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RESEARCH ARTICLE – Pharmaceutical Nanotechnology

Development and Characterization of an In Vitro Release Assayfor Liposomal Ciprofloxacin for Inhalation

DAVID CIPOLLA,1,2 HUIYING WU,2 SIMON EASTMAN,3 TOM REDELMEIER,3 IGOR GONDA,2 HAK-KIM CHAN1

1Faculty of Pharmacy at the University of Sydney, The University of Sydney, Sydney, NSW 2006, Australia2Aradigm Corporation, Hayward, California 945453Northern Lipids Inc., Burnaby, British Columbia V5J 5J1, Canada

Received 1 October 2013; revised 26 October 2013; accepted 30 October 2013

Published online 25 November 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23795

ABSTRACT: A novel method was developed and optimized to measure the in vitro release (IVR) of two liposomal ciprofloxacin formulationsunder development to treat lung infection. The release agent, bovine serum, has components that interact with liposomes to cause theencapsulated drug to be released. The precision and accuracy of the method were characterized. The method has a nearly linear releasephase initially, which then approaches a plateau value after 2–4 h. The robustness of the method was verified over a range of release agentand liposomal concentrations, and in response to changes in incubation temperature, buffer pH, and storage containers of serum. For this“sample and separate” IVR method, there is less than 2% release at the T = 0 time point, indicating negligible artifactual release beforeanalysis. For both inhaled liposomal ciprofloxacin products, the plateau value represents 100% release of the encapsulated drug. The keyelements of this IVR method may prove useful for characterization of other liposomal products as well. C© 2013 Wiley Periodicals, Inc. andthe American Pharmacists Association J Pharm Sci 103:314–327, 2014Keywords: liposomes; nanoparticles; pulmonary drug delivery; dissolution; in vitro release; controlled release; ciprofloxacin; Pulmaquin R©;Lipoquin R©

INTRODUCTION

Liposome-formulated drugs can provide a number of compellingadvantages over treatment with the unencapsulated drug, in-cluding the ability to target specific cells or tissues, promote in-tracellular uptake, improve the safety profile, and reduce sideeffects. Traditionally, the primary motivation is to modify thedrug’s release profile.1,2 Liposomes are phospholipid vesiclescomposed of one or more lipid bilayers surrounding an aqueouscore and can vary in size ranging from approximately 20 nm to10 :m. In addition to phospholipids, liposomes can also incorpo-rate surfactants or cholesterol to alter the membrane rigidity,and surface modification with polyethylene glycol or antibodiesto produce “stealth” liposomes or to target specific receptors, re-spectively. When designing a liposomal formulation, one shouldconsider the route of delivery (e.g., inhalation, injection, infu-sion), the frequency of administration (e.g., once per day) andthe desired in vivo release rate to ensure that drug levels re-main within the “window” of adequate therapeutic effects andsafety. The focus of this paper is on in vitro methodology tocharacterize the release rate of drug from liposomes; the twoliposomal ciprofloxacin formulations used to characterize thisin vitro release (IVR) method are in late stage clinical develop-ment to treat lung infections.3–5

The in vivo release rate of drug from liposomal formulationsdepends on the design of the liposomal formulation, the routeof delivery into the body including potential interactions be-tween the liposomal formulation and any delivery technologythat is used, and finally the interaction of the liposomes with

Correspondence to: David Cipolla (Telephone: +61-2-9351-2516; E-mail:[email protected])

Journal of Pharmaceutical Sciences, Vol. 103, 314–327 (2014)C© 2013 Wiley Periodicals, Inc. and the American Pharmacists Association

the fluid and tissue once in the body. With respect to the for-mulation, the key factors affecting the release rate include thespecific composition and relative proportion of the componentsin the vesicles (e.g., surfactants can fluidize the membrane in-creasing the rate of release, whereas cholesterol can have theopposite effect), the vesicle size distribution, the lamellarity ofthe liposomes (i.e., faster release from unilamellar liposomes),the nature of the drug (e.g., molecular weight, lipophilicity, andcharge), the physical state of the drug (e.g., precipitated or insolution), and the release mechanism.6 For inhaled liposomalformulations, the delivery device can also affect the releaserate. Nebulizers used to generate aerosols have the potential todisrupt liposomes, causing an increased “burst” of drug, or mod-ify the liposome vesicle size distribution or lamellarity, whichcan also affect the rate of release.2,7 For aerosolized liposomes,the site of deposition in the lung (e.g., bronchial airways versusalveolar space) can influence the release rate due to differencesin biological fluid volume and composition. The presence of air-way disease can confound the situation by influencing the siteof aerosol deposition due to changes in airway dimensions, air-way patency, and the ability of the patient to correctly inhalethe aerosol. The drug transport rates can also be affected dueto alterations in lung clearance and the composition of the lungfluid and tissue; for example, mucus and biofilms can interactwith liposomes and impair mucociliary clearance and absorp-tion.

A qualified IVR assay could be useful during product devel-opment to provide confidence in the robustness of the liposomalformulation to changes in manufacturing conditions or duringstorage by examining the reproducibility of a key functionalityof the product—rate of drug release. A number of recent work-shops have been conducted with a focus on defining the currentstate of IVR methodology for sustained release pharmaceuticalproducts including the Controlled Release Society in 20076 and

314 Cipolla et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:314–327, 2014

RESEARCH ARTICLE – Pharmaceutical Nanotechnology 315

the American Association of Pharmaceutical Sciences cospon-sored with both the FDA and the International PharmaceuticalFederation (FIP) in 2009.8 Regulatory agencies have also statedthat an IVR test may be useful to assess the properties of li-posomal products (United States Food and Drug AssociationDraft Guidance on Liposome Drug Products, 2002).9

A number of IVR methods have been evaluated for liposome-encapsulated compounds in the published literature and mostof them fall into three general categories. The most straight-forward are the “in situ” methods, which measure the releaseof drug in real time using an analytical method which can dif-ferentiate between encapsulated and released drug in the IVRsample. For drugs which fluoresce, the change in fluorescenceupon release from the liposomes due to dequenching can betranslated into a drug release rate. In practice, this techniquehas mostly been limited to model compounds such as calcein10

and carboxyfluorescein11, but has also been found to be use-ful for doxorubicin.12,13 The “in situ” IVR methodology has ad-vantages of rapid data output combined with limited samplemanipulation, but, unfortunately, it is not applicable to mostdrugs.

A second category of IVR methods involves membrane dial-ysis to physically separate the released drug from the encapsu-lated drug in the IVR vessel. This has been performed in thestandard dialysis mode,14 the reverse dialysis mode,15,16 or us-ing diffusion cells.17 A dialysis adapter was created for use withthe USP apparatus 4, which demonstrated improved discrim-inating power and less variability over standard and reversedialysis techniques.15 These dialysis-based methods are use-ful when the rate of release from the liposome is relatively slowcompared with the time frame for the free drug to diffuse acrossthe membrane. However, even in the presence of surfactant inthe medium, the transfer of 90% of the free drug across themembrane can take 2 h.16 Thus, these dialysis methods maynot be appropriate for systems where the rate of release of thedrug from the liposomes is comparable to or exceeds the rate ofthe drug transport through the dialysis membrane.

A third category of IVR methods involves sampling from theIVR vessel and using a second method to separate the free drugfrom the encapsulated drug followed by drug quantitation. Theseparation methods that have been used include chromatog-raphy (gel-filtration18 or cation exchange19), centrifugation orfiltration. The downside to these methods has historically beenthe additional sample manipulation, which can lead to artifactsincluding additional drug release during the separation step.One advantage to the published “sample and separate” methodsis that they have frequently used more physiologically relevantincubation medium to affect release; for example, plasma orserum for parenterally administered formulations.

