Nghiên cứu đánh dấu kháng thể đơn dòng kháng CD20 với đồng vị phóng xạ i 131 dùng trong điều trị bệnh ung thư máu

BANG VIET TAT RIT Lieu phap mien dich phdng xa radioimmunotherapy RJS Chup hinh nhap nhay mien dich Radioimmunoscintigraphy CD Cum biet hoa cluster differentiation MAb Khang the don ddng

BO KHOA HOC VA CONG NGHE VIEN NANG Ll/ONG NGUYEN TlT VIET NAM

VlfN NGHIEN ctru HAT NHAN

BAO CAO TONG KET THlTC HIEN DE TAI c A P BO NAM 2007 - 2008

TEN DE TAI

K H A N G CD20 V 6 l DONG VI PHONG XA 1-131 DUNG

TRONG DIEU TRI BENH UNG THU* M A U

Ma s6: 05/07-09/NLNT

Chu nhifm de tai: ThS Nguyen Thj Thu

THANG 12/2009

DANH SACH NHlTNG NGircn THAM GIA THlTC HIEN DE TAI

Nguyen Thi Thu ThS NCV

Duong Van Dong ThS NCVC

V6 Thi Cam Hoa CN, NCV

Chu Van Khoa

Mai Phudc Tho

ThS, NCV

Biii Van Cucmg CN, NCV

CN, NCV

Pham Ngoc Dien CN, NCV

Mai Trong Khoa PGS TS

Vien Nghien ciru Hat nhan

Trung Tam Nghien cuu va dieu che Dong vi Phong xa,

Vien Nghien cuu Hat nhan Trung Tam Nghien ciiu va dieu che Dong vj Phong xa,

Vien Nghien cim Hat nhan Trung Tam Nghien ctiu va dieu che Dong vj Phong xa,

Vien Nghien cuu Hat nhan Trung Tam Nghien cuu va dieu che Dong vi Phong xa,

Vien Nghien cuu Hat nhan Trung Tam Nghien cuu va dieu che Dong vi Phong xa,

Vien Nghien ciiu Hat nhan Trung Tam Y hoc hat nhan va Dieu tri ung budu,

Benh vien Bach Mai, Ha Noi Vien Cong Nghe Sinh hoc, Vien Khoa hoc Viet Nam Benh Vien 108, Ha Noi

Cac ca quan, don vi phoi hap thye hien

1 Vien Cong nghe sinh hoc Ha Noi

2 Benh vien Bach Mai, Ha Noi

3 BenhvienlOS, HaNoi

^^n

MUC LUC

• •

Trang Trang bia

Danh sach nhimg ngudi tham gia thuc hien de tai 1

Muc luc 2 Abstract 5 Tom tat 6

Md dau 7

I Tom tat tinh hinh nghien cuu trong va ngoai nudc 8

II Muc tieu cua de tai 9

III Tom tat noi dung nghien ctju 10

Phan I: TONG QUAN

I Gioi thieu nhirng tien bo moi trong dieu tri mien djch phong xa RIT 11

1.1 Cac Idiai niem 11

1.2 Khang the dugc sir dung dieu tri nham dich 12

1.3 Cac tac nhan chup hinh mdi trong RIT 18

1.4 L/ng dung RIT trong dieu tri NHL 18

II Cac thanh phan co* ban trong RIT 21

II 1 Khang nguyen CD20 vai tro va tiem nang ung dung 21

11.2 Khang the va cong nghe tao khang the 23

11.3 Dong vi phdng xa 28

III Cac phirong phap danh dau khang the voi dong vj phong xa 30

III 1 Dac diem chung 30

111.2 Qua trinh danh dau phong xa 31

111.3 Phuang phap danh dau khang the vdi '^'l 32

111.4 Phuang phap danh dau khang the vdi '^'^'"Tc va ' "in 35

IV Nghien cihi du-ffc dong hoc 37

Phan II: VAT LIEU VA PHT/ONG PHAP NGHIEN ClTU 38

I Nguyen lieu, hoa chat, thiet bi

II Nghien ciru cac dieu kien toi ini danh dau khang the voi phong xa 39

II 1 Xac djnh ham'lugng ChT ' 39

11.2 Xac dinh ham lugng khang the, hoat do, thdi gian phan ung 42

11.3 Xac dinh pH 43

11.4 Quy trinh danh dau khang the 44

11.5 Danh dau theo hoat do rieng 46

11.6 Phan ung mien dich 47

11.7 Phuong phap iodogen 48

U.S Danh dau khang the don chuoi 49

III Kiem tra chat Iirong san pham 49

III 1 Kiem tra do sach hda phdng xa 49

111.2 Kiem tra do vd khuan 50

111.3 Kiem tra ngi doc td vi khuan 51

111.4 Kiem tra do dn dinh trong huyet thanh 53

111.5 Kiem tra do dn dinh theo thdi gian bao quan 53

111.6 Kiem tra phan bd sinh hoc tren chupt 53

III.7 Kiem tra dao thai thudc tren thd 53

IV Chup hinh phan bo thuoc phase I tren benh nhan ung thir NHL 54

Phan III: KET QUA NGHIEN CUtl

I Ket qua danh dau khang the 56

1.1 Ket qua kiem tra chat lugng '^'l danh dau 56

1.2 Ket qua khao sat ham lugng ChT 58

1.3 Ket qua khao sat ham lugng, hoat dp, thdi gian 61

1.4 Kit qua khao sat pH 62

1.5 Ket qua dieu che san pham 63

1.6 Ket qua dieu che san pham theo hoat dp rieng 64

1.7 Ket qua kiem tra hoat tinh mien dich 65

1.8 Ket qua danh dau khang the bang phuong phap iodogen 66

1.9 Ket qua danh dau khang the dcm chudi 67

II Ket qua kiem tra chat Iirong dirtfc chat phong xa 68

II 1 Kiem tra dp sach hoa phdng xa 68

11.2 Ket qua kiem tra dp vd khuan 71

11.3 Ket qua kiem tra npi doc td vi khuan 72

11.4 Kiem tra dp dn dinh trong huyet thanh ngudi 73

11.5 Kiem tra dp dn dinh theo thdi gian 74

11.6 Kiem tra phan bd sinh hpc 76

11.7 Kiem tra dao thai sinh hpc 78

III Ket qua chup hinh phan bo thuoc phase I tren benh nhan ung thir 80

Phan IV: KET LUAN 85

Tai lieu tham khao 86 Giai trinh cac khoan chi 87 Phu luc 1: Tdng hgp khang the don chuoi

Phu luc 2: Kiem tra chat lugng

Phu luc 3: Lam sang

Phu luc 4: Thdng tin de tai

BANG VIET TAT

RIT Lieu phap mien dich phdng xa (radioimmunotherapy)

RJS Chup hinh nhap nhay mien dich (Radioimmunoscintigraphy)

CD Cum biet hoa (cluster differentiation)

MAb Khang the don ddng (monoclonal antibody)

NHL U lympho khdng Hogdkin (non-Hodgkin's lymphoma)

FDA Cue quan ly thudc va thuc pham Hoa Ky (Food and Drug Administration) BCA Tac nhan chelat hda hai chuc nang (bifunctional chelating agent)

CR Dap ling toan phan (complete response)

PFS Tiep tuc sdng sdt (progression-free survival)

PR Dap ung rieng phan (partial response)

ADCC Doc tinh vdi te bao qua khang the (Antibody Dependent Cellular

Cytotoxicity) CDC Ddc tinh vdi te bao qua bd the (Complement Dependent Cytotoxicity) CDCC Ddc tinh vdi te bao phu thudc bd the (Complement Dependent Cellular

Cytotoxicity) CLL Benh mau trang dang te bao lympho man tinh (Chronic Lymphocytic

Leukemia) AML Ung thu bach cau dang tuy (Acute Myeloid Leukemia)

ALL Ung thu nguyen bao tuy cap (Acute Lymphoblastic Leukemia)

RAIT Dieu tri phdng xa mien dich

HACA Khang khang the lai d ngudi (Human Anti-Chimeric Antibody)

HAMA Khang khang the chupt (Human Anti-Murine Antibody)

HAHA Khang khang the nhan hoa d ngudi (Human Anti-humanized Antibody) IL-6 Interleukin 6

Rf Retardation factor (retention factor)

NSB Phan gan khdng dac hieu (non specific binding)

EGFR Epidermal Growth Factor Receptor

HER 2 Herceptin, khang the chira ung thu vii

HAT Hypoxanthin Aminopterin Thymidin

VEGF Vascular Endothelial Growth Factor

CDR Viing trinh dien khang nguyen

FACS phan loai te bao phat huynh quang (Fluorescence activated cell sorting) IgG Immunoglobulin

Sc-FvAb Khang the don chuoi (Single-Chain Fragment Variable Antibody)

cfii Don vi do mat dp vi sinh (Colony forming unit)

ChT Chloramin T

ITLC Sac ky lop mdng ttic thdi (Instant Thin Layer Chromatography)

FPLC Fast Protein Liquid Chromatography

STUDY ON THE RADIOLABELING OF MONOCLONAL ANTIBODY

FOR BLOOD CANCER THERAPY

Nguyen Thi Thu

Abstract

In recent years, radioimmunotherapy (RIT) has become a highly promising oncologic therapeutic modality with established clinical efficacy, particularly in the therapy of haematological malignancies

Anti CD20 monoclonal antibody was labelled with '^'l used in the treatment of B cell non Hodgkin's Lymphoma (NHL), B cell leukemia In this study, the monoclonal antibody Rituximab was labelled with '^'l using chloramin T method (ChT) The optimized ChT concentration for the oxidation of 185 MBq of Na'^'l solution and 750|ag of Rituximab was

50\igl50n\ The reaction time was 5 minutes at room temperature The labeling reaction has

stopped using natrimetabisulphite (SMB) Labelling efficacy was controlled by ITLC The reaction mixture was purified through the Sephadex G-25 PDIO Pharmacia column The collected '^'l-CD20MAb Rituximab was filtered through a 0.20^m milipore sterile filter The radiochemical labeling yield was more than 95% Radiochemical purity of the radiopharmaceutical after purification was more than 99% The product has passed the test for sterility, bacterial endotoxins, to be sufficiency invivo and invitro stable after labeling, ready for clinical use In the phase I trial, a radiation absorbed dose escalation and protein mass dose escalation were undertaken '^'l-CD20MAb be used on two patients with blood cancer type NHL, has recorded immunoscintigraphy biodistribution

The radiolabeled antibody used in clinical is safe and efficacy It's the radiopharmaceutical used in cancer therapy with RIT This study has the goal of research and develop and clinical testing It has the potential to be a new method of treatment for RIT domestically

NGHIEN ClTU DANH DAU KHANG THE DON DONG V6l DONG VI PHONG XA DUNG TRONG DIEU TRI

BENH UNG THU* MAU

Nguyen Thi Thu

Tom tat

Trong nhirng nam gan day lieu phap mien dich phdng xa (RIT) da trd thanh phucmg thiic dieu tri ung thu day hiia hen vdi hieu qua lam sang ro ret dac biet la trong dieu tri cac ung thu mau

Khang the don ddng khang CD20 dugc danh dau vdi ddng vi phdng xa '^'l dimg trong dieu tri benh u lympho khdng Hodgkin (NHL), benh ung thu te bao B Trong nghien ciiu nay, khang the don ddng Rituximab dugc danh dau vdi '^'l bang phuong phap Chloramin T Ham lugng ChT tdi uu de oxy hoa 185MBq '^'l va 750ng Rituximab la 50p.g/50|il Thdi gian phan iing la 3 phiit d nhiet dp phdng Phan ung dugc dimg bang each them chat khu natri metabisulphite (SMB) Hieu suat danh dau dugc kiem tra bang phuong phap sac ky lop mdng ITLC Hdn hgp phan iing dugc Ipc qua cdt Sephadex G25 PDIO San pham '^'l-CD20MAb Rituximab dugc Ipc qua phii Ipc vd triing 0,20fxm Hieu suat danh dau phdng xa hon 95% Dp sach hda phdng xa cua san pham sau khi Ipc qua cot la hem 99% San pham dat tieu chuan vd trung, dat tieu chuan kiem tra npi ddc td vi khuan, dn dinh invitro va invivo cd the sir dung cho lam sang Trong phase I thir nghiem, lieu hap thu biic xa va lieu lugng protein dugc thuc hien '^'l-CD20MAb Rituximab dugc sir dung lam sang tren hai benh nhan ung thu mau dang NHL, da ghi hinh nhap nhay mien dich phan bd thudc

Khang the danh dau phdng xa cd the sir dung tren lam sang an toan va hieu qua Day

la dugc chat phdng xa diing trong dieu tri ung thu bang ky thuat mien dich phdng xa RIT De tai da dat ra muc tieu nghien ctiu dieu che va thir nghiem va cd tiem nang md dau cho hudng dieu tri mien dich phdng xa trong nudc

MOfDAu

Trong thap nien qua, ciing vdi su ra ddi va ngay cang hien dai hda cac thiet bi chup hinh chan doan nhu may chup cat Idp dien toan SPECT va may chup cat Idp diing ddng vi phdng xa phat positron PET-CT, nhieu the he thudc phdng xa mdi cQng da dugc nghien ciiu dieu che va phat trien Su phat trien ddng bp nay da lam ddi mdi bp mat cua y hpc hat nhan bdi chat lugng chan doan va hieu qua dieu tri Ben canh nhiing chan doan chinh xac de phat hien benh, viec nghien ciiu chiia tri benh la nhiem vu hang dau cua y hpc Mot trong nhiing noi bat cua y hpc hat nhan trong tri lieu la viec sir dung khang the don ddng danh dau vdi ddng vi phdng xa dung trong dieu tri ung thu

