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PDF file - 417K, NT compounds are allosteric inhibitors of the full-length IGF1R, and induce translocation of IRS1 to the cytoplasm (S1); The anti-proliferative effects of the NT inhibitors are maintained long after the inhibitors are washed out (S2); NT157 inhibits IGF2-induced and insulin-induced PKB activation, without inhibiting the autophosphorylation of IR and IGF1R (S3); The NT-induced Ser-phosphorylation occurs in an IGF1-independent manner (S4); NT compounds inhibit human ovarian cancer and hormonerefractory prostate cancer in xenograft models in mice (S5); Combined treatment with NT157 and PLX4032 induces a synergistic cytotoxic effect in 451-Lu melanoma cells, and NT157 reverses the overexpression of IRS1/2 in Vemurafenib-resistant Mel1617-BR cells (S6).

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PDF file - 306K, Structure-activity relationship analysis that led to design of NT compounds (S1); Anti-proliferative effects of the NT compounds on various types of cancer cells (S2).

PDF file - 102K, Additional data regarding experimental procedures, kinetic experiments and anti-tumor activity of NT 157.

Pridopidine is a small molecule in clinical development for the treatment of Huntington’s disease. It was recently found to have high binding affinity to the sigma-1 receptor, a chaperone protein involved in cellular defense mechanisms and neuroplasticity. Here, we have evaluated the neuroprotective and neurorestorative effects of pridopidine in a unilateral 6-hydroxydopamine (6-OHDA) lesion model of parkinsonism in mice. By 5 weeks of daily administration, a low dose of pridopidine (0.3 mg/kg) had significantly improved deficits in forelimb use (cylinder test, stepping test) and abolished the ipsilateral rotational bias typical of hemiparkinsonian animals. A higher dose of pridopidine (1 mg/kg) significantly improved only the rotational bias, with a trend towards improvement in forelimb use. The behavioral recovery induced by pridopidine 0.3 mg/kg was accompanied by a significant protection of nigral dopamine cell bodies, an increased dopaminergic fiber density in the striatum, and striatal upregulation of GDNF, BDNF, and phosphorylated ERK1/2. The beneficial effects of pridopidine 0.3 mg/kg were absent in 6-OHDA-lesioned mice lacking the sigma-1 receptor. Pharmacokinetic data confirmed that the effective dose of pridopidine reached brain concentrations sufficient to bind S1R. Our results are the first to show that pridopidine promotes functional neurorestoration in the damaged nigrostriatal system acting via the sigma-1 receptor. Electronic supplementary material The online version of this article (10.1007/s13311-018-00699-9) contains supplementary material, which is available to authorized users.

BACKGROUND Pridopidine, in development for Huntington's disease, may modulate aberrant l-dopa-induced effects including l-dopa-induced dyskinesia (LID). OBJECTIVE This study investigated whether pridopidine could reduce LID in the MPTP macaque model of Parkinson's disease and characterized the observed behavioral effects in terms of receptor occupancy. METHODS The pharmacokinetic profile and effects of pridopidine (15-30 mg/kg) on parkinsonism, dyskinesia, and quality of on-time, in combination with l-dopa, were assessed in MPTP macaques with LID. Pridopidine receptor occupancy was estimated using known in vitro binding affinities to σ1 and dopamine D2 receptors, in vivo PET imaging, and pharmacokinetic profiling across different species. RESULTS Pridopidine produced a dose-dependent reduction in dyskinesia (up to 71%, 30 mg/kg) and decreased the duration of on-time with disabling dyskinesia evoked by l-dopa by 37% (20 mg/kg) and 60% (30 mg/kg). Pridopidine did not compromise the anti-parkinsonian benefit of l-dopa. Plasma exposures following the ineffective dose (15 mg/kg) were associated with full σ1 occupancy (>80%), suggesting that σ1 engagement alone is unlikely to account for the antidyskinetic benefits of pridopidine. Exposures following effective doses (20-30 mg/kg), while providing full σ1 occupancy, provide only modest dopamine D2 occupancy (

