Anti-Inflammatory and Anti-Quorum Sensing Effect of Camellia sinensis Callus Lysate for Treatment of Acne.

Cutibacterium acnes (C. acnes) is involved in the pathogenesis of acne by inducing inflammation and biofilm formation, along with other virulence factors. A Camellia sinensis (C. sinensis) callus lysate is proposed to reduce these effects. The aim of the present work is to study the anti-inflammatory properties of a callus extract from C. sinensis on C. acnes-stimulated human keratinocytes and the quorum-quenching activities. Keratinocytes were stimulated with thermo-inactivated pathogenic C. acnes and were treated with the herbal lysate (0.25% w/w) to evaluate its anti-inflammatory effect. C. acnes biofilm was developed in vitro and treated with 2.5 and 5% w/w of the lysate to evaluate quorum sensing and the lipase activity. The results showed that the lysate was able to reduce the production of interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), and C-X-C motif chemokine ligand 1 (CXCL1), and decrease the nuclear translocation of nuclear factor kappa light chain enhancer of activated B cells (NF-κB). The lysate did not show bactericidal activity but showed diminished biofilm formation, the lipase activity, and the production of autoinducer 2 (AI-2), a member of a family of signaling molecules used in quorum sensing. Therefore, the proposed callus lysate could have the potential to reduce acne-related symptoms without the eradication of C. acnes, which is part of the natural skin microbiome.

Alginate Hydrogel Formulation with Ketorolac for the Treatment of Pain Related Sialolithiasis.

Sialolithiasis mainly affects the oral salivary glands due to the presence of small stones that obstruct the secretion of saliva. The treatment and control of pain and inflammation during the course of this pathology is essential to guarantee the patient's comfort. For this reason, a ketorolac calcium cross-linked alginate hydrogel was developed, and it was then applied in the area of the buccal cavity. The formulation was characterized (swelling and degradation profile, extrusion, extensibility, surface morphology, viscosity, and drug release). The drug release was studied ex vivo in static Franz cells and with a dynamic ex vivo method under artificial saliva continuous flow. The product exhibits adequate physicochemical properties considering the intended purpose, and the drug concentrations retained in the mucosa were high enough to deliver a therapeutic local concentration able to reduce the pain associated with the patient's conditions. The results confirmed the suitability of the formulation for application in the mouth.

Repurposing Disulfiram as an Antifungal Agent: Development of a New Disulfiram Vaginal Mucoadhesive Gel.

Alternative formulations need to be developed to improve the efficacy of treatments administered via the vaginal route. Mucoadhesive gels with disulfiram, a molecule that was originally approved as an antialcoholism drug, offer an attractive alternative to treat vaginal candidiasis. The aim of the current study was to develop and optimize a mucoadhesive drug delivery system for the local administration of disulfiram. Such formulations were composed of polyethylene glycol and carrageenan to improve the mucoadhesive and mechanical properties and to prolong the residence time in the vaginal cavity. Microdilution susceptibility testing showed that these gels had antifungal activity against Candida albicans, Candida parapsilosis, and Nakaseomyces glabratus. The physicochemical properties of the gels were characterized, and the in vitro release and permeation profiles were investigated with vertical diffusion Franz cells. After quantification, it was determined that the amount of the drug retained in the pig vaginal epithelium was sufficient to treat candidiasis infection. Together, our findings suggest that mucoadhesive disulfiram gels have the potential to be an effective alternative treatment for vaginal candidiasis.

Monitoring dexamethasone skin biodistribution with ex vivo MALDI-TOF mass spectrometry imaging and confocal Raman microscopy.

