Statistical testing of drug accumulation in skin tissues by linear regression versus contents of stratum corneum lipids.

This investigation is a contribution to standardization in in vitro drug penetration measurements using excised human skin and to statistical treatment of the observations. The wide variations observed in measurements of drug accumulation in and drug permeation through the stratum corneum are caused not only by analytical errors but also by the variability of the horny layer lipid composition. The last-mentioned systematic influence can be compensated for by stepwise (multiple) linear regression using the contents of the main lipid classes as independent variables. In consequence, the S.E. of estimate given by the regression calculation is lower than the S.E. of the means of the observations. Significant differences in drug quantities accumulated in skin tissues (stratum corneum and dermis) are sensitively detected by Chow's F-test of structural change. Accumulation data of flufenamic acid and hydrocortisone penetrated from different bases are given as examples. The calculation mode is exemplarily explained and discussed. The results of the test for structural change, two-independent-groups t-test and paired-samples t-test are compared. The F-test of structural change proves to be a helpful statistical method suitable to the assessment of biopharmaceutical quality parameters and to measurements using biological materials.

Methoxy-polyethoxy side-chain silastomers as materials controlling drug delivery by diffusion flux.

The density of a diffusion medium and the solubility of the diffusant in this material are predominant parameters which control the diffusion flux. Side-chain silastomers can be structurally modified in such a manner that density and polarity are widely varied and adjustable to distinct conditions. The synthesis of cross-linked methoxy-polyethoxy side-chain polysiloxanes is performed by reaction of alpha, omega-bis-(trimethyl-silyloxy)-poly-(methyl-hydrogen-siloxane), alpha, omega-divinyl-poly-(dimethyl-siloxane), and 4-propenyloxy-(4'-methoxy-polyethoxy)-biphenyl. Silastomer foils were obtained with side-chains having up to 9 ethoxy groups. Various silastomer types were characterized by DSC-measurements, polarization microscopy, uptake of water and salicylic acid (as model drug), and by permeation measurements. The polarity of the polymers depends on their contents of ethoxy groups influencing the uptake of polar substances. Polymers with penta-ethoxy and hepta-ethoxy groups at the side-chains take up water under swelling. The solubility and the distribution coefficients of salicylic acid are linearly correlated to the weight fractions of the methoxy-polyethoxy groups in all silastomer types. The temperature dependence of the distribution coefficients of the penta-ethoxy and hepta-ethoxy polymer types shows deviations from the Arrhenius equation. As the side-chains occupy considerable volumes, the density of the molecular packing within the cross-linked polysiloxane matrices is high. The arrangement of the side-chain domains, therefore, depends on the chain length of the cross-linker; the diffusion coefficients are influenced by this parameter. Evidence for the existence of a lyotropic liquid-crystalline phase was observed for one type of these polymers only. Methoxy-polyethoxy side-chain silastomers as membranes or matrices are suited for controlled drug delivery. Drug liberation rates and swelling by uptake of water can be widely altered by variations of the side-chain structures.

Protein profiles in rabbit pancreatic juice analyzed by HPLC after stimulation of secretion by secretin and cerulein.

This study analyzed the secretory pattern of pancreatic proteins released from the rabbit pancreas after acute stimulation of secretion by the cholecystokinin analog cerulein. To facilitate this, a new analytical approach utilizing high performance liquid chromatography (HPLC) was considered. Secretin (0.1 CU/kg x h) was intravenously infused in anaesthetized rabbits in combination with cerulein (0.05, 0.2 or 0.05 followed by 0.2 ug/kg x h) over 3 hours. Pancreatic juice was collected from the main pancreatic duct. The release of protein, amylase, trypsin and chymotrypsin was measured by conventional photometric methods, and the protein profiles were analyzed by reversed phase HPLC. Separation of pancreatic juice proteins by HPLC (Nucleosil 300-7 RP column; injection of 50 ul aliquots of samples normalized to 10 mg/ml protein concentration) resulted in a resolution of up to 16 peaks. Peaks representing amylase, prolipase, prophospholipase A2, procarboxypeptidases, chymotrypsinogen, trypsinogen, and glycoproteins were identified with some certainty by SDS-gel electrophoresis. Secretin infusion produced a small and short lasting rise in total protein secretion but lead to a persistent increase of fluid flow. The release of enzymes followed a mainly parallel pattern according to the photometric measurements. The resolution of the whole profile of pancreatic juice proteins by HPLC demonstrated only minor variations without a consistent or increasing tendency towards a preferential release of individual enzymes. Since even microheterogenities in the samples became apparent after HPLC, this approach would be sensitive enough to mirror effects like nonparallel release of enzymes.

Penetration and enrichment of flufenamic acid in calf skin from patients with stasis dermatitis.

