Indian Jou rnal of Experimental Biology Vol. 38, February 2000, pp. I 13- 1 I X

Prolonged release biodegradable vesicular carriers for rifampicin-Formulation and kinetics of release

M P Kamath , B D Shenoy, S B Tiwari , R Karki & N Udura*

Department of Pharmaceutics, Co ll ege of Pharmaceut ical Sciences. Manipal 57(1 I I 1J. I ndi :1

and

M Koti an Department of Mi crobiology, Kasturba Medical College, Manipal 576 II t.>. India

Received I January 1999; revised 16 September 1999

An attempt has been made to design suitab le li posome and ni oso me-encapsul ated drug de livery system for riL1111pi cin and evaluated the same in vit ro and in vil'u. A mod ified lipid layer hydration method was employed w pre pare ihC,l' vesicular carriers. The formul ated systems were characteri zed in l'itru for size distribut ion analysi s. dru g cnl rap!nclll. dru g release profiles and vesicu lar stahi lil y at diffe rent condi1ions or storage. In vivo drug kine1ics w:1s eva lualcd in nurm:il. health y albino rats for ni osomal formul ati on upon subcut aneous injecti on and various phannacokinetic par:11llctcr' wc rl· determined. Niosomes and li posomes ex hibited mean diameter or 9.73 and II .X7 pm wit h entrapment c llici eii L'iL'' ol· .10.'i and 34.2% respecti ve ly. Both the products exh ibited sustai ned release characteristics in Film wi th zero urdn dru !C rclc:N' kinetics up to initi al 10 hr. Stab ilit y evaluation indicated that both formulat ions were not sig nificantl y IL:ak) tl\·cr a pn iod t>t' one month . Niosomal formulation elevated plasma el imin ation half life and decreased elimin:11ion ra te cu ll \ lalll ' lur rifampicin in l'ivo suggested th at encapsu latio n retarded the removal or the dru g from ci rcula ti on compared tu free drti !C due to slow drug release into systemic circulat ion. A fi ve- fold increase in the area under plasma rifampi cin l'< lllcc ntr:lllt >n-timc curve for ni osomal rifampici n as compared to free drug indicated better bioavai lability of encapsu l:llcd dru g. It is cvid c111 from thi s study that niosomes and liposomes cou ld he promising delivery systems for rifampic in with prolonged dru g re lt.::1'c profiles and reasonably good stability characteri stics.

Behaviour of drug in vivo can often be modifi ed in dramatic fashion by coupling the drug to a carrier moiety . Plasma clearance kineti cs, ti ssue di stributi on, metaboli sm and cellul ar interac ti on of the drug will be dictated, or atleast strongly influenced, by the behaviour of the carrier. In some cases manipul ati on of these changes in pharmacodynamic behaviour can lead to a hi gher therapeuti c index of the drug. Encapsulation of a drug in ves icul ar structures can be predicted to prolong the ex istence of the drug in the systemic circulation and thu s, enhance ti ssue penetrati on and perhaps tox icity. Phys ica l approaches to the drug de li very consist of microparticu late carriers (liposomes, ni osomes, microspheres, nano­particles) , magnet ic microcapsules, implantable pumps and reservo irs which arc bei ng acti ve ly considered as potenti al systems fo r the site-spec ific delivery of drugs foll owing intra venous admini s­tration 1 -s.

*Correspo ndent author Tel: +t.> 1-8252-7 120 I (Ext. 24X2); Fax : +9 1-8252-70061: E. mail : info @mahe.erncl.ill

Liposomes are micropa rti culate or cn ll oidal carri ers, usuall y 0.05-5 .0 pm in diamete r whi ch form spontaneously when certain lipids arL' hydrat ed in aqueous media with input of ene rgy in the form or phys ical agitat ion or heat. Liposo mes arc co mposed of relati vely biocompati ble and h i odcgr~Kiahlc

materi al, and they consist of an aqueous vo lume entrapped by one or more bilaye rs of natural and/or synthetic lipidsu . Drugs with wi de ly varytng lipophiliciti es can be encapsul ated in liposomes. either in the aqueous vo lume or at the bilayer interface. Liposo mes have been in ves ti ga ted as carriers of various pharmaco logica ll y act ive agents such as antineoplasti c and antimicroh i ~ tl drugs. chelat in g agent s. steroid s. vacc tnc\ ;tml ge neti c

