COVID-19 Infection in Fingolimod- or Siponimod-Treated Patients: Case Series.

BACKGROUND AND OBJECTIVES: A descriptive analysis of COVID-19 infection in patients with multiple sclerosis (MS) receiving fingolimod or siponimod. METHODS: We reviewed the cases of COVID-19 from postmarketing or ongoing clinical trials reported to Novartis through December 27, 2020. RESULTS: As of December 27, 2020, 283 cases had been reported in fingolimod-treated patients. The mean age was 44 years (from n = 224; range 11-69 years), and 190 were women. Of 161 cases with available information, 138 were asymptomatic (6), mild (100), or moderate (32); 50 cases required hospitalization. At the last follow-up, 140 patients were reported as recovered/recovering, condition was unchanged in 22, and deteriorated in 3 patients; 4 patients had a fatal outcome. Information was not available for 114 patients. Of the 54 cases of COVID-19 reported in siponimod-treated patients, 45 were from the postmarketing setting and 9 from an ongoing open-label clinical trial. The mean age was 54 years (from n = 45; range 31-70), and 30 were women. Of 28 cases with available information, 24 were asymptomatic (2), mild (17), or moderate (5); 9 cases required hospitalization. At the last follow-up, 27 patients were reported as recovered/recovering, condition remained unchanged for 1, and 3 patients had a fatal outcome. Information was not available for 23 patients. DISCUSSION: Based on a review of available information, the risk of more severe COVID-19 in patients receiving fingolimod or siponimod seems to be similar to that reported in the general population and the MS population with COVID-19. However, limitations of spontaneous reporting, especially missing data, should be considered in the interpretation of these observations.

Autonomic nervous system abnormalities predict cardiovascular changes after initiation of siponimod in secondary progressive multiple sclerosis.

OBJECTIVE: The aim of this study was to identify whether autonomic nervous system (ANS) dysfunction identified prior to treatment initiation can predict siponimod related decrease in heart rate (HR) after treatment initiation. METHODS: In 26 people with secondary progressive multiple sclerosis (SPMS) the following ANS testing protocol was applied: 10-min supine resting position, Valsalva maneuver, deep breathing test, 10 min tilt-up table test, 5-min supine resting period, ingestion of siponimod, followed by 180-min supine resting period recordings. Heart rate variability (HRV) parameters were investigated as possible predictors of decrease in HR (ΔHR) after treatment initiation. RESULTS: After treatment initiation, there was a statistically significant drop in HR (71.1 ± 9.2 to 66.3 ± 8.1, p < 0.001) and elevation of systolic blood pressure (sBP) (113.2 ± 12.4 to 117.1 ± 10.8, p = 0.04). Values of the diastolic BP (dBP) followed similar trend as did sBP, however not reaching statistical significance (72.8 ± 9.6 to 74.9 ± 8.3, p = 0.13). In a multivariable regression model, disease duration and standard deviation of NN intervals (SDNN) were identified as independent predictors for ΔHR, where increase in SDNN and longer disease duration predict smaller ΔHR. CONCLUSION: ANS abnormalities may predict cardiovascular abnormalities associated with treatment initiation with siponimod. SIGNIFICANCE: Results of this study may help mitigate risks associated with siponimod treatment.

The rodent model of impaired gastric motility induced by allyl isothiocyanate, a pungent ingredient of wasabi, to evaluate therapeutic agents for functional dyspepsia.

