B Y T

TRNG I HC DC H NI

LNG TH THU HNG

TNG QUAN V PHNG PHP

NH GI SINH KH DNG V

TNG NG SINH HC CA

THUC NGOI DA DA TRN

K THUT THM TCH MICRO

KHA LUN TT NGHIP DC S

H NI 2014

B Y T

TRNG I HC DC H NI

LNG TH THU HNG

TNG QUAN V PHNG PHP

NH GI SINH KH DNG V

TNG NG SINH HC CA

THUC NGOI DA DA TRN

K THUT THM TCH MICRO

KHA LUN TT NGHIP DC S

Ngi hng dn:

1. PGS.TS. Thi Nguyn Hng Thu

2. TS. Nguyn Thnh Hi

Ni thc hin:

1. B mn Ha phn tch v c cht

2. B mn Dc lm sng

Trng i hc Dc H Ni

H NI 2014

LI CM N

Vi lng bit n su sc, ti xin gi li cm n chn thnh ti:

PGS.TS. Thi Nguyn Hng Thu

TS. Nguyn Thnh Hi

L nhng ngi thy trc tip hng dn v tn tnh ch bo ti hon

thnh kha lun ny.

Ti xin cm n cc thy c ti B mn Ha phn tch v c cht v b mn

Dc lm sng to iu kin thun li cho ti trong thi gian thc hin kha

lun ny.

Ti xin cm n Ban gim hiu, cc phng ban, cc thy c gio v cn b

nhn vin trng i hc Dc H Ni - nhng ngi dy bo v gip ti

trong sut 5 nm hc tp di mi trng ny.

Cui cng, ti xin gi nhng tnh cm thn thng nht ti gia nh, bn b,

tp th lp A7K64 v N1K64 lun bn quan tm, chia s v ng vin ti vt

qua mi kh khn trong qu trnh hc tp v thc hin kha lun.

H Ni, ngy 12 thng 5 nm 2014

Sinh vin

Lng Th Thu Hng

MC LC

LI CM N

DANH MC CC K HIU, CC CH VIT TT

DANH MC CC BNG

DANH MC CC HNH V, TH

T VN ............................................................................................................ 1

Chng 1. TNG QUAN V VAI TR NH GI SINH KH DNG V

TNG NG SINH HC CA THUC ....................................................... 3

1.1. KHI NIM SINH KH DNG V TNG NG SINH HC .......... 3

1.2. VAI TR CA SINH KH DNG V TNG NG SINH HC

TRONG QUN L CHT LNG THUC TRN TH TRNG ................... 5

1.3. TH NGHIM TNG NG SINH HC .............................................. 6

1.4. MT S QUY NH TI CC QUC GIA V TNG NG SINH

HC .......................................................................................................................... 7

Chng 2. TNG QUAN V K THUT THM TCH MICRO TRN DA

V U NHC IM CA K THUT .......................................................... 14

2.1. TM TT V LCH S PHT TRIN CA K THUT THM TCH

MICRO .................................................................................................................... 14

2.2. NGUYN L CA K THUT THM TCH MICRO ........................... 16

2.2.1. Nguyn l ................................................................................................... 16

2.2.2. Kim thm d .............................................................................................. 18

2.2.3. Dch truyn vo .......................................................................................... 21

2.2.4. nh lng cc phn t thuc .................................................................... 22

2.2.4.1. Sc k lng hiu nng cao (HPLC) ....................................................... 22

2.2.4.2. Phng php sc k lng khi ph (LC-MS) ........................................ 25

2.2.5. Hiu chnh kt qu thu c ...................................................................... 25

2.2.5.1. Phng php No- net - flux (NNF) ........................................................ 27

2.2.5.2. Phng php ngoi suy tc dng chy (ZFR).................................. 28

2.2.5.3. Phng php thm tch ngc (RD) .................................................... 29

2.3. QUY TRNH CHUNG CA K THUT THM TCH MICRO TRONG

NGHIN CU Y DC HC .............................................................................. 30

2.4. U NHC IM CA K THUT THM TCH MICRO ................. 30

2.4.1. u im ..................................................................................................... 30

2.4.2. Nhc im ................................................................................................ 31

Chng 3. TNG QUAN V PHNG PHP NH GI SINH KH

DNG V TNG NG SINH HC CA THUC NGOI DA ............ 33

3.1. S CN THIT PHI O NNG THUC T DO TI DCH K M

DA ........................................................................................................................ 33

3.2. TRIN KHAI NH GI SINH KH DNG V TNG NG SINH

HC CA THUC NGOI DA VI K THUT THM TCH MICRO ....... 34

3.2.1. i tng nghin cu ................................................................................ 35

3.2.2. Cc thuc c s dng trong nghin cu ................................................ 35

3.2.3. Thit k nghin cu ................................................................................... 37

3.2.4. Chun b kim thm d ................................................................................ 38

3.2.5. Cy kim thm d trn da ............................................................................ 39

3.2.6. nh lng bng phng php thch hp .................................................. 40

3.2.7. Thng k, tnh ton v bo co kt qu thu c ...................................... 41

3.3. MT S NGHIN CU NH GI SINH KH DNG V TNG

NG SINH HC CA THUC NGOI DA DA VO K THUT THM

TCH MICRO ........................................................................................................ 42

3.3.1. Xc nh sinh kh dng ca clobetasol propionat ..................................... 42

3.3.2. nh gi tng ng sinh hc ................................................................. 44

3.3.2.1. nh gi tng ng sinh hc ca cc ch phm lidocain ti ch .... 44

3.3.2.2. nh gi tng ng sinh hc ti ch ca cc loi kem acyclovir .... 46

3.3.2.3. nh gi tng ng sinh hc cc ch phm oxytetracyclin HCl v so

snh vi phng php bng tc ....................................................................... 50

KT LUN V KIN NGH ................................................................................ 55

1. KT LUN ....................................................................................................... 55

2. KIN NGH ...................................................................................................... 56

TI LIU THAM KHO

DANH MC CC K HIU, CC CH VIT TT

AUC

BA

BE

C

CI

Cmax

Da

EF

HPLC

kDa

kl

LC MS

MD

DMD

MS

PD

PK

RD

RR

SD

SFR

TLTK

Tmax

tt

Din tch di ng cong (Area Under the Curve)

Sinh kh dng (Bioavailability)

Tng ng sinh hc (Bioequivalence)

Nng (Concentration)

Khong tin cy (Confidence Interval)

Nng nh

Dalton

Hiu sut in vivo ca mng bn thm trn kim thm d

(extraction fraction)

Sc k lng hiu nng cao (High Performance Liquid

Chromatography)

Kilo Dalton

Khi lng

Sc k khi ph (Liquid Chromatography - Mass Spectrometry)

Thm tch micro (Microdialysis)

Thm tch micro da (Dermal Microdilysis)

Khi ph (Mass Spectrometry)

Dc lc hc (Pharmacodynamic)

No-net-flux

Dc ng hc (Pharmacokinetic)

Thm tch ngc (Retrodialysis)

Hiu sut tng i (Relative Recovery)

lch chun (Standard Deviation)

Tc dng chy rt chm (Very Slow Flow Rate)

Ti liu tham kho

Thi gian t nng nh

Th tch

DANH MC CC BNG

Bng 2.1. Cc dung dch truyn s dng trong k thut thm tch micro ................ 21

Bng 3.1. Cc nhm thuc ngoi da c s dng trong cc nghin cu nh gi

tng ng sinh hc da trn k thut thm tch micro ........................................ 36

Bng 3.2. Thnh phn cc dung dch truyn ngoi vi ............................................... 37

Bng 3.3. Vng cy v su kim thm d ............................................................. 39

Bng 3.4. Thnh phn cc cng thc ACV v su cy kim thm d tng

ng.. ....................................................................................................................... 46

Bng 3.5. Cc thng s dc ng hc ca cc loi kem Acyclovir dng ti

ch.... ..................................................................................................................... 48

Bng 3.6. Tng hp mt s nghin cu nh gi tng ng sinh hc ca thuc

ngoi da da trn k thut thm tch micro .............................................................. 49

DANH MC CC HNH V, TH

Hnh 1.1. th biu din nng ca hai ch phm A, B c cng hot cht, cng

hm lng ca hai hng sn xut c coi l tng ng sinh hc ......................... 4

Hnh 1.2. Nng ciclosporin trong mu theo thi gian nhng ngi tham gia

dng CicloMulsion hoc Sandimmune (n = 52) ....................................................... 11

Hnh 1.3. th biu din nng thuc Cefuroxim trung bnh theo thi gian ca

thuc th v thuc chng .......................................................................................... 12

Hnh 2.1. Cc m ch tc dng ca thuc trn ngi c thc hin nghin cu

lm sng da trn k thut thm tch micro ............................................................. 15

Hnh 2.2. Kt qu tm kim trn medline vi cc t kha lin quan n

microdialysis ............................................................................................................. 15

Hnh 2.3. Nguyn tc ca k thut thm tch micro vi mt kim thm d dng

ng tm .................................................................................................................... 16

Hnh 2.4. S nguyn tc thm tch micro thuc ngoi da ............................. 17

Hnh 2.5. Ba kim thm d t song song trong mi khu vc th nghim (3 khu

vc, mi khu cho mt thuc th) trn mt nga ca cnh tay tri ..................... 17

Hnh 2.6. Kim thm d thm tch micro dng ng thng ..................................... 18

Hnh 2.7. Cc dng kim thm d A: ng thng, B: vng mc, C: sng i, D:

ng tm .................................................................................................................... 19

Hnh 2.8. Cm ng dn sau s lun kim thm d qua ng dn ny ..................... 20

Hnh 2.9. S h thng HPLC ............................................................................... 24

Hnh 2.10. M hnh nh gi hiu sut mng bn thm kim thm d in vitro ......... 26

Hnh 2.11. Phng php zero - net - flux trong hiu chnh in vivo .......................... 28

Hnh 2.12. Cy kim thm d no (tri) v b vai (phi) ..................................... 31

Hnh 3.1. M t kim thm d dng ng thng ...................................................... 38

Hnh 3.2. Cy kim thm d vo da ........................................................................... 39

Hnh 3.3. Cy kim thm d vi 2, 3, 4 cp im ...................................................... 40

Hnh 3.4. H thng sc k HPLC (tri) v LC-MS (phi) ........................................ 41

Hnh 3.5. Kim thm d MD tuyn tnh c s dng nghin cu MD in vivo vi

chiu di c nh 3 cm ca mng thm tch c t trong da ................................ 43

Hnh 3.6. Khoang cha PVC gn vo cc v tr pha mt trong cng tay ca cc i

tng to thnh thng cha cho cng thc CP 4% (kl/tt) .................................... 43

Hnh 3.7. Cc th tc cy kim thm d MD trn da trong mt nghin cu lm sng

c i chng .............................................................................................................. 45

Hnh 3.8. Cc v tr s dng ca ba cng thc ACV trn lng ln ...................... 47

Hnh 3.9. Biu gii phng ACV (3%) in vitro t 3 cng thc dng ngoi da ..... 47

Hnh 3.10. M t k thut bng tc ........................................................................ 50

Hnh 3.11. Cy kim thm d thm tch micro vo lp h b mt trong cnh tay ca

tnh nguyn vin khe mnh ..................................................................................... 52

Hnh 3.12. Hiu chun in vitro kim thm d thm tch micro cho oxytetracyclin

HCl ............................................................................................................................ 52

Hnh 3.13. Nng oxytetracyclin HCl trong lp h b cho thuc th v thuc

chng 10 tnh nguyn vin khe mnh (kt qu MD) ........................................... 53

Hnh 3.14. Lng oxytetracyclin HCl trung bnh trong lp sng ca 12 tnh nguyn

vin khe mnh (kt qu bng tc) ......................................................................... 53

1

T VN

Thuc ngoi da l mt dng ch phm rt tin li trong iu tr cc bnh tn

thng da. Thuc bi c nhiu tc dng tt nh bt sng, bt vy, to sng, dng

da, chng nga, chng vim, dit khun kt hp vi thuc ton thn cha khi

c nhiu loi bnh ngoi da. Thuc ngoi da l mt phn khng nh trong kho

tng thuc qu bu ca nhn loi.

