Introduction

Bosshardt et al. (1993) first reported the antifilarial activity of tetracycline against filarial nematode (Brugia pahangi) which instigated new insights in the antifilarial therapeutics. Subsequent studies employing this antibi-otic therapy highlighted the presence of an intracellular bacterium Wolbachia (belonging to Rickettsia), which was discovered earlier in mid-1970s in arthropods and nematodes (McLaren et al., 1975). Studies with a variety of animal models of filariasis demonstrated that tetracycline inversely affects Wolbachia and has profound effect on the development, viability and fertility of filarial parasites (Bandi et al., 1998; McCall, Jun, & Bandi, 1999; Taylor & Hoerauf, 1999; Hoerauf et al., 2001). Later on, in vitro and in vivo evaluations of several other antirickettsial antibiot-ics were carried out for their antiwolbachail efficacy, dem-onstrating that doxycycline and rifampicin were the most effective antibiotics in depleting Wolbachia (Hermans,

Hart, & Trees, 2001; Hoerauf et al., 2001; Rao, Moussa, & Weil, 2002; Volkmann et al., 2003; Gilbert et al., 2005).

The potential results obtained by multitudes of animal studies led to perform clinical trials against major human filariids like Onchocarca volvulus, Wuchereria bancrofti, and Brugia malayi as well with different treatment regimens of doxycycline (4, 6, or 8 weeks) in combination of ivermectin or DEC (Hoerauf et al., 2003; Taylor et al., 2005; Debrah et al., 2007; Supali et al., 2008). These studies illustrated very promising results and appropriate for individual treatment nevertheless, long-term treatment schedule is practically not feasible especially for massive treatment coverage in hyper-endemic regions (Debrah et al., 2007). Therefore, further treatment regimens and/or approaches are still required to make it more compatible at mass level. In addition, the clinical application of doxycycline and par-ticularly rifampicin in filaria endemic area would also be helpful concomitantly, in eradicating other infectious dis-eases like tuberculosis and leprosy (Ridtitid et al., 2006).

Journal of Drug TargetingJournal of Drug Targeting, 2010; 18(5): 343–350

2010

343

350

Address for Correspondence: S. Misra-Bhattacharya, Division of Parasitology, Central Drug Research Institute, P. B. 173, Lucknow 226001, India. Tel: 91 522 2612411-18. PABX Ext. 4224, 4221. Fax: 91 522 2623938/2623405. E-mail: shailja_cdri@rediffmail.com; shailjacdri@yahoo.com

11 June 2009

23 October 2009

25 October 2009

1061-186X

1029-2330

© 2010 Informa UK Ltd

10.3109/10611860903450007

R E S E A R C H A R T I C L E

Improvement in the antifilarial efficacy of doxycycline and rifampicin by combination therapy and drug delivery approach

Anil Dangi1, Varun Dwivedi2, Satish Vedi1, Mohammad Owais2, and Shailja Misra-Bhattacharya1

1Division of Parasitology, Central Drug Research Institute, Lucknow, India, and 2Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, India

AbstractThe present investigation deals with the evaluation of antifilarial efficacy of liposome entrapped antiwol-bachial antibiotics doxycycline and rifampicin (5 doses at 10 mg/kg, subcutaneously for 15 days) alone and/or in combination with standard filaricide diethylcarbamazine (DEC) against human lymphatic filariid Brugia malayi in rodent host Mastomys coucha. The delivery system maintained the sustained release of antibiot-ics up to 48 h and significantly (P<0.05) augmented the antifilarial potential of these antibiotics over their free administration. A combination of DEC with each entrapped antibiotics significantly (P<0.05) improved microfilaricidal efficacy, while marginal enhancement was noticed in adulticidal activity. Combination of both antibiotics formulation with DEC demonstrated marginal increase in macrofilaricidal efficacy; however, it was highest (∼75%).

Keywords: Lymphatic filariasis; Brugia malayi; Wolbachia; doxycycline; rifampicin; liposome; drug delivery; antifilarial activity

DRT

445361

(Received 11 June 2009; revised 23 October 2009; accepted 25 October 2009)

ISSN 1061-186X print/ISSN 1029-2330 online © 2010 Informa UK LtdDOI: 10.3109/10611860903450007 http://www.informahealthcare.com/drt

Jour

nal o

f D

rug

Tar

getin

g D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y N

atio

nal L

ibra

ry o

f H

ealth

Sci

ence

s on

10/

05/1

1Fo

r pe

rson

al u

se o

nly.

344 Anil Dangi et al.

We have earlier demonstrated that liposome drug delivery approach significantly reduced the scheduled doses of DEC or tetracycline with a marked enhancement in their antifilarial efficacies (Owais et al., 2003; Bajpai et al., 2005). The present study was, therefore, intended to evaluate antifilarial efficacy of liposomal formulations of the other two antiwolbachial antibiotics (doxycycline and rifampicin) alone and in combination with standard antifilarial drug DEC against human lymphatic filariid B. malayi in rodent model to find out if they provide better antifilarial activities at shortened treatment schedule.