Ky thuat dieu tri benh bang phdng xa da ra ddi tir nhiing nam 1950 tir khi y hpc biet dung '•"l trong dieu tri tuyen giap Tir do den nay, cac dugc chat phdng xa danh dau vdi ddng

vi ' 'l khdng ngimg nghien ciiu va phat trien Dac biet la trong nhirng nam gan day, khang the don ddng danh dau phdng xa da dugc diing trong chan doan va dieu tri lam sang Phuong phap chiia benh dung khang the don ddng gpi la phuong phap dieu tri nham dich Phuong phap dieu tri nay cdn dugc gpi la lieu phap phdng xa mien dich RIT (Radioimmunotherapy) Nhd nhimg tien bd cua cdng nghe sinh hpc va nhiing hieu biet ve sinh hpc phan tir va sinh hpc te bao, RIT da trd thanh lieu phap chiia tri ung thu day hiia hen vdi nhirng hieu qua lam sang ro ret

U lympho khdng Hodgkin (NHL) la benh ung thu cac lympho bao B trong mau, khi

bi ung thu, tren be mat cac lympho bao B nay bieu hien khang nguyen la, bang ky thuat sinh hpc phan tir hien dai, cac nha khoa hpc tren the gidi da xac dinh dd la khang nguyen CD20 va cau tnic phan tii ciing da xac dinh Phan tir khang the don ddng khang CD20 (Rituximab) dugc su dung lam sang tir nam 1997, phan tti khang the thuc hien chiic nang nham dich cua minh bang each gan dac hieu len khang nguyen dich, diet te bao ung thu bang cac co che biic

xa ion hda (khi gan vdi chat phdng xa), co che gay ddc tinh vdi te bao ung thu qua khang the

va gay ddc tinh vdi te bao ung thu qua bd the Vdi thdi gian ban ra 8 ngay va phat tia gamma vdi nang lugng la 364 keV, va tia beta vdi nang lugng trung binh la 192 keV, '^'l la ddng vi phdng xa ly tudng cho viec chup hinh va dieu tri benh khi gan vdi phan tir khang the

Khang the khang CD20 da dugc gan vdi ddng vi phdng xa '^'l bang phuong phap danh dau phdng xa sir dung chloramin T lam chat oxy hda Khang the danh dau dugc tinh che

va kiem tra chat lugng theo tieu chuan cua thudc phdng xa de su dung lam sang Day la dugc chdt phdng xa dimg trong dieu tri ung thu bang ky thuat RIT D6 tai da dat ra muc tieu nghien ciiu dieu che, thu nghiem va md dau cho hudng dieu tri mien dich phdng xa trong nudc

I Tinh hinh nghien ciru trong va ngoai niro^c:

Ky thuat san xuat khang the don ddng da dugc hai nha bac hgc Cesar Milstein va Georges Kohler de ra vao nam 1975 da dugc giai thudng Nobel vao nam 1984 bdi nhieu iing dung cd gia tri vugt bac trong y hpc

Hien nay, tren the gidi vdi hon 200 Cdng ty nghien ciiu va san xuat khang the, da cd

23 khang the don ddng dugc FDA phe duyet cho sir dung lam sang, hang nghin loai dang trong qua trinh nghien ciiru lam sang Nhieu loai khang the don ddng mdi da dugc nghien ciiru

va cai tien, ddi mdi phuc vu cho cac muc dich chan doan va dieu tri trong y hpc Nhiing ndi bat gan day nhat la sau hon 1/4 the ky ke tir khi ra ddi, viec sii dung khang the dcm ddng cho dieu tri ung thu mdi bat dau sir dung hieu qua tren lam sang Bdi vi, trudc kia ngudi ta san xuat khang the don ddng theo phuong phap tao ra cac khang the tren chupt, do vay khi dua vao CO the con ngudi, he thdng mien dich se phat hien ra dd la chat ngoai lai, va se bi dap ling mien dich chdng lai va dao thai khdi vat chu Do vay ma viec dieu tri van cdn gap nhieu han che Trong nhirng nam 1986-1990, cac nha khoa hpc Michael Neuberger va Greg Winter

da tao ra mot biit pha khi dua ra ky thuat cd ten gpi la "humanise" Nhd ky thuat nay, ddng khang the san xuat tir chupt dugc chuyen thanh ddng khang the ngudi, thich hgp cho viec sii dung tren co the con ngudi va md ra trien vgng mdi trong dieu tri ung thu Trong vai nam gan day viec san xuat va sir dung khang the don ddng trong dieu tri ung thu la van de thdi sir tren toan the gidi

Dac biet la, mdt sd ddng vj phdng xa nhu ^^""Tc, '**Re, '"Sm, '"in, ^"Y, '^'I cd thl gan vdi khang the don ddng, dan dudng mot each dac hieu den cac te bao ung thu, cac chat phdng xa phat tia gamma ('y), giiip cho viec phat hien chan doan va theo doi ung thu, cac chat phdng xa phat tia beta (P) cd the tieu diet te bao ung thu Sir phat trien nay ket hgp vdi su phat trien cdng nghe may tinh, cdng nghe thong tin lam cho nganh y hpc hat nhan ngay cang dat den dinh cao cua nen y hpc hien dai

Khang the don ddng khang CD20 da dugc dua vao su dung lam sang de chua benh

ung thu tk bao lympho B Day la khang the gan dac hieu len khang nguyen CD20, khang

nguyen nay bieu hien vdi mat dp cao te bao lympho B ung thu Tren the gidi, ngudi ta da dua khang the khang CD20 vao sir dung dieu tri lam sang tii nam 1997 vdi ten thucmg mai Rituximab Viec tao conjugate vdi ddng vi phdng xa chi nghien ciiu trong vai nam gan day

va chinh thiic dugc phep sir dung lam sang tir nam 2003, dd la cac san phdm Bexxar, Tositumomab

Trung tam Nghien cim va Di6u ch6 Ddng vi Phdng xa thudc Vien Nghien ciiu hat nhan, Vien Nang lugng nguyen tir Viet Nam la don vi duy nh4t trong nudc hien nay dang san xuat cac loai ddng vi phdng xa va dugc chdt phdng xa phuc vu cho viec chin doan va dieu tri benh Mot sd ddng vi phdng xa chi san xuit dugc tren Id phan ring hat nhan va cd gia tri sir dung cao trong y hpc nhu ' 'l, ^^""Tc, •'^P, '"Sm Hien nay cac ddng vj phdng xa nay dugc san xudt va cung cdp thudng xuyen den cac co sd y hpc hat nhan trong nudc Ciing vdi trang

thik bi phuc vu cho viec san xudt va kilm tra chdt lugng cac dugc chdt phdng xa theo tieu

chuan QA/QC/GMP cua qudc te, Trung tam ciing dugc ddu tu mdt sd trang thilt bi cho san xudt cac kit dinh lugng phdng xa mien dich (RIA), san xudt chdt danh ddu cac hocmon vdi ddng vi phdng xa, danh dau cac khang the vdi dong vi phdng xa, thuc hien cac nghien ciiu tdi

uu qua trinh danh dau va dinh lugng

•' Cac nghien ciiru san xuat khang the don ddng danh ddu vdi ddng vi phdng xa hoac cac

chdt khang ung thu cho muc dich chan doan va dieu tri ung thu ciing da dugc dat ra tir nhieu nam nay, nhung chua cd dieu kien de thuc hien Trong cac benh ung thu mau, theo thdng ke, ung thu mau khdng Hodgkin la dang phd bien va xay ra ca d liia tudi thanh thieu nien Viec nghien ciiu va san xuat cac dugc chat phdng xa khang the don ddng tai Viet Nam, phuc vu cho chan doan va dieu tri trong nudc la viec lam cd y nghia ldn cho xa hdi, cho cac benh nhan ung thu

Hien nay, trong nudc cac phdng thi nghiem tai cac trudng dai hpc Y, tai Vien Cdng Nghe Sinh Hpc Viet Nam dang thuc hien cac nghien ciiu ve san xuat khang the don ddng cho chan doan va dieu tri benh ung thu Vi vay ITnh vuc nghien ciiu danh dau khang the don ddng trong nudc se cd dieu kien nghien ciiu va iing dung cho y hpc

II Muc tieu nghien cihi

Muc tieu cua de tai la hoan thanh quy trinh dieu che va kiem tra chat lugng dugc chat

ca khang the dcm ddng khang CD20 danh dau vdi ddng vi phdng xa '^'l:

Hoan thanh quy trinh danh dau khang the don ddng khang CD-20 vdi ddng vi phdng

xa '^'I

- Hoan thanh cac quy trinh kiem tra chat lugng nhu kiem tra hieu suat danh dau, dp sach hda phdng xa, kiem tra dp dn dinh thudc, dp vd khuan, chi nhiet td, ndi ddc td vi khuan, cac kiem tra chat lugng sinh hpc nhu su phan bd thudc, su dao thai

- U'ng dung lam sang chup hinh phan bd thudc tren benh nhan ung thu mau khdng Hodgkin (NHL) phase I

III NQI dung nghien cihi: Thuc hien 4 ngi dung chinh sau

1) Tinh che khang the don dong

Khang the don ddng don ddng khang CD20 dugc mua d dang tinh khiet, san sang dimg trong y hpc, dugc phan tich kiem tra bang sac ky Idng cao ap FPLC

2) Danh dau khang the doti dong vdi dong vj phong xa ''''l

Day la cdng doan quan trgng nhat de tao ra san pham chinh trong de tai, gdm cac budc sau:

- Chpn phuong phap chloramin T (ChT): Diing chloramin T lam chdt oxy hda va natri metabisulphit (SMB) lam chat khu

- Khao sat hieu suat danh dau va cac yeu td anh hudng den hieu sudt danh ddu: Khao sat cac dieu ki?n tdi uu de dat hieu suat danh ddu hon 90%

- Khao sat ham lugng Chloramin T can thilt trong qua trinh danh ddu vdi phdng xa

- Khao sat thdi gian phan iing tdi uu dk gdn khang th6 vdi phdng xa

- Ty le khang the va hoat dp phdng xa '^'l

- Tach hon hgp phan iing qua cdt Ipc gel Sephadex G-25: Hgp chdt danh ddu se cd nhiSu thanh phdn, do vay hon hgp dugc tach phan doan tinh khilt khang th8 gdn vdi ddng vi

phdng xa ra khdi cac thanh phan khac Day la cdng viec rat quan trgng trong viec tao ra san pham tinh khiet dap iing yeu cau cho sir dung lam sang

3) Kiem tra chat Iirong khang the danh dau phong xa

Thuc hien kiem tra chat lugng nhu dp sach hoa phdng xa, hieu suat danh dau, tinh che san phdm danh ddu qua cdt Sephadex, kiem tra tinh dn dinh cua san pham, kiem tra chat lugng sinh hpc dugc chdt phdng xa gdm vd trimg, chi nhiet td, phan bd sinh hpc tren ddng vat

de san phdm dat tieu chuan chat lugng cua dugc chdt phdng xa

4) iTng dung chup hinh phan bo thuoc tren benh nhan ung thir

Thuc hien phase I lam sang, sir dung che pham khang the don ddng danh dau phdng

xa tren benh nhan ung thu mau dang ung thu lympho bao B khdng Hodgkin cho muc dich chup hinh phan bd thudc, chan doan va dieu tri benh ung thu mau khdng Hodgkin

Phan I TONG QUAN TAI LIEU

I Gidi thieu nhung tien bo moi trong dieu trj mien dich phong xa RIT

Da qua hon hai thap ky, kl tir khi bao cao dau tien cua khang the dugc sir dung danh

ddu vdi ^""Tc dk chup hirih vi tri di can ung thu true trang, viec chan doan va chira benh bang

khang thl khdng ngimg phat triln va ddi mdi Lieu phap dieu tri mien dich phdng xa RIT (radioimmunotherapy) da tiln hda vugt bac nhd sir phat trien sinh hgc phan tir va cdng nghe sinh hpc Khang thl don ddng dugc san xuat vdi miic tinh khiet cao cd the diing tren lam sang [1] Ngay nay, ciing vdi cac phuong phap chiia ung thu truyen thdng, RIT gdp phan dang kl trong viec chiia tri cac ung thu tai phat hoac khdng dap iing vdi hda chat Sau day la vai khai niem trong linh vuc dieu tri mien dich phdng xa RIT

1.1 Cac khai niem

Khang the: La cac phan tir dugc tao ra bdi he thdng mien dich cua co the, hinh 1.1 cho

thdy khang thl cd thl nhan bilt va tieu diet cac chdt ngoai lai nhu la vi khudn, virus Khoa hpc ngay nay cd thl san xudt ra khang thl dl nhan bilt nhiing bia dac hieu hoac nhirng khang nguyen dac hieu hien dien tren cac te bao ung thu dac biet [2]