The “sample and separate” IVR method reported in this pa-per utilizes bovine serum in pH 7.4 buffer as the release agentand uses centrifugal filtration devices to separate the releaseddrug from the encapsulated drug. Serum was chosen as the re-lease agent because there is experience using this agent withother liposomal systems and the components in serum, whichare responsible for release, may also be representative of the re-lease mechanisms in lung fluid. The main purpose of this studyis to demonstrate that this IVR method is precise, reproducibleand discriminatory. To this end, the sources of variability havebeen characterized, which would allow for subsequent methodvalidation for the liposomal ciprofloxacin formulations evalu-ated in this study.

MATERIALS AND METHODS

Materials

Lipoquin R©, liposomal ciprofloxacin for inhalation (CFI), emptyliposomes, and free ciprofloxacin (FCI), 20 mg/mL in an acetate-buffered aqueous formulation at pH 3.3, were provided byAradigm Corporation (Hayward, California). Pulmaquin R©, dualrelease ciprofloxacin for inhalation (DRCFI), is an equivolumemixture of CFI and FCI. Donor Adult Bovine Serum was ob-tained from HyClone (Logan, Utah). Polysorbate 80 was a giftof Croda, Inc. (Edison, New Jersey). HEPES, free acid was pur-chased from Avantor (Center Valley, Pennsylvania). Sodiumchloride was purchased from Amresco (Solon, Ohio). Sodium ac-etate was purchased from Sigma–Aldrich (St. Louis, Missouri).HPLC grade methanol was purchased from Fisher Scientific(Fair Lawn, New Jersey) and triethylamine (TEA) was pur-chased from JT Baker (Center Valley, Pennsylvania) . Nanosepcentrifugal filtration devices, 10K and 30K molecular weight,were obtained from Pall Corporation (Ann Arbor, Michigan).Deionized water was used for all studies.

Preparation of Lipoquin (CFI)

The details of the preparation of Lipoquin have been reportedpreviously.20–22 Briefly, hydrogenated soy phosphatidylcholineand cholesterol in a 7:3 ratio (by weight) were dissolved in at-butanol:ethanol:water mixture (49:49:2) and agitated at 70◦Cfor 1 h to ensure complete dissolution. This preparation wassubsequently mixed 1:10 (by volume) with 500 mM ammoniumsulfate and agitated at 70◦C to form multilamellar liposomes.The multilamellar vesicles were extruded through 80 nm poly-carbonate filters to yield unilamellar liposomes, approximately80 nm in diameter. The solvents were removed by diafiltra-tion with ten volumes of 5 mM histidine, 145 mM NaCl, pH6.0 buffer. Ciprofloxacin was added to the unilamellar lipo-somes at 50◦C and the preparation was agitated to facilitateactive loading of drug.21 After loading and removal of unen-capsulated ciprofloxacin by diafiltration with 25 mM histidine,145 mM NaCl, pH 6.0 buffer, greater than 99% of theciprofloxacin was encapsulated, at a target concentration of50 mg/mL total ciprofloxacin, expressed as ciprofloxacin hy-drochloride, and 100 mg/mL total lipids. Empty liposomes weremanufactured with a comparable size and composition to CFI,but without loading of drug.

EXPERIMENTAL METHODS

Recovery of Free Drug from Nanosep Centrifugal Devicesand Selection of the Centrifugation Time

Nanosep centrifugation devices were used to separate the freeciprofloxacin from the liposomally encapsulated drug (Fig. 1).In preliminary experiments, membrane filters with 10,000 and30,000 molecular weight cutoffs were found to retain the li-posomes without causing artifactual leakage of encapsulateddrug. For control CFI samples, less than 1% of free drug wasrecovered in the filtrate, consistent with greater than 99% ofthe drug being encapsulated. Samples from the IVR assay wereexpected to contain increasing amounts of free drug after longerincubation times and so control experiments were conducted toconfirm that the released “free” ciprofloxacin can be quantita-tively recovered in the filtrate. Three control experiments wereperformed using a centrifugation time of 18 min: FCI diluted

DOI 10.1002/jps.23795 Cipolla et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:314–327, 2014

316 RESEARCH ARTICLE – Pharmaceutical Nanotechnology

NN

NH

O

OHF

O

Ciprofloxacin structure

Filtrate containing free ciprofloxacin (red dots)

IVR sample containing encapsulated and free ciprofloxacin in serum

Centrifuge for 10 min at 10,000 rpm (8100g)

Membrane filter

Figure 1. Schematic of the use of the centrifugation filters to separate free ciprofloxacin from the IVR mixture. The IVR samples composedof free ciprofloxacin (red dots) and liposome-encapsulated ciprofloxacin (blue circles containing red dots) in serum are aliquoted (400 :L) intofiltration devices and centrifuged at 10,000 rpm (8100 g) for 10 min. After centrifugation, the encapsulated drug is retained above the membranefilter, whereas the free drug in the filtrate can be subsequently quantified.

into HEPES buffered saline (HBS: 20 mM HEPES, 145 mMNaCl, pH 7.4) to determine the recovery in the absence of lipo-somes or serum, FCI in HBS buffer diluted 1:1 with serum todetermine if the serum interferes with the recovery, and FCIand empty liposomes in HBS diluted 1:1 with serum to de-termine if the combination of liposomes and serum affects therecovery of free drug. All samples were further diluted 1:1 withHBS buffer before filtration recovery of free drug to improvethe filterability. In all three studies, five free drug concentra-tions were evaluated in triplicate, 0.125, 0.5, 2.5, 6.25, and12.5 :g/mL (to cover the range of 1%, 4%, 20%, 50%, and100% drug release, respectively). In the third study, the amountof liposomes added was identical to the amount of liposomespresent in CFI when diluted to 25 :g/mL ciprofloxacin, thestandard concentration in the IVR assay, before the final 1:1dilution with HBS buffer to aid in the filtration recovery step.The third control experiment was then repeated across a rangeof centrifugation times: 5, 10, 12, 15, and 18 min, to determineif a shorter centrifugation time could be used, which would stilllead to acceptable and repeatable recovery of free drug.

IVR Methodology

The IVR assay is designed to measure the release of encapsu-lated drug, in this case ciprofloxacin, from a liposomal prepa-ration over a period of time that is convenient for analysis; forexample, 2–4 h. Exploratory studies using a range of liposomalciprofloxacin concentrations and percent serum were conductedto identify target values which resulted in 100% release over 2–4 h incubation at 37◦C. Target values of 25 :g/mL ciprofloxacinand 50% serum were identified (data not shown). Liposomalciprofloxacin samples, either CFI or DRCFI, were diluted to

50 :g/mL ciprofloxacin with HBS and chilled at 2◦C–8◦C be-fore assay. The 50 :g/mL ciprofloxacin in HBS preparationwas mixed one-to-one with chilled (2◦C–8◦C) bovine serum andplaced in a shaking water bath [Techne, TSBS40 (Staffordshire,UK)] at 37◦C and 150 rpm to initiate the release of drug. Whilethe T = 0 samples remained at 2◦C–8◦C, the other time pointsamples were loaded into the 37◦C water bath. Samples, typi-cally in triplicate, were removed periodically; for example, 30,60, 120, and 240 min, and immediately placed in an ice-waterbath to terminate any further release of encapsulated drug fromthe liposomes. These chilled 25 :g/mL ciprofloxacin samples(including the T = 0 samples) were then diluted 1:1 with chilledHBS to 12.5 :g/mL ciprofloxacin to reduce the concentration ofserum proteins and liposomes. This step prepares the solutionfor filtration by reducing the load on the filter. The rate of filtra-tion is faster for more dilute solutions and increases the volumeof permeate recovered during centrifugation. An even larger di-lution step would have the potential to improve the filterabilityeven further but would increase the analytical challenges ofquantifying a more dilute concentration of ciprofloxacin. Thus,the 1:1 dilution step was chosen to balance these competingconsiderations. To separate the “released” ciprofloxacin fromthe liposome-encapsulated ciprofloxacin, 400 :L of each chilledsample was transferred to a Nanosep centrifugal device andcentrifuged at 10,000 rpm (8100g) for 10 min. The filtrate wasremoved for subsequent quantitation of the released or ’free’ciprofloxacin by HPLC. The original sample was diluted into80% methanol to dissolve the liposomes and allow for quantita-tion of the total amount of ciprofloxacin by HPLC. The percentrelease was calculated by comparing the free drug to the totaldrug in each sample.