Tuy nhin, da c cu to v chc nng ring nn vic s dng thuc trn da c

nhiu khc bit so vi nhng ng dng khc. Hn na, c nhiu dng thuc c

dng trn da (dung dch, kem, m), mi dng dng c s dng ty vo loi da,

mc cp tnh v v tr ca tn thng. Cng vi s pht trin ca k thut bo

ch, hin nay thuc dng ngoi khng ch dng li tc ng ngay ti b mt da m

ngm qua lp sng, vo cc khe gian bo, cc nang lng, vt qua lp y (lp su

nht ca thng b) ngm vo tn trung b hoc su hn, ni c nhiu u dy thn

kinh v mch mu nh ri vo h tun hon chung. Vic xc nh sinh kh dng

cng nh tng ng sinh hc gia cc cng thc bo ch bit c hiu qu

tc dng ca thuc l ht sc cn thit. Trc kia, nghin cu dc ng hc b hn

ch trong vic o nng thuc t cc t chc sinh hc nh cc m tc dng, nc

tiu, nc bt hoc cc cht dch t cc ch phng rp da... Gn y, mt phng

php ng dng mi c th p dng nh gi tng ng sinh hc v sinh kh

dng ca cc thuc ngoi da, l k thut thm tch micro (Microdialysis - MD).

K thut thm tch micro l mt cng c ly mu rt hu ch c th c s

dng trn in vivo bit c s thay i nng ca cc phn t t do nm trong

khong k hoc ngoi bo da trn s khuch tn th ng cc cht qua mng thm

tch theo gradient nng . K thut ny c th c s dng trn in vivo o

nng thuc t do trong dch k m, ri sau tnh ton cc thng s dc ng

hc (PK) kt hp vi nhng quan st dc lc hc (PD) d on hiu qu lm

sng. Kh nng o nng t do ti v tr tc dng ca thuc theo thi gian lm cho

k thut thm tch micro tr thnh mt cng c rt c gi tr trong vic nh gi

2

sinh kh dng v tng ng sinh hc ca thuc. Phng php ny c cng

nhn bi cc nh sn xut Dc phm v cc c quan qun l nh C quan qun l

Thuc v Thc phm Hoa K (FDA) [92].

Vi nhng u im vt tri ca k thut thm tch micro trong vic nghin

cu dc hc ni chung v thuc ngoi da ni ring, chng ti tin hnh thc hin

ti Tng quan v phng php nh gi sinh kh dng v tng ng sinh

hc ca thuc ngoi da da trn k thut thm tch micro vi 3 mc tiu:

1. Tng quan v vai tr nh gi sinh kh dng v tng ng sinh hc ca

thuc.

2. Tng quan v k thut thm tch micro trn da v u nhc im ca k

thut.

3. Tng quan v phng php nh gi sinh kh dng v tng ng sinh

hc ca thuc ngoi da da trn k thut thm tch micro.

Trn c s kt qu thu c ca ti ny, chng ti hy vng s gip ch cho

cc nh nghin cu Dc c th tin hnh trin khai vic nh gi sinh kh dng v

tng ng sinh hc ca cc ch phm thuc ngoi da ti Vit Nam.

3

Chng 1. TNG QUAN V VAI TR NH GI SINH KH DNG

V TNG NG SINH HC CA THUC

1.1. KHI NIM SINH KH DNG V TNG NG SINH HC

Trong dc hc, sinh kh dng (bioavailability - BA) l thng s biu th t l

thuc vo c tun hon chung (tun hon mu) dng cn hot tnh so vi liu

dng (F%), tc (Tmax) v mc (Cmax) thuc hp thu vo vng tun hon

chung [1, 3]. T nh ngha ny, thuc theo ng tim tnh mch c sinh kh dng

l 100%. Tuy vy, khi thuc c dng bng cc cch thc khc nhau (nh ng

ngoi da) th sinh kh dng ca thuc thng gim (do hp thu khng hon ton).

Sinh kh dng c xem l mt cng c thit yu trong sinh dc hc, y l i

lng quan trng xc nh v tnh ton liu dng cho cc dng bo ch khng

theo ng tnh mch.

Sinh kh dng tuyt i (Fabs): xc nh c sinh kh dng tuyt i ca

mt loi thuc ngi ta cn xc nh c mi lin h gia thi gian v nng

ca thuc lu hnh trong huyt tung. bit c mi tng quan trn trc ht

phi bit c liu dng theo ng tim tnh mch (Div) v c ng ngoi tnh

mch (Dpo). Sinh kh dng tuyt i (Fabs) l t l gia din tch di ng cong

thu c khi a thuc ngoi ng tnh mch (AUCpo) v din tch di ng

cong a qua ng tnh mch (AUCiv) ca cng mt thuc.

Nh vy nu thuc c a theo ng tnh mch (I.V) th F = 1. Cn nu

thuc a ngoi ng tnh mch F s lun nh hn 1 do lun c mt lng nht

nh b tn hao trong qu trnh hp thu vo mu hoc b chuyn ha khi i qua gan.

Sinh kh dng tng i (FA/B): Sinh kh dng tng i l t l gia 2 gi

tr sinh kh dng ca cng mt hot cht, cng mt ng a thuc, cng mt

mc liu nhng ca 2 nh sn xut khc nhau hoc ca 2 dng bo ch khc nhau.

4

Sinh kh cdng tng i thng c s dng so snh thuc ca mt nh

sn xut no vi mt thuc ang lu hnh c uy tn trn th trng [1]. Trong

ti ny, chng ti nh gi v sinh kh dng ca cc thuc ngoi da l dng khi

nim v sinh kh dng tng i.

Tng ng sinh hc (Bioequivalence - BE): Hai ch phm ca cng mt

hot cht, cng liu dng, cng ng a thuc c coi l tng ng sinh hc

khi 3 i lng: F%, Tmax (tc ) v Cmax (cng ) dao ng mc cho php

(thng trong khong t 80 n 125%); tuy nhin, quy nh thng ch i vi

AUC cn 2 i lng Tmax, Cmax khng bt buc, tr nhng thuc c phm vi iu

tr hp, yu cu ny l bt buc i vi c 3 i lng [2]. Tng ng sinh hc

cn c nh ngha nu 2 thuc l tng ng bo ch (cha cng loi dc cht

vi cng hm lng trong cng dng bo ch, c cng ng dng v t cng mt

mc tiu chun cht lng [3]) hay l th phm bo ch (cng loi dc cht nhng

khc nhau v dng ho hc ca dc cht nh base, mui hay ester... hay khc nhau

v hm lng hoc dng bo ch [3]) v sinh kh dng ca chng sau khi dng

cng mt mc liu trong cng iu kin th nghim l tng t nhau dn n hiu

qu iu tr ca chng v c bn c coi l s tng ng nhau [3].

Hnh 1.1. th biu din nng ca hai ch phm A, B c cng hot cht, cng

hm lng ca hai hng sn xut c coi l tng ng sinh hc [29]

5

1.2. VAI TR CA SINH KH DNG V TNG NG SINH HC

TRONG QUN L CHT LNG THUC TRN TH TRNG

Th trng dc phm trn th gii hin nay c hng trm mu m thuc

mi v bit dc khc nhau. Cc nh iu tr s dng th thy c mt s thuc

khng cho tc dng iu tr v hiu qu lm sng ging nh bit dc gc (tc l

thuc c pht minh u tin v c cp php lu hnh trn th gii). Mt thuc

generic c th tng ng bo ch (c cng cng thc, hm lng dc cht,

cng dng bo ch, cng cch dng, liu lng) vi bit dc gc nhng li khng

t c hiu qu iu tr nh thuc bit dc gc th hin. t tin cy cn

thit trong th trng dc phm, mt thuc generic cn phi c chng minh tnh

hiu qu v an ton trong iu tr ca n bng th nghim chng minh tng ng

sinh hc (bioequivalence - BE) vi thuc bit dc gc.

Vi s pht trin ca cng nghip dc phm trn th gii hin nay, nht l

cc quc gia pht trin cho ra i hng ngy ln n hng trm mu m thuc

mi v hng chc loi bit dc khc nhau. Tuy nhin, kh c th bit hoc cha

nh gi ht liu cc thuc do nh sng ch sn xut ban u vi thuc generic c

hiu qu nh nhau v iu tr hay khng, ngha l c tnh sinh kh dng v tng

ng sinh hc gia cc thuc c nh nhau hay khng, l cha k n hiu

lc v tnh an ton ca thuc. Hn na, hng ngy trn cc trang tin trong v ngoi

nc a nhng tin v loi thuc A hay thuc B khng tiu chun, trong cng

cp n tng ng sinh hc.

Do vy, vai tr nh gi sinh kh dng v tng ng sinh hc va c

ngha khoa hc, va l hnh lang php l cho nh chuyn mn v qun l Dc

phm thc hin chm sc sc khe con ngi. iu cng i hi cc nh sn

xut, phn phi, qun l bt buc phi hi nhp cng sm cng tt bnh nhn an

tm, vic qun l v k n thuc hp l hn v c thc o chung l tng

ng sinh hc.

6

Hin nay trn th trng ton cu, thuc ngy cng a dng v phong ph. Rt

nhiu thuc ca nc ngoi a vo Vit Nam vi gi r t n , Pakistan, Hn

Quc, Thi Lan... thm ch r hn thuc trong nc sn xut, trong khi mt s

loi thuc nhp t chu u v chu M hoc lin doanh vi cc quc gia nh ,

Php, Canada li c gi rt t. Vn t ra l ngi bnh s chn dng thuc gi

r hay gi t v c bit l c th dng thuc an ton-hiu qu m cn kinh t

na. iu ny c th l gii v a li khuyn hp l nht ch khi c kt qu hoc

bng chng chng minh c vn sinh kh dng v tng ng sinh hc ca

cc ch phm thuc.

V kha cnh sinh kh dng ca thuc cn phi c 2 i lng l tc (Tmax)

v mc (Cmax) cn phi t c ca mt thnh phn hot cht c gii phng

t mt dng bo ch, c xc nh bng th nng - thi gian ca hot cht

trong h tun hon hoc s bi tit hot cht trong nc tiu. Sinh kh dng ca

thuc s khc nhau trong cc trng hp l cng mt dc cht nhng ng s

dng khc nhau, cng ng s dng nhng dng thuc khc nhau, cng dng

thuc, cng ng s dng nhng cng thc khc nhau.

1.3. TH NGHIM TNG NG SINH HC

Cc th nghim tng ng sinh hc gia thuc c bng sng ch v thuc

gc thng c nghin cu trn i tng l nhng ngi tnh nguyn khe mnh

(trn 12 ngi), nhng i khi c th c th nghim trn c nhng bnh nhn.

Sau , tin hnh so snh v cc thng s dc ng hc c trch xut t bng d

liu nng thuc trong huyt tng theo thi gian nh din tch di ng cong

(AUC: area under the curve), nng nh (Cmax), thi gian t c nng nh

(Tmax). Th nghim nn tin hnh trn nhiu mc nng khc nhau, c bit khi

thuc cho kt qu v dc lc hc khng theo ng tuyn tnh.

Th tng ng sinh hc (TSH) l nghin cu tn km v cn phi thc

hin trn s lng ngi tnh nguyn (trn 12 ngi kho mnh), phi tin hnh

vic o nng thuc trong mu (phi c thit b hin i nh my o HPLC c

7

thm nh trc khi o). Nng trong mu ca 2 thuc: thuc generic v thuc

bit dc gc c so snh chng minh tnh hiu qu ca hai thuc l tng

ng.

Mt th nghim TSH bao gm nhiu bc:

- Thit k nghin cu (study design): chn m hnh nghin cu ph hp

nh dng n liu, ngu nhin, cho i, nhn m, m n, m i

C 2 giai on nghin cu: giai on 1 dng thuc generic, giai on 2

dng thuc i chng, gia 2 giai on l thi gian ngh khng dng

thuc gi l thi gian lm sch thuc thng l 7 ngy.

- Chn lc ngi tnh nguyn tham gia nghin cu: phi chn, sng lc

nh t 30-40 ngi chn ra ti thiu 12 ngi tnh nguyn khe mnh,

tui t 18 n 40-45, t tiu chun ca FDA v khm lm sng (nh o

huyt p, nhp tim, thn nhit, ch s BMI) v cc xt nghim ha

sinh (nh o cng thc mu ).

- Tin hnh th sinh kh dng in vivo vi hai thuc th nghim (generic)

v thuc i chng: c c cc thng s dc ng hc Cmax, Tmax,

AUC. Trong thi gian nghin cu, ngi tnh nguyn cn c theo di

tc dng ph, an ton ca thuc.

- X l thng k cc tr s Cmax, Tmax, AUC: cc tr s o c c s sai

lch s c phn tch bng phng php ANOVA hoc cc phng

php khc tnh ton khong tin cy 90% v kim tra gii hn sai lch

l 80-125% nhm xem hai thuc th nghim v i chng c tng

ng sinh hc hay khng.

