NGHIEN c u t ; DIEU CHE THUOC PHONG XA '"V-DTPA-RITUXIMAB CHO DIEU TRI BENH U LYMPHO KHONG HODGKIN NGUYEN THI THU"', DU'ONG VAN D O N G ' , PHAN QUOC THONG^
BUI VAN CU"6NG', VO THI CAM H O A ' , CHU VAN KHOA' ' Viin Nghien cuu hgt nhdn, Dd Lgt. Viet Nam
^ Trudng Cao Ddng Su Phgm Nha Trang, Viet Nam
Abstract: Yttrium is one of the most useful radionuclides for radioimmunotherapeutic applications, especially labelling with monoclonal antibodies. Rituximab was bound to the DTPA chelating agent using Hnatowich methods. Cyclic anhydride DTPA (cDTPAa, 0.1 mg/mL) was dissolved in chloroform and was degassed under a stream of nitrogen for 30 minutes. Rituximab solution in 0.05 M bicarbonate buffer was immediately added and mixed for one minute at room temperature. The antibody Rituximab at different concentration (5 mg/mL and 10 mg/mL) was coupled with the cDTPAa, at molar ratios (cDTPAa : Rituximab) of 1:1, 3:1, 5:1, 10:1 and 20:1. The conjugation of DTPA-Rhuximab mixture was labelled with Y-90, then using Sephadex 025 in order to determine coupling efficiency. Coupling efficiency at a 3:1 mole ratio was 70%. After purification, the conjugation DTPA-Rituximab was labeled with Y-90 in O.SM acetate buffer, pH 5, at room temperature. The labeling yield was about 99%.
The radiochemical purity of Y-DTPA-Rituximab was more than 98 % which determined by ITLC in 0.1 M acetate at pH 6 as mobile phase. The radiopharmaceuticals have been test for sterility, apyrogenicity and biodistribution. This is a potential radiopharmaceutical for clinical application in therapeutic Non Hodgkin Lymphoma treatments.
Tie khda: Ytrium-90, ^"Y-DTPA-Rituximab, radiopharmaceuticals, Non Hodgkin Lymphoma
1. MCJ DAU
Trong nhung nam qua, buoTig dieu tri ung thu nham dich dang dugc su quan tam idn cua y hpc, dac biet la ky thudt dilu tn phdng xa mien dich. Ben canh nhirng thiet bi hien dgi chup hinh chan doan trong y hpc hat nhdn, nhieu the hf thudc phdng xa moi cung da dugc nghien cim vd phdt trien cho muc dich dieu tri. Su phat trien ddng bp nay da lam ddi mdi bd mat Clia y bpc hgt nhdn bdi chdt lugng chan doan va hieu qua dieu tri. Khang the dcm ddng Rituximab la khdng the dac hieu len te bdo lympho B ung thu, bdi khdng nguyen CD20 bieu hien vdi mat dp cao len ddng tl bao ung thu nay. Dac biet la khi gan vdi ddng vi phdng xa phat tia beta se phat huy hieu qud dieu tri.
Ngay nay, Yltrium-90 da tra thanh mpt nhdn phdng xa dugc sir dung rpng rai trong dieu tri benli ung thu nhd cac tinh chdt vdt ly ciia nd nhu nang lugng phat tia beta cao (Ejj'max =^
2.280 KcV) vdi quang chgy trong md ddi hon tdt cd cac hgt phat tia (3" khdc ( 4 - 1 1 mm), khdng phat tia gamma, thdi gian ban ra cua nd Id 64,14 gid. Mgt thuan lgi khdc nira la ^"Y luon cd san nhd su phan ra cua ddng vi me ^^Sr. Khi danh ddu khang the Rituximab vdi ddng vi phong xa ^^Y bdng phuang phdp tgo phiic trung gian khang the vdi DTPA, thudc phdng xa nham dich ^V-DTPA-Rituximab dugc dieu che va cd tiem nang ung dung trong dieu trj benh u lympho bao B khong Hodgkin. Trong bdo cao nay voi muc tieu nghien cim dieu che thudc khang the gdn phdng xg diing trong dilu tri y hgc, chiing tdi trinh bay phuang phap tap phuc hpp khang the dan ddng Rituximab vdi DTPA, phucmg phap danh dau vai ddng vj phdng xg
592 Hdi nghi Khoa hgc vd Cdng nghe hgt nhdn todn qudc idn thir DC, Ninh Thugn 18-19/8/2011
^^Y va cac phuong phap kiem tra chdt lugng de sdn phdm dat tieu chudn chdt lugng thudc phdng xa.