Khang nguyen

Hinh 1.1 Khang the tao ra bdi he thdng mien dich cua co the

X

Khang the don dong: La nhirng khang the mien dich, true tiep chdng lai mdt yeu td

khang nguyen don ddc, hoac cac bieu vi tren phan tii khang nguyen [3] Tir khi dugc san xuat, khang the don ddng da cd nhieu iimg dung trong cac ITnh vuc nhu y hpc, chan nudi, cdng nghiep Dac biet la trong chan doan va dieu tri benh, khang the don ddng khi gan vdi chat phdng xa hoac hoa chat, sau khi tiem vao ngudi cd the phat hien sdm benh ung thu, phat hien sir lan truyen ung thu, phan biet md lanh tir nhiing md ac tinh, ngan chan va loai trir sir phat trien cua ung thu, dieu tri cac ung thu ac tinh [4]

Dieu trj bang khang the don dong: La phuong phap dieu tri hieu qua ddi vdi te bao

ung thu nhung miic ddc thap nhat ddi vdi te bao binh thudng Cach dieu tri la tiem khang the vao tTnh mach benh nhan Trong mau, khang the di khap noi trong co the va gan vao cac khang nguyen dac hieu tren te bao ung thu [5] Hoat ddng nay ciing bao ddng he thdng mien dich CO the nhan biet va tieu diet cac te bao cd dinh hudng Mac dii mdt vai tl bao binh thudng cung bi tac ddng dac hieu gan khang the don ddng, nhung co thl se dilu chinh va

11

thay thl cac tl bao nay sau khi dieu tri Cach dieu tri bang khang the don ddng la mdt dang dilu tri mien dich thu ddng, bdi vi khang the tao ra sd lugng ldn ben ngoai co the chii khdng phai do dap iing ciia he thdng mien dich co the [6]

Dieu tri mien djch phong xa RIT (Radioimmunotherapy) la gi? RIT la phuong

phap dieu tri ung thu gdm ca viec gan khang the don ddng vdi ddng vi phdng xa [7] Khi mdt khang the don ddng gan vdi ddng vi phdng xa gpi la danh dau phdng xa Vdi RIT, khang the gan phdng xa se nham dich vao khang nguyen dac hieu tren te bao ung thu, phat lieu phdng

xa true tiep vao ung thu, lieu biic xa khdng chi diet te bao nd gan vao, ma cdn diet ca te bao xung quanh gpi la hieu iing "crossfire" [8]

1.2 Khang the dirtfc sv dung chira benh nham dich va chup hinh chan doan

Vao dau the ky 20, Pau Ehrlich, nha hoa hpc tri lieu ngudi Diic da dat ra ten gpi la "hat

ky dieu" de giai thich y tudng diing he mien dich ciia co the de nham dich dac hieu vdi cac receptor tren vi khuan [9] Ngay tir nhiing nam 1950 cac nha khoa hpc ngudi My nhu tien sy David Pressman, William Bale, Irving Spar da nghien ciiu y tudng dinh vi ung thu cd chpn loc bang each tao khang the khang cac receptor tren ung thu d loai gam nham Hp da danh dau phdng xa ' 'l vdi cac khang the nay va thay dupe ung thu bang each do hoat dp phdng xa tap trung cao trong khdi u, sir dung may dem phdng xa va so sanh vdi md binh thudng [9]

Cac khang the la cac glycoprotein sinh ra bdi cac te bao B (hinh 1.2) de chdng cac benh viem nhiem va cac the sinh vat ngoai lai nhd he thdng tien hoa qua hang trieu nam de bao ve ddng vat theo mdi trudng sdng [2]

Khang the ^=~^_</ s ^

Te bao B i^d^fe, ^"^/

•Dai thu'c bao

Hinh 1.2 Dap img mien dich cua co thl

Nhu la mdt phan ciia he dieu hoa miln dich, khang thl cQng dugc sir dung nhu vacxin

de dieu tri, ngan chan cac viem nhiem va dugc phat triln dl chdng lai cac benh khac, kl ca b?nh ung thu Khang the dugc md ta la phan tir cd hinh dang nhu chtr Y, hai canh tay cua chii

Y la vi tri gan cua cac thu the (receptor), hoac cac khang nguyen dac hieu Dilu nay da ggi len y nghT la cac khang the nay se gan vdi khang nguyen ciing loai khi dua vao trong co thl [2] Cai dudi cua khang the tuyen cac te bao miln dich hoac cac lymphocyte va cac bd thl,

cac the nay ndi tiep vdi cac khang the tuan hoan di den cac chd viem nhiem hoac true xuat cac the ngoai lai [9]

Cho den nhung nam 1980, cac nghien ciiu diing khang the danh dau phdng xa '^'l, '^'l cdn bi han che, nhung ITnh vuc nay da trd nen hieu qua hon nhd nhiing ddng gdp ciia cac nha hoa hpc nhu Hnatowich (trudng dai hpc Massachusset) da sir dung phdi tir hoac hda chat gan kim loai de gan khang the vdi cac ddng vi phdng xa, each nay tao ra nhieu kieu nhan phdng

xa gan vdi khang the de phat hien hoac dieu tri benh nhu la cac nhan phdng xa '"in, ' " Y [10]

Sau khi chimg minh dugc la cac khang the tao ra tir ddng vat (la khang the da ddng) cd the phan biet cac ung thu phat trien trong chupt vdi cac cac md binh thudng, van de thii thach

la chuyen iing dung nay ddi vdi benh nhan ung thu, tiic la sir dung khang the khang khang nguyen ung thu tren co the ngudi va cd the dinh vi tren cac khdi u nay

Nam 1972, cac nha khoa hpc quan sat mot ung thu rudt d ngudi cd bieu hien khang nguyen ung thu bieu md phdi (CEA: carcinoma embryo antigen) [9] CEA dugc phat hien dau tien nam 1965 bdi Phil Gold va Samuel O Sam Freedman (trudng dai hpc McGill, Montreal, Canada) tir dich chiet md ung thu rudt d ngudi Cac nha khoa hpc suy luan rang khang nguyen nay phai la mdt dich chpn loc trong qua trinh dua chat phdng xa den u rudt khi khang the khang CEA dugc gan vdi nhan phdng xa Vi the nam 1973, ciing vdi F James Primus va Hans J Hansen, PhD., cac nha nghien ciru tren lan dau tien thay rang, ung thu ngudi cd the phat trien trong co the ddng vat thi nghiem, bang each dinh vi ung thu diing khang the khang CEA gan phdng xa [10, 11] Dieu nay dua den giai doan nghien ciiu lam sang, sau khi phat trien va tinh sach CEA tdi miic lam sang, va danh dau vdi '^'l (mdt ddng vi phdng xa dimg cho con ngudi de phat hien va dieu tri benh tuyen giap), cac nha nghien ciiu

da bat tay vao chiing minh rang ung thu trong benh nhan dugc phat hien va that sir dinh vi vdi khang the gan phdng xa [9]

Cac bao cao ve sau (1978) cho thay, cac ung thu tao ra CEA va nhirng ung thu khac nhu la ung thu vii, bang quang, phdi ciing san sinh ra CEA dii de cd the phat hien bdi cac khang the phdng xa Quan sat nay da cho ra ddi phuong phap sir dung khang the phdng xa de phat hien benh, cac tac gia dat ten la Radioimmunodetection hoac Radioimmunotherapy [9]

Trong nhirng nam ke tiep, nhieu khang the don ddng mdi dugc phat hien, cac ddng vi phdng xa khac ciing dugc dimg, cac khang the cd dang nhd hon ciing dugc sir dung td ra thich hgp cho con ngudi Tir khi nguyen tac dinh vi va nhin thay CEA bieu hien ung thu dugc chiing minh, ky thuat phat hien mien djch phdng xa (radioimmunodetection) dugc iing dung phat hien cac ung thu va cac benh khac ngay ca benh ddng tu mau, nhdi mau co tim, viem nhiem Khi gan vdi ^^""Tc, cho thay vi tri viem nhilm, viem tuy xuong, viem rudt thira,

da cung cap thdng tin chan doan cang nhanh hon [9]

Vao nam 1986, lan dau tien FDA phe chuan cho sir dung 0KT3 ma ban chat la khang the IgG2a chupt khang CD3 diing trong phdng thai ghep d nhung benh nhan ghep npi tang Den nam 2009, da cd 23 san pham cho phep dieu tri (bang 1.1) va hang tram san pham dang thvrc hien giai doan lam sang [9, 12]

13

Bang 1.1 Cac Khang thl don ddng dugc FDA phe chuan

(Johnson & Johnson

and Schering Plough

Fab lai

IgG lai

IgGl lai

IgG 1 nhan hoa IgGl lai IgG 1 nhan hoa

IgGl lai

IgGl nhan h6a

IgG4 lai,

gan

calicheami cin IgG 1 nhan hoa IgGl chuot, danh dau

IgG 1 nhan hoa

IgG 1 nhan h6a

IgG2a, chupt,

Khdng nguyen

CD20

CD25 CD25

5

-8.0

0.3 0.1

sir dung trong dieu tri

Cache hieu irng Khoa chuc ndng te bdo T bieu hien CD3; chong thai loai cay ghep Gdn thu the va doi khdng; kim ham ket tu tieu huyet cau

Gdn thu the vd doi khdng

Lam te bdo nhay vai hoa cdm ung apoptosis, ADCC va CDC

Gan thu the vd doi khdng

Gdn thu the vd doi khdng Gdn vd trung hoa RSV;

kim ham sy tdi bdn vd dung hop

Gdn phoi tii vd doi khdng thy the

Ldm te bdo nhay vai hoa trj; kim hdm hinh thanh mach vd tdng sinh, cdm ung ADCC

Cam ung phd va DNA mach kep vd ldm chet te bdo (do calicheamicin) Cdm ung ADCC va CDC

Gay chet te bdo bdng phong xa; cdm ung apoptosis

Gdn phoi tu vd doi khdng thy th6; cam ung CDC

Gdn phoi tu vd doi khdng thy the; gidm gidi phong chat hieu ung vdi dap ung di iing tir te bdo bach cau ua kiem va duOng bdo

Gay chet t§ bdo bdng phong xa; cam ung

Tri benh

Phong thai loai ghep than cap tinh Ngdn dong mdu

do tieu huyet cau trong phdu thuat dong mach vdnh

Ung thu rupt ket

U lympho khong Hodgkin vd viem khap dang thdp

Phong thdi loai ghep than cap tinh Phong thai loai ghep than cap tinh Chong nhiem

RSV a tre c6

nguy ca cao Benh Crohn, thdp kh6p, vdy n6n, viem lo6t rupt ket

vd viem d6t song cung

Ung thu vu di cdn dang sieu bieu hien

ERBB2 Ung thu bach cdu dang tuy bieu hien

C'D33

Ung thu bach cdu lypho mdn tinh te bdoB

U lympho khong Hodgkin

Thdp khdp vd vay nen

Hen suyen lau ddi

U lympho khong Hodgkin

(Biogen Idee Inc vd

Elan Corporation, pic)

IgGl lai

IgG 1 nhdn h6a

IgG4 nhan h6a

Gdn thy the vd doi khdng; ngdn bach cau gdn vai cdc te bdo khdc Gdn thy the vd doi khdng; kim ham te bao tdng sinh; cam irng apoptosis; ldm te bdo nhay vai hoa trj va xa tri; kim ham hinh thdnh mach, sy xam lan va di cdn; cdm ung ADCC Gdn thu the vd doi khdng phoi tu; kim ham sy hinh thdnh mach vd sy tien trien cua benh di cdn Gdn thy the va doi khdng; kim hdm bach cau bam dinh vai cdc thy the doi lap ciia chung

epidermal growth factor receptor

vascular endothelial growth factor

T cell VLA4 receptor

complement system protein C5

Vay nen dang mdng

Ung thu rupt ket

Cac kieu siir dung khang the trong dieu tri ung thvt

De dieu tri ung thu, khang the dugc sir dung cd the gan ket hoac khdng gan ket Tuy theo each gan ma tao ra nhieu kieu dieu tri ung thu khac nhau Hinh 1.4 minh hga cac kilu sii dung khang the trong dieu tri

Khang the tran: La dung khang the khdng gan vdi mdt chat nao trong dieu tri ung

thu Cac CO che true tiep tieu diet te bao ung thu khi diing khang thl tr4n la co chi lie chi tac dung sinh hgc cua khang nguyen be mat, co che kich thich sir chit cd chuong trinh ciia tl bao (apoptosis) Co che gian tiep la co che miln dich, dd la gay ddc tl bao phu thudc bd the (CDCC), gay ddc tl bao phu thudc khang thl (ADCC), co chi gay ddc phu thudc bd thl (CDC) Vi du cac khang the tran dugc FDA cho phep sir dung nhu Rituximab la khang thl khang CD20, Trastuzumab (Herceptin) la khang thl khang protein HER2, Alemtuzumab (Campath) la khang the khang CD52, Cetuximab (Erbitux) la khang thl khang EGFR protein, Bevacizumab (Avastin) la khang the khang protein VEGF [13]

Nhirng diem h p che cua khang thl khdng danh dau: Tat ca cac tl bao ung thu diu cSn gin vdi khang thl Co chi dap iing miln djch cua benh nhan khdng thl khdng bi anh

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hudng khi tuong tac bdi khang the Cac te bao ung thu cd thl de khang vdi cac co che khang ung thu true tiep ciing nhu co chi mien dich ciia khang thl [4]

do, khang the gan phdng xa ciing dugc diing de phat hien sir lan rdng ciia ung thu trong co the nhu OncoScint (ung thu true trang va ung thu budng tning), ProstaScin (ung thu tuyen tilnliet)[15]