Cipolla et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:314–327, 2014 DOI 10.1002/jps.23795


Characterization of the Accuracy and Precision of the IVR Assay

The IVR profiles for both CFI and DRCFI were character-ized using the standard IVR conditions described above in theIVR Methodology. Both CFI and DRCFI were diluted with HBSbuffer and bovine serum to a final concentration of 25 :g/mLciprofloxacin and 50% serum. In order to measure the IVRmethod precision, six replicates of each sample were evaluatedat each time point: T = 0, 30, 60, 120, and 240 min. The inter-mediate precision of the IVR assay was evaluated by repeatingthe experiment using two different analysts on each of 3 days.

Robustness: Effect of Ciprofloxacin Concentrationon the IVR Profile

The target concentration after dilution was 25 :g/mLciprofloxacin in the IVR assay for both the 50 mg/mL CFI and 35mg/mL DRCFI formulations. Typical drug products have spec-ification limits on the active drug concentration of ±5% and±10% at release and on stability, respectively. Thus, acceptableproduct could vary by up to ±10% around the target concentra-tion in the IVR assay, in this case, 25 :g/mL ciprofloxacin. TheIVR profiles for CFI at 22.5, 25.0, and 27.5 :g/mL ciprofloxacinconcentration were characterized using the standard IVR con-ditions described in IVR Methodology. Three replicates of eachsample were evaluated at each time point: T = 0, 30, 60, 120,and 240 min. A wider range of ±25% of the target concentration(18.75, 25.0, and 31.25 :g/mL) was also evaluated for both CFIand DRCFI in order to understand the sensitivity of the IVRassay to larger variations in drug concentration.

Robustness: Effect of Percent Serum on the IVR Profile

The addition of bovine serum caused release of encapsulateddrug in the IVR assay in a concentration dependent manner(data not shown). Based on exploratory studies, a value of50% serum was selected to yield 100% release of encapsulatedciprofloxacin in a time period of 2–4 h. Additional IVR exper-iments were conducted to evaluate the effect of a deviation inthe serum concentration by ±2% (of target) to account for typ-ical laboratory variability; for example, due to pipetting. TheIVR profiles for CFI using 49%, 50%, and 51% serum werecharacterized using the standard IVR conditions described inIVR Methodology. Three replicates of each sample were eval-uated at each time point: T = 0, 30, 60, 120, and 240 min. Awider range of ±20% of the target value (40%, 50%, and 60%serum) was also evaluated for both CFI and DRCFI in order tounderstand the sensitivity of the IVR assay to larger changes inserum concentration. The initial rate of release, T30 min − T0 min,was defined as the amount of released drug at the 30 min timepoint minus that present at the initial time point.

Robustness: Effect of HBS pH on the IVR Profile

The target pH for the HBS buffer in the IVR assay is 7.4.The effect of variation in the HBS buffer pH was examinedfor deviations of ±0.2 pH unit: 7.2, 7.4, and 7.6. The IVR pro-files for both CFI and DRCFI at 25 :g/mL ciprofloxacin werecharacterized using the standard IVR conditions described inIVR Methodology. Three replicates of each sample were evalu-ated at each time point: T = 0, 30, 60, 120, and 240 min. ForCFI only, a narrower pH range of 7.3, 7.4, and 7.5 was also eval-uated in order to understand the sensitivity of the IVR assayto smaller changes in the HBS buffer pH.

Robustness: Effect of Incubation Temperature on the IVR Profile

The target incubation temperature in the IVR assay is 37◦C.While the temperature of the shaking water bath can be con-trolled to 0.1◦C, it is important to understand the effect of tem-perature variations on the IVR profile. Thus, the IVR assay wasrepeated at 35◦C, 37◦C, and 39◦C. The IVR profiles for both CFIand DRCFI at 25 :g/mL ciprofloxacin were characterized us-ing the standard IVR conditions, except for the variation in theincubation temperature. Three replicates of each sample wereevaluated at each time point: T = 0, 30, 60, 120, and 240 min.

Robustness: Effect of Different Serum Containerson the IVR Profile

Each 500 mL serum container was subaliquoted into 10 mLplastic vials and stored frozen at −20◦C before use. Theseserum containers were all from one batch of serum; contain-ers of serum from a different batch were not available whenthe purchase was made. It is expected that the “activity” ofserum may vary across batches of serum, or even across differ-ent 500 mL containers from the same batch of serum. Whilethis source of variability can be mitigated to some extent byordering multiple containers of serum from a single serum lot,mixing them together to homogeneity, and freezing the serumin small aliquots in plastic vials until needed, thought must begiven to how to transition from one batch to another, even if thevariability across containers in one batch is acceptable. To un-derstand the variability in serum “activity,” samples of serumfrom four individual containers from the same serum batch,numbered 256, 257, 282, and 283, were evaluated in the IVRassay. The IVR profiles for both CFI and DRCFI at 25 :g/mLciprofloxacin concentration were characterized using the stan-dard IVR conditions described in the IVR Methodology. Threereplicates of each sample were evaluated at each time point:T = 0, 30, 60, 120, and 240 min.

Additional Characterization of the Discriminatory Abilityof the IVR Assay

While CFI and DRCFI differ in the amount of ciprofloxacinpresent in the formulations, the liposomal compositions areidentical. To evaluate if the IVR assay can discriminate be-tween different liposomal compositions, CFI was diluted to 12.5mg/mL ciprofloxacin in 0.2% polysorbate 80 and equilibrated for30 min to allow for the surfactant molecules to associate withthe liposomes. The IVR assay was performed with a CFI andDRCFI control, and a further CFI control which was dilutedwith serum containing polysorbate 80. This second control CFIsample had an equivalent amount of surfactant to the experi-mental CFI sample, 0.0004% polysorbate 80 after final dilution,and thus could determine whether the presence of surfactantin the release agent at this level alters the release rate. Allsamples were diluted with HBS to 25 g/mL ciprofloxacin be-fore mixing with serum. Two replicates of each sample wereevaluated at each time point: T = 0, 20, 40, 60, 90, 120, and240 min.

Ciprofloxacin Quantitation

The amount of ciprofloxacin in each sample was quantified us-ing an HPLC method as described previously.23 Briefly, HPLCwas performed using a Nucleosil C-18 column (5 :m, 4.6 ×150 mm2; Canadian Life Science (Peterborough, Ontario)) pro-tected with a Nucleosil C-18 guard column (4 × 3.0 mm2;



Phenomenex (Torrance, California)) both at 35◦C. The mobilephase was a mixture of 0.5% TEA in water, pH 3.0 and 100%methanol (83:17, v/v) and the isocratic elution was performedat a flow rate of 0.9 mL/min. Ciprofloxacin was detected at awavelength of 277 nm.

RESULTS

Recovery of Free Drug from Nanosep Centrifugal Devices

In the absence of serum or liposomes, the recovery of free drugranged from 102% to 106% over the 100-fold concentrationrange for both the 10K and 30K filtration devices, indicatingthat there is no hold-up of free drug across the membrane filter(Table 1, Group 1). In the presence of serum, the recovery wasslightly lower ranging from 93% to 99% and 96% to 100% forthe 10K and 30K filtration devices, respectively (Table 1, Group2). In the presence of empty liposomes and serum, the meanrecovery was approximately 95% for both the 10K and 30Kfiltration devices with an overall range of 89%–99% (Table 1,Group 3A). The recovery of free drug using this procedure ap-peared to be independent of free drug concentration except forthe lowest concentration condition, which had a slightly lowerrecovery. However, this small error would not have a meaning-ful impact on calculation of release values as the lowest concen-tration, 0.125 :g/mL, equates to only 1% release from the target25 :g/mL value after 1:1 dilution with HBS.

