Microemulsions for intravesical delivery of gemcitabine. 

Urinary bladder cancer is among the most common

cers. In 2008, an estimated 69000 new cases of bladder

can-cer were diagnosed in the United States (51000 in men and

18000 in women) and approximately 14000 deaths from

bladder cancer were reported.

Its prevalence in the U.S.A.

and worldwide is approximately 490000 and over one

mil-lion, which makes bladder cancer a significant global public

health issue.

_{Approximately 70% of all urothelial bladder}

cancer cases are classified as superficial bladder cancer

(SBC), i.e., non-muscle-invasive.

The current strategy for treating superficial bladder cancer

is transurethral tumor resection followed by intravesical

adju-vant treatment (chemo- and/or immunotherapy) to reduce

re-currence risk.

Although Bacillus Calmette-Guerin (BCG)

immunotherapy is currently the most potent topical

treat-ment, it often has local or systemic adverse effects, and 30%

of high-risk patients who respond poorly to BCG still

ulti-mately require cystectomy.

Therefore, more active

chemotherapy agents are needed for patients who respond

poorly to BCG.

Gemcitabine, a novel deoxycytidine analogue, is an

in-hibitor of DNA synthesis with a broad spectrum of antitumor

activity. This agent, which has overall response rates ranging

from 22.5 to 28%, is highly effective and well tolerated as a

first- or second-line single-agent therapy for treating

metasta-tic transitional cell carcinoma.

Moreover, gemcitabine

in-duces apoptosis through Fas upregulation without activating

nuclear factor-kappa B (NF-

kB). Hence, it may be more

ef-fective than other anticancer drugs such as doxorubicin,

mit-omycin C and cisplatin for reducing undesirable side effects

such as proliferation, migration, immortality, and inhibition

of apoptosis. Therefore, gemcitabine is a strong candidate for

intravesical therapy in SBC patients who are refractory to

BCG.

However, after intravesical administration of high

doses (40 mg/ml) of gemcitabine in saline solution,

signifi-cant systemic absorption can still cause gastrointestinal,

bladder, and bone marrow toxicity, which limits its clinical

value.

Therefore, this study attempted to develop a

deliv-ery vehicle, which can increase gemcitabine accumulation in

bladder tissue and/or decrease systemic exposure for

effec-tive intravesical administration with minimal side effects.

Microemuslions are dispersions of oil in water (o/w) or of

water in oil (w/o) that are thermodynamically stable due to

the significant reduction of interfacial tension by adsorption

of surface amphiphiles. Microemulsions have been studied

intensively in recent years because of their ease of production

and their unique properties, including thermodynamic

stabil-ity, drug solubilstabil-ity, and drug permeability.

Previous

studies

_{pointed that microemulsions formulated with}

non-irritant components such as non-inonic surfactant with a

very low topical LD

21)

can be applied throughout the body,

which enables significant epidermal localization of the drug.

Therefore, the microemulsion system proposed in this study

was designed to be an intravesical vehicle for delivering

gemcitabine to the bladder. Microemulsions were prepared

using a mixture of polyoxyethylene sorbitan monooleate

(Tween) and sorbitan monolaurate (Span) as surfactant,

ethanol as cosurfactant, isopropyl myristate (IPM) as oil

phase, and distilled water as aqueous phase. The

physico-chemical properties of the microemulsions, including their

electrical conductivity, droplet size, and viscosity as well as

in vitro drug release properties were evaluated. In vivo

stud-ies including drug concentration in plasma, drug

accumula-tion in bladder tissue, and histological changes in tissue were

performed in a rat model to evaluate the effectiveness and

safety of the microemulsion delivery system.

Experimental

Materials Gemcitabine hydrochloride was purchased from Scinopharm (Taiwan). Sorbitan monolaurate (Span) was from Tokyo Chemical Industry (Japan). Polyoxyethylene sorbitan monooleate (Tween) was acquired from Showa Corporation (Japan). Sodium pentanesulfonic acid was from Wako Pure Chemical (Japan). Isopropyl myristate (IPM), perchloric acid and sodium phosphate were obtained from Merck Chemicals (Germany). Aceta-minophen and rhodamine B base were purchased from Sigma-Aldrich (U.S.A.). Tetrahydrouridine was obtained from Calbiochem (U.S.A.). All other chemicals and solvents were analytical reagent grade.

