Free Fraction Of Drug Research Articles

Pharmacokinetics, Tolerability, and Safety of Esmethadone in Subjects with Chronic Kidney Disease or Hepatic Impairment.

Esmethadone (dextromethadone; d-methadone; S-methadone (+)-methadone; REL-1017) is a low potency N-methyl-D-aspartate (NMDA) receptor channel blocker that showed a rapid and sustained adjunctive antidepressant effects in patients with major depressive disorder with inadequate response to ongoing serotonergic antidepressant treatment. Previous studies indicated that esmethadone is partially excreted by the kidney (53.9% of the dose) and by the liver (39.1% of the dose). Here we studied the pharmacokinetics and safety of esmethadone after a single oral dose of 25 mg in subjects with different stages of kidney and liver impairment. In subjects with a mild and moderate decrease in glomerular fraction rate (GFR), esmethadone Cmax and AUC0-inf values did not differ compared with healthy subjects. In patients with severe renal impairment, the ratios of Cmax and AUC0-inf values compared with healthy subjects were above 100% (138.22-176.85%) and, while modest, these increases reached statistical significance. In subjects with end stage renal disease (ESRD) undergoing intermittent hemodialysis (IHD), Cmax and AUC0-inf values were not statistically different compared with healthy subjects. IHD did not modified plasma total esmethadone concentrations in blood exiting versus entering the dialyzer. Dose adjustment is not warranted in subjects with mild-to-moderate impaired renal function. Dose reduction may be considered for select patients with severe renal disfunction. In subjects with mild-or-moderate hepatic impairment, Cmax and AUC0-inf were approximately 20-30% lower compared with healthy controls. The drug free fraction increased with the severity of hepatic impairment, from 5.4% in healthy controls to 8.3% in subjects with moderate hepatic impairment. Mild and moderate hepatic impairment has a minimal to modest impact on exposure to total or unbound esmethadone and dose adjustments are not warranted in subjects with mild and moderate hepatic impairment. Administration of esmethadone was well tolerated in healthy adult subjects, in subjects with mild or moderate hepatic impairment, and in subjects with mild moderate or severe renal impairment, including patients with ESRF undergoing dialysis.

In vitro biodistribution studies on clinically approved FGFR inhibitors ponatinib, nintedanib, erlotinib and the investigational inhibitor KP2692

Binding towards human serum albumin (HSA) and α1-acid glycoprotein (AGP) of three approved fibroblast growth factor receptor (FGFR) inhibitors ponatinib (PON), nintedanib (NIN) and erdafitinib (ERD), as well as the experimental drug KP2692 was studied by means of spectrofluorometric and UV–visible spectrophotometric methods. Additionally, proton dissociation processes, lipophilicity, and fluorescence properties of these four molecules were investigated in detail. The FGFR inhibitors were predominantly presented in their single protonated form (HL+) at pH 7.4 (at blood pH). At gastric pH (pH 1–2) the protonated forms (+1 – +3) are present, which provide relatively good aqueous solubility of the drugs. All of the four inhibitors are highly or extremely lipophilic at pH 7.4 (logD7.4 ≥ 2.7). At acidic pH 2.0 PON and ERD are rather lipophilic, NIN is amphiphilic, while KP2692 is highly hydrophilic. All four compounds bind to HSA and AGP. Moderate binding of PON, KP2692 and NIN was found towards albumin (logK’ = 4.5–4.7), while their affinity for AGP was about one order of magnitude higher (logK’ = 5.2–5.7). ERD shows a larger affinity for both proteins (logK’HSA ≈ 5.2, logK’AGP ≈ 7.0). The computed constants were used to model the distribution of the FGFR inhibitors in blood plasma under physiological and pathological (acute phase) conditions. The changing levels of the two proteins under pathological conditions compensate each other for PON and NIN, so that the free drug fractions do not change considerably. In the case of ERD the higher AGP levels distinctly reduce the free available fraction of the drug. Comparison with clinical pharmacokinetic data indicates that the here presented solution distribution studies can very well predict the conditions in cancer patients.

