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Background: Congenital myasthenic syndromes (CMS) are a group of inherited neuromuscular junction (NMJ) disorders arising from gene variants encoding diverse NMJ proteins. Recently, the VAMP1 gene, responsible for encoding the vesicle-associated membrane protein 1 (VAMP1), has been associated with CMS., Methods: This study presents a characterization of five new individuals with VAMP1-related CMS, providing insights into the phenotype., Results: The individuals with VAMP1-related CMS exhibited early disease onset, presenting symptoms prenatally or during the neonatal period, alongside severe respiratory involvement and feeding difficulties. Generalized weakness at birth was a common feature, and none of the individuals achieved independent walking ability. Notably, all cases exhibited scoliosis. The clinical course remained stable, without typical exacerbations seen in other CMS types. The response to anticholinesterase inhibitors and salbutamol was only partial, but the addition of 3,4-diaminopyridine (3,4-DAP) led to significant and substantial improvements, suggesting therapeutic benefits of 3,4-DAP for managing VAMP1-related CMS symptoms. Noteworthy is the identification of the VAMP1 (NM_014231.5): c.340delA; p.Ile114SerfsTer72 as a founder variant in the Iberian Peninsula and Latin America., Conclusions: This study contributes valuable insights into VAMP1-related CMS, emphasizing their early onset, arthrogryposis, facial and generalized weakness, respiratory involvement, and feeding difficulties. Furthermore, the potential efficacy of 3,4-DAP as a useful therapeutic option warrants further exploration. The findings have implications for clinical management and genetic counseling in affected individuals. Additional research is necessary to elucidate the long-term outcomes of VAMP1-related CMS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

4-aminopyridine (4AP) is a potassium (K + ) channel blocker used clinically to improve walking in people with multiple sclerosis (MS). 4AP binds to exposed K + channels in demyelinated axons, reducing the leakage of intracellular K + and enhancing impulse conduction. Multiple derivatives of 4AP capable of blocking K + channels have been reported including three radiolabeled with positron emitting isotopes for imaging demyelinated lesions using positron emission tomography (PET). However, there remains a demand for novel molecules with suitable physicochemical properties and binding affinity that can potentially be radiolabeled and used as PET radiotracers. In this study, we introduce 3-fluoro-5-methylpyridin-4-amine (5Me3F4AP) as a novel trisubstituted K + channel blocker with potential application in PET. 5Me3F4AP has comparable potency to 4AP and the PET tracer 3-fluoro-4-aminopyridine (3F4AP). Compared to 3F4AP, 5Me3F4AP exhibits comparable basicity (pK a  = 7.46 ± 0.01 vs. 7.37 ± 0.07, P-value = 0.08), greater lipophilicity (logD = 0.664 ± 0.005 vs. 0.414 ± 0.002, P-value < 0.0001) and higher permeability to an artificial brain membrane (P e  = 88.1 ± 18.3 vs. 31.1 ± 2.9 nm/s, P-value = 0.03). 5Me3F4AP is also more stable towards oxidation in vitro by the cytochrome P450 enzyme CYP2E1 (IC 50  = 36.2 ± 2.5 vs. 15.4 ± 5.1, P-value = 0.0003); the enzyme responsible for the metabolism of 4AP and 3F4AP. Taken together, 5Me3F4AP has promising properties as a candidate for PET imaging warranting additional investigation., (© 2024. The Author(s).)

Checkpoint kinase 1 (CHK1), a serine/threonine kinase, plays a crucial role in cell cycle arrest and is a promising therapeutic target for drug development against cancers. CHK1 coordinates cell cycle checkpoints in response to DNA damage, facilitating repair of single-strand breaks, and maintains the genome integrity in response to replication stress. In this study, we employed an integrated computational and experimental approach to drug discovery and repurposing, aiming to identify a potent CHK1 inhibitor among existing drugs. An e-pharmacophore model was developed based on the three-dimensional crystal structure of the CHK1 protein in complex with CCT245737. This model, characterized by seven key molecular features, guided the screening of a library of drugs through molecular docking. The top 10% of scored ligands were further examined, with procaterol emerging as the leading candidate. Procaterol demonstrated interaction patterns with the CHK1 active site similar to CHK1 inhibitor (CCT245737), as shown by molecular dynamics analysis. Subsequent in vitro assays, including cell proliferation, colony formation, and cell cycle analysis, were conducted on gastric adenocarcinoma cells treated with procaterol, both as a monotherapy and in combination with cisplatin. Procaterol, in synergy with cisplatin, significantly inhibited cell growth, suggesting a potentiated therapeutic effect. Thus, we propose the combined application of cisplatin and procaterol as a novel potential therapeutic strategy against human gastric cancer. The findings also highlight the relevance of CHK1 kinase as a drug target for enhancing the sensitivity of cytotoxic agents in cancer.

Demyelination, the loss of the insulating sheath of neurons, causes failed or slowed neuronal conduction and contributes to the neurological symptoms in multiple sclerosis, traumatic brain and spinal cord injuries, stroke, and dementia. In demyelinated neurons, the axonal potassium channels K v 1.1 and K v 1.2, generally under the myelin sheath, become exposed and upregulated. Therefore, imaging these channels using positron emission tomography can provide valuable information for disease diagnosis and monitoring. Here, we describe a novel tracer for K v 1 channels, [ 11 C]3-methyl-4-aminopyridine ([ 11 C]3Me4AP). [ 11 C]3Me4AP was efficiently synthesized via Pd(0)-Cu(I) comediated Stille cross-coupling of a stannyl precursor containing a free amino group. Evaluation of its imaging properties in rats and nonhuman primates showed that [ 11 C]3Me4AP has a moderate brain permeability and slow kinetics. Additional evaluation in monkeys showed that the tracer is metabolically stable and that a one-tissue compartment model can accurately model the regional brain time-activity curves. Compared to the related tracers [ 18 F]3-fluoro-4-aminopyridine ([ 18 F]3F4AP) and [ 11 C]3-methoxy-4-aminopyridine ([ 11 C]3MeO4AP), [ 11 C]3Me4AP shows lower initial brain uptake, which indicates reduced permeability to the blood-brain barrier and slower kinetics, suggesting higher binding affinity consistent with in vitro studies. While the slow kinetics and strong binding affinity resulted in a tracer with less favorable properties for imaging the brain than its predecessors, these properties may make 3Me4AP useful as a therapeutic.

Since it was shown that silicone rubber production can unintentionally release PCBs, these production sites have become a focus of investigation. The use of the cross-linking agent bis(2,4)-dichlorobenzoylperoxide (2,4-DCBP) can lead to emissions of the PCB congeners PCB 47, PCB 51 and PCB 68 into the environment and cause their accumulation e. g. in food crops. To determine the presence and extent of this uptake, we used the newly developed method dandelion screening. Samples were taken from wild dandelion plants near nine production sites in North Rhine-Westphalia, Germany, and analysed for PCBs. In some cases, the regional orientation values for the maximum background level (OMB) were exceeded by up to nine times. Overall, background levels were exceeded at seven of the nine sites investigated and mitigation measures were initiated at the production sites. In order to validate the dandelion screening results, kale was exposed, which allowed for a health assessment. A wide-ranging consumption recommendation was then issued in four out of nine study areas. At this point in the investigations, risk reduction measures had already been implemented at all production sites investigated, so it can be assumed that the exposures at sites not yet in focus are significantly greater. This is a globally relevant problem, as 2,4-DCBP is used in many countries., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This work investigated the modification of low-methoxy pectin with stearic anhydride through microwave action with 4-dimethylaminopyridine as catalyst. Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) analyses indicated that stearic acid was grafted on the pectin through esterification reaction, with the maximum stearic acid grafting ratio (SGR) of 10.7% for the modified pectin. The introduction of stearic acid was shown to significantly improve the emulsifying activity and stability of pectin. Composite films were prepared by blending the modified pectins and chitosan, and compared with the contact angle of 65.3° for the film with native low-methoxy pectin (PC0), the films with modified pectins showed a significant angle increase, with the highest contact angle reaching 101.9°, indicating a hydrophobic surface. Moreover, an appropriate amount of aliphatic chains could improve the tensile strength and elongation at break of the composite films due to the "anchoring effect"., (Copyright © 2021. Published by Elsevier B.V.)

