Do we need to optimize plasma protein and tissue binding in drug discovery?

It is a commonly accepted assumption that only unbound drug molecules are available to interact with their targets. In order to achieve high unbound plasma drug concentration, it seems obvious to design compounds with low plasma protein binding. Similarly to achieve high unbound tissue concentration, we apparently need compounds with low tissue binding. Our theoretical analysis and experimental data demonstrate that unbound plasma concentration is not determined by plasma protein binding but by hepatic intrinsic clearance after oral dose, and unbound tissue concentration is not determined by tissue binding but determined by unbound plasma concentration and transport properties at the blood-tissue barrier. Reduction of plasma and tissue protein binding for a compound will increase the unbound concentration in vitro but may not increase its unbound plasma or tissue concentration in vivo after oral administration. We conclude that plasma protein and tissue binding are essential parameters to understand pharmacokinetics and pharmacodynamics but they should not be optimized independently in drug discovery. Instead we should focus on reducing clearance and efflux at the blood-tissue barrier to increase in vivo plasma and tissue unbound concentration.     

Plasma protein binding and blood-free concentrations: which studies are needed to develop a drug?

INTRODUCTION: The plasma protein binding of drugs and metabolites is known to influence their pharmacokinetics and, therefore, their effects. Evaluating the extent and the linearity of protein binding is an essential piece of information that has to be generated during drug development. Blood cell partitioning has a similar relevance. AREAS COVERED: This paper summarizes the regulatory requirements and focuses particularly on two questions pertaining to the drug development process. The first of these questions asks when is it necessary to perform detailed clinical studies on protein binding while the second asks whether the in vitro studies presently performed in plasma produce biased information. EXPERT OPINION: The authors propose that clinical ex vivo protein-binding studies should be performed on highly bound compounds (a definition of highly bound is suggested as > 95%). They also propose that in vitro studies, to measure the free drug, should be performed in whole blood, rather than in plasma, particularly if binding to proteins or blood cells is nonlinear.     

Tb drug resistance: is it really a threat to Africa?

In most of the world and particularly in Eastern Europe, China and India, drug resistance is increasingly seen as a major threat to tuberculosis (TB) control and even to public health and health security. What about in Africa? The conditions for creation of drug resistance exist in most, if not all, African countries, as a result of underinvestment in basic TB control, poor management of anti-TB drugs and virtual absence of infection control measures. The severity of drug resistance is increasing--following outbreaks all over the world of multi-drug resistant TB (MDR) in the 1990's, extensive drug resistant (XDR) TB has now been found in 37 countries, including South Africa. (MDR is, in essence, resistance to the most powerful first-line drugs, and XDR-TB is TB resistant to the most powerful second-line drugs as well.) Worse still, the impact of XDR-TB is magnified among those with HIV infection, giving rise to a remarkably high mortality, and exposing significant weaknesses in both HIV and TB control. In particular, the lack of laboratories capable of carrying out culture and drug susceptibility testing severely limits the capacity of countries even to detect the problem in Africa. This paper analyses the threat of TB drug resistance to health and to TB control in Africa, and puts forward measures to diminish this threat.     

What is the precise role of human MDR 1 protein in chemotherapeutic drug resistance?

