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1.  Brain extracellular γ-hydroxybutyrate concentrations are decreased by L-lactate in rats: Role in the treatment of overdoses 
Pharmaceutical research  2013;30(5):1338-1348.
Purpose
L-lactate represents a potential treatment for GHB overdose by inhibiting GHB renal reabsorption mediated by monocarboxylate transporters. Our objective was to assess the dose-dependence of L-lactate treatment, with and without D-mannitol, on GHB toxicokinetics/toxicodynamics (TK/TD).
Methods
Rats were administered GHB 600 mg/kg i.v. with L-lactate (low and high doses), D-mannitol, or L-lactate (low dose) with D-mannitol. GHB-induced sleep time and GHB plasma, urine and brain extracellular fluid (ECF) concentrations (by LC/MS/MS) were determined. The effect of L-lactate and D-mannitol on the uptake and efflux of GHB was assessed in rat brain endothelial RBE4 cells.
Results
L-lactate treatment increased GHB renal clearance from 1.4 ± 0.1 ml/min/kg (control) to 2.4 ± 0.2 and 4.7 ± 0.5 ml/min/kg after low and high doses, respectively, and reduced brain ECF AUC values to 65 and 25% of control. Sleep time was decreased from 137 ± 12 minutes (control) to 91 ± 16 and 55 ± 5 minutes (low and high L-lactate, respectively). D-mannitol did not alter GHB TK/TD and did not alter L-lactate’s effects on GHB TK/TD. L-lactate, but not D-mannitol, inhibited GHB uptake, and increased GHB efflux from RBE4 cells.
Conclusions
L-lactate decreases plasma and brain ECF concentrations of GHB, decreasing sedative/hypnotic effects.
doi:10.1007/s11095-013-0973-z
PMCID: PMC3618608  PMID: 23319173
γ-hydroxybutyrate; monocarboxylate transporter; microdialysis; lactate; overdose
2.  Monocarboxylate Transporter Inhibition with Osmotic Diuresis Increases γ-Hydroxybutyrate Renal Elimination in Humans: A Proof-of-Concept Study 
Journal of clinical toxicology  2011;1(2):1000105-.
Background and objective
The purpose of the current study was to demonstrate proof-of-concept that monocarboxylate transporter (MCT) inhibition with L-lactate combined with osmotic diuresis increases renal clearance of γ-hydroxybutyrate (GHB) in human subjects. GHB is a substrate for human and rodent MCTs, which are responsible for GHB renal reabsorption, and this therapy increases GHB renal clearance in rats.
Methods
Ten healthy volunteers were administered GHB orally as sodium oxybate 50 mg/kg (4.5 gm maximum dose) on two different study days. On study day 1, GHB was administered alone. On study day 2, treatment of L-lactate 0.125 mmol/kg and mannitol 200 mg/kg followed by L-lactate 0.75 mmol/kg/hr was administered intravenously 30 minutes after GHB ingestion. Blood and urine were collected for 6 hours, analyzed for GHB, and pharmacokinetic and statistical analyses performed.
Results
L-lactate/mannitol administration significantly increased GHB renal clearance compared to GHB alone, 439 vs. 615 mL/hr (P=0.001), and increased the percentage of GHB dose excreted in the urine, 2.2 vs. 3.3% (P=0.021). Total clearance was unchanged.
Conclusions
MCT inhibition with L-lactate combined with osmotic diuresis increases GHB renal elimination in humans. No effect on total clearance was observed in this study due to the negligible contribution of renal clearance to total clearance at this low GHB dose. Considering the nonlinear renal elimination of GHB, further research in overdose cases is warranted to assess the efficacy of this treatment strategy for increasing renal and total clearance at high GHB doses.
doi:10.4172/2161-0495.1000105
PMCID: PMC3999665  PMID: 24772380
γ-hydroxybutyrate; Pharmacokinetics; Renal clearance; Monocarboxylate transporter
3.  Dietary Phenethyl Isothiocyanate Alters Gene Expression in Human Breast Cancer Cells 
Phenethyl isothiocyanate (PEITC), a component in cruciferous vegetables, can block chemical carcinogenesis in animal models. Our objective was to determine the effect of treatment with PEITC on gene expression changes in MCF-7 human breast cancer cells in order to evaluate potential mechanisms involved in its chemopreventive effects. MCF-7 cells were treated for 48 hours with either PEITC (3 μM) or the vehicle. Total RNA was extracted from cell membrane preparations, and labeled cDNA's representing the mRNA pool were reverse-transcribed directly from total RNA isolated for use in the microarray hybridizations. Two specific human GE Array Kits (Superarray Inc.) that both contain 23 marker genes, related to signal transduction pathways or cancer/tumor suppression, plus 2 housekeeping genes (β-actin and GAPDH), were utilized. Arrays from treated and control cells (n = 4 per group) were evaluated using a Student's t-test. Gene expression was significantly induced for tumor protein p53 (p53), cyclin-dependent kinase inhibitor 1C (p57 Kip2), breast cancer Type 2 early onset (BRCA2), cAMP responsive element binding protein 2 (ATF-2), interleukin 2 (IL-2), heat shock 27 KD protein (hsp27), and CYP19 (aromatase). Induction of p57 Kip2, p53, BRCA2, IL-2, and ATF-2 would be expected to decrease cellular proliferation and increase tumor suppression and/or apoptosis. PEITC treatment produced significant alterations in some genes involved in tumor suppression and cellular proliferation/apoptosis that may be important in explaining the chemopreventive effects of PEITC.
doi:10.1155/2011/462525
PMCID: PMC2952307  PMID: 20953429
4.  Structure–Activity Relationships and Quantitative Structure–Activity Relationships for Breast Cancer Resistance Protein (ABCG2) 
The AAPS Journal  2009;11(3):541-552.
