Clearance and clearance inhibition of the HIV-1 protease inhibitors ritonavir and saquinavir in sandwich-cultured rat hepatocytes and rat microsomes.

The metabolism and active transport of ritonavir and saquinavir were studied using sandwich-cultured rat hepatoyctes and rat liver microsomes. For ritonavir four comparable metabolites were observed in the sandwich-culture and in microsomes. For saquinavir eight metabolites were observed in sandwich-culture and 14 different metabolites in microsomes. Ketoconazole did not affect the metabolism of ritonavir in sandwich-culture or microsomes and slightly inhibited the metabolism of saquinavir in sandwich-culture. This inhibition resulted in a different metabolite profile for saquinavir in microsomes. Ritonavir had a pronounced inhibiting effect on the metabolism of saquinavir and affected the hydroxylation of 6beta-testosterone negatively. In the active transport studies, cyclosporin A and PSC833 enhanced the metabolism of ritonavir, suggesting that ritonavir is normally excreted into the bile canaliculi. Verapamil, showed no effect on the metabolism of ritonavir. The intrinsic clearance was estimated at 1.65 and 67.5 microl/min/1 x 10(6) cells and the hepatic metabolism clearance at 0.017 and 6.83ml/min/SRW for ritonavir and saquinavir respectively. In conclusion, for saquinavir the metabolism rate and the amount of metabolites produced was higher than for ritonavir. Ritonavir had a strong inhibitory effect on the metabolism of saquinavir and seemed to be excreted into the bile.

Spectral EMG Changes in Cervical Dystonia Patients and the Influence of Botulinum Toxin Treatment.

Botulinum toxin (BoNT) injections in the dystonic muscles is the preferred treatment for Cervical Dystonia (CD), but the proper identification of the dystonic muscles remains a challenge. Previous studies showed decreased 8-14 Hz autospectral power in the electromyography (EMG) of splenius muscles in CD patients. Cumulative distribution functions (CDF's) of dystonic muscles showed increased CDF10 values, representing increased autospectral powers between 3 and 10 Hz, relative to power between 3 and 32 Hz. In this study, we evaluated both methods and investigated the effects of botulinum toxin. Intramuscular EMG recordings were obtained from the splenius, semispinalis, and sternocleidomastoid muscles during standardized isometric tasks in 4 BoNT-naïve CD patients, 12 BoNT-treated patients, and 8 healthy controls. BoNT-treated patients were measured 4-7 weeks after their last BoNT injections and again after 11-15 weeks. We found significantly decreased 8-14 Hz autospectral power in splenius muscles, but not in the semispinalis and sternocleidomastoid muscles of CD patients when compared to healthy controls. CDF10 analysis was superior in demonstrating subtle autospectral changes, and showed increased CDF10 values in all studied muscles of CD patients. These results did not change significantly after BoNT injections. Further studies are needed to investigate the origin of these autospectral changes in dystonia patients, and to assess their potential in muscle selection for BoNT treatment.

Dystonic neck muscles show a shift in relative autospectral power during isometric contractions.

OBJECTIVE: To identify effects of a deviant motor drive in the autospectral power of dystonic muscles during voluntary contraction in cervical dystonia patients. METHODS: Submaximal (20%) isometric head-neck tasks were performed with the head fixed, measuring surface EMG of the sternocleidomastoid, splenius capitis and semispinalis capitis in CD patients and controls. Autospectral power of muscle activity, and head forces was analyzed using cumulative distribution functions (CDF). A downward shift between the theta/low alpha-band (3-10Hz) and the high alpha/beta-band (10-30Hz) was detected using the CDF10, defined as the cumulative power from 3 to 10Hz relative to power from 3 to 30Hz. RESULTS: CDF10 was increased in dystonic muscles compared to controls and patient muscles unaffected by dystonia, due to a 3-10Hz power increase and a 10-30Hz decrease. CDF10 also increased in patient head forces. CONCLUSIONS: Submaximal isometric contractions with the head fixed provided a well-defined test condition minimizing effects of reflexive feedback and tremor. We associate shifts in autospectral power with prokinetic sensorimotor control. SIGNIFICANCE: Analysis of autospectral power in isometric tasks with the head fixed is a promising approach in research and diagnostics of cervical dystonia.

