Dose-Time-Response Modeling Using Negative Binomial Distribution

People exposed to certain diseases are required to be treated with a safe and effective dose level of a drug. In epidemiological studies to find out an effective dose level, different dose levels are applied to the exposed and a certain number of cures is observed. Negative binomial distribution is considered to fit overdispersed Poisson count data. This study investigates the time effect on the response at different time points as well as at different dose levels. The point estimation and confidence bands for ED _100p (t) and LT _100p (d) are formulated in closed form for the proposed dose-time-response model with the negative binomial distribution. Numerical illustrations are carried out in order to check the performance level of the proposed model.     

Development of a Generalized,Quantitative Physicochemical Model of CYP3A4 Inhibition for Use in Early Drug Discovery

Purpose. To examine the structure–activity relationships for the inhibition of the activity of recombinant human CYP3A4 and to establish a generalized, quantitative physicochemical model for use in early drug discovery. Methods. Inhibition of the activity of recombinant human CYP3A4 (erythromycin N–demethylase) by 30 diverse chemicals was studied using enhanced throughput methodology. Results. There was a general, strong correlation between the IC₅₀ value determined against erythromycin N–demethylase activity and lipophilicity (LogD_7.4) (r ² = 0.68, p <0.0001). This relationship was strengthened further by subdividing the structures studied into two distinct subpopulations of chemistry within the dataset. These could be identified by the absence (r ² = 0.80, p <0.0001) or presence (r ² = 0.69, p <0.0001) of a sterically uninhindered N–containing heterocycle, more specifically a pyridine, imidazole, or triazole function. The presence of these structural motifs increased the potency of CYP3A4 inhibition by approximately 10–fold for a given lipophilicity (LogD_7.4.value). More detailed analyses of AstraZeneca compounds demonstrated that the inhibitory potency of the pyridine structure can be attenuated through direct steric effects or electronic substitution resulting in a modulation of the pKa of the pyridine nitrogen, thereby influencing its ability to interact with the CYP heme. Conclusions. A generalized, quantitative model is proposed for the inhibition of the major drug metabolizing enzyme, CYP3A4. This model indicates the importance of lipophilicity and rationalizes increased potency arising through additional interactions with the heme iron. These general relationships were shown to be applicable to a selection of compounds of interest to several early research projects.     

Effect of the test media and toxicity of LAS on the growth of <Emphasis Type="Italic">Isochrysis galbana</Emphasis>

In this paper, the toxicity of linear alkylbenzene sulfonate (LAS) was evaluated in the marine microalga Isochrysis galbana using data of growth inhibition toxicity tests at 96-h exposure time. Toxicity was examined in standard conditions and by means of the modification of two variables of the test media: (1) the dilution water and (2) the content of nutrients in the test medium. For this purpose, a total of 10 toxicity test were designed: five dilution waters, four natural marine waters and one synthetic seawater; each in two different nutritive conditions, saturated nutrient concentration (SC) by the addition of modified f/2 nutritive medium, and natural nutrient concentration (NC), i.e., without the addition of f/2. At threshold toxicity levels, the dilution waters used in the test and the nutrient concentrations did not affect the toxicity of LAS. At IC₅₀ concentrations, the toxicity of LAS is influenced by both variables: under SC conditions, the toxic effect of LAS diminishes, obtaining in all the tests IC₅₀ > 10 mg/L LAS. Under NC conditions, IC₅₀ concentrations ranging between 3.15 and 9.26 mg/L LAS have been obtained.     

