Bridging population pharmacokinetic and semimechanistic absorption modeling of APX3330.

APX3330 ((2E)-2-[(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)methylene]-undecanoic acid), a selective inhibitor of APE1/Ref-1, has been investigated in treatment of hepatitis, cancer, diabetic retinopathy, and macular edema. APX3330 is administered orally as a quinone but is rapidly converted to the hydroquinone form. This study describes the pharmacokinetics of APX3330 and explores effect of food on absorption. Total plasma quinone concentrations of APX3330 were obtained following oral administration from studies in healthy Japanese male subjects (single dose-escalation; multiple-dose; food-effect) and patients with cancer patients. Nonlinear mixed effects modeling was performed using Monolix to estimate pharmacokinetic parameters and assess covariate effects. To further evaluate the effect of food on absorption, a semi-physiologic pharmacokinetic model was developed in Gastroplus to delineate effects of food on dissolution and absorption. A two-compartment, first order absorption model with lag time best described plasma concentration-time profiles from 49 healthy Japanese males. Weight was positively correlated with apparent clearance (CL/F) and volume. Administration with food led to an 80% higher lag time. CL/F was 41% higher in the cancer population. The semi-physiologic model indicates a switch from dissolution-rate control of absorption in the fasted-state to gastric emptying rate determining absorption rate in the fed-state. Oral clearance of APX3330 is higher in patients with cancer than healthy Japanese males, possibly due to reduced serum albumin in patients with cancer. Delayed APX3330 absorption with food may be related to higher conversion to the more soluble but less permeable hydroquinone form in the gastrointestinal tract. Future work should address pharmacokinetic differences between APX3330 quinone and hydroquinone forms.

Characterization of the Stereoselective Disposition of Bupropion and Its Metabolites in Rat Plasma and Brain.

BACKGROUND AND OBJECTIVES: Bupropion is an atypical antidepressant and smoking cessation aid; its use is associated with wide intersubject variability in efficacy and safety. Knowledge of the brain pharmacokinetics of bupropion and its pharmacologically active metabolites is considered important for understanding the cause-effect relationships driving this variability. METHODS: Brain concentrations from rats administered a 10 mg/kg subcutaneous dose of racemic bupropion were analyzed using a stereoselective LC/MS-MS method. A 2 mg/kg dose of (S,S)-hydroxybupropion, which has comparable pharmacologic potency to bupropion, was administered to a separate group of rats. Plasma exposure and unbound concentrations in both matrices from companion equilibrium dialysis experiments were determined to assess potential carrier-mediated transport at the blood-brain barrier. RESULTS: Exposures to unbound forms of bupropion enantiomers were similar in plasma; this was also true in brain. This trend held for reductive diastereomer metabolite pairs in the two matrices. Unbound (R,R)-hydroxybupropion exposure was 1.5-fold higher than (S,S)-hydroxybupropion exposure in plasma and brain following bupropion administration. Unbound concentration ratios (Kp,uu) of a given molecular form decreased over time: between 4 and 6 h, these were < 1 for the two bupropion enantiomers, and they were ~ 1 for metabolites that formed. Administration of preformed (S,S)-hydroxybupropion also demonstrated a declining Kp,uu. CONCLUSIONS: The temporal shift in Kp,uu among the different molecular forms provides evidence regarding the operation of carrier-mediated transport and/or within-brain metabolism of bupropion, and, thereby, fresh insight regarding the causes of intersubject variability in the safety and efficacy of bupropion therapy.

Multiscale Model of Antiviral Timing, Potency, and Heterogeneity Effects on an Epithelial Tissue Patch Infected by SARS-CoV-2.