Elucidating the molecular function of hu MDR 1 protein (also called P-glycoprotein or P-gp 1) and the precise role this protein plays in clinically relevant tumor drug resistance remains a perplexing problem. Hundreds of reports over the past decades summarize a dizzying array of observations relevant to hu MDR 1 protein function. A dominant model in the MDR literature that is used to explain many observations is the well known "drug pump" model first suggested by Keld Dano in 1973 [1]. Although this model has proved useful in conceptualizing additional experiments, it violates fundamental laws of biology and chemistry and in well over a decade of intense effort, active outward drug pumping via hu MDR 1 protein has still never been unequivocally measured. Also, in recent years it has become clear that the drug pump model cannot explain several important phenomena that are highly relevant to the cancer clinic. Thus, other models have also proved increasingly popular. One is the altered partitioning model, which does not violate fundamental laws, is consistent with the vast majority of available data, and has important predictive ability. This newer model has several novel facets that are relevant for cancer pharmacology, and that help explain phenomena not explained by the drug pump model. The basic principle of this model is that MDR proteins do not directly transport drugs, but that their altered expression leads to altered regulation of ion transport or signal transduction that is critical for setting key biophysical parameters of the cell (e.g. compartmental pH and membrane potentials) that dictate relative passive diffusion of drugs as well as important signal transduction linked to the cytotoxic actions of these drugs. Along with debate over the molecular details of hu MDR 1 function, additional controversy surrounds the precise role of hu MDR 1 in the clinic. Many investigators now debate the significance of its function (regardless of precise mechanism) with regard to "real" drug resistance phenotypes exhibited in the clinic. I believe that thorough debate on the pros and cons of various molecular models for hu MDR 1 function will help to address confusion over the clinical relevance of hu MDR1. In the current atmosphere of disappointment over the relative success of clinical trials based in large part on the logic of the drug pump model, it is important that we not lose sight of critical points. Namely, hu MDR 1 protein remains an extremely important window in on the complex pathways that lead to induced chemotherapeutic drug resistance. Exploring the rationale behind newer models for hu MDR 1 function leads to key predictions that can be tested.     

Acylation stimulating protein and triacylglycerol synthesis: potential drug targets?

Triacylglycerol storage in adipose tissue is mediated by a host of transporters, enzymes and binding proteins. Additionally, several hormones (both autocrine and endocrine) are known to interact with cell surface receptors and modulate triacylglycerol synthesis (such as acylation stimulating protein, ASP). The many proteins involved contribute to the robustness of the system and, in most cases, deletion of a single gene is not deleterious and adipose tissue is preserved. On the other hand, this does not mean that gene disruption is not without effect, and in fact often results in a leaner, and presumably "healthier" mouse. These insights provide valuable indications for potential drug tools to delay and/or reverse obesity. In this review we examine the potential of ASP as a candidate target. ASP deficiency in mice decreases adipose tissue mass, increases insulin sensitivity and energy expenditure even in obese ob/ob mice, suggesting that partial interference of ASP action could be advantageous. ASP interacts with a specific cell surface receptor present in adipose tissue and certain structural components, such as the tightly folded core region, are implicated in activity. We propose that interference of the ASP-receptor interaction using an antagonist offers future prospect for an anti-obesity target.     

The 4′-O-benzylated doxorubicin analog WP744 overcomes resistance mediated by P-glycoprotein,multidrug resistance protein and breast cancer resistance protein in cell lines and acute myeloid leukemia cells

Summary Background: The synthetic 4’-O-benzylated doxorubicin analog WP744 was designed to abrogate transport by the multidrug resistance (MDR)-associated ATP-binding cassette (ABC) proteins P-glycoprotein (Pgp) and multidrug resistance protein (MRP-1). We compared its uptake and cytotoxicity with those of doxorubicin and daunorubicin in cell lines overexpressing Pgp, MRP-1 or breast cancer resistance protein (BCRP) and in acute myeloid leukemia (AML) cells. Methods: Cellular uptake was studied by flow cytometry and cytotoxicity in 96-h 96-well cultures in cell lines overexpressing Pgp, MRP-1 or wild type (BCRP^R482) or mutant (BCRP^R482T, BCRP^R482G) BCRP and in pre-treatment AML marrow cells. Results: Uptake and cytotoxicity of WP744 were consistently greater than those of doxorubicin and daunorubicin at equimolar concentrations in all cell lines studied and in AML cells. Conclusion: WP744 overcomes transport by Pgp, MRP-1 and BCRP in cell lines and AML cells and is a promising agent for clinical development in AML and other malignancies with broad-spectrum multidrug resistance.     

Do drug metabolism and pharmacokinetic departments make any contribution to drug discovery?