Automated and reproducible read-across like models for predicting carcinogenic potency

Several qualitative (hazard-based) models for chronic toxicity prediction are available through commercial and freely available software, but in the context of risk assessment a quantitative value is mandatory in order to be able to apply a Margin of Exposure (predicted toxicity/exposure estimate) approach to interpret the data. Recently quantitative models for the prediction of the carcinogenic potency have been developed, opening some hopes in this area, but this promising approach is currently limited by the fact that the proposed programs are neither publically nor commercially available. In this article we describe how two models (one for mouse and one for rat) for the carcinogenic potency (TD₅₀) prediction have been developed, using lazar (Lazy Structure Activity Relationships), a procedure similar to read-across, but automated and reproducible. The models obtained have been compared with the recently published ones, resulting in a similar performance. Our aim is also to make the models freely available in the near future thought a user friendly internet web site.     

Development of quantitative structure-activity relationship (QSAR) models to predict the carcinogenic potency of chemicals: I. Alternative toxicity measures as an estimator of carcinogenic potency

Determining the carcinogenicity and carcinogenic potency of new chemicals is both a labor-intensive and time-consuming process. In order to expedite the screening process, there is a need to identify alternative toxicity measures that may be used as surrogates for carcinogenic potency. Alternative toxicity measures for carcinogenic potency currently being used in the literature include lethal dose (dose that kills 50% of a study population [LD₅₀]), lowest-observed-adverse-effect-level (LOAEL) and maximum tolerated dose (MTD). The purpose of this study was to investigate the correlation between tumor dose (TD₅₀) and three alternative toxicity measures as an estimator of carcinogenic potency. A second aim of this study was to develop a Classification and Regression Tree (CART) between TD₅₀ and estimated/experimental predictor variables to predict the carcinogenic potency of new chemicals. Rat TD₅₀s of 590 structurally diverse chemicals were obtained from the Cancer Potency Database, and the three alternative toxicity measures considered in this study were estimated using TOPKAT^®, a toxicity estimation software. Though poor correlations were obtained between carcinogenic potency and the three alternative toxicity (both experimental and TOPKAT) measures for the CPDB chemicals, a CART developed using experimental data with no missing values as predictor variables provided reasonable estimates of TD₅₀ for nine chemicals that were part of an external validation set. However, if experimental values for the three alternative measures, mutagenicity and logP are not available in the literature, then either the CART developed using missing experimental values or estimated values may be used for making a prediction.     

Mechanistic insights into antitumor effects of new dinuclear cis Pt complexes containing aromatic linkers

The primary objective was to understand more deeply the molecular mechanism underlying different antitumor effects of dinuclear Pt^II complexes containing aromatic linkers of different length, {[cis-Pt(NH₃)₂Cl]₂(4,4′-methylenedianiline)}²⁺ (1) and {[cis-Pt(NH₃)₂Cl]₂(α,α′-diamino-p-xylene)}²⁺ (2). These complexes belong to a new generation of promising polynuclear platinum drugs resistant to decomposition by sulfur nucleophiles which hampers clinical use of bifunctional polynuclear trans Pt^II complexes hitherto tested. Results obtained with the aid of methods of molecular biophysics and pharmacology reveal differences and new details of DNA modifications by 1 and 2 and recognition of these modifications by cellular components. The results indicate that the unique properties of DNA interstrand cross-links of this class of polynuclear platinum complexes and recognition of these cross-links may play a prevalent role in antitumor effects of these metallodrugs. Moreover, the results show for the first time a strong specific recognition and binding of high-mobility-group-domain proteins, which are known to modulate antitumor effects of clinically used platinum drugs, to DNA modified by a polynuclear platinum complex.     

Poly(ethyleneimines) in dermal applications: Biocompatibility and antimicrobial effects

