Itraconazole

Lanosterol 14α-Demethylase (CYP51/ERG11) Inhibition

Lanosterol 14α-demethylase, also known as CYP51 or ERG11, is a cytochrome P450 enzyme that catalyzes the removal of the 14α-methyl group from lanosterol, a precursor in the ergosterol synthesis pathway. mdpi.comfrontiersin.orguniprot.org This demethylation step is essential for the conversion of lanosterol to ergosterol. drugbank.compatsnap.comnih.gov this compound effectively blocks this enzymatic reaction. nih.gov

Stereoselective Interaction with Fungal Cytochrome P450 Enzymes

This compound is a chiral drug composed of four cis-diastereoisomers. nih.govnih.gov Studies have investigated the stereoselective interactions of these isomers with cytochrome P450 enzymes, including fungal CYP51. nih.gov While this compound is known to inhibit human CYP3A4, its antifungal activity is attributed to its affinity for fungal CYP51. wikipedia.orgnih.gov The selective inhibition of fungal enzymes over mammalian counterparts minimizes toxicity to human cells. patsnap.comnih.gov

Binding Site Characterization (e.g., Heme Iron Coordination)

Azole antifungals, including this compound, inhibit fungal CYP51 by coordinating with the heme iron atom in the enzyme's active site. drugbank.commdpi.comnih.govsld.cu The nucleophilic nitrogen atom of the triazole ring in this compound forms a stable complex with the ferric (Fe3+) ion of the heme group, acting as the sixth ligand. drugbank.comnih.govsld.cuasm.org This coordination disrupts the enzyme's ability to bind its natural substrate, lanosterol, and catalyze the demethylation reaction. nih.govasm.org The side chains of the this compound molecule also interact with hydrophobic residues within the active site and the substrate access channel of fungal CYP51, contributing to the binding affinity and specificity. asm.org

Downstream Effects on Fungal Cell Membrane Homeostasis

The inhibition of CYP51 by this compound leads to a depletion of ergosterol and the accumulation of methylated sterol precursors, profoundly affecting the fungal cell membrane. sonwuapi.com

Disruption of Membrane Structural Integrity and Permeability

Ergosterol is a vital component of the fungal cell membrane, playing a role analogous to cholesterol in mammalian cells. patsnap.commdpi.com It is essential for maintaining membrane structural integrity, fluidity, and permeability. patsnap.comsonwuapi.commdpi.comcreative-biolabs.com By inhibiting ergosterol synthesis, this compound compromises these critical membrane functions. patsnap.comnih.govnih.govfrontiersin.org The resulting deficiency in ergosterol leads to increased membrane permeability, causing leakage of essential cellular components and ions. patsnap.comnih.govresearchgate.net This disruption ultimately contributes to fungal cell death. patsnap.comnih.gov

Accumulation of Toxic 14-Methylated Sterols

The blockade of the 14α-demethylase step by this compound results in the accumulation of 14α-methylated sterol precursors, such as lanosterol, eburicol, and obtusifoliol, within the fungal cell membrane. drugbank.compatsnap.comfrontiersin.orgnih.govpsu.edunih.gov These accumulated methylated sterols are unable to functionally replace ergosterol and are considered toxic to the fungal cell. patsnap.comfrontiersin.orgresearchgate.net Their presence further disrupts membrane structure and function, impairing the activity of membrane-bound enzymes and transport proteins essential for nutrient uptake and waste elimination. patsnap.comnih.govsonwuapi.comresearchgate.net This accumulation of aberrant sterols, in conjunction with ergosterol depletion, creates a multifaceted attack on fungal cell physiology, leading to growth inhibition and cell death. patsnap.comsonwuapi.comfrontiersin.org Studies have shown the accumulation of various 14α-methyl sterols in different fungal species upon this compound exposure. For example, in Sporothrix schenckii and Sporothrix brasiliensis, this compound treatment promoted the accumulation of nine 14α-methyl sterols, including obtusifoliol and eburicol. nih.gov In Candida albicans, this compound treatment led to the accumulation of 3,6-diol, eburicol, lanosterol, and obtusifoliol, among others. nih.gov

Impairment of Membrane-Bound Enzyme Function

The principal mechanism by which this compound disrupts fungal cell function is through the inhibition of lanosterol 14α-demethylase (also known as CYP51), a key enzyme in the ergosterol biosynthesis pathway drugbank.compatsnap.comaocd.orgnih.govdroracle.aisonwuapi.comfda.govasm.orgmdpi.commedchemexpress.comnih.govnih.gov. This fungal cytochrome P450 enzyme is responsible for the demethylation of lanosterol, a crucial step in converting it to ergosterol drugbank.compatsnap.comnih.govsonwuapi.com.

This compound binds to the heme iron in the active site of the fungal CYP51 enzyme, impeding its catalytic activity drugbank.comresearchgate.netnih.gov. This specific interaction prevents the conversion of lanosterol, leading to a depletion of ergosterol within the fungal cell membrane patsnap.comnih.govsonwuapi.comasm.orgnih.gov. Concurrently, this inhibition results in the accumulation of methylated sterol precursors, such as 14-methylated sterols, which are thought to be toxic to the fungal cell drugbank.compatsnap.comnih.govsonwuapi.comasm.orgnih.gov.

The altered sterol composition of the fungal membrane, characterized by reduced ergosterol and increased methylated sterols, compromises its structural integrity and increases its permeability patsnap.comnih.govsonwuapi.com. This disruption extends to the function of various membrane-bound enzymes, which rely on the proper lipid environment provided by ergosterol for optimal activity drugbank.comnih.govsonwuapi.comsquarepharma.com.bd. The impaired function of these enzymes contributes to a cascade of cellular dysfunctions, further inhibiting fungal growth and viability sonwuapi.comsquarepharma.com.bd.

Research findings highlight the potent inhibitory effect of this compound on fungal CYP51. Studies have shown tight binding of this compound to Candida albicans CYP51 (CaCYP51), with low IC50 values indicating significant inhibition of enzyme activity nih.gov.

Table 1: Inhibitory Activity of this compound Against Fungal and Human CYP51 nih.gov

This data illustrates the selective toxicity of this compound, demonstrating a higher potency against fungal CYP51 compared to the human homologue (Δ60HsCYP51), although some interaction with the human enzyme is noted at higher concentrations nih.gov. Molecular dynamics simulations further support the strong binding affinity of this compound to fungal CYP51, driven significantly by hydrophobic interactions within the enzyme's active pocket mdpi.comfrontiersin.org.

Inhibition of Fungal Cytochrome c Oxidative and Peroxidative Enzymes

One proposed secondary mechanism involves the inhibition of fungal cytochrome c oxidative and peroxidative enzymes drugbank.com. This inhibition is suggested to contribute to the disruption of fungal cell membranes drugbank.com. Research indicates that this compound can inhibit the expression of peroxidases, which aligns with the concept of induced oxidative stress in fungal cells treated with antibiotics asm.org. While the precise link between ergosterol biosynthesis inhibition and effects on cytochrome c oxidase is still being explored, some studies suggest a connection, potentially due to the presence of sterols like ergosterol in fungal mitochondrial membranes nih.gov.

Disruption of Membrane-Bound Transport Proteins

This compound is also understood to disrupt the function of various membrane-bound transport proteins in fungi patsnap.comsonwuapi.com. These transporters are essential for critical cellular processes such as nutrient uptake and the efflux of waste products and potentially the antifungal agent itself patsnap.comsonwuapi.com.

Amino Acid Changes and Mutations in ERG11/CYP51 Gene

Point mutations and other genetic alterations in the ERG11 (or CYP51) gene, which encodes the Cyp51p enzyme, can lead to amino acid substitutions in the enzyme. These substitutions can alter the shape and chemical properties of the active site where this compound binds, reducing the drug's ability to inhibit the enzyme effectively.

Several specific amino acid substitutions in Cyp51p have been linked to this compound resistance in various fungal species. For example, mutations at Gly54 (G54) and Met220 (M220) in Aspergillus fumigatus Cyp51A have been associated with reduced susceptibility to this compound. Studies have reported specific substitutions such as G54E, G54K, G54R, G54V, and M220I, M220K, M220T, M220V conferring resistance. In Candida krusei, polymorphisms in the ERG11 gene have been observed in this compound-resistant isolates. While not all mutations in ERG11 directly confer resistance, those that affect the binding affinity of this compound to the enzyme are particularly significant.

