A Comprehensive Molecular and Clinical Investigation of Approved Anti-HCV Drugs Repurposing against SARS-CoV-2 Infection: A Glaring Gap between Benchside and Bedside Medicine.

The limited availability of effective treatment against SARS-CoV-2 infection is a major challenge in managing COVID-19. This scenario has augmented the need for repurposing anti-virals for COVID-19 mitigation. In this report, the anti-SARS-CoV-2 potential of anti-HCV drugs such as daclatasvir (DCV) or ledipasvir (LDP) in combination with sofosbuvir (SOF) was evaluated. The binding mode and higher affinity of these molecules with RNA-dependent-RNA-polymerase of SARS-CoV-2 were apparent by computational analysis. In vitro anti-SARS-CoV-2 activity depicted that SOF/DCV and SOF/LDP combination has IC50 of 1.8 and 2.0 µM, respectively, comparable to remdesivir, an approved drug for COVID-19. Furthermore, the clinical trial was conducted in 183 mild COVID-19 patients for 14 days to check the efficacy and safety of SOF/DCV and SOF/LDP compared to standard of care (SOC) in a parallel-group, hybrid, individually randomized, controlled clinical study. The primary outcomes of the study suggested no significant difference in negativity after 3, 7 and 14 days in both treatments. None of the patients displayed any worsening in the disease severity, and no mortality was observed in the study. Although, the post hoc exploratory analysis indicated significant normalization of the pulse rate showed in SOF/DCV and SOF/LDP treatment vs. SOC. The current study highlights the limitations of bench side models in predicting the clinical efficacy of drugs that are planned for repurposing.

Discovery of potential multi-target-directed ligands by targeting host-specific SARS-CoV-2 structurally conserved main protease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has resulted in the current COVID-19 pandemic. Worldwide this disease has infected over 2.5 million individuals with a mortality rate ranging from 5 to 10%. There are several efforts going on in the drug discovery to control the SARS-CoV-2 viral infection. The main protease (MPro) plays a critical role in viral replication and maturation, thus can serve as the primary drug target. To understand the structural evolution of MPro, we have performed phylogenetic and Sequence Similarity Network analysis, that depicted divergence of Coronaviridae MPro in five clusters specific to viral hosts. This clustering was corroborated with the comparison of MPro structures. Furthermore, it has been observed that backbone and binding site conformations are conserved despite variation in some of the residues. These attributes can be exploited to repurpose available viral protease inhibitors against SARS-CoV-2 MPro. In agreement with this, we performed screening of ∼7100 molecules including active ingredients present in the Ayurvedic anti-tussive medicines, anti-viral phytochemicals and synthetic anti-virals against SARS-CoV-2 MPro as the primary target. We identified several natural molecules like δ-viniferin, myricitrin, taiwanhomoflavone A, lactucopicrin 15-oxalate, nympholide A, afzelin, biorobin, hesperidin and phyllaemblicin B that strongly binds to SARS-CoV-2 MPro. Intrestingly, these molecules also showed strong binding with other potential targets of SARS-CoV-2 infection like viral receptor human angiotensin-converting enzyme 2 (hACE-2) and RNA dependent RNA polymerase (RdRp). We anticipate that our approach for identification of multi-target-directed ligand will provide new avenues for drug discovery against SARS-CoV-2 infection.Communicated by Ramaswamy H. Sarma.

Discovery of Potent and Selective A2A Antagonists with Efficacy in Animal Models of Parkinson's Disease and Depression.

Adenosine A2A receptor (A2AAdoR) antagonism is a nondopaminergic approach to Parkinson's disease treatment that is under development. Earlier we had reported the therapeutic potential of 7-methoxy-4-morpholino-benzothiazole derivatives as A2AAdoR antagonists. We herein described a novel series of [1,2,4]triazolo[5,1-f]purin-2-one derivatives that displays functional antagonism of the A2A receptor with a high degree of selectivity over A1, A2B, and A3 receptors. Compounds from this new scaffold resulted in the discovery of highly potent, selective, stable, and moderate brain penetrating compound 33. Compound 33 endowed with satisfactory in vitro and in vivo pharmacokinetics properties. Compound 33 demonstrated robust oral efficacies in two commonly used models of Parkinson's disease (haloperidol-induced catalepsy and 6-OHDA lesioned rat models) and depression (TST and FST mice models).