Interactions of polybrominated diphenyl ethers with the aryl hydrocarbon receptor pathway.

Polybrominated diphenyl ethers (PBDEs) are brominated flame retardants that have been in use as additives in various consumer products. Structural similarities of PBDEs with other polyhalogenated aromatic hydrocarbons that show affinity for the aryl hydrocarbon receptor (AhR), such as some polychlorinated biphenyls, raised concerns about their possible dioxin-like properties. We studied the ability of environmentally relevant PBDEs (BDE-47, -99, -100, -153, -154, and -183) and the "planar" congener BDE-77 to bind and/or activate the AhR in stably transfected rodent hepatoma cell lines with an AhR-responsive enhanced green fluorescent protein (AhR-EGFP) reporter gene (H1G1.1c3 mouse and H4G1.1c2 rat hepatoma). 7-Ethoxyresorufin-O-deethylation (EROD) was used as a marker for CYP1A1 activity. Dose- and bromination-specific inhibition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced responses was measured by their ability to inhibit the induction of AhR-EGFP expression and EROD activity. Individual exposure to these PBDEs did not result in any increase in induction of AhR-EGFP or CYP1A1 activity. The lower brominated PBDEs showed the strongest inhibitory effect on TCDD-induced activities in both cell lines. While the highest brominated PBDE tested, BDE-183, inhibited EROD activity, it did not affect the induction of AhR-EGFP expression. Similar findings were observed after exposing stably transfected human hepatoma (xenobiotic response element [XRE]-HepG2) cells to these PBDEs, resulting in a small but statically significant agonistic effect on XRE-driven luciferase activity. Co-exposure with TCDD resulted again in antagonistic effects, confirming that the inhibitory effect of these PBDEs on TCDD-induced responses was not only due to direct interaction at receptor level but also at DNA-binding level. This antagonism was confirmed for BDE-99 in HepG2 cells transiently transfected with a Gal4-AhR construct and the corresponding Gal4-Luc reporter gene. In addition, a chromatin immunoprecipitation assay further confirmed that BDE-99 could bind to the AhR and activate the AhR nuclear translocation and dioxin responsive element (DRE) binding in the context of the CYP1A1 promoter. However, the transactivation function of the BDE-99-activated AhR seems to be very weak. These combined results suggest that PBDEs do bind but not activate the AhR-AhR nuclear translocator protein-XRE complex.

Metabolism-mediated Ochratoxin A genotoxicity in the single-cell gel electrophoresis (Comet) assay.

The mycotoxin Ochratoxin A (OTA) is a contaminant of food and feed commodities in many countries. Long-term exposure to OTA in humans has been associated with an increased incidence of a progressive nephropathy (BEN). Bio-activation of OTA has been implicated in the OTA-mediated toxicity, although inconsistent results have been reported. The aim of the present study was to investigate the genotoxic potency of OTA and its metabolites in NIH/3T3 cells stably expressing the human cytochrome P450 iso-enzymes CYP2C9 and CYP3A4, by using the single-cell gel electrophoresis (SCGE/Comet) assay, which detects single strand DNA breaks. The obtained results confirm the hypothesis that biotransformation processes mediate OTA toxicity, and differences in response were observed in CYP2C9-hOR and CYP3A4-hOR expressing cells, respectively. Results showed that biotransformation of OTA increased the genotoxicity. Measurement of reactive oxygen species (ROS) production showed that the OTA-induced ROS production corresponded to the OTA-induced genotoxicity in the used NIH/3T3 cell lines.

The role of oxidative stress in the ochratoxin A-mediated toxicity in proximal tubular cells.

