IN VITRO RELEASE

1. IN VITRO RELEASE

1.1. Basis of Dissolution Testing Dissolution testing is an in vitro procedure designed to discri-

minate important differences in components, composition, and =or method of manufacture between dosage forms (1). A dissolution test for solid oral dosage forms, utilizing a rotating basket apparatus, was first included in the United States Pharmacopeia (USP) 18 in 1970. The current USP (2) includes general methods for disintegration, dissolution, and drug

126 Clark et al.

release. The disintegration and dissolution tests are intended primarily for immediate release solid oral dosage forms, the former controlling the time taken for a tablet or capsule to break down and the latter controlling release of the active ingredient(s). The drug release test is intended for application to modified release articles including delayed and extended release tablets; seven apparatus are described, the choice being based on knowledge of the formulation design and actual dosage form performance in the in vitro test system. Specific guidance is given with regard to the utility of Appara- tus 1 (basket) or 2 (paddle) at higher rotation frequencies, Apparatus 3 (reciprocating cylinder) for bead-type delivery systems, Apparatus 4 (flow cell) for modified release dosage forms containing active ingredients of very limited solubility, Apparatus 5 (paddle over disc) or Apparatus 6 (cylinder) for transdermal patches, and Apparatus 7 (reciprocating disc) for transdermal systems and non-disintegrating oral modified release dosage forms. The usage of the various apparatus across all modified release dosage forms described in the current USP is shown in Fig. 1.

The state of science is such that in vivo testing is neces- sary in the development and evaluation of dosage forms. It is

Figure 1 Modified release dosage forms—usage of USP apparatus for drug release, as indicated in USP27-NF22.

In Vitro=In Vivo Release 127

a goal of the pharmaceutical scientist to find a relationship between an in vitro characteristic of a dosage form and its in vivo performance (refer to the chapter on In Vitro–In Vivo Correlation for Modified Release Parenteral Drug Delivery Systems in this book).

1.2. General Considerations Whatever dissolution apparatus is used, close control must

be applied to several parameters, including geometry, dimen- sions, materials of construction, environment, temperature, and time, in order that reliable results may be obtained. Con- sideration should be given to the potential for extraction of interferants from the equipment, or adsorption of the active substance, and method validation should include an assess- ment of recovery for a completely dissolved dosage unit.

The dissolution test is carried out at constant temperature (normally 37

C, body temperature, for oral and parenteral dosage forms, or 32

C, skin temperature, for transdermal sys- tems), although other temperatures may be used with justifica- tion. Temperature should be controlled with a tolerance of

C and should be measured and verified as being within limits over the duration of the test. The dissolution test may be carried out using water, or

a medium chosen to mimic physiological conditions, and may include a buffer system to maintain pH, additives such as surfactants or albumin (to mimic protein binding of lipo- philic drugs when administered intravenously). A bacterio- static agent may be incorporated to control microbiological growth, which can be a particular problem for real-time release testing of extended release formulations. Deaeration of dissolution media should be considered, as degassing resulting in the formation of air bubbles on the surface of the dosage form will significantly affect surface area and hence rate of release. The use of non-aqueous media is not normally recommended, as a meaningful in vitro–in vivo correlation is unlikely.

The test is normally carried out on a unit dose of the formulation. The dose may be dispersed in the dissolution medium, contained within a cell, or for solid dosage forms

128 Clark et al.

retained by a sinker, for example, a platinum wire, designed to minimally occlude the dosage form.

The composition and volume of dissolution medium should be chosen to ensure that, when all of the drug substance has dissolved, the concentration of the resulting solution will

be less than one-third of that of a saturated solution; thus, the dissolution medium acts as a sink, in which the concentra- tion of dissolved drug will be low enough not to inhibit ongoing release. The usual volume of dissolution medium is 500–1000 mL, but other volumes may be used with justification.

Under certain circumstances, usually to mimic the change in environment as an enteric-coated or extended release tablet or capsule moves through the gastrointestinal tract, the disso- lution medium may be modified =changed at a predetermined intermediate time-point.

