Y Hoc TP. Ho Chi Minh * Tap 21 * So 5 * 2017 Nghien cuii Y hoc
NGHIEN CUtJ THANH PHAN CONG THU'C LIPOSOME PIROXICAM DU'OC BAO CHE BANG P H U O N G PHAP HYDRAT HOA
MANG PHIM LIPID
Le Thaiih Diem*, Trinh Thi Thu Loan**, Trdn Vdn Thdnh**
TOMTAT
Dat van (&: Dang bdo cheliposome diing ngodi da co nhitng uu diem so v&i dqng bdo che truyen thdng nhu khdng gay kich ieng, gia tdng hiiu qua hdp thu qua da. Piroxicam la mot khdng viim duac chi dinh trong dieu tri binh viim da kh&p... Tuy nhiin tdc dung phu cua piroxicam trin hi tiiu hoa la rat l&n. Vi vay muc tiiu cua di tdi Id bdo che dugc liposome piroxicam bang phucmg phdp hydrat hoa mdng phim lipid nham sie dung tiong che pham diing ngodi.
Doi tuang vd phuang phdp nghien edu: Cdng thiec bdo cheliposome piroxicam dugc khdo sdt dua tien su thay ddi cua labrasol, transcutol, tween 80 vd cholesterol. Cdc cdng thiec tqo thdnh dugc ddnh gid dua tiin hieu suat bat give, kich thu&c tieu phdn vd khd ndng gidi phong hoqt chai.
Kei qud vd bdn ludn: Su hiin dien ciia Cholesterol vd cdc chat dien hoqt trong cdng thiec (Labrasol, Trancustol, Tween 80) deu ldm tdng hieu sudi bat giii piroxicam vd ldm gidm kich thu&c-phdn bo Mch thu&c tieu phdn liposome. Trong do, Tween 80 ti le 0,2% vd CholesterolPhospholipon ti li82 (mokmol) cho kei qud tot nhSi.
Ket luqn: Cdng thiec bdo che bao gom piroxicam, phospholipon BOG, cholesterol, tween 80 Tit khoa: Liposome, piroxicam, Hydrat hoa mdng phim lipid.
ABSTRACT
RESEARCH OF PIROXICAM LIPOSOME COMPOSTTION FORMULATED VmH THIN LIPID FILM HYDRATATION METHOD
Le Thanh Diem, Trinh Thi Thu Loan, Tran Van Thanh
* Y Hoe TP. Ho Chi Minh * VoL 21 - No 5 - 2017:219 - 226
Introduction: Topical applied liposomal formulations have advantages over conventional dosage forms such as non-irritating, increased absorption efficiency, prolonged duration of action and reduced toxicity Piroxicam is an analgesic that is indicated for the treatment of multiple arthritis... However, the side effects of piroxicam on the digestive tract are huge. Thus, the aim of the study was to prepare the liposome of piroxicam by lipid film hydration method which can be in topical formulation.
Materials and methods: The piroxicam liposome formulation was investigated based on changes in labrasol, transcutol, tween 80 and cholesterol Formulations formulated are evaluated based on encapsulation efficiency, particle size, and the drug release.
Results and discussion: The presence of Cholesterol and other excipients (Labrasol, Trancustol, Tween 80) both increased the capture efficacy of piroxicam and reduced the size-size distribution of liposomes where as Tween 80 at 0.2% and Cholesterol: Phospholipon 8:2 (mol.mol) gives the best result.
Conclusion: The final formulation was determined as piroxicam, phospholipon 90G, cholesterol, tween
* Cong ty Dupe pham Imexpharm ** Khoa Dupe, Dai hpc Y Dupe TP. Ho Chi Minh Tdc gid lien lac: TS. Tran Van Thanh DT: 0919.000.008 Email: H-anvanthanhffMiphrm pdu.vn.
Nghiin ciru Y h9c Y Hoc TP. nh Chi Minh * T^p 21 * So 5 " i"*'
Keywords: piroxicam, liposome, thin-film hydratation.
