Antibiotics in the environment and their stereochemistry: from

N Cl

N molecules to the Cl

* O

O * catchment perspective O O

F COOH

N

N N

Cl N *

H O Barbara Kasprzyk-Hordern H 2N Contributors: E. Castrignano, F. Elder, D. Kibbey, K. Proctor, J. Rice >6700 drugs <200 drugs studied in the environment

WHY PHARMA? ■ Biological activity ■ Non-targeted action ■ Pseudo-persistence ■ Synergistic effects ■ Endocrine disruption ■ Antimicrobial resistance ■ Increased use with growing and ageing population

WHY CHIRAL PHARMA? ■ >50% of pharmaceuticals are chiral ■ Evidence on enantioselective toxicity in humans ■ No tests at enantiomeric level required in ERA (+)-enantiomer (-)-enantiomer Sedative Teratogen Importance of chirality: Investigation of HIGH Chiral Active Substances MANUFACTURE LOW EU Directive 75/318/EEC

DEVELOPMENT DISTRIBUTION PHARMA LIFE CYCLE MATERIALS USE (HUMAN & VET)

ERA for Vet MPs EU Directive 81/852/EEC DISPOSAL ERA for Human MPs EMEA/CHMP/203831/2005 Chiral pharma in the environment CONSUMPTION

WASTEWATER ENVIRONMENT Racemic chiral drug TREATMENT DEGRADATION

EXCRETION DISCHARGE Non-racemic Non-racemic drug & drug & by-

metabolism metabolites products I Stereoselective

DRINKING WATER TREATMENT CONSUMPTION WATER WITHDRAWAL Non-racemic Non-racemic drug & drug & by- by-products III products II

The enantiomeric signature of a chiral molecule can change throughout its environmental life-cycle affecting its fate and toxicity ■ Antidepressants: Fluoxetine, venlafaxine, mirtazapine, citalopram and their metabolites ■ Stimulants: Ephedrine/pseudoephedrine ■ NSAIDs, analgesics: Ibuprofen, tramadol CHIRAL ■ Beta-blockers: Atenolol, propranolol, metoprolol, betaxolol, pindolol, sotalol, timolol, carazolol, alprenolol, PHARMA ■ Beta-adrenergic agonists: Salbutamol, clenbuterol, terbutaline ■ Antiarrhythmic agents: Mexiletine ■ Illicit drugs: Amphetamine, methamphetamine, MDMA, MDA, MDEA OUR OBSERVATIONS:  Chiral drugs are enriched with one enantiomer EXPOSURE  Stereoselective fate of ERA chiral drugs in the HAZARD environment is observed OH

* CH3 Enantioselective transformation * HN of ephedrine in water and its CH 3 ecotoxicological relevance Ephedrine/ Pseudoephedrine

OH H OH H 1S, 2S(+)- H H 1R,2R(-)-Pseudoephedrine N N Pseudoephedrine • Synthetic enantiomer CH3 CH3 • Natural enantiomer H H • Humans: similar activity to its enantiomer • Humans: weak CNS CH3 CH3 effects, decongestant 1S,2S(+)-pseudoephedrine 1R,2R(-)-pseudoephedrine

OH H OH H 1S,2R(+)-Ephedrine H H 1R, 2S(-)-Ephedrine N N • Synthetic enantiomer • Natural enantiomer CH3 CH3 • Humans: weak activity on H H • Humans: bronchodilator β1,2 and 3 adrenoceptors CH3 CH3 and abused stimulant 1S,2R(+)-ephedrine 1R,2S(-)-ephedrine

1R,2S(-)-ephedrine Enantioselective transformation Department of Chemistry of ephedrine in water and its 1S,2S(+)-pseudoephedrine ecotoxicological relevance OH

* CH3 * HN CH3 1S,2R(+)-ephedrine Ephedrine/ Pseudoephedrine 1R,2S(-)-Ephedrine and 1S,2S(+)-pseudoephedrine loads in sewage (sum of loads from 7 WWTPs) RP-chiral (CBH) LC-QqQ (H2O, 1 mM NH4OC, 10% IPA) RP (C18)-UPLC-QqQ

1R,2S(-)-ephedrine is more toxic to Daphnia Magna and Selenastrum Capricornutum and Tetrahymena Thermophila than 1S,2S(+)- pesudoephedrine

B. Kasprzyk-Hordern, D. Baker, STOTEN, 423 (2012) 142 OH H OH H H H N N CH CH EnantioselectiveH 3 transformationH of 3 pseudoephedrineCH3 in river simulatingCH3 microcosms 1S,2S(+)-pseudoephedrine 1R,2R(-)-pseudoephedrine

