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Experimental Therapeutics, Preclinical Pharmacology |
College of Pharmacy [D. C., D. S., M. G. W., J. L-S. A.], James Cancer Hospital and Solove Research Institute [M. G. W., J. L-S. A.], The Ohio State University, Columbus, Ohio 43210
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ABSTRACT |
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INTRODUCTION |
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Paclitaxel shows higher activity than other antimicrotubule compounds, such as vinblastin, against human bladder cancer cells (7) . A Phase II study has shown that 24 h i.v. infusion of paclitaxel produced a 42% partial and complete response rate of advanced and/or metastatic bladder cancer (8) . In histocultures of human bladder tumors, 2 h of treatment with paclitaxel was sufficient to cause inhibition of tumor cell proliferation and induction of apoptosis, with a greater apoptotic effect in the more rapidly proliferating tumors (9) . In addition, paclitaxel, because of its lipophilicity, can penetrate the urothelium more readily than other commonly used drugs such as doxorubicin and mitomycin C (10) . These characteristics make paclitaxel an attractive candidate for intravesical therapy.
The paclitaxel formulation approved by the FDA3 for human use (Taxol®) employs Cremophor to solubilize the drug. Our laboratory has shown in dogs that Cremophor, by entrapping paclitaxel in micelles, reduces the free fraction of paclitaxel and consequently lowers the drug penetration into the bladder tissue (11) . Thus, we hypothesized that an increase of the free fraction of paclitaxel would lead to higher drug penetration. Increase of paclitaxel free fraction could be accomplished by using a surface-active agent, such as DMSO, that is capable of disrupting the micelle structure. DMSO is a widely used solvent with pharmacological actions including anti-inflammatory and bacteriostatic activity, analgesia, nerve blockade, diuresis, cholinesterase inhibition, vasodilation, and muscle relaxation (12) . DMSO also has the unique capability of penetrating living tissues without causing significant damage; it has been used as a carrier to enhance the bladder absorption of other chemotherapeutic drugs such as cisplatin, pirarubicin, and doxorubicin (13, 14, 15) . In addition, DMSO is approved by the FDA for the treatment of interstitial cystitis, and up to 50% (v/v) DMSO can be safely instilled in the bladders of human patients (13) .
The present study evaluated the use of DMSO to disrupt the Cremophor micelles, increase the free fraction of paclitaxel, and enhance paclitaxel penetration into bladder tissue.
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MATERIALS AND METHODS |
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Determination of Free Fraction of Paclitaxel.
Stock solutions of paclitaxel were prepared by dissolving paclitaxel in 100% ethanol and serially diluted using water. The final concentration of ethanol was <1% (range, 0.01–0.05%). We have previously shown that ethanol at a concentration of up to 1% had no effect on the free fraction of paclitaxel (11)
. The free fraction of paclitaxel was determined by equilibrium dialysis at 37°C, using a Spectrum side-by-side equilibrium dialysis apparatus (Millipore Corp., Bedford, MA). The donor compartment contained Cremophor, ethanol, and a mixture of unlabeled and tritium-labeled paclitaxel dissolved in 1 ml of distilled water with or without DMSO. The receiver compartment contained only distilled water or different concentrations of DMSO. A regenerated cellulose membrane, with a molecular weight cutoff of 1000, separated the two compartments. The membrane material is resistant to DMSO. The molecular weight is 853 for paclitaxel and >1680 for Cremophor. A preliminary study was performed to establish the time required to achieve equilibrium; the free fraction of paclitaxel showed no significant increase from 36 to 48 h, indicating the attainment of equilibrium at 48 h. Subsequent experiments used the 48 h time point. One hundred µl of aliquots were taken from donor and receiver compartments and analyzed for paclitaxel concentration. The free fraction of paclitaxel (Ffree) was calculated from the following equation:
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Determination of Cremophor Micelle Size.
The change in Cremophor micelle size was monitored using a BI-90 particle sizer (Brookhaven Instruments Inc., Ronkonkoma, NY) that measures the size of colloidal particles in a size range of 0.005–5 µm. The random motion of small particles in solution gives rise to fluctuations in the average intensity of the scattered light and thereby provides a measurement of the translational diffusion coefficient (D). The particle diameter (d) is calculated using the following equation (16)
:
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is the viscosity of liquid in which the particles are suspended.