DATVAN€)E
Dang bao ehe hposome dimg ngoai da eo nhiing uu diem so voi detng bao che tmyen tiiong nhu khong gay kieh ling, gia tang hieu qua hap thu qua da..."'*^'. Piroxicam la mpt khang viem thupc dan chat oxieam dupe ehi dinh trong dieu tri benh viem da khop, cimg khop... Tuy nhien tac dyng phu cua piroxicam tren he tieu hoa la rat lon. Vi vay mue tieu cua de tai la bao che dupe hposome piroxicam bang phuong phap hydrat hoa mang phim lipid CO the dupe dimg nhu mpt thanh phan hoat chat trong che pham diing ngoai.
Labrasol, Transcutol, Tween 80, Cholesterol la nhiing ta dupe thuong dupe sii dung trong cong thuc bao ehe Uposome'^-^' va dupe khao sat trong cong thiic bao che Uposome piroxicam.
NGUYfeN UEU, THIEr BI VA IHl/ONG PHAP NGMENCUtr
Nguyen lieu va thiet hi
Piroxicam (Tnmg Quoc USP 31), Piroxicam chuan (Vien kiem nghiem thuoc TP.HCM), PhosphoUpon 90G (Diic-USP 32), Cholesterol (Diie-USP 32), Labrasol (Phap), Transcutol (Phap) tieu ehuah nha san xuat, Tween 80 va eae ta dupe khae dat ehuan nha san xuat.
Kinh hien vi quang hpc Olympus CX21- FSl (Nhat), Kinh hi&i vi dien tir truyen qua Jem - 1400 (Nhat), May do quang pho UV - Vis UV 1800 (Nhat), May co quay chan khong 1 ht Buchi R210S (Thyy Sy), May do phan bo kich tiiuoc hat Horiba LA - 920 (Nhgt), May dong nhat hoa Ultra Turrax IKA-T25 (Diic), May sieu am dau do Sonic VCX - 130PB (My), May phan tich nhiet vi sai Mettler Toledo DSC (Thuy Sy), Tiii tham tach Spectra/Por 4, I Molecular weight cut off (MWCO): 12 -14 Kd I (Spectrum Labs - My).
Phirong phap nghien cmi
Trong de tai nay, su anh huong cua chat dien boat va cholesterol den tinh chat ciia Uposome piroxicam nhu kich thuoc-phan bo kieh thuoc tieu phan Uposome, hieu suat bat giii hoat chat, dp phong thieh hoat chat qua mang tiiain tach, tinh on dinh ciia Uposome se dupe khao sat.
Ket qua nghien ciiu tir eong trinh bruoc da xae dirth thanh phan eong thiic co b ^ va thong so ky thuat bao ehe nhu sau:
Thanh phian cong ihufc
Piroxicam 0,25 g Phospholipon 90 G 1 g Cloroform 50 ml
Dung dich dem p H 7,4 50 ml
Thong so 1 ^ ihuat
Nhiet dp CO quay 40 °C Thoi gian c6 quay 2 gio Toe dp CO quay miie 5 Nhiet dp hydrat hoa 45 "C Thoi gian hydrat hoa 1 gio Toe dp hydrat hoa miie 8 Toe dp khuay tren thiet hi Ultra Turrax:
19000 vong/phut
Thoi gian khuay tren thiet bi Ultra Turrax:
15 phiit
Thoi gian sieu am: 6 phiit Tir thanh phan cong tinie ca ban nhu tren, cholesterol va nhiing chat dien hoeit nhu Labrasol, Transcutol, Tween 80 dupe them vao cong tiiiie voi nhiing ti \% khae nhau nham tao dupe Uposome piroxicam eo tinh chat tot nhat.
Cac bu6c tien hanh bao che liposome a/ Hoa tan PhosphoUpon 90G, cholesterol (neu CO), chat dien hoat (neu eo), piroxicam trong eloroform trong binh qua le.
