Ind ian Journal of Experimenta l Biology Vol. 42. June 2004. pp. 562-566

Lung specific stealth liposomes as antitubercular drug carriers in guinea pigs

Rajesh Pandey, Sad hna Sharma & G K Khuller*

Department of Biochemistry, Postgradua le Institute of M cdica l Education & Research, Chandigarh 1600 I 2. I ndia

Recei l'ed 15 Decelllber 2003: rel'i.led 10 March 2004

The problem of patient non-compliance in the managemcnl of tuberculosis (TI3 ) can be overcome by )educing the dosing frequency of ant itubercular drugs (ATD) employ ing drug carriers. Th is study reports on the intmvenous ( iv) adm inistmt ion of lung specific stea lth liposomes encapsulating ATD (rifampicin and isoniazid in combination) to guinea pigs and the deta i led pharmacokinetic/chemotherapcutic studi cs. Fol low ing a single iv adm inistrat ion of liposomal drugs. the latter were found to exhibit sustaineu therapeul ic levels in plasma for 96- 168 hI' wilh ha l f- li ves of 24-70 hr. mean residence time (MRT) of 35-8 1 hI' and organ drug leve ls up 10 day 7. The relati ve bioava i labi l ilY (as compared to oral frec drugs) was increascd by 5.4-8 .9 folds. whereas the absol ute bioava ilabi lity (as compared to i v frce drugs) was increased by 2.9-4.2 folds. Week ly therapy w ith liposomal ATD fu r 6 weeks produced eq ui valent clearance o f Mycobacterilllll tllberclliosis from organs as did dail y thcrapy wilh oral free drugs. Hcnce. intravenous li posomal ATD offer the therapeutic advantage of rcducing Ihc dos ing frcquency and improv ing the patienl compliance in the management o fTI3.

Keywords: Liposomes. Rifampicin. Isoniazid. Pharmacokineti cs. Chcmotherapy.

I PC Code: Int. CI7 A6 ! K 47/00

Tuberculosis (TB) continues to be a major infectiou s burden worldwide, claiming nearly 1.5 million lives annual ly'. B:1sed on potent :1ntitubercu lar drugs (ATO) such as rifampicin (RlF) and isoniazid ( IN H), an effect ive chemother:1peutic regimen is available for TB management. However, mUltiple drugs need to be admin istered dail y which frequently results in patient non-compliance and therapeutic failure. The frontline ATO are al so known to bc hepatotox ic. These problems may be largely overcome by using drug carriers such as liposomes that release the drug in a slow and contro lled manner. Liposome-encapsulated drugs are espec ially effecti ve against intrace llular pathogens" and their demonstrated advantages include" ) - (i) the abi lity to formulate biologically active molecules; (ii ) the abi l ity to encapsulate hydrophilic compounds: (iii) reduction in toxicity of the acti ve agent: (iv) increased therapeutic index : (v) increased stabi l ity of labile drugs; (v i) improved pharmacokineti cs; and (v ii ) increased delivery to target ti ssues. Improved pharmacoki netics and therapeu t ic efficacy of once/twice week ly admini stered liposomal drugs as compared to free drugs in mice have been reported~ · 6. However, gUinea pigs are the best animal

"'Correspondent author Phone: 0 172-2747 5X5. Ex t. 5!7~ - 7 5

Fax: 0 172-274440 I. 27~ 5 078. E-mail: gkkhuller@yahoo.co. in

model for TB research because of their high susceptibility to MycobacteriulII Iuberculosis. There is as yet no information available on the detail ed pharmacokineti c and chemotherapeutic effect of liposome-encapsulated ATO in guinea pig model of TB in which progress ion of the di sease mimics human TB infec tion. Hence, the present tudy has been planned to evaluate the same.

