Biological barriers, and the influence of protein binding on the passage of drugs across them.

Drug-protein binding plays a key role in determining the pharmacokinetics of a drug. The distribution and protein binding ability of a drug changes over a lifetime, and are important considerations during pregnancy and lactation. Although proteins are a significant fraction in plasma composition, they also exist beyond the bloodstream and bind with drugs in the skin, tissues or organs. Protein binding influences the bioavailability and distribution of active compounds, and is a limiting factor in the passage of drugs across biological membranes and barriers: drugs are often unable to cross membranes mainly due to the high molecular mass of the drug-protein complex, thus resulting in the accumulation of the active compounds and a significant reduction of their pharmacological activity. This review describes the consequences of drug-protein binding on drug transport across physiological barriers, whose role is to allow the passage of essential substances-such as nutrients or oxygen, but not of xenobiotics. The placental barrier regulates passage of xenobiotics into a fetus and protects the unborn organism. The blood-brain barrier is the most important barrier in the entire organism and the skin separates the human body from the environment.

An LC-MS/MS method for quantification of HR011303, a novel highly selective urate transporter 1 inhibitor in beagle dogs and the application to a pharmacokinetic study.

HR011303 is a novel and highly selective urate transporter 1 (URAT1) inhibitor. In this study, a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantification of HR011303 in beagle dog plasma. Plasma samples were pretreated with protein-precipitation extraction by acetonitrile and added with a trifluoromethyl substituted analog of HR011303 as internal standard. The chromatographic separation was performed on a Shiseido C18 column (100 × 4.6 mm, i.d., 5 µm) by mobile phases consisting of 5 mm ammonium-formic acid (100:0.1) and acetonitrile-formic acid (100:0.1) solutions in gradient elution. The MS detection was conducted in electrospray positive ionization with multiple reactions monitoring at m/z 338 → 240 for HR011303 and m/z 328 → 230 for the internal standard using 25 eV argon gas collision induced dissociation. The established LC-MS/MS method showed good selectivity, sensitivity, precision and accuracy. The plasma pharmacokinetics of HR011303 in beagle dogs following both oral and intravenous administration were then successfully evaluated using this LC-MS/MS method.

Transporters in Drug Development: Scientific and Regulatory Considerations.

Transporters can affect a drug's pharmacokinetics (PK) by controlling absorption, distribution, and elimination processes. They can also affect a drug's pharmacodynamics (PD) by influencing its access to the site of action. More recently, transporters have become important as drug targets (e.g., urate transporter inhibitors as treatment for gout and sodium/glucose cotransporter-2 inhibitors for treating type 2 diabetes). As such, it is important to consider the role of transporters during drug development.

Physiologically Based Pharmacokinetic Modeling in Pregnancy: A Systematic Review of Published Models.

During recent years there has been a surge in developing and applying physiologically based pharmacokinetic (PBPK) models in pregnant women to better understand and predict changes in drug pharmacokinetics throughout pregnancy. As a consequence, the number of publications focusing on pregnancy PBPK models has increased substantially. However, to date these models, especially across various platforms, have not been systematically evaluated. Hence, this review aims to assess published PBPK models in pregnancy used for therapeutic purposes.

Exploring in vivo metabolism and excretion of QO-58L using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry.

QO-58 lysine (QO-58L) as a new potassium channel opener, reported to have a potential activity to cure neuropathic pain. The aim of this research is to develop and validate a high-performance liquid chromatography with tandem spectrometry (LC-MS/MS) method for the quantification of QO-58L in rat urine, feces and bile. In addition, analyze and identify the metabolites in urine and bile. The assay for this compound in samples detected with multiple reaction monitoring mode (MRM), and take nimodipine as internal standards (IS). To better understand the biotransformation of QO-58L, metabolites in urine and bile were identified by using ultra high performance liquid chromatography tandem quadrupole/time of flight mass spectrometry (UHPLC-Q-TOF-MS) in the positive and negative ion mode. Urine, feces and bile were quantified by three new methods. The results showed that: QO-58L was mainly eliminated through fecal route (92.94%), a small amount of it via biliary excretion (2.05%), and rarely through urinary excretion (0.024%). As a result, there are 11 metabolites were identified, including 8 phase I metabolites resulting from elimination, hydroxylation and dihydroxylation, and 3 phase II metabolites originating from sulfation, N-acetylcysteine conjugation and glucuronidation. Furthermore, the newly discoveries of excretion and metabolism significantly expanded our understanding and was going to be greatly helpful for QO-58L's further pharmacokinetic study in vivo.