Table 1: Examples of Amino Acid Substitutions in Aspergillus fumigatus Cyp51A Associated with this compound Resistance

Overexpression of ERG11/CYP51 (Lanosterol 14α-Demethylase)

Another significant mechanism of resistance involves the overexpression of the ERG11/CYP51 gene, leading to an increased production of the Cyp51p enzyme. With a higher intracellular concentration of the target enzyme, a greater amount of this compound is required to achieve the same level of inhibition of ergosterol synthesis. This effectively raises the minimum inhibitory concentration (MIC) of the drug. Overexpression of ERG11 has been reported in this compound-resistant clinical isolates of various Candida species, including Candida dubliniensis and Candida krusei. In Aspergillus fumigatus, increased expression levels of both CYP51A and CYP51B have been observed in some resistant strains. Overexpression can be a consequence of various genetic events, including the presence of a tandem repeat in the promoter region of the gene, as seen with the TR/L98H mutation in A. fumigatus.

Table 2: Examples of Fungal Species and Genes Involved in Target Overexpression Leading to this compound Resistance

Gene Duplication of Target Enzymes (e.g., Mfcyp51A2 in Madurella fahalii)

Gene duplication, particularly of the CYP51 gene, can also lead to increased resistance by effectively increasing the gene copy number and thus the amount of target enzyme produced. This is a form of target overexpression. A significant example of this mechanism has been observed in Madurella fahalii, a fungal species known for its intrinsic resistance to this compound. Research has shown that M. fahalii possesses an additional copy of the CYP51 gene, designated Mfcyp51A2, in addition to the conserved Mfcyp51A1. Both genes are actively transcribed and upregulated in response to this compound exposure. Heterologous expression studies in Saccharomyces cerevisiae have demonstrated that the presence of Mfcyp51A2 leads to reduced susceptibility to this compound compared to strains with only Mfcyp51A1. This gene duplication and the functional differences in the encoded enzyme contribute significantly to the observed this compound resistance in M. fahalii. Gene duplication of cyp51A has also been potentially linked to this compound and posaconazole resistance in an Aspergillus fumigatus isolate.

Table 4: Example of Gene Duplication Leading to this compound Resistance

Reduced Intracellular Accumulation of this compound

A significant mechanism of fungal resistance to this compound is the reduction of intracellular drug accumulation. nih.govoup.comencyclopedia.pubmdpi.com This is often mediated by the overexpression of efflux pumps located in the fungal cell membrane. nih.govoup.comencyclopedia.pubmdpi.comresearchgate.net These pumps actively transport the antifungal drug out of the cell, thereby lowering its concentration at the site of action, the Erg11 enzyme. nih.govoup.com

In Candida species, two main superfamilies of efflux pumps are involved: the ATP-binding cassette (ABC) transporters and the Major Facilitator Superfamily (MFS) transporters. nih.govoup.combiomedpharmajournal.org Genes encoding these pumps, such as CDR1, CDR2 (ABC transporters), and MDR1 (MFS transporter), have been shown to be upregulated in azole-resistant Candida albicans isolates. oup.comnih.gov Overexpression of CDR1 and CDR2 is particularly associated with resistance to a broad range of azoles, including this compound, while MDR1 overexpression primarily confers resistance to fluconazole. nih.govoup.comasm.org Studies have demonstrated that increased expression of CDR1 and CDR2 leads to enhanced drug efflux and reduced azole accumulation within the fungal cell. nih.gov

In Aspergillus fumigatus, decreased intracellular accumulation of this compound has also been observed in resistant strains, linked to the overexpression of MDR efflux transporter genes from both the ABC and MFS classes. nih.govasm.orgresearchgate.net Research indicates that azole-resistant A. fumigatus strains can exhibit a significant increase in efflux pump expression compared to susceptible strains. researchgate.net

Altered Membrane Properties Affecting Efflux

Alterations in fungal membrane properties can indirectly affect drug efflux and contribute to this compound resistance. Changes in membrane composition, particularly in the sterol content, can influence the activity and localization of efflux pumps. encyclopedia.pubmdpi.comnih.gov

While azoles target ergosterol synthesis, mutations in the ergosterol biosynthesis pathway, such as in the ERG3 gene encoding sterol C5,6-desaturase, can lead to the accumulation of aberrant sterols and a reduction in ergosterol. asm.orgnih.govmdpi.comnih.gov These changes in membrane sterol composition can affect membrane fluidity and the proper functioning of efflux pumps, potentially leading to altered drug transport and reduced intracellular accumulation of this compound. encyclopedia.pubmdpi.comnih.gov For instance, studies in Candida dubliniensis have suggested that altered membrane properties, consistent with a defective sterol C5,6-desaturase, were associated with decreased rhodamine 6G efflux, a marker for efflux pump activity, in this compound-resistant derivatives. nih.gov

Solid Lipid Nanoparticles (SLNs) Development and Evaluation

Solid Lipid Nanoparticles (SLNs) are colloidal carriers composed of a solid lipid matrix at room and body temperature. researchgate.net They have been investigated for this compound delivery to improve its solubility and stability. impactfactor.orgresearchgate.net The development and evaluation of this compound-loaded SLNs involve careful selection of lipid matrices and optimization of carrier characteristics. nih.govresearchgate.net

Lipid Matrix Selection and Carrier Optimization

The selection of the lipid matrix is crucial for the performance of this compound-loaded SLNs. Lipids such as palmitic acid and stearic acid have been explored. nih.govimpactfactor.orgresearchgate.net The solubility of this compound in various solid lipids is examined to identify suitable candidates. impactfactor.org For instance, stearic acid showed a stronger affinity for this compound compared to palmitic acid, indicated by partition coefficient studies. impactfactor.orgresearchgate.net

Carrier optimization involves varying factors such as the drug-lipid ratio and the ratio of surfactant to co-surfactant (Km ratio) to achieve desired formulation characteristics like maximized drug loading, modulated release, and minimized particle size. nih.govresearchgate.net Studies have shown that optimizing the ratio of excipients can lead to high drug loading (around 80%) and a narrow size distribution. nih.govresearchgate.net For example, optimized formulations have been achieved with a lipid-drug ratio of 1.5:1 and a Km ratio of 1:2. nih.govresearchgate.net

Impact on Solubility and Stability

This compound's low water solubility is a limiting factor in drug development. impactfactor.org Incorporating this compound into SLNs, where it is embedded in a lipid matrix, has been shown to improve its solubility and stability. impactfactor.orgresearchgate.net The small particle size of SLNs increases the surface area, which contributes to enhanced dissolution. impactfactor.org

Studies evaluating this compound-loaded SLNs have demonstrated improved solubility and extended-release profiles compared to conventional formulations. nih.govresearchgate.net Physicochemical stability studies at different temperatures have also indicated the stability of optimized SLN formulations over time. nih.govresearchgate.net The crystalline nature of this compound can be assessed using techniques like Differential Scanning Calorimetry (DSC) and X-Ray Diffraction (XRD) in both the raw drug and the SLN formulations to understand the impact of encapsulation on its solid state. nih.govimpactfactor.orgresearchgate.net

Nanocrystals and Nanosuspensions

Nanocrystals and nanosuspensions represent another approach to address the solubility challenges of poorly water-soluble drugs like this compound. researchgate.net These systems consist of drug particles reduced to the nanoscale, typically below one micrometer. This size reduction leads to a significant increase in surface area, which enhances the dissolution rate and saturation solubility of the drug. nih.gov

This compound nanosuspensions have been prepared using techniques such as wet pearl milling and microprecipitation-high-pressure homogenization. nih.gov Stabilizers like Poloxamer F127, Poloxamer 407, cholic acid, hydroxypropyl cellulose (HPC-SL), sodium dodecyl sulfate (SDS), and polysorbate 80 (PS80) are used to prevent particle aggregation and maintain stability. nih.govnih.govacs.org

