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Discussione
Il trattamento DIT è risultato efficace nel ridurre la carica batterica iniziale di almeno 5 log su tutti i ceppi batterici esaminati con almeno uno dei protocolli testati. Tra i parametri di processo valutati, la tempe- ratura T2sembra avere influenzato notevolmente l’efficacia del tratta-
mento, unitamente alla differenza tra temperatura iniziale e tempera- tura finale (ΔT) del processo DIT. Infatti, gli obiettivi DIT con T2=60°C
e ΔT=30°C (DIT1 e DIT3) non si sono dimostrati efficaci su nessuna delle specie batteriche testate; al contrario, gli obiettivi DIT con T2=65°C e ΔT=35°C (DIT2 e DIT4), hanno determinato un abbattimen-
to della carica batterica > 5 log per tutte le specie, ad eccezione di E.
hirae, la cui resistenza ai trattamenti termici è nota in letteratura.3,4E.
hirae è risultato sensibile ai trattamenti DIT 2 e DIT4 solo se seguiti dal
mantenimento della temperatura T2e in presenza di saccarosio. Per
questo microrganismo, in particolare, sarebbe opportuno ottimizzare il trattamento DIT 2 e 4 (ΔT=35°C) cercando di ridurre al massimo il tempo di mantenimento della T2. La velocità di riscaldamento (θ) e le
due sostanze interferenti utilizzate nella preparazione della sospensio- ne test non hanno generalmente influenzato la suscettibilità delle spe- cie batteriche ai trattamenti DIT.
Conclusione
In conclusione, questi test preliminari suggeriscono che il processo DIT può essere efficace nell’ottenere l’abbattimento della carica batte- rica in un substrato liquido. I parametri che influenzano maggiormente l’efficacia del processo sembrano essere quelli puramente termici: ΔT e T2. Ulteriori sperimentazioni tenderanno ad adattare i parametri di
processo alle esigenze della produzione a livello industriale.
Bibliografia
1. Sterilisation of liquids in hermetically closed vessels; PCT Patent No. 2008/114136; China Patent No. 200880014495; United States Patent Application No. 20110123690
2. UNI EN 1276:2009 Quantitative suspension test for the evaluation of bactericidal activity of chimica disinfectants and antiseptics used in food, industrial, domestic and institutional areas. 3. Spinks AT, Dunstan RH, Harrison T, et al. Thermal inactivation of
water-borne pathogenic and indicator bacteria at sub-boiling tem- peratures. Water Res 2006;40:1326-32.
4. Aguirre JS, Pin C, Rodríguez MR, García de Fernando GD. Analysis of the variability in the number of viable bacteria after mild heat treatment of food. Appl Environ Microbiol 2009;75:6992-7.
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Journal of Biological Research 2015; volume 88:5161
Epilepsy, affecting at least 50 million persons worldwide, is one of the most common neurological disorders. Despite the significant advances in understanding epileptogenic mechanisms and in counteracting their pathological consequences, this clinical condition still has to be faced of treating more effectively the symptoms (epileptic seizures) and of pre- venting their unfavourable evolution. So far, research has been unsuc- cessful involved in developing effective antiepileptic drugs (AEDs) capa- ble of preventing the development of the pathogenic process, set in motion by different etiological factors, that leads ultimately to chronic epilepsies.1,2So, a substantial need remains to develop new AEDs with
better safety, less toxicity, and higher efficacy.3,4Valproic acid, VPA, is
one of the four most widely prescribed AEDs. Besides its wide use in both generalized and partial epilepsies, VPA has also gained widespread use in recent years for the treatment of bipolar disorders, neuropathic pain and for prophylactic treatment of migraine.5,6However the use of
VPA is limited by two rare but potentially life-threatening side effects, hepatotoxicity, induced from the formation of metabolite(s) with a ter- minal double bond, specifically 4-ene-VPA,7and teratogenicity, associat-
ed with the parent compound itself.8In a previous work we reported the
synthesis of aminoacidic ester derivatives of VPA as resulted of chemical conjugation of VPA with esters of essential neutral aminoacids, with the aim of modifying the physicochemical properties relevant to bioavail- ability, such as solubility or lipophilicity, improving the efficacy and reducing unwanted side or toxic effects of VPA.9We had reported also
the synthesis of N-valproyl-L-tryptophan, that has shown adequate physicochemical characteristics to permeate biological membranes and antiepileptic activity at lower concentration than VPA.10,11In this paper,
