The chick embryo and its chorioallantoic membrane (CAM) for the in vivo evaluation of drug delivery systems☆
Introduction
Nowadays, the pharmaceutical field faces an ever growing demand for making innovative formulations that are able to “intelligently” deliver modern active compounds. The development of high-throughput strategies for the discovery, synthesis, and screening of drugs, as well as the advances in genomics and proteomics, have resulted in a huge amount of new drug candidates for which formulations need to be designed. In this context, drugs, such as peptides, proteins, monoclonal antibodies, and nucleic acids, are particularly difficult to formulate. Progress in both polymer and biomaterial sciences offers the possibility of using new excipients for developing improved drug carriers. Ideally, delivery systems should have adequate drug loading, remain stable during storage and biodistribution, accumulate selectively at the target site, be able to release the drug in a controlled manner, be biodegradable, and perfectly biocompatible. To achieve these requirements, formulations have become increasingly complex. Although the use of high-throughput systems may accelerate both the formulation phases and the in vitro evaluation of drug delivery systems (DDS) [1], preclinical assays using mammalian models are still time-consuming. Furthermore, limits from ethical and legal points of view for working with those models are already very restrictive and are increasing steadily [2].
The chick embryo is a well-known animal model, which has been extensively studied from Aristotle's time—who opened hen's eggs daily to examine progressive stages of embryogenesis [3]—until the modern molecular era. The increasing interest in the chick embryo as a model in biological and pharmaceutical research is related to its simplicity and low cost compared with mammalian models. Current laws regulating animal experimentation in the USA, the European Union, and Switzerland allow experimentation with chick embryos without authorization from animal experimentation committees, on the grounds that experiments begin and end before hatching. Nevertheless, experimentation with chick embryos must be refined to reduce the number of embryos used through adequate experimental design.
The present review outlines the potential use of the chick embryo, and specifically its chorioallantoic membrane (CAM), as an alternative to mammalian models for the evaluation of DDS. The CAM of the developing chick embryo is an extraembryonic membrane mediating gas and nutriment exchanges until hatching. Since the CAM has a very dense capillary network, it is commonly used to study in vivo both new vessel formation (angiogenesis) and its inhibition in response to different factors. In the literature, the use of CAM in research is referred to as the CAM assay or as the CAM model.
Although chick embryos are not widely used for the evaluation of new drug carriers, the United States Food and Drug Administration (FDA) has approved products preclinically evaluated with chick embryos. Indeed, a FDA guidance for industry, published in June 2006, regarding the development of products for the treatment of chronic cutaneous ulcer and burn wounds, considered the CAM model as an alternative for preclinical testing. Various papers summarized the different applications of the CAM model in areas of interest for the pharmaceutical community, such as angiogenesis and antiangiogenesis [4], [5], [6], wound healing [7], tissue engineering [8], biomaterials and implants [9], [10], [11], and biosensors [12].
The scope of the present review has been restricted to studies regarding the evaluation of DDS with the CAM model. The biological and physiological characteristics of the CAM are presented, as well as the protocols for embryo cultivation and administration of formulations. The use of chick embryos for the evaluation of drug activity, toxicity, biocompatibility, pharmacokinetics, and biodistribution are also summarized, concluding with an outline on the advantages and limitations, as well as some perspectives in the use of this model in pharmaceutical research.
Section snippets
Chick embryo and its chorioallantoic membrane
Chick embryo development lasts 21 days before hatching. Hamburger and Hamilton classified the embryo development depending on a series of stages designed based on the external characteristics of the embryo [13]. The 21-day incubation period corresponds to 46 stages, known as the HH stages, which are not uniformly distributed over the time of development. However, in most studies on DDS, a different classification is followed: the first day of incubation is considered as the first day of
Chick embryos for drug delivery systems evaluation
Pharmaceutical technology scientists make huge efforts to produce new delivery systems capable of regulating the rate of drug delivery, sustaining the duration of the therapeutic action, and/or targeting the delivery of drugs. During the development of DDS, chick embryos can be used to evaluate the activity or toxicity of a drug on both the CAM and CAM-grafted tumors, as well as on the development of the body of the embryo. Toxicity of drugs or carriers on chick embryos can be evaluated in
Advantages and limitations of chick embryos for evaluating drug delivery systems
The interest in using chick embryos for the early evaluation of DDS has been outlined in this overview. However, the chick embryo is still a model and presents some advantages and limitations that are summarized in Table 4. The use of the chick embryo, as a whole living organism, overcomes some limitations encountered when working with simple in vitro systems frequently used in the pharmaceutical field. Although, in vitro drug release in simulated biological media and in vitro cellular models
Conclusions and perspectives
Although the chick embryo and its chorioallantoic membrane have been extensively used to study both angiogenesis and antiangiogenesis processes, its potential use has not been fully exploited in pharmaceutics and biopharmaceutics. Most of the DDS evaluated with the CAM model incorporated drugs modifying the vasculature, such as pro-, antiangiogenic molecules and PS. The strategy frequently used is to optimize the formulations in terms of excipients, drug loading, and other properties by in vitro
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This review is part of the Advanced Drug Delivery Reviews theme issue on “Prediction of Therapeutic and Drug Delivery Outcomes using Animal Models”.