Study on the interaction between erythrosine B and the cardiac drug amiodarone using fluorescence, scattering, and absorbance spectra and their analytical application.

In an acidic buffered solution, erythrosine B can react with amiodarone to form an association complex, which not only generates great enhancement in resonance Rayleigh scattering (RRS) spectrum of erythrosine B at 346.5 nm but also results in quenching of fluorescence spectra of erythrosine B at λemission = 550.4 nm/λexcitation = 528.5 nm. In addition, the formed erythrosine B-amiodarone complex produces a new absorbance peak at 555 nm. The spectral characteristics of the RRS, absorbance, and fluorescence spectra, as well as the optimum analytical conditions, were studied and investigated. As a result, new spectroscopic methods were developed to determine amiodarone by utilizing erythrosine B as a probe. Moreover, the ICH guidelines were used to validate the developed RRS, photometric, and fluorimetric methods. The enhancements in the absorbance and the RRS intensity and the decrease in the fluorescence intensity of the used probe were proportional to the concentration of amiodarone in ranges of 2.5-20.0, 0.2-2.5, and 0.25-1.75 µg/mL, respectively. Furthermore, limit of detection values were 0.52 ng/mL for the spectrophotometric method, 0.051 µg/mL for the RRS method, and 0.075 µg/mL for the fluorimetric method. Moreover, with good recoveries, the developed spectroscopic procedures were applied to analyze amiodarone in its commercial tablets.

Crucial Role of PLGA Nanoparticles in Mitigating the Amiodarone-Induced Pulmonary Toxicity.

BACKGROUND: Amiodarone (AMD) is a widely used anti-arrhythmic drug, but its administration could be associated with varying degrees of pulmonary toxicity. In attempting to circumvent this issue, AMD-loaded polymeric nanoparticles (AMD-loaded NPs) had been designed. MATERIALS AND METHODS: AMD was loaded in NPs by the nanoprecipitation method using two stabilizers: bovine serum albumin and Kolliphor® P 188. The physicochemical properties of the AMD-loaded NPs were determined. Among the prepared NPs, two ones were selected for further investigation of spectral and thermal analysis as well as morphological properties. Additionally, in vitro release patterns were studied and kinetically analyzed at different pH values. In vitro cytotoxicity of an optimized formula (NP4) was quantified using A549 and Hep-2 cell lines. In vivo assessment of the pulmonary toxicity on Sprague Dawley rats via histopathological and immunohistochemical evaluations was applied. RESULTS: The developed NPs achieved a size not more than 190 nm with an encapsulation efficiency of more than 88%. Satisfactory values of loading capacity and yield were also attained. The spectral and thermal analysis demonstrated homogeneous entrapment of AMD inside the polymeric matrix of NPs. Morphology revealed uniform, core-shell structured, and sphere-shaped particles with a smooth surface. Furthermore, the AMD-loaded NPs exhibited a pH-dependent and diffusion-controlled release over a significant period without an initial burst effect. NP4 demonstrated a superior cytoprotective efficiency by diminishing cell death and significantly increasing the IC50 by more than threefold above the pure AMD. Also, NP4 ameliorated AMD-induced pulmonary damage in rats. Significant downregulation of inflammatory mediators and free radicle production were noticed in the NP4-treated rats. CONCLUSION: The AMD-loaded NPs could ameliorate the pulmonary injury induced by the pure drug moieties. Cytoprotective, anti-fibrotic, anti-inflammatory, and antioxidant properties were presented by the optimized NPs (NP4). Future studies may be built on these findings for diminishing AMD-induced off-target toxicities.

Appraisal of amiodarone-loaded PLGA nanoparticles for prospective safety and toxicity in a rat model.

