Comprehensive preclinical evaluation of human-derived anti-poly-GA antibodies in cellular and animal models of C9ORF72 disease

Hexanucleotide G4C2 repeat expansions in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Dipeptide repeat proteins (DPRs) generated by translation of repeat-containing RNAs show toxic effects in vivo as well as in vitro and are key targets for therapeutic intervention. We generated human antibodies that bind DPRs with high affinity and specificity. Anti-GA antibodies engaged extra- and intracellular poly-GA and reduced aggregate formation in a poly-GA over-expressing human cell line. However, antibody treatment in human neuronal cultures synthesizing exogenous poly-GA resulted in the formation of large extracellular immune complexes and did not affect accumulation of intracellular poly-GA aggregates. Treatment with antibodies was also shown to directly alter the morphological and biochemical properties of poly-GA and to shift poly-GA/antibody complexes to more rapidly sedimenting ones. These alterations were not observed with poly-GP and have important implications for accurate measurement of poly-GA levels including the need to evaluate all centrifugation fractions and disrupt the interaction between treatment antibodies and poly-GA by denaturation. Targeting poly-GA and poly-GP in two mouse models expressing G4C2 repeats by systemic antibody delivery for up to 16 months was well-tolerated and led to measurable brain penetration of antibodies. Long term treatment with anti-GA antibodies produced improvement in an open field movement test in aged C9ORF72450 mice. However, chronic administration of anti-GA antibodies in AAV-(G4C2)149 mice was associated with increased levels of poly-GA detected by immunoassay and did not significantly reduce poly-GA aggregates or alleviate disease progression in this model.

Long-lasting rescue of schizophrenia-relevant cognitive impairments via risperidone-loaded microPlates

Schizophrenia is a disorder characterized by cognitive impairment and psychotic symptoms that fluctuate over time and can only be mitigated with the chronic administration of antipsychotics. Here, we propose biodegradable microPlates made of PLGA for the sustained release of risperidone over several weeks. Two microPlate configurations – short: 20 × 20 × 10 μm; tall: 20 × 20 × 20 μm – are engineered and compared to conventional ~ 10 μm PLGA microspheres in terms of risperidone loading and release. Tall microPlates realize the slowest release documenting a 35% risperidone delivery at 100 days with a residual rate of 30 ng/ml. Short microPlates and microspheres present similar release profiles with over 50% of the loaded risperidone delivered within the first 40 days. Then, the therapeutic efficacy of one single intraperitoneal injection of risperidone microPlates is compared to the daily administration of free risperidone in heterozygous knockout mice for dysbindin-1, a clinically relevant mouse model of cognitive and psychiatric liability. In temporal order object recognition tasks, mice treated with risperidone microPlates outperform those receiving free risperidone up to 2, 4, 8, and 12 weeks of observation. This suggests that the sustained release of antipsychotics from one-time microPlate deposition can rescue cognitive impairment in dysbindin mice for up to several weeks. Overall, these results demonstrate that risperidone-loaded microPlates are a promising platform for improving cognitive symptoms associated to schizophrenia. Moreover, the long-term efficacy with one single administration could be of clinical relevance in terms of patient’s compliance and adherence to the treatment regimen. Graphical abstract Single injection of long-acting risperidone-loaded µPL ameliorates the dysbindin-induced deficit in a clinically relevant mouse model of cognitive and psychiatric liability for up to 12 weeks

Drug-drug interaction between cannabidiol and phenobarbital in healthy dogs

OBJECTIVE To assess drug-drug interactions between cannabidiol (CBD) and phenobarbital (PB) when simultaneously administered to healthy dogs. ANIMALS 9 healthy, purpose bred Beagles. PROCEDURES A 3-phase prospective, randomized pharmacokinetic (PK) interaction study of CBD and PB was performed as follows: phase 1, CBD PK determination and evaluation of CBD tolerability by 3 single-dose CBD (5 mg/kg, 10 mg/kg, and 20 mg/kg) protocols followed by 2-week CBD dosing; phase 2, a single-dose, 3-way, crossover PK study of CBD (10 mg/kg), PB (4 mg/kg), or CBD (10 mg/kg) administration plus PB (4 mg/kg); and phase 3, evaluation of chronic PB (4 mg/kg, q 30 d) administration followed by single-dose CBD (10 mg/kg) PK study. RESULTS Although there were variations in CBD PK variables in dogs receiving CBD alone or in conjunction with PB, significance differences in CBD PK variables were not found. No significant difference was observed in PB PK variables of dogs receiving PB alone or with CBD. During chronic CBD administration, mild gastrointestinal signs were observed in 5 dogs. At daily CBD doses of 10 to 20 mg/kg/d, hypoxia was observed in 5 dogs and increased serum alkaline phosphatase (ALP) activities (range, 301 to 978 U/L) was observed in 4 dogs. A significant increase in ALP activity was observed with chronic administration of CBD during phase 1 between day 0 and day 14. CONCLUSIONS AND CLINICAL RELEVANCE No significant PK interactions were found between CBD and PB. Dose escalation of CBD or adjustment of PB in dogs is not recommended on the basis of findings of this study.

