Traditional Herb (Moxa) Modified Zinc Oxide Nanosheets for Quick, Efficient and High Tissue Penetration Therapy of Fungal Infection.

Fungal infection possesses the characteristics of high invasion depth and easy formation of a biofilm under the skin, which greatly hinders the treatment process. Here, traditional Chinese medicine moxa is carbonized and modified with zinc oxide (ZnO) nanosheets to synthesize carbonized moxa@ZnO (CMZ) with the dual response properties of yellow light (YL) and ultrasound (US) for synergistic antifungal therapy. CMZ with narrow bandgap can respond to long-wavelength YL that is highly safe and helpful for skin repair. Simultaneously, CMZ with a piezoelectric effect can further enhance the photocatalytic efficiency under the stimulation of US with high tissue penetration. Gene mechanism investigation indicates that when exposed to US and YL irradiation, CMZ-based therapy can adjust the expression of genes associated with fungal virulence, metabolic activity, mycelial growth and biofilm development, thus efficaciously eradicating planktonic Candida albicans (C. albicans) and mature biofilm. Importantly, despite the 1.00 cm thick tissue barrier, CMZ can rapidly eliminate 99.9% of C. albicans within 4 min, showing a satisfactory deep fungicidal efficacy. The in vivo therapeutic effect of this strategy is demonstrated in both open wound and deep cutaneous infection tests, speaking of dramatically better efficacy than the traditional fungicide ketoconazole (KTZ).

Novel anti-dandruff shampoo incorporated with ketoconazole-coated zinc oxide nanoparticles using green tea extract.

BACKGROUND: Dandruff caused by Malassezia furfur is a prevailing fungal infection. Although ketoconazole (KTZ) is widely intended for anti-dandruff treatment, poor solubility, and epidermal permeability limits its use and the marketed KTZ shampoo adversely effects scalp and hair. OBJECTIVE: To prepare a novel shampoo loaded with KTZ-coated zinc oxide nanoparticles using green tea extract and evaluate its antifungal activity. METHODS: The KTZ-coated zinc oxide nanoparticles was prepared by green synthesis and was characterized by UV, FTIR, XRD, and the drug entrapment efficiency was investigated. The antifungal activity of the nanoparticles with respect to standard drug, KTZ was tested against Malassezia furfur. Further, a novel antidandruff shampoo was developed by incorporating the prepared nanoparticles into the shampoo base. RESULTS: The formation of KTZ-coated ZnO nanoparticles was confirmed by UV and FTIR analysis. XRD analysis confirmed the amorphous phase of KTZ in nanoparticles. The drug entrapment efficiency was found to be 91.84%. The prepared nanoparticles showed enhanced activity against Malassezia furfur compared to drug of choice, KTZ (1%). The evaluation of shampoo showed an ideal result. CONCLUSION: KTZ-coated ZnO nanoparticles loaded novel shampoo in comparison to marketed anti-dandruff shampoo could be an effective alternate for the treatment of dandruff.

An integrated One Health framework for holistic evaluation of risks from antifungal agents in a large-scale multi-city study.

A new framework for retrospective mass spectral data mining for antifungal agents (AFs) and Wastewater-Based Epidemiology (WBE) was developed as part of One Health framework to tackle risks from AFs. A large scale, multi-city study was undertaken in South-West England. Key drivers of AFs in the catchment were identified with communal wastewater discharges being the main driver for human AFs (fluconazole, ketoconazole) and agricultural runoff being the main driver for pesticide AFs (prochloraz, prothioconazole and tebuconazole). Average WBE-estimated human used fluconazole and ketoconazole PNDIs (population normalised daily intake) exceeded 300 mg day-1 1000 inh-1 and 2000 mg day-1 1000 inh-1. This is much higher than PNDPs (population normalised daily prescriptions <40 mg day-1 1000 inh-1 and <80 mg day-1 1000 inh-1 for fluconazole and ketoconazole respectively). This was expected due to both prescription and over-the-counter usage, and both oral and topical applications. Pesticide AF, prothioconazole had PNDIs <40,000 mg day-1 1000 inh -1, which gave intake: 0.43, 0.26, 0.07 mg kg-1 in City A, B, and C, likely due to accounting for external/non-human sources. This is higher than the acceptable daily intake (ADI) of 0.01 mg kg-1bw day-1, which warrants further study. Intake per kg of body weight estimated using tebuconazole was 0.86, 1.39, 0.12, 0.13, and 2.7 mg kg-1 in City A-E respectively and is likely due to external/non-human sources. Intake calculated using its metabolite was 0.02 and 0.01 mg kg-1 in City B and C respectively, which aligned with ADI (0.03 mg kg-1bw day-1). The environmental risk assessment of AFs indicated low/medium risk from fluconazole, prochloraz, and tebuconazole, medium risk from epoxiconazole, prothioconazole's metabolite, and tebuconazole, and high risk for prothioconazole in river water. High risk was estimated from fluconazole, epoxiconazole, prothioconazole and its metabolite, tebuconazole, ketoconazole in wastewater samples, which is important during raw sewage discharge events via sewer overflows.