Interpreting permeability as a function of free drug fraction: The case studies of cyclodextrins and liposomes

In order to solubilize poorly soluble active pharmaceutical ingredients, various strategies have been implemented over the years, including the use of nanocarriers, such as cyclodextrins and liposomes. However, improving a drug's apparent solubility does not always translate to enhanced bioavailability. This work aimed to investigate to which extent complexation with cyclodextrins and incorporation into liposomes influence drug in vitro permeability and to find a mechanistic description of the permeation process. For this purpose, we investigated hydroxypropyl-β-cyclodextrin (HP-β-CD) and phosphatidylcholine liposomes formulations of three chemically diverse compounds (atenolol, ketoprofen and hydrocortisone). We studied drug diffusion of the formulations by UV-localized spectroscopy and advanced data fitting to extract parameters such as diffusivity and bound-/free drug fractions. We then correlated this information with in vitro drug permeability obtained with the novel PermeaPadⓇ barrier. The results showed that increased concentration of HP-β-CD leads to increased solubilization of the poorly soluble unionized ketoprofen, as well as hydrocortisone. However, this net increment of apparent solubility was not proportional to the increased flux measured. On the other hand, normalising the flux over the empirical free drug concentration, i.e., the free fraction, gave a meaningful absolute permeability coefficient. The results achieved for the liposomal formulation were consistent with the finding on cyclodextrins. In conclusion, we proved the adequacy and usefulness of our method for calculating free drug fractions in the examined enabling formulations, supporting the validity of the established drug diffusion/permeation theory that the unbounded drug fraction is the main driver for drug permeation across a membrane.

Biased computation of probability of target attainment for antimicrobial drugs.

The medical literature is replete with articles in which there is confusion between "free concentration" and "unbound fraction" (fu ), which is the ratio of free to total plasma concentration. The lack of clarity in distinguishing between these two terms has led to biased computations, erroneous interpretations, and misleading recommendations. The problems are highlighted in this paper, taking the example of calculation of Probability of Target Attainment (PTA). This metric is used to propose pharmacokinetic/pharmacodynamic (PK/PD) breakpoints required for the interpretation of Antimicrobial Susceptibility Testing. Based on Monte Carlo simulations of the PK/PD index, area under the unbound concentration time curve/minimum inhibitory concentration (fAUC/MIC), computation of PTA from total plasma concentrations scaled by fu ineluctably leads to biased estimates. The bias is greater if the variability associated with fu is added, instead of removing it during this scaling. The explanation for the bias is that total plasma drug concentrations are intrinsically more variable than the corresponding free concentrations. This is due to the variability of antimicrobial binding for total, but not for free plasma concentrations. In consequence, the greater variability always leads to underestimation of the PK/PD cutoff (i.e., the critical MIC that is guaranteed for a given percentile of the population). A further consequence is an increase in calculated dosage required to attain the targeted quantile. This erroneous approach, of using free antimicrobial drug fraction, is not limited to the derivation of PK/PD cutoff, but may also have consequences for antimicrobials drug safety in clinical patients.

Drug binding and drug-drug interaction considerations in individuals with obesity before and after bariatric surgery: A retrospective cross-sectional study

Obesity impacts pharmacokinetic and pharmacodynamic parameters, changing the drug-binding state via plasma proteins, which is critical for drug effectiveness and toxicity. Albumin and α1-acid glycoprotein (AAG) are the two main proteins accountable for drug binding in humans. Previous studies have shown higher values of AAG in individuals with obesity, but the interference of AAG in the pharmacokinetics of drugs remains unclear. We, therefore, aimed to analyze the pharmacotherapeutic profile and interfering factors in patients undergoing bariatric surgery in the pre-operative period and after 12 months. A retrospective cross-sectional study design was conducted and serum AAG was determined, and potential drug-drug interactions were monitored in patients with severe obesity who underwent bariatric surgery. The drug classes most used were antidepressants, cardiovascular agents, and lipid-modifying agents, with a decrease in use after 12 months of surgery. Before surgery, it was found that more than half of the patients had at least one drug interaction in their therapeutic regimen. Of these, most were classified as moderate. After surgery, a decrease in drug interaction was observed. Serum AAG showed a significant reduction after 12 months, which can influence the free fraction of drugs with a high-affinity rate and how they impact therapeutic efficacy.