Materials and methods

Chemicals used

Phosphatidylcholine (PC) was prepared in laboratory by egg yolk, while cholesterol, doxycycline, and rifampicin were purchased from Sigma Chemicals Co., USA. Culture media, Luria Broth was also purchased from Sigma Chemicals Co., USA. The solvents (chloroform and methanol) were purchased from Ranbaxy, India.

Preparation of liposomal formulation of doxycycline and rifampicin

Egg phosphatidylcholine was isolated and purified by published procedures (Singleton et al., 1965). Cholesterol was re-crystallized three times with methanol. Unilamellar liposomes were prepared from egg phosphatidylcholine, cholesterol (7:3 of molar ratio) and drug as described earlier (Owais et al., 2003; Bajpai et al., 2005). Briefly, the mixture of lipids and doxycycline or rifampicin (in a ratio of 20:1 w/w) along with cholesterol was dissolved in a mixture of chloroform and methanol, and the solvent was evaporated to get a thin lipid film deposited on wall of the round bottom flask. The dried lipid film was hydrated with sterile PBS (Phosphate buffer saline, pH 7.2). This preparation was sonicated for 15 min in ice (30 sec pulse with 30 sec rest) in a probe sonicator (Soniprep 150, MSE, UK). The sonicated preparation was extensively dialyzed against PBS in dark for 24 h at 4°C to remove free drug. Finally, it was centrifuged (Biofuge, Thermo, Germany) at 10,000 g for 1 h at 4°C to remove the undispersed lipids (aggregates). Stock solutions of both the drugs were pre-pared to final concentration of 10 mg/mL for liposomal formulation and stored at −20°C in aliquots until use. The empty liposomes without any drug (sham) were also pre-pared in the same manner for control studies.

Determination of drug encapsulation in liposomes

The entrapment of doxycycline and rifampicin in lipo-somes was determined by the spectrophotometric

method as described earlier (Bajpai et al., 2005). Briefly, liposomes were lyzed using 1% (w/v) Triton X-100 in 0.1 M PBS to release the entrapped antibiotics. To remove any possibility of suspended particulates before spectro-photometric analysis, liposome preparations were centri-fuged (at 10,000 g for 15 min at 4°C) and the supernatants were collected. Absorbance values in the supernatant of both doxycycline and rifampicin were determined at 274 nm and 231 nm, respectively. The amount of drug entrapped in both the formulations was calculated using standard curve plot of doxycycline and rifampicin, pre-pared using various known concentrations of both the antibiotics through same protocol.

Determination of in vivo drug release

The formulated and free doxycycline and rifampicin were subcutaneously (s.c.) injected in four separate groups of mastomys (males of ∼100 gm body weight), each con-sisting of five animals. A control group of five animals was also included which received sham liposomes only (without drug). For serum collection, animals of each were bled (∼50 µL) through retro-orbital plexus asepti-cally at different time intervals (0, 6, 12, 18, 24, 30, 42, and 48 h). Blood samples collected at one time frame from each animal group were pooled. In vivo release of drug at various time intervals was determined in serum sam-ples as described elsewhere (Billová et al., 2003). Briefly, sterile 96 wells plastic microplates were filled with 100 µL Luria Broth and each well was inoculated with 5 μL of Escherichia coli suspension prepared by the direct colony suspension method and diluted to reach turbidity that matched the optical density corresponding to 1.5 × 108 colony forming units per milliliter (CFU/mL). Twenty-five microliters of each serum sample was inoculated separately with the same growing suspension. Finally, after overnight incubation of plates at 37°C the optical density (OD600) of each well was measured (Tecan M200, Switzerland). More the inhibition of bacterial growth (i.e., lowered OD600 value) in wells containing serum of drug treated animals was indicative of higher drug concen-tration in the serum. Finally, antibiotic concentration in serum was determined with the help of a standard curve plot for both antibiotics, which were prepared employ-ing the same protocol with known amount of these drugs mixed with control animal sera.

Animal model and infection

Male mastomys of same age (6–8 weeks) were used as experimental animal model. These animals were fed on rodent diet and water ad libitum and housed in hygienic and standard conditions of light (12 h light/12 h dark) and temperature (∼28°C). The infective larvae (L3) were recovered from Aedes aegypti fed on infected donor

Jour

nal o

f D

rug

Tar

getin

g D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y N

atio

nal L

ibra

ry o

f H

ealth

Sci

ence

s on

10/

05/1

1Fo

r pe

rson

al u

se o

nly.

Improved antifilarial efficacy of liposome formulated doxycycline and rifampicin 345

mastomys 9 ± 1 day earlier (Singh et al., 1997). Infection in mastomys was given by inoculation of 100 L3 of sub-periodic strain of human lymphatic filariid B. malayi in the back region subcutaneously. All the animals and the experimental procedures included in the present study were duly approved by the Institutional Animal Ethics Committee.

