Paclitaxel gelatin nanoparticles for intravesical bladder cancer therapy by Francisco Alvarez

Investigative Urology

Paclitaxel Gelatin Nanoparticles for Intravesical Bladder Cancer Therapy Ze Lu,* Teng-Kuang Yeh, Jie Wang,* Ling Chen, Greg Lyness, Yan Xin, M. Guillaume Wientjes,* Valerie Bergdall, Guillermo Couto, Francisco Alvarez-Berger, Carrie E. Kosarek and Jessie L.-S. Au*,† From Optimum Therapeutics, L. L. C. (ZL, TKY, JW, LC, GL, YX) and Colleges of Pharmacy (MGW, JLA) and Veterinary Medicine (VB, GC, FAB, CEK), Ohio State University, Columbus, Ohio

Abbreviations and Acronyms AUC ⫽ area under concentration time curve ELISA ⫽ competitive inhibition enzyme immunoassay HPLC ⫽ high performance liquid chromatography MMC ⫽ mitomycin C PBS ⫽ phosphate buffered saline PNP ⫽ paclitaxel gelatin nanoparticle Submitted for publication June 28, 2010. Study received approval from the Ohio State University institutional animal care and use committee, and the Veterinary Medicine Hospital institute review board. Supported by Research Grants R43 CA107743 and R44 CA107743 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services. Presented at annual meeting of American Urological Association, Chicago, Illinois, April 2530, 2009. * Financial interest and/or other relationship with Optimum Therapeutics. † Correspondence: College of Pharmacy, Ohio State University, 496 West 12th Ave., Columbus, Ohio 43210 (telephone: 614-292-3494; FAX: 614688-3223; e-mail: au.1@osu.edu).

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Purpose: We have noted that inadequate drug delivery to tumor cells is a major cause of failed intravesical therapy for nonmuscle invading bladder cancer, partly due to the dilution of drug concentration by urine production during treatment. To address this problem we developed gelatin nanoparticles of paclitaxel designed to yield constant drug concentrations. The hypothesis that a constant, therapeutic concentration in urine, bladder tissue and tumors can be attained was evaluated in dogs. Materials and Methods: We studied drug release from paclitaxel gelatin nanoparticles in culture medium in vitro. In vivo studies were performed in tumor-free dogs and in pet dogs with naturally occurring transitional cell carcinoma, in which the pharmacokinetics of paclitaxel gelatin nanoparticles were determined in plasma, urine and tumors. Results: Paclitaxel release from paclitaxel gelatin nanoparticles in vitro and in vivo was rate limited by the drug solubility in aqueous medium. This property yielded constant drug concentrations independent of changes in urine volume during the 2-hour treatment. Intravesical paclitaxel gelatin nanoparticles showed low systemic absorption, and favorable bladder tissue/tumor targeting and retention properties with pharmacologically active concentrations retained in tumors for at least 1 week. Conclusions: Constant drug release from paclitaxel gelatin nanoparticles may overcome the problem of drug dilution by newly produced urine and the sustained drug levels in tumors may decrease treatment frequency. Key Words: urinary bladder; carcinoma, transitional cell; paclitaxel; dogs; nanoparticles PATIENTS presenting with nonmuscle invading tumors, ie Ta tumors located in the urothelium, T1 tumors located in the lamina propria and/or carcinoma in situ Tis, are typically treated with transurethral tumor resection plus neoadjuvant or adjuvant intravesical immunotherapy or chemotherapy.1 The most commonly used agents are bacillus Calmette-Guérin and MMC. We

have identified inadequate drug delivery and chemoresistance as the 2 major causes of the highly variable, incomplete response of intravesical therapy in patients. Our data further indicate that a major source of drug exposure variability and, hence, variable treatment efficacy is dilution of the instilled drug by residual and newly produced urine.2– 4

Vol. 185, 1478-1483, April 2011 Printed in U.S.A. DOI:10.1016/j.juro.2010.11.091

AND

RESEARCH, INC.

