Treatments for COVID-19: emerging drugs against the coronavirus.

The Coronavirus disease 19 (COVID-19) outbreak has been recognized as a global threat to public health. It is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and no effective therapies currently exist against this novel viral agent. Along with extensive public health measures, an unprecedented global effort in identifying effective drugs for the treatment is being implemented. Potential drug targets are emerging as the result of a fast-evolving understanding of SARS-CoV-2 virology, host response to the infection, and clinical course of the disease. This brief review focuses on the latest and most promising pharmacological treatments against COVID-19 currently under investigation and discuss their potential use based on either documented efficacy in similar viral infections, or their activity against inflammatory syndromes. Ongoing clinical trials are also emphasized.

Relaxing effects of clenbuterol, ritodrine, salbutamol and fenoterol on the contractions of horse isolated bronchi induced by different stimuli.

ß2-adrenoceptor agonists are considered the most effective drugs to counteract bronchoconstriction in horses with asthma, but only clenbuterol is commonly employed in clinical practice. We evaluated the effects of different selective ß2 agonists: clenbuterol, ritodrine, salbutamol, and fenoterol on the contractions of isolated bronchial muscle of horses induced by electrical field stimulation (EFS), carbachol, histamine, and KCl. All ß2 agonists reduced the amplitude of contraction induced by the different stimuli but with variable efficacy and potency. Fenoterol and salbutamol were more effective than clenbuterol in relaxing the bronchial contractions induced by EFS and histamine, and were able to completely abolish carbachol-induced contractions, unlike clenbuterol and ritodrine. The respective potency values (pEC50) of clenbuterol, ritodrine, salbutamol, and fenoterol were 7.74 ±â€¯0.20, 7.77 ±â€¯0.17, 7.30 ±â€¯0.23, 8.01 ±â€¯0.13, for EFS-induced contractions; 8.39 ±â€¯0.26, 5.49 ±â€¯0.28, 6.63 ±â€¯0.14, 7.68 ±â€¯0.11, for carbachol-induced contraction; 7.39 ±â€¯0.27, 7.04 ±â€¯0.28, 6.45 ±â€¯0.34, 7.34 ±â€¯0.22, for histamine-induced contraction; 7.15 ±â€¯0.06, 6.07 ±â€¯0.20, 6.48 ±â€¯0.14, 6.70 ±â€¯0.18, for KCl-induced contraction. Salbutamol and fenoterol showed a higher efficacy than clenbuterol in relaxing horse bronchial muscle pre-contracted by most stimuli. Clenbuterol displayed a good potency but a rather low efficacy, and this may be due to its partial agonist nature; ritodrine showed lower or not significantly different efficacy and potency compared to the other agonists. An evaluation of the clinical efficacy by fenoterol and salbutamol in horses with asthma could be of great interest to assess if they could represent more effective bronchodilators compared to clenbuterol.

Role of muscarinic receptors in the contraction of jejunal smooth muscle in the horse: An in vitro study.

Nonselective antimuscarinic drugs are clinically useful in several pathologic conditions of horses, but, blocking all muscarinic receptor (MR) subtypes, may cause several side effects. The availability of selective antimuscarinic drugs could improve therapeutic efficacy and safety. We aimed to enlighten the role of different MR subtypes by evaluating the effects of nonselective, and selective M1, M2 and M3 MR antagonists on the contractions of horse jejunum. Segments of circular muscle of equine jejunum, were put into organ baths, connected to isotonic transducers, and the effects on ACh concentration-response curves, and on electrical field stimulation (EFS)-evoked contractions of intestinal preparations, induced by nonselective or selective MR antagonists, compared to pre-drug level, were studied. Atropine (nonselective MR antagonist), pirenzepine (selective M1 antagonist), and p-FHHSiD (selective M3 antagonist) competitively antagonized ACh (pA2=9.78±0.21; 7.14±0.25 and 7.56±0.17, respectively). Methoctramine (selective M2 antagonist) antagonized ACh in a concentration-unrelated fashion; however, it competitively antagonized carbachol, a nonselective muscarinic agonist (pA2=6.42±0.23). Atropine dose-dependently reduced EFS-evoked contractions, reaching a maximal effect of -45.64±6.54%; the simultaneous block of neurokinin receptors, almost completely abolished the atropine-insensitive contractions. p-FHHSiD dose-dependently reduced EFS-induced contractions, while pirenzepine caused a minor decrease. Methoctramine, ineffective up to 10-7M, enhanced the contractions at 10-6M; the block of neurokinin receptors abolished the increase of contraction. Cholinergic contractions of horse jejunum are mainly mediated by M3 receptors; M2 selective antagonists seem to scarcely affect cholinergic, and to enhance neurokininergic contractions of equine jejunum, thus their use entails a lower risk of causing intestinal hypomotility, compared to nonselective drugs.