Perinatal exposure to the fungicide ketoconazole alters hypothalamic control of puberty in female rats.

Introduction: Estrogenic endocrine disrupting chemicals (EDCs) such as diethylstilbestrol (DES) are known to alter the timing of puberty onset and reproductive function in females. Accumulating evidence suggests that steroid synthesis inhibitors such as ketoconazole (KTZ) or phthalates may also affect female reproductive health, however their mode of action is poorly understood. Because hypothalamic activity is very sensitive to sex steroids, we aimed at determining whether and how EDCs with different mode of action can alter the hypothalamic transcriptome and GnRH release in female rats. Design: Female rats were exposed to KTZ or DES during perinatal (DES 3-6-12µg/kg.d; KTZ 3-6-12mg/kg.d), pubertal or adult periods (DES 3-12-48µg/kg.d; KTZ 3-12-48mg/kg.d). Results: Ex vivo study of GnRH pulsatility revealed that perinatal exposure to the highest doses of KTZ and DES delayed maturation of GnRH secretion before puberty, whereas pubertal or adult exposure had no effect on GnRH pulsatility. Hypothalamic transcriptome, studied by RNAsequencing in the preoptic area and in the mediobasal hypothalamus, was found to be very sensitive to perinatal exposure to all doses of KTZ before puberty with effects persisting until adulthood. Bioinformatic analysis with Ingenuity Pathway Analysis predicted "Creb signaling in Neurons" and "IGF-1 signaling" among the most downregulated pathways by all doses of KTZ and DES before puberty, and "PPARg" as a common upstream regulator driving gene expression changes. Deeper screening ofRNAseq datasets indicated that a high number of genes regulating the activity of the extrinsic GnRH pulse generator were consistently affected by all the doses of DES and KTZ before puberty. Several, including MKRN3, DNMT3 or Cbx7, showed similar alterations in expression at adulthood. Conclusion: nRH secretion and the hypothalamic transcriptome are highly sensitive to perinatal exposure to both DES and KTZ. The identified pathways should be exploredfurther to identify biomarkers for future testing strategies for EDC identification and when enhancing the current standard information requirements in regulation.

In Vitro and In Vivo Metabolic Activation of Tolterodine Mediated by CYP3A.

Tolterodine (TOL) is an antimuscarinic drug used for the treatment of patients with overactive bladder presenting urinary frequency, urgency, and urge incontinence. During the clinical use of TOL, adverse events such as liver injury took place. The present study aimed at the investigation of the metabolic activation of TOL possibly associated with its hepatotoxicity. One GSH conjugate, two NAC conjugates, and two cysteine conjugates were found in both mouse and human liver microsomal incubations supplemented with TOL, GSH/NAC/cysteine, and NADPH. The detected conjugates suggest the production of a quinone methide intermediate. The same GSH conjugate was also observed in mouse primary hepatocytes and in the bile of rats receiving TOL. One of the urinary NAC conjugates was observed in rats administered TOL. One of the cysteine conjugates was found in a digestion mixture containing hepatic proteins from animals administered TOL. The observed protein modification was dose-dependent. CYP3A primarily catalyzes the metabolic activation of TOL. Ketoconazole (KTC) pretreatment reduced the generation of the GSH conjugate in mouse liver and cultured primary hepatocytes after TOL treatment. In addition, KTC reduced the susceptibility of primary hepatocytes to TOL cytotoxicity. The quinone methide metabolite may be involved in TOL-induced hepatotoxicity and cytotoxicity.

The molecular docking and molecular dynamics study of flavonol synthase and flavonoid 3'-monooxygenase enzymes involved for the enrichment of kaempferol.