The alignment of drug metabolism and pharmacokinetic departments with drug discovery has not produced a radical improvement in the pharmacokinetic properties of new chemical entities. The reason for this is complex, reflecting in part the difficulty of combining potency, selectivity, water solubility, metabolic stability and membrane permeability into a single molecule. This combination becomes increasingly problematic as the drug targets become more distant from aminergic seven-transmembrane-spanning receptors (7-TMs). The leads available for aminergic 7-TMs, like the natural agonists, are invariably small molecular weight, water soluble and potent. Even moving to 7-TMs for which the agonist is a peptide invariably produces lead matter that is less drug-like (higher molecular weight and lipophilic). The role of drug metabolism departments, therefore, has been to guide chemistry to obtaining adequate, rather than optimal, pharmacokinetic properties for these 'difficult' drug targets. A consistent belief of many researchers is that a high value is placed on optimal, rather than adequate, pharmacokinetic properties. One measure of value is market sales, and when these are examined no clear pattern emerges. Part of the success of amlodipine in the calcium channel antagonist sector must be due to its excellent pharmacokinetic profile, but the best-selling drugs among the angiotensin antagonists and beta-blockers have a much greater market share than other agents with better pharmacokinetic properties. Clearly, many other factors are important in the successful launch of a medicine, some reflected in the manner the compound is developed and the subsequent structure of the labelling. Overall, therefore the presence of drug metabolism in drug discovery has probably contributed most by allowing 'difficult' drug targets to be prosecuted, rather than by guiding medicinal chemists to optimal pharmacokinetics. These 'difficult' target candidates become successful drugs when skilfully developed. There is no doubt that skilful development relies heavily on drug metabolism and pharmacokinetic departments, in this case those with a clinical rather than a preclinical orientation.     

Analysis of drug resistance and virulence‐factor genotype of Irish Helicobacter pylori strains: is there any relationship between resistance to metronidazole and cagA status?

Background  Helicobacter pylori infection is eradicated with antimicrobial agents and drug-resistant strains make successful treatment difficult. Geographical variations in virulence-factor genotype also exist.
Aim  To evaluate prevalence of drug resistance and virulence-factor genotype in Irish H. pylori strains and to investigate if there is any relationship between drug resistance and genotype.
Methods  Helicobacter pylori strains isolated from 103 patients were examined. Antimicrobial susceptibilities were tested by Etest. The virulence-factor genotypes were determined using PCR. Frequencies of spontaneous metronidazole-resistance were measured in vitro .
Results  Metronidazole resistance was present in 37.9% of strains examined. 16.5% of strains were clarithromycin-resistant and resistance to both agents observed was found in 12.6% of strains. 68% of strains were cagA ⁺. The dominant vacA type was s1/m2, followed by s1/m1 and s2/m2. The metronidazole resistance rate in cagA ⁻ group was significantly higher than in cagA ⁺ ( P  =   0.0089). Spontaneous resistance to metronidazole in cagA ⁻ occurred in higher frequency when compared with cagA ⁺.
Conclusions  cagA ⁺ and vacA s1/m2 type was the dominant genotype in Irish H. pylori strains. Significant rates of metronidazole resistance were observed in cagA ⁻ group. cagA ⁻ strains tend to acquire metronidazole resistance in vitro . Absence of cagA might be a risk factor in development of metronidazole resistance.     

Global analytical strategy to measure drug–plasma protein interactions: from high-throughput to in-depth analysis

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Is the oral route possible for peptide and protein drug delivery?

Oral delivery of peptides and proteins remains an attractive alternative to parenteral delivery and has challenged various attempts at delivery development. Incorporation of new tools into the delivery systems that can raise membrane permeability of macromolecules is essential to attain high oral bioavailability that is acceptable in clinical applications. In developing oral protein delivery systems with high bioavailability, three practical approaches might be most helpful: (1) modification of the physicochemical properties of macromolecules; (2) addition of novel function to macromolecules; or (3) use of improved delivery carriers. Clearly, it is essential that these approaches maintain the biological activity of the proteins.     