Functional dyspepsia (FD) is thought to be mainly based on gastric motility dysfunction and chronic hypersensitivity, yet FD animal models has been reported a few. We studied to establish the mouse model of impaired gastric motility induced by a pungent ingredient of wasabi allyl isothiocyanate (AITC), which is reliable to evaluate prokinetic agents. Male ddY mice were used. Gastric motility was measured by 13C-acetic acid breath test in conscious mice. AITC (80 mM) was given 60 min before the measurement of motility. Prokinetic agents including itopride (30, 100 mg/kg), mosapride (0.1-1 mg/kg), neostigmine (30 µg/kg), acotiamide (10-100 mg/kg), and daikenchuto (100-1000 mg/kg) were given 40 min before the measurement. AITC impaired gastric motility without mucosal damages, which reverted 24 h after AITC treatment. The decreased motility induced by AITC was restored by prokinetic agents such as itopride, mosapride, neostigmine, and acotiamide. In separate experiment, daikenchuto recovered the decreased motility induced by AITC, although daikenchuto had no effect on motility in normal condition. In conclusion, it is considered that the AITC-induced impaired gastric motility mouse model is useful to develop new prokinetic agents for treatment of FD, and to re-evaluate traditional Japanese herbal medicines.

Siponimod: A Review in Secondary Progressive Multiple Sclerosis.

Oral siponimod (Mayzent®), a next-generation, selective sphingosine 1-phosphate receptor (S1PR) 1 and 5 modulator, is approved in several countries for the treatment of secondary progressive multiple sclerosis (SPMS), with specific indications varying between individual countries. In the pivotal EXPAND trial (median duration double-blind treatment 18 months) in a broad spectrum of patients with SPMS, once-daily oral siponimod 2 mg (initial dose titration over 6 days) was significantly more effective than placebo in reducing clinical and MRI-defined outcomes of disease activity and disability progression, including 3-month confirmed disability progression on the Expanded Disability Status Scale (EDSS), and was generally well tolerated in the core phase of the study. These beneficial effects of siponimod appeared to be sustained during up to 5 years of treatment in the ongoing open-label extension phase of EXPAND. The safety profile of siponimod is similar to that of other agents in its class, including adverse events of special interest (i.e. those known to be associated with S1PR modulators). No new safety signals were identified during up to 5 years' treatment in the open-label extension phase. Albeit further long-term efficacy and safety data from the real-world setting are required to fully define its role, given the paucity of current treatment options and its convenient dosage regimen, siponimod represents an important emerging option for the treatment of adult patients with SPMS with active disease evidenced by relapses or imaging-features of inflammatory activity.

Nanoparticle BAF312@CaP-NP Overcomes Sphingosine-1-Phosphate Receptor-1-Mediated Chemoresistance Through Inhibiting S1PR1/P-STAT3 Axis in Ovarian Carcinoma.

PURPOSE: Platinum/paclitaxel-based chemotherapy is the strategy for ovarian cancer, but chemoresistance, inherent or acquired, occurs and hinders therapy. Therefore, further understanding of the mechanisms of drug resistance and adoption of novel therapeutic strategies are urgently needed. METHODS: In this study, we report that sphingosine-1-phosphate receptor-1 (S1PR1)-mediated chemoresistance for ovarian cancer. Then we developed nanoparticles with a hydrophilic PEG2000 chain and a hydrophobic DSPE and biodegradable CaP (calcium ions and phosphate ions) shell with pH sensitivity as a delivery system (CaP-NPs) to carry BAF312, a selective antagonist of S1PR1 (BAF312@CaP-NPs), to overcome the cisplatin (DDP) resistance of the ovarian cancer cell line SKOV3DR. RESULTS: We found that S1PR1 affected acquired chemoresistance in ovarian cancer by increasing the phosphorylated-signal transduction and activators of transcription 3 (P-STAT3) level. The mean size and zeta potential of BAF312@CaP-NPs were 116 ± 4.341 nm and -9.67 ± 0.935 mV, respectively. The incorporation efficiency for BAF312 in the CaP-NPs was 76.1%. The small size of the nanoparticles elevated their enrichment in the tumor, and the degradable CaP shell with smart pH sensitivity of the BAF312@CaP-NPs ensured the release of BAF312 in the acidic tumor niche. BAF312@CaP-NPs caused substantial cytotoxicity in DDP-resistant ovarian cancer cells by downregulating S1PR1 and P-STAT3 levels. CONCLUSION: We found that BAF312@CaP-NPs act as an effective and selective delivery system for overcoming S1PR1-mediated chemoresistance in ovarian carcinoma by inhibiting S1PR1 and P-STAT3.