. 1h-x '--"

matena . The ves icles formed , when a mixtmc or clw lesternl

and a single-alkyl cha in . non-ioni c smf~t ctant ts hydrated, are termed ni oso mes . can entr;tp so lutes. are os motica lly active and stabl e. In addi ti on. handling and storage of surfac tant requtres nu special conditi ons9

. Niosomes appear to be s imilar in terms

114 INDIAN J EXP BIOL, FEBRUARY 2000

of their physical properties to liposomes 10. Niosomes behave in vivo like liposomes, prolonging the circulation of entrapped drug and altering its organ distribution and metaboli c stability11 . Properties of niosomes depend on composition and method of their production. Niosomes may be regarded either as inexpensive alternat ives, of non-bi ologica l origin , to liposomes, or perhaps in vivo as a carrier system h . II . .I I. " ' 11 p ystca y Simi ar to tposomes· · -· · .

Liposomes and ni osomes could be used as implantable drug deli very devices if given intramuscularly (im) or subcutaneously (sc) 14 . They form a drug depot at the site of inj ec ti on, releasi ng the entrapped drug slowly, initi al ly by diffusion and then by bioerosion of the components of the delivery 5ystem. Rate of drug absorption would be slower in case of s.c. admini strati on than i.m. due to less perfusion of the subcutaneous tissues. Though the release rate would be faster than other nove l implan table systems like microspheres, nanoparticles, films, hydrogels etc., liposomes and ni osomes can serve the purpose of slow and contro ll ed drug release than many other conventi onal depot forming inj ec­tables1 5.

In the present study , the main objecti ve has been to encapsulate rifampicin, a first line antitubercular drug in liposomes and niosomes and utilize thi s drug deli very system fo r opt imum therapeutic use.

Materials and Methods Rifamp icin was a gift sample from M/s Cadi la

Pharmaceuticals Ltd. Ahmedabad. Cholesterol, span 60, dicetyl phosphate, egg phosphatidyl choline and Triton X-1 00 were purchased from Sigma Chemicals Co., USA and were used as obtai ned. All the other chemicals and reagents were of ana lytica l grade.

Preparation of niosomes and liposomes by lipid hydration method17 -Niosomes were prepared by adopting the procedure of Azmin et a/11 with slight modificati on. Known quantity of rifampicin was di ssolved in minimal volume of chl oroform in a round bottommed flask. Cholesterol, span 60 and dicety l phosphate (47.5:47 .5:5 mg of eac h) were added to thi s drug concentrate which was diluted with diethyl ether ( 10 ml ). The organic so lvent was evaporated complete ly under contro lled conditions of temperature (60°±2°C) and pressure ( I 5±2 psi) with gentle agitat ion to obtain a thin film on the wa ll of the flask . This was hyd rated with I 0 ml of phosphate buffered sa line (PBS ; pH 7.4) and was vortexed to get a uniform suspension of niosomes .

A similar method was employed for preparati on of liposomes taking cholesterol, egg phosphatidyl choline and dicetyl phosphate (50: 50: 12.5 mg or each) in the round bottommed flask.

Size of the ves ic les was determined hy li ght microscopy using calibrated eyepiece micrometer. One hundred vesicles were counted in triplicates and size range with mean diameter were computed. Entrapment efficiency of the vesi cula r carri ers was determined by column chrom<1togr;q1hy us1ng Sephadex G-5010. One ml of the ves icular suspension was trick led down the colu mn (20 cm length ) with PBS as the eluent. The vesic les e lut e first as milky suspension depending on the si ze foll owed hy the free drug. The eluent was co ll ected ill fracti ons and unentrapped drug was estimated by U V spectroscopy. Amount of drug still remaining within the ves icles was further confirmed by incub<ttin g a know n quantity of separated ni osomes/lipo~ mes wit h Triton X-1 00 ( I %v/v) to disrupt the ves i cle~ . The resultin g mixture was centrifuged and the supe rnatan t assayed for drug content spectrophotometnGtll y at 334 nm after suitab le dilution with PBS .