Life emerges from complicated and sophisticated chemical networks comprising numerous biomolecules (e.g., nucleic acids, proteins, sugars, and lipids) and chemical reactions catalyzed by enzymes. Dysregulation of these chemical networks is linked to the emergence of diseases. Our research goal is to develop abiotic chemical catalysts that can intervene into life's chemical networks by complementing, surrogating, or exceeding enzymes in living cells or multicellular organisms such as animals or plants. Mending dysregulated networks in pathological states by the chemical catalysts will lead to a new medicinal strategy, catalysis medicine. This research direction will also advance catalysis science, because highly active and selective chemical catalysts must be developed to promote the intended reactions in a complex mixture of life in aqueous solution at body temperature.Epigenetics exists at the crossroads of chemistry, biology, and medicine and is a suitable field to pursue this idea. Post-translational modifications (PTMs) of histones epigenetically regulate chromatin functions and gene transcription and are intimately related to various diseases. Investigating the functions and cross-talk of histone PTMs is crucial for mechanistic elucidation of diseases and their treatments. We launched a program to develop chemical catalysts enabling endogenous histone modifications in living cells without relying on enzymes. We reported two types of chemical catalyst systems so far for synthetic histone acylation. The first system comprised a DNA-binding oligo-4-dimethylaminopyridine (DMAP) catalyst and a phenyl ester acyl donor, PAc-gly. This system promoted histone hyperacetylation in Xenopus laevis sperm chromatin. Using the thus-synthesized hyperacetylated sperm chromatin, we found a novel relationship between histone acetylation and DNA replication. The second system involved a histone-binding catalyst, LANA-DSH, composed of a catalytic motif (DSH) and a histone-binding peptide ligand (LANA), and thioester acyl donors, including endogenous acyl-CoA. This system regioselectively (i.e., selectively to a lysine residue at a specific position) acylated lysine 120 of histone H2B (H2BK120), a lysine residue proximal to the DSH motif defined by binding of the LANA ligand to a nucleosome substrate. This catalyst system was optimized to achieve H2BK120-selective acetylation in living cells without genetic manipulation. The synthetically introduced H2BK120Ac inhibited enzyme-catalyzed ubiquitination at the same lysine residue, acting as a protecting group. H2BK120Ub is a mark recognized by methyltransferase that plays an essential role in mixed-lineage leukemia (MLL)-rearranged leukemia, suggesting the potential of the catalyst system as an epigenetic tool and a cancer therapy. We also discuss the prospects of chemical catalyst-promoted synthetic epigenetics for future PTM studies and therapeutic uses.

Leukemia, a malignant hematological disease, has poor therapeutic outcomes due to chemotherapeutic resistance. Increasing evidence has confirmed that the elevated capacity for DNA damage repair in cancer cells is a major mechanism of acquired chemotherapeutic resistance. Thus, combining chemotherapy with inhibitors of DNA damage repair pathways is potentially an ideal strategy for treating leukemia. Checkpoint kinase 1 (CHK1) is an important component of the DNA damage response (DDR) and is involved in the G2/M DNA damage checkpoint. In the present study, we demonstrated that shRNA‑mediated CHK1 silencing suppressed cell proliferation and enhanced the cytotoxic effects of etoposide (VP16) in the chronic myeloid leukemia (CML) cell line K562 through the results of CCK‑8, and comet assay. The results demonstrated that shRNA‑induced CHK1 silencing can override G2/M arrest and impair homologous recombination (HR) repair by reducing breast cancer susceptibility gene 1 (BRCA1) expression. Cells had no time, and thus limited ability, to repair the damage and were thus more sensitive to chemotherapy after CHK1 downregulation. Second, we tested the therapeutic effect of VP16 combined with CCT245737, an orally bioavailable CHK1 inhibitor, and observed strong synergistic anticancer effects in K562 cells. Moreover, we discovered that CCT245737 significantly prevented the G2/M arrest caused by acute exposure to VP16. Interestingly, CCT245737 inhibited both BRCA1 and Rad51, the most important component of the HR repair pathway. In conclusion, these results revealed that CHK1 is potentially an ideal therapeutic target for the treatment of CML and that CCT245737 should be considered a candidate drug.

4-Aminopyridine (4AP) is a specific blocker of voltage-gated potassium channels (K V 1 family) clinically approved for the symptomatic treatment of patients with multiple sclerosis (MS). It has recently been shown that [ 18 F]3F4AP, a radiofluorinated analog of 4AP, also binds to K V 1 channels and can be used as a PET tracer for the detection of demyelinated lesions in rodent models of MS. Here, we investigate four novel 4AP derivatives containing methyl (-CH 3 ), methoxy (-OCH 3 ) as well as trifluoromethyl (-CF 3 ) in the 2 and 3 position as potential candidates for PET imaging and/or therapy. We characterized the physicochemical properties of these compounds (basicity and lipophilicity) and analyzed their ability to block Shaker K + channel under different voltage and pH conditions. Our results demonstrate that three of the four derivatives are able to block voltage-gated potassium channels. Specifically, 3-methyl-4-aminopyridine (3Me4AP) was found to be approximately 7-fold more potent than 4AP and 3F4AP; 3-methoxy- and 3-trifluoromethyl-4-aminopyridine (3MeO4AP and 3CF 3 4AP) were found to be about 3- to 4-fold less potent than 4AP; and 2-trifluoromethyl-4-AP (2CF 3 4AP) was found to be about 60-fold less active. These results suggest that these novel derivatives are potential candidates for therapy and imaging.

The combination of computational design and laboratory evolution is a powerful and potentially versatile strategy for the development of enzymes with new functions 1-4 . However, the limited functionality presented by the genetic code restricts the range of catalytic mechanisms that are accessible in designed active sites. Inspired by mechanistic strategies from small-molecule organocatalysis 5 , here we report the generation of a hydrolytic enzyme that uses N δ -methylhistidine as a non-canonical catalytic nucleophile. Histidine methylation is essential for catalytic function because it prevents the formation of unreactive acyl-enzyme intermediates, which has been a long-standing challenge when using canonical nucleophiles in enzyme design 6-10 . Enzyme performance was optimized using directed evolution protocols adapted to an expanded genetic code, affording a biocatalyst capable of accelerating ester hydrolysis with greater than 9,000-fold increased efficiency over free N δ -methylhistidine in solution. Crystallographic snapshots along the evolutionary trajectory highlight the catalytic devices that are responsible for this increase in efficiency. N δ -methylhistidine can be considered to be a genetically encodable surrogate of the widely employed nucleophilic catalyst dimethylaminopyridine 11 , and its use will create opportunities to design and engineer enzymes for a wealth of valuable chemical transformations.

