Clinical and Experimental Pharmacology and Physiology

(2003)

30

,

153–156

DISPOSITION KINETICS OF KETOTIFEN FROM LIPOSOMAL DRY POWDER FOR INHALATION IN RAT LUNG

Mayank Joshi and Ambikanandan Misra

Pharmacy Department, Faculty of Technology and Engineering, Kalabhavan, MS University of Baroda, Baroda, Gujarat, India

SUMMARY

1. The aim of the present study was to understand thebenefit of liposomal dry powder for inhalation (LDPI) ofketotifen fumarate (KF) over plain drug dry powder forinhalation as a pulmonary targetted drug-delivery system.

2. The KF liposomes, composed of egg phosphatidyl cholineand cholesterol, were prepared by the lipid film hydrationtechnique. The liposomal dispersion was freeze dried andformulated to a dry powder for inhalation. Values of 89.0–65.3% drug entrapment of freeze-dried liposomes wereestimated in prepared batches.

3. Rehydrated KF liposomes formed by the hydration ofLDPI or the plain KF solution was delivered to rat lungs byintratracheal instillation. Simultaneous monitoring of druglevels in the bronchoalveolar lavage and lung tissue enabledassessment of pulmonary drug disposition.

4. Cumulative drug levels in lung tissue after intratrachealadministration revealed that with liposomes targetting factorswere between 1.36 and 1.54. The maximal drug concentrationin lung homogenate for LDPI was 42.0

�

g compared with73.6

�

g for plain drug solution.5. Similarly, the time to reach maximum drug concentration

in the lung homogenate for liposomal dry powder was 9–12 hcompared with 3 h for plain drug.

6. Hence, the use of LDPI of KF was found to providedesired drug levels in the lung for a long time and therebyincreased pulmonary targetting

7. This is expected to enhance the therapeutic index of thedrug and probably reduce the dose administered and the costof therapy.

Key words: dry powder, inhalation, ketotifen fumarate,liposomes, pulmonary.

INTRODUCTION

Inhalation therapy is an effective means of delivering relativelysmall doses of an active ingredient directly to the respiratorysystem. Lung localization of drug maximizes the therapeutic effect

while minimizing unwanted systemic activity or toxicity. Althoughthe onset of action is very rapid, duration is short lived because thedrug is rapidly removed from the lung.

1

Hence, regardless of thetype of aerosol used (i.e. meter-dose inhaler or nebulizer), mostpatients require dosing every 6–8 h and often more frequently.

2

Drug distribution limited to the intended site of action in the lung,maintenance of prolonged therapeutic levels of drug and slowsystemic dilution of the drug are the necessary characteristics to bedeveloped pharmaceutically in pulmonary drug-delivery systemsfor maximizing the therapeutic index of a drug, reducing the dose,duration of therapy and systemic side-effects and, thereby,reducing the cost of therapy.

The use of liposomes for pulmonary delivery was first investi-gated as a potential treatment for respiratory distress syndrome.

3

Subsequent studies have indicated that liposomes have an inherentcapacity to act as a drug carrier system for local pulmonarytreatment. McCullough and Juliano

4

demonstrated that

�

-cystosinearabinoside was cleared rapidly from the lung when administeredas a solution, whereas a liposomal formulation of the drugdisplayed little redistribution to other tissues and was retainedthroughout all bronchial spaces. Similarly, Woolfrey

et al

.

5

foundthat liposomal encapsulation prolonged absorption of 6-carboxy-flurescin in the rat lung while decreasing its rate of systemicabsorption when compared with a solution.

These findings highlight the potential benefits of liposomes as apulmonary drug-delivery system (i.e. selective and prolongedpharmacological activity of liposome-encapsulated drugs). Thedifficulty in the application of liposomes had been their long-termstability. However, Gursoy and Akbuga

6

demonstrated that lyophi-lization improved the retention of liposome-associated indometh-acin during long-term storage. Beaulac

et al

.

7

have further addedto optimizing this delivery system by demonstrating the feasibilityof delivering liposomal tobramycin as a dry powder aerosol. Thepresent investigation was an attempt to continue these studies. Aliposomal dry powder for inhalation (LDPI) formulation of keto-tifen fumarate (KF) was developed to specifically target the lungsand to reduce the rate of systemic availability. Herein we show, inan albino rat model, the efficacy of LDPI of KF in the preventivemanagement of asthma.