Amiodarone, a multi-channel blocker, enhances anticonvulsive effect of carbamazepine in the mouse maximal electroshock model.

Cardiac arrhythmia may occur in the course of epilepsy. Simultaneous therapy of the two diseases might be complicated by drug interactions since antiarrhythmic and antiepileptic agents share some molecular targets. The aim of this study was to evaluate the influence of amiodarone, an antiarrhythmic drug working as a multi-channel blocker, on the protective activity of four classical antiepileptic drugs in the maximal electroshock test in mice. Amiodarone at doses up to 75 mg/kg did not affect the electroconvulsive threshold in mice. Acute amiodarone at the dose of 75 mg/kg significantly potentiated the anticonvulsive effect of carbamazepine, but not that of valproate, phenytoin or phenobarbital in the maximal electroshock-induced seizures in mice. The antiarrhythmic agent and its combinations with antiepileptic drugs did not impair motor performance or long-term memory in mice, except for the combination of amiodarone and phenobarbital. Brain concentrations of antiepileptic drugs were not changed. Despite favourable impact of amiodarone on the anticonvulsive action of carbamazepine in the maximal electroshock, co-administration of the two drugs should be carefully monitored in clinical conditions. Further studies are necessary to evaluate effects of chronic treatment with amiodarone on seizure activity and the action of antiepileptic drugs.

Discovery and Characterization of 1H-Pyrazol-5-yl-2-phenylacetamides as Novel, Non-Urea-Containing GIRK1/2 Potassium Channel Activators.

The G protein-gated inwardly-rectifying potassium channels (GIRK, Kir3) are a family of inward-rectifying potassium channels, and there is significant evidence supporting the roles of GIRKs in a number of physiological processes and as potential targets for numerous indications. Previously reported urea containing molecules as GIRK1/2 preferring activators have had significant pharmacokinetic (PK) liabilities. Here we report a novel series of 1H-pyrazolo-5-yl-2-phenylacetamides in an effort to improve upon the PK properties. This series of compounds display nanomolar potency as GIRK1/2 activators with improved brain distribution (rodent Kp > 0.6).

Decrease in Long-Chain Acylcarnitine Tissue Content Determines the Duration of and Correlates with the Cardioprotective Effect of Methyl-GBB.

Ischaemia in the heart is accompanied by the accumulation of long-chain acylcarnitines (LCACs) which is one of the multiple factors that contribute to the ischaemia-reperfusion damage development. Long-term pre-treatment that decreases carnitine and LCAC contents also reduces ischaemia-reperfusion (IR) damage; however, the duration of the post-treatment effects is not known. The aim of the study was to assess the post-treatment effects of the carnitine transport (OCTN2) inhibitor, methyl-GBB, on LCAC content and the duration of its cardioprotective effect. Male Wistar rats received methyl-GBB (5 mg/kg for 28 days), and the anti-infarction effects on Langendorff-perfused hearts and the acylcarnitine profile in cardiac tissues were measured up to 28 days following the end of the treatment. Methyl-GBB pre-treatment for 28 days decreased LCAC heart tissue content by 87%, and the infarct size was decreased by 57%. Fourteen days post-treatment, the LCAC content was still decreased by 69%, and the infarct size was decreased by 32% compared to Control. A significant Pearson correlation (r = 0.48, p = 0.026) was found between infarct size and LCAC tissue content in the methyl-GBB-treated rat hearts. The addition of 2 mM carnitine to isolated heart perfusate significantly diminished the methyl-GBB-induced decrease in LCACs and infarct size. In conclusion, the anti-infarction effect of methyl-GBB continues for at least 2 weeks post-treatment. No less than a 70% decrease in LCAC content is required to protect ischaemic heart tissues, and the decrease in LCAC levels defines the duration of the post-treatment cardioprotective effect of the OCTN2 inhibitor, methyl-GBB.

Lithium-Responsive Seizure-Like Hyperexcitability Is Caused by a Mutation in the Drosophila Voltage-Gated Sodium Channel Gene paralytic.

