Finite dose skin mass balance including the lateral part: comparison between experiment, pharmacokinetic modeling and diffusion models.

This work investigates in vitro finite dose skin absorption of the model compounds flufenamic acid and caffeine experimentally and mathematically. The mass balance in different skin compartments (donor, stratum corneum (SC), deeper skin layers (DSL), lateral skin parts and acceptor) is analyzed as a function of time. For both substances high amounts were found in the lateral skin compartment after 6h of incubation, which emphasizes not to elide these parts in the modeling. Here, three different mathematical models were investigated and tested with the experimental data: a pharmacokinetic model (PK), a detailed microscopic two-dimensional diffusion model (MICRO) and a macroscopic homogenized diffusion model (MACRO). While the PK model was fitted to the experimental data, the MICRO and the MACRO models employed input parameters derived from infinite dose studies to predict the underlying diffusion process. All models could satisfyingly predict or describe the experimental data. The PK model and MACRO model also feature the lateral parts.

Non steady-state descriptions of drug permeation through stratum corneum. I. The biphasic brick-and-mortar model.

PURPOSE: The diffusion equation should be solved for the non-steady-state problem of drug diffusion within a two-dimensional, biphasic stratum corneum membrane having homogeneous lipid and corneocyte phases. METHODS: A numerical method was developed for a brick-and-mortar SC-geometry, enabling an explicit solution for time-dependent drug concentration within both phases. The lag time and permeability were calculated. RESULTS: It is shown how the barrier property of this model membrane depends on relative phase permeability, corneocyte alignment, and corneocyte-lipid partition coefficient. Additionally, the time-dependent drug concentration profiles within the membrane can be observed during the lag and steady-state phases. CONCLUSIONS: The model SC-membrane predicts, from purely morphological principles, lag times and permeabilities that are in good agreement with experimental values. The long lag times and very small permeabilities reported for human SC can only be predicted for a highly-staggered corneocyte geometry and corneocytes that are 1000 times less permeable than the lipid phase. Although the former conclusion is reasonable, the latter is questionable. The elongated, flattened corneocyte shape renders lag time and permeability insensitive to large changes in their alignment within the SC. Corneocyte/lipid partitioning is found to be fundamentally different to SC/donor partitioning, since increasing drug lipophilicity always reduces both lag time and permeability.

Identification of the promoter sequences involved in the interleukin-6 dependent expression of the rat alpha 2-macroglobulin gene.

The 5'-region of the rat alpha 2-macroglobulin gene has been characterized. A 5.6 kb Sal I - Xba I fragment containing the first 4 exons of the alpha 2-macroglobulin gene and 1.3 kb of its 5'-flanking region was sequenced. The putative transcriptional start site was determined by RNase protection and primer extension analysis. TATA- and CAAT-box equivalent sequences were found. A potential glucocorticoid receptor binding site was located on the antisense strand. DNA sequences containing the 5'-flanking region of the rat alpha 2-macroglobulin gene were linked to the gene coding for the bacterial chloramphenicol acetyltransferase and introduced into Hep G2 cells. In these transfected Hep G2 cells CAT activity could be induced by recombinant human interleukin-6. Deletion analyses have shown that the sequences between -852 and -777 as well as between -404 and -165 relative to the cap site, contain regulatory elements involved in the interleukin-6 dependent induction of the alpha 2-macroglobulin gene.

Regulation of alpha 2-macroglobulin gene expression by interleukin-6 (BSF-2/HSF).

