Differential Effects of Clotrimazole on X-Ray Crystal Structures of Human Cytochromes P450 3A5 and 3A4.

Cytochromes P450 CYP3A5 and CYP3A4 exhibit differential plasticity that underlies differences in drug metabolism and drug-drug interactions. To extend previous studies, CYP3A4 and CYP3A5 were cocrystallized with clotrimazole, a compact ligand that binds to the heme iron in the catalytic center of the active site. Binding studies indicate that clotrimazole exhibits tight binding to CYP3A5 with a binding affinity (Kd) of <0.01 µM like that of CYP3A4. A single clotrimazole is bound to the heme iron in CYP3A4 that triggers expansion of active site cavity that reflects a loss of aromatic interactions between phenylalanine sidechains in the distal active site and increased conformational entropy for the F-F' connector due to reorientation of Phe-304 to accommodate clotrimazole. In contrast to CYP3A4, the CYP3A5 Phe-304 exhibits an induced fit along with Phe-213 to form edge-to-face aromatic interactions with heme-bound clotrimazole. These aromatic interactions between aromatic amino acids propagate by induced fits with a second clotrimazole residing in the distal active site and a third clotrimazole bound in an expanded entrance channel as well as between the three clotrimazoles. The large, expanded entrance channel surrounded by the C-terminal loop and the F' and A' helices in CYP3A5 suggests conformational selection for the binding of clotrimazole due to its large girth, which may also cause the entrance channel to remain open after the binding of the first clotrimazole to the heme iron. The additional binding sites suggest a path for sequential binding of one molecule to reach and bind to the heme iron. SIGNIFICANCE STATEMENT: Clotrimazole binds to the heme iron of CYP3A5 and CYP3A4. In CYP3A5, two clotrimazoles also bind in the distal active site and in an expanded entrance channel. Aromatic interactions between clotrimazoles and phenylalanine sidechains including Phe-304 indicate induced fits for each clotrimazole. In contrast to CYP3A5, displacement of the CYP3A4 Phe-304 rotamer by clotrimazole leads to extensive disruption of phenylalanine interactions that limit the space above the heme, to an expanded active site cavity, and to increased CYP3A4 conformational heterogeneity.

Unravelling the antifungal mode of action of curcumin by potential inhibition of CYP51B: A computational study validated in vitro on mucormycosis agent, Rhizopus oryzae.

Like human, fungi too are known to share lot of structural similarities amongst their CYPs (Cytochrome P450 super family of enzymes) which allows antifungal 'azole' compounds to interact with CYPs of human. Clotrimazole, an 'azole' antifungal drug, is a known inhibitor of fungal CYP named CYP51B. Curcumin, a phytochemical obtained from Curcuma longa has the ability to interact with several different human CYPs to induce inhibition. The sequence and the structural similarities amongst both human and fungal CYPs suggest a strong possibility for curcumin to interact with fungal CYP51B to behave like an antifungal agent. To test this hypothesis a study was designed involving mucormycosis agent, Rhizopus oryzae. The ability of curcumin to interact with fungal CYP51B was analysed computationally through molecular docking, MM-GBSA and Molecular Dynamics (MD) simulation assessment. Further, interaction profile for fungal CYP51B-curcumin was compared with human CYP3A4-curcumin, as there are published evidence describing curcumin as an inhibitor of human CYPs. Additionally, to validate in silico findings, an in vitro assay was performed to examine the antifungal potentials of curcumin on the R. oryzae. Conclusive results allow us to determine a plausible mode of action of curcumin to act as an antifungal against a mucormycosis agent.

Clotrimazole Loaded Ufosomes for Topical Delivery: Formulation Development and In-Vitro Studies.

