PROJECT CODE PN-II-RU-TE-2014-4-1670
Grant number 342/01/10/2015
Title: Sortase A inhibitors. New therapeutic approach for the management of infections with gram-positive multiresistant cocci
Project Leader, Niţulescu George Mihai
FUNDING AUTHORITY - THE NATIONAL AUTHORITY FOR SCIENTIFIC RESEARCH OF ROMANIA – UEFISCDI
Budget: 517.000 lei
REASERCH PROJECTS for YOUNG RESEARCH TEAMS
The project tries to resolve the problem of the limited number of therapeutic resources for the management of gram-positive multiresistant cocci infections. Based on the project leader's expertise in targeted-drug design and in antimicrobial agents’ development, the project has the objectives to synthesize original molecules designed to target sortase A, to validate their effectiveness as anti-enterococcal agents, to assess their safety profile and to extend the research on other pathogens within the group of positive cocci. The compounds will be subjected to physicochemical and biopharmaceutical analyses, will be tested on susceptible/resistant bacterial strains. Toxicity assessment will use alternative methods including the use of Jurkat cell cultures and invertebrate models, like Artemia salina and Daphnia magna, with the purpose of reducing the number of animals used further. Structure-activity relationship analysis will be used for structural optimization. The results of the present project can be used to obtain original Romanian drugs with antimicrobial activity and to decrease the morbidity and mortality of affected patients. A special emphasis was put in promoting young, but experienced, researchers in a well-balanced interdisciplinary team.
STAGE I (2015)
Budget: 44.000 lei
The activities planned for this phase of the contract were:
• In silico analysis of the optimal parameters characterizing ligand - sortase A interactions
• Evaluation of biopharmaceutical profile by identifying physico-chemical molecular descriptors, molecular weight, partition coefficient octanol/water, number of hydrogen bonds donors/acceptors
All activities under the work plan have been completed, obtaining the following results:
• a structural basis of compounds containing 71 sortase inhibitors structures
• assessment of the main biopharmaceutical parameters for the sortase inhibitors using computational methods
• a structural profile of the typical sortase A inhibitor for virtual screening
STAGE II (2016)
Budget: 295.692 lei
The activities planned for this phase of the contract were:
- developing a strategy and methodology for synthesis, determination of optimal parameters for the synthesis process and for the extraction of compounds from vegetable products to provide substantial quantities necessary for microbiology and toxicology subsequent testing
- physico-chemical characterization and assessment of the purity of the synthesized compounds: 1H and 13C NMR, elemental analysis, IR spectra
- microbial susceptibility testing against certain Gram-positive strains using disk diffusion method; quantitative evaluation of the antimicrobial effect using the micro-dilution technique bullion; determination of minimum inhibitory concentration
- assessment of toxicity by bioassays using cell culture, invertebrates species, and rodents
- elaborating articles, communications, posters at conferences, symposia, national and international congresses, patent application
All activities under the work plan have been completed, obtaining the following results:
- several synthesis methods were elaborated and the synthesis processes were used to obtain 3 series of new compounds
- a large variety of plant products were used to obtain vegetable extracts or essential oils
- both synthetic compounds and the plant products were physico-chemical characterizated using several methods: 1H and 13C NMR, elemental analysis, IR spectra
- microbial susceptibility was determined against certain Gram-positive strains using the disk diffusion method
- the toxicity of both synthetic compounds and the plant products was determined using Daphnia and/or Artemia species
- several article manuscripts were prepared, two of them were published and two are under evaluation
- a number of 16 communications and posters were presented at national and international conferences, symposia, and congresses.
- a national patent application is underwork
The results of this research stage were capitalized by the following articles published in ISI journals
- Nitulescu, G. M.; Margina, D.; Juzenas, P.; Peng, Q.; Olaru, O. T.; Saloustros, E.; Fenga, C.; Spandidos, D. Α.; Libra, M.; Tsatsakis, A. M. Akt inhibitors in cancer treatment: The long journey from drug discovery to clinical use (Review). Int. J. Oncol. 2016, 48, 869–85. (Impact factor: 3.03).
- Nitulescu, G.; Zanfirescu, A.; Olaru, T. O.; Nicorescu, M. I.; Nitulescu, M. G.; Margina, D. Structural Analysis of Sortase A Inhibitors. Mol. 2016, 21. (Impact factor: 2.46).
The results of this research stage were capitalized by the following presentations at international of national congresses:
1. Nitulescu, G. M., Draghici, C., Velescu, B. S., Nitulescu, G., Olaru, O. T. Synthesis of new pyrazole derivatives as AKT inhibitors, MÆDICA - a Journal of Clinical Medicine, Volum 11, Supplement, 2016, p. 54, ISSN 2501-6903.
2. Nitulescu, G. M., Olaru, O. T., Socea L. I., Barbuceanu, S. F. Prediction targeted combinatorial library design, 15th Belgian Organic Synthesis Symposium, 10-16 iulie 2016, Antwerp, Belgia, Book of Abstracts, p. 278
3. Nitulescu, G. M., Olaru, O. T., Nitulescu, G., Margina, D. Synthesis of new 1,5-dimethyl-2-phenylpyrazol-3-one derivatives targeted towards bacterial sortase A, 15th Belgian Organic Synthesis Symposium, 10-16 iulie 2016, Antwerp, Belgia, Book of Abstracts, p. 279.