Evaluation of this compound nanosuspensions includes particle size analysis, polydispersity index (PDI), zeta potential measurements, and solid-state characterization using techniques like DSC and XRD. nih.govnih.gov Studies have reported obtaining this compound nanoparticles with sizes in the range of approximately 225.7 nm to 369 nm with low PDI values, indicating a narrow size distribution. nih.gov DSC and XRD analyses have confirmed that this compound often remains in its crystalline state within the nanocrystals, although some studies suggest conversion to an amorphous form within a solid matrix upon lyophilization. nih.govresearchgate.net

In vitro dissolution studies have shown that this compound nanosuspensions exhibit significantly higher dissolution rates compared to the bulk drug and even marketed formulations, primarily due to the increased surface area. nih.gov

Integration into Hydrogels and Other Systems

This compound nanocrystals and nanosuspensions can be integrated into various drug delivery systems, including hydrogels, to create advanced formulations for different routes of administration. researchgate.net Hydrogels, due to their biocompatibility and ability to provide a sustained release, are suitable vehicles for incorporating nanocrystals. researchgate.net

For example, this compound nanocrystals have been successfully integrated into hydrogel contact lenses for ophthalmic drug delivery. acs.orgresearchgate.net This approach aims to increase the precorneal residence time of the drug. acs.orgresearchgate.net In these formulations, nanocrystals stabilized with agents like Poloxamer 407 were prepared and then printed onto hydrogel contact lenses. acs.orgresearchgate.net The resulting formulations demonstrated a dual drug release profile in simulated tear fluid. acs.orgresearchgate.net

Furthermore, this compound nanosuspensions have been incorporated into hydrogel formulations for topical administration, such as nanogels using Carbopol 940 as a gelling agent. pharmascigroup.us These formulations are hypothesized to treat fungal infections topically, potentially avoiding systemic side effects associated with oral administration. pharmascigroup.us Nanoemulsion-based hydrogels of this compound have also been developed for transdermal drug delivery. researchgate.net

Self-Nano Emulsifying Drug Delivery Systems (SNEDDS) Formulation

The formulation of this compound SNEDDS involves the careful selection and optimization of various excipients to ensure the formation of a stable and effective nanoemulsion upon dilution. This process typically involves identifying suitable oil phases, surfactants, and co-surfactants that can efficiently solubilize this compound and promote spontaneous emulsification. impactfactor.orgjneonatalsurg.comresearchgate.netjazindia.comjazindia.comijcrt.org

Optimization of Excipients (Oil Phase, Surfactant, Co-Surfactant)

The selection of the oil phase is crucial as it needs to effectively solubilize this compound. Studies have investigated the solubility of this compound in various oils. For instance, high solubility was observed in anise oil (235.18 ± 8.56 mg/gm) and clove oil (220.58 ± 5.45 mg/gm) compared to other oils like sesame pure oil, olive oil, isopropyl myristate, Capmul MCM C8 NF, and Capmul PG 8 NF. impactfactor.org Another study indicated high solubility in Capmul MCM (8.7 mg/ml). jneonatalsurg.com

Surfactants play a critical role in reducing the interfacial tension between the oil and water phases, facilitating the formation of a nanoemulsion. The choice of surfactant impacts the emulsification efficiency, the self-nanoemulsification region, and the resulting droplet size. globalresearchonline.net Co-surfactants are often included to further reduce interfacial tension and improve the flexibility of the interfacial film, leading to a larger nanoemulsion region in the phase diagram. globalresearchonline.net Common surfactants and co-surfactants explored for this compound SNEDDS include Tween 20, Tween 80, Kolliphor CS 20, PEG 400, and PEG 200. impactfactor.orgjneonatalsurg.comresearchgate.netjazindia.comjazindia.comijcrt.orgresearchgate.netinsightsjhr.com The ratio of surfactant to co-surfactant (Smix) is also optimized to achieve the desired nanoemulsion characteristics. jneonatalsurg.comrjptonline.org

Research findings highlight the importance of solubility studies in selecting appropriate excipients. Table 1 presents some examples of this compound solubility in different oils based on reported studies.

Similarly, the solubility of this compound in various surfactants and co-surfactants is evaluated to select the most effective ones. PEG 200, for instance, demonstrated high solubility among co-surfactants (31.04 ± 0.88 mg/mL). researchgate.net

Pseudo-Ternary Phase Diagram Approach

Pseudo-ternary phase diagrams are indispensable tools in the development of SNEDDS. They are constructed to identify the concentration ranges of oil, surfactant, and co-surfactant (and sometimes water) that result in the formation of a clear, monophasic nanoemulsion upon aqueous dilution. impactfactor.orgjneonatalsurg.comresearchgate.netjazindia.comjazindia.comresearchgate.netinsightsjhr.comrjptonline.org By systematically varying the ratios of the components, researchers can map the nanoemulsion region, which represents the compositions that will spontaneously self-emulsify into a nano-sized dispersion. impactfactor.orgjneonatalsurg.comrjptonline.org

The phase diagrams help in optimizing the component concentrations to achieve a wide nanoemulsion region, indicating robust self-emulsifying efficiency. impactfactor.orgjneonatalsurg.comrjptonline.org Different surfactant to co-surfactant ratios are often investigated to determine their impact on the microemulsion area. impactfactor.org The optimal component concentrations for SNEDDS are determined based on the extent of the emulsification area generated by each system. impactfactor.org

Particle Size and Droplet Size Analysis

Particle size, or droplet size in the case of nanoemulsions, is a critical parameter that influences drug absorption and bioavailability. Smaller droplet sizes lead to a larger surface area, which can enhance the dissolution and absorption of poorly soluble drugs like this compound. globalresearchonline.net Techniques such as photon correlation spectroscopy and Zeta sizer are commonly used to measure the droplet size distribution of the reconstituted nanoemulsion. globalresearchonline.netjneonatalsurg.comresearchgate.netjazindia.comjazindia.comijcrt.orgresearchgate.netjneonatalsurg.comnih.gov The polydispersity index (PDI) is often reported alongside the mean droplet size to indicate the uniformity of the droplet size distribution. A low PDI suggests a narrow size distribution. jneonatalsurg.comjneonatalsurg.com

Reported studies show varying droplet sizes for this compound SNEDDS formulations, typically in the nanometer range. For example, an optimized solid SNEDDS formulation (SF1) showed a mean particle size of 95.2 nm with a PDI of 0.115. jneonatalsurg.comjneonatalsurg.com Other formulations have shown droplet sizes ranging from 120 nm to 505 nm. researchgate.netjazindia.comjazindia.com Polymeric nanoparticles encapsulating this compound have also shown particle sizes, such as 265 ± 5.8 nm for ITZ-loaded TPP NPs. nih.gov

Drug Entrapment Efficacy and Release Profiles

Drug entrapment efficacy (EE) quantifies the amount of drug successfully incorporated into the delivery system. For SNEDDS, this refers to the amount of this compound solubilized within the oil phase and the formed nanoemulsion droplets. High entrapment efficiency is desirable to ensure consistent dosing and minimize drug loss during formulation. Studies report high entrapment efficiency for optimized this compound SNEDDS formulations. For instance, an optimized solid SNEDDS formulation (SF1) showed an entrapment efficiency of 89.56% ± 0.12. jneonatalsurg.comjneonatalsurg.com Polymeric nanoparticles encapsulating this compound have also demonstrated high entrapment efficiency, such as 95% for ITZ-loaded TPP NPs. nih.gov

In vitro drug release studies are conducted to evaluate the rate and extent of this compound release from the advanced delivery systems in a simulated physiological environment. These studies provide insights into how the formulation might perform in vivo. The release profiles of this compound from SNEDDS and nanoparticles are often compared to that of the pure drug or conventional formulations to demonstrate improved dissolution and release characteristics. jneonatalsurg.comresearchgate.netinsightsjhr.comjneonatalsurg.comnih.govafjbs.com Optimized SNEDDS formulations have shown significantly improved dissolution profiles and faster drug release compared to conventional formulations. researchgate.netinsightsjhr.com For example, one optimized SNEDDS formulation achieved complete dissolution in 180 minutes, while a commercial capsule released only 67% of the drug after 600 minutes. researchgate.netinsightsjhr.com Polymeric nanoparticles have also shown extended drug release profiles. nih.gov

Table 2 summarizes some reported data on entrapment efficiency and drug release for this compound advanced delivery systems.