We extend our established agent-based multiscale computational model of infection of lung tissue by SARS-CoV-2 to include pharmacokinetic and pharmacodynamic models of remdesivir. We model remdesivir treatment for COVID-19; however, our methods are general to other viral infections and antiviral therapies. We investigate the effects of drug potency, drug dosing frequency, treatment initiation delay, antiviral half-life, and variability in cellular uptake and metabolism of remdesivir and its active metabolite on treatment outcomes in a simulated patch of infected epithelial tissue. Non-spatial deterministic population models which treat all cells of a given class as identical can clarify how treatment dosage and timing influence treatment efficacy. However, they do not reveal how cell-to-cell variability affects treatment outcomes. Our simulations suggest that for a given treatment regime, including cell-to-cell variation in drug uptake, permeability and metabolism increase the likelihood of uncontrolled infection as the cells with the lowest internal levels of antiviral act as super-spreaders within the tissue. The model predicts substantial variability in infection outcomes between similar tissue patches for different treatment options. In models with cellular metabolic variability, antiviral doses have to be increased significantly (>50% depending on simulation parameters) to achieve the same treatment results as with the homogeneous cellular metabolism.

Novel Quantification of Extracellular Vesicles with Unaltered Surface Membranes Using an Internalized Oligonucleotide Tracer and Applied Pharmacokinetic Multiple Compartment Modeling.

PURPOSE: We developed an accessible method for labeling small extracellular vesicles (sEVs) without disrupting endogenous ligands. Using labeled sEVs administered to conscious rats, we developed a multiple compartment pharmacokinetic model to identify potential differences in the disposition of sEVs from three different cell types. METHODS: Crude sEVs were labeled with a non-homologous oligonucleotide and isolated from cell culture media using a commercial reagent. Jugular vein catheters were used to introduce EVs to conscious rats (n = 30) and to collect blood samples. Digital PCR was leveraged to allow for quantification over a wide dynamic range. Non-linear mixed effects analysis with first order conditional estimation - extended least squares (FOCE ELS) was used to estimate population-level parameters with associated intra-animal variability. RESULTS: 86.5% ± 1.5% (mean ± S.E.) of EV particles were in the 45-195 nm size range and demonstrated protein and lipid markers of endosomal origin. Incorporated oligonucleotide was stable in blood and detectable over five half-lives. Data were best described by a three-compartment model with one elimination from the central compartment. We performed an observation-based simulated posterior predictive evaluation with prediction-corrected visual predictive check. Covariate and bootstrap analyses identified cell type having an influence on peripheral volumes (V2 and V3) and clearance (Cl3). CONCLUSIONS: Our method relies upon established laboratory techniques, can be tailored to a variety of biological questions regarding the pharmacokinetic disposition of extracellular vesicles, and will provide a complementary approach for the of study EV ligand-receptor interactions in the context of EV uptake and targeted therapeutics.

Inhibiting Fatty Acid Synthase with Omeprazole to Improve Efficacy of Neoadjuvant Chemotherapy in Patients with Operable TNBC.

PURPOSE: Fatty acid synthase (FASN) is overexpressed in 70% of operable triple-negative breast cancer (TNBC) and is associated with poor prognosis. Proton pump inhibitors selectively inhibit FASN activity and induce apoptosis in TNBC cell lines. PATIENTS AND METHODS: Patients with operable TNBC were enrolled in this single-arm phase II study. Patients began omeprazole 80 mg orally twice daily for 4-7 days prior to neoadjuvant anthracycline-taxane-based chemotherapy (AC-T) and continued until surgery. The primary endpoint was pathologic complete response (pCR) in patients with baseline FASN overexpression (FASN+). Secondary endpoints included pCR in all surgery patients, change in FASN expression, enzyme activity, and downstream protein expression after omeprazole monotherapy, safety, and limited omeprazole pharmacokinetics. RESULTS: Forty-two patients were recruited with a median age of 51 years (28-72). Most patients had ≥cT2 (33, 79%) and ≥N1 (22, 52%) disease. FASN overexpression prior to AC-T was identified in 29 of 34 (85%) evaluable samples. The pCR rate was 72.4% [95% confidence interval (CI), 52.8-87.3] in FASN+ patients and 74.4% (95% CI, 57.9-87.0) in all surgery patients. Peak omeprazole concentration was significantly higher than the IC50 for FASN inhibition observed in preclinical testing; FASN expression significantly decreased with omeprazole monotherapy [mean change 0.12 (SD, 0.25); P = 0.02]. Omeprazole was well tolerated with no grade ≥ 3 toxicities. CONCLUSIONS: FASN is commonly expressed in early TNBC. Omeprazole can be safely administered in doses that inhibit FASN. The addition of omeprazole to neoadjuvant AC-T yields a promising pCR rate that needs further confirmation in randomized studies.