V i ng tham gia nghin cu gm bc s lm sng, vi vai tr lin tc gim

st y khoa v khng c b qua bt k tc dng ph no, d nh nht. Mt nh

dc ng lc hc chuyn xc nh thi gian ly mu mu v mt nh ha hc phn

tch lng thuc trong mu. Nghin cu phi c tin hnh ti bnh vin kp

8

thi x l cc tnh hung cp cu c th xy ra. Cc mu c phn tch bng thit

b chun ha. Sau hng lot cc nghin cu thc hin, h s bo co ca ch

phm s c trnh ln Cc qun l dc phm quc gia quyt nh xem thuc

generic va qua thc nghim c c xem l tng ng sinh hc hay khng. Ch

phm phi t yu cu v tng ng sinh hc, mi c a ra th trng.

1.4. MT S QUY NH TI CC QUC GIA V TNG NG SINH

HC

Ti Australia:

Theo c quan qun l v iu tr ca Australia (Therapeutics Goods

Administration - TGA), cc ch phm dc c xem l tng ng sinh hc nu

vi khong tin cy CI 90% s chuyn dng sinh hc t nhin tng ng gia 2

ch phm, nng thuc ti a (Cmax) v din tch di ng cong (AUC) nm

trong khong 0,80-1,25. Thi gian t nng nh (Tmax) cng cn tng ng

gia cc sn phm.

Ti chu u:

Theo quy nh chun ca cng ng chu u EMEA-CPMP v hng dn

sinh kh dng v tng ng sinh hc ca thuc (London, thng 7.2001): 2 ch

phm c xem l c tng ng sinh hc nu sinh kh dng v hiu qu tc dng

ca chng sau khi dng cng liu phn t gam l nh nhau, c v hiu lc ca thuc

v tnh an ton. Khong tin cy 90% s chuyn dng sinh hc t nhin tng ng

gia 2 ch phm, nng thuc ti a (Cmax) v vng di ng cong (AUC) nm

trong khong 0,80-1,25.

Ti M:

C quan FDA ca M xem 2 ch phm tng ng v mt sinh hc nu

khong tin cy 90% ca Cmax, AUC(0-t) v AUC(0-) ca thuc th v thuc chng

nn dao ng trong khong 80% - 125% [99] khi tnh trng bnh nhn i.

9

Ti Vit Nam:

Thng tin ti hi tho "Thuc cht lng = tng ng sinh hc" t chc ti

H Ni 2006, ti Vit Nam khong hn 80% thuc ang lu hnh l thuc generic

(thuc thng mi, c cng gc ha hc vi cc thuc ht thi gian c bo h

c quyn). Cc thuc ny c gi thnh thp hn so vi cc thuc pht minh, gip

gim chi ph iu tr. Tuy nhin, s thuc generic c th tng ng sinh hc

cn rt t, phn ln cha c thc hin v Vit Nam cha c quy nh ny. Ti hi

tho, cc chuyn gia cho rng, thuc generic gip gim chi ph 40-60% so vi thuc

pht minh, v vy ngi bnh c thm c hi tip cn thuc iu tr. Tuy nhin,

khng nh c cht lng ca cc thuc generic, c quan qun l cn c cc quy

nh th nghim tng ng sinh hc. Th nghim ny cho bit mc tng t

gia thuc generic v thuc pht minh v dc hc, hiu qu, an ton. Vic cp

s ng k cho thnh phm thuc mi dng li vic kim tra tng ng dc

hc (tng ng bo ch): cng dng thuc, cng loi thuc v lng dc cht,

cng ng s dng, cng t cc tiu chun cht lng quy nh, c th khc nhau

v t dc, mu, mi, v, tui th, nhn nhng tr liu c th ging hoc khng

ging nhau... Cn tin ti l trnh ca vic cp s ng k s phi c kt qu kim

tra tng ng sinh hc, ch c nh vy chng ta mi thc s bit c hiu lc

ca thuc (tng ng tr liu). Tng ng tr liu bao gm cc dc phm

c ng k lu hnh, cc dc phm l tng ng dc hc, cc dc phm l

tng ng sinh hc, c nhn v ng, sn xut tun theo quy nh v thc

hnh sn xut tt. p ng yu cu ca thc tin, ngy 26/04/2010 B Y t ban

hnh Thng t 08/2010/TT-BYT hng dn bo co s liu nghin cu sinh kh

dng/tng ng sinh hc trong ng k thuc. Theo Thng t ny nguyn tc la

chn cc dc cht a vo Danh mc cc dc cht yu cu bo co s liu

nghin cu tng ng sinh hc khi ng k thuc phi p ng mt hoc mt s

cc nguyn tc u tin sau [3]:

10

1) C trong cc thuc thuc Danh mc thuc cha bnh ch yu s dng ti cc c

s khm cha bnh do B Y t ban hnh km theo Quyt nh s 05/2008/Q-BYT

ngy 01/02/2008 v thuc mt trong cc nhm tc dng dc l c u tin sau:

- Cc thuc tim mch- huyt p

- Cc thuc chng co git, chng ng kinh

- Cc thuc h ng huyt

- Cc thuc khng sinh

- Cc thuc tc dng trn ng tiu ho lm gim tit acid dch v

- Cc thuc chng ri lon tm thn

- Cc thuc khng vim (khng steroid v steroid)

- Cc thuc khng virus.

2) C trong cc thuc thuc danh mc cc thuc c s dng trong cc chng

trnh quc gia:

- Thuc lao

- Thuc st rt

- Thuc khng HIV

- Thuc trnh thai...

3) C khong iu tr hp v/ hoc c vn v sinh kh dng.

Theo mt s nghin cu khoa hc:

Nghin cu ca Ehinger KH v cc cng s (2013, Clinical Drug

Investigation) tin hnh nh gi tng ng sinh hc ca 2 ch phm tim

truyn tnh mch l CicloMulsion (NeuroVive, Thy in) v Sandimmune

(Novartis, USP) cng c hot cht chnh l Ciclosporin-mt cht c ch min dch.

Nghin cu c thc hin trn 52 tnh nguyn vin khe mnh, theo phng php

m i, n liu, ngu nhin, nghin cu cho, 2 giai on. Kt qu thu c cho

thy khong tin cy 90% ca AUC(0-) l 0,90 (0,88-0,92) v ca Cmax l 0,95 (0,92-

0,97) nm trong khong 0,80-1,25, c chp nhn l tng ng sinh hc [93].

11

Hnh 1.2. Nng ciclosporin trong mu theo thi gian nhng ngi tham gia

dng CicloMulsion hoc Sandimmune (n = 52) [93]

Nhiu nghin cu v iu tra, cc b phn ng k cc nc pht trin ln

nh M, Australia hoc chu u nhn thy rng tng ng sinh hc l cch thch

hp nht chng t s tng thch tr liu gia thuc generic v thuc sng ch.

Mt thuc generic c cht lng l mt thuc c chng minh tng ng sinh

hc so vi thuc sng ch. Trong thc t sn xut, cc tiu chun thc hnh sn xut

thuc tt ca trang thit b sn xut cho thuc generic v thuc sng ch phi hon

ton ging nhau.

Thc t trn th trng c qu nhiu bit dc, nh gi thuc no tt,

ngi ta cn phi xem xt ti sinh kh dng v tng ng sinh hc. Vi vic p

dng dc ng hc, dc lc hc, quan st v th nghim lm sng, k c cc th

nghim in vitro: o nng huyt tng, o lng thuc trong cc dch bi tit

(nc tiu), o phn ng dc lc cp, nh gi lm sng, ha tan, phng

thch thuc (tnh tan, tnh thm) khng nh thuc no l tt.

Vit Nam c rt nhiu nghin cu v tng ng sinh hc trc v sau

khi Thng t 08/2010 ra i c nhng doanh nghip dc phm ln n cc c s

sn xut dc phm cp tnh, cc x nghip dc phm qun i. Mt nghin cu

12

gn y ti Vit Nam [4] nh gi tng ng sinh hc vin nn bao phim

Cefuroxime 500 mg do X nghip Dc phm 150 sn xut. Nghin cu tin hnh

trn 12 ngi trng thnh khe mnh tnh nguyn theo phng php nghin cu

cho, n liu, ngu nhin, 2 thuc, 2 giai on nhm nh gi tng ng sinh

hc gia thuc th l vin nn bao phim Cefuroxime 500 mg (X nghip Dc

phm 150, ARMEPHACO) v thuc i chng l vin nn bao phim Zinnat 500

mg (Glaxo, UK).

Kt qu cho thy thuc th so vi thuc i chng c khong tin cy 90% ca

AUC0-t t t 95,76-106,53%; ca AUC0- t t 93,71-105,75%; ca Cmax t t

93,76-112,94%; phn tch ANOVA cc thng s AUC0-t, AUC0- v Cmax v s

khc nhau gia 2 trnh t, 2 thuc, 2 giai on khng c ngha thng k (p >

0,05); gi tr Tmax khc nhau khng c ngha thng k theo phng php thng k

phi tham s. Vin nn bao phim Cefuroxim 500 mg c kt lun tng ng sinh

hc in vivo vi vin nn bao phim Zinnat 500 mg [4].

Hnh 1.3. th biu din nng thuc Cefuroxim trung bnh theo thi

gian ca thuc th v thuc chng [4]

Dc phm l hng ha c bit dng phng, cha bnh, thay i chc

nng sinh l ca con ngi. Chnh v nh hng trc tip ti c th nh vy nn cc

nh khoa hc v cc nh qun l cn gii quyt v iu chnh mi quan h gia gi

c v hiu lc iu tr sao cho hp l, an ton, hiu qu, kinh t. Tng ng sinh

13

hc s l cng c sc bn gii quyt tt mi quan h ny, vic thc hin th

nghim tng ng sinh hc gip cho vic s dng thuc theo cch tt nht va

an ton - hp l - hiu qu - kinh t. Tuy nhin nhng nghin cu c mi ch tp

trung nh gi sinh kh dng v tng ng sinh hc ca cc thuc ng tim v

ng ung m t cp n vic nh gi sinh kh dng v tng ng sinh hc

ca cc thuc dng ngoi da, c bit ti Vit Nam, trong khi nhng thuc ny ngy

cng c s dng ph bin vi cc tc dng nhanh ti ch m t tc dng ph ton

thn. Vic tm c mt phng php gip h tr nh gi sinh kh dng v tng

ng sinh hc ca cc thuc dng ngoi da ny l ht sc cn thit. K thut thm

tch micro c gii thiu di y s l mt cng c c lc gip gii quyt

vn ny.

14

Chng 2. TNG QUAN V K THUT THM TCH MICRO

TRN DA V U NHC IM CA K THUT

2.1. TM TT V LCH S PHT TRIN CA K THUT THM TCH

MICRO

Xut hin t nhng nm 60 ca th k XX, k thut thm tch micro

(microdialysis) da trn s khuch tn th ng cc cht qua mng thm tch theo

gradient nng . Ban u, thit b thm tch micro s dng mt ti thm tch c

kch thc l mng rt ln thu c cc phn t acid amin trong cc t chc m

trn ng vt thc nghim [15, 21]. n nm 1974, Ungerstedt v cng s nh s

h tr ca cc phng php nh lng mi ci tin li k thut di dng kim

thm d c u l mt mng bn thm v a ra cng trnh nghin cu u tin v

nh lng nng dopamin trong no chut thc nghim [94].

Cui nhng nm 80, k thut ny bt u c dng trong cc nghin cu trn

ngi [64]. u tin, k thut ny c p dng nghin cu nh lng phn t

glucose trong dch k t bo ca ngi tnh nguyn. Tuy nhin, phi n nm 1991,

nhng bo co u tin v thm tch micro trong nghin cu dc ng hc ca

thuc trn lm sng mi c cng b [84]. Nm 1994, Palsmeier v cng s s

dng k thut ny xc nh s phn b ca thuc trong m ung th [69] v rt

nhiu cc nghin cu khc c thc hin hu ht cc m trong c th ngi cho

kt qu rt kh quan nh m no, m tim, m phi, m gan, m v, m di da [5,

21, 55, 64]... (Hnh 2.1). Ngoi ra, thm tch micro cn l cng c tt nht nh

gi sinh kh dng (bioavailability BA) v tng ng sinh hc (bioequivalence -

BE) ca thuc ti m ch [17, 68, 77, 78, 90], c bit l cc thuc ngoi da khi

vic o nng thuc dch k m da s phn nh ng hn mc phn b thuc

ti m da.