Preparation of Gemcitabine or Rhodamine Microemulsion

Formula-Microemulsions for Intravesical Delivery of Gemcitabine

Yi-Hung T

,

Yi-Hang H

,

Yaw-Bin H

,

Jui-Sheng C

,

Chi-Te H

,

and

Pao-Chu W

a_{Graduate Institute of Clinical Pharmacy, Kaohsiung Medical University; and} b_{School of Pharmacy, Kaohsiung Medical}

University; 100 Shih-Chuan 1st Road, Kaohsiung 80708, Taiwan.

Received May 22, 2010; accepted August 13, 2010; published online August 13, 2010

The objective of this work was to develop a safe and effective delivery vehicle for topical treatment of gem-citabine. The physicochemical properties, drug release rate, drug level in plasma and bladder, and histological changes of tissue after drug administration were investigated. The electrical conductivity, mean size, and viscos-ity of drug-loaded microemulsions were 0.8—102.0mS/cm, 116.8—322.5 nm, and 42.9—105.0 cpsⴛ103, respec-tively. Gemcitabine loaded microemulsions showed a slower and sustained release. After intravesical administra-tion of aqueous control and microemulsions treated, the drug concentraadministra-tions in plasma were 15.11mg/ml and 2.81—12.82mg/ml, respectively, and the accumulation in bladder were 18.27 mg and 9.12—64.16 mg, respectively. Microemulsions slightly decreased the systemic absorption and significantly enhanced the accumulation in blad-der tissue. Moreover, the preliminary toxicity studies revealed no overt adverse histological changes or tissue irri-tation by the microemulsion application. Therefore, the microemulsions were suggested to be a promising drug carrier for intravesical chemotherapy.

Key words gemcitabine; microemulsion; intravesical administration

© 2010 Pharmaceutical Society of Japan ∗ To whom correspondence should be addressed. e-mail: pachwu@kmu.edu.tw

tions The component rations of microemulsion formulations are listed in Table 1. The aqueous phase consisted of double-distilled water containing 40% of ethanol (cosurfactant) was prepared. The surfactant mixture of Tween/Sapn⫽3/2 and IPM was mixed well. Then the aqueous phase was added to the oily phase drop by drop. The clear and transparent microemul-sions were obtained under a vortex shaken at room temperature. Gemc-itabine and rhodamine were dissolved in the final microemulsion formula-tions to obtain concentraformula-tions of 1% and 0.5%, respectively.

Microemulsion Characterization The electrical conductivity of the microemulsions was measured by a handheld conductivity meter (WTW Cond 315i, SUNTEX, Germany) at 25⫾2 °C. Average particle sizes of gem-citabine microemulsions were determined by photo correlation spectroscopy by laser light scattering (Zetasizer 3000HSA, Malvern, U.K.) using a he-lium-neon laser with a l of 633 nm. Samples were loaded into 1 cm2

cylin-drical cuvettes and placed in a thermostated scattering chamber. Light scat-tering was monitored at a fixed angle of 90° and a fixed temperature of 25 °C.

The viscosity of the microemulsions was measured using a cone-and-plate viscometer (Brookfield, Model LVDV-II, U.S.A.) maintained at 37 °C. The x was read 30 s after y was measured, at which time the level of z had stabi-lized. The sample was sheared at a rate of 20 rpm. All experiments were re-peated three times, and the average results were recorded.

In-Vitro Gemcitabine Release Gemcitabine release rates from the mi-croemulsions were measured through a cellulose membrane (CelluSep®_T2

with a molecular weight cutoff of 6000—8000, Sartorious, Goettingen, Ger-many). Franz diffusion cells with a diffusion area of 3.46 cm2_{and 20 ml of}

receptor volume of pH 7.4 phosphate-citrate buffer were used. One milliliter of drug-loaded microemulsion was dosed in the donor compartment. The system was kept in a temperature-controlled water bath to maintain the donor compartment temperature at 37 °C, and the receptor phase was stirred continuously at 600 rpm. At predetermined time intervals, 0.5 ml samples were taken and replaced by the same volume of fresh preheated receptor medium. Gemcitabine concentrations were determined by HPLC. Each ex-periment was done in triplicate.