Application of machine learning to predict reduction in total PANSS score and enrich enrollment in schizophrenia clinical trials.

Clinical trial efficiency, defined as facilitating patient enrollment, and reducing the time to reach safety and efficacy decision points, is a critical driving factor for making improvements in therapeutic development. The present work evaluated a machine learning (ML) approach to improve phase II or proof-of-concept trials designed to address unmet medical needs in treating schizophrenia. Diagnostic data from the Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) trial were used to develop a binary classification ML model predicting individual patient response as either "improvement," defined as greater than 20% reduction in total Positive and Negative Syndrome Scale (PANSS) score, or "no improvement," defined as an inadequate treatment response (<20% reduction in total PANSS). A random forest algorithm performed best relative to other tree-based approaches in model ability to classify patients after 6 months of treatment. Although model ability to identify true positives, a measure of model sensitivity, was poor (<0.2), its specificity, true negative rate, was high (0.948). A second model, adapted from the first, was subsequently applied as a proof-of-concept for the ML approach to supplement trial enrollment by identifying patients not expected to improve based on their baseline diagnostic scores. In three virtual trials applying this screening approach, the percentage of patients predicted to improve ranged from 46% to 48%, consistently approximately double the CATIE response rate of 22%. These results show the promising application of ML to improve clinical trial efficiency and, as such, ML models merit further consideration and development.

Novel bone-targeted parathyroid hormone-related peptide antagonists inhibit breast cancer bone metastases.

Patients with advanced breast cancer often develop bone metastases. Treatment is limited to palliative care. Parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP) antagonists for bone metastases failed clinically due to short half-life and inadequate concentration in bone. We synthesized two novel PTHrP antagonists fused to an inert bacterial collagen binding domain (CBD) that directs drugs to bone. PTH(7-33)-CBD is an N-terminal truncated PTHrP antagonist. [W2]PTH(1-33)-CBD is an PTHrP inverse-agonist. The aim of this study was to assess PTH(7-33)-CBD to reduce breast cancer bone metastases and prevent osteolytic destruction in mice and to assess both drugs for apoptosis of breast cancer cells in vitro and inhibition of PTH receptor (PTHR1). PTH(7-33)-CBD (1000 µg/kg, subcutaneous) or vehicle was administered 24 h prior to MDA-MB-231 breast cancer cell inoculation into the tibia marrow. Weekly tumor burden and bone density were measured. Pharmacokinetic analysis of PTH(7-33)-CBD in rat serum was evaluated. Drug effect on cAMP accumulation in SaOS-2 osteosarcoma cells and apoptosis of MDA-MB-231 cells was assessed. PTH(7-33)-CBD reduced MDA-MB-231 tumor burden and osteolytic destruction in mice 4-5 weeks post-treatment. PTH(7-33)-CBD (1000 µg/kg i.v. and subcutaneous) in rats was rapidly absorbed with peak concentration 5-min and terminal half-life 3-h. Bioavailability by the subcutaneous route was 43% relative to the i.v. route. PTH(7-33)-CBD was detected only on rat periosteal bone surfaces that stained positive for collagen-1. PTH(7-33)-CBD and [W2]PTH(1-33)-CBD (10-8M) blocked basal and PTH agonist-induced cAMP accumulation in SaOS-2 osteosarcoma cells. Both drugs induced PTHR1-dependent apoptosis of MDA-MB-231 cells in vitro. Novel bone-targeted PTHrP antagonists represent a new paradigm for treatment of breast cancer bone metastases.

Population model analysis of chiral inversion and degradation of bupropion enantiomers, and application to enantiomer specific fraction unbound determination in rat plasma and brain.