Experimentation and treatment schedule

Fifty mastomys with patent infection (4 months postin-fective larvae inoculation) showing progressive rise in microfilaraemia, were divided into ten groups each comprising of five animals. Of these, two separate groups received five doses of free doxycycline (Fr-Dox) or rifampicin (Fr-Rif) subcutaneously at every third day (at 72 h interval). Similarly two other groups were treated individually with five doses of liposomal formulations (Li-Dox and Li-Rif). In addition, two more groups were included each receiving liposomal antibiotic along with an oral dose of DEC (50 mg/kg) (Lip-Dox, Dec and Lip-Rif, Dec). One separate group received a combination of two antibiotic liposome formulations followed by an oral dose of DEC (Li-Dox,Li-Rif,Dec). One more group treated with five doses of free doxycycline plus rifampicin followed by an oral dose of free DEC was kept as control for comparison (Fr-Dox,Fr-Rif,Dec). The two group’s one receiving five doses of sham liposome s.c. and the other receiving only PBS were also kept as controls. One more group of five mastomys received free DEC to serve as DEC control. All the treatment regimens are summarized in Table 1.

Evaluation of microfilaraemia and macrofilaricidal efficacy

Microfilarial counts of treated and control mastomys were evaluated in 10 µL of tail blood collected by needle prick between 12:00 and 12:45 h (microfilaraemia at its peak in peripheral circulation) as described earlier (Bajpai et al., 2005) just before start of treatment and at various time points (Table 2) after treatment. Briefly, the blood was

spread into a thick smear on a clean glass slide with a needle; air dried for 24 h, dehaemoglobinized with tap water and stained with 0.15% Leishman stain for 20 min. The slides were washed with tap water, air dried and examined under the compound microscope for counting the number of microfilariae per slide to assess percent change in mf density at different periods before and after treatment.

At the end of observation period, animals from all the treated and control groups were euthanized on day 120 and their visceral organs namely, lungs, testes, heart, and peripheral lymph nodes were isolated and dissected carefully in PBS to recover the adult parasites. The adult female worms were examined for their uterine contents to assess embryostatic effects of both the formulations.

Statistical analysis

Data were analyzed employing nonparametric Student’s t-test (Mann–Whitney test) and one-way analysis of vari-ance (ANOVA) as appropriate. Individual comparisons following ANOVA were made using a posttest (Bonferroni) with the help of statistical software PRISM 3.0. Results of mf density, adult parasites mortality have been presented as mean ± SEM. The criterion for statistical significance (P < 0.05) between antibiotic treated and their relevant controls was considered as significant.

Results

Encapsulation of doxycycline and rifampicin and in vivo release

The encapsulation efficiency of doxycycline and rifampicin in neutral liposomes was found to be 45 ± 5% and 80 ± 2% or 0.35 and 0.61 mg/mg lipid, respectively. Figure 1 shows that the peak concentration of doxycy-cline (8.8 µg/mL) or rifampicin (8.6 µg/mL) was observed by 12 h postadministration of the antibiotic formulation. Both the formulations showed sustained release of drugs in the circulation up to the end of observation period

Table 1. Animal grouping, antibiotic formulations, dose and treatment schedules, and the day of animal autopsy.

Group Doxycycline 10 mg/kg, s.c. Rifampicin 10 mg/kg, s.c. DEC 50 mg/kg, oralDay of treatment with

antibiotic/DEC Day of autopsy

Fr-Dox Free – – 1, 4, 7, 10, 13 120

Li-Dox Formulated – – 1, 4, 7, 10, 13 120

Li-Dox,Dec Formulated – Free 1, 4, 7, 10, 13/14 120

Fr-Rif – Free – 1, 4, 7, 10, 13 120

Li-Rif – Formulated – 1, 4, 7, 10, 13 120

Li-Rid,Dec – Formulated Free 1, 4, 7, 10, 13/14 120

Fr-Dox,Fr-Rif,Dec Formulated Formulated Free 1,4,7,9,13/14 120

Li-Dox,Li-Rif,Dec Free Free Free 1,4,7,9,13/14 120

Fr-DEC – – Free –/14 120

Control – – – – 120

Jour

nal o

f D

rug

Tar

getin

g D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y N

atio

nal L

ibra

ry o

f H

ealth

Sci

ence

s on

10/

05/1

1Fo

r pe

rson

al u

se o

nly.

346 Anil Dangi et al.

(48 h). The concentrations of doxycycline and rifampicin at 48 h were 6.9 µg/mL and 6.7 µg/mL, respectively which were still quite higher than the amount of antibiotic released by 6 h postsubcutaneous administration of for-mulations and their minimal inhibitory concentrations (MIC, 4 µg/mL). Apart from this, when free form of these was administered via same route, rapidly attained their peak and decreased below their MICs after 18 and 6 h, respectively, and these are plasma half lives of both the antibiotics.