15

Hnh 2.1. Cc m ch tc dng ca thuc trn ngi c thc hin

nghin cu lm sng da trn k thut thm tch micro

Qua nhng thp k s dng, k thut thm tch micro ngy cng c ng

dng rng ri, c bit cho nghin cu dc phm v y hc lm sng. T kha

Microdialysis xut hin trn cc cng c tra cu medline vi hn 15000 kt qu,

trong c gn 1500 kt qu lin quan n vic s dng k thut ny trn da vi

cc ch phm dng ti ch. ng dng chnh ca k thut ny trong giai on u

pht trin thuc nhm la chn nhng thuc mi c hiu qu tt v xc nh liu

lng ti u an ton v hiu qu.

Hnh 2.2. Kt qu tm kim trn medline vi cc t kha lin quan n

microdialysis

16

2.2. NGUYN L CA K THUT THM TCH MICRO

2.2.1. Nguyn l

K thut thm tch micro da trn s khuch tn th ng cc cht qua mng

thm tch theo gradient nng [24, 84, 87, 93]. Sau khi mt kim thm d c

cy ghp vo cc v tr ch ly mu [20, 107] (thng l mt mch mu hoc

m), dch truyn bao gm cc dung dch m sinh l chy chm qua mng bn

thm thu li cc phn t nh t khoang ngoi bo pha bn kia mng thm tch

[42, 51, 82]. Dch thm tch thu c c th c phn tch xc nh cc phn

t thuc hoc cht cn phn tch trong mu thm tch micro bng sc k lng hoc

cc k thut phn tch ph hp khc [53, 54, 70, 98].

Ngoi ra, k thut ny cn c th c p dng truyn mt cht vo

khoang ngoi bo bi kim thm d thm tch micro, k thut ny c gi thm

tch ngc [23, 26, 34, 92]. Bng cch ny, vic dng thuc ti ch v ly mu

ng thi cc hp cht ni sinh trong cc khoang ngoi bo c th cng mt lc

c thc hin.

Hnh 2.3. Nguyn tc ca k thut thm tch micro vi mt kim thm d

dng ng tm

Sau khi cy kim thm d, dung mi thm tch i vo kim thm d thng qua

17

dy truyn dch vo (ng bn trong), i xung mi kim thm d, chy ln trn

khong khng gian gia ng bn trong v bn ngoi mng lc, v ri kim thm d

thng qua ng ra cnh sn, t c ly vo mt l ng mu [92].

i vi phng php thm tch micro ca thuc ngoi da trong cc mu sinh

hc, nguyn tc thm tch micro l kim thm d c a vo lp h b v c

truyn thng qua my bm truyn vi tc rt chm (Hnh 2.4). Thuc t ni s

dng s thm vo qua lp biu b v cui cng khuch tn th ng qua mng bn

thm [6]. Cc cht thm tch c ly mu ti cc khong thi gian c nh [16,

38, 101].

Hnh 2.4. S nguyn tc thm tch micro thuc ngoi da [101]

Hnh 2.5. Ba kim thm d t song song trong mi khu vc th nghim (3 khu vc,

mi khu cho mt thuc th) trn mt nga ca cnh tay tri [32]

18

2.2.2. Kim thm d

Kim thm d l phn khng th thiu ca k thut thm tch micro. Qu trnh

thm tch l qu trnh khuch tn th ng ca cc phn t theo gradient nng t

m ch c nng cao vo dch truyn. Mng bn thm cho php cc phn t c

kch thc nh hn l mng khuch tn qua mng v gi li cc phn t c kch

thc ln.

Kim thm d c cu to gm 3 b phn: (1) mt dy truyn dch vo, (2)

mng bn thm v (3) mt dy ly dch ra (Hnh 2.6).

Hnh 2.6. Kim thm d thm tch micro dng ng thng [16]

Kim thm d dng ly mu cc dch sinh hc hin nay c chia lm 4 loi

c bn [20, 93]: ng thng (linear), vng mc (loop), sng i (side - by - side)

v ng tm (concentric) (Hnh 2.7), khc nhau v kch thc, hnh dng v cht

liu, ph thuc vo v tr t chc m ch cn nghin cu [93]. Trong nghin cu

thm tch micro da, kim thm d dng ng thng (linear probe) c dng ph

bin nht [13, 39, 101].

19

Hnh 2.7. Cc dng kim thm d

A: ng thng, B: vng mc, C: sng i, D: ng tm [39]

(1) Dy truyn dch vo

Dy dch truyn vo (inlet tube) c ni vi bm thm tch truyn vo

c th dch truyn vi tc nh sn [77, 101].

Dch truyn vo phn ln c truyn vi tc chm 0,1-5 l/pht [60]

qua my truyn tc chm (CMA100, Thy in) [6]. Thnh phn ca dch

truyn vo s c m t chi tit bng 2.1.

(2) Mng bn thm

Mng bn thm (semi-permeable membrane) chnh l b phn c vai tr

thm tch, ch cho php cc phn t c kch thc nh hn kch thc l mng

i qua mng theo s chnh lch gradient nng nn c im quan trng nht

l kch thc l mng [60, 74, 92]. Trng lng phn t nm trong khong 5-

100kDa [9, 107], do c th thm tch nhiu cht khc nhau bng k thut

thm tch micro [18, 39, 76, 80].

Mng bn thm thng c ng knh ngoi t 0,2-0,5 mm [60], chiu di

t 0,7-3 cm ty thuc v tr cy kim [74]. Mng bn thm u c lm bng

vt liu c tng hp sinh hc cao nh cellulose acetat, polyether sulfonat,

polycarbonat - ether, polyamid, polyacrylonitril (PAN)... [9, 74] c kh nng

ht nc v khng c tnh chn lc ha hc.

20

(3) Dy truyn dch ra

Dy dch truyn ra (outlet tube) c ni vi b phn cha mu. Dch thu

c cha cc cht cn phn tch dng t do c thm tch t dch ngoi

bo khong k theo c ch khuch tn th ng i qua mng bn thm, tc

theo gradient nng t m ch c nng cht cn thm tch cao vo dch

truyn.

Dch thu c s c chuyn vo cc ng nghim trong khay cha ng,

sau em nh lng ngay (thm tch micro online) [22, 73] hoc bo qun

lnh ch nh lng tng mu (thm tch micro offline) [75, 105].

Kim thm d thng c lun qua mt kim dn (guide cannula) tin

cho vic cy kim do kim thm d mnh, d gy [27, 37, 39] (Hnh 2.8)

Hnh 2.8. Cm ng dn sau s lun kim thm d qua ng dn ny [39]

Kim thm d phi c cy trong iu kin v khun v vic cy kim thm d

trong iu kin khng v khun c th gy nhim khun ti v tr cy v to iu

kin cho vi khun pht trin. Mt khc, n cn lm nh hng n kt qu hiu

chnh ca mng bn thm trn kim thm d. Trong cc nghin cu tin hnh trn

ngi tnh nguyn, yu cu v khun i vi kim thm d v dch truyn vo l bt

buc.

21

2.2.3. Dch truyn vo

Dch truyn vo khi s dng k thut thm tch micro phi v khun, c thnh

phn, p sut thm thu v pH tng t nh dch ti m cy kim thm d [83, 85].

Thnh phn ca cc dung dch hay dng cho k thut thm tch micro c ghi

trong bng 2.1.

Bng 2.1. Cc dung dch truyn s dng trong k thut thm tch micro

Thnh phn Nh snhu xut

Dch truyn cho m no

Dung dch CNS

MgCl2 0,85 mmol/l

CaCl2 1,2 mmol/l

NaCl 147 mmol/l

KCl 2,7 mmol/l

Hng CMA, Thy

in

Dch truyn cho m ngoi vi

(m da v cc m t chc ca

c th)

Dung dch T1 [14, 35]

Na+

147 mmol/l

Ca2+

2,3 mmol/l

K+

4 mmol/l

Cl- 156 mmol/l

Hng CMA, Thy

in

Dung dch Ringer [44]

Na+

147 mmol/l

Ca2+

2,25 mmol/l

K+

4 mmol/l

Cl- 156 mmol/l

Baxter, Utrecht, H

Lan

22

Nh tng v trng

Intralipid (IL) [10]

Du u nnh 200 g

Phospholipid trng 12 g

Glycerol anhydrid 22 g

NaOH v H2O (lng

va iu chnh

pH=8)

Fresenius Kabi,

Midrand, Nam M

2.2.4. nh lng cc phn t thuc

Sau khi ly mu, vic nh lng mu l khng th thiu, trong k thut

thm tch micro, lng mu thu c rt nh (c vi chc microlit) nn i hi cc

phng php nh lng phi c nhy cao nh sc k lng hiu nng cao

(HPLC), sc k lng kt hp khi ph (LC - MS)

2.2.4.1. Sc k lng hiu nng cao (HPLC)

Sc k lng hiu nng cao (HPLC) l k thut phn tch da trn s phn tch

cc cht trn mt pha tnh c c nh trong ct, nh dng di chuyn ca pha ng

lng di p sut cao [2]. Cc thuc c nghin cu bng k thut thm tch

micro hu ht c bn cht thn nc nn sc k phn b pha o thng c dng,

ngoi ra c th s dng ct trao i ion. Cc thng s khc ca ct sc k (chiu di

ct, kch thc ht, ng knh trong) c xc nh da theo khong cch ly mu

mong mun v yu cu v nhy.

Phn ln cc my sc k lng cn lng mu phn tch ti thiu l 10l. Nu

tc truyn dch vo l 1l/pht th sau khong thi gian phn tch khong 15

30 pht, chng ta s thu c mt lng dch t 15 n 30l, lng ny phn

tch bng cc my sc k lng hiu nng cao. Tc truyn dch cng chm th hiu

sut kim thm d cng cao, tuy nhin thi gian phn tch s ko di. Ct mao dn

c s dng tng nhy v rt ngn thi gian phn tch [70].

23

Mu sau khi ly c th bo qun ng lnh vi nhit thch hp trong khi

ch nh lng (offline microdialysis) [75, 105]. Ngy nay, vi nhng tin b trong

phn tch ha hc cho php thit k mt h thng ly mu v nh lng trc tip

(online microdialysis) [22, 73] nng thuc trong dch k m mt cch nhanh

chng, gip cho vic gim st nng thuc ti m, to ra kh nng iu chnh tc

thi liu dng ca thuc trn tng bnh nhn c th [22].

Do bn cht ha hc ca cc cht phn tch rt khc nhau nn c nhiu k

thut tch khc nhau [2]. Tuy vy, mt my sc k lng lun c cu to gm cc

b phn c bn sau:

1. H thng cp pha ng

Pha ng thng l hai dung mi ha tan vo nhau c kh nng tch vi

phn gii ph hp. Cn phi c b phn kh kh ui kh ha tan trong pha ng

bng cc cch nh lc (mng lc 0,45m), chy siu m, sc kh trdo kh ha

tan c th lm bin dng pic, gim hiu lc ct, nhiu ng nn.

Pha ng thng c cha trong bnh thy tinh. C 2 kiu thc hin chng

trnh dung mi: trn cc dung mi p sut thp hoc p sut cao.

2. H thng bm

H thng bm trong sc k lng cn p ng cc yu cu:

- C kh nng hot ng p sut u vo khong 5000 psi tr ln.

- m bo bm lu lng lp li trong khong 2-3l/pht n 20

mL/pht.

- Cn ch to t vt liu thch hp m bo chu c tc ng ca

nhiu loi dung mi (khng b n mn).

3. H tim mu

H tim mu l mt van tim c vng cha mu dung tch t 0,5-20l, d dng

t ng ha, c iu khin v kim sot bng phn mm my tnh.

4. Ct v pha tnh

24

Ct c ch to bng thp khng g, thy tinh hoc cht do c chiu di 10-

30 cm, ng knh 4-10mm, bao gm ct nhi, ct bo v v iu nhit ct.

5. Detector

Pht hin cht phn tch da trn tnh cht ca cht phn tch nh hp th bc

x UV, hunh quang, ch s khc x, dn in Detector trong sc k lng cn

p ng cc yu cu:

- p ng nhanh v lp li,

- nhy cao, c th pht hin cht phn tch khi lng hoc nng

thp,

- Vn hnh n nh, s dng d dng,

- Khong hot ng tuyn tnh rng,

- t thay i theo nhit v tc dng.