Shudderer (Shu) is an X-linked dominant mutation in Drosophila melanogaster identified more than 40 years ago. A previous study showed that Shu caused spontaneous tremors and defects in reactive climbing behavior, and that these phenotypes were significantly suppressed when mutants were fed food containing lithium, a mood stabilizer used in the treatment of bipolar disorder (Williamson, 1982). This unique observation suggested that the Shu mutation affects genes involved in lithium-responsive neurobiological processes. In the present study, we identified Shu as a novel mutant allele of the voltage-gated sodium (Nav) channel gene paralytic (para). Given that hypomorphic para alleles and RNA interference-mediated para knockdown reduced the severity of Shu phenotypes, Shu was classified as a para hypermorphic allele. We also demonstrated that lithium could improve the behavioral abnormalities displayed by other Nav mutants, including a fly model of the human generalized epilepsy with febrile seizures plus. Our electrophysiological analysis of Shu showed that lithium treatment did not acutely suppress Nav channel activity, indicating that the rescue effect of lithium resulted from chronic physiological adjustments to this drug. Microarray analysis revealed that lithium significantly alters the expression of various genes in Shu, including those involved in innate immune responses, amino acid metabolism, and oxidation-reduction processes, raising the interesting possibility that lithium-induced modulation of these biological pathways may contribute to such adjustments. Overall, our findings demonstrate that Nav channel mutants in Drosophila are valuable genetic tools for elucidating the effects of lithium on the nervous system in the context of neurophysiology and behavior.

Generation and Characterization of a Breast Cancer Resistance Protein Humanized Mouse Model.

Breast cancer resistance protein (BCRP) is expressed in various tissues, such as the gut, liver, kidney and blood brain barrier (BBB), where it mediates the unidirectional transport of substrates to the apical/luminal side of polarized cells. Thereby BCRP acts as an efflux pump, mediating the elimination or restricting the entry of endogenous compounds or xenobiotics into tissues and it plays important roles in drug disposition, efficacy and safety. Bcrp knockout mice (Bcrp(-/-)) have been used widely to study the role of this transporter in limiting intestinal absorption and brain penetration of substrate compounds. Here we describe the first generation and characterization of a mouse line humanized for BCRP (hBCRP), in which the mouse coding sequence from the start to stop codon was replaced with the corresponding human genomic region, such that the human transporter is expressed under control of the murineBcrppromoter. We demonstrate robust human and loss of mouse BCRP/Bcrp mRNA and protein expression in the hBCRP mice and the absence of major compensatory changes in the expression of other genes involved in drug metabolism and disposition. Pharmacokinetic and brain distribution studies with several BCRP probe substrates confirmed the functional activity of the human transporter in these mice. Furthermore, we provide practical examples for the use of hBCRP mice to study drug-drug interactions (DDIs). The hBCRP mouse is a promising model to study the in vivo role of human BCRP in limiting absorption and BBB penetration of substrate compounds and to investigate clinically relevant DDIs involving BCRP.

[ABSOLUTE AND RELATIVE BIOAVAILABILITY OF GLUTARON--A NEW DERIVATIVE OF GLUTAMIC ACID].

The pharmacokinetics of studies of 3-phenylglutamic acid hydrochloride (glutaron) has been studied in rats. The main pharmacokinetic parameters show low values of the half-life (T1/2 = 3.75 h), mean retention time in the body (MRT = 5.77 h). The medium rate of drug concentration decrease in the blood plasma leads to a low value of the area under pharmacokinetic curve (AUC = 41.18 mg · h/mL). The general volume of distribution (Vd = 3.42 L/kg) is 3.5 times greater than the volume of extracellular fluid in the rat body. These data indicate a high ability of the glutaron to be distributed and accumulated in animal tissues. The value of absolute bioavailability is 84%, and the relative bioavailabity is 100%.

Development of a new photochromic ion channel blocker via azologization of fomocaine.

Photochromic blockers of voltage gated ion channels are powerful tools for the control of neuronal systems with high spatial and temporal precision. We now introduce fotocaine, a new type of photochromic channel blocker based on the long-lasting anesthetic fomocaine. Fotocaine is readily taken up by neurons in brain slices and enables the optical control of action potential firing by switching between 350 and 450 nm light. It also provides an instructive example for "azologization", that is, the systematic conversion of an established drug into a photoswitchable one.