Global incidence of superficial fungal infections caused by dermatophytes is high and affects around 40 million people. It is the fourth most common cause of infection. Clotrimazole, a broad spectrum imidazole antifungal agent is widely used to treat fungal infections. Conventional topical formulations of clotrimazole are intended to treat infections by effective penetration of drugs into the stratum corneum. However, drawbacks such as poor dermal bioavailability, poor penetration, and variable drug levels limit the efficiency. The present study aims to load clotrimazole into ufosomes and evaluate its topical bioavailability. Clotrimazole loaded ufosomes were prepared using cholesterol and sodium oleate by thin film hydration technique and evaluated for size, polydispersity index, and entrapment efficiency to obtain optimized formulation. Optimized formulation was characterized using scanning electron microscopy (SEM), X-ray diffraction (XRD), and differential scanning calorimetry (DSC). Skin diffusion studies and tape-stripping were performed using human skin to determine the amount of clotrimazole accumulated in different layers of the skin. Results showed that the optimized formulation had vesicle size <250 nm with ~84% entrapment efficiency. XRD and DSC confirmed the entrapment of clotrimazole into ufosomes. No permeation was observed through the skin up to 24 h following the permeation studies. Tape-stripping revealed that ufosomes led to accumulation of more clotrimazole in the skin compared to marketed formulation (Perrigo). Overall, results revealed the capability of ufosomes in improving the skin bioavailability of clotrimazole.

Biodegradation of clotrimazole and modification of cell properties after metabolic stress and upon addition of saponins.

Azole fungicides constitute an extensive group of potential emerging pollutants which can be found in natural environment. This study focuses on the biodegradation of clotrimazole and the characterization of cell surface properties of microorganisms capable of degradation of this compound. The influence of long-term contact of bacteria with clotrimazole and the impact of the addition of Saponaria officinalis extract on cell surface modification was also checked. The biodegradation of clotrimazole did not exceed 70%. The presence of plant extract increased biodegradation of fungicide. The cells metabolic activity after one-month exposure to clotrimazole was the highest for each tested strain. Moreover, metabolic stress led to a strong modification of cell surface properties. The results are promising for determining the impact of clotrimazole on environmental microorganisms.

Clotrimazole-cyclodextrin based approach for the management and treatment of Candidiasis - A formulation and chemistry-based evaluation.

Clotrimazole (CT) is a poorly soluble antifungal drug that is most commonly employed as a topical treatment in the management of vaginal candidiasis. The present work focuses on a formulation approach to enhance the solubility of CT using cyclodextrin (CD) complexation. A CT-CD complex was prepared by a co-precipitation method. Various characterization techniques such as differential scanning calorimetry, infrared (IR) and X-ray spectroscopy, scanning electron microscopy and nuclear magnetic resonance (NMR) spectroscopy were performed to evaluate the complex formation and to understand the interactions between CT and CD. Computational molecular modeling was performed using the Schrödinger suite and Gaussian 09 program to understand structural conformations of the complex. The phase solubility curve followed an AL-type curve, indicating formation of a 1:1 complex. Molecular docking studies supported the data obtained through NMR and IR studies. Enthalpy changes confirmed that complexation was an exothermic and enthalpically favorable phenomenon. The CT-CD complexes were formulated in a gel and evaluated for release and antifungal activity. The in vitro release studies performed using gels demonstrated a sustained release of CT from the CT-CD complex with the complex exhibiting improved release relative to the un-complexed CT. Complexed CT-CD exhibited better fungistatic activity toward different Candida species than un-complexed CT.

Clotrimazole causes membrane depolarization and induces sub G0 cell cycle arrest in Leishmania donovani.

