Tolbutamide

Synthesis and Chemical Properties

Tolbutamide, with the chemical formula C₁₂H₁₈N₂O₃S, is a white crystalline powder. wikipedia.orgpharmacy180.com It is practically insoluble in water but finds solubility in organic solvents like acetone and alcohol, as well as in dilute alkali hydroxide solutions. pharmacy180.com

The synthesis of this compound can be achieved through a single-step reaction involving the interaction of p-toluenesulfonylamide (in its sodium salt form) with butyl isocyanate. chemicalbook.comchemicalbook.com Another synthetic route starts from 4-methylbenzene sulphonamide. pharmacy180.com

Table 1: Chemical Properties of this compound

Mechanism of Action

The primary mechanism of action of this compound involves its interaction with the ATP-sensitive potassium (K-ATP) channels on the surface of pancreatic β-cells. patsnap.com These channels are a complex of two proteins: the sulfonylurea receptor 1 (SUR1) and the inwardly rectifying potassium channel subunit Kir6.2. patsnap.com

This compound specifically binds to the SUR1 subunit. patsnap.com This binding leads to the closure of the K-ATP channels. patsnap.com The closure of these channels prevents the efflux of potassium ions from the β-cell, causing the cell membrane to depolarize. patsnap.com This depolarization, in turn, triggers the opening of voltage-gated calcium channels. patsnap.com The subsequent influx of calcium ions into the cell is a critical signal that promotes the fusion of insulin-containing granules with the cell membrane and the subsequent exocytosis of insulin into the bloodstream. patsnap.comjustintimemedicine.com For this reason, the presence of functional pancreatic β-cells is essential for this compound's action. patsnap.comdrugbank.com

Beyond its primary effect on insulin secretion, some research suggests that this compound may have secondary mechanisms, such as enhancing the sensitivity of peripheral tissues to insulin and potentially decreasing glucose production by the liver. patsnap.comdrugbank.com

Pharmacokinetics and Metabolism

Absorption, Distribution, and Elimination

Following oral administration, tolbutamide is readily absorbed from the gastrointestinal tract. drugbank.com It can be detected in the plasma within 30 to 60 minutes, with peak plasma concentrations typically reached within 3 to 5 hours. drugbank.com The absorption of this compound is not significantly affected by food. drugbank.com

Once in the bloodstream, this compound is extensively bound to plasma proteins, with approximately 95-96% being protein-bound. wikipedia.orgdrugbank.com This high degree of protein binding is a significant pharmacokinetic characteristic. nih.gov Studies have investigated the binding of this compound to human serum albumin (HSA), indicating that it binds to multiple sites on the protein. nih.govresearchgate.net

The elimination half-life of this compound is relatively short, ranging from 4.5 to 6.5 hours. wikipedia.org The primary route of excretion is through the kidneys, with its metabolites being eliminated in the urine. drugbank.com

Metabolism

This compound is primarily metabolized in the liver. drugbank.com The main metabolic pathway is the oxidation of the p-methyl group, a reaction catalyzed by the cytochrome P450 enzyme CYP2C9. researchgate.netdrugbank.com This process results in the formation of an inactive carboxyl metabolite, 1-butyl-3-p-carboxyphenylsulfonylurea. drugbank.com Another metabolite that may be formed is hydroxythis compound. researchgate.netdrugbank.com Unlike some other sulfonamides, this compound does not have a p-amino group and therefore does not undergo acetylation. drugbank.com

Table 2: Pharmacokinetic Parameters of this compound

Key Structural Moieties and Their Contribution to Activity

The sulfonylurea group is the cornerstone of Tolbutamide's pharmacological action and is essential for its primary mechanism. firsthope.co.inhumanjournals.com This moiety is responsible for binding to the sulfonylurea receptor 1 (SUR1), a subunit of the ATP-sensitive potassium (K-ATP) channels located on pancreatic β-cells. firsthope.co.indiabetesjournals.org The interaction between the sulfonylurea group and the receptor is a critical step that initiates the cascade of events leading to insulin secretion. firsthope.co.in Specifically, the sulfonyl group can modify the molecule's conformation within receptor complexes, influencing binding. mdpi.com Studies have identified that a specific residue, serine 1237, in the SUR1 subunit is crucial for the high-affinity binding of the sulfonylurea moiety of drugs like this compound. diabetesjournals.org

