Curing cats with Feline Infectious Peritonitis with an oral multi-component drug containing GS-441524

Feline infectious peritonitis (FIP) caused by feline coronavirus (FCoV) is a common dis-ease in cats, fatal if untreated, and no effective treatment is currently legally available. The aim of this study was to evaluate efficacy and toxicity of the multi-component drug Xraphconn$^{®}$ in vitro and as oral treatment in cats with spontaneous FIP by examining survival rate, development of clinical and laboratory parameters, viral loads, anti-FCoV antibodies, and adverse effects. Mass spectrometry and nuclear magnetic resonance identified GS-441524 as an active component of Xraphconn$^{®}$. Eighteen cats with FIP were prospectively followed up while being treated orally for 84 days. Values of key parameters on each examination day were compared to values before treatment initiation using linear mixed-effect models. Xraphconn$^{®}$ displayed high virucidal activity in cell culture. All cats recovered with dramatic improvement of clinical and laboratory parameters and massive reduction in viral loads within the first few days of treatment without serious adverse effects. Oral treatment with Xraphconn$^{®}$ containing GS-441524 was highly effective for FIP without causing serious adverse effects. This drug is an excellent option for the oral treatment of FIP and should be trialed as potential effective treatment option for other severe coronavirus-associated diseases across species

In vitro studies of the hepatotoxic and hepatoprotective potential and metabolism of chalcones and a tacrine-silibinin codrug