Animal Protocol.
The animal protocol was approved by the Institutional Review Board. Twelve male or female beagle dogs weighing 8.95 ± 0.6 kg (mean ± SD) were used. All animals were fasted overnight and allowed access to water. The surgical procedures were as described elsewhere (17)
. In brief, a jugular vein was catheterized for the collection of systemic blood samples, and a cephalic vein was catheterized for the administration of anesthetics. A urethral catheter was inserted for the collection of urine samples and the administration of drug solution. All experiments were conducted in the morning between 7 and 10 a.m. Animals were anesthetized for the duration of the experiment. After emptying urine from the bladder, an intravesical dose of paclitaxel in DMSO was instilled through the catheter. One dog was given only the vehicle (50% DMSO solution), and its bladder tissue was used to prepare the standard curve samples. Because of the uncertainty of the effects of DMSO, the first group of five dogs was given paclitaxel (1120 µg) in 50% DMSO (referred to henceforth as paclitaxel/DMSO). Subsequent experiments in the remaining six dogs used a lower dose of paclitaxel (500 µg) so that the results could be compared with our previous results obtained in animals treated with paclitaxel in water, where the maximal solubility was 25 µg/ml. This second group of six dogs was given Taxol® in 50% DMSO solution. The dosing solution was prepared by diluting Taxol® (6 mg/ml; 50% (w/v) Cremophor, and 50% (v/v) ethanol] with physiological saline solution containing DMSO to yield a final concentration of 500 µg of paclitaxel per 20 ml of 0.22% (w/v) Cremophor, 0.21% (v/v) ethanol, and 50% DMSO (referred to henceforth as the paclitaxel/Cremophor/DMSO group).
We have shown, using mitomycin C and human patient bladders, a linear relationship between the drug concentration in dosing solution or urine and the drug penetration in bladder tissues (18) . Hence, to compare data obtained from animals that received different paclitaxel doses, the drug concentrations in biological samples were normalized to a paclitaxel dose of 500 µg.
Serial blood and urine samples were collected before and during instillation and immediately before surgical removal of the bladder. At 120 min, urine was collected through the urethral catheter, and the bladder was removed. The bladder tissue was cut into three sections i.e., left and right lateral sides and dome. The tissue sections were snap-frozen in liquid nitrogen on a flat stainless steel plate cooled on dry ice. The procedures between removing the urine and freezing the bladder tissue were completed as rapidly as possible (i.e., less than 5 min). The rapid removal and freezing was necessary to maintain the concentration gradient between urine and tissue and to avoid washout of the drug during tissue processing. Animals were euthanized with an overdose of i.v. pentobarbital immediately after removal of the bladder.
Tissue Extraction.
Frozen transurethral bladder wall tissue samples were cut in parallel to the urothelial surface into 10-µm slices using a cryotome, as described previously (10
, 17)
. The first one or two sections were discarded to avoid contamination of tissue by urine, which contained high drug concentration. The first 10 samples contained 4 slices each. The next 10 samples contained 8 slices each, and the remaining samples contained 16 slices each. After weighing, the frozen tissue samples were spiked with 40 µl of the internal standard (cephalomannine, 10 µg/ml) and homogenized with 4 ml of ethyl acetate. The homogenizer probe was washed with a second portion of ethyl acetate to recover residual adhering tissue. The two ethyl acetate fractions were combined and centrifuged at 2000 x g for 5 min. The supernatant was transferred and evaporated to dryness under a stream of nitrogen. The residue was reconstituted and injected into the HPLC system.
To ascertain that the presence of DMSO did not alter the extraction of paclitaxel, the standard curves for the concentrations of paclitaxel in bladder tissues were constructed using the bladder removed from the control dog treated with only 50% DMSO. Briefly, the bladders of the DMSO-treated dogs were sectioned. The samples were mixed with known amounts of paclitaxel and processed as described above (10 , 17) .
Determination of Paclitaxel Concentration.