Two of the most promising techniques in terms of ex vivo skin imaging and quantifying are confocal Raman microscopy and MALDI-TOF mass spectrometry imaging (MALDI-TOF MSI). Both techniques were set up, and the semiquantitative skin biodistribution of previously developed dexamethasone (DEX) loaded lipomers was compared using Benzalkonium chloride (BAK) as a tracer of the nanoparticles. In MALDI-TOF MSI, DEX was derivatised with GirT (DEX-GirT) and the semiquantitative biodistribution of both DEX-GirT and BAK was successfully obtained. The amount of DEX measured by confocal Raman microscopy was higher than that measured by MALDI-TOF MSI, but MALDI-TOF MSI proved to be a more suitable technique for tracing BAK. An absorption-promoting tendency of DEX loaded in lipomers versus a free-DEX solution was observed in confocal Raman microscopy. The higher spatial resolution of confocal Raman microscopy (350 nm) with respect to MALDI-TOF MSI (50 µm) allowed to observe specific skin structures like hair follicles. Nevertheless, the faster sampling rate of MALDI-TOF-MSI, permitted the analysis of larger tissue regions. In conclusion, both techniques allowed to simultaneously analyze semiquantitative data together with qualitative images of biodistribution, which is a very helpful tool when designing nanoparticles that accumulate in specific anatomical regions.

Latanoprost-Loaded Nanotransfersomes Designed for Scalp Administration Enhance Keratinocytes Proliferation.

Latanoprost (LAT) has been shown to have a hypertrichotic effect, which makes it a promising candidate for alopecia treatments. For the first time, LAT has been encapsulated in nanotransfersomes in order to increase its efficacy. Ex vivo skin biodistribution was studied by confocal laser microscopy both in human scalp and pig skin. Results showed that nanotransfersomes increase the penetration of two different fluorochromes, with similar patterns in both species, compared with fluorochrome solutions containing no nanotransfersomes. Nanotransfersomes were stable under accelerated conditions (40 °C/75% RH) and long-term conditions (25 °C/60% RH) for up to 1 year, with no differences in vesicle size and polydispersity when LAT was loaded. Nanotransfersomes increased the LAT cell proliferation effect in HaCaT cell via MAPK signaling pathway. Collectively, our results demonstrate LAT-nanotransfersomes formulation could be a promising therapy for hair growth disorders.

Repurposing Disulfiram as an Antimicrobial Agent in Topical Infections.

Antimicrobial drugs applied topically offer several advantages. However, the widespread use of antibiotics has led to increasing antimicrobial resistance. One interesting approach in the drug discovery process is drug repurposing. Disulfiram, which was originally approved as an anti-alcoholism drug, offers an attractive alternative to treat topical multidrug resistance bacteria in skin human infections. This study aimed to evaluate the biopharmaceutical characteristics of the drug and the effects arising from its topical application in detail. Microdilution susceptibility testing showed antibacterial activity against Gram-positive bacteria Staphylococcus aureus and Streptococcus pyogenes. Dermal absorption revealed no permeation in pig skin. The quantification of the drug retained in pig skin demonstrated concentrations in the stratum corneum and epidermis, enough to treat skin infections. Moreover, in vitro cytotoxicity and micro-array analyses were performed to better understand the mechanism of action and revealed the importance of the drug as a metal ion chelator. Together, our findings suggest that disulfiram has the potential to be repurposed as an effective antibiotic to treat superficial human skin infections.

The Efficacy and Biopharmaceutical Properties of a Fixed-Dose Combination of Disulfiram and Benzyl Benzoate.

Scabies and hair lice are parasitic diseases that affect human skin and hair, respectively. The incidence and resistances of these infections are increasing. Tenutex® (disulfiram and benzyl benzoate emulsion) is an alternative to standard insecticides to avoid resistances. The aim of the work is to evaluate the transdermal absorption and the in vitro efficacy against scabies and hair lice after different exposition times. Dermatomed human skin was used to assess the dermal absorption using a validated High Performance Liquid Chromatography (HPLC) method. HEK001 keratinocytes were used to evaluate the cytotoxicity of benzyl benzoate. Only benzyl benzoate was able to cross the skin, but it did not show cytotoxicity at any of the tested concentrations. The product efficacy was tested on Psoroptes ovis after direct contact and after administration on sheep skin explants at different contact times. Permethrin/malathion-resistant strains of Pediculus humanis capitis adults and eggs were directly exposed to Tenutex, and the vitality and hatchability, respectively, were evaluated. The anti-scabies study demonstrated that exposure for 6 or 24 h completely eradicated the parasite. The pediculicidal activity of Tenutex exhibited superior efficacy than standard treatment on resistant lice. The positive results obtained suggest that Tenutex® is a good treatment option, especially in drug resistance situations.