Pharmacologic effects elicited by drugs most directly relate to their unbound concentrations. Measurement of binding in blood, plasma and target tissues are used to estimate these concentrations by determining the fraction of total concentration in a biological matrix that is not bound. In the case of attempting to estimate R- and S-bupropion concentrations in plasma and brain following racemic bupropion administration, reversible chiral inversion and irreversible degradation of the enantiomers were hypothesized to confound attempts at unbound fraction estimation. To address this possibility, a kinetic modeling approach was used to quantify inversion and degradation specific processes for each enantiomer from separate incubations of each enantiomer in the two matrices, and in pH 7.4 buffer, which is also used in binding experiments based on equilibrium dialysis. Modeling analyses indicated that chiral inversion kinetics were two to four-fold faster in plasma and brain than degradation, with only inversion observed in buffer. Inversion rate was faster for S-bupropion in the three media; whereas, degradation rates were similar for the two enantiomers in plasma and brain, with overall degradation in plasma approximately 2-fold higher than in brain homogenate. Incorporation of degradation and chiral inversion kinetic terms into a model to predict enantiomer-specific binding in plasma and brain revealed that, despite existence of these two processes, empirically derived estimates of fraction unbound were similar to model-derived values, leading to a firm conclusion that observed extent of plasma and brain binding are accurate largely because binding kinetics are faster than parallel degradation and chiral inversion processes.

Simultaneous Pharmacokinetic Analysis of Nitrate and its Reduced Metabolite, Nitrite, Following Ingestion of Inorganic Nitrate in a Mixed Patient Population.

PURPOSE: The pharmacokinetic properties of plasma NO3- and its reduced metabolite, NO2-, have been separately described, but there has been no reported attempt to simultaneously model their pharmacokinetics following NO3- ingestion. This report describes development of such a model from retrospective analyses of concentrations largely obtained from primary endpoint efficacy trials. METHODS: Linear and non-linear mixed effects analyses were used to statistically define concentration dependency on time, dose, as well as patient and study variables, and to integrate NO3- and NO2- concentrations from studies conducted at different times, locations, patient groups, and several studies in which sample range was limited to a few hours. Published pharmacokinetic studies for both substances were used to supplement model development. RESULTS: A population pharmacokinetic model relating NO3- and NO2- concentrations was developed. The model incorporated endogenous levels of the two entities, and determined these were not influenced by exogenous NO3- delivery. Covariate analysis revealed intersubject variability in NO3- exposure was partially described by body weight differences influencing volume of distribution. The model was applied to visualize exposure versus response (muscle contraction performance) in individual patients. CONCLUSIONS: Extension of the present first-generation model, to ultimately optimize NO3- dose versus pharmacological effects, is warranted.

Pharmacological, Mechanistic, and Pharmacokinetic Assessment of Novel Melatonin-Tamoxifen Drug Conjugates as Breast Cancer Drugs.

Tamoxifen is used to prevent and treat estrogen receptor-positive (ER+) breast cancer (BC); however, its chronic use can increase uterine cancer risk and induce tamoxifen resistance. Novel melatonin-tamoxifen drug conjugates may be promising to treat BC and may help offset the adverse effects of tamoxifen usage alone due to the presence of melatonin. We synthesized and screened five drug conjugates (C2, C4, C5, C9, and C15 linked) for their effects on BC cell (MCF-7, tamoxifen-resistant MCF-7, mouse mammary carcinoma, MDA-MB-231, and BT-549) viability, migration, and binding affinity to melatonin receptor 1 (MT1R) and estrogen receptor 1 (ESR1). C4 and C5 demonstrated the most favorable pharmacological characteristics with respect to binding profiles (affinity for ESR1 and MT1R) and their potency/efficacy to inhibit BC cell viability and migration in four phenotypically diverse invasive ductal BC cell lines. C4 and C5 were further assessed for their actions against tamoxifen-resistant MCF-7 cells and a patient-derived xenograft triple-negative BC cell line (TU-BcX-4IC) and for their mechanisms of action using selective mitogen-activated protein kinase kinase MEK1/2, MEK5, and phosphoinositide 3-kinase (PI3K) inhibitors. C4 and C5 inhibited tamoxifen-resistant MCF-7 cells with equal potency (IC50 = 4-8 µM) and efficacy (∼90% inhibition of viability and migration) but demonstrated increased potency (IC50 = 80-211 µM) and efficacy (∼140% inhibition) to inhibit migration versus cell viability (IC50 = 181-304 mM; efficacy ∼80% inhibition) in TU-BcX-4IC cells. Unique pharmacokinetic profiles were observed, with C4 having greater bioavailability than C5. Further assessment of C4 and C5 demonstrates that they create novel pharmacophores within each BC cell that is context specific and involves MEK1/2/pERK1/2, MEK5/pERK5, PI3K, and nuclear factor κB. These melatonin-tamoxifen drug conjugates show promise as novel anticancer drugs and further preclinical and clinical evaluation is warranted.