Cumulative release of gemcitabine was plotted against square root of time:

Q(t)⫽K⫻t(1/2)

where Q(t) is the cumulative amount (mg/cm2_{) of gemcitabine released in}

time t (⬍60%), K (mg/(h1/2_cm2_{)) is the kinetic constant indicating}

gemc-itabine release rate, and t(1/2)_{is square root of time.}

In Vivo Intravesical Administration of Gemcitabine Sprague-Dawley female rats weighing 200—250 g were used in this study according to the care and use protocol for experimental animals approved by the Institutional Review Board at this institution. Animals were housed in a temperature-con-trolled room with free access to food and water until use.

Each animal was anesthetized by isoflurane. The residual urine was evac-uated by pressing the lower abdomen. A polyurethane catheter (25 gauge, BD Angiocath Plu®_{, Becton Dickinson Korea, Gyeongbuk, Korea) was}

in-serted into the bladder through the urethra. The bladder was washed twice with 0.5 ml normal saline. An 0.8 ml quantity of gemcitabine microemulsion or saline solution (as control) was then instilled into the bladder and main-tained for 1 h by ligating the urethra orifice using a cotton thread under isoflurane anesthesia. One hour after the instillation, the cotton thread was

cut off and the isoflurane was moved out. The animals will promptly recover consciousness, and then evacuate the residual formulation by urine mic-turate. The animals were euthanized at 0.5, 1, and 2 h after drug instillation, and blood samples were collected in heparinised tubes containing 10ml tetrahydrouridine (1 mg/ml saline) to prevent ex vivo degradation of the gemcitabine by cytidine deaminase in the serum. Blood samples from the jugular vein were examined for systemic exposure of gemcitabine during and after intravesical administration of gemcitabine. The heparinised tubes were centrifuged for 10 min at 4000 rpm at 4 °C. A pipette was used to trans-fer the top 0.2 ml plasma layer into another tube containing 0.05 ml internal standard of acetaminophen 200mg/ml and 0.1 ml of 1M perchloric acid.

After 5 s vortex, the mixture was incubated in ice bath for 10 min then cen-trifuged at 16000 g for 5 min at 4 °C. The 0.02 ml of clear supernatant was analyzed by HPLC method as reported previously with some modifica-tions.22)

At the end of the intravesical administration experiment, the gemcitabine accumulation in the bladder was also determined by a homogenization method. After wash, the excised bladder cut to small pieces and place into a glass tube containing 2 ml saline in an ice bath. The sample was homoge-nized at 17800 rpm for 1 min. The homogenizer probe was washed with 2 ml of saline to recover residual adhering tissues. The two saline fractions were combined, and then shaken horizontally for 10 min. The resulting solution was centrifuged at 3000⫻g for 5 min. The supernatant was used for the assay by HPLC.22)

HPLC Analysis The HPLC analysis was performed using an Agilent 1200 series HPLC system. A Merck Lichrospher®_{C18 column (250⫻4 mm}

i.d., particle size 5mm) was used. The mobile phase was a mixture of aque-ous phase containing 3 mMpentanesulfonic acid and 50 mMsodium phos-phate (adjusted to pH 3.0 by phosphoric acid) and acetonitrile at a 95 : 5 ratio and flow rate was 0.9 ml/min. The UV detection was performed at 278 nm. The detection limits for drug concentration in plasma and drug ac-cumulation in the bladder were 25 ng/ml and 250 ng/bladder, respectively.

Penetration Depth Measurement by Confocal Laser Scanning Mi-croscopy (CLSM) Permeation of bladder tissue by rhodamine-loaded mi-croemulsions was investigated using CLSM (FV 500, Olympus, Tokyo, Japan). Rats were sacrificed at 1 h after drug administration, and bladders were removed intact. Connective tissue, lipoid tissue, and drug residues were removed from the bladder walls. The bladders were then sectioned into 1 mm2_{specimens to compare the penetration depths of the microemulsion}

formulations. Bladder wall thickness was measured by CLSM through the z axis at ca. 20mm increments. Optical excitation was performed using a 500 nm argon laser, and fluorescence emission was detected at 540 nm. Two different sites were evaluated in each bladder. Fluorescence emissions were measured in darkness to avoid errors caused by ambient light.