Hepatorenal Effects of Diclofenac and Ciprofloxacin in Rats

The toxic effect of diclofenac (DCF) sodium and Ciprofloxacin (CIP) on gene expression of cytochrome P450 oxidase (CYPs) and the histology of liver and kidney of male albino rat has been evaluated in this study. DCF and CIP were chosen since they are inhibitors for specific CYP enzymes. Thirty-five adult male albino rats were divided into 7 groups of 5 animals each (A, B, C, D, E, F and G) and were treated orally with drugs for 21 consecutive days. Group A served as the control while B and C were treated with 5.3, 10.6 mg/kg body weight (bw) DCF sodium and groups D and E were treated with 40 and 80 mg/kg bw CIP, respectively. Groups F and G were treated with a mixture of the low and the high doses of both drugs, respectively. Both drugs significantly downregulated the mRNA expression of CYP1a2, CYP3a4 and CYP2c9. They caused hepatorenal histological changes. In the liver, massive fibrosis, necrosis, inflammatory cell infiltration with hemorrhages and hydrophilic degeneration have been observed. A massive tissue injury with glomerular and tubular damages due to sever necrosis, degeneration of concomitant inflammatory cells and blood vessels congestion have been shown in renal tissues. Although DCF and CIP are still used as therapeutic drugs, their use should be limited as their chronic administration induces a toxic effect on human health.

Hypothalamic regulation of reproduction under the chronic influence of toluene and melatonin

Experiments on chronic administration of melatonin with and without chronic inhalation of toluene dosed at both maximal permissible concentration (50 mg/ml) and limited chronical range (500 mg/m3) have been carried out on female rats to discover their effects on biogenic amines system in hypothalamic structures related to gonadoliberin synthesis and secretion - preoptic area (PA) and median eminence (ME). Contents of biogenic amines in ME and especially in PA have been shown to have circadian variations with maximum in the morning in control group of rats.The chronic effect of synchronizing agent melatonin (administered dissolved in drinking water in concentration of 10 pg/m l, at night during 2 months) on neotransmitters and their circadian variations in both hypothalamic structures proved surprisingly to be much alike the effect of toluene. Both chemicals cause the disturbances of normal circadian variations o f norepinephrine, dopamine and serotonine in PA and dopamine in ME. The simultaneous administration of toluene and melatonin showed likewise no synchronizing ability of the latter under the conditions described.

The antidepressants effects on microbiota: unobvious possibilities Research article

Despite the emergence of new antidepressants with different mechanisms of action, a large number of problems in antidepressant therapy remain. Considering the known antimicrobial activity of antidepressants, the role of the microbiota in the thymoanaleptic activity of these drugs is of high interest. In recent years, important data have been obtained on the role of the gut microbiota in the regulation of behavior and the pathophysiology of a number of mental disorders, including depression. Of particular interest is the assessment of the normal intestinal microbiota role in the course of the therapeutic process. The emerging bi-directional interactions between drugs and microorganisms may be critical for personalized drug selection and future drug development. However, at the present time, this problem remains poorly understood. The proposed manuscript articulates the main directions that are of clinical importance and can become an object for further study in this area.The research results indicate that the effect of antidepressants on the microbiota is a promising area, the study of which could provide many important findings for clinical practice. This type of therapeutic manipulation can provide an opportunity for intervention in order to potentiate the activity of antidepressants or to minimize side effects. The problem with this method of intervention is enormous complexity, when manipulations can have both positive and negative effects simultaneously, depending on different strains of microorganisms influencing different therapeutic effects. More research is needed to understand what changes occur in the microbiome with acute and chronic administration of specific antidepressants. Perhaps this will contribute to the development of microbiomodulatory tactics for individualized interventions.

On the Antioxidant Properties of L-Kynurenine: An Efficient ROS Scavenger and Enhancer of Rat Brain Antioxidant Defense

