Vitro Models

Both brain tissue slice preparation and dissociated primary cultures of brain cells are convenient models to investigate the functions of ascorbic acid in the nervous system. These systems lose their ascorbic acid when they are incubated in ascorbic acid-free media, thus affording models of scurvy. In both preparations, ascorbic acid has been proposed to be a protective agent against several forms of brain insult. 

In the case of primary cultures, the cellular ascorbic acid content drops sharply to undetectable levels when no ascorbic acid is added to the medium. For example, ascorbic acid is not detected in chick forebrain cell cultures, although the chick forebrains contain appreciable levels of ascorbic acid (Makar et al, 1994). Al- 

Ascorbic acid has also been suggested to be neuroprotective by inhibiting NMDA receptor activity via a redox modulatory site on the receptor (Section 4). Theoretically, high levels of ascorbic acid in the central nervous system could be neuroprotective, as inhibition of the NMDA receptor protects neurons from ischemic damage (Simon et al., 1984). In fact, ascorbic acid protects against NMDA-mediated neurotoxicity in cortical cell cultures (Majewska and Bell, 1990), although the required concentration is relatively high (1-3 mM). 

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Chemical Antioxidant Systems. The antioxidant activity of tea extracts and tea polyphenols have been determined using in vitro model systems which are based on hydroxyl-, peroxyl-, superoxide-, hydrogen peroxide-, and oxygen-induced oxidation reactions (109—113). The effectiveness of purified tea polyphenols and cmde tea extracts as antioxidants against the autoxidation of fats has been studied using the standard Rancimat system, an assay based on air oxidation of fats or oils. A direct correlation between the antioxidant index of a tea extract and the concentration of epigallocatechin gallate in the extract was found (107). 

It was agreed at the workshop that endocrine disrupting activity could only be adequately defined in terms of effects in intact animals, be they juvenile or adult, or in the offspring of exposed parents. For many chemicals, evidence of endocrine disrupting activity has been obtained only by the use of in vitro models, such as hormone binding assays. It was accepted, therefore, that chemicals active in such models should be considered only as potential EDs and should be distinguished from those established as active in vivo. For such chemicals, an alternative definition was recommended ... 

Although in vitro models clearly show that MDR transporters can protect tumor cells, their relevance in clinical oncology remains controver sial. As is the case for most potentially useful cancer biomarkers, no universally embraced guidelines for analytical or clinical validation of MDR transporters exist. Evidence linking ABCB1 Pgp/MDRl expression with poor clinical outcome is most conclusive for breast cancer, sarcoma, and certain types of leukemia. The relevance of the other MDR transporters in clinical MDR is still unclear. The prognostic implication of ABCCl/ MRPl remains controversial and very little is known clinically about ABCG2. 

In-vitro models can provide preliminary insights into some pharmacodynamic aspects. For example, cultured Caco 2 cell lines (derived from a human colorectal carcinoma) may be used to simulate intestinal absorption behaviour, while cultured hepatic cell lines are available for metabolic studies. However, a comprehensive understanding of the pharmacokinetic effects vfill require the use of in-vivo animal studies, where the drug levels in various tissues can be measured after different dosages and time intervals. Radioactively labelled drugs (carbon-14) may be used to facilitate detection. Animal model studies of human biopharmaceutical products may be compromised by immune responses that would not be expected when actually treating human subjects. 

European Centre for the Validation of Alternative Methods (ECVAM), six in vitro systems for chronic neurotoxicity testing are recommended for further consideration (Worth and Balls 2002). These are described as in vitro models that may be suitable for long-term toxicity testing. The systems are... 

In the summary of the aforementioned report, the authors recommend, as did earlier reviewers of this subject, the development and evaluation of a tiered testing strategy for neurotoxicity. The further development of in vitro models for establishing mechanisms of neurotoxicity should be part of this strategy. Full consideration should also be given to advances in the omics and other technological fields. 