To investigate the hypothesis that the application of medicaments to skin from the lower leg of patients with stasis dermatitis might lead to their enhanced enrichment, compared with uninvolved skin from the same region, a penetration study was performed with flufenamic acid. In 5 patients with pronounced changes of chronic venous insufficiency and in 5 control patients without chronic venous insufficiency the flufenamic acid content in skin sections parallel to the surface was determined by HPLC. In chronic venous insufficiency-skin, the flufenamic acid concentration was higher in all skin levels compared to control skin. This enrichment of the substance could lead to a prolonged and more intense contact with antigen-presenting cells in this region, thus promoting the development of contact allergies observed so frequently in this pathologic condition.

Skin permeability.

The histochemical and physicochemical aspects of skin permeability and transdermal drug permeation are reviewed under preponderant consideration of the past years' literature supplied by relevant investigations from the author's laboratories. The chemical composition and the physical order of the horny layer lipids as well as the interactions of drugs and vehicle components with these lipids are the basis of understanding drug penetration, its influencing by vehicles and penetration enhancement. By means of measuring drug concentration profiles in human skin the mechanisms of vehicle effects and penetration enhancement are demonstrated. The consequences of increased permeability and xenobiotic enrichment in pathologically altered skin are discussed.

Characterization of phospholipase A2 activity in aspirates of human pancreatic pseudocysts after isolation by reversed-phase high performance liquid chromatography.

Phospholipase A (PLA) is able to attack membrane phospholipids and thereby plays a putative role in the pathogenesis of pancreatic pseudocysts. We looked for PLA2-like activity in aspirates from human pancreatic pseudocysts. In material originating from one cyst which occurred shortly after an acute pancreatitis attack, hydrolyzing enzymatic activity measured by a sensitive bioassay system for PLA2 activity was found without prior trypsin activation (67 x 10(3) U/min/100 microliters). A biochemical characterization of this hydrolyzing enzymatic activity was provided after resolution of the respective proteins contained in the cyst fluid by HPLC. High hydrolyzing activities were found in correspondence to one specific, early eluting peak. The purified enzyme had pH optima at 3.5 and 6. Addition of EDTA (5 mM) to the test system abolished the enzymatic activity which mirrored the requirement for calcium ions. The activity was optimal at calcium concentrations ranging from 1-2 mM. Higher calcium concentrations reduced the enzymatic activity. The enzyme showed high heat stability. SDS-gel analysis of the peak showed one single band with a molecular weight of about 20,000 Daltons. Our findings demonstrate the possibility of activated, PLA-like activity in human pancreatic pseudocyst fluid. We speculate that an inappropriate activation of this enzyme in peri- or intrapancreatic "fluid collections" could account for pseudocyst formation after an acute pancreatitis attack.

DNCB skin test in cases of malignant melanoma.

Two hundred sixty-nine patients suffering from malignant melanoma were sensitized with DNCB acetone solution. The intensity of the skin reaction was normally independent of both the current stage and the further progression of the disease.

Comparative nuclear and cellular incorporation of daunorubicin, doxorubicin, carminomycin, marcellomycin, aclacinomycin A and AD 32 in daunorubicin-sensitive and -resistant Ehrlich ascites in vitro.

The kinetics of cellular and nuclear incorporation of a number of new anthracyclines into daunorubicin-sensitive and -resistant Ehrlich ascites cells were determined in vitro. For comparative quantitative analyses the substances were extracted with a 0.3 N HCl/50% ethanol (v/v) solution from either whole cells or purified citric acid nuclei after various intervals of in vitro incubation. At steady state the intracellular and intranuclear concentrations of daunorubicin and doxorubicin were reduced by about 50% in the resistant cell line. Marcellomycin and carminomycin concentrations were only reduced by 9% and 11%, respectively, and no differences between sensitive and resistant cells were seen in the case of aclacinomycin A and AD 32. When the ratios of nuclear to cellular drug were determined at steady state lowest value was found for AD 32 (0.26). In contrast, aclacinomycin A and carminomycin were mainly (78% and 74%) and marcellomycin almost exclusively (95%) concentrated in the nucleus. When the total amounts of drug incorporated per cell were compared, the highest values were measured for aclacinomycin A and the lowest for AD 32 both in the sensitive and the resistant tumor. Additional determinations of the 50% inhibitory concentrations for thymidine uptake showed similar differences between these anthracyclines which were not related to the potency of the drugs in vivo. It is concluded that apart from nuclear incorporation and inhibition of DNA synthesis other factors may be decisive for anthracycline-induced cytotoxicity.

[Treatment of malignant melanoma with dinitrochlorobenzene ointment]. / Die Behandlung des malignen Melanoms mit Dinitrochlorbenzol-Salbe.

35 patients with malignant melanomas in different stages were treated with dinitrochlorobenzene according to Malek-Mansour in the last three years. Nine typical cases are demonstrated. The treatment is more effective when the tumor lies close to skin surface and has not yet spread. Distant metastases may react--but normally these metastases cannot be removed. Regarding effects and side effects of DNCB, the methods of treatment and the range of indications are described.