the case of the placebo sample.

High magnification images (Fig. 5) show that extensive porosity is also apparent in the active sample, pores generally being small in comparison with those in the placebo sample.

SSIMS and IR spectroscopy showed that the drug was still distributed throughout the bulk of these samples and that the coating of mannitol was no longer present.

Figure 5 SEM images as for Fig. 4, but

548 Clark et al.

Further investigations after 21 days of the preliminary in vitro release test established that the remaining undissolved material was semi-liquid and therefore not amenable to SEM analysis. A combination of NMR, IR, SSIMS, and GPC was used to investigate chemical changes in the polymer that may have occurred during in vitro testing. GPC clearly shows

a reduction in its molecular weight after 7 days of in vitro test- ing and this reduction was greater for the placebo than for the samples containing the drug. After 21 days of the preliminary in vitro release test, only low molecular weight oligomeric species were found by GPC (Fig. 6 and Fig. 7 ).

NMR revealed that the lactide content had increased, in comparison to glycolide, but no monomeric species was detected. Furthermore SSIMS did not detect degradation pro- ducts or additional contamination on the ‘‘day 7’’ samples.

IR spectroscopy showed that some of the peaks in the IR spectra of the drug occurred at slightly different positions when the drug was incorporated into the microspheres (specifically a shift in the peptide backbone carbonyl signal from 1645 to 1670

Figure 6 GPC volume-elution curves for active microspheres ‘‘as manufactured’’ and after 7 and 21 days of the preliminary in vitro release test (isotonic PBS, pH 7.4, 37 C).

In Vitro=In Vivo Release from Injectable Microspheres 549

Figure 7 GPC volume-elution curves for placebo microspheres ‘‘as manufactured’’ and after 7 and 21 days of the preliminary in vitro release test (isotonic PBS, pH 7.4, 37 C).

cm ). This indicates an interaction (possibly hydrogen bond- ing) between the drug and the PLGA copolymer.

The glass transition temperature, T g , of the active microspheres was found to vary from 43.3 to 45.6

C for freshly manufactured microspheres. After exposure to dissolution med- ium for 24 h, the T g was re-measured and values were found to vary from 25.3 to 27.7

C, indicating extensive hydration. In summary, it appears that active microspheres are che- mically homogeneous save for a discontinuous surface coating of mannitol enriched with drug. The loss of this coating is rapid, and the surface drug is likely to contribute significantly to the initial burst in the in vitro release profile. It is consid- ered likely that the surface voids apparent in the SEM images were formed in the later stages of the microsphere-hardening process due to the loss of solvent from the globule surface, and are absent from the placebo due to reduced viscosity. The alternative hypothesis that voids are due to loss of active drug into the aqueous phase during hardening is not supported, as IR spectroscopy gave no indication of surface drug depletion.

550 Clark et al.

Figure 8 Annotated in vitro release profile for the experimental microspheres in the preliminary in vitro release test (isotonic PBS, pH 7.4, 37 C), indicating the predominant factors for each phase of drug release.

Drug release in the preliminary in vitro release test is characterized by agglomeration =amalgamation of micro- spheres into a continuous mass with a network of voids. Continuous release of drug occurs by a process involving hydrolytic degradation, reduction in molecular weight, ero- sion, channel formation and diffusion, as indicated in Fig. 8.

2.2. Stability Studies Stability studies on active microspheres (pre- and post-

irradiated) were carried out and the results are summarized in Tables 1 and 2 .

