IN VIVO RELEASE

3. IN VIVO RELEASE

3.1. Introduction The following section discusses the preclinical in vivo

evaluation of injectable dispersed systems particularly the

In Vitro=In Vivo Release 143

evaluation of extended release systems intended for intra- muscular or subcutaneous administration and should be read in conjunction with the chapters in this book by Oussoren et al. (Biopharm) and Young et al. (IVIVC).

Preclinical testing of parenteral modified release formu- lations is performed for two major reasons:

chemical entities and formulations, clinically with regard to safety and efficacy.

lations with the predicted desired clinical performance (which is usually assessed by pharmacokinetic perfor- mance).

This section will focus on the design of in vivo preclinical experiments aimed at supporting the pharmaceutical develop- ment of modified release particulate drug delivery systems with special emphasis on those in which the excipient modify- ing release is a poly-lactic acid or poly-lacticco-glycolic acid ester polymer (PLA =PLGA). When performing preclinical in vivo evaluations, a fundamental assumption is that the model = species chosen is likely to be predictive of the clinical situation. There is, however, a lack of systematic investigations to estab- lish which animal species are the most predictive of the clinical situation. Indeed an AAPS, FDA, and USP co-sponsored work- shop on ‘‘Assuring Quality and Performance of Sustained and Controlled Release Parenterals’’ recommended the initiation of research in this area (33). Notwithstanding this lack of sys- tematic research, from a knowledge of first principles and review of the literature, it is possible to draw conclusions about the relative merits of different animal models.

During the pharmaceutical development of PLGA-based dispersed systems, the primary aim of preclinical experiments, in common with in vitro dissolution testing, is to characterize the release of drug from the delivery system. Therefore, the pri- mary requirement for the preclinical model is that the absorp- tion =injection site is sufficiently similar to that in humans such that the release mechanism and release kinetics of drug from the PLA =PLGA system are qualitatively equivalent to that

144 Clark et al.

which will be experienced in the clinic. There is a substantial body of evidence supporting the hypothesis that the release of drug from PLA =PLGA-based systems is predominately con- trolled by the characteristics of the delivery system and depen- dent mainly on a combination of diffusion (early phase) and hydrolytic erosion (later phase) (34). For PLA =PLGA-dispersed systems, it is clear that this release is also the rate-determining step for the pharmacokinetics (35), otherwise there would be no pharmacokinetic driver to produce such a complicated system. It is therefore not unreasonable to assume that if the injection sites of preclinical species do not differ too markedly for human tissue in terms of biochemistry and tissue reaction then the release profiles are likely to be comparable across species. Exhaustive comparative analysis of the subcutaneous and intramuscular tissue interstitial fluid has apparently not been performed; however, we know that interstitial fluid is in equili- brium with serum = plasma and the serum data for preclinical species and man are available ( Table 1 ).

For larger molecular weight species (plasma proteins), the equilibrium point between plasma and interstitial fluid is dependent on the endothelial properties of each tissue (37) and, for both small and large molecular weight species, the metabolic fate within the tissue (38). Lymph to plasma (interstitial) concentration ratios are available for different species. While somewhat variable, and dependent upon mea- surement technique (39), they are broadly comparable across species although for albumin, dogs and rabbits may exhibit a lower ratio than seen in humans while rats show the most similar ratios. It should also be noted that tissue differences in concentration ratios also exist with interstitial albumin concentration being higher in skeletal muscle than subcuta- neous tissue under some conditions (37,40). Thus it can be concluded that in terms of biochemistry interstitial fluid is likely to be broadly similar across species.