The dissolution medium should be stirred, or the sample compartment rotated or oscillated, to ensure homogeneity of solution. Other than this, the dissolution apparatus should not contribute and should be isolated from, any vibration or other motion which could affect the rate of release.

Test duration is normally 30–60 min for immediate release formulations, but may be much longer for extended release products. Evaporative losses during the test must be minimized or compensated for. For tests longer than 24 hr, measures (sanitization of equipment and =or inclusion of anti- microbial additive) must be taken to prevent microbiological proliferation.

The release profile may be characterized by determining the concentration of drug released at each of a minimum of three time-points—an early time-point to determine ‘‘dose dumping,’’ a late time-point to evaluate completeness of release, and an intermediate time-point to define the in vitro release profile. Measurement may be continuous (e.g., by use of a flow cell or fiber-optic probe) or discrete, and if a sample of dissolution medium is withdrawn for analysis, it should be replaced (if the assay method is non-destructive), or an equal volume of fresh dissolution medium added and the amount of drug removed corrected for in subsequent calculations, or the test may be continued with diminished volume. If a

In Vitro=In Vivo Release 129

sample-and-replace approach is used, care should be taken to minimize any perturbation to temperature, perhaps by pre- heating the replacement medium. For products containing two or more active ingredients, release should be measured for each active ingredient.

The analytical methodology used to determine drug con- centration should be selective for the active ingredient. Where the formulation is dispersed in the dissolution medium, separation of dissolved from undissolved drug may be accom- plished by filtration, centrifugation or by the use of an analy- tical technique sensitive only to dissolved drug. Care must be taken to ensure that the sampling and subsequent analysis does not influence the distribution of drug between undis- solved and dissolved forms. Degradation of the drug sub- stance under the conditions of the test should be evaluated during method development (3,4); if significant degradation is apparent, it may be appropriate to sum active and degra- dants, or to utilize a non-specific method, such that the reported results are indicative of release. It is normal practice to report results as cumulative release, as a percentage of the labeled content of drug (Q).

The drug release test is normally performed in replicate, initially using 6 units but with the scope for additional testing (up to a total of 24 units) if acceptance criteria are not met. Acceptance criteria should control mean release and the range of individual values for a batch of the formulation. The drug release test is normally considered to be stability indicating.

1.3. Applicability to Injectable Dispersed Systems Current guidance is that no product where a solid phase

exists, including suspensions and chewable tablets, should

be developed without dissolution or drug release characteriza- tion. In the context of injectable dispersed systems, it is there- fore appropriate to apply a drug release test to suspensions and microspheres. Drug release characterization is also relevant for emulsion and liposomal products where the formulation is designed to control the release of the active substance(s).

130 Clark et al.

1.4. Mechanistic Studies The development of in vitro drug release methodology should

be underpinned by an understanding of the mechanism of drug release. This requires knowledge of the drug substance, the release-controlling excipients, and any interactions in the formulation. Depending on the characteristics of the dosage form and the route of administration, in vitro drug release may involve hydration, swelling, aggregation, disintegration, diffusion, hydrolysis, and =or erosion. In vivo release may be additionally complicated by enzymatic action, encapsulation by tissue, complexation, or partitioning into tissue.

1.4.1. Emulsions Submicron emulsions are typically used for parenteral nutri-

tion or the intravenous administration of a hydrophobic, lipo- philic drug substance. Characterization studies should include investigation of particle size distribution (particles

> 5 mm are likely to cause pulmonary embolism), zeta (surface) potential, which is a key indicator of the physical stability of the emulsion, pH, which is a determinant factor for surface potential and which is liable to decrease on storage due to the formation of free fatty acids, and drug substance content. The drug substance will partition between the dis- perse (oil) phase, the continuous (aqueous) phase, and the oil–water interface where the drug may associate with the emulsifying agent(s). A quantitative assessment of drug dis- tribution is required if the mechanism of release is to be understood, and this may be determined using a combination of ultrafiltration and ultracentrifugation techniques (5).