We report a novel approach for generating nanosized DNA hollow spheres (HSs) using enzymatically produced DNA microsponges in a self-templating manner. In previous studies, preparation of DNA nanostructures with specified functions required multiple complicated steps. In this study, however, a simple treatment with the nucleophilic agent 4-dimethylaminopyridine (DMAP) enabled a gradual disentanglement of DNA in microsponges by electrostatic interactions between DMAP and DNA, and the DNA underwent a reassembly process to generate hollow shell structures without denaturation/annealing by thermal cycling. In addition, this synthetic process was conducted in a water-based system without organic solvents, enabling the synthesis of biologically and environmentally friendly products. Based on the benefits of hollow shell structures, which include their high surface-to-volume ratio and ability to encapsulate small molecules, we envision that this simple approach for synthesizing DNA HSs will provide a new platform for maximizing their potential use in drug delivery and bio-imaging.

Four series of novel compounds based on 4-aminopyridine, glatiramer acetate, pyrone, and coumarin backbones were sufficiently synthesized and identified by spectroscopic methods. CYP enzyme inhibition assays of five predominate human P450 isozymes indicate that all compounds, except for 4-hydrazide pyridine 1c, seem to be less toxic than 4-aminopyridine. Further investigation of the compounds using molecular docking experiments revealed different, the same, or stronger binding modes for most of the synthesized compounds, with both polar and hydrophobic interactions with the 1WDA and 1J95 receptors compared to benzoyl l-arginine amide and 4-aminopyridine, respectively. These results introduce the synthesized compounds as K + channel blockers that could be considered for in vivo CNS disease studies., (© 2019 Deutsche Pharmazeutische Gesellschaft.)

3,4-Diaminopyridine (34DAP) is a presynaptic transmission enhancer. Its efficacy for Lambert-Eaton myasthenic syndrome (LEMS) and myasthenia gravis (MG) was demonstrated. However, there are cases sharing the characteristics of both disease and the effect of 34DAP in "gray zone" patients is sparse. Recently, we prescribed 34DAP to five anti-acetylcholine receptor antibody-positive MG patients with electrophysiological LEMS patterns and three LEMS patients, and carefully monitored the responses. Sero-positive MG patients exhibited more favorable responses than LEMS patients. The combination of 34DAP and pyridostigmine resulted in the best outcomes. No significant side effects were recorded during the follow-up period. In conclusion, this study results provide evidence that 34DAP could be effective in sero-positive MG patients with pre-synaptic dysfunction., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Acquired brain injury can cause eye movement disorders which may include: strabismus, gaze deficits and nystagmus, causing visual symptoms of double, blurred or 'juddery' vision and reading difficulties. A wide range of interventions exist that have potential to alleviate or ameliorate these symptoms. There is a need to evaluate the effectiveness of these interventions and the timing of their implementation., Objectives: We aimed to assess the effectiveness of any intervention and determine the effect of timing of intervention in the treatment of strabismus, gaze deficits and nystagmus due to acquired brain injury. We considered restitutive, substitutive, compensatory or pharmacological interventions separately and compared them to control, placebo, alternative treatment or no treatment for improving ocular alignment or motility (or both)., Search Methods: We searched the Cochrane Central Register of Controlled Trials (CENTRAL) (containing the Cochrane Eyes and Vision Trials Register) (2017, Issue 5), MEDLINE Ovid, Embase Ovid, CINAHL EBSCO, AMED Ovid, PsycINFO Ovid, Dissertations & Theses (PQDT) database, PsycBITE (Psychological Database for Brain Impairment Treatment Efficacy), ISRCTN registry, ClinicalTrials.gov, Health Services Research Projects in Progress (HSRProj), National Eye Institute Clinical Studies Database and the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP). The databases were last searched on 26 June 2017. No date or language restrictions were used in the electronic searches for trials. We manually searched the Australian Orthoptic Journal, British and Irish Orthoptic Journal, and ESA, ISA and IOA conference proceedings. We contacted researchers active in this field for information about further published or unpublished studies., Selection Criteria: We included randomised controlled trials (RCTs) of any intervention for ocular alignment or motility deficits (or both) due to acquired brain injury., Data Collection and Analysis: Two review authors independently selected studies and extracted data. We used standard methods expected by Cochrane. We employed the GRADE approach to interpret findings and assess the quality of the evidence., Main Results: We found five RCTs (116 participants) that were eligible for inclusion. These trials included conditions of acquired nystagmus, sixth cranial nerve palsy and traumatic brain injury-induced ocular motility defects. We did not identify any relevant studies of restitutive interventions.We identified one UK-based trial of a substitutive intervention, in which botulinum toxin was compared with observation in 47 people with acute sixth nerve palsy. At four months after entry into the trial, people given botulinum toxin were more likely to make a full recovery (reduction in angle of deviation within 10 prism dioptres), compared with observation (risk ratio 1.19, 95% CI 0.96 to 1.48; low-certainty evidence). These same participants also achieved binocular single vision. In the injection group only, there were 2 cases of transient ptosis out of 22 participants (9%), and 4 participants out of 22 (18%) with transient vertical deviation; a total complication rate of 24% per injection and 27% per participant. All adverse events recovered. We judged the certainty of evidence as low, downgrading for risk of bias and imprecision. It was not possible to mask investigators or participants to allocation, and the follow-up between groups varied.We identified one USA-based cross-over trial of a compensatory intervention. Oculomotor rehabilitation was compared with sham training in 12 people with mild traumatic brain injury, at least one year after the injury. We judged the evidence from this study to be very low-certainty. The study was small, data for the sham training group were not fully reported, and it was unclear if a cross-over study design was appropriate as this is an intervention with potential to have a permanent effect.We identified three cross-over studies of pharmacological interventions for acquired nystagmus, which took place in Germany and the USA. These studies investigated two classes of pharmacological interventions: GABAergic drugs (gabapentin, baclofen) and aminopyridines (4-aminopyridines (AP), 3,4-diaminopyridine (DAP)). We judged the evidence from all three studies as very low-certainty because of small numbers of participants (which led to imprecision) and risk of bias (they were cross-over studies which did not report data in a way that permitted estimation of effect size).One study compared gabapentin (up to 900 mg/day) with baclofen (up to 30 mg/day) in 21 people with pendular and jerk nystagmus. The follow-up period was two weeks. This study provides very low-certainty evidence that gabapentin may work better than baclofen in improving ocular motility and reducing participant-reported symptoms (oscillopsia). These effects may be different in pendular and jerk nystagmus, but without formal subgroup analysis it is unclear if the difference between the two types of nystagmus was chance finding. Quality of life was not reported. Ten participants with pendular nystagmus chose to continue treatment with gabapentin, and one with baclofen. Two participants with jerk nystagmus chose to continue treatment with gabapentin, and one with baclofen. Drug intolerance was reported in one person receiving gabapentin and in four participants receiving baclofen. Increased ataxia was reported in three participants receiving gabapentin and two participants receiving baclofen.One study compared a single dose of 3,4-DAP (20 mg) with placebo in 17 people with downbeat nystagmus. Assessments were made 30 minutes after taking the drug. This study provides very low-certainty evidence that 3,4-DAP may reduce the mean peak slow-phase velocity, with less oscillopsia, in people with downbeat nystagmus. Three participants reported transient side effects of minor perioral/distal paraesthesia.One study compared a single dose of 4-AP with a single dose of 3,4-DAP (both 10 mg doses) in eight people with downbeat nystagmus. Assessments were made 45 and 90 minutes after drug administration. This study provides very low-certainty evidence that both 3,4-DAP and 4-AP may reduce the mean slow-phase velocity in people with downbeat nystagmus. This effect may be stronger with 4-AP., Authors' Conclusions: The included studies provide insufficient evidence to inform decisions about treatments specifically for eye movement disorders that occur following acquired brain injury. No information was obtained on the cost of treatment or measures of participant satisfaction relating to treatment options and effectiveness. It was possible to describe the outcome of treatment in each trial and ascertain the occurrence of adverse events.