These characterization and evaluation methodologies are essential for ensuring the quality, stability, and performance of this compound advanced drug delivery systems, paving the way for potentially improved therapeutic outcomes.

Viscosity and Robustness to Dilution Assessments

Viscosity and robustness to dilution are critical parameters for evaluating the physical stability and performance of liquid or semi-solid advanced drug delivery systems containing this compound, such as nanoemulsions, microemulsions, and gels. impactfactor.orgijcrt.orgijprajournal.comnih.govpharmascigroup.usnih.govresearchgate.netmdpi.com These assessments provide insights into the formulation's ability to maintain its structural integrity and drug solubilization capacity upon administration and subsequent dilution in biological fluids. nih.govresearchgate.net

Studies on this compound-loaded self-nanoemulsifying drug delivery systems (SNEDDS) have included robustness to dilution assessments to confirm the absence of phase separation or drug precipitation upon dilution with aqueous media. researchgate.netresearchgate.netresearchgate.net For instance, one study evaluated the robustness of SNEDDS formulations upon 1000-fold dilution, confirming no phase separation. researchgate.net Another investigation into this compound-comprising SMEDDS assessed the impact of dilution volume and the pH of the dilution medium on physical integrity and drug solubilization capacity. researchgate.netimpactfactor.org

For microemulsion-based formulations, robustness of dilution tests are performed to screen self-dispersed emulsions. nih.gov An emulsion size exceeding a certain threshold (e.g., 200 nm) or the appearance of phase separation can lead to the exclusion of a formulation from further study. nih.gov Dilution with media like artificial saliva is used to simulate physiological conditions. nih.gov

In the context of topical nanogels containing this compound nanoparticles, viscosity is a key parameter evaluated for stability. pharmascigroup.us The viscosity of the nanogel formulation is assessed over time under different storage conditions to ensure it remains within an acceptable range, indicating physical stability. pharmascigroup.us Similarly, the viscosity of nanoemulsion intermediate gels of this compound has been evaluated, with a selected system showing a viscosity of 1583.47 cp. nih.gov

Research into solid lipid nanoparticles (SLNs) for this compound delivery has also considered viscosity, particularly in the context of optimizing formulations for sustained drug delivery. impactfactor.org The viscosity of SLN formulations can influence drug absorption and bioavailability. impactfactor.org

Data from a study on this compound-loaded microemulsion gel formulations illustrates the evaluation of viscosity and robustness to dilution for different formulations. nih.gov

Note: Data extracted from a study on sustained release gel of this compound-loaded microemulsion for oral candidiasis treatment. nih.gov

Spectroscopic Analysis (e.g., FTIR Spectroscopy) for Excipient Compatibility

Fourier Transform Infrared (FTIR) spectroscopy is a widely used technique to assess the chemical compatibility between this compound and the various excipients used in advanced drug delivery systems. researchgate.netjddtonline.infojpsionline.comscispace.comglobalresearchonline.netijprajournal.cominnovareacademics.inresearchgate.netnih.govacs.orgresearchgate.netsphinxsai.comnih.govscienceopen.comresearchgate.net This analysis helps to detect any potential interactions, such as shifts in characteristic peaks or the appearance of new peaks, which could indicate chemical reactions or complex formation between the drug and the excipients, potentially affecting the stability and performance of the formulation. globalresearchonline.netijprajournal.cominnovareacademics.inresearchgate.net

Numerous studies on this compound formulations have employed FTIR spectroscopy to confirm drug-excipient compatibility. researchgate.netjddtonline.infojpsionline.comscispace.comglobalresearchonline.netijprajournal.cominnovareacademics.innih.govsphinxsai.comnih.govresearchgate.net For example, FTIR spectroscopy confirmed compatibility between this compound and formulation excipients in self-nanoemulsifying drug delivery systems. researchgate.net In the development of this compound ocular in-situ gels, FTIR studies indicated no interaction between this compound and polymers such as hydroxypropyl-beta-cyclodextrin (HPBCD), Carbopol, hydroxypropyl methylcellulose (HPMC), and Benzalkonium chloride, with major peaks of the drug remaining almost at the same wavenumbers. globalresearchonline.net Similar findings of compatibility have been reported in studies involving this compound transdermal patches, solid dispersions with various carriers (e.g., porous calcium silicate, skimmed milk, Eudragit E100, weak organic acids), and nanoemulsions. jpsionline.comscispace.cominnovareacademics.inresearchgate.netsphinxsai.comnih.gov The retention of characteristic peaks of this compound in the presence of excipients is a key indicator of compatibility. ijprajournal.comnih.govsphinxsai.com

FTIR analysis has also been used to investigate changes in the molecular arrangement of this compound–polymer solid dispersions, for instance, examining the disruption in hydrogen bonding upon compression. acs.org While FTIR primarily confirms the absence of significant chemical interactions, it can also provide insights into the physical state of the drug, such as the amorphization of this compound in solid dispersions, although this is often complemented by techniques like Powder X-ray Diffraction (PXRD) and Differential Scanning Calorimetry (DSC). scispace.comsphinxsai.comnih.gov

A study on this compound solid dispersions prepared with weak organic acids used ATR-FTIR (Attenuated Total Reflectance-FTIR) and observed possible weak interactions, such as hydrogen bonding, between the drug and the acid, but no salt formation was indicated. nih.gov

Inhibition of Human Cytochrome P450 Isozymes

This compound is known to inhibit several human CYP isozymes. nih.govmedsafe.govt.nz

Potent Inhibition of CYP3A4 and CYP3A5

This compound is recognized as a potent inhibitor of CYP3A4, the major drug-metabolizing enzyme in humans. tandfonline.comnih.govnih.govresearchgate.netmedsafe.govt.nzmdpi.comselleckchem.comresearchgate.net This inhibition is a significant factor in the drug-drug interactions associated with this compound. tandfonline.comnih.govacs.org Studies using recombinant CYP3A4 and CYP3A5, as well as human liver microsomes, have demonstrated that this compound inhibits both isoforms. nih.govresearchgate.net The inhibitory potency can vary depending on the substrate used in the assay. nih.gov For instance, IC50 ratios between recombinant CYP3A5 and CYP3A4 for this compound were reported as 8.8 for midazolam and 9.1 for testosterone, suggesting substrate-dependent selectivity between these two isoforms. nih.gov

Inhibition of Other CYPs (e.g., CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1)

Beyond its potent effects on CYP3A4 and CYP3A5, this compound has also been investigated for its inhibitory effects on other CYP isoforms. Studies examining the inhibitory potencies of this compound's individual optical isomers towards nine drug-metabolizing cytochrome P450 enzymes, including CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1, have been conducted. nih.govmedsafe.govt.nz While potent inhibition is primarily observed for CYP3A, moderate inhibition of CYP2C19 has also been noted, albeit without large differences between the individual optical isomers. nih.govnih.gov Inhibition of other listed CYPs by this compound is generally less pronounced compared to its effect on CYP3A isoforms. tandfonline.com

Stereoselective Inhibition of Cytochrome P450s by this compound Diastereoisomers

This compound is a chiral drug composed of four cis-diastereoisomers: (+)-2R,4S,2′R-ITZ-A, (+)-2R,4S,2′S-ITZ-B, (−)-2S,4R,2′S-ITZ-C, and (−)-2S,4R,2′R-ITZ-D. tandfonline.comresearchgate.netnih.gov These diastereoisomers can exhibit different pharmacokinetic and pharmacodynamic properties, including their inhibitory effects on CYP enzymes. tandfonline.comnih.gov All four cis-diastereoisomers have been shown to inhibit CYP3A activity dose-dependently. tandfonline.comnih.gov Significant differences in inhibitory potencies towards CYP3A have been observed among the individual diastereoisomers. tandfonline.comnih.gov For example, studies using testosterone hydroxylation as a probe reaction for CYP3A activity reported varying Ki values for the diastereoisomers. tandfonline.comnih.gov Similarly, when using midazolam hydroxylation, different Ki values were determined for each isomer. tandfonline.comnih.gov This stereoselective inhibition contributes to the complex pharmacokinetic profile of this compound, which is administered as a mixture of these stereoisomers. tandfonline.comscispace.compsu.edu

