Exploring the potential of minoxidil tretinoin liposomal based hydrogel for topical delivery in the treatment of androgenic alopecia.

Purpose: Androgenic alopecia (AGA) is a condition of progressive hair loss and involves follicular miniaturization triggered mainly due to varying levels of androgen besides environmental and genetic factors, which may also play some role. Minoxidil (MXD) has been considered as most effective therapeutic moiety to treat this disorder. Another drug Tretinoin (TRET) is known for its comedolytic activity and is reported to enhance percutaneous absorption of MXD. Presently both these drugs are being utilized for treatment of androgenic alopecia (AGA) in solution form which poses several problems in terms of poor solubility of drug, frequency of application and side effects.Materials and methods: Current work investigates liposomal hydrogel system for simultaneous delivery of MXD and TRET to overcome the limitations of existing formulation. Successful development of liposomes was commenced by thin film hydration method and various parameters affecting desired characteristics like size, morphology, entrapment efficiency; stability and ex vivo permeation were optimized. The formulated liposomes were further characterized for various physicochemical properties and evaluated for in vivo irritancy study in animals.Results and discussion: Results suggested prepared liposomes to be stable, homogenous and capable to hold both the drugs within. Association with hydrogel enhanced the permeation of MXD through skin ex vivo but TRET retained on the skin. Liposome loaded hydrogel was found to be non-irritant to skin.Conclusion: Overall developed system showed potential for effective and simultaneous delivery of both the drugs.

Controlled Release of Salicylic Acid from Biodegradable Cross-Linked Polyesters.

The purpose of this work was to develop a family of cross-linked poly(xylitol adipate salicylate)s with a wide range of tunable release properties for delivering pharmacologically active salicylic acid. The synthesis parameters and release conditions were varied to modulate polyester properties and to understand the mechanism of release. Varying release rates were obtained upon longer curing (35% in the noncured polymer to 10% in the cured polymer in 7 days). Differential salicylic acid loading led to the synthesis of polymers with variable cross-linking and the release could be tuned (100% release for the lowest loading to 30% in the highest loading). Controlled release was monitored by changing various factors, and the release profiles were dependent on the stoichiometric composition, pH, curing time, and presence of enzyme. The polymer released a combination of salicylic acid and disalicylic acid, and the released products were found to be nontoxic. Minimal hemolysis and platelet activation indicated good blood compatibility. These polymers qualify as "bioactive" and "resorbable" and can, therefore, find applications as immunomodulatory resorbable biomaterials with tunable release properties.

The effect of retinoic acid on mouse mandibular molar development in vitro, using alkaline phosphatase as a molecular indicator of differentiation.

INTRODUCTION: An excellent model system that links evolutionary biology and developmental biology in seeking to understand evolutionary diversity is the study of tooth development in mammals. These studies reflect the diversity of mammalian radiations which bear on the interpretation of South African fossil hominids. Tooth development occurs via epithelio-mesenchymal interactions and involves the production of many substances, including alkaline phosphatase, which is necessary for dentine and enamel formation. Retinoic acid is a known morphogen and is important in tooth development. In excess, retinoic acid has been found to alter the formation of teeth. OBJECTIVES: In order to determine whether retinoic acid has any effect on tooth morphology, exogenous retinoic acid was administered to developing mouse molar teeth in vitro, and alkaline phosphatase was utilized as an indicator of differentiation. METHODS: Molars were microdissected from 15.5 day mouse embryo mandibles and cultured at the air: medium interface with or without retinoic acid for seven days. Following fixation and embedding, the explants were sectioned for morphological analysis. Alkaline phosphatase activity was detected using a modified Gomori's histochemical method. RESULTS AND CONCLUSION: Retinoic acid appeared to retard the growth and differentiation of the molar explants. This was coincident with reduced alkaline phosphatase.

Differential effects of all-trans retinoic acid on the growth of human keratinocytes and mouth carcinoma epidermoid cultures. Involvement of GRIM-19 and complex I of the respiratory chain.