4. Barbuceanu, S. F., Nitulescu, G. M., Socea L. I., Barbuceanu, F., Nitulescu, G., Saramet, G., Draghici, C. Synthesis and characterization of some 1,3,4-thiadiazoles derivatives as potential biological agents, 15th Belgian Organic Synthesis Symposium, 10-16 iulie 2016, Antwerp, Belgia, Book of Abstracts, p. 281.
5. Socea L. I., Draghici, C., Nitulescu, G. M., Olaru, O. T., Nitulescu, G., Barbuceanu, S. F. Synthesis of novel hydrazinecarbothioamides and products of their cyclization, 15th Belgian Organic Synthesis Symposium, 10-16 iulie 2016, Antwerp, Belgia, Book of Abstracts, p. 282.
6. Olaru, O.; Ivopol, M.; Nitulescu, G.; Dune, A.; Calinescu, I.; Ivopol, G.; Popescu, M.; Nitulescu, G. M. Toxicity of various antimicrobial essential oils assessed using Artemia salina and Daphnia magna. 52st Congress of the European-Societies-of-Toxicology (EUROTOX), 4-7 septembrie 2016, Sevilia, Spania, Toxicology Letters, 258(2), S68. ISSN: 0378-4274.
7. Ivopol, M.; Olaru, O.; Nitulescu, G. M.; Dune, A.; Popescu, M.; Spinu, C.; Nitulescu, G.; Calinescu, I. Zingiberaceae extracts and essential oils with potential sortase inhibitory effects. Preparation, characterization and toxicity evaluation. 52st Congress of the European-Societies-of-Toxicology (EUROTOX), 4-7 septembrie 2016, Sevilia, Spania, Toxicology Letters, 258(2), S69. ISSN: 0378-4274.
8. Nitulescu, G.; Olaru, O.; Nitulescu, G. M.; Ungurianu, A.; Margina, D. Toxicity assessment of sortases inhibitors. 52st Congress of the European-Societies-of-Toxicology (EUROTOX), 4-7 septembrie 2016, Sevilia, Spania, Toxicology Letters, 258(2), S75. ISSN: 0378-4274.
9. Ivopol, M.; Nitulescu, G. M.; Calinescu, I.; Dune, A.; Popescu, M.; Ivopol, G.; Nitulescu, G.; Olaru, O. GC/MS analysis and toxicity assessment of essential oils and extracts with antimicrobial activity obtained from plants belonging to Pinaceae and Cupressaceae. 52st Congress of the European-Societies-of-Toxicology (EUROTOX), 4-7 septembrie 2016, Sevilia, Spania, Toxicology Letters, 258(2), S68. ISSN: 0378-4274.
10. Nitulescu, G. M.; Nitulescu, G.; Olaru, O.; Margina, D. Structure-based prediction of side effects of sortase A inhibitors. 52st Congress of the European-Societies-of-Toxicology (EUROTOX), 4-7 septembrie 2016, Sevilia, Spania, Toxicology Letters, 258(2), S121. ISSN: 0378-4274.
11. Nitulescu, G. M.; Drăghici, C.; Nitulescu, G.; Olaru, O. T. Design and synthesis of new pyrazole derivatives as sortases inhibitors. Congresul Național de Farmacie din România, 28 sept. - 1 oct. 2016, București, România, Volum de rezumate, p. 55, ISBN 2537-2823.
12. Nitulescu, G.; Olaru, O. T.; Nitulescu, G. M.; Margina, D.; Potolea, I. M.; Pirvu, O. M. Effect of ultrasounds on triticum root elongation. Congresul Național de Farmacie din România, 28 sept. - 1 oct. 2016, București, România, Volum de rezumate, p. 223, ISBN 2537-2823.
13. Nitulescu, G. M.; Draghici, C; Barbuceanu, S. F.; Socea, L. I.; Olaru, O. T. Synthesis of new phenazone derivatives as potential bacterial sortase a inhibitors. International Symposium: Priorities of Chemistry for a Suitable Development - PRIOCHEM, 27-28 oct. 2016, Bucuresti, Volum de rezumate, p. 50, ISSN: 2285-8334.
14. Olaru, O. T.; Guțu, C. M.; Chirita, C.; Iordache, A. A.; Ionita, I. M.; Ilie, M.; Zanfirescu, A.; Nitulescu, G. M. The obtaining and analysis of Salvia glutinosa L. (Lamiaceae) volatile oil. International Symposium: Priorities of Chemistry for a Suitable Development - PRIOCHEM, 27-28 oct. 2016, Bucuresti, Volum de rezumate, p. 51, ISSN: 2285-8334.