Deficiency of O-linked-glycosylation regulates activation of T cells and aggravates Concanavalin A-induced liver injury.

OBJECTIVE: Protein glycosylation is involved in immunological recognition and immune cell activation. The role of O-glycosylation in Concanavalin A (Con A)-induced autoimmune hepatitis (AIH) was elucidated in the present study. METHODS: Mice were intravenously injected with Con A (10 mg/kg) to establish an AIH mouse model. Here, 24 h prior to administration of Con A, experimental mice were intragastrically administrated with O-glycosylation inhibitor (benzyl-α-GalNAc) at doses of 1 and 5 mg/kg, respectively, while control mice were administrated with the same volume of saline. Before and after administration of Con A for 6 and 12 h, mice were sacrificed and their plasma and livers were collected to score liver injury. Peripheral blood, spleen, and thymus were collected for flow cytometry analysis. The expression levels of neutrophilic alkaline phosphatase-3 (NALP3) and NALP6 in liver were evaluated as well. RESULTS: Pre-treatment with benzyl-α-GalNAc increased the serum transaminase levels and induced more infiltration and necrosis in livers of Con A administrated mice. The levels of some pro-inflammation cytokines also increased in administrated mice. In addition, pretreatment with benzyl-α-GalNAc up-regulated the expression levels of NALP3 and NALP6. And benzyl-α-GalNAc inhibited the levels of apoptosis of thymus cells and influenced activation of T cells in peripheral blood and spleen of Con A administrated mice, especially that accelerated the physiological progression of CD4+CD25-CD69+ subset. CONCLUSION: The present research demonstrated that benzyl-α-GalNAc aggravated Con A-induced AIH, and the role of the O-glycosylation inhibitor as the aggravation may be related to regulation of the levels of cytokines, as well as influencing proliferation of T cells.

Safety, Tolerability, Pharmacodynamics and Pharmacokinetics of Intravenous Siponimod: A Randomized, Open-label Study in Healthy Subjects.

PURPOSE: The goal of this study was to assess the safety, tolerability, pharmacodynamics (PD) and pharmacokinetics (PK) of intravenous (IV) siponimod in healthy subjects. METHODS: This randomized, open-label study was conducted in 2 parts. In Part 1, a total of 16 eligible subjects received either a single oral dose of siponimod (0.25 mg) followed by a single IV infusion (0.25 mg/3 h) in Sequence 1, or vice versa in Sequence 2. In Part 2, a total of 17 eligible subjects received single IV infusions of siponimod (1 mg/24 h). FINDINGS: No clinically relevant effect on mean 5-minute or hourly average heart rate was observed following the siponimod IV dosing regimens and both remained above 50 beats/min. Observed atrioventricular blocks and sinus pauses were asymptomatic. The mean change in absolute lymphocyte count from baseline was comparable for the siponimod 0.25 mg oral regimen and the two IV siponimod regimens. Oral siponimod displayed a good absolute bioavailability of 84%. The mean peak exposure of oral siponimod was approximately 48% lower than that of IV siponimod. The M17 metabolite was found to be the most prominent systemic metabolite of siponimod in humans. IMPLICATIONS: Siponimod IV infusions were well tolerated, with safety and PD (absolute lymphocyte count) profiles similar to those of oral siponimod. The PD/PK findings supported the development of an innovative rapid IV titration regimen for patients with intracerebral hemorrhage.

Altered dopamine signalling in chronic epigastric pain syndrome.