(1) Kava kava, traditionally used by the Pacific Islanders as a socio-ceremonial beverage and remedy, was approved as an anxiolytic drug in Western countries. However, the reports of severe hepatotoxic side effects led to the withdrawal of kava-containing products in most of these countries. Since then, responsible constituents and possible mechanisms of hepatotoxicity are controversially discussed. To evaluate the hepatotoxic potential of single compounds, six kavalactones were isolated from an acetonic kava root extract and the three kava-chalcones (flavokawains) were obtained by synthesis. Subsequently, these major and minor constituents were tested towards their effects on cell viability and proliferation of human hepatoma cell lines. Whereas the toxicity of five tested kavalactones was found to be very low (K, M, DMY) or completely absent (DHK, DHM), distinct effects on both cell viability and proliferation were observed for the kavalactone Y and the kava-chalcones, FKB being the most toxic (IC50 ~ 30 µM on HepG2 and IC50 ~ 40 µM on HuH-7) of the test compounds. The results of this study suggest that the flavokawains might contribute to the hepatotoxic potential of kava. Based on obtained in vitro toxicity data, further testing of long-term toxicity of Y and flavokawains is recommended preferably on primary human hepatocytes or in vivo in a suitable animal model. (2) Therapy options for the treatment of Alzheimer’s disease are still constricted to a few approved drugs. Facing the therapy limiting hepatotoxic side effects of tacrine in a new approach, a tacrine-silibinin codrug was synthesized that showed promising pharmacological properties such as AChE and BChE inhibitory activity in vitro and pro-cognitive effects in vivo in rats. Driven by these promising results, the in vitro hepatotoxic potential of the codrug was assessed and compared to tacrine and an equimolar mixture of tacrine and silibinin. Therefore, the compounds’ effects on cell number and fluorescence intensity of mitochondria of hepatic stellate cells were investigated. For both, the codrug and the physical mixture, reduced mitotoxicity compared to tacrine was observed. Additionally, the codrug did not show any influence on the cell number up to concentrations of 200 µM. The results correlated well with the findings of the in vivo hepatotoxicity studies in rats. Furthermore, to find first hints of the active principle of the codrug, in vitro stability and microsomal metabolism studies were performed. In the presence of microsomes, the codrug was rapidly cleaved by esterases into tacrine hemi succinamide and silibinin. In phase II metabolism, codrug- and silibininglucuronides were detected. As these metabolites are likely also formed in vivo, they may constitute the active principles of the codrug. Indeed the tacrine hemi succinamide showed pronounced AChE inhibition. Noteworthy, for the codrug cleavage products tacrine hemi succinamide and silibinin, toxicity in the in vitro HSC model was completely absent. (3) Chalcones possess an interesting pharmacological profile for the treatment of hepatic fibrosis, as they comprise several favorable biological activities such as anti-inflammatory, anti-oxidant, antiviral, and apoptosis-inducing effects. The activation and the perpetuation of this activiation of hepatic stellate cells are considered as the key steps in the pathogenesis of liver fibrosis. Hence, the inhibitory activity of structurally related prenylated and non-prenylated chalcones on activated human hepatic stellate cells was investigated. With exception of the dihydrochalcones, P, and 3OHHeli, the investigated chalcones had a significant impact on cell viability, proliferation, cell organelles, and cytochrome c release. For the metabolites 4’AcXAN, XANC, and dhXANC, similar activities were found as for the parent compound XAN whereas 4MeXAN and XANH behaved differently. The induction of apoptosis via mitochondrial pathways is suggested as the test chalcones provoked a release of cytochrome c together with altered fluorescence intensity of mitochondria at low concentrations of 10 µM. Several structure elements were identified which enhance or attenuate the activity of chalcones. The double bond in α,β position was essential for the activity. The unprotected hydroxyl group in position C-6’ led to a complete loss of activity in case of P. Prenylation of C-3’ and methoxy-groups in the A ring enhanced the activity whereas substituents in the B ring mostly attenuated the chalcones’ effects. 4’MeXAN, A, and FKB showed the strongest inhibitory effects on HSC. Based on the findings of this study, the most effective chalcones may be chosen to investigate the mechanisms on the molecular level. In addition, toxicity studies on primary human hepatocytes are needed. (4) For studying the in vitro metabolism of flavokawains and alpinetin chalcone, different microsomal incubation systems were established. LC–HRESIMS was applied to identify phase I and II metabolites and to compile a metabolic profile for these compounds. Obtained data can be used to predict the in vivo metabolism and provide first information about main biotransformation pathways. Demethylation (C-4, C-4’) and hydroxylation (mainly C-4) constitute the major phase I reactions in flavokawain and A metabolism. FKC is generated as major metabolite of both FKA by demethylation and FKB by hydroxylation in position C-4. Heli is formed as major metabolite of FKC and A after demethylation in position C-4’ and hydroxylation of C-4 respectively. In phase II metabolism, extensive conjugation to glucuronic acid was observed. In combined metabolism, the conjugated metabolites clearly predominated also. Glucuronidation of the parent chalcones is consequently considered as major reaction in the microsomal metabolism of investigated chalcones. The structures of major flavokawain-glucuronides were additionally elucidated using HPLC–NMR techniques. According to the results of this study, the chalcone monoglucuronides may constitute the major metabolites in vivo. It is not known if these metabolites contribute to pharmacological activities of chalcones. Regarding the observed in vitro hepatotoxicity of flavokawains, the biotransformation may also constitute a detoxifying reaction

Biotransformation of Flavokawains A, B, and C, Chalcones from Kava (Piper methysticum), by Human Liver Microsomes

The in vitro metabolism of flavokawains A, B, and C (FKA, FKB, FKC), methoxylated chalcones from Piper methysticum, was examined using human liver microsomes. Phase I metabolism and phase II metabolism (glucuronidation) as well as combined phase I+II metabolism were studied. For identification and structure elucidation of microsomal metabolites, LC-HRESIMS and NMR techniques were applied. Major phase I metabolites were generated by demethylation in position C-4 or C-4′ and hydroxylation predominantly in position C-4, yielding FKC as phase I metabolite of FKA and FKB, helichrysetin as metabolite of FKA and FKC, and cardamonin as metabolite of FKC. To an even greater extent, flavokawains were metabolized in the presence of uridine diphosphate (UDP) glucuronic acid by microsomal UDP-glucuronosyl transferases. For all flavokawains, monoglucuronides (FKA-2′-<i>O</i>-glucuronide, FKB-2′-<i>O</i>-glucuronide, FKC-2′-<i>O</i>-glucuronide, FKC-4-<i>O</i>-glucuronide) were found as major phase II metabolites. The dominance of generated glucuronides suggests a role of conjugated chalcones as potential active compounds in vivo