Breast cancer resistance protein (ABCG2), the newest ABC transporter, was discovered independently by three groups in the late 1990s. ABCG2 is widely distributed in the body with expression in the brain, intestine, and liver, among others. ABCG2 plays an important role by effluxing drugs at the blood–brain, blood–testis, and maternal–fetal barriers and in the efflux of xenobiotics at the small intestine and kidney proximal tubule brush border and liver canalicular membranes. ABCG2 transports a wide variety of substrates including HMG-CoA reductase inhibitors, antibiotics, and many anticancer agents and is one contributor to multidrug resistance in cancer cells. Quantitative structure–activity relationship (QSAR) models and structure–activity relationships (SARs) are often employed to predict ABCG2 substrates and inhibitors prior to in vitro and in vivo studies. QSAR models correlate in vivo biological activity to physicochemical properties of compounds while SARs attempt to explain chemical moieties or structural features that contribute to or are detrimental to the biological activity. Most ABCG2 datasets available for in silico modeling are comprised of congeneric series of compounds; the results from one series usually cannot be applied to another series of compounds. This review will focus on in silico models in the literature used for the prediction of ABCG2 substrates and inhibitors.
doi:10.1208/s12248-009-9132-1
PMCID: PMC2758125  PMID: 19629710
ABC transporter; ABCG2; breast cancer resistance protein; quantitative structure–activity relationships; structure–activity relationships
5.  Mechanistic Models Describing Active Renal Reabsorption and Secretion: A Simulation-Based Study 
The AAPS Journal  2012;15(1):278-287.
The objective of the present study was to evaluate mechanistic pharmacokinetic models describing active renal secretion and reabsorption over a range of Michaelis–Menten parameter estimates and doses. Plasma concentration and urinary excretion profiles were simulated and renal clearance (CLr) was calculated for two pharmacokinetic models describing active renal reabsorption (R1/R2), two models describing active secretion (S1/S2), and a model containing both processes. A range of doses (1–1,000 mg/kg) was evaluated, and Vmax and Km parameter estimates were varied over a 100-fold range. Similar CLr values were predicted for reabsorption models (R1/R2) with variations in Vmax and Km. Tubular secretion models (S1/S2) yielded similar relationships between Michaelis–Menten parameter perturbations and CLr, but the predicted CLr values were threefold higher for model S1. For both reabsorption and secretion models, the greatest changes in CLr were observed with perturbations in Vmax, suggesting the need for an accurate estimate of this parameter. When intrinsic clearance was substituted for Michaelis–Menten parameters, it failed to predict similar CLr values even within the linear range. For models S1 and S2, renal secretion was predominant at low doses, whereas renal clearance was driven by fraction unbound in plasma at high doses. Simulations demonstrated the importance of Michaelis–Menten parameter estimates (especially Vmax) for determining CLr. Km estimates can easily be obtained directly from in vitro studies. However, additional scaling of in vitro Vmax estimates using in vitro/in vivo extrapolation methods are required to incorporate these parameters into pharmacokinetic models.
Electronic supplementary material
The online version of this article (doi:10.1208/s12248-012-9437-3) contains supplementary material, which is available to authorized users.
doi:10.1208/s12248-012-9437-3
PMCID: PMC3535105  PMID: 23196805
kidney transport parameters; models; reabsorption; renal clearance; secretion
6.  Effects of L-Lactate and D-Mannitol on γ-Hydroxybutyrate Toxicokinetics and Toxicodynamics in Rats 
Overdoses of γ-hydroxybutyrate (GHB), a drug of abuse, result in coma, respiratory arrest, and death. The objective of this study was to evaluate a potential GHB detoxification strategy by inhibiting the monocarboxylate transporter (MCT)-mediated renal reabsorption of GHB in rats, using the MCT substrate L-Lactate. The use of the osmotic diuretic D-mannitol alone or combined with L-Lactate was also explored. GHB (208 mg/h/kg) was infused i.v. for 3 h in the absence or presence of L-Lactate (60.5, 121, and 302.5 mg h−1 kg−1), D-mannitol (0.5 g/kg), or L-Lactate (60.5 mg h−1 kg−1) combined with D-mannitol (0.5 g/kg). GHB in plasma and urine samples was determined along with blood pH, electrolytes, glucose, and L-Lactate. Administration of L-Lactate, or the combination of L-Lactate and D-mannitol, but not D-mannitol alone, significantly increased the renal and total clearances of GHB in rats. Blood pH and electrolyte concentrations exhibited small changes with GHB, GHB/lactate, and GHB/mannitol treatments, although most values remained within their normal range. The concomitant administration of lactated Ringer's solution (28 mM L-Lactate) at 300 µl/min with mannitol (0.5 g/kg) resulted in a significant increase in GHB clearance and a decrease in sleep time after an i.v. dose of 1 g/kg. Overall, our results indicated the following: 1) the use of the MCT inhibitor L-Lactate can increase the renal and total clearances of GHB, and 2) the combination of lactated Ringer's solution and D-mannitol significantly alters GHB toxicokinetics and toxicodynamics and represents a potential clinical detoxification strategy for the treatment of GHB overdoses.