C nhiu loi detector nh detector hp th UV-VIS, detector hunh quang,

detector ch s khc x, detector tn x bay hi, detector o dng, detector dn

6. H thu nhn v x l d liu

H thng my tnh c phn mm ghi nhn tn hiu, x l d liu v iu khin

h thng; my in

Hnh 2.9. S h thng HPLC

25

2.2.4.2. Phng php sc k lng khi ph (LC-MS)

Vi s tin b tin b ca khoa hc k thut, phng php sc k lng khi

ph ang dn chim u th v c s dng rng ri trong 10 nm tr li y. Sc

k lng (liquid chromatography-LC) l mt k thut c bn dng phn tch cc

cht. Khi ph (mass spectrometry MS) tr thnh mt detector ngy cng c

dng ph bin cho phn tch online [22, 73] hoc offline [75, 105] cht thm tch

micro thu c. Phng php ny cung cp nhng thng tin v khi lng phn t,

cu trc ha hc, tn hp cht, s lng hp cht v tinh khit ca mu phn

tch. Nhng d liu ny lm chc chn thm cc kt qu nh tnh v nh lng.

Nguyn l hot ng ca khi ph l o trc tip t s khi lng v in tch

ca ion (m/z) c to thnh trong pha kh t phn t hoc nguyn t ca mu, qua

xc nh cc ng v ha hc m khng dng bc x in t nh cc k thut

quang ph. T s m/z c biu th bng n v khi lng nguyn t (1 n v

khi lng nguyn t bng 1/12 khi lng ca Carbon 12C) hoc bng Dalton (1

dalton bng khi lng ca nguyn t Hydro) [2]. Khi ph cung cp cho ta nhng

thng tin v khi lng phn t, cu trc ha hc ca hp cht, s lng hp cht

v tinh khit ca mu phn tch. Nhng d liu ny lm tng thm tin cy ca

cc kt qu nh tnh v nh lng [2].

Sc k lng kt ni khi ph hay khi ph ghp (tandem mass spectrometry)

lm tng ng k tnh chn lc v nhy ca phng php phn tch. Gii hn

pht hin c th n 10-14 gam [2]. Nng cc cht trong mu thm tch micro rt

nh, thnh phn tng i phc tp, bao gm c thuc, cc cht chuyn ha ca

thuc v cc cht khc c trong dch ngoi bo. S dng LC MS gip cho kt qu

thu c chnh xc hn v c gi tr hn [48]. V vy rt nhiu nghin cu v thm

tch micro s dng phng php ny nh lng [53, 59, 71, 72].

2.2.5. Hiu chnh kt qu thu c

K thut thm tch micro da vo s khuch tn cc phn t thuc theo

gradient nng qua mng bn thm, chu nh hng bi kh nng thm tch ca

26

mng kim thm d, nn nng thuc trong dch thu c cha phn nh c

nng thuc thc cht ti m ch. Trong a s cc trng hp, nng o c

thp hn nng thc t ti dch k m. V th, ngoi vic chng minh kh nng

ng dng k thut thm tch micro trong nghin cu thuc, cc nghin cu cn

cp n s cn thit phi c phng php hiu chnh li lng thuc thu c ti

m [56].

T l gia nng trong dch thu c v nng thc trong dch k m ca

mt cht c gi l hiu sut tng i, cn lng cht thu c trong mt n

v thi gian gi l hiu sut tuyt i. Xc nh hiu sut tuyt i l so snh

gia cc gi tr o c. Tuy nhin, mun bit chnh xc nng trong dch k m

th cn phi tnh hiu sut tng i bng cc phng php in vivo hay in vitro

[106].

Hnh 2.10. M hnh nh gi hiu sut mng bn thm kim thm d in vitro

Hiu sut tng i in vitro c xc nh mt cch n gin bng cch c

nh gi tr nng dch xung quanh mng bn thm trn kim thm d v o gi tr

nng thu c [22]. T l gia nng thuc trong dch thu c ly ra t u

thu ca kim thm d (Cout) v nng thuc bit khi t kim thm d vo dung

dch ny (Cin) chnh l hiu sut tng i in vitro (Hnh 2.10).

Tnh hiu sut kim thm d in vivo phc tp hn v gp phi nhiu kh khn

do nh hng ca nhiu yu t dc ng hc, dc lc hc nh c im sinh l

ca m v tng tc gia m ch vi phn t thuc [22]. Hin nay, cc phng

27

php tnh hiu sut in vivo thng c s dng gm c: No - net - flux (NNF) [39],

ngoi suy tc dng chy (ZFR, extrapolation to zero-flow rate) [20], thm tch

ngc (RD, retrodialysis) [91], tc dng chy rt chm (SFR, very slow flow

rate) [12] Tuy phc tp v gp nhiu kh khn trong qu trnh tnh ton, kt qu

thu c t phng php in vivo li ng tin cy hn v gip xc nh nng thc

ca thuc trong dch k m mt cch chnh xc.

2.2.5.1. Phng php No- net - flux (NNF)

Hay cn gi l Zero-net-flux, phng php ny s dng thuc dch k m,

da trn nguyn l cc cht khuch tn thm tch ngc (RD, retrodialysis) qua

mng bn thm theo gradient nng [50, 57]. Truyn vo cc dch truyn c nng

phn t thuc khc nhau bit qua kim thm d (Cin), sau s o nng

mu thu c Cout. Dch c truyn vi tc n nh sao cho nng thuc

trong dch k m l mt hng s. Khi , hiu sut in vivo ca kim thm d (EF,

extraction fraction) ti dch k m c tnh theo cng thc [21, 57, 106]:

trong :

Cin l nng ca dch truyn vo

Cout l nng ca dch thu c

Cm l nng cht phn tch trong dch k m bao quanh kim thm d.

Trng hp c bit khi Cin= 0, EF chnh bng hiu sut tng i (RR,

relativerecovery).

28

Hnh 2.11. Phng php no - net - flux trong hiu chnh in vivo

th trn th hin quan h tuyn tnh gia = Cout Cin v Cin. dc ca

th chnh l hiu sut tng i (Hnh 2.11) [39, 66].

2.2.5.2. Phng php ngoi suy tc dng chy (ZFR)

Hiu sut tng i ph thuc vo tc dng chy, s thay i ca tc

truyn dch dn ti s thay i ca hiu sut tng i, phng php ny da trn

nguyn tc: tc truyn dch vo thay i, trong khi nng dch truyn vo Cin

bng 0, cn nng cht phn tch trong dch k m l hng nh. Lc ny, hiu

sut ca kim thm d ph thuc vo tc truyn dch v c tnh theo cng thc

[20, 40]:

Vi

trong :

Cout l nng dch thu c

Cm l nng dch xung quanh kim thm d

r l h s chuyn khi

29

A l din tch mng bn thm

F l tc truyn dch.

Ngi ta gi nh rng tnh thm v din tch ca mng bn thm l khng i

trong sut qu trnh th nghim. Nng thc ca cht phn tch trong dch k m

bao quanh kim thm d c xc nh bng cch dng php ngoi suy trn th

khi tc truyn dch F = 0 v h thng t cn bng, lc Cout = Cm.

Phng php ny c nhc im chnh l tn nhiu thi gian do tc dng

chy nh, c trng hp cn n 12h ly lng mu cn thit [20].

2.2.5.3. Phng php thm tch ngc (RD)

Phng php thm tch ngc (RD, retrodialysis) hin l phng php c

s dng c nhiu nht [3, 21], l trng hp c bit ca phng php No - net

flux, s dng chnh cht c nghin cu hoc dng mt cht chun ni thm vo

dch truyn vo kim thm d [95, 97]. Cht chun ni l tng phi ging thuc

khng nhng v tnh cht vt l (kh nng khuch tn qua mng, tan...) m cn

c v c tnh sinh hc (chuyn ha, lin kt protein...) [20, 49]. Gi nh rng h s

khuch tn qua mng bn thm theo c hai chiu l nh nhau, nng dch k m

Cm = 0, th hiu sut ca thuc chnh l hiu sut ca cht chun ni. Hiu sut ny

c tnh bng t l gia nng b mt i ( = Cin Cout) v nng ban u Cin

ca cht chun ni theo cng thc [99, 106]:

Cht chun ni cn phi c phn tn, dn truyn tng t nh cht

phn tch, v th ngi ta thng chn cht chun ni l ng phn phng x hoc

ng phn deuteri (2H) vi cht phn tch.

Cc phng php hiu chnh u c nhng u im v nhc im ring. Ty

vo tng nghin cu c th, vic la chn phng php cn c cn nhc sao cho

c th tnh c chnh xc hiu sut ca kim thm d.

30

2.3. QUY TRNH CHUNG CA K THUT THM TCH MICRO TRONG

NGHIN CU Y DC HC

K thut thm tch micro trong nghin cu y dc hc c tin hnh theo

mt quy trnh chung gm 5 bc [7]:

Bc 1: Chun b cc kim thm d, dung dch truyn c thnh phn v nng

tng ng vi dch ti m ch cn nghin cu v cc i tng nghin cu

(ngi, chut, th, ln, kh, mo).

Bc 2: Cy kim thm d vo m ch tc dng ca thuc (no, cnh tay, bp

i, lng.)

Bc 3: Truyn dch c thnh phn v nng tng ng vi dch ti m ch

vi tc rt chm (t 0,1 - 5 l/pht), dch thm tch thu c s c cha cc

phn t thuc/cht chuyn ha.

Bc 4: nh lng nng phn t thuc/cht chuyn ha/cht ni sinh

trong dch thm tch thu c bng phng php phn tch thch hp (HPLC,

UPLC [89, 91, 102], LC MS).

Bc 5: Hiu chnh li nng thuc/cht chuyn ha/cht ni sinh thng qua

hiu sut in vitro hoc in vivo ca mng bn thm.

2.4. U NHC IM CA K THUT THM TCH MICRO

2.4.1. u im

Cho php xc nh nng ca phn t thuc dng t do [7, 9], dng ny

thuc mi c tc dng v c mi tng quan thun vi hiu qu iu tr, t

cung cp nhng thng tin v dc ng hc ca thuc [7, 21].

K thut thm tch micro c th dng ly mu nhiu cht khc nhau (dao

ng t 5 - 100 kDa), ch yu l cc phn t c trng lng thp (thng t

10 30 kDa) [106].

K thut ny t xm ln [94], dng c trn ngi, ngay c trong trng

hp cp cu hoc chm sc tch cc [21].

31

Kim thm d c dng nghin cu trn nhiu m khc nhau [30, 31, 67, 96,

103] (Hnh 2.12).

Hnh 2.12. Cy kim thm d no (tri) v b vai (phi) [79]

C th cy nhiu kim thm d vo cng mt m ch [25, 61, 108].

ng dng k thut thm tch micro trong iu tr ti m ch, ni m cc

phn t thuc khng th phn b n cc m bnh [7, 62].

C th kt hp vi nhiu phng php phn tch ha hc nh sc k lng

hiu nng cao HPLC, sc k lng siu hiu nng UPLC, sc k lng khi

ph LC MS [33, 37, 86, 104].

Ly c mu sch trong cht phn tch khng b nh hng bi hot tnh

ca enzym. Mu ly c c th nh lng trc tip ngay, khng mt thi

gian chun b hay lm sch [52].

Khng lm mt cn bng ni mi, khng lm mt dch ca c th v ch c

phn t thuc c ly ra

2.4.2. Nhc im

Th tch mu rt nh, nng thuc thp (c l) nn i hi cc phng

php phn tch phi c nhy cao, chng hn nh sc k lng hiu nng

cao HPLC, sc k lng, khi ph LC MS [33, 37].

Vic cy kim thm d s gy ra mt s thay i nht nh ti m v do

nh hng n kt qu nghin cu. hn ch nn bt u truyn dch t

32

nht 30 pht trc khi hiu chnh cc cht trung gian ca phn ng vim

gim xung ti mc n nh [45, 65, 100].

K thut ny c mt s kh khn khi ly mu cc cht thn du hoc cc cht

c i lc cao vi protein. hn ch c th b sung cht din hot [58] hoc

thay th dch truyn nc bng nh tng [10, 19, 47].

Nng thuc thc t ti m ch rt kh xc nh chnh xc, v vy cn

c cc phng php hiu chnh in vitro v in vivo.

Thi gian ly mu ko di, trung bnh 15-20 pht.

Qu trnh cy kim thm d i hi phi v khun, ngi lm thc nghim

phi kho lo, chnh xc [7] nn cn ngi thc hin c trnh k thut, tay

ngh cao.

