The CAM-LDPI method: a novel platform for the assessment of drug absorption Stephanie Li Mei Tay, Paul Wan Sia Heng and Lai Wah Chan Department of Pharmacy, National University of Singapore, Singapore Keywords blood perfusion; CAM; drug absorption; LDPI Correspondence Lai Wah Chan, Department of Pharmacy, National University of Singapore, No.18 Science Drive 4, Block S4, Singapore 117543. E-mail: phaclw@nus.edu.sg Received August 26, 2011 Accepted November 21, 2011 doi: 10.1111/j.2042-7158.2011.01431.x Abstract Objectives This study aimed to explore the use of the chicken chorioallantoic mem- brane (CAM) with laser doppler perfusion imaging (LDPI) as a platform to assess absorption of vasoactive drugs. Methods The optimal age of the CAM to be employed in the test and the indicator of vasoactivity were first established.Test substances that included common solvents and vasoactive drugs were tested on the CAM surface to determine their irritancy and blood perfusion effects. Key Findings Insignificant changes in blood perfusion were observed with deion- ized water, 0.9% w/v soldium chloride and 5% w/v glucose monohydrate, as well as theophylline and glucagon. Complex changes in blood perfusion were detected with ethanol, N-methyl-2-pyrrolidone, glycerin and propranolol. Both caffeine and glyc- eryl trinitrate resulted in a drop in blood perfusion. Conclusions It was concluded that the LDPI offers a rapid and non-invasive method to measure blood perfusion in the CAM. The latter provides a potentially useful platform in formulation studies to evaluate the effects of additives on drug absorption using caffeine or glyceryl trinitrate as model drugs. Introduction In vivo drug absorption is one of the critical parameters used to determine the bioavailability of a drug. Drug absorption is a complex process that is affected by various drug properties, formulation factors and physiological variables. In formula- tion studies, there is ongoing investigation on the effects of additives on drug absorption. The traditional method, which involves the use of animals to assess drug absorption, is not only expensive but also time-consuming. In the pursuit of alternatives that employ living tissues but avoid the use of whole animals, it would be ideal to have an in-vivo method that is sensitive, inexpensive and capable of high throughput. Chick chorioallantoic membrane The chick chorioallantoic membrane (CAM) is a potential alternative to animal investigation of the effects of additives on drug absorption. The use of the CAM does not pose ethical issues and does not require ethics committee approval for use in countries such as the USA and Switzerland. Another attrac- tive factor is its reasonable price (a few dollars). The CAM, also known as the chorioallantois, is derived from two extra- embryonic membranes: chorion and allantois. The chorion and allantois start to fuse together to form CAM at about 4 days after the egg is laid by a hen. [1] The incubation period of the egg is 21 days. The day that the egg is incubated, which may not coincide with the day that it is laid, is known as embryo age 0 day (EA 0). The following days are then known as embryo age 1 day (EA 1), embryo age 2 days (EA 2) so on and so forth. Histologically, the CAM consists of three layers: ectoderm, mesoderm and endoderm. [2] Besides being a respi- ratory and excretory organ, it provides support to the under- lying extraembryonic blood vessels such as the vitelline vessels over the surface of the yolk. The CAM is also involved in the transport of sodium and chloride from the allantoic sac, and calcium from the eggshell to the vasculature. Through dilation of the associated blood vessels (known as chorioallantoic vessels), the embryo is able to avoid overheat- ing for a relatively long time. [3] Advantages and limitations of the CAM The egg is disinfected and an opening is made in the shell to expose the CAM for investigation. The CAM can be viewed without disruption through the opening, which is sealed with transparent tape. [3] No restraint of the CAM is necessary in comparison with animal models. In the UK, fertilised eggs up to 10 days old can be used without the need for a licence for animal experimentation. [4] This is in line with the British And Pharmacology Journal of Pharmacy Research Paper © 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society 2012 Journal of Pharmacy and Pharmacology, 64, pp. 517–529 517 Downloaded from https://academic.oup.com/jpp/article/64/4/517/6135300 by guest on 11 March 2023