Assessment of intestinal peristalsis in vitro.

The protocol detailed in this unit is designed to assess intestinal peristaltic motility in the isolated small intestine in vitro and to measure the effects of drugs able to interfere with gut propulsive activity. The procedure is based on Trendelenburg's classic technique, described at the beginning of the 20th century in the isolated guinea pig ileum and, later on, extended to other intestinal preparations from the same animal and other animal species. This unit illustrates the basic procedures for setting up the intestinal preparation, recording peristalsis under near-physiologic conditions, and testing the pharmaco-toxicological effects of drugs and pollutants on the contractile behavior of the gut wall. The protocol allows evaluating the action of drugs affecting sensory and/or motor neurons of the enteric nervous system and how these neurons control the development of the motor program of the gut wall. This model can be exploited to investigate novel compounds undergoing preclinical development and both inhibitors and stimulants of gastrointestinal peristaltic activity, as well as environmental or alimentary pollutants, like xenobiotics and naturally-occurring toxins, endowed with noxious activity with regard to digestive functions.

Diazoxide attenuates indomethacin-induced small intestinal damage in the rat.

ATP-sensitive potassium (K(ATP)) channel openers have been shown to protect against cellular damage in neurons, cardiac muscle, and kidney and to effectively reduce nonsteroidal anti-inflammatory drug (NSAID)-induced gastric damage in rats. We investigated the effects of K(ATP) channel opener diazoxide on small intestinal injury induced in rats by indomethacin administration. The effect of glibenclamide, a K(ATP) channel blocker, was also evaluated. Diazoxide (15, 45 and 135mg/kg) or glibenclamide (18mg/kg), were given by oral gavage 1h before and 6h after indomethacin treatment (20mg/kg p.o.). After 24h, macroscopic and histologic lesions, myeloperoxidase (MPO) activity and lipid peroxidation levels were evaluated. Diazoxide at 15mg/kg was ineffective, while at doses of 45mg/kg and 135mg/kg was able to significantly improve all damage parameters. Glibenclamide administration enhanced intestinal injury. These results show for the first time a beneficial effect of diazoxide in indomethacin-induced enteritis in the rat. Several mechanisms, such as oxidative phosphorylation uncoupling and hypermotility seem particularly important in NSAID-induced intestinal injury. Such events lead to increased mucosal permeability and to penetration of noxious lumen components, which ignite the inflammatory response. Since K(ATP) channel openers were shown to protect against mitochondrial damage, to reduce intercellular permeability and to relax smooth muscle, we suggest that diazoxide could exert its beneficial effects by one or more of these actions.

Effects of α2-adrenergic drugs on small intestinal motility in the horse: an in vitro study.

The effects of selective α(2)-agonists (xylazine, detomidine and medetomidine) and antagonists (yohimbine and atipamezole) on in vitro small intestine motility in the horse were evaluated. Samples of equine jejunum were placed in isolated organ baths and drug-induced modifications of motility were measured by means of an isotonic transducer. All tested α(2)-agonists dose-dependently reduced both spontaneous and electrically-evoked phasic contractions. Conversely, α(2)-antagonists were ineffective when tested alone, and showed a heterogeneous and dose-independent ability to inhibit agonist activity. In particular, the antagonism exerted by higher concentrations of both yohimbine and atipamezole against α(2)-agonists was weaker than when lower concentrations were used. The data are indicative of the presence of both pre- and post-synaptic α(2)-adrenoceptors with inhibitory activity on equine jejunum motility, and support a possible therapeutic utility of these drugs in horse intestinal disorders associated with hypermotility.

Assessment of gastrointestinal motility using three different assays in vitro.

The protocols detailed in this unit are designed to assess the motor activity of different gastric and intestinal muscle preparations in vitro and the effects of drugs that modulate gastrointestinal motility. The preparations described are characterized by different contractile behaviors, consisting of spontaneous (duodenum), neurogenic (ileum), and drug-stimulated (fundus, ileum) motility; these reproduce motility patterns occurring in the gut wall in vivo. These protocols document the variety of factors that can influence the responses of isolated tissues and describe how such tissues can be used for testing substances that affect gut movements. These preparations allow evaluation of direct interactions with the processes that control contractile machinery, as well as indirect effects resulting from the modification of neurotransmitter release from myenteric neurons. These models can be exploited to assay novel compounds undergoing preclinical development or to evaluate the functional toxicity exerted by environmental or alimentary pollutants, like xenobiotics and naturally occurring toxins, as well as the mechanisms underlying these effects.

