Synthesis and evaluation of dopaminergic prodrugs designed for transdermal iontophoretic drug delivery:highly water-soluble amino acid ester prodrugs applicable for the treatment of Parkinson's disease

Dopamine agonisten worden toegepast als farmacotherapie voor patienten met de ziekte van Parkinson. In dit onderzoek beschrijven we het chemisch bereiden en de evaluatie van zeer wateroplosbare varianten van de dopamine agonist 5-OH-DPAT. Deze varianten zijn chemisch aan elkaar gekoppelde stoffen van 5-OH-DPAT met andere moleculen. In dit geval zijn dat aminozuren. In de farmacie noemt men deze stoffen prodrugs. Een prodrug is een niet werkzame stof die na omzetting in het lichaam het actieve geneesmiddel vrijgeeft. Ons onderzoek heeft geleid tot een aantal bevindingen. Ten eerste blijkt het mogelijk te zijn zeer wateroplosbare prodrugs te maken die voldoende stabiel zijn in de iontoforese pleister (voldoende houdbaarheid), maar eenmaal in de bloedbaan het geneesmiddel direct vrijgeven. Ten tweede, door het koppelen van 5-OH-DPAT aan dipeptiden (twee gecombineerde aminozuren), is het mogelijk de stabiliteit van de prodrug in de pleister nog groter te maken. Ten derde is het mogelijk om 5-OH-DPAT gecontroleerd toe te dienen aan het lichaam wat de ontwikkeling van een feedback systeem in principe mogelijk zou maken. En als laatste vonden we dat de iontoforetische toediening van een zeer wateroplosbare prodrug van 5-OH-DPAT een verlenging van het effect van het geneesmiddel geeft in vergelijking met 5-OH-DPAT zelf. Deze resultaten laten zien dat de toepassing van prodrugs voordelig kan zijn bij de toediening van dopamine agonisten of andere geneesmiddelen met behulp van transdermale iontoforese. Verder onderzoek naar deze farmaceutische toedieningsvorm zou uiteindelijk kunnen leiden tot een nieuwe farmacotherapie voor patiënten met de ziekte van Parkinson

Identification of Srp9 as a febrile seizure susceptibility gene

Objective: Febrile seizures (FS) are the most common seizure type in young children. Complex FS are a risk factor for mesial temporal lobe epilepsy (mTLE). To identify new FS susceptibility genes we used a forward genetic strategy in mice and subsequently analyzed candidate genes in humans. Methods: We mapped a quantitative trait locus (QTL1) for hyperthermia-induced FS on mouse chromosome 1, containing the signal recognition particle 9 (Srp9) gene. Effects of differential Srp9 expression were assessed in vivo and in vitro. Hippocampal SRP9 expression and genetic association were analyzed in FS and mTLE patients. Results: Srp9 was differentially expressed between parental strains C57BL/6J and A/J. Chromosome substitution strain 1 (CSS1) mice exhibited lower FS susceptibility and Srp9 expression than C57BL/6J mice. In vivo knockdown of brain Srp9 reduced FS susceptibility. Mice with reduced Srp9 expression and FS susceptibility, exhibited reduced hippocampal AMPA and NMDA currents. Downregulation of neuronal Srp9 reduced surface expression of AMPA receptor subunit GluA1. mTLE patients with antecedent FS had higher SRP9 expression than patients without. SRP9 promoter SNP rs12403575(G/A) was genetically associated with FS and mTLE. Interpretation: Our findings identify SRP9 as a novel FS susceptibility gene and indicate that SRP9 conveys its effects through endoplasmic reticulum (ER)-dependent synthesis and trafficking of membrane proteins, such as glutamate receptors. Discovery of this new FS gene and mechanism may provide new leads for early diagnosis and treatment of children with complex FS at risk for mTLE

Synthesis and evaluation of dopaminergic prodrugs designed for transdermal iontophoretic drug delivery : highly water-soluble amino acid ester prodrugs applicable for the treatment of Parkinson's disease