MaUk AA, Radhakiishnan N, Reddy K, Smith AD, Singhal PC (2002) Morphine-induced macrophage apoptosis modulates migration of macrophages use of in vitro model of urinary tract infection. J Endourol 16 605-610... 

The use of mierobial systems as in vitro models for dmg metabohsm in humans has been proposed sinee there are many similarities between certain microbial enzyme systems and mammalian liver enzyme systems. The major advantages of using miero-organisms is their ability to produce significant quantities of metabohtes that would otherwise be diffieult to obtain from animal systems or by chemical synthesis, and the considerable reduetion in operating costs compared with animal studies. 

Stelzer, A, Slater, NT and Bruggencate, G (1987) Activation of NMD A receptors blocks GABA ergic innervation in an in vitro model of epilepsy. Nature 326 698-701. 

Of course, the term proven efficacy is central to any resource investment in this area. Basic information on time and dose responses in humans to complex foods rich in carotenoids (and other phytochemicals) is pitifully small. Much of our information is based upon inadequate databases derived from chemical analysis, in vitro models that have not been properly evaluated or validated, and short-term, high-dose human studies. Future research progress requires much more rigorous debate on the experimental systems employed... 

Based on the limitations of using human subjects, simple alternative in vitro models were developed to investigate mechanisms involved in the intestinal absorption process of a compound of interest and to screen the relative bioavailability of a compound from various food matrices. However, the data generated from in vitro approaches must be taken with caution because they are obtained under relatively simplified and static conditions compared to dynamic physiological in vivo conditions. Indeed, the overall bioavailability of a compound is the result of several complex steps that are influenced by many factors including factors present in the gastrointestinal lumen and intestinal cells as described later. Nevertheless, these in vitro approaches are useful tools for guiding further smdies in humans. 

In contrast to previous in vivo models, this in vitro model provides the possibility of dissociating experimentally two important processes of intestinal absorption cellular uptake and secretion. Under conditions mimicking the postprandial state (taurocholate/oleic acid supplementation), differentiated Caco-2 cells were able to (1) take up carotenoids at the apical sides and incorporate them into CMs and (2) secrete them at the basolateral sides associated with CM fractions. Using this approach, the extent of absorption of P-carotene through Caco-2 cell monolayers after 16 hr of incubation was 11.2%, a value falling within the in vivo range (9 to 22%). ° - Of the total amount of P-carotene secreted, 78% was associated with the two CM fractions and 10% with the VLDL fraction. ... 

Reglinski, J., Hoey, S. and Smith, W.E. (1988) Exchange reactions between disulfides and myocrisin an in vitro model for a mechanism in chrysotherapy. Inorganica Chimica Acta, 152, 261—264. 

Isab, A.A., Shaw, C.E. Ill and Locke, J. (1988) GC-MS and oxygen-17 NMR tracer studies of triethylphosphine oxide formation from auranofin and water- O in the presence of bovine serum albumin an in vitro model for auranofin metabolism. Inorganic Chemistry, 27, 3406-3409. 

Avdeef, A., Tsinman, O. PAM PA -a drug absorption in vitro model 13. Chemical selectivity due to membrane hydrogen bonding in comho comparisons of HDM-, DOPC-, and DS-PAMPA models. Eur. J. Pharm. Set. 2006, 28, 43-50. 

Avdeef, A., Strafford, M., Block, E., Balogh, M. P., Chambliss, W., Khan, I. Drug absorption in vitro model filter-immobilized artifidal membranes 2. Studies of the permeability properties of lactones in Piper methysticum Forst. Eur.J. Pharm. Sci. 2001, 14, 271-280. 

McLean, A. and Nuttal, L. (1978). An in vitro model of liver injury using paracetamol treatment of liver slices and prevention of injury by some antioxidants. Biochem. Pharmacol. 27, 425-430. 