METHODS

Materials

Ketotifen fumarate was a gift from Torrent Pharmaceuticals (Ahmedabad,India). Other materials used were egg phosphatidyl choline (EPC; SigmaChemicals, Mumbai, India), cholesterol (SD Fine Chemicals, Baroda,India),

�

-tocopherol (E Merck India, Mumbai, India), lactose (Sorbolac-

Correspondence: Ambikanandan Misra, Pharmacy Department, Facultyof Technology and Engineering, Kalabhavan, MS University of Baroda,Baroda 390 001, Gujarat, India. Email: misraan@hotmail.com

Received 7 March 2002; revision 20 September 2002; accepted23 September 2002.

154

M Joshi and A Misra

400; Meggle, Wasserburg, Germany) and sucrose (Pokkie OdcaynnlkiChemicals, Warsaw, Poland). All other reagents and chemicals used wereof analytical or pharmacopoeia grade.

Albino rats, weighing 200–240 g, were procured from Deep Biolabs(Ahmedabad, India). This study was performed in accordance with theGuidelines for the Care and Use of Laboratory Animals as adopted andpromulgated by the Animal Ethics Committee.

Preparation

Multilamellar vesicles (MLV) of KF were prepared by the modifiedBangham method, as reported by Juliano and Daoud.

8

Ketotifen fumarate(20 mg), EPC, cholesterol and

�

-tocopherol (1% of EPC) were coprecipi-tated to a thin film in the ratios shown in Table 1, using 15 mL solventsystem (CHCl

3

: CH

3

OH = 2 : 1) in a rotary flash evaporator, under vacuumand with a bleed of nitrogen. The dried film was hydrated at 25

±

2

�

C for1 h with 5 mL phosphate-buffered saline (PBS), pH 7.4 (0.17

�

ionicstrength) containing 1 mmol/L EDTA and 500 mmol/L sucrose. The lipo-somal dispersion so formed was subjected to ultrasonic downsizing undera nitrogen atmosphere in an ice bath for 30 min. Sonicated vesicles werestabilized by hydration for 2 h at ambient temperature and separated fromfree drug by dialysis through a cellophane membrane (MW cutoff12 000 Da).

9

The dispersion was analysed for percentage drug entrapmentin liposomes and the receptor solvent was analysed for the free drug,removed during the dialysis of the liposomal dispersion (Table 1). In thepreparation

9

of LDPI, the liposomal dispersion was diluted with sufficienthydrating medium to obtain a lipid : sugar ratio of 1 : 12. An equivalentproportion of sorbolac calculated to have a final strength of 100

�

g entrapeddrug (in purified liposomal dispersion) per 200 mg formulation wasdispersed into the diluted liposomal dispersion. The paste so formed wasfrozen at –70

�

C overnight and dried under negative displacement pressure(model DW1 0-60E; Heto Drywinner, Birkerod, Denmark) for 24 h. Theporous cake thus formed was sized successively through #120 and #240sieves and rotated in a planetary ball mill (200 r.p.m) without balls for10 min for further deaggregation of the particles. Capsules (size ‘2’) werefilled with individually weighed powder (200 mg) containing 100

�

g KFand packed under nitrogen atmosphere in high-density polyethylene(HDPE) bottles containing silica bags as desiccant. The bottle with desic-cant was sealed with polyvinyl chloride-coated aluminium foils and storedin a refrigerator (2

�

– 8

�

C) until further use. A fraction of the powder wasrehydrated with triple-distilled water with gentle, occasional agitation. Therehydrated liposomal dispersion was separated from the leaked drug bydialysis

9

and analysed for percentage drug entrapment and percentage freedrug in the LDPI formulation. The results are given in Table 1.

Characterization

Ketotifen fumarate loading, described as percentage drug entraped, wasdetermined by reverse-phase HPLC

10

following solubilization of liposomeswith 10% Triton X-100 in methanol. The mean vesicle size of rehydratedliposomes was determined by a laser light scattering technique using

Mastersizer (Malvern Instruments, London, UK) operating at a beam lengthof 2.40 mm and range of lens at 300 mm (Table 1).

