Development of glycine-α-methyl-proline-containing tripeptides with neuroprotective properties.

Herein is described the synthesis of novel glycine-α-methyl-proline-containing tripeptides (GP(Me)X tripeptides namely GP(Me)R, GP(Me)K, and GP(Me)H) with the aim of obtaining derivatives highly stable in human plasma and able to counteract neuroinflammatory processes that are distinctive of neurodegenerative pathologies. The syntheses of GP(Me)R, GP(Me)K, and GP(Me)H were all achieved both by introducing the Pro(Me) residue into the Gly-Pro-Arg (GPR) sequence in place of the native Pro in P2 position and replacing the basic amino acid Arg in P3 position by Lys or His. Results showed that all novel GP(Me)X tripeptides are stable in human plasma (t1/2 > 51 h) and that GP(Me)H - generating stable intramolecular H-bond in a C11-turn by interaction of His imidazole ring and Gly carbonyl group - restored physiological levels of nitric oxide deriving from neuronal NOS (nNOS) activity, thus preventing the inflammatory response by suppression of the NF-kB activity and, consequently, the expression of inflammatory genes such as inducibile NOS (iNOS). Therefore, GP(Me)H could be a lead compound for further development of peptidomimetics able to contrast neuroinflammatory processes.

Carvacrol codrugs: a new approach in the antimicrobial plan.

OBJECTIVE: The increasing prevalence of antibiotic-resistant bacterial infections led to identify alternative strategies for a novel therapeutic approach. In this study, we synthesized ten carvacrol codrugs - obtained linking the carvacrol hydroxyl group to the carboxyl moiety of sulphur-containing amino acids via an ester bond - to develop novel compounds with improved antimicrobial and antibiofilm activities and reduced toxicity respect to carvacrol alone. METHOD: All carvacrol codrugs were screened against a representative panel of Gram positive (S. aureus and S. epidermidis), Gram negative (E. coli and P. aeruginosa) bacterial strains and C. albicans, using broth microdilution assays. FINDINGS: Results showed that carvacrol codrug 4 possesses the most notable enhancement in the anti-bacterial activity displaying MIC and MBC values equal to 2.5 mg/mL for all bacterial strains, except for P. aeruginosa ATCC 9027 (MIC and MBC values equal to 5 mg/mL and 10 mg/mL, respectively). All carvacrol codrugs 1-10 revealed good antifungal activity against C. albicans ATCC 10231. The cytotoxicity assay showed that the novel carvacrol codrugs did not produce human blood hemolysis at their MIC values except for codrugs 8 and 9. In particular, deepened experiments performed on carvacrol codrug 4 showed an interesting antimicrobial effect on the mature biofilm produced by E. coli ATCC 8739, respect to the carvacrol alone. The antimicrobial effects of carvacrol codrug 4 were also analyzed by TEM evidencing morphological modifications in S. aureus, E. coli, and C. albicans. CONCLUSION: The current study presents an insight into the use of codrug strategy for developing carvacrol derivatives with antibacterial and antibiofilm potentials, and reduced cytotoxicity.

Effect of 17ß-estradiol on striatal dopaminergic transmission induced by permethrin in early childhood rats.

A positive effect of estrogen treatment has been observed in neurodegenerative diseases such as Parkinson's disease. Since 17ß-estradiol can modulate positively dopaminergic system, here we sought to evaluate the effect of 17ß-estradiol supplementation on an animal model developing dopaminergic alterations on nucleus of striatum after neonatal exposure to permethrin pesticide. The goal of the study was to verify if the co-treatment with 17ß-estradiol could protect against the damage induced by pesticide exposure in early life. Permethrin treated rats showed a decrease of dopamine and Nurr1 gene expression in striatum, while a more pronounced decrease of dopamine was observed in rats co-administered with permethrin+17ß-estradiol. No difference between control and permethrin treated rats was observed in both mRNA of ERα and ERß, whereas the rats co-administered with permethrin+17ß-estradiol showed a down-regulation of ERα expression. The in vitro studies showed that permethrin, at high concentration may have an antagonist effect on ERα and even more pronounced in ERß, thus suggesting that permethrin may block the estrogen neuroprotective effects. In conclusion, in male rats, the administration of estrogen further enhanced the impairment of dopaminergic transmission due to exposure to permethrin.

Modulation of monoaminergic transporters by choline-containing phospholipids in rat brain.