Cationic polyamines, such as poly(ethyleneimines) (PEIs), may recommend themselves for antimicrobial applications as they can interact with microbial membranes resulting in their disruption. The purpose of the study was the assessment of biocompatibility and antibacterial activity of PEIs with different architectures (branched (b) and linear (l)) and molar masses (0.8–750 kDa). lPEI and bPEI exhibited a strong antibacterial activity against Staphylococcus aureus and Escherichia coli with a more pronounced effect on the Gram-positive bacteria. lPEIs further demonstrated a higher antibacterial efficacy compared to bPEIs but no significant differences between 5 and 25 kDa were observed. In accordance, antibacterial activity of bPEI did not specifically depend on molar mass. Only slightly lower minimal inhibitory concentrations (MIC) were observed at 5 kDa (S. aureus) and 25 kDa (E. coli) in the tests. As PEIs are compelling candidates for use in antimicrobial treatment, two basic aspects have to be investigated: treatment effectiveness and safety. PEIs clearly induced molecular weight dependent cytotoxic effects in vitro. PEIs with low molecular weight (0.8 and 5 kDa) exhibited higher biocompatibility. Nonetheless, the results confirmed a low genotoxic potential of lPEI and bPEIs.     

DNA interactions of new cytotoxic tetrafunctional dinuclear platinum complex trans,trans-[{PtCl2(NH3)}2(piperazine)

A new tetrafunctional dinuclear platinum complex trans,trans-[{PtCl2(NH3)}2(piperazine)] with sterically rigid linking group was designed, synthesized and characterized. In this novel molecule, the DNA-binding features of two classes of the platinum compounds with proven antitumor activity are combined, namely trans oriented bifunctional mononuclear platinum complexes with a heterocyclic ligand and polynuclear platinum complexes. DNA-binding mode of this new complex was analyzed by various methods of molecular biology and biophysics. The complex coordinates DNA in a unique way and interstrand and intrastrand cross-links are the predominant lesions formed in DNA in cell-free media and in absence of proteins. An intriguing aspect of trans,trans-[{PtCl2(NH3)}2(piperazine)] is that, using a semi-rigid linker, interstrand cross-linking is diminished relative to other dinuclear platinum complexes with flexible linking groups and lesions that span several base pairs, such as tri- and tetrafunctional adducts, become unlikely. In addition, in contrast to the inability of trans,trans-[{PtCl2(NH3)}2(piperazine)] to cross-link two DNA duplexes, the results of the present work convincingly demonstrate that this dinuclear platinum complex forms specific DNA lesions which can efficiently cross-link proteins to DNA. The results substantiate the view that trans,trans-[{PtCl2(NH3)}2(piperazine)] or its analogues could be used as a tool for studies of DNA properties and their interactions or as a potential antitumor agent. The latter view is also corroborated by the observation that trans,trans-[{PtCl2(NH3)}2(piperazine)] is a more effective cytotoxic agent than cisplatin against human tumor ovarian cell lines.     

DNA interactions of new antitumor platinum complexes with trans geometry activated by a 2-metylbutylamine or sec-butylamine ligand

The global modification of mammalian and plasmid DNAs by novel platinum compounds, trans-[PtCl(2)(NH(3))(Am)], where Am=2 -methylbutylamine or sec-butylamine was investigated in cell-free media using various biochemical and biophysical methods. These modifications were analyzed in the context of the activity of these new compounds in several tumor cell lines including those resistant to antitumor cis-diamminedichloroplatinum(II) (cisplatin). The results showed that the replacement of one amine group by 2-methylbutylamine or sec-butylamine ligand in clinically ineffective trans-diamminedichloroplatinum(II) (transplatin) resulted in a radical enhancement of its activity in tumor cell lines so that they are more cytotoxic than cisplatin and exhibited significant antitumor activity including activity in cisplatin-resistant tumor cells. Importantly, this replacement also markedly altered DNA binding mode of transplatin and reduced the efficiency of repair systems to remove the adducts of the new analogues from DNA. The results support the view that one strategy to activate trans geometry in bifunctional platinum(II) compounds including circumvention of resistance to cisplatin may consist in a chemical modification of the ineffective transplatin which results in an increased efficiency to form DNA interstrand cross-links.     

