Preclinical development of kinetin as a safe error-prone SARS-CoV-2 antiviral able to attenuate virus-induced inflammation.

Orally available antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are necessary because of the continuous circulation of new variants that challenge immunized individuals. Because severe COVID-19 is a virus-triggered immune and inflammatory dysfunction, molecules endowed with both antiviral and anti-inflammatory activity are highly desirable. We identified here that kinetin (MB-905) inhibits the in vitro replication of SARS-CoV-2 in human hepatic and pulmonary cell lines. On infected monocytes, MB-905 reduced virus replication, IL-6 and TNFα levels. MB-905 is converted into its triphosphate nucleotide to inhibit viral RNA synthesis and induce error-prone virus replication. Coinhibition of SARS-CoV-2 exonuclease, a proofreading enzyme that corrects erroneously incorporated nucleotides during viral RNA replication, potentiated the inhibitory effect of MB-905. MB-905 shows good oral absorption, its metabolites are stable, achieving long-lasting plasma and lung concentrations, and this drug is not mutagenic nor cardiotoxic in acute and chronic treatments. SARS-CoV-2-infected hACE-mice and hamsters treated with MB-905 show decreased viral replication, lung necrosis, hemorrhage and inflammation. Because kinetin is clinically investigated for a rare genetic disease at regimens beyond the predicted concentrations of antiviral/anti-inflammatory inhibition, our investigation suggests the opportunity for the rapid clinical development of a new antiviral substance for the treatment of COVID-19.

Preclinical safety evaluation of IQG-607 in rats: Acute and repeated dose toxicity studies.

In the present study, we evaluated the safety and the possible toxic effects of IQG-607 after acute and 90-day repeated administrations in rats. Single oral administration of IQG-607 (300 or 2000 mg/kg) on female rats did not result in any mortality. No gross lesions were observed in the animals at necropsy. Ninety-day administration test resulted in 20% of deaths, in both male and female rats administered with the highest dose of IQG-607, 300 mg/kg. Repeated administration of the IQG 607 (25, 100 and 300 mg/kg) did not result in any significant body mass alteration, or changes in food and water consumption. The most important clinical sign observed was salivation in both sexes. Importantly, long-term treatment with IQG-607 did not induce alterations in any hematological (for both sex) and serum biochemical (for female) parameters evaluated, even at the highest dose tested. Treatment of male rats with 100 or 300 mg/kg of IQG-607 decreased total cholesterol levels, while animals treated with 100 mg/kg also presented reduction on triglyceride levels. Of note, no treatment induced significant histopathological alterations in tissues of all organs and glands analyzed, even in that group that received the highest dose of IQG-607.

Role of CXCR2 and TRPV1 in functional, inflammatory and behavioural changes in the rat model of cyclophosphamide-induced haemorrhagic cystitis.

BACKGROUND AND PURPOSE: Cyclophosphamide induces urotoxicity characterized by the development of cystitis, which involves bladder overactivity and inflammation. Here, we investigated the roles of chemokine receptor 2 (CXCR2) and transient receptor potential vanilloid 1 (TRPV1) channels in a rat model of cyclophosphamide-induced cystitis. EXPERIMENTAL APPROACH: Cystitis induced by cyclophosphamide in rats was assessed by gross morphology, histology and immunohistochemistry of bladder tissue. mRNA for CXCR2 and TRPV1 channels were measured by RT-PCR. Nociceptive responses in paw and abdomen, along with cystometric measures were recorded. KEY RESULTS: Cyclophosphamide, i.p., induced pain behaviour, bladder inflammation and voiding dysfunction. The CXCR2 antagonist, SB225002, the TRPV1 channel antagonist, SB366791 or their combination reduced the mechanical hypersensitivity of paw and abdominal area and nociceptive behaviour after cyclophosphamide. Cyclophosphamide-induced cystitis was characterized by haemorrhage, oedema, neutrophil infiltration and other inflammatory changes, which were markedly decreased by the antagonists. Up-regulation of CXCR2 and TRPV1 mRNA in the bladder after cyclophosphamide was inhibited by SB225002, SB366791 or their combination. Expression of CXCR2 and TRPV1 channels was increased in the urothelium after cyclophosphamide. Bladder dysfunction was shown by increased number of non-voiding contractions (NVCs) and bladder pressures and a reduction in bladder capacity (BC), voided volume (VV) and voiding efficiency (VE). SB225002 or its combination with SB366791 reduced bladder pressures, whereas SB225002, SB366791 or their combination increased BC, VV and VE, and also reduced the number of NVCs. CONCLUSIONS AND IMPLICATIONS: CXCR2 and TRPV1 channels play important roles in cyclophosphamide-induced cystitis in rats and could provide potential therapeutic targets for cystitis.