Histopathological Evaluation Rats were sacrificed at 1 h after blank microemulsion instillation was performed. The bladders were removed in-tact, cleaned to remove connective and lipoid tissue from around the wall, and weighed to test for presence of edema. The bladders were then fixed in 10% buffered formaldehyde for 24 h. Each bladder was cut into three equal sections from the dome to the bottom. Each piece was dehydrated using ethanol and embedded in paraffin. At least three cross sections 20mm thick were taken from each section of bladder for hematoxylin–eosin staining.

Statistical Analysis Group comparisons were performed using analysis of variance (ANOVA) tests. A p value less than 0.05 was considered statisti-Table 1. Composition, Particle Size, Polydispersity Index (PI), Electrical Conductivity (EC) and Viscosity of Blank and Gemcitabine Loaded Microemul-sions

IPM W S Particle size

PI EC Viscosity (%) (%) (%) (nm) (mS/cm) (cps⫻103₎ Aa) _{13 35 52 299.7⫾22.1 0.78⫾0.06 28.6 42.2⫾0.7} Ba) _{50 10 40 122.7⫾1.8 0.37⫾0.01 0.5 44.3⫾0.5} Ca) _{20 20 60 323.8⫾27.7 0.79⫾0.09 2.3 108.8⫾3.4} Ab) _{13 35 52 176.1⫾10.7 0.62⫾0.04 102.0 42.9⫾1.0} Bb) _{50 10 40 322.5⫾9.6 0.75⫾0.06 0.8 45.1⫾1.0} Cb) _{20 20 60 116.8⫾2.5 0.30⫾0.01 6.6 105.0⫾1.7} Ac) _{13 35 52 163.7⫾13.7 0.68⫾0.11 103.6 49.8⫾0.4} Bc) _{50 10 40 323.4⫾10.6 0.68⫾0.05 0.9 43.9⫾3.5} Cc) _{20 20 60 120.3⫾3.8 0.45⫾0.04 6.3 102.3⫾2.4}

Microemulsion composited of water (W) containing 40% cosurfactant, isopropyl myristate (IPM), and mixture surfactant (S) of Tween/Span⫽3/2. a) Blank microemul-sions. b) Gemcitabine loaded microemulsions. c) After 2 months storage of gemcitabine loaded microemulsions.

cally significant. Tukey test was then performed to analyze two groups con-secutively.

Results and Discussion

Physicochemical Characterization of Microemulsions

Translucent and stable microemulsions were formed by

mix-ing different components of the oil phase with the aqueous

phase containing cosurfactant and surfactant. The

physico-chemical parameters of blank and drug loaded

microemul-sions are shown in Table 1. The electrical conductivity of all

blank microemulsions was 0.5 to 28.6

mS/cm, which

ex-ceeded the electrical conductivities of oil phase (0.0

mS/cm),

aqueous phase with cosurfactant (0.4

mS/cm), and surfactant

(0.40

mS/cm). The results were consistent with a previous

re-port

_{that microemulsions, even w/o type, can increase the}

electrical conductivity of formulations. The w/o

microemul-sion B with higher content of oil had lower electrical

conduc-tivity than the o/w type microemulsions A and C with lower

content of oil. As expected, addition of gemcitabine

hy-drochloride significantly increased the electrical conductivity

of both type microemulsions. Electrical conductivity of the

w/o type microemulsion B increased from 0.5 to 0.8

mS/cm,

a 1.9 fold increase, whereas those of o/w type

microemul-sions A and C increased from 2.3—28.6

mS/cm to 6.6—

102.0

mS/cm, a 2.9—15.5 fold increase. These data indicate

that gemcitabine dissolution was fastest in aqueous phase.

Moreover, electrical conductivity of both blank and

drug-loaded microemulsions correlated with that of proportion of

aqueous phase in formulation. The increased electrical

con-ductivity may have been caused by increased dissociation of

surfactant as a function of water content.

Table 1 shows that, at 37 °C, blank and drug-loaded

mi-croemulsions exhibited viscosities of 42.2—108.8 cps

⫻10

and 42.9—105.0 cps

⫻10

_{, respectively. Viscosity was}

unaf-fected by drug incorporation (p

⬎0.05), whereas increased

surfactant correlated with increased viscosity (A vs. C,

p

⬍0.05).