Development of a Semimechanistic Pharmacokinetic-Pharmacodynamic Model Describing Dextroamphetamine Exposure and Striatal Dopamine Response in Rats and Nonhuman Primates following a Single Dose of Dextroamphetamine.

Acute central nervous system exposure to dextroamphetamine (d-amphetamine) elicits a multitude of effects, including dual action on the dopamine transporter (DAT) to increase extracellular dopamine, and induction of a negative feedback response to limit the dopamine increase. A semimechanistic pharmacokinetic and pharmacodynamic (PK/PD) model with consideration of these multiple effects as a basis was developed. Integrated pharmacokinetics of d-amphetamine in plasma, brain extracellular fluid (ECF) via microdialysis, and cerebrospinal fluid were characterized using a population approach. This PK model was then linked to an indirect-response pharmacodynamic model using as a basis the measurement of extracellular striatal dopamine, also via microdialysis. In both rats and nonhuman primates (NHPs), d-amphetamine stimulation of dopamine outflow (reverse transport) through DAT was primarily responsible for the dose-linear increase in dopamine. As well, in both species a moderator function was needed to account for loss of the dopamine response in the presence of a relatively sustained d-amphetamine ECF exposure, presumptive of an acute tolerance response. PK/PD model structure was consistent between species; however, there was a 10-fold faster return to baseline dopamine in NHPs in response to an acute d-amphetamine challenge. These results suggest preservation from rodents to NHPs regarding the mechanism by which amphetamine increases extracellular dopamine, but a faster system response in NHPs to tolerate this increase. This microdialysis-based PK/PD model suggests greater value in directing preclinical discovery of novel approaches that modify reverse transport stimulation to treat amphetamine abuse. General value regarding insertion of an NHP model in paradigm rodent-to-human translational research is also suggested.

Development of a non-human primate model to support CNS translational research: Demonstration with D-amphetamine exposure and dopamine response.

BACKGROUND: Challenges specific to the discovery and development of candidate CNS drugs have led to implementation of various in silico, in vitro and in vivo approaches to improve the odds for commercialization of novel treatments. NEW METHOD: Advances in analytical methodology and microdialysis probe design have enabled development of a non-human primate model capable of measuring concentrations of drugs or endogenous chemicals in brain extracellular fluid (ECF) and cerebrospinal fluid (CSF). Linking these to population modeling reduces animal numbers to support predictive translational sciences in primates. Application to measure D-amphetamine exposure and dopamine response in ECF and CSF demonstrate the approach. RESULTS: Following a 0.1 mg/kg intravenous bolus dose of D-amphetamine, a population approach was used to build a plasma compartmental-based and brain physiologic-based pharmacokinetic (PK) model linking drug concentrations in plasma to brain ECF and CSF concentrations. Dopamine was also measured in brain ECF. The PK model was used to simulate the relationship between D-amphetamine exposure and dopamine response in ECF over a wide dose range. COMPARISONS WITH EXISTING METHODS: Ability to co-sample and measure drug and endogenous substances in blood, brain ECF and/or CSF, coupled with population modeling, provides an in vivo approach to evaluate CNS drug penetration and effect in non-human primates. CONCLUSIONS: A method to measure drug and endogenous neurochemicals in non-human primate brain fluids is demonstrated. Its basis in non-human primates merits improved confidence regarding predictions of drug exposure and target engagement in human CNS.