New Formulations Loading Caspofungin for Topical Therapy of Vulvovaginal Candidiasis.

Vulvovaginal candidiasis (VVC) poses a significant problem worldwide affecting women from all strata of society. It is manifested as changes in vaginal discharge, irritation, itching and stinging sensation. Although most patients respond to topical treatment, there is still a need for increase the therapeutic arsenal due to resistances to anti-infective agents. The present study was designed to develop and characterize three hydrogels of chitosan (CTS), Poloxamer 407 (P407) and a combination of both containing 2% caspofungin (CSP) for the vaginal treatment of VVC. CTS was used by its mucoadhesive properties and P407 was used to exploit potential advantages related to increasing drug concentration in order to provide a local effect. The formulations were physically, mechanically and morphologically characterized. Drug release profile and ex vivo vaginal permeation studies were performed. Antifungal efficacy against different strains of Candida spp. was also evaluated. In addition, tolerance of formulations was studied by histological analysis. Results confirmed that CSP hydrogels could be proposed as promising candidates for the treatment of VVC.

Pilot Equivalence Study Comparing Different Batches of Topical 0.025% Capsaicin Emulsion: Product Microstructure, Release, and Permeation Evaluation.

The European Medical Agency (EMA) has issued a draft guideline on the quality and equivalence of topical products. The equivalence for complex semisolid formulations involves several steps: the same quantitative content, the same microstructure, the same release, and permeation profile. In this paper, several batches of a low strength topical product, which we used as a reference/comparator product, were evaluated according to the recommendations of the EMA draft guideline. The batches were 0.025% capsaicin emulsions from the same manufacturer that were evaluated in terms of droplet size, X-ray diffraction patterns, rheology, release, and permeation profile. The generated data revealed a large batch-to-batch variability, and if the EMA guideline was applied, these batches would not be considered equivalent, although they were produced by the same manufacturer. The result of this work illustrates the difficulties in obtaining equivalence according to the current draft guidelines. It also highlights that the equivalence guidelines should consider the variability of the comparator product, and in our opinion, the guidelines should allow for claiming equivalence by comparing the limits in the variability of the data generated for the comparator product with the limits in the variability of the data generated for the intended equivalence product.

Polymeric Nanoparticles and Chitosan Gel Loading Ketorolac Tromethamine to Alleviate Pain Associated with Condyloma Acuminata during the Pre- and Post-Ablation.

This study describes the preparation and evaluation of two formulations, a hydrogel and a nanostructured system, containing ketorolac tromethamine as an anti-inflammatory agent for the local therapy against the inflammatory process derived from the surgical excision of Condyloma acuminata. Both formulations were physicochemically characterized. In vitro release profiles show that the nanoparticles release 92% ± 2.3 of the total ketorolac tromethamine encapsulated, while the chitosan gel releases 18.6% ± 0.2. The ex vivo permeation and distribution through human skin were also assayed and was observed how the main amount of ketorolac tromethamine is retained in the epidermis. In vivo studies were accomplished to evaluate the anti-inflammatory efficacy in mice which also involved the histological analysis to confirm the in vivo results. The nanoparticles present a significantly higher anti-inflammatory efficacy than chitosan gel. The tolerability of developed formulations was assessed by monitoring the biomechanical properties of the skin before and after application of both formulations. No statistical differences in trans-epidermal water loss and skin hydration with respect to the basal values were observed and the formulations exhibited higher anti-inflammatory activity compared to a reference ketotorlac tromethamine solution. Therefore, it can be concluded that both formulations can be proposed as outstanding candidates for offering a local anti-inflammatory therapeutical tool with potential clinical application.