Background: Pharmaceutical compounding preparations, produced by (hospital) pharmacies, usually do not have marketing authorization. As a consequence, some of these pharmaceutical compounding preparations can be picked-up by a pharmaceutical company to obtain marketing authorization, often leading to price increases. An example is the 3,4-diaminopyridine slow release (3,4-DAP SR) tablets for Lambert-Eaton Myasthenic Syndrome (LEMS). In 2009 marketing authorization was given for the commercial immediate release phosphate salt of the drug, including a fifty-fold price increase compared to the pharmaceutical compounding preparation. Obtaining marketing authorization for 3,4-DAP SR by academia might have been a solution to prevent this price increase. To determine whether the available data of a pharmaceutical compounding preparation with long-term experience in regular care are adequate to obtain marketing authorization, 3,4-DAP SR is used as a case study., Methods: A retrospective qualitative case-study was performed. Initially, document analysis was executed by collecting the required data for marketing authorization in general and whether data of Firdapse® and 3,4-DAP SR met these requirements. Secondly, the (non-) available data of the two formulations were compared with each other to determine the differences in availability., Results: At the time of approval, almost all data were available for both Firdapse® and 3,4-DAP SR. Conversely, much of the data used for the approval of Firdapse® originated from the 3,4-DAP immediate release (3,4-DAP IR) formulation. Only two bioequivalence studies and one pharmacology safety study was performed with Firdapse® before marketing authorization application., Conclusions: In conclusion, at time Firdapse® obtained approval, the data available did not differ substantially from 3,4-DAP SR, indicating that approval with 3,4-DAP SR would have been possible. We make a plea for approval of orphan medicinal products developed and manufactured by academic institutions as to keep utilization of these products affordable., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In this independent clinical study, we analyzed retrospectively the clinical features of 9 cases (6 male and 3 female) of Lambert-Eaton myasthenic syndrome that were administered 3,4-diaminopyridine (3,4-DAP). Four cases showed no cancer and 5 cases had small cell lung carcinoma. Seven cases were positive for anti voltage-gated calcium channel antibodies. Activities of daily living (ADL) were improved by 3,4-DAP in 8 cases that showed mainly weakness of the extremities, but did not improve ADL in 1 case with cerebellar ataxia of paraneoplastic cerebellar degeneration (PCD). Seven cases showed autonomic symptoms, and 6 cases were improved with 3,4-DAP. The maintenance dose varied widely among individuals, with a single dose ranging from 10 to 40 mg. Each patient was prescribed a maintenance dose 3 to 7 times a day. The daily dosage ranged from 36 to 100 mg. Two cases showed adverse effects to the treatment. Of those 2 cases, 1 case treated at 45 mg/day discontinued treatment, but another case treated at 100 mg/day reduced the dosage and continued treatment. The administration period was 1 to 149 months. Three cases have continued 3,4-DAP for more than 10 years. Four cases have discontinued 3,4-DAP, with 2 cases discontinuing due to death, 1 case discontinuing due to progression of cancer, and 1 case discontinuing due to an adverse reaction. Our results suggest that 3,4-DAP treatment is effective for weakness and autonomic symptoms, but may be ineffective for ataxia of PCD. Treatment with 3,4-DAP can be tolerated for a long period, but the optimal dosage varies widely among individuals.

Botulinum neurotoxins (BoNTs) are exceedingly potent neurological poisons that prevent neurotransmitter release from peripheral nerve terminals by cleaving presynaptic proteins required for synaptic vesicle fusion. The ensuing neuromuscular paralysis causes death by asphyxiation. Although no antidotal treatments exist to block toxin activity within the nerve terminal, aminopyridine antagonists of voltage-gated potassium channels have been proposed as symptomatic treatments for botulism toxemia. However, clinical evaluation of aminopyridines as symptomatic treatments for botulism has been inconclusive, in part because mechanisms responsible for reversal of paralysis in BoNT-poisoned nerve terminals are not understood. Here we measured the effects of 3,4-diaminopyridine (DAP) on phrenic nerve-elicited diaphragm contraction and end-plate potentials at various times after intoxication with BoNT serotypes A, B, or E. We found that DAP-mediated increases in quantal content promote neurotransmission from intoxicated nerve terminals through two functionally distinguishable mechanisms. First, DAP increases the probability of neurotransmission at non-intoxicated release sites. This mechanism is serotype-independent, becomes less effective as nerve terminals become progressively impaired, and remains susceptible to ongoing intoxication. Second, DAP elicits persistent production of toxin-resistant endplate potentials from nerve terminals fully intoxicated by BoNT/A, but not serotypes B or E. Since this effect appears specific to BoNT/A intoxication, we propose that DAP treatment enables BoNT/A-cleaved SNAP-25 to productively engage in fusogenic release by increasing the opportunity for low-efficiency fusion events. These findings have important implications for DAP as a botulism therapeutic by defining conditions under which DAP may be clinically effective in reversing botulism symptoms., (Published by Elsevier Inc.)

Ecto-nucleotidase members i.e., ecto-5'-nucleotidase and alkaline phosphatase, hydrolyze extracellular nucleotides and play an important role in purinergic signaling. Their overexpression are implicated in a variety of pathological states, including immunological diseases, bone mineralization, vascular calcification and cancer, and thus they represent an emerging drug targets. In order to design potent and selective inhibitors, new derivatives of 4-aminopyridine have been synthesized (10a-10m) and their structures were established on the basis of spectral data. The effect of nature and position of substituent was interestingly observed and justified on the basis of their detailed structure activity relationships (SARs) against both families of ecto-nucleotidase. Compound 10a displayed significant inhibition (IC 50  ± SEM = 0.25 ± 0.05 µM) that was found ≈168 fold more potent as compared to previously reported inhibitor suramin (IC 50  ± SEM = 42.1 ± 7.8 µM). This compound exhibited 6 times more selectivity towards h-TNAP over h-e5'NT. The anticancer potential and mechanism were also established using cell viability assay, flow cytometric analysis and nuclear staining. Molecular docking studies were also carried out to gain insight into the binding interaction of potent compounds within the respective enzyme pockets and herring-sperm DNA., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Catalyst-mediated protein modification is a powerful approach for the imaging and engineering of natural proteins. We have previously developed affinity-guided 4-dimethylaminopyridine (AGD) chemistry as an efficient protein modification method using a catalytic acyl transfer reaction. However, because of the high electrophilicity of the thioester acyl donor molecule, AGD chemistry suffers from nonspecific reactions to proteins other than the target protein in crude biological environments, such as cell lysates, live cells, and tissue samples. To overcome this shortcoming, we here report a new acyl donor/organocatalyst system that allows more specific and efficient protein modification. In this method, a highly nucleophilic pyridinium oxime (PyOx) catalyst is conjugated to a ligand specific to the target protein. The ligand-tethered PyOx selectively binds to the target protein and facilitates the acyl transfer reaction of a mild electrophilic N-acyl-N-alkylsulfonamide acyl donor on the protein surface. We demonstrated that the new catalytic system, called AGOX (affinity-guided oxime) chemistry, can modify target proteins, both in test tubes and cell lysates, more selectively and efficiently than AGD chemistry. Low-background fluorescence labeling of the endogenous cell-membrane proteins, carbonic anhydrase XII and the folate receptor, in live cells allowed for the precise quantification of diffusion coefficients in the protein's native environment. Furthermore, the excellent biocompatibility and bioorthogonality of AGOX chemistry were demonstrated by the selective labeling of an endogenous neurotransmitter receptor in mouse brain slices, which are highly complicated tissue samples.

