Identification of small peptides and glycinamide that inhibit melanin synthesis using a positional scanning synthetic peptide combinatorial library.

BACKGROUND: Antimelanogenic peptides are potentially useful to treat hyperpigmentation, but many peptides have limited application because of high cost and/or low activity. OBJECTIVES: To identify small and potent peptide inhibitors of cellular melanin synthesis that are useful for cosmetic and medical applications. METHODS: A positional scanning synthetic tetrapeptide combinatorial library was used for screening of potentially active peptides. Antimelanogenic activities of the peptide pools and individual peptides were evaluated in B16-F10 melanoma cells and human epidermal melanocytes treated with alpha-melanocyte-stimulating hormone (α-MSH). RESULTS: Predicted active tetrapeptide sequences were R-(F/L)-(C/W)-(G/R)-NH2 . Of the individual tetrapeptides tested, D3 (RFWG-NH2 ) and D5 (RLWG-NH2 ) exhibited high antimelanogenic activities. Tetrapeptide D9 (FRWG-NH2 ) with a sequence identical to that of a portion of α-MSH also showed antimelanogenic activity. Of the tripeptides tested, E5 (FWG-NH2 ), E6 (LWG-NH2 ) and E7 (RWG-NH2 ) were relatively more active. Dipeptide F1 (WG-NH2 ) and monopeptide G1 (G-NH2 , glycinamide) retained activity, but G2 (Ac-G-NH2 ) and G3 (glycine) did not. The antimelanogenic activities of peptides D3, E5, F1 and G1 were verified in α-MSH-stimulated human epidermal melanocytes. Commercially available G-NH2 ·HCl suppressed the phosphorylation levels of cAMP-responsive element binding protein, protein levels of microphthalmia-associated transcription factor and tyrosinase, l-tyrosine hydroxylase activity of tyrosinase, and the melanin levels in stimulated cells. CONCLUSIONS: Small peptides, including glycinamide and tryptophanyl glycinamide, are potent antimelanogenic agents with potential value for the treatment of skin hyperpigmentation.

Synthesis and Biological Evaluation of Thalidomide Derivatives as Potential Anti-Psoriasis Agents.

Several thalidomide derivatives were synthesized and evaluated for their anti-inflammatory activity. Introduction of the benzyl group to the parent thalidomide is unfavorable in which 2-(1-benzyl-2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (4a) was inactivated. However, the inhibitory activities on TNF-α and IL-6 expression in HaCaT cells were improved by the substitution of a chloro- or methoxy- group at the phenyl position of 4a. The IL-6 inhibitory activity decreased in an order of 5c (69.44%) > 4c (48.73%) > 6c (3.19%) indicating the 3-substituted derivative is more active than the 4-substituted counterpart, which in turn is more active than the 2-substituted counterpart. Among them, 2-[1-(3-chlorobenzyl)-2,6-dioxopiperidin-3-yl]isoindoline-1,3-dione (5c) was found to inhibit TNF-α and IL-6 expression in HaCaT cells with a higher potency than thalidomide and no significant cell cytotoxicity was detected at 10 µM. In psoriasis, Compound 5c reduced IL-6, IL-8, IL-1ß and IL-24 in imiquimod-stimulated models. Our results indicated that compound 5c is a potential lead of novel anti-psoriasis agents. Structural optimization of compound 5c and its in vivo assay are ongoing.

Novel Furocoumarin Derivatives Stimulate Melanogenesis in B16 Melanoma Cells by Up-Regulation of MITF and TYR Family via Akt/GSK3ß/ß-Catenin Signaling Pathways.