Choline-containing phospholipids were proposed as cognition enhancing agents, but evidence on their activity is controversial. CDP-choline (cytidine-5´-diphosphocholine, CDP) and choline alphoscerate (L-alpha-glycerylphosphorylcholine, GPC) represent the choline-containing phospholipids with larger clinical evidence in the treatment of sequelae of cerebrovascular accidents and of cognitive disorders. These compounds which display mainly a cholinergic profile interfere with phospholipids biosynthesis, brain metabolism and neurotransmitter systems. Dated preclinical studies and clinical evidence suggested that CDP-choline may have also a monoaminergic profile. The present study was designed to assess the influence of treatment for 7 days with choline-equivalent doses (CDP-choline: 325 mg/Kg/day; GPC: 150 mg/Kg/day) of these compounds on brain dopamine (DA), and serotonin (5-HT) levels and on DA plasma membrane transporter (DAT), vesicular monoamine transporters (VMAT1 and VMAT2), serotonin transporter (SERT), and norepinephrine transporter (NET) in the rat. Frontal cortex, striatum and cerebellum were investigated by HPLC with electrochemical detection, immunohistochemistry, Western blot analysis and ELISA techniques. CDP-choline did not affect DA levels, which increased after GPC administration in frontal cortex and cerebellum. GPC increased also 5-HT levels in frontal cortex and striatum. DAT was stimulated in frontal cortex and cerebellum by both CDP and GPC, whereas VMAT2, SERT, NET were unaffected. VMAT1 was not detectable. The above data indicate that CDP-choline and GPC possess a monoaminergic profile and interfere to some extent with brain monoamine transporters. This activity on a relevant drug target, good tolerability and safety of CDP-choline and GPC suggests that these compounds may merit further investigations in appropriate clinical settings.

(R)-α-lipoyl-glycyl-L-prolyl-L-glutamyl dimethyl ester codrug as a multifunctional agent with potential neuroprotective activities.

The (R)-α-lipoyl-glycyl-L-prolyl-L-glutamyl dimethyl ester codrug (LA-GPE, 1) was synthesized as a new multifunctional drug candidate with antioxidant and neuroprotective properties for the treatment of neurodegenerative diseases. Physicochemical properties, chemical and enzymatic stabilities were evaluated, along with the capacity of LA-GPE to penetrate the blood-brain barrier (BBB) according to an in vitro parallel artificial membrane permeability assay for the BBB. We also investigated the potential effectiveness of LA-GPE against the cytotoxicity induced by 6-hydroxydopamine (6-OHDA) and H2O2 on the human neuroblastoma cell line SH-SY5Y by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. Our results show that codrug 1 is stable at both pH 1.3 and 7.4, exhibits good lipophilicity (log P=1.51) and a pH-dependent permeability profile. Furthermore, LA-GPE was demonstrated to be significantly neuroprotective and to act as an antioxidant against H2O2- and 6-OHDA-induced neurotoxicity in SH-SY5Y cells.

Designing prodrugs for the treatment of Parkinson's disease.

INTRODUCTION: Current Parkinson's disease (PD) therapy is essentially symptomatic, and l-Dopa (LD), is the treatment of choice in more advanced stages of the disease. However, motor complications often develop after long-term treatment, and at this point physicians usually prescribe adjuvant therapy with other classes of antiparkinsonian drugs, including dopamine (DA) agonists, catechol-O-methyl transferase (COMT) or monoamine oxidase (MAO)-B inhibitors. In order to improve bioavailability, the prodrug approach appeared to be the most promising, and some antiparkinsonian prodrugs have been prepared in an effort to solve these problems. AREAS COVERED: This review discusses the evidence of progress in PD therapy, mainly focused on prodrug approach for treatment of this neurological disorder. Several derivatives were studied with the aim of enhancing its chemical stability, water or lipid solubility, as well as diminishing the susceptibility to enzymatic degradation. Chemical structures mainly related to LD, DA and dopaminergic agonists are also reviewed in this paper. EXPERT OPINION: In order to strengthen the pharmacological activity of antiparkinsonian drugs, enhancing their penetration of the blood-brain barrier (BBB), different approaches are possible. Among these, the prodrug approach appeared to be the most promising, and many prodrugs have been prepared in an effort to optimize physicochemical characteristics. In addition, novel therapeutic strategies based on formulations linking dopaminergic drugs with neuroprotective agents, increasing LD striatal levels and offering sustained release of the drug without any fluctuation of brain concentration, offer promising avenues for development of other effective new treatments for PD.

