Drug release and skin penetration from solid lipid nanoparticles and a base cream: a systematic approach from a comparison of three glucocorticoids.

Solid lipid nanoparticles (SLNs) can enhance drug penetration into the skin, yet the mechanism of the improved transport is not known in full. To unravel the influence of the drug-particle interaction on penetration enhancement, 3 glucocorticoids (GCs), prednisolone (PD), the diester prednicarbate (PC) and the monoester betamethasone 17-valerate (BMV), varying in structure and lipophilicity, were loaded onto SLNs. Theoretical permeability coefficients (cm/s) of the agents rank BMV (-6.38) ≥ PC (-6.57) > PD (-7.30). GC-particle interaction, drug release and skin penetration were investigated including a conventional oil-in-water cream for reference. Both with SLN and cream, PD release was clearly superior to PC release which exceeded BMV release. With the cream, the rank order did not change when studying skin penetration, and skin penetration is thus predominantly influenced by drug release. Yet, the penetration profile for the GCs loaded onto SLNs completely changed, and differences between the steroids were almost lost. Thus, SLNs influence skin penetration by an intrinsic mechanism linked to a specific interaction of the drug-carrier complex and the skin surface, which becomes possible by the lipid nature and nanosize of the carrier and appears not to be derived by testing drug release. Interestingly, PC and PD uptake from SLN even resulted in epidermal targeting. Thus, SLNs are not only able to improve skin penetration of topically applied drugs, but may also be of particular interest when specifically aiming to influence epidermal dysfunction.

Innovative agents for actinic keratosis and nanocarriers enhancing skin penetration.

Actinic keratosis and cutaneous squamous cell carcinoma are of increasing importance with aging and increased ultraviolet light exposure in Western societies. Efficient and well-tolerated therapy is still a matter of concern. As with tumours of other organs, new target sites and innovative drugs selectively addressing them are widely looked for. Due to the relevance for DNA synthesis and thus cell proliferation, human DNA polymerase alpha should be such a target, the more so as the three-dimensional structure of the active site has been proposed based on the application of molecular modelling methods and molecular dynamics simulations. The modelled structure of the active site was used for docking nucleotide analogues in order to design selective inhibitors. Consequently, well-fitting thymidine and guanosine analogues were synthesized and tested in vitro for their influence on normal and transformed human keratinocytes. In fact, the combination of modelling studies and in vitro tests allowed us to design antiproliferative and cytotoxic agents which are new drug candidates for the therapy of skin tumours, given the agents are no relevant substrates of nucleotide transporters (MRP-4, MRP-5) expressed by skin cancer cells. Essential kinases for nucleoside activation were detected, too, corresponding with the observed effects of nucleoside analogues. Due to the rather high molecular weight and poor solubility, however, skin penetration should be poor and thus topical therapy may require carriers to improve the uptake. This becomes feasible by lipidic and non-lipidic nanoparticles which can enhance the uptake of lipophilic agents up to 13-fold.

Identification of inhibitors of the nicotine metabolising CYP2A6 enzyme--an in silico approach.

The compulsive nature of tobacco use is attributable to nicotine addiction. Nicotine is eliminated by metabolism through the cytochrome P450 2A6 (CYP2A6) enzyme in liver. Inhibition of CYP2A6 by chemical compounds may represent a potential supplement to anti-smoking therapy. The purpose of this study was to rationally design potent inhibitors of CYP2A6. 3D-QSAR models were constructed to find out which structural characteristics are important for inhibition potency. Specifically located hydrophobic and hydrogen donor features were found to affect inhibition potency. These features were used in virtual screening of over 60,000 compounds in the Maybridge chemical database. A total of 22 candidate molecules were selected and tested for inhibition potency. Four of these were potent and selective CYP2A6 inhibitors with IC(50) values lower than 1 muM. They represent novel structures of CYP2A6 inhibitors, especially N1-(4-fluorophenyl)cyclopropane-1-carboxamide. This compound can be used as a lead in the design of CYP2A6 inhibitor drugs to combat nicotine addiction.