AIMS: Amiodarone (AM) is a highly efficient drug for arrhythmias treatment, but its extra-cardiac adverse effects offset its therapeutic efficacy. Nanoparticles (NPs)-based delivery system could provide a strategy to allow sustained delivery of AM to the myocardium and reduction of adverse effects. The primary purpose was to develop AM-loaded NPs and explore their ameliorative effects versus off-target toxicities. MATERIALS AND METHODS: Polymeric NPs were prepared using poly lactic-co-glycolic acid and their physicochemical properties were characterized. Animal studies were conducted using a rat model to compare exposure to AM versus that of the AM-loaded NPs. Biochemical evaluation of liver enzymes, lipid profile, and thyroid hormones was achieved. Besides, histopathological changes in liver and lung were studied. KEY FINDINGS: Under optimal experimental conditions, the AM-loaded NPs had a size of 186.90 nm and a negative zeta potential (-14.67 mV). Biochemical evaluation of AM-treated animal group showed a significant increase in cholesterol, TG, LDL, T4, and TSH levels (ρ < 0.05). Remarkably, the AM-treated group exhibited a significant increase of liver enzymes (ρ < 0.05) coupled with an obvious change in liver architecture. The AM-loaded NPs displayed a reduction of liver damage and enzyme levels. Lung sections of the AM-treated group demonstrated thickening of interalveolar septa, mononuclear cellular infiltration with congested blood vessels, and heavy collagenous fibers deposition. Conversely, less cellular infiltration and septal thickening were observed in the animal lungs treated with the AM-loaded NPs-treated. SIGNIFICANCE: Our findings demonstrate the competence of the AM-loaded NPs to open several exciting avenues for evading the AM-induced off-target toxicities.

Assessment of practices for suspended oral drugs by tablet crushing in pediatric units.

The aim of this study was to assess the impact of suspended drug by tablet crushing in our pediatric hospital in term of targeted dose and to identify parameters involved in the potential variability. Four usually crushed pediatric drug substances were selected: amiodarone, warfarin, hydrocortisone and captopril. Each tablet was crushed in a bag using a crusher device. Once crushed, a pre-determined volume of water was added using oral syringes before taking the necessary volume to obtain the targeted drug amount. For each drug, operators among pharmacy technicians and nurses investigated 2 targeted doses (high and low). Each suspension was assayed 3 times using the corresponding validated HPLC procedure. Statistical analysis was performed (GraphPad Prism®) to evaluate the impact of operators, the level of suction in bag, and actual drug doses. To investigate the impact of formulation change on syringe drug content, five generic drugs of amiodarone were selected. Syringes contents were compared using one-way ANOVA. Drug loss in syringe ranged from 8.1% to 54.1%. The drug loss represented 18.9% to 30.5% for amiodarone, 0.1% to 5.5% for captopril, 5.6% to 19.7% for warfarin and 5.0% to 30.7% for hydrocortisone. The comparison of level sampling of suspensions presented significant differences for amiodarone, hydrocortisone, and warfarin. Comparison of operators demonstrated significant difference between pharmacy technician and nurse (p = 0.0251). Finally, comparison of 5 generic drugs for amiodarone showed some statistical difference between the syringes content obtained when using the original medicine as compared to the generics. The physicochemical properties of each drug substance and the formulation of the drug product may both factor that should be considered. As a result, crushing tablets in water for oral administration needs a case by case assessment. Although appropriate pediatric formulations are lacking, suspend the crushed material in a given volume of water should be discouraged and not recommended because far from good practice.

Identification of hub genes and discovery of promising compounds in gastric cancer based on bioinformatics analysis.

Aim: To explore the mechanism of gastric carcinogenesis by mining potential hub genes and to search for promising small-molecular compounds for gastric cancer (GC). Materials & methods: The microarray datasets were downloaded from Gene Expression Omnibus database and the genes and compounds were analyzed by bioinformatics-related tools and software. Results: Six hub genes (MKI67, PLK1, COL1A1, TPX2, COL1A2 and SPP1) related to the prognosis of GC were confirmed to be upregulated in GC and their high expression was correlated with poor overall survival rate in GC patients. In addition, eight candidate compounds with potential anti-GC activity were identified, among which resveratrol was closely correlated with six hub genes. Conclusion: Six hub genes identified in the present study may contribute to a more comprehensive understanding of the mechanism of gastric carcinogenesis and the predicted potential of resveratrol may provide valuable clues for the future development of targeted anti-GC inhibitors.

Access to Multifunctionalized Benzofurans by Aryl Nickelation of Alkynes: Efficient Synthesis of the Anti-Arrhythmic Drug Amiodarone.