Developmental changes in the extent of drug binding to rat plasma proteins

Binding of therapeutics to proteins in blood plasma is important in influencing their distribution as it is their free (unbound) form that is able to cross cellular membranes to enter tissues and exert their actions. The concentration and composition of plasma proteins vary during pregnancy and development, resulting in potential changes to drug protein binding. Here, we describe an ultrafiltration method to investigate the extent of protein binding of six drugs (digoxin, paracetamol, olanzapine, ivacaftor, valproate and lamotrigine) and two water soluble inert markers (sucrose and glycerol) to plasma proteins from pregnant and developing rats. Results showed that the free fraction of most drugs was lower in the non-pregnant adult plasma where protein concentration is the highest. However, plasma of equivalent protein concentration to younger pups obtained by diluting adult plasma did not always exhibit the same extent of drug binding, reinforcing the likelihood that both concentration and composition of proteins in plasma influence drug binding. Comparison between protein binding and brain drug accumulation in vivo revealed a correlation for some drugs, but not others. Results suggests that plasma protein concentration should be considered when using medications in pregnant and paediatric patients to minimise potential for fetal and neonatal drug exposure.

Pharmacokinetics of Novel Furoxan/Coumarin Hybrids in Rats Using LC-MS/MS Method and Physiologically Based Pharmacokinetic Model.

Novel furoxan/coumarin hybrids were synthesized, and pharmacologic studies showed that the compounds displayed potent antiproliferation activities via downregulating both the phosphatidylinositide 3-kinase (PI3K) pathway and the mitogen-activated protein kinase (MAPK) pathway. To investigate the preclinical pharmacokinetic (PK) properties of three candidate compounds (CY-14S-4A83, CY-16S-4A43, and CY-16S-4A93), liquid chromatography, in tandem with the mass spectrometry LC-MS/MS method, was developed and validated for the simultaneous determination of these compounds. The absorption, distribution, metabolism, and excretion (ADME) properties were investigated in in vitro studies and in rats. Meanwhile, physiologically based pharmacokinetic (PBPK) models were constructed using only in vitro data to obtain detailed PK information. Good linearity was observed over the concentration range of 0.01−1.0 μg/mL. The free drug fraction (fu) values of the compounds were less than 3%, and the clearance (CL) values were 414.5 ± 145.7 mL/h/kg, 2624.6 ± 648.4 mL/h/kg, and 500.6 ± 195.2 mL/h/kg, respectively. The predicted peak plasma concentration (Cmax) and the area under the concentration-time curve (AUC) were overestimated for the CY-16S-4A43 PBPK model compared with the experimental ones (fold error > 2), suggesting that tissue accumulation and additional elimination pathways may exist. In conclusion, the LC-MS/MS method was successively applied in the preclinical PK studies, and the detailed information from PBPK modeling may improve decision-making in subsequent new drug development.

Analysis of the binding of warfarin to glyoxal- and methylglyoxal-modified human serum albumin by ultrafast affinity extraction