Clotrimazole is an FDA approved drug and is widely used as an antifungal agent. An extensive body of research is available about its mechanism of action on various cell types but its mode of killing of Leishmania donovani parasites is unknown. L. donovani causes Visceral Leishmaniasis which is a public health problem with limited treatment options. Its present chemotherapy is expensive, has adverse effects and is plagued with drug resistance issues. In this study we have explored the possibility of repurposing clotrimazole as an antileishmanial drug. We have assessed its efficacy on the parasites and attempted to understand its mode of action. We found that it has a half-maximal inhibitory concentration (IC50) of 35.75 ± 1.06 µM, 12.75 ± 0.35 µM and 73 ± 1.41 µM in promastigotes, intracellular amastigotes and macrophages, respectively. Clotrimazole is 5.73 times more selective for the intracellular amastigotes as compared to the mammalian cell. Effect of clotrimazole was reduced by ergosterol supplementation. It leads to impaired parasite morphology. It alters plasma membrane permeability and disrupts plasma membrane potential. Mitochondrial function is compromised as is evident from increased ROS generation, depolarized mitochondrial membrane and decreased ATP levels. Cell cycle analysis of clotrimazole treated parasites shows arrest at sub-G0 phase suggesting apoptotic mode of cell death.

Expression and characterization of Mycobacterium tuberculosis CYP144: common themes and lessons learned in the M. tuberculosis P450 enzyme family.

CYP144 from Mycobacterium tuberculosis was expressed and purified. CYP144 demonstrates heme thiolate coordination in its ferric form, but the cysteinate is protonated to thiol in both the carbon monoxide-bound and ligand-free ferrous forms (forming P420 in the former). Tight binding of various azole drugs was shown, with affinity for miconazole (K(d)=0.98 µM), clotrimazole (0.37 µM) and econazole (0.78 µM) being highest. These azoles are also the trio with the highest affinity for the essential CYP121 and for the cholesterol oxidase CYP125 (essential for host infection), and have high potency as anti-mycobacterial drugs. Construction of a Mtb gene knockout strain demonstrated that CYP144 is not essential for growth in vitro. However the deletion strain was more sensitive to azole inhibition in culture suggesting an important role for CYP144 in cell physiology and/or in mediating azole resistance. The biophysical and genetic features of CYP144 are compared to those of other characterized Mtb P450s, identifying both commonality in properties (including thiolate protonation in ferrous P450s) and intriguing differences in thermodynamic and spectroscopic features. Our developing knowledge of the Mtb P450s has revealed unusual biochemistry and gene essentiality, highlighting their potential as drug targets in this human pathogen.

QSAR model for human pregnane X receptor (PXR) binding: screening of environmental chemicals and correlations with genotoxicity, endocrine disruption and teratogenicity.

The pregnane X receptor (PXR) has a key role in regulating the metabolism and transport of structurally diverse endogenous and exogenous compounds. Activation of PXR has the potential to initiate adverse effects, causing drug-drug interactions, and perturbing normal physiological functions. Therefore, identification of PXR ligands would be valuable information for pharmaceutical and toxicological research. In the present study, we developed a quantitative structure-activity relationship (QSAR) model for the identification of PXR ligands using data based on a human PXR binding assay. A total of 631 molecules, representing a variety of chemical structures, constituted the training set of the model. Cross-validation of the model showed a sensitivity of 82%, a specificity of 85%, and a concordance of 84%. The developed model provided knowledge about molecular descriptors that may influence the binding of molecules to PXR. The model was used to screen a large inventory of environmental chemicals, of which 47% was found to be within domain of the model. Approximately 35% of the chemicals within domain were predicted to be PXR ligands. The predicted PXR ligands were found to be overrepresented among chemicals predicted to cause adverse effects, such as genotoxicity, teratogenicity, estrogen receptor activation and androgen receptor antagonism compared to chemicals not causing these effects. The developed model may be useful as a tool for predicting potential PXR ligands and for providing mechanistic information of toxic effects of chemicals.

Cavamax W7 composite ethosomal gel of clotrimazole for improved topical delivery: development and comparison with ethosomal gel.