STI571/doxorubicin concentration-dependent switch for diverse caspase actions in CML cell line K562

We examined the response of the apoptosis-reluctant CML cell line K562 to doxorubicin alone or in combination with the tyrosine kinase inhibitor STI571. We found that at clinically relevant concentrations, doxorubicin induced differentiation and senescence, but did not induce apoptosis. Doxorubicin induced G(2)/M arrest and mitochondrial transmembrane potential dissipation. Interestingly, drug-induced differentiation could be diminished by caspase inhibitors. STI571 caused a graded response characterized by differentiation at low concentrations and apoptosis at higher. STI571 was not observed to induce senescence. Combination of STI571 and caspase inhibitors protected cells from apoptosis but did not influence differentiation. The diverse mode of action of both drugs contributed to the response observed during combination treatment. An additive effect on proliferation was obtained. The mechanisms contributing to inhibition of cellular proliferation were complex and strongly dependent on the applied drug concentrations. Differentiation or apoptosis were enhanced by combined treatment only in narrow ranges of concentrations. Conclusion: DOX and STI571 along diverse mechanisms contributed to elevated levels of activated caspases which might be then responsible for a switch from differentiation to apoptosis.     

A critical review of the data related to the safety of quercetin and lack of evidence of in vivo toxicity, including lack of genotoxic/carcinogenic properties.

Quercetin is a naturally-occurring flavonol (a member of the flavonoid family of compounds) that has a long history of consumption as part of the normal human diet. Because a number of biological properties of quercetin may be beneficial to human health, interest in the addition of this flavonol to various traditional food products has been increasing. Prior to the use of quercetin in food applications that would increase intake beyond that from naturally-occurring levels of the flavonol in the typical Western diet, its safety needs to be established or confirmed. This review provides a critical examination of the scientific literature associated with the safety of quercetin. Results of numerous genotoxicity and mutagenicity, short- and long-term animal, and human studies are reviewed in the context of quercetin exposure in vivo. To reconcile results of in vitro studies, which consistently demonstrated quercetin-related mutagenicity to the absence of carcinogenicity in vivo, the mechanisms that lead to the apparent in vitro mutagenicity, and those that ensure absence of quercetin toxicity in vivo are discussed. The weight of the available evidence supports the safety of quercetin for addition to food.     

Similarities and differences in the coupling of human beta1- and beta2-adrenoceptors to Gs(alpha) splice variants

The human beta1-adrenoceptor (beta1AR) and beta2-adrenoceptor (beta2AR) couple to Gs-proteins to activate adenylyl cyclase (AC). There are differences in desensitization between the beta2AR and the originally cloned Gly389-beta1AR, but with respect to ternary complex formation, constitutive activity, and AC activation the picture is unclear. To learn more about the similarities and differences between the beta1AR and beta2AR, we analyzed coupling of the Gly389-beta1AR to the G(s(alpha)) splice variants Gs(alpha)L and Gs(alpha)S using beta1AR-Gs(alpha) fusion proteins expressed in Sf9 cells and compared the data with previously published data on beta2AR-Gs(alpha) fusion proteins (Seifert et al., J Biol Chem 1998;273:5109-16). Fusion ensures defined receptor/G-protein stoichiometry and efficient coupling. The agonist (-)-isoproterenol stabilized the ternary complex at beta1AR-Gs(alpha)S, beta1AR-Gs(alpha)L, beta2AR-Gs(alpha)S, and beta2AR-Gs(alpha)L with similar efficiency. beta1AR-Gs(alpha)L but not beta1AR-Gs(alpha)S showed the hallmarks of constitutive activity as assessed by increased potencies and efficacies of partial agonists and AC activation by the agonist-free receptor. Similar differences were observed previously for beta2AR-Gs(alpha)S and beta2AR-Gs(alpha)L. beta1AR-Gs(alpha)S and beta2AR-Gs(alpha)S were similarly efficient at activating AC, but beta1AR-Gs(alpha)L was approximately 4-fold more efficient at activating AC than beta2AR-Gs(alpha)L. Our data show that (i) the beta1AR and beta2AR are similarly efficient at stabilizing the ternary complex with Gs(alpha) splice variants, (ii) Gs(alpha)L confers constitutive activity to the beta1AR and beta2AR, and (iii) the beta1AR coupled to Gs(alpha)L is more efficient at activating AC than the beta2AR coupled to Gs(alpha)L. These data help us understand some of the discrepancies regarding similarities and differences between the beta1AR and beta2AR.     