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Y Hoc TP. Hh Chi Minh * Tap 21 * So 5 * 2017 Nghiin cihi Y hoc
b/ C6 quay 6 nhiet dp 40 °C, toe d6 quay 6 miic 5 trong thoi gian 2 gia de tao mang Upid m6ng va trong suot.
c/ Them 50 ml dung dich dem p H 7,4 vao binh qua le, eo quay, giii nhiet dp 45 °C, toe dp quay 6 miic 8 trong 1 gio.
d/ Cho hon hpp vao ly, khuay tS'c d6 eao bang may Ultra Turrax vai tSe d0 19.000 vong/phiit trong 15 phiit.
e/ Sieu am bang may sieu am dau do trong 6 phiit.
f/ Mau liposome dupe de 6 nhiet dp phong trong 2 gio.
g/ Bao quan Uposome 6 nhiet dp 2-8 °C trong Ip thliy tinh kin, tranh anh sang.
Fhuong phap danh gia tinh chat liposome Cdmquan
Quan sat bang mat duoi anh sang tu nhien hon dich liposome phai dong nhat eo mau vang, khong eo hien tupng ket bong, noi kem, Iang can.
Kich thu6c, phdn ho kich thuac tieu phan Kich thuoe va phan bo kieh thuac tieu phan Uposome dupe xac dinh bang phuorng phap tan xa laser trong moi trucmg phan tan:
nuoc eat.
Hmh thehcfc
Cau tnic ciia Uposome dupe xac djnh sa bp bang kinh hien vi quang hpe va xac dinh chi tiet cau tnic bang kinh hien vi di&n tii truyen qua (TEM).
Hieu suat hat giii/piroxicam
Piroxicam tu do se dupe tach ra khoi he liposome bang tui fham tach, piroxicam dupe bat giu trong tieu phan liposome dupe dinh lupng bang phucmg phap quang pho UV - Vis.
Xae djnh thoi gian piroxicam tiiain tach qua mang: hiit chinh xac 1 ml dung dieh piroxicam 0,5 % trong dem pH 7,4 cho vao tui tham tach da duac hoat hoa, eho tiii tham tach
%EE =
vao moi trucmg 300 ml dung dich dem p H 7,4.
Dinh lupng nong dp piroxicam 6 moi truong ben ngoai theo thai gian. Ghi nhan Igi thai gian piroxicam phong thich hoan toan ra moi trucmg ben ngoai.
Danh gia hieu suat bat giii piroxicam: hut chinh xae 1 ml Uposome piroxicam cho vao tiii thaim tach, cho tiii tham tach vao moi trucmg 300 ml dung djch dem pH 7,4 trong khoang thai gian phii hpp. Piroxicam toan phan, tu do va bat giu trong liposome dupe dinh lupng bang phuong phap quang pho UV - Vis tai bu6e song hap thu cue dai.
Hieu suat bat giu piroxicam dupe tinh theo eong tiuie:
m ^ - m | d _ jj^QQ mtp Trong d o :
mw: lupng piroxicam tu do (mg) mtp: lupng picroxieam toan phan (mg)
%EE: hieu suat bat giii
Bg phong thich hoqt ch^t cua liposome piroxicam
Hut 1 ml che pham cho vao ttii tham tach, dat tiii vao trong ly eo ehiia 300 ml dung dich dem pH 7,4. Khuay tir miic 1, duy tri nhiet dp eiia mau la 37 »C. Dinh lupng piroxicam dupe phong thieh tai cac fhoi diem sau: 15 phiit, 30 phiit, 1 gio, 2 gio, 3 gio, 4 gia, 5 gio. Moi Ian lay 10 ml dung djch va bo sung bang 10 ml dung dich dem. Do dp hap thu eiia cac mau tai buoc song 353 nm. Ti le % piroxicam phong thieh dupe tinh theo cong tiiiic sau :
Trong do:
%PT: Ti le % piroxicam phong thieh ra moi ben ngoai.
Nghien cuu Y hoc Y Hgc TP. Ho Chi Minh * Tap 21 * Stt a " -^"^
G : Nong dp piroxicam giai phong hieu chinh tai thai diein lan hiit thii i tinh theo cong thiic Nelson (fjg/ml).
CTp: Ham lupng piroxicam toan phan ciia mau (^lg/ml).
Ci, Cj: Nong dp piroxicam 6 dung dieh ben ngoai tiii tham tach tai thoi diem lan hiit thii i, j (^ig/ml).
Ai: Dp hap thu ciia mau thii trong Tan do thiii.
AC: Dp hap thu ciia mau chuan.