1.4.2. Liposomes Liposomes may be used for drug delivery to confer sustained

release, for tumor targeting, to increase bioavailability or expand the therapeutic window. The characterization techniques described above for emulsions may also be applied to liposomes, although the partitioning of the drug substance is complicated by the existence of internal and external aqueous phases.

In Vitro=In Vivo Release 131

1.4.3. Suspensions Injectable suspensions may be used for drug delivery primarily

for insoluble drug substances; for intravenous administration,

a submicron particle size distribution is essential. Characteri- zation studies should encompass particle size distribution, partitioning of the drug substance between solid and solution; and the potential for Ostwald ripening should be considered.

1.4.4. Microspheres Microsphere drug delivery systems are usually based on biode-

gradable polymers (6) such as poly(lactic acid), poly(lactide-co- glycolide), polyanhydrides, cross-linked polysaccharides, gela- tin or serum albumin, and are intended for subcutaneous or intramuscular administration. Drug loading is determined by potency, duration of release, and other factors, but is gener- ally in the range 0.1–15% by weight. Characterization studies should include the particle size distribution, drug distribution within the formulation (solid solution, drug polymer salt, dis- crete domains of drug in the polymer matrix), and the surface and bulk morphology. Solid state imaging techniques are important in elucidating structural information.

1.5. Methodology Experimental methodology for the determination of in vitro

release from injectable disperse systems may be considered to fall into four categories (7): membrane diffusion, sample and separate, in situ, and continuous flow methods.

1.5.1. Membrane Diffusion Techniques These techniques are characterized by their use of a dialysis

membrane to partition the sample and test media, thereby facilitating the determination of concentration of released drug. The membrane is selected to have a molecular weight cut-off allowing permeation of the drug substance, and it is assumed that diffusion of drug through the membrane is not a rate-limiting step. The dialysis membrane must be con- ditioned by soaking in dissolution medium prior to use, in

132 Clark et al.

order to remove extractables which may interfere in the subsequent analysis.

The dialysis sac diffusion technique involves placing a suitably sized sample (unit dose if possible), along with a sui- table carrier medium (continuous phase, suspending medium or dissolution buffer), into a dialysis sac or tube. This is sealed and placed in a large volume of dissolution buffer, which is stirred to ensure uniform mixing, and the concentration of drug arising from diffusion through the membrane is deter- mined at an appropriate frequency. The dialysis sac diffusion technique has been used to measure in vitro release from lipo- somes (8), submicron emulsions (9,10), and microspheres (11).

In a variation of the method, release is determined by assay of microspheres remaining within a dialysis tube at each test time-point (12); this approach also allows measure- ment of mass loss, hydration, and polymer degradation. The technique is simple to apply, separates the sample from the dissolution medium simplifying subsequent assay, and is applicable to a wide range of formulation types, but suffers the significant disadvantage that the sample within the dialy- sis sac is largely undiluted and therefore sink conditions do not apply. In the example of an emulsion formulation of a lipo- philic drug substance, release rate measured using this tech- nique will be determined largely by the partition coefficient between disperse and continuous phases within the dialysis bag and will not be indicative of release in the blood stream, which can be considered a true sink due to binding of the lipo- philic drug substance to blood proteins. This issue may be resolved by the inclusion of a solubilizing agent, in the form of a hydrophilic b-cyclodextrin derivative, in the dissolution medium to maintain sink conditions (13). Further applica- tions may include the study of depot formulations adminis- tered by subcutaneous or intramuscular injection, where the depot may become encapsulated by tissue leading to membrane-mediated release.

A modification to the above approach, the bulk equili- brium reverse dialysis sac technique (5,10), avoids this pro- blem by placing the sample directly into an appropriate volume of dissolution buffer in equilibrium with several

In Vitro=In Vivo Release 133

dialysis sacs each containing 1 mL of the same dissolution buffer. At appropriate intervals, one dialysis sac and a 1 mL sample from the bulk dissolution buffer are removed and the drug contents of the dialysis sac and the bulk solution are assayed. In this approach, release may be studied under sink conditions. If the active substance is chemically stable under the conditions of the test, and if the sample is accu- rately dispersed, analysis of the bulk solution is unnecessary as the percentage release can be calculated from the assay of the dialysis sac alone. In this approach, the formulation is diluted in a large volume of dissolution medium and sink con- ditions may be considered to apply. The technique may there- fore have utility in the study of intravenous emulsions and liposomes.