15. Guțu, C. M.; Olaru, O. T.; Iordache, A. A.; Chirita, C.; Ionita, I. M.; Velescu, B. S.; Seremet, O. C.; Nitulescu, G. M. Variation in composition of essential oil from Tanacetum vulgare L. Flowers from different locations. International Symposium: Priorities of Chemistry for a Suitable Development - PRIOCHEM, 27-28 oct. 2016, Bucuresti, Volum de rezumate, p. 40, ISSN: 2285-8334.
16. Barbuceanu, S. F., Socea, L. I., Nitulescu, G. M., Apostol, T. V., Barbuceanu, F., Olaru, O. T. Cytotoxicity evaluation of some compounds from triazole and acylthiosemicarbazide class with potential antimicrobial activity. International Congress of the Medical Sciences, Behavioral Sciences, Education and Physical Rehabilitation from the perspective of Quality of Life, 27-29 mai 2016, Craiova, Volum de rezumate, p. 104-106, ISSN: 2457-2772.
Financing source: Executive Unit for Financing Higher Education, Research, Development and Innovation
Project code: PN-II-ID-PCE-2012-4-0651.
Contract no.: 25/29.08.2013.
In vitro screening of biopharmaceutical limitations and pharmacodynamic/toxicodynamic consequences for oximes
Projects description - abstract: The project addresses a high interest framework from both toxicological and biopharmaceutical perspective. Either tertiary or quaternary oxime structures, the reactivators role in the increase of survival rate for OP-exposed organisms is undoubted, but lately the biopharmaceutical characteristics have been reconsidered as the key features in improving the clinical outcome of the antidotal approach. The results from in-silico prediction of values for the main biorelevant molecular descriptors, solubility experiments in simulated gastro-intestinal fluids, hydrophobicity/lipophilicity determination by various methods (shake-flask, chromatographic and diffusion tests using vertical or side-by-side diffusion cells), partition at various biorelevant interfaces and available pharmaco-/toxicokinetic data modeling will be correlated with the in-vivo effect. The in-silico and kinetic modeling approaches will be also applied to the OP series, underlining the significance of difference and possible correlation with corresponding values predicted for antidotes. The final goal will be an unitary characterization and classification of currently available and future potential new antidotal tools, generated either by single or combined effect of optimal structures. Updated drug products starting from the existing therapeutic tools, possibly associating two oximes will be developed in a biopharmaceutical and toxicokinetic-based attempt to generate a wider if not broad spectrum antidote.
Contractor: University of Medicine and Pharmacy “Carol Davila” Bucharest.
Project Coordinator: Acad.Prof.Dr.Victor A. Voicu.
Prof.Dr.Andrei Valentin Medvedovici, Prof.Dr.Constantin Mircioiu, Prof.Dr.Dumitru Lupuleasa, CPI Dr.Constantin Draghici, Dr.Dalia Simona Miron, Dr.Flavian Stefan Radulescu, Dr.Mirela Adriana Mitu, Dr.Ana Andreea Stanescu, Dr.George Traian Alexandru Burcea Dragomiroiu, Vlad Negulescu, Dr.Dragos Florian Ciolan, Mirona Alexandra Botescu, Oliwer Constantin Hrapciuc, Adrian Gosav, Elena Gosav, Maria Gabriela Puiu.
The final goal will be an accurate, unitary characterization and classification of the currently available and potential new antidotal tools, generated either by single or combined effect of optimal structures, within accurate reactivating triad (including anticholinergic antimuscarinic agents). Secondary, updated drug products starting from the existing therapeutic tools (the Romanian patent OSIM 79914/1974), possibly associating two oximes will be considered in a biopharmaceutical and toxicokinetic-based attempt to generate a wider if not broad spectrum antidote (subject to patent requests developed within project framework).
The project will assess the validation of several in-silico and in-vitro screening tools, integrated in unitary experimental protocols, and intends to offer a set of scientific criteria for the development and selection of new, candidate molecules. Several characteristics of toxicants will be included, since the global toxic processes and the antidotism seem to be characterized by a structural specificity.
The experimental plan will be applied on the most promising series of oximes, further adding biorelevant evaluation to the drug design concept that leaded to their development. Another key issue is the fact that several entities belong to an analog series, thus varying by position of several groups. This will generate important structure solubility or permeability quantitative relationships. Simulating specific condition for several absorption or distribution sites, the project will finally offer information on optimal administration pathway, thus guiding the pharmaceutical formulation process. The solubility, stability and quantification methodologies could also serve as future basis for future quality control monographs.
Nevertheless, the experimental models, classification systems and in vitro in vivo correlations developed throughout the project implementation timeframe could be extended to other low permeability new molecular entities.
Date projects starts: 02/09/2013.
Date projects ends: 30/09/2016.
Projects duration (months): 37.
Total financed value: 1.125.000,00 lei.
Phase 1 - Scientific report, dated 29.11.2013.