Simple Summary Nowadays, the importance of breastfeeding has been very well recognized not only by the scientific world but also by public opinion. Such awareness has nonetheless put a lot of pressure on women under chronic pharmacological medication, or that simply need to alleviate common post-partum health issues, due to the lack of scientific data regarding the potential transfer to the offspring during lactation. In such a scenario, the ConcePTION project aims at creating a trusted ecosystem that can efficiently generate and disseminate reliable evidence-based information regarding the effects of medications used during pregnancy and breastfeeding to women and their healthcare providers. Due to the need for a reliable animal species to obtain scientific data, the present review summarizes the main features contributing to the lactation process in the most commonly used laboratory animal species. Abstract The present review aims to summarize the main features of mammary gland anatomy, and the physiology of lactation and colostrum/milk in the most commonly used animal species for regulatory toxicity. The final goal is the selection of a preferred animal species to be enrolled in studies investigating the potential transfer of drugs and exogenous molecules through milk, within the Innovative Medicines Initiative (IMI) funded project ConcePTION. Reference data regarding humans were also collected and analyzed in order to highlight critical similarities and differences with the studied species. Additional practical considerations were also taken into account, such as ethical consideration regarding the chosen species which affects the group size, financial implications and technical feasibility of lactation trials (e.g., ease of sampling, volume of sampling, husbandry requirements and scientific recognition). In conclusion, the present analysis of the literature confirms the complexity of the decisional process behind the choice of an animal model for in vivo trials. For some of the evaluated species, data were either poor or missing, highlighting the necessity to generate more physiological background studies for species that are routinely used in laboratory settings. Overall, when taking into consideration ethical factors, feasible group size, milk volume and ease of milk collection, and physiological similarities with humans, minipigs seem to represent the most appropriate choice.

Microneedle (MN) patches consist of a hydrogel-forming MN array and a drug-containing reservoir. Drug-containing reservoirs documented in the literature include polymeric films and lyophilized wafers. While effective, both reservoir formulations are aqueous based, and so degradation can occur during formulation and drying for drugs inherently unstable in aqueous media. The preparation and characterization of novel, nonaqueous-based, directly compressed tablets (DCTs) for use in combination with hydrogel-forming MN arrays are described for the first time. In this work, a range of drug molecules are investigated. Precipitation of amoxicillin (AMX) and primaquine (PQ) in conventional hydrogel-forming MN arrays leads to use of poly(vinyl alcohol)-based MN arrays. Following in vitro permeation studies, in vivo pharmacokinetic studies are conducted in rats with MN patches containing AMX, levodopa/carbidopa (LD/CD), and levofloxacin (LVX). Therapeutically relevant concentrations of AMX (≥2 µg mL-1 ), LD (≥0.5 µg mL-1 ), and LVX (≥0.2 µg mL-1 ) are successfully achieved at 1, 2, and 1 h, respectively. Thus, the use of DCTs offers promise to expand the range of drug molecules that can be delivered transdermally using MN patches.