Kaempferol is a natural flavonol that shows many pharmacological properties including anti-inflammatory, antioxidant, anticancer, antidiabetic activities etc. It has been reported in many vegetables, fruits, herbs and medicinal plants. The enzyme flavonol synthase (FLS, EC 1.14.20.6) catalyses the conversion of dihydroflavonols to flavonols. Whereas flavonoid 3'-monooxygenase (F3'H, EC 1.14.14.82) catalyses the hydroxylation of dihydroflavonol, and flavonol. FLS is involved in the synthesis of the kaempferol whereas F3'H causes degradation of kaempferol. The present study aimed to analyse the binding affinity, stability and activating activity of enzyme FLS as well as inhibitory activity of enzyme F3'H involved in the enrichment of the kaempferol using the in-silico approaches. Computational study for physico-chemical properties, conserved domain identification, 3-D structure prediction and its validation, conservation analysis, molecular docking followed by molecular dynamics analysis of FLS and F3'H, protein-activator (FLS-LIG Complex) and protein-inhibitor (F3'H-LIG Complex) complexes have been performed. Other structural analyses like root mean square fluctuation (RMSF), root mean square deviation (RMSD), surface area solvent accessibility (SASA), radius of gyration (Rg), hydrogen bond analysis, principal component analysis (PCA), Poisson-Boltzmann analysis (MM_PBSA) and the dynamic cross correlation map (DCCM) analysis to explore the structural, functional and thermodynamic stability of the proteins and the complexes were also studied. The molecular docking result showed that FLS binds strongly with the activator ascorbate (CID _54670067) while F3'H binds with the inhibitor ketoconazole (CID_456201). The most powerful inhibitor (ketoconazole for F3'H) and activator (ascorbate for FLS) is determined by computing the thermodynamic binding free energy through MM_PBSA analysis. The current work provides wide-ranging structural and functional information about FLS and F3'H enzymes showing detailed molecular mechanism of kaempferol biosynthesis and its degradation and hence kaempferol enrichment. Finding of the present work opens up new possibilities for future research towards enrichment of kaempferol by using activator (ascorbate) for FLS and inhibitor (ketoconazole) for F3'H as well as for its large-scale production using in vitro approaches.Communicated by Ramaswamy H. Sarma.

Effect of CYP3A inducer/inhibitor and licorice on hepatotoxicity and in vivo metabolism of main alkaloids of Euodiae Fructus based on UPLC-Q-Exactive-MS.

ETHNOPHARMACOLOGICAL RELEVANCE: As a traditional Chinese medicine, Euodiae Fructus (EF) has been used to treat stomachache, belching, and emesis for more than a thousand years. Ancient records and modern research have shown that EF has mild toxicity, which needs to be processed with licorice juice to reduce its toxicity. Research suggested that the toxicity of EF can be caused by in vivo metabolism, but whether its metabolites are related to hepatotoxicity and whether licorice can affect the metabolism of EF have not been reported, which needed an effective strategy to clarify the correlation between metabolites and toxicity and the attenuation mechanism of licorice processing. AIM OF THE STUDY: The poisonous substances and metabolic pathways were clarified by comparing the mechanism in vivo process of the main alkaloids of EF in normal rats and rats treated with dexamethasone (DXMS), ketoconazole (KTC), and EF processed with licorice (EFP). MATERIALS AND METHODS: Rats were given EF and EFP by oral administration, respectively. The EF + DXMS and EF + KTC groups were pretreated with DXMS and KTC, respectively, by i. p. for seven days, and their toxicity differences were compared. The comprehensive strategy based on UPLC-Q-Exactive-MS and Orthogonal Partial Least Squares Discriminant Analysis was developed to compare the types and contents of metabolites and clarify the metabolic pathways of alkaloids among EF, EFP, EF + KTC, and EF + DXMS groups. RESULTS: EF + DXMS group significantly increased the hepatotoxicity, whereas the EF + KTC and EFP groups reduced the hepatotoxicity compared with the EF group. One hundred and thirty-five metabolites were detected, and the metabolic pathways of the main alkaloid components related to toxicity were inferred in the plasma, urine, feces, and bile of rats. KTC and licorice similarly inhibited the production of toxic metabolites, changed metabolism in vivo, and produced many new II and a few phases I metabolites, while the contents of toxic metabolites increased in the DXMS group. CONCLUSION: Licorice and KTC could inhibit the production of metabolites of EF related to toxicity, increase the production of other metabolites and promote the excretion of alkaloids, which may be why licorice and KTC can minimize EF toxicity.

Cytochrome P450 inhibition activities of non-standardized botanical products.