Ultrafast affinity extraction (UAE) and affinity microcolumns containing immobilized human serum albumin (HSA) were employed to evaluate the effect of advanced stage glycation on HSA and its binding to warfarin, a common site-specific probe for Sudlow site I of this protein. The modification of HSA by glyoxal (GO) and methylglyoxal (MGO) was considered, where GO and MGO are known to be important in the formation of many types of advanced glycation end products. Free drug fractions were measured by UAE for warfarin in solutions containing normal HSA or HSA that had been modified by GO or MGO at levels seen in serum during diabetes. The free fractions measured with the GO-modified HSA gave association equilibrium constants that ranged from 2.42–2.63 × 105 M−1 at pH 7.4 and 37 °C. These values were not significantly different from a value of 2.33 (±0.15) × 105 M−1 that was determined by the same method for warfarin with normal HSA. Similar studies using MGO-modified HSA gave association equilibrium constants for warfarin in the range of 3.07–3.31 × 105 M−1, which were 1.32- to 1.42-fold higher than the value seen for normal HSA (differences that were significant at the 95% confidence level). These results will be valuable in future binding studies based on affinity chromatography or other methods that employ warfarin as a probe to examine drug interactions at Sudlow site I of HSA and modified forms of this protein. This work also illustrates how UAE can be used, with analysis times of only minutes, to detect and measure small changes in the binding by drugs with unmodified or modified forms of a soluble binding agent or protein.

D3S-001, a highly potent, selective, and differentiated covalent inhibitor of KRAS G12C: Human dose prediction and first‐in‐human (FIH) trial design.

e15087 Background: Effective inhibition of G12C mutated KRAS is believed to be critical for anti-tumor efficacy in KRAS-driven cancers. D3S-001 is a potent and highly differentiated KRAS G12C inhibitor, which demonstrates rapid and near complete target inhibition and strong anti-tumor activity in preclinical cell line and animal models [Preclinical characterization of D3S-001, a highly potent, selective, and differentiated covalent inhibitor of KRAS G12C, ASCO 2022 Abstract #378099]. The current study utilizes pharmacokinetic (PK) modeling to predict the extent of KRAS G12C target inhibition at the proposed clinical dose range. Methods: Allometric scaling and non-linear fixed effect modeling were used to predict human PK profiles from DMPK data in animals. Human in vivo target inhibition was calculated based on human PK predictions, free drug fraction and results from in vitro target engagement assay. A First-in-Human (FIH) study was designed to investigate D3S-001 monotherapy in subjects with advanced solid tumors having a KRAS G12C mutation. Six dose escalation cohorts will be investigated, starting from 50mg QD. Results: Based on a GLP toxicology study in dogs, the highest non-severely toxic dose (HNSTD) was determined to be 20 mg/kg/day. Per ICH S9, a 20 mg/kg/day dose in dog supports a FIH starting dose of 110 mg. To ensure an additional safety margin, a lower starting dose of 50 mg was proposed. At 50 mg QD, the predicted human exposure (Cmax 60 nM, Caverage 49 nM, Ctrough 31 nM) is expected to achieve ̃70% target inhibition at 2 hours after dosing. In NCI-H358 mouse xenograft model, this exposure is associated with 94% tumor growth inhibition (TGI). Safety margins based on Cmax and AUC comparing to dog HNSTD are 17-fold and 9-fold, respectively. At 100 mg QD, the predicted human exposure (Cmax 120 nM, Caverage 98 nM, Ctrough 63 nM) is expected to achieve ̃95% target inhibition. This exposure is associated with tumor regression of 72% in the NCI-H358 mouse xenograft model. At dose levels higher than 100 mg QD, predicted human exposures are expected to consistently achieve >95% target inhibition starting 1 hour after dosing. These exposures are associated with deeper levels of tumor regression of 97%. Moreover, even at 400 mg QD, predicted human plasma concentrations are still below dog plasma concentration of 443 nM, Caverage at HNSTD. Conclusions: D3S-001 is predicted to achieve rapid and consistently efficacious KRAS G12C target inhibition in humans at proposed clinical dose range. This high degree of KRAS G12C inhibition over the dosing interval may translate into greater clinical benefit to patients with KRAS G12C mutations comparing with other clinical-stage KRAS G12C inhibitors. Clinical data from the FIH study will be evaluated and compared to preclinical predictions.