Attached to the urea portion of the core is an alkyl side chain, which in the case of this compound is a butyl group. firsthope.co.in The nature and size of this chain are crucial for activity. slideshare.net The butyl group in this compound enhances the molecule's lipophilicity, which contributes to its binding affinity at the receptor site. firsthope.co.in Structure-activity relationship studies indicate that optimal activity is achieved when this alkyl group (referred to as R2) is between three to six carbons in length (C3H7 to C6H13). researchgate.netannualreviews.org If the chain is shorter, activity declines, and it becomes inactive if the chain is as long as twelve carbons. annualreviews.org The nature of this side chain is a key determinant of the compound's inhibitory activity. nih.gov

Relationship between Structure and Pharmacokinetic Profiles

The specific structural features of this compound directly influence its absorption, metabolism, and duration of action. It is readily absorbed from the gastrointestinal tract, with peak plasma levels occurring within 3 to 5 hours. drugbank.commims.com

A key structural element dictating this compound's pharmacokinetics is the para-methyl group on the benzene ring. drugs.com This group makes the molecule susceptible to rapid hepatic metabolism, primarily through oxidation by the CYP2C9 enzyme. mims.commdpi.com The methyl group is first hydroxylated to form hydroxymethylthis compound, which is then further oxidized to the inactive metabolite, 1-butyl-3-p-carboxyphenylsulfonylurea. drugs.commdpi.com This rapid metabolic degradation is the reason for this compound's relatively short duration of action, which is between 6 to 12 hours. drugbank.comwikipedia.org This contrasts with a compound like Chlorpropamide, where the methyl group is replaced by a chlorine atom, making it more resistant to metabolism and thus extending its duration of action. researchgate.net

The alkyl side chain also undergoes minor metabolic oxidation at the terminal (ω) and penultimate (ω-1) carbons, but the resulting alcohol metabolites have minimal hypoglycemic activity. mdpi.com

Comparative SAR with Other Sulfonylureas

This compound is classified as a first-generation sulfonylurea. wikipedia.org Comparing its structure to other sulfonylureas, particularly the second-generation agents, reveals key differences that account for variations in potency and receptor selectivity.

Second-generation drugs like Glibenclamide (Glyburide) are significantly more potent than this compound. annualreviews.orgwikipedia.org This enhanced activity is largely attributed to the nature of the para-substituent on the benzene ring. While this compound has a simple methyl group, second-generation agents feature a much larger p-acylamino-alkyl group. annualreviews.org For instance, the complex side chain in Glibenclamide increases its activity by approximately 100-fold compared to this compound. annualreviews.org

Furthermore, substitutions in the sulfonylurea moiety itself create differences in receptor affinity and selectivity. The replacement of this compound's small methyl group with a large, lipophilic cyclohexyl ring, as seen in Glibenclamide, increases the binding affinity for the SUR1 receptor by over 2000-fold. nih.gov This highlights that selectivity for SUR1 is achieved by introducing a lipophilic substituent into the sulfonylurea portion of the molecule. nih.gov

This also leads to differences in selectivity for sulfonylurea receptor subtypes. This compound and Gliclazide are selective for the pancreatic SUR1 subtype, whereas Glibenclamide and Glimepiride block both SUR1 and the SUR2 subtypes found in cardiac and smooth muscle. diabetesjournals.org

Molecular Docking and Computational Chemistry Studies

Computational methods have provided significant insights into this compound's molecular behavior. Molecular docking studies have been employed to simulate the interaction of this compound with various biological targets. These studies have explored its binding within the active sites of enzymes such as Aldose Reductase and Acetylcholinesterase, predicting binding affinities and interaction modes. nih.govndpublisher.inresearchgate.net Docking studies have also confirmed the inhibitory mechanisms of compounds on human cytochrome P450 isoforms, such as the mixed-type inhibition observed for this compound 4-hydroxylation by CYP2C9. nih.gov

Computational chemistry has also been used to investigate the physical properties of this compound. Studies have explored how the molecule's conformation changes in different solvents. For example, in non-polar solvents, this compound is predicted to assume an intramolecularly hydrogen-bonded conformation, which presents an additional energy barrier to crystal nucleation. approcess.comresearchgate.net Molecular simulation has been used to systematically investigate how molecular conformation and the formation of supramolecular clusters influence phenomena like cosolvency. acs.org