II. PHU'ONG P H A P NGHIEN c u t )
Nguyen lieu, hoa chat: ^'^Y dugc tach chiet tir mdy phat ddng vi '"Sr/^Y bdng phuong phap cd djnh mdng Supported Liquid Membrane (SLM), ngudn phdng xa me ^Sr nhdn dugc tir tdi trg nghien cuu cua IAEA, ^ Y dugc sdn xudt tgi Vien Nghien cuu hat nhdn, cd ndng dp phong xg 40 mCi/ml. Duoi dang ^'^Yttrium- acetat. Rituximab chai 500mg/50ml, hang ROCHE, cot sac ky Sephadex G25 PDIO mua tir hang Amersham Bioscences. Cac hod chit la loai hod chdt tinh khiet.
Phuong phap gan khang thi voi tac nhan tao phuc DTPA: Thuc nghiem dugc tiln hanh theo phuang phap Hnatowich. Hda tan DTPA (cDTPAa, 0.1 mg/mL) trong chloroform sau dd cho vao dng thuy tinh danh dau theo ti le moi (cDTPAa : Rituximab) can dimg la 1:1, 3:1, 5:1, 10:1 vd 20:1. Thdi khi nito vdo cac dng cho den khi khd. Pha loang Rituximab trong df m bicarbonate thanh cac ndng dp 5 mg/mL and 10 mg/mL va cho vao dng cd DTPA. Lac trdn nhe tai nhiet dp phdng.
Phuong phap sac ky loc gel: Phiic hgp DTPA-Rituximab dugcn pha loang va nap cot Sephadex G25 de tdch phuc vd phdn cdn thira. Dung dich rira gidi Id nude mudi sinh ly 0,9%.
PhuoTig phap danh dau phong xa: Phiic hgp DTPA-Rituximab tinh khiet dugc danh ddu vdi ddng vi phdng xa Y trong dfm acetat 0,5 M, pH 5, tai nhift dp phdng. Thdi gian danh dau la 5 phut.
Phuong phap sac ky lop mong: Phuang phdp sdc ky ITLC dugc dimg dl kilm tra dd sach hda phdng xg cua san pham va kiem tra hifu sudt ciia hgp chdt danh ddu. Kich thudc gidy ITLC la 1 x 10 cm, he dung moi khai trien Id acetat 0,1 M, pH 6. Chdm 5 |il mdu len gidy ITLC tai vi tri centimet thu hai, khi gidy kho, ddt bang gidy vao binh chua sdn hon hgp dung mdi. Thdi gian sac ky la 2 phiit. Khi dung mdi di chuyen din centimet thii 10, ldy bdng giay ra, de khd rdi cdt thanh tung bang nhd, moi bang rdng 1 cm vuong gdc vai chilu di chuyen cua dung mdi, do dem hogt do phdng xa. '°Y tu do di chuyin vl tuyin tren dung moi (Rf ^ 1).
Phiic ^"l-DTPA-Rituximab ndm tgi dilm gdc (Rf = 0). Tinh ti le hoat dO phdng xa cua cac vimg tucmg irng vdi cac hgp chat cua '"Y .