doi:10.1124/dmd.108.022996
PMCID: PMC2593404  PMID: 18719239
7.  Transport of γ-Hydroxybutyrate in Rat Kidney Membrane Vesicles: Role of Monocarboxylate Transporters 
Intoxication with γ-hydroxybutyrate (GHB) is associated with coma, seizure, and death; treatment of overdoses is symptomatic. Previous studies in our laboratory have demonstrated that L-lactate and pyruvate treatment can increase the renal clearance of GHB and increase its elimination in rats, suggesting that GHB may undergo renal reabsorption mediated by monocarboxylic acid transporters (MCTs). The goals of this study were to characterize the renal transport of GHB in rats and to determine the role of MCT in its renal transport. Brush-border membrane (BBM) and basolateral membrane (BLM) vesicles were isolated from rat kidney cortex, and the uptake of L-lactate and GHB was characterized. L-Lactate and GHB undergo both pH- and sodium-dependent transport in BBM vesicles and pH-dependent transport in BLM vesicles. A simple Michaelis-Menten equation best described the pH-dependent uptake of GHB in BBM (Km, 8.0 ± 1.8 mM; Vmax, 838 ± 45 pmol/mg/s) and in BLM vesicles (Km, 10.5 ± 2.6 mM; Vmax, 806 ± 253 pmol/mg/s). mRNA of MCT1 and MCT2 was determined in rat kidney cortex using reverse transcriptase-polymerase chain reaction; using Western blot, the protein expression of MCT1 was present mainly in BLM vesicles, with weak expression in BBM vesicles, whereas that of MCT2 was exclusively in BLM vesicles. Studies with rat MCT1 gene-transfected MDA-MB231 cells demonstrated that GHB was a substrate of MCT1. The data suggest that rat MCT1 may represent an important transporter for GHB in renal tubule cells. This investigation provides evidence for the importance of MCTs in the reabsorption of the monocarboxylic acids L-lactate and GHB in the kidney.
doi:10.1124/jpet.106.105965
PMCID: PMC1635030  PMID: 16707723
GHB, γ-hydroxybutyrate; MCT, monocarboxylate transporter; BBM, brush-border membrane; BLM, basolateral membrane; AA, acetoacetate; BHB, β-hydroxybutyrate; BTD, 1,4-butanediol; CHC, α-cyano-4-hydroxycinnamate; DIDS, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt; TEA, tetraethylammonium; MES, 2-(N-morpholino)ethanesulfonic acid; ALP, alkaline phosphatase; GGT, γ-glutamyl transferase; RT, reverse transcriptase; PCR, polymerase chain reaction; ANOVA, analysis of variance; bp, base pair(s); GBL, γ-butyrolactone
8.  Renal Clearance of γ-Hydroxybutyric Acid in Rats: Increasing Renal Elimination as a Detoxification Strategy 
Intoxication with γ-hydroxybutyric acid (GHB) is associated with coma, seizure, and death; treatment of overdoses is symptomatic. The objectives of this investigation were to characterize the renal clearance and total clearance of GHB in rats and to evaluate potential strategies for increasing the elimination of GHB after drug overdoses. GHB was administered by i.v. infusion at low (108 mg/h/kg), medium (128 mg/h/kg), or high (208 mg/h/kg) doses. Crossover studies were performed under steady-state conditions using the medium dose in the absence or presence of l-lactate, pyruvate, d-mannitol, sodium bicarbonate, or normal saline. GHB in plasma and urine samples was assayed using liquid chromatography-tandem mass spectrometry. Infusion of the low, medium, and high doses of GHB produced steady-state plasma concentrations of 0.22 ± 0.04, 0.43 ± 0.05, and 0.68 ± 0.11 mg/ml. The renal clearance of the medium (51.8 ± 13.0 ml/h/kg) and high (97.1 ± 43.1 ml/h/kg) doses was significantly higher than that of the low dose (14.9 ± 5.1 ml/h/kg), whereas the total clearance values were significantly lower than that of the low dose. The renal clearance was significantly increased by the concomitant administration of l-lactate, pyruvate, d-mannitol, or sodium bicarbonate with GHB but was not altered by normal saline. The total and metabolic clearance values were significantly increased by all treatments except normal saline. Overall, our results indicated that the renal clearance of GHB is dose-dependent, involving capacity-limited reabsorption. Monocarboxylate transport inhibitors, osmotic diuresis using d-mannitol, or the administration of sodium bicarbonate can increase the renal and total clearances of GHB. The approaches used in this investigation may offer potential detoxification strategies for the treatment of GHB overdoses.