An unconventional nickel-catalyzed reaction was developed for the synthesis of multifunctionalized benzofurans from alkyne-tethered phenolic esters. The transformation involves the generation of a nucleophilic vinyl NiII species by the regioselective syn-aryl nickelation of an alkyne, which then undergoes an intramolecular cyclization with phenol ester to yield highly functionalized 1,1-disubstituted alkenes with 3-benzofuranyl and (hetero)aryl substituents. The methodology can be used for the late-stage benzofuran incorporation of various drug molecules and natural products, such as 2-propylvaleric acid, gemfibrozil, biotin, and lithocholic acid. Furthermore, this arylative cyclization method was successfully applied for the efficient synthesis of the anti-arrhythmic drug amiodarone.

Live Imaging and Quantitation of Lipid Droplets and Mitochondrial Membrane Potential Changes with Aggregation-Induced Emission Luminogens in an in Vitro Model of Liver Steatosis.

High specificity, low background, good biocompatibility and photostability are common properties of aggregation-induced emission luminogens (AIEgens). In this study, an AIEgen FAS was used in live HepG2 cells, an in vitro model of liver steatosis, to quantify lipid droplet number and size instead of the traditional method of only measuring fluorescence intensity emitted from fluorescence dye stained in lipid droplet. In parallel, another AIEgen, TPE-Ph-In, was used to perform continuous monitoring and quantitation of mitochondrial membrane potential in the same batch of live HepG2 cells. The data show a significant increase in lipid droplet numbers after 24 h treatment by amiodarone and a significant increase in both lipid droplet numbers and size after 48 h amiodarone treatment. Moreover, the data suggest a significant increase in mitochondria membrane potential in cells treated with amiodarone for 24 and 48 h, with restoration to pre-treatment level 24 h after removal of the amiodarone. Further investigation is needed to fully understand the underlying mechanism.

Physicochemical Compatibility of Amiodarone with Parenteral Nutrition.

BACKGROUND: Y-site administration of total parenteral nutrition (TPN) and drugs is frequently required in the intensive care setting. Amiodarone is commonly administered by continuous intravenous infusion and subject to be co-administered via a Y-site with TPN. The aim of this study is to determine the physicochemical stability of amiodarone Y-site administered with TPN. METHODS: Two standard TPN and 2 amiodarone solutions were designed. The 2 TPN differed in the lipid source (Lipofundin MCT/LCT® 20% or SMOFlipid® 20%). The 2 amiodarone solutions were prepared at different concentrations (900 mg and 1200 mg in 250 mL of dextrose 5% in water). Each TPN and amiodarone solutions ran at a rate that simulated a 24-hour Y-site infusion to obtain different admixture samples. Each sample was then visually examined and further tested to determine the mean lipid droplet size distribution by dynamic light scattering and amiodarone concentrations by HPLC. RESULTS: No alterations were detected by visual inspection. Average droplet size remained below 500 nm (252.5 ± 5.9 nm for Lipofundin MCT/LCT® TPN and 327.7 ± 14.4 nm for SMOFlipid® TPN). For the samples obtained after running 900 mg and 1200 mg amiodarone solutions with TPN, the concentrations observed at 24 hours were 0.4491 ± 0.0111 mg/mL and 0.5773 ± 0.0214 mg/mL, respectively. These results represent approximately 100% of the zero-time concentrations and are within ±15% of the predicted values. No degradation products were observed in the chromatograms. CONCLUSION: Amiodarone is physicochemically compatible with standard TPN via a Y-site administration at the tested amiodarone concentrations.

Investigations on small molecule inhibitors targeting the histone H3K4 tri-methyllysine binding PHD-finger of JmjC histone demethylases.

Plant homeodomain (PHD) containing proteins are important epigenetic regulators and are of interest as potential drug targets. Inspired by the amiodarone derivatives reported to inhibit the PHD finger 3 of KDM5A (KDM5A(PHD3)), a set of compounds were synthesised. Amiodarone and its derivatives were observed to weakly disrupt the interactions of a histone H3K4me3 peptide with KDM5A(PHD3). Selected amiodarone derivatives inhibited catalysis of KDM5A, but in a PHD-finger independent manner. Amiodarone derivatives also bind to H3K4me3-binding PHD-fingers from the KDM7 subfamily. Further work is required to develop potent and selective PHD finger inhibitors.

Biomimetic Synthesis of Drug Metabolites in Batch and Continuous-Flow Reactors.