In vitro release studir!s o( dm,r..: .fimn niosonll'sl liposomes-N iosoma l/1 i posoma l s u ~. pen s ion conta­ining known amount of drug was dialyzed using a Sigma dialysis sac against PBS ( 100 ml ) which was under constant magnetic stirring. Samples were peri odica ll y withdrawn from receptor co mpartment with replacement of fresh medium and drug cont ent was determined spectrophotometri c; tll y at 334 11111 .

Study of drug leakagr! .fimn uio.\nt/1 !'.1'/lit ){J.I'ollt C.I'

and stabili~y evaluat ion- iosomes/lipuso mes conta­ining a known quantity of rifampicin were stored in vials at 37°C, room temperatu re <llld under refri geration for one month. Samp les \Nere withdra wn weekly and entrapment effici ency was determined by co lumn chromatography .

In vivo pharmacokinetic sllalics-These were carried ou t on normal health y rat s or albino strain weighing about 225-250 g obta ined from central animal house facility of Department of Pharma­co logy , Kasturba Medical Co llege. Manipal. The animals were divided in to four gr<,ups containing eight animals each and were housed m pol ypropylene cages with sterile paddy husk as th e hedd in g and maintained under cont ro ll ed conditi ons of tempera­ture (23°±2°C). humidity (50±Yk l and li ght ( 10 14 hr of light and dark respecti ve ly) . They were fed balanced pellet diet and water wl/ihilll tll .

KAMATH eta /.: BIODEGRADABLE VES ICULAR CARRIERS FOR RIFAMPICIN 115

Group I was injected with I 0 mg/kg body weight of free rifampicin suspended in 0.5% w/v sodium carboxy methyl cellulose; Group 2 with empty niosomes (0.5 ml); Group 3 with I 0 mg/kg body weight of drug loaded ni osomes. Group 4 served as control. All the injections were given subcutaneously using a 24 guage needl e. Blood samples were collected periodically (at 2, 4, 6, 8, I 0, I 2 and 24 hr post treatment) from retro-orbital plexus using sterile heparinized capill aries tak ing appropriate precautions. Samples thus obtained were centrifuged at 4000 rpm for I 0 min and plasma co ll ected was transferred to sterile plastic vials and stored under refrigeration pending analys is.

Microbiological assay- This was done using Kirby Bauer di sc diffusion technique1

R. Zone of inhibition observed with Staphylococcus aureus was used for quantitating the drug in the pl asma by extrapolation from the calibration curve. From the data obtained, plasma drug concentration-time curve was drawn and area under thi s curve (AUC) was calculated by the trapezo idal rule and vanous pharmacokinetic parameters were assessed.

Results Size and entrapment e.fficiency-Niosomes

prepared were of the size 3. 14 to 15.7 !Jm, the mean diameter being 9.73 11m whereas liposomes formul ated had the size range 6.28 to 18.4 11m with mean diameter of 11 .87 ~Jm . Both the ves icul ar carriers were spherical in shape with occasional ova l ones as characterized by I ight microscopy. Entrapment efficiencies of 30.5 and 34.2% were obtained for niosomes and liposomes respecti vely.

In vitro release studies-Both the vesicul ar carriers exhibited sustained release characteri stics over a prolonged period compared to control (drug dispersed in PBS). It was observed that, fo r liposome­encapsulated rifampicin , 98% of the drug was released within 20 hr whereas ni osomes however, could hold on the drug load up to 18 hr at whi ch 97% of the drug was released. In case of free dru g suspended in PBS, 94% of the drug was diffused within I 0 hr. The drug release fo ll owed zero order kinetics upto initial I 0 hr with both the ves icul ar carriers after which a burst effec t was observed (Fig. 1).