The recovery of free ciprofloxacin in the presence of emptyliposomes and serum was repeated for various centrifugationtimes, ranging from 5 to 18 min, to determine if a shorter cen-trifugation time would be acceptable (Table 1, Group 3B). Therecovery was close to 95% for centrifugation times between 10and 18 min but was slightly lower for the 5 min centrifugationtime. Thus, a centrifugation time of 10 min was selected for in-corporation into the standard IVR procedure. To correct for thissmall but reproducible loss of free drug in the filtration devices,the free drug concentration in each IVR sample is normalizedby dividing by 0.949, the overall mean recovery factor using the10 min centrifugation time.

Characterization of the Accuracy and Precision of the IVR Assay

The IVR profiles for both CFI and DRCFI were characterizedusing the standard IVR conditions described in IVR Method-ology by two different analysts on each of three separatedays (Figs. 2a and 2b). CFI represents liposomal encapsulatedciprofloxacin, whereas DRCFI represents approximately 70%liposomal encapsulated ciprofloxacin and approximately 30%unencapsulated ciprofloxacin. The mean (±SD) time zero re-lease values (T0 min) for the six IVR experiments with CFI andDRCFI were 1.1% (0.3) and 31.4% (1.1), respectively. These re-sults are close to the expected values of <1% and approximately30% and indicate that there is negligible additional release offree drug during the sampling and separation steps prior todrug quantitation by HPLC. Chilling the samples at 2◦C–8◦Cappears to adequately inhibit further release of free drug be-fore or during the 10 min centrifugation. Furthermore, theseresults confirm that the centrifugation and filtration step doesnot damage the liposomes which could lead to significant leak-age of drug.

The release of drug from CFI and DRCFI in the IVR as-say proceeds in a nearly linear fashion initially, and eventuallyplateaus in a 2–4 h time frame at a value corresponding to com-plete release of drug for these liposome preparations (Figs. 2aand 2b). The range in mean release at the 240 min time pointwas 97.5%–104.7% (100.9 ± 3.1%, n = 6) for CFI and 97.1%–103.2% (100.3 ± 2.4%, n = 6) for DRCFI. In addition to having atime zero point, (T0 min), to characterize any burst, and a plateauvalue at 240 min (T240 min) to determine the overall extent of re-lease, another time point was sought to represent the midpointof release. During IVR method optimization, the 30 min timepoint (T30 min) was chosen as a “midpoint” value. In character-ization studies, the rate of release, equal to the slope of thecurve, at the 30 and 40 min time points was 92% and 90%,respectively, of the rate of release at the 10 min time point,suggesting that the value of T30 min is also a reasonable esti-mate of the initial rate of release (data not shown). The 30 minperiod is also adequately long to ensure that any variabilityin the timing of removal of multiple samples from the incu-bation bath, and subsequent transfer to an ice water bath toterminate further reaction will not have a meaningful impact

Table 1. Percent Recovery of Free Drug (Ciprofloxacin) from the Filtrate of the Nanosep Filtration Devices

Ciprofloxacin Concentration (:g/mL)

Filter TypeCentrifugeTime (min) 0.125 0.5 2.5 6.25 12.5

Overall MeanRecovery (%)

Group 1 10K 18 105.6 ± 3.3 103.2 ± 0.5 102.2 ± 0.6 103.1 ± 0.6 102.2 ± 0.3 103.330K 105.9 ± 0.4 106.4 ± 2.8 101.7 ± 0.4 102.9 ± 0.5 101.9 ± 0.9 103.7

Group 2 10K 18 92.8 ± 0.5 98.5 ± 0.9 95.6 ± 0.1 97.1 ± 0.1 95.6 ± 0.1 95.930K 97.1 ± 0.6 99.7 ± 0.5 96.0 ± 0.1 96.8 ± 0.1 96.6 ± 0.1 97.2

Group 3A 10K 18 93.9 ± 0.6 97.6 ± 0.8 95.4 ± 0.4 95.5 ± 0.2 95.7 ± 0.1 95.630K 89.3 ± 1.5 99.3 ± 1.4 95.5 ± 0.2 96.2 ± 0.3 96.0 ± 0.1 95.3

Group 3B 30K 5 89.6 ± 2.1 93.1 ± 1.2 92.8 ± 0.3 92.6 ± 0.8 92.9 ± 0.3 92.210 93.6 ± 1.6 96.6 ± 0.9 94.3 ± 1.5 94.7 ± 0.1 95.3 ± 0.3 94.912 90.4 ± 2.9 96.5 ± 0.3 94.7 ± 0.7 95.3 ± 0.3 95.7 ± 0.4 94.515 90.9 ± 3.2 98.7 ± 0.8 96.4 ± 0.2 95.3 ± 0.5 96.8 ± 0.1 95.618 89.3 ± 3.2 99.3 ± 1.5 95.5 ± 1.3 96.2 ± 0.2 96.0 ± 0.3 95.3

The recoveries represent mean ± SD of triplicate determinations. Group 1 represents recovery of ciprofloxacin diluted in HBS. Group 2 represents the recoveryof ciprofloxacin in HBS diluted 1:1 with serum and then 1:1 with HBS. Groups 3A and 3B represent the recovery of ciprofloxacin in empty liposomes diluted 1:1 withserum and then 1:1 with HBS. For Groups 1, 2 and 3A, either 10K or 30K filtration devices were used. For Group 3B, the centrifugation time ranged from 5 to 18 minfor the 30K centrifugation device.



Figure 2. Evaluation of the intermediate precision of the IVR assay across three nonconsecutive days and two different analysts. Release ofciprofloxacin from CFI (a) and DRCFI (b) in 50% bovine serum and 10 mM HEPES buffered saline, pH 7.4 after incubation at 37◦C for 4 h. Eachvalue represents the mean ± SD (n = 6). There was one outlier value removed, according to predefined criteria, out of a total of 360 data points.The “No Serum Control” sample was diluted into 10 mM HEPES buffered saline, pH 7.4, without serum, and incubated at 37◦C for 4 h (n = 3per time point).

on the release values. The range in mean release values atT30 min was 45.3% to 50.7% (48.1 ± 2.1%, n = 6) for CFI and59.1 to 64.8% (61.8 ± 2.3%, n = 6) for DRCFI. These valuesrepresent release of 47.4% and 44.3%, respectively, of the en-capsulated drug present at T0 min (98.9% and 68.6% for CFIand DRCFI, respectively) indicating that T30 min is well chosento represent the midpoint and that the rate of release fromboth formulations is comparable after the initial burst: Whilethe initial rate of release is comparable for CFI and DRCFI, theIVR assay discriminates between the two formulations due tothe 30% “burst”in the DRCFI formulation.

Six replicates were conducted for each sample at each timepoint to assess the method precision (repeatability). The repli-cates were very repeatable with a mean precision of 1.4%, 1.3%,0.5%, and 0.9% RSD for the 30, 60, 120, and 240 min timepoints, respectively, for CFI. The mean precision for DRCFIwas also comparable with values of 1.1%, 1.1%, 0.9%, and 0.7%RSD at the 30, 60, 120, and 240 min time points, respectively.

The T0 min release values are also very repeatable, in terms ofstandard deviation, but because the release values are all closeto zero for CFI it is not sensible to report relative standard devi-ations. The intermediate precision was evaluated by repeatingthe IVR assay using two different analysts on three days to as-sess the analyst-to-analyst and day-to-day variability (Figs. 2aand 2b). The day-to-day variability was a greater source of errorthan the analyst-to-analyst variability.