Discovery of liver-directed glucokinase activator having anti-hyperglycemic effect without hypoglycemia.

Glucokinase activators (GKAs) are among the emerging drug candidates for the treatment of type 2 diabetes (T2D). Despite effective blood glucose lowering in clinical trials, many pan-GKAs "acting both in pancreas and liver" have been discontinued from clinical development mainly because of their potential to cause hypoglycemia. Pan-GKAs over sensitize pancreatic GK, resulting in insulin secretion even at sub-normoglycemic level which might be a possible explanation for hypoglycemia. An alternative approach to minimize the risk of hypoglycemia is to use liver-directed GKAs, which are reported to be advancing well in clinical development. Here, we report the discovery and structure-activity relationship (SAR) studies on a novel 2-phenoxy-acetamide series with the aim of identifying a liver-directed GKA. Incorporation of a carboxylic acid moiety as an active hepatocyte uptake recognizing element at appropriate position of 2-phenoxy-acetamide core led to the identification of 26, a potent GKA with predominant liver-directed pharmacokinetics in mice. Compound 26 on oral administration significantly reduced blood glucose levels during an oral glucose tolerance test (oGTT) performed in diet-induced obese (DIO) mice, while showing no sign of hypoglycemia in normal C57 mice over a 10-fold dose range, even when dosed at fasted condition. Together, these data demonstrate a liver-directed GKA has beneficial effect on glucose homeostasis with reduced risk of hypoglycemia.

Design and synthesis of novel xanthine derivatives as potent and selective A2B adenosine receptor antagonists for the treatment of chronic inflammatory airway diseases.

Adenosine induces bronchial hyperresponsiveness and inflammation in asthmatics through activation of A2B adenosine receptor (A2BAdoR). Selective antagonists have been shown to attenuate airway reactivity and improve inflammatory conditions in pre-clinical studies. Hence, the identification of novel, potent and selective A2BAdoR antagonist may be beneficial for the potential treatment of asthma and Chronic Obstructive Pulmonary Disease (COPD). Towards this effort, we explored several prop-2-ynylated C8-aryl or heteroaryl substitutions on xanthine chemotype and found that 1-prop-2-ynyl-1H-pyrazol-4-yl moiety was better tolerated at the C8 position. Compound 59, exhibited binding affinity (Ki) of 62 nM but was non-selective for A2BAdoR over other AdoRs. Incorporation of substituted phenyl on the terminal acetylene increased the binding affinity (Ki) significantly to <10 nM. Various substitutions on terminal phenyl group and different alkyl substitutions on N-1 and N-3 were explored to improve the potency, selectivity for A2BAdoR and the solubility. In general, compounds with meta-substituted phenyl provided better selectivity for A2BAdoR compared to that of para-substituted analogs. Substitutions such as basic amines like pyrrolidine, piperidine, piperazine or cycloalkyls with polar group were tried on terminal acetylene, keeping in mind the poor solubility of xanthine analogs in general. However, these substitutions led to a decrease in affinity compared to compound 59. Subsequent SAR optimization resulted in identification of compound 46 with high human A2BAdoR affinity (Ki = 13 nM), selectivity against other AdoR subtypes and with good pharmacokinetic properties. It was found to be a potent functional A2BAdoR antagonist with a Ki of 8 nM in cAMP assay in hA2B-HEK293 cells and an IC50 of 107 nM in IL6 assay in NIH-3T3 cells. Docking study was performed to rationalize the observed affinity data. Structure-activity relationship (SAR) studies also led to identification of compound 36 as a potent A2BAdoR antagonist with Ki of 1.8 nM in cAMP assay and good aqueous solubility of 529 µM at neutral pH. Compound 46 was further tested for in vivo efficacy and found to be efficacious in ovalbumin-induced allergic asthma model in mice.