Squamous cell carcinoma (SSC) is the most frequent malignant tumor of the oral cavity. A study on the effect of all-trans retinoic acid (RA) on cell growth, expression of GRIM-19 and content and activity of complex I of the mitochondrial respiratory chain in normal human keratinocytes (NHEK) and mouth carcinoma cells with low (HN) and high (KB) transformation grade was carried out. In NHEK cells, RA treatment resulted in growth suppression, significant overexpression of GRIM-19 protein, enhanced content of complex I but depressed activity of NADH-UQ oxidoreductase activity of the complex. In HN cells, RA treatment depressed cell growth, inhibited the enzymatic activity of complex I but had no significant effect on the levels of GRIM-19 and complex I. In KB cells RA had no effect on cell growth, GRIM-19 expression, content and activity of complex I.

Utilizing combination therapy for ethnic skin.

A major issue in treating acne in individuals of color is the need to treat and prevent postinflammatory hyperpigmentation (PIH), which is common in this population. This subset analysis reports the pigmentary changes in subjects of color with acne who were enrolled in a community-based trial comparing 3 different topical therapeutic regimens. All subjects received combination clindamycin 1%-benzoyl peroxide (BPO) 5% topical gel containing glycerin and dimethicone. Subjects were randomized to receive this combination therapy in addition to either a tretinoin microsphere (RAM) gel at concentrations of either 0.04% or 0.1% or adapalene (AP) gel 0.1%. There was a trend toward better resolution of hyperpigmentation in the subjects receiving the clindamycin-BPO topical gel in combination with RAM gel 0.04%.

[Induced cleft palat by Retinoic acid through altering the cell proliferation and apoptosis at the key stages of palatal development].

Objective: To investigate the effects of retinoic acid (RA) on the proliferation and apoptosis of mesenchyme and epithelium at the key stages of palatal development, and to determine the embryonic alterations associated with cleft palate. Methods: 100 pregnant C57 females (E12) were equally divided into two groups. The experimental group was administered RA once (70 mg/kg) while the control group was administered corn oil only, and the heads of the embryos at E13.5-17.5 were collected and processed to paraffin. Sections were stained with hematoxylin and eosin for the morphological assessment of anterior,posterior, left and right palate, and BrdU and TUNEL assays for the detection of proliferation and survival of palatal mesenchyme and epithelium. Results: Simple treatment of RA at 70 mg/kg caused an incidence of 100％ cleft palate. During E13.5-15.5,cell proliferation was significantly promoted on the anterior palatal mesenchyme in the RA group, while no difference for the posterior palatal mesenchyme. Besides, the cell proliferation on anterior and posterior epithelium was comparable between the control and RA group. Less apoptotic cells were observed on epithelium during El3.5-14.5 in the RA group. Conclusions: RA induces excessive cell proliferation of palatal mesenchyme, causing abnormal vertical palatal growth and failure of palatal shelves contact and fusion, which finally lead to cleft palate. RA also increases epithelium apoptosis.

Formulation of tretinoin-loaded topical proniosomes for treatment of acne: in-vitro characterization, skin irritation test and comparative clinical study.

Tretinoin (TRT) is a widely used retinoid for the topical treatment of acne, photo-aged skin, psoriasis and skin cancer which makes it a good candidate for topical formulation. Yet side effects, like redness, swelling, peeling, blistering and, erythema, in addition to its high lipophilicity make this challenging. Therefore, the aim of this study was the development of TRT-loaded proniosomes to improve the drug efficacy and to increase user acceptability and compliance by reducing its side effects. Nine formulae were prepared according to 3(2) factorial design and were evaluated for their morphology, vesicle size, entrapment efficiency (EE %), and% of drug released after 5 h. Hydrogel of the candidate formula, N8G (proniosomes prepared with 0.025% TRT, and Span60: cholesterol molar ratio of 3:1 and incorporated in 1% carbopol gel) was developed and evaluated for skin irritation test and clinical study in acne patients compared to marketed product. Candidate formula showed higher efficacy and very low irritation potential when compared to marketed product in human volunteers.

A composites theory predicts the dependence of stiffness of cartilage culture tissues on collagen volume fraction.

The tensile stiffness of tissue grown from chondrocyte culture was both measured experimentally and predicted using a composites model theory relating tissue microstructure to macroscopic material stiffness. The tissue was altered by several treatment protocols to provide a wide range of collagen fibril volume fraction (0.015-0.15). The rate of change of tissue modulus with change in collagen volume fraction predicted by the theory was within 14% of the slope of the linear fit through the experimental data, without the use of fitting parameters for the theoretical value of the slope. Use of the model to simulate cytokine mediated tissue digestion suggests that the action of IL-1beta and retinoic acid is mainly removal of proteoglycans and some removal of collagen. The model also indicates that the matrix and collagen remaining in the tissue has the same elastic properties as the untreated tissue, and is not damaged due to the alteration. Young's modulus of the collagen fibrils is predicted to be 120 MPa, a value in the range of previous studies. This value is dependent mainly on the matrix modulus and collagen fibril volume fraction and not on Poisson's ratio of either matrix or fibril. Poisson's ratio of the tissue depends primarily on the Poisson's ratio of the matrix.