The extracts of Ficuscarica L. and Psoralen corylifolia L. are traditional Uygur medicines for the treatment of vitiligo, and its active ingredients furocoumarins, were are found to be the most effective agents against this skin disorder nowadays. Therefore, a series of novel easter derivatives (8a-8p) of furocoumarin were designed and synthesized based on our previous research to improve this activity in the present study. The synthesized derivatives were biologically evaluated for melanin synthesis in murine B16 cells and the SAR (structure-activity relationship) was summarized. Eight derivatives were more potent than positive control (8-MOP, 8-methoxypsoralan), especially compounds 8n (200%) and 8o (197%), which were nearly 1.5-fold potency when compared with 8-MOP (136%). Furthermore, the signaling pathway by which 8n activates the melanin biosynthesis was defined. Our results showed that it not only elevated the melanin content, but also stimulated the activity of tyrosinasein a concentration-dependent manner. Increasing of phosphorylation of Akt (also named PKB, protein kinase B) and non-activated GSK3ß (glycogen synthase kinase 3 beta), which inhibited the degradation of ß-catenin were observed through Western blot analysis. The accumulation of ß-catenin probably led to the activation of transcription of MITF (microphthalmia-associated transcription factor) and TYR (tyrosinase) family, as well as the subsequent induction of melanin synthesis.

Promotion of hair growth by newly synthesized ceramide mimetic compound.

Based on the crucial roles of ceramides in skin barrier function, use of ceramides or their structural mimetic compounds, pseudoceramides, as cosmetic ingredients are getting more popular. While currently used pseudoceramides are intended to substitute the structural roles of ceramides in stratum corneum, development of bioactive pseudoceramides has been repeatedly reported. In this study, based on the potential involvement of sphingolipids in hair cycle regulation, we investigated the effects of newly synthesized pseudoceramide, bis-oleamido isopropyl alcohol (BOI), on hair growth using cultured human hair follicles and animal models. BOI treatment promoted hair growth in cultured human hair follicles ex vivo and induced earlier conversion of telogen into anagen. Although we did not find a significant enhancement of growth factor expression and follicular cell proliferation, BOI treatment resulted in an increased sphinganine and sphingosine contents as well as increased ceramides contents in cultured dermal papilla (DP) cells. Taken together, our data strongly suggest that biologically active pseudoceramide promotes hair growth by stimulating do novo synthesis of sphingolipids in DP cells.

[New developments in extemporaneous formulations]. / Neue Entwicklungen der Magistralrezepturen.

Dermatology is in a state of flux, and systemic therapies have changed the prescription practice in the past few years. Nevertheless, topical therapy for dermatological illnesses is still the mainstay of dermatologists. Pharmaceutically manufactured drugs have a wide spectrum and allow for variability. Additionally, there are therapeutic niches that can be bridged by prescribing extemporaneous formulations. This is also true for the newly established basic therapies for many chronic dermatological illnesses which have become essential and are needed in large amounts. Unfortunately, neither during medical school, nor during residency training, not even the basic knowledge or the complexity of these extemporaneous formulations for topical therapy in dermatology is taught. This emphasizes why standardized, proven extemporaneous formulations are vital for physicians to achieve optimal and goal-oriented therapy for their patients. Sensible and effective prescriptions enhance the quality of formulations and the maintenance and well-being of our patients.

Rosmarinus officinalis L. extract and some of its active ingredients as potential emulsion stabilizers: a new approach to the formation of multiple (W/O/W) emulsion.

Nowadays, novel topical formulations loaded with natural functional actives are under intense investigations. Therefore, the aim of our study was to evaluate how the rosemary extract and some of its active ingredients [rosmarinic acid (RA), ursolic acid (UA) and oleanolic acid (OA)] affect technological characteristics of multiple emulsion. Formulation has been prepared by adding investigated solutions (10%) in water/oil/water (W/O/W) multiple emulsion consisting of different lipophilic phases: olive oil and liquid paraffin, with 0.5% emulsifying agent (complex of sodium polyacrylate and polysorbate 20) under constant stirring with mechanical stirrer at room temperature. The emulsion parameters were evaluated using centrifugation test, freeze-thaw cycle test, microscopical and texture analyses. Rosemary's triterpenic saponins UA and OA showed the highest emulsion stabilizing properties: they decreased CI from 3.26% to 10.23% (p < 0.05). According to obtained interfacial tension data, the effect of rosemary active ingredients is not surfactant-like. Even though emulsifier itself at low concentration intends to form directly the multiple emulsion, the obtained results indicate that rosemary extract containing active ingredients does not only serve as functional cosmetic agent due to a number of biological activities, but also offer potential advantages as a stabilizer and an enhancer of W/O/W emulsions formation for dermopharmaceutical and cosmetic preparations.

