Re-discover the value of protein binding assessments in hepatic and renal impairment studies and its contributions in drug labels and dose decisions.

One of the key pharmacokinetic properties of most small molecule drugs is their ability to bind to serum proteins. Unbound or free drug is responsible for pharmacological activity while the balance between free and bound drug can impact drug distribution, elimination, and other safety parameters. In the hepatic impairment (HI) and renal impairment (RI) clinical studies, unbound drug concentration is often assessed; however, the relevance and impact of the protein binding (PB) results is largely limited. We analyzed published clinical safety and pharmacokinetic studies in subjects with HI or RI with PB assessment up to October 2022 and summarized the contribution of PB results on their label dose recommendations. Among drugs with HI publication, 32% (17/53) associated product labels include PB results in HI section. Of these, the majority (9/17, 53%) recommend dose adjustments consistent with observed PB change. Among drugs with RI publication, 27% (12/44) of associated product labels include PB results in RI section with the majority (7/12, 58%) recommending no dose adjustment, consistent with the reported absence of PB change. PB results were found to be consistent with a tailored dose recommendation in 53% and 58% of the approved labels for HI and RI section, respectively. We further discussed the interpretation challenges of PB results, explored treatment decision factors including total drug concentration, exposure-response relationships, and safety considerations in these case examples. Collectively, comprehending the alterations in free drug levels in HI and RI informs treatment decision through a risk-based approach.

Pharmacokinetics and Abuse Potential of Benzhydrocodone, a Novel Prodrug of Hydrocodone, After Intranasal Administration in Recreational Drug Users.

Objective: Developing an acetaminophen-free, immediate-release hydrocodone product remains an unmet medical need; however, new opioid analgesics should not introduce new abuse risks. Benzhydrocodone is a prodrug of hydrocodone that must be metabolized into hydrocodone by enzymes in the intestinal tract to optimally deliver its pharmacologic effects. This study evaluated the intranasal pharmacokinetics and abuse potential of benzhydrocodone active pharmaceutical ingredient (API) compared with hydrocodone bitartrate (HB) API. Design: Single-center, randomized, double-blind, crossover study. Setting: Clinical research site. Subjects: Healthy adult, nondependent, recreational opioid users. Methods: Subjects (N = 51 Completers) were randomized to receive 13.34 mg of intranasal benzhydrocodone API and 15.0 mg of intranasal HB API (molar-equivalent doses of hydrocodone). Blood samples were taken, and Drug Liking scores (assessed on a bipolar visual analog scale) were obtained throughout each dosing interval. Nasal irritation and safety were assessed. Results: Peak hydrocodone plasma concentration (Cmax) was 36.0% lower, and total hydrocodone exposures (AUClast and AUCinf) were 20.3% and 19.5% lower, respectively, for benzhydrocodone API compared with HB API (P < 0.0001). All partial AUC values were lower for benzhydrocodone API, with a ≥ 75% reduction in hydrocodone exposure at all time intervals up to one hour postdose (P < 0.0001). Median Tmax of hydrocodone following benzhydrocodone API was delayed by more than one hour compared with HB. Drug Liking score, as assessed by maximal liking (Emax), was significantly lower for benzhydrocodone API vs HB API (P = 0.004), with 45% of subjects showing a ≥ 30% reduction in Drug Liking Emax. Conclusion: Reductions in hydrocodone exposure and associated decreases in Drug Liking relative to HB suggest that the prodrug benzhydrocodone may deter intranasal abuse.

Relative Bioavailability, Intranasal Abuse Potential, and Safety of Benzhydrocodone/Acetaminophen Compared with Hydrocodone Bitartrate/Acetaminophen in Recreational Drug Abusers.