The present research work was aimed to formulate clotrimazole encapsulated Cavamax W7 composite ethosomes by injection method for improved delivery across epidermis. 3(2) factorial design was used to design nine formulations (F1-F9) and compared with ethosomal formulations (F10-F12). F9 with vesicle size of 202.8 ± 4.8 nm, highest zeta potential (-83.6 ± 0.96 mV) and %EE of 98.42 ± 0.15 was selected as optimized composite ethosome and F12 as reference ethosomal formulation. As revealed by transmission electron microscopy F9 vesicles were more condensed, uniformly spherical in shape than F12 vesicles. Vesicular stability studies indicated F9 to be more stable as compared to F12. Both F9 and F12 were incorporated in carbopol 934 gel base to get G1-G8 gel formulations and evaluated for in vitro skin permeability. Cavamax W7 composite ethosomal optimized gel (G5) showed higher in vitro percent cumulative drug permeation (88.53 ± 2.10%) in 8 h and steady state flux (J(ss)) of 3.39 ± 1.45 µg/cm(2)/min against the J(ss) of 1.57 ± 0.23 µg/cm(2)/min for ethosomal gel (G1) and 1.13 ± 0.06 µg/cm(2)/min for marketed formulation. The J(ss) flux of G5 was independent of amount of drug applied/unit area of skin. In vivo confocal laser scanning microscopic study of G5 depicted uniform and deeper penetration of rhodamine B (marker) in epidermis from Cavamax W7 composite ethosomal gel in comparison to G1. Finally, G5 demonstrated better (p < 0.05) antifungal activity against Candida albicans and Aspergillus niger than G1 thus, signifying that Cavamax W7 composite ethosomes present a superior stable and efficacious vesicular system than ethosomal formulation for topical delivery of clotrimazole.

Structural basis of drug binding to CYP46A1, an enzyme that controls cholesterol turnover in the brain.

Cytochrome P450 46A1 (CYP46A1) initiates the major pathway of cholesterol elimination from the brain and thereby controls cholesterol turnover in this organ. We determined x-ray crystal structures of CYP46A1 in complex with four structurally distinct pharmaceuticals; antidepressant tranylcypromine (2.15 Å), anticonvulsant thioperamide (1.65 Å), antifungal voriconazole (2.35 Å), and antifungal clotrimazole (2.50 Å). All four drugs are nitrogen-containing compounds that have nanomolar affinity for CYP46A1 in vitro yet differ in size, shape, hydrophobicity, and type of the nitrogen ligand. Structures of the co-complexes demonstrate that each drug binds in a single orientation to the active site with tranylcypromine, thioperamide, and voriconazole coordinating the heme iron via their nitrogen atoms and clotrimazole being at a 4 Å distance from the heme iron. We show here that clotrimazole is also a substrate for CYP46A1. High affinity for CYP46A1 is determined by a set of specific interactions, some of which were further investigated by solution studies using structural analogs of the drugs and the T306A CYP46A1 mutant. Collectively, our results reveal how diverse inhibitors can be accommodated in the CYP46A1 active site and provide an explanation for the observed differences in the drug-induced spectral response. Co-complexes with tranylcypromine, thioperamide, and voriconazole represent the first structural characterization of the drug binding to a P450 enzyme.

Muscle-type 6-phosphofructo-1-kinase and aldolase associate conferring catalytic advantages for both enzymes.

6-Phosphofructo-1-kinase (PFK) and aldolase are two sequential glycolytic enzymes that associate forming heterotetramers containing a dimer of each enzyme. Although free PFK dimers present a negligible activity, once associated to aldolase these dimers are as active as the fully active tetrameric conformation of the enzyme. Here we show that aldolase-associated PFK dimers are not inhibited by clotrimazole, an antifungal azole derivative proposed as an antineoplastic drug due to its inhibitory effects on PFK. In the presence of aldolase, PFK is not modulated by its allosteric activators, ADP and fructose-2,6-bisphosphate, but is still inhibited by citrate and lactate. The association between the two enzymes also results on the twofold stimulation of aldolase maximal velocity and affinity for its substrate. These results suggest that the association between PFK and aldolase confers catalytic advantage for both enzymes and may contribute to the channeling of the glycolytic metabolism.

Inhibition of the gastric H,K-ATPase by clotrimazole.