doi:10.1124/jpet.105.083253
PMCID: PMC1482796  PMID: 15722403
GHB, γ-hydroxybutyric acid; MCT, monocarboxylate transporter; LC/MS/MS, liquid chromatography-tandem mass spectrometry; GFR, glomerular filtration rate; LLOQ, lower limit of quantitation; CV%, coefficient of variation
9.  Chemopreventive and anti-angiogenic effects of dietary phenethyl isothiocyanate in an N-methyl nitrosourea-induced breast cancer animal model 
The effect of phenethyl isothiocyanate (PEITC), a component of cruciferous vegetables, on the initiation and progression of cancer was investigated in a chemically induced estrogen-dependent breast cancer model. Breast cancer was induced in female Sprague Dawley rats (8 weeks old) by the administration of N-methyl nitrosourea (NMU). Animals were administered 50 or 150 µmol/kg oral PEITC and monitored for tumor appearance for 18 weeks. The PEITC treatment prolonged the tumor-free survival time and decreased the tumor incidence and multiplicity. The time to the first palpable tumor was prolonged from 69 days in the control, to 84 and 88 days in the 50 and 150 µmol/kg PEITC-treated groups. The tumor incidence in the control, 50 µmol/kg, and 150 µmol/kg PEITC-treated groups was 56.6%, 25.0% and 17.2%, while the tumor multiplicity was 1.03, 0.25 and 0.21, respectively. Differences were statistically significant (p < 0.05) from the control, but there were no significant differences between the two dose levels. The intratumoral capillary density decreased from 4.21 ± 0.30 vessels per field in the controls to 2.46 ± 0.25 in the 50 µmol/kg and 2.36 ± 0.23 in the 150 µmol/kg PEITC-treated animals. These studies indicate that supplementation with PEITC prolongs the tumor-free survival, reduces tumor incidence and burden, and is chemoprotective in NMU-induced estrogen-dependent breast cancer in rats. For the first time, it is reported that PEITC has anti-angiogenic effects in a chemically induced breast cancer animal model, representing a potentially significant mechanism contributing to its chemopreventive activity.
doi:10.1002/bdd.1826
PMCID: PMC3705647  PMID: 23138465
phenethyl isothiocyanate; estrogen-dependent breast cancer model; anti-angiogenic effects; N-methyl nitrosourea; PEITC; NMU
10.  Comparison of the Effects of Phenethyl Isothiocyanate and Sulforaphane on Gene Expression in Breast Cancer and Normal Mammary Epithelial Cells 
Phenethyl isothiocyanate (PEITC) and sulforaphane (SF) exhibit tumor preventive activity in lung, prostate, breast and colon cancers. Our objective was to examine the effect of these two isothiocyanates on estrogen receptor-related genes, and genes related to apoptosis and cell cycle in the estrogen-dependent breast cancer cell line MCF7 and in normal human epithelial breast (HME) cells. We treated cells with 0.3 μM or 3.0 μM concentrations of PEITC or SF. In HME cells, gene expression was significantly altered for 23 genes by PEITC at a concentration of 0.3 μM and 4 genes at 3.0 μM. SF altered the expression of 16 genes at a concentration of 0.3 μM and 2 genes at 3.0 μM. In HME cells, genes altered by both PEITC and SF exhibited changes in gene expression that were similar in extent as well as direction of change. In MCF-7 cells, PEITC did not produce any significant changes in the gene expression at both treatment levels. SF produced significant changes in 7 genes, but only at the higher treatment level of 3.0 μM. Normal mammary cells exhibited more changes in the expression of estrogen receptor related genes than did breast cancer cells, and significantly these changes occurred predominantly at the low concentration of 0.3 μM, a concentration achievable by dietary input of isothiocyanates. Novel findings were the upregulation of the pro-apoptotic gene BAD and estrogen receptor beta gene in normal human mammary cells. These gene alterations observed, along with upregulation of tumor suppressors p21 and p27, may provide a protective effect to mammary cells against breast cancer.
doi:10.3181/0808-RM-241
PMCID: PMC3698577  PMID: 19144873
phenethyl isothiocyanate; sulforaphane; human mammary epithelial cells; breast cancer MCF-7 cells; breast cancer prevention; gene expression
11.  In vivo modulation of 4E binding protein 1 (4E-BP1) phosphorylation by watercress: a pilot study 
The British journal of nutrition  2010;104(9):1288-1296.
Dietary intake of isothiocyanates (ITC) has been associated with reduced cancer risk. The dietary phenethyl ITC (PEITC) has previously been shown to decrease the phosphorylation of the translation regulator 4E binding protein 1 (4E-BP1). Decreased 4E-BP1 phosphorylation has been linked to the inhibition of cancer cell survival and decreased activity of the transcription factor hypoxia-inducible factor (HIF), a key positive regulator of angiogenesis, and may therefore contribute to potential anti-cancer effects of PEITC. In the present study, we have investigated the in vitro and in vivo effects of watercress, which is a rich source of PEITC. We first demonstrated that, similar to PEITC, crude watercress extracts inhibited cancer cell growth and HIF activity in vitro. To examine the effects of dietary intake of watercress, we obtained plasma and peripheral blood mononuclear cells following the ingestion of an 80 g portion of watercress from healthy participants who had previously been treated for breast cancer. Analysis of PEITC in plasma samples from nine participants demonstrated a mean maximum plasma concentration of 297 nM following the ingestion of watercress. Flow cytometric analysis of 4E-BP1 phosphorylation in peripheral blood cells from four participants demonstrated significantly reduced 4E-BP1 phosphorylation at 6 and 8 h following the ingestion of watercress. Although further investigations with larger numbers of participants are required to confirm these findings, this pilot study suggests that flow cytometry may be a suitable approach to measure changes in 4E-BP1 phosphorylation following the ingestion of watercress, and that dietary intake of watercress may be sufficient to modulate this potential anti-cancer pathway.
doi:10.1017/S0007114510002217
PMCID: PMC3694331  PMID: 20546646
Isothiocyanates; Cancer; 4E binding protein 1 (4E-BP1); Phosphorylation
12.  ABC Transporters and Isothiocyanates: Potential for Pharmacokinetic Diet–Drug Interactions 
Isothiocyanates, a class of anti-cancer agents, are derived from cruciferous vegetables such as broccoli, cabbage and watercress, and have demonstrated chemopreventive activity in a number of cancer models and epidemiologic studies. Due to public interest in cancer prevention and alternative therapies in cancer, the consumption of herbal supplements and vegetables containing these compounds is widespread and increasing. Isothiocyanates interact with ATP-binding cassette (ABC) efflux transporters such as P-glycoprotein, MRP1, MRP2 and BCRP, and may influence the pharmacokinetics of substrates of these transporters. This review discusses the pharmacokinetic properties of isothiocyanates, their interactions with ABC transporters, and presents some data describing the potential for isothiocyanate-mediated diet–drug interactions.