2.2.1. Summary of Stability Results

A preliminary stability study encompassing light exposure [ >1.2 million lux-hours, alongside a light-protected (dark) control to assess the effect of temperature variations within the light cabinet], postulated long-term storage conditions (4

C, 25 C =60% RH), accelerated conditions (30 C =60% RH)

In Table 1 Stability Results for Non-Irradiated Active Microspheres (6 min Time-Point)

Vitro

In Parameter = Initial 4 C Light Dark control % RH % RH 40 C 50 C Vivo Appearance

25 C =60

30 C =60

An off-white aggregate Active agent

A homogeneous, white, free-flowing solid

98.7 94.7 Release Total impurities, %

4.81 4.36 4.45 4.74 4.29 4.53 5.68 10.9 Water content, %w =w

0.7 0.6 1.0 1.0 0.7 0.9 0.5 0.4 Residual solvents, %w =w

1.9 2.0 1.8 1.7 1.5 0.3 1.5 1.6 from Weight-average molecular

11.2 10.7 7.5 4.9 weight (Mw), kDa

N =T

10.0 N =T

N =T

Injectable Burst, %

0.5 0.1 19.1 65.3 N =T: not tested.

Microspheres Table 2 Stability Results for g-Irradiated Active Microspheres (6 min Time-Point)

40 C 50 C Appearance

Initial

4 C Light

Dark control

% RH

% RH

An off-white aggregate Active agent

A homogeneous, white, free-flowing solid

96.1 97.4 90.8 98.7 96.1 94.7 92.1 84.2 Total impurities, %

6.80 6.53 6.84 6.25 6.72 9.43 8.05 13.8 Water content,

0.8 0.7 1.0 0.8 0.8 1.0 0.7 0.4 %w =w Residual solvents,

1.8 1.8 1.7 1.6 1.3 0.2 1.0 1.1 %w =w Weight-average molecular

9.4 9.1 6.7 4.4 551 weight (Mw), kDa Burst, %

N =T

10.5 N =T

N =T

0.5 3.2 27.5 78.4 N =T: not tested.

© 2005 by Taylor & Francis Group, LLC

552 Clark et al.

and stress conditions (40

C, 50 C), was conducted over a per- iod of 6 months. Samples were stored in unopened vials, within a secondary aluminum container except for the light exposure samples. All vials were stored inverted with the exception of the control samples at 4

C and 25 C =60% RH, in order to assess the potential for interaction with the closure system. A summary of the results is presented in Table 1 (non-irradiated active microspheres) and Table 2 (g-irradiated microspheres). All active agent results are expressed as a percentage of the initial assay for the non-irradiated micro- sphere so that degradation due to g-irradiation and storage can be assessed.

The results for active microspheres demonstrate that terminal sterilization by g-irradiation at a standard dose of

25 kGy gives rise to immediate degradation leading to an approximate 4% loss of active agent and a corresponding approximate 2% increase in total impurities; the poor mass- balance was not investigated but is assumed to be due to chro- mophore degradation and =or the formation of drug-polymer adducts not eluted by the chromatographic assay procedure. Non-irradiated microspheres appear to be stable at conditions

up to and including 30 C =60% RH, and to be unaffected by light exposure, whereas g-irradiated microspheres exhibit some degradation in light and at 30 C =60% RH. At higher temperatures, degradation is significant and temperature related, and is more pronounced for g-irradiated microspheres. Degradation is indicated by aggregation,

a reduction in active agent assay, a reduction in polymer mole- cular weight, an increase in total impurities, and an increase, very marked for the 40

C storage conditions, in the in vitro burst. The subsequent release profile is much less affected by storage conditions, as shown in Figs. 9 and 10 . The increase in the in vitro burst correlates with the observed decrease in weight average molecular weight, and is considered to arise partly due to an increase in erosion (loss of polymer and associated drug from the surface of the dissoluting material) and partly due to an increase in drug mobility arising from the reduction in polymer viscosity.

C and 50

In Vitro=In Vivo Release from Injectable Microspheres 553

Figure 9 In vitro release profiles (isotonic PBS, pH 7.4, 37 C) for the non-irradiated stability batch after 6 months storage at 4 C, 25 C =60% RH (‘25=60’), 30 C =60% RH (‘30=60’), 40 C =75% RH (‘40 =75’), and 50 C.

Figure 10 In vitro release profiles (isotonic PBS, pH 7.4, 37 C) for the g-irradiated stability batch after 6 months storage at 4 C, 25 C =60% RH (‘25=60’), 30 C =60% RH (‘30=60’), 40 C =75% RH (‘40 =75’) and 50 C.