A medium-throughput screening (MTS) of biomimetic drug metabolite synthesis is developed by using an iron porphyrin catalyst. The microplate method, in combination with HPLC-MS analysis, was shown to be a useful tool for process development and parameter optimization in the production of targeted metabolites and/or oxidation products of forty-three different drug substances. In the case of the biomimetic oxidation of amiodarone, the high quantity and purity of the isolated products enabled detailed HRMS and NMR spectroscopic studies. In addition to identification of known metabolites, several new oxidation products of the drug that was studied were characterized. Fast degradation and poor recovery of the catalyst under batch conditions was overcome by immobilization of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin iron(III) chloride (FeTSPP) on the surface of 3-aminopropyl-functionalized silica by electrostatic interaction. The supported catalyst was successfully applied in a packed-bed reactor under continuous-flow reaction conditions for the large-scale synthesis of amiodarone metabolites.

Minimally Invasive Delivery of Hydrogel-Encapsulated Amiodarone to the Epicardium Reduces Atrial Fibrillation.

BACKGROUND: Atrial fibrillation (AF) is the most common cardiac arrhythmia. Although treatment options for AF exist, many patients cannot be maintained in normal sinus rhythm. Amiodarone is an effective medication for AF but has limited clinical utility because of off-target tissue toxicity. METHODS: Here, we use a pig model of AF to test the efficacy of an amiodarone-containing polyethylene glycol-based hydrogel. The gel is placed directly on the atrial epicardium through the pericardial space in a minimally invasive procedure using a specially designed catheter. RESULTS: Implantation of amiodarone-containing gel significantly reduced the duration of sustained AF at 21 and 28 days; inducibility of AF was reduced 14 and 21 days post-delivery. Off-target organ drug levels in the liver, lungs, thyroid, and fat were significantly reduced in animals treated with epicardial amiodarone gel compared with systemic controls in small-animal distribution studies. CONCLUSIONS: The pericardium is an underutilized therapeutic site and may be a new treatment strategy for AF and other cardiovascular diseases.

Molecular interaction of some cardiovascular drugs with human serum albumin at physiological-like conditions: A new approach.

In the present study, the interaction of human serum albumin (HSA) with some cardiovascular drugs (CARs) under physiological conditions was investigated via the fluorescence spectroscopic and Fourier transform infrared spectroscopy. The CAR included Captopril, Timolol, Propranolol, Atenolol, and Amiodarone. Cardiovascular drugs can effectively quench the endogenous fluorescence of HSA by static quenching mechanism. The fluorescence quenching of HSA is mainly caused by complex formation of HSA with CAR. The binding reaction of CAR with HSA can be concluded that hydrophobic and electrostatic interactions are the main binding forces in the CAR-HSA system. The results showed that CAR strongly quenched the intrinsic fluorescence of HSA through a static quenching procedure, and nonradiation energy transfer happened within molecules. Fourier transform infrared spectroscopy absorption studies showed that the secondary structure was changed according to the interaction of HSA and CAR. The binding reaction of CAR with HSA can be concluded that hydrophobic and electrostatic interactions are the main binding forces in the CAR-HSA system. The results obtained herein will be of biological significance in pharmacology and clinical medicines.

Anti-Trypanosoma cruzi action of a new benzofuran derivative based on amiodarone structure.

Chagas disease is a neglected tropical affection caused by the protozoan parasite Trypanosoma cruzi. There is no current effective treatment since the only two available drugs have a limited efficacy and produce side effects. Thus, investigation efforts have been directed to the identification of new drug leads. In this context, Ca2+ regulating mechanisms have been postulated as targets for antiparasitic compounds, since they present paramount differences when compared to host cells. Amiodarone is an antiarrhythmic with demonstrated trypanocidal activity acting through the disruption of the parasite intracellular Ca2+ homeostasis. We now report the effect of a benzofuran derivative based on the structure of amiodarone on T. cruzi. This derivative was able to inhibit the growth of epimastigotes in culture and of amastigotes inside infected cells, the clinically relevant phase. We also show that this compound, similarly to amiodarone, disrupts Ca2+ homeostasis in T. cruzi epimastigotes, via two organelles involved in the intracellular Ca2+ regulation and the bioenergetics of the parasite. We demonstrate that the benzofuran derivative was able to totally collapse the membrane potential of the unique giant mitochondrion of the parasite and simultaneously produced the alkalinization of the acidocalcisomes. Both effects are evidenced by a large increase in the intracellular Ca2+ concentration of T. cruzi.