Dopamine agonisten worden toegepast als farmacotherapie voor patienten met de ziekte van Parkinson. In dit onderzoek beschrijven we het chemisch bereiden en de evaluatie van zeer wateroplosbare varianten van de dopamine agonist 5-OH-DPAT. Deze varianten zijn chemisch aan elkaar gekoppelde stoffen van 5-OH-DPAT met andere moleculen. In dit geval zijn dat aminozuren. In de farmacie noemt men deze stoffen prodrugs. Een prodrug is een niet werkzame stof die na omzetting in het lichaam het actieve geneesmiddel vrijgeeft. Ons onderzoek heeft geleid tot een aantal bevindingen. Ten eerste blijkt het mogelijk te zijn zeer wateroplosbare prodrugs te maken die voldoende stabiel zijn in de iontoforese pleister (voldoende houdbaarheid), maar eenmaal in de bloedbaan het geneesmiddel direct vrijgeven. Ten tweede, door het koppelen van 5-OH-DPAT aan dipeptiden (twee gecombineerde aminozuren), is het mogelijk de stabiliteit van de prodrug in de pleister nog groter te maken. Ten derde is het mogelijk om 5-OH-DPAT gecontroleerd toe te dienen aan het lichaam wat de ontwikkeling van een feedback systeem in principe mogelijk zou maken. En als laatste vonden we dat de iontoforetische toediening van een zeer wateroplosbare prodrug van 5-OH-DPAT een verlenging van het effect van het geneesmiddel geeft in vergelijking met 5-OH-DPAT zelf. Deze resultaten laten zien dat de toepassing van prodrugs voordelig kan zijn bij de toediening van dopamine agonisten of andere geneesmiddelen met behulp van transdermale iontoforese. Verder onderzoek naar deze farmaceutische toedieningsvorm zou uiteindelijk kunnen leiden tot een nieuwe farmacotherapie voor patiënten met de ziekte van Parkinson. Dopamine agonists are used as pharmacotherapy for patients with Parkinson's disease. In this research we describe the preparation and evaluation of highly water-soluble derivatives of the dopamine agonist 5-OH-DPAT. These derivatives are chemically coupled compounds of 5-OH-DPAT with other molecules, i.e. amino acids. In pharmacy these compounds are named prodrugs. A prodrug is a non-pharmacologically active compound which will release the active drug after conversion in the body. Our research has led to several findings. First, highly water-soluble prodrugs were synthesized which showed sufficient stability while in solution in the iontophoretic patch (sufficient sustainability), but released the drug on entry in the bloodstream. Second, by coupling 5-OH-DPAT to dipeptides (two combined amino acids) we obtained even more stable prodrugs. Third, 5-OH-DPAT could be administered in a controlled manner, which would facilitate the development of a feedback system. Finally, we found that the iontophoretic delivery of a highly water-soluble prodrug of 5-OH-DPAT led to a prolongation of the pharmacological effect in comparison to 5-OH-DPAT itself. These results show that the application of prodrugs can be advantageous for the administration of dopamine agonists and other drugs by transdermal iontophoresis. Further research of this pharmaceutical dosage form could eventually lead to a new pharmacotherapy of patients with Parkinson's disease.

Mechanistic Studies of the Transdermal Iontophoretic Delivery of 5-OH-DPAT In Vitro

A characterization and optimization of the in vitro transdermal iontophoretic transport of 5-hydroxy-2-(N,N,-di-n-propylamino)tetralin (5-OH-DPAT) is presented. The utility of acetaminophen as a marker of electroosmotic flow was studied as well. The following parameters of iontophoretic transport of 5-OH-DPAT were examined: drug donor concentration, electroosmotic contribution, influence of co-ions, current density, and composition of the acceptor phase. The steady-state flux (Flux(ss)) of acetaminophen was linearly correlated with the donor concentration and co-iontophoresis of acetaminophen did not influence the iontophoretic flux of 5-OH-DPAT, indicating that acetaminophen is an excellent marker of electroosmotic flow. Lowering the Na(+) concentration from 78 to 10 mM in the donor phase, resulted in a 2.5-fold enhancement of the Flux(ss). The Flux(ss) showed a nonlinear relation with the drug donor concentration and an excellent linear correlation with the current density. Reducing the pH of the acceptor phase from 7.4 to 6.2 resulted in a dramatic decrease of the Flux(ss) of 5-OH-DPAT, explained by a reduced electroosmotic flow and an increased counter-ion flow. Optimization of the conditions resulted in a maximum Flux(ss) of 5-OH-DPAT of 1.0 mu mol . cm(-2) h(-1) demonstrating the potential of the iontophoretic delivery of this dopamine agonist for the symptomatic treatment of Parkinson's disease. (C) 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:275-285, 201

Transdermal iontophoretic delivery of a novel series of dopamine agonists in vitro:physicochemical considerations