Comparison of In Vitro Stereoselective Metabolism of Bupropion in Human, Monkey, Rat, and Mouse Liver Microsomes.

BACKGROUND AND OBJECTIVES: Bupropion is an atypical antidepressant and smoking cessation aid associated with wide intersubject variability. This study compared the formation kinetics of three phase I metabolites (hydroxybupropion, threohydrobupropion, and erythrohydrobupropion) in human, marmoset, rat, and mouse liver microsomes. The objective was to establish suitability and limitations  for subsequent use of nonclinical species to model bupropion central nervous system (CNS) disposition in humans. METHODS: Hepatic microsomal incubations were conducted separately for the R- and S-bupropion enantiomers, and the formation of enantiomer-specific metabolites was determined using LC-MS/MS. Intrinsic formation clearance (CLint) of metabolites across the four species was determined from the formation rate versus substrate concentration relationship. RESULTS: The total clearance of S-bupropion was higher than that of R-bupropion in monkey and human liver microsomes. The contribution of hydroxybupropion to the total racemic bupropion clearance was 38%, 62%, 17%, and 96% in human, monkey, rat, and mouse, respectively.  In the same species order, threohydrobupropion contributed 53%, 23%, 17%, and 3%, and erythrohydrobupropion contributed 9%, 14%, 66%, and 1.3%, respectively, to racemic bupropion clearance. CONCLUSION: The results demonstrate that phase I metabolism in monkeys best approximates that observed in humans, and support the preferred use of this species to investigate possible pharmacokinetic factors that influence the CNS disposition of bupropion and contribute to its high intersubject variability.

Drug conjugates-an emerging approach to treat breast cancer.

Breast cancer treatment using a single drug is associated with a high failure rate due, in part, to the heterogeneity of drug response within individuals, nonspecific target action, drug toxicity, and/or development of resistance. Use of dual-drug therapies, including drug conjugates, may help overcome some of these roadblocks by more selective targeting of the cancer cell and by acting at multiple drug targets rather than one. Drug-conjugate approaches include linking drugs to antibodies (antibody-drug conjugates), radionuclides (radioimmunoconjugates), nanoparticles (nanoparticle-drug conjugates), or to other drugs (drug-drug conjugates). Although all of these conjugates might be designed as effective treatments against breast cancer, the focus of this review will be on drug-drug conjugates because of the increase in versatility of these types of drugs with respect to mode of action at the level of the cancer cell either by creating a novel pharmacophore or by increasing the potency and/or efficacy of the drugs' effects at their respective molecular targets. The development, synthesis, and pharmacological characteristics of drug-drug conjugates will be discussed in the context of breast cancer with the hope of enhancing drug efficacy and reducing toxicities to improve patient quality of life.

Cellular Pharmacokinetic Model-Based Analysis of Genistein, Glyceollin, and MK-571 Effects on 5 (and 6)-Carboxy-2',7'-Dichloroflourescein Disposition in Caco-2 Cells.

Pharmacokinetic modeling was used to describe 5 (and 6)-carboxy-2',7'-dichloroflourescein (CDF) disposition in Caco-2 cells following CDF or CDFDA (CDF diacetate) dosing. CDF transcellular flux was modeled by simple passive diffusion. CDFDA dosing models were based on simultaneous fitting of CDF levels in apical, basolateral, and intracellular compartments. Predicted CDF efflux was 50% higher across the apical versus the basolateral membrane. This difference was similar following apical and basolateral CDFDA dosing, despite intracellular levels being 3-fold higher following basolateral dosing, thus supporting nonsaturable CDF efflux kinetics. A 3-compartment catenary model with intracellular CDFDA hydrolysis described CDF disposition. This model predicted that apical CDF efflux was not altered in the presence of MK-571, and that basolateral membrane clearance was enhanced to account for reduced intracellular CDF in the presence of this multidrug resistance-associated protein (MRP) inhibitor. Similar effects were predicted for glyceollin, while genistein exposure had no predicted effects on CDF efflux. These modulator effects are discussed in the context of model predicted intracellular CDF concentrations relative to reports of CDF affinity (measured by Km) for MRP2 and MRP3. This model-based analysis confirms the complexity of efflux kinetics and suggests that other transporters may have contributed to CDF efflux.