Pyrazolinone analgesics prevent the antiplatelet effect of aspirin and preserve human platelet thromboxane synthesis.

BACKGROUND: Anti-inflammatory analgesics, including ibuprofen and naproxen, are known to interfere with the antiplatelet effect of aspirin, presumably as a result of a drug-drug interaction at the level of platelet cyclooxygenase-1 (COX-1). OBJECTIVE: We studied whether dipyrone, which has recently been reported to inhibit COX isoforms by a mechanism different from conventional non-steroidal anti-inflammatory drugs (NSAIDs), also interferes with the antiplatelet effect of aspirin. METHODS: Arachidonic acid- and collagen-induced aggregation, as well as thromboxane formation, were measured in human platelet-rich plasma. Platelet P-selectin expression was determined by flow cytometry and cell-free COX enzyme activity was quantified by luminol-enhanced luminescence of human platelet microsomes. In addition, computerized docking was performed based on the crystal structure of COX-1. RESULTS: 4-Methylaminoantipyrine (MAA), the active metabolite of dipyrone, largely attenuated or even completely abolished the inhibition of arachidonic acid-induced platelet aggregation, thromboxane formation and P-selectin expression by aspirin. Similar results were obtained for other pyrazolinones, as well as for the conventional NSAIDs ibuprofen and naproxen. Moreover, MAA attenuated the effect of aspirin on COX activity of platelet microsomes, suggesting a competition with aspirin at the COX-1 enzyme. This was confirmed by docking studies, which revealed that MAA forms a strong hydrogen bond with serine 530 within the COX-1, thereby preventing enzyme acetylation by aspirin. CONCLUSION: This study demonstrates for the first time that dipyrone and other pyrazolinones have a high potential to attenuate or prevent the antiplatelet effect of aspirin. This should be considered if pyrazolinone analgesics are administered to patients with cardiovascular disease requiring antiplatelet aspirin therapy.

Molecular model of an alpha-helical prion protein dimer and its monomeric subunits as derived from chemical cross-linking and molecular modeling calculations.

Prions are the agents of a series of lethal neurodegenerative diseases. They are composed largely, if not entirely, of the host-encoded prion protein (PrP), which can exist in the cellular isoform PrP(C) and the pathological isoform PrP(Sc). The conformational change of the alpha-helical PrP(C) into beta-sheet-rich PrP(Sc) is the fundamental event of prion disease. The transition of recombinant PrP from a PrP(C)-like into a PrP(Sc)-like conformation can be induced in vitro by submicellar concentrations of SDS. An alpha-helical dimer was identified that might represent either the native state of PrP(C) or the first step from the monomeric PrP(C) to highly aggregated PrP(Sc). In the present study, the molecular structure of these dimers was analyzed by introducing covalent cross-links using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. Inter- and intramolecular bonds between directly neighboured amino groups and carboxy groups were generated. The bonds formed in PrP dimers of recombinant PrP (90-231) were identified by tryptic digestion and subsequent mass spectrometric analysis. Intra- and intermolecular cross-links between N-terminal glycine and three acidic amino acid side chains in the globular part of PrP were identified, showing the N-terminal amino acids (90-124) are not as flexible as known from NMR analysis. When the cross-linked sites were used as structural constraint, molecular modeling calculations yielded a structural model for PrP dimer and its monomeric subunit, including the folding of amino acids 90-124 in addition to the known structure. Molecular dynamics of the structure after release of the constraint indicated an intrinsic stability of the domain of amino acids 90-124.

Development of models for cytochrome P450 2A5 as well as two of its mutants.