Microfilaricidal efficacy

The entrapped doxycycline treatment regimen (Li-Dox) with highly reduced number of dosing frequency (total five doses, once every 72 h) at a low dose of 10 mg/kg was capable of reducing microfilaraemia. Of course, the reduction in mf density was prolonged, however, mf level started gradually decreasing from day 20, where 15.18% reduction in pretreatment level was observed which sub-sequently decreased further and microfilariae completely disappeared by day 110. Conversely, the regimen of free doxycycline (Fr-Dox) using the same dose and route demonstrated a gradual increase in microfilaraemia, however, this increase was significantly less (P<0.001) from day 80 to the end of observation period posttreat-ment in comparison to untreated infected controls. Liposomal doxycycline treatment followed by a single dose of DEC (Li-Dox, Dec) further improved the micro-filaricidal efficacy of the formulated antibiotic thereby depleting mf density completely within 80 days poston-set of the regimen. A single dose of DEC alone on day 14 also caused significant (P<0.001) reduction (∼41%) in

microfilaraemia which remained below pre-treatment level up to day 50 (Table 2).

In addition, five doses of liposomal rifampicin (Li-Rif) at 10 mg/kg also exhibited antimicrofilarial efficacy. Postonset of this regimen, mf started decreasing gradu-ally below pretreatment level from day 60 (30.64%), reducing further and totally disappeared from blood on day 120. In contrast, mastomys treated with free rifampicin via subcutaneous route at 10 mg/kg (Fr-Rif) revealed no effect on mf density and mf pattern was almost similar to that of untreated infected controls. The microfilaraemia in mastomys treated with entrapped rifampicin plus DEC (Li-Rif,Dec) reduced by 57.75% on day 20 and remained below the pre-treatment level and later on microfilariae disappeared completely by day 100 (Table 2). Multi drug treatment with liposome formulated doxycycline and rifampicin plus DEC (Li-Dox,Li-Rif,Dec) demonstrated significantly (P<0.01) higher reduction in mf density on day 20 (88.1%) with total disappearance of microfilaraemia from blood on day 80 following initia-tion of treatment. In contrast, a similar treatment with free antibiotic combination followed by DEC treatment (Fr-Dox,Fr-Rif,Dec), did not bring about any significant microfilaricidal action (Table 2). The data of microfila-raemia pattern in both the controls (normal and sham) were almost similar and therefore displayed as pooled data (Table 2).

Macrofilaricidal efficacy and effects on intrauterine contents

Only five doses of encapsulated doxycycline alone or in combination with DEC (Li-Dox or Li-Dox,Dec) also caused significant (P<0.001) mortality of adult worms (55.93% and 62.71%, respectively). Besides retarding the worm endurance, both of these regimens affected the fecundity of worms as well causing sterilization in 86% of the surviving females. Conversely, free doxycycline at similar doses failed to impart any significant detri-mental effects on adult worms; however, low degree of worm mortality (18.64%) could be observed in this group (Figure 2). DEC also did not show any significant adulti-cidal or embryostatic action just like free doxycycline.

Sustained release of rifampicin using liposomal for-mulation (Li-Rif) was also found effective which rendered 49.15% worm death, while 90% of the surviving females were absolutely deprived of developmental stages. In contrast, free rifampicin proved completely ineffective. DEC in a single dose when given a day after completion of liposomal rifampicin therapy (Li-Rif,Dec) significantly augmented (P<0.001) the worm mortality (61.02%) with no further improvement in female worm sterility (88%). Maximal worm mortality (74.58%) was observed in mastomys treated with a combination of two antibiotic liposomal formulations (doxycycline and rifampicin)

12

Ant

ibio

tic c

onc.

(µg/

mL)

10

8

6

4

2

0

0 h 6 h

Li-Dox Li-Rif Fr-Dox Fr-Rif

12 h 18 h 24 h 30 h 42 h 48 h

Figure 1. In vivo release of entrapped and free form of doxycycline and riampicin in permissive rodent model M. coucha. Liposomal for-mulation and free form of both the antibiotics was administered at 10 mg/kg and blood was collected at 6 h interval from retro-orbital plexus and samples from each group at each time point were pooled. Highest concentration of both the antibiotics in blood circulation was reached at 12 h which was maintained above their MICs (4 µg/mL) up to 48 h postadministration of the formulations. Peak concentration of antibiotics in serum when administered subcutaneously in free form last only for 18 and 6 h for doxycycline and rifampicin, respectively.

Jour

nal o

f D

rug

Tar

getin

g D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y N

atio

nal L

ibra

ry o

f H

ealth

Sci

ence

s on

10/

05/1

1Fo

r pe

rson

al u

se o

nly.

Improved antifilarial efficacy of liposome formulated doxycycline and rifampicin 347

Tabl

e 2.

Mic

rofi

lari

cid

al e

ffica

cy o

f diff

eren

t reg

imen

s of

free

an

d li

pos

omiz

ed fo

rmu

lati

ons

of d

oxyc

yclin

e, r

ifam

pic

in a

nd

thei

r co

mb

inat

ion

wit

h s

tan

dar

d fi

lari

cid

e D

EC

.