PACLITAXEL GELATIN NANOPARTICLES FOR INTRAVESICAL BLADDER CANCER THERAPY

The American Urological Association recommends adjuvant intravesical treatment as the standard of care in patients at high risk. However, a recent study based on the Surveillance, Epidemiology and End Results database showed that only 42% of eligible patients receive intravesical therapy.5 The conclusion of that group that overcoming underuse is a priority was echoed by others.6 Potential reasons for underuse include inconvenience due to multiple weekly treatments, the required pharmacokinetic interventions, such as dehydration and ultrasound guided bladder emptying, and unfamiliarity or difficulty with dose administration. We propose that the problems of inadequate drug delivery, chemoresistance and underuse of intravesical therapy may be overcome by using chemotherapy that 1) has activity equal to or greater than that of MMC, 2) penetrates bladder tissue better than MMC, 3) requires less frequent treatment and 4) is easy to administer, eg without exhaustive bladder emptying or patient dehydration. We identified paclitaxel as a candidate since it showed higher activity against human bladder cancer cells7 and produced a 42% response in cases of advanced and/or metastatic bladder cancer in a phase II trial.8 Also, due to its lipophilicity paclitaxel can penetrate the urothelium more readily than MMC.9 Paclitaxel is tightly bound to intracellular macromolecules such as tubulin and microtubule, resulting in significant drug accumulation (70 to 1,500-fold) and retention in tumor cells.10 It thereby offers the possibility of extending drug action beyond the typical 2-hour treatment duration. Finally, paclitaxel induces apoptosis through p53 dependent and independent pathways, which in view of the high frequency of p53 mutations in bladder cancer11 is an advantage over agents such as MMC, which depend on functional p53 pathways for apoptosis.12 Accordingly we studied the paclitaxel formulation approved for intravenous administration, ie paclitaxel dissolved in Cremophor® and ethanol.13 Results showed that during intravesical therapy paclitaxel remains entrapped in Cremophor micelles, which decreases the free drug fraction and consequently lowers drug penetration into bladder tissue. We then explored using the surface-active agent dimethyl sulfoxide to disrupt micelle structure. Results showed that while dimethyl sulfoxide increased the free drug fraction, it had other counteracting effects, such as increased urine production and increased drug removal by perfusing capillaries, that decreased tissue drug concentrations.14 We eventually developed a new formulation, PNPs, that rapidly released paclitaxel, had activity in vitro and yielded a high paclitaxel concentration in bladder tissue in 3 tumor-free dogs in vivo.15 In the current study we evaluated whether intravesical PNPs provide constant drug concentrations in urine and yield favorable concentration-time-

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depth profiles in bladder tissues and tumors. The study was done in tumor-free dogs and in pet dogs with naturally occurring bladder tumors.

MATERIALS AND METHODS Chemicals Cephalomannine was obtained from the National Cancer Institute, Bethesda, Maryland. Paclitaxel was obtained from Hande Tech, Houston, Texas. HPLC supplies were obtained from Fisher Scientific, Fairlawn, New Jersey. Porcine skin type A gelatin (175 bloom), Sephadex™ G50, pronase and glutaraldehyde (25% in water), and all other chemicals were obtained from Sigma®. ELISA kits were obtained from Hawaii Biotech, Aiea, Hawaii. All chemicals were used as received.

PNP Preparation and Paclitaxel Release PNPs were prepared using the desolvation method, as described previously.15,16 Mean ⫾ SD drug loading in 4 preparations was 0.52% ⫾ 0.14%. Mean particle size in 4 preparations was 638 ⫾ 61 nm, as determined using a Nano ZS90 particle size analyzer (Malvern Instruments, Malvern, United Kingdom). For in vitro release PNP (1 to 50 ␮g/ml) was incubated with 20 ml PBS at 37C. The in vivo drug release study was done in tumor-free dogs, as described.