- Khang the gan vdi ddc td gpi la ddc td mien dich (immunotoxin), chat ddc diing de g5n vdi khang the thudng cd ngudn gdc tir thuc vat hoac vi khuan Vai loai ddc td vi khuan nhu diphtherial toxin (DT) hoac pseudomonal exotoxin (PE 40), hoac ddc td tir thuc vat nhu ricin A, saponin Cac nghien ciiu lam sang da cho thay cd su co lai trong vai ung thu, nhung nhieu van de chu yeu can giai quyet trudc khi sir dung ddc td mien dich rdng rai tren lam sang Chi duy nhat gemtuzumab ozogamicin (Mylotarg) la ddc td mien dich dugc FDA phe chuan cho sir dung lam sang, chat nay chiia ddc td calicheamicin, gan vdi khang the khang CD33, dimg trong dieu tri ung thu bach cau tiiy cap (acute myelogenous leukemia AML)

Ddc td mien dich cdn dugc iing dung trong dieu tri cac ung thu lympho, u nao va cac ung thu khac [16]

Cai tien dieu tri bang khang the don ddng: Hieu qua ciia dieu tri cdn nhieu han che bdi khang the san xuat tir tap doan te bao lai ciia chupt Sau thdi gian dieu tri, he thdng mien dich cua CO the phat hien khang the la chat ngoai lai va tieu diet ngay khi dua vao co the Vi ly do nay ma cac nha khoa hpc da gan gen mang doan hoat tinh ciia khang the chupt vdi gen mang khang the ngudi Viec san xuat gen khang the chupt - ngudi gpi la khang thl don ddng kham

"chimeric" hoac "humanized" [17] Khang the mdi tao ra nay gidng nhu khang the ngudi, vi vay khdng bi he thdng mien dich co the phat hien Cach dieu tri mdi hon niia la diing mdt doan khang the thay vi diing ca phan tir Cac doan khang the nhd nay cd the di den ung thu tdt hon, vi vay viec chira tri se hieu qua hon

Khang the gan nhieu bu'd'c (multistep targeting): La phuong phap mdi dat ra nhim khac phuc su ehiu lieu cua nhiing te bao lanh binh thudng trong qua trinh luu thdng cua khang the gan phdng xa, ddng thdi ciing lam gia tang hieu qua dieu tri Cd nhieu phuong phap de tien hudng dich, dd la diing khang the gan vdi biotin va streptavidine (Ab/SA) Hgp chat nay tiem vao cd kha nang dinh vi len te bao ung thu Sau khi tich liiy cue dai, Ab/SA di vao vi tri khdi u Tiep theo la cho lieu ^"Y-DOTA-Biotin ^°Y-DOTA-biotin nay cd kich thudc nhd nen xam nhap, Ipt vao khdi u nhanh chdng, tai dd nd gan vdi phiic SA-Ab, phat lilu phdng xa den ung thu Phan tir DOTA biotin khdng phdng xa nhanh chdng dao thai ra khdi mau va qua nudc tieu ra ngoai Phuong phap nay hieu qua va giam thdi gian ehiu lieu phdng xa cua co quan lanh [8]

E3l4u trj b a bu'cS-c t l 6 n hu-6-no Ctfch ( p r e t a r g e t i n g )

I \ ^ Bu'ffc I: Tiem Bio-IgG

BifoclliTiemStAv, dao thai StAv-IgG ra ktioi mau

BLro-c III: Tiem Biotini deinh d i u phong xa Hoat do phong xa loc qua th^n Vci th^i ra kh6i cff thS i ^ B ^ "

Hinh 1.4 Khang the tien hudng dich Mdt phuong phap tiln hudng dich khac nira la dimg enzym gin khang thl dac hieu, sau khi tiem dua thudc tien than (prodrug) chua hoat hoa vao, khi vao dich, thudc tiln than dugc enzym hoat hoa, chuyin thanh thudc, tieu diet ung thu Phuong phap nay gpi la lieu phap khang the kit hgp enzym va thudc tiln than chdng ung thu ADEPT (antibody-directed enzyme prodrug therapy) [9]

17

L3 Cac tac nhan chup hinh moi trong dieu trj RIT

Tir khi dugc gan vdi cac chat phdng xa, cac khang the, va ca nhirng doan khang the, khang cac khang nguyen khac nhau lien quan din cac loai benh tat da dugc phat triln dl hien hinh dac biet cac loai benh nay [17,18] Cac benh diing khang thl danh diu phdng xa dl chup hinh nhu ung thu true trang (satumomab pendetic, arcitumomab), ung thu tuyin tiln liet (capromab, pendetide), u lympho khdng Hodgkin, ung thu gan, nhdi mau co tim (imciromab penlefate) nhiem triing va viem (sulesomab, fanolesomab), nhiem trimg dac hieu va tic mach mau Dong vi phdng xa phd biin la ^'""Tc, '"in, '^''l, ^^Ga, ^^Cu va '^F la tac nhan cho chup hinh PET va phat triln phuong phap immunoPET (hinhl.5), se cho ra nhiing hinh anh cd dp phan giai cai tiln dang kl khi diing vdi ddng vj ciia PET Steven M Larson MD, trung tam ung thu Memorial Sloan - Kettering (My) la ngudi diu tien dua ra phuong phap nhim dich

cac ung thu diing khang thl danh diu vdi ddng vi phdng xa phat positron dimg I [9]

Hinh 1.5 May PET va chup hinh immunoPET

1.4 Ung dung RIT trong dieu trj benh u lymphoma khong Hodgkin

Benh u lympho khdng Hodgkin la dang ung thu cac te bao lympho B trong he mien dich Cimg vdi benh Hodgkin, la cac benh ve cac hach bach huyet, benh khdng Hodgkin la b$nh ciing gidng nhu benh ung thu bach cau dang nguyen bao lympho cap Acute Lymphobllastic Leukaemia (ALL), cac te bao trong he bach huyet khiem khuyet va phat triln

vd dp xam chiem tuy xuong va ngan chan hoat ddng san xuat cac te bao quan trpng khac trong mau, gay thieu mau, dau xuong va nhiem triing Day la mdt loai benh phd biin, xay

ra theo mpi liia tudi Cac te bao ung thu nay mang nhiing dau hieu dac trung bat thudng, dd

la khang nguyen Khang nguyen CD20 dac biet xuat hien 50.000 - 200.000 vi tri tren phan tii lympho bao B ac tinh (hinh 1.6) Do vay, khang the don ddng khang CD20 la cdng cu ly tudng de nhan biet nhirng khang nguyen tren nhirng te bao ac tinh dd va tieu diet

Khai niem ve dieu tri nham dich da dugc cac nha khoa hgc dua ra tir nhieu nam trudc Vao nam 1953, Pressman va Komgold da chiing minh rang cac khang the cd the nham dich dSc hieu len te bao ung thu [9] Tuy vay, phai tdi nam 1975, khi Kohler va Milstein nhan giai thudng Nobel cho cdng trinh nghien ciiru ciia hg ve ky thuat tao te bao lai hybridoma, viec san xuit khang the don ddng nham dich khang nguyen dac hieu mdi bat dau di vao hien thuc [9]

Nam 1979, Nadler va cdng sir da dieu tri benh nhan u lymphd ac tinh dau tien bang khang the don ddng [19]

Hinh 1.6 Te bao B ung thu cd bieu hien khang nguyen CD20

Sir dap ling dieu tri phu thudc vao nhieu yeu td nhu the tich khdi u, tdc do phat trien, muc bieu hien khang nguyen, lieu hap thu biic xa vao khdi u, dp nhay cua tia biic xa Vi vay, cin chup hinh RIT vdi '^'l cho NHL Ndi chung, viec dieu tri ung thu dugc che ngu bdi

3 hinh thiic la giai phiu, biic xa va hoa tri Neu ung thu dugc phat hien sdm khi nd dugc dinh vi, giai phau hoac hire xa, cd the dii de kiem soat va loai trir triet de benh Dieu tri biic

xa diln hinh gom chilu xa ngoai len viing dac biet ciia co the cd khdi u, hoac "hat gidng" biic

xa se phat tia trong viing ap sat ung thu (brachytherapy) dang tiec la ung thu thudng dugc phat hien sau khi nd da di can khdi viing ung thu gdc den cac md va co quan khac Dieu tri bing hoa chat la phuong phap dieu tri khdi dau, khang the dugc dimg de gan vdi thudc khang ung thu, hoac chat phdng xa, de di sudt co the va phan phdi lieu phdng xa den cac te bao ung thu

Trong 30 nam qua, ti le mac phai benh NHL ngay cang tang d My, trd thanh mot ung thu diing hang thii 5 va nguy co tir vong diing hang thii sau d My Vdi gan 270.000 ngudi

My hien nay, udc tinh cd 60.000 ca mdi dugc chan doan va gan mdt nira trong sd dd dugc dilu tri Lymphocyte binh thudng va ac tinh bieu hien cac receptor khac nhau, chiing la dich cho cac khang the don ddng Gerald DeNardo, MD, Sally DeNardo, MD, trudng dai hpc California, Davis, la nhimg ngudi dau tien da chiing minh viec tri lieu thanh cdng NHL vdi khang the phdng xa [5] Oliver Press, da tien phong trong viec diing phdng xa lieu cao ciia '^'l gin khang the khang CD20, de dieu tri NHL Ong va cdng su da chiing minh tinh kha thi

va thuan lgi cua phdi hgp giiia RIT va hda tri Thuc ra la dieu tri hudng dich NHL dimg khang thl khang CD 19, CD20 chupt (khdng gan phdng xa) da dugc nghien ciiu tir hon 20 nSm nay, nhung tir khi phat trien ky thuat khang the khang CD20 kham (chimeric) giiia ngudi

va chupt, gpi la rituximab, da tao ra mdt cudc each mang trong dieu tri NHL va lam nen tang cho nhung nghien ciiu xa hon trong viec diing khang the don ddng de dieu tri cac ung thu khac [5]

Dieu tri NHL diing Rituximab cho thay su thuyen giam mdt each tdng the trong mdt mia sd benh nhan NHL cd dp ac thap (indolent) trong phac dd phdi hgp dieu tri vdi hda chat,

nd cd the cai thien hieu qua hda tri, ket qua la ung thu bien mat hoan toan trong nhieu benh

19

nhan NHL Dac biet hip dan la khdng cd cac ddc tinh nhu vdi cac thudc khang ung thu thudng thay nhu su iirc che (suppression) cac te bao mau Id mieng va rung tdc [9]

Cac nha nghien ciiu nhu Thomas Witzig, Gregory Wiseman, Christine White la nhung ngudi da cd cdng trong nghien ciiu lam sang cho thay nhiing uu diem cua viec dieu tri khang the danh diu phdng xa khi so sanh vdi khang thl khdng danh dau phdng xa, va dugc FDA cho phep sir dung lam sang san pham dilu tri ung thu ten la ^''V-ibritumomab tiuxetan Cimg vdi phac do phdi hgp dilu tri rituximab vdi ^°Y-ibritumomab tiuxetan, mdt khang the khang CD20 ban chit chupt gin vdi ddng vi phdng xa kim loai ^"Y da dugc dieu che San pham nay da thuong mai tir nam 2002 dimg dilu tri NHL [51] Mdt san pham khac diing de dieu tri phdng xa mien dich dugc phep sir dung lam sang tir nam 2003 la khang the khang CD20 gan '•"l, ten la '^' I-tositumomab diing trong dieu tri NHL Mac dii khang the nay la khang the ed ban chat chupt, ca hai san phim deu cho thay budc cai tien dang ke de tieu diet ung thu khi so sanh vdi khang the khdng gan phdng xa (bang 1.2) [9]

Bang 1.2 Cac khang the danh dau phdng xa dang dugc nghien ciiu dieu tri

^"Y-epratuzumab anti-CD22 IgG

^'^Bi-HuM195 anti-CD33 IgG

Benh bach cau (leukemia) T-cell lymphomas; non-Hodgkin's and Hodgkin's lymphomas

Ung thu budng triing (ovarian cancer) Ung thu tuyen tien liet (prostate cancer) Ung thu tuyen tuy (pancreatic cancer) Ung thu rudt ket (colorectal cancer) Ung thu rudt ket (colorectal cancer) Cac ung thu cd khang nguyen CEA (CEA producing cancers)

Ung thu phdi (lung cancer) Ung thu gan (hepatocellular cancer) Ung thu than (renal cancer)

CNS or leptomeningeal cancer Ung thu dau va cd (head and neck cancer) Ung thu nao (brain cancers) (gliomas)

U than kinh dem (glicomas)

Bang 1.2 tdm tit nhilu loai khang thl danh diu phdng xa trong nghien ciiu lam sang, cho thay rang RIT ddng vai trd tich cue trong dieu tri da dang ung thu Viec lira chpn chat danh dau thich hgp cho dilu tri nhim dich la mdt thach thiic chu yeu, trong khi chgii lira nhan phdng xa thich hgp, phuong phap danh diu thich hgp va ngay ca dang khang the sir dung trong ky thuat nhim dich la tit ca nhiing thay ddi cin xem xet can than tiiy theo tinh trang lam sang

Ngoai ra, cac phucmg phap cai tiln de gia tang lieu biic xa chpn Ipc den khdi u can dugc tinh den Mdt phuong phap mdi cd hieu qua ro ret la khu tni phdng xa vao khu vuc ung thu nhu la viing ung thu hoac trong khdi u That vay, ca hai quan diem nay da cho thay nhirng ket qua lam sang dang khich le khi phat lilu phdng xa danh dau khang the den cac viing phlu thuat trong dieu tri ung thu nao va ung thu vii da dugc md ta bdi Pietro Riva, Giovanni Paganelli, Vien Nghien ciiu ung thu Chau Au, Milan, Italy Hoac la dua phdng xa true tiep vao khoang bung (Ruby M Meredith, MD, PhD, trudng dai hpc Alabama, Birmingham) [9]