Magistral formulations and pruritus therapy - What is established, what is confirmed, what is new?

Pruritus is a common symptom encountered by many different specialties. One must clinically differentiate between pruritus associated with skin disease or inflammation and pruritus with normal skin. Searching for possible underlying diseases is indispensable, because pruritus can be very chronic and has multiple pathogenetic mechanisms. Therapy ­ especially topical therapy ­ is difficult and often not successful. Very often systemic treatment has to be combined with topical approaches, considering both the active ingredients and appropriate vehicles. There are still open therapeutic gaps in the pharmaceutical product market, which can partially be resolved by using standard prescriptions for formulations. Due to the new pharmacy practice order, standardized compounded formulations should be given preference, since individual formulations often do not pass the plausibility check of the compounding pharmacist. Also the use of cosmetic ingredients (by example, commercial cold creams) is no longer permitted, since only ingredients with pharmaceutical quality can be used in compounding. We will show ­ based on practical cases ­ different therapeutic options for treatment with standardized magistral formulations from the NRF (New German Pharmacopoeia for compounded medications).

Dimeric cinnamoylamide derivatives as inhibitors of melanogenesis.

Dimeric cinnamoylamide derivatives were synthetized and tested as inhibitors of tyrosinase activity and melanin formation. The most active dimeric cinnamoylamide derivatives was dimeric compound of p-coumaric acid (compound 1) that inhibited tyrosinase activity more efficiently than p-coumaric acid. It also inhibited melanin production by B16 melanoma cell line and normal human melanocytes more efficiently than kojic acid. We next investigated the potential mutagenic and skin sensitization effect of compound 1. Compound 1 was found to induce no mutagenic activity, no irritation and no delayed contact hypersensitivity at the maximum concentration of 10%. In vitro percutaneous absorption studies exhibited that compound 1 could diffuse across the skin till its site of action. All these results lead us to propose that compound 1 may be a safe and effective candidate for treating skin hyperpigmentation related disorders.

A Chemical Switch for Transforming a Purine Agonist for Toll-like Receptor 7 to a Clinically Relevant Antagonist.

Toll-like receptor 7 (TLR7) is an established therapeutic target in myriad autoimmune disorders, but no TLR7 antagonist is available for clinical use to date. Herein, we report a purine scaffold TLR7 antagonist, first-of-its-kind to our knowledge, which was developed by rationally dissecting the structural requirements for TLR7-targeted activity for a purine scaffold. Specifically, we identified a singular chemical switch at C-2 that could make a potent purine scaffold TLR7 agonist to lose agonism and acquire antagonist activity, which could further be potentiated by the introduction of an additional basic center at C-6. We ended up developing a clinically relevant TLR7 antagonist with favorable pharmacokinetics and 70.8% oral bioavailability in mice. Moreover, the TLR7 antagonists depicted excellent selectivity against TLR8. To further validate the in vivo applicability of this novel TLR7 antagonist, we demonstrated its excellent efficacy in preventing TLR7-induced pathology in a preclinical murine model of psoriasis.

Potent and selective nonpeptidic inhibitors of procollagen C-proteinase.

6-Cyclohexyl-N-hydroxy-3-(1,2,4-oxadiazol-5-yl)hexanamides were previously disclosed as inhibitors of procollagen C-proteinase (PCP) culminating in the identification of amide 1. Our objective was to discover a second inhibitor that would have improved affinity for PCP and to optimize properties for transepidermal delivery (TED) to intact skin. Further investigation of this template identified a number of potent PCP inhibitors (IC50 values of 2-6 nM) with improved TED flux. Sulfonamide 56 had excellent PCP enzyme activity when measured with a peptide substrate (Ki 8.7 nM) or with the endogenous substrate procollagen (IC50 3.4 nM) and demonstrates excellent selectivity over MMPs involved in wound healing (>10 000-fold). In the fibroplasia model, 56 inhibited deposition of insoluble collagen by 76 +/- 2% at 10 microM and was very effective at penetrating human skin in vitro with a TED flux of 1.5 microg/cm2/h, which compares favorably with values for agents that are known to penetrate skin well in vivo. Based on this profile, 56 (UK-421,045) was selected as a candidate for further preclinical evaluation as a topically applied, dermal anti-scarring agent.

