A unified algorithm for predicting partition coefficients for PBPK modeling of drugs and environmental chemicals

The algorithms in the literature focusing to predict tissue:blood PC (P_tb) for environmental chemicals and tissue:plasma PC based on total (K_p) or unbound concentration (K_pu) for drugs differ in their consideration of binding to hemoglobin, plasma proteins and charged phospholipids. The objective of the present study was to develop a unified algorithm such that P_tb, K_p and K_pu for both drugs and environmental chemicals could be predicted. The development of the unified algorithm was accomplished by integrating all mechanistic algorithms previously published to compute the PCs. Furthermore, the algorithm was structured in such a way as to facilitate predictions of the distribution of organic compounds at the macro (i.e. whole tissue) and micro (i.e. cells and fluids) levels. The resulting unified algorithm was applied to compute the rat P_tb, K_p or K_pu of muscle (n = 174), liver (n = 139) and adipose tissue (n = 141) for acidic, neutral, zwitterionic and basic drugs as well as ketones, acetate esters, alcohols, aliphatic hydrocarbons, aromatic hydrocarbons and ethers. The unified algorithm reproduced adequately the values predicted previously by the published algorithms for a total of 142 drugs and chemicals. The sensitivity analysis demonstrated the relative importance of the various compound properties reflective of specific mechanistic determinants relevant to prediction of PC values of drugs and environmental chemicals. Overall, the present unified algorithm uniquely facilitates the computation of macro and micro level PCs for developing organ and cellular-level PBPK models for both chemicals and drugs.     

A structured approach to Exposure Based Waiving of human health endpoints under REACH developed in the OSIRIS project

Exposure Based Waiving (EBW) is one of the options in REACH when there is insufficient hazard data on a specific endpoint. Rules for adaptation of test requirements are specified and a general option for EBW is given via Appendix XI of REACH, allowing waiving of repeated dose toxicity studies, reproductive toxicity studies and carcinogenicity studies under a number of conditions if exposure is very low. A decision tree is described that was developed in the European project OSIRIS (Optimised Strategies for Risk Assessment of Industrial Chemicals through Integration of Non-Test and Test Information) to help decide in what cases EBW can be justified. The decision tree uses specific criteria as well as more general questions. For the latter, guidance on interpretation and resulting conclusions is provided. Criteria and guidance are partly based on an expert elicitation process. Among the specific criteria a number of proposed Thresholds of Toxicological Concern are used. The decision tree, expanded with specific parts on absorption, distribution, metabolism and excretion that are not described in this paper, is implemented in the OSIRIS webtool on integrated testing strategies.     

Effect of P-glycoprotein-mediated efflux on cerebrospinal fluid concentrations in rhesus monkeys

Brain penetration of drugs which are subject to P-glycoprotein (Pgp)-mediated efflux is attenuated, as manifested by the fact that the cerebrospinal fluid concentration (C_CSF), a good surrogate of the unbound brain concentration (C_ub), is lower than the unbound plasma concentration (C_up) for Pgp substrates. In rodents, the attenuation magnitude of brain penetration by Pgp-mediated efflux has been estimated by correlating the ratio of CSF to plasma exposures (C_CSF/C_p) with the unbound fraction in plasma (f_u) upon the incorporation of the in vivo or in vitro Pgp-mediated efflux ratios (ERs). In the present work, we investigated the impact of Pgp-mediated efflux on C_CSF in monkeys. Following intravenous administration to cisterna magna ported rhesus monkeys, the CSF and plasma concentrations were determined for 25 compounds from three discovery programs. We also evaluated their f_u in rhesus plasma and ER in human and African green monkey MDR-transfected LLC-PK1 cells. These compounds varied significantly in the f_u (0.025-0.73), and 24 out of 25 are considered Pgp substrates based on their appreciable directional transport (ER > 2). The C_CSF/C_p was significantly lower than the corresponding f_u (≥3-fold) for 16 compounds regardless of a significant correlation (R² = 0.59, p = 4 × 10⁻⁵) when the C_CSF/C_p was plotted against the f_u. When the f_u was normalized to the ER (f_u/ER) the correlation was improved (R² = 0.75, p = 8 × 10⁻⁸). More importantly, only one compound showed the C_CSF/C_p that exceeded 3-fold of the normalized f_u. The results suggest that the impact of Pgp-mediated efflux in monkeys, similar to the case in rodents, is reasonably reflected by the gradient between the free concentrations in plasma and in CSF. Therefore, f_u and Pgp ER may serve as useful measurements in estimating in vivo C_CSF/C_p ratios in monkeys, and potentially in humans.     