Chronic epigastric pain syndrome (CEPS) is an important diagnostic problem, especially in patients without macroscopic and microscopic changes in gastric mucosa. The cause of this ailment is unclear. The aim of this study was the assessment of coexistence between symptoms of this syndrome and secretion level of dopamine (DA), as well as the efficacy of peripheral and central D2 receptors antagonist. Sixty depressive patients with CEPS occurring independently of the diet and with no Helicobacter pylori infection and 30 healthy subjects were enrolled in this study. Plasma DA and urinary homovanilic acid (HVA) concentration were measured by ELISA, and the mRNA expression of dopa decarboxylase (DDC) in gastric mucosa was evaluated by RT-PCR in 30 patients with CEPS and 30 controls. Severity of epigastric pain before and after 12 weeks 2 x 50 mg itopride or sulpiride treatment was evaluated using the modified 10-point Visual Analogue Scale. Higher average levels of plasma DA and urinary HVA levels in CEPS patients than controls 129.5 ± 22.0 versus 109.1 ± 18.4 pg/ml (p < 0.001) and 6.82 ± 1.55 versus 5.39 ± 1.04 mg/24 h, respectively were obtained. Moreover, the expression of DDC in gastric mucosa of CEPS patients was higher than in healthy subjects (p < 0.01). Sulpiride subsided epigastric pain in 73.3%, but itopride reduced it only in 6.6% of CEPS patients. We concluded that altered dopamine signalling may affect locally-and-centrally mediated chronic epigastric pain.

Novel benzyl phenyl sulfide derivatives as antibacterial agents against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infection is a major threat to human health due to its resistance to almost all classes of antibiotics. Discovery of novel antibacterial agents with new structures which combat the pathogens responsible for MRSA is urgent. In this study, three series of benzyl phenyl sulfide derivatives were designed and synthesized, and their antibacterial activity against eleven MRSA strains were evaluated. The results showed that two series of the synthetic compounds (5a-5l and 12p-12u) exhibit potent antibacterial activity against S. aureus and MRSA, with minimum inhibitory concentrations of 2-64 µg/mL. The structure-activity relationships are discussed and the mechanism of the antibacterial activity was shown to involve the destruction of the bacterial cell membrane. Finally, the MTT assay results suggest that the toxicity of compounds 5f and 5h is selective between bacteria and mammalian cells.

Siponimod in the treatment of multiple sclerosis.

Introduction: Multiple sclerosis (MS) causes focal lesions of immune-mediated demyelinating events followed by slow progressive accumulation of disability. Over the past 2 decades, multiple medications have been studied and approved for use in MS. Most of these agents work by modulating or suppressing the peripheral immune system. Siponimod is a newer-generation sphingosine 1 phosphate (S1P) receptor modulator that internalizes S1P1 receptors, thereby inhibiting efflux of lymphocytes from lymph nodes and thymus. There are promising data suggesting that it may also have a direct neuroprotective property independent of peripheral lymphocytopenia.Areas covered: We reviewed the pharmacology and the clinical and radiological effects of siponimod.Expert opinion: The selective effect of siponimod on the S1P1 and S1P5 receptors offers a favorable side-effect profile and transient bradycardia can be avoided by dose titration. A phase-II study showed that siponomod has dose-dependent beneficial effects in patients with relapsing remitting disease. The results of a phase-III study suggest that siponimod may be beneficial in secondary progressive MS, at least in patients with disease activity.

Efficacy and safety of siponimod for multiple sclerosis: Protocol for a systematic review and meta-analysis.

BACKGROUND: Multiple sclerosis is the most common demyelinating disease of the central nervous system with serious social and economic burden. Siponimod is a sphingosine-1-phosphate receptor agonist, and clinical trials in the past decade have shown good prospects for the treatment of multiple sclerosis. But there is a lack of comprehensive understanding of the dose-effect relationship and safety in different subtypes of multiple sclerosis at present. METHODS: We will perform a systematic review and meta-analysis of clinical randomized controlled trials to evaluate the efficacy and safety of siponimod in multiple sclerosis. We will search PubMed, EMBASE, Cochrane Library, Clinical Trials, Cochrane Central Register of Controlled Trials (CENTRAL) using a comprehensive strategy. The reference lists of the articles we select for inclusion will be checked to identify additional studies for potential inclusion. Two reviewers will review all literature independently. Upon inclusion of articles, another 2 reviewers will extract available data using a standardized form and assess the potential bias. Review Manager will be used to conduct data synthesis. There is no requirement of ethical approval and informed consent. RESULT: This is the first systematic assessment of siponimod for the treatment of multiple sclerosis. We predict it will provide high-quality synthesis of existing evidence for the efficacy and safety of siponimod for multiple sclerosis and a relatively comprehensive reference for clinical practice and clinical trials about siponimod to be conducted. CONCLUSION: The results of the systematic review and meta-analysis will provide updated evidence for the use of siponimod for multiple sclerosis. REGISTRATION: The systematic review and meta-analysis is registered in the PROSPERO international prospective register of systematic review (PROSPERO#CRD42018112721).