Financed value: 166.667 lei
1.1. Abstract on the research activities
1.1.1. Activity 1.1 - Initiation of the database including organophosphorous compounds and reactivating oximes
An evaluation of the molecular descriptors was performed, as well as the screening of the biopharmaceutical properties for a group of drug substances used as antidotes in the intoxication woth organophosphorous compounds. The results of the predictions indicated a limited or absent absorption through the biological membranes for the classical reactivators (Hagedron series), independent on the structural variations. The source of these biopharmaceutical limitations of oximes is their polar structure, a requirement imposed by the necessity to bind the inhibited enzyme, in order to induce its reactivation (nucleophilic attack).
1.1.2. Activity 1.2 - In-silico evaluations of the biorelevant molecular descriptors of the theoretical biopharmaceutical risks
With few exceptions, negative values were observed for n-octanol - water partition coefficient, doubled by high polar surface areas. Moreover, the molecular volume was high, by including at least one pyridinic nucleus. Several structures with increased lipophilicity were noted within the group, a result of adding a hydrocarbon side chain. Even though this design approach leads to a consecutive increase of the molecular volume (limiting, at least theoretically, the partition and absorption), the logP values overpass the threshold value of 3, imposed by the distribution across the blood-brain barrier (octadecyl derivatives having logP values higher than 5).
By applying the Fagerholm’s Permeability Classification System, based on the effective permeability at the intestinal level, most of the oximes seemed to belong to the third class. The antidotes and the toxicants belong to different chemical and structural spaces. The values of the molecular descriptors can be considered an argument supporting the hypothesis that reactivator oximes have a compartment specific biological distribution. Nevertheless, the phosphyl-oximes seem to display a distinct toxicokinetic behavior, similar to organophophorous compounds, having a major contribution to the toxicodynamic profile and correlated with their in-vivo stability.
1.1.3. Activity 2.1 - Evaluation of the chemical stability in experimental conditions simulating the critical phases of the biological evolution
The stability profile of the reactivators with an oxime structure was also analyzed, confirming the accentuated instability of the HI6 type entities, as well as the dependence of the degradation process on the pH or temperature.
1.2.1. Submitted articles:
Voicu V, Sârbu C, Tache F, Micăle F, Rădulescu ŞF, Sakurada K, Ohta H, Medvedovici A. Lipophilicity indices derived from the liquid chromatographic behavior observed under bimodal retention conditions (reversed phase/hydrophilic interaction): application to a representative set of pyridinium oximes. Talanta (2013).
1.2.2. Book chapters (accepted for publication):
Voicu VA, Medvedovici AV, Miron DS, Rădulescu FȘ. Development of an in-vitro assay of apparent permeability across artificial membranes for antidotal oximes (2013)
Phase 2 - Scientific report, dated 03.12.2014.
Financed value: 252.603 lei
2.1. Abstract on the research activities
2.1.1. Activity 1.1 - The evaluation of the partition coefficient in n-octanol / cyclohexane // phosphate buffer pH=7.4 10 mM systems
The partition coefficients in the two binary systems have been experimentally evaluated by the shake-flask method. A group of 41 oximes reactivators of human acethylcholinesterase were assessed in this study, including reference entities (2PAM iodide and chloride, HI6, obidoxime, tertiary structures i.e. DAM and MINA), but also new derivatives with pyridinic nucleus and a side chain, linear or branched, aliphatic or aromatic, with or without the presence of a heterocycle. The biorelevant organic solvents were saturated with phosphate buffers for 24 hours. Concentrations of 40 ppm were used for each analyte, which was added to the aqueous phase. The equilibrium was attained after 48 hours, of each 24 hours under stirring. The quantitative analysis was performed baseon spectrophotometric methods, develop for the concentration interval of 0.2-40 ppm. The results confirmed the increased hydrophilic character of the quanternary structures, but the values of the partition coefficients were lower than previously predicted. The use of cyclohexane induced the concentration into the aqueous phase, since the organic solvent was not able to generate hydrogen bonding. The presence of a linear side hydrocarbon chain was associate with the maximum values of the logD (1.7 for 4PAOd), correlated with its tensioactive bahvior, not with the lipophilicity. The use of buffers with higher concentration induced significant precipitation phenomenon.
2.1.2. Activity 1.2 – The evaluation of the partition coefficient in isopropyl-myristate // phosphate buffer pH=5.4 10 mM systems
The applied experimental procedures were similar to those previously described, the only differences being the composition of the buffer system and its pH value (relevant for the application at the level of the skin). The values of the partition coefficients were negative, in the vast majority of instances, confirming an increased hydrophilic character. The 3PAH type structures or the tertiary oximes have displayed a higher potential of penetration across the simulated biological interface, being of particular interest for formulation designed for local application.
2.1.3. Activity 2.1 – Development of alternative experimental models, using artificial membrane interfaces within vertical diffusion cells
The developed experimental models represented adoption of the protocols currently recommended by the USP in the new chapter 1724 for semisolid dosage forms. The hydrophilic membranes (mean pore diameter 0.45 microns; cellulose acetate) have been soaked for 2 hours in the three biorelevant organic solvents. The protocol was based on vertical diffusion cells system, type Hanson Microette, Hanson Research Inc. (net volume, 12 mL). The experimental results corresponded to the low values of the partition coefficients. A higher permeability was demonstrated in case of the tertiary oximes. The lipophilic derivatives with side chain group at the level of the hydrophobicized interface, due to their tensioactive behavior.