Additionally, molecular modeling has been instrumental in studying the formation of inclusion complexes, for instance, with cyclodextrins. These studies have confirmed that the most energetically favorable position occurs when the aromatic ring of this compound is included within the cyclodextrin cavity, which aligns with experimental NMR data. jst.go.jpacs.org

Genetic Control of Tolbutamide Metabolism

This compound is primarily metabolized in the liver through the oxidation of its p-methyl group, leading to the formation of 4-hydroxythis compound, an inactive metabolite, which is then further oxidized to carboxythis compound g-standaard.nlresearchgate.netontosight.aidrugbank.com. The main enzyme responsible for this initial hydroxylation is Cytochrome P450 2C9 (CYP2C9) g-standaard.nlresearchgate.netontosight.airesearchgate.netgbcbiotech.comdrugbank.com. While other enzymes like CYP2C8 and CYP2C19 have also been shown to metabolize this compound in vitro, CYP2C9 is considered the primary and most significant contributor to its metabolism drugbank.comnih.gov.

Early studies highlighted the substantial genetic control over this compound disposition. A ninefold variation in the rate of this compound disappearance from plasma (Kd) has been observed, exhibiting a trimodal frequency distribution. This pattern strongly suggests monogenic inheritance, with rapid and slow inactivation of this compound being transmitted autosomally. A high heritability value of 0.995 for Kd indicates minimal influence of environmental factors on this metabolic rate researchgate.netnih.govdiabetesjournals.orgresearchgate.netimrpress.com. The microsomal oxidation of this compound to hydroxythis compound has been identified as the primary site of this genetic control researchgate.netnih.govdiabetesjournals.orgresearchgate.net.

Polymorphisms in Drug-Metabolizing Enzymes (e.g., CYP2C9)

CYP2C9 is a highly polymorphic enzyme, with numerous genetic variants identified that can significantly impact its activity ontosight.aifrontiersin.orgaafp.orgnih.govhmdb.ca. Among these, the CYP2C92 (Arg144Cys) and CYP2C93 (Ile359Leu) alleles are the most well-studied and have been consistently associated with reduced enzymatic activity researchgate.netimrpress.comaafp.orgnih.govpharmgkb.orgmdpi.comnih.govdrugbank.com.

CYP2C92 (Arg144Cys): This variant leads to decreased clearance of this compound compared to the wild-type CYP2C91 allele pharmgkb.org.

CYP2C93 (Ile359Leu): This variant results in a more profound decrease in this compound metabolism compared to CYP2C91 and CYP2C92 nih.govpharmgkb.orgnih.govdrugbank.comdovepress.com. Individuals carrying the CYP2C93 allele have significantly reduced intrinsic clearance for this compound nih.gov.

In vitro studies using recombinant CYP2C9 variants have demonstrated that the intrinsic clearance of this compound is significantly lower for CYP2C92 and even more so for CYP2C93 compared to the wild-type CYP2C91 pharmgkb.org. For instance, one study reported mean oral clearances of this compound as 0.97 l/h for CYP2C91/1 carriers, decreasing to 0.56 l/h for CYP2C91/3 carriers and further to 0.15 l/h for CYP2C93/*3 carriers, indicating a co-dominant mode of inheritance for elimination researchgate.netnih.gov.

While CYP2C19 was initially suggested to be involved in this compound metabolism based on in vitro studies, clinical pharmacogenomic studies have generally indicated no significant influence of CYP2C19 polymorphism on this compound pharmacokinetics researchgate.netnih.gov.

Impact of Genetic Variations on Pharmacokinetic and Pharmacodynamic Parameters

Genetic variations in CYP2C9 significantly affect the pharmacokinetic parameters of this compound, particularly its clearance and half-life. Carriers of reduced-function CYP2C9 alleles, such as CYP2C92 and CYP2C93, exhibit decreased oral clearance of this compound researchgate.netaafp.orgnih.govdovepress.com. This reduced clearance leads to higher plasma concentrations and a longer half-life of the drug g-standaard.nlresearchgate.netnih.govdovepress.com.