Assessment of gastrointestinal propulsive activity using three different models of peristalsis in vivo in the mouse.

The protocols described in this unit are designed to assess the acute effects of drugs on the propulsive activity of the gastrointestinal muscles in the conscious mouse. These protocols are currently applied to investigate the pharmacological activity of novel compounds undergoing preclinical development and to obtain predictive data needed to advance drugs into clinical trials. Moreover, these methods could be useful in evaluating the functional toxicity by environmental or alimentary pollutants, like xenobiotics and naturally occurring toxins endowed with noxious activity in the control of physiologic peristalsis. The three models detailed-the measurement of gastric emptying, ileal transit, and colonic propulsion-are substantially non-invasive and do not require analgesic pretreatments or the induction of general anesthesia. In contrast to an in vitro approach, these in vivo studies provide a unified understanding of drug effects on gut functionality, in particular when the central nervous system, the extrinsic nerves, or the (neuro)endocrine system is targeted by the test drugs.

Stereospecific effects of benzo[d]isothiazolyloxypropanolamine derivatives at beta-adrenoceptors: synthesis, chiral resolution, and biological activity in vitro.

We performed the asymmetric synthesis of four enantiopure benzo[d] isothiazo-3-or 5-yloxypropanolamine derivatives, previously described as competitive antagonists at beta-adrenoceptors. The chemical characterization of each enantiomer was accomplished by (1)H NMR and HPLC/DAD/CD. The direct chromatographic separation of the enantiomers via chiral HPLC was investigated. The best resolutions were achieved using cellulose tris (3,5-dimethylphenyl carbamate) (Chiralcel OD-H) and amylose tris (3,5-dimethylphenyl carbamate) (Chiralpak AD). The enantiomers obtained had enantiomeric purities suitable for biological assays. Tested in isolated rat cardiac and intestinal tissues to evaluate their effects at beta(1)- and beta(3)-adrenoceptors, the (S)-enantiomers revealed a higher degree of antagonism than (R)-enantiomers at both subtypes, even though their activity was greater at the cardiac beta(1)-subtype. The potent and cardiospecific antagonistic effect exerted by the compounds tested suggests that the benzisothiazole moiety could be an interesting scaffold for discovering new chiral beta-blocking drugs.

Functional effects of nitric oxide-releasing aspirin on vein conduits of diabetic patients undergoing CABG.

BACKGROUND: Type 2 diabetes mellitus (DM) is known to negatively affect biological properties of venous vasculature, and, particularly, to reduce endothelium-derived nitric oxide release. This condition might influence venous graft function following coronary artery bypass surgery (CABG). The aim of this study was to evaluate the functional effects of a NO-releasing aspirin (NORA) on vein grafts (VG) of diabetics and control patients undergoing elective CABG. METHODS: In 40 consecutive ischemic heart disease patients, the effects of NORA were tested on segments of saphenous vein conduits harvested during elective CABG. Twenty patients had type-2 DM (mean age 69+/-2), whereas 20 patients had no DM (NDM) and represented the control group (mean age 67+/-4). Functional responses were tested by exposing VGs to NORA and to standard vasoactive agents in an organ-bath preparation. Histological features of VGs were also assessed by light and electronic microscopy. RESULTS: Significant impairment of endothelial-dependent vasodilation (acetylcholine induced) was documented in VGs of DM subjects. NORA induced a significant and comparable vascular relaxation in all venous segments of NDM and DM patients (56+/-12% of maximal relaxation vs 61+/-11% in the control group, respectively). Histology showed variable extent of vascular layer and cellular abnormalities in VGs of diabetics (intimal hyperplasia, calcific deposition, endothelial cell degeneration) likely responsible of the endothelial functional impairment, whereas control group VG showed preserved structures. CONCLUSIONS: This preliminary study confirms the impairment of endothelium-dependent vasodilative property of VGs in DM patients. It also indicates that NORA effectively induces vasodilation of VGs which was effective also in DM patients thereby representing a promising therapy for diabetics undergoing CABG with the use of VGs, although further studies are mandatory to conclusively assess the safety and benefits of this pharmacological agent.

Bovine herpesvirus 4 based vector interaction with liver cells in vitro and in vivo.

