**9.10.** *In vivo* **animal study**

This is one of the stages for preclinical study on the new formulation which can also be a new type of polymer or material used [19]. The safety and efficacy of this formulation need to be established. Before starting the study, the animal ethic committee needs to be consulted to get approval to start the study. Most of the studies are to prove that the pharmacokinet‐ ics of the drug delivered is appropriate as stipulated. The ADME (Absorption, Distribution, Metabolism, and Excretion) of the drug delivered by the system is important at this stage, especially the absorption and distribution.

For the technique in determining the absorption and the distribution of the active in a formulation, a researcher may in his or her study use optical *in vivo* imaging technique for monitoring the distribution of the drug in question the proposed delivery system in comparison with the conventional dosage form available. This technique is able to image the whole body of small animals and body cells. This technique includes both fluorescence *in vivo* imaging and fluorescence microscopy, and a low‐light camera and proper filters were also used to collect fluorescence excitation and emission light from samples. In fluo‐ rescence microscopy, the objects of imaging are cells, slides, or culture dishes, while the whole body of small animals is pictured with optical *in vivo* imaging system. However, *in vivo* imaging is technically a more challenging process, as the animal tissues are opaque or/and thick, therefore, they absorb scatters photons and generate strong autofluorescence. Furthermore, it is essential to apply an appropriate imaging probe, which provides biolog‐ ically stable distribution and preferential accumulation at the intended target site. Loading near‐infrared (NIR) fluorophores with drug delivery agents would be a great opportunity to follow medicine distribution with optical *in vivo* imaging system without using specific conjugated antibodies. Near‐infrared excitable fluorescent agents (NIR) provided deep tis‐ sue penetration and low tissue autofluorescence.

Researcher performing the animal study should also take the opportunity to do plasma level drug monitoring, urine metabolite level, and histological studies on heart, lungs, kidneys, spleen, and the liver.

#### **9.11. The human bioavailability study**

This is a regulatory requirement as to prove that the new system will make the drug avail‐ able as the conventional system. It indirectly also determines if the drug pharmacokinetic parameters in human are the same as for the original available formulation. This is different from bioequivalence, which is used to evaluate the predictable *in vivo* biological equivalence of two proprietary preparations of a drug. Two pharmaceutical products are bioequivalent if they are pharmaceutically equivalent and their bioavailabilities after administration in the same dose are similar to such a degree that their effects, with respect to both efficacy and safety.

Bioavailability measures the extent of a drug reaching the systemic circulation and is there‐ fore available for action at the expected site. For most drugs that are taken orally, the drug is released in the gastrointestinal (GI) tract and arrives at their site of action via the systemic circulation. Plasma concentrations of the drug or its metabolite would provide a marker for the concentration at the site of action and a valid measure of bioavailability. The researcher needs to build a plasma blood concentration time curve to prove the release of the drug from the preparation and its absorption from the GI tract, but also other factors including presystemic metabolism, distribution, and elimination. Bioavailability is proven through the area under the blood drug concentration versus time curve (AUC), the maximum blood concentration (*C*max) and the time to reach maximum concentration (*T*max). Clearly, bioavailability studies of the new delivery systems compared to the conventional ones need to be done so as to be assured that the new delivery system is not inferior compared to the existing systems.