554 Clark et al.

The results of the exploratory stability study are consid- ered to support a storage life, for g-irradiated microspheres, of

6 months when stored at 2–8

C in the dark.

2.3. Comparison of In Vitro Release Profiles Microspheres manufactured at laboratory (F2) and pilot (F1)

scale were compared using the preliminary dissolution test (see Sec. 2.1 ) (Fig. 11). No formal comparison of release pro- files could be made, as the available data were insufficient to support the development of a mathematical model, and the use of different test time-points prohibited the determina- tion of similarity =difference factors. However, the correspon- dence of the middle section (7–21 days) of the release profiles for F1 and F2 was considered sufficient to support the use of pilot scale material for in vivo studies.

2.4. Preliminary In Vitro–In Vivo Correlation The preliminary in vitro release test described in Sec. 2.1 was

considered deficient in that microspheres are uncontained,

Figure 11 Comparison of in vitro release profiles in the prelimin- ary in vitro release test (isotonic PBS, pH 7.4, 37

C) for laboratory (F2) and pilot (F1) scale microsphere batches, showing correspon- dence between 7 and 21 days.

In Vitro=In Vivo Release from Injectable Microspheres 555

leading to sampling difficulties and poor reproducibility at early time-points; release after 28 days was low in comparison with in vivo data, perhaps as a result of sample agglomeration in the non-agitated dissolution vessel, and the test did not dis- criminate between formulation variants expected to exhibit different release profiles.

An improved in vitro release test was developed, using a sequential factorial experimental design (FED) approach to maximize discrimination between two batches known to differ with regard to in vivo release profile.

The factors investigated in the FED studies are shown in Table 3. Temperature and pH of the dissolution medium were identified as being the most important factors, followed by the chemical composition and osmolarity of the buffer system, sample size and mixing by agitation. Other factors were of lesser importance.

The following test parameters were selected for further investigation: 100 mg of microspheres were placed in a 4 cm length of dialysis tubing and the ends clipped. The tubing was placed in a 60 mL amber glass vial, 25 mL of pre-heated

Table 3 Variable Factors Investigated in FED Optimization Studies for the In Vitro Release Method

Variable

Significance Buffer system

Range investigated

Intermediate citrate =phosphate=borate Buffer pH

Acetate, phosphate,

High Buffer osmolarity

Intermediate Osmolarity adjuster

200–500 mOsmol =L

NaCl, Na 2 SO 4 Intermediate Temperature

30–45 C High Catalyst

Low Surfactant

Piperidine 0–1.0%

Polyethylene glycol,

Low

Sample containment None, dialysis membrane Low

(8000 Da cut-off)

Agitation

Intermediate (after day 12) Sample size

Y =N

10–100 mg

Intermediate

556 Clark et al.

isotonic citrate-phosphate-borate buffer (pH 9.5) containing 0.5% surfactant added (this was done to aid wetting of the sample, even though the FED study did not identify surfac- tant as an important variable), the vial sealed, and incubated at 37

C with continuous agitation. At pre-determined time- points (typically 1,4,7,11,14,18,21 and 28 days), 5 mL samples of supernatant liquid were withdrawn and the concentration of the dissolved drug measured by UV spectroscopy, and

5 mL of pre-heated replacement buffer was added to the disso- lution vial to maintain constant volume. Sink conditions may

be assumed, even within the dialysis tubing, as the solubility of the active substance is greater than 100 mg =mL of dissolu- tion medium.

This dialysis sac diffusion method was applied to three active microsphere batches formulated to give low (appro- ximately 1%), intermediate (approximately 10%), and high (approximately 20%) burst in vivo using a rat model; in vivo

and in vitro release profiles are compared in Figs. 12– 14 . The purpose of the comparison was primarily to establish whether the in vitro release method was capable of discrimi-

Figure 12 Comparison of in vivo and in vitro release for a low- burst batch (dialysis sac diffusion method, citrate-phosphate-borate buffer, pH 9.5, 37 C).