Kiem tra tinh on djnh san pham: Khang the ddnh ddu phdng xa dugc kilm tra tinh dn dinh trong huylt thanh ngudi. Ldy san phdm "^Y-DTPA-Rituximab tinh khilt, them vao do huylt thanh sao cho 100.000 CPM/ml. Chia ra cac dng nghiem I ml/dng, cdc dng dugc u d 37 C tgi cac khoang thdi gian 1, 2, 3, 4, 5, 6, 7 ngdy. Sau cdc khoang thdi gian lay ra, ldy sdn phdm phan tich dp sgch hod phdng xg bdng phuang phap ITLC, do hoat dp phdng xg, tinh todn pbdn tram ti le giiia cac phdn tren hoat dp tdng.
Kiem tra phan bo va dao thai tren dpng v^t: Tiem vao vein dudi mdi con chudt 100
^1 ^°Y-DTPA-Rituximab (50 ^Ci), moi nhdm 5 chudt, gilt vd mo theo cac khodng thai gian 1 gid, 6 gid, 24 gid, 72 gid, 120 gid. Ldy cdc ca quan npi tang nhu gan, Idch, than, phdi, tim, mau, da day, xuang, ca, dudi. Can mau va do dim phdng xa, tinh phan b6 theo ID%/g.
III. KET QUA NGHIEN ClTU
1. Ket qua kilm tra chdt lugng ^°Y dimg de ddnh ddu khang thi: ^°Y dimg trong nghien ciru dugc kiem tra chdt lugng bdng phuong phap ddc hieu EPC (Extract Paper Chromatography). Chdt phdng xg ^'^Y dugc kilm tra dp sach hda phong xg bdng sdc ky gidy diing hda chdt PC88A (2-ethylhexyl 2-ethylhexyl phosphonic acid) tdm vdo dilm gdc, sau dd
Hgi nghi Khoa hgc vd Cdng nghe hgt nhdn tgdn qudc lin Ihi DC, Ninh Thugn 18-I9/8/20II 593
chit phdng xa '"Yttrium- acetat duoe cho gjn dac hieu len PC88A, gidy sdc ky duoc phat tri^n trong dimg mdi NaCl 0,9%.
eooonoo c mtm'
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Hinh I: Kiem tra dg bdn ^Sr trong dung dich Hinh 2: Kiem tra dg sgch hoa phdng xg ciia ^"Y-
^'V-acetat bdng phuang phdp EPC acetat bdng phuang phdp sdc ky lap mdng ITLC Kit qua tren hinh 1 cho thdy PCSSA tao phiic ddc hieu vai ''^Y^'^, vi vdy ^"Y^"^ bj giir tgi diem gdc, Sr "^ di chuyen ve tuyen tren cua dung mdi, dp sach hat nhdn phdng xg ciia sdn pham dat ban 99,999%, ddp umg yeu cdu nghiem ngat dat ra khi sir dung chat phdng xg ^^Y trong nghien cuu va dieu tri, vi ^ Sr cd thdi gian bdn ra rdt Idu (28 nam), rat nguy hiem cho bfnh nhdn, '"Sr^^ neu tdn tgi tir do, sg dinh vi trong xuong. Do vdy, kiem tra dp sach hat nhan phong xa ciia Y la can thiet trong qua trinh ddnh ddu vdi phdn tir sinh hpc. Hinh 2 cho thdy n f * tu do di chuyen ve tuyen tren dung mdi tren sac dd.
2. Ket qua khdo sdt hieu sudt gan DTPA va khang the: Dd thi khdo sat hieu sudt gdn khang thi vdi DTPA cho thdy, a cac ti If moi 1:1,3:1,5:1, 10:1 va 20:1. DTPA gin len khang the vdi ndng dp 5 mg/ml cho hieu sudt gan cao hon gan d ndng dp 10 mg/ml.