Vesicle stability- A direct relationship was observed between percentage leaching of the drug out of the ves icles and temperature (Table I). Liposomes

exhibited comparati ve ly hi gher ex tent of dru g leakage than ni osomes. Drug leakage was less from both the carri ers stored at 4°C. In vitro drug release profi le was not altered signi ficantl y under sto rage condition upon storage for one month except fo r 3rC. There was no stati stically significant difference in the dru g content upon storage for one month between the groups as compared aga inst cont ro l (room temperature).

In vivo drug kinetics-Pharmacoki neti c parameters evaluated are depicted in Table 2. It was obse rved that time taken to attain maximum plasm;1 drug concentration (T11.,. ) was 4 hr fo r both free and ni osomal ri fampicin with concent rati ons (C 11 m) being

,-..., Cf) ::i.

..___. 120 <l) tl)

ro ~100

<l) ...... bf)

2 80 'U '+-< 0

60 ..... ::::: ;:; 0 E 40 ro <l)

> 20 ..... ro :::J

E 0 ;:; u

-+-Liposomal rifampicin

----NiosomaJ rifampicin

_._Free rifampicin

-;-----1 --1-1

0 2 4 6 10 12 14 10 IS 70 22 24

Time (hr)

Fig. l- In vitro release patterns ror li posom:il. lliliS!llll:il :llld rree rifampicin in phosphate butTered sa line (Jil-l 7.-1 1

Table !-Effect or temperature on ves icle slahil i1 v 1Jf ri l:unpicin loaded niosome' and I i psomc'

[Values are mean ± SO or three rep I icallll llS I

Time in days Dru~ entrapmenl ('/, l

7

14

2 1

n

7

14

2 1

2R

4"C room 1e111 p \ 7"('

Nioso 1ne'

30.10 ±2.42 2X .XX ±::' . .lll 2X .. \O ±2.:1:1

27.00 ±2.04 24.2() ±2. 1 2 2.\.72 ±2. 17

26.00 ± I.X7 1:2.') I ±2.0'! 2::'. 4(1 :t l .'lh

25.60 ±2.2 1 .22 .00 ± 1.7 11 1'1 .30 ±I.x, ·

Liposon1e'

32.27 ±3.3 1 30.55 ±247 2 ').()') ±2.:15

30.25 ±2.'.H\ 27 .54 ±2HI 27. 1 () ±2.X4

27.97 ±2. 13 25.X2 ±2. 1 5 2.\ (17 ±2.1 1

26.90 ±2.45 2-1 .5:\ :t2.0X .2100 ± 1.77

116 I 'DIAN J EXP BIOL, FEBRUARY 2000

I 0.828 and 14. 17 fl g/ml respecti vely. Trend of the graph of plasma drug concentration vs time revea ls that concentration of ni osomal drug was maintained over a longer peri od than free counterpart at comparatively higher plasma level (Fig. 2). Approxi­mately one and a half fold hi gher plasma elimination half life (tY2) was obtained for ni osomal rifampicin compared to free drug. El imination rate constant (Kc1) was reduced for ni oso mal rifampicin to a consi­derable extent. A notab le increase (approximately five-fold) in the area under plasma rifampicin concentration-time curve (AUC) was observed for niosomal rifampicin than free drug.

Discussion

Purpose of thi s study was to deve lop an easy, rapid and effici ent method for preparing ni osomes and liposomes loaded with rifampic in. Lipid layer hydration method is the most simple and is wide ly used procedure in which a thin film of lipids is hydrated with an aqueous buffer at a temperature above the phase transition temperature of lipids. Niosomes/liposomes prepared by thi s meth od would be a heterogeneous population of multilamellar

Table 2-Phannacokinetics of rifamp icin in normal healthy albino rats

Parameter

c""" (!l g/ml)

T""" (hr) Ko~ (h( 1

)

lY2 (hr)

AUC <11 g hr/ml )

3

.5 <J 'i5. 2.5 E ., .... ·c 2 '-0

t: ,gu ., c t: C) <J t: 0 <J ell 0.5

.3

Free rifampi cin iosomal rifampicin

10.828 14. 174

4.0 4.0

0. 1327 O.OX42

5.22 8.21

64.65 300.15

--Niosomal rifampicin

0·-----~----~~·---+----~----~

0 5 10 15 20 25

Time (hr)

