The effect of repeated administration of cyclophosphamide

on induction of CYP2BI and 2B2 in rat

Parvaneh Afsharianl, Ylva Terelius2, Stefan Lundgren3 and Moustapha Hassanl a

t Department of Medicine, Division of Hematology, Karolinska Institutet, Stockholm,

Sweden

2 Department of Research DMPK, AstraZeneca R& D Sddefialje, Sddertiilje, Sweden

3 Department of Laboratory Medicine, Division of Clinical Pharmacology, Karolinska

Institutet, Sweden

4 Laboratory of Hematology, Karolinska University Hospital, Huddinge, 14l 86

Stockholm, Sweden

Cyclophosphamide is used in high doses as a part of the conditioning regimen prior to

stem cell transplantation. It is usually given for two or four consecutive days,

primarily to facilitate engraftment of donor cells. Cyclophosphamide is a prodrug that

is activated in liver by a 4-hydroxylation reaction catalyzed by cytochrome P450

(CYP) enzymes. Several studies have shown that cyclophosphamide induces its own

metabolism, which affects its pharmacokinetic parameters after repeated dose (Chang

TK, Yu L et al. Cancer Res 199'7 ; 5'7:1946-54 and Schuler U, Ehninger G et al.

Cancer Chemother Pharmacol 198'71' 20:248-52). In the present study, we aimed to

investigate the effect of repeated doses of cyclophosphamide on the CYPs in rat.

mRNA, protein, and enzyme activity levels were investigated. Animals received (200

mg/kg, i.v.) at time 0, 20, 48 h. Additionally another group of animals was treated at

0, 6,20,26,48 and 54 h. At each time point three animals were killed 30 min after the

administration (Xie H, Afsharian P et al. Xenobiotica 2005:35:239-51). mRNAs of

CYP2BI and 282 were significantly induced up to458- and 8.3-fold at6h,983- and

0)

102-fold at 26 h, and 342- and 33-fold at 54 h. CYP2B protein levels were increased

and their peaks was observed at 20 and 48 h. Microsomal activity of CYP2B was

determined at three different concentration of cyclophosphamide (1, 0.5, 0.1 mM) by

measuring the formation rate of 4-hydroxy-cyclophosphamide (4-OH-CPA). The

microsomal activity increased as reflected in an increase in cyclophosphamide 4-

hydroxylation at all concentrations used using microsomes from rats treated at 6, 20

and 48 h. A significant increase of 4-hydroxylation of cyclophosphamide (0.1 mM)

by 2.9-, 4.4- and 4.2- fold, respectively compared to the control rats. However, a

decrease in the hydroxylation rate was observed using microsomes from rats treated at

26 and 54 h and in all concentrations used that might due to hepatoloxicity effect of

cyclophosphamide when administered in short time period of repeated dose. A

significant (p<0.05) decrease in CPA concentration and a significant (p<0.05)

increase in its metabolite (4-OH-CPA) level in plasma were observed with repeated

administration of CPA. In conclusion, an induction effect on CYP2B and a substantral

up regulation of its mRNA expression were observed after repeated administration of

CPA. This information of a high clinical importance when a dosing schedule for

cyclophosphamide considering the polymorphism of CYPs in cancer patient.

Insiint Fiiredrae el,

I am going to present a part of my study about the effect of repeated dose of

cyclophosphami de on CW2BI|2 in rat. The study has a good clinical impact.

Cyclophosphamide is used in high doses as a part of the conditioning regimen prior to

stem cell transplantation. It is usually given for two or four consecutive days,

primarily to facilitate engraftment of donor cells. We observed when we give the

same doses to rats after the second administration there is high induction in gene

expression of CYPs but after the third repeated dose, we observed inhibition. These

results have very important clinical impact since patients sometimes receive

cyclophosphamide for 4 days.