II Cac thanh phan co ban trong RIT

Cd ba thanh phan co ban trong RIT, dd la khang nguyen, khang the va ddng vi phdng

xa Sau day la cac tinh chat va dac diem cua ba thanh phan dimg trong dieu tri RIT:

II.l Khang nguyen CD20, vai tro va tiem nang irng dung

He mien dich thu dugc la mdt co che phdng ve vd ciing hieu qua tdn tai trong hau het cac loai ddng vat cd xuong sdng bac cao He thong nay tao ra cac dap iing bao ve nhanh, dac hieu cao, khang lai mam benh trong mdi trudng sdng De cd the tao ra dap iing khang lai bat cii vi sinh vat nao, dau tien he mien djch can vi sinh vat dd Day la diem ma he mien djch thu dugc khac vdi cac he thdng khac trong co the, vdi kha nang nhan biet cac loai phan tir khac nhau tao thanh vi khuan Cac thanh phan dugc nhan ra nay gpi la khang nguyen Cac co chat, hoac cu the hon la cac phan tir dugc nhan ra bdi mdt hoac nhieu thanh td ciia dap iing mien djch thu dugc gpi la khang nguyen [2]

He mien djch dugc tien hda de nhan ra tat ca cac phan tir ngoai lai khdng phai ciia vat chu, ve mat nguyen ly, cac khang nguyen bao ham mdt phd rdng bao triim tat ca cac nhdm phan tir hiru co, tir cac hgp chat hda hpc don gian nhat cho tdi cac dai phan tir phiic tap nhat

Khang nguyen tren te bao ung thu phan bd khdng ddng nhat, bieu hien vdi mat dp cao, cd the gan dac hieu vdi khang the don ddng, khdng thay ddi hoac dieu chinh sau khi gan vdi khang nguyen, khang nguyen dich khong luu thdng trong he tuan hoan hoac rcri ra he tuan hoan Sau day la vai dac trung ciia khang nguyen CD20

II.l.l Dac trung cau true

CD20, cdn cd ten khac la BI, la mdt khang nguyen dac hieu don ddng te bao lympho

B, bieu hien tren te bao lympho B d mdt sd giai doan nhat djnh CD20 la mdt protein khdng glycosyl hoa cd khdi lugng phan tir khoang 33-35 kDa Gene ma hoa CD20 nam tren nhiem sac the sd 11, canh dai, locus 12-13

Cau tnic va vi tri ciia CD20 dugc md ta trong hinh 1.7:

21

Viiig xuyen dng

CD40

QCXJUOC

Ngoai lio

Ldp phosphol^iid ' kep

-Baotutfng

viaig noi bdo

Hinh 1.7: Cau tnic va vi tri ciia CD20 tren mang te bao

CD20 la mdt protein cd 4 viing xoan xuyen mang, ca 2 dau tan NH2 va COOH deu nam trong bao tuong Hai vdng ngoai bao ciia CD20 rat khac nhau ve kich thudc Vdng nhd hau nhu khdng Id ra khdi mang, vdng ldn cd dp dai khoang 46 axit amin, tir axit amin sd 140 den axit amin sd 185 Tren vdng ldn cd cau disulfid giira axit amin sd 167 va 183 Vdng nay chiia

da sd cac epitope cua cac khang the don ddng [52]

11.1.2 Chirc nang

Den nay, chiic nang ciia CD20 van chua dugc biet mdt each rd rang Tren nhirng con chupt cd te bao B khdng bieu hien CD20 khdng thay cd sir suy giam chiic nang te bao B ro ret Tuy nhien, mdt sd nghien ciiu da chiing minh CD20 cd vai trd trung gian trong nhieu hoat ddng sinh hpc cua te bao B thdng qua sir gan khang the nhu:

Ddi vdi sir di chuyen ciia ddng canxi vao te bao: su chuyen vi tri va gan dac hieu CD20 vao cac "mang" lipid sau khi CD20 gan khang the la dieu kien tien quyet ddi vdi khdi dau su dan truyen tin hieu va/hoac sir tao thanh cac kenh canxi

Ddi vdi chu ki te bao: CD20 cd vai trd quan trpng trong dieu hoa su phat trien chu ki te bao (cell cycle progression), it nhat la d te bao B binh thudng Bang chiing la cac khang the don ddng hudng dich CD20 deu gay thay ddi chu ki te bao, lam day nhanh su chuyen te bao

B tir pha GO sang Gl, cac khang the don ddng cimg nhdm vdi BI lai lic che su chuyen te bao

B tu pha Gl sang pha S/G2 va pha M

11.1.3 Tiem nang irng dung cua khang nguyen CD20

Tai My, moi nam cd khoang 55.000 nguoi dugc chan doan mic benh u lympho khdng Hodgkin, trong dd 65% khdng dap iing vdi dieu tri Benh nay hien diing thii 6 trong cac nguyen nhan gay chet do ung thu Khoang 85% den 90% NHL cd ngudn gdc do tl bao B va bieu hien cac chi thi khang nguyen be mat te bao B Ngudi mac benh nay cd thdi gian sdng trung binh 8-10 nam [20] Tai Viet Nam, theo tai lieu tai Hdi Nghj Ung thu nam 2008 td chiic tai benh vien Ung budu thanh phd Hd Chi Minh, benh NHL la mdt trong 10 loai ung thu thudng gap, xuit do chuin theo tudi la 4,6/100.000 (nam) va 33,2/100.000 (nir) Ghi nhan nhirng benh ung thu hay gap d tre em tai 5 tinh thanh giai doan 2001-2004, NHL diing hang thiir hai d Ha Ndi (15%), Hai Phdng (15,4%), Thai Nguyen (14,3%), Thira Thien Hue (16,4%), diing thii ba d Cin Tho (20,9%) Tudi trung binh la 9, dinh cao nhit 7-10 tudi, dinh tilp theo la cudi tudi thieu nien Ti le nam:nii la 3:1 [51]

U lympho loai nay cd thl chuyin tdi miic do nang hon ve mat md hgc (histologic) va sau dd rat khd dieu trj Benh phat triln cham va thudng thuyen giam trong nhieu nam, nhung cudi ciing se gay tir vong cho ngudi benh

CD20 la mdt phan tir dich tilm nang trong y hpc bdi khang nguyen nay chi bieu hien tren te bao B; miic bieu hien cao (khoang 100.000 phan tir/tl bao); khdng bien dang hay ddng hda khi gan vdi khang thl; va thudng khdng phat hien d dang hda tan [21] 6 chupt binh thudng va chupt chuyin gen CD20 ciia ngudi diu cho thiy CD20 la dich hieu qua cho sir lam giam te bao B Ngoai ra, do CD20 khdng bilu hien trong cac te bao gdc tao mau, khdng de dang hi ddng hda va khdng bj tach rdi khi gin vdi khang the danh dau phdng xa nen la mdt dich hap dan cho lieu phap miln djch phdng xa [3]

II.2 Khang the va cong nghe tao khang the:

Khang the sir dung trong RIT trudc hit phai dap iing tinh dac hieu, va cd ai luc cao vdi antigen, trpng lugng phan tir nhd khdng thay ddi va cd dap iing mien djch nhu HAMA, HAHA, HACA

II.2.1 Cau triic:

Cau tnic chung cua khang the gdm hai chuoi nhe va hai chuoi nang ddng nhat, gan vdi nhau nhd cac lien ket disulfide Cd 5 nhdm globulin mien djch (IgG, IgA, IgM, IgD va IgE) vdi trinh tu va sd mien trong cac viing hang djnh tren chudi nang khac nhau Cd hai

ddng phan chudi nang la (k va K) Globulin mien dich G (IgG) la loai khang the dac tnmg

trong huyet thanh binh thudng va dugc nghien ciiu phd bien nhat (hinh 1.8) Globulin mien djch cau thanh tir cac don vj gap nep gdm 100 axit amin, mdi don vj cd cau tnic ddc lap gidng nhau gpi la kieu gap nep globulin mien djch Mien dau N ciia mdi polypeptide (tren chudi nang va nhe), gpi la vimg bien ddi (V), rat khac nhau Cac mien cdn lai cd trinh tu gidng nhau gpi la viing bat bien (C) So sanh trinh tu viing V cho thay tinh kha bien khdng phan bd deu ma tap trung vao ba viing gpi la cac viing sieu bien [2]

Mofv <ry»t

(b) («)

Hinh 1.8 Cau tnic ciia IgG (a) So dd ciia IgG tieu bilu Cac chudi L va H dugc thl

hien la cac ddng ke dam, vdi cau disulfide ndi phan tir lien kit cac gdc Cys (S-S) (b) Ciu tnic lap the cua IgG

23

11.2.2 Ai lu-c (affinity) va do bam (avidity)

Khi khang the don ddng dugc tao ra bdi ky thuat te bao lai cd hai vj tri gan khang nguyen va huyet thanh chiia cac khang the da ddng (cd kha nang gan vdi nhieu yeu td trinh dien khang nguyen), khang the cd the da trj trong phan iing vdi khang nguyen

Khi trong mdt khang nguyen cd nhieu vj tri vdi khang the gidng nhau (dai phan tir hoac vi sinh vat) phan iing vdi khang the da tri, ai luc gan tang manh vi tat ca cac lien ket khang nguyen-khang the phai bi pha vo ddng thdi trudc khi khang nguyen va khang the tach

ra Do dd, nang lugng gan ciia khang nguyen da tri vdi cac vj tri gan khang nguyen nam tren khang the da tri se ldn hon tdng ai luc cua mdi vi tri gan vdi mdt khang nguyen Luc gan giiia mot khang the da tri va khang nguyen da trj gpi la dp bam (avidity), de phan biet vdi ai lire

Dp bam cua mdt khang the vdi khang nguyen cua nd dugc xac djnh bdi tdng tat ca cac tuong tac ddc lap xay ra giira cac vj tri gan khang nguyen nam khang the va cac yeu td trinh dien nam tren khang nguyen Dp bam ciia mdt khang the vdi khang nguyen cua nd phu thudc ldn vao ai luc cua cac vj tri gan rieng biet vdi cac yeu td trinh dien tren khang nguyen

Nd ldn hon tdng cua cac ai luc nay neu ca hai vj tri gan khang nguyen cua mdt khang the gan vdi khang nguyen Do dd, cho dii moi vj tri gan khang nguyen cd ai luc thap (nhu IgM mdi tao ra trong dap iing mien djch), thi cac khang the van cd chiic nang trong he mien djch

11.2.3 Ban chat va irng dung cua khang the khang CD20 Rituximab

Rituximab la khang the khang CD20 dau tien dugc FDA phe chuan vao cudi nam

1997 su dung cho ngudi Rituximab dugc tao thanh bang ky thuat di truyen, dung hgp vimg bien ddi chudi nang va nhe ciia chupt vdi vimg hang djnh IgGl/K cua ngudi (hinh 1.9)

Khang the lai tao thanh cd kha nang tieu diet cac te bao B in vitro va lam giam lugng te bao

B trong mau ngoai vi in vivo Su giam te bao B trong tiiy xuong, lach va cac hach lympho

chua dugc nghien cim ky [23]

I I = Vung bien 3Si cua chuot

•• Vung hixig dinh CU4 ngujcyi

= Vung hing 3mh Fc cua n g u ^

Vi = Vung bien doi chuoi nhe v = Vung bien d6i chuoi nang

Cl = Vung hing dinh chuoi nhe

Cy = Vung hing dinh chuoi nang

Hinh 1.9 Cau tnic cua khang the Rituximab

FDA phe chuan Rituximab phan ldn dua tren cac thir nghiem ddi vdi u lympho te bao

B khdng Hodgkin tai phat nhe Ty le dap iing trong nhdm khd chiia nay la khoang 50% khi chi sii dung Rituximab Sau dd, tren 380.000 trudng hgp da dugc kiem chimg cho thay dp an

toan tot Mdt sd khdi u ac tinh khac vdi dac trung CD20+ tren tl bao B nhu benh Hodgkin, CLL, u lympho cip cao, hdi chiing Waldenstrom, cung dugc dieu tri bang rituximab vdi cac kit qua dang khich le Kit hgp Rituximab vdi methotrexate hoac cyclophosphamide cd hieu qua dac biet, vdi ACR50 xip xi 50% Cac nghien ciiu vdi benh da thin kinh do IgM, ITP, hdi chiing Wegener, hdi chiing ngung kit to lanh, hdi chiing Waldenstrom va viem da/viem da-

co cung cho thay nhCrng hieu ung dieu tri hiiu ich [24]

II.2.4 Cong nghe tao khang the

Ky thuat te bao lai: Ngay sau khi dugc md ta lin diu tien vao nam 1975, ky thuat dimg te bao lai dl tao khang thl don ddng dac hieu da cho thay day la mdt cdng cu manh de phan tich nhiing van de chua biet tdi trong cac he thdng sinh hpc Tac gia ciia cdng trinh nay dugc nhan giai Nobel y hpc vao nam 1984

D I tao ra khang the don ddng bing ky thuat tl bao lai, tiem khang nguyen vao vat chii thich hgp va "bit tir hda" cac tl bao T tilt khang thl mong mudn bang each dung hgp te bao