Evaluating SGLT2 inhibitors for type 2 diabetes: pharmacokinetic and toxicological considerations.

INTRODUCTION: Inhibitors of sodium-glucose cotransporters type 2 (SGLT2), which increase urinary glucose excretion independently of insulin, are proposed as a novel approach for the management of type 2 diabetes mellitus (T2DM). AREAS COVERED: An extensive literature search was performed to analyze the pharmacokinetic characteristics, toxicological issues and safety concerns of SGLT2 inhibitors in humans. This review focuses on three compounds (dapagliflozin, canagliflozin, empagliflozin) with results obtained in healthy volunteers (including drug-drug interactions), patients with T2DM (single dose and multiple doses) and special populations (those with renal or hepatic impairment). EXPERT OPINION: The three pharmacological agents share an excellent oral bioavailability, long half-life allowing once-daily administration, low accumulation index and renal clearance, the absence of active metabolites and a limited propensity to drug-drug interactions. No clinically relevant changes in pharmacokinetic parameters were observed in T2DM patients or in patients with mild/moderate renal or hepatic impairment. Adverse events are a slightly increased incidence of mycotic genital and rare benign urinary infections. SGLT2 inhibitors have the potential to reduce several cardiovascular risk factors, and cardiovascular outcome trials are currently ongoing. The best positioning of SGLT2 inhibitors in the armamentarium for treating T2DM is still a matter of debate.

Therapeutic peptides: new arsenal against drug resistant pathogens.

Our incessant tug-of-war with multidrug resistant pathogenic bacteria has prompted researchers to explore novel methods of designing therapeutics in order to defend ourselves against infectious diseases. Combined advances in whole genome analysis, bioinformatics algorithms, and biochemical techniques have led to the discovery and subsequent characterization of an abundant array of functional small peptides in microorganisms and multicellular organisms. Typically classified as having 10 to 100 amino acids, many of these peptides have been found to have dual activities, executing important defensive and regulatory functions in their hosts. In higher organisms, such as mammals, plants, and fungi, host defense peptides have been shown to have immunomodulatory and antimicrobial properties. In microbes, certain growth-inhibiting peptides have been linked to the regulation of diverse cellular processes. Examples of these processes include quorum sensing, stress response, cell differentiation, biofilm formation, pathogenesis, and multidrug tolerance. In this review, we will present a comprehensive overview of the discovery, characteristics, and functions of host- and bacteria-derived peptides with antimicrobial activities. The advantages and possible shortcomings of using these peptides as antimicrobial agents and targets will also be discussed. We will further examine current efforts in engineering synthetic peptides to be used as therapeutics and/or drug delivery vehicles.

ABC transporters in multidrug resistance and pharmacokinetics, and strategies for drug development.

Multidrug resistance (MDR) is a serious problem that hampers the success of cancer pharmacotherapy. A common mechanism is the overexpression of ATP-binding cassette (ABC) efflux transporters in cancer cells such as P-glycoprotein (P-gp/ABCB1), multidrug resistance-associated protein 1 (MRP1/ABCC1) and breast cancer resistance protein (BCRP/ABCG2) that limit the exposure to anticancer drugs. One way to overcome MDR is to develop ABC efflux transporter inhibitors to sensitize cancer cells to chemotherapeutic drugs. The complete clinical trials thus far have showen that those tested chemosensitizers only add limited or no benefits to cancer patients. Some MDR modulators are merely toxic, and others induce unwanted drug-drug interactions. Actually, many ABC transporters are also expressed abundantly in the gastrointestinal tract, liver, kidney, brain and other normal tissues, and they largely determine drug absorption, distribution and excretion, and affect the overall pharmacokinetic properties of drugs in humans. In addition, ABC transporters such as P-gp, MRP1 and BCRP co-expressed in tumors show a broad and overlapped specificity for substrates and MDR modulators. Thus reliable preclinical assays and models are required for the assessment of transporter-mediated flux and potential effects on pharmacokinetics in drug development. In this review, we provide an overview of the role of ABC efflux transporters in MDR and pharmacokinetics. Preclinical assays for the assessment of drug transport and development of MDR modulators are also discussed.

SGLT-2 inhibition in patients with kidney disease.