2.1.4. Activity 2.2 – Development of alternative experimental models, using artificial membrane interfaces within horizontal diffusion cells
Evaluations have been performed using horizontal diffusion cells, having stirring elements within both compartments. The membrane conditioning has been performed using the same procedure. The temperature was set at 37, respectively 32oC. The values of the apparent permeability have confirmed the increased potential for distribution at the central level, for both tertiary oximes and lipophilic derivatives.
2.2.1. Published articles
Voicu V, Sârbu C, Tache F, Micăle F, Rădulescu ŞF, Sakurada K, Ohta H, Medvedovici A. Lipophilicity indices derived from the liquid chromatographic behavior observed under bimodal retention conditions (reversed phase/hydrophilic interaction): application to a representative set of pyridinium oximes. Talanta. 2014 May;122:172-9. doi: 10.1016/j.talanta.2014.01.048. Epub 2014 Jan 31.PMID: 24720980.
Voicu V, Rădulescu FS, Medvedovici A. Relationships between the antidotal efficacy and structure, PK/PD parameters and bio-relevant molecular descriptors of AChE reactivating oximes: inclusion and integration to biopharmaceutical classification systems. Expert Opin Drug Metab Toxicol. 2014 Nov 6:1-15. [Epub ahead of print]. PMID: 25373357.
2.2.2. Book chapters
Voicu VA, Medvedovici AV, Miron DS, Rădulescu FȘ. Development of an in-vitro assay of apparent permeability across artificial membranes for antidotal oximes (Chapter 55). In: Dishovsky C, Radenkova-Saeva J. Toxicological Problems. Military Publishing House, Sofia, Bulgaria, 2014: 404-10 (ISBN 978-954-509-509-2).
Rădulescu FS et al. Correlation between rheology, in-vitro release and in-vivo performance of topical dosage forms. Disso Asia 2014 International Symposium, Society for Pharmaceutical Dissolution Science, Mumbai, India, 06.05.2014.
Rădulescu FS et al. Perspectives on in-vitro dissolution/release studies. School of Advanced Studies. Biowaivers, development of in vitro and quality generic drugs. SoAS 2014, Bucharest, Romania, 09.07.2014.
Medvedovici A. Hydrophobicity descriptors resulting from LC bimodal behavior (RP and HILIC). 2nd International Conference on Analytical Chemistry RO-ICAC'2014. Analytical Chemistry for a Better Life, Târgoviște, Romania. 17-21.09.2014.
Stănescu AA, Ciolan DF, Rădulescu FS, Miron DS, Voicu VA. Evaluation of distribution coefficients in biorelevant binary systems for tertiary and quaternary oximes. The National Congress of Pharmacy from Romania, XVth edition, The 10th Symposium of Biopharmacy and Pharmacokinetics. e-Poster, Iasi, Romania, 24-27.09.2014.
Rădulescu FS, Ciolan DF, Minea A, Lupuleasa D, Miron DS. Evaluation of concentration - in-vitro release rate relationship for miconazole nitrate creams. International Symposium on Scientific and Regulatory Advances in Complex Drugs. Poster, Budapest, Hungary, 27-28.10.2014.
Phase 3 - Scientific report, dated 04.12.2015.
Financed value: 305.460 lei
3.1. Abstract on the research activities
3.1.1. Activity 3.1 - Finalizing the assessments of diffusion coefficients across artificial membranes models
The experimental model applied for the in-vitro evaluation of apparent permeability across hydrophilic membranes hydrophobized by soaking into biorelevant organic solvent was optimized. This step included the saturation of n-octanol with saline phosphate buffer pH=7.4, which prevented its elimination from the membrane support. Moreover, the solution of the tested compound was pre-heated at 37°C, whereas the volume was decreased to 2.0 mL. The experimental parameters previously developed were maintained, except for the use of cyclohexane and isopropyl-mirystate. Their relevance was considered by inclusion in the assessment of distribution coefficients. The set of reactivating entities that was previously evaluated was extended by inclusion of 15 newly synthesized substances. Their structure was design after analysis of the available experimental results. It was concluded that the tertiary oximes have an increased permeability through all applied membrane models (e.g. mono-isonitroso-acetone - MINA and diacethyl-monoxime - DAM). The obtained structural variations generated rapid diffusions, the trans-membranar equilibrium being reached rapidly. The apparent permeability varied between 19.19 ± 1.37 10-6 cm/sec for the n-butyl derivative of MINA and 57.48 ± 1.41 10-6 cm/sec. The values of the distribution coefficients (Shake-flask method) were strongly influenced by the nature and position of the substituents placed on the structure of the two reference tertiary oximes. For the cyclohexane - phosphate buffer saline pH=7.4 binary mixture, the lack of hydrogen bonding generated between the organic solvent and the considered entities induced negative values for the LogD, frequently at the borderline of the experimental model’s applicability (mean ± standard deviation: 1.97 ± 0.8; n=3). The potential permeability across biological barriers seems to be increased.