Intratracheal administration

Intratracheal instillation was performed as reported by Shek

et al

.

11

Briefly,six albino rats (220–240 g) were used in each group for each time interval.Rats were housed in individual plastic cages at a constant temperature.Animals were allowed free access to water and rat chow, but were fastedovernight prior to each experiment. Rats of either sex were selectedrandomly and anaesthetized by intraperitoneal administration of pentobar-bitone sodium (40 mg/kg). The trachea was exposed by blunt dissection ofthe sternohyoideus muscle and a small midline incision was made over thetrachea between the fifth and sixth tracheal rings using a 20 guage needle.The trachea was cannulated with PE200 tubing (5–7 cm) with the tippositioned approximately at the tracheal bifurcation. The PE50 (10–15 cm)tubing connected to a glass Hamilton syringe (Waters, Banglore, India) wasinserted into the cannula and advanced to the bifurcation of the trachea.Solutions containing 100

�

g plain KF (PDK) or liposome-encapsulated KFprepared by rehydration (30 min) of powder with 250

�

L triple-distilledwater were instilled slowly over a 1 min period, followed by 50

�

L normalsaline. Animals that were to be killed at 3, 6 and 9 h after administrationhad the cannula secured with sutures and the access cannula excised toleave a 1 cm protrusion. For rats that were to be killed after 12 and 24 h, thetrachea was threaded with sutures and the incision closed with surgicalstaples. Animals were allowed to recover under a heating lamp and, afterrecovery, animals were housed in individual plastic cages with free accessonly to water. Sham animals receiving PBS (pH 7.4) were included alongwith the experimental groups on the day of the experiment as controls.

Biological sampling

Bronchoalveolar lavage (BAL) was performed on anaesthetized and recan-nulated (as necessary) animals with 12 mL PBS, prewarmed to 37

�

C. Toperform the lavage, the Hamilton syringe connected to the PE50 tubing wasreplaced with a three-way stopcock attached to two 20 mL syringes.Approximately 12 mL sterile (0.45

�

m filtered) PBS was injected slowly infractions to fill the lungs. The fluid was withdrawn by gentle aspiration; thisBAL yielded between 7 and 11 mL liquid, which was centrifuged at4.38

�

10

3

g

for 5 min. The supernatant was mixed with 10% Triton X-100in a ratio of 9 : 1

12

and analysed by HPLC to determine unreleased KF. Thelungs and portions of the trachea below the instillation site were excised andhomogenized (= lung homogenate; LH) in 10 mL PBS containing 1%Triton X-100. Deprotenization was performed with 10% sulphosalicylicacid and the KF released was analysed in the supernatant by HPLC.

10

Data analysis

Each batch was prepared six times and data from all experiments areexpressed as the mean

±

SEM. Drug entrapment expressed in Table 1 is a

Table 1

Analytical profiles of liposomal dry powder for inhalation of ketotifen fumarate

Batch KF1 KF2 KF3

Initial drug : lipid (molar ratio) 1 : 15 1 : 150 1 : 15Initial EPC : cholesterol (molar ratio) 1 : 00 1 : 0.5 1 : 10Drug entraped in liposomal DPI* (%) 89.0

±

0.50 76.4

±

1.20 65.3

±

0.90Drug free in liposomal DPI* (%) 0.9

±

0.1 2.3

±

0.2 2.0

±

0.1Free drug removed in the purification of liposomal dispersion (%) 10.1

±

0.60 21.3

±

2.60 32.7

±

1.20Mean vesicle size (

�

m) D [4,3]* 1.62

±

0.01 1.72

±

0.02 2.00

±

0.01Span 1.03

±

0.01 1.60

±

0.01 1.71

±

0.01

Data are the mean

±

SEM (

n

= 6).*Volume mean diameter.EPC, egg phosphatidyl choline; DPI, dry powder for inhalation; D [4,3], volume mean diameter.

Pulmonary disposition of liposomal ketotifen

155

percentage of the drug added initially. Data were compared using

ANOVA

and Student’s

t

-test and

P

< 0.05 was considered significant.The various pharmacokinetic parameters calculated for comparison in

this investigation are defined below and the results are summarized inTable 2.