Keratinocyte growth factor expression in human gingival fibroblasts and stimulation of in vitro gene expression by retinoic acid.

BACKGROUND: Keratinocyte growth factor (KGF) is a stromally derived growth factor of the fibroblast growth factor (FGF) family with paracrine effects targeted to influence the growth and differentiation of epithelia. Regional and temporal changes in KGF expression play important roles in the development and maintenance of epithelial structures and in epithelial wound healing. Differing patterns of expression of KGF by fibroblasts in the gingival region could therefore be related to the observed regional variation in the differentiation and behavior of gingival epithelia. METHODS: The in vitro and in vivo patterns of expression of KGF mRNA in human gingival and periodontal fibroblasts were examined using reverse transcription polymerase chain reactions (RT-PCR) and in situ hybridization with digoxigenin-labeled riboprobes. The patterns observed for human gingiva were compared with those for human skin and for murine tissues. RESULTS: Gingival and periodontal fibroblasts showed expression of KGF transcripts in vitro, and the degree of expression was markedly influenced by the presence of retinoic acid, an agent known to influence patterns of epithelial differentiation. Sections of human and murine gingiva and skin showed regionally variable expression of transcripts with the cells expressing KGF in the subepithelial, rather than the deeper, connective tissues and periodontium. CONCLUSIONS: The results point to a role of KGF in the maintenance of normal growth and differentiation of gingival epithelia. A lack of KGF expression by periodontal fibroblasts in vivo is expected to hinder apical epithelial migration and thus stabilize the epithelial attachment. The effects of retinoic acid (RA) on KGF expression in vitro provide an indirect mechanism by which RA may regulate the growth and differentiation of gingival epithelia.

Expression of connexins and the effect of retinoic acid in oral keratinocytes.

Differential expression of members of the connexin (Cx) gap junction multigene family permits formation of gap junctions with the varied physiological properties required by different tissues. The aim of this study was to characterize connexin expression and the influence of all-trans-retinoic acid (RA) in mouse gingival epithelial cells (GE1). The cells were treated with RA, and expression of Cxs was analyzed by immunofluorescence, reverse transcriptase-polymerase chain reaction (RT-PCR), and real-time PCR. RT-PCR revealed that GE1 cells expressed mRNA for Cx26, Cx30.3, Cx31.1, Cx32, and Cx43. In addition, real-time PCR revealed that RA significantly decreased expression of Cx31.1 as compared with control. These results indicate that GE1 cells are useful in analyzing the expression of connexin molecules in oral keratinocytes from oral mucosal lesions.

Reduced troponin I phosphorylation and increased Ca(2+)-dependent ATP-consumption in triton X-skinned fiber preparations from Galphaq overexpressor mice.

Overexpression of the Galphaq-protein has been shown to result in hypertrophic and dilated cardiomyopathy. This study investigated Ca(2+ )sensitivity of tension and myosin-ATPase activity in skinned fiber preparations of male and female wildtype (WT; n = 12) and transgenic mice with a cardiac specific overexpression of the Galphaq-protein (Galphaq-OE; n = 11). In addition, the phosphorylation status of troponin I was measured. Ca(2+) sensitivity of tension was increased in Galphaq-OE with a significant reduction in the half-maximum Ca(2+) concentration (EC(50)) compared to WT. Similarly, Ca(2+) sensitivity of myosin ATPase activity was increased in Galphaq-OE when comparing Galphaq-OE to WT. Maximum Ca(2+)-dependent tension and ATPase activity were both enhanced in Galphaq-OE compared to WT littermates. Phosphorylation of troponin I was significantly reduced in Galphaq-OE compared to WT. In the above experiments, no gender specific differences were observed in either Gaq-OE or in WT. We conclude that, in mice, increased expression of the Galphaq-protein induces alterations of myofibrillar function and energy consumption, which are also characteristics of human heart failure. This may result from a decreased phosphorylation of troponin I in Galphaq-OE.