The histopathological reaction observed following injec- tion of PLA =PLGA microspheres is typical of a response to an inert foreign body in which the aim of the tissue reaction is the removal of the material from the host without the generation of an antigen-specific immune response. The cells

In Vitro

= In Vivo

Table 1 Mean Values of the Inorganic Components in the Serum of the Male of Each Species Listed Release

Man Sodium

Mice (albino)

Rat (albino)

Rabbit

Dog

141 (135–155) (mEq =L) Potassium

4.1 (3.6–5.5) (mEq =L) Chloride

104 (98–109) (mEq =L) Bicarbonate

27 (22–33) (mEq =L) Phosphorous

3.5 (2.5–4.8) (mg =dL) Calcium

9.8 (8.5–10.7) (mg =dL) Magnesium

2.12 (1.8–2.9) (mg =dL)

The bracketed values indicate the range in literature values. (From Ref. 36.)

© 2005 by Taylor & Francis Group, LLC

146 Clark et al.

involved in this reaction are overwhelmingly those of the macrophage series, but the detailed form of the response is dependent upon the size of the microspheres injected.

Following injection of microspheres of less than approxi- mately 10 mm in diameter, the response is characterized by the progressive invasion and phagocytosis of the mass of micro- spheres by single macrophages. The single macrophage, how- ever, is incapable of phagocytosing larger microspheres, and if these are injected the host reaction includes large numbers of multinucleate giant cells that are formed from the fusion of individual macrophages. The macrophages or giant cells engulf and presumably digest the microspheres. In all instances, a two- to three-cell thick rim of fibrous tissue sur- rounds the invading phagocytic cells and small blood vessels invade alongside the phagocytes ( Fig. 6 ).

This is a subcutaneous injection site and hair follicles are clearly visible ( ). An area of the microsphere tissue reaction is illustrated. There is a thin fibrous capsule (arrow), under which there is the advancing wall of macrophages and giant cells (line) that is engulfing microspheres (arrowhead). To the center of the reaction site, the microspheres are lost as

a tissue processing artifact. The size of the lesion is directly related to the number of microspheres injected. It is the mass of invading phagocytic cells which are palpable at the injection site, which explains the delay between injection of the microspheres and the for- mation of a clinically obvious lump. These lesions are progres- sive, and resolution is complete to a point where there is no histopathological abnormality detected at the injection site.

This tissue reaction is identical in subcutaneous and intramuscular injection sites, and is very similar across spe- cies including rat, mouse, and primate.

For drugs which are non-irritant, the tissue reaction to drug laden microspheres is indistinguishable from that to control microspheres. If the drug is an irritant or has other proinflammatory properties, the cellular infiltrate contains large numbers of lymphocytes and fibrosis is prominent.

To summarize, it seems reasonable to conclude that the absorption site environment across species should be

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Figure 6 Photomicrograph illustrating the typical tissue reaction to PLGA microspheres, showing hair follicles ( ), the fibrous capsule ( #), giant cells (j ) and microspheres (;).

sufficiently similar so as not to affect the release kinetics of drugs from PLA =PLGA microspheres. Review of the literature in which PLA =PLGA-based systems have been administered to more than one species or strain or have been administrated both subcutaneously and intramuscularly seem, to confirm this conclusion. It was shown during the development of a 1-month leuprolide acetate PLGA microsphere formulation (41,42) that the rate of release of leuprolide acetate from PLGA microspheres (as measured by loss from the injection site) was the same after administration to both subcuta- neous and skeletal muscle tissue. Furthermore, the rat strain (Sprague–Dawley vs. Wistar) did not affect the performance of the microspheres as assessed by pharmacodynamic end- points (41). This research group also demonstrated that the plasma concentration–time curves for leuprolide acetate in dogs and rats after intramuscular administration of leuprolide acetate-loaded PLGA microspheres had essentially the same

148 Clark et al.

pattern indicating non-species-specific release of leuprolide acetate from the microspheres (43). Furthermore, the same group also demonstrated similar performance for rat and dog after subcutaneous and skeletal muscle administration for a 3-month leuprolide acetate PLA microsphere formulation (44).