Balkan endemic nephropathy (BEN), a disease characterized by progressive renal fibrosis in human patients, has been associated with exposure to ochratoxin A (OTA). This mycotoxin is a frequent contaminant of human and animal food products, and is toxic to all animal species tested. OTA predominantly affects the kidney and is known to accumulate in the proximal tubule (PT). The induction of oxidative stress is implicated in the toxicity of this mycotoxin. In the present study, primary rat PT cells and LLC-PK(1) cells, which express characteristics of the PT, were used to investigate the OTA-mediated oxidative stress response. OTA exposure of these cells resulted in a concentration-dependent elevation of reactive oxygen species (ROS) levels, depletion of cellular glutathione (GSH) levels and an increase in the formation of 8-oxoguanine. The OTA-induced ROS response was significantly reduced following treatment with alpha-tocopherol (TOCO). However, this chain-braking anti-oxidant did not reduce the cytotoxicity of OTA and was unable to prevent the depletion of total GSH levels in OTA-exposed cells. In contrast, pre-incubation of the cell with N-acetyl-L-cysteine (NAC) completely prevented the OTA-induced increase in ROS levels as well as the formation of 8-oxoguanine and completely protected against the cytotoxicity of OTA. In addition, NAC treatment also limited the GSH depletion in OTA-exposed PT- and LLC-PK(1) cells. From these data, we conclude that oxidative stress contributes to the tubular toxicity of OTA. Subsequently, cellular GSH levels play a pivotal role in limiting the short-term toxicity of this mycotoxin in renal tubular cells.

Improved identification of dystonic cervical muscles via abnormal muscle activity during isometric contractions.

BACKGROUND: The preferred treatment for cervical dystonia (CD) is injection of botulinum toxin in the dystonic muscles. Unfortunately, in the absence of reliable diagnostic methods it can be difficult to discriminate dystonic muscles from healthy muscles acting in compensation. We investigated if dystonic muscle activation patterns could be identified in cervical dystonia patients during a harmonized isometric contraction task. Furthermore, we investigated whether dystonia worsens at higher levels of voluntary contraction, which might further improve the identification of dystonic muscle activity. METHODS: An isometric device was used to investigate muscle activation during voluntary contraction tasks in 10 controls and 10 CD patients. Surface electromyography (EMG) of the sternocleidomastoidus, splenius capitis, and semispinalis capitis muscles was evaluated during a rest task and when performing submaximal (20%) and maximal voluntary contractions for eight head transversal force directions and for head twist. Two measures were developed to identify dystonic activation: 1) Muscle activity in the contraction direction in which the contribution of the muscle was lowest (Minimum EMG), and 2) the average muscle activity over all contraction directions (Total Mean EMG). RESULTS: Patients showed increased dystonic activity in the rest task and during submaximal contractions relative to controls, but not during maximal contractions. Increases in Minimum EMG indicated an inability of patients to deactivate dystonic muscles counteracting the task. Increases in Total Mean EMG indicated dystonic activity in all task directions. During maximal contractions these effects were absent in dystonic muscles. Dystonia is therefore found not to worsen at higher levels of isometric voluntary contraction. The activity of dystonic muscles modulated with different loading directions similar to controls. Using Minimum EMG 54% of the muscles clinically diagnosed as dystonic and 91% of non-dystonic muscles were predicted correctly. CONCLUSIONS: Dystonic muscle activity was found in cervical dystonia patients during submaximal contractions in all task directions using a harmonized isometric task, but no differences were found during maximal contractions. With some adaptation this method may prove useful to identify dystonic muscles.

The regulation of oxidative drug metabolism by growth hormone in the dwarf goat: differences from and similarities to the mechanisms in rats.

The effects of bovine GH (BST), administered in different dose patterns, on in-vivo oxidative drug metabolism, were studied in female dwarf goats. Animals received recombinantly derived methionyl BST at a dose of 500 micrograms/kg body weight per 24 h for 6 days. It was administered to one group of goats as one s.c. injection per day, another group received a similar 24-h dose divided into three s.c. injections given at 8-h intervals, and the third group received 50 micrograms BST/kg body weight every 2.5 h by a pulsative i.v. infusion. Oxidative metabolic capacity was assessed by determining plasma sulphadimidine (SDD) elimination and urinary metabolite excretion. SDD shows a marked sex-dependent plasma elimination in dwarf goats, with male goats having a lower plasma clearance than female goats. When BST was given by daily injection, no clear effects on SDD plasma clearance or urinary metabolite excretion were observed. However, when the total dose was divided into three injections given at 8-h intervals, the plasma SDD elimination rate decreased. This was associated with a decrease in urinary excretion of the two main hydroxy SDD metabolites. When BST was given by discontinuous i.v. infusion, simulating the male endogenous plasma GH pattern, a marked decrease in SDD plasma clearance was observed. In addition, the excretion of the two urinary hydroxy metabolites was considerably reduced. These results suggest that GH can affect drug oxidation in dwarf goats via mechanisms similar to those suggested for rats. However, in the dwarf goat, the sex differences in drug metabolism are opposite to those in rats.