Animal Protocols Two types of dogs were studied. Tumor-free beagle dogs were used to determine in vivo drug release, systemic absorption and pharmacokinetics in bladder tissues under protocols approved by the Ohio State University institutional animal care and use committee. Tumor bearing pet dogs of various breeds with pathologically confirmed transitional cell carcinoma were used to determine paclitaxel/ PNP levels and drug effects in tumors. These dogs were patients seen at Ohio State University Veterinary Medicine Hospital that received intravesical therapy for disease management under protocols approved by the Veterinary Medicine Hospital Institute Review Board and with informed consent from owners. Animals received weekly intravesical PNP (1 mg/20 ml physiological saline) for 3 weeks. We previously reported that this dose would yield pharmacologically active drug concentrations in the urothelium and the lamina propria.13,14

Plasma, Urine and Bladder Tissue Pharmacokinetics in Tumor-Free Dogs After intubation dogs were anesthetized with isoflurane inhalation. A jugular vein was catheterized for blood sample collection and a urethral catheter was used to administer drug solution and collect urine samples. All experiments were done between 7 and 10 a.m. The bladder was emptied and PNPs were instilled into the bladder through the urethral catheter. After 2 hours the bladder was emptied through the catheter. In experiments that required tissue samples at time points beyond 8 hours the dogs were allowed to awaken from anesthesia and return to the housing units until the times when they were re-anesthetized. For tissue harvesting the bladder was emptied and excised at different times, ie at 4, 8, 24, 72 and 168 hours. For example, the

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PACLITAXEL GELATIN NANOPARTICLES FOR INTRAVESICAL BLADDER CANCER THERAPY

Systemic bioavailability of paclitaxel in tumor-free dogs given 1 mg/20 ml PNPs intravesically

Species Mice Rats Humans

% Bioavailability (hrs)

Median Paclitaxel Clearance (range) (1/hr x kg)

0–4

4–8

0–8

0.301 (0.15–0.56)17,18 0.366 (0.243–0.837)19–21 0.366 (0.243–0.837)22

0.60 0.73 0.84

0.35 0.43 0.50

0.95 1.16 1.35

Systemic bioavailability was calculated using dog AUC (0 to 4 hours 119.2, 4 to 8 hours 70.3 and 0 to 8 hours 189.5 ng ⫻ min/ml), and median paclitaxel clearance in rodents and humans.

4-hour time point corresponded to 2 hours after voiding the bladder. Sacrifice was done by pentobarbital overdose. Tissues were processed as described previously.9,15

Intravesical PNP Drug Concentrations in Dog Tumors When medically feasible and with informed consent of the owners, tumor specimens were obtained from pet dog patients before and/or immediately after weekly treatments.

Paclitaxel Analysis Samples were analyzed for free and total (sum of free plus PNP bound) drug concentrations, as described. An aliquot was incubated with the enzyme pronase (1 mg/ml) at 37C for 1 hour to digest the gelatin. The resulting solution was analyzed for the total drug concentration. A second aliquot was centrifuged at 2,500 ⫻ gravity for 30 minutes using a membrane with a molecular weight cutoff of 10,000 Da at room temperature. Filtrates were analyzed for free drug concentrations. Paclitaxel was extracted and analyzed by 2 methods, ie HPLC for urine and drug release medium samples that contained high drug levels, and ELISA for plasma and tissue samples that contained lower drug levels, as described previously.14,15 The lower limit of detection was 5 ng/ml for HPLC. ELISA detects all taxanes, including paclitaxel and its metabolites. The lower detec-

tion limits were 0.1 ng/ml for 1 ml plasma, 2 ng/gm for 50 mg bladder tissue and 100 ng/gm for 1 mg bladder tumor.