This approach has been further developed into a fully automated system, microdialysis sampling, initially applied to tablets (14,15), and subsequently to implants (16). A sche- matic illustration of such an apparatus is shown in Fig. 2.

The test sample is added to a suitable volume of continu- ously stirred dissolution medium and the microdialysis probe, consisting of narrow-bore dialysis tubing, is positioned below the surface. A perfusion medium is continuously pumped through the probe and collected for analysis by high-performance

Figure 2 Microdialysis sampling.

134 Clark et al.

liquid chromatography (HPLC). The perfusion medium may be buffered to ensure compatibility with the HPLC column, and the flow rate and surface area of the microdialysis probe may

be manipulated to ensure that the drug concentration is within the range of the assay method. As for the reverse dialysis sac technique, this approach allows sink conditions to be maintained, and therefore may

be applicable to intravenous formulations. The rotating dialysis cell is a further variation on the membrane diffusion theme. This approach was first used to assess in vitro release from parenteral oil depot formulations (17) and has also been used to assess drug salt release from suspensions (18). The apparatus consists of a small (10 mL) and a large (1000 mL) compartment separated by a dialysis membrane, as shown in Fig. 3.

In use, approximately 5 mL of sample is introduced into the dialysis cell which is placed in a large (typically 1000 mL) volume of dissolution medium. The dialysis cell is rotated at a constant speed, typically 50 rpm, and the concentration of drug arising through diffusion into the sink solution is mea- sured at appropriate intervals.

Figure 3 Rotating dialysis cell.

In Vitro=In Vivo Release 135

It is considered that this approach, in which the apparatus acts as a two-compartment model, may mimic release in vivo where the route of administration is into a small compartment (e.g., intra-articular) or where release into the systemic circu- lation is mediated by passive diffusion through a membrane.

1.5.2. Sample and Separate Techniques This category covers methods in which the sample is diluted

with dissolution medium under sink conditions, a sample is withdrawn at appropriate intervals, and undissolved material removed leaving a solution containing dissolved drug.

This approach has been applied to assess drug release from PLGA microspheres, using USP Apparatus 2 (paddle method); samples were withdrawn, filtered, and the filtrate analyzed by HPLC (19). A variation involved shaking several tubes (one per test time-point) containing sample, taking one tube at each test time-point, and centrifuging to separate free drug in solution from undissolved material then determining dissolved drug concentration using HPLC (20). Tube-to-tube variability may be eliminated by replacing the supernatant removed for assay with an equal volume of fresh dissolution medium, vortexing to resuspend, then continuing the test with the same tube (21).

The centrifugal ultrafiltration technique developed by Millipore (22) in the form of the Ultrafree Õ -MC unit, illu-

strated in Fig. 4 , utilizes an ultrafiltration membrane having

a nominal molecular weight limit (NMWL) of 5000–100,000 Da. A maximum 400 mL sample of dissolution medium con- taining the suspended formulation is withdrawn from the dis- solution vessel at appropriate intervals and transferred to a centrifugal filter unit with an NMWL value chosen to allow passage of the drug. The unit is placed in a microcentrifuge tube and centrifuged at up to 5000 g using a fixed angle micro- centrifuge. The resulting ultrafiltrate is assayed to determine the free drug substance concentration.

The centrifugal ultrafiltration method has been applied to the determination of in vitro release from a submicron emulsion (23).

136 Clark et al.

Figure 4 Centrifugal ultrafiltration apparatus.