Tacrine-Silibinin Codrug Shows Neuro- and Hepatoprotective Effects <i>in Vitro</i> and Pro-Cognitive and Hepatoprotective Effects <i>in Vivo</i>

A codrug of the anti-Alzheimer drug tacrine and the natural product silibinin was synthesized. The codrug’s biological and pharmacological properties were compared to an equimolar mixture of the components. The compound showed potent acetyl- and butyrylcholinesterase inhibition. In a cellular hepatotoxicity model, analyzing the influence on viability and mitochondria of hepatic stellate cells (HSC), the toxicity of the codrug was markedly reduced in comparison to that of tacrine. Using a neuronal cell line (HT-22), a neuroprotective effect against glutamate-induced toxicity could be observed that was absent for the 1:1 mixture of components. In subsequent <i>in vivo</i> experiments in rats, in contrast to the effects seen after tacrine treatment, after administration of the codrug no hepatotoxicity and no induction of the cytochrome P450 system were noticed. In a scopolamine-induced cognitive impairment model using Wistar rats, the codrug was as potent as tacrine in reversing memory dysfunction. The tacrine–silibinin codrug shows high AChE and BChE inhibition, neuroprotective effects, lacks tacrine’s hepatotoxicity <i>in vitro</i> and <i>in vivo</i>, and shows the same pro-cognitive effects <i>in vivo</i> as tacrine, being superior to the physical mixture of tacrine and silibinin in all these regards

Curing Cats with Feline Infectious Peritonitis with an Oral Multi-Component Drug Containing GS-441524

Feline infectious peritonitis (FIP) caused by feline coronavirus (FCoV) is a common dis-ease in cats, fatal if untreated, and no effective treatment is currently legally available. The aim of this study was to evaluate efficacy and toxicity of the multi-component drug Xraphconn® in vitro and as oral treatment in cats with spontaneous FIP by examining survival rate, development of clinical and laboratory parameters, viral loads, anti-FCoV antibodies, and adverse effects. Mass spectrometry and nuclear magnetic resonance identified GS-441524 as an active component of Xraphconn®. Eighteen cats with FIP were prospectively followed up while being treated orally for 84 days. Values of key parameters on each examination day were compared to values before treatment initiation using linear mixed-effect models. Xraphconn® displayed high virucidal activity in cell culture. All cats recovered with dramatic improvement of clinical and laboratory parameters and massive reduction in viral loads within the first few days of treatment without serious adverse effects. Oral treatment with Xraphconn® containing GS-441524 was highly effective for FIP without causing serious adverse effects. This drug is an excellent option for the oral treatment of FIP and should be trialed as potential effective treatment option for other severe coronavirus-associated diseases across species

Curing Cats with Feline Infectious Peritonitis with an Oral Multi-Component Drug Containing GS-441524

Feline infectious peritonitis (FIP) caused by feline coronavirus (FCoV) is a common dis-ease in cats, fatal if untreated, and no effective treatment is currently legally available. The aim of this study was to evaluate efficacy and toxicity of the multi-component drug Xraphconn® in vitro and as oral treatment in cats with spontaneous FIP by examining survival rate, development of clinical and laboratory parameters, viral loads, anti-FCoV antibodies, and adverse effects. Mass spectrometry and nuclear magnetic resonance identified GS-441524 as an active component of Xraphconn®. Eighteen cats with FIP were prospectively followed up while being treated orally for 84 days. Values of key parameters on each examination day were compared to values before treatment initiation using linear mixed-effect models. Xraphconn® displayed high virucidal activity in cell culture. All cats recovered with dramatic improvement of clinical and laboratory parameters and massive reduction in viral loads within the first few days of treatment without serious adverse effects. Oral treatment with Xraphconn® containing GS-441524 was highly effective for FIP without causing serious adverse effects. This drug is an excellent option for the oral treatment of FIP and should be trialed as potential effective treatment option for other severe coronavirus-associated diseases across species