B trong he mien djch vat chu vdi ddng te bao myeloma (mdt ddng te bao ung thu tuy) Ddng

te bao myeloma nay cd kha nang khang vdi purine analogue 6-thioguanine do bat hoat enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT) Dieu nay lam ddng te bao myeloma cd the chet khi cd aminopterin (chat khda budc dau trong qua trinh tdng hgp purine) Te bao B binh thudng khdng nhay vdi aminopterin khi cd hypoxanthine va thymidine; cac con dudng thu nhan sii dung HGPRT la can thiet de suy tri sir sdng

Do dd, chi cac te bao lai (te bao B binh thudng dung hgp vdi te bao ung thu) sdng trong HAT Neu te bao lai tao ra khang the mong mudn dugc xac dinh va tach ddng de tao tinh don ddng, cd the tao ra mAb vdi tinh dac hieu can thiet

Ky thuat nhan hoa khang the tir chuot: Khi cdng nghe san xuat Mab tu chupt bang

te bao lai (hybridoma) ra ddi nam 1975, mdt sd khang the da dugc tao ra tuy nhien rat it thanh cdng bdi dap iing khang khang the tir chupt d ngudi (HAMA) De giam dap ling nay, ngudi ta da tao ra cac khang the lai, trong dd gan viing bien ddi cua chupt vdi cac viing hang djnh cua ngudi bang ky thuat di truyen Tuy nhien mac dii khang the lai it gay dap iing mien djch tu sinh hon so vdi khang the tir chupt, van xuat hien hieu ling khang khang the lai d ngudi (HACA) [2]

De giam thieu hon niia nhiing nhugc diem khang the cua chuot, ngudi ta da tao ra cac khang the nhan hda bang ky thuat protein Trong khang the nhan hda, chi viing CDR chju trach nhiem cho kha nang gan khang nguyen dugc chuyen vao viing khung mien bien ddi cua ngudi Tuy nhien, dieu nay lam mat dang ke ai luc gan khang nguyen Do dd, de phuc hdi ai luc nhu ban dau, cac axit amin quan trgng trong viing khung ciia khang the tir chupt vdi kha nang hd trg cau hinh viing CDR ciing dugc chuyen sang viing khung trong khang the nhan hda Day la phan quan trpng nhat trong phat trien cac khang the nhan hda bang phuong phap nay Ky thuat nhan hda khang the dugc thiet ke vdi sir trg giiip rat nhieu cua may tinh, vdi kha nang md phdng mien bien ddi cua khang the Gan day hon mdt sd each nhan hda khang

the khac da dugc phat trien O dd cac nha nghien ciiu chgn viing khung ciia khang the d

ngudi dua tren tinh tuong ddng ve mat cau tnic giira cac CDR cua ngudi va chuot va xac dinh

bp khung cua khang the d ngudi cd the hd trg tdt nhat cho vimg CDR chuyen sang tir chupt sao cho ai lire gan khang nguyen dat tdi da Mdt nhdm khac dung hgp vimg CDR ciia khang the tir chupt vdi mdt thu vien cac bd khung khang the ddng gdc ciia ngudi, sau dd sang lgc thu vien tao thanh de tim khang the cd ai luc gan khang nguyen cao nhat Mdt each khac la xac djnh cac axit amin quan trpng nhat cho tuong tac khang nguyen - khang the sir dung

25

thdng tin cau tnic va thuc nghiem, sau dd ghep cac goc nay (chii khdng phai toan bd CDR) vao bd khung khang the ciia ngudi

Ky thuat phage display: K I tir khi dugc md ta lan dau tien nam 1985, cac cdng bd

lien quan den phage display diu dua tren mgt nguyen ly chung la lien ket virus (bacteriophage) vdi thuc thl dugc display (peptide, protein, doan khang the) Su ket hgp nay cho phep thue hien chgn lgc protein vdi dac trung mong mudn tir mdt hon hgp chiia hang trieu protein khac, sau dd cd thl khulch dai gen ma hda protein nay nhu mdt phan ciia bd gen phage Thuc the display thudng dugc diing nhit la scFv va Fab, tuy nhien gan day thu vien phage display vdi mdt doan khang thl chuoi nang VH (sdAb) da dugc tao ra va cho thay nhieu uu diem (hinh 1.10) Phin virus trong ky thuat nay thudng dimg filamentous bacteriophage Ml3 va hg hang gin ciia nd (fd), cac peptide va protein ciing dugc bieu hien

va gan tren be mat cua bacteriophage va virus eukaryote khac Ngoai ra cdn mdt sd ky thuat display khac nhu RNA display, ribosome display , mdi ky thuat cd dac trung rieng va dugc sir dung tiiy theo muc dich [27,28]

- Q

Phan tii IgG Doan Fab Don chudi (scFv) Doan (sdAb)

Hinh 1.10 Cau tnic chung ciia mdt sd loai khang the

Ky thuat tao khang the giong ngiroi diing chuot chuyen gen: Mot each tiep can cd

y nghia thuc tien khac de nhan hda khang the la phat trien cac te bao lai d chupt vdi kha nang tao ra khang the hoan toan gidng ngudi Vdi muc dich nay, chupt chuyen gen trong dd thu vien globulin mien dich ciia chiing dugc thay the bang cac gen V d ngudi da dugc tao ra Cac ddng chupt nay se mang VDJ tai td hgp va sieu dot bien soma cua cac gen ma hda khang the ddng gdc cua ngudi, do dd tao ra cac khang the cd ai lire cao vdi trinh tu hoan toan gidng ngudi, chi khac d md hinh glycosyl hda Cd the diing khang nguyen nghien ciru tiem cho chupt chuyen gen, sau dd thu nhan gen ma hda khang the bang each tach ddng va sang Ipc thu vien mien djch, hoac bang ky thuat te bao lai truyen thdng Bang phuong phap nay da thu nhan dugc cac khang the cd ai luc d miic picomolar va cho thay nhiing hira hen trong iing dimg tri lieu Cae ddng chupt nay la mdt tien bd quan trpng ciia cdng nghe te bao lai de san xuat khang the [27]

Ky thuat cell display: Ciing cd the bieu hien cac khang the tren be mat vi khuan nhu

E.coli va Saccharomyces cerevisiae Vdi muc dich sang Ipc, thu vien cac te bao, moi te bao bieu hien nhieu ban sao cua mdt bien the vi khuan khac nhau, dugc ii vdi phdi tir danh dau huynh quan trong dem thich hgp Cac te bao bieu hien khang the gan vdi phdi tir se dugc danh dau huynh quang va dugc tach ra nhd ky thuat phan loai te bao phat huynh quang (fluorescence-activated cell sorting, FACS) Bang ky thuat ddng khdi te bao, cd the djnh lugng moi ddng cd kha nang gan vdi phdi tir nhat djnh trong thu vien Cd the de dang tdi uu cac tham sd nhu ndng do phdi tir hoac thdi gian phan ly ciia phirc he khang the-phdi tir de

d ^ b^o thu dugc khdng thl cd di luc cao nhit Cd thl sdng Ipc thu vi^n qua 1-2 vdng de giam dp da dang ciia thu vifn, sau do diing cac ky thuat bieu hi^n Uen bl m$t tl bao de dam bao tach dugc khang thl bilu hien tdt va cd ai luc cao [27]

Cdc khdng thl vd cdng nghe tao khdng thl dugc FDA cho phep sii dung lam sang (hinh 1.11) [53]:

XoWr • • • Raptfvi

CsmpaMi Mywurg

H H H | Zenapax

1979

Hinh 1.11 Cdc khdng th^ dirge sa d^ing lam sang

K y thu^t ribosome display: Ribosome display dpa tren ndn tang la SI,T tao thanh

phirc he bac ba giira ribosome, mRNA, va polypeptide Cdc phiic h$ chiia cdc khdng the dd gip nip vdi tinh ddc hi^u mong mudn dugc ldm gidu bdng cdch sdng Ipc vdi phdi tir cd dinh mRNA ciia phirc h? ribosome-mRNA-protein dugc phien ma nguge de tao thdnh DNA ma hda khdng thl cd khd ndng gin vdi phoi tii cd dinh Sau dd lai phien md DNA thdnh RNA bdng RNA polymerase dl bit dau chu trinh sdng Ipc tilp theo Mpt cdch khdc, cd the tao lien kit cpng hda tri true tilp gifl-a mRNA vd protein md nd ma hda Cdc phiic h$ cpng hda Ui mRNA-protein dugc tao thanh nhd puromycin, mpt chit cd hoat tinh tuong tu axit amin Cdc lgi thl ciia cdng ngh# ndy la phiic h# mRNA-protein cpng hda tri dn dinh hon vd cd the diing cdc dilu kien sdng lgc khic nghi#t hon de lam gidu cdc khang the hodc cac protein khdc vdi tinh dn djnh tdng len va todn bd qud trinh dugc thuc hifn hodn todn in viuo vd khdng qua budc phien md Day Id mgt cdng ngllf hiia h^n md nhieu nhdm nghien cim dang tap trung vdo nhdm tao ra cac khdng the cd khi ndng dilu tri [25,26]

27

II.3 Dong vi phong xa

Chpn ddng vj phdng xa cd tinh dac trung va dn djnh ddi vdi cac conjugate mien djch phdng xa Ddng vj phdng xa sir dung trong chan doan va dieu tri RIT cd nhirng dac tnmg phat tia biic xa nhu sau:

- Biic xa hat alpha: giet chet te bao dich, te bao ung thu xung quanh dich, do cd khoang chay dai, hieu qua trong dam tl bao dich

- Biic xa hat beta: do cd khoang chay ngan hon, nen it ddc td ddi vdi cac te bao khdng dac hieu, diet te bao lanh va xung quanh te bao ung thu la tdi thieu

- Tia gamma: can tinh toan ve lieu va each ly benh nhan

Cff so cua RIT: Cac co che co ban trong RIT la sir phat lieu hire xa vao trong te bao ung thu

(hinh 1.12) Te bao ung thu chju lieu biic xa ldn hon te bao binh thudng do sir gidi han ve khoang chay cua cac tia biic xa Te bao ung thu tiep xiic vdi biic xa trong khoang thdi gian lau [20]

\'i'^ H-131

•131

Hinh 1.12 Co che tieu diet ung thu bang biic xa

Cac ddng vi phdng xa diing trong dieu trj RIT dugc tdm tat trong bang 1.2 va bang 1.3 Bang 1.2 cho thay cac nhan phdng xa sir dung cho RIT va neu mdt sd van de can xem xet khi danh gia tiem nang sir dung cac nhan phdng xa Hau het la cac chat phat tia beta va khoang chay la vai milimet trong md, mdt khoang each cd kha nang diet te bao bang dudng kinh khoang 50 - 200 te bao Tia beta nhin chung thich hgp tieu diet cac ung thu nhin thay dugc, nhung day rdng cua nd ciing cd the gay ra nhiing thiet hai cho nhiing md binh thudng gan dd Khang the danh diu vdi ddng vi phat beta cd the luu thdng trong mau trong vai ngay, cd the

lam hdng mdt sd te bao tao mau trong tiiy xuong, dieu nay lam cho sd dem trong mau thip

hon d nhirng benh nhan dang dieu tri va gidi han hoat tinh phdng xa nhu vay phai duoc dua

ra [29]

Cac ddng vj phdng xa phat tia alpha cd khoang chay ngan trong md, dudng kinh tieu diet khoang 10 te bao, cd the thich hgp cho dieu trj cac ung thu trong mau, nhu leukemia, vi ung thu nay d dang te bao don hoac chiim nhd [51] Cac ung thu nay sdn sang tim din khang the danh dau khi tiem vao mau Dieu nay rat quan trgng bdi vi phan ra alpha chi trong thdi gian ngan do vay phai nhanh chdng chgn lira, djnh vj ung thu Cac chit phat electron nang lugng thap cd khoang chay nhd hon cac chit phat tia alpha, va cd kha nang tieu diet hau hit cac te bao don ddc va giam bdt di sir de dga tieu diet cac tl bao lanh lan can Tuy nhien hieu qua cao cd y nghia cua kieu phat tia phdng xa la sao cho tang kha nang diet cac tl bao dac

hieu [10]

Cac nha khoa hgc David A Scheinberg va Jules Mattes da phat hien thiy ring tit ca cac ddng vj phat alpha va electron nang lugng thap cd the dugc sir dung gin vdi cac khang

the de giet cac te bao hoac cum tl bao ung thu Nhu vay, ciing cd the md rdng sir lira chgn cac nhan phdng xa vugt ra ngoai quy udc nhu da chgn '^'l va ^'^Y Dudi day la hai ddng vj phdng xa thudng dimg trong danh diu vdi khang thl don ddng de dieu trj la '^'I va ^''Y [29]

Bang 1.3 Nhan phdng xa diing trong RIT

Thoi gian ban ra

8,02 ngay 2,67 ngay 6,73 ngay 3,72 ngay

17 gid

62 gid 0,75 gid 7,2 gid 10,00 ngay 60,00 ngay 2,79 ngay 3,25 ngay

Nang Iirong hat

577

>6000

7450

Nang Iirong gamma, (keV)

0,08-2,3 4,0-11,3 0,04-1,8 5,0 1,9-11,0 0,05-2,1 0,084 0,06

<100nm

<100nm

So Iirong

te bao diet (*)

10-230 400-1100 4-180 9,0 200-1000 5-210

8

6

(1) (1)