Effects of kinin B(1) and B(2) receptor antagonists on overactive urinary bladder syndrome induced by spinal cord injury in rats.

BACKGROUND AND PURPOSE: Kinin B(1) and B(2) receptors have been implicated in physiological and pathological conditions of the urinary bladder. However, their role in overactive urinary bladder (OAB) syndrome following spinal cord injury (SCI) remains elusive. EXPERIMENTAL APPROACH: We investigated the role of kinin B(1) and B(2) receptors in OAB after SCI in rats. KEY RESULTS: SCI was associated with a marked inflammatory response and functional changes in the urinary bladder. SCI resulted in an up-regulation of B(1) receptor mRNA in the urinary bladder, dorsal root ganglion and spinal cord, as well as in B(1) protein in the urinary bladder and B(1) and B(2) receptor protein in spinal cord. Interestingly, both B(1) and B(2) protein expression were similarly distributed in detrusor muscle and urothelium of animals with SCI. In vitro stimulation of urinary bladder with the selective B(1) or B(2) agonist elicited a higher concentration-response curve in the SCI urinary bladder than in naive or sham urinary bladders. Cystometry revealed that treatment of SCI animals with the B(2) selective antagonist icatibant reduced the amplitude and number of non-voiding contractions (NVCs). The B(1) antagonist des-Arg(9) -[Leu(8) ]-bradykinin reduced the number of NVCs while the non-peptide B(1) antagonist SSR240612 reduced the number of NVCs, the urinary bladder capacity and increased the voiding efficiency and voided volume. CONCLUSIONS AND IMPLICATIONS: Taken together, these data show the important roles of B(1) and B(2) receptors in OAB following SCI in rats and suggest that blockade of these receptors could be a potential therapeutic target for controlling OAB.

C-fibers, but not the transient potential receptor vanilloid 1 (TRPV1), play a role in experimental allergic airway inflammation.

The activation of C-fibers in the airways induces coughing, mucus production and bronchoconstriction, which are also symptoms of airway diseases. In this study, we evaluated the role of the C-fibers and the TRPV1 (transient receptor potential vanilloid 1) receptor in an experimental mouse model of allergic airway inflammation. To study the role of C-fibers, we either degenerated the C-fibers persistently (capsaicin administration in neonate mice) or transiently (capsaicin administration in adult mice). No alteration was observed in eosinophil recruitment to the bronchoalveolar lavage fluid in animals treated with capsaicin in the neonatal period. However, in adult animals, capsaicin treatment after the first ovalbumin challenge (in the establishment of the inflammatory process) decreased the eosinophil numbers. This effect was more pronounced in adult animals treated with capsaicin before beginning the ovalbumin immunization (in the development of the inflammatory process). In addition, interleukin (IL)-5 and chemokine ligand 11 (CCL11) levels in the bronchoalveolar lavage fluid, as well as P-selectin expression and p65 nuclear factor κB (NF-κB) activation in the lung were also decreased. No alterations were observed in the IL-10 and IL-13 levels. Next we determined the effect of TRPV1 receptor blockade on allergic airway inflammation. SB366791 administrated in mice by intraperitoneal (500µg/kg) or intranasal (0.1, 1 or 10nmol/site) route failed to decrease eosinophil recruitment to the bronchoalveolar lavage fluid or alter any other metrics cited above. Thus, the present results confirm and extend previous data supporting the involvement of C-fibers, but not the TRPV1 receptor, in allergic airway inflammation.

Antinociceptive activities of the methanol extract of the bulbs of Dioscorea bulbifera L. var sativa in mice is dependent of NO-cGMP-ATP-sensitive-K(+) channel activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Dioscorea bulbifera var sativa is a medicinal plant commonly used in Cameroonian traditional medicine to treat pain and inflammation. AIM: The present work evaluated the effects of the methanol extract of the bulbs of Dioscorea bulbifera in inflammatory and neuropathic models of pain and further investigated its possible mechanism of action. MATERIALS AND METHODS: The effects of Dioscorea bulbifera administered orally at the doses of 250 and 500mg/kg were tested in mechanical hypernociception induced by intraplantar (i.pl.) injection of complete Freund's adjuvant (CFA), lipopolysaccharides (LPS) or prostaglandin-E(2) (PGE(2)), as well as in partial ligation sciatic nerve (PLSN), nociception induced by capsaicin and thermal hyperalgesia induced by i.pl. injection of CFA. The therapeutic effects of Dioscorea bulbifera on PGE(2)-induced hyperalgesia were evaluated in the absence and in the presence of l-NAME, an inhibitor of nitric oxide synthase (NOS) and glibenclamide, an inhibitor of ATP-sensitive potassium channels. RESULTS: The extract showed significant antinociceptive effects in persistent pain induced by CFA and on neuropathic pain induced by PLSN. The effects of Dioscorea bulbifera persisted for 5 days after two administrations in CFA-induced hypernociception. Dioscorea bulbifera significantly inhibited acute LPS-induced pain but failed to reduce thermal hypernociception and capsaicin-induced spontaneous nociception. The antinociceptive effects of this plant extract in PGE(2) model was antagonized by either l-NAME or glibenclamide. CONCLUSION: Present demonstrate the antinociceptive activities of Dioscorea bulbifera both in inflammatory and neuropathic models of pain and these effects may result, at least partially, from its ability to activate the NO-cGMP-ATP-sensitive potassium channels pathway.