3.1.2. Activity 3.2 - Evaluation of ionization constants
The activity was focused on the experimental evaluation of the ionization constants (pKa values), for both tertiary oxime group that was previously mentioned and a set of structures considered as references based on literature data (including the long clinically experimented pralidoxime and HI6) and on correlation with results obtained during the previous activities within the project. A spectrophotometric method was selected, utilizing a universal buffer system able to generate pH values between 2 and 12. The obtained data for the internal references were in line with literature data. For the substances with long, linear or branched hydrocarbon side chain (pralidoxime derivatives), the ionization constants overpassed the 11 or even 12 threshold, therefore reducing the efficacy of their nucleophilic attack of the anionic species, required by the reactivation mechanism. In the same time, this data is correlated with the available preclinical reports. The new entities had pKa1 values between 5.81 and 9.75, respectively higher than 10.02 (pKa2, for BE1-BE4 elements). One of the preliminary conclusions was that the molecular design applied for the new series allows theoretically the generation of structures with optimal pKa values (necessary for the reactivation of acethylcholinesterases inhibited by organophosphorous compounds), while maintaining the adequate permeability for biodistribution in various compartments of the human body, including the central nervous system.
3.1.3. Activity 3.3 - Classification of toxicants and rectivators according to official systems
The biopharmaceutical classification of reactivating oximes was performed using the definitions of solubility and permeability according to the guidance documents for Biopharmaceutics Classification issued by drug regulatory bodies (Food and Drug Administration, European Medicine Agency) or professional organizations. Moreover, the in-silico and in-vitro generated data was analyzed, as well as current extensions of the regulatory framework (Biopharmaceutics Drug Disposition Classification System - BDDCS). The quaternary oximes were previously administered in oral prophylaxis protocols as immediate or modified release dosage forms. Independently on the composition variables or on the resulting mechanisms of release, the absorption was low but rapid. The elimination is facilitated by the accentuated hydrophilic character, with predominance of renal excretion of the un-metabolized form. Except for tertiary oximes (including the entities from the newly synthesized series) and hydrophobic terms lacking the pharmacodynamic activity, the reactivators - quaternary ammonium compounds belong to the class 3 BCS/BDDCS. We issued the hypothesis of the involvement of efflux transporters. For lipophilic structures with increased permeability across phospholipidic barriers, the intense metabolizing may limit the peripheral and / or central exposure, with the consecutive decrease of the antidotal efficacy.
3.2.1. Published articles (final versions)
Voicu V, Sârbu C, Tache F, Micăle F, Rădulescu ŞF, Sakurada K, Ohta H, Medvedovici A. Lipophilicity indices derived from the liquid chromatographic behavior observed under bimodal retention conditions (reversed phase/hydrophilic interaction): application to a representative set of pyridinium oximes. Talanta. 2014 May;122:172-9. doi: 10.1016/j.talanta.2014.01.048. Epub 2014 Jan 31. PMID: 24720980.
Voicu V, Rădulescu FŞ, Medvedovici A. Relationships between the antidotal efficacy and structure, PK/PD parameters and bio-relevant molecular descriptors of AChE reactivating oximes: inclusion and integration to biopharmaceutical classification systems. Expert Opin Drug Metab Toxicol. 2015 Jan;11(1):95-109. doi: 10.1517/17425255.2015.980813. Epub 2014 Nov 6. Review. PMID: 25373357.
3.2.2. Submitted articles
Shah VP, Rădulescu FS, Miron DS, Yacobi A. Commonality between BCS and TCS. Invited Article, AAPS J. manuscript number: AAPSJ-D-15-00274 (25.11.2015).
Rădulescu FS. The BCS-based approaches in the evaluation of oxime reactivators. Plenary Session 1. Multidisciplinary Approach of the Drug Research. The Congress of University of Medicine and Pharmacy Carol Davila Bucharest. 28-30.05.2015, Bucharest, Romania.
Rădulescu FS. 2nd MENA Regulatory Conference on Bioequivalence, Biowaivers, Bioanalysis, Dissolution and Biosimilars. 15-17.09.2015, Amman, Jordan.
Medvedovici AV, Voicu VA. Chromatographic approaches for evaluation of hydrophobic characteristics of chemicals with emphasis on newly developed separation mechanisms. 19-21.10.2015, Bucharest, Romania.
Rădulescu FS. In-vitro dissolution / release testing of novel and special dosage forms. From Science to Guidance and Practice. 19-21.10.2015, Bucharest, Romania.
Rădulescu FS. Evolution of dissolution methodologies. The current status and perspectives on the revision of compendial dissolution chapters. Solutions and Innovations for the Pharmaceutical Industry. 28-29.10.2015, Bucharest, Romania.
Phase 4 - Scientific report, dated 30.09.2016.