doi:10.1002/bdd.668
PMCID: PMC3694332  PMID: 19623673
isothiocyanates; ABC transporters; pharmacokinetics; diet-drug interactions
13.  Mechanism-Based Pharmacodynamic Modeling 
Pharmacodynamic modeling is based on a quantitative integration of pharmacokinetics, pharmacological systems, and (patho-) physiological processes for understanding the intensity and time-course of drug effects on the body. Application of such models to the analysis of meaningful experimental data allows for the quantification and prediction of drug–system interactions for both therapeutic and adverse drug responses. In this chapter, commonly used mechanistic pharmacodynamic models are presented with respect to their important features, operable equations, and signature profiles. In addition, literature examples showcasing the utility of these models to adverse drug events are highlighted. Common model types that are covered include simple direct effects, biophase distribution, indirect effects, signal transduction, and irreversible effects.
doi:10.1007/978-1-62703-050-2_21
PMCID: PMC3684160  PMID: 23007443
Adverse drug effects; Exposure–response relationships; Mathematical modeling; Pharmacodynamics; Pharmacokinetics
14.  Mechanistic Determinants of Biotherapeutics Absorption Following SC Administration 
The AAPS Journal  2012;14(3):559-570.
The subcutaneous (SC) route is of growing interest for the administration of biotherapeutics. Key products on the biotherapeutic market such as insulins, but also several immunoglobulins or Fc-fusion proteins, are administered SC. Despite the importance of the SC route, the available knowledge about the processes involved in the SC absorption of biotherapeutics is limited. This review summarizes available information on the physiology of the SC tissue and on the pharmacokinetic processes after SC administration including “first pass catabolism” at the administration site as well as transport in the extracellular matrix of the SC tissue, followed by absorption into the blood circulation or the lymphatic system. Both monoclonal antibodies and other biotherapeutics are discussed. Determinants of absorption after SC administration are summarized including compound properties such as charge or molecular weight. Scale-up of animal data to humans is discussed, including the current shortcomings of empirical scaling approaches and the lack of suitable mechanistic approaches.
doi:10.1208/s12248-012-9367-0
PMCID: PMC3385825  PMID: 22619041
bioavailability; hypodermis; lymphatic uptake; subcutaneous; therapeutic proteins
15.  A Physiologically Based Pharmacokinetic Model of Mitoxantrone in Mice and Scale-up to Humans: a Semi-Mechanistic Model Incorporating DNA and Protein Binding 
The AAPS Journal  2012;14(2):352-364.
We conducted a pharmacokinetic (PK) study of mitoxantrone (Novantrone®), a clinically well-established anticancer agent, in mice and developed a mechanism-based PBPK (physiologically based pharmacokinetic) model to describe its disposition. Mitoxantrone concentrations in plasma and six organs (lung, heart, liver, kidney, spleen, and brain) were determined after a 5 mg/kg i.v. dose. We evaluated three different PBPK models in order to characterize our experimental data: model 1 containing Kp values, model 2 incorporating a deep binding compartment, and model 3 incorporating binding of mitoxantrone to DNA and protein. Among the three models, only model 3 with DNA and protein binding captured all the experimental data well. The estimated binding affinity for DNA (KDNA) and protein (Kmacro) were 0.0013 and 1.44 μM, respectively. Predicted plasma and tissue AUC values differed from observed values by <19 %, except for heart (60 %). Model 3 was further used to simulate plasma mitoxantrone concentrations in humans for a 12-mg/m2 dose, using human physiological parameters. The simulated results generally agreed with the observed time course of mitoxantrone plasma concentrations in patients after a standard dose of 12 mg/m2. In summary, we reported for the first time a mechanism-based PBPK model of mitoxantrone incorporating macromolecule binding which may have clinical applicability in optimizing clinical therapy. Since mitoxantrone is a substrate of the efflux transporters ABCG2 and ABCB1, the incorporation of efflux transporters may also be necessary to characterize the data obtained in low-dose studies.
doi:10.1208/s12248-012-9344-7
PMCID: PMC3326165  PMID: 22451016
DNA binding; human scale-up; mitoxantrone; physiologically based pharmacokinetic model
16.  Pharmacokinetics, Lymph Node Uptake, and Mechanistic PK Model of Near-Infrared Dye-Labeled Bevacizumab After IV and SC Administration in Mice 
The AAPS Journal  2012;14(2):252-261.