Mechanistic competitive binding interaction study between olmutinib and colchicine with model transport protein using spectroscopic and computer simulation approaches

Drug-drug interactions are due to the binding of a drug to plasma proteins displacing another plasma protein-bound drug. Two ligands, olmutinib (OLM) and colchicine (COL), were explored for their binding and displacement interaction with bovine serum albumin (BSA). Olmutinib is a 3rd-generation tyrosine kinase inhibitor (TKI), inhibits EGFR. COL is an anti-mitotic agent used in the treatment of gouty arthritis and has chemotherapeutic properties. The binding of BSA with OLM and COL was evaluated independently and in the presence of each other with spectroscopic and molecular docking and molecular dynamic simulation. Results based on quenching curves, displacement curves, binding constants, and docking results were evaluated. The presence of either drug (OLM or COL) influenced the binding of OLM and COL to BSA. The OLM had a higher binding capacity to BSA compared to COL. The presence of COL in the BSA-OLM system lowered the binding constants in comparison to its binary system. The OLM in the BSA-COL system increased the binding constant values for the system compared to the BSA-COL system. These findings are essential since increased non-bound fraction or reduced free drug fraction availability in the systemic circulation. The interference in the binding of drugs may lead to adverse events or subtherapeutic responses of the drugs.

Dissolution/Permeation of Albendazole in the Presence of Cyclodextrin and Bile Salts: A Mechanistic In Vitro Study into Factors Governing Oral Bioavailability

We aimed to understand the impact of the interplay between bile salts and cyclodextrins on the dissolution-permeation of poorly soluble drug compounds with a moderate-strong binding constant to cyclodextrin. Phase diagrams were prepared on the chosen model compound albendazole in phosphate buffer, fasted state simulated intestinal fluid (FaSSIF), and a modified fed state simulated intestinal fluid (FeSSIFmod) with (2-hydroxypropyl)-beta-cyclodextrin (HP-β-CD) concentrations of up to 10 % (m/m). Then we investigated the dissolution/permeation interplay of albendazole dissolved/suspended in the different media through a biomimetic barrier on a 96-well in vitro model. The apparent solubility of albendazole was enhanced by HP-β-CD and FaSSIF/FeSSIFmod separately. However, when albendazole was dissolved in HP-β-CD and biomimetic media together, the solubility was significantly lower than the predicted additive solubility from the solubilizing effects. It is postulated that this is due to the sodium taurocholate from the biomimetic media displacing albendazole from the hydrophobic cavity of HP-β-CD. In the permeation experiments, the highest permeation was observed at cyclodextrin concentrations able to solubilize close to the total dose of albendazole without a major surplus of solubilization capacity. Furthermore, an over-proportional permeation enhancement was observed when both, cyclodextrin and biomimetic media were present. These results indicate that the interplay between bile salts and cyclodextrins can enhance the free (molecularly dissolved) fraction of drug in solution to a greater extent than could be obtained with one of the solubilizing components alone. In conclusion, at carefully selected cyclodextrin-concentrations in combination with biomimetic media, obviously, a transient supersaturation is induced, which is made responsible for the observed major permeation enhancement.

Impact of Changes in Free Concentrations and Drug-Protein Binding on Drug Dosing Regimens in Special Populations and Disease States.

Over the last few decades, scientists and clinicians have often focused their attention on the unbound fraction of drugs as an indicator of efficacy and the eventual outcome of drug treatments for specific illnesses. Typically, the total drug concentration (bound and unbound) in plasma is used in clinical trials to assess a compound's efficacy. However, the free concentration of a drug tends to be more closely related to its activity and interaction with the body. Thus far, measuring the unbound concentration has been a challenge. Several mechanistic models have attempted to solve this problem by estimating the free drug fraction from available data such as total drug and binding protein concentrations. The aims of this review are first, to give an overview of the methods that have been used to date to calculate the unbound drug fraction. Second, to assess the pharmacokinetic parameters affected by changes in drug protein binding in special populations such as pediatrics, the elderly, pregnancy, and obesity. Third, to review alterations in drug protein binding in some selected disease states and how these changes impact the clinical outcomes for the patients; the disease states include critical illnesses, transplantation, renal failure, chronic kidney disease, and epilepsy. And finally, to discuss how various disease states shift the ratio of unbound to total drug and the consequences of such shifts on dosing adjustments and reaching the therapeutic target.