Development of a Rat Plasma and Brain Extracellular Fluid Pharmacokinetic Model for Bupropion and Hydroxybupropion Based on Microdialysis Sampling, and Application to Predict Human Brain Concentrations.

Administration of bupropion [(±)-2-(tert-butylamino)-1-(3-chlorophenyl)propan-1-one] and its preformed active metabolite, hydroxybupropion [(±)-1-(3-chlorophenyl)-2-[(1-hydroxy-2-methyl-2-propanyl)amino]-1-propanone], to rats with measurement of unbound concentrations by quantitative microdialysis sampling of plasma and brain extracellular fluid was used to develop a compartmental pharmacokinetics model to describe the blood-brain barrier transport of both substances. The population model revealed rapid equilibration of both entities across the blood-brain barrier, with resultant steady-state brain extracellular fluid/plasma unbound concentration ratio estimates of 1.9 and 1.7 for bupropion and hydroxybupropion, respectively, which is thus indicative of a net uptake asymmetry. An overshoot of the brain extracellular fluid/plasma unbound concentration ratio at early time points was observed with bupropion; this was modeled as a time-dependent uptake clearance of the drug across the blood-brain barrier. Translation of the model was used to predict bupropion and hydroxybupropion exposure in human brain extracellular fluid after twice-daily administration of 150 mg bupropion. Predicted concentrations indicate that preferential inhibition of the dopamine and norepinephrine transporters by the metabolite, with little to no contribution by bupropion, would be expected at this therapeutic dose. Therefore, these results extend nuclear imaging studies on dopamine transporter occupancy and suggest that inhibition of both transporters contributes significantly to bupropion's therapeutic efficacy.

Glyceollin Effects on MRP2 and BCRP in Caco-2 Cells, and Implications for Metabolic and Transport Interactions.

Glyceollins are phytoalexins produced in soybeans under stressful growth conditions. On the basis of prior evaluations, they show potential to treat multiple diseases, including certain cancers, Type 2 diabetes, and cardiovascular conditions. The aim of the present study was to expand on recent studies designed to initially characterize the intestinal disposition of glyceollins. Specifically, studies were undertaken in Caco-2 cells to evaluate glyceollins' effects on apical efflux transporters, namely, MRP2 and BCRP, which are the locus of several intestinal drug-drug and drug-food interactions. 5- (and 6)-carboxy-2',7'-dichloroflourescein (CDF) was used to provide a readout on MRP2 activity, whereas BODIPY-prazosin provided an indication of BCRP alteration. Glyceollins were shown to reverse MRP2-mediated CDF transport asymmetry in a concentration-dependent manner, with activity similar to the MRP2 inhibitor, MK-571. Likewise, they demonstrated concentration-dependent inhibition of BCRP-mediated efflux of BODIPY-prazosin with a potency similar to that of Ko143. Glyceollin did not appreciably alter MRP2 or BCRP expression following 24 h of continuous exposure. The possibility that glyceollin mediated inhibition of genistein metabolite efflux by either transporter was evaluated. However, results demonstrated an interaction at the level of glyceollin inhibition of genistein metabolism rather than inhibition of metabolite transport.

Genistein and Glyceollin Effects on ABCC2 (MRP2) and ABCG2 (BCRP) in Caco-2 Cells.