C

max

: Maximum concentration of drug attained in lung during the study(i.e. the maximum concentration of drug as estimated in the LH).

T

max

: The time point at which maximum drug concentration is attained inthe LH (i.e. the time required to achieve C

max

).

AUC

24h0

: The area under the curve of drug concentration in the LHcompared with time, over the period of study (24 h).

T

1

/

2

: The pulmonary half-life of drug was calculated by calculating thesum of the values of drug concentration in BAL and LH at individualsampling points, regressing the calculated sum over the entire duration ofstudy and deriving the time point at which the sum of drug level is 50%compared to the quantity instilled initially (i.e. the median of the regressionline).

Targeting factor (TF): The targeting factor was calculated using thefollowing equation:

TF = (Mean AUC

024

of liposomal KF)/(Mean AUC

024

of PDK)

RESULTS

The drug encapsulation efficiency varied from 89.0 to 65.3% of thetotal amount of KF, corresponding to a molar drug : lipid ratio of1 : 15 with change in the EPC : cholesterol molar ratio from 1 : 0to 1 : 1 (Table 1). The mean liposomal size determined by laserlight scattering was in the range of 1.6–2.0

�

m and the sizedistribution expressed as the polydispersity index (Span) was in therange of 1.0–1.7 for all batches studied (Table 1).

The pulmonary disposition studies were performed, as reportedearlier by Shek

et al

.,

11

by the estimation of the percentage drug inBAL and LH after intratracheal instillation of rehydrated liposomaldry powder and plain drug (PDK). A drug dose of 100

�

g wasinstilled. The amount of drug present in the LH was considered asdrug absorbed and available for a pharmacological response andthe amount of drug present in the BAL was considered unabsorbeddrug present in the lung and still available for absorption in lungtissue. Mean lung drug concentration–time data following eachindividual treatment are plotted in Fig. 1a,b. The pharmacokineticparameters calculated, as described in the data analysis section, aregiven in Table 2.

After instillation of liposomal drug, 61.91

±

1.13–68.84

±

0.98% of administered drug was recovered in BAL during the first6 h, eventually decreasing to 9.78

±

0.08–17.46

±

0.26% by 24 h;no drug was recovered after instillation of PDK at the end of 6 h in

BAL. When the concentration–time profiles up to 24 h postinstill-ation were examined, a rank order increase in T

max

was observedfrom PDK to the formulation having a 50% molar ratio of choles-terol (molar ratio of EPC : cholesterol = 1 : 0.5; KF2); that is,PDK < KF1 < KF2

�

�

KF3, where KF3 is the formulation withequimolar cholesterol and KF1 is the formulation with no choles-terol. A reverse relationship was observed in the case of C

max

andalmost double C

max

was observed for PDK compared with the C

max

for KF1, KF2 and KF3. Similarly, there was an increase in AUC

24h0

for liposomal formulations compared with the plain drug. Theincrease in the AUC

24h0

value calculated as the TF is given inTable 2. The liposomal formulation with a 50% molar ratio ofcholesterol (KF2) was found to have the highest TF of 1.54 andformulations with maximum cholesterol (KF3) and no cholesterol(KF1) were found to have TF of 1.48 and 1.36, respectively. TheT

max

values for KF2 and KF3 were highest (12 h), thereby con-firming the maintenance of effective drug concentration with KF2and

KF3

in

lung

tissue

for

longer

periods

compared

with

KF1(T

max

9 h).The pulmonary half-life (T

1

/

2

) was calculated and is given inTable 2. When the T

1

/

2

values were compared, a rank order increasewas observed starting from PDK to the LDPI with anEPC : cholesterol molar ratio of 1 : 1 (i.e. PDK < KF1 < KF2

Table 2

Mean pharmacokinetic parameters of the liposomal formulation compared with the plain drug

Formulation AUC

24h0

C

max

(

�

g)T

max

(h)T

1

/

2

(h) TF

PDK 49 741

±

1865 73.56

±

5.52 3 8.10

±

0.30 –KF1 68 011

±

1469 38.94

±

1.86 9 21.04

±

0.50 1.36KF2 79 677

±

1490 41.96

±

1.65 12 27.99

±

0.52 1.54KF3 73 981

±

1132 40.77

±

1.42 12 29.31

±

0.45 1.48

Data are the mean

±

SEM (

n

= 6).PDK, solutions containing 100

�

g plain ketotifen fumarate; KF1, KF2, KF3, liposomal formulations containing no cholesterol, 50% molar ratio choles-terol and equimolar cholesterol, respectively;

AUC24h0

, the area under the curve of drug concentration in the lung homogenate compared with time, over theperiod of study (24 h); C

max

, maximum concentration of the drug; T

max

, time to achieve C

max

; T

1

/

2

, pulmonary half-life of the drug; TF, targeting factor.