Ch

ange

(m

ean

± S

EM

) in

mic

rofi

lari

al d

ensi

ty p

er 1

0 µL

of t

ail b

lood

du

rin

g ob

serv

atio

n p

erio

d, p

ostt

reat

men

t (%

)

Day

10

Day

20

Day

30

Day

40

Day

50

Day

60

Day

70

Day

80

Day

90

Day

100

Day

110

Day

120

Fr-D

ox23

.6±

8.5

66.4

±13

.512

3.6±

13.9

174.

5±11

.024

7.3±

10.1

320.

0±23

.438

5.5±

20.0

440.

0±14

.656

1.8±

37.5

744.

5±65

.780

3.6±

64.7

837.

3±46

.7

Li-D

ox

44.6

±28

.01.

8±35

.8−

14.6

±32

.1−

19.1

±36

.9−

21.8

±30

.3−

44.6

±14

.3−

51.8

±16

.8−

53.6

±15

.0−

75.5

±10

.1−

97.3

±1.

8−

100.

0±0.

0−

100.

0±0.

0

Li-D

ox,D

ec

29.2

±11

.2−

32.6

±12

.9−

73.0

±5.

9−

87.6

±3.

2−

92.1

±5.

2−

93.3

±4.

1−

97.8

±2.

2−

100.

0±0.

0−

100.

0±0.

0−

100.

0±0.

0−

100.

0±0.

0−

100.

0±0.

0

Fr−

Rif

62

.2±

9.2

151.

4±24

.923

7.8±

24.3

296.

4±38

.940

4.5±

44.4

528.

8±52

.865

2.3±

59.1

856.

8±96

.610

04.0

±94

.011

76.0

±99

.413

26.0

±90

.314

50.0

±90

.9

Li-R

if

46.5

±10

.349

.0±

14.4

55.7

±34

.336

.5±

11.2

9.6±

17.5

30.7

±19

.1−

22.1

±11

.9−

37.5

±10

.4−

52.8

±8.

5−

55.7

±5.

5−

88.4

±2.

4−

100.

0±0.

0

Li−

Rif,

Dec

49

.0±

10.6

−57

.7±

5.9

−1.

0±23

.3−

2.9±

32.1

−21

.2±

26.0

−23

.1±

23.5

−62

.5±

9.7

−88

.5±

4.4

−93

.3±

2.8

−10

0.0±

0.0

−10

0.0±

0.0

−10

0.0±

0.0

Fr-D

ox,F

r-R

if,D

ec

31.6

±12

.8−

81.6

±7.

0−

42.7

±6.

2−

26.5

±11

.2−

3.7±

8.5

21.3

±9.

834

.6±

12.7

79.4

±8.

181

.6±

20.4

107.

4±9.

213

0.9±

13.5

150.

7±14

.4

Li−

Dox

,Li-

Rif,

Dec

48

.1±

13.8

−88

.1±

3.8

−73

.8±

4.1

−70

.6±

7.1

−77

.5±

5.0

−81

.3±

3.2

−91

.3±

1.8

−10

0.0±

0.0

−10

0.0±

0.0

−10

0.0±

0.0

−10

0.0±

0.0

−10

0.0±

0.0

Fr−

DE

C60

.5 ±

13.8

−40

.9 ±

6.1

−33

.0 ±

4.5

−20

.1 ±

4.0

−5.

0 ±

2.0

11.7

±4.

835

.4 ±

7.4

64.0

±7.

410

5.6

±10

.216

2.2

±10

.822

1.9

±15

.631

7.7

±21

.8

Con

trol

10

0.0±

25.0

161.

7±31

.623

9.2±

26.2

310.

0±18

.938

1.7±

20.9

469.

2±24

.358

5.0±

26.9

803.

3±65

.110

89.0

±84

.711

97.0

±81

.111

48.0

±93

.415

17.0

±11

3.8

Jour

nal o

f D

rug

Tar

getin

g D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y N

atio

nal L

ibra

ry o

f H

ealth

Sci

ence

s on

10/

05/1

1Fo

r pe

rson

al u

se o

nly.

348 Anil Dangi et al.

along with standard filaricide DEC (Li-Dox,Li-Rif,Dec). Both the antibiotics in free form plus DEC resulted into a marginal (25.42%) adulticidal action with moderate (50%) effect on embryogenesis (Figure 2).

Discussion

Recent advancement and successes in the control of lym-phatic filariasis and onchocerciasis employing antiwol-bachial therapy has offered a comprehensive remedy for these diseases. Although, prolonged treatment course of doxycycline could be carried out by community-directed treatment at good coverage (Specht & Wanji, 2009), nevertheless, involvement of alternative strategies like drug delivery approaches would definitely improve the efficacy as well as the population coverage significantly. An increase in the therapeutic index of the antibiotics after liposomal entrapment has been well documented in various diseases as precisely reviewed by Bakker-Woudenberg et al. (1995). Better efficacy of liposome-encapsulated tetracycline and doxycycline against C. trachomatis in vitro has already been reported (Sangaré et al., 1999).