ETHNOPHARMACOLOGICAL RELEVANCE: R-tab, H-tab and E-cap botanical products are used for the treatment of various ailments. R-tab is traditionally prescribed for improving urination, H-tab is for relieving piles, hemorrhoids, fissures, and rectal inflammation and E-cap is for regulating menstruation. AIMS OF THE STUDY: To extract the botanical products and determine their potential interaction with the cytochrome P450 (CYP1A2, CYP2D6 and CYP3A4) enzymes. MATERIALS AND METHODS: R-tab, H-tab and E-cap botanical products were first extracted using solvents and analyzed using HPLC and LC-MS/MS. The effects of methanol extracts on the cytochrome induction and inhibition activities were determined using a series of in vitro assays, including multiplex RT-qPCR, CYP activity assays (P450-Glo™) and LC-MS/MS-based assays. For the CYP induction assay, omeprazole, rifampicin and dexamethasone were used as CYP1A2, CYP2D6 and CYP3A4 inducers, respectively. Ketoconazole and acetaminophen were used as positive and negative controls for the CYP3A4 inhibition assay, whereas furafylline and ketoconazole were used as positive and negative controls for the CYP1A2 inhibition assay. RESULTS: All three botanical products did not show any significant induction in CYP1A2, CYP2D6 and CYP3A4 mRNA expression. By contrast, R-tab inhibited the mRNA expression of CYP1A2 significantly from the lowest concentration of 0.01 µg/mL, while, H-tab inhibited the mRNA expression of CYP1A2 and CYP3A4 from 0.1 µg/mL. Based on the P450 Glo assays, E-cap extract inhibited the metabolic activity of CYP1A2 with an IC50 value of 37.24 µg/mL. On the other hand, R-tab, H-tab and E-cap showed inhibitory effects on the CYP3A4 enzymatic activity with IC50 values of 17.42, 18.20 and 20.60 µg/mL, respectively. However, using the LC-MS/MS-based methods, the concentration-dependent effects of R-tab and H-tab extracts on the metabolism of testosterone appeared to be more prominent, with IC50 values of 51.90 and 56.90 µg/mL as compared with the rest of the results, which were all above 100 µg/mL CONCLUSION: The CYP3A4 mRNA and enzymatic activity were moderately inhibited by R-tab and H-tab. Methanol extract of botanical products in solid dosage forms can be evaluated for their herb-drug interaction risks using in vitro assays and may provide the minimum data required for safety labeling.

Influence of methylxanthines isolated from Bancha green tea on the pharmacokinetics of sildenafil in rats.

BACKGROUND: Sildenafil is used to treat erectile dysfunction and pulmonary arterial hypertension and is metabolized in the liver mainly by CYP3A4, thus co-administration with drugs or herbal extracts that affect CYP3A4 activity may lead to drug-drug or drug-herb interactions, respectively. The aim of the present study was to evaluate the influence of single and multiple oral doses of methylxanthine fraction, isolated from Bancha green tea leaves on the pharmacokinetics of sildenafil in rats. METHODS: Rats were given sildenafil alone as well as simultaneously with methylxanthines or ketoconazole. The plasma concentrations of sildenafil were measured with high-performance liquid chromatography method with ultraviolet detection. The pharmacokinetic parameters of sildenafil were calculated by non-compartmental analysis. RESULTS: Concomitant use of sildenafil with a single oral dose of methylxanthines resulted in a decrease in Cmax (p > 0.05), AUC0-t (p < 0.05) and AUC0-inf (p < 0.05), while the administration of sildenafil after methylxanthines pretreatment resulted in an increase in Cmax (p < 0.0001), AUC0-t (p < 0.0001) and AUC0-inf (p < 0.001) compared to the sildenafil group. After co-administration of sildenafil and ketoconazole, a significant increase in Cmax, AUC0-t and AUC0-inf was observed in both of the experiments. CONCLUSION: Drug-herb interactions were observed when sildenafil was co-administered with Bancha methylxanthines in rats. Further in vivo studies about the potential drug interactions between sildenafil and methylxanthines, especially caffeine, are needed to clarify mechanisms underlying the observed changes in sildenafil pharmacokinetics.

Screening and verification of CYP3A4 inhibitors from Bushen-Yizhi formula to enhance the bioavailability of osthole in rat plasma.