The concentrations of paclitaxel in bladder tissue and urine samples were analyzed by a previously described HPLC assay using a column-switching method (19)
. In brief, the HPLC stationary phase consisted of a clean-up column (NovaPak C8, 75 x 3.9-mm inner diameter, 4-µm particle size; Waters Associates, Milford, MA) and an analytical column (Bakerbond octadecyl, 250 x 4.6-mm inner diameter, 5 µm particle size; J. T. Baker, Phillipsburg, NJ). Samples were injected onto the clean-up column and eluted with the clean-up mobile phase (37.5% acetonitrile in water) at 1 ml/min. Concurrently, the analytical mobile phase (49% acetonitrile in water) was directed through the analytical column at a flow rate of 1.2 ml/min. The fraction from 6 to 13 min containing paclitaxel and cephalomannine was transferred from the clean-up column onto the analytical column. The limit of detection for paclitaxel was 5 ng/ml for urine samples and 5 ng/injection for bladder tissue samples.
Because of the low levels, the concentrations of paclitaxel in plasma samples were analyzed by the competitive inhibition enzyme immunoassay, which measures all taxanes including paclitaxel and its metabolites. The procedures were as described previously (10) . Briefly, paclitaxel was extracted from plasma (1 ml) using 2 x 5 ml ethyl acetate. The standard curve was prepared using the taxane standards provided in the assay kit. Paclitaxel concentrations ranging from 0.12 to 3 ng/ml were within the linear range.
Analysis of Tissue Concentration-Depth Profiles of Paclitaxel.
Our laboratory has previously shown that drug penetration in bladder follows the distributed model, which describes drug removal by capillary drainage in addition to drug diffusion (17)
. The tissue concentration-depth profile is described by the following equation:
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It is noted that Eq. 3 assumes negligible drug concentrations recycling from the systemic circulation because, as shown below, the drug concentration in plasma is <0.01% and <0.02% of the urine and tissue concentrations, respectively.
Analysis of Concentration-Time Profiles of Paclitaxel in Urine.
The concentration-time profiles of paclitaxel in urine during the 120-min instillation period were analyzed using the following equations:
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Vu is the volume of urine at time t, V0 is the volume of paclitaxel dosing solution (20 ml), k0 is the zero-order rate constant for urine production and was calculated as the difference between final urine volume and volume of dosing solution divided by 120 min, Vres is the volume of residual urine present in bladder at time of dosing, and ka and kd are the first-order rate constants describing drug absorption and degradation.
Because paclitaxel is stable in urine for 120 min at a pH of 5–7 (7)
, drug removal due to degradation is negligible, and drug removal is primarily by absorption into the bladder. The hybridized first-order rate constant (ka + kd) is replaced by ka, and Eq. 4
is simplified to Eq. 5
.
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Statistical Analysis.
Comparison of values between groups was performed using two-tailed Student’s t tests. A value of P < 0.05 was considered statistically significant.
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RESULTS |
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shows the effect of Cremophor and DMSO on the free fraction of paclitaxel. In the absence of Cremophor, DMSO had no effect on the free fraction. Without DMSO, the free fraction of paclitaxel decreased from 100% to 23% and 11%, respectively, in 0.25% and 1% Cremophor. The addition of 50% DMSO completely reversed the Cremophor effect at lower Cremophor concentrations (0.01% and 0.0625%) and partially reversed the Cremophor effect at higher Cremophor concentrations (free paclitaxel fraction of 92% and 75% at 0.25% and 1% Cremophor, respectively).
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shows the concentration-dependent effect of DMSO on the free fraction of paclitaxel dissolved in 0.25% (w/v) Cremophor and 0.25% (v/v) ethanol. DMSO had no effect at 10% concentration but significantly increased the free paclitaxel fraction from 23% to 92% when the DMSO concentration was increased from 10% to 50%.
Effect of DMSO on Cremophor Micelle Sizes.
Fig. 1C
shows that DMSO altered the size of Cremophor micelles. In the presence of 0.22% (w/v) Cremophor and 0.21% (v/v) ethanol, addition of DMSO caused a concentration-dependent, nonlinear increase in micelle size from 13 nm at 0% DMSO to up to 230 nm at 50% DMSO.
Urine and Plasma Pharmacokinetics of Paclitaxel/Cremophor/DMSO and Paclitaxel/DMSO.