The present investigation is based on our previous study in which antifilarial efficacy of liposome encap-sulated tetracycline was evaluated (Bajpai et al., 2005) and this is discussed here for a comparative analysis with other two antibiotics. The entrapment of doxycycline in the present study was 45% higher than tetracycline (25%)

evaluated earlier. Sangaré et al. (1998) had also demon-strated that doxycycline was more readily entrapped in the liposomes than tetracycline. Apart from this, rifampicin illustrated very high encapsulation (80%), due to superior solubility in the solvents used. In the present investigation, the encapsulation of both the antibiotics was higher than the tetracycline therefore, in the in vivo experiment concentrations were monitored up to 48 h. Both the formulations showed sustained release of anti-biotics, although, it starts gradually decreasing after 42 h, however, serum concentrations were still higher than their MICs up to 48 h. On the other hand, serum con-centration of doxycycline (above its MIC) remained up to 18 h when administered freely, whereas, this duration was even less than 12 h for rifampicin. This trend dem-onstrates that entrapment led to slow and continuous release of antibiotics thereby considerably increasing the availability of drugs in the circulation for a longer dura-tion over free drug administration. Thus, both the drugs were administered at an interval of 72 h at 10 mg/kg. In the present study, though, polydispersity index and zeta potential of the liposomes were not determined as this is a conventional way of drug delivery and multitude of studies has already been published these data. However, to be very sure about the preparation, the average size of liposomal formulation was determined. The size of the liposomes was in the range of 50–100 nm. The majority of the liposomes were found to be unilamellar, but some of them were multilamellar.

The data accumulated from the present study unequiv-ocally demonstrates that merely five doses of entrapped doxycycline and rifampicin were found to be effective in complete clearance of mf load, markedly higher death of adult worms and their sterilization in contrast to free drug administration via subcutaneous route. The most noticeable observation was that the entrapped doxycy-cline alone also showed significant (P<0.01) mf reduction on day 30, while entrapped rifampicin demonstrated sig-nificant (P<0.01) mf reduction much later, that is, by day 60. These findings again substantiate earlier observation (Turner et al., 2006; Specht et al., 2008) that doxycycline possesses superior microfilaricidal efficacy as compared to tetracycline and rifampicin. Sangaré et al. (1999, 2001) also demonstrated that entrapped doxycycline imparts a beforehand activity against tetracycline and rifampicin in chlamydial infections as well. A combination of encapsu-lated doxycycline or rifampicin with standard filaricide DEC in the present study was found to be superior in microfilaricidal efficacy over single entrapped antibi-otic. DEC is, principally, a strong microfilaricidal drug and it imparted an additive effect on the microfilaricidal efficacy of both the antibiotic formulations. Moreover, combination of both the antibiotics in free form or lipo-some form with DEC exerted synergistic microfilaricidal action. This synergistic effect was also observed when

100

Fr-D

ox

Li-D

ox

Li-D

ox, F

r-D

ec

Fr-R

if

Li-R

if

Li-R

if, F

r-D

ec

Fr-D

ox, F

r-R

if, F

r-D

ec

Li-D

ox, L

i-Rif,

Fr-

Dec

Fr-D

ec

80

60

40

20

0

Mortality Infertility

Mor

talit

y of

adu

ltw

orm

s an

d fe

mal

e in

ferti

lity

(%)

Figure 2. Adulticidal and sterilization efficacy of different treatment regimens of doxycycline. The brief treatment regimens (only five doses) of the liposomal formulation of both antibiotics alone (Li-Dox and Li-Rif ) and their combination with DEC (Li-Dox,Dec and Li-Rif,Dec) significantly increased these efficacies in contrast to administration of free doxycycline (Fr-Dox) and rifampicin (Fr-Rif ). A combination of both the formulations with DEC (Li-Dox,Li-Rif,Dec) demonstrated markedly higher (P<0.001) mortality of 74.58% of adult worms in con-trast to free antibiotics with DEC.

Jour

nal o

f D

rug

Tar

getin

g D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y N

atio

nal L

ibra

ry o

f H

ealth

Sci

ence

s on

10/

05/1

1Fo

r pe

rson

al u

se o

nly.

Improved antifilarial efficacy of liposome formulated doxycycline and rifampicin 349

both antibiotics administered in either free or formulated form in combination with DEC.

Apart from the microfilaricidal efficacy, the animals treated with these antibiotic formulations also showed remarkable adulticidal as well as female worm steriliza-tion activity over free drug administration. The combina-tion of individual entrapped antibiotic with DEC exhibited marginally better macrofilaricidal efficacy than any of the single formulation. The combined administration of both entrapped antibiotics plus DEC revealed strong macro-filaricidal (adulticidal) activity (74.58%); however, female worm sterilization remained almost unchanged (∼86%). A synergistic effect of both the antibiotics on adult para-sites was observed as also described earlier (Volkmann et al., 2003; Karabay et al., 2004), nevertheless, this was not the case with microfilaricidal efficacy. Free antibiot-ics in combination with DEC displayed low antifilarial efficacy.