In Vitro=In Vivo Release from Injectable Microspheres 557

Figure 13 Comparison of in vivo and in vitro release for an intermediate batch (dialysis sac diffusion method, citrate- phosphate-borate buffer, pH 9.5, 37 C).

Figure 14 Comparison of in vivo and in vitro release for a high- burst batch (dialysis sac diffusion method, citrate-phosphate-borate buffer, pH 9.5, 37 C).

558 Clark et al.

nating between batches differing in initial burst, and there- fore in vitro testing was not continued to 100% release. The in vitro method places the batches in the correct rank order with regard to in vivo release. Pooling data for the low-, inter- mediate-, and high-burst batches and performing a regression analysis resulted in a second-order polynomial fit with an R 2 value of 0.88, as shown in Fig. 15.

The in vitro–in vivo correlation is considered preliminary as the measurement of in vitro release was not continued until 100% release; the in vivo and in vitro sampling regimes differ and it is possible that the in vivo measurements do not adequately model the initial burst release. For the correlation to be useful in assuring the performance of a marketed pro- duct, these issues would need to be addressed and studies repeated in human subjects. However, the preliminary in vitro =in vivo correlation was considered adequate to support the use of the in vitro release method for the selection of batches for toxicological and pharmacokinetic studies in animal models.

Figure 15 Preliminary IVIVC for high-, intermediate-, and low- burst batches (dialysis sac diffusion method, citrate-phosphate-borate buffer, pH 9.5, 37 C).

In Vitro=In Vivo Release from Injectable Microspheres 559

2.5. Suspending Medium The experimental formulation is stored dry and is mixed with

an aliquot of suspending medium immediately prior to admin- istration. The suspending medium is an isotonic solution of sodium carboxymethylcellulose (viscosity improver), polysor- bate 80 (surfactant), and sodium chloride (tonicity adjuster) designed to hold microspheres in a homogeneous suspension to allow withdrawal and administration of an accurate dose.

2.5.1. Investigation of Dose Homogeneity for Repeat Dosing

Microspheres for administration in pre-clinical studies were supplied in multi-dose vials and the viability of sequential withdrawal of individual doses at 5 min intervals was investi- gated. The results are presented in Table 4.

The results exhibit some variability but were considered to support the use of this dosing regimen in pre-clinical studies.

2.5.2. Release of Drug into Suspending Medium It was intended that repeat dosing would take place over a

period of about 30 min; a study to assess the extent of release of drug into the suspending medium at room temperature, prior to injection, was performed and the results are presented in Table 5 .

Table 4 Homogeneity of Suspension Dose number

Nominal dose (%) 1 71.2

7 103.0 Average

91.9 RSD (%)

560 Clark et al. Table 5 Drug Release from Microspheres into Suspending

Medium Drug release (% nominal) 1.0 g =2.5 mL

1.0 g =4.5 mL Months

1.0 g =3.4 mL

The extent of drug release into the suspending medium under the conditions of the study was considered to be accep- table for all dilutions, and this administration procedure was applied in pre-clinical dosing.

2.5.3. Stability of Suspending Medium While the suspending medium was shown to be stable under

normal conditions ( Table 6 ), some degradation was evident at high temperatures (40

C and above) and on exposure to light. Degradation was characterized by a reduction in pH, an increase in osmolality, and a reduction in viscosity. After

C, slight turbidity was observed. These changes are thought to be due to breakdown of sodium carbox- ymethylcellulose and polysorbate 80 leading to the formation of acidic contaminants.

6 months at 50

The data are considered to support an interim storage life of 12 months at 20–25

C in the designated container.

3. IN VIVO STUDY This study was primarily performed to ensure that the larger

(pilot scale) batch and new suspending media produced a for- mulation with a similar release profile in vivo to that pro- duced by an earlier smaller (laboratory scale) batch and using a different suspending medium. The effect of dose volume (microsphere concentration) was also investigated.

