Development of Potential Orphan Drug Therapy of Intravesical

Liposomal Tacrolimus for Hemorrhagic Cystitis Due to Increased

Local Drug Exposure

Jayabalan Nirmal, Pradeep Tyagi,* Michael B. Chancellor,† Jonathan Kaufman,†Michele Anthony,† David D. Chancellor,† Yen-Ta Chen and Yao-Chi Chuang‡From the Department of Urology, Oakland University William Beaumont School of Medicine (JN, MBC), Royal Oak, Michigan,Department of Urology, University of Pittsburgh (PT) and Lipella Pharmaceuticals, Inc. (JK, MA, DDC), Pittsburgh, Pennsylvania, andDepartment of Urology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine (YTC, YCC),Kaohsiung, Taiwan, Republic of China

Purpose: The potent immunosuppressive effect of systemic tacrolimus is limitedby the high incidence of severe adverse effects, including nephrotoxicity andhypertension. Intravesical application of tacrolimus is hindered by its poor aque-ous solubility, justifying the search for novel delivery platforms such as lipo-somes. We evaluated the pharmacokinetics of tacrolimus encapsulated in lipo-somes (lipo-tacrolimus), which is being developed as a potential orphan drugindication for hemorrhagic cystitis.Materials and Methods: A single dose of lipo-tacrolimus was instilled in thebladder with the rat under anesthesia. Also, tacrolimus was instilled intravesi-cally or injected intraperitoneally in other rat groups. The tacrolimus dose wasconstant in all formulations at 200 �g/ml. At different times blood, urine andbladder samples were collected and stored at �80C until analysis. Tacrolimuslevels in samples were analyzed using microparticle enzyme immunoassay II.Results: The AUC of lipo-tacrolimus in serum at 0 to 24 hours was significantlylower than that of tacrolimus instillation or injection. Noncompartmental phar-macokinetic data analysis revealed maximum concentration of lipo-tacrolimusand tacrolimus in serum and urine at 1 and at 2 hours, respectively. UrineAUC(0–24) after intravesical administration was significantly higher than in theintraperitoneal group (p �0.05). Bladder tacrolimus AUC(0–24) did not differsignificantly between the groups.Conclusions: Single dose pharmacokinetics revealed that bladder instillation ofliposome encapsulated tacrolimus significantly decreased systemic exposure toinstilled tacrolimus as well as vehicle related toxicity. Intravesical liposomaltacrolimus may be a promising approach as an orphan drug indication for hem-orrhagic cystitis.

Key Words: urinary bladder, cystitis, tacrolimus,

Abbreviations

and Acronyms

LP � liposome

Tmax � time to peakconcentration

Accepted for publication October 25, 2012.Study received Kaohsiung Chang Gung Me-

morial Hospital animal care and use committeeapproval.

Supported by National Science Council Tai-wan Grant 100-2314-B-182A-021-MY3 and ChangGung Memorial Hospital Grant NMRPG 8A6063.

* Financial interest and/or other relationshipwith Astellas.

† Financial interest and/or other relationshipwith Lipella Pharmaceutical.

‡ Correspondence: Kaohsiung Chang GungMemorial Hospital, Kaohsiung, Taiwan, Republicof China (e-mail: chuang82@ms26.hinet.net).

liposomes, pharmacokinetics

TACROLIMUS (FK506) is a potent hydro-phobic immunosuppressive agent thathinders interleukin-2 dependent T-cellactivation by inhibiting calcineurinphosphatase.1 Tacrolimus has a di-

rect inhibitory effect on cell mediated

0022-5347/13/1894-1553/0THE JOURNAL OF UROLOGY®

© 2013 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION AND RES

immunity but systemic administra-tion is limited by the high incidenceof severe adverse effects, includingnephrotoxicity and hypertension.2,3

Site specific tacrolimus treatment

was efficacious as an ointment or

http://dx.doi.org/10.1016/j.juro.2012.10.123Vol. 189, 1553-1558, April 2013

EARCH, INC. Printed in U.S.A.www.jurology.com 1553

INTRAVESICAL LIPOSOMAL TACROLIMUS FOR HEMORRHAGIC CYSTITIS DUE TO DRUG EXPOSURE1554

lotion formulation for inflammatory skin condi-tions without systemic side effects.4