HPV Lesions and Other Issues in the Oral Cavity Treatment and Removal without Pain.

Due to different oral and dental conditions, oral mucosa lesions such as those caused by the human papilloma virus and temporomandibular joint pathologies often have to be treated by surgical, ablative or extractive procedures. The treatment and control of pain and inflammation during these procedures is essential to guarantee the patient's well-being. For the foregoing reason, a hydrogel based on sodium alginate and hyaluronic acid containing 2% of ketorolac tromethamine has been developed. We characterized it physically, mechanically and morphologically. The rheological results suggest that the formulation can be easily and gently applied. Ex vivo permeation studies show that Ketorolac Tromethamine is able to penetrate through the buccal and sublingual mucosae, in addition to being retained in the mucosae's structure. Through an in vitro test, we were able to evaluate the role that saliva plays in the bioavailability of the drug, observing that more than half of the applied dose is eliminated in an hour. The histological and cytotoxic studies performed on pigs in vivo showed the excellent safety profile of the formulation, as well as its high tolerability. In parallel, a biomimetic artificial membrane (PermeaPad®) was evaluated, and it showed a high degree of correlation with the oral and sublingual mucosa.

The Physicochemical, Biopharmaceutical, and In Vitro Efficacy Properties of Freeze-Dried Dexamethasone-Loaded Lipomers.

Dexamethasone-loaded polymer hybrid nanoparticles were developed as a potential tool to treat alopecia areata due to their follicular targeting ability. Freeze drying (FD) is a common technique used to improve nanoparticle stability; however, there are few studies focused on its effect on ethyl cellulose lipid-core nanoparticles. Nanoparticles were lyophilized with different cryoprotectants. Sucrose was selected because it allowed for a good resuspension and provided acceptable physicochemical parameters (374.33 nm, +34.7 mV, polydispersion 0.229%, and 98.87% encapsulation efficiency). The nanoparticles obtained were loaded into a pleasant xanthan gum hydrogel, and the rheological, release, and skin permeation profiles of different formulations were studied. The FD formulation significantly modified the particle size, and the drug release and permeation properties were also altered. In addition, analyses of the cytotoxicity and anti-inflammatory efficacy of FD and non-FD particles on human keratinocytes indicated no differences.

Dexamethasone-Loaded Lipomers: Development, Characterization, and Skin Biodistribution Studies.

Follicular targeting has gained more attention in recent decades, due to the possibility of obtaining a depot effect in topical administration and its potential as a tool to treat hair follicle-related diseases. Lipid core ethyl cellulose lipomers were developed and optimized, following which characterization of their physicochemical properties was carried out. Dexamethasone was encapsulated in the lipomers (size, 115 nm; polydispersity, 0.24; zeta-potential (Z-potential), +30 mV) and their in vitro release profiles against dexamethasone in solution were investigated by vertical diffusion Franz cells. The skin biodistribution of the fluorescent-loaded lipomers was observed using confocal microscopy, demonstrating the accumulation of both lipomers and fluorochromes in the hair follicles of pig skin. To confirm this fact, immunofluorescence of the dexamethasone-loaded lipomers was carried out in pig hair follicles. The anti-inflammatory (via TNFα) efficacy of the dexamethasone-loaded lipomers was demonstrated in vitro in an HEK001 human keratinocytes cell culture and the in vitro cytotoxicity of the nanoformulation was investigated.

Nano-engineering of ketorolac tromethamine platforms for ocular treatment of inflammatory disorders.