Mean droplet size was small in all blank and drug-loaded

microemulsions (122.7—323.8 nm and 116.8—322.5 nm,

re-spectively). Notably, the drug-loaded formulations had the

smallest droplet size in o/w microemulsions and the largest

droplet size in w/o microemulsions. The phenomenon might

be attributed to that drug is solubilized at the interface of

mi-croemulsion droplets and shrinks the droplets by interacting

with the surfactant.

After 2 months storage at room temperature, none of the

microemulsions in the current study revealed changes in

clar-ity, phase behavior, or particle size (Table 1). All

microemul-sions exhibited gemcitabine concentrations above 98.0

⫾

1.5%, which indicated that no degradation occurred.

In Vitro Gemcitabine Release

It is well known that

mi-croemulsion type, internal structure, size, and viscosity

might influence the drug release from microemulsions.

In

this study, two type microemulsions with different

physico-chemical properties (Table 1) were investigated. Figure 1

shows their release profiles. Gemcitabine release from saline

was studied as a control. Figure 1 shows that drug release

from microemulsions was slower than that from saline (78%

release in 1 h), which indicates the potential effectiveness of

microemulsions as drug delivery vehicles for controlled

re-lease.

To facilitate comparison of different formulations,

the release rate constant was calculated from the slope of the

linear portion of the plots of cumulative drug quantity

re-leased against t

_{and expressed in}

_mg/(cm

_h

_{). The release}

rates of microemulsions A, B, and C were 630.1

⫾55.0,

390.3

⫾96.2, and 141.1⫾9.6

mg/(cm

h

), respectively,

indi-cating that the ratio of ingredients in the microemulsions was

an important factor for modulating drug release. Generally,

increased emulsion viscosity tends to decrease drug release

rate by increasing structural rigidity and droplet size while

reducing total surface area.

The current study revealed

a strong correlation (r

⫽0.886) between viscosity and release

rate, but a weak correlation (r

⫽0.291) between droplet size

and release rate. In addition, surfactant quantity

corre-sponded positively with viscosity but negatively with release

rate and droplet size (A vs. C, p

⬍0.05). The result might be

due to a decreased thermodynamic activity of the drug in

mi-croemulsion with higher concentrations of surfactant.

An-other possibility was that drug diffusion through the double

layer microemulsion might be a rate-determining step, as the

viscosity plays an important role in controlling the release of

the drug into the receptor.

In Vivo Intravesical Instillation of Gemcitabine

The

rats were given intravesical doses of 0.8 ml gemcitabine

saline solution (control) or drug-loaded microemulsions, and

the doses were maintained in the bladder for 1 h by ligating

the urethral orifice. Figures 2 and 3 show the plasma

concen-trations and gemcitabine accumulations in the bladder, which

were determined at 0.5, 1, and 2 h after drug instillation and

used to evaluate the safety and efficacy of the

microemul-sions. Formulations were ranked by measuring the plasma

drug concentration at 1 h after drug instillation as control

⬎

A

⬎B⬎C. In terms of drug accumulation in the bladder, they

were ranked as A

⬎B⬎control⬎C. Strong correlations were

noted between release rate and bladder accumulation

(r

⫽0.9996) and between release rate and plasma

concentra-tion (r

⫽0.9994), which indicated that the permeation and

ac-cumulation of gemcitabine delivered by microemulsion

de-pended on the release rate of drug from the formulation.

Moreover, plasma concentrations corresponded positively

with gemcitabine accumulations in the bladder. These

labora-tory results agree with earlier canine studies,

which

re-ported significant dose-dependent systemic absorption of

Fig. 1. In Vitro Release–Time Profiles of Gemcitabine from

gemcitabine after intravesical administration.

Compared to controls, subjects treated with

microemul-sion C showed lower plasma concentration (p

⬍0.05) and less

bladder accumulation (p

⬎0.05). In subjects treated with

mi-croemulsion B, the plasma concentration and bladder

accu-mulation were comparable to those of controls (p

⬎0.05).

Compared to controls, subjects treated with microemulsion

A, the plasma concentration was slightly decreased (p

⬎0.05)

and the bladder accumulation was significantly increased

(p

⬍0.05). The comparison results showed that the bladder

accumulation was increased without a corresponding

in-crease in plasma drug concentration when compared to the

control. The result was consistent with previous reports

which pointed that microemulsions can efficiently promote

localization without concomitantly increasing systemic side

effects.