Financed value: 400.270 lei
4.1. Abstract on the research activities
Activity 4.1.1 - Evaluation of solubility in biorelevant media
The preliminary assay by equilibrium (thermodynamic) solubility assessment, but also by high-throughput screening confirmed the literature data, through the pH independency of this parameter in fluids simulating the pH of proximal regions of the gastro-intestinal tract. Saturated solution were not achievable within the usual limits of quantitative ratios between the analyte and compendial buffer systems. For the entities with aliphatic side chain, the assessment was difficult because of their increased tendency of supersaturation or of their tensioactive behavior, i.e. the specific outcome at concentration above the micelle level. The isonitrosonacetanilide structures had lower solubility (1.2 to 2.4 mg/mL), representing isolated cases for which the endogenous tensioactives might have a significant impact.
Activity 4.2.1 - Estimation of the impact of pKa values on permeability and protein / AChE binding
The acido-basic character of the oximes has been correlated with their specific reactivating action. The efficacy is rather difficult to prospect through the structural characteristics, especially in case of quaternary substances with polarity increased by the presence of several pyridinic nucleus. The oxamate anion is involved in the nucleophilic attack and finally in the removal of the phosphoryl fragment.
The isonitrosoacetanilide entities newly synthesized benefit from pKa values favourable to the reactivating activity, especially the terms with dissociation constants between 7.57 and 7.79 (slightly higher than the in silico predictions). The protein binding is extremely reduced, but higher compared to quaternary oximes, based on the increase in lipophilicity.
Activity 4.3.1 - Classification according to unofficial classification systems
Before applying the unofficial classification system, the database of compounds has been update, using two complementary procedures. Firstly, the included entities have been seprataed into two distinct categories, based on the rough assessment of their polarity: tertiary oximes, having as leading standard structures such as monoisonitroso-acetone or diacethyl-monoxime, including the subset of isoniotrosoacetone, respectively the quaternary oximes, illustrated by mono- and bis-pyridinium compounds, e.g. 2-pralidoxime, obidoxime, trimedoxime or HI6. Next, a global scaling of the physicochemical parameters, biopharmaceutical characteristics and molecular descriptors was performed, by considering the experimental data generated during the project (logP, logD, pKa, apparent permeability). Secondly, an extensive literature research was conducted, focusing on the new entities with potential application as reactivators (data published after the initial enunciation of the utility of biopharmaceutical screening criteria. We focused on the Extended Clearance Classification System and Permeability Classification System.
The results conformed the pronounced tendency of designing and synthesizing new reactivators with imporved brain distribution, which may in turn have promiscuous spectra of activity, as well as increased metabolic liability.
Activity 4.4.1 - Correlated analysis of pharmaco-toxico kinetic/dynamic database and identification of specific and relevant parameters
The correlated analysis of the database for the identification of specific and biorelevant parameters was performed after preliminary centralizing of data on the physic chemical constants (experimental data from literature or generated in house for the aqueous solubility, boiling or melting point, partition or distribution coefficients in n-octanol - water binary systems etc.) and lethal doses (LD50, for different species, genders and administration pathways).
The interval of the values for biorelevant descriptors, as well as for ADME parameters and toxicity were particularly large. For example, the 550 compounds with potential antidotal activity in the treatment of oprganophosphorous poisoning described an interval of the partition coefficient of 18.35 units (between -12 for entities with four pyridinic nucleus and multiple inserted functional groups and +6.35, for heterodimers of tetrahydroacridine with 3-hydroxy-2-pyridine aldoximes, having a high molecular weight and increased hydrophobic character). The interval for the distribution coefficient at physiologic pH was 1 unit larger (-12.85 - 6.34).
Activity 4.4.2 - Development of a new classification system, based on combined biopharmaceutic and toxicological criteria
New classification criteria was proposed, including the composite CNS MPO score and the molecular weight or the pKa value described previously as optimal for the reactivation and adapted. CNS MPO was calculated by scaling the contribution of the permeability and distribution coefficients (determined at pH=7.4), the molecular weight and topological polar surface area, pKa value and number of hydrogen bond donors (HBD). The limit value associated with the permeability across blood-brain barrier was 5, with an interval of intermediate penetration (both as rate and extent) of 2-5 (according to the proposal of Wager TT et al, 2016). For the dissociation constant, an optimal region was proposed, based on the particularities of tertiary entities with zwitterionic profile. It resulted a classification system with 9 apparent subgroups, combining the BB+ potential with minimal structural requirements for reactivation at the level of the central nervous system.
Activity 4.5.1 - The screening of new reactivating associations, with large spectra
The combination of mono and bis-quaternary oximes have been suggested as an alternative to the considerable efforts in the design of new entities with better activity (improved survival rate, even when prolonged exposure to large doses of toxic agents) and/or enlarged spectra (especially considering as biomarker the reactivation of AChE inhibited by tabun or soman.
The stability of several isonitrosoacetanilide representatives newly synthesized and subject to a preliminary screening, the adequate solubility in aqueous media doubled by a moderate lipophilic character, accurate distribution into the central nervous system (demonstrated in silico and confirmed by experimental evaluation of the ΔlogP parameter in relevant organic solvents) triggered their inclusion into binary mixtures, beside a quaternary oxime with proved reactivating ability (preferably a K-oxime, pralidoxime or HI6; Voicu VA et al, 2016).