Detection of AmpC β-lactamase and drug resistance of Enterobacter cloacae

In order to provide useful information for effective control and clinical therapy of infection, the resistance status and the rate of carrying AmpC β-lactamase of Enterobacter cloacae (E. cloacae) were investigated. By VITEK (Bacterial automatic biochemical analyzer), the isolates of E. cloacae were identified and the drug resistance was measured. The AmpC enzyme was detected by the five-disk diffusion test. Antibiotic sensitivity test showed that the resistance effects of E. cloacae to cefazolin, cefoxitin and ampicillin were more serious, with resistant rates of 80.5%, 75.3% and 70.1%, respectively. However, it was more sensitive to Sulperazone (cefoperazone/sulbactam, 13.0%), amikacin (16.9%) and ciprofloxacin (19.5%). Meanwhile, the phenotype detection showed that 35.06%(27/77) isolates of E. cloacae produced AmpC β-lactamase. Most of E. cloacae are multi-drug resistant strains. Sulperazone (cefoperazone/sulbactam), a kind of component β-lactamase, is a more effective antibiotic for treating infection caused by E. cloacae. Unreasonable application of the third generation cephalosporins plays an important role in leading to emergence of high-yield AmpC β-lactamase strains, so antibiotics should be used wisely.     

β-Adrenoceptors and resistance to stress: old problems and new possibilities

The commentators suggest that our approach may be too limited to account for many of the factors which influence stress-related behaviour, yet too general to help us interpret the neurochemical coding of behavioural responses to stress. In our reply we argue that the strategy described in our Critique both confronts the issues discussed in the Commentaries and is more objective and rigorous than approaches used currently.     

DNA repair protein levels vis-à-vis anticancer drug resistance in the human tumor cell lines of the National Cancer Institute drug screening program

Nucleotide excision repair (NER) is a multi-enzyme DNA repair pathway in eukaryotes. Several NER genes in this pathway including XPB, XPD, XPA and ERCC-1 have been implicated in anticancer drug resistance in human tumor cells. In this study, we assessed the levels of the above-mentioned proteins in the NCI panel of 60 human tumor cell lines in relation to the cytotoxicity patterns of 170 compounds that constitute the standard agent (SA) database. The database consists of drugs used in the clinic for which a mechanism of action has been at least partially defined. The ERCC-1, XPD and XPB protein expression patterns yielded significant negative Pearson correlations with 13, 32 and 17 out of the 170 compounds, respectively (using p<0.05). XPA produced a random assortment of negative and positive correlations, and did not appear to confer an overall resistance or sensitivity to these drugs. Protein expression was also compared with a pre-defined categorization of the standard agents into six mechanism-of-action groups resulting in an inverse association between XPD and alkylating agent sensitivity. Our present data demonstrate that XPD protein levels correlate with resistance to alkylating agents in human tumor cell lines suggesting that XPD is implicated in the development of this resistance. NER activity, using the in vitro cell-free system repair assay, revealed no correlation between NER activity and the level of XPD protein in four cell lines with widely varying XPD protein levels. This lack of correlation may be due to the contribution of XPD to other functions including interactions with the Rad51 repair pathway.     

Validation of cell-based OATP1B1 assays to assess drug transport and the potential for drug–drug interaction to support regulatory submissions

1. Transporters are carrier proteins that may influence pharmacokinetic, pharmacodynamic, and toxicological characteristics of drugs. The development of validated in vitro transporter models is imperative to support regulatory submissions of drug candidates. This study is focused on utilizing human embryonic kidney (HEK) 293 cell cultures genetically transfected with the human organic anion transporting polypeptides (OATP) 1B1 transporter to identify substrates and inhibitors in drug development.