In addition, plasma concentration and bladder

accumula-tion of gemcitabine were also evaluated at 0.5 and 2 h after

instillation of microemulsion A and control. Figure 3 shows

that, in both groups of microemulsion and control,

gemc-itabine was absorbed and distributed to the bladder wall by

0.5 h. After 2 h, most of the drug was eliminated, which

indi-cated its short half-life after intravesical administration,

pos-sibly due to urine maturation and excretion. Previous dog

studies

_{have also reported a short half-life after intravesical}

administration of 350 mg gemcitabine. However, as expected,

bladder accumulation and plasma concentration at 0.5 and

2 h, respectively, were higher in subjects treated with

mi-croemulsions than in controls.

In Vivo CLSM Analysis

To clarify the permeation

depth of microemulsion in the bladder, superficial

distribu-tion of rhodomine-loaded microemulsion was analyzed by

CLSM. Figure 4 depicts the optical scanning results for the

superficial layer at ca. 20

mm increments for 16 fragments

from the surface to the muscle (left to right, top to bottom).

After intravesical administration of microemulsion A, only

pale signals were detected in the first image (ca. 20

mm) and

in the 9—16th images (180—300

mm), and most signals

were in the 140—160

mm range.

Histological Examination of Bladder Tissue

Safety is

a key factor in delivery vehicle formulation. For preliminary

safety evaluation of the experimental formulation,

histologi-cal photographs (Fig. 5) were compared between controls

treated with saline for 1 h and the group treated with blank

microemulsion A for 1 h. Mild signs of inflammatory

re-sponse (subepithelial leukocyte infiltration) and epithelial

cellular nuclear enlargement was seen in drug solution

Fig. 3. Plasma Concentration and Accumulation in Bladder at 0.5, 1 and 2 h after Gemcitabine Microemulsion A and Aqueous Control Solution In-travesical Administration in Rats (n⫽6)

∗ p⬍0.05 compared with control group.

Fig. 4. Confocal Laser Scanning Microscopic (CLSM) Micrographs of the Rhodamine Intensity in Bladder after Intravesical Administration of Mi-croemulsion A for 1 h

The full thickness was divided into 16 fragments from the surface of the bladder mu-cosa (left to right, top to bottom). Images below the photographs of the 16 segments are the sum of all segments.

Fig. 2. Plasma Concentration and Accumulation in Bladder at 1 h after Gemcitabine Microemulsions and Aqueous Solution Intravesical Adminis-trations in Rats (n⫽6)

∗ p⬍0.05 compared with saline control group.

treated group. The photographs revealed no bladder wall

damage in the microemulsion-treated groups, and their

mor-phologies were similar to those of controls.

Conclusion

This study evaluated the potential use of microemulsions

as vehicles for topical delivery of gemcitabine. The results

suggest that microemulsions efficiently promote gemcitabine

localization into the bladder wall. By enhancing gemcitabine

accumulation in the bladder wall, the microemulsions may be

useful for optimizing drug delivery without concomitantly

increasing systemic side effects.

Acknowledgment This work was supported by the National Science Council of Taiwan (NSC 95-2320-B-037-022).

References

1) Jemal A., Siegel R., Ward E., Hao Y., Xu J., Murray T., Thun M. J., CA

Cancer J. Clin., 58, 71—96 (2008).

2) Lerner S. P., Urol. Oncol., 23, 275—279 (2005).

3) Kirkali Z., Chan T., Manoharan M., Algaba F., Busch C., Cheng L., Kiemeney L., Kriegmair M., Montironi R., Murphy W. M., Sesterhenn I. A., Tachibana M., Weider J., Urology, 66, 4—34 (2005).

4) Mugabe C., Hadaschik B. A., Kainthan R. K., Brooks D. E., So A. I., Gleave M. E., Burt H. M., BJU Int., 103, 978—986 (2009).

5) Shen Z., Shen T., Wientjes M. G., O’Donnell M. A., Au J. L., Pharm.

Res., 25, 1500—1510 (2008).