Inhibition Constant (Ki) Determination Methodologies

The inhibitory potency of this compound and its diastereoisomers on CYP enzymes is commonly quantified by determining the inhibition constant (Ki) or the half-maximal inhibitory concentration (IC50). tandfonline.comnih.govselleckchem.comnih.gov Various in vitro methodologies are employed for this purpose, often utilizing human liver microsomes or recombinant CYP enzymes. tandfonline.comnih.govnih.govmdpi.comnih.gov Probe substrates specific to each CYP isoform are used, and the inhibition is assessed by measuring the decrease in the formation rate of the substrate's metabolite in the presence of varying concentrations of the inhibitor. tandfonline.comnih.govmdpi.comnih.govfda.gov

For instance, midazolam hydroxylation is frequently used as a probe reaction for CYP3A4 activity when determining this compound's inhibitory constants. tandfonline.comnih.govnih.govpsu.edufda.govabcam.com Testosterone hydroxylation is another commonly used probe for CYP3A activity. tandfonline.comnih.govfda.gov The determination of Ki values often involves analyzing the enzyme kinetics data using models such as the Lineweaver-Burk plot, which can help establish the mechanism of inhibition (e.g., competitive, non-competitive, mixed). tandfonline.comfrontiersin.org Studies have reported this compound exhibiting mixed-type inhibition of CYP3A4, incorporating both non-competitive and competitive components. frontiersin.org

Reported Ki and IC50 values for this compound and its diastereoisomers against CYP3A4 vary depending on the study, the probe substrate used, and the experimental conditions. For example, IC50 values for the inhibition of CYP3A4-catalyzed midazolam hydroxylation by the four cis-diastereoisomers have been reported in the nanomolar range. researchgate.netpsu.edu Ki values for the individual diastereoisomers inhibiting CYP3A activity, using testosterone and midazolam as substrates, have also been determined, highlighting the stereoselective nature of this inhibition. tandfonline.comnih.gov

Role of CYP3A4 in this compound Metabolism

This compound is primarily metabolized in the liver by the cytochrome P450 system, with CYP3A4 being the main enzyme responsible for its metabolism. tandfonline.comnih.govnih.govselleckchem.comnih.govamazonaws.comresearchgate.net This metabolism leads to the formation of several metabolites, including hydroxy-itraconazole (hydroxy-ITZ), which is an active metabolite, and other inactive metabolites. tandfonline.comnih.govscispace.comamazonaws.comresearchgate.net The metabolism of this compound by CYP3A4 is subject to stereoselectivity. nih.govnih.govresearchgate.netscispace.compsu.edu Specifically, only two of the four cis-diastereoisomers, (+)-2R,4S,2′R-ITZ-A and (+)-2R,4S,2′S-ITZ-B, are significantly metabolized by CYP3A4 to hydroxy-ITZ, keto-itraconazole (keto-ITZ), and N-desalkyl-itraconazole (N-desalkyl-ITZ). tandfonline.comnih.govnih.govresearchgate.netscispace.compsu.eduresearchgate.net The other two diastereoisomers, (−)-2S,4R,2′S-ITZ-C and (−)-2S,4R,2′R-ITZ-D, are not metabolized to these products by CYP3A4. tandfonline.comresearchgate.netpsu.eduresearchgate.net

Contribution of this compound Metabolites to CYP3A4 Inhibition (e.g., Hydroxythis compound, Keto-itraconazole, N-Desalkylthis compound)

This compound is metabolized by the CYP3A4 enzymatic system to form primarily three active metabolites: hydroxythis compound, keto-itraconazole, and N-desalkylthis compound. globalresearchonline.netinnovareacademics.in Hydroxythis compound is considered the main metabolite and is also biologically active, often circulating at higher plasma concentrations than the parent drug at steady state. innovareacademics.inumich.edu Both this compound and its metabolites, including hydroxythis compound, keto-itraconazole, and N-desalkylthis compound, are potent inhibitors of the CYP3A4 isozyme. globalresearchonline.netumich.edu

Studies have demonstrated that these metabolites can contribute substantially to the inhibition of CYP3A4 observed clinically after this compound administration. umich.eduresearchgate.net For instance, in vitro studies using human liver microsomes have shown that hydroxythis compound, keto-itraconazole, and N-desalkylthis compound exhibit unbound IC50 values for CYP3A4 inhibition that are comparable to or even lower (more potent) than that of this compound itself. researchgate.net Specifically, unbound IC50 values when coincubated with human liver microsomes and midazolam (as a substrate) were reported as 6.1 nM for this compound, 4.6 nM for hydroxythis compound, 7.0 nM for keto-itraconazole, and 0.4 nM for N-desalkylthis compound. researchgate.net This indicates that these metabolites are as potent as or more potent CYP3A4 inhibitors than the parent compound. researchgate.net

In vitro Models for P450 Inhibition Studies (e.g., Human Liver Microsomes, Hepatocytes)

In vitro studies investigating the effects of drugs on CYP enzymes, including the inhibition by this compound and its metabolites, commonly utilize human liver microsomes and human hepatocytes. ecancer.orgresearchgate.netfrontiersin.org Human liver microsomes (HLM) are a widely used model system as they contain a high concentration of CYP enzymes. frontiersin.org Studies using HLM have been instrumental in determining the inhibitory potential (e.g., IC50 and Ki values) of this compound and its metabolites against CYP3A4 and other CYP isoforms. researchgate.netnih.gov

Human hepatocytes, which are intact liver cells, represent a more complex and potentially more physiologically relevant model compared to microsomes as they retain cellular structures and metabolic pathways. researchgate.netfrontiersin.org They are also used to assess drug metabolism and enzyme inhibition. researchgate.netfrontiersin.orgnih.gov Comparisons between HLM and hepatocyte models have shown similarities in the inhibition profiles of certain CYP enzymes, although differences can exist, potentially due to factors like transmembrane transport in the cellular model. frontiersin.org Recombinant CYP enzymes are also utilized to study the interaction of specific isoforms with inhibitors like this compound. ecancer.orgnih.govd-nb.info

P-Glycoprotein (P-gp) Inhibition and Substrate Activity

This compound is a potent inhibitor of P-glycoprotein (P-gp), an important efflux transporter also known as MDR1 or ABCB1. ecancer.orgresearchgate.netnih.govumaryland.eduscispace.comasm.org P-gp is located in various tissues, including the intestine, liver, kidney, and blood-brain barrier, and actively pumps many structurally unrelated lipophilic and amphiphilic xenobiotics out of cells. umaryland.eduasm.orgasm.org

In vitro studies have demonstrated that this compound can inhibit P-gp function in cell lines overexpressing the transporter. ecancer.orgasm.org For example, in a cell line with overexpressed human P-gp, this compound inhibited P-gp function with an IC50 of approximately 2 μM. ecancer.orgasm.org This inhibition can lead to increased intracellular concentrations of P-gp substrate drugs. spandidos-publications.com

This compound has also been identified as a substrate of P-gp. umich.eduasm.org Studies have shown that P-gp can transport this compound, and this transporter may play a role in the efflux of this compound from certain tissues, such as the brain. asm.org The dual capacity of this compound as both an inhibitor and a substrate of P-gp contributes to its complex pharmacokinetic interactions. asm.org Clinically, coadministration of this compound has been shown to increase the plasma concentrations of P-gp substrates, likely due to reduced efflux activity. researchgate.netumaryland.edunih.gov

Breast Cancer Resistance Protein (BCRP) Inhibition

In addition to P-gp, this compound has also been shown to inhibit the Breast Cancer Resistance Protein (BCRP), also known as ABCG2. ecancer.orgresearchgate.netnih.govscispace.comnih.gov BCRP is another efflux transporter that contributes to multidrug resistance and influences the disposition of various drugs. ecancer.orgnih.gov

In vitro studies using BCRP-overexpressing cells have demonstrated that this compound can significantly inhibit BCRP-mediated efflux of substrate markers at low micromolar concentrations. ecancer.orgnih.gov This inhibition can reverse BCRP-mediated resistance to certain cytotoxic agents in cell models. ecancer.orgnih.gov While this compound is an inhibitor of BCRP, studies have also indicated that it is not transported by BCRP. nih.gov The inhibition of BCRP by this compound can impact the pharmacokinetics of coadministered drugs that are substrates for this transporter. nih.gov

In vitro Characterization of Serum Protein Binding

This compound is highly bound to serum proteins. In vitro studies have consistently shown that this compound exhibits very high protein binding, typically reported as greater than 99%. spandidos-publications.commdpi.comdrugbank.comnih.govnih.govresearchgate.netresearchgate.net This extensive binding is primarily to albumin and alpha 1-acid glycoprotein (AAG).