Assessing the risk of ballast water treatment to human health

Ballast water management systems utilizing noxious chemicals have to be approved according to the Ballast Water Convention by the International Maritime Organization (IMO). The approval procedure requires human health risk assessment. Our objective was to evaluate the existing human health risk assessment process for ballast water management systems. Towards this end, we analyzed the available applications for IMO approval. Since the majority of active substances currently in use are oxidative compounds the corresponding treatment systems generate and release a large number of disinfection by-products. The application dossiers only select a number of by-products for risk assessment. We propose a more comprehensive approach based on the type of ballast water management system, the quality of water treated and the toxicity of compounds discharged into the environment. Subsequent to effects assessment we propose to classify substances according to a hazard evaluation procedure, based on an approach used for maritime transport. We identified a need for better exposure assessment. This requires knowledge of exposure situations. We provide a comprehensive listing of occupational and non-occupational exposure settings and quantification models for exposure assessment.     

Kidney and liver distribution of ochratoxin A in male and female F344 rats

The impact of age and gender on Ochratoxin A (OTA) distribution in kidney and liver were studied. OTA was quantified in kidney and liver of young and mature rats of both sexes. Data was fit simultaneously using the population approach with NONMEM program. Fed and fasted mature males showed a 30% decrease and an 11% increase in relative bioavailability, respectively, in comparison with the rest of the groups. The OTA concentrations reached in kidney and liver were very similar between both organs. The models that best fit to data were the ones that considered that distribution of OTA to kidney and liver occurs from the central compartment and that elimination occurs mainly from the liver compartment. The kinetic analysis revealed that both, the apparent volume of distribution of the central compartment (V/F) and the apparent volume of distribution of the liver and kidney compartments (V_L,K/F) increased significantly with body weight. Thus, the sex differences observed in organs distribution are a reflection of the differences in relative bioavailability observed in adult males, as a consequence of the fed and fasted conditions and to the significant higher body weight of mature males which directly affected the V/F and V_L,K/F.     

Validation of an HPLC-UV method for sorafenib determination in human plasma and application to cancer patients in routine clinical practice

Sorafenib, a new oral multikinase inhibitor with antiangiogenic properties, has demonstrated preclinical and clinical activity against several tumor types. The aims of this study were to validate a method for the measurement of sorafenib in plasma from cancer patients, then to test this method in clinical practice. Following liquid–liquid extraction, the compounds were separated with gradient elution (on a C18 ultrasphere ODS column using a mobile phase of acetonitrile/20 mM ammonium acetate), then detected at 255 nm. The calibration was linear in the range 0.5–20 mg/L. Intra- and inter-assay precision was lower than 7 and 10%, respectively, at 0.5, 3 and 20 mg/L. Plasma sorafenib concentrations were measured in 22 cancer patients (99 samples). The mean trough sorafenib concentration (C_min) and concentration at peak were 4.3 ± 2.5 mg/L (n = 68, CV = 57.5%) and 6.2 ± 3.0 mg/L (n = 31, CV = 47.5%), respectively. Mean sorafenib C_min in eight patients who experienced grade 3 drug-related adverse events was approximately 1.5-fold greater than that observed in the remaining patients (7.7 ± 3.6 mg/L vs. 4.4 ± 2.4 mg/L, P = 0.0083). In conclusion, the method was successfully used in routine practice to monitor plasma concentrations of sorafenib in cancer patients. Finally, large interindividual variability and higher exposure in patients experiencing severe toxicity support the need for therapeutic drug monitoring to ensure an optimal exposure to sorafenib.     

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.     

Role of MAP kinases in regulating expression of antioxidants and inflammatory mediators in mouse keratinocytes following exposure to the half mustard, 2-chloroethyl ethyl sulfide