Molecular dynamics of retinoic acid-induced craniofacial malformations: implications for the origin of gnathostome jaws.

BACKGROUND: Intake of retinoic acid (RA) or of its precursor, vitamin A, during early pregnancy is associated with increased incidence of craniofacial lesions. The origin of these teratogenic effects remains enigmatic as in cranial neural crest cells (CNCCs), which largely contribute to craniofacial structures, the RA-transduction pathway is not active. Recent results suggest that RA could act on the endoderm of the first pharyngeal arch (1stPA), through a RARbeta-dependent mechanism. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that RA provokes dramatically different craniofacial malformations when administered at slightly different developmental times within a narrow temporal interval corresponding to the colonization of the 1(st) PA by CNCCs. We provide evidence showing that RA acts on the signalling epithelium of the 1(st) PA, gradually reducing the expression of endothelin-1 and Fgf8. These two molecular signals are instrumental in activating Dlx genes in incoming CNCCs, thereby triggering the morphogenetic programs, which specify different jaw elements. CONCLUSIONS/SIGNIFICANCE: The anatomical series induced by RA-treatments at different developmental times parallels, at least in some instances, the supposed origin of modern jaws (e.g., the fate of the incus). Our results might provide a conceptual framework for the rise of jaw morphotypes characteristic of gnathostomes.

Protective effects of prenatal administration of folic acid on retinoic acid-induced cellular damages of Meckel's cartilage in rats.

Craniofacial malformations are among the most common congenital deformities. Meckel's cartilage plays a major role in the development of the mandible and is highly susceptible to maternal teratogenic drug use. We therefore investigated possible protective effects of prenatal administration of folic acid on a retinoic-acid induced maxillofacial defect model. Sprague-Dawley pregnant female rats (n=36) were used in this study. Retinoic acid was administered orally at the dose of 40, 60, or 80 mg/kg respectively on gestational day 8. Folic acid of 4.0 mg/kg was injected intraperitoneally on 7th, 8th and 9th days of pregnancy. Animals were sacrificed on the day 17th. Administration of retinoic acid at all doses resulted in statistically significant decreases in mean fetal weight and mean fetal height and the increase in mortality rate, and caused severe ultrastructural damages in Meckel's cartilage. Folic acid administration prevented the decrease in mean fetal weight and height of the embryos treated with retinoic acid of 40 mg/kg. In addition, there was a marked decrease in the number of degenerated chondrocytes and an improvement in the structure of granular endoplasmic reticulum along with intact nuclei. We conclude that folic acid has protective effects on retinoic acid-induced intracellular damages in Meckel's cartilage.

Transcription regulatory elements of the first intron control human transglutaminase type I gene expression in epidermal keratinocytes.

Expression of the transglutaminase type1 gene (TGM1), which encodes an epithelial cell-specific protein cross-linking enzyme, is limited to particular stages of epidermal development and keratinocyte differentiation. As a result, transglutaminase type 1 (TGase1) enzyme activity in epidermal cells increases with the onset of keratinization in vivo and in vitro. We determined, by functional mapping of deletion mutations in the TGM1 5' untranslated region, that an element in first intron of the human TGM1 gene, in addition to the 5' proximal promoter, initiates transcription and upregulates transcriptional activity. These two transcription control elements function interdependently to regulate the expression of the human TGM1 gene in keratinocytes. We also identified distinct regulatory elements that cooperatively modify the 5' proximal and intron 1 promoter activities in response to environmental variations in retinoic acid and calcium ion concentrations. In conclusion, we report that TGM1 differential gene expression is controlled by two distinct elements, proximal and intronic, which function cooperatively to initiate and modulate TGM1 gene transcription in response to regulatory signals. We propose that in nonexpressing cells these regulatory signals repress a default mechanism that operates in their absence. The specificity of their function is integrated into the default mechanism and consists of the tissue-, developmental-, and differentiation-specific interplay of 5' URR and intron 1 elements tuned to physiological status.

The Microsponge Delivery System (MDS): a topical delivery system with reduced irritancy incorporating multiple triggering mechanisms for the release of actives.