Gene transfer into hepatocytes is highly desirable for the long-term goal of replacing deficient proteins and correcting metabolic disorders. Bovine herpesvirus 4 (BoHV-4) based vector capability to transduce rat liver cells in vitro and in vivo was assessed. For the in vitro study, a buffalo rat liver cell line was successfully transduced by BoHV-4 and although did not show toxicity, the immediate early two viral gene was transcribed and cells harboring the intact viral genome could be pharmacologically selected, but no viral replication took place. For the in vivo study, adult male rats were inoculated intraportally and intraparenchimally with a BoHV-4 expressing enhanced green fluorescent protein and liver sections were analyzed through fluorescent microscopy. Although the liver parenchyma could not be transduced, the endothelial layer of the liver vasculature showed a robust transgene expression without toxicity. Successful BoHV-4 based vector transduction of primary cultures of rat hepatocytes suggests that extrinsic factors, and not hepatocytes per se, are the cause of such lack of transducibility. The present study serves as a starting point for study of the use of BoHV-4 based vectors to target gene delivery to vascular endothelial cells.

Synthesis of 1,2-benzisothiazolyloxypropanolamine derivatives and investigation of their activity at beta-adrenoceptors.

The synthesis of 3-methoxy-1,2-benzisothiazole derivatives, substituted in position 5- (compounds 1-7) or 7- (compounds 8-14), with oxypropanolaminic side chains and the pharmacological investigation on their activity at beta-adrenoceptors are described. Compounds were prepared in an attempt to explore the ability of the benzisothiazole ring to interact with the beta-adrenoceptor site and to establish whether oxypropanolaminic derivatives recognise the beta3-adrenoceptor subtype. All the products were tested on rat atria, bladder and small intestine, which preferentially (but not exclusively) express beta1-, beta2- and beta3-adrenoceptors, respectively. When compared with the reference, non-specific, beta-adrenoceptor agonist isoprenaline, the products tested did not show any consistent beta-adrenoceptor agonistic activity in the different models. Most compounds relaxed smooth muscle preparations, but such effect was resistant to the blockade by propranolol (1 micromol/l), ICI 118,551 (1 micromol/l) or bupranolol (1-10 micromol/l), thus excluding that the spasmolytic effect involves any beta-adrenoceptors. When tested as antagonists, some of these products showed a concentration-dependent attenuation of the isoprenaline-induced effects in rat atria, without affecting beta-adrenoceptor-mediated relaxation in smooth muscle. These data confirm the ability of the benzisothiazole ring to interact with beta-adrenoceptors, but the substitution in 5- or 7-positions with oxypropanolaminic groups does not generate compounds endowed with specific activity at beta3-adrenoceptors. Conversely, most of these compounds behave as (specific) antagonists at beta1- (cardiac) adrenoceptors. At the maximum concentrations tested (1-100 micromol/l), these compounds also exert direct spasmolytic and negative chronotropic effects, which could be related to a blockade of Ca2+-dependent mechanisms at an intracellular level and/or an anaesthetic-like activity at plasma membranes.

Effect of the macrolide antibacterial drug, tylosin, on TNBS-induced colitis in the rat.

Bacterial antigens, such as intestinal microflora, are known to play a role in the pathogenesis of human inflammatory bowel disease (IBD). Tylosin, a macrolide antimicrobial agent, has proven to be effective in cat and dog chronic colitis, but the reasons underlying this efficacy are still unclear. In the present study we evaluated the effects of tylosin on 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in the rat, in comparison with the antibacterial drug metronidazole and the corticosteroid budesonide. Colitis was induced by a single intrarectal administration of 10 mg TNBS under light ether anesthesia. Tylosin (20 mg/kg twice a day), metronidazole (160 mg/kg twice a day) and budesonide (500 microg/kg once a day) were given orally for up to 6 days to separate groups of rats. The animals were sacrificed after 6 days and colonic lesions evaluated (colon weight, macroscopic and histologic damage, myeloperoxidase activity). Tylosin and metronidazole significantly lowered macroscopic lesion score, reduced colon weight, the severity of histologic lesions and myeloperoxidase activity; budesonide did not significantly change the parameters of colonic inflammation. These data indicate a protective effect of tylosin against intestinal inflammation, suggesting a major role for bacteria, anaerobes in particular, in the development of TNBS-induced mucosal damage.

N-substituted piperidinyl alkyl imidazoles: discovery of methimepip as a potent and selective histamine H3 receptor agonist.