Materials and Methods Chelll icals alld drugs~gg phosphatidylcholine

(PC) W:1S isolated in the laboratory. Cholesterol (CH), dicetylphosphate (OCP), amy lopecti n. polyethy lene glycol-2000 (PEG2000), distearylphosphatidyleth a­nolamine (OSPE), RIF and INH were purchased from Sigma Chemical Co., St. Loui, MO, USA. Conjugation of OSPE t.o PEG2000, and deri vati zation of amy lopectin to O-stearylamylopecti n (O-SAP) \Vcre carried out in the laboratory. A ll other chemicals \Vere of analyt ical grade.

Allill1ols--Dunkin-Hart ley guinea pigs of either sex (300-400 g) obtained from Hisar Agricultural University, Hi sar. India were used for this study. The animals were fed standard pellet diet and water ad Iibitull1 . The study was approved by the Institute' s Anima l Ethics Committee.

Cultllre-Th e cu lture of Mw:o!Jacterilllll Illberclliosis H.17 Rv ori gi nally obtained from National

•

PANDEY e/. 01.: LUNG SPECIFIC STEALTH L1POSOMES 563

Collection of Type Cultures (NCTC, London) was maintained on Youman's modified medium.

Preparation of liposollles---M uitilamel lar liposomes using PC, CH, OCP, OSPE-PEG (molar ratio 2.0: 1.5 :0.2:0.2) and coated with O-SAP (1:3 w/w with respect to PC) for lung targeting were prepared as described before6

. Briefl y, the lipids were di ssolved in chloroform/methanol (2 : I v/v) and dried under nitrogen. After adding DSPE-PEG and the aq ueous drug so lution, microtip probe sonicat ion (Misonix Inc. , Farmingdale, NY ; Model XL 2020, 120W, 20 KHz) was carri ed out fo r 10 cycles, each cycle compri sing of Imin 'on' fo llowed by 30 sec 'off'. The liposomes recovered by cen trifugation (25000 g) were mixed with O-SAP, stirred (30 min) and recentri fuged to obtain the final pell et. After lys ing the liposomes with Triton X-IOO ( I % v/v) , drug encapsul ation efficiency was fou nd to be 40-45 % fo r RIF and 8-12% for INH. RIF was es timated by microbiological assa/ whi le INH was es timated spectrofluorimetri ca ll l . The sensiti vities of the methods were 0.2 and 0.1 IAg/ml respectively. Separate preparations were made for RIF and I H, and then mi xed ( I: I v/v) for in vivo studies.

Dmg disposition studies- Animals were di vided into three groups with 6-8 animals each. Group I was ad mini stered free (unencapsu lated) drugs once orally, group II received free drugs once via iv route and group III received liposome-encapsulated drugs once via iv route. The lateral leg ve in was used for iv dosing. Therapeuti c drug doses, i.e. RTF 10 mg/kg and INH 12mg/kg body weight were used. Animals were bled via the retro-orbital plexus and drug level s assayed in pla.·ma and 20% organ homogenates. The results were expressed as lAg drug/ml plasma or organ homogenates.

PhannC/cokinetic onalysis-The plasma drug concentrat ion versus time data was used to determine various pharmacokinetic parameters. Peak plasma concen trati on (Cma,) and ti me taken to reach ella, (Tma, ) were obtained by vi sual data in spection. Eliminati on rate constant (Ke!) was calculated by regression analys is whereas el iminat ion half life (t 1/2) was calcul ated from the form ul a 0.693/KcJ . The area under concentration over time curve (AUCo.t) was detennincd by trapezoidal rul e. The terminal AUC t .

was obta ined by dividi ng the las t measurable plasma drug co ncentration by Kci . AUMC/AUC, i.e. area under moment curve (AUMC)/area under curve (A UC), gave the mean residence ti me (M RT).