OH H SYNTHETICOH H H H OH H OH EphedrineH Pseudoephedrine NATURALOH H OH H N H N 1.6H 1.0 0.9 1.0 H H CH3 N CH3 N N N H H CH CH 0.9 0.8 0.9 CH CH 3 1.4 3 H 3 H 3 CH3 CH3H H 0.8 0.7 0.8 CH CH CH 1.2 CH 3 3

3 ephedrine 3 1S,2S(+)-pseudoephedrine 1R,2R(-)-pseudoephedrine - 0.7 0.7

0.6 pseudoephedrine

g/L] g/L]

- 1S,2S(+)-pseudoephedrine 1R,2R(-)-pseudoephedrine

µ ) 1S,2R(+)-ephedrine 1R,2Sµ 1.0 (-)-ephedrine

0.6 - 0.6 0.5 0.8 0.5 0.5 OH H OH H 0.4 0.4 SYNTHETIC H H 0.6 OH 0.4H OH H

N N 0.3 H H

Concentration[

Concentration[ EnantiomericFraction 0.3 EnantiomericFraction 0.3N N CH3 CH3 0.4 H H 0.2 CH CH 0.2 0.2H 3 H 3 CH3 CH3 0.2 0.1 0.1 C0.1H3 CH3 1S,2R(+)-ephedrine 1R,2S(-)-ephedrine 0.0 0.0 0.0 0.0

Enrichment with 1S,2R(+) Enrichment 1S,2S(+)-pseudoephedrine 1R,2R(-)-pseudoephedrine 0 1 4 5 6 7 8 11 12 13 14 0 1 4 5 6 7 8 11 12 13 14 NATURAL Time [day] Time [day] with 1R,2R( Enrichment

Stereoselective degradation of 1R,2S-(-)-ephedrine and 1S,2S-(+)-pseudoephedrine under biotic conditions in river simulating microcosms

1R,2R(-)-pseudoephedrine 20 x more toxic to Tetrahymena Thermophila

Rice et al, J Hazard Materials 348 (2018) 39 Enantioselective transformation of fluoxetine in wastewater and Activated sludge simulating microcosms -1 120 FL 100 µg L - DBR 1.0 NFL 100 µg L-1 - DBR its ecotoxicological relevance 8.0 1.0

100 7.0 0.8 0.8

6.0

) 1

- 80

)

1 - 0.6 5.0 0.6 60 4.0 0.4 0.4

3.0 Concentration (µg L

40 EnantiomericFraction

Concentration (µg L EnantiomericFraction 2.0 0.2 20 0.2 1.0

0 0.0 0.0 0.0 0 0.5 1 1.5 2 3 5 8 12 24 0 0.5 1 1.5 2 3 5 8 12 24 Time (hours) Time (hours) EC5028h (T. thermophila)

Despite the overall decrease in FL

concentration accumulation of toxic

FL

NFL

-

-

) -

(R)-FL and formation of toxic (S)-NFL (

-

(+)

- R

in activated sludge leads to higher than S Enrichment with Enrichmentwith estimated toxicological effects Enrichmentwith

MJ Andres et al., Sci Reports 7 (2017) 15777 Pan-European monitoring of quinolones (ABs: ciprofloxacin, ofloxacin, lomifloxacin, norfloxacin, nalidixic acid, moxifloxacin, ulifloxacin and flumequine) and qnrS gene in wastewater.

N Cl

N Cl

* O

O *

O

O

F COOH

N

N N

Cl N *

H O H 2N

• Spatial distribution of quinolones • Temporal trends • Daily loads and potency • Consumption vs direct disposal (by considering human metabolism)

Castrignanò, et al., Enantiomeric profiling of quinolones and quinolones resistance gene qnrS in European wastewaters, Water Research, 2019, submitted. NERC AMR in the Real World Impact of stereochemistry of antimicrobial agents on their environmental fate, biological potency and the emergence of resistance Our hypothesis: Stereochemistry of AAs determines their environmental fate and biological effects

RR-(-)- chloramphenicol was marketed in 1950s as the most potent isomer

Enterococcus faecalis RR-(-)- with chloramphenicol chloramphenicol is acetyltransferase not the most potent selective towards RR- isomer anymore! CAP only

[email protected] ACKNOWLEDGMENTS NETWORKS EnvironmentalAcknowledgments Chemistry @ Bath

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