6) Nseyo U. O., Lamm D. L., Semin. Surg. Oncol., 13, 342—349 (1997). 7) Nadler R. B., Catalona W. J., Hudson M. A., Ratliff T. L., J. Urol., 152,

367—373 (1994).

8) Lorusso V., Pollera C. F., Antimi M., Luporini G., Gridelli C., Frassineti G. L., Oliva C., Pacini M., De Lena M., Eur. J. Cancer, 34, 1208—1212 (1998).

9) Moore M. J., Tannock I. F., Ernst D. S., Huan S., Murray N., J. Clin.

Oncol., 15, 3441—3445 (1997).

10) Serretta V., Galuffo A., Pavone C., Allegro R., Pavone-MacAluso M.,

Urology, 65, 65—69 (2005).

11) Gardmark T., Carringer M., Beckman E., Malmstrom P. U., Urology, 66, 527—530 (2005).

12) Dalbagni G., Russo P., Bochner B., Ben-Porat L., Sheinfeld J., Sogani P., Donat M. S., Herr H. W., Bajorin D., J. Clin. Oncol., 24, 2729— 2734 (2006).

13) Mohanty N. K., Nayak R. L., Vasudeva P., Arora R. P., Urol. Oncol., 26, 616—619 (2008).

14) Cozzi P. J., Bajorin D. F., Tong W., Nguyen H., Scott J., Heston W. D., Dalbagni G., Clin. Cancer Res., 5, 2629—2637 (1999).

15) Hendricksen K., Witjes J. A., Curr. Opin. Urol., 16, 361—366 (2006). 16) Paolino D., Ventura C. A., Nistico S., Puglisi G., Fresta M., Int. J.

Pharm., 244, 21—31 (2002).

17) Peltola S., Saarinen-Savolainen P., Kiesvaara J., Suhonen T. M., Urtti A., Int. J. Pharm., 254, 99—107 (2003).

18) Yuan Y., Li S. M., Mo F. K., Zhong D. F., Int. J. Pharm., 321, 117— 123 (2006).

19) Grundmann-Kollmann M., Behrens S., Peter R. U., Kerscher M.,

Pho-todermatol. Photoimmunol. Photomed., 15, 87—89 (1999).

20) Baroli B., Lopez-Quintela M. A., Delgado-Charro M. B., Fadda A. M., Blanco-Mendez J., J. Controlled Release, 69, 209—218 (2000). 21) Kibbe A. H., “Handbook of Pharmaceutical Excipients,” 3rd ed.,

Phar-maceutical Press, London, 2000.

22) Kirstein M. N., Hassan I., Guire D. E., Weller D. R., Dagit J. W., Fisher J. E., Remmel R. P., J. Chromatogr. B Analyt. Technol. Biomed. Life

Sci., 835, 136—142 (2006).

23) Bumajdad A., Eastoe J., J. Colloid Interface Sci., 274, 268—276 (2004).

24) Baker R. C., Florence A. T., Ottewill R. H., Tadros T. H. F., J. Colloid

Interface Sci., 100, 332—349 (1984).

25) Spernath A., Aserin A., Ziserman L., Danino D., Garti N., J.

Con-trolled Release, 119, 279—290 (2007).

26) Madhusudhan B., Rambhau D., Apte S. S., Gopinath D., J. Drug

Tar-get., 15, 154—161 (2007).

27) Nornoo A. O., Osborne D. W., Chow D. S., Int. J. Pharm., 349, 108— 116 (2008).

28) Chung H., Kim T. W., Kwon M., Kwon I. C., Jeong S. Y., J. Controlled

Release, 71, 339—350 (2001).

29) Spernath A., Aserin A., Adv. Colloid Interface Sci., 128—130, 47—64 (2006).

30) Wu H., Ramachandran C., Bielinska A. U., Kingzett K., Sun R., Weiner N. D., Roessler B. J., Int. J. Pharm., 221, 23—34 (2001). 31) Rhee Y. S., Choi J. G., Park E. S., Chi S. C., Int. J. Pharm., 228, 161—

170 (2001).

32) Ho H. O., Hsiao C. C., Sheu M. T., J. Pharm. Sci., 85, 138—143 (1996).

33) Grundmann-Kollmann M., Behrens S., Krahn G., Leiter U., Ochsendorf F., Kaufmann R., Peter R. U., Kerscher M., Arch.