Our objective was to determine the pharmacokinetics, bioavailability and lymph node uptake of the monoclonal antibody bevacizumab, labeled with the near-infrared (IR) dye 800CW, after intravenous (IV) and subcutaneous (SC) administration in mice. Fluorescence imaging and enzyme-linked immunosorbent assay (ELISA) assays were developed and validated to measure the concentration of bevacizumab in plasma. The bevacizumab–IRDye conjugate remained predominantly intact in plasma and in lymph node homogenate samples over a 24-h period, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and size exclusion chromatography. The plasma concentration vs. time plots obtained by fluorescence and ELISA measurements were similar; however, unlike ELISA, fluorescent imaging was only able to quantitate concentrations for 24 h after administration. At a low dose of 0.45 mg/kg, the plasma clearance of bevacizumab was 6.96 mL/h/kg after IV administration; this clearance is higher than that reported after higher doses. Half-lives of bevacizumab after SC and IV administration were 4.6 and 3.9 days, respectively. After SC administration, bevacizumab–IRDye800CW was present in the axillary lymph nodes that drain the SC site; lymph node uptake of bevacizumab–IRDye 800CW was negligible after IV administration. Bevacizumab exhibited complete bioavailability after SC administration. Using a compartmental pharmacokinetic model, the fraction absorbed through the lymphatics after SC administration was estimated to be about 1%. This is the first report evaluating the use of fluorescent imaging to determine the pharmacokinetics, lymphatic uptake, and bioavailability of a near-infrared dye-labeled antibody conjugate.
Electronic supplementary material
The online version of this article (doi:10.1208/s12248-012-9342-9) contains supplementary material, which is available to authorized users.
doi:10.1208/s12248-012-9342-9
PMCID: PMC3326166  PMID: 22391791
bevacizumab; bioavailability; fluorescence imaging; lymphatic absorption; pharmacokinetics
17.  Use of a Local Sensitivity Analysis to Inform Study Design Based on a Mechanistic Toxicokinetic Model for γ-Hydroxybutyric Acid 
The AAPS Journal  2011;13(2):240-254.
γ-Hydroxybutyric acid (GHB), a drug of abuse, demonstrates complex toxicokinetics with capacity-limited metabolism and active renal reabsorption. The objectives of the present study were to conduct a local sensitivity analysis of a mechanistic model for the active renal reabsorption of GHB and to use the results to inform the design of future studies aimed at developing therapeutic strategies for treating GHB overdoses. A local sensitivity analysis was used to assess the influence of parameter perturbations on model outputs (plasma concentrations and urinary excretion of GHB). Further, a sensitivity index was calculated for each perturbed parameter to assess the specific segments of the time course that are critical to parameter estimation. Model outputs were simulated for rats dosed with 200, 400, 600, and 1,000 mg/kg GHB intravenously and individual parameters were perturbed by two-, five-, and tenfold higher and lower than the nominal value. Model outputs were sensitive to perturbations in clearance and volume parameters. In contrast, model outputs were found to be insensitive to changes in distributional parameters suggesting that additional tissue distribution data is required. Based on the sensitivity analysis the 1,000-mg/kg GHB dose can be eliminated from future studies as the parameters can be adequately estimated from the lower doses. To further validate the use of this model, dose-specific sampling schedules were designed based on model predictions for doses of 600 and 1,500 mg/kg. These sampling schedules were able to adequately capture the inflection point and terminal elimination phase of the plasma concentration–time profiles obtained.
doi:10.1208/s12248-011-9264-y
PMCID: PMC3085705  PMID: 21387146
active renal reabsorption; gamma-hydroxybutyric acid; monocarboxylate transporters; sensitivity analysis
18.  Effect of orally administered phenethyl isothiocyanate on hepatic gene expression in rats 
Molecular nutrition & food research  2010;54(12):1802-1806.
Phenethyl isothiocyanate (PEITC) is a constituent of cruciferous vegetables that has demonstrated cancer preventive activity in a number of cancer models including lung, prostate, and breast cancer. Our objective was to examine the effects of the oral administration of PEITC for 7 days on the hepatic expression of genes important in drug metabolism and toxicity in Sprague Dawley rats. The liver is the major site for the metabolism of various xenobiotics and carcinogens, and determining the effects of PEITC on the gene expression of hepatic enzymes may provide insight into mechanisms underlying the cancer preventive activity of PEITC. Using a microarray containing 282 genes, we observed that PEITC significantly up-regulated UDP-glucuronosyltransferase UGT1A6 and strongly down-regulated nicotinamide N-methyltransferase (NNMT). We also confirmed the down-regulation of NNMT by real-time quantitative RT-PCR. NNMT was recently shown to be elevated in the serum of tumor bearing patients with pancreatic, lung, and colorectal cancer, and may be involved in cell migration. Other genes that were significantly up-regulated were the drug metabolizing enzyme cyp2b15, the anti-apoptotic gene bcl2l2, and the stress regulators Gadd45b, Dnajb9, Dnajb5 and Hspb1. Our results indicate new targets that may be important in the mechanisms of the anticancer effects of PEITC. Of particular significance was the down-regulation of NNMT which may represent a new target for the treatment of a variety of cancers.
doi:10.1002/mnfr.200900607
PMCID: PMC3017723  PMID: 20626002
19.  Mechanistic Toxicokinetic Model for γ-Hydroxybutyric Acid: Inhibition of Active Renal Reabsorption as a Potential Therapeutic Strategy 
The AAPS Journal  2010;12(3):407-416.