Objectives The transdermal iontophoretic delivery of a novel series of 2- aminotetralins and chromanamine-based dopamine agonists was investigated in corn. Methods Systematic structural modifications allowed us to investigate their effect on solubility in the donor phase and iontophoretic delivery across human skin. Transport profiles were analysed with nonlinear mixed effect modelling, utilizing an extension to an existing compartmental model. Furthermore, relationships between physicochemical properties and transport parameters were addressed. Key findings A solubility increase was observed: 5,6-di-OH-DPAT <5-OH-MPAT <5-OH-EPAT <8-OH-DPAC. The structure significantly affected the iontophoretic delivery across human stratum corneum and dermatomed human skin with the highest flux for 5-OH-EPAT and 5-OH-MPAT. The extended model with two skin release constants (K(R1), K(R2)) described more adequately iontophoretic transport profiles than the existing model with one release constant. The extended model suggested two parallel transport pathways during current application. Across human stratum corneum, the electrophoretic mobility. measured with capillary electrophoresis, showed a linear relationship with the electromigrative flux and the zero-order iontophoretic mass input into the skin (I(0)). Conclusions Combining transport parameters (I(0), K(R1) and K(R2)). predicted from physicochemical properties, with compartmental modelling provided a powerful tool to simulate iontophoretic transport profiles for screening potential candidates and designing experiments

The Pharmacokinetics and Pharmacological Effect of (S)-5-OH-DPAT Following Controlled Delivery with Transdermal Iontophoresis

The pharmacokinetic (PK) and pharmacodynamic (PD) properties of the active (S)-enantiomer of the potent dopamine (DA) agonist 5-hydroxy-2-(N,N,-di-n-propylamino)tetralin (5-OH-DPAT) were investigated in a novel anesthetized animal model. First, the relationship between current density, in vivo transport, and plasma profile was characterized. Second, the effect of the anesthetic mixture, transdermal iontophoresis, and blood sampling on the striatal DA release (PD end point) was investigated. Third, the PK-PD relationship following transdermal iontophoresis was investigated during a controlled reversible pharmacological response. Given that striatal DA levels are unaltered during experimental procedures, this rat model can be used to investigate the PK-PD relationship. The in vivo flux was linearly correlated with the current density, indicating that drug delivery can be titrated by the current density. Following transdermal iontophoresis and intravenous infusion, a strong reversible effect was observed. Compartmental modeling showed that the relationship between plasma concentration and biomarker response is best characterized by an effect compartment, rather than an indirect response model. In addition, covariate analysis suggested that the delivery rate can affect the PD efficiency. Finally, PK-PD analysis revealed that steady delivery rates are translated into continuous dopaminergic stimulation. This can be of benefit for reducing side effects in the symptomatic treatment of Parkinson's disease with 5-OH-DPAT

Antagonizing Increased miR-135a Levels at the Chronic Stage of Experimental TLE Reduces Spontaneous Recurrent Seizures

Mesial temporal lobe epilepsy (mTLE) is a chronic neurological disease characterized by recurrent seizures. The antiepileptic drugs currently available to treat mTLE are ineffective in one-third of patients and lack disease-modifying effects. miRNAs, a class of small noncoding RNAs which control gene expression at the post-transcriptional level, play a key role in the pathogenesis of mTLE and other epilepsies. Although manipulation of miRNAs at acute stages has been reported to reduce subsequent spontaneous seizures, it is uncertain whether targeting miRNAs at chronic stages of mTLE can also reduce seizures. Furthermore, the functional role and downstream targets of most epilepsy-associated miRNAs remain poorly understood. Here, we show that miR-135a is selectively upregulated within neurons in epileptic brain and report that targeting miR-135a in vivo using antagomirs after onset of spontaneous recurrent seizures can reduce seizure activity at the chronic stage of experimental mTLE in male mice. Further, by using an unbiased approach combining immunoprecipitation and RNA sequencing, we identify several novel neuronal targets of miR-135a, including Mef2a Mef2 proteins are key regulators of excitatory synapse density. Mef2a and miR-135a show reciprocal expression regulation in human (of both sexes) and experimental TLE, and miR-135a regulates dendritic spine number and type through Mef2. Together, our data show that miR-135a is target for reducing seizure activity in chronic epilepsy, and that deregulation of miR-135a in epilepsy may alter Mef2a expression and thereby affect synaptic function and plasticity.SIGNIFICANCE STATEMENT miRNAs are post-transcriptional regulators of gene expression with roles in the pathogenesis of epilepsy. However, the precise mechanism of action and therapeutic potential of most epilepsy-associated miRNAs remain poorly understood. Our study reveals dramatic upregulation of the key neuronal miRNA miR-135a in both experimental and human mesial temporal lobe epilepsy. Silencing miR-135a in experimental temporal lobe epilepsy reduces seizure activity at the spontaneous recurrent seizure stage. These data support the exciting possibility that miRNAs can be targeted to combat seizures after spontaneous seizure activity has been established. Further, by using unbiased approaches novel neuronal targets of miR-135a, including members of the Mef2 protein family, are identified that begin to explain how deregulation of miR-135a may contribute to epilepsy