A Comprehensive Spectroscopic Analysis of the Ibuprofen Binding with Human Serum Albumin, Part II

Human serum albumin (HSA) is the most abundant human plasma protein. HSA plays a crucial role in many binding endos- and exogenous substances, which affects their pharmacological effect. The innovative aspect of the study is not only the interaction of fatted (HSA) and defatted (dHSA) human serum albumin with ibuprofen (IBU), but the analysis of the influence of temperature on the structural modifications of albumin and the interaction between the drug and proteins from the temperature characteristic of near hypothermia (308 K) to the temperature reflecting inflammation in the body (312 K and 314 K). Ibuprofen is a non-steroidal anti-inflammatory drug. IBU is used to relieve acute pain, inflammation, and fever. To determine ibuprofen’s binding site in the tertiary structure of HSA and dHSA, fluorescence spectroscopy was used. On its basis, the fluorescent emissive spectra of albumin (5 × 10−6 mol/dm3) without and with the presence of ibuprofen (1 × 10−5–1 × 10−4 mol/dm3) was recorded. The IBU-HSA complex’s fluorescence was excited by radiation of wavelengths of λex 275 nm and λex 295 nm. Spectrophotometric spectroscopy allowed for recording the absorbance spectra (zero-order and second derivative absorption spectra) of HSA and dHSA under the influence of ibuprofen (1 × 10−4 mol/dm3). To characterize the changes of albumin structure the presence of IBU, circular dichroism was used. The data obtained show that the presence of fatty acids and human serum albumin temperature influences the strength and type of interaction between serum albumin and drug. Ibuprofen binds more strongly to defatted human serum albumin than to albumin in the presence of fatty acids. Additionally, stronger complexes are formed with increasing temperatures. The competitive binding of ibuprofen and fatty acids to albumin may influence the concentration of free drug fraction and thus its therapeutic effect.

Prediction of Free Drug Absorption in Cyclodextrin Formulation by a Modified Physiologically Based Pharmacokinetic Model and Phase Solubility 3-D Surface Graph

Cyclodextrin (CD) is commonly used to enhance the solubility of oral drugs. However, with the increase of CD concentrations, the fraction of free drug molecules decreases, which may potentially impede drug absorption. This study aims to predict the optimal ratio between drug and CD to achieve the best absorption efficiency by computational simulation. First, a physiologically based pharmacokinetic (PBPK) model was developed. This model can continuously adjust absorption according to free drug fraction and was validated against two model drugs, progesterone (PG) and andrographolide (AG). The further analysis involves 3-D surface graphs to investigate the relationship between free drug amount, theoretically absorbable concentration, and contents of drug and CD in the formulation. The PBPK model predicted the PK behavior of two drugs well. The concentration ratio of drug to CD, leading to maximal free drug amount and the best absorption efficiency, is nearly the same as the slope determined in the phase solubility test. The new modified PBPK model and 3-D surface graph can easily predict the absorption difference of formulations with various drug/CD ratios. This PBPK model and 3-D surface graph can predict the absorption and determine the optimal concentration ratio of CD formulation, which could accelerate the R&D of CD formulation.

Characterization of drug binding with alpha1-acid glycoprotein in clinical samples using ultrafast affinity extraction