In Vitro

= In Vivo

Release

Table 6 Stability Results for Suspending Medium (6 m Time-Point) from

Injectable Parameter

Dark

Initial 4 C Light control 25 C =60% RH 30 C =60% RH 40 C 50 C Appearance

Clear colorless solution free from extraneous matter

Slightly opaque solution Microspheres

pH 5.4 5.3 5.1 5.3 5.2 5.0 4.8 4.5 Volume average (mL)

5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 Osmolality (mOsm =kg)

312 Viscosity (cP)

© 2005 by Taylor & Francis Group, LLC

562 Clark et al.

3.1. Experimental Procedures

3.1.1. Formulations Larger batch manufacture (F1) in suspending medium 1 (lower

viscosity): Injection volume ¼ 0.62 mL

32 mg mL drug concentration (40% w =v microspheres) Injection volume ¼ 0.83 mL

24 mg mL drug concentration (31% w =v microspheres) Injection volume ¼ 1.25 mL

16 mg mL drug concentration (21% w =v microspheres)

Smaller scale manufacture (F2) in suspending medium 2 (higher viscosity): Injection volume ¼ 1.5 mL

13.3 mg mL drug concentration (19% w =v microspheres)

3.1.2. Study Design Four dosing groups of 12 male Sprague–Dawley rats (48 in total,

390–453 g) received a single subcutaneous injection in the scruff of the neck containing 20 mg of drug formulated as described in Sec. 3.1.1. Each dosing group was sub-divided into four sam- pling groups for removal of blood samples at various times up to 42 days post-dose. On termination, a representative number of injections sites were dissected and any formulation remaining assayed for drug. Four sampling groups were employed to ensure that sample volumes did not exceed accepted limits as discussed in Sec. 3.2.2 of Chapter 4 . This approach means that only mean data can be reported. Rats were chosen as the least neurophysiological-sensitive animal suitable to meet the aims of the study. The use of this rat data in an attempt to start to establish IVIVCs suffers from the fact that mean data must be used and the reference formulation must be dosed to a sepa- rate group of animals (see Chapter 5 ). Blood was collected into tubes containing protease inhibitors that had previously been stored in iced water; the plasma was then immediately sepa- rated by centrifugation at 4

C and the plasma transferred to tubes and frozen immediately. Drug plasma concentrations were determined by high per- formance liquid chromatography and electrospray ionization

In Vitro=In Vivo Release from Injectable Microspheres 563

tandem mass spectroscopy after solid phase extraction of the drug. The amount of drug remaining encapsulated at the injec- tion site was determined by HPLC-UV after extraction. All data manipulations were carried out using standard methods. The concentrations were adjusted for drug dose and rat weight as appropriate. When the drug concentration was below the limit of quantification it was considered to be 0 ng mL in ani- mals which received a full dose and for animals that did not receive the full dose, that data point was removed from the cal- culation of mean concentration for that time-point. The area under the plasma concentration (AUC) time curve was deter- mined using the linear trapezoidal rule on mean data due to sparse sampling points for individual animals. The burst, defined as the release over 1.5 days as a proportion of the release over 30 days, was approximated by comparing AUC for these time intervals. The fraction absorbed vs. time was also approximated by comparing the AUC for the time interval to the AUC from 0 to 42 days for that formulation or to the AUC from 0 to 42 days measured for the F1 injection volu- me ¼ 0.83 mL formulation. It should be noted that approximating the absorption rate using AUC, for these data, might underesti- mate the rate of absorption at the earlier time-points (days 1– 3). As this was an early development study, sophisticated IVIVC approaches (inclusion of reference formulation) were not pursued. Later studies did incorporate these approaches.

3.2. Results and Discussion Mean drug plasma concentration times curves after adminis-

tration of drug encapsulated in microspheres from the larger and smaller batches are shown in Fig. 16 . Pharmacokinetic parameters for the data are given in Table 7 .