For example, a study in healthy volunteers demonstrated a substantial reduction in mean oral clearances of this compound across different CYP2C9 genotypes:

researchgate.netnih.gov

This table illustrates a clear gene-dose effect, where the presence of more reduced-function alleles (like *3) leads to progressively lower clearance rates. The elimination rate constants for the six CYP2C9 genotype groups could be expressed as a linear combination of constants specific to the *1, *2, and *3 alleles (0.05, 0.04, 0.01 h-1 respectively), supporting a co-dominant inheritance pattern researchgate.netnih.gov.

Despite these pronounced differences in pharmacokinetic parameters, some studies in healthy volunteers have not found a significant effect of CYP2C9 genetic variations on plasma insulin and glucose concentrations following this compound administration researchgate.netnih.gov. However, other research indicates that individuals carrying the CYP2C93 allele may experience a greater therapeutic response to lower initial doses of sulfonylureas, including this compound, suggesting an impact on pharmacodynamic parameters in a clinical setting g-standaard.nlimrpress.commdpi.com. For instance, a study found that the increase in plasma glucose concentration in individuals with CYP2C91/3 was half that in CYP2C91/*1 carriers after this compound and glucose administration g-standaard.nl.

Implications for Personalized Medicine and Therapeutic Response

The significant inter-individual variability in this compound metabolism due to CYP2C9 polymorphisms has important implications for personalized medicine drugbank.comnih.govhmdb.camdpi.comjneonatalsurg.com. Pharmacogenomic insights can potentially lead to more tailored therapeutic strategies, aiming to optimize treatment outcomes and minimize variability in drug response nih.govpersonalizedmedicinecoalition.orgjaypeedigital.com.

For patients with genotypes predicting lower CYP2C9 activity (e.g., those carrying CYP2C92 or CYP2C93 alleles), there is a risk of higher accrued blood levels of this compound if fixed dosage regimens are used nih.govdiabetesjournals.org. This could potentially lead to an enhanced therapeutic effect or, in the case of other sulfonylureas, an increased risk of hypoglycemia mdpi.com. Although this compound is less commonly prescribed now, the principles apply to other sulfonylureas largely metabolized by CYP2C9 mdpi.com.

Genetic testing for CYP2C9 polymorphisms could, in theory, help identify individuals who are slow metabolizers of this compound, allowing for potential dose adjustments to prevent overdose or to optimize the starting dose g-standaard.nlnih.govdiabetesjournals.orgmdpi.com. While some studies have suggested that genotyping might help in preventing overdose at the initiation of drug treatment, particularly for the choice of the starting dose g-standaard.nl, the KNMP Pharmacogenetics Working Group has concluded that therapy adjustment is not currently required for these gene-drug interactions based on available clinical effect data g-standaard.nlpharmgkb.org. Nevertheless, the concept of using pharmacogenomic biomarkers to guide drug therapy, including for drugs like this compound, remains a key aspect of personalized medicine, aiming for more effective and safer treatment nih.govpersonalizedmedicinecoalition.orgjaypeedigital.comresearchgate.net.

Alcohol Interactions

The co-ingestion of alcohol with tolbutamide can lead to significant drug interactions, primarily manifesting as a disulfiram-like reaction and altered glucose homeostasis. These interactions involve both pharmacodynamic and pharmacokinetic mechanisms.

In Vitro Studies

In vitro methodologies have provided detailed insights into the cellular and enzymatic interactions of tolbutamide.

Animal Models

Animal models have been instrumental in evaluating the in vivo efficacy of this compound and its systemic effects on glucose homeostasis and pancreatic hormone secretion.

Early Clinical Trials and Efficacy in Type 2 Diabetes

Early clinical investigations established tolbutamide as an effective oral hypoglycemic agent for the treatment of type 2 diabetes. This compound functions by stimulating the release of insulin from the beta cells of the pancreas, an effect contingent upon the presence of functioning beta cells. It also contributes to blood glucose reduction by enhancing the body's efficiency in utilizing insulin. researchgate.netpgkb.orgdrugbank.commayoclinic.org