CC: Nong dp cua dung dieh piroxicam chuan ben ngoai tiii tham tach.
KETQUA V A B A N L U A N
Ket qua danh g|a sir anh hirdng cua chat dien ho^t
Thanh phan cong fhiic eae mau Uposome voi nhiing chat dien hoat khac nhau (Bang 1) duoc bao che theo cae buoc da dupe xae dinh.
Sau khi bao ehe, cae cong thiic Upsome dupe danh gia ve hieu suat bat giii piroxicam va kieh thuoc-phan bo kich thuoc tieu phan.
Ket qua hieu suat bat giii eua cae mau Uposome dupe trinh bay trong Bang 2 eho tiiay cac chat dien hoat Labrasol, Transcutol va Tween 80 deu lam tang hieu suat bat giu piroxicam so vdi mau khong co (iiat dien hoat (p < 0,05). Hieu suat bat giii piroxicam eiia mau vcri Tween 80 la eao nhat.
Bdnf( 1. Cdc cong thitc hdo cheliposome vdi cdc chat dien hoat khdc nhau Cdng thirc
Phospholipon (g) Labrasol (mg) Transcutol (mg) Tween 80 (mg) Piroxicam (mg) Dung dich d^m (ml)
CTNE 1
250 50
CT1E 1 50
-
250 60
CT2E 1 100
250 50
CT3E 1 50
-
250
-
50 CT4E
1 100 250 50
CT5E 1
50 250 50
CT6E 1
100 250 50 Bang 2. Hiiu suat bdt giie piroxicam cita cdc cong
thiec v&i chai diin hoat khdc nhau Cdng thijc
CTNE (Khdng c6 chit dien hoat) CT1E (Labrasol) CT2E (Labrasol) CT3E CTranscutol) CT4E (Transcutol) CT5E (Tween 80) CT6E (Tween 80)
Hieu suit bit giOCii) (n=3) 16,6 ± 0,4 22,7 ± 0,6 17,2 ±0,2 23,1 ±o,g 19.7 ±0,8 19,1 ±0,2 23,4 ± 0,6
SU anh hucmg ciia ti le Tween 80 den hieu suat bat giir piroxicam.
Bdng 3. Kit qud phdn tich bieu do phan boMch thu&c tieu phdn cita cdc cong thiec v&i chdi diin hoqt khdc nhau
Ket qua kich thuac va phan bo kich tiiuoc tieu phan ciia eae mau dupe trinh bay trong Hinh 1 va Bang 3 eho thay su hien dien ciia Labrasol (CTIE) va Tween 80 (CT6E) giiip lam giam kieh thuoc va day phan bo kich thuoe tieu phan. Transcutol (CT3E) co day phan bo kich thuoe va kieh thuoe tieu phan trung binh lon hon so v6i cae cong cong thiic con lai. Vi vay, Tween 80 se dupe ehpn va dupe khao sat
C&c cdng thtpc Kieu phdn bo Day phdn bo Dm (Median) Dmean (Mean)
Dm Mode CTNE tdinh 38,7- 339,3 104,6 121,5 93,7
CTIE tdinh 38,6- 396,3 106,5 116,2 106,8
CT3E Idinh 38,7- 509,8 120,7 137.2 122,4
CT6E talnh 33,7- 388,6 101.2 117.4 93,6 Thanh phan cong thiic cac m l u liposome diroc bao che voi cac ti le Tween 80 khac nhau duoc trinh bay trong Bang 4. Sau khi bao che, cac mau lipsome dugc danh gia ve hieu suat bat giii piroxicam (Bang 5). Hieu suaft bSt giu piroxicam tang khi ti le Tween 80 tang tif 0,1%
len 0,2% va hieu suat bat giii giam khi ti le Tween 80 tir 0,4% tra len. VI v$y Tween SO voi
Y Hoc TP, nh Chi Minh * Tap 21 * So 5 * 2017 Nghien cuu Y hoc
ti le 0,2% se dupe chpn de them vao cong thiie bao che Uposome piroxicam.
