In vivo system

The mode of action has been a subject for research for a number of years. While it was originally thought that maleic hydrazide replaced uracil in the RNA sequence, it has been deterrnined that the molecule may be a pyrimidine or purine analogue and therefore base-pair formation is possible with uracil and thymine and there exists the probabiHty of base-pair formation with adenine however, if maleic hydrazide occurs in an in vivo system as the diketo species, then there remains the possibiHty of base-pairing with guanine (50). Whatever the mechanism, it is apparent that the inhibitory effects are the result of a shutdown of the de novo synthesis of protein. 

The ineubation of slices directly in the parent compound (PCA) did not induce 5-HT depletion, and the 5-HT innervation in these slices was indistinguishable from that in control animals. Moreover, incubation of slices from PCA-treated animals in PCA-containing buffer did not prevent the recovery of 5-HT immunoreactive axons. The absence of 5-HT depletion after immersion of hippocampal slices in PCA strongly supports the proposition that PCA and related drugs are not directly neurotoxic. Thus, in vivo systemic administration of the drug appears necessary for the formation of a neurotoxic compound, such as a metabolite of the drug or of 5-HT, which is released. 

Whereas the relationship of solute permeability with lipophilicity has been studied in a large number of in vivo systems (including intestinal absorption models [54,55], blood-brain [56 58] and blood nerve [59] barrier models, and cell culture models [60 62], to name just a few), numerous in vitro model systems have been developed to overcome the complexity of working with biological membranes [63-66]. Apart from oil-water systems that are discussed here, the distribution of a solute between a water phase and liposomes is... 

The analysis of carotenoid identity, conformation, and binding in vivo should allow further progress to be made in understanding of the functions of these pigments in the photosynthetic machinery. One of the obvious steps toward improvement could be the use of continuously tuneable laser systems in order to obtain more detailed resonance Raman excitation profiles (Sashima et al 2000). This technique will be suitable for the investigation of in vivo systems with more complex carotenoid composition. In addition, this method may be applied for the determination of the energy of forbidden Sj or 2 Ag transition. This is an important parameter, since it allows an assessment of the energy transfer relationship between the carotenoids and chlorophylls within the antenna complex. 

The reaction conditions should be optimized prior to scale-up. As the in vivo system is mainly used for initial metabolite characterization, the goal of reaction optimization is finding suitable conditions (not necessary the optimal conditions) to enable rapid synthesis of small amounts of the desired metabolite at a reasonable cost. 

Figure 3. Structure of major DNA adduct detected in many in vivo systems as a result of metabolic activation of benzo[a]pyrene or the reaction of anti-B[alPDE with DNA jji vitro dR=deoxyribose moiety.

Benz[alanthracenes. The parent hydrocarbon is a weak carcinogen and the extent of modification of DNA in most in vivo systems is low. 

The mechanism of prooxidant effect of a-tocopherol in aqueous lipid dispersions such as LDLs has been studied [22], This so-called tocopherol-mediated peroxidation is considered in detail in Chapter 25, however, in this chapter we should like to return once more to the question of possible prooxidant activity of vitamin E. The antioxidant effect of a-tocopherol on lipid peroxidation including LDL oxidation is well established in both in vitro and in vivo systems (see, for example, Refs. [3,4] and many other references throughout this book). However, Ingold et al. [22] suggested that despite its undoubted high antioxidant efficiency in homogenous solution a-tocopherol can become a chain transfer agent in aqueous LDL... 

From chemical point of view, efficient free radical scavengers must contain substituents with the very weak C—H, O—H, or S—H bonds, from which reactive free radicals are able to abstract a hydrogen atom. It can be seen that the antioxidants discussed above (ascorbic acid, a-tocopherol, ubihydroquinones, glutathione, etc) fall under this category. However, many other compounds manifest free radical scavenging activity in in vitro and in vivo systems. 

Sankaranarayanan, K. 1991a. Ionizing radiation and genetic risks II. Nature of radiation-induced mutations in experimental mammalian in vivo systems. Mutat. Res. 258 51-73. 

It is difficult to simulate in vitro the interaction of all of the various cell types and modulators of immune function that make up the in vivo system. 

In vivo systems may assess only short-term site of application or immediate structural alterations produced by agents. Note, however, that tests may be intended to evaluate only local effects. 

Rinkus SJ, Legator MS. 1980. The need for both in vitro and in vivo systems in mutagenicity screening. In de Serres FJ, Hollaender A, eds. Chemical mutagens Principles and methods for their detection. New York, NY Plenum Press, 365-473. 

Lu et al. (1992) performed a comparison of water and solute uptake in the in situ single-pass perfusion model and the isolated loops conscious rat model. Water flux in both experimental set-ups was found to be comparable. It was found that the solute (i.e. acetaminophen and phenytoin) membrane permeabilities (Pm) were consistently higher in the chronically isolated loops compared to the in situ perfusion. It was suggested that this was as a result of greater luminal fluid mixing in the in vivo system. A key advantage of the in vivo approach was that each animal can act as its own control for drug absorption studies. 

Cultured nasal cells are reliable models for drug transport and metabolism studies, since they are known to express important biological features (e.g. tight junctions, mucin secretion, cilia, and various transporters), resembling those found in vivo systems. Moreover, easy control of experimental conditions as well as separation of the permeation step from the subsequent absorption cascade is also possible. A relatively simple primary culture condition using human nasal epithelial cells for in vitro drug transport studies has been established and applied in transport and metabolism studies of drugs. It is known that the culture condition and/or selection of culture media are critical in the recapitulation of well-differentiation features of in vivo nasal mucosal epithelium [46],... 

In addition to screening molecules for intestinal absorption, Caco-2 cells have also been used to study mechanisms of drug transport. For many compounds, intestinal permeation involves a transporter to either aid or limit transepithelial transport. The value of Caco-2 cells in this type of studies is due to the fact that these cells express various membrane transporters relevant to drug absorption.1719-23,28,30 However, when interpreting results of studies that involve carrier-mediated transport, discretion, and scaling factors may be required because of the difference in expression level of transporters between in vitro and in vivo systems.12 Another important consideration in carrier-mediated transport studies is that some transport systems in Caco-2 cells may achieve maximal expression level at different days in culture.17,21,38,74 Thus, validation of Caco-2 cells for mechanistic studies should include the identification of the time for optimal expression of transporters as well as the qualitative evaluation of the transporters to establish that they are representative of the native intestinal transporters. 

While it is relatively easy to show that the two calculations are equivalent in simple systems, it is not so easy with more comjj plex in vivo systems, as when these equilibria are studied with P NMR spectra from perfused or intact organs. We recently (3) became involved in a controversy where a 4-fold difference in magnesium ion level was calculated from substantially identical NMR spectra as a result of such differences in definition. Our experience indicates that an intelligent program to supervise such calculations would be quite useful. 

---