Rationale: To report our experience on 7 patients (4 males and 3 females), affected by nonparaneoplastic Lambert-Eaton myasthenic syndrome, treated with 3,4-diaminopyridine phosphate (3,4-DAPP) either alone or in combination with other immunosuppressants or steroids., Patient Concerns: Patients have been evaluated at specific timepoints (ie, baseline and last 5 year follow-up), with neurological examination, autoantibodies against presynaptic voltage-gated Cav2.1 (P/Q type) calcium ion channel (VGCC) dosage, neurophysiological evaluation focusing on the increased amplitude of the compound muscle action potential (cMAP) after maximum voluntary effort, quantitative myasthenia gravis (QMG) and activities of daily living scales, and autonomic nervous system involvement evaluation., Outcomes: Five out of 7 patients presented a clinical improvement persisting at last 5-year follow-up; 2 out of them improved taking only 3,4-DAPP at the maximal dosage, whereas the remaining received concomitant medications, such as prednisone and azathioprine. However, the clinical amelioration was not statistically significant. No one of the patients reported severe adverse events, except one, complaining of transient chin and perioral paresthesias. A significant association between QMG and the type of pharmacological drugs therapy (P = .028) emerged. Indeed, we observed an improvement of the clinical condition in all 3 subjects treated with 3,4-DAPP and prednisone., Conclusions: In this study, we confirm 3,4-DAPP treatment efficacy on muscle strength, but minor evidence of drug effectiveness have been demonstrated on the autonomic nervous system involvement and on the deep tendon reflexes reappearance, a part from patients who received 3,4-DAPP associated to prednisone.

Lambert-Eaton myasthenia (LEM) is a rare autoimmune disorder associated with debilitating muscle weakness. There are limited treatment options and 3,4-diaminopyridine (3,4-DAP) free base is an investigational orphan drug used to treat LEM-related weakness. We performed a population pharmacokinetic/pharmacodynamic (PK/PD) analysis using 3,4-DAP and metabolite concentrations collected from a phase II study in patients with LEM. The Triple Timed Up & Go (3TUG) assessment, which measures lower extremity weakness, was the primary outcome measure. A total of 1,270 PK samples (49 patients) and 1,091 3TUG data points (32 randomized patients) were included in the PK/PD analysis. A two-compartment and one-compartment model for parent and metabolite, respectively, described the PK data well. Body weight and serum creatinine partially explained the variability in clearance for the final PK model. A fractional inhibitory maximum effect (E max ) model characterized the exposure-response relationship well. The PK/PD model was applied to identify a suggested dosing approach for 3,4-DAP free base., (© 2017 The Authors CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.)

The application of six novel α,β-dipeptides as chiral organocatalysts in the asymmetric Michael addition reaction between enolizable aldehydes and N -arylmaleimides or nitroolefins is described. With N -arylmaleimides as substrates, the best results were achieved with dipeptide 2 as a catalyst in the presence of aq. NaOH. Whereas dipeptides 4 and 6 in conjunction with 4-dimethylaminopyridine (DMAP) and thiourea as a hydrogen bond donor proved to be highly efficient organocatalytic systems in the enantioselective reaction between isobutyraldehyde and various nitroolefins., Competing Interests: The authors declare no conflict of interest and the founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Purpose: The purpose of this study is to evaluate safety, tolerability, and pharmacokinetic (PK) properties of amifampridine phosphate (Firdapse™) and its major inactive 3-N-acetyl metabolite in renally impaired and healthy individuals with slow acetylator (SA) and rapid acetylator (RA) phenotypes., Methods: This was a Phase I, multicenter, open-label study of the PK properties and safety profile of amifampridine phosphate in individuals with normal, mild, moderate, or severely impaired renal function. Amifampridine phosphate was given as a single 10 mg (base equivalent) dose, and the plasma and urine PK properties of amifampridine and its 3-N-acetyl metabolite were determined. The safety profile was evaluated by monitoring adverse events (AEs), clinical laboratory tests, and physical examinations., Findings: Amifampridine clearance was predominantly metabolic through N-acetylation, regardless of renal function in both acetylator phenotypes. In individuals with normal renal function, mean renal clearance represented approximately 3% and 18% of the total clearance of amifampridine in RA and SA, respectively. Large differences in amifampridine exposure were observed between acetylation phenotypes across renal function levels. Mean amifampridine exposure values of AUC 0-∞ and C max were up to 8.8-fold higher in the SA group compared with the RA group across renal function levels. By comparison, mean AUC 0-∞ was less affected by renal function within an acetylator group, only 2- to 3-fold higher in individuals with severe renal impairment (RI) compared with those with normal renal function. Exposure to amifampridine in the SA group with normal renal function was higher (AUC 0-∞, approximately 1.8-fold; C max, approximately 4.1-fold) than the RA group with severe RI. Exposure to the inactive 3-N-acetyl metabolite was higher than amifampridine in both acetylator groups, independent of renal function level. The metabolite is cleared by renal excretion, and exposure was clearly dependent on renal function with 4.0- to 6.8-fold increases in AUC 0-∞ from normal to severe RI. No new tolerability findings were observed., Implications: A single dose of 10 mg of amifampridine phosphate was well tolerated, independent of renal function and acetylator status. The results indicate that the PK profile of amifampridine is affected by metabolic acetylator phenotype to a greater extent than by renal function level, supporting Firdapse™ administration in individuals with RI in line with current labeling recommendations. Amifampridine should be dosed to effect per the individual patient need, altering administration frequency and dose in normal through severe RI. The therapeutic dose of amifampridine phosphate should be tailored to the individual patient needs by gradual dose titration up to the present maximum recommended dose (60-80 mg/day) or until dose-limiting AEs intervene to avoid overdosing and underdosing. EudraCT identifier: 2013-005349-35., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Chromium(III) chlorido amine-bis(phenolate) complexes paired with nucleophilic co-catalysts are a promising family of catalysts for the copolymerization of CO 2 and epoxides to selectively produce polycarbonates with a very high degree of carbonate linkages. Single-component catalyst systems can be prepared, where the neutral nucleophile, 4-dimethylaminopyridine (DMAP), is coordinated to the metal site to provide a stable octahedral Cr III complex. These complexes possess the potential for both anionic (from the chlorido ligand) or neutral (DMAP) nucleophilic epoxide ring-opening during the proposed rate-determining initiation step. Concentration effect studies support a first-order dependence of the polymerization rate on the concentration of single-component catalyst. End-group analysis of polycarbonates by MALDI-TOF MS indicate the presence of predominantly DMAP-initiated chains as well as the occurrence of chain-transfer events resulting in ether linkages, likely from the presence of cyclohexene diol formed by the reaction of cyclohexene oxide and adventitious water., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: We investigated the effects of 3,4-diaminopyridine (3,4-DAP) and its acetylated metabolite, N-(4-amino-pyridin-3-yl) acetamide (3-Ac), at the mammalian neuromuscular junction., Methods: Quantal release of acetylcholine was studied in diaphragm muscles of mice, using in vitro intracellular microelectrode recordings., Results: Under conditions of low probability of release, 3,4-DAP produced a 1,000% increase in quantal release, but 3-Ac had no effect. Under conditions of normal probability of release, the effect of 3,4-DAP was modest and limited by concurrent depletion of synaptic vesicles, especially with high concentrations of 3,4-DAP and high frequencies of nerve stimulation., Conclusions: These findings predict 3,4-DAP is most effective in conditions with low probability of quantal release, such as Lambert-Eaton myasthenic syndrome. A beneficial effect is also expected in disorders of neuromuscular transmission in which the effect of 3,4-DAP on quantal release is not limited by depletion of synaptic vesicles, such as postsynaptic congenital myasthenic syndromes. Muscle Nerve, 2016 Muscle Nerve 55: 223-231, 2017., (© 2016 Wiley Periodicals, Inc.)