Studies investigating the binding of this compound to human serum albumin (HSA) and bovine serum albumin (BSA) have utilized spectroscopic methods, such as fluorescence and UV-Vis spectroscopies, to characterize the interaction. These studies suggest that the binding involves interactions like electrostatic and hydrophobic forces. The binding constant values indicate a strong affinity between this compound and serum albumin.

While protein binding is generally high, variations in serum protein levels, such as decreased albumin or elevated AAG levels observed in certain patient populations (e.g., cancer patients or those with diabetes mellitus), can potentially influence the unbound fraction of this compound. Studies have shown that the unbound percentage of this compound can be higher in patients with certain conditions compared to healthy volunteers, and correlations between this compound protein binding and albumin or free fatty acid concentrations have been observed. Despite the high protein binding, some in vitro studies suggest that the antifungal activity of this compound may not be diminished as significantly as might be predicted based solely on the free drug hypothesis, particularly in the presence of albumin. drugbank.comnih.gov

Here is a summary of some research findings related to this compound interactions:

Quantification of Unbound Fraction of this compound and Metabolites

The unbound fraction of a drug in plasma is considered pharmacologically active, as only the unbound drug can readily distribute into tissues and interact with its target. researchgate.netbg.ac.rs Determining the unbound fraction of this compound and its metabolites is crucial for understanding their pharmacokinetics and pharmacodynamics.

Studies have employed methods such as ultracentrifugation and equilibrium dialysis to quantify the unbound fractions. Due to potential non-specific binding to filters and membranes, ultracentrifugation has been used to overcome these complications in measuring the free fraction of this compound and its metabolites in plasma. nih.gov

Research indicates that this compound is highly protein-bound in plasma, with reported binding exceeding 99%. mims.combg.ac.rsnih.govasm.orgdrugbank.com Its major metabolite, hydroxythis compound, also exhibits high protein binding, reported at 99.6%. mims.com

However, some studies using methods like ultracentrifugation have reported a higher unbound fraction for this compound than previously indicated. One study measured the free fraction of this compound in plasma to be 3.6% ± 0.3%. nih.gov The unbound fractions for its metabolites were also quantified: hydroxythis compound (OH-ITZ) at 0.5% ± 0.2%, keto-itraconazole (keto-ITZ) at 5.3% ± 0.7%, and N-desalkyl-itraconazole (ND-ITZ) at 1.2% ± 0.2%. nih.gov These unbound fractions were found to be independent of the total concentration within the tested range. nih.gov

The unbound fractions of the metabolites are approximately 5-fold to 10-fold higher than that of this compound, which is consistent with decreasing lipophilicity. researchgate.net

Influence of Serum Components (e.g., Albumin, Fatty Acids, Globulins) on Binding and Antifungal Activity

Serum protein binding significantly influences the distribution and activity of antifungal agents like this compound. bg.ac.rsnih.gov this compound is primarily bound to albumin in plasma. mims.comdrugbank.com

Studies have investigated the impact of serum components, including albumin, fatty acids, and globulins, on this compound's binding and antifungal activity. While intense albumin binding might be expected to diminish antifungal activity based on the free-drug hypothesis, studies exposing Candida albicans to this compound serum concentration-time profiles showed that antifungal activity was not reduced despite significant albumin binding. nih.govasm.orgdrugbank.com

In vitro susceptibility testing of C. albicans demonstrated that this compound activity was not diminished by the presence of albumin, contrary to the expected reduction based on its high protein binding. nih.govasm.orgdrugbank.com The expected ratio of IC30 (inhibitory concentration 30%) with and without serum protein was significantly higher than the observed ratio for this compound in the presence of albumin. nih.govasm.orgdrugbank.com

However, the influence of other serum components has also been examined. Alpha-globulin, but not gamma-globulin, induced a major loss in the anti-Candida activity of this compound in some studies. nih.govdrugbank.com The effect of plasma on this compound activity paralleled the decline observed with alpha-globulin. nih.govdrugbank.com

Furthermore, the protein binding of this compound can be affected by physiological conditions. In patients with insulin-dependent and non-insulin-dependent diabetes mellitus, the unbound percentage of this compound in serum was found to be significantly higher compared to healthy volunteers. researchgate.netpsu.edunih.gov This increase in the unbound fraction in diabetic patients showed a negative correlation with albumin concentration and a positive correlation with free fatty acid concentration. researchgate.netpsu.edunih.govoup.com Elevated free fatty acid concentrations, which can occur in diabetic patients, may contribute to the observed increase in the unbound fraction of this compound. oup.com

Significance of Triazole Moiety for Ergosterol Biosynthesis Inhibition

The triazole moiety stands out as a critical structural feature essential for this compound's antifungal potency. acs.orgnih.govdrugbank.comnih.govmdpi.comnih.govnih.govnih.govgoogle.comnih.gov The nitrogen atoms within the triazole ring are capable of coordinating with the heme iron situated in the active site of fungal CYP51. drugbank.comnih.gov This coordination effectively blocks the binding of molecular oxygen, which is a necessary cofactor for the demethylation of lanosterol, a pivotal step in the ergosterol biosynthetic pathway. drugbank.comnih.govgoogle.com The consequence of this enzymatic inhibition is the accumulation of methylated sterols within the fungal cell membrane, leading to disruptions in membrane permeability and fluidity. This ultimately impairs fungal growth and can result in cell death. drugbank.comnih.gov While the triazole ring is paramount for this direct interaction and antifungal activity, other parts of the molecule, such as the extended linker and the triazolone/side chain regions, contribute through interactions with amino acid residues located in the substrate access channel of the enzyme, rather than directly at the catalytic center. acs.orgnih.govgoogle.com

Importance of Specific Structural Regions (e.g., Triazolone/Side Chain, Dioxolane Ring)

Research into the SAR of this compound for its non-antifungal activities has underscored the significance of specific structural regions beyond the triazole moiety. For instance, modifications within the triazolone/side chain region have shown considerable impact on anti-Hedgehog activity, with certain structural alterations leading to enhanced potency compared to the parent compound. nih.govnih.govresearchgate.netacs.org The sec-butyl side chain attached to the triazolone ring has been identified as a key determinant for activity against VEGFR2 and Hedgehog signaling. nih.gov The dioxolane ring and the chemical groups attached to it also contribute to these non-antifungal activities. acs.orgnih.gov

Stereochemical Influence on Biological Activity

The stereochemistry of the this compound molecule exerts a notable influence on its various biological activities. With three chiral centers, this compound can exist as eight possible stereoisomers. nih.govnih.govdrugbank.comresearchgate.netwikipedia.orgnih.govresearchgate.net Studies comparing the activities of individual stereoisomers have revealed distinct profiles for antifungal versus antiangiogenic effects, suggesting that different molecular mechanisms are at play. researchgate.netresearchgate.net Regarding anti-Hedgehog activity, the stereochemical orientation of the dioxolane ring, particularly the 2S,4R-cis configuration, appears to be favored for potent inhibition. nih.gov However, pharmacokinetic investigations suggest that the 2R,4R-trans orientation might be more advantageous in terms of metabolic stability and clearance. nih.gov While the absolute stereochemistry of the sec-butyl group may have a lesser impact on anti-Hedgehog activity, the presence of the methyl group in this position is crucial for maintaining potent inhibition. nih.gov