One notable early multi-center, placebo-controlled clinical trial, the University Group Diabetes Program (UGDP), commenced in 1960 with the objective of assessing the efficacy of various treatments for type 2 diabetes, including this compound. nih.govoup.com Prior to the full UGDP findings, other studies provided insights into this compound's effectiveness. For instance, a randomized double-blind study involving 121 ketoacidosis-resistant diabetic males demonstrated that this compound successfully lowered fasting blood sugar levels in 60% of trials, in contrast to a 26% success rate for placebo. diabetesjournals.org This study also compared this compound with chlorpropamide, noting that chlorpropamide was more frequently effective. diabetesjournals.org

Another early trial conducted by Harry Keen, a British diabetologist-epidemiologist, involved 250 subjects with type 2 diabetes randomized to either this compound or placebo. This study reported a significantly lower incidence of cardiovascular events in the this compound-treated group over a five-year period compared to the placebo group. oup.com These initial findings underscored this compound's potential in managing blood glucose levels in type 2 diabetes.

Table 1: Summary of Early Clinical Trial Efficacy (Selected Data)

Long-Term Outcomes and Observational Studies

The long-term effects of this compound therapy have been a subject of extensive research, most notably through the UGDP study, which brought significant controversies and concerns regarding cardiovascular mortality.

Chromatographic Techniques

Chromatographic methods are widely utilized for the separation and quantification of tolbutamide and its metabolites due to their high selectivity and sensitivity.

HPLC is a predominant technique for the determination of this compound and its metabolites, including 4-hydroxythis compound and carboxythis compound, in various matrices such as human plasma and urine, as well as in pharmaceutical formulations. Several HPLC methods have been developed, often employing reversed-phase columns and UV detection.

Another validated RP-HPLC method for this compound in pure and pharmaceutical formulations used a Zodiac C18 column (250 mm × 4.6 mm, 5 µm particle size) with a mobile phase of Methanol: 0.1% Orthophosphoric acid: Acetonitrile (10:30:60) and UV detection at 231 nm. This method demonstrated linearity in the range of 20-120 µg/mL. metabolomicsworkbench.orgnih.gov

For the determination of this compound and 4-hydroxythis compound in human plasma, a sensitive and selective HPLC method employed a ZORBAX SB-C18 column (150 × 4.6 mm, 5 µm) with gradient elution using water, acetonitrile, and 0.1% trifluoroacetic acid in water as the mobile phase, detected by UV at 230 nm. The calibration curves were linear over 0.5-100 µg/mL for this compound and 0.01-2.0 µg/mL for 4-hydroxythis compound. Intra-day and inter-day relative standard deviations (RSD) were less than 9.30% for this compound and less than 7.55% for 4-hydroxythis compound. researchgate.net

A solid-phase extraction HPLC method for this compound and its metabolites in human plasma and urine used a Waters Spherisorb Phenyl column (4.6 mm × 250 mm, 5 µm) with a mobile phase of methanol-0.02 mmol·L⁻¹ pH 3.3 NaAc buffer (28:72) and UV detection at 230 nm. The linear range for this compound in human plasma was 2-100 µmol·L⁻¹ with recoveries of 105.1%-103.9%. For carboxythis compound in human urine, the linear range was 2-50 µmol·L⁻¹ with recoveries of 98.8%-100.1%. For 4-hydroxythis compound in human urine, the linear range was 1-50 µmol·L⁻¹ with recoveries of 95.4%-103.5%. guidetopharmacology.org

Table 1: Summary of HPLC Methodologies for this compound and Metabolites

Gas chromatography has been employed for the analysis of this compound, though detailed methodologies are less frequently reported compared to HPLC or LC-MS. One study described the use of GC for this compound after treatment with dimethyl sulfate, on a stainless steel column (6 ft. x 0.125 in.) packed with 1% OV-1 on Gas-Chrom Q. The temperature was programmed from 150 to 190°C at 20°C per minute, using helium as the carrier gas (30 mL/min). sigmaaldrich.com Another application involved GC-MS for this compound and its metabolites in human serum, following the formation of thermally stable N-methyl-N-trifluoroacetyl derivatives. The GC was performed on a glass column (1.5 m x 2 mm) operated at 170°C with helium as the carrier gas. sigmaaldrich.com

Spectrometric Techniques

Spectrometric techniques, particularly mass spectrometry, offer high sensitivity and specificity for the identification and quantification of this compound and its metabolites.