ETHNOPHARMACOLOGICAL RELEVANCE: With the features of multiple-components and targets as well as multifunction, traditional Chinese medicine (TCM) has been widely used in the prevention and treatment of various diseases for a long time. During the application of TCM, the researches about bioavailability enhancement of the bioactive constituents in formula are flourishing. Bushen-Yizhi formula (BSYZ), a TCM prescription with osthole (OST) as one of the main bioactive ingredients, have been widely used to treat kidney deficiency, mental retardation and Alzheimer's disease. However, the underlying biological mechanism and compound-enzyme interaction mediated bioavailability enhancement of OST are still not clearly illuminated. AIM OF THE STUDY: The aim of this study is to explore the material basis and molecular mechanism from BSYZ in the bioavailability enhancement of OST. Screening the potential CYP3A4 inhibitors using theoretical prediction and then verifying them in vitro, and pharmacokinetics study of OST in rat plasma under co-administrated of screened CYP3A4 inhibitors and BSYZ were also scarcely reported. MATERIALS AND METHODS: Screening of CYP3A4 inhibitors from BSYZ was performed with molecular docking simulation from systems pharmacology database. The screened compounds were verified by using P450-Glo Screening Systems. A multiple reaction monitoring (MRM) mass spectrometry method was established for OST quantification. Male Sprague-Dawley rats divided into four groups and six rats in each group were employed in the pharmacokinetics study of OST. The administrated conditions were group I, OST (20 mg/kg); group II, BSYZ (containing OST 1 mg/mL, at the dose of 20 mg/kg OST in BSYZ); group III, co-administration of ketoconazole (Ket, 75 mg/kg) and OST (20 mg/kg); group IV, co-administration of CYP3A4 inhibitor (10 mg/kg) and OST (20 mg/kg). They were determined by using HPLC-MS/MS (MRM) and statistical analysis was performed using student's t-test with p < 0.05 as the level of significance. RESULTS: 21 potential CYP3A4 inhibitors were screened from BSYZ compounds library. From the results of verification in vitro, we found 4 compounds with better CYP3A4 inhibition efficiency including Oleic acid, 1,2,3,4,6-O-Pentagalloylglucose, Rutin, and Schisantherin B. Under further verification, Schisantherin B exhibited the best inhibitory effect on CYP3A4 (IC50 = 0.339 µM), and even better than the clinically used drug (Ket) at the concentration of 5 µM. In the study of pharmacokinetics, the area under the curve (AUC, ng/L*h) of OST after oral administration of BSYZ, Ket and Schisantherin B (2196.23 ± 581.33, 462.90 ± 92.30 and 1053.03 ± 263.62, respectively) were significantly higher than that of pure OST treatment (227.89 ± 107.90, p < 0.01). CONCLUSIONS: Schisantherin B, a profoundly effective CYP3A4 inhibitor screened from BSYZ antagonized the metabolism of CYP3A4 on OST via activity inhibition, therefore significantly enhanced the bioavailability of OST in rat plasma. The results of this study will be helpful to explain the rationality of the compatibility in TCM formula, and also to develop new TCM formula with more reasonable drug compatibility.

Impact of Swelling of Spray Dried Dispersions in Dissolution Media on their Dissolution: An Investigation Based on UV Imaging.

Impact of SDD-dissolution medium interactions on the swelling and dissolution of spray dried dispersions (SDDs) was investigated using UV imaging by monitoring SDD swelling in situ, along with correlating of the swelling with the micro-dissolution and intrinsic dissolution of SDDs. SDDs of ketoconazole or indomethacin with three polymers: polyvinylpyrrolidone (PVP), hydroxypropyl cellulose (HPC), and hydroxypropyl methylcellulose acetate succinate (HPMC-AS) were prepared for the study. Dissolution media employed for assessing swelling and dissolution include water, acetate buffer, phosphate buffer, fasted state simulated intestinal fluid (FaSSIF), and fed state simulated intestinal fluid (FeSSIF), in which influence of polymers and drugs together with the physical-chemical properties of dissolution media (pH, and the presence of sodium taurocholate and lecithin) on SDD swelling and dissolution was evaluated. It appears that hydrophilic and hydrophobic properties of polymers can significantly impact SDD swelling and thus the dissolution. Furthermore, properties of dissolution media such as pH as well as presence of bile salts and lecithin seems to affect SDD swelling and dissolution as well. Throughout the text, thermodynamic swelling of polymers was used to interpret SDD dissolution behavior. Finally, practical implication of polymer swelling on dissolution was discussed.

The Influence of Tea Tree Oil on Antifungal Activity and Pharmaceutical Characteristics of Pluronic® F-127 Gel Formulations with Ketoconazole.