Tiamulin inhibits human CYP3A4 activity in an NIH/3T3 cell line stably expressing CYP3A4 cDNA.

Tiamulin is an antibiotic frequently used in veterinary medicine. The drug has been shown to produce clinically important interactions with other compounds that are administered simultaneously. An NIH/3T3 cell line, stably expressing human cytochrome P450 (EC 1.14.14.1) cDNA (CYP3A4), was used to study the effect of tiamulin on CYP3A4 activity. The 6 beta-hydroxylation activity of testosterone, which is increased in CYP3A4-expressing cells compared to vector-transfected cells, showed reduced activity after incubation with 1 microM tiamulin and was completely reduced to background level after incubation with 2, 5 and 10 microM tiamulin. The CYP3A4-expressing cell line was used in combination with a shuttle vector containing the bacterial lacZ' gene to study the effect of tiamulin on CYP3A4-mediated mutagenicity of aflatoxin B1. The mutation frequency of aflatoxin B1 could be completely inhibited by tiamulin in CYP3A4-expressing cells, but no effect was observed on the mutation frequency of the direct mutagen ethylmethanesulphonate. Western blotting of homogenates of the CYP3A4-expressing cell line showed stabilization of CYP3A4 protein after incubation with tiamulin, supporting the hypothesis that the mechanism of inhibition is by binding of tiamulin to the cytochrome.

Differential effects of pentoxifylline on the hepatic inflammatory response in porcine liver cell cultures. Increase in inducible nitric oxide synthase expression.

Pentoxifylline (PTX) has been shown to exert hepatoprotective effects in various liver injury models. However, little information is available about the effect of PTX on the hepatic acute phase response. In the present study, the effect of PTX on a lipopolysaccharide (LPS)-induced acute phase response in primary porcine liver cell cultures was examined. During 72 hr of incubation with or without LPS, the ability of PTX to influence the secretion of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), acute phase proteins, and nitric oxide (NO) was assessed. PTX completely inhibited LPS-induced TNF-alpha production and attenuated IL-6 only after 48 hr of incubation. In contrast, PTX potentiated NO production and the expression of inducible nitric oxide synthase (iNOS) in hepatocytes after stimulation with LPS. The increased expression of iNOS and concurrent production of NO was also observed when liver cell cultures were incubated with dibutyryl cyclic adenosine monophosphate. No effect of PTX on acute phase protein secretion was observed during 72 hr of incubation. The present results show that PTX differentially affects the endotoxin-induced inflammatory response in primary porcine liver cell cultures by suppressing TNF-alpha and IL-6 while potentiating NO production.

Characterization of cytochrome P450 isoenzymes in primary cultures of pig hepatocytes.

Despite the fact that pigs are increasingly used in pharmacological and toxicological studies, knowledge on the enzymes which metabolize xenobiotics, in particular cytochrome P450 (CYP) enzymes, in pigs is still very limited. Primary cultures of pig hepatocytes were used to characterize CYP enzymes. The characterization was performed at the level of enzymatic activities, apoprotein and mRNA analyses. Enzyme inducers investigated were beta-naphthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX) and rifampicin (RIF). After 48hr of BNF treatment, CYP1A protein and mRNA levels were increased, and ethoxyresorufin O-deethylation and caffeine 3-demethylation were strongly induced. PB and RIF increased the levels of CYP3A apoprotein and mRNA, whereas BNF down-regulated CYP3A and related activities. PB and RIF treatment resulted in increased ethylmorphine N-demethylation and testosterone hydroxylation, which appears to be the result of CYP3A induction. Hybridization of pig RNA with a human CYP2C9 cDNA probe showed a PB and RIF inducible CYP, which was down-regulated by BNF. Similar inducing effects were observed for tolbutamide, a marker substrate for CYP2C. DEX was not a potent inducer, although some induction of CYP3A mRNA was observed. The present results indicate the absence of CYP2B and probably CYP2D enzymes and activities in pig liver. Despite some dissimilarities, the results indicate that pigs, apart from their very human-like physiology, might represent a more appropriate model species for oxidative drug metabolism in humans than rats.