Design and synthesis of novel, potent and selective hypoxanthine analogs as adenosine A1 receptor antagonists and their biological evaluation.

Multipronged approach was used to synthesize a library of diverse C-8 cyclopentyl hypoxanthine analogs from a common intermediate III. Several potent and selective compounds were identified and evaluated for pharmacokinetic (PK) properties in Wistar rats. One of the compounds 14 with acceptable PK parameters was selected for testing in in vivo primary acute diuresis model. The compound demonstrated significant diuretic activity in this model.

A2B adenosine receptor antagonists: Design, synthesis and biological evaluation of novel xanthine derivatives.

A2BAdoR is a low affinity adenosine receptor that functions by Gs mediated elevation of cAMP and subsequent downstream signaling. The receptor has been implicated in lung inflammatory disorders like COPD and asthma. Several potent and selective A2BAdoR antagonists have been reported in literature, however most of the compounds suffer from poor pharmacokinetic profile. Therefore, with the aim to identify novel, potent and selective A2BAdoR antagonists with improved pharmacokinetic properties, we first explored more constrained form of MRS-1754 (4). To improve the metabolic stability, several linker modifications were attempted as replacement of amide linker along with different phenyl or other heteroaryls between C8 position of xanthine head group and terminal phenyl ring. SAR optimization resulted in identification of two novel A2BAdoR antagonists, 8-{1-[5-Oxo-1-(4-trifluoromethyl-phenyl)-pyrrolidin-3-ylmethyl]-1H-pyrazol-4-yl}-1,3-dipropyl-xanthine (31) and 8-(1-{2-Oxo-2-[4-(3-trifluoromethyl-phenyl)-piperazin-1-yl]-ethyl}-1H-pyrazol-4-yl)-1,3-dipropyl-xanthine (65), with high binding affinity (Ki = 1 and 1.5 nM, respectively) and selectivity for A2BAdoR with very good functional potency of 0.9 nM and 4 nM, respectively. Compound 31 and 65 also displayed good pharmacokinetic properties in mice with 27% and 65% oral bioavailability respectively. When evaluated in in vivo mice model of asthma, compound 65 also inhibited airway inflammation and airway reactivity in ovalbumin induced allergic asthma at 3 mpk dose.

Discovery of pyrazole carboxylic acids as potent inhibitors of rat long chain L-2-hydroxy acid oxidase.

Long chain L-2-hydroxy acid oxidase 2 (Hao2) is a peroxisomal enzyme expressed in the kidney and the liver. Hao2 was identified as a candidate gene for blood pressure (BP) quantitative trait locus (QTL) but the identity of its physiological substrate and its role in vivo remains largely unknown. To define a pharmacological role of this gene product, we report the development of selective inhibitors of Hao2. We identified pyrazole carboxylic acid hits 1 and 2 from screening of a compound library. Lead optimization of these hits led to the discovery of 15-XV and 15-XXXII as potent and selective inhibitors of rat Hao2. This report details the structure activity relationship of the pyrazole carboxylic acids as specific inhibitors of Hao2.

Discovery of a potent and selective small molecule hGPR91 antagonist.