Fig. 2
shows the decline of urine paclitaxel concentrations, as a function of time, in dogs treated with either paclitaxel/DMSO or paclitaxel/Cremophor/DMSO. Table 1
summarizes the urine pharmacokinetic parameters. The concentration at zero time is the concentration of paclitaxel in the dosing solution, and the first sample was obtained at 5 min. In both groups, the 11–15% concentration decline during the first 5 min was due to the dilution of the 20-ml dosing solution by the 3-ml residual urine. From 5 to 120 min, the drug concentration was further reduced by >2.3-fold due to dilution by newly produced urine and due to removal by absorption into the bladder tissue and systemic circulation.
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Bladder Tissue Pharmacokinetics of Paclitaxel/Cremophor/DMSO and Paclitaxel/DMSO.
Fig. 3
shows the concentration-depth profile of paclitaxel in bladder tissues, and Table 2
summarizes the tissue pharmacokinetic parameters. The partition ratio of paclitaxel across the urothelium was 45% for the paclitaxel/Cremophor/DMSO group and 105% for the paclitaxel/DMSO group, as indicated by the ratio of concentrations in urothelium and urine. After penetrating the urothelium, paclitaxel diffused through the capillary-perfused tissues, and the drug concentrations declined exponentially, reaching a minimum plateau level of about 0.34 and 0.9 µg/g at a depth of 2000 µm for paclitaxel/Cremophor/DMSO and paclitaxel/DMSO groups, respectively. The half-width, or the distance for the drug concentration to decrease by 50%, was not statistically different between the two groups.
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Most of the Cremophor dose (>99%) was recovered in the urine at 120 min. The Cremophor concentrations ranged from 0.07 to 0.09% (w/v), levels that are well above the critical concentration for micelle formation [i.e., 0.01% (w/v)]. This is consistent with our previous finding of a negligible penetration of Cremophor into the bladder (i.e., less than 0.001% of the dose; Ref. 11 ).
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DISCUSSION |
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Effect of DMSO on Cremophor Micelles.
Results of the present study show that the free fraction of paclitaxel in solution depended on two factors, Cremophor concentration and solvent environment. The free fraction of paclitaxel declined with increasing concentrations of Cremophor, whereas the addition of DMSO increased the fraction of the organic phase in the solution, altered the structure of Cremophor micelles, and enhanced the partition of paclitaxel out of Cremophor micelles (Fig. 1, B and C)
. Hence, DMSO was effective in reversing the undesired entrapment of paclitaxel in Cremophor micelles.
The release of paclitaxel from Cremophor micelles depended on the DMSO concentration (Fig. 1B)
. Hence, changes in the DMSO concentrations would result in changes in the free fraction of paclitaxel. DMSO concentrations in urine over time were not measured. Under the assumption that DMSO was not rapidly absorbed and remained in the urine, the DMSO concentration in urine would decline from 50% at time 0 to about 18% at 120 min (calculated based on the change in urine volume over 120 min) and would result in a decrease of free fraction of 92% at time 0 to about 50% at 120 min. However, it is likely that a significant fraction of DMSO was absorbed into bladder tissues because DMSO is known to readily penetrate bilipid membrane (12
, 21)
. In this case, DMSO concentration at 120 min would be <18%, and the free fraction of paclitaxel would be <50%.
Effect of DMSO on Urine Pharmacokinetics of Paclitaxel.
Table 1
shows comparable residual urine volumes at the time of drug instillation, but different urine production rate constants and different final urine volumes for each group. The urine production rate constant increased from 0.07 ml/min for paclitaxel/water to 0.13 ml/min for paclitaxel/Cremophor, to 0.22 ml/min for paclitaxel/DMSO, and to 0.27 ml/min for paclitaxel/Cremophor/DMSO. This indicates that DMSO enhanced the urine production rate, consistent with the diuretic effect of DMSO in rats, where topical administration of DMSO led to a 10-fold increase in urine volume (21)
, and in newborn rabbits, where an i.v. dose of 1% solution of DMSO led to >1.5-fold increase in urine volume (22)
. The mechanism of the diuretic effect of DMSO is unknown.
As discussed below, the enhanced urine production by DMSO resulted in decreased delivery of paclitaxel to bladder tissues.
Effect of DMSO on Paclitaxel Penetration into Bladder Tissues.