Objectives: Benzhydrocodone is a hydrocodone prodrug that has been combined with acetaminophen (APAP) in a novel immediate-release analgesic. This study evaluated the relative bioavailability, intranasal abuse potential, and safety of benzhydrocodone/APAP compared with commercially available hydrocodone bitartrate (HB)/APAP. Design: Single-center, randomized, double-blind, double-dummy, two-part study comprising a Dose Selection (Part A) phase and a Main Study (Part B) phase. Setting: Clinical research site. Subjects: Healthy adult, nondependent, recreational opioid users with a history of intranasal abuse. Methods: Subjects (N = 42) in Part B received five in-clinic treatments consisting of intranasal and oral benzhydrocodone/APAP (13.34/650 mg), intranasal and oral hydrocodone/APAP (15/650 mg), and placebo, with four or more days of washout between treatments. Pharmacodynamic assessments included subjective effects of Drug Liking, Overall Drug Liking, and Take Drug Again (assessed on visual analog scale [VAS]), as well as nasal irritation. Pharmacokinetics and safety were also assessed. Results: Hydrocodone Cmax was 11% lower for intranasal benzhydrocodone/APAP vs intranasal HB/APAP (P = 0.0027). Early cumulative hydrocodone exposures for intranasal benzhydrocodone/APAP through 0.5, 1, and 2 hours were reduced by approximately 50%, 29%, and 15%, respectively (P ≤ 0.0024). Correspondingly, Drug Liking VAS values up to two hours postdose were significantly lower for intranasal benzhydrocodone/APAP vs intranasal HB/APAP (P ≤ 0.0079), although peak Drug Liking VAS (Emax) scores were not different (P = 0.2814). Adverse nasal effects were more frequent for intranasal benzhydrocodone/APAP vs intranasal HB/APAP. Conclusions: Reduced hydrocodone exposure and drug liking at early time intervals, coupled with adverse nasal effects, can be expected to provide a level of deterrence to the intranasal route of abuse for benzhydrocodone/APAP.

Lack of significant effect of bilastine administered at therapeutic and supratherapeutic doses and concomitantly with ketoconazole on ventricular repolarization: results of a thorough QT study (TQTS) with QT-concentration analysis.

The effect of bilastine on cardiac repolarization was studied in 30 healthy participants during a multiple-dose, triple-dummy, crossover, thorough QT study that included 5 arms: placebo, active control (400 mg moxifloxacin), bilastine at therapeutic and supratherapeutic doses (20 mg and 100 mg once daily, respectively), and bilastine 20 mg administered with ketoconazole 400 mg. Time-matched, triplicate electrocardiograms (ECGs) were recorded with 13 time points extracted predose and 16 extracted over 72 hours post day 4 dosing. Four QT/RR corrections were implemented: QTcB; QTcF; a linear individual correction (QTcNi), the primary correction; and a nonlinear one (QTcNnl). Moxifloxacin was associated with a significant increase in QTcNi at all time points between 1 and 12 hours, inclusively. Bilastine administration at 20 mg and 100 mg had no clinically significant impact on QTc (maximum increase in QTcNi, 5.02 ms; upper confidence limit [UCL] of the 1-sided, 95% confidence interval, 7.87 ms). Concomitant administration of ketoconazole and bilastine 20 mg induced a clinically relevant increase in QTc (maximum increase in QTcNi, 9.3 ms; UCL, 12.16 ms). This result was most likely related to the cardiac effect of ketoconazole because for all time points, bilastine plasma concentrations were lower than those observed following the supratherapeutic dose.

High-performance liquid chromatographic analysis of pterostilbene in biological fluids using fluorescence detection.

A method of analysis of pterostilbene [trans-3,5-dimethoxy-4'-hydroxystilbene] is necessary to study the kinetics of in vitro and in vivo metabolism and determine its concentration in foodstuffs. A novel and simple high-performance liquid chromatographic method was developed for determination of pterostilbene in rat serum. Serum proteins (0.1 mL) are precipitated with cold acetonitrile after addition of the internal standard, pinosylvin. Separation was achieved on a Phenomenex C18 column (250 mm x 4.60 mm) with fluorescence excitation at 330 nm and emission at 374 nm. The calibration curves were linear ranging from 0.5 to 100 microg/mL. The mean extraction efficiency was >99%. Precision of the assay was <15% (CV), and was within 14% at the limit of quantitation (0.5 microg/mL). Bias of the assay was lower than 14%, and was within 9% at the limit of quantitation. The assay was applied successfully to the study of pterostilbene pharmacokinetics in rats.

Pharmacokinetics of selected stilbenes: rhapontigenin, piceatannol and pinosylvin in rats.