The restriction of immune response mechanismsto the targeted site or organ5 by topical therapyusing potent immunosuppressive drugs prompted usto investigate bladder instillation of tacrolimus forimmunogenic cystitis or sterile hemorrhagic cystitis.There is a reported 5.35% incidence of hemorrhagiccystitis in patients treated with cyclophosphamideand ifosfamide, and a lower incidence of hemor-rhagic cystitis due to radiation requiring therapy.The estimated prevalence of hemorrhagic cystitis isless than 80,000 cases in the United States.6

However, tacrolimus delivery in the bladder ishindered due to its poor aqueous solubility. LPswere used in the past as pharmaceutical nanocarri-ers to deliver poorly water soluble drugs.7,8 LPs arevesicles composed of concentric phospholipid bilay-ers separated by aqueous compartments.9 LPs canserve as vehicles for drug and gene delivery becausethey adsorb to cell surfaces and fuse with cells.10,11

The ability of LPs to form a molecular film on cellsurfaces led to their use in healing wounds andinjured uroepithelium.11–13

An earlier report from our laboratory demon-strated local immunosuppression produced by intra-vesical delivery of tacrolimus using LPs in a ratmodel of sterile hemorrhagic cystitis.5 From thetherapeutic point of view it is important to quantifythe drug concentration in urine and drug penetra-tion in the site of action. In the current study wehypothesized that the improved therapeutic effi-ciency of liposomal tacrolimus is due to a sustainedtherapeutic concentration in urine and bladder tis-sue, and decreased systemic absorption. We tested thehypothesis by evaluating the single dose pharmacoki-netics of lipo-tacrolimus after intravesical administra-tion compared with the pharmacokinetics of a plaintacrolimus formulation administered intravesicallyand systemically.

MATERIALS AND METHODS

Preparation of LPs,

Lipo-Tacrolimus and Plain TacrolimusA 5 mg/ml stock solution of tacrolimus (Sigma®) wasprepared in methanol (Thermo Fisher Scientific, Pitts-burgh, Pennsylvania). Dehydrated LPs (1.8 mg in 1 ml)were dispersed in physiological saline, in which dispersionis in liposomal form. LPs encapsulating tacrolimus (lipo-tacrolimus) were prepared by loading a dried film of 200�g tacrolimus (Sigma) into the LP dispersion. Briefly, astock methanolic solution of tacrolimus (80 �l) was driedby nitrogen in a glass tube to remove methanol. After thetube was dry with tacrolimus sticking to the inside wall,3.6 mg lyophilized lipid powder were added to the tube.Deionized water (2 ml) was then added. The tube was

vortexed vigorously for 15 minutes, frozen at �20C for 1

hour and vortexed again to achieve a turbid LP suspension.For the plain tacrolimus formulation the stock solution oftacrolimus in methanol was diluted 25-fold in normal salineto 200 �g/ml and used in animal experiments.

Drug AdministrationAll experimental procedures were reviewed and approvedby the Kaohsiung Chang Gung Memorial Hospital insti-tutional animal care and use committee. Rats were di-vided into group 1—6 treated with intravesical lipo-ta-crolimus, group 2—5 treated with intravesical tacrolimusand group 3—4 treated with intraperitoneal tacrolimus.An intravesical catheter was tied in place by a ligaturearound the urethral orifice with the rat under isofluraneanesthesia. The bladder was emptied of urine and in-stilled with 1 ml lipo-tacrolimus or tacrolimus (containing200 �g tacrolimus) for 1 hour via the catheter. The intra-peritoneally injected group received the same tacrolimusdose (200 �g) while under isoflurane anesthesia. The ta-crolimus dose was uniform in all groups at 200 �g.

Serum, Urine and Bladder PharmacokineticsBaseline urine and blood samples were collected from allexperimental rats. After drug administration, the ratswere sacrificed at 1, 2, 4, 8, 12 and 24 hours, respectively,to collect venous blood. Urine samples were collected fromawake rats kept in a metabolic cage and at sacrifice by aurethral catheter. For tissue harvesting the bladder wasemptied and excised at 1, 2, 8, 12 and 24 hours, respec-tively. The rats were deeply anesthetized using pentobar-bital and sacrificed by transcardiac perfusion with Krebsbuffer, followed by 4% paraformaldehyde fixative. Therats were then dissected to harvest the bladder.5 To esti-mate tacrolimus levels, samples were stored at �80C untilanalysis.