Systemic Drug Absorption Calculation and Statistical Analysis Calculating the systemic bioavailability of intravesical PNP requires the AUC and the plasma clearance of paclitaxel. Bioavailability is the multiplication product of AUC and clearance. AUC was calculated using the linear trapezoidal rule. Due to the severe hypersensitivity of dogs to Cremophor paclitaxel clearance in dogs has not been studied to our knowledge. Hence, the calculation was done using drug clearance values in other species instead, ie mice, rats and humans (see table 1).17–22 Values were compared between groups using the 2-tailed Student t test with p ⬍0.05 considered statistically significant.

RESULTS PNP Solubility Limited Drug Release Figure 1, A shows paclitaxel release from PNPs in PBS at 37C. The free paclitaxel concentration released into medium increased only 2.5-fold and attained a plateau when the starting PNP concentration was increased 50-fold from 1 to 50 ␮g/ml. This lack of dose-

Figure 1. Mean ⫹1 SD paclitaxel release. A, in 3 in vitro preparations of 1 to 50 ␮g/ml paclitaxel equivalents from PNPs in PBS. SDs were smaller than symbols. B, in urine of 8 tumor-free dogs given PNP dose of 1 mg/20 ml paclitaxel equivalents intravesically. Note break in y axis. During 2-hour treatment urine volume increased from 20 to 28 ml, and total PNP bound and free drug decreased from 50 to 34 ␮g/ml but free paclitaxel remained relatively constant at 1.18 to 1.36 ␮g/ml.

PACLITAXEL GELATIN NANOPARTICLES FOR INTRAVESICAL BLADDER CANCER THERAPY

concentration proportionality indicated a nonlinear process that was rate limited by a saturable property. The similarity between the plateau free concentration of paclitaxel (between 1.3 and 1.4 ␮g/ml) and its aqueous solubility (about 1 ␮g/ml)23 suggests that its release from PNP was rate limited by drug dissolution. Figure 1, B shows the results of in vivo drug release in 8 tumor-free dogs. Urine results revealed that during the 2-hour treatment urine volume increased 40% from 20 ml to a mean of 28 ⫾ 3 and total paclitaxel concentrations in the urine (the sum of free and PNP bound drug) decreased about proportionally from 50 ␮g/ml in the dosing solution to a mean of 34 ⫾ 4 ␮g/ml. In contrast, the free drug concentration remained almost constant at a mean of 1.29 ⫾ 0.07 ␮g/ml (range 1.18 to 1.36) or almost identical to the free plateau concentration obtained under in vitro release. Intravesical PNP Pharmacokinetics In plasma in tumor-free dogs. In dogs treated with intravesical PNPs paclitaxel was detectable in plasma at all time points, that is up to 8 hours after the initiation of the 2-hour treatment. The mean total concentration was 0.54 ⫾ 0.25 ng/ml (range 0.1 to 1.72) in 8 dogs (fig. 2, A). The table shows AUC and systemic bioavailability at different intervals. Bioavailability was about 0.60% to 0.84% of the administered dose during 0 to 4 hours and 0.35% to 0.50% during 4 to 8 hours with a cumulative value of 0.95% to 1.35% during 0 to 8 hours. In bladder tissues in tumor-free dogs. Figure 2, B shows drug concentration-depth profiles 4 to 168 hours after initiation of the 2-hour treatment in 3 animals per time point. Total drug concentrations (the sum of free and PNP bound concentrations) throughout the bladder were the highest at the first