Larger batch formulations produced a greater initial drug plasma concentration than that from the smaller scale formu- lation. The plasma concentration then declined to a nadir at days 4–5 which was earlier and lower than for the smaller scale batch. The plasma concentration rose to a ‘‘plateau’’ value at days 7–9 and then tracked the smaller scale formula- tion. Comparison across the three F1 formulations suggests that, as injection volume increased (from 0.62 to 1.25 mL) the

Clark Figure 16 Mean drug plasma concentration time curves after administration of 20 mg of drug encapsulated

in microspheres manufactured on two different scales (n ¼ 3, mean et total).

al.

© 2005 by Taylor & Francis Group, LLC

In Vitro

= In Vivo

Table 7 Mean PK Parameters after SC Administration of 20 mg of Drug Encapsulated in Microspheres Release Pharmacokinetic

Formulation F1

Formulation F2

parameter Inj. vol. ¼ 0.627 mL Inj. vol. ¼ 0.83 mL Inj. vol. ¼ 1.25 mL Inj. vol. ¼ 1.5 mL from AUC (0–1.5d) (ng mL

Injectable AUC (0–4d) (ng mL

day) a 64.8 85.8 99.9 61.23

78.9 AUC (0–28d) (ng mL

day)

196.4 AUC (0–30d) (ng mL

day) a 191.0

212.1 AUC (0–42d) (ng mL

day) a 213.6

day) a 265.5

73.5 t max (day)

C max (ng mL ) a 133.5

0.25 0.25 0.25 0.5 C ss,av 7–28 days (ng mL ) a 5.56 7.60 4.83 5.11 Drug remaining at injection site (mg)

1.54 b 0.38 b n.d. c 0.34 b Burst (%)

30.3 29.7 42.7 28.9 a Values normalized for 272 g rat weight.

b Mean n ¼ 2. c Not determined.

© 2005 by Taylor & Francis Group, LLC

566 Clark et al.

release rate (absorption rate) of the drug increased ( Fig. 17 ). That is, F1 injection volume 1.25 mL gave the greatest burst and then the lowest plateau concentration (C ss,av 7–28 days) suggesting that the rapid early release meant that there was less drug for release later on ( Table 7 ). The total amount released was similar to that for the F1 injection volume

0.62 mL as AUCs (0–42d) were comparable for both formulations. The F1 injection volume 0.83 mL gave the same percent burst as the F1 injection volume 0.62 mL solids formulation; how- ever, the C ss,av 7–28 days was higher as was the AUC (0–42d) sug- gesting prolonged faster release (Table 7). This is supported by the apparently lower quantity of drug left at the injection site for the F1 injection volume 0.83 mL formulation relative to the F1 injection volume 0.62 mL formulation (Table 7), although the small sample size should be noted. These observa- tions are probably explained by the injection volume which led to different extents of dispersion within the subcutaneous tis- sue (see Sec. 3.3). That is, the larger injection volumes pro- duced greater dispersion and therefore greater formulation surface area, which might be expected to produce greater drug release. The relative surface area differences due to dispersion extent may have been accentuated by the agglomeration of microspheres, which was also seen in the in vitro tests ( Fig.

4 ). The nature of the injection vehicle may be important as, although the F2 injection volume 1.5 mL formulation was dosed in a volume most similar to the F1 injection volume

1.25 mL formulation, the subcutaneous dispersion and phar- macokinetic parameters were most similar to the F1 injection volume 0.83 mL formulation. Possibly the greater viscosity of the F2 media reduced dispersion in the subcutaneous tissue.

3.3. Injection Site Injection of the formulation produced the expected tissue

reaction (see Sec. 3 of Chapter 4). For the F1 injection volume

0.62 mL formulation, the subcutaneous mass was well defined ( Fig. 18 ), the formulation being encapsulated within a foreign body reaction. As the injection volume increased for the larger batch microspheres, more, smaller masses were found dis-

persed within the subcutaneous tissue ( Figs. 19 and 20 ).

In Vitro

= In Vivo

Release

from Injectable

Microspheres

Figure 17 Fraction of drug absorbed relative to total absorbed at day 42 for the F1 injection volume

567 spheres manufactured on two different scales.