Pogostone, isolated from Pogostemon cablin, has many biological activities such as potential antibacterial, anticandida, and antifungal. Traditional extraction leads to low output of PO about 17.6 mg/kg from Herba Pogostemonis. The previous literature had reported a synthetic study and the yield had reached 4.48% with strictly controlled reaction conditions. The two methods above cannot meet the large demand of PO; we report a new synthesis method. 4-hydroxy-6-methyl-2-pyrone (1) was added in toluene, with the existence of acylation catalyst 4-dimethylaminopyridine (DMAP), 4-methylvaleric acid (2), and condensing agent dicyclohexylcarbodiimide (DCC), PO was synthesized after the combination of 3-carbon of (1) with 1-OH of (2) in the acylation reaction. The purity had reached 98%, determined by HPLC. The structure was confirmed by spectroscopic methods including infrared, electron ionization mass spectrometry, and nuclear magnetic resonance spectroscopy. PO was totally synthesized in one step including cyclization, with total yield of 27.2%.

This paper reports on improved conventional thermal silylation (CTS) and microwave-assisted silylation (MAS) methods for simultaneous determination of tocopherols and sterols by gas chromatography. Reaction parameters in each of the methods developed were systematically optimized using a full factorial design followed by a central composite design. Initially, experimental conditions for CTS were optimized using a block heater. Further, a rapid MAS was developed and optimized. To understand microwave heating mechanisms, MAS was optimized by two distinct modes of microwave heating: temperature-controlled MAS and power-controlled MAS, using dedicated instruments where reaction temperature and microwave power level were controlled and monitored online. Developed methods: were compared with routine overnight derivatization. On a comprehensive level, while both CTS and MAS were found to be efficient derivatization techniques, MAS significantly reduced the reaction time. The optimal derivatization temperature and time for CTS found to be 55°C and 54min, while it was 87°C and 1.2min for temperature-controlled MAS. Further, a microwave power of 300W and a derivatization time 0.5min found to be optimal for power-controlled MAS. The use of an appropriate derivatization solvent, such as pyridine, was found to be critical for the successful determination. Catalysts, like potassium acetate and 4-dimethylaminopyridine, enhanced the efficiency slightly. The developed methods showed excellent analytical performance in terms of linearity, accuracy and precision., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The coding of sound level by ensembles of neurons improves the accuracy with which listeners identify how loud a sound is. In the auditory system, the rate at which neurons fire in response to changes in sound level is shaped by local networks. Voltage-gated conductances alter local output by regulating neuronal firing, but their role in modulating responses to sound level is unclear. We tested the effects of L-type calcium channels (Ca L : Ca V 1.1-1.4) on sound-level coding in the central nucleus of the inferior colliculus (ICC) in the auditory midbrain. We characterized the contribution of Ca L to the total calcium current in brain slices and then examined its effects on rate-level functions (RLFs) in vivo using single-unit recordings in awake mice. Ca L is a high-threshold current and comprises ∼50% of the total calcium current in ICC neurons. In vivo, Ca L activates at sound levels that evoke high firing rates. In RLFs that increase monotonically with sound level, Ca L boosts spike rates at high sound levels and increases the maximum firing rate achieved. In different populations of RLFs that change nonmonotonically with sound level, Ca L either suppresses or enhances firing at sound levels that evoke maximum firing. Ca L multiplies the gain of monotonic RLFs with dynamic range and divides the gain of nonmonotonic RLFs with the width of the RLF. These results suggest that a single broad class of calcium channels activates enhancing and suppressing local circuits to regulate the sensitivity of neuronal populations to sound level., (Copyright © 2016 the American Physiological Society.)

Lambert-Eaton myasthenic syndrome (LEMS) is an uncommon autoimmune idiopathic or paraneoplastic syndrome producing antibodies against voltage presynaptic calcium channels. According to previous studies, many patients with LEMS experience remission in both the clinical symptoms of muscle weakness and the electrophysiologic abnormalities after successful treatment of lung SCC. However, some patients might not respond to conventional therapy and eventually require palliative care. Hereby, we reported a LEMS patient with advanced lung malignancy was referred for palliative care. He was benefited from multidisciplinary approach even with limited survival. In this case, use of 3,4-diaminopyridine (3,4-DAP) had other roles apart from symptomatic treatment.

Organocatalytic site-selective diversification of 10-deacetylbaccatin III, a key natural product for the semisynthesis of taxol, has been achieved. Various acyl groups were selectively introduced into the C(10)-OH of 10-deacetylbaccatin III. The C(10)-OH selective acylation was also applied to acylative site-selective dimerization of 10-deacetylbaccatin III to provide the structurally defined dimer.

The organocatalytic site-selective monoacylation of avermectin B2a, an insecticidal and anti-parasitic drug, was accomplished. Although an acetylation of avermectin B2a using a 4-dimethylaminopyridine (DMAP) as a catalyst gave poor site-selectivity, use of our organocatalyst increased site-selectivity of the acylation at the C-5-OH as well as the yield of monoacetate. This catalyst was also effective in other acylations. Interestingly, trihaloacetylation under same conditions gave poor site-selectivity. However, the use of an enantiomer of our organocatalyst provided the C-4″-O-trihaloacetyl avermectin B2a with excellent site-selectivity. These results indicate that the site-selective acylation of avermectin B2a can be controlled by the combination of a suitable organocatalyst and an acid anhydride.

