Mannitol marker

Fig. 5.5. Example of using the Caco-2 monolayers to evaluate paracellular transport of an unknown compound. Two markers, mannitol and propranolol are added together with the unknown compound to the apical side and transport of each of the molecules is measured both in the absence and in the...

Internal standards Used to demonstrate method reproducibility. Reference materials, such as the low permeability markers mannitol and atenolol and the high permeability markers metoprolol and ketoprofen, may be used to compare values between laboratories [3, 50]... 

Evaluation of the epithelial integrity can be performed by measuring the transepithelial electrical resistance (TEER). TEER values ranging from 150 ohms.cm2 up to 600 ohms.cm2 have been reported. An alternative method for assessing the monolayer integrity is to monitor the flux of hydrophilic marker molecules that pass the monolayers by the paracellular route (e.g., mannitol, Na-fluorescein, or atenolol). 

Extracellular marker molecules, such as mannitol, inulin, Na-fluorescein, and PEG-400, have been used to verify a tight epithelium they have also been used for testing the effects of enhancers and increased fluid absorption [159],... 

An alternative method for assessing cell layer integrity is through the use of hydrophilic paracellular transport markers (e.g., radiolabeled D-mannitol or fluorescein-Na+), which passively traverse cells by the paracellular route. Small amounts of compound required for in vitro conjunctival cell culture transport experiments make this approach well suited for screening purposes. Relative absorption index of a series of pharmacologically active molecules can be ranked against known markers for the identification of candidates with potential absorption problems, which is a reliable tool to select drug candidates with optimal characteristics. 

An example of RUI analysis method using [3H]adenosine is shown in Figure 14.3B. The RUI value of [3H]adenosine is greater than that of [3H]d-mannitol (used as a paracellular transport marker) and significantly reduced by 30% in the presence of 2 mM unlabeled adenosine and thymidine, while 2 mM cytidine has no effect. Thus, the nature of the inhibition shown by this approach confirms the carrier-mediated transport of adenosine from the blood to the retina across the BRB in vivo [27],... 

Another study found that 5% Tween 80 and HP-(3-CD were effective as solubilizing compounds with poor aqueous solubility without causing an increase in the transepithe-lial permeability of the monolayer integrity marker mannitol. The effect of these excipients on the biochemical integrity of the cells was not reported. 

Measurements of the changes in the permeability to various markers (e.g., inulin, mannitol, and FlTC-dextran) [73], and... 

FIGURE 14.6 Iontophoretic fluxes (mean SD, n > 4) of the cationic drug propranolol, and of the electroosmotic marker mannitol, as a function of propranolol concentration in the anode solution. It is observed that (a) drug flux does not increase linearly with increasing concentration, and (b) electroosmosis is significantly impeded with increasing propranolol concentration. (Data from Marro, D. et al., Pharm. Res., 18 (12), 1701, 2001.)... 

Ilium and coworkers (1994) reported at first that chitosan is able to promote the transmucosal absorption of small polar molecules as well as peptide and protein drugs across nasal epithelia. Immediately afterwards Artursson and collaborators (1994) reported that chitosan can increase the paracellular permeability of [14C]mannitol (a marker for the paracellular route) across Caco-2 intestinal epithelia. 

Control measurements of the monolayer include measurements of transepithelial resistance with an Endohm Meter (World Precision Instruments, New Haven) and permeability measurements of mannitol and polyethylenglycol 4000 (marker for low... 

Standard markers should be included in all experiments. Usually marker compounds for different permeability classes are used like metoprolol for high permeability and radioactive mannitol for low permeability. Quality assurance criteria define accepted upper permeability values for mannitol (in the case of mannitol many laboratoratories use 1.0 x E-06 cm/sec). Permeability values higher than upper limit should lead to rejection of the test. 

Integrity control of the monolayer is based on measurements of both TEER (transepithelial resistance measurements) and permeability for a standard marker (radioactive mannitol or Lucifer yellow as non-radioactive alternative). 

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