Analog Design and Synthesis for Enhanced Potency and Physicochemical Properties

The insights gained from SAR studies of this compound have been instrumental in guiding the design and synthesis of novel analogs. The primary goals of this research are often to enhance specific biological activities and improve physicochemical properties such as aqueous solubility and metabolic stability. nih.govacs.org Modifications have been strategically introduced into various parts of the molecule, including the triazolone/side chain and the triazole moiety itself. google.comnih.gov For example, replacing the triazolone/side chain with different functional groups, such as hydrazine carboxamides and meta-substituted amides, has resulted in the creation of analogs exhibiting improved anti-Hedgehog potency. nih.gov Analog design efforts also prioritize improving drug-like characteristics. nih.gov For instance, substituting the phenyl group with a pyridine or a fluorine-substituted benzene ring in some analogs has led to significantly enhanced solubility and a reduction in the inhibition of CYP3A4, a major drug-metabolizing enzyme that is a known target of this compound. google.comacs.org

Hedgehog (Hh) Signaling Pathway Inhibition

The Hedgehog signaling pathway plays a crucial role in embryonic development and tissue homeostasis, but its aberrant activation is linked to the development and progression of various cancers. frontiersin.orgdovepress.comreya-lab.org this compound has been identified as a potent antagonist of the Hh pathway. nih.govdovepress.comnih.govresearchgate.netmdpi.com Unlike some other Hh inhibitors that bind to the same site as cyclopamine, this compound appears to act on Smoothened (SMO), a key component of the pathway, through a distinct mechanism. reya-lab.orgnih.govresearchgate.netmdpi.comnih.gov

This compound's inhibition of the Hh pathway leads to the suppression of downstream targets, including GLI transcription factors, which are responsible for regulating genes involved in cell growth and survival. frontiersin.orgdovepress.com This inhibition has been shown to reduce cancer cell proliferation and induce apoptosis in various cancer models. frontiersin.orgdovepress.com Studies have demonstrated that this compound can suppress Hh pathway activity and tumor growth in preclinical models, such as medulloblastoma and basal cell carcinoma. nih.govresearchgate.netnih.gov

Inhibition of SMO Accumulation

A key aspect of this compound's mechanism in inhibiting the Hedgehog pathway involves preventing the accumulation of Smoothened (SMO) in the primary cilium. reya-lab.orgnih.govresearchgate.netmdpi.com In the presence of Hedgehog ligands, SMO translocates to the primary cilium, leading to the activation of downstream signaling. dovepress.comreya-lab.org this compound interferes with this process, thereby blocking pathway activation. reya-lab.orgnih.govresearchgate.netmdpi.com This mechanism is distinct from that of other SMO antagonists like cyclopamine. reya-lab.orgnih.govresearchgate.netmdpi.comnih.gov

Antiangiogenic Activity

Angiogenesis, the formation of new blood vessels, is essential for tumor growth, invasion, and metastasis. aacrjournals.orgnih.gov this compound has been found to possess potent antiangiogenic activity, inhibiting multiple aspects of endothelial cell function critical for blood vessel formation. aacrjournals.orgnih.govresearchgate.net This activity was initially discovered through screens for inhibitors of endothelial cell proliferation. aacrjournals.orgnih.govresearchgate.net this compound has shown dose-dependent inhibition of endothelial cell proliferation, migration, and tube formation in response to angiogenic stimuli like VEGF and bFGF. aacrjournals.orgnih.gov In vivo studies using tumor xenograft models have also demonstrated that oral administration of this compound can inhibit tumor vascularity and enhance the efficacy of chemotherapy. aacrjournals.orgnih.gov

Inhibition of VEGFR2 Phosphorylation and Glycosylation

A significant mechanism contributing to this compound's antiangiogenic effect is its interference with Vascular Endothelial Growth Factor Receptor 2 (VEGFR2). nih.govresearchgate.netaacrjournals.orgnih.gov VEGFR2 is a key receptor in mediating VEGF-induced angiogenesis. wikipedia.org this compound has been shown to inhibit the phosphorylation of VEGFR2, which is necessary for its activation and downstream signaling. nih.govaacrjournals.org This inhibition of phosphorylation is associated with a decrease in the activation of downstream signaling molecules like phospholipase C γ1 (PLCγ1). nih.gov

Furthermore, this compound affects the glycosylation of VEGFR2. nih.govresearchgate.netaacrjournals.orgnih.govaacrjournals.org It induces the accumulation of immature N-glycans on VEGFR2, leading to incomplete glycosylation. nih.govresearchgate.netaacrjournals.orgnih.gov This altered glycosylation is linked to defects in VEGFR2 trafficking and a reduction in its cell surface expression, ultimately impairing VEGF binding and signaling. nih.govresearchgate.netaacrjournals.orgnih.gov

Inhibition of Glycosylation and Trafficking

Beyond its effects on VEGFR2, this compound appears to have a broader impact on protein glycosylation and intracellular trafficking in endothelial cells. nih.govresearchgate.netaacrjournals.orgnih.gov It has been shown to globally reduce certain types of N-glycans and can induce hypoglycosylation of other receptors, such as the epidermal growth factor receptor (EGFR) in certain cell lines. nih.govresearchgate.netnih.gov This suggests a more general interference with glycosylation processes and protein trafficking pathways, which can affect the function and localization of various proteins essential for cellular processes, including angiogenesis. nih.govresearchgate.netaacrjournals.orgnih.gov The disruption of intracellular trafficking is proposed as a potential mechanism underlying the observed defects in VEGFR2 signaling and glycosylation, as well as effects on cholesterol trafficking and mTOR signaling. aacrjournals.org

Influence on Cholesterol Biosynthesis Pathways

This compound's antiangiogenic activity is also linked to its influence on cholesterol biosynthesis and trafficking pathways. nih.govanticancerfund.orgresearchgate.netaacrjournals.orgaacrjournals.orgiiarjournals.org While its primary antifungal mechanism involves inhibiting fungal ergosterol synthesis, in human cells, it affects cholesterol metabolism and trafficking. nih.goviiarjournals.org this compound has been shown to inhibit intracellular cholesterol trafficking, leading to the accumulation of cholesterol in late endosomes and lysosomes. nih.govresearchgate.netiiarjournals.org This disruption of cholesterol homeostasis can influence various signaling pathways, including mTOR and VEGFR2 signaling, both of which are critical for endothelial cell function and angiogenesis. nih.govanticancerfund.orgnih.govresearchgate.netaacrjournals.org The antiangiogenic effect of this compound has been shown to be reduced in the presence of excess cholesterol in vitro. nih.gov

Data Table: Mechanisms of this compound's Anticancer Activity

Broad-Spectrum Antiviral Activity (e.g., against Enteroviruses, Cardioviruses, Hepatitis C Virus)

This compound has been identified as a broad-spectrum inhibitor of several RNA viruses, including members of the Picornaviridae family, such as enteroviruses (e.g., poliovirus, coxsackievirus, enterovirus-71, rhinovirus) and cardioviruses (e.g., encephalomyocarditis virus, Saffold virus), as well as Hepatitis C Virus (HCV) nih.govnih.govmdpi.com. Studies have shown that this compound can inhibit the replication of these viruses in vitro nih.govnih.govresearchgate.net. For instance, it has demonstrated effectiveness against Enterovirus 71 (EV71), a key causative agent of hand, foot, and mouth disease, as well as coxsackievirus A16, coxsackievirus B3, poliovirus 1, and enterovirus 68 nih.gov. The antiviral activity against enteroviruses and cardioviruses appears to occur at the viral RNA replication stage nih.gov. Furthermore, this compound has been shown to inhibit in vitro HCV replication nih.govnih.gov.

Interactive Data Table: In Vitro Antiviral Activity of this compound Against Selected Viruses

This broad antiviral activity is distinct from this compound's known antifungal targets nih.govnih.gov.

Targeting Oxysterol-Binding Protein (OSBP)

A key mechanism underlying this compound's antiviral activity against these viruses involves the targeting of host cellular proteins, specifically Oxysterol-Binding Protein (OSBP) and OSBP-related protein 4 (ORP4) nih.govnih.govresearchgate.net. OSBP is a cellular lipid shuttling protein that plays a crucial role in maintaining lipid homeostasis at membrane contact sites between the endoplasmic reticulum (ER) and the trans-Golgi apparatus nih.govmdpi.com. This protein is involved in the non-vesicular transport of cholesterol and phosphatidylinositol-4-phosphate (PI4P) between membranes nih.govmdpi.com.