Fungal skin infections are currently a major clinical problem due to their increased occurrence and drug resistance. The treatment of fungal skin infections is based on monotherapy or polytherapy using the synergy of the therapeutic substances. Tea tree oil (TTO) may be a valuable addition to the traditional antifungal drugs due to its antifungal and anti-inflammatory activity. Ketoconazole (KTZ) is an imidazole antifungal agent commonly used as a treatment for dermatological fungal infections. The use of hydrogels and organogel-based formulations has been increasing for the past few years, due to the easy method of preparation and long-term stability of the product. Therefore, the purpose of this study was to design and characterize different types of Pluronic® F-127 gel formulations containing KTZ and TTO as local delivery systems that can be applied in cases of skin fungal infections. The influence of TTO addition on the textural, rheological, and bioadhesive properties of the designed formulations was examined. Moreover, the in vitro release of KTZ, its permeation through artificial skin, and antifungal activity by the agar diffusion method were performed. It was found that obtained gel formulations were non-Newtonian systems, showing a shear-thinning behaviour and thixotropic properties with adequate textural features such as hardness, compressibility, and adhesiveness. Furthermore, the designed preparations with TTO were characterized by beneficial bioadhesive properties. The presence of TTO improved the penetration and retention of KTZ through the artificial skin membrane and this effect was particularly visible in hydrogel formulation. The developed gels containing TTO can be considered as favourable formulations in terms of drug release and antifungal activity.

Activity of antifungal drugs and Brazilian red and green propolis extracted with different methodologies against oral isolates of Candida spp.

BACKGROUND: Oral candidiasis is an opportunistic disease caused by fungi of the Candida genus. The occurrence of Candida spp. resistance to the commercial antifungal drugs points to the search for alternative treatments. Propolis has been successfully used in the treatment of infectious diseases for centuries. It has been proposed that an ultrasound pretreatment in the propolis extraction protocol can enhance the concentrations of molecules with antimicrobial activities in the final extract. Thus, this study aimed to compare the antifungal activity against oral Candida spp. isolates of green and red propolis extracts submitted or not to an ultrasound pretreatment before the extraction procedure. METHODS: Candida spp. were isolated from denture stomatitis lesions and identified by sequencing. Oral Candida spp. isolates and reference strains were submitted to broth microdilution assays using commercial antifungals and Brazilian green and red propolis extracts submitted or not to an ultrasound pretreatment. Minimal Inhibitory Concentrations (MIC) and Minimal Fungicide Concentrations (MFC) were determined and biofilm formation interference was evaluated for resistant isolates. RESULTS: C. albicans, Candida tropicalis and Candida dubliniensis were isolated from denture stomatitis lesions. Growth inhibition was observed in all Candida isolates incubated with all green and red propolis extracts. At lower doses, red propolis extracts presented significant antifungal activity. The ultrasound pretreatment did not promote an increase in the antifungal activity of green or red propolis. Three isolates, which were highly resistant to fluconazole and itraconazole, were susceptible to low doses of red propolis extracts. These same three specimens had their biofilm formation inhibted by red propolis ethanolic extract. CONCLUSIONS: Thus, red propolis can be faced as a promising natural product to be used in the auxiliary antifungal therapy of denture stomatitis.

In Vitro-In Vivo Correlation in Cocrystal Dissolution: Consideration of Drug Release Profiles Based on Coformer Dissolution and Absorption Behavior.

This study assessed the in vitro-in vivo correlation in cocrystal dissolution based on the coformer behavior. 4-Aminobenzoic acid (4ABA) was used as a coformer. Cocrystals of poorly water-soluble drugs with 4ABA, ketoconazole cocrystal (KTZ-4ABA), posaconazole cocrystal (PSZ-4ABA), and itraconazole cocrystal (ITZ-4ABA) were used. These three cocrystals generated supersaturated solutions in fasted state simulated intestinal fluid (FaSSIF) in a small-scale, 8 mL dissolution vessel. The time profile of the dissolved amount of 4ABA, an indicator of cocrystal dissolution, was significantly different among the three cocrystals. Under the conditions utilized, half of the KTZ-4ABA cocrystal solid rapidly dissolved within 5 min and the dissolved amount (% of applied amount) of KTZ and 4ABA was the same. Then, even though the residual solid cocrystal gradually dissolved, KTZ precipitated with time. The PSZ-4ABA cocrystal dissolved in a linear fashion with time but the dissolved concentration of PSZ reached a plateau in the supersaturated state and was maintained for at least 2 h. The dissolution rate of ITZ-4ABA was very slow compared to those of the other cocrystals, but a similar tendency was observed between cocrystal dissolution and the dissolved amount of ITZ. The rank order of the cocrystal dissolution rate based on the conformer concentration was KTZ-4ABA > PSZ-4ABA > ITZ-4ABA. Furthermore, cocrystallization of the three drugs with 4ABA significantly enhanced the oral drug absorption in rats. The rank order of the in vivo cocrystal dissolution rate by a deconvolution analysis with the plasma concentration-time profile of 4ABA was KTZ-4ABA > PSZ-4ABA > ITZ-4ABA, which corresponded well with the in vitro dissolution profiles of the cocrystals. These results indicate that analysis of cocrystal dissolution based on the coformer behavior may be useful to evaluate the in vitro and in vivo cocrystal dissolution.

