Vitro Transport

The delivery of LHRH through human skin in vitro was measured before, during, and alter the application of direct current (0.5 mA/cm for 30 min), either without or with (Fig. 3) a single exponential electrical pulse, applied at the initiation of the direct-current treatment (Bommannan et al, 1994). Prior to electrical treatment, the passive flux of LHRH was near 0.05 g/(cm2. hr) for both pulsed and nonpulsed samples. The application of 

Electnqsoration ( 500V 6.7msec at 0 houi) + tontopborasls (0.39mA/cm 2 1 hoiH) 

4x10 cm/hr. When the skin was pretreated with ethanol for 2 hr, however, these investigators measured a value of 6 x lO cm/hr. Thus, the passive transport of LHRH through human skin is very small, with a permeability coefficient near 10-5 cm/hr in the absence of a penetration enhancer. 

In a separate experiment, the LHRH flux was measured for 24hr during and following electrotreatment. The results (Fig. 5) were obtained using a direct current ofO.SOmA/cm for 1 h, with or without an exponential pulse (500-V initial amplitude and 6.7-msec time constant). The results again showed a significant increase in LHRH flux following the application of an electrical pulse. Moreover, the flux decreased after treatment to values near that obtained prior to electrotreatment, or in the absence of a pulse. 

A plot of the LHRH transport data (Fig. 4a,b) shows a linear dependence of flux upon current density for both treatment protocols (Fig. 6). The flux of an ionic peptide (J ) of charge Z, through skin is related to the applied current (i) by 

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SNAPs is an acronym for soluble NSF attachment proteins. They were originally discovered as cofactors for NSF that mediate the membrane binding of NSF in in vitro transport assays. Several isoforms of SNAPs exist in mammalian cells. SNAPs are also highly conserved proteins. Crystallographic studies indicated that the proteins form a very stiff and twisted sheet that is formed by a series of antiparallel and tightly packed helices connected by short loops. 

Buur A, N Mprk. (1992). Metabolism of testosterone during in vitro transport across Caco-2 cell monolayers Evidence for beta-hydroxysteroid dehydrogenase activity in differentiated Caco-2 cells. Pharm Res 9 1290-1294. 

The effects of D-glucose observed in vivo are not well reproduced in vitro. Madara [203] reported that cytoskeletal contraction and enhanced paracellular permeability were observed only in an in situ perfusion preparation and not in an isolated tissue preparation. Although its in vivo effect was not tested, 25 mM D-glucose, an effective concentration in the jejunum [47], failed to enhance the in vitro transport of sotalol (log PC = -0.62), atenolol (log PC = 0.16), or nadolol (log PC = 0.93) across the isolated conjunctiva [213], For a similar reason and possibly due to the absence of a Na+-glucose cotransporter in the cornea, 25 mM D-glucose was ineffective in increasing the corneal transport of these three drugs. 

It is important to establish an in vitro system which will allow in vivo transport across the bile canalicular membrane to be predicted quantitatively. By comparing the transport activity between in vivo and in vitro situations in isolated bile canalicular membrane vesicles, it has been shown that there is a significant correlation for nine types of substrates [90]. Here, in vivo transport activity was defined as the biliary excretion rate, divided by the unbound hepatic concentration at steady-state, whereas in vitro transport activity was defined as the initial velocity for the transport into the isolated bile canalicular membrane vesicles divided by the medium concentration [90]. Collectively, it is possible to predict in vivo canalicular transport from in vitro experiments with the isolated bile canalicular membrane vesicles. 

Aoki, J., Suzuki, H., Sugiyama, Y., Quantitative prediction of in vivo biliary excretion clearance across the bile canalicular membrane from in vitro transport studies with isolated membrane vesicles. Abstract of Millennial World Congress of pharmaceutical Sciences, San Francisco, April 16-20, 2000, p. 92. 

Shen Q, Lin Y, Handa T, Doi M, Sugie M, Wakayama K, Okada N, Fujita T, Yamamoto A (2006) Modulation of intestinal P-glycoprotein function by polyethylene glycols and their derivatives by in vitro transport and in situ absorption studies. Int J Pharm 313 49-56. 

Schaefer, M., Roots, 1., and Gerloff, T. (2005) In vitro transport characteristics discriminate wildtype mdrl (abcbl) from ala893ser and ala893thr polymorphisms. Eur. J. Clin. Pharmacol. 61,718. 

Honeywell-Nguyen, P.L., and J.A. Bouwstra. 2003. The in vitro transport of pergolide from surfactant-based elastic vesicles through human skin A suggested mechanism of action. J Control Release 86 145. 

Table 2 Possible Sites for Drug-Drug Interaction and the In Vitro Transport Models... 

Tissue Process From To In vitro transport experiment... 

Figure 1 The schematic diagram for the prediction of drug-drug interactions involving membrane transport from in vitro transport experiments.

A. In Vitro Transport Systems Using Tissues, Cells, and Membrane Vesicles... 

Figure 2 Comparison between the uptake clearance obtained in vivo and that extrapolated from the in vitro transport study of endothelin antagonists. In vivo uptake clearance of endothelin antagonists (BQ-123, BQ-518, BQ-485, compound A) was evaluated by integration plot analysis using the plasma concentration-time profile after intravenous administration (500 nmol/kg) and the amount of drug in the liver and that excreted in the bile. In vitro hepatic uptake clearance was measured using isolated rat hepatocytes and was extrapolated to the in vivo uptake clearance assuming the well-stirred model. Source From Ref. 5.

As mentioned earlier, the underlying mechanisms for many of the transporter-mediated interactions are not fully understood and remain elusive at the present time. With the limited knowledge on the molecular mechanisms of transporter-mediated interaction and the fact that many inhibitors and inducers can simultaneously affect both drug transporters and CYP enzymes, it is difficult to quantitatively differentiate transporter-mediated interactions from CYP-mediated interactions. From the literature, it becomes clear that evidence of transporter-mediated dmg interactions, with few exceptions, is often indirectly derived from in vitro transport studies with cellular culture models and heterologous expression systems. 

Masereeuw R, Jaehde U, Langemeijer MWE, De Boer AG, Breimer DD (1994) In vivo and in vitro transport of zidovudine (AZT) across the blood-brain barrier and the effects of transport inhibitors. Pharm Res 11 324—330. 

HoneyweU-Nguyen PL et al (2002) Transdermal delivery of pergolide from sm-factant-based elastic and rigid vesicles Characterization and in vitro transport studies. Pharm Res 19 991-997... 

Hanisch G, Utter L, Ungell A-L, and Lucas M. Surface pH Measurements During in vitro Transport Studies Across Intestinal Epithelia Thickness of Acid Microclimate. AAPS annual meeting in San Diego, California, 6-10 Nov, 1994. Pharm Res.l994 ,vo 11 10 suppl. 

The first clue that dissociation of SNARE complexes required the assistance of other proteins came from in vitro transport reactions depleted of certain cytosolic proteins. The observed accumulation of vesicles in these reactions indicated that vesicles could form but were unable to fuse with a target membrane. Eventually two proteins, designated NSF and a-SNAP, were found to be required for ongoing vesicle fusion in the in vitro transport reaction. The function of NSF in vivo can be blocked selectively by N-ethylmaleimide (NEM), a chemical that reacts with an essential -SH group on NSF (hence the name, AlEM-sensitive /actor). 

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