M. Guill Wientjes

Paclitaxel-Loaded Gelatin Nanoparticles for Intravesical Bladder Cancer Therapy

Purpose: The present report describes the development of paclitaxel-loaded gelatin nanoparticles for use in intravesical therapy of superficial bladder cancer. The commercial formulation of paclitaxel contains Cremophor, which forms micelles and thereby entraps the drug and reduces its partition across the urothelium. Experimental Design: Paclitaxel-loaded gelatin nanoparticles were prepared using the desolvation method, and their physicochemical and biological properties were characterized. Results: The size of the particles ranged from 600 to 1,000 nm and increased with the molecular weight of the gelatin polymer. Under optimal conditions, the yield was >80%, and the drug loading was 0.7%. Wide-angle X-ray diffraction analysis showed that the entrapped paclitaxel was present in an amorphous state, which has higher water solubility compared with the crystalline state. Identical, rapid drug release from nanoparticles was observed in PBS and urine, with ∼90% released at 37°C after 2 hours. Treatment with a protease (i.e., Pronase) rapidly degraded the nanoparticles, with half-lives of 23.8 minutes, 0.6 minute, and 0.4 minute in the presence of 0.01, 0.05, and 0.25 mg/mL Pronase, respectively. The paclitaxel-loaded nanoparticles were active against human RT4 bladder transitional cancer cells; the IC50 paclitaxel-equivalent concentrations were nearly identical to those of aqueous solutions of paclitaxel, i.e., ∼30 nmol/L (equivalent to ∼25 ng/mL) for 2-hour treatments and ∼4 nmol/L for 96-hour treatments. In dogs given an intravesical dose of paclitaxel-loaded particles, the drug concentrations in the urothelium and lamina propria tissue layers, where Ta and T1 tumors would be located, were 7.4 ± 4.3 μg/g (mean ± SD; 3 dogs; 9 tissue sections), which were 2.6× the concentrations we reported for dogs treated with the Cremophor formulation. Conclusions: Paclitaxel-loaded gelatin nanoparticles represent a rapid release, biologically active paclitaxel formulation that can be used for intravesical bladder cancer therapy.

Clinical aspects of drug delivery to tumors

This report describes our experience on enhancement of drug delivery to solid tumors. Results of our preclinical and clinical studies including a randomized prospective phase III trial have validated the concept that enhanced drug delivery can significantly improve the treatment efficacy of intravesical mitomycin C therapy of superficial bladder cancer. The report further describes the roles of interstitial space, drug removal by capillaries, tissue structure and tissue composition on drug distribution. In general, drug distribution favors interstitial space and vasculature, with little penetration in muscles. The transport of highly protein-bound drugs such as paclitaxel and doxorubicin in a solid tumor is retarded by a high tumor cell density and enhanced by drug-induced apoptosis. Results of in vitro studies using solid tumor histocultures and in vivo studies using tumor-bearing animals demonstrate that the delivery of highly protein-bound drugs to tumor can be enhanced using a pretreatment that induces apoptosis and reduces cell density, and by using treatment schedules designed to take advantage of these drug-induced changes in tumor tissue composition.

Time- and concentration-dependent penetration of doxorubicin in prostate tumors.

The penetration of paclitaxel into multilayered solid tumors is time- and concentration-dependent, a result of the drug-induced apoptosis and changes in tissue composition. This study evaluates whether this tissue penetration property applies to other highly protein-bound drugs capable of inducing apoptosis. The penetration of doxorubicin was studied in histocultures of prostate xenograft tumors and tumor specimens obtained from patients who underwent radical prostatectomy. The kinetics of drug uptake and efflux in whole tumor histocultures were studied by analyzing the average tumor drug concentration using high-pressure liquid chromatography. Spatial drug distribution in tumors and the drug concentration gradient across the tumors were studied using fluorescence microscopy. The results indicate that drug penetration was limited to the periphery for 12 hours in patient tumors and to 24 hours in the more densely packed xenograft tumors. Subsequently, the rate of drug penetration to the deeper tumor tissue increased abruptly in tumors treated with higher drug concentrations capable of inducing apoptosis (i.e., >5 μm), but not in tumors treated with lower concentrations. These findings indicate a time- and concentration-dependent penetration of doxorubicin in solid tumors, similar to that of paclitaxel. We conclude that doxorubicin penetration in solid tumors is time- and concentration-dependent and is enhanced by drug-induced cell death.

Relative value of prostate-specific antigen and prosttic acid phosphatase in diagnosis and management of adenocarcinoma of prostate Ohio State University Experience

Serum concentrations of prostate-specific antigen (PSA), prostate-specific acid phosphatase (PAP), and transrectal prostatic ultrasound were utilized in the evaluation of 193 men with various urologic disorders. Of the 193 patients, 48 had prostate cancer, and the other 145 included 5 with genitourinary neoplasms, 69 with benign prostatic hypertrophy, and 71 with other non-neoplastic genitourinary disease. PSA levels were elevated in 35 patients with prostate cancer and in 25 of the 145 without prostate cancer. PAP levels were elevated in 15 with prostate cancer and in 2 of the 145 without prostate cancer. The data indicate that PSA is a more sensitive but less specific tumor marker than PAP in the detection of prostate cancer. PSA appears to be more sensitive than PAP in monitoring the response to treatment. The use of PSA and PAP jointly to detect and to monitor prostate cancer did not appear to enhance the clinical utility over that of PSA alone.

