Can DBS stabilize ester pro-drugs and glucuronide metabolites? Dr. Katja Heinig, Almudena Gajate Pérez, Thomas Wirz & Franz Bucheli F. Hoffmann-La Roche Ltd, Non-Clinical Safety, Bioanalytical Section, Basel, Switzerland
Background • Enzymes naturally present in biological samples degrade unstable pro-drugs and metabolites. • Accurate determination is necessary for pharmacokinetic / toxicokinetic studies in Drug Discovery & Development or later in therapeutic drug monitoring. • Conventional analyses need the addition of inhibitors or cooling the samples. • Can DBS be a solution? Can new treated cards containing agents which degrade enzymes stop the degradation? Almudena Gajate Perez 2
Compounds under study • Two ester pro-drugs: Valganciclovir (Valcyte), and Oseltamivir (Tamiflu), both anti-viral drugs. • One glucuronide: Mycophenolic acid acylglucuronide a metabolite from Cellcept, an immunosuppressant drug. • One dihydropyrimidinone drug, sensitive to hydrolysis. • LC-MS/MS was used for compound quantification. • Experiments were performed in duplicate or triplicate. • At least partial method validation was carried out for Valcyte, Tamiflu, and Cellcept. Almudena Gajate Perez 3
Valcyte • The pro-drug “Valganciclovir” is rapidly converted into an active metabolite “Ganciclovir” by intestinal and hepatic esterases. O N O H N N Enzymes H N N H 2 N N N H 2 N O N OH O OH O H O * H 2 N O Valganciclovir Ganciclovir • Conventional analysis implies working on ice. • What about DBS? Almudena Gajate Perez 4
Stability in blood versus DBS DBS Stability in rat blood Stability of Valganciclovir 16 100 14 Rat blood Conversion (%) 12 80 Conversion (%) Human blood T=0 10 60 24 hours 8 6 40 4 2 20 0 0 Ahlstrom DMPK-A DMPK-B 0 15 30 60 120 240 360 FTA Time / min Card type • Valganciclovir is not stable in whole blood. • Not stable on Ahlstrom & DMPK-A. • Good stability in FTA Elute & DMPK-B during 24h. • Treated cards clearly improve short-term stability. Almudena Gajate Perez 5
Long-term stability • Valganciclovir is not stable over a long term on treated or untreated cards in rat blood. • Do “fast drying” (cool air stream) or additives improve stability? • Either the addition of acid or speeding-up the drying process reduces the instability of the pro-drug in a long-term. • Nevertheless the turn-over remains significant due to chemical hydrolysis. Rat blood stability on Ahlstrom cards Rat blood stability on FTA cards 60 60 58 49 51 46 50 50 Untreated blood Conversion (%) Conversion (%) 40 1% Acetic A 40 40 38 35 Fast dried cards 30 30 24 Untreated blood 26 22 16 20 1% Acetic A 20 16 11 14 10 2% Acetic A 15 10 10 10 10 3 10 Fast dried cards 7 5 0 0 2 0 5 10 15 20 25 30 35 0 5 10 15 20 Time / days Time / days Almudena Gajate Perez 6
Tamiflu • Tamiflu, an ethyl ester pro-drug, degrades rapidly into its carboxylic metabolite in rat blood. O O OH O Enzymes NH 2 NH 2 O O O NH O NH Tamiflu Metabolite • Conventional analysis needs the addition of dichlorvos. • Is DBS a solution? Almudena Gajate Perez 7
Stability in blood versus DBS Short & long term DBS stability in rat blood Stability of Tamiflu 50 100% 45 40 Conversion (%) Rat blood Drying process (T=0) 80% Conversion (%) 35 Human Blood 9 days 30 60% 25 21 days 20 40% 15 10 20% 5 0 0% m A o - B r K t - A s K P 0 15 30 60 180 l h T M P A F M D D Time (min) Card type • Tamiflu is not stable in rat blood. • Not stable on Ahlstrom. Good stability during the drying process on FTA Elute & DMPK-B & A. • Tamiflu remains stable in FTA & DMPK-B cards for at least 3 weeks, less than 5% conversion. Almudena Gajate Perez 8
MPA Acyl-glucuronide • Acyl-glucuronide metabolite converts back into the parent drug MPA. Critical when the metabolite is formed above 15% of MPA. • Cooling the samples or the addition of acid is necessary to avoid its back – conversion to MPA. OH OH H O OH OH O O O OH O O O O OH O O O O MPA Acylglucuronide (metabolite) Mycophenolic acid (MPA) Almudena Gajate Perez 9
Stability in blood versus DBS • The metabolite is unstable in human blood. After 15 min the response decreases by 12%. • On DBS it is stable for only 1 day on treated cards and up to 5 days on plain cards. • Significant instability after 6 weeks: 25% conversion on untreated and almost complete conversion on treated cards. DBS stability in treated blood and/or cool storage 14 DBS stability in human blood 100 12 T=0 90 Conversion (%) 1 week 10 80 two weeks Conversion (%) 8 70 Ahsltrom 60 6 DMPK-B 50 4 40 30 2 20 0 10 d d C C i i C C c c ° ° a a 4 ° ° 4 4 4 Conditions 0 % % ; ; m B . d d 5 5 - i o i c . . K c 0 0 r a a t P T=0 s 1 day m % M % 2 days B l h 5 days 14 days 42 days o 5 - A D 5 K r . t . 0 l P 0 s M h m B A Time D - o K r t P l s M h A D Almudena Gajate Perez 10
Reactive drug • The hydrolysis of a dihydropyrimidinone drug is studied in human / mouse blood and in human plasma • Not stable in mouse blood, stable in DBS? • Stable in human blood but not in plasma, stable in DPS? Stability in human plasma versus DPS Stability in mouse blood versus DBS 100 110 100 81 80 84 80 % Response % Response 65 60 64 60 Whole blood 53 Human plasma 40 Ahlstrom 40 Ahlstrom DPS DMPK-B DPS 20 20 0 0 0 5 10 15 20 25 30 0 5 10 15 20 Time / hours Time / hours Stability is enhanced on Ahlstrom cards. Stable on untreated cards. Unstable on treated cards. Almudena Gajate Perez 11
Conclusions • DBS technique generally improves stability of compounds over a short term. A long-term stability was proven to be compound dependant. • Stability of reactive compounds cannot be taken for granted on DBS and each case should be studied independently. • Recommendations in case of stability issues: select appropriated card type, use fast drying and consider low temperature storage or the addition of stabilization agents. • The coating of treated cards may stop enzymatic activity but may not stop chemical degradation. • Fast drying and additives improves stability, but does it nullify DBS advantages? Almudena Gajate Perez 12
Acknowledgments • Berthold Lausecker • Katja Heinig, Thomas Wirz and Franz Bucheli • Andreas Gloge & Justyna Menzel • Hoffmann- La Roche Almudena Gajate Perez 13
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