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time point of 4 hours. Concentrations then decreased with time and attained a relatively constant level, which was sustained between 24 to 168 hours. The average total paclitaxel concentration in tissue sections comprising the urothelium and the lamina propria (a 300 ␮m depth from the inner surface of the bladder in dogs) was 15.1 ␮g/gm at 4 hours and 0.18 ␮g/gm at 1 week. Average drug concentrations in deeper tissues were 7.9 ␮g/gm at 4 hours and 0.24 ␮g/gm after 1 week in the first mm, and 4.4 ␮g/gm at 4 hours and 0.19 ␮g/gm after 1 week in the entire bladder. These data indicate significant drug retention throughout the bladder for at least 1 week. In bladder tumors in pet dogs. A total of 13 tumor bearing dogs were studied. One dog without transitional cell carcinoma was excluded from analysis. The remaining 8 females and 4 males received 3 weekly treatments. Tumor biopsy samples were obtained from 7 female dogs immediately after the first treatment, and again on days 7 and 14 before administering the second and third weekly doses, respectively. One dog was re-treated 3 months later, again with 3 weekly treatments. Figure 3 shows drug concentrations in tumors. As expected, there were substantial intersubject and intrasubject variations. Total paclitaxel concentrations showed a 13-fold range immediately after the first treatment, a 7-fold range on day 7 and a 20-fold range on day 14. Time dependent variations were observed in all animals, ie none had consistently high or consistently low drug concentrations, indicating random variations. Mean drug concentrations at the 3 time points remained relatively constant at about 40 ␮g/gm and median concentrations were within 20% to 40% of mean concentrations,

Figure 2. Pharmacokinetics of paclitaxel in tumor-free dogs given single PNP dose of 1 mg/20 ml paclitaxel equivalents intravesically, as analyzed by ELISA for total paclitaxel concentration. A, mean ⫹1 SD paclitaxel in 3 plasma samples at 3 at 240 and 480 minutes, and in 8 at other times. Some SDs were smaller than symbols. B, mean paclitaxel in 3 tissue samples per group from bladders removed at predetermined times, frozen and cryosectioned. Initial 2, 40 ␮m sections were discarded to avoid tissue contamination by urine with high drug concentration. Total paclitaxel as function of tissue depth was determined 4, 8, 24, 72 and 168 hours after initiation of 2-hour treatment. Average SD was 68% (median 61%, range 6% to 180%) of mean (data not shown). g, gm.

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PACLITAXEL GELATIN NANOPARTICLES FOR INTRAVESICAL BLADDER CANCER THERAPY

Figure 3. Bladder tumor paclitaxel concentration in dogs with naturally occurring tumors after 3 weekly PNP doses of 1 mg/20 ml paclitaxel equivalents intravesically. Tumor samples were obtained immediately after treatment 1, and on days 7 and 14, latter before weekly treatments 2 and 3, that is day 7 samples 1 week after treatment 1, and day 14 samples 2 weeks after treatment 1 and 1 week after treatment 2. ELISA revealed mean (solid lines) ⫾ SD drug concentration of 36.3 ⫾ 29.4 ␮g/gm (g) (median [dotted lines]28.8, range 6.4 to 76.6) 2 hours after treatment start in 7 preparations, 43.9 ⫾ 35.9 ␮g/gm (median 30.3, range 16.8 to 117.6) 1 week after treatment 1 start in 6, and 35.5 ⫾ 37.8 ␮g/gm (median 19.7, range 5.5 to 98.8) 2 weeks after treatment 1 start and 1 week after treatment 2 start in 6.

indicating relatively constant drug exposure in individual animals upon repeat treatments. Comparing results in figures 2, B and 3 shows a 360-fold higher average total paclitaxel concentration in bladder tumors than in the urothelium of tumor-free dogs, ie the first 80 ␮m tissue sections, 1 week after treatment. This indicates greater drug delivery/retention in tumor tissues.

DISCUSSION We noted several major findings. 1) Drug release data indicate that drug solubility limited release from PNPs in vivo, yielding constant drug concentrations irrespective of changes in urine volume. This feature would minimize the substantial intersubject and intrasubject variability in drug exposure due to post-void residual urine at dose administration and to newly formed urine, eg 20fold variations for MMC,4 thereby eliminating the need to dehydrate the patient and exhaustively empty the bladder before treatment. Hence, it may improve the efficacy and enhance the ease of intravesical therapy. 2) Only about 1% of the intravesical PNP dose (1 mg paclitaxel equivalent) was absorbed, yielding average plasma paclitaxel concentrations of less than 2 ng/ml. These levels are 1,000 times below the threshold levels associated with clinical toxicity,22 indicating that intravesical PNP is not likely to result in systemic toxicity.