It is known that small changes in the amino acid sequence can change the catalytic activity of cytochromes towards substrates dramatically. With the aim to broaden our knowledge about the structural properties of cytochromes and their relation with substrate specificity a model of CYP2A5 was built by homology modelling based on the crystal structure of CYP2C5. Model stability was evaluated by subjection of the model to a free molecular dynamics simulation in a waterbox under almost physiological conditions using the GROMACS program. The protein folding remains stable over 1.5 ns under these conditions. The modelling procedure was repeated for two mutated forms of CYP2A5 with known differing substrate selectivities towards corticosterone and desoxycorticosterone. A detailed analysis of the models and their behaviour in long running molecular dynamics simulations allows an understanding of the requirements for enzyme activity as well as an explanation of respective experimental data on the molecular level.

Glucocorticoids for human skin: new aspects of the mechanism of action.

Topical glucocorticoids have always been considered first-line drugs for inflammatory diseases of the skin and bronchial system. Applied systemically, glucocorticoids are used for severe inflammatory and immunological diseases and the inhibition of transplant rejection. Owing to the progress in molecular pharmacology, the knowledge of the mechanism of action has increased during the last years. Besides distinct genomic targets, which are due to the activation of specific cytoplasmatic receptors resulting in the (trans-) activation or (trans-) repression of target genes, there are non-genomic effects on the basis of the interference with membrane-associated receptors as well as with membrane lipids. In fact, various glucocorticoids appear to differ with respect to the relative influence on these targets. Thus, the extended knowledge of glucocorticoid-induced cellular signalling should allow the design and development of even more specifically acting drugs - as it has been obtained with other steroids, e.g. estrogens for osteoporosis prevention.

Investigations on the predictability of the formation of glassy solid solutions of drugs in sugar alcohols.

A prerequisite for the formation of glassy solid solutions prepared by the melting method is the miscibility of the respective drug and the carrier in the molten state. As could be shown experimentally, all investigated drug/sugar alcohol combinations miscible in the molten state form to some extent glassy solid solutions, dependent on their tendency to recrystallize during preparation. Therefore, the present study focuses on the evaluation of factors that govern the miscibility of molten drugs and sugar alcohols as carriers. In this context, solubility parameters are discussed as a means of predicting miscibility in comparison to a new approach, using calculated interaction parameters derived from molecular dynamics (MD) studies. There is evidence that a Coulomb interaction term C(SR), comprising short-range electrostatic interactions and hydrogen bonding energy is essential for the miscibility of drug and carrier in the molten state. To relate C(SR) to the molecular volume, a non-dimensional parameter P(i) is defined. For this parameter, a limiting value for miscibility exists. Contrary, calculated solubility parameter differences between drug and sugar alcohol in the range of 8-15 MPa(1/2) are not suitable for a prediction of miscibility or immiscibility, since the mixtures deviate from regular solution behavior. In irregular mixtures of drugs and sugar alcohols, an excess entropy and the formation of hydrogen bonds between unlike molecules favor miscibility, that cannot be predicted by regular solution theory.

Charge-oriented modelling in charge-transfer systems.

Various mechanisms are often used toxplain the charge-transfer interactions between various electron donors and acceptors. Commonly accepted mechanisms are those in witch the acceptors forms a weak interaction of the Lewis acid--Lewis base type. A modelling approach is applied in this study to examine this mechanism as well as other mechanism. A charge-oriented mechanism is proposed in solvent systems that may not favour this mechanism of interaction.

Molecular modelling of 17 alpha-hydroxylase-17,20-lyase.

New methods in treatment of hormone-dependent diseases like prostate or breast cancer have become a major subject in medical and pharmaceutical research. Because of the direct correlation of cancer growth and hormone concentration inhibition of hormone biosynthesis reveals a promising strategy in cancer therapy. The key enzyme of androgen biosynthesis is the cytochrome P450 system 17 alpha-hydroxylase-17,20-lyase. To gain deeper insights into the structure and function of this enzyme, whose crystal structure is still unknown we present in this paper a theoretical 3D-model of the human 17 alpha-hydroxylase-17,20-lyase. The model was built by homology modelling using the crystal structure of the P450 CYPeryF as a template. After energy minimisation followed by molecular dynamics simulation the refined model exhibits reasonable protein geometry and a good protein folding quality. For evaluation of protein stability the structure was subjected to molecular dynamics in a waterbox under almost physiological conditions using the GROMACS program. The protein structure and folding remains stable even after 300 ps of free molecular dynamics simulation. The calculation of interaction fields employing the program GRID was used to characterise the active site of the protein. Subsequent docking studies with the natural substrate pregnenolone and further molecular dynamics of the protein-substrate-complexes enabled us to propose a putative binding-site for the physiological substrates.