Influence of frying fat on the formation of heterocyclic amines in fried beefburgers and pan residues

The influence of six frying fats (butter, margarine, margarine fat phase, liquid margarine, rapeseed oil and sunflower seed oil) on the formation of mutagenic/carcinogenic heterocyclic amines (HAs) during the frying of beefburgers was investigated. Frying was performed at 165 and 200°C (i.e. under conditions that represented normal household cooking practices). The fried beefburgers and their corresponding pan residues were purified using solid-phase extraction and analysed for HAs using HPLC with photodiode array UV and fluorescence detection. The HAs 2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline (MelQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 9H-pyrido[3,4-b]indole (norharman) and 1-methyl-9H-pyrido[3,4-b]indole (harman) were recovered. The amount increased with the temperature, and the content of HAs in the pan residue was much higher than in the corresponding beefburger. The amounts of MeIQx ranged from 0.2 to 1.6 ng/g in the beefburgers and from 0.8 to 4.3 ng/g in the pan residues. DiMeIQx ranged from undetectable to 0.4 ng/g in the beefburgers and from 0.4 to 1.3 ng/g in the residues. PhIP ranged from 0.08 to 1.5 ng/g in the meat and from 0.4 to 13.3 ng/g in the residues. The total amount of HAs in meat and pan residue combined was significantly lower after frying in sunflower seed oil or margarine than after frying with the other fats. The observed differences in MeIQx and DiMeIQx formation could be explained in terms of oxidation status (peroxide and anisidine value) and antioxidant content (vitamin A, vitamin E and tocopherols/tocotrienols) using partial least squares analysis.     

Evaluation of the binding of the tricyclic isoxazole photoaffinity label LY475776 to multidrug resistance associated protein 1 (MRP1) orthologs and several ATP- binding cassette (ABC) drug transporters

Several of the ATP-binding cassette (ABC) transporters confer resistance to anticancer agents and/or antiviral agents when overexpressed in drug-sensitive cells. Recently a MRP1 (ABCC1) tricyclic isoxazole inhibitor, LY475776 was shown to be a glutathione-dependent photoaffinity label of human MRP1 and showed poor labeling of murine mrp1, an ortholog that does not confer anthracycline resistance. In the present study, the specificity of LY475776 was examined for its ability to modulate or photolabel orthologs of MRP1 and several other drug efflux transporters of the ABC transporter family. LY475776 modulated MRP1 and Pgp-mediated resistance (MDR, ABCB1) in, respectively, HeLa-T5 and CEM/VLB(100) cells to both vincristine and doxorubicin. LY475776 photolabeled 170kDa Pgp and was inhibited by the potent Pgp inhibitor LY335979 (Zosuquidar.3HCl). The labeling of the 190kDa MRP1 protein in membranes of HeLa-T5 cells was inhibited by substrates of MRP1 such as leukotriene C(4), vincrisine, and doxorubicin and by the inhibitor, MK571. LY475776 did not photolabel human MRP2 (ABCC2), MRP3 (ABCC3), MRP5 (ABCC5) or breast cancer resistance protein (ABCG2). Because LY475776 photolabels murine mrp1 less well than human MRP1 and binds to a region believed important for anthracycline binding, studies were conducted with monkey and canine MRP1 which also show a reduced ability to confer resistance to anthracyclines. Unlike murine mrp1, both orthologs were photolabeled well by LY475776. These studies indicate that the specificity of LY475776 is fairly limited to Pgp and MRP1 and further studies will help to define the binding regions.     