It is a general belief that reduction in microfilaraemia, loss of female worm fecundity and adult worm mortal-ity after a course of antirickettsial antibiotic is followed by the depletion of endosymbiont Wolbachia (Chirgwin et al., 2003a,b; Bajpai et al., 2005; Shakya et al., 2008). In the present study, though, we did not determine the wolbachial status in recovered worms, the significant female worm infertility and mortality of adults directly substantiates the antiwolbachial activity of the anti-biotics used. It is established by several workers that antifilarial efficacy of these antibiotics is the secondary outcome with primary being wolbachial death. However, the results obtained in the present study indicate that doxycycline exhibited beforehand microfilaricidal activ-ity over tetracycline and rifampicin, if administered as entrapped with standard filaricide DEC. Second, this approach significantly reduced the antifilarial treatment schedule.

Acknowledgements

Authors SMB and AD conceived the idea, designed experimentation and written the manuscript, AD and SV executed all the experiments, MO and VD helped in preparation of liposomal formulations. We are thank-ful to Mr. A. K. Roy and R. N. Lal for their excellent technical assistance in infection maintenance. The financial assistance in the form of the research fel-lowship from the Council of Scientific and Industrial Research, New Delhi to two of us (AD, and SV) is grate-fully acknowledged.

Declaration of interest

The authors have declared no conflict of interest.

References

Bajpai P, Vedi S, Owais M, Sharma SK, Saxena PN, Misra-Bhattacharya S. (2005). Use of liposomized tetracycline in elimination of Wolbachia endobacterium of human lymphatic filariid Brugia malayi in a rodent model. J Drug Target, 13: 375–381.

Bakker-Woudenberg IA, ten Kate MT, Stearne-Cullen LE, Woodle MC. (1995). Efficacy of gentamicin or ceftazidime entrapped in lipo-somes with prolonged blood circulation and enhanced localiza-tion in Klebsiella pneumoniae-infected lung tissue. J Infect Dis, 171: 938–947.

Bandi C, Anderson TJ, Genchi C, Blaxter ML. (1998). Phylogeny of Wolbachia in filarial nematodes. Proc Biol Sci, 265: 2407–2413.

Billová S, Kizek R, Jelen F, Novotná P. (2003). Square-wave voltammetric determination of cefoperazone in a bacterial culture, pharmaceu-tical drug, milk, and urine. Anal Bioanal Chem, 377: 362–369.

Bosshardt SC, McCall JW, Coleman SU, Jones KL, Petit TA, Klei TR. (1993). Prophylactic activity of tetracycline against Brugia pahangi infection in jirds (Meriones unguiculatus). J Parasitol, 79: 775–777.

Chirgwin SR, Coleman SU, Porthouse KH, Nowling JM, Punkosdy GA, Klei TR. (2003a). Removal of Wolbachia from Brugia pahangi is closely linked to worm death and fecundity but does not result in altered lymphatic lesion formation in Mongolian gerbils (Meriones unguiculatus). Infect Immun, 71: 6986–6994.

Chirgwin SR, Nowling JM, Coleman SU, Klei TR. (2003b). Brugia pahangi and Wolbachia: the kinetics of bacteria elimination, worm viability, and host responses following tetracycline treat-ment. Exp Parasitol, 103: 16–26.

Debrah AY, Mand S, Marfo-Debrekyei Y, Batsa L, Pfarr K, Buttner M, Adjei O, Buttner D, Hoerauf A. (2007). Macrofilaricidal effect of 4 weeks of treatment with doxycycline on Wuchereria bancrofti. Trop Med Int Health, 12: 1433–1441.

Gilbert J, Nfon CK, Makepeace BL, Njongmeta LM, Hastings IM, Pfarr KM, Renz A, Tanya VN, Trees AJ. (2005). Antibiotic chemo-therapy of onchocerciasis: in a bovine model, killing of adult par-asites requires a sustained depletion of endosymbiotic bacteria (Wolbachia species). J Infect Dis, 192: 1483–1493.

Hermans PG, Hart CA, Trees AJ. (2001). In vitro activity of antimi-crobial agents against the endosymbiont Wolbachia pipientis. J Antimicrob Chemother, 47: 659–663.

Hoerauf A, Mand S, Adjei O, Fleischer B, Büttner DW. (2001). Depletion of Wolbachia endobacteria in Onchocerca volvulus by doxycycline and microfilaridermia after ivermectin treatment. Lancet, 357: 1415–1416.

Hoerauf A, Mand S, Fischer K, Kruppa T, Marfo-Debrekyei Y, Debrah AY, Pfarr KM, Adjei O, Büttner DW. (2003). Doxycycline as a novel strategy against bancroftian filariasis-depletion of Wolbachia endosymbionts from Wuchereria bancrofti and stop of microfilaria production. Med Microbiol Immunol, 192: 211–216.

Karabay O, Sencan I, Kayas D, Sahin I. (2004). Ofloxacin plus rifampicin versus doxycycline plus rifampicin in the treatment of brucellosis: a randomized clinical trial (ISRCTN11871179). BMC Infect Dis, 4: 18.