The antimycotic drug clotrimazole inhibits the function of the gastric H,K-ATPase in a manner similar to that observed for the Na,K-ATPase. Because of the high hydrophobicity of the compound, the interaction between clotrimazole and the ion pump occurs at the membrane domain in the apolar core of the membrane. The enzymatic activity was inhibited with a half-saturating concentration of 5.2 microM. Various partial reactions of the pump cycle were analyzed with the electrochromic styryl dye RH421 that has been widely used to study the transport mechanism of P-type ATPases. We discovered that the interaction of clotrimazole with the H,K-ATPase introduces a single "dead-end" branch added to the Post-Albers scheme in the E(1) state of the pump. In this inhibiting state, the ion binding sites have a significantly enhanced affinity for protons and bind up to two protons even at pH 8.5. Inhibition of the pump can be reversed by a decreased pH or increased K(+) concentrations. The mechanistic proposal that allows an explanation of all experiments presented is similar to that published for the Na,K-ATPase.

Expression, purification, and characterization of Aspergillus fumigatus sterol 14-alpha demethylase (CYP51) isoenzymes A and B.

Aspergillus fumigatus sterol 14-α demethylase (CYP51) isoenzymes A (AF51A) and B (AF51B) were expressed in Escherichia coli and purified. The dithionite-reduced CO-P450 complex for AF51A was unstable, rapidly denaturing to inactive P420, in marked contrast to AF51B, where the CO-P450 complex was stable. Type I substrate binding spectra were obtained with purified AF51B using lanosterol (K(s), 8.6 µM) and eburicol (K(s), 22.6 µM). Membrane suspensions of AF51A bound to both lanosterol (K(s), 3.1 µM) and eburicol (K(s), 4.1 µM). The binding of azoles, with the exception of fluconazole, to AF51B was tight, with the K(d) (dissociation constant) values for clotrimazole, itraconazole, posaconazole, and voriconazole being 0.21, 0.06, 0.12, and 0.42 µM, respectively, in comparison with a K(d) value of 4 µM for fluconazole. Characteristic type II azole binding spectra were obtained with AF51B, whereas an additional trough and a blue-shifted spectral peak were present in AF51A binding spectra for all azoles except clotrimazole. This suggests two distinct azole binding conformations within the heme prosthetic group of AF51A. All five azoles bound relatively weakly to AF51A, with K(d) values ranging from 1 µM for itraconazole to 11.9 µM for fluconazole. The azole binding properties of purified AF51A and AF51B suggest an explanation for the intrinsic azole (fluconazole) resistance observed in Aspergillus fumigatus.

Clotrimazole potentiates the inhibitory effects of ATP on the key glycolytic enzyme 6-phosphofructo-1-kinase.

Clotrimazole (CTZ) has been proposed as a potential anti-neoplastic agent, which inhibits glucose metabolism. The present work aimed to evaluate the effects of CTZ on the kinetic mechanism of 6-phosphofructo-1-kinase (PFK). We show that CTZ promotes a dose-dependent inhibition of PFK, presenting a K(i) of 28 +/- 2 microM. Inhibition occurs through the dissociation of the enzyme tetramers, as demonstrated through fluorescence spectroscopy and gel filtration chromatography. Moreover, the affinities of the enzyme for ATP and fructose-6-phosphate are reduced 50% and 30%, respectively. Furthermore, the affinity of PFK for ATP at the inhibitory site becomes 2-fold higher. Altogether, the results presented here suggest that PFK inhibition by CTZ involves a decrease in the affinity of PFK for its substrates at the catalytic site with the concomitant potentiation of the inhibitory properties of ATP.

Assessment of clotrimazole gels for in vitro stability and in vivo retention in the frontal sinus of dogs.