Toxicological evaluation of a dietary supplement formulated for male sexual health prior to market release

The dietary supplement, 112 Degrees™, was formulated with the goal of supporting sexual functioning in men. Due to rampant problems with drug adulteration for this category of products, a comprehensive screening for active pharmaceutical agents, with an emphasis on drugs prescribed for erectile dysfunction such as type 5 phosphodiesterase (PDE-5) inhibitors, and known unapproved PDE-5 drug analogues, was performed along with preclinical toxicology studies prior to the introduction of this product into the marketplace. 112 Degrees™ was found to be free of all pharmaceutical adulterants tested, and was not mutagenic, clastogenic, or genotoxic as demonstrated by the Ames test, chromosomal aberration assay, and mouse micronucleus assay, respectively. The LD₅₀ in the 14-day acute oral toxicity study was greater than 5000 mg/kg, the highest dose tested.     

Pharmaceutical applications of vibrational chemical imaging and chemometrics: a review

The emergence of chemical imaging (CI) has gifted spectroscopy an additional dimension. Chemical imaging systems complement chemical identification by acquiring spatially located spectra that enable visualization of chemical compound distributions. Such techniques are highly relevant to pharmaceutics in that the distribution of excipients and active pharmaceutical ingredient informs not only a product's behavior during manufacture but also its physical attributes (dissolution properties, stability, etc.). The rapid image acquisition made possible by the emergence of focal plane array detectors, combined with publication of the Food and Drug Administration guidelines for process analytical technology in 2001, has heightened interest in the pharmaceutical applications of CI, notably as a tool for enhancing drug quality and understanding process. Papers on the pharmaceutical applications of CI have been appearing in steadily increasing numbers since 2000. The aim of the present paper is to give an overview of infrared, near-infrared and Raman imaging in pharmaceutics. Sections 2 and 3 deal with the theory, device set-ups, mode of acquisition and processing techniques used to extract information of interest. Section 4 addresses the pharmaceutical applications.     

Alpha-naphthylisothiocyanate modulates hepatobiliary transporters in sandwich-cultured rat hepatocytes

Alpha-naphthylisothiocyanate (ANIT) induces intra-hepatic cholestasis mixed with hepatocellular injury mainly by bile ductular damage. However, its direct effect on hepatic parenchymal cells (hepatocytes) is unclear. Sandwich-cultured rat hepatocytes (SCRH) were applied to clarify this question. Though cytotoxicity was not observed (0–180 μM) in ANIT-treated SCRH, metabonomics analysis of the hepatocytes revealed a shift in the metabolic pattern and a decrease in cellular cholesterol level, accompanied by an increase in total bile acids after 48 h ANIT (5–45 μM) treatment. To assess the function of major hepatic bile acid transporters, the accumulation and efflux of [D-Pen^2,5]-enkephalin (DPDPE), 5 (and 6)-carboxy-2′,7′-dichlorofluorescein (CDF) diacetate promoiety and deuterium-labeled sodium taurocholate (d8-TCA) were measured. ANIT incubation for either 30 min or 48 h led to dose-dependent decreases in the biliary excretion index (BEI) of DPDPE and CDF, as well as the intracellular accumulation of d8-TCA, CDF and DPDPE. The basolateral efflux of d8-TCA was also decreased with its BEI barely changed. mRNA expression of multiple uptake transporters and bile acid synthesizing enzymes was down-regulated after 48 h incubation. In conclusion, ANIT could directly induce retention of bile acids in hepatocytes by inhibiting the function of bile acid transporters, which might contribute to its cholestatic effect.     

Application and potential of capillary electroseparation methods to determine antioxidant phenolic compounds from plant food material

Antioxidants are one of the most common active ingredients of nutritionally functional foods which can play an important role in the prevention of oxidation and cellular damage inhibiting or delaying the oxidative processes. In recent years there has been an increased interest in the application of antioxidants to medical treatment as information is constantly gathered linking the development of human diseases to oxidative stress.