Many positive-sense RNA viruses, including enteroviruses, cardioviruses, and HCV, remodel intracellular membranes to form replication organelles (ROs) where viral genome replication occurs nih.govplos.org. These ROs are often enriched in PI4P and cholesterol, and OSBP is recruited to these sites nih.govplos.org. OSBP's function in shuttling lipids, particularly the exchange of PI4P and cholesterol, is essential for the formation and function of these viral replication organelles nih.govplos.org.

This compound binds to OSBP and inhibits its lipid shuttling function nih.govnih.govresearchgate.net. This disruption of OSBP activity perturbs the lipid environment necessary for the formation of viral replication organelles, thereby inhibiting viral RNA replication nih.govnih.govresearchgate.net. Evidence suggests that this compound directly binds to OSBP nih.govmdpi.comresearchgate.net. Inhibition of OSBP by this compound leads to an increase in PI4P levels at the Golgi or ROs and blocks the accumulation of cholesterol at ROs nih.govmdpi.com.

Studies using OSBP antagonists like OSW-1 have further supported the role of OSBP as a target for inhibiting enterovirus replication nih.gov. Knockdown of OSBP has also been shown to inhibit virus replication, while overexpression of OSBP or ORP4 can counteract the antiviral effects of this compound nih.govnih.gov. The antiviral activity mediated through OSBP is independent of this compound's established antifungal targets nih.govnih.gov.

High-Performance Liquid Chromatography (HPLC)

Optimization of Chromatographic Conditions (Columns, Mobile Phase, Flow Rate, Detection Wavelength)

High-Performance Liquid Chromatography (HPLC) and Ultra-Performance Liquid Chromatography (UPLC) are widely used for the analysis of Itraconazole, requiring careful optimization of chromatographic parameters to achieve adequate separation, sensitivity, and peak characteristics.

Columns: Reversed-phase C18 columns are commonly employed for this compound analysis. Examples include Enable C-18G (250 × 4.6 mm, 5 µm) globalresearchonline.net, HiQSil C18-HS (250 × 4.6 mm) scholarsresearchlibrary.com, Thermo Hypersil BDS C18 (150mm X 4.6 mm, 5μm and 100 mm x 4.6 mm, 3 μm) jocpr.com, Agilent Zorbax Eclipse XDB C18 (4.6 × 50 mm, 1.8 µm) oup.comtandfonline.com, Zodiac C18 (250mm x 4.6mm, 5µm and 100x4.6mm, 5μm) ijrrjournal.com, Dionex C18 (4.6 X 250mm, 5µm) ajpaonline.com, and Inertsil C18 (250 x 4.6 mm, 5 µm) ijpbs.com. The choice of column can depend on the specific application, such as assay or related substances determination, and the desired analysis time. jocpr.comoup.com

Mobile Phase: The composition of the mobile phase is critical for optimizing the separation of this compound and potential impurities or metabolites. Common mobile phases utilize mixtures of organic solvents (such as acetonitrile or methanol) and aqueous components, often with buffers or additives to control pH and improve peak shape. Examples include:

The addition of ion-pairing agents like tetrabutylammonium hydrogen sulfate (TBAHS) is sometimes used in USP and China Pharmacopoeia methods to improve retention and peak shape, although these mobile phases may not be compatible with mass spectrometry. fishersci.com

Flow Rate: Typical flow rates for HPLC methods range from 1.0 mL/min to 1.5 mL/min. globalresearchonline.netscholarsresearchlibrary.comjocpr.comajrconline.orgajpaonline.comijpsr.com UPLC methods, utilizing smaller particle size columns, often employ lower flow rates, such as 0.40 mL/min, to achieve faster analysis times. researchgate.net

Detection Wavelength: this compound exhibits UV absorbance, and detection is commonly performed using UV detectors. The optimal detection wavelength is typically in the range of 225 nm to 306 nm, with specific wavelengths reported including 264 nm globalresearchonline.net, 263 nm scholarsresearchlibrary.com, 225 nm jocpr.comijpsr.com, 235 nm oup.com, 306 nm ijrrjournal.comajpaonline.com, 257 nm ijpbs.com, and 262 nm ajrconline.org. The chosen wavelength often corresponds to the maximum absorbance of this compound or allows for optimal detection of both this compound and co-eluting substances.

Liquid Chromatography-Mass Spectrometry (LC-MS/MS)

LC-MS/MS is a powerful technique widely used for the analysis of this compound, particularly in complex matrices like biological samples, due to its high sensitivity and selectivity. ingentaconnect.compsu.edushimadzu.comnih.govingentaconnect.comnih.govresearchgate.netshimadzu.frnih.govlcms.cznih.govnih.gov

Ionization: Electrospray ionization (ESI) in positive ion mode is commonly used for this compound and its metabolites. ingentaconnect.comshimadzu.comingentaconnect.comnih.govnih.gov

Mass Transitions: LC-MS/MS methods typically utilize multiple reaction monitoring (MRM) to quantify this compound and its metabolites by monitoring specific precursor-to-product ion transitions. shimadzu.comnih.govnih.govnih.govlcms.cznih.govinnovareacademics.in

Note: Specific m/z values may vary slightly between methods and instruments. ingentaconnect.comshimadzu.comnih.govingentaconnect.comnih.govnih.govlcms.cznih.govinnovareacademics.in

Monitoring the halogen isotopic peak (e.g., [M+2]+) for this compound can help reduce interference from co-eluting peaks. nih.gov

Applications: LC-MS/MS is frequently applied for the quantification of this compound and its active metabolite, hydroxythis compound, in biological matrices like human plasma for pharmacokinetic and bioequivalence studies. ingentaconnect.compsu.edushimadzu.comnih.govingentaconnect.comnih.govresearchgate.netshimadzu.frnih.govlcms.cznih.goveurofins-viracor.com Methods have also been developed for the simultaneous determination of this compound and multiple metabolites, including ketothis compound and N-desalkyl this compound. nih.govnih.gov

Gas Chromatography (GC) and GC-MS

While HPLC and LC-MS/MS are the predominant techniques for this compound analysis, Gas Chromatography (GC) and GC-Mass Spectrometry (GC-MS) are less commonly used for the intact molecule due to its low volatility. However, GC-MS can be applied for the determination of volatile impurities or degradation products in this compound active pharmaceutical ingredient (API). omicsonline.orginnovareacademics.inresearchgate.net

A GC-MS method using Selective Ion Monitoring (SIM) has been developed and validated for the trace level determination of methane sulfonyl chloride, a genotoxic impurity, in this compound API. omicsonline.orginnovareacademics.inresearchgate.net This method utilized a ZB-5 ms column (30 m × 0.25 mm × 0.25 µm) with helium as the carrier gas. innovareacademics.in GC-MS offers advantages in sensitivity and specificity compared to some HPLC-UV methods for certain impurities. omicsonline.org

High-Performance Thin-Layer Chromatography (HPTLC)

High-Performance Thin-Layer Chromatography (HPTLC) offers a rapid, selective, and economical approach for the analysis of this compound, particularly in quality control settings and for the analysis of pharmaceutical formulations. researchgate.netresearchgate.net

Stationary Phase: HPTLC plates precoated with silica gel 60 F-254 are typically used. researchgate.netresearchgate.net

Mobile Phase: Various mobile phase compositions have been reported for HPTLC analysis of this compound, often involving mixtures of organic solvents. An example includes Toluene: Chloroform : Methanol in a ratio of 5:5:1.5 (v/v). researchgate.net Another method used Toluene: Ethyl acetate: Ammonia (1:5:0.1 v/v). researchgate.net

Detection: Densitometric analysis is commonly performed using a TLC scanner at a suitable UV wavelength, such as 260 nm or 266 nm, where this compound absorbs. researchgate.netresearchgate.net

HPTLC methods for this compound have been developed and validated for parameters such as linearity, LOD, LOQ, recovery, and precision, demonstrating their suitability for quantitative analysis in bulk drug and pharmaceutical formulations. researchgate.netresearchgate.net HPTLC can also be used as a stability-indicating method by separating this compound from its degradation products. researchgate.netresearchgate.net