Selective changes in oxidative xenobiotic metabolism in vivo and in vitro after parenteral administration of recombinant bovine somatotrophin to rats.

Effects of recombinant bovine somatotrophin (rBST) on in vivo and in vitro oxidative drug metabolism were studied in male rats. rBST was given subcutaneously at a dose of 250 or 500 micrograms 100 g-1 bodyweight 24 h-1 in different dosage patterns. Sulphadimidine (SDD) plasma clearance, urinary excretion of 6-hydroxy-SDD and the in vitro microsomal SDD-hydroxylations were only inhibited when rBST was given in three injections per 24 hours. The hepatic microsomal ethylmorphine N-demethylation rate and the testosterone hydroxylation rate at the 6 beta position were significantly reduced after one rBST injection per 24 hours. Microsomal testosterone hydroxylation rates at the 16 alpha and 2 alpha-positions were reduced depending on the frequency of rBST administration. It is concluded that the inhibition of in vivo and in vitro drug oxidation in rats by rBST is associated with selective changes in activity of cytochrome P450 enzymes in the liver.

Effect of trenbolone and testosterone on the plasma elimination rates of sulfamethazine, trimethoprim, and antipyrine in female dwarf goats.

Plasma elimination rates of sulfamethazine (100 mg/kg of body weight, IV), trimethoprim (20 mg/kg, IV), and antipyrine (35 mg/kg, IV) were studied in adult female dwarf goats (n = 5) before and after implantation with trenbolone acetate (5 mg/kg). Pretreatment with trenbolone caused a significant decrease in the elimination rate of the drugs tested: for sulfamethazine, 5 times; for antipyrine, 3 times; and for trimethoprim, 2 times. After treatment with testosterone (1 mg/kg, SC, twice weekly for 2.5 weeks), female goats (n = 5) had a similar decrease in the elimination rate of sulfamethazine. Other induced effects included a change in social behavior, a lower voice, and the development of a typical billy goat-like odor. Plasma creatinine concentrations after androgen administration were significantly higher than those before androgen administration; changes were not observed in plasma urea values. Because of the differences observed, we believe that more attention should be paid to the effects of androgenic agents on drug kinetic properties, with particular reference to studies on clinical efficacy, side effects, and drug residues in food products.

Effects of triiodothyronine treatment on pharmacokinetic properties and metabolite formation of antipyrine in dwarf goats.

The influence of triiodothyronine (5 micrograms/kg of body weight, sc, q 12 h for 7 days) on antipyrine (AP, 25 mg/kg, IV) plasma elimination and urinary metabolite excretion was studied in castrated male dwarf goats. After triiodothyronine treatment, a significant increase in AP elimination was found. However, the observed changes in clearances for production of AP metabolites (nor-AP, 3-hydroxymethyl-AP; 4-hydroxy-AP, and 4,4'-dihydroxy-AP) do not suggest a clear selectivity of triiodothyronine toward any of the metabolic pathways of AP.

Effects of Ehrlichia phagocytophila infection on serum thyroid hormone concentrations and on antipyrine clearance and metabolite formation in dwarf goats.

The influence of infection with Ehrlichia phagocytophila (EP) on serum thyroid hormone concentrations and on antipyrine (25 mg/kg of body weight, IV) plasma elimination and urinary metabolite excretion was studied in castrated male dwarf goats. Mean thyroid hormone concentrations moderately decreased in EP-infected goats, with maximal decrease in total and free triiodothyronine and thyroxine serum concentrations of 56, 64, 23, and 19%, respectively. The estimated pharmacokinetic values of antipyrine (AP) in EP-infected goats were similar to those in the goats when healthy. However, glucuronidation of the AP-metabolites 3-hydroxymethyl-AP, 4,4'-dihydroxy-AP, and 4-hydroxy-AP was reduced during the febrile episode of the acute-phase response to EP infection.