L-kynurenine (L-KYN) is an endogenous metabolite, that has been used as a neuroprotective strategy in experimental models. The protective effects of L-KYN have been attributed mainly to kynurenic acid (KYNA). However, considering that L-KYN is prone to oxidation, this redox property may play a substantial role in its protective effects. The aim of this work was to characterize the potential impact of the redox properties of L-KYN, in both synthetic and biological systems. First, we determined whether L-KYN scavenges reactive oxygen species (ROS) and prevents DNA and protein oxidative degradation in synthetic systems. The effect of L-KYN and KYNA (0.1–100 µM) on redox markers (ROS production, lipoperoxidation and cellular function) was compared in rat brain homogenates when exposed to FeSO4 (10 µM). Then, the effect of L-KYN administration (75 mg/kg/day for 5 days) on the GSH content and the enzymatic activity of glutathione reductase (GR) and glutathione peroxidase (GPx) was determined in rat brain tissue. Finally, brain homogenates from rats pretreated with L-KYN were exposed to pro-oxidants and oxidative markers were evaluated. The results show that L-KYN is an efficient scavenger of ●OH and ONOO−, but not O2●– or H2O2 and that it prevents DNA and protein oxidative degradation in synthetic systems. L-KYN diminishes the oxidative effect induced by FeSO4 on brain homogenates at lower concentrations (1 µM) when compared to KYNA (100 µM). Furthermore, the sub-chronic administration of L-KYN increased the GSH content and the activity of both GR and GPx, and also prevented the oxidative damage induced by the ex vivo exposure to pro-oxidants. Altogether, these findings strongly suggest that L-KYN can be considered as a potential endogenous antioxidant.

The Effects of Separate and Combined Treatment of Male Rats with Type 2 Diabetes with Metformin and Orthosteric and Allosteric Agonists of Luteinizing Hormone Receptor on Steroidogenesis and Spermatogenesis

In men with type 2 diabetes mellitus (T2DM), steroidogenesis and spermatogenesis are impaired. Metformin and the agonists of luteinizing hormone/human chorionic gonadotropin(hCG)-receptor (LH/hCG-R) (hCG, low-molecular-weight allosteric LH/hCG-R-agonists) can be used to restore them. The aim was to study effectiveness of separate and combined administration of metformin, hCG and 5-amino-N-tert-butyl-2-(methylsulfanyl)-4-(3-(nicotinamido)phenyl)thieno[2,3-d]pyrimidine-6-carboxamide (TP3) on steroidogenesis and spermatogenesis in male rats with T2DM. hCG (15 IU/rat/day) and TP3 (15 mg/kg/day) were injected in the last five days of five-week metformin treatment (120 mg/kg/day). Metformin improved testicular steroidogenesis and spermatogenesis and restored LH/hCG-R-expression. Compared to control, in T2DM, hCG stimulated steroidogenesis and StAR-gene expression less effectively and, after five-day administration, reduced LH/hCG-R-expression, while TP3 effects changed weaker. In co-administration of metformin and LH/hCG-R-agonists, on the first day, stimulating effects of LH/hCG-R-agonists on testosterone levels and hCG-stimulated expression of StAR- and CYP17A1-genes were increased, but on the 3–5th day, they disappeared. This was due to reduced LH/hCG-R-gene expression and increased aromatase-catalyzed estradiol production. With co-administration, LH/hCG-R-agonists did not contribute to improving spermatogenesis, induced by metformin. Thus, in T2DM, metformin and LH/hCG-R-agonists restore steroidogenesis and spermatogenesis, with metformin being more effective in restoring spermatogenesis, and their co-administration improves LH/hCG-R-agonist-stimulating testicular steroidogenesis in acute but not chronic administration.

Angiotensin-(3–4) (Val-Tyr) Normalizes the Elevated Arterial Blood Pressure and Abnormal Na+/Energy Handling Associated With Chronic Undernutrition by Counteracting the Effects Mediated by Type 1 Angiotensin II Receptors

Purpose To investigate the mechanisms by which chronic administration of a multideficient diet after weaning alters bodily Na+ handling, and culminates in high systolic blood pressure (SBP) at a juvenile age.Methods From 28 to 93 days of age, weaned male Wistar rats were given a diet with low content and poor-quality protein, low lipid, without vitamin supplementation, which mimics the diets consumed in impoverished regions worldwide. We measured food, energy and Na+ ingestion, together with urinary Na+ excretion, Na+ density (Na+ intake/energy intake), plasma Na+ concentration, SBP), and renal proximal tubule Na+-transporting ATPases. Results Undernourished rats aged 93 days had only one-third of the control body mass, lower plasma albumin, higher SBP, higher energy intake, and higher positive Na+ balance accompanied by decreased plasma Na+ concentration. SBP was normalized with Losartan and with Ang-(3–4), and the combination of the 2 substances induced an accentuated negative Na+ balance as a result of strong inhibition of Na+ ingestion. Na+ density in undernourished rats was higher than in control, irrespective of the treatment, and they had downregulated (Na++K+)ATPase and upregulated Na+-ATPase in proximal tubule cells, which returned to control levels after Losartan or Ang-(3–4).Conclusions Na+ density, not only Na+ ingestion, plays a central role in the pathophysiology of elevated SBP in chronically undernourished rats. The observations that Losartan and Ang-(3–4) normalized SBP together with Na+ distribution and handling give support to the proposal that Ang IIÞAT1R and Ang IIÞAT2R axes have opposite roles within the renin-angiotensin-aldosterone system of undernourished juvenile rats.