Antifungal Activity of Melaleuca alternifolia Essential Oil (TTO) and Its Synergy with Itraconazole or Ketoconazole against Trichophyton rubrum.

Over the past 20-30 years, Trichophyton rubrum represented the most widespread dermatophyte with a prevalence accounting for 70% of dermatophytosis. The treatment for cutaneous infections caused by Trichophyton spp. are imidazoles (ketoconazole (KTZ)) and triazoles (itraconazole (ITZ)). T. rubrum can develop resistance to azoles after prolonged exposure to subinhibitory concentrations resulting in therapeutic failures and chronic infections. These problems have stimulated the search for therapeutic alternatives, including essential oils, and their potential use in combination with conventional antifungals. The purpose of this study was to evaluate the antifungal activity of tea tree oil (TTO) (Melaleuca alternifolia essential oil) and the main components against T. rubrum and to assess whether TTO in association with KTZ/ITZ as reference drugs improves the antifungal activity of these drugs. We used a terpinen-4-ol chemotype (35.88%) TTO, and its antifungal properties were evaluated by minimum inhibitory and minimum fungicidal concentrations in accordance with the CLSI guidelines. The interaction between TTO and azoles was evaluated through the checkerboard and isobologram methods. The results demonstrated both the fungicide activity of TTO on T. rubrum and the synergism when it was used in combination with azoles. Therefore, this mixture may reduce the minimum effective dose of azole required and minimize the side effects of the therapy. Synergy activity offered a promise for combination topical treatment for superficial mycoses.

Establishment of a Novel Human Fetal Adrenal Culture Model that Supports de Novo and Manipulated Steroidogenesis.

CONTEXT: Disorders affecting adrenal steroidogenesis promote an imbalance in the normally tightly controlled secretion of mineralocorticoids, glucocorticoids, and androgens. This may lead to differences/disorders of sex development in the fetus, as seen in virilized girls with congenital adrenal hyperplasia (CAH). Despite the important endocrine function of human fetal adrenals, neither normal nor dysregulated adrenal steroidogenesis is understood in detail. OBJECTIVE: Due to significant differences in adrenal steroidogenesis between human and model species (except higher primates), we aimed to establish a human fetal adrenal model that enables examination of both de novo and manipulated adrenal steroidogenesis. DESIGN AND SETTING: Human adrenal tissue from 54 1st trimester fetuses were cultured ex vivo as intact tissue fragments for 7 or 14 days. MAIN OUTCOME MEASURES: Model validation included examination of postculture tissue morphology, viability, apoptosis, and quantification of steroid hormones secreted to the culture media measured by liquid chromatography-tandem mass spectrometry. RESULTS: The culture approach maintained cell viability, preserved cell populations of all fetal adrenal zones, and recapitulated de novo adrenal steroidogenesis based on continued secretion of steroidogenic intermediates, glucocorticoids, and androgens. Adrenocorticotropic hormone and ketoconazole treatment of ex vivo cultured human fetal adrenal tissue resulted in the stimulation of steroidogenesis and inhibition of androgen secretion, respectively, demonstrating a treatment-specific response. CONCLUSIONS: Together, these data indicate that ex vivo culture of human fetal adrenal tissue constitutes a novel approach to investigate local effects of pharmaceutical exposures or emerging therapeutic options targeting imbalanced steroidogenesis in adrenal disorders, including CAH.

The tangential flow absorption model (TFAM) - A novel dissolution method for evaluating the performance of amorphous solid dispersions of poorly water-soluble actives.

There is a substantial demand for absorptive dissolution tests, as single vessel dissolution experiments were originally not designed for testing supersaturating systems. Current approaches suffer from inadequate mass transfer of the dissolved active from the dissolution site, discrepancies in the fluid volume compared to in vivo intestinal fluid volumes or the dilution of functional excipients. In this work a novel dissolution apparatus was developed that enables adjustable mass transfer of the active through a membrane, while retaining the functional polymeric excipients at the dissolution site. Using this setup the dissolution behavior of various spray dried amorphous solid dispersions containing carbamazepine, hydrochlorothiazide and ketoconazole as model actives at intermediate and high supersaturation levels was evaluated. Compared to non-absorptive dissolution experiments, differences in the concentration-time profiles were noted. The experiments with a high supersaturation of ketoconazole revealed a concentration decrease over time under absorptive conditions. Additionally, it was observed that the difference between "spring" as well as "spring and parachute" formulations was less pronounced with increasing drug efflux. Further, the apparatus was also tested with Fasted State Simulated Intestinal Fluid as dissolution medium and results were compared to phosphate buffer pH6.8. As major benefits of the new TFAM apparatus the easy experimental procedure and sample preparation for drug concentration measurements using spectroscopy in the permeate, without the necessity for additional filtration and/or centrifugation to remove precipitated drug molecules, could be highlighted. This TFAM approach seems to be a promising tool for identifying formulations for amorphous solid dispersions with optimal in vitro performance.