The pharmacokinetics of piceatannol, pinosylvin and rhapontigenin were characterized in male Sprague-Dawley rats after single intravenous doses of 10 mg kg(-1) of each stilbene. Serial blood samples were collected via a catheter inserted into the right jugular vein and plasma samples were analysed for the selected stilbenes concentrations using reverse phase HPLC methods. After an acute intravenous dose of piceatannol, plasma AUC, urine t(1/2), CL and V(d) were 8.48+/-2.48 micro g h mL(-1), 19.88+/-5.66 h, 2.13+/-0.92 Lh(-1) kg(-1) and 10.76+/-2.88 L kg(-1)(mean+/-s.e.m.), respectively. The acute intravenous dose of pinosylvin yielded the plasma AUC, urine t(1/2), CL and V(d) values of 5.23+/-1.20 micro g h mL(-1), 13.13+/-2.05 h, 1.84+/-0.44 Lh(-1) kg(-1) and 2.29+/-0.56 L kg(-1)(mean+/-s.e.m.), respectively. Rhapontigenin intravenous dosing yielded the plasma AUC, urine t(1/2), CL and V(d) values of 8.39+/-0.10 micro g h mL(-1), 25.31+/-1.46 h, 1.18+/-0.035 Lh(-1) kg(-1) and 11.05+/-0.17 L kg(-1)(mean+/-s.e.m.), respectively. Each stilbene was extensively glucuronidated. These stilbenes were predominantly eliminated via non-urinary routes. All three stilbenes were highly distributed into tissues and were highly extracted by the liver. The detectable plasma half-lives of these xenobiotics appear to be relatively short. However, utilizing urinary concentration-time data, much longer elimination half-lives were evident. The estimates of oral bioavailability characterize these stilbenes as poorly bioavailable compounds.

Pharmacometrics of stilbenes: seguing towards the clinic.

Stilbenes are small molecular weight (approximately 200-300 g/mol), naturally occurring compounds and are found in a wide range of plant sources, aromatherapy products, and dietary supplements. These molecules are synthesized via the phenylpropanoid pathway and share some structural similarities to estrogen. Upon environmental threat, the plant host activates the phenylpropanoid pathway and stilbene structures are produced and subsequently secreted. Stilbenes act as natural protective agents to defend the plant against viral and microbial attack, excessive ultraviolet exposure, and disease. One stilbene, resveratrol, has been extensively studied and has been shown to possess potent anti-cancer, antiinflammatory and anti-oxidant activities. Found primarily in the skins of grapes, resveratrol is synthesized by Vitis vinifera grapevines in response to fungal infection or other environmental stressors. Considerable research showing resveratrol to be an attractive candidate in combating a wide variety of cancers and diseases has fueled interest in determining the disease-fighting capabilities of other structurally similar stilbene compounds. The purpose of this review is to describe four such structurally similar stilbene compounds, piceatannol, pinosylvin, rhapontigenin, and pterostilbene and detail some current pharmaceutical research and highlight their potential clinical applications.

Direct high-performance liquid chromatographic analysis of D-tocopheryl acid succinate and derivatives.

A method of analysis of a Vitamin E derivative D-tocopheryl acid succinate (TS) in biological fluids and commercially available products is necessary to study the kinetics of in vitro and in vivo metabolism, tissue distribution, and content uniformity. A simple and inexpensive high-performance liquid chromatographic method was developed for the direct determination of D-tocopheryl acid succinate in commercially available products, rat serum, and rat tissues. This method can also be applied to the determination of 15 Vitamin E derivatives. Rat serum (0.1 ml) was extracted with sodium dodecyl sulfate, ethanol, hexane, and then dried under nitrogen gas after addition of the internal standard, DL-alpha-tocopherol acetate. Separation was achieved on a C18 column with UV detection at 205 nm. The calibration curve for D-tocopheryl acid succinate was linear ranging from 0.025 to 100 microg/ml. The mean extraction efficiency was >92%. Precision of the assay was <5% (CV), and was within 5% at the limit of quantitation (0.025 microg/ml). Bias of the assay was lower than 5%, and was within 5% at the limit of quantitation. The assay was applied successfully to the serum and tissue distribution of D-tocopheryl acid succinate in rats, various Vitamin E derivatives, and content uniformity in commercially available products containing D-tocopheryl acid succinate.

Determination and assay validation of pinosylvin in rat serum: application to drug metabolism and pharmacokinetics.