Although a case has been made for the similarity of all ani- mal models including humans for the evaluation of the release (pharmacokinetic) behavior of preformed PLA =PLGA-based sys- tems, a few cautionary points should be considered. If there is likely to be a specific immune interaction in a species that is not present in other species then this may make this species inap- propriate for the pharmacokinetic evaluation of the PLGA sys- tem. This has been highlighted previously for liposomal systems (45). The foregoing discussion can only be considered to apply to ‘‘preformed’’ controlled release systems. For other for- mulations which are dependent on the formation of the rate-con- trolling structure in vivo (for instance precipitation), there may

be sufficient difference between species for the structure forming step, which is likely to be rapid, to be sufficiently different to give different formulation behavior. For this type of formulation, the identification of the most appropriate preclinical species may need to be performed empirically as part of the development program. Finally, it should be remembered that rabbits, and possibly dogs, have a slightly higher body temperature (rabbit 38.5–39.5 C; dog 37.5–39.0

C) than other preclinical species (mouse 36.5–38.0 C; rat 35.9–37.5 C; non-human primate 37.0–39.0

C) which maybe an important consideration depend- ing on the glass transition temperature of the formulation.

3.2. Choice of Animal Species As all preclinical species are likely to be equally predictive for

preformed PLA =PLGA delivery systems other selection cri- teria become important. These are discussed briefly below.

3.2.1. Ethical Considerations Within the European Union and United Kingdom in particu-

lar, there is an ethical and legal obligation to use the species

In Vitro=In Vivo Release 149

with the lowest neurophysiological sensitivity to meet the objectives of the experiment.

3.2.2. Dose Volume and Sample Volume Considerations

Both ethically and scientifically, it is desirable not to give dose volumes or take blood volumes that will unduly change the physiology of the animal and therefore cause unnecessary dis- comfort or invalidate the scientific integrity of the study. Therefore, there is a balance to be struck between delivering sufficient drug to give quantifiable systemic plasma drug con- centrations (see Sec. 4) or volumes of complex formulations that can be practically administered (see Sec. 5 ) and what can be ethically and scientifically justified. Currently accepted European good practice guide on dosing and sampling volumes are reported (46). A strategy to allow experimental design to meet these limits is discussed in the case study.

3.2.3. Toxicological Species Where possible, it would seem appropriate that the formula-

tion development program is performed in species that are to be used in the safety assessment evaluation of the com- pound and formulation as this should reduce the number of studies that need to be performed (for instance avoidance of pharmacokinetic sighting studies prior to the start of a full safety assessment study). Choice of toxicological species is dri- ven by the regulatory requirement to provide data in a rodent and non-rodent, demonstration of pharmacological activity in the chosen species, and the ethical consideration to use ani- mals of the lowest neurophysiological sensitivity to meet the objectives of the experiment.

4. BIOANALYSIS There is little value in taking such care to choose species and

design the live phase of any preclinical evaluation if the drug blood =plasma concentration analysis is then lacking. Many

150 Clark et al.

compounds are considered for parenteral-controlled release due to their poor oral bioavailability and usually short elimination half-life (in many cases, these compounds are peptides and pro- teins). This presents the bioanalyst with considerable challenges as these compounds are usually unstable in blood =plasma and also difficult to resolve from endogenous material in plasma. The advent of quantitative HPLC–MS–MS has made this task easier (47,48); however, radioimunnoassay may still need to be considered as an analytical method. Development of the assay and assessment of assay performance should meet accepted cri- teria, for instance those proposed (49) and documented in FDA guidance documents (50). For biopharmaceuticals, special atten- tion should be given to the stability of the drug in blood =plasma which is likely to be relatively poor and requires special steps to make the stability manageable, for example, inclusion of protease inhibitors; special care to avoid hemolysis on plasma collection; storage on ice and specialized collection procedures. At the planning stage of an in vivo study, it is particularly important to talk through these aspects of sample collection with the animal technicians who will perform the study as this is likely to be somewhat different to the procedures they usually follow.