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Hinh 3: Khdo sdt hi?u sudt gdn DTPA vd Rituximab Hmh 4: Tdch ' Y-DTPA-Rituximab qua cdt Sephadex
Nghien cuu cho thdy trong dem bicarbonat hogt tinli gin cua khang the vdi DTPA cao hon trong dem citrate (hinh 3). Kit qud gdn tai cdc tl le moi DTPA-Rituximab 1:1 cho ket qua 88,0% va 84% tlieo hai ndng dp. Tuong tu voi ti le moi DTPA-Rituximab 20:1 cho kit qua la 28,4% vd 22,0%. 6 ti le 3:1 hifu suat g i n dat khoang 70%, ket qua tinli toan cho tlidy trung
594 Hdi nghi Khoa hgc vd Cdng nghe hgt nhdn todn qudc idn thir IX, Ninh Thudn 18-19/8/2011
binli 2 nhdm DTPA tren mpt phan tir khang the thudng dugc chpn d l ldy san phdm sir dung cho dieu tri.
Phuc cdng hgp DTPA-Rituximab dugc tach qua cgt Sephadex vd san phdm tinh khilt thu dugc d phdn doan 4 vd 5 vdi tdng the tich la 2 ml (hinh 4), ndng dp khang thi la 100
^ig/ml. Phiic DTPA-Rituximab Id sdn phdm tinh khiet dugc dimg de nghien cuu khdo sat cac dilu kien danh dau voi dong vi phdng xa ^*'Y, thu dugc dugc chdt phdng xg ^°Y-DTPA- Rituximab diing cho cdc nghien cim kiem tra chdt lugng tiSp theo.
3. Ket qud khdo sat thoi gian phdn iing
Phiic hgp DTPA khdng the dugc ddnh ddu vdi ddng vi Y trong dem acetat vdi hieu suat danh ddu cao, ket qua ban 99% Y dugc danh ddu trong thdi gian tir 1 den 150 phiit diu cho hieu sudt danh ddu cao vai ket qua hon 99% (bdng 1)
Bdng i ThM
gian (phiit) Hieu sudt
(%)
; Ket qud ddnh ddu DTPA-Rituximab vai Y theo thdi gian 1
99.5 3
99.5 5
99.6 7
99.7 10
99.8 20
99.8 60
99.8 120
99.8 150
99.8
4. Ket qud kiem tra dp sach hda phdng xa va dp dn dinh ciia sdn phdm
Sdn phdm ^°Y-DTPA-Rituximab dat dp sach hda phdng xa ban 99% va dn dinh trong huyet thanh ngudi sau 6 ngdy d nhift dp u la 37°C (hinh 5 vd hinh 6).
9 0 0 0 • 8 0 0 0 7 0 0 0 6 0 0 0 5 5 0 0 0 • 6 4 0 0 0
3 0 0 0 ^ 2 0 0 0 1 0 0 0 -
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Hinh 5: Do sgch hda phong xg cua Y-DTPA- Rituximab
Hinh 6: Do on dinh cua "Y-DTPA- Rituximab
Lien kit '°Y-DTPA-khang tha b i n invitro co le do cDTPAa la mot anhydrid rit manh, d l tao phiic voi khang t h i qua lien kit amin tren phan hi lysin. ' ° Y d i tao phurc cation kim loai ho lantaniab va actinide, tao lien kit cpng hoa tri voi phtie cang cua eDTPAa vung bin vi mat hoa hpc. Do vay sau khi danh d t o , phan tir phuc hpp b i n vdi d6ng vi phong xa kim loai
(hinh 7). ' . . .