OBJECTIVE: To evaluate the stability and retention of viscous formulations of the antifungal drug clotrimazole in vitro and to evaluate retention times, absorption, and histologic response to these compounds when placed in the frontal sinus of dogs. ANIMALS: 6 male Beagles. PROCEDURES: 1% clotrimazole gels were formulated with hydroxypropyl cellulose, poloxamer, and carboxymethylcellulose sodium bases. Commercially available 1% clotrimazole creams were also evaluated. Each compound was incubated at 37 degrees C in a funnel. Volume retained and clotrimazole stability were evaluated for 4 weeks. Six compounds were then chosen for in vivo evaluation. The frontal sinuses of 6 dogs were filled with 1 of the 6 compounds. Computed tomographic evaluation was performed weekly for up to 4 weeks to evaluate gel retention. Blood samples were collected to evaluate clotrimazole absorption. Following euthanasia, sinuses were examined histologically. RESULTS: Commercially available clotrimazole creams were not retained in funnels in vitro. In vivo, hydroxypropyl cellulose- and carboxymethylcellulose-based gels resulted in the most severe inflammatory response and were retained the longest. Poloxamer-based gels had a shorter retention time and were associated with less inflammation. Clotrimazole was minimally absorbed. Despite a marked inflammatory response to several of the clotrimazole-containing gels, no notable adverse clinical responses were observed. CONCLUSIONS AND CLINICAL RELEVANCE: Poloxamer gels had the most promise for improving drug contact within the frontal sinus of dogs, while limiting the inflammatory response. Poloxamer gels have the additional benefit of improved handling as a result of reverse gelation (ie, they gel when warmed to 37 degrees C).

Charge compensation for NADPH oxidase activity in microglia in rat brain slices does not involve a proton current.

The membrane properties of isolated cultured microglia have been extensively studied but it is important to understand their properties in situ, where they protect the brain against infection, but also contribute to neurodegenerative diseases. Microglia and macrophages attack bacteria by generating reactive oxygen species, a process which involves NADPH oxidase pumping electrons out across the cell membrane. The resulting inward current evokes a depolarization, which would inhibit the activity of the NADPH oxidase if there were no charge-compensating current which moves positive charge out across the membrane. The mechanism of this charge compensation is controversial. In neutrophils and in cultured microglia a depolarization-activated H(+) conductance has been proposed to provide charge compensation, and also to remove protons generated intracellularly by the NADPH oxidase. Alternatively, a depolarization-activated K(+) conductance has been proposed to mediate charge compensation. Here we show that in microglia, either in the resting state or when activated by the bacterial coat component lipopolysaccharide, both in acute and in cultured hippocampal slices, no significant H(+) current is detectable. This implies that the membrane properties of microglia in their normal cellular environment differ from those of cultured microglia (similarly, microglia generated a current in response to ATP but, unlike in culture, not to glutamate or GABA). Furthermore, the K(+) current (Kv1.3) that is activated by lipopolysaccharide is inactivated by depolarization, making it unsuitable for mediating charge compensation on a long time scale at positive voltages. Instead, charge compensation may be mediated by a previously undescribed non-selective cation current.

Preparation and evaluation of buccal bioadhesive films containing clotrimazole.

Buccal bioadhesive films, releasing topical drugs in the oral cavity at a slow and predetermined rate, provide distinct advantages over traditional dosage forms. The aim of present study was to prepare and evaluate buccal bioadhesive films of clotrimazole for oral candidiasis. The film was designed to release the drug at a concentration above the minimum inhibitory concentration for a prolonged period of time so as to reduce the frequency of administration of the available conventional dosage forms. The different proportions of sodium carboxymethylcellulose and carbopol 974P (CP 974P) were used for the preparation of films. Carbopol was used to incorporate the desired bioadhesiveness in the films. The films were prepared by solvent casting method and evaluated for bioadhesion, in vitro drug release and effectiveness against Candida albicans. In vitro drug release from the film was determined using a modified Franz diffusion cell while bioadhesiveness was evaluated with a modified two-arm balance using rabbit intestinal mucosa as a model tissue. Films containing 5% CP 974P of the total polymer were found to be the best with moderate swelling along with favorable bioadhesion force, residence time and in vitro drug release. The microbiological studies revealed that drug released from the film could inhibit the growth of C. albicans for 6 h. The drug release mechanism was found to follow non-Fickian diffusion.