L-Dopa prodrugs: an overview of trends for improving Parkinson's disease treatment.

L-Dopa is the mainstay of Parkinson's disease therapy; this drug is usually administered orally, but it is extensively metabolized in the gastrointestinal tract, so that relatively little arrives in the bloodstream as intact L-Dopa. The peripheral conversion of L-Dopa by amino acid decarboxylase to dopamine is responsible for the typical gastrointestinal and cardiovascular side effects. To minimize the conversion to dopamine outside the central nervous system, L-Dopa is usually given in combination with peripheral inhibitors of amino acid decarboxylase. In spite of that, other central nervous side effects such as dyskinesia, on-off phenomenon and end-of-dose deterioration still remain. The main factors responsible for the poor bioavailability are the drug's physical-chemical properties: low water and lipid solubility, resulting in unfavorable partition, and the high susceptibility to chemical and enzymatic degradation. Starting from these considerations the prodrug approach has been applied to L-Dopa in order to overcome its metabolism problems and to improve its bioavailability. The goal of this paper is to provide the reader with a critical overview on L-Dopa prodrugs here classified according to the nature of the main chemical modification on L-Dopa backbone that led to the formation of the desired derivative.

Effect of choline-containing phospholipids on brain cholinergic transporters in the rat.

The influence of one week treatment with the choline-containing phospholipids cytidine-5'-diphosphocholine (CDP-choline) and choline alphoscerate (L-alpha-glyceryl-phosphorylcholine) at choline-equivalent doses (CDP-choline: 325 mg/kg/day; choline alphoscerate: 150 mg/kg/day) on vesicular acetylcholine transporter (VAChT), on choline transporter (CHT) and on acetylcholine (ACh) concentrations was investigated in rat frontal cortex, striatum and cerebellum. ACh was assayed by HPLC with electrochemical detection, VAChT by Western blot, ELISA and immunohistochemistry, CHT by Western blot and immunohistochemistry. After CDP-treatment, ACh levels were slightly increased in the frontal cortex, not substantially different in the striatum, and reduced significantly in the cerebellum compared to controls. Choline alphoscerate stimulated significantly the neurotransmitter concentration in the frontal cortex, however, the levels were similar to the controls in both the striatum and cerebellum. In comparison to the controls, VAChT expression following either CDP-choline or choline alphoscerate treatment, was enhanced greatly in the striatum and cerebellum. Also, ELISA measurements for VAChT showed significant increases in all choline alphoscerate treated brain areas. In contrast, in the CDP-choline treated rats the vesicular transporter amount was greater than the control only in the striatum. The cholinergic presynaptic transporters VAChT and CHT play a relevant role in sustaining new ACh synthesis and release. To sum up, CDP-choline and choline alphoscerate stimulated to a different extent the expression of VAChT and CHT primarily in a cognitive area such as frontal cortex. In the lack of novel therapeutic strategies, safe compounds developed since a long time such as the choline-containing phospholipids investigated would merit to be further investigated by new and adequate clinical studies. This for assessing their place if any in pharmacotherapy of dementia disorders characterized by diminished cholinergic tone.

New drug delivery strategies for improved Parkinson's disease therapy.

Increasing interest has been addressed toward the introduction of new therapeutic approaches to obtaining continuous dopaminergic stimulation (CDS). The goal of this therapeutic strategy is to reduce the occurrence and severity of L-DOPA (LD)-associated motor fluctuations and dyskinesia, and provide good long-term safety and tolerability. CDS can be achieved by the administration of oral dopamine (DA) agonists with a long half-life, transdermal or subcutaneous delivery of DA agonists, or intestinal LD infusion. To allow higher concentrations of LD to reach the brain and to reduce peripheral side effects, the therapeutic approach provides the concomitant administration of LD, carbidopa and entacapone that have been developed in tablet form, standard LD/carbidopa, LD/benserazide, LD/entacapone, LD/tolcapone associations or long-acting controlled release formulations, LD/carbidopa and LD/benserazide. Alternatively to solid formulations, LD/carbidopa liquid forms have been developed. Furthermore, the authors examine a series of new LD codrugs and non-dopaminergic drugs for Parkinson's disease treatment, together with a variety of experimental delivery strategies including transdermal therapeutic systems, liposomes, solid lipid nanoparticles and biocompatible microparticles. This review provides an overview of progress in anti-Parkinson therapy, mainly focused on delivery strategies and codrug approach for treatment of this neurological disorder.