3) Tissue pharmacokinetic data in tumor-free dogs indicate that the average drug concentration in the urothelium and the lamina propria at 4 hours was at least 13 times the free drug concentration in urine at 2 hours and a single dose of intravesical PNP yielded substantial drug levels that were sustained at least 1 week. These results indicate preferential drug accumulation in tissues. 4) Compared to concentrations previously observed with Cremophor micelle formulation at 2 hours, average drug concentrations derived from PNP at 4 hours were 5.4 times higher in tissue sections comprising the urothelium plus the lamina propria (300 ␮m deep from the inner surface of the bladder in dogs) and 4.4 times higher throughout the entire bladder (15.1 vs 2.8 and 4.4 vs 1.0 ␮g/gm, respectively, dose adjusted to 1 mg/20 ml). These results indicate superior drug delivery by PNPs. 5) Comparison of urine, tissue and plasma pharmacokinetics indicates the fate of the drug in tissues with time. For example, the sum of drug amounts in all bladder tissue sections equaled about 0.49% of the dose at 4 hours and 0.16% at 8 hours (fig. 2, B), indicating a loss of 0.33% during the 4-hour interval. This fraction is comparable to the sum of the amount of drug recovered in urine during the same interval, that is 0.09% at 4 hours and 0.12% at 8 hours or a 0.03% gain, plus the amount of drug absorbed into the systemic circulation, a 0.4% gain. Hence, the drug in tissue was removed primarily by systemic absorption and to a lesser extent by diffusion back to urine. 6) We observed significant drug retention in bladder tissues from 24 to 168 hours, likely due to drug binding, since we previously found a 100:1 ratio between bound and free extracellular concentrations.24 We further observed 360-fold higher drug concentrations in tumors relative to normal tissues at the same tissue depth, indicating greater delivery/retention of PNPs in tumors. A possibility is the loss of intact urothelium in tumors, resulting in enhanced drug penetration, as we previously observed for MMC.25 Another possibility is preferential adsorption and/or trapping of PNPs on or in tumors, eg papillary tumors in humans show a cauliflower-like structure with grooves and creases. This would have resulted in higher, more sustained drug concentrations. Drug concentrations in bladder tissues, which were above 100 ng/gm or 120 nM at all tissue depths, were pharmacologically active. For example, in tumors in the urothelium and the lamina propria of tumor bearing dog bladders the calculated AUC of paclitaxel from 2 to 168 hours, derived from a single dose of intravesical PNPs, was about 6,660 ␮g ⫻ hour per gm. This value is about 60 times higher than the pharmacologically active AUC of paclitaxel in tumor bearing mice given an intravenous dose of paclitaxel dissolved in Cremo-

PACLITAXEL GELATIN NANOPARTICLES FOR INTRAVESICAL BLADDER CANCER THERAPY

phor/ethanol or formulated as albumin coated nanoparticles (AUC 110 and 145 â?Žg âŤť hour per gm, respectively, for a 20 mg/kg dose26). Based on these tissue/ tumor pharmacokinetic data we propose that a single intravesical PNP dose would yield pharmacologically active drug levels in the urothelium and the lamina propria that would be sustained 1 week or longer.

CONCLUSIONS Intravesical PNP shows the desired properties for intravesical therapy, ie favorable bladder tissue/tumor targeting, penetration and retention properties,

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and low systemic absorption. We propose that clinical evaluation of intravesical PNPs is warranted. We are pursuing computational studies to integrate the pharmacokinetic data described and our previously reported pharmacodynamic data on paclitaxel in human bladder tumor histocultures27 to define the clinical trial design, ie dose, dosing frequency, number of patients and statistical power. We successfully applied a similar computational approach to design a phase III trial of intravesical mitomycin C, in which the clinical outcome closely aligned with the computation predicted outcome.28,29

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