Successful co-encapsulation of benzoyl peroxide and chloramphenicol in liposomes by a novel manufacturing method - dual asymmetric centrifugation.

Encapsulation of more than one active pharmaceutical ingredient into nanocarriers such as liposomes is an attractive approach to achieve a synergic drug effect and less complicated dosing schedules in multi-drug treatment regimes. Liposomal drug delivery in acne treatment may improve drug efficiency by targeted delivery to pilosebaceous units, reduce adverse effects and improve patient compliance. We therefore aimed to co-encapsulate benzoyl peroxide (BPO) and chloramphenicol (CAM) into liposomes using the novel liposome processing method - dual asymmetric centrifugation (DAC). Liposomes were formed from soybean lecithin, propylene glycol and distilled water (2:1:2w/v/v ratio), forming a viscous liposome dispersion. Liposomes containing both drugs (BPO-CAM-Lip), single drug (BPO-Lip and CAM-Lip), and empty liposomes were prepared. Drug entrapment of BPO and CAM was determined by a newly developed HPLC method for simultaneous detection and quantification of both drugs. Encapsulation of around 50% for BPO and 60% for CAM respectively was obtained in both single-drug encapsulated formulations (BPO-Lip and CAM-Lip) and co-encapsulated formulations (BPO-CAM-Lip). Liposome sizes were comparable for all liposome formulations, ranging from 130 to 150nm mean diameter, with a polydispersity index <0.2 for all formulations. CAM exhibited a sustained release from all liposomal formulations, whereas BPO appeared retained within the liposomes. BPO retention could be attributed to its poor solubility. However, HaCaT cell toxicity was found dependent on BPO released from the liposomes. In the higher concentration range (4%v/v), liposomal formulations were less cytotoxic than the corresponding drug solutions used as reference. We have demonstrated that DAC is a fast, easy, suitable method for encapsulation of more than one drug within the same liposomes.

Synthesis and in vitro and in vivo structure-activity relationships of novel antifungal triazoles for dermatology.

In search for new compounds with potential for clinical use as antifungal agents in dermatology, a series of 12 azole compounds were synthesized stereospecifically and investigated specifically for their activity against dermatophyte fungal infections in animal models. This panel of azoles was studied in vitro and compared with itraconazole and terbinafine for their antifungal activity using a panel of 24 Candida spp. and 182 dermatophyte isolates. Three azoles (1c, 2c, and 4c) showed in vitro antifungal potency equivalent to itraconazole, but superior to terbinafine, against a panel of 24 Candida spp. with comparable or lower activity than that of itraconazole and terbinafine against 182 dermatophyte isolates and only rare activity against other pathogenic fungi. However, in vivo 1c and 4c, both given orally, demonstrated antifungal activity at least three times greater than itraconazole and were superior compared to terbinafine in M. canis infected guinea pigs. In a mouse model infected by T. mentagrophytes, again 4c, but not 1c, showed 5-fold superior activity over itraconazole and terbinafine. Compound 2c was effective in both models but less effective than itraconazole in these models. On the basis of these promising results, 4c is currently being clinically investigated for its potential as a novel antifungal agent against dermatophytosis.

Norpiperidine imidazoazepines as a new class of potent, selective, and nonsedative H1 antihistamines.

Clinical doses of available H(1) antihistamines are limited mainly by sedative side effects. However, higher doses are often required to obtain optimal therapeutic activity, especially in dermatology. We report the synthesis of three norpiperidine imidazoazepines representative of a new class of selective and nonsedating H(1) antihistamines. The compounds were at least as potent as cetirizine and loratadine as measured by H(1) receptor binding affinity, by protection against compound 48/80- and histamine-induced lethality in rats and guinea pigs, respectively, and by skin reaction tests in rats, guinea pigs, and dogs. The compounds, in particular 3a, were less prone than the reference compounds to penetrate the brain and to occupy central H(1) receptors, suggesting absence of sedative side effects. In vitro and in vivo cardiovascular safety tests showed that 3a had no intrinsic potential to prolong ventricular repolarization or induce cardiac arrhythmias. Compound 3a has been selected for further clinical development, mainly for application in dermatology.