Fig. 1

Comparative pulmonary drug release from liposomal dry powderfor inhalation (LDPI) of ketotifen fumarate (KF) following intratrachealinstillation. (a) Percentage drug in lung homogenate (LH). (b) Percentagedrug in bronchoalveolar lavage (BAL). (

�

), plain drug; (

�

), liposomalformulation KF1 (no cholesterol); (�), liposomal formulation KF2 (50%molar ratio cholesterol); (�), liposomal formulation KF3 (maximumcholesterol). Data are the mean±SD.

156 M Joshi and A Misra

< KF3). Thus, the free drug was absorbed rapidly from lung tissueand then into the systemic circulation, whereas the liposomal-encapsulated drug remained in the lung for a prolonged period oftime.13

DISCUSSION

Liposomal encapsulation prolonged the drug residence time in thelung (i.e. drug absorption). Recovery of drug from lung tissue, afterinstillation of KF liposomes, increased with time until Cmax wasachieved. The drug mass balance between the percentage drugabsorbed (drug analysed in the LH) and percentage drug remainingentraped within liposomes (estimated in BAL) was initially foundto be close to 100%. It was assumed that the amount of drug thatcould not be accounted for in later stages may have been eithermetabolized, systemically absorbed or both.

The liposomal membrane permeability in vivo was reduced inproportion to cholesterol incorporated in the liposomal bilayer.Cholesterol is known to protect liposomes from in vivo destabiliz-ation,14 but only up to an optimum concentration, after which itcontributes to the destabilization of the liposomes in vivo, asevidenced by the reduction in the AUC24h

0 for formulations with a1 : 1 molar ratio of EPC : cholesterol. The destabilization causedby the increased level of cholesterol is expected to be more for ahydrophobic drug because both compete for the interlamellaespaces.9 Thus, the kinetics of the LDPI formulation of a drug inlung is found to be dependent on the physicochemical properties ofthe drug and on the bilayer composition of the liposomes. Theinclusion of cholesterol is necessary for physical stability of theliposomes; however, biological stability requires the proportion ofcholesterol to be optimal and a further increase in its proportionmay lead to a relatively rapid release of the drug.

These studies were conducted in order to evaluate the possibilityof obtaining localized actions of KF (an anti-asthmatic drug)using the LDPI drug-delivery system. Liposome-encapsulated KFadministered via the respiratory system persists within the lung fora prolonged period of time; in contrast, free drug is lost rapidlyfrom the lung. Animal studies reported up until now have used theinstillation of liquid formulations in order to obtain accuratedosimetry.11,13 Such results depend upon the spread of the instilleddose within the lung. The distribution and absorption of inhaledaerosols in the lungs and airways are different from those ofinstilled liquids15,16 and it is possible that the release kinetics ofLDPI formulations in humans may differ from the release kineticsof instilled formulations in rats.

In summary, the present study has provided evidence that it ispossible to confine the anti-asthmatic drugs, like KF, primarily tolung using an LDPI delivery system and, hence, to provide thedesired pharmacological effect proportional to the drug level avail-able at the target site. An essential requirement for any slow-releaseor long-acting pharmaceutical preparation is the ability to remainat or near the absorptive interface and supply the drug continuouslyto its site of action. The superiority of liposomal KF in this respect

was demonstrated by drug concentrations that were maintained inthe lung tissue and the less rapid disappearance of KF from theBAL. However, whether LDPI will ultimately result in superiorclinical activity in pathological states in humans cannot be ascer-tained, but deserves further study.

ACKNOWLEDGEMENT

The authors gratefully acknowledge Ambalal Sarabhai Enterprises(Baroda, India) for providing research funds for this investigation.

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