Tacrolimus AssaySerum and urine samples, and the bladders of rats in-stilled intravesically with lipo-tacrolimus or tacrolimusand rats injected intraperitoneally with tacrolimus weremaintained at �80C until analysis. The microparticle en-zyme immunoassay II for tacrolimus was performed on anIMX Analyzer (Abbott Laboratories, Abbott Park, Illinois)according to manufacturer instructions.5

HistopathologyThe bladder was fixed in 10% buffered formaldehyde for24 to 48 hours and embedded in paraffin. Bladder tissuefor histology was cut in 3 �m pieces and stained withhematoxylin and eosin.

Statistical AnalysisAUC was calculated using the linear trapezoidal rule.Quantitative data are shown as the mean � SEM. Statis-tical analysis was performed using the Student t test withp �0.05 considered significant using the Bonferroni cor-rection for multigroup comparison.

RESULTS

Pharmacokinetics

Serum. Serum levels were detectable at all time

points until 24 hours after the initiation of treat-

INTRAVESICAL LIPOSOMAL TACROLIMUS FOR HEMORRHAGIC CYSTITIS DUE TO DRUG EXPOSURE 1555

ment in rats administered lipo-tacrolimus, and in-travesical and intraperitoneal tacrolimus. The timevs concentration graph of tacrolimus showed thatthe serum tacrolimus concentration after a singleintraperitoneal administration was significantlyhigher at 2, 4 and 12 hours than that in rats instilledintravesically with tacrolimus or lipo-tacrolimus(p �0.05, fig. 1, A). The table shows the AUC ofserum tacrolimus in the groups. The intravesicaltacrolimus AUC was 4.5-fold lower than the intra-

Figure 1. Concentration-time profiles in rats in serum (A), urine (lipo-tacrolimus (solid line, diamonds) and tacrolimus (dashedpoints represent mean � SEM.h, hours. Asterisk indicates intraindicates intravesical vs intraperitoneal tacrolimus p �0.05.

Tacrolimus AUC(0–24)

Mean � SEM AUC(0–24) (ng/ml/gm/hr)*

Serum Urine Bladder

Intravesicallipo-tacrolimus

25.72 � 4.02 384.87 � 16.91 20,033.81 � 2,621.6

Tacrolimus:Intravesical 42.55 � 4.26 399.76 � 19.77 22,381.67 � 792.4Intraperitoneal 192.57 � 11.75 114.27 � 3.09 17,412.86 � 1,233.0

* Serum lipo-tacrolimus vs intravesical and intraperitoneal tacrolimus, serumintravesical vs intraperitoneal tacrolimus, urine lipo-tacrolimus vs intraperitonealtacrolimus, urine intravesical vs intraperitoneal tacrolimus and bladder intravesical

vs intraperitoneal tacrolimus p �0.05.

peritoneal tacrolimus AUC. Lipo-tacrolimus showedlower concentration at all points and the level wassignificantly lower at 1 hour than that of intravesi-cal tacrolimus (p �0.05). For lipo-tacrolimus andintravesical tacrolimus Tmax was attained at 1 hour,while for intraperitoneal tacrolimus it was attainedat 2 hours.

Urine. Figure 1, B shows urine tacrolimus levels inthe groups. After intraperitoneal injection, a signif-icantly lower tacrolimus concentration was achievedin urine than in the groups treated intravesicallywith tacrolimus and lipo-tacrolimus (p �0.05). TheAUC in urine for intraperitoneal injection was 3.5-fold less than in the groups treated with instillation.At the 2-hour point comparison of the intravesicalinstillation groups revealed a significantly lowerurine drug concentration in the lipo-tacrolimusgroup than in the tacrolimus group (p �0.05). How-ever, at later time points levels in the lipo-tacroli-mus group were higher, including significantlyhigher at 8 hours (p �0.05), suggesting sustaineddrug delivery. The table shows that the AUC of the

bladder tissue (C) measured after administration of intravesical), and intraperitoneal (dashed line, triangles) tacrolimus. Datal lipo-tacrolimus vs intravesical tacrolimus p �0.05. Dollar sign

B) andline, �vesica

intravesical lipo-tacrolimus and tacrolimus groups

guisha

INTRAVESICAL LIPOSOMAL TACROLIMUS FOR HEMORRHAGIC CYSTITIS DUE TO DRUG EXPOSURE1556

was significantly higher than the AUC of the intra-peritoneal tacrolimus group (p �0.05), although nosignificant difference was found between the intra-vesical lipo-tacrolimus and tacrolimus groups.