The Microsponge Delivery System (MDS) is a unique technology for the controlled release of topical agents and consists of macroporous beads, typically 10-25 microns in diameter, loaded with active agent. When applied to the skin, the MDS releases its active ingredient on a time mode and also in response to other stimuli (rubbing, temperature, pH, etc). MDS technology is being used currently in cosmetics, over-the-counter (OTC) skin care, sunscreens and prescription products. By delivering the active gradually to the skin, MDS-benzoyl peroxide formulations, for example, have excellent efficacy with minimal irritation. These are typical benefits from the use of the MDS.

Influence of retinoic acid on the expression of cytokeratins, vimentin and ICAM-1 in human gingival epithelia in vitro.

Phenotypic differences exist in vivo between junctional (JE) and oral gingival (OGE) epithelia and an in vitro system has been developed that maintains phenotypic differences. This system, which permits in vitro studies of factors that may influence the epithelial phenotype, was used to investigate the effects of retinoic acid (RA) on epithelial expression of various markers known to distinguish JE from OGE. Primary cultures of JE and OGE were initiated from defined gingival regions and were subcultured and grown for 48 h in 96-well plates or on multiple-well slides. Control cultures were grown in medium supplemented with delipidized serum and all-trans RA was added to experimental groups. Other cultures were grown in a defined RA-free medium. Cultures were examined using monoclonal antibodies against cytokeratins, vimentin, and ICAM-1 and binding displayed by indirect immunocytochemical staining. Staining reactions were assessed by direct microscopic observation and assayed by spectrophotometric quantitation. The results showed that RA had minor effects on the marker expression of JE but markedly enhanced expression of cytokeratins 8, 18, 19, vimentin and ICAM-1 in OGE. These markers, which normally distinguish JE from OGE, were expressed at levels approaching or exceeding those of control JE cultures. These observations indicate that RA responsive mechanisms affect the phenotypes expressed by epithelia in vitro and suggest that such mechanisms may be related to the different phenotypic patterns expressed by gingival epithelia in vivo.

Keratolytic activity of microemulsions.

OBJECTIVE: To compare the keratolytic activities of a drug-free hydrophilic microemulsion (ME) and a drug-free lipophilic ME with water, and with regard to the hydrophilic ME also with a 5% salicylic acid gel on the sole of the foot. METHODS: Twenty healthy volunteers had their plantar forefoot, midfoot, and rearfoot stratum corneum blackened with silver nitrate and a photographic developer, and a chromameter was used to determine the extent of removal of this black dye by a* value and L value measurement at 24 and 48 h. RESULTS: Both drug-free MEs produced significantly greater increases in a* value and L value than water, and the hydrophilic ME was also more effective than 5% salicylic acid gel. CONCLUSION: The irritating effect of MEs is rather negligible on the sole of the foot because of the thick plantar stratum corneum. Both MEs therefore appear suitable for the elimination or prevention of plantar desquamative and hyperkeratotic skin changes.

Retinoic acid disintegrated desmosomes and hemidesmosomes in stratified oral keratinocytes.

BACKGROUND: Although it is known that retinoic acid (RA) regulates the cellular differentiation of skin keratinocytes, the effects of RA on the anchoring junction have not been clarified. The effects of all-trans RA on cell-cell and cell-matrix connections of gingival epithelial (GE)1 cells in a multilayered culture were investigated. METHODS: Ultrastructures of GE1 cells were observed and immunohistochemistry was used to detect keratin 4, keratin 13, and desmoglein expression. Reverse transcription-polymerase chain reaction was performed to detect expression of desmosome and hemidesmosome-associating adhesion molecules, keratin 13, and keratin14. RESULTS: Retinoic acid caused immunohistochemical diminution of keratin 4, keratin 13, and desmoglein. Ultrastructurally, RA induced drastic loss of typical desmosomes and complete loss of hemidesmosomes. RA significantly decreased the transcript levels of keratin 13, keratin 14, desmoglein 1, and desmocollin 1 in a dose-dependent manner. The 230-kD bullous pemphigoid antigen (BPAG1) gene expression was also reduced by RA, whereas transcript levels of integrin alpha6, integrin beta4, the 180-kD bullous pemphigoid antigen (BPAG2), and laminin 5 were not affected. CONCLUSION: These results indicated that RA disintegrated not only desmosomes by depriving the cells of desmoglein 1, desmocollin 1, keratin 13, and keratin 4, but also hemidesmosomes by reducing the expression of BPAG1 and keratin 14 in basal keratinocytes.