U.(ll]
I
90 IAS
r u
IMQ UNOO
Dmettrlan) 100 V
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—-do tow
I;M - -
I
00 100
4t-
100 a 10000 DnaScr (oa)
- - 100.0
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— ^ . 0 0 iOOO
Hinh 1. Bieu do phdn bo'kich thuac ciia cdc cdng thuc v&i chat dim hoqt khdc nhau: (A) CTNE, (B) CTIE, (C) CT3E, (D) CT6E.
Bdng 4. Cdc cdng thiec bdo cheliposome v&i cdc ti le Tween 80 khdc nhau
knh hudng cua
Cdng thi>c Phospholipon gOG (g)
Tween 80 (mg) Piroxicam (mg) Dung djch dem (ml)
CT1T 1 60 250 50
CT2T 1 100 250 50
CT3T 1 200 250 50
CT4T 1 250 250 60 Bang 5. Hiiu sudi bat gia piroxicam ciia cdc mau v&i ti le Tween 80 khdc nhau
Cdng ttlifC CT1T CT2T CT3T CT4T
%EE 19,7 ±0,8 23,4 ± 0,5 15,4 ±0,4 13,7 ±0,5
Ket q u i khao sat cholesterol
Thanh phan eong thiic voi ti le cholesterol khae nhau dupe trinh bay trong Bang 6. KH qud hiiu sudi bat giie piroxicam dupe trinh bay trong Bang 7 cho thay ti Ie cholesterol co anh hucmg den hieu suat bat giii piroxicam (p
<0,05). Khi ti le cholesterol tang d&i 7:3 (mol:mol) so vol phosphoUpon thi hieu suat hit giii giam, nguyen nhan eo the do ti Ie cholesterol cao lam thay doi tinh on dinh eiia lap mang kep va piroxicam bi giai phong ra ben ngoai.
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Nghien cihi Y hoc Y Hoc TP. H^ Chi Minh * T a p 21 * So 5 * 201?
Bdng 6. Thdnh cdng thiec bdo cheliposome v&i ti li cholesterol khdc nhau
Cdng t h i ^ Phospholipon 90G
(mg) Cholesteidl (mg)
Tween 80 (mg) Piroxicam (mg) Dung djch d§m (ml)
CT1CH (9:1 )•
847 53 100 250 60
CT2CH (8:2)*
887 113 100 250 50
CT3CH (7:3)'
821 179 100 250 50
CT4CH (6:4)-
746 254 100 250 60
*: TI li Phospholipon 90G:Cholesterol (molmol) dugc qui ra mg.
Bdng 7. Hiiu sudi bat giie piroxicam ciia cdc mdu v&i tl li cholesterol khdc nhau
Cdng thijc CT1CH CT2CH CT3CH CT4CH HM
1 - 1
M f a ^ B lOD 100 0
%EE (n = 3) 22,4 ± 0,4 26,06 ± 0.5 19,46 ±0,6 18,86 ±0,4
^ - - -lOOO
B L ti 10009 6000
K^ qud ve kich thu&c vd phdn bo'kich thu&c tieu phan dupe trinh bay trong Bang 8 va Hinh 2 eho thay ti Ie Cholesterol so voi PhosphoUpon cang lon tiii kich thuoe tieu phan eang lon va day phan bo kich thuoe tieu phan cang rpng.
Bdng 8. Kei qud phdn tich bieu do phdn bo kich thu&c tieu phdn cua cdc mau voi ti le cholesterol khdc nhau
C d n g thiJC KiSu phSn bo Day phan b6 Oso (Median) Dmean (Mean) Dm Mode
CT1CH 1 dinh 33,8- 296,2 96,9 103,6 106,2
CT2CH Idinh 3 3 , 8 - 388,6 101,2 117,4 93.6
CT3CH Idinh 38,7- 339,3 110,7 119 107.5
CT4CH Idinh 50,7- 766 163,7 192,4 160,3
1>»
'
e . t — — ^ lao
y'
m» 1000 D im
I
tm
Hijth 2. Bieu do phdn bo kich thuac tieu phdn cua cdc cong thitc vai ti le cholesterol khdc nhau Kei qua ve phong thich hoat chat cua cac mSu thay ti le phong thich boat chat giiia cac mau Uposome du(jc h-inh bay trong Bang 9 cho l*6ng khac nhau co y nghia (P - 0,99). Tuy
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Y Hpc TP. Ho Chi Minh * Tap 21 * S6 5 * 2017 Nghien cvu Y hoc
nhien, ti le hoat chat qua mang tir mau CT4CH (ti le cholesterol cao) tai nhimg thai diem dau cao hon so voi nhiing mau eon lai. Dieu nay co the do khi ti le cholesterol eao hieu suat bat giii piroxicam thap, piroxicam ton tai tren mang nen piroxicam t u do khuech tan qua mang nhanh hon.