Drug penetration into bladder tissues consists of two processes. The first process is to partition across the bladder urothelium, which is an absorption barrier (23
, 24)
. The extent of partitioning is reflected by the ratio of paclitaxel concentrations in urothelium and urine (Curo:Cu ratio) and the apparent absorption rate constant ka. The major difference between the two measurements is that the Curo:Cu ratio is a single time point measurement (i.e., at 120 min, when the bladder was removed from the animal) whereas the ka value is the averaged rate constant over the 120 min. The second process is drug transport through the capillary-perfused tissue layers; this process is determined by the drug diffusion driven by the concentration gradient and the drug removal by the perfusing capillaries. This process is reflected by the half-width measurement.
Compared with the paclitaxel/water group, the paclitaxel/DMSO group showed a 2.2-fold higher Curo:Cu ratio and a 2.6-fold higher ka value. Likewise, the paclitaxel/Cremophor/DMSO group showed a 2.8-fold higher Curo:Cu ratio and a 5.6-fold higher ka value, compared with the paclitaxel/Cremophor group. The higher Curo:Cu ratios and ka values, in the absence or presence of Cremophor, indicate that DMSO enhanced drug absorption into the bladder. This is consistent with the known effect of DMSO as a penetration enhancer, through its action on the disruption of the urothelium (13) . Furthermore, the greater increase in the ka value by DMSO in the presence of Cremophor, as compared with that without Cremophor, is likely a consequence of the additional DMSO effect on increasing the free fraction of paclitaxel.
We previously showed that entrapment of paclitaxel in Cremophor micelles reduced partition of paclitaxel across the urothelium (11) . This effect was also observed in the presence of DMSO; the Curo:Cu ratio in the paclitaxel/Cremophor/DMSO group is 2.3-fold lower than that in the paclitaxel/DMSO group.
Similar half-widths were found for the paclitaxel/water and paclitaxel/Cremophor groups, indicating no significant net changes in the drug diffusion and absorption via the capillaries by Cremophor. In contrast, the half-widths in the paclitaxel/DMSO and paclitaxel/Cremophor/DMSO groups were significantly shorter than their corresponding control groups without DMSO, indicating that DMSO treatment significantly reduced the half-width. A likely cause is the enhanced drug removal by the capillaries due to enhanced capillary permeability by DMSO, as reported previously (15) .
Net Effect of DMSO on Paclitaxel Concentration in Bladder Tissues.
As discussed above, DMSO produced opposing effects on the penetration and retention of paclitaxel in bladder tissues. To determine the net effect of DMSO on paclitaxel delivery to bladder tissue during intravesical therapy, we compared the total amount of drug in the bladder tissue. Table 2
shows that the amount of paclitaxel in bladder was highest in the paclitaxel/water and paclitaxel/DMSO groups. In the presence of Cremophor/ethanol, this amount was reduced by about 70%. Further addition of DMSO partially reversed the decrease due to Cremophor, such that the paclitaxel delivery was about 50% of the delivery using water as the solvent.
Conclusions.
Results of the present study confirm that paclitaxel is entrapped in Cremophor micelles. During systemic therapy, Cremophor is metabolized and thereby releases paclitaxel from the micelles. However, in regional therapy such as intravesical therapy for bladder cancer, Cremophor is localized in the bladder cavity and is not metabolized. The resulting entrapment of paclitaxel in Cremophor micelles then leads to diminish drug delivery to tissues. This may limit the efficacy of the FDA-approved Cremophor-containing paclitaxel formulation in the regional therapy setting. The present study shows that disruption of Cremophor micelles by a surface-active agent such as DMSO can reverse the undesirable effect of Cremophor on the free fraction of paclitaxel and partially restore the favorable bladder delivery of intravesical paclitaxel.
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FOOTNOTES |
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1 Supported in part by MERIT Grant R37CA49816 from the National Cancer Institute, NIH. 
2 To whom requests for reprints should be addressed, at College of Pharmacy, James Cancer Hospital and Solove Research Institute, The Ohio State University, 500 West 12th Avenue, Columbus, OH 43210. Phone: (614) 292-4244; Fax: (614) 688-3223.
3 The abbreviations used are: FDA, Food and Drug Administration; HPLC, high-performance liquid chromatography.
Received 5/30/02; accepted 8/27/02.
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) are shown together with the results of our previous studies 
), and Cremophor/ethanol (i.e., 
) as solvents. Means ± SE (n = 5 for the three groups in the previous studies). Note that for the paclitaxel/DMSO group, the values are normalized to the paclitaxel dose of 500 µg/20 ml (see "Materials and Methods").