Aim: The development and optimization of Ketorolac tromethamine-loaded polylactic-co-glycolic acid nanoparticles (KT-NPs) for the treatment of inflammatory processes of the eye. Materials & methods: KT-NPs were developed by factorial design and characterized by assessing their physicochemical properties. Biopharmaceutical behavior studies, ocular tolerance, anti-inflammatory efficacy and bioavailability tests were performed on pigs. Results: Optimized KT-NPs of 112 nm, narrow distribution with encapsulation efficiency near 100% were obtained. KT release followed the Weibull model and there was significantly greater retention in the cornea and sclera than in the commercial reference. KT-NPs showed no signs of ocular irritancy and similar anti-inflammatory efficacy to the commercial reference. Conclusion: KT-NPs were a suitable alternative for the treatment of inflammatory disorders of the anterior and posterior segments of the eye as an alternative to conventional topical formulations.

Topical Mucoadhesive Alginate-Based Hydrogel Loading Ketorolac for Pain Management after Pharmacotherapy, Ablation, or Surgical Removal in Condyloma Acuminata.

Condyloma acuminata is an infectious disease caused by the human papilloma virus (HPV) and one of the most common sexually transmitted infections. It is manifested as warts that frequently cause pain, pruritus, burning, and occasional bleeding. Treatment (physical, chemical, or surgical) can result in erosion, scars, or ulcers, implying inflammatory processes causing pain. In this work, a biocompatible topical hydrogel containing 2% ketorolac tromethamine was developed to manage the painful inflammatory processes occurring upon the removal of anogenital condylomas. The hydrogel was physically, mechanically, and morphologically characterized: it showed adequate characteristics for a topical formulation. Up to 73% of ketorolac in the gel can be released following a one-phase exponential model. Upon application on human skin and vaginal mucosa, ketorolac can permeate through both of these and it can be retained within both tissues, particularly on vaginal mucosa. Another advantage is that no systemic side effects should be expected after application of the gel. The hydrogel showed itself to be well tolerated in vivo when applied on humans, and it did not cause any visible irritation. Finally, ketorolac hydrogel showed 53% anti-inflammatory activity, suggesting that it is a stable and suitable formulation for the treatment of inflammatory processes, such as those occurring upon chemical or surgical removal of anogenital warts.

Amoxicillin and Clarithromycin Mucoadhesive Delivery System for Helicobacter pylori Infection in a Mouse Model: Characterization, Pharmacokinetics, and Efficacy.

Helicobacter pylori is the main pathogen responsible for gastric ulcers and a predisposing factor of stomach cancer. Although current treatment is usually successful, it requires high doses and frequent administration. An innovative mucoadhesive system (Mucolast®) loaded with amoxicillin and clarithromycin is proposed to improve the efficacy of treatment against H. pylori. The drug product was optimized based on its viscoelastic properties to obtain long-term stability of the vehicle. The drug release mechanisms were different for both antibiotics based on their solubilization status. A systemic and stomach pharmacokinetic profile was obtained after three different doses were administered to mice, obtaining similar systemic exposure levels but an increase in drug concentration in the stomach. The efficacy results in mice infected with H. pylori also demonstrated the superiority of the antibiotics when administered in Mucolast®, as shown by the bacterial count in stomach tissue and under histopathological and biochemical evaluation. The proposed treatment was efficacious and safe and is presented as a realistic alternative to current treatment options to improve patient compliance and to reduce bacterial resistance.

Transungual Delivery, Anti-Inflammatory Activity, and In Vivo Assessment of a Cyclodextrin Polypseudorotaxanes Nail Lacquer.

A new cyclodextrin polypseudorotaxanes nail lacquer (Regenail®) containing biotin, methyl sulphonyl methane (MSM), and dimethylsilanediol salicylate was developed and evaluated in vitro and in vivo. The product was developed to improve nail status and diminish signs of pathological nail alterations. A reference product (Betalfatrus®) was used for comparative purposes. An in vitro permeation experiment in hooves showed high MSM and biotin absorption. The content of sulfur and silicon in hooves was also found to be higher compared with the reference product. MSM was tested in human keratinocytes, exhibiting a good cytotoxicity profile and anti-inflammatory activity by the reduction in IL-8 and TNF-α under LPS stimuli. A clinical study was performed to check product safety and efficacy against nail brittleness and alterations such as Beau's lines and onychorrhexis. A reduction in both alterations and in surface roughness without alteration of nail structure was observed, with a good level of patient acceptance and satisfaction.