2. The kinetics of OATP1B1-mediated uptake of [³H]-oestradiol 17β-glucuronide and inhibition of uptake by rifamycin SV were used to determine K_m, V_max, and IC₅₀ values over a range of passage numbers to investigate accuracy and precision. The mean K_m and V_max values were found to be 6.3?±?1.2 μM and 460?±?96 pmol min^?1 mg^?1, respectively. The mean IC₅₀ value for rifamycin SV was 0.23?±?0.07 μM on uptake of 1 μM [³H]-oestradiol 17β-glucuronide. These data were similar to previously reported values (accuracy greater than 82%), reproducible (precision less than 29%) and exhibited low standard deviations (SDs) obviating the need to study test compounds on more than one occasion.

3. [³H]-oestrone 3-sulfate and [³H]-pravastatin exhibited concentration-dependent OATP1B1 uptake, and statistically significant differences were observed at each concentration between uptake rates of HEK293-OATP1B1 and HEK293-MOCK cells (uptake ratios greater than or equal to 3). Propranolol showed no positive uptake ratio. Bezafibrate and gemfibrozil exhibited concentration-dependent inhibition of OATP1B1-mediated uptake of [³H]-oestradiol 17β-glucuronide with mean IC₅₀ values of 16 and 27 μM, respectively.

4. Based on the validation results, acceptance criteria to identify a test compound as a substrate and/or inhibitor using these specific cell lines were determined. These validated OATP1B1 assays were robust, reproducible, and suitable for routine in vitro evaluation of candidate drugs.

     

Cryptotanshinone switches target from estrogen receptor α to breast cancer resistance protein(BCRP/ABCG2) to reverse multidrug resistance in breast cancer

OBJECTIVE Targeting individual molecule is not only difficult to cure multigenic cancers, but also prone to inducing resistance. The so-cal ed multidrug resistance(MDR) in breast cancer is highly associated with estrogen receptor α induction and the overexpression of breast cancer resistance protein(BCRP) transporter.Thus, one of the strategies to overcome MDR is to inhibit the expression of the transporter by small molecule inhibitors. METHODS Western blotting and RT-PCR were used respectively for quantification of protein expression and m RNA, non-reducing gradientgel electrophoresis and fluorescence resonance energy transfer(FRET) microscopy imaging for determination of BCRP dimer. RESULTS Cryptotanshinone(CPT) a natural anti-cancer compound,was found to bind BCRP and inhibit its membrane dimerization to attenuate its transport function. And this process is dependent on estrogen receptor α(ERα) in breast cancer. Furthermore, the resistant breast cancer cells with high BCRP expression are also sensitive to CPT followed with the inhibition of membrane dimer of BCRP although they are ERα-negative, suggesting that BCRP expression is essential to CPT reversing the resistance.Meanwhile, the combination of CPT and chemotherapy drugs could obviously enhance the chemotherapeutic effect in vitro. CONCLUSION Totally, we thought that CPT is a novel natural BCRP inhibitor via blocking the formation of BCRP membrane dimer. At the presence of ERα, it functions via ERα regulation, but directly propels BCRP at the absence of ER in resistant cells. CPT can target multi molecules and switch the target from ERα to BCRP in ERα-negative breast cancer, contributing to decrease the occurrence of resistance and reverse multidrug resistance in breast cancer.     

Prospects of multitarget drug designing strategies by linking molecular docking and molecular dynamics to explore the protein–ligand recognition process

The designing of drugs that can simultaneously affect different protein targets is one novel and promising way to treat complex diseases. Multitarget drugs act on multiple protein receptors each implicated in the same disease state, and may be considered to be more beneficial than conventional drug therapies. For example, these drugs can have improved therapeutic potency due to synergistic effects on multiple targets, as well as improved safety and resistance profiles due to the combined regulation of potential primary therapeutic targets and compensatory elements and lower dosage typically required. This review analyzes in-silico methods that facilitate multitarget drug design that facilitate the discovery and development of novel therapeutic agents. Here presented is a summary of the progress in structure-based drug discovery techniques that study the process of molecular recognition of targets and ligands, moving from static molecular docking to improved molecular dynamics approaches in multitarget drug design, and the advantages and limitations of each.