)foduct Support,09:24 2005-08-10 -0500, ASH Abstract Received

)ate: Wed. 10 Auo 2005 09:24:03 -0500= io m : P rb d utt-S u [p-rt . s u p po rt @ m a rat h on m u lti m e d i a. c om >3ubject: ASH Abstract Receivedfo: Parvaneh Afsharian <Parvaneh.Afsharian@medhs.ki.se>(-PMX-Version: 4.7.1.128O75, Antispam-Engine'. 2.O.3.2,Antisoam-Data: 2005.8. 1 0. 1 2(-PerlMx-Spam: Gauge=lllllll, Probability=7%, Report='-C230066-P5 0,

-HAS-MSGID O,

-MIME_TEXT_ONLY O,

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_dnsbl.njabl.org_T|MEOUT')riginal-recipie nt'. rlc822;P arvaneh.Af sharian @ med hs. ki. se

)ear Dr. Afsharian:

Page 1 of I

/our Online abstract submission, entitled The effect of repeated administration of cyclophosphamide onnduction of CYP2B1 and 2B.2 in rat, has been received by the American Society for Hematology.

/our confirmation number is 5 137.

f you need additional assistance, please feel free to contact our technical support staff by:

)hone: 507-334-5212i-mail: support @ marathonmultimedia.com.

3incerely,

Vlarathon Multimedia'or the American Society for Hemaiology\bstract Processing

"l-u2005-08-27?rinted for Parvaneh Afsharian <Parvaneh.Afshanan @ medhs.ki.se>

Dear Sir/ Madam

I have submitted my abstract entitled:"The effect of repeated administration of cyclophosphamide on induction of CYP2B Iand 282 in rut"on August 9, 2005. Unfortunately, the result for acceptance of the abstracts is notready and according on the ASH schedule it will be announced in the end ofSeptember. Since the congress will be on December 3, 2005 and the deadline for thegrant in your institute is 5'n of September, I am sending this application before theresult of abstract acceptance, but as soon as possible I receive the result I will send itto your office.

Thank you so much in

gLrS P&r9l

Travel plan

The conference is started on December 3, 2005. As the final programme has not been

announced yet andjust we know the program will start on Saturday, December 3 at

9:15 am, we plan to go one day earlier on.

Our group plan to leave Sweden on December 2 early moming, 2005 by plane from

Arlanda to be in New Orleans, USA in Saturday evening (December 2). The

conference will last 4 days until December 6, 2005. We plan to travel back to Sweden

the day after the conference is finished. The final program for the conference is not

published yet and I enclosed some information from ASH home page.

Your sincerelv.

Parvaneh Afsharian

\tnerican Society of Hematology: Annual Meeting Page I of 2

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KAROLINSKA INSTITUTETDepartment of MedicineDivivion of HematologyKarolinska University Hospital, HuddingeMoustapha Hassan, Professor

Stockholm 2005-08-22

Intyg

Angiende Parvaneh Afsharian ansdkan om bidrag till ASH 2005, USA

Parvaneh Afsharian (660321-2041), hon ar registrerad doktorand vid Institutionen f<jr Medicin,

Karolinska lnstitutet, Karolinska universitets sjukhus, Huddinge sedanjuni 2003. Hennes

forskningsproj ekt bertir cltostatika och dess farmakokinetik-farmakogenetik hos cancer patienter

som genomgir benmargstransplantation.

Parvaneh kommer att presentera ett arbete som handlar om effekten av upprepad dosering avcyklofosfamide pi cyokrom P450 hos refta "The effect of repeated dose of cyclophosphamide onCYP 2Bl/2 in rat". ASH iir den stcirsta hematologiska kongress, dessutom iir Parvanehs deltagandetav stor betydelse ftir hennes utbildning. Aktivt kongress deltagande kan utvidga hennessamarbetskontakts niit och diirmed hennes framtida projekts utveckling. Parvaneh ar mycketenergisk och person och diirltir kan jag rekommendera henne varmt ftjr detta ansdkan.

M

Professor, f6r medicin

Lab chel H ogiska laboratoriet

Karolinska universitets sjukhus, Huddinge

Tel:08-58583862

E-mail: Moustapha.Hassan@medhs.ki.se

Mailing addressLaboratory ofHematology, KFC, NowmDivision ofHematologyKarolinska Universiry Hospital, HuddingeS-l4l 86 Stockholm, Sweden

+46 8 58580000Fax+ 46 8 585810?0