Many drugs bind to serum transport proteins, which can affect both drug distribution and activity in the body. α1-Acid glycoprotein (AGP) is a key transport protein for basic and neutral drugs. Both elevated levels and altered glycosylation patterns of AGP have been seen in clinical conditions such as systemic lupus erythematosus (SLE). This study developed, optimized, and used the method of ultrafast affinity extraction (UAE) to examine whether these changes in AGP are associated with changes in the binding by some drugs to this transport protein. This approach used affinity microcolumns to capture and measure, in serum, the free fractions of several drugs known to bind AGP. These measurements were made with pooled normal control serum and serum samples from individuals with SLE. Immunoaffinity chromatography was used to obtain the content of AGP and HSA in these samples, and CE was used to examine the glycoform pattern for AGP in each serum sample. The free drug fractions measured for normal control serum ranged from 3.5 to 29.1%, in agreement with the results of ultrafiltration, and provided binding constants of ~105–106 M−1 for the given drugs with AGP at 37⁰C. Analysis of a screening set of SLE serum samples by UAE gave decreased free fractions (relative change, 12-55%) vs normal serum when spiked with the same types and amounts of drugs. These changes were related in some cases to AGP content, with some SLE samples having AGP levels 1.3- to 2.1-fold above the upper end of the normal range. In other cases, the changes in free fractions appeared to be linked to alterations in the glycoforms and binding constants of AGP, with some affinities differing by 1.2- to 1.5-fold vs normal AGP. This approach can be employed with other solute-protein systems and to investigate binding by other drugs or transport proteins directly in clinical samples.

Canine orosomucoid (alpha-1 acid glycoprotein) variants and their influence on drug plasma protein binding.

Orosomucoid polymorphisms influence plasma drug binding in humans; however, canine variants and their effect on drug plasma protein binding have not yet been reported. In this study, the orosomucoid gene (ORM1) was sequenced in 100 dogs to identify the most common variant and its allele frequency determined in 1,464 dogs (from 64 breeds and mixed-breed dogs). Plasma protein binding extent of amitriptyline, indinavir, verapamil, and lidocaine were evaluated by equilibrium dialysis using plasma from ORM1 genotyped dogs (n=12). Free and total drug plasma concentrations were quantified by liquid chromatography-mass spectrometry. From the five polymorphisms identified in canine ORM1, two were nonsynonymous. The most common was c.70G>A (p.Ala24Thr) with an allele frequency of 11.2% (n=1464). Variant allele frequencies varied by breed, reaching 74% in Shetland Sheepdogs (n=21). Free drug fractions did not differ significantly (p>.05; Mann-Whitney U) between plasma collected from dogs with c.70AA (n=4) and those with c.70GG (n=8) genotypes. While c.70G>A did not affect the extent of plasma protein binding in our study, the potential biological and pharmacological implication of this newly discovered ORM1 variant in dogs should be further investigated.

Strategy for Determining the Free Fraction of Labile Covalent Modulators in Plasma Using Equilibrium Dialysis

Determination of free drug fraction (fu) in plasma can be challenging for labile covalent modulators due to the off-target reactivity of chemical warheads to matrix proteins. The resulting poor drug recovery yields low confidence in fu. Two approaches using diluted plasma and low temperature (4 & 20 °C) for equilibrium dialysis (ED) have been investigated using covalent modulators including osimertinib, ibrutinib, rociletinib, afatinib, neratinib and acalabrutinib. Our data indicate that stability of covalent modulators in plasma varies in different species, and drug depletion may lead to overestimation of fu if true equilibrium is not reached. Additionally, although ED at low temperature improves the recovery of covalent modulators, the impact of low temperature may lead to underestimate of fu. Overall, ED using diluted plasma is a preferred method because of its faster equilibrium, improved recovery and free of temperature effect on fu. If low temperature ED must be used for extremely labile compounds, precaution must be taken to ensure no temperature dependence of fu in plasma. Nevertheless, an orthogonal ED approach is recommended for labile covalent modulators to confirm the true equilibrium and impact of temperature on fu. Additionally, this strategy can be used for determining fu of other liable compounds.

A model-based analysis of phenytoin and carbamazepine toxicity treatment using binding-competition during hemodialysis