Bdng 9. Piroxicam dugc phong thich tie cdc mdu v&i ti li cholesterol khdc nhau
eau tnic mpt I6p mang kep, piroxicam phan bo ca trong lop mang kep va trong loi than nuac.
Ti 1$ piroxicam difgv phdng thich (%) Thoi gian
15 phut 30 phllt
I g i d 2gi&
3 gid 4 gid 5 gid
CT1CH 23.6 34,4 54,2 72,4 84,4 94,3 98,2
CT2CH 20,2 32,9 55,9 75,2 84,9 91,3 95,7
CT3CH 23,6 34,1 59.8 74.2 89.6 98.3 97.2
CT4CH 27.2 39.7 58.3 77.2 91.3 98.6 97,2 Ket qua danh gia tinh chat cua liposome piroxicam
Voi thanh phan eong thiic nhu tren, liposome piroxicam dupe bao ehe theo cac buoc da dupe xac dinh va dupe danh gia ve cam quan, kieh thuoc-phan bo kieh thuoe tieu phan, hinh the hpc, hieu suat bat giii va dinh luong.
Camquan
Hon dich Uposome dong nhat eo mau vang xanh nhat, khong co hien tupng ket bong, noi kem, Iang c§n trong qua trinh bao quan.
Kich thudt, phdn bo kich thuac tieu phdn Liposome co kich thuoc va phan bo kich thuac tieu phan d^t yeu cau de ra, cy the n h u sau:
Day phan bo kich thuac : 51 - 445 nm Dso (Median): 107,4 nm Hinh thehcfc
Ket quei dupe trinh bay trong Hinh 3 eho tfiay Uposome tao thanh la loai Uposome co
Hinh 3. Hinh chup be mat cat ngang cua liposome Hi^u suiit bat giii piroxicam
Hieu suat bat giii piroxicam trong tieu phan Uposome:
Dinh lupng
Ket qua piroxicam toan phan la 100,2%.
KETLUAN
Su hien dien eiia Cholesterol va cae chat dien hoat nhu Labrasol, Trancustol, Tween 80 deu lam tang hieu suat bat giii piroxicam va lam giam kich thuoc tieu phan Uposome.
Trong do, Tween 80 ti le 0,2% va ChoIesterol:PhosphoUpon ti le 8:2 (mokmol) eho ket qua tot nhat. Liposome tao thanh la loai Uposome co cau tnic mpt 16p mang k^p, piroxicam phan bo ca trong lop mang kep va trong loi than nuoc.
TAIUiUTHAMKHAO
1 Baneriee R. (2001), "Liposomes: Applications m medicme", journal ofBiomaterials applications, vol. 16, p p . 3 - 2 1 .
2 C a d d e o C, Manconi M, et al (2012), "The role of Labrasol in the enhanacement of the cutaneous bioavailability of Minoxidil in phospholipid vesicles", Researcli J. Pharm and Tech, vol.5, p p . 1563 - 1569.
3 Clerc S., Barenholz Y (1995), "Loading of amphipathic weak acid into hposome in response to transmembrance calcium acetate gradients", Biochem el Biophys Acta. p p . 257 - 265.
4 Dua JS, Rana AC, Bhandri AK (2010), "Liposome methods of preparahon an appUcation", lntemal,a«al
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Nghien cihi Y hoc Y Hgc TP. Hh Chi Minh * Tap 21 * So 5 * ^"*'
Journal of Pharmaceutical Studies and Reaearctia, vol. 3:14 - Ngdy nhan bdi hdo: 1510712017 5 Koynova R, Caffrey M (1998), "Phase and phase Ngdy phdn biin nhan xet bdi hoo: 2010712017
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