Adsorption and Leachable Contamination of Flucloxacillin, Cyclosporin and Amiodarone Following Delivery Through an Intravenous Administration Set.

PURPOSE: Interactions between a pharmaceutical drug and its delivery device can result in changes in drug concentration and leachable contamination. Flucloxacillin, amiodarone and cyclosporin were investigated for drug concentration changes and leachable contamination after delivery through an intravenous administration set. METHODS: Flucloxacillin, amiodarone and cyclosporin were delivered through an intravenous administration set and the eluate analysed by HPLC-UV and HPLC-MS. RESULTS: The average recovery of flucloxacillin was 99.7% and no leachable compounds were identified. The average recovery of cyclosporin was 96.1%, which contrasts previous findings that have reported up to 50% loss of cyclosporin. This is likely due to the use of DEHP-free administration sets in this study, as adsorption of cyclosporin is linearly related to DEHP content. The average recovery of amiodarone was 91.5%. 5-hydroxymethylfurfural was identified in the amiodarone solution following delivery through the administration set as well as the 5% glucose solution used for delivery. CONCLUSIONS: Drug/administration set interactions may modify pharmaceuticals during delivery. In this study, only 90% of the amiodarone was delivered through a generic administration set. Given the growing use of generic administration sets in hospital settings, validation of the suitability of their use is required to ensure patient safety and expected levels of efficacy.

Effects of dronedarone, amiodarone and their active metabolites on sequential metabolism of arachidonic acid to epoxyeicosatrienoic and dihydroxyeicosatrienoic acids.

Cardiac enzymes such as cytochrome P450 2J2 (CYP2J2) metabolize arachidonic acid (AA) to cardioprotective epoxyeicosatrienoic acids (EETs), which in turn are metabolized by soluble epoxide hydrolase (sEH) to dihydroxyeicosatrienoic acids (DHETs). As EETs and less potent DHETs exhibit cardioprotective and vasoprotective functions, optimum levels of cardiac EETs are paramount in cardiac homeostasis. Previously, we demonstrated that dronedarone, amiodarone and their main metabolites, namely N-desbutyldronedarone (NDBD) and N-desethylamiodarone (NDEA), potently inhibit human cardiac CYP2J2-mediated astemizole metabolism in vitro. In this study, we investigated the inhibition of recombinant human CYP450 enzymes (rhCYP2J2, rhCYP2C8, rhCYP2C9)-mediated AA metabolism and human recombinant sEH (rhsEH)-mediated EET metabolism by dronedarone, amiodarone, NDBD and NDEA. A static model describing sequential metabolism was further developed to predict the aggregate effect of dual-inhibition of rhCYP2J2 and rhsEH on the fold-of 14,15-EET level (CEET'/CEET). Dronedarone, amiodarone and NDBD inhibit rhCYP2J2-mediated metabolism of AA to 14,15-EET with Ki values of 3.25, 5.48, 1.39µM respectively. Additionally, dronedarone, amiodarone, NDBD and NDEA inhibit rhsEH-mediated metabolism of 14,15-EET to 14,15-DHET with Ki values of 5.10, 13.08, 2.04, 1.88µM respectively. Based on static sequential metabolism modelling, dronedarone (CEET'/CEET=0.85), amiodarone (CEET'/CEET=0.48) and NDBD (CEET'/CEET=0.76) were predicted to decrease cardiac 14,15-EET level whereas NDEA (CEET'/CEET>35.5) was predicted to elevate it. Based on our novel findings, we postulate the differential cardiac exacerbation potential of dronedarone and amiodarone is partly associated with their differential inhibition potencies of cardiac CYP2J2 and sEH.

Cardiac drug-drug interaction between HCV-NS5B pronucleotide inhibitors and amiodarone is determined by their specific diastereochemistry.