Mechanistic appraisal of the charge-transfer complexes of promethazine with chloranil: a modelling approach.

Various mechanisms are often used to explain the interaction between electron donors and acceptors. Commonly proposed mechanisms are those in which the acceptor interacts with the aromatic pi-systems in the donor molecule or the acceptor forms a weak interaction of the Lewis acid with Lewis base type. In this study, the above mechanisms were examined as well as other possible mechanisms. Promethazine was chosen as the model drug containing aromatic systems capable of pi-pi interaction as well as N-methyl group capable of forming a complex with the weak Lewis acid, p-chloranil. Our modelling studies revealed that the situation where the p-chloranil interacts with a protonated N-methyl group is the most significant mechanism of interaction, based on the calculated energies for the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO), the Tripos force field energy terms and also the stability of the complexes during molecular dynamics simulations.

Molecular dynamics simulations of stratum corneum lipid models: fatty acids and cholesterol.

We report the results of an investigation on stratum corneum lipids, which present the main barrier of the skin. Molecular dynamics simulations, thermal analysis and FTIR measurements were applied. The primary objective of this work was to study the effect of cholesterol on skin structure and dynamics. Two molecular models were constructed, a free fatty acid bilayer (stearic acid, palmitic acid) and a fatty acid/cholesterol mixture at a 1:1 molar ratio. Our simulations were performed at constant pressure and temperature on a nanosecond time scale. The resulting model structures were characterized by calculating surface areas per headgroup, conformational properties, atom densities and order parameters of the fatty acids. Analysis of the simulations indicates that the free fatty acid fraction of stratum corneum lipids stays in a highly ordered crystalline state at skin temperatures. The phase behavior is strongly influenced when cholesterol is added. Cholesterol smoothes the rigid phases of the fatty acids: the order of the hydrocarbon tails (mainly of the last eight bonds) is reduced, the area per molecule becomes larger, the fraction of trans dihedrals is lower and the hydrophobic thickness is reduced. The simulation results are in good agreement with our experimental data from FTIR analysis and NIR-FT Raman spectroscopy.

Investigations on inhibitors of human 17 alpha-hydroxylase-17,20-lyase and their interactions with the enzyme. Molecular modelling of 17 alpha-hydroxylase-17,20-lyase, Part II.

New methods in treatment of hormone-dependent diseases like prostate or breast cancer have become a major subject in medical and pharmaceutical research. Because of the direct correlation of cancer growth and hormone concentration, inhibition of hormone biosynthesis presents a promising strategy in cancer therapy. The key enzyme in androgen biosynthesis is the 17 alpha-hydroxylase-17,20-lyase a cytochrome P450 system, which specifically converts gestagens to androgens. Because the 3D-structure of the enzyme is still unknown most recently a ligand-based design was used to gain deeper insights into protein structure and function. In this paper we present molecular modelling studies on compounds acting as competitive inhibitors of the human 17 alpha-hydroxylase-17,20-lyase. The compounds developed by Hartmann et al. belong to two different structural classes and show a wide range of inhibitory potency. The physico-chemical properties of the molecules were investigated and compared by studying structural flexibility and by calculating molecular interactions fields. The superimposition of all inhibitors in a low energy conformation yielded in the common pharmacophore. In the second part of the paper individual inhibitors were docked into the active site of the enzyme model of CYP17 developed in our group. The dynamic behaviour and stability of the protein-inhibitor-complexes was studied. The protein ligand interactions observed in course of the molecular dynamics simulations correspond well with the experimental data.

Construction of a full three-dimensional model of the transpeptidase domain of Streptococcus pneumoniae PBP2x starting from its Calpha-atom coordinates.