Relati ve bioavailability was computed by the fo rmula :

AVCi.v.liposollles Dose oral Fee -----.:...---x ,whereas

AVC oral free Dose . I ' I.V. Iposo/n es

AVe. I ' Dose. F. I. V. lpOSOll1es x 1. 11 . .Jree yielded the

AVC . F. Dose. I' I.V . .Jree l.V.lpOSOl1les

absolute bioavailability. Hepatotoxicity sludies-Drug-free liposomes

(\iposomes containing PBS) as well as liposome containing (RIF+INH) at therapeutic doses were ad mini stered iv to guinea pigs weekly for 6 weeks. After 5 days of the last dose, plasma was analyzed for total bilirubin, alanine transaminase (AL T) and alkaline phosphatase (ALP) using standard kits.

Experimental injection al/.d chelllotherapy-Guinea

pigs were infected im with I x 105 bacilli of M. tuberculosis H37Rv. After 20 days, the animal s were grouped into 3 for chemotherapy studies-group I served as untreated controls, group II was administered free drugs , orally, daily for 46 days and group III was admini stered liposome-encapsulated drugs, iv weekly for 6 weeks (7 shots). Drug doses were RIF 10 mg/kg + INH 12 mg/kg. At day 46, animals were sacrificed and lungs/spleen were homogenized in 3 ml sterile PBS . 50 ).lL of I : 100 and I: 1000 diluted homogenates were inocul ated on Lowenstein Jensen medi a. Colony forming units (CFUs) were counted on day 30 post inocul at ion .

Statistical analysis-The CFUs data was analyzed by one-way analys is of variance (ANOV A) to compare the control and treated groups.

Results Drug dispositioll--Following the iv 'admini stration

of liposo me-encapsulated drugs , RIF was detectabl e in the plasma from 6 hr onwards up to 96 hI'. On the other hand , INH started appearing at 12 hr and therapeuti c concen trati ons were maintained till 168 h ' (Fig. I). Following the admini stration of free drugs orall y, drugs were detectab le as early as I hr. however, plasma was completel y cleared of the drugs by 12 hI'. Similarly, in case of iv free drugs, no drug could be detected in plasma beyond 10 hI' (Fig . 2).

In i v Ii posomal drugs. therapeutic drug level s \.vere present in the ti ssues at day 7 except for RI F which was present at below therapeutic concentrati on in lungs (Fig. 3). Free drugs g iven ora lly/i v were undetectab le in the tissues beyond 48 hr.

564 INDI AN J EX P BIOL, JUN E 2004

Pha nllacokillelics--Liposome-encapsulated drugs on iv admini strati on attained a lower c'nax as compared to animals receiving free drugs (iv/oral) and a longer time was required to achi eve the same (T max) (Tabl e 1). Because of a slower rate of elimination, liposomal drugs exhibited a longer t 1/2 as we ll as a

longer MRT. These fac tors ensured a higher AUCo_~

for liposome-encapsulated as compared to oralli v free

drugs, resulting in a 8.9 fold and 5 .4 fold increase in the relati ve bioavailability of RTF and TN H respecti ve ly. In addition, the absolute bioava il abil ity was also increased by 2.9 fo ld for RIF and 4.2 fold fo r INH .

E rn 2 3 c o

.~

C ~ 1.5 c o u 010 .5 2 "0

~ RIF O INH

E 0 +-"="'--r-

~ 6 9 a:

12 18 24

Hepatotoxicity-Total bili rubin (normal range 0 . 1 - 0 .42 mg/dl) , ALT (normal range 17-50 U/L) and A LP (normal range 19-70 U/L) ranged from 0.22-

72 96 120 168

hr

Fi g . I- Plasma drug pro fil e fo ll ow in g the iv ad mini stra ti o n of liposo me e ncapsul ated a ntitu be rc u la r d rugs to g ui nea p igs .

lIJ RIF 0 INH

0 .017 0 .083 0 .25 0 .5 2 3 6 10

hr

Fig. 2- Plasma drug pro fil e fo llow ing the iv adm in istra tio n o f free drugs to guinea pi gs.

