Can DBS stabilize ester pro-drugs and glucuronide metabolites? Dr. - - PowerPoint PPT Presentation

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

Almudena Gajate Perez 2

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 3

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 4

Valcyte

• The pro-drug “Valganciclovir” is rapidly converted into an active

metabolite “Ganciclovir” by intestinal and hepatic esterases.

• Conventional analysis implies working on ice.
• What about DBS?

N N N H N O O O N H2 O N H2 OH N N N N H O O N H2 OH O H * Valganciclovir Ganciclovir Enzymes



Almudena Gajate Perez 5

Stability in blood versus DBS

Stability of Valganciclovir 20 40 60 80 100 15 30 60 120 240 360

Time / min Conversion (%)

Rat blood Human blood

• 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.

Ahlstrom DMPK-A DMPK-B FTA

2 4 6 8 10 12 14 16

Conversion (%) Card type

DBS Stability in rat blood

T=0 24 hours



Almudena Gajate Perez 6

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

51 10 35 38 49 46 10 40 16 15 10 24 22 3

10 20 30 40 50 60 5 10 15 20

Time / days

Conversion (%)

Untreated blood 1% Acetic A 2% Acetic A Fast dried cards

Rat blood stability on FTA cards

10 14 58 26 7 5 16 11 2

10 20 30 40 50 60 5 10 15 20 25 30 35

Time / days Conversion (%)

Untreated blood 1% Acetic A Fast dried cards

Almudena Gajate Perez 7

Tamiflu

• Tamiflu, an ethyl ester pro-drug, degrades rapidly into its

carboxylic metabolite in rat blood.

• Conventional analysis needs the addition of dichlorvos.
• Is DBS a solution?

O O O NH NH2 O O O OH NH NH2 O Tamiflu Metabolite Enzymes



Almudena Gajate Perez 8

Stability in blood versus DBS

• Tamiflu is not stable in rat blood.
• Not stable on Ahlstrom. Good stability during the drying process
• n FTA Elute & DMPK-B & A.
• Tamiflu remains stable in FTA & DMPK-B cards for at least 3

weeks, less than 5% conversion.

A h l s t r

• m

D M P K

• A

D M P K

• B

F T A

5 10 15 20 25 30 35 40 45 50

Conversion (%) Card type

Short & long term DBS stability in rat blood

Drying process (T=0) 9 days 21 days



Stability of Tamiflu

0% 20% 40% 60% 80% 100% 15 30 60 180 Time (min)

Conversion (%)

Rat blood Human Blood

Almudena Gajate Perez 9

MPA Acyl-glucuronide

O OH O O O O O OH OH O H OH O O OH O O O OH Mycophenolic acid (MPA) MPA Acylglucuronide (metabolite)

• 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.



Almudena Gajate Perez 10

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

• n

untreated and almost complete conversion on treated cards.

T=0 1 day 2 days 5 days 14 days 42 days 10 20 30 40 50 60 70 80 90 100

Conversion (%) Time DBS stability in human blood

Ahsltrom DMPK-B

A h s l t r

• m

. 5 % a c i d D M P K

• B

. 5 % a c i d A h l s t r

• m

; 4 ° C D M P K

• B

; 4 ° C A h s l t r

• m

. 5 % a c i d . 4 ° C D M P K

• B

. 5 % a c i d 4 ° C 2 4 6 8 10 12 14 Conversion (%)

Conditions DBS stability in treated blood and/or cool storage

T=0 1 week two weeks

Almudena Gajate Perez 11

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 is enhanced on Ahlstrom cards.

Stability in mouse blood versus DBS

65 84 81

20 40 60 80 100 5 10 15 20 25 30

Time / hours % Response Whole blood Ahlstrom

Stable on untreated cards. Unstable on treated cards.

Stability in human plasma versus DPS

110 53 64

20 40 60 80 100 5 10 15 20

Time / hours % Response Human plasma Ahlstrom DPS DMPK-B DPS



Almudena Gajate Perez 12

Conclusions

• DBS technique generally improves stability of compounds
• ver a short term. A long-term stability was proven to be

compound dependant.

• Stability of reactive compounds cannot be taken for granted
• n DBS and each case should be studied independently.
• Recommendations

in case

• f

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 13

Acknowledgments

• Berthold Lausecker
• Katja Heinig, Thomas Wirz and Franz Bucheli
• Andreas Gloge & Justyna Menzel
• Hoffmann- La Roche