0.83 mL formulation, as approximated by AUC, after administration of 20 mg of drug encapsulated in micro-

© 2005 by Taylor & Francis Group, LLC

568 Clark et al.

Figure 18 Subcutaneous masses removed from a rat on day 42 which had received the F1 injection volume 0.62 mL formulation.

Figure 19 Subcutaneous masses removed from a rat on day 42 which had received the F1 injection volume 0.83 mL formulation.

In Vitro=In Vivo Release from Injectable Microspheres 569

Figure 20 Subcutaneous masses removed from a rat on day 42 which had received the F1 injection volume 1.25 mL formulation.

Figure 21 Subcutaneous masses removed from a rat on day 42 which had received the F2 injection volume 1.5 mL formulation.

570 Clark et al.

The F2 formulation produced masses similar to the F1 injection volume 0.83 mL formulation ( Fig. 21 ).

4. CONCLUSIONS Based on the similarity of the in vitro and in vivo release profiles

for microspheres manufactured at pilot scale to those manufac- tured at laboratory scale, alongside manufacturing perfor- mance, the manufacture of active microspheres was considered to have been successfully scaled up from laboratory- to pilot-scale. Terminal sterilization by g-irradiation at a dose of

25 kGy is feasible, provided the product is stored under appro- priate conditions. Particular care must be taken to avoid even short-term temperature excursions, such as may be experienced during transportation of material, as a significant increase in burst release may be expected to arise. An in vitro release method capable of discriminating between low-, intermediate-, and high-burst batches has been developed, and the preliminary in vitro =in vivo correlation for the rat model supports the use of the in vitro release method for batch selection. The use of multi- dose vials in which microspheres are suspended in an appropri- ate medium, and repeat doses withdrawn for injection, is accep- table for pre-clinical studies of these formulations.

It has been shown elsewhere (Sec. 3 of Chapter 4 ) that pre-clinical species are likely to give similar release pharma- cokinetics as those seen in the clinic for ‘‘preformed’’ con- trolled release systems and should be sufficiently good for formulation development work. However, this case study has shown that, even within a species, differences can occur depending on the dosing technique =dose volume. Therefore, care should be taken when extrapolating to the clinical situa- tion and development of the optimal or absolute pre-clinical model will require some empirical development based on feed- back from initial clinical studies.

REFERENCE 1. Tice TR, Gilley RM. Microencapsulation process and products

therefrom. United States Patent Number 5,407,609 (1995).

Case Study: Biodegradable Microspheres for the Sustained Release of Proteins

Guidelines for Formulating and Processing Dry Powder Pharmaceutical Products

MARK A. TRACY Formulation Development, Alkermes, Inc.,

Cambridge, Massachusetts, U.S.A.

1. INTRODUCTION In developing dry powder pharmaceutical products, there are

important general principles that provide valuable guidance in formulating and processing the product. This article will review two important guidelines and provide examples using the devel- opment of biodegradable microsphere products as a case study.

572 Tracy

The first guideline is to maximize product stability by minimizing molecular mobility. Minimizing molecular mobi- lity can be achieved by selecting formulation components and processing unit operations and conditions that reduce the probability that formulation components will interact and change from the desired form. Examples include proces- sing proteins at reduced temperature to prevent denaturation and reducing solvent levels in the product to prevent drug reactions or polymer relaxation.

The second guideline is to understand the role of particle structure and morphology in product function and stability. Particle properties can vary tremendously depending on vari- ables such as size, porosity, and morphology. All too often the

performance of a pharmaceutical powder formulation depends on the powder microstructure which can be con- trolled by formulation and processing. Examples include the effect of drug particle size on initial release from microspheres and the effects of protein lyophilizate particle size and mor- phology on protein stability.

Biodegradable microspheres for the controlled release of drugs, including proteins, provide an excellent illustration of the importance of understanding and utilizing these guide- lines. Microspheres provide a means of delivering drugs for periods of days, weeks, or months with a single injection. They consist primarily of a biodegradable polymer, for exam- ple poly(lactide-co-glycolide) (PLG), and the encapsulated drug as well as appropriate stabilizers or release modifiers.