(*) Gid thiet Id 1 te bdo ung thu cd dudng kinh Id lOpm

Bang 1.4 So sanh nhan phdng xa '^'l va ^°Y dimg trong RIT

192 keV 0,8 mm

Re

90Y

2,7 ngay Khd, phai tao phiic Xuong

Beta

934 keV

5 mm Dit

29

Sir phan bo sinh hoc ciia '^'l va '"Y

Ca hai ddng vj phdng xa '^'l va ^°Y khi danh diu vdi khang the don ddng va sir dung lam sang, cac dugc chat phdng xa nay tap trung trong gan, lach va trong tuy xuong va dinh vi

131

trong ung thu Vdi I-khang the nhan phdng xa se dao thai ra khdi md, cdn vdi Y- khang the nhan phdng xa se duy tri trong md '^'l tu do se djnh vi trong tuyen giap (cd the dugc khoa bdi KI) bai fiet theo dudng tilu 90%, ^''Y tu do se djnh vi trong xuong, va bai tilt theo

dudng tieu \0% Hinh 1.13 dudi day so sanh thl tich diet tl bao bang nang luong beta cua

A

120 tebao (~ 5mm)

213

Hinh 1.13 So sanh the tich giet chet te bao bang nang lugng hat beta ciia cac nhan

phdng xa khac nhau

HI Cac phvong phap danh dau khang the vol dong vj phong xa

III.l Dac diem chung

Tir khi ky thuat san xuat khang the don dong phat trien, ngudi ta da danh diu vdi cac dong vj phdng xa nhu '^'l, ^^Ga, '"in, ' " l , ''"'"Tc, *Y Viec chgn dong vj phdng xa dua tren nhirng dac diem thdi gian ban ra phai tir 6 - 200 gid, nang lugng gamma tir 100 - 300 keV, do giau ganuna don nang tren mdi phan ra phai cao, khdng cd nhirng biic xa dac biet khac, ddng vi con phai dn djnh va san pham d dang khdng cd chat mang [7] Phiic cua nhan phdng xa va protein phai dn djnh hda hgc invivo Gia ca hgp ly va thuan lgi trong thuong mai

Viec chgn nhan phdng xa thich hgp cho qua trinh danh dau ngoai tinh chit rieng ciia timg nhan phdng xa thich hgp cho chup hinh hay dieu trj cdn phu thudc vao tinh chit dugc ddng hgc cua phan tir khang the Vi du cac nhan phdng xa cua ' ^I, "'""Tc, vdi thdi gian ban ra ngan, chi thich hgp cho viec danh dau vdi cac phan doan ciia khang the cd thl dao thai nhanh

99m

nhu F(ab) hoac Fab', Tc thich hgp cho viec danh dau vdi Fab va Fab' trong khi I thich hgp cho viec danh dau vdi F(ab)2 Ket hgp vdi thdi gian ban ra vat ly ngin va tinh chit dao thai nhanh cua khang the, cho phep thdi gian chup hinh cd the lam sdm, sau khi tiem cac dugc chat phdng xa loai nay Trong de tai thuc hien,

Sodd phan ra '^'l ("hinh 1.14) nhu sau [30]:

131

I dugc chgn danh dau vdi khang the

Hinh 1.14 So do phan ra cua I 'Xe

(Nang lugng phat tia gamma cho chup hinh cua ''"l la 364 keV, Emax: 0,25, 0,33, 0,47, 0,61, 0,81 MeV,'Eav 192 keV)

III.2 Qua trinh danh dau phong xa [30]

- Khdng lam thay ddi boat tinh mien djch ciia khang the Mdt san pham cd boat tinh mien dich thap se lam cham qua trinh dao thai ra khdi mau va viing khdng ung thu, nhung lai lam tang qua trinh dao thai cua khang the danh dau ra khdi ung thu, vi the ti le gan dich va khdng gan dich thap

- Khau cudi ciing cua qua trinh danh dau nen don gian, va khdng can tinh sach nira trudc khi tiem, de cd the thuc hien tai cac khoa y hgc hat nhan Cac khang the danh dau phai cd tinh dac hieu cao de ed the giam thieu cac thanh phan khdng phdng xa ciia dung djch tiem

- Cd hai ky thuat co ban de gan phdng xa vdi khang the do la iod hda va gan ket phdng

xa kim loai qua phiic hai chiic nang

Khang the danh dau phdng xa phai duy tri dugc tinh mien dich, ben invitro va invivo Khang the phdng xa khdng ben se lam giam di chat lugng hinh anh va giam gia trj dieu trj

Nd cd the lam tang hoac giam siic chju lieu biic xa cho benh nhan, phu thudc vao cd hay khdng qua trinh su phan buy cac thanh phan mang nhan phdng xa gan vdi protein huyet tuong hoac ndi bao hoac bai tiet qua than

Dau tien '^'l dugc dimg trong chan doan vi nd cd nhieu thuan lgi la gia thanh thip, de san xuat, nhung cdn nhieu nhugc diem la cdn tdn tai nhirng iod tu do, lieu biic xa cao cho benh nhan, thdi gian ban ra dai '"in cd tinh chat hoan hao hon, thdi gian ban ra la 68 gid, nang lugng 171 keV, dn dinh invivo, tuy nhien van cd nhieu nhugc diem la gia ca cao, tap trung khdng dac hieu trong cac co quan (phdng d gan, than, lach cao), khd san xuat hon, chi

de chan doan """"Tc cd nhieu uu diem hon, tinh chat hat nhan hoan hao, de tim, gia thanh thap, chi cd dieu la thdi gian ban ra qua ngan (6 gid), va tinh phiic tap vl mat hda hgc, chi

31

dvmg trong chan doan Sau day la cac phuong phap danh dau khang the vdi cac ddng vj khac nhau:

III.3 Phu-ong phap danh dau khang the voi '^'l [31]:

Khang the gin vdi '^^I va '^'l cd thl chup hinh trong khi '^'l cdn diing cho dilu trj

Cd hai each de gan phdng xa vao '^'l dd la phuong phap thay the true tiep iot phdng xa vao trong vj tri ortho da boat hoa tren vdng therm, hiu hit la tyrosine, each khac la cdng hgp phan

tu huu CO da iod hoa vdi nhdm chiic nang tren day chuyen phan iing ciia phan tir khang the

Phuomgphdp thay the true dip [31]: Phuong phap nay bao gom diing mdt chat oxy

hoa nhe de cd the bao tdn boat tinh miln djch ciia khang thl, dd la diing cac chat chloramin

T, tetrachlorodiphenylglycouril (iodogen) va hydrogenperoxidase (lactoperoxidase) lam chat oxy hod nhe de iod hoa phan tir khang the Cac van de chu ylu lien quan den ky thuat nay nhu sau:

• Su khdng gan dugc ciia tyrosine du thira co the lam duoi phan tu protein Phan tir duoi ra nay se lam thay ddi cau triic toan ven ciia protein trong mdi trudng phan ring

• Sir xa phan cd the se xay ra, lam giai phdng Iod tu do va cd thl se tich liiy trong tuyIn giap hoac trong da day Tuy nhien trong nhirng nam gan day, cac nha nghien ciiu van tien hanh nghien ciiu danh dau khang the vdi '^'l cho muc dich dieu trj benh ngay cang nhieu

Phucmg phdp cong hap khdng the vd '^'h Trong phuong phap nay gdm 2 budc vi du

nhu la iod hoa dimg mdt phan tir hiiu co nhd bang each thay the true tiep va sau dd cdng hgp vdi khang the: iod hoa N-hydroxysuccinimide ester, mdt dan xuat ciia ortho-iodohippurate va succinimidyl para-iodobenzoate diing lien ket cdng hoa trj vdi khang the Diing ky thuat cdng hgp nay cd the tao kit de [30] Tuy nhien cac ban che cua phuong phap nay nhu sau:

• Cac chat cdng hgp vdi phan tir khang the khi gan vdi phdng xa de bj phan hiiy invivo Tiiy thudc vao tac ddng sinh hpc ciia phan tir chat hiru co, no c6 the nam trong tuan hoan hoac gan trong te bao, hoac bai tiet qua than

• Viec tdng hgp va tinh sach chat cdng hgp khang the phirc tap

• Hieu suat gan nhin chung la thap hon so vdi danh dau true tiep

Gidi thieu cdc phucmg phdp ddnh ddu cdc chdt cd bdn chdt protein vdi iod phdng xa:

De cd san pham protein danh dau vdi iod phdng xa, phuong phap dugc sii dung rdng rai nhat la oxy hda phan tii Nal'^' trong sir cd mat ciia protein hoac nhiing phan tir cd chiia gdc tyrosine, iod phdng xa se gan vao phan tir tyrosine Trong vai trudng hgp iod se gan vdi phan tir histidine, tryptophan hoac nhdm sulphydryl Sau day la cac phuong phap thudng dimg:

Phirong phap do hoa iod (iodine monocloride) [31]:

Day la phuong phap dau tien dugc sir dung de iod hda cac phan tir protein ICl dugc can bdng vdi iod phdng xa, sau dd phan iing vdi protein can danh dau Vi du nhu de danh dau insulin vdi ' 'l, ngudi ta pha loang '^'l vdi ICl, them insulin vao, sau dd them natri methabisulphit de khir I, thdi gian phan iing xay ra la 1 phut Sau khi danh dau xong san pham dugc lam sach Tuy nhien, phuong phap nay khdng dugc sir dung rdng rai do su de phan huy ciia cac protein bdi '^'l

Phvong phap iodogen

Dimg Iodogen (1,3,4,6- tetrachloro-3a,6a-diphenylglycoluril) hoa tan trong methyl chloride, cho bay hcri, tao nen dng trang phu ddng nhat Chat danh dau dugc trdn vao dng iodogen trong khoang 10-15 phiit, lay hdn hgp phan iing ra, iod tu do dugc tach ra bang sac

ky lgc gel dimg cdt sephadex hoac vat lieu trao ddi ion DEAL Theo phuong phap nay, su bien tinh ciia protein la tdi thieu, vi phan iing xay ra tren pha ran va iodogen khdng hda tan trong nudc Hieu suat danh dau khoang 70 - 80%

Phirong phap chloramin T

Day la phuong phap dugc sir dung rdng rai nhat de iod hda mdt khdi lugng rat be phan tu protein de san xuat cac chat danh dau cho RIA '^^I bj oxy hda bdi chloramin T trong sir cd mat ciia protein can danh dau Iod phdng xa gan vao tyrosine tren phan tii protein vdi hieu suat cao Chloramin T thira bi khir bdi natri metabisulphite va iod phdng xa bj khu ve dang iodide Sau khi them vao mdt lugng chat mang KI hoac Nai, hgp chat danh dau dugc tach ra khdi iod phdng xa tu do bang ky thuat thich hgp, thudng la lgc gel Lugng chat oxy hda them vao vdi ndng do tdi thieu nham ban che sir phan buy protein Thdi gian phan img xay ra nhanh San pham thu dugc cd hieu suat cao va boat tinh rieng cao Phuong phap chloramin T cd the gay phan buy protein nhung phuong phap nay ky thuat don gian, de lam,

vi vay, dugc chgn de san xuat chat danh dau cho RIA va RIT

Phirong phap diing chat oxy hoa hypochloride

D I khac phuc vin de phan buy protein trong qua trinh iod hda bdi phuong phap chloramin T, mdt vai phuong phap oxy hda khac dugc dua ra Dien hinh la diing natri hypochloride (NaOCl) lam chat oxy hda Iod phong xa phan iing vdi protein trong sir cd mat cua chat oxy hda nay, phan iing cho phep 30 giay, diing natri metabisulphite de dimg phan ling, sau dd dung chat mang Nai hoac KI Ket qua cho ra san pham danh dau cd tinh dn djnh cao Tuy nhien, phuong phap nay ciing cd nhirng ban che nhat djnh bdi sir chju dung ddc hai true tiep ciia protein vdi chat hda hgc

De ban che su tiep xue true tiep cua protein vdi chat oxy hda, ngudi ta dung protein trdn vdi Na'^^I Sau dd, nhd boat ddng ciia chloramin T, cio sinh ra do sue NaCl, di vao hon hgp tren lam giai phdng '^'l khdi Na'^^I va iod hda phan tir protein Nhung each nay lai cd nhugc diem la protein de bj phan buy bdi iod phdng xa, vi thdi gian iod hda dien ra rat lau (khoang 10 phiit)

Phirong phap dien ly (electrolytic iodination)

Phuong phap nay dugc dua ra bdi Penirisi va Rosa diing ddng dien de chuyen Y ve

dang r Day la ky thuat diing dl iod hda insulin Cho insulin vao dung dich NaCl 0,9% va Na^ I Dien ly hon hgp 40 phiit vdi ddng dien 6 - 7 mA San phim thu dugc cd hieu suit cao, boat tinh sinh hgc, boat tinh mien dich hpc cao U'u dilm ciia phuong phap nay la protein khdng tilp xiic true tilp vdi chit oxyhda, chit khir Nhung do thdi gian tiep xiic vdi phdng xa lau nen protein de bj phan buy va dien ly d nhiet dp phdng dl lam hdng protein Hon niia, thiet bj ddi hdi kha phiic tap