Bladder. There was no significant difference in thetissue distribution profile in the intravesical lipo-tacrolimus and tacrolimus groups. The bladder dis-tribution of tacrolimus in the intraperitoneally in-jected group showed rapid absorption at 1 hour,significantly greater than in the intravesical group(p �0.05, fig. 1, C). From hour 2 it showed a clear-ance phase, which continued until the 24-hour timepoint. AUC calculations revealed no significant dif-ference between the groups except for intravesical vsintraperitoneal tacrolimus (see table).

Histopathology

The safety of tacrolimus instilled locally in the blad-der using different formulations was assessed byhistopathology. No visible differences in histopathol-ogy across the groups were noted in rats sacrificed at1 hour but differences were identified in the blad-ders of rats sacrificed at 24 hours (fig. 2). Infiltrationof a large number of mononuclear inflammatory

Figure 2. Histopathology in rats after intraperitoneal injection (instillation (C and F) in rats sacrificed 1 (A to C) and 24 (D to F)inflammatory cells suggested that bolus delivery of tacrolimus dtaken at 24 hours in other 2 groups were histologically indistin

cells was noted between the tacrolimus and lipo-

tacrolimus groups (fig. 2, E and F), suggesting thatbolus delivery of the tacrolimus dose using alcoholas the carrier induced inflammation. Photographstaken at 24 hours in other 2 groups were histologi-cally indistinguishable from rat bladders harvestedat 1 hour. Since the tacrolimus dose was constant inall formulations, improved histopathology empha-sized the safety and biocompatibility of LPs as atacrolimus delivery platform (fig. 2, F).

DISCUSSION

Tacrolimus is a clinically effective immunosuppres-sive agent for inflammatory disorders.1,4 However,its efficacy after systemic administration is limitedby drug related side effects, such as hypertension,renal failure and neurotoxicity.2,3 Previous studiesshowed that topical administration of tacrolimus didnot compromise its anti-inflammatory efficacy, whiledecreasing its systemic side effects.4,14

Such encouraging reports prompted us to developthe local administration of LP encapsulated tacroli-mus as a potential treatment for immunogenic cys-titis or hemorrhagic cystitis.5 Sterile hemorrhagic

D) and instillation (B and E) of tacrolimus, and lipo-tacrolimusafter instillation. Infiltration of large numbers of mononuclear

th alcohol induced bladder inflammation (E and F). Photographsble from rat bladder harvested at 1 hour. Reduced from �100.

A andhours

ose wi

cystitis is a serious condition with significant unmet

INTRAVESICAL LIPOSOMAL TACROLIMUS FOR HEMORRHAGIC CYSTITIS DUE TO DRUG EXPOSURE 1557

medical needs. It is found in patients who received 1or each of 2 specific chemotherapy agents (cyclophos-phamide and ifosfamide) or pelvic radiation. In aprevious study our group observed that intravesicallipo-tacrolimus attenuated hemorrhage, inflamma-tory reaction and an overactive micturition patternin cyclophosphamide induced hemorrhagic cystitis.5

In the current study we investigated the mechanismof improved efficacy of a liposomal formulation oftacrolimus by studying its pharmacokinetics in se-rum, bladder tissue and urine. In addition, we eval-uated lipo-tacrolimus local tissue toxicity and bio-compatibility in normal rats by histopathology.

Although tacrolimus after bladder instillationshowed higher drug exposure in serum, liposomaltacrolimus inverted this phenomenon in favor of anincreased drug concentration in urine and bladdertissue. Decreased systemic exposure caused by LPencapsulation was also evident in the 2.5-fold lowerserum levels at 1 hour for lipo-tacrolimus relative totacrolimus (mean 2.93 � 0.25 vs 7.44 � 2.18 ng/ml,fig. 1, A). Furthermore, the AUC of lipo-tacrolimuswas 1.7-fold lower than that of tacrolimus (mean