A control CFI sample diluted into HBS buffer alone, withoutserum, was incubated at 37◦C for 4 h and subjected to the same“sample and separate” methodology as for the experimentalsamples. Only 0.8% additional release of encapsulated drugwas observed over the 4 h incubation (Fig. 2a) demonstratingthat serum is required for release. This result also confirmsthat there is negligible nonspecific release due to the “sampleand separate” methodology itself; that is, incubation for 4 h at37◦C in pH 7.4 HBS buffer followed by the centrifugal filtrationprocessing step.



Figure 3. Evaluation of the effect of changes in ciprofloxacin concentration on the IVR assay. The release of (a) 22.5, 25, and 27.5 :g/mLciprofloxacin from CFI and (b) 18.75, 25, and 31.25 :g/mL ciprofloxacin from CFI and DRCFI in 50% bovine serum and 10 mM HEPES bufferedsaline, pH 7.4 after incubation at 37◦C for 4 h. Each value represents the mean ± SD (n = 3). There were no outlier values out of a total of 135data points.

Robustness: Effect of Ciprofloxacin Concentrationon the IVR Profile

In order to evaluate the robustness of the assay to changes indrug concentration of ±10%, the IVR profiles for CFI at 22.5,25.0, and 27.5 :g/mL ciprofloxacin were characterized (Fig. 3a).All three profiles were comparable with no meaningful initial“burst,” followed by a linear release for 30–60 min, which ap-proached a plateau within 2–4 h corresponding to 100% releaseof drug. There was not a meaningful change in the initial re-lease values over this concentration range with the T30 min val-ues varying between 43.3%–45.4%. These results suggest thatthe acceptable variability in drug concentration, up to ±10%,that might be expected between vials, across batches or on sta-bility will not influence the performance in the IVR assay.

A wider range of ±25% of the target ciprofloxacin concen-tration (18.75, 25.0, and 31.25 :g/mL) was also evaluated forboth CFI and DRCFI (Fig. 3b). The IVR profiles again sharethe same features as in Figure 3a with a limited spread in

the T30 min values for CFI (40.6%–43.2%) and DRCFI (58.1%–61.8%). This suggests that the IVR assay is robust to changes inthe target concentration of up to ±25%. The differences in theCFI T30 min values between the two sets of data (40.6%–43.2%vs. 43.3%–45.4%) reflect the day-to-day assay variability. Thehighest ciprofloxacin concentration (31.25 :g/mL) does appearto have a slightly faster rate of release by the 60 min time pointfor both CFI and DRCFI but reaches the same plateau value(T240 min) corresponding to 100% release.

Robustness: Effect of Percent Serum on the IVR Profile

The robustness of the IVR assay to changes in serum concen-tration of ±2% and ±20% of the target value of 50% serumare reported in Figures 4a and 4b, respectively. As might beexpected, changes in the serum concentration over both rangeshad no effect on the T0 min value or the overall extent of re-action as the plateau values all leveled off at approximately100% release. However, higher serum concentrations resulted



Figure 4. Evaluation of the effect of changes in serum concentration on the IVR assay. After incubation at 37◦C for 4 h, the release of 25 :g/mLciprofloxacin from (a) CFI in 49%, 50%, and 51% bovine serum and 10 mM HEPES buffered saline, pH 7.4 and (b) CFI and DRCFI in 40%, 50%,and 60% bovine serum and 10 mM HEPES buffered saline, pH 7.4. Each value represents the mean ± SD (n = 3). There were no outlier valuesout of a total of 135 data points. (c) The initial release rate, equivalent to the release value for T30 min – T0 min, is plotted versus the percent serumfor both CFI and DRCFI.



Figure 5. Evaluation of the effect of changes in the pH of HEPESbuffered saline (HBS) on the IVR assay. The release of 25 :g/mLciprofloxacin from (a) CFI in 50% bovine serum and 10 mM HBS, pH7.3, 7.4, and 7.5 and (b) CFI and DRCFI in 50% bovine serum and10 mM HBS, pH 7.2, 7.4, and 7.6 after incubation at 37◦C for 4 h. Eachvalue represents the mean ± SD (n = 3). There were no outlier valuesout of a total of 135 data points.

in a faster initial rate of release and an earlier plateau, whereaslower serum concentrations resulted in slower initial rates anda later plateau for CFI and DRCFI (Fig. 4b). For small varia-tions in serum of ±2% that might be expected during routineuse of the IVR assay, the change in the T30 min values was rel-atively small, ranging from 44.1% to 47.1% release for CFI,similar to the spread in T30 min values due to day-to-day as-say variation (section IVR Methodology). The initial reactionrate, T30 min – T0 min, is directly proportional to the concentra-tion of serum used in the assay over the 40%–60% serum range(Fig. 4c).

Robustness: Effect of HBS pH on the IVR Profile

A deviation of 0.1 pH unit in the HBS buffer from the 7.4 targetvalue resulted in changes in the release values for CFI at T30 min

(Fig. 5a) that exceeded the day-to-day assay variability (Fig. 2):the range in the T30 min values was 43.7%–55.6% with the lowestrelease at pH 7.3 and the highest release at pH 7.5. There wasno effect on the “burst” value at T0 min, or the plateau value of100% release at T240 min. However, the lower buffer pH resultedin a delay in the time to reach the plateau. This finding was alsoconfirmed for DRCFI over the HBS buffer pH range of 7.2–7.6with the lower HBS buffer pH (7.2) having a greater degree of

impact on the initial rate of release than the higher buffer pH(7.6) (Fig. 5b).

Robustness: Effect of Incubation Temperature on the IVR Profile

While the target IVR incubation temperature is 37.0◦C, and theshaking water bath temperature set-point can be controlled to0.1◦C, if the assay is conducted across multiple labs using dif-ferent shaking water baths the actual temperature may varyto a greater degree. Thus, it is important to understand theeffect of incubation temperature on the performance of the IVRassay. The effect of a ±2◦C change in incubation temperatureon the IVR profile is shown in Figure 6a for both CFI and DR-CFI. Changing the temperature did not affect the “burst” value(T0 min), as would be expected as these samples were not placedin the water bath, or the ultimate extent of release (T240 min) foreither formulation. However, the rate of release was acceler-ated by an increase in temperature and reduced by a decreasein temperature from the target value of 37.0◦C. For CFI, theinitial rate of release, T30 min − T0 min, was 28.5, 44.0, and 68.8%ciprofloxacin at 35◦C, 37◦C, and 39◦C, respectively. For DRCFI,the initial release rate, T30 min − T0 min, was lower due to the“burst” at time zero: 18.4%, 28.7%, and 41.0% ciprofloxacin at35◦C, 37◦C, and 39◦C, respectively. The initial rate of releaseof ciprofloxacin from both the CFI and DRCFI formulations isconsistent with Arrhenius kinetics over the 35◦C–39◦C temper-ature range as shown in Figure 6b. The values for the slopeswere within 10% of each other (r2 = 0.9999 and 0.9964 forCFI and DRCFI, respectively). An activation energy of approx-imately 170 kJ/mol was imputed from the mean slope values.

Robustness: Effect of Different Serum Containerson the IVR Profile

The IVR activity of serum from four different containers wasevaluated for both CFI and DRCFI (Fig. 7). All four contain-ers of serum produced comparable IVR profiles with no sig-nificant changes in the “burst” value (T0 min) or the plateauvalue (T240 min) within the variability of the assay, for both CFIand DRCFI. When comparing the release at T30 min, serum con-tainer 256 was slightly more active, whereas serum container257 was slightly less active than containers 282 and 283, forboth CFI and DRCFI. For CFI, the T30 min values were 43.8%and 40.1% for containers 256 and 257, respectively, comparedwith 42.9 and 41.4 for containers 282 and 283, respectively. ForDRCFI, there was a slightly greater spread in the T30 min valueswith 70.3% and 60.1% recorded for containers 256 and 257, re-spectively, versus 65.3% and 65.2% for containers 282 and 283,respectively. The relative activity of the serum containers is inthe order 256 > 282 = 283 > 257.