Development of a cream from a self-emulsifying base and moisturizing actives.

This research study is based on the design and development of a semisolid emulsion system whose novel self-emulsifying base and preferentially moisturizing actives were investigated to find out whether the system mentioned could be used as a dermatological treatment for highly sensitive skins, including atopic ones. Thus, one of the main objectives of the present study consisted of in vivo evaluation of its effectiveness by means of non-invasive assessment techniques currently employed in cosmetology. Due to the fact that the new formula is, in principle, designed for skins that could present any kind of alteration, the current study was focused on rheological parameters of viscosity, thixotropy, and extensibility to guarantee not only an accurate assessment of composition but also a comfortable and safe application on skin.

[Synthesis of triterpene 2-deoxy-alpha-D-hexopyranosides on the basis of glycals and their effect on reparative processes]. / Sintez triterpenovykh 2-dezoksi-alpha-D-geksopiranozidov na osnove glikale'i i ikh vliianie na reparativnye protsessy.

Triterpene 2-deoxy-alpha-D-hexopyranosides were synthesized by the glycosylation of oleanane triterpene alcohols with D-glucal and D-galactal acetates in the presence of di(sym-collidine)iodonium perchlorate with subsequent deiodination and deacetylation of the resulting 2-deoxy-2-iodo-alpha-D-glycosides. 2-Deoxy-alpha-D-arabino- and -lyxo-hexopyranosides of methyl glycyrrhetinate demonstrated pronounced antiulcer activity and stimulated reparative skin regeneration in rats more effectively than glycyrrhizic acid and methyluracil.

Sequential release of salidroside and paeonol from a nanosphere-hydrogel system inhibits ultraviolet B-induced melanogenesis in guinea pig skin.

Melanin is the one of most important pigments for skin color in mammals. Excessive biosynthesis of melanin induces various pigment disorders. Much effort has been made to develop regulators to minimize skin pigmentation abnormalities. However, only a few of them are used, primarily because of safety concerns and low efficiency. In this study, we aimed to construct a novel nanosphere-gel for sequential delivery of salidroside and paeonol, to investigate the synergistic effects of these drugs in anti-melanogenesis, and to decrease their potential for toxicity in high dosage. Nanospheres were prepared and characterized for their particle size, polydispersity index, zeta potential, and morphological properties. The optimized nanospheres were incorporated in carbomer hydrogel with both paeonol and salidroside entrapped to form a dual drug-releasing nanosphere-gel. With this nanosphere-gel, rapid release of salidroside from the hydrogel followed by sustained release of paeonol from the nanosphere was achieved. Using a classical model of the melanogenesis response to ultraviolet exposure, it was shown that the anti-melanogenesis effects of the dual drug-releasing system, in which the doses of the individual drugs were decreased by half, was obviously enhanced when compared with the effects of the single drug preparations. Mechanistically, the burst release of salidroside from the hydrogel may enable prompt suppression of melanocyte proliferation on exposure to ultraviolet B radiation, while the paeonol released in a sustained manner can provide continuous inhibition of tyrosinase activity in melanocytes. Combined delivery of salidroside and paeonol was demonstrated to be a promising strategy for enhancing the therapeutic efficacy of these agents in anti-melanogenesis and reducing their toxicity, so may have great potential in nanomedicine.

Generic development of topical dermatologic products, Part II: quality by design for topical semisolid products.

The emergence of quality by design as a relatively new systematic science and risk-based approach has added a new dimension to pharmaceutical development and manufacturing. This review attempts to discuss the quality by design elements and concepts applied for topical semisolid products. Quality by design begins with defining a quality target product profile as well as critical quality attributes. Subsequently, this is followed by risk identification/risk analysis/risk evaluation to recognize critical material attributes and critical process parameters, in conjunction with design of experiments or other appropriate methods to establish control strategies for the drug product. Several design-of-experiment examples are included as practical strategies for the development and optimization of formulation and process for topical drug products.