Codrugs linking L-dopa and sulfur-containing antioxidants: new pharmacological tools against Parkinson's disease.

A series of multifunctional codrugs (1-6) were synthesized to overcome the pro-oxidant effect associated with L-dopa (LD) therapy. Target compounds release LD and dopamine (DA) in human plasma after enzymatic hydrolysis, displaying an antioxidant effect superior to that of N-acetylcysteine (NAC). After intracerebroventricular injection of codrug 4, the levels of DA in the striatum were higher than those in LD-treated groups, indicating that this compound has a longer half-life in brain than LD.

New L-dopa codrugs as potential antiparkinson agents.

This paper reports the synthesis and preliminary evaluation of new L-dopa (LD) conjugates (1 and 2) obtained by joining LD with two different natural antioxidants, caffeic acid and carnosine, respectively. The antioxidant efficacy of compounds 1 and 2 was assessed by evaluating plasmatic activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) in the rat. Rat striatal concentration of LD and dopamine (DA), and central nervous effects were evaluated after oral administration of the codrugs 1 and 2. The results suggest that, though our codrugs are devoid of significant antioxidant activity, they are able to induce sustained delivery of DA in rat striatum and can improve LD and DA release in the brain.

Characterization of alkanoyl-10-O-minocyclines in micellar dispersions as potential agents for treatment of human neurodegenerative disorders.

Minocycline is a widely used antibacterial agent. Moreover, it is also demonstrated to be effective in several neurodegenerative disorders, due to its antioxidant and anti-inflammatory activities. However, the last activity is only apparent at very high doses. In fact, minocycline poorly crosses the blood-brain barrier (BBB) due to its low lipophilicity and half-life. The present work details the physicochemical characterization of a series of alkanoyl-10-O-minocycline derivatives (2-6), which are able to produce self-assembled aggregates in aqueous solution. The n-octanol/aqueous phase lipophilicity of minocycline and its derivatives were assessed by theoretical calculation, by shake-flask method, and by reversed-phase HPLC. Moreover, we determined their affinity for membrane phospholipids measuring their HPLC retention on phospholipid-based stationary phases, the so-called "Immobilized Artificial Membranes" (IAMs). Our results indicate high lipophilicity values for the minocycline derivatives (compounds 2-6); these values and the corresponding phospholipid affinities increase with the length of the hydrocarbon moiety substituent. Furthermore, the ability of the investigated alkanoyl-10-O-minocycline derivatives to self-assemble could allow a direct administration by oral and intraperitoneal routes as supramolecular systems. The advantages are an enhancement of drug solubilization, a sustained release, and the consequent less frequent drug administration. Moreover, we can hypothesize the potential solubilization in the micellar core of other poorly water soluble drugs which could improve the therapeutic effects of the pharmaceutical formulation in a combined therapy. Given the high lipophilicity of the title derivatives, they can be supposed to offer higher half-life and a better BBB penetration than minocycline. Since the new derivatives retain the structural features related to the antioxidant and anti-inflammatory effects of minocycline, they can be regarded not only as long-acting antimicrobial agents but also as candidate drugs for a targeted treatment of mental illness.

Antiparkinson prodrugs.

Parkinson's disease (PD) is a progressive, neurodegenerative disorder which involves the loss of dopaminergic neurons of the substantia nigra pars compacta. Current therapy is essentially symptomatic, and L-Dopa (LD), the direct precursor of dopamine(DA), is the treatment of choice in more advanced stages of the disease. Substitution therapy with LD is, however, associated with a number of acute problems. The peripheral conversion of LD by amino acid decarboxylase (AADC) to DA is responsible for the typical gastrointestinal (nausea, emesis) and cardiovascular (arrhythmia, hypotension) side effects. To minimize the conversion to DA outside the central nervous system (CNS) LD is usually given in combination with peripheral inhibitors of AADC (carbidopa and benserazide). In spite of that, other central nervous side effects such as dyskinesia, on-off phenomenon and end-of-dose deterioration still remain. The main factors responsible for the poor bioavailability and the wide range of inter- and intra-patient variations of plasma levels are the drug's physical-chemical properties: low water and lipid solubility, resulting in unfavourable partition, and the high susceptibility to chemical and enzymatic degradation. In order to improve the bioavailability, the prodrug approach appeared to be the most promising and some LD prodrugs have been prepared in an effort to solve these problems. We report here a review of progress in antiparkinson prodrugs, focusing on chemical structures mainly related to LD, DA and dopaminergic agonists.