ANSWER Actually we used synthesized (-l-)-fenfluramine. The fluoxetine story is not clear. It does not block the discriminative cue, but other workers have shown that it blocks the neurotoxicity. We have not looked at it in enough detail or at any of the in vitro models to see whether it blocks or releases serotonin. 

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... 

Abbott NJ (2006) In vitro models for examining and predicting brain uptake of drugs. In Taylor JB, Triggle DJ (eds) Comprehensive medicinal chemistry II. Elsevier, Oxford, Sect 5.13... 

Harper JD, Wong SS, Lieber CM, Lansbury PT Jr. Assembly of A amyloid protofibrils an in vitro model for a possible early event in Alzheimer s disease. Biochemistry 1999 38 8972-8980. 

We define this permeability as apparent, to emphasize that there are important but hidden assumptions made in its derivation. This equation is popularly (if not nearly exclusively) used in culture cell in vitro models, such as Caco-2. The sink condition is maintained by periodically moving a detachable donor well to successive acceptor wells over time. At the end of the total permeation time f, the mass of solute is determined in each of the acceptor wells, and the mole sum mA (t) is used in Eq. (7.10). Another variant of this analysis is based on evaluating the slope in the early part of the appearance curve (e.g., solid curves in Fig. 7.14) ... 

The in vivo environment of the GIT is characterized by a pH gradient the pH value is constant at 7.4 in the receiving compartment (blood), and varying in the donor compartment (lumen) from 5 to 8 from the start to the end of the small intestine. In contrast, the BBB has a constant iso-pH 7.4. Modeling the two environments requires proper pH adjustment in the in vitro model, as indicated in Table 7.22. 

The GIT has about 13% wt/wt negatively charged lipid-to-zwitterionic phospholipid ratio. It is about twice as large in the BBB. Factoring this into the in vitro model is expected to be important. 

The white fat content of the GIT is higher than that of the BBB. The use of triglycerides and cholesterol in in vitro modeling seems important. 

The strategy for the development of the oral absorption model at pION is illustrated in Fig. 7.58. The human jejunal permeabilities reported by Winiwarter et al. [56] were selected as the in vivo target to simulate by the in vitro model. In particular, three acids, three bases and two nonionized molecules studied by the University of Uppsala group were selected as probes, as shown in Fig. 7.58. They are listed in the descending order of permeabilities in Fig. 7.58. Most peculiar in the ordering is that naproxen, ketoprofen, and piroxicam are at the top of the list, yet these three acids are ionized under in vivo pH conditions and have lipophilicity (log Kj) values near or below zero. The most lipophilic molecules tested, verapamil and carbamazepine... 

Ruell, J. A. Tsinman, K. L. Avdeef, A., PAMPA—a drug absorption in vitro model. 4. Unstirred water layer in iso-pH mapping assays in pKR flux-optimized design (pOD-PAMPA) (submitted). 

Avdeef, A., PAMPA—a drug absorption in vitro model. 16. Gradient-pH permeability (in preparation). 

Walter,E. Janich, S. Roessler, B.J. Hilfinger,J.M. Amidon, G. L., HT29-MTX/Caco-2 cocultures as an in vitro model for the intestinal epithelium In vitro-in vivo correlation with permeability data from rats and humans, J. Pharm. Sci. 85, 1070-1076 (1996). 

This section discusses potential health effects from exposures during the period from conception to maturity at 18 years of age in humans, when all biological systems will have fully developed. Potential effects on offspring resulting from exposures of parental germ cells are considered, as well as any indirect effects on the fetus and neonate due to maternal exposure during gestation and lactation. Relevant animal and in vitro models are also discussed. 

In the pharmaceutical sciences, the nonequilibrium thermodynamics approach has been particularly important in the design of osmotic drug delivery devices, as discussed in Chapter 11. It has also been used to describe the convective transport of a binding antibody in an in vitro model of a solid tumor [8], As our appreciation of the roles of convection and osmosis in drug delivery increases, the nonequilibrium thermodynamics approach may find wider appeal. 

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