Fig. 2- Plasma concentrati on of free and niosomal rifampi cin upon sc injec ti on

• • I ' I 4J '() vestcles (MLYs) as reported earlier- ' · - and MLV ~

offer greater encapsulati on efficiency for hydro­phobic drugs. The ves icl e s ize is a crit ica l p<Jrameter in determining the rate and ex tent of biodegrada ti on and hence half life of the carri ers besides influ e n ci n ~

the ex tent of drug encaps ulati on ~ ' . Ninso mes and liposomes ex hibited a drug load ing nf 0 .~0) and 0.342 mg/ml respectively. Howeve r. the encapsu­lation effici ency was quite low for hoth ves icular carriers which could be because or zw itter ioni c nature of the drug at p H 7.-l (re lat ed to 3-piperazine nitrogen). Hi gher en tra p.nent efficiencies could be obtained for both liposomes and ni osomes by altering lipid compos iti o n/s i z.e 23 ~.: For hydro­phobic drugs like rifampi c in .. the entrapment efficiency depends On the tOtal amount of lipid present in the di spersion and also the properties or bilayer (gel or fluid state)12

.

In vitro release studies--A relati ve ly slovv release pattern of the entrapped drug from til L' ves icles prepared by the lipid hyd rati on method indicated an enhanced stabilit y of the system Prese nce of cholesterol in the formulati on affect s the membrane fluidity by making it more rigid 2

' . Thi s el'kct is evident as liposomcs. co ntaining h1ghe r cholesterol concentration, ex hibited a sl ower release pattern than niosomes. The drug be in g lipophill ic. was bound within the hydrophobic regions of the lipid bil aye rs or the multilamellar vesicles. and was leached out slowly at a steady rate. It was observed from Hi guc hi plot that drug release followed ze ro order kineti cs upto 10 hr and then first order later <l n2

.j. The rlu x of free rifampi cin (control) was linear and w~ 1 s

proportional to the dru g concen trati on 111 the reservoir. Comparative data of rif;unp ic in . both l'ree and upon encapsul ati on into ni osumes/liposo mes. indicated that , by encapsulati on. it co uld be poss ible to sustain and control the drug re lease !'or a longer period of time. MLYs may have the added ad vantage of not leaking all of their co ntents when outer bilayer is ex posed to biologica l tissue fluid s. Si mil ar res ults

I 7 ' . have been reported by D'Souz<J et ul . Vya~ ('/ u!-' . As the drug is bound to li pid s tra t ~ 1. it is released slowly, layer by layer and t oward ~ the tailing of the release study , relative ly hi gher rate or release wa~ observed indicating rupture and co llapse of the vesicles due to constant erosi on uf the bi layer membrane.

Vesicle stability--One of the maj or problems limiting the widespread use or ni osomes/liposo rnes is

KAMATH el a/.: BIODEGRADABLE VESICULAR CARRIERS FOR RIFAMPICIN 11 7

stability-both physical and chemical. Depending on their composition, the final formulation may have short shelf-life partly due to chemical (hydrolysis and/or oxidation) and physical instability (aggrega­tion or fusion of vesic les)26. A stable niosome/ liposome suspension should ex hibit a constant particle size and a constant level of entrapped drug. Assessment of degree of leakage of rifampicin from the vesicles and alteration in in vitro drug release profile were used as the parameters to ascertain the stabi lity of the vesicles at various temperatures with which the vesicles would come in contact either during storage or on administration.