γ-Hydroxybutyric acid (GHB), a drug of abuse, exhibits saturable renal clearance and capacity-limited metabolism. The objectives of this study were to construct a mechanistic toxicokinetic (TK) model describing saturable renal reabsorption and capacity-limited metabolism of GHB and to predict the effects of inhibition of renal reabsorption on GHB TK in the plasma and urine. GHB was administered by iv bolus (200–1,000 mg/kg) to male Sprague-Dawley rats and plasma and urine samples were collected for up to 6 h post-dose. GHB concentrations were determined by LC/MS/MS. GHB plasma concentration and urinary excretion were well-described by a TK model incorporating plasma and kidney compartments, along with two tissue and two ultrafiltrate compartments. The estimate of the Michaelis-Menten constant for renal reabsorption (Km,R) was 0.46 mg/ml which is consistent with in vitro estimates of monocarboxylate transporter (MCT)-mediated uptake of GHB (0.48 mg/ml). Simulation studies assessing inhibition of renal reabsorption of GHB demonstrated increased time-averaged renal clearance and GHB plasma AUC, independent of the inhibition mechanism assessed. Co-administration of GHB (600 mg/kg iv) and l-lactate (330 mg/kg iv bolus plus 121 mg/kg/h iv infusion), a known inhibitor of MCTs, resulted in a significant decrease in GHB plasma AUC and an increase in time-averaged renal clearance, consistent with the model simulations. These results suggest that inhibition of renal reabsorption of GHB is a viable therapeutic strategy for the treatment of GHB overdoses. Furthermore, the mechanistic TK model provides a useful in silico tool for the evaluation of potential therapeutic strategies.
doi:10.1208/s12248-010-9197-x
PMCID: PMC2895455  PMID: 20461486
gamma-hydroxybutyrate; kidney reabsorption; pharmacokinetic model; renal clearance; toxicokinetics
20.  Prediction of Biliary Excretion in Rats and Humans Using Molecular Weight and Quantitative Structure–Pharmacokinetic Relationships 
The AAPS Journal  2009;11(3):511-525.
The aims were (1) to evaluate the molecular weight (MW) dependence of biliary excretion and (2) to develop quantitative structure–pharmacokinetic relationships (QSPKR) to predict biliary clearance (CLb) and percentage of administered dose excreted in bile as parent drug (PDb) in rats and humans. CLb and PDb data were collected from the literature for rats and humans. Receiver operating characteristic curve analysis was utilized to determine whether a MW threshold exists for PDb. Stepwise multiple linear regression (MLR) was used to derive QSPKR models. The predictive performance of the models was evaluated by internal validation using the leave-one-out method and external test groups. A MW threshold of 400 Da was determined for PDb for anions in rats, while 475 Da was the cutoff for anions in humans. MW thresholds were not present for cations or cations/neutral compounds in either rats or humans. The QSPKR model for human CLb showed a significant correlation (R2 = 0.819) with good prediction performance (Q2 = 0.722). The model was further assessed using a test group, yielding a geometric mean fold-error of 2.68. QSPKR models with significant correlation and good predictability were also developed for CLb in rats and PDb data for anions or cation/neutral compounds in rats and humans. Both CLb and PDb data were further evaluated for subsets of MRP2 or P-glycoprotein substrates, and significant relationships were derived. QSPKR models were successfully developed for biliary excretion of non-congeneric compounds in rats and humans, providing a quantitative prediction of biliary clearance of compounds.
doi:10.1208/s12248-009-9124-1
PMCID: PMC2758117  PMID: 19593675
biliary clearance; humans; in silico; molecular weight cutoff; molecular volume; QSPKR; rats
21.  Pharmacokinetic Interaction between the Flavonoid Luteolin and γ-Hydroxybutyrate in Rats: Potential Involvement of Monocarboxylate Transporters 
The AAPS Journal  2008;10(1):47-55.
Monocarboxylate transporter 1 (MCT1) has been previously reported as an important determinant of the renal reabsorption of the drug of abuse, γ-hydroxybutyrate (GHB). Luteolin is a potent MCT1 inhibitor, inhibiting the uptake of GHB with an IC50 of 0.41 μM in MCT1-transfected MDA-MB231 cells. The objectives of this study were to characterize the effects of luteolin on GHB pharmacokinetics and pharmacodynamics in rats, and to investigate the mechanism of the interaction using model-fitting methods. GHB (400 and 1,000 mg/kg) and luteolin (0, 4 and 10 mg/kg) were administered to rats via iv bolus doses. The plasma or urine concentrations of luteolin and GHB were determined by HPLC and LC/MS/MS, respectively. The pharmacodynamic parameter sleep time in rats after GHB administration was recorded. A pharmacokinetic model containing capacity-limited renal reabsorption and metabolic clearance was constructed to characterize the in vivo interaction. Luteolin significantly decreased the plasma concentration and AUC, and increased the total and renal clearances of GHB. Moreover, luteolin significantly shortened the duration of GHB (1,000 mg/kg)-induced sleep in rats (161 ± 16, 131 ± 14 and 121 ± 5 min for control, luteolin 4 and 10 mg/kg groups, respectively, p < 0.01). An uncompetitive inhibition model, with an inhibition constant of 1.1 μM, best described the in vivo pharmacokinetic interaction. The results of this study indicated that luteolin significantly altered the pharmacokinetics of GHB by inhibiting its MCT1-mediated transport. The interaction between luteolin and GHB may offer a potential clinical detoxification strategy to treat GHB overdoses.
doi:10.1208/s12248-007-9001-8
PMCID: PMC2751446  PMID: 18446505
γ-hydroxybutyrate; luteolin; MCT; pharmacokinetic interactions
22.  Overview of the Proton-coupled MCT (SLC16A) Family of Transporters 
The AAPS journal  2008;10(2):311-321.