Anti-inflammatory effect of quercetin-loaded microemulsion in the airways allergic inflammatory model in mice.

Quercetin is a plant-derived flavonoid widely known by its anti-oxidant and anti-inflammatory properties, but its oral bioavailability is very poor and this becomes difficult to assess its therapeutic potential. Here we have compared the anti-inflammatory effect of quercetin-loaded microemulsion (QU-ME) and quercetin suspension (QU-SP) in an experimental model of airways allergic inflammation. Mice received daily oral doses of QU-ME (3 or 10mg/kg; in an oil-in-water microemulsion content 0.02:0.2:1 of lecithin:castor oil:Solutol HS15((R))), QU-SP [10mg/kg, in carboxymethylcellulose (CMC) 0.5% in water] or vehicle from the 18th to the 22nd day after the first immunization with ovalbumin (OVA). Dexamethasone was used as positive control drug. Every parameter was evaluated in the 22nd day (24h after the second OVA-challenge). We have also tried to assess by HPLC-MS a quercetin metabolite in the blood of rats treated with QU-SP or QU-ME. QU-ME was better orally absorbed when compared with QU-SP. Furthermore, oral administration of QU-SP failed to interfere with leukocyte recruitment, while QU-ME inhibited in a dose-dependent way, the eosinophil recruitment to the bronchoalveolar lavage fluid (BALF). QU-ME also significantly reduced both IL-5 and IL-4 levels, but failed to interfere with CCL11, IFN-gamma and LTB(4) levels. In addition, QU-ME oral treatment inhibited the nuclear transcription factor kappa B (NF-kappaB) activation, P-selectin expression and the mucus production in the lung. The present results show that QU-ME exhibits pronounced anti-inflammatory properties in a murine model of airways allergic inflammation and suggest that it might present therapeutic potential for the airways inflammatory diseases management.

Preventive and therapeutic anti-inflammatory properties of the sesquiterpene alpha-humulene in experimental airways allergic inflammation.

BACKGROUND AND PURPOSE: alpha-Humulene and trans-caryophyllene are plant sesquiterpenes with pronounced anti-inflammatory properties. Here, we evaluated the effects of these compounds in an experimental model of airways allergic inflammation. EXPERIMENTAL APPROACH: Female BALB/c mice, sensitized to and challenged with ovalbumin received daily alpha-humulene or trans-caryophyllene (50 mg.kg(-1), orally) or alpha-humulene (1 mg.mL(-1), by aerosol) as either a preventive (for 22 days) or therapeutic (from the 18th to the 22nd day) treatment. Dexamethasone or budesonide was used as a positive control drug. Inflammation was determined on day 22 post-immunization by leukocyte recruitment, interleukin-5 (IL-5), CCL11, interferon-gamma (IFN-gamma) and leukotriene (LT)B(4) levels in bronchoalveolar lavage fluid (BALF). In addition, transcription factors [nuclear factor kappaB (NF-kappaB), activator protein 1 (AP-1)] and P-selectin in lung tissue were measured by immunohistochemistry and mucus secretion by histochemistry. KEY RESULTS: Preventive or therapeutic treatments with alpha-humulene, but not with trans-caryophyllene, significantly reduced the eosinophil recruitment to the BALF. In addition, alpha-humulene recovery INF-gamma and reduced the IL-5, CCL11 and LTB(4) levels in BALF, as well as the IL-5 production in mediastinal lymph nodes (in vitro assay). Furthermore, alpha-humulene decreased the NF-kB and the AP-1 activation, the expression of P-selectin and the increased mucus secretion in the lung. CONCLUSIONS AND IMPLICATIONS: alpha-Humulene, given either orally or by aerosol, exhibited marked anti-inflammatory properties in a murine model of airways allergic inflammation, an effect that seemed to be mediated via reduction of inflammatory mediators, adhesion molecule expression and transcription factors activation.