Treatment resistant depression is, by definition, difficult to treat using standard therapeutic interventions. Recently, esketamine has been shown as a viable rescue treatment option in patients in depressive crisis states. However, IV administration is associated with a number of drawbacks and advanced delivery platforms could provide an alternative parenteral route of esketamine dosing in patients. Hydrogel-forming microneedle arrays facilitate transdermal delivery of drugs by penetrating the outer layer of the skins surface, absorbing interstitial skin fluid and swelling. This subsequently facilitates permeation of medicines into the dermal microcirculation. This paper outlines the in vitro formulation development for hydrogel-forming microneedle arrays containing esketamine. Analytical methods for the detection and quantitation of esketamine were developed and validated according to International Conference on Harmonisation standards. Hydrogel-forming microneedle arrays were fully characterised for their mechanical strength and skin insertion properties. Furthermore, a series of esketamine containing polymeric films and lyophilised reservoirs were assessed as drug reservoir candidates. Dissolution testing and content drug recovery was carried out, followed by permeation studies using 350 μm thick neonatal porcine skin in modified Franz cell apparatus. Lead reservoir candidates were selected based on measured physicochemical properties and brought forward for testing in female Sprague-Dawley rats. Plasma samples were analysed using reverse phase high performance liquid chromatography for esketamine. Both polymeric film and lyophilised reservoirs candidate patches achieved esketamine plasma concentrations higher than the target concentration of 0.15–0.3 μg/ml over 24 h. Mean plasma concentrations in rats, 24 h post-application of microneedle patches with drug reservoir F3 and LW3, were 0.260 μg/ml and 0.498 μg/ml, respectively. This developmental study highlights the potential success of hydrogel-forming microneedle arrays as a transdermal drug delivery platform for ESK and supports moving to in vivo tests in a larger animal model., Graphical abstract Unlabelled Image, Highlights • Esketamine may be a therapeutic option in treatment-resistant depression. • Novel drug delivery methods are needed to reduce dosing frequency. • Microneedle technology has proven benefits as a method of minimally invasive drug delivery. • Hydrogel-forming microneedles enhance transdermal delivery of esketamine in vivo. • Therapeutic plasma levels of >0.15–0.3 μg/ml were achieved in rats over 24 h.

Aims Pridopidine is an oral drug in clinical development for treatment of patients with Huntington's disease. This study examined the interactions of pridopidine with in vitro cytochrome P450 activity and characterized the effects of pridopidine on CYP2D6 activity in healthy volunteers using metoprolol as a probe substrate. The effect of food on pridopidine exposure was assessed. Methods The ability of pridopidine to inhibit and/or induce in vitro activity of drug metabolizing enzymes was examined in human liver microsomes and fresh hepatocytes. CYP2D6 inhibition potency and reversibility was assessed using dextromethorphan. For the clinical assessment, 22 healthy subjects were given metoprolol 100 mg alone and concomitantly with steady-state pridopidine 45 mg twice daily. Food effect on a single 90 mg dose of pridopidine was evaluated in a crossover manner. Safety assessments and pharmacokinetic sampling occurred throughout the study. Results Pridopidine was found to be a metabolism dependent inhibitor of CYP2D6, the main enzyme catalysing its own metabolism. Flavin-containing monooxygenase heat inactivation of liver microsomes did not affect pridopidine metabolism-dependent inhibition of CYP2D6 and its inhibition of CYP2D6 was not reversible with addition of FeCN3. Exposure to metoprolol was markedly increased when coadministered with pridopidine; the ratio of the geometric means (90% confidence interval) for maximum observed plasma concentration, and area under the plasma concentration–time curve from time 0 to the time of the last quantifiable concentration and extrapolated to infinity were 3.5 (2.9, 4.22), 6.64 (5.27, 8.38) and 6.55 (5.18, 8.28), respectively. Systemic exposure to pridopidine was unaffected by food conditions. Conclusions As pridopidine is a metabolism-dependent inhibitor of CYP2D6, systemic levels of drugs metabolized by CYP2D6 may increase with chronic coadministration of pridopidine. Pridopidine can be administered without regard to food.

Aims Pridopidine is an oral drug in clinical development for treatment of patients with Huntington's disease. This study examined the interactions of pridopidine with in vitro cytochrome P450 activity and characterized the effects of pridopidine on CYP2D6 activity in healthy volunteers using metoprolol as a probe substrate. The effect of food on pridopidine exposure was assessed. Methods The ability of pridopidine to inhibit and/or induce in vitro activity of drug metabolizing enzymes was examined in human liver microsomes and fresh hepatocytes. CYP2D6 inhibition potency and reversibility was assessed using dextromethorphan. For the clinical assessment, 22 healthy subjects were given metoprolol 100 mg alone and concomitantly with steady‐state pridopidine 45 mg twice daily. Food effect on a single 90 mg dose of pridopidine was evaluated in a crossover manner. Safety assessments and pharmacokinetic sampling occurred throughout the study. Results Pridopidine was found to be a metabolism dependent inhibitor of CYP2D6, the main enzyme catalysing its own metabolism. Flavin‐containing monooxygenase heat inactivation of liver microsomes did not affect pridopidine metabolism‐dependent inhibition of CYP2D6 and its inhibition of CYP2D6 was not reversible with addition of FeCN3. Exposure to metoprolol was markedly increased when coadministered with pridopidine; the ratio of the geometric means (90% confidence interval) for maximum observed plasma concentration, and area under the plasma concentration–time curve from time 0 to the time of the last quantifiable concentration and extrapolated to infinity were 3.5 (2.9, 4.22), 6.64 (5.27, 8.38) and 6.55 (5.18, 8.28), respectively. Systemic exposure to pridopidine was unaffected by food conditions. Conclusions As pridopidine is a metabolism‐dependent inhibitor of CYP2D6, systemic levels of drugs metabolized by CYP2D6 may increase with chronic coadministration of pridopidine. Pridopidine can be administered without regard to food.