GPR91, a 7TM G-Protein-Coupled Receptor, has been recently deorphanized with succinic acid as its endogenous ligand. Current literature indicates that GPR91 plays role in various pathophysiology including renal hypertension, autoimmune disease and retinal angiogenesis. Starting from a small molecule high-throughput screening hit 1 (hGPR91 IC(50): 0.8 µM)-originally synthesized in Merck for Bradykinin B(1) Receptor (BK(1)R) program, systematic structure-activity relationship study led us to discover potent and selective hGPR91 antagonists e.g. 2c, 4c, and 5 g (IC(50): 7-35 nM; >1000 fold selective against hGPR99, a closest related GPCR; >100 fold selective in Drug Matrix screening). This initial work also led to identification of two structurally distinct and orally bio-available lead compounds: 5g (%F: 26) and 7e (IC(50): 180 nM; >100 fold selective against hGPR99; %F: 87). A rat pharmacodynamic assay was developed to characterize the antagonists in vivo using succinate induced increase in blood pressure. Using two representative antagonists, 2c and 4c, the GPR91 target engagement was subsequently demonstrated using the designed pharmacodynamic assay.

RBx-0597, a potent, selective and slow-binding inhibitor of dipeptidyl peptidase-IV for the treatment of type 2 diabetes.

Dipeptidyl peptidase IV (DPP-IV) inhibiton is a well recognized approach to treat Type 2 diabetes. RBx-0597 is a novel DPP-IV inhibitor discovered in our laboratory. The aim of the present study was to characterize the pharmacological profiles of RBx-0597 in vitro and in vivo as an anti-diabetic agent. RBx-0597 inhibited human, mouse and rat plasma DPP-IV activity with IC(50) values of 32, 31 and 39nM respectively. RBx-0597 exhibited significant selectivity over dipeptidyl peptidase8 (DPP-8), dipeptidyl peptidase9 (DPP-9) (150-300 fold) and other proline-specific proteases (>200-2000 fold). Kinetic analysis revealed that RBx-0597 is a competitive and slow binding DPP-IV inhibitor. In ob/ob mice, RBx-0597 (10mg/kg) inhibited plasma DPP-IV activity upto 50% 8h post-dose and showed a dose-dependent glucose excursion. RBx-0597 (10mg/kg) showed a significant glucose lowering effect (∼25% AUC of △ blood glucose) which was sustained till 12h, significantly increased the active glucagon-like peptide-1(GLP-1) and insulin levels. It showed a favourable pharmacokinetic profile (plasma clearance:174ml/min/kg; C(max) 292ng/ml; T(1/2) 0.28h; T(max) 0.75h and V(ss) 4.13L/kg) in Wistar rats with the oral bioavailability (F(oral)) of 65%. In summary, the present studies indicate that RBx-0597 is a novel DPP-IV inhibitor with anti-hyperglycemic effect and a promising candidate for further development as a drug for the treatment of type 2 diabetes.

Potent and Selective Inhibitors of Long Chain l-2-Hydroxy Acid Oxidase Reduced Blood Pressure in DOCA Salt-Treated Rats.

l-2-Hydroxy acid oxidase (Hao2) is a peroxisomal enzyme with predominant expression in the liver and kidney. Hao2 was recently identified as a candidate gene for blood pressure quantitative trait locus in rats. To investigate a pharmacological role of Hao2 in the management of blood pressure, selective Hao2 inhibitors were developed. Optimization of screening hits 1 and 2 led to the discovery of compounds 3 and 4 as potent and selective rat Hao2 inhibitors with pharmacokinetic properties suitable for in vivo studies in rats. Treatment with compound 3 or 4 resulted in a significant reduction or attenuation of blood pressure in an established or developing model of hypertension, deoxycorticosterone acetate-treated rats. This is the first report demonstrating a pharmacological benefit of selective Hao2 inhibitors in a relevant model of hypertension.

Ratiometric Ca+2 measurement in human recombinant muscarinic receptor subtypes using the Flexstation scanning fluorometer.