Development and evaluation of a biorelevant medium simulating porcine gastrointestinal fluids.

Simulated human intestinal media, have proved to be a useful biopharmaceutics tool as a dissolution media for predicting in vivo dissolution and pharmacokinetic profile in humans. During drug product development preclinical animal models are also required to assess drug product performance, and there is a need to develop species specific intestinal media to similarly predict in vivo pharmacokinetic profiles in each preclinical model. Pigs, are increasingly being used in preclinical drug development, however to date there is a lack of quantitative information about the composition of porcine gastrointestinal (GI) fluids. As a result, a porcine biorelevant medium has not yet been developed, which is essential to improve interpretation and forecast of preclinical results using biorelevant in vitro dissolution studies. GI fluid samples, were collected from landrace pigs, and characterized. Fasted State Simulated Intestinal Fluid of pigs (FaSSIFp) was developed based on the physiological composition of the GI fluids in terms of pH, buffer capacity, osmolality, surface tension, as well as the bile salt, phospholipid and free fatty acid content. This study demonstrated that FaSSIFp was superior at predicting the solubility of the six model drugs in porcine intestinal fluids (PIF). A markedly high correlation (r2 0.98) was observed between the solubility obtained in PIF and FaSSIFp, whereas poor correlation (r2 0.12) was found for the solubility of the model drugs between human FaSSIF and PIF. This confirms that species specific biorelevant intestinal media are crucial to provide more accurate predictions of pharmacokinetic studies in preclinical models. Additionally, the availability of a species specific intestinal medium offers the potential to improve in vitro-in silico approaches to predict in vivo absorption and to reduce the overall number of animals needed in oral drug product development testing.

Evaluation of the Effects of Maytenus ilicifolia on the Activities of Cytochrome P450 3A and P-glycoprotein.

BACKGROUND: Maytenus ilicifolia is a Brazilian popular medicine commonly used to treat ulcer and gastritis. Despite the absence of toxicity regarding its consumption, possible interactions when co-administrated with conventional drugs, are unknown. OBJECTIVE: This study aimed to evaluate the effects of M. ilicifolia extracts on Cytochrome P450 3A (CYP3A) and P-glycoprotein (P-gp) activities. METHODS: The extracts were obtained by infusion (MI) or turbo-extraction using hydro-acetonic solvent (MT70). The content of polyphenols in each extract was determined. To assess the modulation of M. ilicifolia on P-gp activity, the uptake of fexofenadine (FEX) by Caco-2 cells was investigated in the absence or presence of MI or MT70. The effect on CYP3A activity was evaluated by the co-administration of midazolam (MDZ) with each extract in male Wistar rats. The pharmacokinetic parameters of the drug were determined and compared with those from the control group. The content of total phenolic compounds, tannins, and flavonoids on MT70 extract was about double of that found in MI. RESULTS: In the presence of the extracts, the uptake of the P-gp marker (FEX) by Caco-2 cells increased from 1.7 ± 0.4 ng.mg-1 protein (control) to 3.5 ± 0.2 ng.mg-1 protein (MI) and 4.4 ± 0.5 ng.mg-1 protein (MT70), respectively. When orally co-administrated with MDZ (substrate of CYP3A), the extracts augmented the AUC(0-∞) (Control: 911.7 ± 215.7 ng.h.mL-1; MI: 1947 ± 554.3 ng.h.mL-1; MT70: 2219.0 ± 506.3 ng.h.mL-1) and the Cmax (Control: 407.7 ± 90.4 ng.mL-1; MI: 1770.5 ± 764.5 ng.mL-1; MT70: 1987.2 ± 544.9 ng.mL-1) of the drug in rats indicating a 50% reduction of the oral Cl. No effect was observed when midazolam was given intravenously. CONCLUSION: The results suggest that M. ilicifolia can inhibit the intestinal metabolism and transport of drugs mediated by CYP3A and P-gp, respectively, however, the involvement of other transporters and the clinical relevance of such interaction still need to be clarified.