Phu-ong phap enzym

Mdt phuong phap nhe nbang dl iod hda protein la dimg enzym lactoperoxidase Phuong phap nay dugc Marchalonis gidi thieu dl iod hda cac phan tir globulin miln djch Protein dugc trdn vdi '^^I va lactoperoxidase Phan iing bit dau khi them mdt lugng nhd hydrogen peroxide va thdi gian duy tri la 10 phiit Them cystein vao de ket thiic qua trinh iod

33

hda va sau do lgc gel Ky thuat nay it manh hon so vdi cac phuong phap hda hpc khac va chi sir dung cho nhung protein de bj diit gay khi diing chloramin T

Trong cac phuong phap ke tren, ngudi ta thuong chpn phuong phap chloramin T Chloramin^ T la mudi natri cua dan xuit n-mono chloro cua p-toluene sulphonamide Trong nudc nd de bj phan buy thanh hypochlorous acid va boat ddng nhu la chit oxy hda:

Cau au tnic cua chloramin T: [cHa V 7 ) \ _ S 0 2 - N - C M Na^

O pH 7,5 chloramin T oxy hda Nai vl dang L va T nay se thay vao vj tri ortho trong vdng phenol cua tyrosine Ciu tnic mono, diiodo tyrosine nhu sau:

la chat oxy hda nhe, trong nudc de tao thanh acid hypochlorous theo phan iing sau:

3 - ( r ~ ^ y S 0 2 -N <; + H2O > HOCl

Cl CH3- " ^ ' ^

^Na (N-Chloro-4-methylbenzen sulfonamide natri)

Acid hypochlorous la chat oxy hda thuc hien phan ung chuyen V thanh T :

HOCl + *v > HO*i + c r

HO*I > OH"" + *l"^

r la chat oxy hda manh, phan iing thuan va nhanh tai pH trung tinh, T thay vao vj tri ortho ciia hydro tren vdng phenol Viec them natri metabisulfite vao se dimg phan iing oxy hda:

Na2S205 + NaOH + I2 > Na2S04 + H2O + Nai

NIU pH > 8 thi se xay ra su thay the ciia nhdm khac vao thyroxin hoac phan iing trd nen mat hieu qua do su tao thanh 10 ' :

I2 + OH' > 10^" + 1 + H2O

Phan ling oxy hda xay ra nhanh, hon 90% Ab dugc tao thanh, mdt nguyen tir iod phdng xa (I*) dugc gin vao vdng phenol tai vj tri 3' ddng thdi ciing cd mdt sd it nguyen tir I* gin vao cav j tri 5'

III.4 Phu-ong phap danh dau khang the voi '^""Tc va "'in

Danh dau khang the vol ""Tc: De chan doan sdm benh ung thu, ngudi ta ciing gan

khang the vdi ""Tc Cd hai phuong phap gan khang the vdi ^"^"^Tc, dd la phuong phap true

tiep va phuong phap gian tiep Khi gan true tiep khang the vdi ^^""Tc, ngudi ta diing cac tac

nhan khir nhu 2-mercaptoethanol, dithiothreitol, Sn dam dac, bicarbonate de khir disulfides

ve dang sulflivdryl tren phan tir khang the de dl tao phiic vdi ^^"^Te Phuong phap gian tiep la

tao phiic vdi "^Tc trudc rdi sau dd gan vdi phan tir khang the Vi du diing methallothionin, mdt protein giau nhdm sulfliydryl nhu la mdt phiic [66], hoac la nen kit hgp khang the vdi

phiic cd hai nhdm chiic, phiic nay xuat phat tir viec kit hgp DTPA hoac EDTA vdi nhdm

amin cua phan tir protein Hoac sir dung deferoxamin lam tac nhan tao phiic, nd gan len nhdm

cacboxyl ciia phan tii protein Ngoai ra cdn diing phuong phap "pretinning" de danh dau

khang the doan F(ab)2 vdi ""Tc Theo each nay, diing enzyme pepsin hoac papain cat khang

the thanh cac phan doan, sau dd danh dau vdi ^^'^Tc Sau day la cac so dd tao phiic vdi khang

thl khi danh diu vdi ^^"^Tc:

So dd phiic ^^"^Tc tao thanh trudc khi danh dau vdi khang the

Danh diu ^^""Tc vdi McAb theo phirc N2S2

Danh dau khang the vdi In: Phiic khang the va 111, In tao thanh bang phan iing true

tiep hoac gian tiep giira In^^ va khang the Trong phan iing true tiep, phiic ylu '"in - khang the tao thanh Khi tiem vao co the, nd phan ly va giai phdng '"in, "in tu do se gin vdi protein huyet tuong nhu la transferrin Trong phuong phap danh dau gian tilp, khang thl dugc cdng hgp qua mdt phdi tir hai nhdm chiic, trong dd cd mdt nhdm chiic gan kim loai manh tai doan cudi va mdt nhdm chiirc cd boat tinh gan vdi protein tai doan diem cuoi khac Vai phiic da chiic da dugc tdng hgp va danh gia, nhung mdt hgp chat thdng dung nhat la aminopolycarboxylate cua DTPA, mdt hdn hgp ciia anhydride hoac mdt anhydride vdng va isothianate Viec gan ket cua phiic hai chiic nang vdi phan tir khang the dugc thuc hien qua nhdm amino cua lysin tren phan tir protein, Vdi anhydride hoac anhydride vdng, mdt carboxyl ciia DTPA gan cdng hoa trj vdi khang the, trong khi bdn nhdm cacboxyl cdn lai gan vdi In^* (xem hinh) [49]

protein

i l l ,

Phirc I g G - D T P A - " T n

; Tuy nhien cau gan nay yeu, dl bj phan ly In bi phan ly tuong ddi nhanh hon'"in gan DTPA De khac phuc van de nay, isothianate dugc them vao, trong dd 5 nhdm carboxyl

tu do gan vdi ion In^^, cdn thiocarbamoyl gan vdi amino tren phan tir protein Theo each nay

"'in gin vdi DTPA, tao nen phiic DTPA - " 'in - Thiocyanate - Ab bin invivo [50]

Khang thl gin sau dd dugc loai DTPA tu do bing each thim tich hoac sic ky cdt Khang thl gin DTPA nay cd thl tao thanh dudi dang kit, khi danh diu chi them vao ion

In * Viec danh diu vdi '"in cin cac ylu to nhu ti le moi cua DTPA va khang thl thich hgp, qua trinh chuyin boa cua phiic trong co thl, tdc do dao thai ciia phiic, boat tinh mien dich cua phiic Thuan lgi chu ylu cua phiic nay la de tao kit va thdi gian ban ra hgp ly Dilm khdng thuan lgi la phiic tap trung cao trong gan, lach, tuy xuong va tinh hoan Vi du eae khang the gan In nhu antimyosin, CEA va cac chit khang ung thu gin glycoprotein

IV Cac nghien ciru du-ffc dong hoc cua '^'l-CD20MAb

Cac nghien ciiu phase I ciia '^'l-CD20MAb da xac djnh la viec tiem tiln lilu 475 mg khang the khdng danh dau phdng xa lam giam lilu di din lach va lam tang thdi gian sdng cua khang the danh dau trong co the Sir dao thai ra khdi mau sau khi tiem lilu 485 mg tren benh nhan NHL la 68,2 mg/gid Su dao thai ra khdi mau dugc theo ddi bing chup gamma toan than I dugc dao thai bang phan ra va bai tiet qua dudng tilu Sau 5 ngay co thl thai 67%

so vdi lieu tiem Viec fiem lieu dieu trj se lam tieu buy dudng chit ciia cac tl bao CD20 duong tinh trong mau Liic 7 tuan, sd te bao tuan hoan trong mau la bing 0 (day 0 - 490 tl bao/mm^) Cac lymphocyte phuc hdi sau 12 tuan dieu trj [19]

Viec danh gia lieu hap thu '•"l-CD20Mab dugc tien hanh bing each chup timg phin mdi CO quan trong co the va diing phan mem xir ly MIRDOS E 3 Cac benh nhan dugc chgn

de phan tich lieu Ket qua danh gia dugc tdng ket tren bang 1.5

Bang 1.5 Danh gia lieu hap thu

Co* quan

Tuyen giap Than

Co tim

Lach Tinh hoan Gan Phdi Tuy Thanh da day

Trung binh mGy/MBq

2,71

1,96 1,25

0,14 0,83 0,82 0,79 0,65 0,40

Dai mGy/MBq

1,4-6,2 1,5-2,5 0,5-1,8 0,7 - 5,4 0,3-1,3 0,6-1,3 0,5-1,1 0,5-1,1 0,2 - 0,8

37

Phan II: VAT LIEU VA PHlTONG PHAP NGHIEN ClTU

I Nguyen lieu, hoa chat, thiet bj

1.1 Hoa chat: Cac hda chat deu la nhirng hoa chit tinh khi It dugc sii dung trong nghien ciiu

ve sinh hgc phan tir va danh diu phdng xa

131,

I dang Na I san xuat tai Vien Nghien ciiu hat nhan Chit lugng diing dl danh diu vdi khang the dat cac chi fieu sau (bang 2.1):

8ang 2.1 Tdm tat chat lugng san pham ' I

Thong so Idem tra

' ^ \ ' " I < 0 , 1 %

'3ii > 99 904

Dat vd triing Khdng chira chi nhiet to

Phirong phap

Cam quan

Do tren may CAPINTEC Theo nha san xuat cung cap

Giay do pH

Do phd gamma Sac ky giay Sac ky Idp mdng Dien di

Thir vd triing Thir chi nhiet td

- ' 'l dang Na'^'l mua ciia Indonexia de so sanh

- Khang thl khang CD20 Rituximab (ten khac la MabThera, chai 500mg/50ml, hang ROCHE, cac Id sd MF 0072005B2074 va EU/1/98/067/002 B5057 Thdng fin vl khang thl

do nha san xuat cung cap (bang 2.2):

Bang 2.2 Tdm tat chat lugng san pham khang the don ddng khang CD20

Thong so

Ten thuong mai

Cdng thiic phan tir

Trgng lugng phan tir

Bia dich

Thdi gian ban hiiy

Hieu luc sinh hgc

30 - 400 gid 100%

Khang thl khang CD20 sc-Fv, Vien Cdng nghe Sinh hgc (xem phu luc 1)

Khang nguyen CD20, Vien Cdng nghe Sinh hgc

- Sephadex G25 PDIO mua tir hang Amersham Bioscences, Id sd 170851-01

- Cac hoa chit Chloramin T (ChT), natrimetabisulphit (SMB) mua tir hang Sigma

- Huyet thanh ngudi

- Natri Photphat va Natri Dihydro Photphat

- Kit LAL

- Ong iodogen

1.2 Cac thiet bj, dung cu

- May do LTI, My (GENESYS, 5000 Series)

- May do boat do phdng xa (Dose Calibrator CAPINTEC CR 127)

- Be dn nhiet

- Til hiit vd trimg

-Tuam

- Ndi hap vd triing

- May lay phan doan

- May ly tam

- May lac trdn vortex

- Pipet ty ddng 2^1, 5^1, 10^1 50^1, 100^1, 1000^1

- Giiy sac ky Whatman No 1, giiy ITLC

- Ong thuy tinh danh dau

II Nghien ciru cac dieu kien toi iru de danh dau khang the vdi phong xa

Cac nghien ciiu danh dau khang the don ddng khang CD20 vdi ddng vj phdng xa '^'l

da dugc thuc hien va dugc FDA phe chuan sir dung lam sang tir nam 2003 Viec nghien ciiu danh dau khang the don ddng khang CD20 vdi ddng vj phdng xa '^'l dugc thuc hien bing phuong phap chloramin T Day la phuong phap dugc gidi thieu bdi Hunter va Greenwood dl danh dau cdc hgp chat sinh hgc nhu protein, peptid vdi chat phdng xa iod '^'l dugc chgn lam chat danh dau vi sir cd mat ciia nd khdng gay anh hudng den tinh chat ciia khang the sir dung danh dau Hon niia tren phan tir khang the co cac axit amin tyroxin cd vdng phenol nen

I cd the thay the true tiep, va vdi biic xa y phat ra cd the do, chup hinh tren may chuyen dimg Khang the dugc danh dau vdi '^'l trong cac dieu kien tdi uu nhu ndng do cac chat tham gia phan iing, boat do phdng xa, thdi gian phan img, pH danh dau, san pham sau khi danh dau dugc tach qua cdt Hieu suat danh dau dugc tinh toan bang cac phuong phap nhu sic ky lgc gel, dien di tren giay va sac ky Idp mdng San pham dugc tach ra bang phuong phap sac ky lgc gel diing sephadex G-25

II.l Xac dinh ham Iirong Chloramin T

Chloramin T tham gia trong phan iing danh dau, vdi vai trd la chat oxy hda nhe De phan ling gin iod phdng xa len khang thl vdi ham lugng Chloramin T tham gia trong phan iimg la tdi thilu, cin thilt khao sat ham lugng va hieu suit danh diu Sau day la phuong phap khao sat:

Chuan bi vat lieu, hoa chat

- Khang the cd ndng do 10 mg/ml

- Dung dich natri metabisulphite Na2S205 cd ndng do 2 mg/ml: Can 10 mg Na2S205 hda tan trong 5 ml dem photphate 0,2 M

- Dung djch chloramin T cd ndng do 2000 mg/ml: Can 2000 mg chloramin T hda tan trong

1ml dem photphat 0,2 M Pha chloramin T (bang 2.3) theo cac ndng dg khac nhau theo

phuong phap pha loang nhilu bac Chuin bj cac chai thuy tinh sach, danh dau ghi ten chai A,

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