Dermal exposure to sulfur mustard causes inflammation and tissue injury. This is associated with changes in expression of antioxidants and eicosanoids which contribute to oxidative stress and toxicity. In the present studies we analyzed mechanisms regulating expression of these mediators using an in vitro skin construct model in which mouse keratinocytes were grown at an air-liquid interface and exposed directly to 2-chloroethyl ethyl sulfide (CEES), a model sulfur mustard vesicant. CEES (100-1000 µM) was found to cause marked increases in keratinocyte protein carbonyls, a marker of oxidative stress. This was correlated with increases in expression of Cu,Zn superoxide dismutase, catalase, thioredoxin reductase and the glutathione S-transferases, GSTA1-2, GSTP1 and mGST2. CEES also upregulated several enzymes important in the synthesis of prostaglandins and leukotrienes including cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-2 (mPGES-2), prostaglandin D synthase (PGDS), 5-lipoxygenase (5-LOX), leukotriene A₄ (LTA₄) hydrolase and leukotriene C₄ (LTC₄) synthase. CEES readily activated keratinocyte JNK and p38 MAP kinases, signaling pathways which are known to regulate expression of antioxidants, as well as prostaglandin and leukotriene synthases. Inhibition of p38 MAP kinase suppressed CEES-induced expression of GSTA1-2, COX-2, mPGES-2, PGDS, 5-LOX, LTA₄ hydrolase and LTC₄ synthase, while JNK inhibition blocked PGDS and GSTP1. These data indicate that CEES modulates expression of antioxidants and enzymes producing inflammatory mediators by distinct mechanisms. Increases in antioxidants may be an adaptive process to limit tissue damage. Inhibiting the capacity of keratinocytes to generate eicosanoids may be important in limiting inflammation and protecting the skin from vesicant-induced oxidative stress and injury.     

Subchronic mycotoxicoses in rats. Histopathological changes and modulation of the sphinganine to sphingosine (Sa/So) ratio imbalance induced by Fusarium verticillioides culture material, due to the coexistence of aflatoxin B1 in the diet.

Mycotoxicoses are diseases caused by consumption of diets contaminated with mycotoxins, a special class of fungal secondary metabolites. Fumonisin B1 (FB1) and aflatoxin B1 (AFB1), the main toxins synthesized by toxicogenic stocks of Fusarium spp. and Aspergillus spp., respectively, can coexist in grains and in its by-products. We investigated a probable synergism of a fumonisins-containing Fusarium verticillioides culture material and AFB1 in the induction of hepatocyte apoptosis in rats subchronically fed on a mixture of them. Furthermore, the possibility of modifications in the fumonisins-induced Sa/So ratio imbalance in tissues and urine from rats poisoned with this mycotoxin, due to the presence of AFB1 in the diet, was evaluated. The co-exposure to fumonisins and AFB1 produced a higher liver toxicity, with respect to their individual administration, inducing apoptosis and mitotic hepatocytes. There was an inversion of the typical Sa/So ratio in rats fed on the culture material as well as in those subjected to a diet co-contamined with fumonisins and AFB1. Moreover, the later had a synergistic effect in the induction of Sa/So variations in kidneys. Therefore, the mixture of fumonisins and AFB1 induced toxic responses which could not be considered a sum of the effects caused individually by these mycotoxins.     

The environmental carcinogen 3-nitrobenzanthrone and its main metabolite 3-aminobenzanthrone enhance formation of reactive oxygen intermediates in human A549 lung epithelial cells

The environmental contaminant 3-nitrobenzanthrone (3-NBA) is highly mutagenic and a suspected human carcinogen. We aimed to evaluate whether 3-NBA is able to deregulate critical steps in cell cycle control and apoptosis in human lung epithelial A549 cells. Increased intracellular Ca(2+) and caspase activities were detected upon 3-NBA exposure. As shown by cell cycle analysis, an increased number of S-phase cells was observed after 24 h of treatment with 3-NBA. Furthermore, 3-NBA was shown to inhibit cell proliferation when added to subconfluent cell cultures. The main metabolite of 3-NBA, 3-ABA, induced statistically significant increases in tail moment as judged by alkaline comet assay. The potential of 3-NBA and 3-ABA to enhance the production of reactive oxygen species (ROS) was demonstrated by flow cytometry using 2',7'-dichlorofluorescein-diacetate (DCFH-DA). The enzyme inhibitors allopurinol, dicumarol, resveratrol and SKF525A were used to assess the impact of metabolic conversion on 3-NBA-mediated ROS production. Resveratrol decreased dichlorofluorescein (DCF) fluorescence by 50%, suggesting a role for CYP1A1 in 3-NBA-mediated ROS production. Mitochondrial ROS production was significantly attenuated (20% reduction) by addition of rotenone (complex I inhibition) and thenoyltrifluoroacetone (TTFA, complex II inhibition). Taken together, the results of the present study provide evidence for a genotoxic potential of 3-ABA in human epithelial lung cells. Moreover, both compounds lead to increased intracellular ROS and create an environment favorable to DNA damage and the promotion of cancer.     