Tabl e I- Sa lie nl pharm acoki neli c para me le rs fo ll ow ing iv ad mini slra li o n o f liposo me- e ncapsul aled a nli luberc ular dru gs lo g uinc a pigs . as co mpa red to o ra l/ iv free d rugs.

c"':'" ( ~t g/m l ) T ",,,, . ( I1r)

Kcl t '/2. ( I1r)

MRT. (hr)

AUCo~. (Il g.hr/ml ) Relati ve bioava ilab il ity Absolute bioavai labi li lY

Free (oral )

1.8 ± 0.3 I ± 0

-0.404 ± 0 .06

1. 72 ± 0.2

2.9 ± 0.4 6.96 ± 0.8 1

0 .3

[Va lu es are mea n ± SO fro m 6-8 animal s in eac h gro up]

Rifampic in

Free (iv) Liposo ma l (iv) Free (ora l)

25 ± 4 1.48 ± 0. 3 3.02 ± 0 .5 I

0.017 ± 0 24 ± 0 I ± 0

-0.35 ± 0.04 -0.028 ± 0.005 -0.295 ± 0.07

1.98 ± 0 .2 23 .5 ± 4 2 .35 ± 0 .22

2. 18 ±0.7 35 ± 4. 1 3.69 ± 0.4

20.9 ± 3.0 62 .1 ± 5 9.9 ± 0 .9 8.9

2 .9 0.7

Isoni azid

Free (iv) Liposo lll ~d (iv)

24. 1 ±4 0.68 ± 0. 1 0 .0 17 ± 0 72 ± 0

-0.32 ± 0.04 -0.009 ± 0.00 I

2. 17 ± 0. 3 70 ± 8 1.74 ± 0.5 8 1 ± 5. 1 12.7 ± 2.7 53.9 ± 4.2

5.4

4.2

PANDEY el. al.: LUNG SPECIFIC STEALTH LIPOSOMES 565

0.29 mg/dl , 19.1-22.9 U/L and 24.7-38.4 U/L respectively in case of empty-liposome treated animals. For iv liposomal-drug treated animals, the values ranged from 0.21-0.28 mgldl , 22.4-31.8 U/L

1

~ Lung 0 Uver • Spleen

Q) -ro c Q)

Ol 0 0 E 0 ..c

E 2 -Ol :i.

Day5 Day7 Fig . 3-Tissue drug leve ls fo ll ow in g the iv administration of liposo me-encapsu lated antitubercular drugs to g uin ea pigs . No drug was det ec table in the ti ssues beyo nd 24-48 hI' following the oral / iv administration o f free drugs. [*Below th e rape uti c concentration].

Table 2--Colony forming units (CFU) in organ hOlllogenates of guinea pi gs following chemotherapy with free and liposome­

encapsulated drugs (RIP + INH)

lVa lues are mean ± SD from 3-4 animals in eac h group]

Lungs Spleen

Log CFUlml homogenate

Untreated Oral free drugs daily IV liposolllal drugs control (46 doses) weekly (7 shots)

6.8S±O.03 6.86±O.02

S.IO±O.17 * S.13±O.21 *

4 .9S±O.09* 4.7±O.S8*

* P<O.OO I according to ANOVA (F values: 344.8 and 37.76 for lungs and splee ns respectively).

and 24-37.4 U/L respectively. Thus, in either group, there was no evidence of biochemical hepatotoxicity .

Chemotherapeutic efficacy-There was a significant reduction (approximately 1.7 fold) in the log CFUs in tissues obtained from treated (oral free drugs or iv liposomal drug) animals as compared to untreated controls (Table 2).

Discussion The current TB therapy entails the daily

administration of A TO, which is the commonest cause of patient non-compliance and therapeutic failure. Liposome-encapsulated drugs offer a poss ible solution to this problem firstly by reducing the dosing frequency and secondly by selective lung targeting.