Additional Discrimination Across Liposomal CiprofloxacinFormulations

CFI and DRCFI only differ in the initial “burst” of drug at T0 min.Excluding the initial value, the rate of release of ciprofloxacinwas comparable (Fig. 2a vs 2b) as would be expected becausethe liposomal composition in CFI and DRCFI are identical. Toevaluate if the IVR assay can differentiate between differentcompositions that might be expected to have different releaserates, 0.2% polysorbate 80 was added to 12.5 mg/mL CFI andallowed to equilibrate for 30 min before dilution into HBS andserum. After dilution, the final concentration of polysorbate80 was only 0.0004%. The IVR profile of this formulation was



Figure 6. Evaluation of the effect of change in incubation temperature on the IVR assay. (a) The release of 25 :g/mL ciprofloxacin from CFIand DRCFI in 50% bovine serum and 10 mM HEPES buffered saline, pH 7.4 after incubation at 35◦C, 37◦C, or 39◦C for 4 h is reported. Eachvalue represents the mean ± SD (n = 3). There were no outlier values out of a total of 90 data points. (b) The natural logarithm (ln) of the initialrelease rates (k = T30 min – T0 min) for each of the six curves is plotted versus the inverse of temperature in ◦K.

substantially different from CFI and DRCFI (Fig. 8). The“burst” (T0 min) increased from <1% (c.f. CFI) to 29% and theremaining encapsulated drug had a much faster release ratethan for the CFI and DRCFI controls with more than 90%released after 20 min (Fig. 8). This result could not simplybe explained by the presence of the surfactant in the releasebuffer: the IVR profiles for CFI when diluted into serum, withor without 0.0004% polysorbate 80, were comparable (Fig. 8).The time points in this IVR experiment were not chosen toprovide a complete IVR profile for the CFI sample containingsurfactant—otherwise earlier time points would have been se-lected given its faster release profile (e.g., 5 and 10 min); butinstead simply to determine whether the IVR assay can differ-entiate between the samples.

DISCUSSION

Numerous drugs have been evaluated in liposomes for in-halation delivery24 and a subset has been delivered to hu-mans using nebulizers including imaging agents, anti-asthmadrugs, analgesics, immunosuppresives, cytotoxic agents, an-

tifungals, and antimicrobials.1,2 There are now two inhaledliposomal products in late-stage clinical development target-ing lung infections, liposomal amikacin (ARIKACE R©; Insmed(Monmouth Junction, New Jersey)) and liposomal ciprofloxacin(Pulmaquin R©; Aradigm Corporation). Our goal was to developand qualify an IVR assay for the inhaled liposomal ciprofloxacinformulations in order to have a laboratory assay to assess akey functional aspect of the product—the rate of drug release.Therefore, the robustness of the IVR assay was investigatedwith respect to the variation of the key parameters of the assay:drug concentration, serum concentration, buffer pH, incubationtemperature, and serum container.

For intravenously administered liposomal products, it is rea-sonable to use serum as a release vehicle because the lipo-somes will be rapidly diluted into serum in the blood stream.However, it is less clear that serum would be an appropriaterelease fluid for products delivered to the lung. It is thus impor-tant to understand the components in lung fluid which wouldlikely interact with liposomes and contribute to the releaseof encapsulated drug. It is well recognized that lipoproteinsand apolipoproteins in serum can have a destabilizing effect on



Figure 7. Evaluation of the effect of using different serum containers (#256, 257, 282, and 283) from the same serum batch on the IVR assay.The release of 25 :g/mL ciprofloxacin from CFI and DRCFI in 50% bovine serum and 10 mM HEPES buffered saline, pH 7.4 after incubation at37◦C for 4 h is reported. Each value represents the mean ± SD (n = 3). There were no outlier values out of a total of 120 data points.

Figure 8. Evaluation of the effect of liposomal composition on the IVR assay. The release of 25 :g/mL ciprofloxacin from CFI, CFI containing0.0004% polysorbate 80, and DRCFI in 50% bovine serum and 10 mM HEPES buffered saline, pH 7.4 after incubation at 37◦C for 4 h is reported.An additional control CFI formulation was diluted into 50% bovine serum, 0.0004% polysorbate 80, and 10 mM HEPES buffered saline, pH 7.4.Each value represents the mean (n = 2). There was one outlier value out of a total of 56 data points.

liposomes causing drug release.11,25 Scherphof et al.18 reportthat the phosphatidylcholine in liposomes is found to rapidlyassociate with lipoprotein-like particles after incubation withserum and this correlates with massive destruction of the li-posome structures and release of drug. Allen and Cleland10

confirmed and expanded on that finding by suggesting thatthe presence of cholesterol in the liposomes may inhibit, butnot eliminate, the exchange of phospholipid with high den-sity lipoprotein. Weinstein et al.11 studied the interaction ofproteins with lipid bilayers and found that most serum pro-teins did not induce release of encapsulated drug, includingtrypsin, albumin and ovalbumin, but that all serum lipopro-teins and apolipoproteins do induce rapid release. Lipoproteins

and apolipoproteins are also present in lung fluid, but at abouthalf the concentration in serum.26 So the IVR assay should ide-ally incorporate those elements into the release medium. Lungfluid also contains surfactant and lipids, which may also have acontributory effect. While there are a number of simulated lungfluid preparations published in the literature, no recognizedstandard exists.27 Furthermore, of the six listed preparations,only one contains lipid and none contain surfactant or lipopro-teins. Confounding the situation further, there is considerablevariability in the composition of the lung fluid throughout thelung, and additional variability among individuals, withouteven taking into account the presence of disease on lung fluidcomposition. Thus, there is not a straightforward choice for



release vehicle which simulates the release mechanisms in thelung. Our goal was to identify a release medium that wouldbe biological in composition, relatively homogeneous, easy toobtain, and relevant to the release mechanism in the lung. Pu-rified lipoprotein or apolipoprotein is not easily obtained butthey are both present in serum. For that reason, bovine serumwas chosen as the release medium.

The studies reported in this paper demonstrate that the se-lected filtration devices are able to separate free ciprofloxacinfrom the serum and liposomes reproducibly with an efficiencyof approximately 95% across a 100-fold range in ciprofloxacinconcentration (Table 1). The liposomes containing encapsulatedciprofloxacin are not compromised by the centrifugation and fil-tration process (Fig. 1). The utility of this filtration techniquemay be significantly affected by the characteristics of the en-capsulated agent and therefore may not be broadly applicableto other liposomal formulations. Its usefulness will need to beassessed on a formulation by formulation basis. The IVR assay,which involves incubation with bovine serum to cause release ofencapsulated drug from the liposomes, was also shown to havegood precision and reproducibility. The IVR profiles for our li-posomal ciprofloxacin formulations are characterized by threedistinct phases of release, the initial “burst” of drug (wheresuch “burst” was designed into each formulation), followed bya nearly linear release of drug that proceeds until a majorityof the drug is released (60%–80%), and then a slower releaseto reach a plateau value which defines the total extent of re-lease (Fig. 2). The IVR assay appears to be relatively robustto changes in ciprofloxacin concentration over a range of ±25%with respect to quantifying the amount of “burst,” the amountof drug released after 30 min, and the extent of release (Fig. 3)making these parameters suitable to monitor the release prop-erties. The IVR assay is sensitive to intentional changes in theamount of serum (±20%), with higher serum concentrationscausing an increase in the release rate. However, the normalvariability due to pipette technique (±2%) did not have a mean-ingful impact on the reproducibility of the assay (Fig. 4).