we focused our research on synthesis and characterization of new aminoacidic compounds with potential antiepileptic activity: N-Valproyl- L-Leucine (ValLeu), N-Valproyl-L-methionine (ValMet) and N-Valproyl- L-Histidine (ValHist). The conjugation could consent to obtain VPA derivatives, lacking of structural characteristics usually implicated on VPA teratogenicity, and avoiding formation of possible hepatotoxic
metabolites. The aminoacidic derivatives of VPA was successfully obtained covalent linking carboxyl group of drug with aminic group of L- aminoacids, by synthesis involving two main steps. The first step, described in our previous work9was modified by adding DMAP as fur-
ther coupling agent together with DCC. The structures of obtained com- pounds were assigned on the basis of respective analytical data-sets, FT- IR, MS and 1H and 13C-NMR spectral data. Since the drug lipophilicity is
an important factor conditioning brain uptake, the apparent partition coefficient (Papp) could be used as simple descriptor of ability to cross the
BBB: values of log Pappwithin -0.2 to 1.3 have been described as optimal
for cerebral transport; on the other hand higher values than these could reduce the rate of transport inside the membrane.12,13Apparent partition
coefficient (Papp) of ValLeu, ValMet and ValHist were determined in n- octanol/phosphate buffer pH 7.4 solution and expressed as Log Papp. The determined Log Papp resulted -0.11,- 1,02 and -1,61 respectively. The Log DpH7.4values indicate that ValLeu, ValMet are adequate to cross bio-
logical membranes and in particular BBB barrier while ValHist value is too low, probably due to the fact that was obtained as hydrochloride.
Compared to others drug administration routes, the oral one remains the most preferred as it implies ease of administration as well as high patient compliance. However, the transit through the gastrointestinal tract could constitute a limiting step to bioavailability as a consequence of degradation correlated to the environmental pH. In view of a possible administration of ValLeu, ValMet and ValHist by oral route, studies on their chemical stability were performed in simulated gastro-intestinal buffer (37°C, pH 1.2 to 8.0) and monitored by HPLC analysis. The exper- iments demonstrated that ValLeu, ValMet and ValHist remained unchanged up to 24 h, and did not produce degradation products or potential metabolites. This behaviour indicates high stability at pH con- ditions of gastro-intestinal tract.
Since compounds containing amide functional group could be suscep- tible of hydrolysis by plasma and/or cerebral enzymes, our experiments were focused on the evaluation of stability of ValLeu, ValMet and ValHist in these biological environments. Otherwise, plasma stability of drug candidates plays an important role in drug discovery and development; it is essential for maintaining acceptable drug concentration and half-life in order to achieve desirable pharmacological effects.14
Experimental data highlighted that ValLeu, ValMet and ValHist remained unmodified up to 24 h in plasma environment. In rat brain homogenate ValLeu, ValMet and ValHist did not undergo cleavage after 24 h, indicating that synthetized compounds have also good stability to cerebral enzymes.
References
1. Arroyo S, Brodie MJ, Avanzini G, et al. Is refractory epilepsy pre- ventable? Epilepsia 2002;43:437-44.
Correspondence: Anna Lisa Scaturro, Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of Palermo, via Archirafi 32, 90123, Palermo, Italy.
E-mail: [email protected]
©Copyright V. De Caro et al., 2015 Licensee PAGEPress, Italy
Journal of Biological Research 2015; 88:5161
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License (by-nc 3.0) which permits any noncom- mercial use, distribution, and reproduction in any medium, provided the orig- inal author(s) and source are credited.
N-Valproyl-aminoacids as new potential antiepileptic drugs:
synthesis, characterization and in vitro
studies on stability
V. De Caro, A.L. Scaturro, F.M. Sutera, L.I. Giannola
Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, University of
Palermo, Italy
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Article
2. Walker MC, White HS, Sander JWAS. Disease modification in par- tial epilepsy. Brain 2002;125:1937-50.
3. Bialer M, Yagen B. Valproic acid: second generation. Neurotherapeutics 2007;4:130-7.
4. Shimshoni JA, Bialer M, Yagen B. Synthesis and anticonvulsant activity of aromatictetramethylcyclopropanecarboxamide deriva- tives. Bioorg Med Chem 2008;16:6297-305.
5. Lennkh C, Simhandl C. Current aspects of valproate in bipolar dis- order. Int Clin Psychopharmacol 2000;15:1-11.
6. Guidotti A, Dong E, Kundakovic M, et al. Characterization of the action of antipsychotic subtypes on valproate-induced chromatin remodelling. Trends Pharmacol Sci 2008;30:55-60.