Inhibition of phosphate transport in rat heart mitochondria by 3'-azido-3'-deoxythymidine due to stimulation of superoxide anion mitochondrial production

In order to gain some insight into the mechanism by which 3'-azido-3'-deoxythymidine (AZT) damages mitochondria, we investigated whether externally added AZT can stimulate reactive oxygen species (ROS) production by rat heart mitochondria (RHM). An increase in superoxide anion ((O(2)(.-)) production was measured in RHM added with AZT, by using a photometrically method which allows an early O(2)(.-) detection by following the absorbance increase at 550 nm due to the ferricytochrome c reduction. Such an increase was found to be prevented from externally added superoxide dismutase. The stimulation of O(2)(.-) mitochondrial production induced by AZT was found to occur under conditions in which mitochondrial oxygen consumption was prevented by both inhibitors of electron flow and ATP synthesis. Since ROS can cause mitochondrial carrier impairment, we investigated whether AZT can affect mitochondrial permeability in virtue of its capability to stimulate ROS production. In this regard, we studied the transport of phosphate (P(i)), by measuring the mitochondrial shrinkage that takes place as a result of P(i) uptake by RHM previously swollen in a calcium acetate medium. As a result of the AZT-dependent O(2)(.-) production, uncompetitive inhibition of the rate of P(i) transport in RHM was found (K(i) of about 10 microM), consistently, such an inhibition was found to prevent by certain known ROS scavengers, i.e. superoxide dismutase, the antioxidant Vitamin C and reduced gluthatione.     

A quantitative structure-activity approach for lipophilicity estimation of antitumor complexes of different metals using microemulsion electrokinetic chromatography

Microemulsion electrokinetic chromatography (MEEKC) offers a valuable tool for the rapid and highly productive determination of lipophilicity for metal-based anticancer agents. In this investigation, the MEEKC technique was applied for estimation of n-octanol-water partition coefficient (logP(oct)) of a series of antiproliferative complexes of gallium(III) and iron(III) with (4)N-substituted α-N-heterocyclic thiosemicarbazones. Analysis of relationships between the experimental logP(oct) and the retention factors of compounds showed their satisfactory consistency in the case of single metal sets, as well as for both metals. Since none of available calculation programs allows for evaluating the contribution of central metal ion into logP(oct) (i.e. ΔlogP(oct)) of complexes of different metals, this parameter was measured experimentally, by the standard 'shake-flask' method. Extension of the logP(oct) programs by adding ΔlogP(oct) data resulted in good lipophilicity predictions for the complexes of gallium(III) and iron(III) included in one regression set. Comparison of metal-thiosemicarbazonates under examination in terms of logP(oct) vs. antiproliferative activities (i.e. 50% inhibitory concentration in cancer cells) provided evidence that their cytotoxic potency is associated with the ability to cross the lipid bilayer of the cell-membrane via passive diffusion.     

Non-clinical safety assessment and toxicokinetics of voriconazole and anidulafungin in the juvenile rat: A combination study design in support of a Paediatric Investigation Plan

Anidulafungin and voriconazole are potent antifungal agents that may provide a powerful therapeutic option over current therapies when coadministered. A non-clinical combination toxicity study was required as part of the voriconazole Paediatric Investigation Plan. Rats received anidulafungin or voriconazole alone or in combination once daily from postnatal day (PND) 21–56 with a recovery period to PND 84. Doses used were based upon the approximate adult rat no observed adverse-effect level (NOAEL). Transient and reversible reductions in bodyweight, haematology, serum chemistry, liver weight and minimal liver changes were associated with anidulafungin. Voriconazole caused an increase in gamma-glutamyltransferase in female rats only. No increased toxicity was observed with the combination. Toxicokinetics were determined using a validated dual-analyte bioanalytical method. Systemic exposure at juvenile rat NOAELs was comparable to that found with adult rats in previous studies. There were no drug–drug interactions affecting exposure of either drug. Juvenile rats were not more sensitive to each drug dosed alone compared with adult rat data on the single drugs. No novel, additive or synergistic toxicities were noted with the combination in juvenile rats. This study will support future studies of the combination of voriconazole and anidulafungin in children with invasive fungal infection.     