Epidermal Delivery of Retinyl Palmitate Loaded Transfersomes: Penetration and Biodistribution Studies.

The alteration of retinoids levels in the skin can cause different disorders in the maturation of epithelial skin cells. Topical administration of these lipophilic molecules is a challenge that can be addressed by encapsulation into drug delivery systems. In this study, retinyl palmitate transferosomes formulated in cream were developed and the increases in the penetration of the active ingredients as well as the biodistribution were evaluated in vitro and in vivo. Transfersomes demonstrated a significant increase in the administration of retinyl palmitate to the epidermis by quantification of the active ingredients in the different layers of the skin, as well as by fluorescence microscopy of biopsies of non-dermatomized pig-ear skin. These results suggest that transfersomes may be an efficient vehicle for the delivery of retinoids to inner layers of the skin, such as the epidermis.

Ex-Vivo and In-Vivo Assessment of Cyclamen europaeum Extract After Nasal Administration.

Rhinosinusitis is a prevalent disorder with a severe impact on the health-related quality of life. Saponins of Cyclamen europaeum exert a clinically proven curative effect on rhinosinusitis symptoms when instilled into the nasal cavity, however, more extensive preclinical assessment is required to better characterize the efficacy of this botanical extract. This work evaluates the potential use of a natural freeze-dried extract of C. europaeum given as topical nasal administration. Permeation experiment on porcine nasal mucosa was performed with Franz diffusion cells. Experiments in rabbits were performed to test for any toxicological, hematological, biochemical or histological evidence of systemic action. No theoretical levels of saponins were found in the receptor chamber of Franz diffusion cells. Hematological data did not show significant differences between control and experimental animals (p > 0.05). Histological studies also showed that enhanced secretory activity in response to intranasal administration was not accompanied by any visible signs of injury. An examination of the brain, lungs, liver, kidneys, spleen, and gastrointestinal organs did not reveal any abnormality. The absence of mucosal permeation of saponins and negligible probability of C. europaeum saponins absorption in the course of a therapeutic application was demonstrated.

Developing Transdermal Applications of Ketorolac Tromethamine Entrapped in Stimuli Sensitive Block Copolymer Hydrogels.

PURPOSE: In order to obtain dermal vehicles of ketorolac tromethamine (KT) for the local treatment of inflammation and restrict undesirable side effects of systemic levels hydrogels (HGs) of poloxamer and carbomer were developed. METHODS: KT poloxamer based HG (KT-P407-HG) and KT carbomer based HG (KT-C940-HG) were elaborated and characterized in terms of swelling, degradation, porosity, rheology, stability, in vitro release, ex vivo permeation and distribution skin layers. Finally, in vivo anti-inflammatory efficacy and skin tolerance were also assessed. RESULTS: HGs were transparent and kept stable after 3 months exhibiting biocompatible near neutral pH values. Rheological patterns fitted to Herschel-Bulkley for KT-C940-HG and Newton for KT-P407-HG due to its low viscosity at 25°C. Rapid release profiles were observed through first order kinetics. Following the surface the highest concentration of KT from C940-HG was found in the epidermis and the stratum corneum for P407-HG. Relevant anti-inflammatory efficacy of KT-P407-HG revealed enough ability to provide sufficient bioavailability KT to reach easily the site of action. The application of developed formulations in volunteers did not induce any visual skin irritation. CONCLUSIONS: KT-P407-HG was proposed as suitable formulation for anti-inflammatory local treatment without theoretical systemic side effect.