Increased degree of drug leaching with time at al l storage conditions indicate that there is constant partitioning of the hydrophobic drug from the bilayer into solvent on standing2. The bilayers also remain fluid to certain extent and hence cause leakage. Degree of drug leakage upon storage was not significant in both vesicular carriers which was due to the presence of dicetyl phosphate which imparts negative charge, thereby preventing fusion/aggre­gation of the ves icl es. This result confirms earlier results17·25·27·2s. Niosomes appeared to be better than liposomes in terms of physical stability though there was no significant difference between both in terms of drug leachi ng. Inferior chemical stabi lity profile of liposomes compared to niosomes could be attributed to the presence of phospholipids, which render liposomes much vu lnerable for thermal degradation

b ~ as o served by Case ttes et at- . Choles terol and fatty acids of egg lec ithin are more susceptible for oxidation catalyzed by temperature. Further, egg phosphatidyl choline has lower phase transiti on temperature than span 60, thus making the bilayer of liposome more fluid and leaky. Niosomes, on the other hand, were less sensitive to this effect.

In vitro studies of niosomes/liposomes stored at different temperatures for a period of one month revealed that drug release at 4°C and room temperature was less compared to that at 3]CC.

In vivo drug kinetics-Pharmacokinetic evaluation of rifampicin niosomes was carried out in hea lthy male albino rats using di sc diffusion sensitivity tes t. Niosomes were selected for in vivo kinetic studies as they ex hibited superior size di stribution charac­teristics, adequate drug loading, reasonable in vitro drug release profiles and improved stability upon storage. Further, the cost niosomes preparation was lower than liposomes for vesicu lar components and

mosomes were less leaky, hence offered the possibility of holding the drug load for longer duration . Microbiologica l assay proced ure was followed because it was sensiti ve up to nanogram levels of rifampicin , reproducible. accurat e and cost effective compared to HPLC estimation '° Ca librati on curve in rat plasma has show n a linear dose-response (growth inhibition) relati onship in concentrati on range studied with correlati on coeffi c ient of 0.9797 (Ref. 18) . Dose selected ( I 0 mg/kg body we ight ) was chosen according to the publi shed repmt s :1. Route of administration was subcutaneous whi ch would favou r prolong drug release than intravenous route because of diffused vascul arity of ad ipose ti ssue. It is hypothesized that liposomes/ni oso mcs are neither absorbed intact into circulati on from the sit e of inj ection, nor cleared by li ver and spleen (due to size >200 nm) , but , formed a dru g depot at inj ec ti on site thereby releasing the dru g slow ly into neighbouring tissues 14. Blood samples were co ll ected upt o 24 hr on ly as predicted from the ni osomal compos iti on. size and in vitro experiments. Ni oso mes arc negati \'e ly charged because of the presence of dicetyl phosphate and release their contents ex trace llul arl y aft er interaction with blood components and ti ss ues:2.

Cnax attained for niosomal rifampicin was hi gher than the free drug after 4 hr post treatment was due to the presence of unentrapped drug in the ni osomal suspension injected which prov ided the loadin g dose of the drug. Trend of graph of concentrati on vs time showed that the concentrati on of the nioso mal drug was maintained over a longer period than free counterpart at comparati ve ly hi gher pla s 111 ~ 1 le ve l. Encapsulation coupl ed with sc inj ec ti on could exe rt thi s effect with drug release from ves icular aggregate at the site of inj ec tion as it s surface heing the rate­limiting step in drug absorpti on. This was furth er supported by e levated t\12 and reduc ·d Kc1 for niosomal rifampicin compared to free dru g. Both these parameters suggested that encapsulation within ni osomes retarded remova l of drug from the circulation compared to free drug. Further. the vesic les were not availabl e for phagocyti c ca pture and were not in the systemic circulati on. A fi ve-fo ld increase in AUC for niosomal rifamp icin as compared to free drug refl ected better bi oa vai labi I it y of niosomal encapsulated drug. Dicetyl phosphate produced ani onic niosomes. thereby influencing the extent of vesic le interaction with ce ll s leading to increase in intracellular uptake.

11 8 I DIAN J EXP BIOL, FEBR UAR Y 2000

The present study suggested the poss ibility of better application of niosomes/1 iposomes encapsu­lated rifampicin in the treatment of tuberculosis.

Acknowledgement

The authors are grateful to M/s Cadila Pharma­ceuticals Ltd ., Ahmedabad for the gift sample of rifampicin .

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