The transport of monocarboxylates, such as lactate and pyruvate, is mediated by the SLC16A family of proton-linked membrane transport proteins known as monocarboxylate transporters (MCTs). Fourteen MCT-related genes have been identified in mammals and of these seven MCTs have been functionally characterized. Despite their sequence homology, only MCT1-4 have been demonstrated to be proton-dependent transporters of monocarboxylic acids. MCT6, MCT8 and MCT10 have been demonstrated to transport diuretics, thyroid hormones and aromatic amino acids, respectively. MCT1-4 vary in their regulation, tissue distribution and substrate/inhibitor specificity with MCT1 being the most extensively characterized isoform. Emerging evidence suggests that in addition to endogenous substrates, MCTs are involved in the transport of pharmaceutical agents, including γ-hydroxybuytrate (GHB), 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors (statins), salicylic acid, and bumetanide. MCTs are expressed in a wide range of tissues including the liver, intestine, kidney and brain, and as such they have the potential to impact a number of processes contributing to the disposition of xenobiotic substrates. GHB has been extensively studied as a pharmaceutical substrate of MCTs; the renal clearance of GHB is dose-dependent with saturation of MCT-mediated reabsorption at high doses. Concomitant administration of GHB and l-lactate to rats results in an approximately two-fold increase in GHB renal clearance suggesting that inhibition of MCT1-mediated reabsorption of GHB may be an effective strategy for increasing renal and total GHB elimination in overdose situations. Further studies are required to more clearly define the role of MCTs on drug disposition and the potential for MCT-mediated detoxification strategies in GHB overdose.
doi:10.1208/s12248-008-9035-6
PMCID: PMC2574616  PMID: 18523892
butyrate; gamma-hydroxybutyrate; lactate; monocarboxylate transporters; SLC16A
24.  Overview of the Proton-coupled MCT (SLC16A) Family of Transporters: Characterization, Function and Role in the Transport of the Drug of Abuse γ-Hydroxybutyric Acid 
The AAPS Journal  2008;10(2):311-321.
The transport of monocarboxylates, such as lactate and pyruvate, is mediated by the SLC16A family of proton-linked membrane transport proteins known as monocarboxylate transporters (MCTs). Fourteen MCT-related genes have been identified in mammals and of these seven MCTs have been functionally characterized. Despite their sequence homology, only MCT1–4 have been demonstrated to be proton-dependent transporters of monocarboxylic acids. MCT6, MCT8 and MCT10 have been demonstrated to transport diuretics, thyroid hormones and aromatic amino acids, respectively. MCT1–4 vary in their regulation, tissue distribution and substrate/inhibitor specificity with MCT1 being the most extensively characterized isoform. Emerging evidence suggests that in addition to endogenous substrates, MCTs are involved in the transport of pharmaceutical agents, including γ-hydroxybuytrate (GHB), 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors (statins), salicylic acid, and bumetanide. MCTs are expressed in a wide range of tissues including the liver, intestine, kidney and brain, and as such they have the potential to impact a number of processes contributing to the disposition of xenobiotic substrates. GHB has been extensively studied as a pharmaceutical substrate of MCTs; the renal clearance of GHB is dose-dependent with saturation of MCT-mediated reabsorption at high doses. Concomitant administration of GHB and l-lactate to rats results in an approximately two-fold increase in GHB renal clearance suggesting that inhibition of MCT1-mediated reabsorption of GHB may be an effective strategy for increasing renal and total GHB elimination in overdose situations. Further studies are required to more clearly define the role of MCTs on drug disposition and the potential for MCT-mediated detoxification strategies in GHB overdose.
doi:10.1208/s12248-008-9035-6
PMCID: PMC2574616  PMID: 18523892
butyrate; gamma-hydroxybutyrate; lactate; monocarboxylate transporters; SLC16A
25.  Pharmacokinetics and bioavailability of the isoflavone biochanin A in rats 
The AAPS Journal  2006;8(3):E433-E442.
Biochanin A(BCA) is a dietary isoflavone present in legumes, most notably red clover, and in many herbal dietary supplements. BCA has been reported to have chemopreventive properties and is metabolized to the isoflavone genistein (GEN), BCA conjugates, and GEN conjugates. The metabolites may contribute to the chemopreventive effects of BCA. The absorption, metabolism, and disposition of BCA have not been determined in rats. Our objective was to evaluate the pharmacokinetics and metabolism of BCA in rats. Male Sprague-Dawley rats were administered BCA by intravenous injection (1 and 5 mg/kg), by intraperitoneal injection (5 and 50 mg/kg), and orally (5 and 50 mg/kg). Plasma and bile samples were enzymatically hydrolyzed in vitro to determine conjugate concentrations for BCA and GEN. Equilibrium dialysis was used to determine protein binding. The BCA and GEN concentrations in plasma, urine, and bile were determined by liquid chromatography-tandem mass spectrometry (LC/MS/MS). The pharmacokinetic parameters of BCA were analyzed by noncompartmental analysis. Significant levels of BCA conjugates and GEN conjugates were detected in plasma and bile. Both BCA and GEN were found to have a high clearance and a large apparent volume of distribution; the bioavailability of both was poor (<4%). Reentry peaks were evident after oral administration of both BCA and GEN, suggesting enterohepatic cycling. The free fraction of BCA in rat plasma was 1.5%. A2-compartment model that included both linear and nonlinear clearance terms and enterohepatic recirculation best described the plasma data. This represents the first evaluation of the dose-dependent pharmacokinetics and metabolism of BCA in rats.
doi:10.1208/aapsj080351
PMCID: PMC2761049  PMID: 17025260
Biochanin A; pharmacokinetics; intraperitoneal administration; enterohepatic recirculation; rat; genistein

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