Insulin receptor substrates 1 and 2 (IRS1/2) mediate mitogenic and antiapoptotic signaling from insulin-like growth factor 1 receptor (IGF-IR), insulin receptor (IR), and other oncoproteins. IRS1 plays a central role in cancer cell proliferation, its expression is increased in many human malignancies, and its upregulation mediates resistance to anticancer drugs. IRS2 is associated with cancer cell motility and metastasis. Currently, there are no anticancer agents that target IRS1/2. We present new IGF-IR/IRS-targeted agents (NT compounds) that promote inhibitory Ser-phosphorylation and degradation of IRS1 and IRS2. Elimination of IRS1/2 results in long-term inhibition of IRS1/2-mediated signaling. The therapeutic significance of this inhibition in cancer cells was shown while unraveling a novel mechanism of resistance to B-RAFV600E/K inhibitors. We found that IRS1 is upregulated in PLX4032-resistant melanoma cells and in cell lines derived from patients whose tumors developed PLX4032 resistance. In both settings, NT compounds led to the elimination of IRS proteins and evoked cell death. Treatment with NT compounds in vivo significantly inhibited the growth of PLX4032-resistant tumors and displayed potent antitumor effects in ovarian and prostate cancers. Our findings offer preclinical proof-of-concept for IRS1/2 inhibitors as cancer therapeutics including PLX4032-resistant melanoma. By the elimination of IRS proteins, such agents should prevent acquisition of resistance to mutated-B-RAF inhibitors and possibly restore drug sensitivity in resistant tumors. Cancer Res; 73(14); 4383–94. ©2013 AACR.

The insulin-like growth factor-1 receptor (IGF-1 receptor) is a receptor tyrosine kinase, highly homologous to the insulin receptor. In contrast to the insulin receptor, which is mostly involved in metabolic pathways, the IGF-1 system plays a pivotal role in normal and neoplastic cell growth through anti-apoptotic, proliferative and metastatic pathways. Furthermore, IGF-1 receptor over-activation is found to correlate with a variety of tumors, such as breast cancer, prostate cancer, hematological malignancies, colorectal cancer and other proliferative diseases, such as psoriasis and papilloma. In addition, accumulating evidence implies that blockade of IGF-1 receptor activity causes reversal of tumor progression in cell lines as well as in animal tumor models. Because of the central role the IGF-1 receptor plays in oncogenic maintenance and metastatic processes, it is a highly appropriate target for anti-cancer agents. Here we report on a novel substrate-mimic family of IGF-1 receptor inhibitors. These compounds are tertiary aromatic amines, non-competitive with ATP and possess high affinity towards the IGF-1 receptor. The most potent compound, SBL02 inhibited the IGF-1 receptor with an IC(50) of 170 nM in a cell-free kinase assay and was found to inhibit IGF-1 receptor auto-phosphorylation and substrate phosphorylation at the low micromolar range in cellular assays. SBL02 also blocks the formation of colonies in soft agar by cancer cells and inhibits the growth of keratinocytes and of HPV16 immortalized keratinocytes. This new family of non-ATP competitive, IGF-1 receptor inhibitors can serve as a lead for the development of anti-cancer, anti-psoriatic and anti-papilloma agents.