Developmental toxicity and alteration of gene expression in zebrafish embryo exposed to 6-benzylaminopurine.

6-benzylaminopurine (6-BA) is widely used in agriculture and horticulture as plant growth regulator. Its excessive use may pose a potential risk to both environment and human health, which is causing great concern. This study was undertaken to assess the acute developmental toxicity of 6-BA to zebrafish embryos based on OECD protocols and mortality, hatching rate and malformation were investigated. Results showed that the 96 h-LC50 and 96 h- EC50 values were 63.29 mg/L and 41.86 mg/L, respectively. No mortality or teratogenic effects were found at concentrations lower than 10 mg/L 6-BA at concentrations higher than 50 mg/L significantly inhibited hatchability and embryo development, induced serious toxicity characterized by morphologic abnormalities (elongated pericardium, heart and yolk sac edema, spine curvature) and functional failure (slow spontaneous movement and heart rate, growth retardation, yolk sac absorption retention). Moreover, 6-BA-induced apoptosis was observed in embryos by the acridine orange staining and confirmed by the apoptotic-related genes, all of which p53 was significantly up-regulated at concentrations higher than 10 mg/L, bax at concentrations higher than 12.5 mg/L, while bcl2 was down-regulated at concentrations higher than 25 mg/L. As for genes of cardiac development, qPCR results demonstrated that nkx2.5, gata5, and amhc were significantly down-regulated at concentrations higher than 25 mg/L, vmhc and atp2a2a at concentration of 50 mg/L, in contrast, hand2 was up-regulated at concentration of 50 mg/L. Our data indicate that 6-BA induces a dose-dependent toxicity resulting in apoptosis through the involvement of p53-dependent pathways and hindering normal heart development in zebrafish embryos.

Effect of Fluconazole Coadministration and CYP2C9 Genetic Polymorphism on Siponimod Pharmacokinetics in Healthy Subjects.

OBJECTIVES: The aim of this study was to assess the pharmacokinetics (PK) and safety/tolerability of siponimod in healthy subjects when coadministered with (1) the moderate cytochrome P450 (CYP) 2C9 and CYP3A inhibitor fluconazole (Study A), and (2) with three different CYP2C9 genotype variants (Study B). METHODS: Study A was an open-label, single-dose study comprising periods 1 (14 days; day 1: siponimod 4 mg) and 2 (20 days; day 1: fluconazole 200 mg twice daily; days 2-19: fluconazole 200 mg once daily; day 3: siponimod 4 mg) in healthy subjects (n = 14) with the wild-type CYP2C9 genotype (CYP2C9*1/*1). Study B was a multicentre, open-label study comprising parts 1 (day 1: siponimod 0.25 mg once daily in the CYP2C9*1/*1, CYP2C9*2/*3 and CYP2C9*3/*3 genotypes) and 2 (days 1-2: 0.25 mg once daily; day 3: 0.5 mg once daily in the CYP2C9*2/*3 and CYP2C9*3/*3 genotypes only) in healthy subjects with polymorphic variants of CYP2C9 (n = 24). Pharmacokinetic parameters were calculated using noncompartmental methods. RESULTS: In Study A, coadministration with fluconazole produced an approximately twofold increase in mean area under the curve (AUC) versus siponimod alone (from 1110 to 2160 h*ng/mL), and an increase in maximum plasma concentration (Cmax; from 31.2 to 34.0 ng/mL) and elimination half-life (T½; from 40.6 to 61.6 h). In Study B, the AUCs of siponimod were approximately two to fourfold greater in subjects with the CYP2C9*2/*3 and CYP2C9*3/*3 genotypes, with a minor increase in Cmax versus the CYP2C9*1/*1 genotype. The mean T½ was prolonged in the CYP2C9*2/*3 (51 h) and CYP2C9*3/*3 (126 h) genotypes versus the CYP2C9*1/*1 (28 h) genotype. Siponimod did not result in increased adverse events in healthy subjects in both studies. CONCLUSIONS: Changes in siponimod PK, when coadministered with fluconazole at steady-state and in subjects with different CYP2C9 genotypes, indicate that the reduced CYP2C9 enzymatic activity does not affect the absorption phase of siponimod but prolongs the elimination phase. These results confirm the relevance of CYP2C9 activity on siponimod metabolism in humans.