A new method is described for generating all-atom protein structures from Calpha-atom information. The method, which combines both local structural trace alignments and comparative side chain modeling with ab initio side chain modeling, makes use of both the virtual-bond and the dipole-path methods. Provided that 3D structures of structurally and functionally related proteins exist, the method presented here is highly suitable for generating all-atom coordinates of partly solved, low-resolution crystal structures. Particularly the active site region can be modeled accurately with this procedure, which enables investigation of the binding modes of different classes of ligands with molecular dynamics simulations. The method is applied to the trace of Streptococcus pneumoniae, in order to construct an all-atom structure of the transpeptidase domain. Since after generation of full coordinates of the transpeptidase domain the structure had been solved to 2.4 A resolution, new X-ray coordinates for the worst modeled loop (residues T370 to M386; 17 out of a total number of 351 residues constituting the transpeptidase domain) were incorporated, as kindly provided by Dr. Dideberg. The structure was relaxed with molecular dynamics simulations and simulated annealing methods. The RMS deviation between the 144 aligned Calpha-atoms and the corresponding ones in the originally solved 3.5 A resolution crystal structure was 0.98. The 351 Calpha-atoms of the whole transpeptidase domain of the final model showed an RMS deviation of 1.58. The Ramachandran plot showed that 79.3% of the residues are in the most favored regions, with only 1.0% occurring in disallowed regions. The model presented here can be used to investigate the three-dimensional influences of mutations around the active site of PBP2x.

Skin penetration and metabolism of topical glucocorticoids in reconstructed epidermis and in excised human skin.

PURPOSE: To investigate pharmacokinetic differences between the nonhalogenated double ester prednicarbate (PC) and the fluorinated monoester betamethasone 17-valerate (BM17V) their metabolism in human keratinocytes and fibroblasts as well as their permeation and biotransformation in reconstructed epidermis and excised human skin was compared. Special attention was given to the 17-monoesters because of their high receptor affinity and antiproliferative effects. METHODS: Glucocorticoid penetration was determined using Franz diffusion cells, quantifying metabolite concentrations by HPLC. Chemical stability and reactivity of the monoesters was determined by molecular modeling analysis. RESULTS: PC accumulated in the stratum corneum. A considerable amount of penetrating PC was hydrolyzed by viable keratinocytes to prednisolone 17-ethylcarbonate (PI7EC), P17EC permeated the skin very rapidly when compared to BM17V. Overall P17EC concentrations in viable tissue were low. Inside of the acceptor fluid, but not within the tissue, P17EC was converted to the more stable prednisolone 21-ethylcarbonate (P21EC). CONCLUSIONS: The inactivation of highly potent, but also cell toxic, 17-monoesters to almost inactive 21-congeners seen with isolated cell monolayers appears less important in the skin. In vitro determination of the dermal 17-monoesters concentrations may allow the prediction of the atrophogenic risk in man. BM17V levels exceeding P17EC concentration about 6-fold may contribute to its lower tolerance when compared to PC.

Relaxant activity in rat aorta and trachea, conversion to a muscarinic receptor antagonist and structure-activity relationships of new K(ATP) activating 6-varied benzopyrans.

To characterize ATP-sensitive channels (K(ATP) channels) benzopyrans with different substituents at position 6 were synthesized as new K(ATP)-activators. Their relaxant potencies were determined in rat aorta and trachea. In aorta, pEC50-values (-log, M) ranged from 7.37 to 5.43; in trachea, pEC50-values were 0.3 to 0.8 log units lower. Functional data were compared with binding data obtained in calf tracheal cells using the cyanoguanidine [3H]P1075 (N-cyano-N'-1,1-dimethyl[2,3(n)-3H]propyl)-N11-(3-pyridinyl)guanidine) as radioligand. A high correlation (r = 0.96) between pEC50- and pKD-values indicated that tracheal relaxation produced by benzopyrans is mediated via K(ATP) channels without signal amplification. The permanently charged trimethylammonium derivative designed as a probe for the membrane site of action completely lost its affinity for K(ATP) channels, but converted to an antagonist for muscarinic acetylcholine receptors (pK(B) = 6.12+/-0.10), as confirmed in radioligand binding studies (pK(D) = 5.77+/-0.04). Structure-activity analyses revealed that the 6-substituent influences biological activity by a direct receptor interaction of its own and not indirectly by withdrawing electrons from the benzopyran nucleus. The variance of the biological activity is primarily determined by electrostatic properties, but desolvation energies additionally contribute.