Hgi nghi Khoa hgc vd Cdng nghe hgt nhdn todn qudc idn thu DC, Ninh Thudn 18-I9/8/20I1 595
i-cmmer E= 2.25 MeV
Kinh 7: Sa dd ddnh ddu tgo phuc cita ^°Y-DTPA-Rituximab 5. Ket qud khdo sat pbdn bd sinh hpc tren chupt
Thong ke phan bd thudc ciia ^*'Y-DTPA-Rituximab tren chudt dugc liet ke trong bang 2 Bdng 2. Phdn bd sinh hgc ciia ^°Y-DTPA-Rituximab tren chudt (n=5) Mo/Cff
quan Gan Lach Than Phii Tim Mau Da day Xuong Co
Igifr 0,72 ±0.14 0,09± 0,04 0,51±0,17 0,17± 0,02 0,25±0,18 3,20±1,09 0,66±0,15 4,68± 0,79 4,23±1,62
6gi6' 4,45 ±1,15 0,24± 0,23 2,66± 1,40 0,70± 0,06 0,40± 0,13 13,59± 1,08
0,5 8± 0,08 6,64± 2,32 8,40± 3,22
24gia' 5,31 ±0,76 0,34±0,12 1,95±0,72 1,75±1,15 0,43± 0,07 15,16±2,86 1,08±0,18 23,92± 7,93 12,86± 3,58
72glor 4,23± 1,81 0,22± 0,07 1,26±0,56 0,41+0,20 0,23± 0,06 9,56±3,61 1,27±0,27 38,3717,68 9,93±3,15
120 gior 6,22 ±
3,17 0,30±0,16 1,37±0,75 1,93±2,47 1,27± 1,60 3,01±1,37 3,19±0,54 29,52±
9,17 7,63± 2,48 Ket qud cho thdy rdng khang the danh ddu vol ddng vi phat beta cd the luu thdng trong mau trong vai ngdy, sau dd cd tap trung trong xuong (sau 3 ngay), cd le do mot phdn bi phdn hiiy do he emzym hoac do thuc bdo, ^"Y tu do hdp thu vdo xuang, dinh vj trong xucmg va sau do bai tiet it theo dudng tilu.
596 Hdi nghi Khoa hgc vd Cdng nghe hgt nhdn todn qudc idn thir IX, Ninh Thudn 18-19/8/2011 IV. KET LUAN
Phiic hgp DTPA khang the dugc dieu che, tgo dieu kien thuan lgi cho viec danh ddu vdi dong vj phdng xa '"Y ndi rieng va vdi cdc ddng vi phdng xa kim loai ndi chung, KJ? thudt gan DTPA vdi khdng thi cd thi dp dung voi cdc chdt sinh hgc khac cd irng dyng trong hda dugc.
Dugc chdt phdng xg khdng the don ddng ^'*Y-DTPA-Rituximab qua dd dugc dilu chi vdi hifu suit danh ddu cao, do sach hda phdng xa ddm bdo, sdn pham on dinh, cd tiem ndng iing dung ldm sdng trong dilu tri benh u lympho bao B khong Hodgkin.
TAI LIEU THAM KHAO
[1] IAEA, (2009). Therapeutic radionuclide generators *°Sr/^Y and '^V/'^^Re. Technical Reports Series Number 470.
[2] Fisher, R. I. (2003). Overview of non-Hodgkin's lymphoma: biology, staging, and treatment. Seminars in oncology. 30:3-9
[3] Knox, S. J., Goris, M. L., Trisler, K., Negrin, R., Davis, T., et al. (1996). Yttrium-9a- labeled anti-CD20 monoclonal antibody therapy of recurrent B-cell lymphoma. Clin Cancer Res. 2:457-470
[4] Hnatowich DJ, Layne WW, Childs RL, et al. (19S3). Radioactive labeling of antibody: A simple and efficient method. Science. 220:613-615
[5] Goldenberg, D. M. (2001). The role of radiolabeled antibodies in the treatment of non- Hodgkin's lymphoma: the coming of age of radioimmimotherapy. Critical reviews in oncology/hematology. 39:195-201
[6] Witzig, T. £., Gordon, L. I., Cabanillas, F., Czuczman, M. S., Emmanouilides, C, et al.
(2002). Randomized controlled trial of yttrium-90-labeled ibritumomab tiuxetan radioimmunotherapy versus rituximab immunotherapy for patients with relapsed or refractory low-grade, follicular, or transformed B-cell non-Hodgkin's lymphoma. J Clin Oncol. 20:2453-2463