Multiparameter optimization of a series of 5-((4-aminopyridin-2-yl)amino)pyrazine-2-carbonitriles resulted in the identification of a potent and selective oral CHK1 preclinical development candidate with in vivo efficacy as a potentiator of deoxyribonucleic acid (DNA) damaging chemotherapy and as a single agent. Cellular mechanism of action assays were used to give an integrated assessment of compound selectivity during optimization resulting in a highly CHK1 selective adenosine triphosphate (ATP) competitive inhibitor. A single substituent vector directed away from the CHK1 kinase active site was unexpectedly found to drive the selective cellular efficacy of the compounds. Both CHK1 potency and off-target human ether-a-go-go-related gene (hERG) ion channel inhibition were dependent on lipophilicity and basicity in this series. Optimization of CHK1 cellular potency and in vivo pharmacokinetic-pharmacodynamic (PK-PD) properties gave a compound with low predicted doses and exposures in humans which mitigated the residual weak in vitro hERG inhibition.

Boron-doped nanocrystalline diamond (BNCD) films exhibit outstanding electrochemical properties that make them very attractive for the fabrication of electrodes for novel neural interfaces and prosthetics. In these devices, the physicochemical properties of the electrode materials are critical to ensure an efficient long-term performance. The aim of this study was to investigate the relative contribution of topography and doping to the biological performance of BNCD films. For this purpose, undoped and boron-doped NCD films were deposited on low roughness (LR) and high roughness (HR) substrates, which were studied in vitro by means of protein adsorption and fibroblast growth assays. Our results show that BNCD films significantly reduce the adsorption of serum proteins, mostly on the LR substrates. As compared to fibroblasts cultured on LR BNCD films, cells grown on the HR BNCD films showed significantly reduced adhesion and lower growth rates. The mean length of fibronectin fibrils deposited by the cells was significantly increased in the BNCD coated substrates, mainly in the LR surfaces. Overall, the largest influence on protein adsorption, cell adhesion, proliferation, and fibronectin deposition was due to the underlying sub-micron topography, with little or no influence of boron doping. In perspective, BNCD films displaying surface roughness in the submicron range may be used as a strategy to reduce the fibroblast growth on the surface of neural electrodes.

Altholactone exhibited the anti-fungal activity with a high MIC value of 128 μg ml(-1) against Cryptococcus neoformans and Saccharomyces cerevisiae. Fifteen ester derivatives of altholactone 1-15 were modified by esterification and their structures were confirmed by spectroscopic methods. Most of the ester derivatives exhibited stronger anti-fungal activities than that of the precursor altholactone. 3-Bromo- and 2,4-dichlorobenzoates (7 and 15) exhibited the lowest minimal inhibitory concentration (MIC) values against C. neoformans at 16 μg ml(-1), while the 4-bromo-, 4-iodo-, and 1-bromo-3-chlorobenzoates (11-13) displayed potent activity against S. cerevisiae with MIC values of 1 μg ml(-1). In conclusion, this analysis indicates that the anti-fungal activity of altholactone is enhanced by addition of halogenated benzoyl group to the 3-OH group.

Objective: We evaluated the efficacy and safety of amifampridine phosphate (Firdapse(®)) for symptomatic treatment in Lambert-Eaton myasthenic syndrome (LEMS)., Methods: Phase 3, randomized, double-blind, study. Patients were treated initially with amifampridine phosphate for 7-91 days, followed by randomization to continue amifampridine phosphate for 14 days or placebo (7-day taper, 7-day placebo). The primary efficacy endpoints were changes from baseline at day 14 in Quantitative Myasthenia Gravis and Subject Global Impression scores., Results: The coprimary efficacy end points and 1 of the secondary efficacy end points were met, showing a significant benefit of aminfampridine phosphate over placebo at Day 14. All 5 primary, secondary, and tertiary endpoints achieved statistical significance at Day 8. Amifampridine phosphate was well tolerated; the most common adverse events were oral and digital paresthesias, nausea, and headache., Conclusions: This study provides Class I evidence of efficacy of amifampridine phosphate as a symptomatic treatment for LEMS., (© 2016 Wiley Periodicals, Inc.)

3,4-Diaminopyridine has shown promise in reversing botulinum intoxication, but poor pharmacokinetics and a narrow therapeutic window limit its clinical utility. Thus, we developed a pH-dependent oral delivery platform using club moss spore exines. These exine microcapsules slowed 3,4-diaminopyridine absorption, limited its seizure activity, and enabled delivery of doses which prolonged mouse survival after botulism neurotoxin A intoxication.

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase belonging to the insulin receptor superfamily. Expression of ALK in normal human tissues is only found in a subset of neural cells, however it is involved in the genesis of several cancers through genetic aberrations involving translocation of the kinase domain with multiple fusion partners (e.g., NPM-ALK in anaplastic large cell lymphoma ALCL or EML4-ALK in non-small cell lung cancer) or activating mutations in the full-length receptor resulting in ligand-independent constitutive activation (e.g., neuroblastoma). Here we are reporting the discovery of novel and selective anaplastic lymphoma kinase inhibitors from specific modifications of the 2,4-diaminopyridine core present in TAE684 and LDK378. Synthesis, structure activity relationships (SAR), absorption, distribution, metabolism, and excretion (ADME) profile, and in vivo efficacy in a mouse xenograft model of anaplastic large cell lymphoma are described., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Rapsyn (RAPSN) mutations are a common cause of postsynaptic congenital myasthenic syndromes. We present a comprehensive description of the clinical and molecular findings of ten patients with CMS due to mutations in RAPSN, mostly with a long-term follow-up. Two patients were homozygous and eight were heterozygous for the common p.Asn88Lys mutation. In three of the heterozygous patients we have identified three novel mutations (c.869T > C; p.Leu290Pro, c.1185delG; p.Thr396Profs*12, and c.358delC; p.Gln120Serfs*8). In our cohort, the RAPSN mutations lead to a relatively homogeneous phenotype, characterized by fluctuating ptosis, occasional bulbar symptoms, neck muscle weakness, and mild proximal muscle weakness with exacerbations precipitated by minor infections. Interestingly, episodic exacerbations continue to occur during adulthood. These were characterized by proximal limb girdle weakness and ptosis, and not so much by respiratory insufficiency after age 6. All patients presented during neonatal period and responded to cholinergic agonists. In most of the affected patients, additional use of 3,4-diaminopyridine resulted in significant clinical benefit. The disease course is stable except for intermittent worsening., (Copyright © 2015 Elsevier B.V. All rights reserved.)

CCT245737 is the first orally active, clinical development candidate CHK1 inhibitor to be described. The IC50 was 1.4 nM against CHK1 enzyme and it exhibited>1,000-fold selectivity against CHK2 and CDK1. CCT245737 potently inhibited cellular CHK1 activity (IC50 30-220 nM) and enhanced gemcitabine and SN38 cytotoxicity in multiple human tumor cell lines and human tumor xenograft models. Mouse oral bioavailability was complete (100%) with extensive tumor exposure. Genotoxic-induced CHK1 activity (pS296 CHK1) and cell cycle arrest (pY15 CDK1) were inhibited both in vitro and in human tumor xenografts by CCT245737, causing increased DNA damage and apoptosis. Uniquely, we show CCT245737 enhanced gemcitabine antitumor activity to a greater degree than for higher doses of either agent alone, without increasing toxicity, indicating a true therapeutic advantage for this combination. Furthermore, development of a novel ELISA assay for pS296 CHK1 autophosphorylation, allowed the quantitative measurement of target inhibition in a RAS mutant human tumor xenograft of NSCLC at efficacious doses of CCT245737. Finally, CCT245737 also showed significant single-agent activity against a MYC-driven mouse model of B-cell lymphoma. In conclusion, CCT245737 is a new CHK1 inhibitor clinical development candidate scheduled for a first in man Phase I clinical trial, that will use the novel pS296 CHK1 ELISA to monitor target inhibition.