In vitro Callus Induction of Sipahutar Pineapple (Ananas comosus L.) from North Sumatra Indonesia.

BACKGROUND AND OBJECTIVE: Sipahutar pineapple (Ananas comosus L.) is a indigenous of pineapple grown in Sipahutar district, North Sumatra, Indonesia. Propagation of Sipahutar pineapple that being done traditionally is less effective, because the number of seeds that produced is very limited and requires a long time. Propagation through in vitro culture is an alternative solution to solve this problem. It is necessary to add plant growth regulator (PGR) to stimulate callus formation in Sipahutar pineapple explants (Ananas comosus L.). Callus induction of pineapple from Sipahutar was carried out by PGR treatment on MS medium. The purpose of this study was to determine the effect MS medium treatment with added dichlorophenoxyacetic acid (2,4-D) and benzyl amino purin (BAP) PGR on Sipahutar pineapple callus formation (Ananas comosus L.) with light and dark treatment. MATERIALS AND METHODS: This callus induction research used a completely randomized design (CRD) with 2 factors, the first factor was treatment 2,4-D (0, 1, 2) ppm. The second factor is BAP (0, 0.5, 1) ppm. RESULTS: Nine combinations of treatments are obtained. Each combination of treatments is treated in both light and dark conditions. The parameters of this study were the percentage (%) of explants that formed callus, the time of callus formed, callus texture, callus biomass, callus surface height and callus surface area. Data were analyzed with two-way ANOVA, followed by Duncan Multiple Rate Test (DMRT). CONCLUSION: The study showed that the interaction between 2,4-D and BAP significantly affected the time of callus formed but 2,4-D and BAP did not significantly affect callus biomass, callus surface height and callus surface area. All explants can form callus, except explants without the addition of 2,4-D and BAP. The callus formed on 10 days after induction (DAI) and 12 DAI with the treatment of light and dark. The color of the produced callus were white, yellowish white, greenish white, brown, brownish yellow, brownish white, brownish green, yellowish green and greenish white. The callus formed is generally compact textures, except for explants which by giving 1 ppm 2,4-D produce friable callus.

Siponimod Chips Away at Progressive MS.

Progressive multiple sclerosis (PMS) causes slow accumulation of neurologic disability and has been refractory to treatment with the immunomodulatory medications that effectively control relapsing MS. Siponimod modestly slowed the rate of disability progression among PMS patients who had inflammatory disease activity, evidenced by new or gadolinium-enhancing MRI lesions. To view this Bench to Bedside, open or download the PDF.

Formulation and in vitro evaluation of a thermoreversible mucoadhesive nasal gel of itopride hydrochloride.