Changes in the HBS buffer pH had an effect on the releaserate greater than the day-to-day assay variations, with devi-ations below pH 7.4 having a greater effect than deviationsabove pH 7.4 (Fig. 5). It is unclear why relatively small devia-tions in buffer pH of 0.2 units have a significant influence onthe rate of release. While the exact mechanism is unknown,the apolipoproteins and lipoproteins in the serum likely inter-act with the phospholipid in the liposomes to cause release.11

One explanation may be that a change in pH affects the chargeof ionizable groups on these lipoproteins and thus modulatestheir affinity for the liposomes or their affinity for each other.Another explanation may be that the serum lipoproteins in-crease the permeability of the liposomes, without destroyingtheir structure, and dissipates the pH gradient across theirliposome bilayers. Only the neutral, uncharged ciprofloxacinmolecules are able to diffuse across the liposome bilayer so therate of release could be proportional to the fraction of neutral,uncharged ciprofloxacin molecules. It is possible that the pro-portion of neutral, uncharged ciprofloxacin molecules withinthe liposomes increases for the pH 7.6 buffer and decreases forthe pH 7.2 buffer. There may be other explanations as well. Re-gardless of the exact mechanism causing drug release, in orderto minimize this source of variability, it is essential to imple-ment laboratory procedures to ensure that the HBS buffer pHis as close to 7.40 as practicable.

While the details of the experiments have not been reportedin this paper, there was no influence of shaking speed over therange of 100–200 rpm on the IVR assay. A shaking speed of 150rpm, representing the midpoint of this range, has been usedthroughout these studies. In contrast, the incubation tempera-ture over the range of 35◦C–39◦C does affect the release rate inline with Arrhenius kinetics indicating that no phase transitionis taking place over this temperature range (Fig. 6). It is un-known if the liposomes retain their structure in the presence ofserum. Changes in temperature affect the diffusion rate acrossbiological membranes for large molecules to a greater degreethan for small molecules.28 They report that the activation en-ergy associated with diffusion of molecules within polyisobuty-lene increased from 33 to 75 kJ/mol as the diffusant molecularweight increased from 2 to 72 g/mol. Thus, the imputed ac-tivation energy of approximately 170 kJ/mol for ciprofloxacinwhich, being a larger molecule (MW 368) would require largerpores to diffuse through, is not inconsistent with this model.Other explanations are also possible. In any case, to reducevariability in the IVR assay, for example, across laboratories, itis thus essential that the temperature control of the water bathbe closely calibrated and monitored.

Each 500 mL container of bovine serum was divided into10 mL aliquots and stored frozen until the day of use. Serumsamples from all four containers produced similar IVR profiles,and had comparable effect in terms of the initial rate of release,T30 min (Fig. 7), but there is no assurance that subsequent con-tainers from this batch, or containers from a different batch,will not have larger differences in activity which could be prob-lematic when transitioning from one container to the next. Weplan to investigate in the future the impact of the variation inthe serum batches on the IVR assay, once they become avail-able.

For liquid aerosols produced by nebulizers, there is the po-tential for the aerosol droplets to dry during laboratory collec-tion, which could disrupt the liposome vesicles, in contrast tothe in vivo situation in which the droplets are unlikely to dryin the moist and humid environment of the lungs.7 The lipo-somal ciprofloxacin formulations evaluated in this paper werealso characterized for their stability to the nebulization processby collecting the aerosol in an SKC BioSampler that preventsthe artifactual drying.23 In addition, the residual solution inthe nebulizer at the end of nebulization was analyzed. Bothwere shown to retain the liposome size distribution and drugencapsulation properties. The IVR profiles of these liposomalciprofloxacin formulations were also shown to be unaffected bynebulization using three different nebulizers.7 Thus, this IVRmethod may also be amenable to characterization of liposomalaerosols if they are collected by a method that does not disruptthem.

The IVR assay was also shown to be discriminating for threeliposomal ciprofloxacin formulations including two that differedin the amount of ‘burst’ but had similar release rates (Figs. 2avs. 2b) and one with both a different “burst” and release rate(Fig. 8). The presence of surfactant in the latter liposomal for-mulation caused an increase in the amount of free drug as wellas an increase in the IVR release rate; however, no change to ei-ther parameter was observed for CFI when the surfactant wasadded to the release medium only. While high surfactant con-centrations can solubilize liposomes, at lower concentrationsthe surfactant can associate with the phospholipid bilayersof the liposome and lead to increased permeability without



general loss of structure to the liposome.29 This may explainthe increase in release rate. The increase in “burst” at T0 min

has also been explained by either of two mechanisms: the for-mation of transient pores during the initial surfactant-liposomeassociation process which allows some of the encapsulated drugto escape before the channels close, or the rupture of a subset ofliposomes which results in an increase in free drug.30 The pres-ence of surfactant in the release medium alone had no effect onthe IVR profile indicating that the surfactant concentration wastoo low to compromise the liposome structure or permeability.While these formulations all approach a plateau by the 2 h timepoint, the inclusion of a 4 h time point (T240 min) is to providediscrimination power for formulations having a slower releaseprofile which may occur for liposomes with differing composi-tions from those reported here (unpublished data). This IVRtest may also be useful to compare different batches of productor after product is aged or subjected to various stress condi-tions; for example, light, heat, acid, base, temperature cycling,and so on.

The pharmacokinetic profiles of CFI and DRCFI liposomalciprofloxacin formulations have been compared in an inhala-tion study in healthy subjects and both were found to have aprolonged systemic uptake, with a half-life of approximately10 h.31–33 However, the DRCFI formulation also achieved muchhigher systemic levels of ciprofloxacin over the first two hours,consistent with the “burst” of 30% ciprofloxacin observed in theIVR assay. The in vivo release is longer than the release ob-served in the IVR assay reported here, which was by intention,so that the assay would be rapid and convenient. The IVR as-say could have been designed to more closely simulate the invivo release rate by using a lower concentration of serum, or ef-fecting other changes to the methodology, but that was not thegoal of the present study: to develop and qualify a laboratorymethod for routine testing during product development.

CONCLUSIONS

This study describes an accurate and precise IVR method thathas been used to characterize the release of drug from two li-posomal ciprofloxacin formulations undergoing evaluation inclinical trials to treat lung infections in cystic fibrosis (CF) andnon-CF bronchiectasis patients. The robustness of the assaywas characterized with respect to changes in: (a) drug concen-tration, (b) serum (releasing agent) concentration, (c) bufferpH, (d) incubation temperature, and (e) serum containers. TheIVR profile provides a measurement of the initial “burst” fromthe liposomal formulation when it is designed to be presentin the formulation (and observed following administration tohumans), followed by a nearly linear release of drug initially,ultimately reaching a plateau value representing the full ex-tent of release. Selection of a convenient time point during theinitial release phase, which is 30 min in the case of these li-posomal ciprofloxacin preparations, allows for comparison be-tween different lots of product or product from various tem-poral stages in its shelf-life, to provide assurance that theproduct retains reproducible drug release characteristics. Thismethod was discriminatory between formulations of liposomalciprofloxacin that differed in composition. Although this IVRassay could be useful in principle for other liposomal products,its performance may be significantly affected by the charac-teristics of the drug and the formulation. Its usefulness will

therefore need to be assessed on a formulation by formulationbasis.

ACKNOWLEDGMENTS

The authors recognize and appreciate the support of CindyOuyang from Northern Lipids, Inc. who contributed to the de-velopment of a precursor IVR assay for liposomal ciprofloxacin.The authors thank Francis Dayton, Sujata Mudumba, and JimBlanchard of Aradigm Corporation for helpful discussions.