33

Chng 3. TNG QUAN V PHNG PHP NH GI SINH KH

DNG V TNG NG SINH HC CA THUC NGOI DA

3.1. S CN THIT PHI O NNG THUC T DO TI DCH K M

DA

Da c cu to v chc nng ring nn vic s dng thuc trn da c nhiu

khc bit so vi nhng ng dng khc. Hn na, c nhiu dng thuc c dng

trn da, mi dng dng c s dng ty vo loi da, mc cp tnh v v tr ca

tn thng. Cng vi s pht trin ca k thut bo ch, hin nay thuc dng ngoi

khng ch dng li tc ng ngay ti b mt da m thm su qua da vo h tun

hon chung. Vic xc nh sinh kh dng cng nh tng ng sinh hc gia cc

cng thc bo ch bit c hiu qu tc dng ca thuc l ht sc cn thit [13,

36], trong vic xc nh tng ng sinh hc ti ch l rt quan trng xc

nh tng ng iu tr in vivo ca thuc gc v cc dng bo ch ti ch c cha

cng cc thnh phn hot cht [99].

Cho n nhng nm gn y, nghin cu v dc ng hc (PK) vn b hn

ch v cc php o nng thuc t cc t chc nh mu v cc mu m ton

phn. Tuy nhin, nhng cch tip cn trc kia gy ra s nhm ln ng k bi

mt s quan nim cha ph hp [92]:

1. Cc m hnh nh gi da trn nng thuc trong huyt tng cho

thy qu trnh thm nhp vo cc m l cha ng, v n khng tnh n s phc

tp ca qu trnh thm nhp thuc vo cc m v cc qu trnh sinh l bnh.

2. Quan nim cha ng na l s gi nh rng m l mt t chc ng nht.

Vic o nng khng sinh ton phn t cc mu sinh thit c th gy nhm ln

bi nhiu l do. iu quan trng nht l phi ch n khng gian ch thc t cho

cc khng sinh, tr mt vi ngoi l, chnh l dch k. Nu ch c nng thuc

ton phn m c xc nh, nng ti v tr tc dng ca cc loi khng sinh

ngoi bo, v d nh -lactam, c th b nh gi thp. Ngc li, s dn n mt

34

nh gi qu cao nng ti v tr tc dng ca cc thuc tch ly trong t bo,

chng hn nh cc quinolon hoc macrolid.

3. Quan nim cha ng th ba l quan im cho rng ton b phn thuc hin

c trong cc khng gian m khc nhau u c tc dng dc l. Trong thc t, ch

c nng thuc t do ti v tr tc dng mi c kh nng pht huy hiu qu tt

nht, c in vitro v in vivo v cng ch c thuc t do mi c kh nng phn b

c ti v tr ch. iu ny ng vi nhiu loi thuc, c bit l khng sinh.

T 3 l do trn, nng thuc m trong a s trng hp nn c nh

ngha l nng thuc t do trong dch k. Phng php c coi l tiu chun

vng o nng thuc m th cn phi o lng trc tip c nng thuc

trong dch k. K thut thm tch micro p ng yu cu ny.

3.2. TRIN KHAI NH GI SINH KH DNG V TNG NG SINH

HC CA THUC NGOI DA VI K THUT THM TCH MICRO

K thut thm tch micro l mt cng c ly mu rt hu ch c th c s

dng in vivo thy c s thay i nng thuc t do trong khong k hoc

ngoi bo. K thut ny da trn s khuch tn th ng cc cht qua mng thm

tch theo gradient nng . Sau khi mt kim thm d thm tch micro c cy

ghp vo cc v tr ch ly mu, thng l mt mch mu hoc m, mt dch

truyn bao gm cc dung dch m sinh l chy chm qua mng thm tch, thu li

cc phn t nh t khoang ngoi bo pha bn kia mng. Dch thm tch thu c

c th c phn tch xc nh cc phn t thuc hoc cht cn phn tch trong

mu thm tch micro bng sc k lng khi ph LC-MS, sc k lng hiu nng cao

HPLC [99] hoc cc k thut phn tch ph hp khc. Chnh v th, k thut thm

tch micro l mt phng php hu hiu nh gi nng thuc cc b ti bt

k v tr no c cy thm d. Thm tch micro da cung cp mt la chn y

ha hn nghin cu cc loi thuc dng ti ch, qua ph hp nghin cu

tng ng sinh hc ti ch. nh hng ca nhng thay i trong cc c tnh ro

35

cn ca da trn s thm thuc c th nghim bng cch s dng phng php

thm tch micro [92, 99].

3.2.1. i tng nghin cu

K thut thm tch micro dng trong nh gi sinh kh dng hoc tng

ng sinh hc ch yu c thc hin trn ngi (nhng tnh nguyn vin khe

mnh, theo quy nh ca FDA, thng trn 12 ngi) [10, 13, 32, 63], ngoi ra

c th tin hnh trn ng vt (th, chut, ln) [11, 60, 81] to tin cho th

nghim lm sng trn ngi sau . Ty theo c tnh da ca tng i tng nghin

cu m c th cho p ng khc nhau vi cc thuc, tc mc hp thu, phn b

thuc.

Tnh trng sc khe ca i tng nghin cu lun c kim tra m bo

trong thi gian nghin cu thc nghim theo cc tiu chun c quy nh bi B

Y t.

3.2.2. Cc thuc c s dng trong nghin cu

Thuc th

Cc thuc th tng ng sinh hc l cc thuc generic c dng bo ch s

dng ngoi da.

- Thuc generic (generic product) c nh ngha l mt thuc thnh phm

nhm thay th mt thuc pht minh c sn xut khng c giy php nhng

quyn ca cng ty pht minh v c a ra th trng sau khi bng pht minh v

cc c quyn ht hn.

- Thuc pht minh (Innovator pharmaceutical product): l thuc c cp

php lu hnh u tin, trn c s c y cc s liu v cht lng, an ton v

hiu qu.

Cc thuc dng ngoi da c nh gi sinh kh dng v tng ng sinh

hc thng l cc thuc khng sinh, chng vim corticoid, cc thuc khng nm

virus, gim au, chng vim khng steroid (NSAID) liu iu tr.

36

Bng 3.1. Cc nhm thuc ngoi da c s dng trong cc nghin cu nh

gi tng ng sinh hc da trn k thut thm tch micro

Nhm thuc Dc cht Dng bo ch Liu TLTK

Khng sinh Oxytetracyclin

M Oxytetracyclin

HCl 3% kl/kl 4ml/12cm

2 [91]

Metronidazol Kem 1% 4mg/cm2

[32]

Chng vim

corticoid Clobetasol

Dung dch cn

Clobetasol

propionat 4% kl/tt

4ml/8cm2

[10]

Chng nm virus Acyclovir Kem 3% 0.2g/cm2 [99]

NSAID Ketoprofen Gel 2.5% kl/kl Mt lp

mng/3cm2

[88,

89]

Thuc i chng

- Thuc i chng (Comparator product): l thuc m thuc generic s c

dng thay th n trong iu tr. Thng thng, thuc i chng l thuc pht

minh vi cc s liu v hiu qu, an ton v cht lng c thit lp [3].

i vi cc thuc generic c s phi hp mt s dc cht: Thuc i chng

s dng trong nghin cu phi l thuc pht minh c s phi hp tng t v thnh

phn dc cht v t l phi hp gia cc thnh phn hoc l thuc i chng n

thnh phn tng ng.

Dch truyn vo (perfusate)

Dch truyn vo c th t mua ca cc cng ty CMA (Thy in), hoc t

pha ch theo cng thc trong bng 3.2.

37

Bng 3.2. Thnh phn cc dung dch truyn ngoi vi

Dung dch Thnh phn Nng

Nc mui sinh l [41, 91, 99] NaCl 0,9%

Dung dch T1 [14]

Na+

Ca2+

K+

Cl-

147 mM

2,3 mM

4 mM

156 mM

Dung dch Ringers [44]

Na+

Ca2+

K+

Cl-

147 mM

2,25 mM

4 mM

156 mM

Thuc gy t ti ch

Mt s th nghim cn s dng thuc gy t ti ch trc khi tin hnh cy

kim thm d gip qu trnh thao tc c thun tin v gim au cho qu trnh th

nghim nh tim mng bng urethan 30% vi liu 3 ml/kg vo thi im 30 pht

trc mi th nghim [99], xylocain [32, 46], lidocain [46], ti nc [10]

Kem chng vim sau khi cy kim thm d

C th dng thm cc thuc chng vim ngoi da ti v tr cy sau khi tin

hnh th nghim cy kim thm d thm tch micro nh kem Biocort (hydrocortison

acetat 1% (kl/kl); Adcock Ingram Ltd, Bryanston, Nam Phi) [10, 89]nhm mc

ch phng vim da sau khi cy kim thm d.

3.2.3. Thit k nghin cu

Chn m hnh nghin cu ph hp: dng n liu (single dose) [28, 78], ngu

nhin (randomized) [13, 28, 32, 43, 46, 78, 89], cho i (crossover) [28, 43, 90],

vi 2 giai on nghin cu: giai on 1 dng thuc generic, giai on 2 dng thuc

i chng l bit dc gc, gia 2 giai on l thi gian ngh khng dng thuc gi

38

l thi gian lm sch thuc (washout) thng l 7 ngy [43, 78] hoc 14 ngy [21,

28, 78].

3.2.4. Chun b kim thm d

Kim thm d c vai tr chnh l ly mu thuc, cht chuyn ha, cht ni

sinh ti m cy da trn s khuch tn theo gradient nng , c thit k thnh

nhiu loi khc nhau v kch thc, hnh dng v vt liu s dng ty thuc v tr

cy.

Trong k thut thm tch micro da (Dermal microdialysis-DMD) kim thm

d loi ng thng c s dng ph bin nht [39, 60]. Kim thm d ny c th

c lm t vt liu si rng, v d nh ly t mt xylanh chy thn nhn to, trong

c hng ngn si. a s cc kim thm d c a vo da thng qua mt kim

dn nh v cc kim thm d thng c lm bng vt liu mnh v mm.

Hnh 3.1. M t kim thm

d dng ng thng

Vi kim thm d dng ng thng, dch truyn ch chy theo mt dng c

hng v v th kim thm d dng ng thng c kch thc rt nh, ng knh

ch khong 200 m. Trong khi cc loi kim thm d in hnh c s dng trong

cc nghin cu lm sng c lin quan n thuc c tc dng ton thn, nh kim

thm d dng sng i v dng ng tm, c phn u vo v u ra c t song

song vi nhau nn cc loi kim thm d ny yu cu c kch thc ln hn (khong

500m) so vi kim thm d dng tuyn tnh dng cho cc nghin cu lin quan n

39

thuc dng ti ch. Tuy nhin, kim thm d dng ng thng, cn phi thm nhp

vo da gp i so vi cc loi kim thm d khc, tnh t im u vo ti im u

ra.

3.2.5. Cy kim thm d trn da

V tr cy kim thm d trong k thut thm tch micro da (Dermal

microdialysis-DMD) kh a dng, c th cnh tay, bp i, bng, lng Cc v

tr ny u cn c lm v sinh, o v, nh du cn thn trc khi cy kim thm

d [10]. V c mt im u vo v mt im u ra nn cc v tr cy u c

nh du di dng nhng cp im, s lng cc cp im ty thuc tng nghin

cu (2, 3, 4 cp im).

Hnh 3.2. Cy kim thm d vo da [89]

Bng 3.3. Vng cy v su kim thm d

Vng cy S cp im su (mm) TLTK

Cnh tay

2 1,5 0,3 [91]

4 ~ 0,7 [89]

9 0,96 0,10 [32]

Lng 6 ~ 1,86 [99]

40

Hnh 3.3. Cy kim thm d vi 2, 3, 4 cp im [89, 108]

3.2.6. nh lng bng phng php thch hp

Phng php phn tch hay c s dng nht trong thm tch micro da l

sc k lng hiu nng cao (HPLC), sc k lng siu hiu nng (UPLC) [89, 91,

102], sc k lng kt hp khi ph (LC-MS) Mu thm tch micro c th c

bo qun lnh trc khi em phn tch (phn tch offline [75, 105]) hoc phn tch

ngay sau khi ly mu nhng thi im cch nhau 30 pht, 1 gi thy c

s bin thin nng mt cch r nt.