Comparative pharmacology of human dopamine D(2)-like receptor stable cell lines coupled to calcium flux through Galpha(qo5)

The goal of this study was to develop a new approach to study the pharmacology of the dopamine D(4) receptor that could be used in comparative studies with dopamine D(2) and D(3) receptors. Stable HEK-293 cell lines co-expressing recombinant human D(2L), D(3) or D(4) receptors along with Galpha(qo5) cDNA were prepared. Dopamine induced a robust, transient calcium signal in these cell lines with EC(50)s for D(2L), D(3) and D(4) of 18.0, 11.9 and 2.2 nM, respectively. Reported D(4)-selective agonists CP226269 and PD168077 were potent, partial D(4) agonists exhibiting 31-1700-fold selectivity for D(4) over D(3) or D(2). Non-selective D(2)-like agonists apomorphine and quinpirole showed full efficacy but did not discriminate across the three receptors. D(3)-selective agonists 7-hydroxy-DPAT and PD128907 were potent but non-selective D(2)-like agonists. The reported D(3) partial agonist BP-897 exhibited minimal agonist activity at D(3) but was a potent D(3) antagonist and a partial D(4) agonist. Other D(2)-like antagonists, haloperidol, clozapine, and domperidone showed concentration-dependent inhibition of dopamine responses at all three receptors with K(i) ranging from 0.05 to 48.3 nM. The D(3) selective antagonist S33084 and D(4)-selective antagonist L-745870 were highly selective for D(3) and D(4) receptors with K(b) of 0.7 and 0.1 nM, respectively. Stable co-expression of D(2)-like receptors with chimeric Galpha(qo5) proteins in HEK-293 cells is an efficient method to study receptor activation in a common cellular background and an efficient method for direct comparison of ligand affinity and efficacy across human D(2L), D(3) and D(4) receptors.     

The role of reactive oxygen species in WP 631-induced death of human ovarian cancer cells: A comparison with the effect of doxorubicin

In the present study, we investigated the anticancer activity of WP 631, a new anthracycline analog, in weakly doxorubicin-resistant SKOV-3 ovarian cancer cells. We studied the time-course of apoptotic and necrotic events: the production of reactive oxygen species (ROS) and changes in the mitochondrial membrane potential in human ovarian cancer cells exposed to WP 631 in the presence and absence of an antioxidant, N-acetylcysteine (NAC). The effect of WP 631 was compared with the activity of doxorubicin (DOX), the best known first-generation anthracycline.Cytotoxic activity was determined by the MTT assay. The morphological changes characteristic of apoptosis and necrosis in drug-treated cells were analyzed by double staining with Hoechst 33258 and propidium iodide (PI) using fluorescence microscopy. The production of reactive oxygen species and changes in mitochondrial membrane potential were studied using specific fluorescence probes: DCFH₂-DA and JC-1, respectively.The experiments showed that WP 631 was three times more cytotoxic than DOX in the tested cell line. It was found that the new anthracycline analog induced mainly apoptosis and, marginally, necrosis. Apoptotic cell death was associated with morphological changes and a decrease in mitochondrial membrane potential. In comparison to DOX, the novel bisanthracycline induced a significantly higher level of ROS and a greater drop in the membrane potential.The results provide direct evidence that the novel anthracycline WP 631 is considerably more cytotoxic to human SKOV-3 ovarian cancer cells than doxorubicin. The drug can produce ROS, which are immediately involved in the induction of apoptotic cell death.     

Organic copper complexes as a new class of proteasome inhibitors and apoptosis inducers in human cancer cells

Here we report that organic copper complexes can potently and selectively inhibit the chymotrypsin-like activity of the proteasome in vitro and in vivo. Several copper compounds, such as NCI-109268 and bis-8-hydroxyquinoline copper(II) [Cu(8-OHQ)(2)], can inhibit the chymotrypsin-like activity of purified 20S proteasome. In human leukemia cells, proteasome inhibition occurs within 15min after treatment, followed by apoptosis. Neither proteasome inhibition nor apoptosis occurs in non-transformed, immortalized human natural killer cells under the same treatment. Furthermore, proteasome inhibition and apoptosis induction were detected in prostate cancer cells treated with the ligand 8-OHQ alone following pre-treatment with copper(II) chloride. None of these events occurred in cells treated with copper(II) chloride alone, 8-OHQ alone (without growth in copper-enriched media), or nickel(II) chloride pre-treatment followed by 8-OHQ. Furthermore, we found that copper-mediated inhibition of purified 20S proteasome cannot be blocked by a reducing agent and that organic copper compounds do not generate hydrogen peroxide in the cells, suggesting that proteasome inhibition and apoptosis induction are not due to copper-mediated oxidative damage of proteins. Our results suggest that certain types of organic ligands could bind to tumor cellular copper, forming potent proteasome inhibitors and apoptosis inducers at copper concentrations found in tumor tissues.     

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.