A single iv dose of liposomal-(RIF+INH) was ab le to maintain therapeutic drug levels in the plasma for 4-7 days (Fig. 1) as against free drugs administered iv/orally which were cleared by 10-1 2 hr (Fig. 2). Drugs are released from the liposomes in a slow and controlled manner as evident by a lower c'nax and longer T lllox in case of liposomal drugs (Table I ). For the same reason , liposomal drugs exhibit a lower K el

and a prolonged t Y2 thereby ensuring a sustained drug stay in the plasma. Indeed, MRT values were approximately 12-21 times higher for encapsulated drugs versus oral free drugs and 16-46 times higher as compared to iv free drugs. Another reason for the enhancement in MRT was the use of steal th liposomes because surface coating of liposomes with PEG is known to reduce their uptake by the reticuloendothelial sys tem (RES) thereby prolonging the circulation time4

. On the other hand, free drugs when administered orally, fail to maintain effective therapeutic levels in plasma for prolonged periods because of gastrointestinal losses as well as rapid hepatic uptake and metabolism. Thus, significantly higher AUC values were obtained for liposomal drugs, which explains the marked enhancement in the relative as well as absolute bioavailability of liposomal drugs. Similar results were observed in mice dosed with iv liposomal AT05. Liposomc encapsulation improves the pharmacokinetic characteristics as also observed for other drugs such

h .. 8 9 d . fl . 10 F h as amp otenclll an clpro oxacll1. urt er, liposome-encapsulated drugs as we ll as empty liposomes administered weekly for 6 weeks showed no evidence of biochemical hepatotoxicity as assessed by total bilirubin, AL T and ALP. This supports the general notion that liposome-encapsulation minimi zes I . . f h d 4 II t le tOXtCtty o ' t e . rugs '

566 INDIAN J EXP BIOL. JUNE 2004

In case of iv liposome-encapsulated drugs, effecti ve ti ssue drug levels cou ld be maintained in all the organs for 5-7 days, except for RIF whose levels fell below therapeutic concentrat ion in the lungs at day 7 (Fig 3). To evaluate the chemotherapeuti c efficacy of iv I iposomal A TO, as the tissue distributi on profi Ie predicted, a once weekly schedule was followed in infected guinea pigs.

Treatment w ith oral free drugs (daily for 46 days) or iv liposome-encapsu lated drugs (week ly for 6 weeks) resu lted in an approximately 1.7 fold reduction in log CFUs in the ti ssues as compared to untreated controls (Table 2) . A lthough free drugs and liposomal drugs produced equi valent/comparable resu lts, the data provide w ith a clear message, i.e. 46 doses of free drugs could be rep laced w ith just 7 doses of liposome­cncapsulated drugs. This could be achieved because of: (i) a slow but sustained drug release from the liposomes; ( ii ) reducing liposome uptake by RES: (iii ) targeting of liposomes towards the lungs; and (iv) ready uptake of liposomes by infected macrophages (the abode of M. tuberculosis). Other workers have also shown the ability of liposomes to enhance the therapeutic effi cacy of drugs against intracellular pathogens such as leishmania 12

, M. {fviufI / ' and M. tllberculosis1 4. rhe

present results substantiate previous reports documenting the effi cacy of l iposome-encapsulated ATO in ll1ice4

.5

. However, the detailed pharmacoki nctic and chemotherapeutic studies have bcen canied out for the first time in guinea pigs in which disease progress ion rcsembles that of human TB.

Liposoll1es rcq uire the iv route for effective delivery and th is route is the least preferred by the pati ents. Howevcr, 7 iv doses arc li kely to be more ancllor equa ll y acceptable than 46 oral doses. In conclusion, liposome-cncapsulated ATD hold promi se in terms of cnhanci ng thc pharmacoki netics and therapeutic efficacy as well as a reduction in dosing frequcncy. Co llectively, these factors wou ld certainl y improve the patient compliance and aid in better managcment of

TB.

Acknowledgement The authors are thankful to CS IR , New Delhi, India

for financial support.

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