41

Hnh 3.4. H thng sc k HPLC (tri) v LC-MS (phi)

Sc k lng hiu nng cao c th thc hin theo nhiu k thut khc nhau ty

thuc vo c im cu to ca cht phn tch nh:

- Sc k phn b (bao gm c sc k cp ion)

- Sc k hp ph

- Sc k trao i ion

- Sc k loi c

- Sc k i lc

- Sc k ng phn quang hc

3.2.7. Thng k, tnh ton v bo co kt qu thu c

S bin i nng thuc th v thuc chng trong dch thm tch thu c

c phn tch thng k theo phng php ANOVA 1 bin (one-way) hoc 2 bin

(two-way) [8, 10, 28]; hoc cc phn mm 3P97 (Trung Quc), phn mm SPSS

12.0 cho h iu hnh Windows [99]; phn mm WinNonlin Professional V.5.2.1

(Pharsight, M) [91]

Cc thng s dc ng hc (PK parameter) quan trng cn bo co trong

nh gi sinh kh dng v tng ng sinh hc l din tch di ng cong theo

thi gian (AUC), nng nh (Cmax) ti m da, thi gian t nng nh (Tmax),

khong tin cy (CI) 90% i vi AUC, Cmax v Tmax

42

3.3. MT S NGHIN CU NH GI SINH KH DNG V TNG

NG SINH HC CA THUC NGOI DA DA VO K THUT THM

TCH MICRO

Cc nghin cu thm tch micro v nh gi sinh kh dng v tng ng

sinh hc ca thuc ngoi da c thc hin ti nhiu quc gia trn th gii, tuy

nhin mi nghin cu ph thuc vo nhiu yu t, iu kin th nghim cc nhau.

Sau y l tng hp mt s nghin cu c cng b c th nhn mt cch

tng th v ng dng ny ca k thut thm tch micro v nh hng cho vic p

dng ti Vit Nam.

3.3.1. Xc nh sinh kh dng ca clobetasol propionat

Thm tch micro da c s dng nh gi sinh kh dng ca mt s

corticosteroid, nh clobetasol propionat (CP). Nghin cu c thc hin trn 10

tnh nguyn vin khe mnh (4 nam. 6 n). S thm nhp ca clobetasol propionat

t mt dung dch ethanol 4% (kl/tt) p dng 4 cp im trn mt cnh tay ca

nhng ngi tnh nguyn khe mnh c nghin cu [10].

Tng cng c 4 v tr c s dng trn mt tay ca mi i tng v mt kim

thm d tuyn tnh c a vo mi im di da bng cch s dng kim dn c

23 (Crown, Pretoria, Nam Phi) to iu kin cho vic a kim thm d vo d

dng. V tr vo v ra c nh du m bo rng chiu di 30 mm ca mng

thm tch ca kim thm d s c t trong da. Mt ti nc (Medac (Pty) Ltd,

Cape Town, Nam Phi) c t trc tip trn mi v tr trong vi pht gy t cc

b v sau b ra trc khi chn kim dn. Cc kim thm d trc c tit

trng trong ethanol 70% (tt/tt) c a vo thng qua kim dn v kim dn sau

s c g b khi kim thm d c t ng v tr.

43

Hnh 3.5. Kim thm d tuyn tnh c s dng nghin cu MD in vivo vi chiu

di c nh 3 cm ca mng thm tch c t trong da [10]

IL (Intralipid, mt nh tng lipid v trng), c s dng lm dch truyn

vo vi tc 1,0 l/pht trong khong 1 gi cho php cn bng v cho chn

thng chn du bt. Trong thi gian cn bng, cc u d c m bo ti ch

vi mt gel keo epoxy (Keo, Genkem, Johannesburg, Nam Phi) v khoang cha

PVC (3,52,32,5cm) s dng nh thng cha cho dung dch CP, c gn vo cc

v tr cy trn da bng cch s dng keo dnh tt (Bostik, Cape Town, Nam Phi) nh

trong hnh 3.6.

Hnh 3.6. Khoang cha PVC gn vo cc v tr pha mt trong cng tay ca

cc i tng to thnh thng cha cho cng thc CP 4% (kl/tt) [10]

Ngay sau giai on cn bng, nghin cu ny c bt u bng cch thm

vo khoang PVC 4 ml dung dch CP ethanol 4% (kl/tt) v truyn vi IL bt u vi

44

tc 1,0 l/pht. Mu thm tch c thu thp 30 pht/ln trong 4 gi v cc mu

lp tc c phn tch ngay bng phng php HPLC.

Vo cui thi gian ly mu, thit b thm d c tch ra t cc my bm

(nhng khng loi b khi da) v cc dung dch CP ethanol cn li cng vi cc

ng cha PVC c g b mt cch cn thn. Cc v tr da sau nh nhng lau

bng gc cn v trc khi loi b cc kim thm d khi da, su ca kim thm d

c nh gi ba ln ( gia, gn v tr vo v ra ca kim) cho mi kim thm d

bng cch siu m 20 MHz bng mt my qut siu m Amode Dermascan C

(cng ngh Cortex, Hadsund, an Mch). Cc kim thm d sau c cn thn

loi b khi cc v tr cy da v c lm sch bng lau nh nhng vi gc cn v

ming bng. Sau , cc v tr cy trn cnh tay ca tng i tng c x l bng

kem Biocort (hydrocortison acetat 1% kl/kl; Adcock Ingram Ltd, Bryanston, Nam

Phi) phng vim da v c kim tra y t 2 ln/tun trong mt thng.

Kt qu ca nghin cu cho thy gi tr AUC0-4h gia cc i tng dao ng

trong khong t 0,74 0,90 g/ml.h n 3,30 1,24 g/ml.h. Khng c s khc

bit gia cc i tng, khng c s khc bit gia 2 gii v khng c s khc bit

gia cc v tr cy kim thm d.

Nhng pht hin trong nghin cu ny cho thy mt cch r rng rng c

th nh sinh kh dng ca cc cng thc clobetasol propionat (CP) dng ngoi da

da trn k thut thm tch micro v mt phng php phn tch nhy pht

hin v o c CP nng rt thp ngm vo da.

3.3.2. nh gi tng ng sinh hc

3.3.2.1. nh gi tng ng sinh hc ca cc ch phm lidocain ti ch

Nhiu nghin cu nh gi tng ng sinh hc ca cc ch phm ti ch

cho thy tnh kh thi ca vic s dng phng php thm tch micro cho mc ch

ny. Cc nh gi tng ng sinh hc ca hai dng bo ch lidocain dng kem

v thuc m c xc nh bng cch s dng k thut thm tch micro v so

snh kt qu vi k thut bng tc [13]. Trong khi k thut thm tch micro xc

45

nh c AUC ca lidocain dng kem cao hn 4,8 ln so vi dng thuc m th k

thut bng tc li ch cho thy mc khc bit khng ng k, cho thy k thut

thm tch micro cho kt qu c chnh xc cao hn.

Cc nghin cu so snh 2 cng thc lidocain trn c thc hin trn 18 i

tng nu cy ba kim thm d vo mt cnh tay (Hnh 3.7) hoc 27 i tng nu

cy hai kim thm d. S khc bit v tnh cht ro cn ca da gia cc i tng l

nguyn nhn ch yu gy ra s khc bit c th cao.

Kreilgaard v cng s [46] nh gi tng ng sinh hc gia lidocain

dng vi nh tng v mt dng bo ch dng ngoi khc. Tham s dc lc hc

y chnh l tc dng gim au. Kt qu, vi nh tng c AUC cao gp 4,3 ln so

vi thuc m v thi gian thi gian t nng nh (Tmax) ngn hn 20 pht. c

tnh h s hp thu ca vi nh tng cao hn 2,9 ln so vi thuc m. Nhng ngay

c khi vi nh tng c AUC cao hn (tc l nhiu thuc t do c mt ti v tr tc

dng hn), khng c s khc bit gia iu tr hai thuc v hiu qu ti a u

t c ti cc si thn kinh ch. Mt nhn t ng gp ch yu n s bin thin

c th trong cc php o l do nhng khc bit trong tnh cht da. Khi nh gi liu

su cy thm d c l mt yu t nh hng n s thay i, cc tc gi nhn

thy khng c s khc bit ng k. Kt qu trong da ngi sau c so snh vi

th nghim thc hin trong da chut. , ngi ta thy rng da chut c thi

gian t tc dng ngn hn, t l hp thu cng c s khc bit.

Hnh 3.7. Cc th tc cy kim thm d MD trn da trong mt nghin cu

lm sng c i chng [102]

46

3.3.2.2. nh gi tng ng sinh hc ti ch ca cc loi kem acyclovir

Huilin Wei v cc cng s tin hnh nghin cu nh gi tng ng

sinh hc ca cc loi kem Acyclovir (ACV) dng ti ch bng vic s dng thm

tch micro da trn da ln [99]. Ba kem ACV (3%), ACV1, ACV2 v ACV3 (ang

trong qu trnh nghin cu thuc, cha c sn ngoi th trng) c dng ti ch

trn lng ln v cc k thut ly mu thm tch micro da (DMD) c s dng

thu thp lin tc mu thm tch.

Bng 3.4. Thnh phn cc cng thc ACV v su cy kim thm d tng ng

Cng thc Thnh phn su cy kim thm d (mm)

ACV1 Glycerol monostearat, glycerol,

albolen, natri dodecyl sulfat, parafin

lng, triethanolamin v ethylparaben

1,85 0,37

ACV2 Glycerol monostearat, glycerol,

albolen, sodium dodecyl sulfat,

octadecanol v benzencarbinol

1,88 0,32

ACV3 Glycerol monostearat, glycerol,

albolen, natri dodecyl sulfat, paraffin

lng, simethicone v ethylparaben

1,84 0,33

Ln c gy t bng cch tim mng bng urethan 30% vi liu 3 ml/kg

vo thi im 30 pht trc mi th nghim. Cc v tr s dng ACV1, ACV2 v

ACV3 c ch nh. Ba kim thm d c cy vo da trn cng mt bn cho mi

th nghim.

47

Hnh 3.8. Cc v tr s dng ca ba cng thc ACV trn lng ln

Ba loi kem ACV (3%) c dng trn lng ln vi liu 0,2 g/cm2. Trong

DMD, tc dng chy 1 l/ pht c s dng thu thp lin tc cht thm tch.

Mu thm tch lin tc c thu thp trong vng 30 pht mt ln trong thi gian 4

gi. Nng ACV mi ln c o bng HPLC. S liu c trnh by di dng

nng trung bnh SD (n = 12) (Hnh 3.9).

Hnh 3.9. Biu gii phng ACV (3%) in vitro t 3 cng thc dng ngoi da

48

Bng 3.5. Cc thng s dc ng hc ca cc loi kem Acyclovir dng ti ch

Thng s ACV1 ACV2 ACV3

Cmax (g/mL) 1,21 0,53 0,89 0,48 0,82 0,27

Tmax (h) 0,33 0,19 0,42 0,25 0,38 0,23

AUC0- (gh/mL) 1,74 0,65 1,35 0,58 1,37 0,35

AUC0-4h (gh/mL) 1,73 0,66 1,33 0,58 1,35 0,52

Cmax v AUC thng c s dng nh gi tng ng sinh hc cho cc

cng thc ti ch theo quy nh trong cc hng dn ca FDA. Trong nghin cu

ny, c Cmax v AUC0-4h ca ACV1 trong lp di da cao hn ng k so vi cho

ACV2 v ACV3 (Bng 3.5, Hnh 3.9), cho thy ACV1 l khng tng ng sinh

hc vi ACV2 hoc ACV3. Do , ACV2 v ACV3 tip tc c nh gi tng

ng sinh hc. Kt qu cho thy khong tin cy 90% ca t l AUC0-4h ACV2 v

ACV3 l 88,2 - 105,7%, nm trong phm vi chp nhn (80-125%). Khong tin cy

90% ca t l Cmax gia ACV2 v ACV3 l 87,4 - 124,4%, cng nm trong phm vi

chp nhn (80-125%). Nhng s liu ny cho php kt lun rng ACV2 v ACV3

c s dng trong nghin cu ny l tng ng sinh hc trn ln, t pht

trin hng nghin cu trn ngi.

Nghin cu ny cho thy rng cc mu ln kt hp vi ly mu thm tch

micro da c th cung cp mt bin php hiu qu nh gi phn b thuc ngoi

da v nghin cu cc thng s dc dc ng hc, t nh gi sinh kh dng

v tng ng sinh hc ca cc thuc ngoi da.