This mixtures were subject of a patent request, submitted to the Romanian authorities (Voicu VA, Medvedovici AV, Rădulescu FS, Miron DS, Drăghici C. Association of tertiary and quaternary oximes as large spectra reactivators of acethylcholinesterase; A/00689/30.09.2016).
4.2.1. Published articles
Voicu VA, Medvedovici AV, Sakurada K, Ohta H, Rădulescu FȘ, Miron DS. The forgotten or underestimated relevance of biopharmaceutical-based assessments for the oral absorption studies of oxime reactivators. Expert Opin Drug Metab Toxicol. 2016 Jul;12(7):743-52. doi: 10.1080/17425255.2016.1179282. Epub 2016 May 4. PMID: 27144662.
Shah VP, Rădulescu FŞ, Miron DS, Yacobi A. Commonality between BCS and TCS. Int J Pharm. 2016 Jul 25;509(1-2):35-40. doi: 10.1016/j.ijpharm.2016.05.032. Epub 2016 May 18. PMID: 27208656.
Iorgulescu E, Voicu VA, Sârbu C, Tache F, Albu F, Medvedovici A. Experimental variability and data pre-processing as factors affecting the discrimination power of some chemometric approaches (PCA, CA and a new algorithm based on linear regression) applied to (+/-)ESI/MS and RPLC/UV data. Talanta. 2016 Aug 1;155:133-44. doi: 10.1016/j.talanta.2016.04.042. Epub 2016 Apr 21. PMID: 27216666
Rădulescu FŞ. In vitro methods and classification systems – an open discussion about the future. Plenary Session 1. Pharmaceutical Sciences - A key Stone of Drug Development. Congress of the University of Medicine and Pharmacy Carol Davila Bucharest, Interdisciplinary Perspectives, 4th Edition, Palace of the Parliament, 2-4 June 2016, Bucharest, Romania.
Rădulescu FȘ, Miron DS. 2nd Sotax Congress 2016. Testing Innovations for the Pharmaceutical Industry. 20-21 September 2016, Basel, Switzerland.
Rădulescu FȘ, Voicu VA. Drug Release Tests for Special Dosage Forms. The Second Edition of the International Conference From Science to Guidance and Practice. Library of the Romanian Academy, 6-7 June 2016, Bucharest, Romania.
4.2.3. Patent requests
Voicu VA, Medvedovici AV, Rădulescu FS, Miron DS, Drăghici C. Asocieri de oxime terțiare și cuaternare ca reactivatori cu spectru larg ai acetilcolinesterazei. A/00689/30.09.2016.
Dezvoltarea unui medicament pe bază de plante cu administrare topică în dermatoze alergice
Development of a herbal medicinal product with topical administration in allergic dermatitides
DIRECTOR PROIECT: PROF. DR. MIHAELA DINU
- UNIV.DE MEDICINA SI FARMACIE “CAROL DAVILA”, BUCUREŞTI
- HOFIGAL EXPORT IMPORT SA
- AGROECO BIOTERRA SRL
- UNIVERSITATEA DE STIINTE AGRONOMICE SI MEDICINA VETERINARA
Titlul proiectului (Romana): Studii privind corelaţiile sol-plantă-aliment-om în vederea obţinerii unui supliment alimentar cu un conţinut crescut în fier de origine vegetală
Acronimul proiectului: SALIFEROVEG
Titlul proiectului (Engleza): Studies on soil-plant-food-man correlations to obtain a food supplement with an increased content in iron of herbal origin
Abstract (Romana): Anemia feriprivă (AF) este cea mai răspândită formă de anemie din lume şi cea mai frecventă formă a anemiei la femeile gravide. Se estimează că AF are o prevalenţă de 2% la bărbaţii adulţi şi 9–20% la femeile adulte. Numeroasele efecte negative ale anemiei asupra sănătăţii şi calităţii vieţii justifică intervenţiile destinate să prevină şi să controleze anemia, iar cea mai importantă dintre acestea este utilizarea de suplimente alimentare cu fier.
Obiectivul principal al proiectului constă în dezvoltarea unui supliment alimentar cu fier, utilizând fier în forma sa naturală, sub forma unui extract din plante bogat în acest oligoelement, iar în acest scop se vor explora corelaţiile dintre sol, plante şi procesele de extracţie care vor sta la baza suplimentului alimentar dezvoltat, pentru a asigura o utilizare optimă la om. Există foarte puţine suplimente alimentare de origine vegetală care conţin fier pe piaţa românească (majoritatea celor disponibile conţin săruri de fier). Există doar un supliment alimentar de origine chineză cu fier (10 mg/comprimat) şi acid folic, fierul provenind dintr-un aşa-numit "praf negru de fungi" (fără nici o identificare taxonomică a speciilor de fungi) şi unul fabricat la nivel local (cu două formule uşor diferite), dar cu o cantitate foarte mică de fier ("Fier biologic", care asigură un aport de Fe mai mic de 1,0 mg pe capsulă).