A method of analysis of pinosylvin in biological fluids is necessary to study the kinetics of in vitro and in vivo metabolism and determine its concentration in natural products. A novel and simple high-performance liquid chromatographic method was developed for simultaneous determination of pinosylvin and products of its metabolism in rat serum and liver microsomes. Serum, or microsomes (0.1 mL) were precipitated with acetonitrile after addition of the internal standard, 7-ethoxycoumarin. Separation was achieved on an amylose tris 3,5 dimethylphenylcarbamate column (150 mm x 4.6 mm, ID, 5 microm) with UV detection at 308 nm. The calibration curves were linear ranging from 0.5 to 100 microg/mL. The mean extraction efficiency was >99%. Precision of the assay (coefficient of variation) was <10%, including the limit of quantitation (0.5 microg/mL). Bias of the assay was lower than 15%. The limit of detection was 100 ng/mL for a 0.1 mL sample. The assay was successfully applied to both the in vitro and in vivo metabolic kinetic study of pinosylvin. Three metabolites of pinosylvin, two oxidative and one glucuronidated, have been identified. The two oxidative metabolites of pinosylvin have been identified as E- and Z-resveratrol.

Stereospecific high-performance liquid chromatographic analysis of hesperetin in biological matrices.

A method of analysis of hesperetin (+/--3,5,7-trihydroxy-4'-methoxyflavanone) in biological fluids is necessary to study the kinetics of in vitro and in vivo metabolism and tissue distribution. A simple high-performance liquid chromatographic method was developed for simultaneous determination of hesperetin enantiomers in rat serum, and rat and human urine. Serum and urine (0.1 ml) were precipitated with cold acetonitrile after addition of the internal standard, 7-methoxycoumarin. Separation was achieved on a Chiralpak AD-RH column with UV detection at 298 nm. The calibration curve was linear ranging from 0.5 to 100 microg/ml for each enantiomer. The mean extraction efficiency was >98%. Precision of the assay was <5% (CV), and was within 5% at the limit of quantitation (0.5 microg/ml). Bias of the assay was lower than 5%, and was within 5% at the limit of quantitation. The assay was applied successfully to the urinary excretion of hesperetin in rats and humans.

Preparative enzymatic synthesis and HPLC analysis of rhapontigenin: applications to metabolism, pharmacokinetics and anti-cancer studies.

PURPOSE: A facile method was established to enzymatically synthesize rhapontigenin from the glycosylated parent compound rhaponticin. A novel and simple high-performance liquid chromatographic method was developed for the determination of rhapontigenin. The assay was successfully applied to both the in vitro and in vivo metabolic kinetic study of rhapontigenin. METHODS: Serum, or microsomes (0.1 mL) was precipitated with acetonitrile after addition of the internal standard, daidzein. Separation was achieved on an amylose tris 3,5 dimethylphenylcarbamate column (150 x 4.6 mm, ID, 5m) with UV detection at 324 nm. Hep G2 hepatoma cells were treated with rhapontigenin or rhaponticin (0-250 microg/mL) and cell viability was measured. RESULTS: The calibration curves were linear ranging from 0.5 to 100 micromg/mL. The mean extraction efficiency was > 99%. Precision of the assay (coefficient of variation) was < 5%, including the limit of quantitation (0.5 microg/mL). Bias of the assay was lower than 5%. The limit of detection was 100 ng/mL for a 0.1 mL sample. One glucuronidated metabolite of rhapontigenin has been identified. Preliminary pharmacokinetic data revealed the presence of a glucuronidated metabolite in the serum and a terminal elimination t1/2 of approximately 6 h. Rhapontigenin demonstrated concentration-dependent anti-cancer activity with an IC50 115 microg/mL in HEP G2 cells while rhaponticin showed no activity across the concentrations tested in vitro. CONCLUSIONS: The preparative enzymatic synthesis method has demonstrated utility to provide sufficient rhapontigenin for pharmaceutical studies. Rhapontigenin is an active anti-cancer compound. The developed HPLC assay is sensitive, reproducible and accurate and can be applied to pharmacokinetic and metabolism studies.

Determination of piceatannol in rat serum and liver microsomes: pharmacokinetics and phase I and II biotransformation.