5. INJECTABILITY In vitro measurements of content and dose uniformity should

be reviewed in the light of in vivo (clinical) behavior. It is important to note that under clinical conditions, this behavior may be substantially compromised. Injectability has been identified as an important performance parameter. Injection into tissue differs in two ways from that experienced when using standard in vitro techniques due to changes in fluid dynamics and the potential for ‘‘coring’’ of tissue within the needle bore ( Fig. 7 ).

Both these factors increase the potential for needle block- age possibly preceded by filtering out of the microspheres. That is if the microsphere size, morphology, and suspending agent characteristics are not optimal then the suspending fluid is able to pass into the tissue while the microspheres

In Vitro=In Vivo Release 151

Figure 7 Example of tissue coring that can partially occlude the needle and lead to filtering of the microspheres and eventually blockage (example shown is a wide bore needle for clarity).

are trapped in the needle and syringe luer. The trapped microspheres eventually reach a critical mass and on further pressure to the syringe plunger compress to form a ‘‘plug’’ which blocks the needle =syringe. When there is a potential for sieving to occur, for instance with earlier development for- mulations, it is important to note that injection volume may not necessarily equate to microsphere dose and a prudent step maybe to assay for drug remaining in the syringe even if a correct volume has been injected. A useful model to qualita- tively assess in vivo injectability is injection into meat. We have found that subcutaneous injection into chicken carcasses produced for the food industry mimics subcutaneous inject- ability in preclinical species.

6. CONCLUSIONS In vivo and in vitro studies are essential components of the

drug development process. The objective of such studies is to determine a relationship between an in vitro characteristic of a dosage form and its in vivo performance, such that the in vitro test may be used to predict in vivo performance. In vitro testing is used in early development to select batches for in vivo pharmacokinetic =pharmacodynamic studies, but the

152 Clark et al.

ultimate objective is a test capable of distinguishing, prior to medical use, clinically effective batches from those which would be ineffective and =or unsafe if used.

In vitro dissolution tests were first developed for immedi- ate release solid oral dosage forms then extended to modified release formulations. In recent years, the application of disso- lution testing has been extended to ‘‘special’’ dosage forms including injectable dispersed systems, and for such products administered by a non-oral route the term ‘‘drug release’’ or ‘‘in vitro release’’ test is preferred. Due to the significant dif- ferences in formulation and hence in physicochemical and release characteristics, it is not possible to specify a generally applicable apparatus or method, rather different techniques are employed on a case-by-case basis (51).

Preclinical in vivo studies are performed to provide safety, efficacy, and pharmacokinetic data to support formulation development and clinical use. Animal models are selected on the basis of their relevance to humans, with reference to the for- mulation and route of administration. For depot formulations in particular, it is important to study histopathological reactions at the injection site, as this may mediate drug release and is important in assessing the tissue compatability of both the for- mulation and the drug substance. Injectability may be an issue for injectable dispersed systems, and should be assessed prior to the commencement of in vivo studies. Ethical considerations govern the choice of animal species (that with the lowest neuro- physiological sensitivity, other factors being equal), dosing, and sampling regimes; where possible the scale of in vivo testing should be minimized by, for example, use of the same species for toxicological and pharmacokinetic studies, and by the early development of a predictive in vitro release test.

A thorough understanding of the in vivo drug release mechanism of the dosage form, underpinned by comprehen- sive physicochemical characterization of the drug substance and delivery system, is a necessary foundation both for the development of a discriminatory in vitro release test and for the development of a high-quality product. Application of the principles outlined in this chapter should lead to a biorelevant release test and facilitate the development of a meaningful

In Vitro=In Vivo Release 153

in vitro–in vivo correlation using techniques described else- where in this publication. This information should be consid- ered an essential component of the Chemistry and Manufacturing Controls section of a New Drug =Marketing Authorization Application for Injectable Dispersed Systems.

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In Vitro=In Vivo Correlation for Modified Release Injectable Drug Delivery Systems

DAVID YOUNG, COLM FARRELL, and THERESA SHEPARD

GloboMax Division of ICON plc, Hanover, Maryland, U.S.A.