Hemodialysis (HD) has limited efficacy towards treatment of drug toxicity due to strong drug-protein binding. In this work, we propose to infuse a competitor drug into the extracorporeal circuit that increases the free fraction of a toxic drug and thereby increases its dialytic removal. We used a mechanistic model to assess the removal of phenytoin and carbamazepine during HD with or without binding-competition. We simulated dialytic removal of (1) phenytoin, initial concentration 70 mg/L, using 2000 mg aspirin, (2) carbamazepine, initial concentration 35 mg/L, using 800 mg ibuprofen, in a 70 kg patient. The competitor drug was infused at constant rate. For phenytoin (~ 13% free at t = 0), HD brings the patient to therapeutic concentration in 460 min while aspirin infusion reduces that time to 330 min. For carbamazepine (~ 27% free at t = 0), the ibuprofen infusion reduces the HD time to reach therapeutic concentration from 265 to 220 min. Competitor drugs with longer half-life further reduce the HD time. Binding-competition during HD is a potential treatment for drug toxicities for which current recommendations exclude HD due to strong drug-protein binding. We show clinically meaningful reductions in the treatment time necessary to achieve non-toxic concentrations in patients poisoned with these two prescription drugs.

Simultaneous determination of free and total paclitaxel in blood in a three-phase laminar flow microchip

In this study, a three-phase laminar flow microfluidic chip (TPL chip) combined with HPLC was developed for monitoring free and total concentrations of paclitaxel (PTX) in blood simultaneously. A diluted whole blood sample (aqueous phase) was introduced into the chip, ethyl acetate (organic phase) was introduced into the chip for extraction, and an interphase was used to prevent the blood sample from coming into direct contact with the organic phase. Because only free drug can quantitatively diffuse into the organic extraction phase and the free drug fraction has a linear relationship with the dilution factor of blood, both the free and total drug concentrations can be obtained by detecting the concentration of paclitaxel in the organic extraction phase. The governing factor such as flow rate for extraction was optimized. Docetaxel was used as an internal standard. The reliability of the quantitative diffusion of molecules in the TPL chip was proved by the methodological investigation of PTX in PBS sample, which showed a good linearity in the concentration range of 0.5 - 100µg/mL and a detection limit of 7ng/mL. Good repeatibilities for retention time (RSD of PTX is 1.23%, docetaxel is 1.14%, n=5) and peak area ratio of PTX to docetaxel (RSD is 4.38%) were obtained. For blood sample analysis, only 100 µL of sample was needed and whole pretreatment was finished in 35min, and a recovery of 94~117% were obtained. The provided method showed advantages in fast analysis speed, minimum sample handing, and potential ability of automation, and integration.

Biopharmaceutic In Vitro In Vivo Extrapolation (IVIV_E) Informed Physiologically-Based Pharmacokinetic Model of Ritonavir Norvir Tablet Absorption in Humans Under Fasted and Fed State Conditions.

Ritonavir is a well-known CYP3A4 and CYP2D6 enzyme inhibitor, frequently used to assess the drug-drug interaction (DDI) liability of susceptible drugs. It is also used as a pharmacokinetic booster to increase exposure to CYP3A4 substrates. This study aimed to develop a mechanistic absorption and disposition model to describe exposure to ritonavir following oral dosing of the commercial amorphous solid dispersion tablet, Norvir, under fasted and fed conditions. A mechanistic description of ritonavir absorption from Norvir tablets may help to improve the design of DDI studies. Key parameters of amorphous ritonavir including free base solubility (solubility of the unbound, un-ionized species), bile micelle partition coefficients, formulation wetting/disintegration, and in vivo precipitation parameters were either obtained from the literature or estimated by modeling in vitro biopharmaceutic experiments. Based on variety of in vitro evidence, a main assumption of the model is that ritonavir does not form a crystalline precipitate while resident in the gastrointestinal tract. In the model, if simulated luminal concentration exceeds the amorphous solubility limit, then precipitation to an amorphous form is immediate. Simulated and observed Cmax and AUC0-t parameters were well captured (within 1.5-fold) for both fasted and fed states in healthy volunteers. By accounting for luminal fluid viscosity differences in the different prandial states (affecting drug diffusivity) as well as the effect of drug free fraction on gut wall permeation rates, it was possible to explain the negative food effect observed for Norvir tablets in humans. In summary, a biopharmaceutic in vitro in vivo extrapolation approach provides confidence in (verification of) key input parameters of the physiologically-based pharmacokinetic ritonavir model which resulted in successful simulation of observed plasma profiles.