A peptide sensor that integrates the 4-dimethylaminophthalimide (4-DMAP) fluorophore in a short cyclin A binding sequence displays a large fluorescence emission increase upon interacting with the cyclin A Binding Groove (CBG). Competitive displacement assays of this probe allow the straightforward identification of peptides that interact with the CBG, which could potentially block the recognition of CDK/cyclin A kinase substrates.

An improved Helferich method is presented. It involves the glycosylation of 4-methyl-umbelliferone with glycosyl acetates in the presence of boron trifluoride etherate combined with triethylamine, pyridine, or 4-dimethylaminopyridine under mild conditions, followed by deprotection to give fluorogenic 4-methylumbelliferyl glycoside substrates. Due to the use of base, the glycosylation reaction proceeds more easily, is uncommonly α- or β-stereoselective, and affords the corresponding products in moderate to excellent yields (51%-94%) under appropriate conditions.

Objectives: To describe the clinical and electrophysiologic features of synaptotagmin II (SYT2) mutations, a novel neuromuscular syndrome characterized by foot deformities and fatigable ocular and lower limb weakness, and the response to modulators of acetylcholine release., Methods: We performed detailed clinical and neurophysiologic assessment in 2 multigenerational families with dominant SYT2 mutations (c.920T>G [p.Asp307Ala] and c.923G>A [p.Pro308Leu]). Serial clinical and electrophysiologic assessments were performed in members of one family treated first with pyridostigmine and then with 3,4-diaminopyridine., Results: Electrophysiologic testing revealed features indicative of a presynaptic deficit in neurotransmitter release with posttetanic potentiation lasting up to 60 minutes. Treatment with 3,4-diaminopyridine produced both a clinical benefit and an improvement in neuromuscular transmission., Conclusion: SYT2 mutations cause a novel and potentially treatable complex presynaptic congenital myasthenic syndrome characterized by motor neuropathy causing lower limb wasting and foot deformities, with reflex potentiation following exercise and a uniquely prolonged period of posttetanic potentiation., (© 2015 American Academy of Neurology.)

Objective: 3,4-diaminopyridine (3,4-DAP) is commonly used for treating neuromuscular diseases, such as the Lambert-Eaton myasthenic syndrome, but the pharmacokinetics of 3,4-DAP base have not been investigated. We therefore studied 3,4-DAP base pharmacokinetics in healthy Japanese volunteers., Materials and Methods: In this crossover study, we administered a single oral dose of 10 or 20 mg 3,4-DAP base to healthy Japanese volunteers (n = 5) after food intake, or 10 mg 3,4-DAP to fasting individuals. We measured serum 3,4-DAP concentrations, performed electrocardiography (ECG), and administered questionnaires., Results: After administration of 10 or 20 mg 3,4-DAP following food intake, the maximum serum concentrations (Cmax) were 8.09 ± 4.47 ng/mL and 35.8 ± 15.7 ng/mL, respectively (mean ± standard deviation; SD), and the areas under the serum concentration-time curve (extrapolated to infinity) were 639 ± 213 ng x min/mL and 2,097 ± 936 ng x min/mL (mean ± SD), respectively. Administration to fasted individuals indicated that food intake did not significantly alter 3,4-DAP pharmacokinetics. ECG showed no clinically significant changes, but PR intervals were prolonged in all cases. Two out of 5 subjects showed perioral paresthesia symptoms after administration of 20 mg 3,4-DAP., Conclusion: This study indicated that 3,4-DAP base pharmacokinetics were non-linear. Although no clinically significant changes in ECG were observed, it is advisable to perform ECG periodically during 3,4-DAP administration in order to monitor cardiac function. Moreover, the development of perioral paresthesia may be dependent on the dose of 3,4-DAP used.

Magnetic gold nanoclusters (MGNCs) functionalized with 4-dimethylaminopyridine (DMAP) enables the trace detection of tetrabromobisphenol A (TBBPA), an environmental pollutant, using surface-enhanced Raman scattering (SERS) spectroscopy. The synthesis, cleansing, and functionalization of MGNCs are conducted in aqueous solutions; SERS samples are prepared by magnetic precipitation in the presence of trace analyte. The limit of detection (LOD) for TBBPA is greatly increased by the use of DMAP as a reporter molecule: DMAP-modified MGNCs can detect TBBPA at 10 pM in water, whereas the LOD for TBBPA by unfunctionalized Au is 1 nM. The reproducibility of picomolar TBBPA detection with DMAP-modified MGNCs is confirmed by two-dimensional correlation analysis. The high SERS sensitivity for TBBPA can be attributed to its capacity to modulate the Raman spectrum of adsorbed DMAP. This indirect mode of detection can also be applied toward the detection of other hydrophobic analytes, each identifiable by its characteristic SERS identity.

Purpose: Amifampridine (3,4-diaminopyridine) has been approved in the European Union for the treatment of Lambert-Eaton myasthenic syndrome. Amifampridine has a narrow therapeutic index, and supratherapeutic exposure has been associated with dose-dependent adverse events, including an increased risk for seizure. This study assessed the effect of food on the relative bioavailability of amifampridine in healthy subjects and informed on conditions that can alter exposure., Methods: This randomized, open-labeled, 2-treatment, 2-period crossover study enrolled 47 healthy male and female subjects. Subjects were randomly assigned to receive 2 single oral doses of amifampridine phosphate salt (20 mg base equivalents per dose) under fed or fasted conditions separated by a washout period. Blood and urine samples for pharmacokinetic analyses were taken before and after dosing. Plasma concentrations of amifampridine and an inactive 3-N-acetyl metabolite were determined. The relative bioavailability values of amifampridine and metabolite were assessed based on the plasma PK parameters AUC0-∞, AUC0-t, and Cmax in the fed and fasted states using noncompartmental pharmacokinetic analysis. Parent drug and metabolite excretion were calculated from urinary concentrations. A food effect on bioavailability would be established if the 90% CI of the ratio of population geometric mean value of AUC0-∞, AUC0-t, or Cmax between fed and fasted administration was not within the bioequivalence range of 80% to 125%. Tolerability was assessed based on adverse-event reporting, clinical laboratory assessments, physical examination including vital sign measurements, 12-lead ECG, and concurrent medication use., Findings: Food slowed and somewhat decreased the absorption of amifampridine. There was a decrease in exposure (Cmax, 44%; AUC, 20%) after oral administration of amifampridine phosphate salt in the presence of food, and mean Tmax was 2-fold longer in the fed state. The extent of exposure and plasma elimination half-life of the major metabolite was greater than those of amifampridine in the fed and fasted conditions. Mean AUCs in the fed and fasted states were slightly greater in women than men, with no difference in mean Cmax. Orally administered amifampridine was renally eliminated (>93%) as the parent compound and metabolite within 24 hours. Single oral doses of 20 mg of amifampridine phosphate salt were considered well tolerated in both the fed and fasted conditions. High intersubject variability (%CVs, >30%) in amifampridine pharmacokinetic parameter values was observed., Implications: At the intended dose under fasting conditions, amifampridine exposure may be increased. European Union Drug Regulating Authorities Clinical Trials identifier: 2011-000596-13., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)