A method of analysis of piceatannol in biological fluids is necessary to study the kinetics of in vitro and in vivo metabolism and determine its concentration in foodstuffs. A novel and simple high-performance liquid chromatographic method was developed for simultaneous determination of piceatannol and products of its metabolism in rat serum and liver microsomes. Serum, or microsomes (0.1 mL), were precipitated with acetonitrile after addition of the internal standard, 4-methylumbelliferone. Separation was achieved on a phenomenex C(18) column (250 x 4.6 mm i.d., 5 microm) equipped with a phenomenex C(18) (4 x 3.0 mm i.d., 5 microm) guardcolumn with fluorescence excitation at 320 nm and emission at 420 nm. Separation was also possible with UV detection at 310 nm. The fluorescent calibration curves were linear ranging from 0.05 to 100 microg/mL. The mean extraction efficiency was >95%. Precision of the assay was <10% (coefficient of variation), and was within 10% at the limit of quantitation (0.05 ng/mL). Bias of the assay was lower than 7%. The limit of detection was 50 ng/mL for a 0.1 mL sample. The assay was applied successfully to the in vitro kinetic study of metabolism of piceatannol in rat liver microsomes and pharmacokinetics in rats. Three metabolites of piceatannol have been identified. .

Cyclooxygenase-3: axiom, dogma, anomaly, enigma or splice error?--Not as easy as 1, 2, 3.

A continued need to develop safe and effective analgesics and anti-inflammatory drugs fuels the ongoing investigations of cyclooxygenase (COX). In addition, a long unanswered question in the biomedical arena revolves around the mechanism of action of acetaminophen leading to its analgesic and antipyretic activity. Upon the discovery of COX in 1971, alternative enzyme forms were initially suggested. With the development of molecular biology, the discovery of two major cyclooxygenase genes (COX-1 and COX-2) was heralded in 1990, which has subsequently led to the clinical use and development of selective COX-2 inhibitors. Splice variants of both COX-1 and COX-2 were first encountered in the early 1990s, as were single nucleotide polymorphisms of COX-1 and 2. There have been some recently well-publicized investigations of COX-1 and -2 enzyme variants that may assist in our eventual conceptual understanding of the mechanisms of action of acetaminophen. The term "COX-3" has been utilized by some scientists and in the media. The evidence for "COX-3" in terms of various COX-variants and possible scientific and therapeutic implications of COX variant enzymes are described.

Chemotherapy induced gastrointestinal toxicity in rats: involvement of mitochondrial DNA, gastrointestinal permeability and cyclooxygenase -2.

PURPOSE: The gastrointestinal damage induced by xenobiotics is occurring more frequently and with greater toxicological significance than previously thought. Although there are some preliminary clinical studies and reports, there does not appear to be an extensive examination of gastrointestinal toxicity of various chemotherapeutic agents in the rat. This study was undertaken to examine the suitability of a rat model to detect the gastrointestinal damage after administration of various anti-neoplastic agents including etoposide, teniposide, melphalan, 5-fluorouracil, methotrexate and cisplatin. METHODS: Acute toxic doses of indomethacin and chemotherapeutic agents were administered to rats. The urinary excretion of orally administered sucrose and 51(Cr)-EDTA were measured as markers of gastroduodenal and intestinal permeability, respectively. Cyclooxygenase-2 messenger RNA and mitochondrial DNA damage were measured as toxicological endpoints. RESULTS: Each anti-neoplastic agent examined induced appreciable and significant dose-dependent increase in gastrointestinal permeability that correlated with gross toxicological and pathological changes to the gastrointestinal tract including ulceration and bleeding. COX-2 mRNA was upregulated > 2 fold in intestinal mucosa with enteropathy and dose-dependent mitochondrial oxidative damage was apparent in gastric and intestinal mucosa. After administration of each drug, the rats presented with histological evidence of drug-induced gastroenteropathy, ulceration and increased cecal hemoglobin. CONCLUSIONS: The rat appears to be a suitable model to study gastrointestinal toxicity of chemotherapeutic agents and non-steroidal anti-inflammatory drugs. Damage to mitochondrial DNA occurs in both the gastric and intestinal epithelium after the administration of these agents and may be an important factor in the pathogenesis and resolution of gastrointestinal toxicity.