In modern drug discovery, numerous assay formats are available to screen and quantitate receptor-ligand interactions. Radioactive assays are "gold standard" because they are fast, easy, and reproducible; however, they are hazardous, produce radioactive waste, require special lab conditions, and are expensive on a large scale. Thus, it provides a lot of importance to the "mix & measure" assays that have an optical readout. Fluorescence techniques are likely to be among the most important detection approaches used for high throughput screening due to their high sensitivity and amenability to automation. The aim of the present study was to determine the functional antagonistic affinities of standard muscarinic antagonists in CHO cells over expressing m1, m3, and m5 receptors and to compare them with the respective binding affinities. This study was further extended to elucidate that Ca+2 measurement assays can serve as a functional screening tool for GPCRs. For this purpose, standard muscarinic receptor antagonists, namely, tolterodine, oxybutynin, and atropine were used. We determined and compared the IC50 values of these three standard inhibitors in fura 2 AM loaded m1, m3, and m5 overexpressing CHO cells and in radioligand binding assay. Both the assays exhibited comparable rank order potencies of the standard inhibitors. This study suggests that Ca+2 mobilization assays can be an alternate to radioligand binding assays.

RBx 6198: a novel alpha1-adrenoceptor antagonist for the treatment of benign prostatic hyperplasia.

The present study, investigates the effect of RBx 6198, 2-{3-[4-(2-Isopropoxy-phenyl)-piperazin-1-yl]-propyl}-3a, 4, 7, 7a-tetrahydro-isoindole-1, 3,-dione, a novel alpha(1)-adrenoceptor antagonist, in both in vitro and in vivo test systems. RBx 6198 is a potent (nanomolar affinity) alpha(1A)-adrenoceptor antagonist with demonstrable uroselectivity in anaesthesized dog model. In radioligand binding studies using human recombinant receptors, RBx 6198 exhibited high selectivity (approximately 50 fold) for the alpha(1A)-adrenoceptor subtype as compared to alpha(1B)-adrenoceptor subtype. In order to assess tissue selectivity, the antagonistic effect of RBx 6198 on the phenylephrine induced contractile response of isolated rat prostate, spleen and aorta was characterized. RBx 6198 was 8 fold more potent in inhibiting phenylephrine-evoked contractions of isolated tissues compared to tamsulosin. However, the compound was non-selective for alpha(1A) vs. alpha(1D)-adrenoceptor like tamsulosin. In anaesthetized beagle dogs RBx 6198 suppressed the intraurethral pressure response to phenylephrine to a greater extent than the mean arterial pressure response thereby demonstrating uroselectivity consistent with in vitro binding and functional data. RBx 6198 was 6.4 fold more uroselective as compared to tamsulosin after i.v. route dose administration. Taken together all results from preclinical studies, it is suggested that RBx 6198 is a novel alpha(1)-adrenoceptor antagonist that exhibited improved pharmacological profile over tamsulosin in both in vitro and in vivo.

Discovery of conformationally rigid 3-azabicyclo[3.1.0]hexane-derived dipeptidyl peptidase-IV inhibitors.

The induction of conformationally restricted N-(aryl or heteroaryl)-3-azabicyclo[3.1.0]hexane derivatives at P(2) region of compounds of 2-cyanopyrrolidine class was explored to develop novel DPP-IV inhibitors. The synthesis, structure-activity relationship, and selectivity against related proteases are delineated.

Synthesis and optimization of novel and selective muscarinic M(3) receptor antagonists.

A series of constrained piperidine analogues were synthesized as novel muscarinic M(3) receptor antagonists. Evaluation of these compounds in binding assays revealed that they not only have high affinity for the M(3) receptor but also have high selectivity over the M(2) receptor.

Comparative in vivo uroselectivity profiles of anticholinergics, tested in a novel anesthetized rabbit model.