Antagonizing Increased miR-135a Levels at the Chronic Stage of Experimental TLE Reduces Spontaneous Recurrent Seizures

Mesial temporal lobe epilepsy (mTLE) is a chronic neurological disease characterized by recurrent seizures. The antiepileptic drugs currently available to treat mTLE are ineffective in one-third of patients and lack disease-modifying effects. miRNAs, a class of small noncoding RNAs which control gene expression at the post-transcriptional level, play a key role in the pathogenesis of mTLE and other epilepsies. Although manipulation of miRNAs at acute stages has been reported to reduce subsequent spontaneous seizures, it is uncertain whether targeting miRNAs at chronic stages of mTLE can also reduce seizures. Furthermore, the functional role and downstream targets of most epilepsy-associated miRNAs remain poorly understood. Here, we show that miR-135a is selectively upregulated within neurons in epileptic brain and report that targeting miR-135a in vivo using antagomirs after onset of spontaneous recurrent seizures can reduce seizure activity at the chronic stage of experimental mTLE in male mice. Further, by using an unbiased approach combining immunoprecipitation and RNA sequencing, we identify several novel neuronal targets of miR-135a, including Mef2a Mef2 proteins are key regulators of excitatory synapse density. Mef2a and miR-135a show reciprocal expression regulation in human (of both sexes) and experimental TLE, and miR-135a regulates dendritic spine number and type through Mef2. Together, our data show that miR-135a is target for reducing seizure activity in chronic epilepsy, and that deregulation of miR-135a in epilepsy may alter Mef2a expression and thereby affect synaptic function and plasticity.SIGNIFICANCE STATEMENT miRNAs are post-transcriptional regulators of gene expression with roles in the pathogenesis of epilepsy. However, the precise mechanism of action and therapeutic potential of most epilepsy-associated miRNAs remain poorly understood. Our study reveals dramatic upregulation of the key neuronal miRNA miR-135a in both experimental and human mesial temporal lobe epilepsy. Silencing miR-135a in experimental temporal lobe epilepsy reduces seizure activity at the spontaneous recurrent seizure stage. These data support the exciting possibility that miRNAs can be targeted to combat seizures after spontaneous seizure activity has been established. Further, by using unbiased approaches novel neuronal targets of miR-135a, including members of the Mef2 protein family, are identified that begin to explain how deregulation of miR-135a may contribute to epilepsy

Antagonizing Increased miR-135a Levels at the Chronic Stage of Experimental TLE Reduces Spontaneous Recurrent Seizures

Mesial temporal lobe epilepsy (mTLE) is a chronic neurological disease characterized by recurrent seizures. The antiepileptic drugs currently available to treat mTLE are ineffective in one-third of patients and lack disease-modifying effects. miRNAs, a class of small noncoding RNAs which control gene expression at the post-transcriptional level, play a key role in the pathogenesis of mTLE and other epilepsies. Although manipulation of miRNAs at acute stages has been reported to reduce subsequent spontaneous seizures, it is uncertain whether targeting miRNAs at chronic stages of mTLE can also reduce seizures. Furthermore, the functional role and downstream targets of most epilepsy-associated miRNAs remain poorly understood. Here, we show that miR-135a is selectively upregulated within neurons in epileptic brain and report that targeting miR-135a in vivo using antagomirs after onset of spontaneous recurrent seizures can reduce seizure activity at the chronic stage of experimental mTLE in male mice. Further, by using an unbiased approach combining immunoprecipitation and RNA sequencing, we identify several novel neuronal targets of miR-135a, including Mef2a Mef2 proteins are key regulators of excitatory synapse density. Mef2a and miR-135a show reciprocal expression regulation in human (of both sexes) and experimental TLE, and miR-135a regulates dendritic spine number and type through Mef2. Together, our data show that miR-135a is target for reducing seizure activity in chronic epilepsy, and that deregulation of miR-135a in epilepsy may alter Mef2a expression and thereby affect synaptic function and plasticity.SIGNIFICANCE STATEMENT miRNAs are post-transcriptional regulators of gene expression with roles in the pathogenesis of epilepsy. However, the precise mechanism of action and therapeutic potential of most epilepsy-associated miRNAs remain poorly understood. Our study reveals dramatic upregulation of the key neuronal miRNA miR-135a in both experimental and human mesial temporal lobe epilepsy. Silencing miR-135a in experimental temporal lobe epilepsy reduces seizure activity at the spontaneous recurrent seizure stage. These data support the exciting possibility that miRNAs can be targeted to combat seizures after spontaneous seizure activity has been established. Further, by using unbiased approaches novel neuronal targets of miR-135a, including members of the Mef2 protein family, are identified that begin to explain how deregulation of miR-135a may contribute to epilepsy