[Coexpression of PXRLBD with SRC88 and construction of equilibrium dialysis model of screening PXR ligands].

The aim of this study was to obtain the soluble protein of human pregnane X receptor ligand binding domain (PXRLBD) through the coexpression of PXRLBD and 88 amino acids of steroid receptor coactivator-1 (SRC88) and apply the protein to constructing a new model of screening PXR ligands. Expression plasmid of pETDuet-1-SRC88-PXRLBD was constructed and transformed into Escherichia coli Rosetta (DE3) to coexpress PXRLBD and SRC88 via induction by IPTG at low temperature. Then an equilibrium dialysis model was constructed to study the interaction between PXRLBD and drugs including clotrimazole and dexamethasone, using HPLC as the analysis method. The results showed that the soluble protein of PXRLBD was obtained and the HPLC data indicated that clotrimazole bound to PXRLBD, while dexamethasone did not bind to PXRLBD, which indicated the successful establishment of a new method for studying the interaction between PXR and drugs. The new method may be useful in the screening of PXR ligands in vitro.

Silver-loaded graphene as an effective SERS substrate for clotrimazole detection: DFT and spectroscopic studies.

Vibrational infrared, Raman and surface-enhanced Raman scattering (SERS) spectra of clotrimazole (CTZ) were documented and evaluated. Density-functional theory, B3LYP/6-311++G(d,p), approach was implemented to identify the possible conformations, develop the electrostatic potential map, evaluate frontier molecular orbitals and calculate the vibrational spectra of the target compound. The silver-loaded graphene was shown to be an effective SERS substrate for CTZ trace detection. The SERS spectrum showed two enhanced bands at 670 cm-1 and 700 cm-1 which confirmed the absorption of the silver substrate through chlorine and nitrogen atoms. A detection limit as low as 5 nM could be reached with a determination coefficient of 0.9988 using the band at 670 cm-1. The protein-ligand interaction with Secreted Aspartic Proteinase 2 (SAP2) of C. albicans showed that the four stable forms of CTZ maintain a free energy of binding of 6-7 kcal/mol, which could give insights into the mode of action in treating Candidiasis.

Self-assembled nanomicelles of amphiphilic clotrimazole glycyl-glycine analogue augmented drug delivery, apoptosis and restrained melanoma tumour progression.

In present investigation, self-assembled nanomicelles of amphiphilic clotrimazole glycyl-glycine (CLT-GG-SANMs) analogue were customized for augmenting drug delivery, permeability and apoptosis in B16F1 mouse melanoma cancer cells both in vitro and in vivo following intratumoral (i.t.) route of administration. The mean particle size of CLT-GG-SANMs was measured to be 35.9 ±â€¯3.4 nm in addition to zeta-potential of -17.1 ±â€¯3.5 mV. The shape of CLT-GG-SANMs was visualized to be smooth and spherical as like nanoparticles. The critical micellar concentration (CMC) of CLT-GG-SANMs was estimated to be 17 µg/ml using DPH (1,6-diphenyl-1,3,5-hexatriene) as a UV probe. Modification of CLT to CLT-GG-SANMs induced the amorphization in therapeutic moiety. Next, CLT suspension released only 9.7% of the drug within 1 h under dissolution testing and further analysis up to 48 h did not display any remarkable effect on the drug release. On the other hand, CLT-GG-SANMs released 46.2% of the drug significantly (P < 0.01) higher than CLT suspension at 4 h. The IC50 of CLT-GG-SANMs was measured to be 15.1-µM significantly (P < 0.05) lower than CLT suspension (IC50 > 20 µM) in B16F1 cells. Western blotting and histopathological analysis also supported the superior therapeutic efficacy of CLT-GG-SANMs in terms of higher extent of apoptosis, tumour regression and exhibition of strong antioxidant potential against B16F1 cells induced tumour in C57BL6J mice. In conclusion, in vitro and in vivo therapeutic efficacy analysis indicated that CLT-GG-SANMs may be a potential candidate for translating in to a clinically viable product.