Figure 3. Possible mechanisms of action of radiation and cyclcystitis and possible role of tacrolimus liposomal formulation. Cactivates urothelial transient receptor potential cation channel (Ttranscription via calcineurin dependent nuclear factor of activatemembrane lipid peroxidation, leading to acrolein formation andfree radical formation, leading to oxidative damage and inflamdephosphorylation of nuclear factor of activated T cells (NF-AT),

suppressing transcription of several cytokine genes. NOS, nitric oxide

42.55 � 4.26 ng/ml per hour). Lower serum tacroli-mus due to LP formulation is expected to causelesser drug related adverse effects (neurotoxicityand neuropathy).2,3 Decreased systemic exposure oftacrolimus can be partly explained by higher drugexposure in bladder tissue. Serum Tmax in the intra-vesical groups was attained at 1 hour, while Tmax intissue and urine was attained at 2 hours in the samegroups. In contrast, serum Tmax in the systemicgroup was achieved at 2 hours. The different timingof Tmax across the groups probably indicates differ-ences in the absorption mechanisms of the 2 routesinvestigated (fig. 1, A).

Since the drug concentration in urine was alsoavailable for bladder uptake, we then determinedurine pharmacokinetics in all groups (fig. 1, B). Ahigher tacrolimus concentration in the groups in-stilled with lipo-tacrolimus persisted up to 12 hoursrelative to that in the group instilled with tacroli-mus. The prolonged higher urinary concentration oftacrolimus in the lipo-tacrolimus group was consis-tent with the slow drug release provided by LP en-capsulation, as previously reported in penile skin.15

phamide (CYP) induced inflammation leading to hemorrhagicherapy agent cyclophosphamide is converted to acrolein, whichand TRPA1) receptors, causing calcium influx and cytokine genels (NF-AT) pathway. Radiation can have similar effect by causingting inflammatory pathways. Radiation can induce cytoplasmicn. Tacrolimus-FK506 binding protein (FKBP) complex inhibitsting formation of nuclear factor of activated T cells complex and

ophoshemotRPV1d T celactivamatio

preven

synthase. NO● � ●O2�, free radicals. P, phosphate.

INTRAVESICAL LIPOSOMAL TACROLIMUS FOR HEMORRHAGIC CYSTITIS DUE TO DRUG EXPOSURE1558

The higher AUC in serum (192.57 � 11.75 ng/ml perhour) was complemented by the lowest AUC in urine(114.2 � 3.09 ng/ml per hour) in rats with systemicinjection of tacrolimus. Serum protein binding oftacrolimus may also explain the lower levels inurine.

Tissue pharmacokinetics revealed that tacroli-mus was retained in bladder tissue up to 24 hours inall groups without a significant difference in theAUC (fig. 1, C). However, there was a significantincrease at 1 hour in the intraperitoneal injectiongroup, which may have been caused by the highconcentration in serum from which the drug wasabsorbed through the systemic circulation. Highertissue levels of tacrolimus in the lipo-tacrolimusgroup may also be related to the protection of tacroli-mus in the LPs from the efflux caused by P-glyco-protein expression in bladder tissue.16 This protec-tive effect of LPs against P-glycoprotein may explainthe supplementary increase in the drug concentra-tion in bladder tissue at the 2 and 8-hour points. Theslight increase in the bladder tacrolimus concentra-tion after intravesical plain tacrolimus administra-tion may have been caused by disruption of theepithelial barrier due to the alcohol in the formula-tion.

Decreased infiltration of mononuclear inflamma-tory cells on histopathology in normal rats after

lipo-tacrolimus instillation further revealed the su-

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CONCLUSIONS

Based on these serum, urine and bladder pharma-cokinetics data we propose that a single intravesicallipo-tacrolimus administration can yield pharma-cologically active drug levels in the bladder withsignificantly decreased systemic levels that aremaintained for at least 24 hours. Intravesical lipo-tacrolimus has the desirable attributes of higherresidence in the bladder and retention in urine aswell as significantly decreased systemic exposure ofinstilled tacrolimus and its resultant toxicity. Furtherstudies are warranted to study pharmacokinetics inrats with induced hemorrhagic cystitis. Intravesicaltacrolimus formulated in LPs may hold promise as anorphan drug therapy for hemorrhagic cystitis inducedby radiation and chemotherapy (fig. 3).

ACKNOWLEDGMENTS

Dehydrated LPs were provided by Lipella Pharma-

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