Boronic acids: a novel class of bacterial mutagen

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Jerome Nash
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1 oronic acids: a novel class of bacterial mutagen Mike Donovan Lhasa virtual International Collaborative Group Meeting 16 th May 2012 M.R. Donovan, C.D.Mee, S.enner, A.Teasdale and D.H.Phillips Mutation Research 2011; 724: 1-6

oronic acids Important building blocks in organic synthesis Key intermediates in the preparation of drugs Main advantages: Can be used in carbon-carbon bond-forming methods Readily available Stable

2 oronic acids Important building blocks in organic synthesis Key intermediates in the preparation of drugs Main advantages: Can be used in carbon-carbon bond-forming methods Readily available Stable in air and water React under mild conditions Inorganic boron by-products easily removed Couplings have high regio- and stereo-selectivity Are potential impurities in drug substances 2 MR D 16 May 2012

3 oronic acid properties + 3H 2 H H H H + H 2 Readily converted to trimeric boroxines by dehydration Rapid reconversion in aqueous medium Monomeric species stabilised by conversion cyclic boronate esters Generally acids, esters and boroxines can be used interchangeably 3 MR D 16 May 2012

4 oronic acids in synthesis Transition metal-catalysed reactions Me + (H) 2 r S Me S S S H Pd() H (H) 2 + S Rh(I) S 4 MR D 16 May 2012

5 oronic acids bind to cis-diols inding is reversible In carbohydrates in general and particularly ribonucleosides Can separate ribonucleoside/deoxyribonucleoside mixtures HCH H H 2 A,U,G or C HCH 2 H H + A,U,G or C 5 MR D 16 May 2012

6 acterial mutagenicity - AZ S.typhimurium Ta1535, TA1537, TA98, TA100 and E.coli WP2uvrA( pkm101) Positive only for TA100 and/or E.coli ± S9 except TA1537 x2.6 +S9 6 MR D 16 May 2012

7 acterial mutagenicity - Pfizer Some also positive for TA1537 usually ± S9 rom poster presented at EMS meeting, ort Worth, 2012 with thanks to Michelle Kenyon 7 MR D 16 May 2012

8 Mechanism of action - bacteria H H o evidence of DA binding by 32 P post-labelling TA100 and E.coli ase-pair substitution in G-C and A-T sequences Dependent on error-prone repair TA1537 Different mechanism? Intercalation? Role of S9 metabolism? Mechanism not understood real genotoxic hazard? Prudent to control boronic acid impurities to TTC levels 8 MR D 16 May 2012

9 oronic acids eukaryotic mutagens? Are boronic acids genotoxic only in bacteria? o need to control impurities if no risk in mammalian cells Initial results showed no activity in the mouse lymphoma TK assay (up to 1 mmol/l) H H 9 MR D 16 May 2012

10 Collaborative data sharing project Participants ayer Healthcare Pharmaceuticals Andreas Sutter GlaxoSmithKline Angela owen, Amanda Giddings, Jim Harvey ovartis Pharmaceuticals Susanne Glowienke Pfizer Inc. igel Greene, Michelle Kenyon, Russell aven Sanofi Aventis Alexander Amberg, Andreas Czich, Hans-Peter Spirkl Hoffmann-La Roche Alessandro rigo, Wolfgang Muster Astra Zeneca Andy Teasdale, Scott oyer, Catrin Hasselgren, Mike Donovan 10 MR D 16 May 2012

11 Proposed studies ayer Healthcare Pharmaceuticals Micronucleus and comet tests in V79 cells ovartis Pharmaceuticals Micronucleus test in TK6 cells. Comet assay in human lymphocytes Pfizer Inc. Micronucleus test in CH cells (possibly also in TK6 cells) Sanofi Aventis Micronucleus test in TK6 or mouse lymphoma cells Hoffmann-La Roche Micronucleus and comet tests in mouse lymphoma cells (possibly also in TK6 cells) Astra Zeneca TK, micronucleus and comet tests in mouse lymphoma cells 11 MR D 16 May 2012

12 AZ test results H H Mouse lymphoma TK assay 3 hours S9 3 hours +S9 24 hours -S9 mmol/l Survival % RTG M X10-6 DMS P mmol/l Survival % RTG M X10-6 DMS P mmol/l Survival % RTG M X10-6 DMS Q P Precipitate at the end of treatment CPA Q MR D 16 May 2012

13 AZ test results H H Mouse lymphoma micronucleus and comet assays 3 hours S9 3 hours +S9 mmol/l Survival % RSG M per 1000 % Tail intensity mmol/l Survival % RSG M per 1000 % Tail intensity DMS P Q DMS P CPA P Precipitate at the end of treatment 13 MR D 16 May 2012

14 AZ test results H H Mouse lymphoma micronucleus assay 24 hours exposure mmol/l Survival % RSG M per 1000 DMS P Q Conclusions o evidence of genotoxicity in mouse lymphoma TK or comet assays ±S9 Micronucleus tests will be repeated and include centromere staining 14 MR D 16 May 2012

15 Questions 15 MR D 16 May 2012

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6 November 2014 EMA/CVMP/VICH/526/2000 Committee for Medicinal Products for Veterinary Use (CVMP) VICH GL23: Studies to evaluate the safety of residues of veterinary drugs in human food: genotoxicity testing