The goal of the present study was to determine the effects of glyceollins on intestinal ABCC2 (ATP Binding Cassette C2, multidrug resistance protein 2, MRP2) and ABCG2 (ATP Binding Cassette G2, breast cancer resistance protein, BCRP) function using the Caco-2 cell intestinal epithelial cell model. Glyceollins are soy-derived phytoestrogens that demonstrate anti-proliferative activity in several sources of cancer cells. 5 (and 6)-carboxy-2',7'-dichloroflourescein (CDF) was used as a prototypical MRP2 substrate; whereas BODIPY-prazosin provided an indication of BCRP function. Comparison studies were conducted with genistein. Glyceollins were shown to inhibit MRP2-mediated CDF transport, with activity similar to the MRP2 inhibitor, MK-571. They also demonstrated concentration-dependent inhibition BCRP-mediated efflux of BODIPY-prazosin, with a potency similar to that of the recognized BCRP inhibitor, Ko143. In contrast, genistein did not appear to alter MRP2 activity and even provided a modest increase in BCRP efflux of BODIPY-prazosin. In particular, glyceollin inhibition of these two important intestinal efflux transporters suggests the potential for glyceollin to alter the absorption of other phytochemicals with which it might be co-administered as a dietary supplement, as well as alteration of the absorption of pharmaceuticals that may be administered concomitantly.

Design and synthesis of N-[6-(Substituted Aminoethylideneamino)-2-Hydroxyindan-1-yl]arylamides as selective and potent muscarinic M1 agonists.

The observation that cholinergic deafferentation of circuits projecting from forebrain basal nuclei to frontal and hippocampal circuits occurs in Alzheimer's disease has led to drug-targeting of muscarinic M1 receptors to alleviate cognitive symptoms. The high homology within the acetylcholine binding domain of this family however has made receptor-selective ligand development challenging. This work presents the synthesis scheme, pharmacokinetic and structure-activity-relationship study findings for M1-selective ligand, LY593093. Pharmacologically the compound acts as an orthosteric ligand. The homology modeling work presented however will illustrate that compound binding spans from the acetylcholine pocket to the extracellular loops of the receptor, a common allosteric vestibule for the muscarinic protein family. Altogether LY593093 represents a growing class of multi-topic ligands which interact with the receptors in both the ortho- and allosteric binding sites, but which exert their activation mechanism as an orthosteric ligand.

Prediction of brain clozapine and norclozapine concentrations in humans from a scaled pharmacokinetic model for rat brain and plasma pharmacokinetics.

BACKGROUND: Clozapine is highly effective in treatment-resistant schizophrenia, although, there remains significant variability in the response to this drug. To better understand this variability, the objective of this study was to predict brain extracellular fluid (ECF) concentrations and receptor occupancy of clozapine and norclozapine in human central nervous system by translating plasma and brain ECF pharmacokinetic (PK) relationships in the rat and coupling these with known human disposition of clozapine in the plasma. METHODS: Unbound concentrations of clozapine and norclozapine were measured in rat brain ECF using quantitative microdialysis after subcutaneous administration of a 10 mg/kg single dose of clozapine or norclozapine. These data were linked with plasma concentrations obtained in the same rats to develop a plasma-brain ECF compartmental model. Parameters describing brain ECF disposition were then allometrically scaled and linked with published human plasma PK to predict human ECF concentrations. Subsequently, prediction of human receptor occupancy at several CNS receptors was based on an effect model that related the predicted ECF concentrations to published concentration-driven receptor occupancy parameters. RESULTS: A one compartment model with first order absorption and elimination best described clozapine and norclozapine plasma concentrations in rats. A delay in the transfer of clozapine and norclozapine from plasma to the brain ECF compartment was captured using a transit compartment model approach. Human clozapine and norclozapine concentrations in brain ECF were simulated, and from these the median percentage of receptor occupancy of dopamine-2, serotonin-2A, muscarinic-1, alpha-1 adrenergic, alpha-2 adrenergic and histamine-1 for clozapine, and dopamine-2 for norclozapine were consistent with values reported in the literature. CONCLUSIONS: A PK model that relates clozapine and norclozapine disposition in rat plasma and brain, including blood-brain barrier transport, was developed. Using allometry and published human plasma PK, the model was successfully translated to predict clozapine and norclozapine concentrations and accordant receptor occupancy of both agents in human brain. These predicted exposure and occupancy measures at several receptors that bind clozapine may be employed to extend our understanding of clozapine's complex behavioral effects in humans.