The aim of this study was to describe a new experimental animal model for simultaneous measurement of carbachol-induced increase in intravesical pressure and salivary secretion in rabbits. Further, we also compared the in vivo potency and urinary bladder versus salivary gland selectivity profiles of Oxybutynin, Tolterodine, Solifenacin and Darifenacin. The intravesical pressure and salivary secretion were evoked by intra-arterial injection of carbachol (1.5 microg/kg). The carbachol-induced increase in intravesical pressure and salivation was simultaneously recorded before and after increasing doses of test drugs administered intravenously. The basal mean changes in intravesical pressure and salivation subsequent to carbachol administration were in the range of 6.7-7.5 mm Hg and 0.5-0.7 g respectively. Repeated administration of vehicle did not elicit any appreciable changes in intravesical pressure and salivary secretion to carbachol administration from the basal values till 3 h. All the test drugs exhibited a dose-dependent inhibition of carbachol-induced increase in intravesical pressure and salivary secretion. Darifenacin demonstrated a greater potency compared to other muscarinic receptor antagonists for inhibiting carbachol-induced increase in intravesical pressure. It also exhibited functional selectivity for the urinary bladder versus salivary gland. In contrast, Oxybutynin was functionally more selective in inhibiting carbachol-induced increase in salivary secretion. The observed urinary bladder versus salivary selectivity values were 0.6+/-0.2, 1.1+/-0.2, 1.7+/-0.5, and 2.3+/-0.5 for Oxybutynin, Tolterodine, Solifenacin and Darifenacin respectively. These results suggest that the functional selectivity of muscarinic receptor antagonists between urinary bladder and salivary glands can be readily detected in this model. Thus rabbits may represent a useful animal model for evaluating putative bladder selective muscarinic receptor antagonists for the treatment of overactive bladder.

Role of peripheral 5-HT1A receptors in detrusor over activity associated with partial bladder outlet obstruction in female rats.

We have investigated the role of peripheral 5-hydroxytryptamine 1A (5-HT(1A)) receptors and their probable up-regulation in rat model of partial bladder outlet obstruction. Bladder outlet obstruction was induced in adult female rats, hypertropic bladders were harvested after 6 weeks and isometric contractions of bladder strips were recorded. A marked spontaneous activity of the bladder was observed in obstructed bladder strips compared to control strips. The effect of alpha(1A/1D)-adrenergic antagonist, tamsulosin, was observed to be inhibitory on the spontaneous contractions albeit at higher doses (10, 30 and 100 nM). As tamsulosin at higher doses also has high affinity for 5-HT(1A) receptors, the role of peripheral 5-HT(1A) receptors in overactive bladder was hypothesized. 8-hydroxy-2-(di-n-propylamino) tetralin [8-OH-DPAT], a selective 5-HT(1A) receptor agonist, dose-dependently induced significant contractions in the obstructed bladder strips, compared to control bladders. N-[2-[4-(2-methoxyphenyl) piperazin-1-yl]ethyl]-N-pyridin-2-yl-cyclohexanecarboxamide dihydrochloride (WAY-100635), a selective 5-HT(1A) receptor antagonist, competitively antagonized the contractile response to 8-OH-DPAT in obstructed bladder strips in a dose-dependent manner. Tamsulosin at a higher dose was also observed to antagonize the responses to 8-OH-DPAT. Taken together, these observations suggest the involvement of peripheral 5-HT(1A) receptors in detrusor over activity associated with bladder outlet obstruction in female rats.

Statins and myotoxicity: a therapeutic limitation.

Hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors represent the most successful class of drugs for the treatment of hypercholesterolaemia and dyslipidaemia implicated in the pathogenesis of coronary heart disease and atherosclerosis. However, the popular profile of statins in terms of efficacy has been maligned by its adverse events. The myotoxicity, ranging from mild myopathy to serious rhabdomyolysis, associated with HMG-CoA reductase inhibitors, during treatment of hypercholesterolaemia is of paramount importance. Rhabdomyolysis is a rare but idiosyncratic muscle wasting disorder of different etiologies. Statin-associated rhabdomyolysis causes skeletal muscle injury by self-perpetuating events leading to fatal irreversible renal damage through a series of biochemical reactions. Preferential distribution and action of statins in liver could be the key to minimise myotoxicity concerns. Hepato-specific distribution of statins is governed by various factors such as physicochemical properties, pharmacokinetic properties and selective transporter-mediated uptake in liver rather in extrahepatic cells. The interactions of statins with concomitant drugs of different classes merit attention for their safety profile. Although pharmacokinetic as well as pharmacodynamic interactions have been implicated in pathophysiology of statin-induced muscle wasting, the underlying mechanism is not clearly understood. Besides, pharmacokinetic and phramcodynamic factors, statin-associated myotoxcity may also implicate pharmacogenomic factors. The pharmacogenomics characterised by CYP polymorphism and other genetic factors is responsible for inter-individual variations to efficacy and tolerability of statins. The pathophysiological mechanisms may include statin-induced differences in cholesterol:phospholipid ratio, isoprenoid levels, small GTP binding proteins and apoptosis. However, the present understanding of pathophysiological mechanisms, does not offer a reliable approach to address the same at preclinical level. Although statin-associated myotoxicity affects compliance, quality of life of patient and discontinuation rate, yet the low incidence of myotoxicty including rhabdomyolysis and less severity of commonly occurring myopathy and myalgia do not raise doubts about the clinical efficacy and tolerability of statins. Medical management of myotoxicity seems to be pivotal for the proper compliance of patients with statin treatment. The appropriate and judicious use of drugs would substantially reduce the likelihood of developing clinically important myopathy.

Molecular cloning, stable expression and cellular localization of human alpha1-adrenergic receptor subtypes: effect of charcoal/dextran treated serum on expression and localization of alpha1D -adrenergic receptor.

The cDNAs encoding for three subtypes of adrenergic receptors, alpha1A-, alpha1B- and alpha1D-ARs, were cloned and expressed in HEK 293 cells. Expression of alpha1A- and alpha1B-AR subtypes in HEK 293 cells was stable even with increased passages but that of alpha1D-AR was not. Cellular localization studies using immunofluorescence and flow cytometry revealed that expression of alpha1A- and alpha1B-ARs was primarily localized on the cell membrane whereas expression of alpha1D-AR was predominantly intracellular. Our studies clearly demonstrated that the culturing of the recombinant cell lines expressing alpha1D-AR in charcoal/dextran treated fetal bovine serum (FBS) resulted in targeting of alpha1D-AR to the cell membrane and thus, significantly improving its stability and availability for ligand binding studies.

Intermittent claudication: an overview.

Intermittent claudication (IC) is defined by leg muscle pain, cramping and fatigue brought on by ambulation/exercise; relieved on rest; and caused by inadequate blood supply and is the primary symptom of peripheral arterial disease (PAD). PAD has a detrimental effect on the quality of life. PAD is a debilitating atherosclerotic disease of the lower limbs and is associated with an increased risk of cardiovascular morbidity and mortality. IC is an extremely important marker of atheroma. Up to 60% patients with IC have significant underlying coronary and/or carotid disease and 40% of all patients suffering from IC die or suffer a stroke within 5 years of presentation. The therapeutic intervention of IC essentially aims at providing symptomatic relief and reducing the systemic cardiovascular complications. Although exercise therapy is one of the most efficacious conservative treatments for claudication, the pharmacotherapeutic goals can be best achieved through an increase in the walking capacity to improve quality of life and a decrease in rates of amputation. In the development of treatment for IC, an aggressive non-pharmacological intervention and pharmacological treatment of the risk factors associated with IC are considered. In the next 2 years, the results of major trials of drugs that stabilize and regress atherosclerosis such as statins and angiotensin converting enzyme inhibitors, and anti-platelet agents, recombinant growth factors and immune modulators will be available for IC. Levocarnitine (l-carnitine) and a derivative, propionyl levocarnitine, are emerging agents that increase the pain-free walking and improve the quality of life in IC patients by working at the metabolism and exercise performance of ischemic muscles. This article provides a comprehensive review of the pathophysiology involved, diagnosis of IC and existing and emerging pharmacotherapies with rationale for their use in its treatment.