49

Bng 3.6. Tng hp mt s nghin cu nh gi tng ng sinh hc ca thuc

ngoi da da trn k thut thm tch micro

Thuc i

tng CI90%(AUC)

CI90%(Cmax)

v/hoc

CI90% (Tmax)

Kt lun TLTK

M Oxytetracyclin

HCl 3% (kl/kl)

(Thuc th: Polimisin,

Koak Farma

Thuc chng:Turkey and

Terramycin, Pfizer)

Ngi 89-135 % 82-161 %

Ngoi khong

80-125%, khng

c cho l

tng ng

sinh hc

[91]

Kem Metronidazol

(Metronidazol 1%, Alpharma

ApS, an Mch v Flagyl

1%, Aventis, an Mch)

Ngi 69,0172,3%

Tng ng

sinh hc khng

c chng

minh

[32]

Kem Metronidazol

(Rozex 0,75%, Galderma,

Thy in v Flagyl 1%,

Aventis, an Mch)

Ngi 49,5135,1%

Tng ng

sinh hc khng

c chng

minh

[32]

Kem Metronidazol

(Rozex 0,75%, Galderma,

Thy in v Metronidazol

1%, Alpharma, an Mch)

Ngi 46,8107,1%

Tng ng

sinh hc khng

c chng

minh

[32]

3 loi kem

Acyclovir 3%

(cng thc ACV1, ACV2,

ACV3 ang trong qu trnh

nghin cu, cha c bn

trn th trng)

Ln 88,2-105,7%

(AUC0-4hACV2-3)

87,4124,4%

(Cmax ACV2-3)

ACV2 v ACV3

l tng ng

sinh hc trn

ln, t pht

trin hng

nghin cu trn

ngi

[32]

50

Thuc i

tng CI90%(AUC)

CI90%(Cmax)

v/hoc

CI90% (Tmax)

Kt lun TLTK

Gel Ketoprofen

2.5% (kl/kl)

(Thuc th: Gel Fastum,

Adcock Ingram, Bryanston,

Nam Phi

Thuc chng:

Gel Ketoprofen,

SigmaAldrich,Atlasville,

Nam Phi)

Ngi,

(c

chia lm

3 nhm

A, B, C

theo v

tr cy)

97,4 15,7%

(AUC0-5h nhm A)

89,9109,1%

(AUC0-5h nhm B)

80,4 93,5%

(AUC0-5h nhm C)

C 3 nhm u

cho kt qu

tng ng

sinh hc, nhng

c s khc nhau

v hp thu gia

cc v tr s

dng

[89]

3.3.2.3. nh gi tng ng sinh hc cc ch phm oxytetracyclin HCl v so

snh vi phng php bng tc

Mt nghin cu khc ca Tuba Incecayir v cng s [91] so snh k thut

thm tch micro v k thut bng tc (Hnh 3.10) trong nh gi tng ng sinh

hc hai loi bit dc ca oxytetracyclin HCl (OTC).

Hnh 3.10. M t k thut bng tc [101]

Nghin cu vi k thut bng tc c tin hnh trn 12 ngi tnh nguyn

khe mnh. Sau khi s dng cc ch phm 30 pht, bng tc c s dng ly

mu tng lp sng cc thi im 0,15; 0,5; 1; 1,5; 2; 3; 4 gi.

Nghin cu thm tch micro c thc hin trn 10 tnh nguyn vin khe

51

mnh vi thi gian 4 tun gia cc th nghim. Kim thm d thm tch micro c

a vo lp di da ca cnh tay. Cht thm tch c thu thp trong 4 gi, 30 pht

mt ln.

Tin hnh:

Vi cc th nghim MD, cc i tng c thc hin trong t th nm nga

nhit phng vi cnh tay tri t trn tay vn. Hai khu vc trn (ng knh 4,3

cm), khong cch gia cc hnh trn l 2,7 cm c v trn mt trong cng tay. Cc

khu vc c kh trng bng cn. Mt kim thm d MD CMA 66 vi chiu di

mng 30 mm (CMA, Thy in) c a vo b mt lp di da ca mi khu vc

vng trn vi s tr gip ca ngi cy kim, sau c rt ra (Hnh 3.11). ng

knh ca mng lc l 0,5 mm vi cho php phn t nh hn 20 kD i qua. Cc l

vo v ra c chn vi mt git keo cyanoacrylat (Pattex , Henkel) phng

nhim trng c th xy ra t cc ch phm ti ch. Vic cy ny c thc hin m

khng cn gy m trong iu kin v trng.

Cc kim thm d c truyn vo vi nc mui sinh l (dung dch NaCl

0,9% v trng v ng trng) vi tc dng chy ca 2l/pht nh bm MD

(CMA 107, CMA, Thy in). Sau khi cy cc kim thm d, n nh 30 pht

lm gim chn thng do cy kim c c gi l giai on cn bng. Sau , 4ml

thuc th v thuc chng c dng ng thi cc khu vc th nghim l

chnh l cc vng trn ng knh 4,3cm. Cht thm tch sau c thu thp

trong nhng ng nh 200l c 30 pht mt ln, ly mi mu 60 l phn tch, cho

n 4 gi sau ln ly mu u tin. Ton b thi gian ca vic nghin cu p dng

phng php MD l 4,5 gi c vi thuc th v thuc chng. Cc mu cht thm

tch thu c c bo qun lnh cho n thi im phn tch trong thi gian

khng qu 1 tun k t sau khi ly mu.

52

Hnh 3.11. Cy kim thm d thm tch micro vo lp h b mt trong

cnh tay ca tnh nguyn vin khe mnh

Nng OTC trong cht thm tch c xc nh bng UPLC-MS/MS (Ultra

Performance Liquid Chromatography Tandem Mass Spectroscopy- Sc k lng

siu hiu nng khi ph) v hiu chnh cho phc hi in vivo.

Kt qu: Trong th nghim ny, c mt s tng quan tuyn tnh gia nng

thuc trong cht thm tch v v nng thuc trong mi trng dng trn mt

khong rng (R2=1,00). Phn tch hi quy tuyn tnh trong cc th nghim phc hi

in vitro c hin th trong hnh 3.12.

Hnh 3.12. Hiu chun in vitro kim thm d thm tch micro cho

oxytetracyclin HCl

53

nh gi dc ng hc:

Hnh 3.13. Nng oxytetracyclin HCl trong lp h b cho thuc th v thuc

chng 10 tnh nguyn vin khe mnh (kt qu MD)

Hnh 3.14. Lng oxytetracyclin HCl trung bnh trong lp sng ca 12 tnh nguyn

vin khe mnh (kt qu bng tc)

Cc th nghim nh gi dc ng hc p dng k thut thm tch micro

cho thy thuc nghin cu Oxytetracyclin HCl khng tng ng sinh hc vi

54

thuc chng vi khong tin cy CI 90%. Tuy nhin th nghim dc ng hc vi

k thut bng tc li cho thy thuc th v thuc chng l tng ng sinh hc.

Mt nghin cu khc ca Garca Ortiz P v cc cng s [32] nh gi tng ng

sinh hc gia hai ch phm kem metronidazol u ca an Mch l Metronidazol

1% (Alpharma ApS) v Flagyl 1% (Aventis) cho thy trong khi k thut thm tch

micro cho kt qu khng tng ng sinh hc th k thut bng tc li khng

pht hin c cc khc bit v nng thuc m da gia cc ch phm, dn n

cho kt qu l tng ng sinh hc, iu ny c th dn n nhng sai lm trong

quyt nh cho php ng k thuc sau .

Nh vy, k thut thm tch cho thy s vt tri hn so vi k thut bng

tc trong vic pht hin nhng khc bit v nng thuc thm nhp vo m da

gia cc ch phm, gip nh gi dc ng hc v tng ng sinh hc chnh

xc hn, l mt k thut c s dng thng xuyn trong cc nghin cu tng

ng sinh hc ti ch.

55

KT LUN V KIN NGH

1. KT LUN

Vic nh gi sinh kh dng v tng ng sinh hc gip cho vic s dng

thuc theo cch tt nht va an ton - hp l - hiu qu - kinh t, va c ngha

khoa hc, va l hnh lang php l cho nh chuyn mn v qun l thc hin chm

sc sc khe con ngi. Vic nh gi sinh kh dng v tng ng sinh hc ca

cc thuc ngoi da khi m cc thuc ny ngy cng c s dng ph bin vi tc

dng nhanh ti ch m t tc dng ph ton thn l ht sc cn thit. K thut thm

tch micro s l mt cng c c lc gip gii quyt vn ny.

K thut thm tch micro m ra mt hng i mi trong nghin cu nh gi

tng ng sinh hc v sinh kh dng ca cc thuc ngoi da. So vi cc phng

php ly mu m nguyn vn hoc dch c th khc, k thut ny cho thy mt s

li th cho ngi th nghim. K thut thm tch micro cho php o c nng

thuc t do trong dch k, l phn c hot tnh sinh hc, do , mi quan h PK-

PD chnh xc hn c th c xy dng gip pht trin thuc v cc phc

iu tr lm sng. Ngoi ra, vic ly mu lin tc ca k thut thm tch micro cho

php thy c s bin thin ca cc thng s dc ng hc gia cc khong thi

gian rt ngn. Hn na, vic o ti ch v cc c im chun b mu ca k thut

thm tch micro cho php thu c cht thm tch tng i sch sn sng phn

tch trnh c vic nhim bn v lm long mu.

K thut thm tch micro ngy cng pht trin v c ng dng su rng

trong nghin cu thuc ngoi da, gip cho php a ra mt quy trnh nh gi

c sinh kh dng v tng ng sinh hc ca thuc ngoi da. Nhng thng tin

c gi tr v dc ng hc - dc lc hc ca thuc ti m ch to iu kin cho

vic la chn thuc, gip ti u ha liu dng v phc iu tr. Tm li, thm

tch micro no l mt k thut ly mu c lc cho vic nh gi dc ng hc v

sinh kh dng ca thuc trn da v m mm.

56

2. KIN NGH

Kha lun tin hnh tng quan phng php nh gi sinh kh dng v

tng ng sinh hc ca cc thuc ngoi da. Sau y, chng ti xin c mt s

xut pht trin ti ny:

Chun b c s vt cht, trang thit b v ngun nhn lc c chuyn mn bc

u trin khai k thut thm tch micro trong nghin cu, pht trin v qun l

dc phm ti Vit Nam.

ng dng k thut ny trn thc t nh gi cc thuc mi xin cp php trn

c s cc thuc i chng trong danh mc quy nh, to c s php l cc

c quan chc nng cho php lu hnh thuc trn th trng.

Tip tc nghin cu cc ng dng ca k thut thm tch micro vi cc dng

thuc khc.

TI LIU THAM KHO

Ti liu bng ting Vit:

1. B mn Dc lm sng (2011), Dc lm sng, Nh xut bn Y hc, H

Ni.

2. B mn Ha phn tch v c cht (2007), Ha phn tch 2, Nh xut bn Y

hc, H Ni.

3. B Y T (2010), Thng t 08/2010/TT-BYT - Hng dn bo co s liu

nghin cu sinh kh dng/ tng ng sinh hc trong ng k thuc.

4. Vn B, Vn Cng Tr, Nguyn Hu Duy, Nguyn Th Kim Hng, Trn

Th Qunh Chi (2012), Nghin cu nh gi tng ng sinh hc vin nn bao

phim Cefuroxime 500 mg do X nghip Dc phm 150 sn xut, Tp ch Y hc

qun s - Bo qun i nhn dn online.

5. Nguyn Thnh Hi, Alain Gardier, Thi Nguyn Hng Thu (2011), Nghin

cu ch tc dng ca esciloparam trong iu tr trm cm da vo k thut thm

tch micro v m hnh t bin gen", Tp ch Dc hc, 8(436), 2-5.

6. Nguyn Thnh Hi, Bruno Guiard, Alain Gardier, Thi Nguyn Hng Thu

(2011), "Nghin cu c ch tc dng mi ca escitalopram trong iu tr trm cm

da vo k thut thm tch micro v m hnh t bin gen", Tp ch Dc hc,

3(431), 37-41.

7. Nguyn Thnh Hi, Bruno Guiard, Alain Gardier, Thi Nguyn Hng Thu

(2012), "Thm tch micro, k thut nh lng thuc dng t do ti m ch tc

dng: Nguyn tc v ng dng trong nghin cu thuc", Tp ch Dc hc,

52(429), 6-8.

8. Nguyn Thnh Hi, Bruno Guiard, Phm Thu H, Thi Nguyn Hng Thu

(2012), Nghin cu tc dng ca morphin trn nng dopamine v serotonin da

vo k thut thm tch micro, Tp ch Dc hc, 11(439), 9-12.

9. Nguyn Sinh Thi, Nguyn Thnh Hi, Thi Nguyn Hng Thu (2012),

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