Pharmacophore and pseudoreceptor modelling of class Ib antiarrhythmic and local anaesthetic lidocaine analogues.

A molecular modelling study was carried out in order to investigate the molecular binding behaviour of antiarrhythmically and local anaesthetically active aminoacylanilide derivatives from the lidocaine type at their specific sodium channel binding site. An examination of relevant X-ray structures and of results derived from systematic and random search conformational analyses yielded information about the spatial requirements of these sodium channel blocking compounds. Common structural elements in combination with their non-covalent interaction potentials were used to generate a rational pharmacophore model. To further support and refine this model an atomistic pseudoreceptor of the Na+ channel binding site was constructed using a training set of eight well-defined lidocaine homologues. With the final pseudoreceptor, composed of tyrosine, phenylalanine, serine, valine and three isoleucine residues, it was possible to correlate experimental versus calculated dissociation constants of the training set with a correlation coefficient of 0.98. To test the accuracy of this model, the affinities of three additional compounds, not used for pseudoreceptor modelling, were predicted. After free relaxation within the binding cavity using a Monte-Carlo minimization the test set yielded a RMS error in the prediction of 0.039 kcal/mol corresponding to an uncertainty factor of 1.06. In addition, this hypothetical receptor model provides evidence for an exceptional binding mode of the lidocaine metabolite glycinexylidide (GX) which could explain its low binding affinity and thereby possibly the minor physiological effects with respect to lidocaine.

Development of a binding site model for histamine H3-receptor agonists.

On the basis of molecular modelling studies the structural and conformational requirements for receptor affinity and activity of histamine H3-receptor agonists were studied. It was shown that the known H3-receptor agonists can be fitted accurately into a common pharmacophoric pattern. Using the YAK pseudoreceptor approach an amino acid model for the H3-receptor agonist binding site was generated which reflects binding properties and biological data of the investigated agonists. The postulated binding site model was validated by predicting biological data for four structures not considered in model construction. The amino acid positions of the pseudoreceptor were found to be in good agreement with calculated GRID interaction fields for the investigated histamine H3-receptor agonists.

Modified cyclodextrins as chiral selectors: molecular modelling investigations on the enantioselective binding properties of heptakis(2,3-di-O-methyl-6-O-tert.-butyldimethylsilyl)-beta-cyclodextri n.

Molecular modelling methods have been used to investigate the enantioselective binding properties of chiral dihydrofuranones on heptakis(2,3-di-O-methyl-6-O-tert.-butyldimethylsilyl)-beta-cyclod extrin in capillary gas chromatography. A conformational analysis of the modified beta-cyclodextrin was performed using annealed molecular dynamics. With the program GRID the molecular interaction potential for each of the received energetically reasonable structures of the beta-cyclodextrin and the dihydrofuranones was evaluated using different probe groups. The results of these computations have been used as starting points for constructing geometrically reasonable host-guest complexes between the beta-cyclodextrin and the dihydrofuranones. The subsequently performed molecular dynamics simulations yielded different complex states reflecting the conformational flexibility of the diastereomeric complexes. Considering the evaluated interaction energy between the beta-cyclodextrin and the dihydrofuranones as a measure of complex stability the results are in close agreement with the experimentally determined elution sequences. The methodology for the construction of the interaction model used in this study is capable of simulating the experimental data. We believe that it may serve as a basis for predictions of hitherto unknown elution sequences at modified cyclodextrins.