7. Silva MFB, Aires CC, Luis PB, et al. Valproic acid metabolism and its effects on mitochondrial fatty acid oxidation: a review. J Inherit Metab Dis 2008;31:205-16.
8. Bojic U, Ehlers K, Ellerbeck U, et al. Studies on the teratogen phar- macophore of Valproic acid analogues: Evidence of interactions at a hydrophobic centre. Eur J Pharmacol 1998;354:289-99. 9. Giannola LI, Lamartina L, De Caro V. Synthesis and characteriza-
tion of aminoacidic pro-drugs of valproic acid. Pharmazie 1998; 53:829-34.
10. De Caro V, Giandalia G, Siragusa MG, et al. N-Valproyl-L- Tryptophan for CNS-targeting: synthesis, characterization and effi- cacy in vitro studies of a new potential antiepileptic drug. Med Chem 2011;7:9-17.
11. Sardo P, Rizzo V, Friscia S, et al. Inhibitory effects of N-valproyl-L- tryptophan on high potassium, low calcium and low magnesium- induced CA1 hippocampal epileptiform bursting activity in rat brain slices. J Neural Transm 2012;119:1249-59.
12. Trojnar MK, Weirzchowska-Cioch E, Krzyzanowski M, et al. New generation of valproic acid. Pol J Pharmacol 2004;56:283-8. 13. Gimenez F, Fernandez C, Mabondzo A. Transport of HIV protease
inhibitors through the bloodbrain barrier and interactions with the efflux proteins, P-glycoprotein and multidrug resistance proteins. J Acquir Immune Defic Syndr 2004;36:649-58.
14. Di L, Kerns EH, HongY, Chen H. Development and application of high throughput plasma stability assay for drug discovery. Int J Pharm 2005;297:110-9.
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Introduction
Recent molecular studies showed that the dimorphic fungus Sporothrix schenckii is no longer the only species able to cause sporotrichosis, a cutaneous lymphatic or systemic mycosis particular- ly frequent in certain geographical areas such as Mexico, Brazil, Peru, and India.1In fact, S. schenckii can now be recognized as a species
complex comprising at least six sibling species: Sporothrix brasilien-
sis, Sporothrix globosa, Sporothrix luriei, Sporothrix mexicana, Sporothrix pallida (formerly Sporothrix albicans) and S. schenckii
sensu stricto.2,3Like S. schenckii, all these new species have been
reported to cause diseases in humans and in other animals1,4,5
although the extent of their impact on human infections is not yet completely known. However, infections due to S. schenckii have also been reported from other parts of the world, including Europe, where sporotrichosis is considered a rare disease.6Nevertheless, in recent
years, several clinical autochthonous cases have been described in patients and animals that live in European countries, showing that this pathogenic fungus is more widespread than is now believed.1,6At
present, there are relatively few works that have evaluated the sus- ceptibility of S. schenckii sensu lato to antifungal agents and the drugs tested so far have shown, in general, poor activity especially against S. pallida, S. globosa, and S. mexicana. Therefore, in this study we decided to evaluate the activities of a panel of antifungal drugs against all members of the S. schenckii complex with particular refer- ence to Italian isolates. To our knowledge this is the first study that evaluates in vitro activities of antifungal agents against a number of
Sporothrix spp. isolates recovered from clinical and environmental
samples in Italy.
Materials and Methods
Fourteen Sporothrix spp. were examined in this study (Table 1). Seven of them were environmental S. pallida isolates that have already been well characterized in our previous study.6The identity of each iso-
late was determined by partial amplification and sequencing of the
calmodulin-encoding gene according to recent studies.2,6 Antifungal
activity of seven drugs (Table 1) was evaluated by disk diffusion method according to the procedures reported in the National Committee for Clinical Laboratory Standards (NCCLS) document M44-A.
In this study, a total of 14 clinical and environmental Sporothrix spp. were examined to evaluate their susceptibility to a panel of antifungal agents. The resulting values of the in vitro susceptibility of S. schenckii
sensu lato isolates are shown in Table 1.
All fungal species were resistant to fluconazole, flucytosine and metronidazole whereas were susceptible to nystatin. An excellent broad-spectrum antifungal activity of miconazole was observed against all examined strains. Regarding ketoconazole, different degree of sus- ceptibility were observed. In particular this drug was active against S.
schenckii, S. brasiliensis and S. mexicana but for S. pallida, S. globosa
and S. luriei was not possible to measure the diameter of the zone of inhibition due to the presence of a high number of resistant colonies.