Mass spectrometry, often coupled with chromatographic separation, is a powerful tool for analyzing this compound. Early applications of MS in conjunction with GC involved obtaining mass spectra of column effluents at 70 eV with a double-focusing instrument linked to a digital data system. sigmaaldrich.com MS analysis has also been used to corroborate findings from other techniques, such as the increased glycation of albumin induced by this compound, observed by AGE-specific fluorescence and confirmed by mass spectrometric analysis. wikipedia.org

LC-MS and LC-MS/MS are highly sensitive and selective methods for the determination of this compound and its metabolites in complex biological matrices.

A sensitive and selective LC-MS method was developed for the simultaneous determination of this compound and its metabolite hydroxythis compound in rat plasma. Analytes were extracted using liquid-liquid extraction with ethyl acetate and separated on a Zorbax SB-C18 column (2.1 mm × 50 mm, 3.5 µm) using acetonitrile-0.1% formic acid as the mobile phase with gradient elution. Electrospray ionization (ESI) in positive ion mode and selected ion monitoring (SIM) mode were used for quantification. The assay was linear over 10–20000 ng/mL for this compound and 5–800 ng/mL for hydroxythis compound, with LLOQs of 10 ng/mL and 5 ng/mL, respectively. uni.lu Another LC-MS method for this compound and hydroxythis compound in rat plasma utilized acetonitrile precipitation for extraction and a Zorbax SB-C18 column (150 × 2.1 mm, 5 µm) with acetonitrile-0.1% formic acid gradient elution. ESI in positive ion mode and SIM were used. Linearity was established from 20-5000 ng/mL for this compound and 5–500 ng/mL for hydroxythis compound, with LLOQs of 20 ng/mL and 5 ng/mL, respectively. lgcstandards.com

LC-MS/MS has been used for proteome profiling in this compound-treated rat primary hepatocytes, employing nano UPLC-MS/MS analysis for label-free protein quantitation. chem960.com It has also been used to confirm the increased glycation of BSA in the presence of this compound, by analyzing glycated peptides using a high-resolution accurate mass spectrometer (Q-Exactive).

A rapid LC-ESI-MS/MS method for the quantification of chrysin using this compound as an internal standard was developed and validated. This method utilized an Atlantis C-18 column with an isocratic mobile phase of 0.2% formic acid in water and acetonitrile (20:80, v/v) at a flow rate of 0.9 mL/min. MS/MS detection was performed in negative ion mode.

Table 2: Summary of LC-MS/MS Methodologies for this compound and Metabolites

LC-RI-MS is a specialized technique particularly useful in drug metabolism studies involving radiolabeled compounds, allowing for comprehensive quantitative and qualitative analysis of metabolites without the need for standard samples.

LC-RI-MS analysis has been used to determine the structures of 12 (four previously unknown) ¹⁴C-tolbutamide (TB) metabolites in rat biological samples, including plasma, urine, bile, feces, and microsomes. The four novel metabolites identified were ω-carboxy TB, hydroxyl TB (HTB)-O-glucuronide, TB-ortho or meta-glutathion, and tolylsulphoaminocarbo-glutathion.

In rat plasma, after oral administration of ¹⁴C-TB at therapeutic (1 mg/kg) and microdose (1.67 µg/kg) levels, the total radioisotope (RI) and six metabolites (HTB, carboxy TB (CTB), M1: desbutyl TB, M2: ω-hydroxyl TB, M3: α-hydroxyl TB, and M4: ω-1-hydroxyl TB) were quantified by LC-RI-MS. Plasma concentrations were calculated using their response factors (MS-RI intensity ratio) without standard samples.

Table 3: Identified this compound Metabolites via LC-RI-MS

Sample Preparation Techniques (e.g., Solid-Phase Extraction, Protein Precipitation)

Effective sample preparation is a crucial step in bioanalytical assays for this compound and its metabolites, aiming to remove interfering substances from the biological matrix and concentrate the analytes. Commonly employed techniques include protein precipitation, liquid-liquid extraction, and solid-phase extraction.

Method Validation and Quantification in Biological Samples

Method validation is a critical process to ensure that an analytical method is suitable for its intended purpose, typically following guidelines from regulatory bodies such as the Food and Drug Administration (FDA). Key validation parameters include linearity, accuracy, precision, recovery, sensitivity (Lower Limit of Quantification, LLOQ, and Limit of Detection, LOD), selectivity, and matrix effects. ingentaconnect.comnih.govnih.govmedcraveonline.com

Q. Q1. What experimental approaches are recommended to elucidate Tolbutamide’s mechanism of action in pancreatic β-cells?