McCall JW, Jun JJ, Bandi C. (1999). Wolbachia and the antifilarial properties of tetracycline: aAn untold story. Italian J Zool, 66: 7–10.

McLaren DJ, Worms MJ, Laurence BR, Simpson MG. (1975). Micro-organisms in filarial larvae (Nematoda). Trans R Soc Trop Med Hyg, 69: 509–514.

Owais M, Misra-Bhattacharya S, Haq W, Gupta CM. (2003). Immunomodulator tuftsin augments antifilarial activity of diethylcarbamazine against experimental brugian filariasis. J Drug Target, 11: 247–251.

Rao RU, Moussa H, Weil GJ. (2002). Brugia malayi: effects of antibac-terial agents on larval viability and development in vitro. Exp Parasitol, 101: 77–81.

Ridtitid W, Wongnawa M, Mahatthanatrakul W, Pattanawongsa A, Bumrungwong N, Sunbhanich M. (2006). Effects of rifampicin and ketoconazole on the pharmacokinetics of a single oral dose of diethylcarbamazine in healthy volunteers. Songkla Med J, 24: 527–536.

Jour

nal o

f D

rug

Tar

getin

g D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y N

atio

nal L

ibra

ry o

f H

ealth

Sci

ence

s on

10/

05/1

1Fo

r pe

rson

al u

se o

nly.

350 Anil Dangi et al.

Sangaré L, Morisset R, Omri A, Ravaoarinoro M. (1998). Incorporation rates, stabilities, cytotoxicities and release of liposomal tetracy-cline and doxycycline in human serum. J Antimicrob Chemother, 42: 831–834.

Sangaré L, Morisset R, Ravaoarinoro M. (1999). In-vitro anti-chlamy-dial activities of free and liposomal tetracycline and doxycycline. J Med Microbiol, 48: 689–693.

Sangaré L, Morisset R, Gaboury L, Ravaoarinoro M. (2001). Effects of cationic liposome-encapsulated doxycycline on experimental Chlamydia trachomatis genital infection in mice. J Antimicrob Chemother, 47: 323–331.

Shakya S, Bajpai P, Sharma S, Misra-Bhattacharya S. (2008). Prior kill-ing of intracellular bacteria Wolbachia reduces inflammatory reactions and improves antifilarial efficacy of diethylcarbamazine in rodent model of Brugia malayi. Parasitol Res, 102: 963–972.

Singh U, Misra S, Murthy PK, Katiyar JC, Agrawal A, Sircar AR. (1997). Immunoreactive molecules of Brugia malayi and their diagnostic potential. Serodiag Immunother Infect Dis, 8: 207–212.

Singleton WS, Gray MS, Brown ML, White JL. (1965). Preparation of egg yolk lecithin. J Am Oil Chem Soc, 42: 53–56.

Specht S, Mand S, Marfo-Debrekyei Y, Debrah AY, Konadu P, Adjei O, Büttner DW, Hoerauf A. (2008). Efficacy of 2- and 4-week rifampicin treatment on the Wolbachia of Onchocerca volvulus. Parasitol Res, 103: 1303–1309.

Specht S, Wanji S. (2009). New insights into the biology of filarial infec-tions. J Helminthol, 83: 199–202.

Supali T, Djuardi Y, Pfarr KM, Wibowo H, Taylor MJ, Hoerauf A, Houwing-Duistermaat JJ, Yazdanbakhsh M, Sartono E. (2008). Doxycycline treatment of Brugia malayi-infected persons reduces microfilaremia and adverse reactions after diethyl-carbamazine and albendazole treatment. Clin Infect Dis, 46: 1385–1393.

Taylor MJ, Hoerauf A. (1999). Wolbachia bacteria of filarial nematodes. Parasitol Today (Regul Ed), 15: 437–442.

Taylor MJ, Makunde WH, McGarry HF, Turner JD, Mand S, Hoerauf A. (2005). Macrofilaricidal activity after doxycycline treatment of Wuchereria bancrofti: a double-blind, randomized placebo-controlled trial. Lancet, 365: 2116–2121.

Turner JD, Mand S, Debrah AY, Muehlfeld J, Pfarr K, McGarry HF, Adjei O, Taylor MJ, Hoerauf A. (2006). A randomized, double-blind clinical trial of a 3-week course of doxy-cycline plus albendazole and ivermectin for the treat-ment of Wuchereria bancrofti infection. Clin Infect Dis, 42: 1081–1089.

Volkmann L, Fischer K, Taylor M, Hoerauf A. (2003). Antibiotic ther-apy in murine filariasis (Litomosoides sigmodontis):comparative effects of doxycycline and rifampicin on Wolbachia and filarial viability. Trop Med Int Health, 8: 392–401.

Jour

nal o

f D

rug

Tar

getin

g D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y N

atio

nal L

ibra

ry o

f H

ealth

Sci

ence

s on

10/

05/1

1Fo

r pe

rson

al u

se o

nly.