Accustomed to managing diabetes with agents that mostly act by modulating the secretion and actions of insulin, with the advent of sodium-glucose linked transporter-2 (SGLT-2) inhibitors, physicians are now aware that the kidney also needs to be considered in the spectrum of action of anti-hyperglycaemic agents. Though familiar with the need for dose adjustment when prescribing many of our current anti-hyperglycaemic drugs in the setting of kidney dysfunction, with the SGLT-2 inhibitors pharmacodynamic as well as pharmacokinetic aspects also need to be considered. Finally, through their ability to reduce intraglomerular pressure, systemic blood pressure and plasma uric acid concentration, the SGLT-2 inhibitors offers the possibility of kidney protection. An hypothesis that will need to be tested with long term studies that address changes in the kidney beyond albuminuria, assessing the rate of decline in glomerular filtration rate and 'hard'kidneyrelated endpoints such as the need for renal replacement therapy (dialysis, transplantation) will be important in this setting.

Adverse effects and safety of SGLT-2 inhibitors.

In type 2 diabetes (T2DM), glycaemic control delays the development and slows the progression of complications. Although there are numerous glucose-lowering agents in clinical use, only approximately half of T2DM patients achieve glycaemic control, while undesirable side-effects, such as hypoglycaemia and body weight gain, often impede treatment in those taking these medications. Thus, there is a need for novel agents and treatment options. Sodium-glucose cotransporter-2 inhibitors (SGLT-2-i) have recently been developed for the treatment of T2DM. The available data suggest a good tolerability profile for the three available drugs - canagliflozin, dapagliflozin and empagliflozin - approved by the US Food and Drug Administration (FDA) for the American market as well as in other countries. The most frequently reported adverse events with SGLT-2-i are female genital mycotic infections, urinary tract infections and increased urination. The pharmacodynamic response to SGLT-2-i declines with increasing severity of renal impairment, requiring dosage adjustments or restrictions with moderate-to-severe renal dysfunction. Most patients treated with SGLT-2-i also have a modest reduction in blood pressure and modest effects on serum lipid profiles, some of which are beneficial (increased high-density lipoprotein cholesterol and decreased triglycerides) and others which are not (increased low-density lipoprotein cholesterol, LDL-C). A number of large-scale and longer-term cardiovascular trials are now ongoing. In patients treated with dapagliflozin, a non-significant excess number of breast and bladder cancers has been reported; considered as due to a bias, this is nevertheless being followed in the ongoing trials. No other significant safety issues have been reported so far. Although there is some benefit for several cardiovascular risk factors such as HbA1c, high blood pressure, obesity and increases in LDL-C, adequately powered trials are still required to determine the effects of SGLT-2-i on macrovascular outcomes.

Coadministration of lapatinib increases exposure to docetaxel but not doxorubicin in the small intestine of mice.

Combination therapy is increasingly being utilized for the treatment of metastatic breast cancer. However, coadministration of drugs, particularly agents that are substrates for or inhibitors of p-glycoprotein, can result in increased tissue toxicity. Unfortunately, determination of levels of chemotherapeutics in human tissues is challenging, and plasma drug concentrations are not always indicative of tissue toxicokinetics or toxicodynamics, especially when tissue penetration is altered. The aim of the present work was to determine whether concomitant administration of compounds currently being combined in clinical trials for metastatic breast cancer treatment alters plasma and tissue pharmacokinetics in mice if both agents are p-glycoprotein substrates and/or inhibitors. Accordingly, we investigated the pharmacokinetic interactions of the classic cytotoxics and p-glycoprotein substrates docetaxel and doxorubicin when administered concurrently with the targeted agent and p-glycoprotein inhibitor lapatinib. Our time-course plasma and tissue distribution studies showed that coadministration of lapatinib with doxorubicin did not appreciably alter the pharmacokinetics of this anthracycline in the plasma or six tissues evaluated in mice, presumably because, at doses relevant to human exposure, lapatinib inhibition of p-glycoprotein did not significantly alter doxorubicin transport out of these tissue compartments. However, combining lapatinib with docetaxel significantly increased intestinal exposure to this chemotherapeutic, which has clinical implications for enhancing gastrointestinal toxicity. The significant lapatinib-docetaxel interaction is likely CYP3A4-mediated, suggesting that caution should be exercised when this combination is administered, particularly to patients with compromised CYP3A activity, and recipients should be monitored closely for enhanced toxicity, particularly for adverse effects on the intestine.