Methodological Answer:

• Step 1 : Use in vitro electrophysiology (e.g., patch-clamp techniques) to confirm this compound’s binding to sulfonylurea receptor 1 (SUR1) and subsequent ATP-sensitive potassium (K$_\text{ATP}$) channel inhibition .
• Step 2 : Measure insulin secretion in isolated islets via ELISA under varying glucose concentrations to correlate channel inhibition with hormonal output.
• Step 3 : Validate findings in animal models (e.g., streptozotocin-induced diabetic rats) to assess glucose-lowering efficacy and tissue specificity .

Basic Research Question

Q. Q2. How does CYP2C9 polymorphism influence this compound pharmacokinetics, and what experimental designs address this variability?

Methodological Answer:

• Approach : Conduct in vitro metabolism assays using recombinant CYP2C9 isoforms (e.g., CYP2C92 and CYP2C93) to quantify 4-hydroxythis compound formation rates .
• Clinical Validation : Perform pharmacokinetic studies in genotyped cohorts, measuring plasma concentration-time profiles (AUC, C$_\text{max}$, t$_{1/2}$) using HPLC-UV (mobile phase: 10 mM sodium acetate buffer [pH 4.4]/acetonitrile, 25:75 v/v) .
• Statistical Analysis : Apply ANOVA to compare metabolic clearance between genotypes, adjusting for covariates like age and liver function .

Advanced Research Question

Q. Q3. How can physiologically based pharmacokinetic (PBPK) modeling resolve contradictions in this compound-drug interaction studies?

Methodological Answer:

• Model Development : Integrate in vitro CYP2C9 inhibition constants (K$_\text{i}$) and clinical PK data into software (e.g., Simcyp®) to simulate this compound exposure with inhibitors (e.g., sulfaphenazole) .
• Verification : Compare simulated AUC ratios (e.g., 2.5–3.5-fold increase with sulfaphenazole) against clinical observations to validate model accuracy .
• Application : Predict interactions with novel CYP2C9 inhibitors (e.g., tasisulam) to guide dose adjustments in cancer patients .

Advanced Research Question

Q. Q4. How should researchers address contradictory efficacy data for this compound across patient subpopulations?

Methodological Answer:

• Meta-Analysis : Pool data from RCTs (e.g., 53-patient cohorts ) to stratify outcomes by covariates (age, BMI, prior insulin use).
• Confounder Adjustment : Use multivariate regression to isolate this compound’s effect from dietary adherence or comorbid conditions .
• In Silico Screening : Apply machine learning to EHR datasets to identify subpopulations with optimal HbA1c reduction .

Methodological Guidance

Q. Q5. What quality control measures ensure reproducibility in this compound batch preparation for preclinical studies?

Methodological Answer:

• Analytical QC :
  • HPLC-UV : Monitor purity (>98%) and stability under storage conditions (e.g., 4°C vs. room temperature) .
  • Mass Spectrometry : Confirm molecular identity (m/z 271.2 for this compound; m/z 287.2 for 4-hydroxythis compound) .
• Batch Consistency :
  • Table 1 : Example QC Parameters

Advanced Research Question

Q. Q6. How can researchers reconcile in vitro CYP2C9 inhibition data with in vivo this compound interaction outcomes?

Methodological Answer:

• Step 1 : Measure unbound inhibitor concentrations in vivo to adjust in vitro K$_\text{i}$ values for protein binding .
• Step 2 : Use static models (e.g., [I]/K$_\text{i}$ >0.1 threshold) to predict clinical relevance of in vitro findings .
• Step 3 : Validate with dynamic PBPK models incorporating hepatic blood flow and enzyme saturation .

Basic Research Question

Q. Q7. What criteria should guide patient selection for this compound trials to minimize confounding variables?

Methodological Answer:

• Inclusion Criteria :
  • Mild type 2 diabetes (HbA1c 6.5–8.0%) .
  • Naïve to insulin therapy, responsive to dietary control .
• Exclusion Criteria :
  • Hepatic impairment (alters CYP2C9 activity) .
  • Concomitant CYP2C9 inhibitors/inducers .