In rat hepatocyte primary cultures recombinant human interleukin-6 (rhIL-6) induced alpha 2-macroglobulin (alpha 2M) synthesis 54-fold. Half-maximal induction was achieved at a rhIL-6 concentration of 30 pM. RhIL-1 beta led only to a 2-fold increase in alpha 2M synthesis, but strongly impaired the action of IL-6. Intraperitoneal injection of rhIL-6 into male rats resulted in a 19.7-fold increase of alpha 2M mRNA already after 4h. In contrast, alpha 2M mRNA levels (50-fold increase) were reached between 16 and 24 h after intramuscular injection of turpentine. Whereas turpentine-induced inflammation resulted in an increased alpha 2M synthesis in male and female rats, rhIL-6 injection had no effect in female rats. The increases after rhIL-6 administration in mRNA concentrations were followed by corresponding changes in alpha 2M levels in serum. By Northern analysis it was demonstrated that LPS-stimulated human monocytes synthesize IL-6 mRNA. The 5'-end of the rat alpha 2M gene has been isolated and the first 3 exons and 166 base pairs of the 5'-flanking region were identified by a combination of oligonucleotide hybridization and DNA sequencing. The transcriptional start site was determined by RNase protection as well as by primer extension experiments. 5'-CATAAAG-3' and 5'-TCAAAA-3' were found as TATA- and CAAT-box equivalent sequences, respectively. Furthermore, a potential glucocorticoid binding site (5'-TGTTCT-3') was localized on the antisense strand of the alpha 2M gene.

Alpha 2-macroglobulin gene expression during rat development studied by in situ hybridization.

The sites of alpha 2-macroglobulin mRNA synthesis during rat development have been localized by in situ hybridization using a rat alpha 2-macroglobulin cDNA probe. Fetal liver was found to be the major site of alpha 2-macroglobulin mRNA synthesis. In addition, alpha 2-macroglobulin mRNA was detected in brain, spinal cord and eye. Alpha 2-Macroglobulin mRNA was quantitated by use of a sensitive RNAse protection assay. Maximal levels of alpha 2-macroglobulin mRNA were found in fetal livers shortly before birth. A rapid decline of alpha 2-macroglobulin mRNA occurred within 1 day after parturition. A similar time course, although at an approximately 20-fold lower level, was observed for alpha 2-macroglobulin mRNA in livers of pregnant rats. Alpha 2-Macroglobulin mRNA could also be detected in placenta. The levels were comparable to those found in maternal livers.

In vitro synthesis of peroxisomal membrane polypeptides.

Peroxisomal membranes containing predominantly integral peroxisome membrane polypeptides were obtained from a highly purified peroxisomal fraction. Following sodium dodecylsulfate polyacrylamide gel electrophoresis three polypeptides with apparent molecular weights of 69, 36, and 22 kDa were isolated and used to raise antibodies in rabbits. Cell-free synthesis of these polypeptides was carried out in an in vitro translational system derived from rabbit reticulocytes. By subjecting peroxisomal membranes to reductive methylation [14C]-radiolabeled mature membrane polypeptides were obtained. The comparison of the three mature integral peroxisome membrane polypeptides with their corresponding in vitro synthesis products revealed no size differences indicating the lack of recognizable presequences for these peroxisomal membrane polypeptides.

Molecular cloning of cDNA sequences for rat alpha 2-macroglobulin and measurement of its transcription during experimental inflammation.

Poly(A)+ RNA enriched in alpha 2-macroglobulin (alpha 2M) mRNA isolated from livers of rats 18 h after injection of turpentine was used for the synthesis of double-stranded cDNA. The double-stranded cDNA was inserted into the PstI site of the plasmid pBR322 by oligo(dG)-oligo(dC)-tailing technique. Clones containing sequences complementary to alpha 2M mRNA were selected by differential colony hybridization using 32P-labeled poly(A)+ RNA and [32P]cDNA from livers of control and turpentine-treated rats and subsequent hybrid-selected translation. The isolated p alpha 2M1 clone had an insert of 657 base pairs. DNA sequence analysis revealed a homology of about 80% to human alpha 2M. Northern analysis showed that the alpha 2M mRNA from rat liver is about 5600 bases in length. The alpha 2M cDNA was used to measure the in vitro transcription of the alpha 2M gene in isolated nuclei. A 4-fold increase in alpha 2M gene activity was found 14 h after turpentine administration. We conclude that alpha 2M transcription is induced during inflammation.