In this study, we continue our efforts toward the development of potent and highly selective histamine H(3) receptor agonists. We introduced various alkyl or aryl alkyl groups on the piperidine nitrogen of the known H(3)/H(4) agonist immepip and its analogues (1-3a). We observed that N-methyl-substituted immepip (methimepip) exhibits high affinity and agonist activity at the human histamine H(3) receptor (pK(i) = 9.0 and pEC(50) = 9.5) with a 2000-fold selectivity at the human H(3) receptor over the human H(4) receptor and more than a 10000-fold selectivity over the human histamine H(1) and H(2) receptors. Methimepip was also very effective as an H(3) receptor agonist at the guinea pig ileum (pD(2) = 8.26). Moreover, in vivo microdialysis (in rat brain) showed that methimepip reduces the basal level of brain histamine to about 25% after a 5 mg/kg intraperitoneal administration.

Benzisothiazoles and beta-adrenoceptors: synthesis and pharmacological investigation of novel propanolamine and oxypropanolamine derivatives in isolated rat tissues.

In an attempt to examine the ability of benzisothiazole-based drugs to interact with beta-adrenoceptors, a series of 1,2-benzisothiazole derivatives, which were substituted with various propanolamine or oxypropanolamine side chains in the 2 or 3 position, were synthesised and tested. The pharmacological activity of these compounds at the beta-adrenoceptors was examined using isolated rat atria and small intestinal segments, which preferentially express the beta1- and beta3-adrenoceptor-mediated responses, respectively. None of these products showed any beta-adrenoceptor agonistic activity. In contrast, the 2- and 3-substituted isopropyl, tert-butyl, benzyl, and piperonyl derivatives 2a-d and 3a-d elicited surmountable inhibition of the isoprenaline-induced chronotropic effects in the atria, suggesting competitive antagonism at the beta1-recognition site. The pA2 values revealed tert-butyl 3b and the isopropyl substituted piperonyl derivatives 3a to be the most effective. Remarkably, many of the 2-substituted propanolamines were less active than the corresponding 3-substituted oxypropanolamines. With the exception of compound 3b, none of these drugs antagonised the muscle relaxant activity of isoprenaline in the intestine, suggesting no effect on the beta3-adrenoceptors. These results confirm the ability of the benzisothiazole ring to interact with the beta-adrenoceptors, and demonstrate that 2-substitution with propanolamine or 3-substitution with oxypropanolamine groups yields compounds with preferential antagonistic activity at the cardiac beta1-adrenoceptors. The degree of antagonism depends strongly on both the nature of the substituent and its position on the benzisothiazole ring.

[3-(1H-imidazol-4-yl)propyl]guanidines containing furoxan moieties: a new class of H3-antagonists endowed with NO-donor properties.

Synthesis and pharmacological characterisation of a series of products obtained by coupling the H(3)-antagonist SKF 91486 through appropriate spacers with the NO-donor 3-phenylfuroxan-4-yloxy and 3-benzenesulfonylfuroxan-4-yloxy moieties, as well as with the corresponding furazan substructures, devoid of NO-donating properties, are reported. All the products were tested for their H(3)-antagonistic and H(2)-agonistic properties on electrically-stimulated guinea-pig ileum segments and guinea-pig papillary muscle, respectively. The whole series of compounds displayed good H(3)-antagonist behaviour and feeble partial H(2)-agonist activity. Among furoxan derivatives, the benzenesulfonyl hybrid 28, a good NO-donor, triggered a dual NO-dependent muscle relaxation and H(3)-antagonistic effect on guinea-pig intestine.

Role of histamine H3 receptors in control of mouse intestinal motility in vivo and in vitro: comparison with alpha2-adrenoceptors.

We tested drugs acting at histamine H3 receptors in mice on the gastrointestinal transit of a charcoal meal in vivo and on neurogenic contractions of isolated ileal preparations. The agonist (R)-alpha-methylhistamine (100 micromol/kg) caused a maximum 25% reduction of gastrointestinal transit, an effect mimicked by immepip (100 micromol/kg) and antagonized by thioperamide (20 micromol/kg) or clobenpropit (20 micromol/kg). In the isolated ileum, (R)-alpha-methylhistamine (10-100 microM) caused a slight, thioperamide-insensitive, reduction (maximum 15%) of electrically evoked cholinergic contractions. In comparison, the alpha2-adrenoceptor agonist clonidine (0.1 micromol/kg) caused a 35.2% inhibition of the gastrointestinal transit and almost completely reduced (maximum 82% at 1 microM) the cholinergic contraction of the isolated ileum, both effects being antagonized by idazoxan (0.4 micromol/kg and 1 microM, respectively). These results suggest that histamine H3 receptors, located outside the myenteric plexus, mediate an inhibition of the gastrointestinal transit in vivo. Conversely, the presence of a2-adrenoceptors in the cholinergic nerve endings and their inhibitory role in the control of gastrointestinal propulsion is confirmed.