1.5.3. In Situ Techniques In this approach, the sample is diluted in the dissolution med-

ium and release is measured in situ, without separation of undissolved material, using a suitable analytical methodology specific to dissolved drug. This approach is little used in the determination of drug release from injectable dispersed sys- tems, as correction for interference from undissolved drug may be problematic. Differential pulse polarography has been successfully used to determine the release of pyroxicam from polymeric nanoparticle dispersions (24).

1.5.4. Continuous Flow Methods This category includes single-pass methods in which dissolu-

tion medium is pumped through a cell containing the sample and the eluant is analyzed continuously or fractions are col- lected for subsequent assay; and loop methods in which the dissolution medium is continuously recirculated.

This technique is mainly applicable to microspheres and other solid dosage forms which may be retained in the flow- through cell by use of an appropriate filter. The sample may

be mixed with glass beads to minimize aggregation as well

In Vitro=In Vivo Release 137

as to alter the flow pattern within the sample bed to help avoid channeling effects that would lead to inaccurate release patterns. The flow-through cell is placed vertically in a thermo-jacketed vessel and dissolution medium pumped from the reservoir, through a delay coil to allow temperature equi- libration, through the flow-through cell from bottom to top, then through an in-line measurement device such as a UV spectrophotometer before being returned to the reservoir. The volume of dissolution medium remains constant through- out. A schematic illustration is shown in Fig. 5.

The flow-through apparatus has been used extensively to evaluate drug release from oral solid dosage forms and has been applied to injectable dispersed systems, mainly microspheres. Release from microwave-treated gelatin microspheres has been investigated under sink and non-sink conditions, using deionized water as dissolution medium (25). In a study of vera- pamil hydrochloride-loaded microspheres intended for oral administration, a surfactant was added to the dissolution med- ium to improve wetting, and the flow rate was controlled to maintain sink conditions in the flow-through cell; different dis- solution media were evaluated, and in the ‘‘half-change’’ method, step changes in pH were introduced at predetermined

Figure 5 USP Apparatus 4 (flow-through cell).

138 Clark et al.

time-points (26). A novel approach was used to investigate release of glial cell line-derived neurotrophic factor (GDNF) from PLGA microspheres; the apparatus utilized an unpacked HPLC column as the sample compartment; dissolution medium was passed through the column to a fraction collector, and pro- tein release determined by gamma counting, ELISA, and =or bioassay methods. A study of the dissolution of a poorly soluble compound in unmicronized and micronized form concluded that homogeneous mixing of the sample with the glass beads in the flow-through cell was effective in achieving maximum dissolution with minimum variability for unmicronized pow- ders, but for micronized powders poor wetting resulted in par- ticles being carried into the filter, resulting in anomalously low release. Presuspending drug in dissolution medium modified to include a suspending medium (0.3% HPMC) and a surfactant (0.2% Tween 80), introducing the sample as a slug below the glass beads, and reducing flow rate, were shown to lead to release profiles in line with particle size (27).

1.6. Method Development Preliminary method development should be based on a knowl-

edge of the dosage form and route of administration, and the in vitro procedure should emulate in vivo conditions so far as is reasonably practical.

For extended release dosage forms, which may be designed to release drug over prolonged periods up to 12 months, the development of an accelerated in vitro release procedure may offer considerable benefits in reducing devel- opment time-lines and, for marketed products, in resource efficiency and enhanced responsiveness to manufacturing problems. In vitro release may be accelerated by the choice of appropriate conditions, in particular increased tempera- ture and extreme pH, but the same requirements for biorelevance must be met.

When in vivo data from exploratory studies are available, method optimization should be carried out with the aim of achieving an in vivo–in vitro correlation for fast, intermedi- ate, and slow-releasing batches. An experimental design

In Vitro=In Vivo Release 139

approach should be utilized; the following are generally considered to be critical parameters for investigation:

Systematic variation of selected parameters to optimize discrimination, duration of release, and release profile for two or more batches or formulation variants known to behave differently in vivo should lead to the definition of a biorele- vant release test.

For detailed information on the development of an in vitro-in vivo correlation, refer to the chapter on In Vitro-In Vivo Correlation for Modified Release Parenteral Drug Deliv- ery Systems in this book.