Itopride hydrochloride (ITO HCl) is a prokinetic agent, used in the treatment of gastrointestinal motility disorders. The aim of the study was to develop stable mucoadhesive thermoreversible nasal gel to avoid first pass effect. ITO HCl was incorporated into the blends of thermoreversible polymers like poloxamer 407 and various mucoadhesive polymers in different concentrations to increase the contact of the formulations with nasal mucosa. The compatibility between the drug and the suggested polymers was studied by Fourier transform infrared and differential scanning calorimetry (DSC). The formulations were evaluated for clarity, pH, gelation temperature, mucoadhesive strength, gel strength, viscosity, and drug content. In addition, the in vitro drug release and the dissolution efficiency (DE)% were measured. The optimized formulations that showed the highest dissolution efficiency% (DE%) in saline phosphate buffer of pH 6.4 at 35 ± 0.5 °C were chosen for stability testing at temperatures of 4 ± 2 and 25 ± 2 °C/60 ± 5% RH. It was found that F1 and F17 that contain 18% w/v poloxamer 407 and 0.5% w/v of hydroxypropylmethyl cellulose K4M or methyl cellulose (MC), respectively, showed higher stability results as indicated by their higher t90 values (days).

Cell dedifferentiation and multiplication of Burdock (Arctium Lappa) as a medicinal plant.

Arctium lappa L. (Burdock) is an important plant with various pharmacological effects. According to the importance of this plant, optimization of its tissue culture will lead to more investigation and application of it. The aim of this study was to develop protocols for callus induction and shoot regeneration of A.  lappa. In order to optimize of tissue culture in A. lappa, callus induction, indirect regeneration and direct regeneration were carried out in factorial experiment based on Completely Randomized Designs (CRDs). Hypocotyl and cotyledon were cultured on the Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of 2,4-Dichlorophenoxyacetic acid (2,4-D) and 6-Benzylaminopurine (BAP) for callus induction. In indirect regeneration experiment various levels of BAP and α-Naphthaleneacetic acid (NAA) and two types of explants (calli derived from cotyledon and hypocotyl) were investigated. In direct regeneration section, various levels of BAP plus 2 mg/l NAA and different explants (cotyledon, hypocotyl and bud) were compared. In both cotyledon and hypocotyl, the maximum callus induction was observed on a media containing 2 mg/l 2,4-D plus 1 mg/l BAP (100% and 76.19% respectively). The highest percentage of indirect regeneration (65%) was observed at 1 mg/l BAP plus 0.5 mg/l NAA on calli from hypocotyl. The highest percentage of direct regeneration (90.33) was observed in hypocotyl with a lateral bud explant on MS medium supplemented with 0.5 mg/l BAP plus 2 mg/l NAA. In this study, optimization of tissue culture protocol for A. lappa was carried out as a research technique, as well as technique for further exploitation of this plant.

Induction of hemangiosarcoma in mice after chronic treatment with S1P-modulator siponimod and its lack of relevance to rat and human.

A high incidence of hemangiosarcoma (HSA) was observed in mice treated for 2 years with siponimod, a sphingosine-1-phosphate receptor 1 (S1P1) functional antagonist, while no such tumors were observed in rats under the same treatment conditions. In 3-month rat (90 mg/kg/day) and 9-month mouse (25 and 75 mg/kg/day) in vivo mechanistic studies, vascular endothelial cell (VEC) activation was observed in both species, but VEC proliferation and persistent increases in circulating placental growth factor 2 (PLGF2) were only seen in the mouse. In mice, these effects were sustained over the 9-month study duration, while in rats increased mitotic gene expression was present at day 3 only and PLGF2 was induced only during the first week of treatment. In the mouse, the persistent VEC activation, mitosis induction, and PLGF2 stimulation likely led to sustained neo-angiogenesis which over life-long treatment may result in HSA formation. In rats, despite sustained VEC activation, the transient mitotic and PLGF2 stimuli did not result in the formation of HSA. In vitro, the mouse and rat primary endothelial cell cultures mirrored their respective in vivo findings for cell proliferation and PLGF2 release. Human VECs, like rat cells, were unresponsive to siponimod treatment with no proliferative response and no release of PLGF2 at all tested concentrations. Hence, it is suggested that the human cells also reproduce a lack of in vivo response to siponimod. In conclusion, the molecular mechanisms leading to siponimod-induced HSA in mice are considered species specific and likely irrelevant to humans.