Oral bioavailability of sulphamethoxydiazine, sulphathiazole and sulphamoxole in dwarf goats.

To get a better insight into the oral bioavailability of sulphonamides in ruminants, sulphamethoxydiazine (pKa 7.0), sulphathiazole (pKa 7.2), and sulphamoxole (pKa 7.4) were administered to dwarf goats (n = 5). The drugs were given at 2-week intervals by the intravenous or intraruminal route at a dose of 100 mg per kg body weight. After IV injection, the mean half-life (t1/2 beta in h +/- SEM) was 0.80 +/- 0.10 h, 2.35 +/- 0.38 h, and 3.36 +/- 1.25 h, for sulphathiazole, sulphamoxole, and sulphamethoxydiazine, respectively and the mean distribution volume (Vd beta) was 0.23 +/- 0.05 l/kg, 0.23 +/- 0.04 l/kg, and 0.33 +/- 0.02 l/kg. After intraruminal administration, the mean bioavailability varied from 86.0 +/- 11.8% for sulphamethoxydiazine to 46.6 +/- 4.3% for sulphamoxole, and 52.6 +/- 7.2% for sulphathiazole. The elimination half-life was significantly prolonged, probably due to a low rate of drug absorption from the gastrointestinal tract. In contrast to chloramphenicol, the sulphonamides studied were stable when incubated in rumen fluid at 39 degrees C.

EMG coherence and spectral analysis in cervical dystonia: discriminative tools to identify dystonic muscles?

OBJECTIVE: Botulinum toxin injections in the dystonic muscles are the preferred treatment for cervical dystonia (CD), but proper selection of the dystonic muscles remains a challenge. We investigated the use of EMG coherence and autospectral analysis as discriminative tools to identify dystonic muscles in CD patients. METHODS: We compared the occurrence of 8-14 Hz autospectral peaks and 4-7 Hz intermuscular coherences between 10 CD patients and 10 healthy controls. Secondly, we compared the muscles with significant 4-7 Hz coherences with the muscles that were selected clinically for botulinum toxin treatment. RESULTS: Autospectral peaks between 8 and 14 Hz were significantly more often absent in the splenius capitis (SPL) muscles of CD patients compared to controls (p<0.01). Contrary to previous findings, there was no significant difference in the occurrence of 4-7 Hz intermuscular coherences between patients and controls and the diagnostic accuracy of coherence analysis to identify the clinically dystonic muscles was low. CONCLUSION: Intermuscular EMG coherence analysis cannot reliably discriminate patients from controls. Autospectral changes in the SPL muscles are a more discriminative feature of CD. In patients, coherence analysis does not seem to be a reliable method to identify dystonic muscles. The clinical relevance and the origin of the autospectral changes need further study.

Cervical dystonia: improved treatment response to botulinum toxin after referral to a tertiary centre and the use of polymyography.

RATIONALE: Cervical dystonia is the most common form of (primary) dystonia. The first line of treatment for cervical dystonia is intramuscular injections with botulinum toxin. To optimise the response to botulinum toxin proper muscles selection is required. Pre-treatment polymyographic EMG in addition to clinical evaluation is hypothesised to be a good tool to improve muscle selection and treatment outcome. OBJECTIVE: To determine the efficacy of botulinum toxin treatment after adjacent polymyographic EMG in cervical dystonia patients referred to our tertiary referral centre with an unsatisfactory response to botulinum toxin treatment elsewhere. METHODS: We performed a retrospective analysis of 40 consecutive second opinion cervical dystonia patients. Standard polymyographic EMG was performed before treatment. We retrieved the Tsui scores and subjective evaluations from the first visit, after 12 weeks and after one year of treatment. In addition, we assessed the final outcome of treatment in our centre based on the records and asked the patients for their personal opinion about the effect of referral to our centre on their treatment response. RESULTS: After one year of treatment there was a significant improvement on both the Tsui scores (p < 0.01) and the subjective treatment evaluation (p < 0.001.) On their last visit 60% of the patients still continued treatment with a reasonable to good response. CONCLUSION: A substantial amount of CD patients with an unsatisfactory response to botulinum toxin improved after polymyography and subsequent treatment with botulinum toxin in a tertiary referral centre.