REFERENCES

1. Cipolla D, Redelmeier T, Eastman S, Bruinenberg P, Gonda I. 2011.Liposomes, niosomes and proniosomes – a critical update of their (com-mercial) development as inhaled products. In Respiratory Drug Deliv-ery Europe 2011; Dalby RN, Byron PR, Peart J, Suman JD, Farr SJ,Young PM, Eds. River Grove, Illinois: Davis Healthcare Int’l Publish-ing, pp 41–54.2. Cipolla D, Gonda I, Chan HK. 2013. Liposomal formulations for in-halation. Ther Deliv 4:1047–1072.3. Cipolla D, Chan HK. 2013. Inhaled antibiotics to treat lung infection.Pharm Pat Analyst 2:647–663.4. Serisier DJ. 2012. Inhaled antibiotics for lower respiratory tract in-fections: Focus on ciprofloxacin. Drugs Today 48:339–351.5. Serisier DJ, Bilton D, De Soyza A, Thompson PJ, Kolbe J, GrevilleHW, Cipolla D, Bruinenberg P, Gonda I. 2013. Inhaled, dual-release li-posomal ciprofloxacin in non-cystic fibrosis bronchiectasis (ORBIT-2)—A randomised, double-blind, placebo-controlled trial. Thorax 68(9):812–817.6. Martinez M, Rathbone M, Burgess D, Huynh M. 2008. In vitro and invivo considerations associated with parenteral sustained release prod-ucts: A review based upon information presented and points expressedat the 2007 controlled release society annual meeting. J Control Release129:79–87.7. Cipolla D, Wu H, Chan J, Chan HK, Gonda I. 2013. Liposomalciprofloxacin for inhalation retains integrity following nebulization. InRespiratory Drug Delivery Europe 2013; Dalby RN, Byron PR, Peart J,Suman JD, Farr SJ, Young PM, Eds. Berlin, Germany: Davis Health-care Int’l Publishing, pp 237–242.8. Brown CK, Friedel HD, Barker AR, Buhse LF, Keitel S, Cecil TL,Kraemer J, Morris M, Reppas C, Stickelmeyer MP, Yomota C, ShahVP. 2011. FIP/AAPS joint workshop report: Dissolution/In vitro releasetesting of novel/special dosage forms. AAPS Pharm Sci Tech 12:782–94.9. US FDA CDER Draft Guidance: Liposome Drug Products. 2002.http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm070570.pdf. Accessed November 14, 2013.10. Allen TM, Cleland LG. 1980. Serum-induced leakage of liposomecontents. Biochim Biophys Acta 597:418–426.11. Weinstein JN, Klausner RD, Innerarity T, Ralston E, BlumenthalR. 1981. Phase transition release, a new approach to the interactionof proteins with lipid vesicles. Applications to lipoproteins. BiochimBiophys Acta 647:270–284.12. Haran G, Cohen R, Bar LK, Barenholz Y. 1993. Transmembraneammonium sulfate gradients in liposomes produce efficient and sta-ble entrapment of amphipathic weak bases. Biochim Biophys Acta1151:201–215.13. Lasic DD, Ceh B, Stuart MCA, Guo L, Frederik PM, Barenholz Y.1995. Transmembrane gradient driven phase transitions within vesi-cles: Lessons for drug delivery. Biochim Biophys Acta 1239:145–156.14. Hunt CA. 1982. Liposomes disposition in vivo: Liposome stabilityin plasma and implications for drug carrier function. Biochim BiophysActa 719:450–463.15. Bhardwaj U, Burgess DJ. 2010. A novel USP apparatus 4 basedrelease testing method for dispersed systems. Int J Pharm 388:287–294.



16. Xu X, Khan MA, Burgess DJ. 2012. A two-stage reverse dialysis invitro dissolution testing method for passive targeted liposomes. Int JPharm 426:211–218.17. Terzano C, Allegra L, Alhaique F, Marianecci C, Carafa M. 2005.Non-phospholipid vesicles for pulmonary glucocorticoid delivery. Eur JPharm Biopharm 59:57–62.18. Scherphof G, Roerdink F, Waite M, Parks J. 1978. Disintegrationof phosphatidylcholine liposomes in plasma as a result of interactionwith high-density lipoproteins. Biochim Biophys Acta 542:296–307.19. Storm G, van Bloois L, Brouwer M, Crommelin DJ. 1985. The in-teraction of cytostatic drugs with adsorbents in aqueous media. Thepotential implications for liposome preparation. Biochim Biophys Acta10:343–351.20. Ong HX, Benaouda F, Traini D, Cipolla D, Gonda I, Forbes B, YoungPM. 2013. In vitro and ex vivo methods predict the enhanced lungresidence time of liposomal ciprofloxacin formulations for nebulisation.Eur J Pharm Biopharm [Epub ahead of print.].21. Webb MS, Boman NL, Wiseman DJ, Saxon D, Sutton K, WongKF, Logan P, Hope MJ. 1998. Antibacterial efficacy against an invivo Salmonella typhimurium infection model and pharmacokinetics ofa liposomal ciprofloxacin formulation. Antimicrob Agents Chemother42:45–52.22. Yim D, Blanchard JD, Mudumba S, Eastman S, Manda K,Redelmeier T, Farr SJ. 2006. The development of inhaled liposome-encapsulated ciprofloxacin to treat cystic fibrosis. In Respiratory DrugDelivery 2006; Dalby RN, Byron PR, Peart J, Suman JD, Farr SJ,Young PM, Eds. River Grove, Illinois: Davis Healthcare Int’l Publishing,pp 425–428.23. Cipolla D, Dayton F, Fulzele SV, Gabatan E, Mudumba S, Wu H,Yim D, Zwolinski R. 2010. Inhaled liposomal ciprofloxacin: In vitroproperties and aerosol performance. In Respiratory Drug Delivery2010; Dalby RN, Byron PR, Peart J, Suman JD, Farr SJ, Young PM,Eds. River Grove, Illinois: Davis Healthcare Int’l Publishing, pp 409–414.

24. Schreier H, Gonzalez-Rothi RJ, Stecenko AA. 1993. Pulmonary de-livery of liposomes. J Control Release 24:209–223.25. Allen TM. 1981. A study of phospholipid interactions between high-density lipoproteins and small unilamellar vesicles. Biochim BiophysActa 640:385–397.26. Forte TM, Cross CE, Gunther RA, Kramer GC. 1983. Characteri-zation of sheep lung lymph lipoproteins: Chemical and physical prop-erties. J Lipid Res 24:1358–1367.27. Marques MRC, Loebenberg R, Almukainzi M. 2011. Simulated bi-ological fluids wih possible application in dissolution testing. DissolutTechnol 18:14–28.28. Lieb WR, Stein WD. 1971. Implications for two different types ofdiffusion for biological membranes. Nat New Biol 234:220–222.29. Lichtenberg D, Robson RJ, Dennis EA. 1983. Solubilization ofphospholipids by detergents. Structural and kinetic aspects. BiochimBiophys Acta 737:285–304.30. Liu Y, Regen SL. 1993. Control over vesicle rupture and leakageby membrane packing and by the aggregation state of an attackingsurfactant. J Am Chem Soc 115:708–713.31. Bruinenberg P, Otulana B, Blanchard J, Cipolla D, Wilson J,Serisier D. 2009. Pharmacokinetics and antibacterial activity of in-haled liposomal ciprofloxacin hydrochloride in healthy volunteers andin cystic fibrosis (CF) patients. 32nd European Cystic Fibrosis Confer-ence, Brest, France. J Cystic Fibrosis 8 (Suppl 2), S49.32. Bruinenberg P, Blanchard J, Cipolla D, Serisier D. 2010. Safety,tolerability and pharmacokinetics of novel liposomal ciprofloxacinformulations for inhalation in healthy volunteers and in non-cysticbronchiectasis patients. American Thoracic Society (ATS) InternationalConference, New Orleans, LA. Am J Respir Crit Care Med 181:A3192.33. Cipolla D, Gonda I, Serisier D, Bruinenberg P. 2011. Dual Re-lease Ciprofloxacin for Inhalation (DRCFI) improves time to firstexacerbation in Bronchiectasis. International Society of Aerosols inMedicine 2011 Congress, Rotterdam, Netherlands, Poster P-014, pg.A-27, JAMPPD, Vol. 24, Number 3, 2011.


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