Severe bradycardia/bradyarrhythmia following coadministration of the HCV-NS5B prodrug sofosbuvir with amiodarone was recently reported. Our previous preclinical in vivo experiments demonstrated that only certain HCV-NS5B prodrugs elicit bradycardia when combined with amiodarone. In this study, we evaluate the impact of HCV-NS5B prodrug phosphoramidate diastereochemistry (D-/L-alanine, R-/S-phosphoryl) in vitro and in vivo. Co-applied with amiodarone, L-ala,SP prodrugs increased beating rate and decreased beat amplitude in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), but D-ala,RP produgs, including MK-3682, did not. Stereochemical selectivity on emerging bradycardia was confirmed in vivo. Diastereomer pairs entered cells equally well, and there was no difference in intracellular accumulation of L-ala,SP metabolites ± amiodarone, but no D-ala,RP metabolites were detected. Cathepsin A (CatA) inhibitors attenuated L-ala,SP prodrug metabolite formation, yet exacerbated L-ala,SP + amiodarone effects, implicating the prodrugs in these effects. Experiments indicate that pharmacological effects and metabolic conversion to UTP analog are L-ala,SP prodrug-dependent in cardiomyocytes.

Molecular modeling, simulation and docking study of ebola virus glycoprotein.

Ebola virus (EBOV) is a filamentous, enveloped, non-segmented, negative-strand ribonucleic acid (RNA) virus which belongs to family Filoviridae. Ebola virus includes different glycoproteins each of which plays their roles in different aspects of viral life cycle. In this study secreted glycoprotein (Q7T9E0) of Ebola virus was acquired from Uniprot. The formation of alpha helix and beta sheets of secondary structures were predicted through online servers. Higher flexibility and disordered regions of proteins were determined through RONN, GLOBPLOT and DISSEMBLE. Three dimensional (3D) structure of the protein was built through homology modeling techniques and MOE software. The validation and evaluation of the refined models were determined with two stereochemical tests i-e RAMPAGE and ERRAT servers. Further docking studies of given protein was performed with different derivatives of two antiviral drugs dronedarone and amiodarone through MOE. Docking score and binding affinity of respective derivatives demonstrate that these might be used as protein receptors.

High-resolution sub-cellular imaging by correlative NanoSIMS and electron microscopy of amiodarone internalisation by lung macrophages as evidence for drug-induced phospholipidosis.

Correlative NanoSIMS and EM imaging of amiodarone-treated macrophages shows the internalisation of the drug at a sub-cellular level and reveals its accumulation within the lysosomes, providing direct evidence for amiodarone-induced phospholipidosis. Chemical fixation using tannic acid effectively seals cellular membranes aiding intracellular retention of diffusible drugs.

Dronedarone Modulates M1 and M3 Muscarinic Receptors with Subtype Selectivity, Functional Selectivity, and Probe Dependence.

We have previously reported that amiodarone interacts with a novel allosteric site on muscarinic receptors. Amiodarone's most striking effect is to enhance the maximal response elicited by muscarinic agonists at the M1, M3, and M5 receptors. Furthermore, the quaternary analog N-ethylamiodarone (NEA) is inhibitory at these receptors and appears to compete with amiodarone at that allosteric site. In the present studies, we show that dronedarone also modulates Gq-mediated responses at M1 and M3, although in a more discriminating manner. For example, dronedarone markedly enhances pilocarpine-stimulated release of arachidonic acid from CHO cells, via the M3 receptor subtype, but does not affect the acetylcholine-stimulated response. Such probe-dependent effects are diagnostic of an allosteric interaction. In comparison to these effects at M3, dronedarone is strongly inhibitory toward both pilocarpine and acetylcholine at the M1 subtype. The effects of dronedarone are consistent with an interaction at the amiodarone site: dronedarone inhibits the enhancement of acetylcholine's response produced by amiodarone at the M3 subtype; also, NEA reverses the enhancement of pilocarpine's response at M3 produced by either dronedarone or amiodarone. In studies with the M1-selective allosteric agonist 4-[3-(4-butylpiperidin-1-yl)-propyl]-7-fluoro-4H-benzo[1,4]oxazin-3-one (AC-260584), amiodarone enhanced the maximal response observed, whereas dronedarone was inhibitory. On the other hand, benzyl quinolone carboxylic acid, the well-known allosteric ligand that dramatically enhances the potency of acetylcholine at the M1 subtype, had no effect on the response profile of AC-260584. In summary, dronedarone acts at M1 and M3 muscarinic receptors in a manner that complements amiodarone and provides an additional tool with which to investigate this novel allosteric site.