Detailed analysis of biased histamine H4 receptor signalling by JNJ 7777120 analogues.

BACKGROUND AND PURPOSE: The histamine H4 receptor, originally thought to signal merely through Gαi proteins, has recently been shown to also recruit and signal via ß-arrestin2. Following the discovery that the reference antagonist indolecarboxamide JNJ 7777120 appears to be a partial agonist in ß-arrestin2 recruitment, we have identified additional biased hH4R ligands that preferentially couple to Gαi or ß-arrestin2 proteins. In this study, we explored ligand and receptor regions that are important for biased hH4R signalling. EXPERIMENTAL APPROACH: We evaluated a series of 48 indolecarboxamides with subtle structural differences for their ability to induce hH4R-mediated Gαi protein signalling or ß-arrestin2 recruitment. Subsequently, a Fingerprints for Ligands and Proteins three-dimensional quantitative structure-activity relationship analysis correlated intrinsic activity values with structural ligand requirements. Moreover, a hH4R homology model was used to identify receptor regions important for biased hH4R signalling. KEY RESULTS: One indolecarboxamide (75) with a nitro substituent on position R7 of the aromatic ring displayed an equal preference for the Gαi and ß-arrestin2 pathway and was classified as unbiased hH4R ligand. The other 47 indolecarboxamides were ß-arrestin2-biased agonists. Intrinsic activities of the unbiased as well as ß-arrestin2-biased indolecarboxamides to induce ß-arrestin2 recruitment could be correlated with different ligand features and hH4R regions. CONCLUSION AND IMPLICATIONS: Small structural modifications resulted in diverse intrinsic activities for unbiased (75) and ß-arrestin2-biased indolecarboxamides. Analysis of ligand and receptor features revealed efficacy hotspots responsible for biased-ß-arrestin2 recruitment. This knowledge is useful for the design of hH4R ligands with biased intrinsic activities and aids our understanding of the mechanism of H4R activation.

Design and pharmacological characterization of VUF14480, a covalent partial agonist that interacts with cysteine 98(3.36) of the human histamine H4 receptor.

BACKGROUND AND PURPOSE: The recently proposed binding mode of 2-aminopyrimidines to the human (h) histamine H4 receptor suggests that the 2-amino group of these ligands interacts with glutamic acid residue E182(5.46) in the transmembrane (TM) helix 5 of this receptor. Interestingly, substituents at the 2-position of this pyrimidine are also in close proximity to the cysteine residue C98(3.36) in TM3. We hypothesized that an ethenyl group at this position will form a covalent bond with C98(3.36) by functioning as a Michael acceptor. A covalent pyrimidine analogue will not only prove this proposed binding mode, but will also provide a valuable tool for H4 receptor research. EXPERIMENTAL APPROACH: We designed and synthesized VUF14480, and pharmacologically characterized this compound in hH4 receptor radioligand binding, G protein activation and ß-arrestin2 recruitment experiments. The ability of VUF14480 to act as a covalent binder was assessed both chemically and pharmacologically. KEY RESULTS: VUF14480 was shown to be a partial agonist of hH4 receptor-mediated G protein signalling and ß-arrestin2 recruitment. VUF14480 bound covalently to the hH4 receptor with submicromolar affinity. Serine substitution of C98(3.36) prevented this covalent interaction. CONCLUSION AND IMPLICATIONS: VUF14480 is thought to bind covalently to the hH4 receptor-C98(3.36) residue and partially induce hH4 receptor-mediated G protein activation and ß-arrestin2 recruitment. Moreover, these observations confirm our previously proposed binding mode of 2-aminopyrimidines. VUF14480 will be a useful tool to stabilize the receptor into an active confirmation and further investigate the structure of the active hH4 receptor.