EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF); Scientific Opinion on Flavouring Group Evaluation 12, Revision 2 (FGE.12Rev2): Primary saturated or unsaturated alicyclic alcohol, aldehyde, acid, and esters from chemical group 7

The Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids of the European Food Safety Authority was requested to evaluate 10 flavouring substances in the Flavouring Group Evaluation 12 (FGE.12), including an additional substance in revision 3, using the Procedure in Commission Regulation (EC) No 1565/2000. This revision is made due to inclusion of one additional flavouring substance, 2,6,6-trimethylcyclohex-2-ene-1-carboxaldehyde [FL-no: 05.182]. None of the substances were considered to have genotoxic potential. The substances were evaluated through a stepwise approach (the Procedure) that integrates information on structure-activity relationships, intake from current uses, toxicological threshold of concern, and available data on metabolism and toxicity. The Panel concluded that all 10 substances [FL-no: 02.134, 02.186, 05.157, 05.182, 05.183, 05.198, 08.135, 09.342, 09.670 and 09.829] do not give rise to safety concerns at their levels of dietary intake, estimated on the basis of the MSDI approach. Besides the safety assessment of these flavouring substances, the specifications for the materials of commerce have also been considered. Specifications including complete purity criteria and identity for the materials of commerce have been provided for all 10 candidate substances

Approaches to soft drug analogues of dihydrofolate reductase inhibitors : Design and synthesis

The main objective of the research described in this thesis has been the design and synthesis of inhibitors of the enzyme dihydrofolate reductase (DHFR) intended for local administration and devoid of systemic side-effects. The blocking of the enzymatic activity of DHFR is a key element in the treatment of many diseases, including cancer, bacterial and protozoal infections, and also opportunistic infections associated with AIDS (Pneumocystis carinii pneumonia, PCP). Recent research indicates that the enzyme also is involved in various autoimmune diseases, e.g., rheumatoid arthritis, inflammatory bowel diseases and psoriasis. Many useful antifolates have been developed to date although problems remain with toxicity and selectivity, e.g., the well-established, classical antifolate methotrexate exerts a high activity but also high toxicity. The new antifolates described herein were designed to retain the pharmacophore of methotrexate, but encompassing an ester group, so that they also would serve as substrates for the endogenous hydrolytic enzymes, e.g., esterases. Such antifolates would optimally comprise good examples of soft drugs because they in a controlled fashion would be rapidly and predictably metabolized to non-toxic metabolites after having exerted their biological effect at the site of administration. A preliminary screening of a large series of simpler aromatic esters as model compounds in a biological assay consisting of esterases from different sources was performed. The structural features of the least reactive ester were substituted for the methyleneamino bridge in methotrexate to produce analogues that were chemically stable but potential substrates for DHFR as well as for the esterases. The new inhibitor showed desirable activity towards rat liver DHFR, being only eight times less potent then methotrexate. Furthermore, the derived metabolites were found to be poor substrates for the same enzyme. The new compound showed good activity in a mice colitis model in vivo, but a pharmacokinetic study revealed that the half-life of the new compound was similar to methotrexate. A series of compounds characterized by a high lipophilicity and thus expected to provide better esterase substrates were designed and synthesized. One of these analogues in which three methoxy groups were substituted for the glutamic residue of methotrexate exhibited favorable pharmacokinetics. This compound is structurally similar to another potent DHFR inhibitor, trimetrexate, used in the therapy of PCP (vide supra). The new inhibitor that undergoes a fast metabolism in vivo is suitable as a model to further investigate the soft drug concept

Synthesis and enzymatic hydrolysis of esters, constituting simple models of soft drugs

One way to minimise systemic side effects of drugs is to design molecules, soft drugs, in such a way that they are metabolically inactivated rapidly after having acted on their pharmacological target. Hydrolases (esterases, peptidases, lipases, glycosidases, etc.) are enzymes well suited to use for drug inactivation since they are ubiquitously distributed. Insertion of ester bonds susceptible to enzymatic cleavage may represent one approach to make the action of a drug more restricted to the site of application.The present study describes the chemical synthesis of fourteen model compounds comprising a bicyclic aromatic unit connected by an ester-containing bridge to another aromatic ring. Initial attempts to define a) the tissue selectivity of the hydrolytic metabolism and b) the molecular structural factors affecting the rate of enzymatic ester cleavage are presented.The data show that human and rat liver fractions were more active than human duodenal mucosa and human blood leukocytes at a hydrolysing the compounds. The rank order of the compounds was, however, very similar in the different biological systems. Commercially available pig liver carboxyl esterase and cholesterol esterase both reasonably well predict the rank order in the tissue fractions

Computational predictions of binding affinities to dihydrofolate reductase: synthesis and biological evaluation of methotrexate analogues

The relative binding affinities to human dihydrofolate reductase of four new potential antifolates, containing ester linkages between the two aromatic systems, were estimated by free energy perturbation simulations. The ester analogue, predicted to exhibit the highest binding affinity to human dihydrofolate reductase, and a reference ester (more structurally related to methotrexate) were synthesized. As deduced from the measured IC(50) values, the calculated ranking of the ligands was correct although a greater difference in affinity was indicated by the experimental measurements. Among the new antifolates the most potent inhibitor exhibited a similar pharmacokinetic profile to methotrexate but lacked activity in a complex antiarthritic model in rat in vivo

Design, synthesis and evaluation of 2,4-diaminoquinazolines as inhibitors of trypanosomal and leishmanial dihydrofolate reductase

This paper describes the design, synthesis and evaluation of a series of 2,4-diaminoquinazolines as inhibitors of leishmanial and trypanosomal dihydrofolate reductase. Compounds were designed by a generating virtual library of compounds and docking them into the enzyme active site. Following their synthesis, they were found to be potent and selective inhibitors of leishmanial dihydrofolate reductase. The compounds were also found to have potent activity against Trypanosoma brucei rhodesiense, a causative organism of African trypanosomiasis and also against Trypanosoma cruzi, the causative organism of Chagas disease. There was significantly lower activity against Leishmania donovani, one of the causative organisms of leishmaniasis. (c) 2005 Elsevier Ltd. All rights reserved.</p