Kinetics of hallmark biochemical changes in paclitaxel-induced apoptosis

Apoptosis is associated with cascades of biochemical changes, including caspase activation, cleavage of poly-ADP-ribose polymerase (PARP), and fragmentation of genomic DNA. Knowledge of the kinetics of these changes in drug-induced apoptosis is important for designing pharmacodynamic studies. We have shown that the slow manifestation of apoptosis contributes to the delayed pharmacological effects of paclitaxel (Cancer Res. 58:2141–2148, 1998). The present study examined the timing of the biochemical changes in paclitaxel-induced apoptosis in human prostate PC3 cancer cells. After treatment with 20 nM paclitaxel, the fraction of cells that detached from the culture flask increased with time to reach 68% at the end of the 96-hour experiment. In contrast, the control samples showed <1% detachment. The attached and detached paclitaxel-treated cells showed different biochemical properties. The detached cells exhibited the full spectrum of apoptotic changes, whereas the attached cells only showed activation of caspase-3-like proteases but not PARP cleavage, DNA fragmentation, nor release of DNA fragments to the cytoplasm. Activation of caspases in the attached cells was several-fold lower and occurred at a later time (ie, 24 vs 12 hours) compared to the detached cells. In the detached cells, caspase activation was first detected at 12 hours and peaked at 36 hours, whereas PARP cleavage was first detected at 24 hours and was completed prior to 72 hours. In contrast, the extent of internucleosomal DNA fragmentation and the release of DNA-histone complex to the cytoplasm (both were first detected at 24 hours) were cumulative over time up to the last time point of 96 hours. In summary, in paclitaxel-induced apoptosis, caspase activation was followed with a 12-hour lag time by PARP cleavage, internucleosomal DNA fragmentation, and release of DNA-histone complex to the cytoplasm. There was no detectable lag time between PARP cleavage and DNA fragmentation. The observation that only the detached cells but not the attached cells showed the full spectrum of apoptotic changes suggests that detachment is either a part of the initiation execution phases of apoptosis and or is required for their completion.

Cytostatic and apoptotic effects of paclitaxel in human breast tumors

Purpose: We have previously reported incomplete cytotoxic responses of other human solid tumors (bladder, head and neck, ovarian and prostate) to paclitaxel. This finding is qualitatively different from the nearly complete response observed in monolayer cultures of human cancer cell lines. The present study examined the pharmacodynamics of paclitaxel in human breast tumors. Methods: Three-dimensional histocultures of patient tumors were used. The cytostatic effect was evaluated by measurement of the inhibition of 48-h cumulative bromodeoxyuridine (BrdUrd) incorporation. The apoptotic effect was evaluated in terms of morphological changes and by in situ DNA end labeling. Results: Paclitaxel produced partial cytostasis (∼30% maximum) and induced apoptosis (maximum apoptotic index of 3.3% to 29%) in all 15 tumors. More than 95% of apoptotic cells were BrdUrd labeled, but not all BrdUrd-labeled cells were apoptotic. The maximal apoptotic indices in the tumors were significantly correlated with the BrdUrd labeling index of untreated controls (r2= 0.63, P < 0.01). The maximum apoptotic effect was observed at a tenfold lower drug concentration (0.1 μM ) compared to the maximum cytostatic effect (1 μM ). Neither of these effects was enhanced by increasing the drug concentration to 10 μM. Conclusions: The pharmacodynamics of paclitaxel in human breast tumors are comparable to those found in other human solid tumors. The labeling of apoptotic cells by BrdUrd and the correlation between the proliferation index and apoptosis suggest that drug-induced apoptosis is linked to cell proliferation and is completed after DNA synthesis. The finding that maximal cytostatic and apoptotic effects of paclitaxel were achieved at or below the clinically achievable concentration of 1 μM suggests further increasing the dose to elevate plasma concentration beyond 1 μM may not improve treatment outcome.

DISTRIBUTION OF DT-DIAPHORASE AND REDUCED NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE: CYTOCHROME P450 OXIDOREDUCTASE IN BLADDER TISSUES AND TUMORS

PURPOSE We previously showed that mitomycin C activity in human bladder tumors is inversely related to tumor stage and muscle invasive tumors are less sensitive than superficial tumors. Because DT-diaphorase and reduced nicotinamide adenine dinucleotide phosphate:cytochrome P450 oxidoreductase (P450R) have a role in mitomycin C bioactivation, we investigated the distribution of these enzymes as a function of depth in the bladder wall and in human bladder tumors located at different parts of the bladder. MATERIALS AND METHODS Gene expression of DT-diaphorase and P450R relative to the expression of beta-actin was measured by reverse transcriptase-polymerase chain reaction in 4 dog and 8 human bladder tissue specimens, and in 46 human bladder tumors. DT-diaphorase activity was measured by enzymatic assay in dog and human bladders. RESULTS Data showed decreasing expression of DT-diaphorase and P450R from urothelium to muscle layer in the normal bladder wall with higher interindividual variation in humans than in dogs (greater than 40-fold versus approximately 3-fold). The expression of DT-diaphorase and P450R in bladder tumors correlated inversely with tumor stage (p <0.05) and was significantly higher in superficial than in muscle invasive bladder disease. DT-diaphorase and P450R expression in bladder tumors was approximately 6-fold higher than in normal bladder tissue. In normal and tumor tissues DT-diaphorase expression correlated significantly with P450R (r2 = 0.33, p <0.01), while DT-diaphorase expression correlated with its enzyme activity (r2 = 0.84, p <0.01). CONCLUSIONS Our results indicate a higher distribution of DT-diaphorase and P450R in superficial bladder tumors and tissues. Preferential enzyme distribution in superficial tumors may be a cause of the higher efficacy of intravesical mitomycin C therapy for superficial versus muscle invasive disease.

Histocultures of Human Prostate Tissues for Pharmacologic Evaluation

This study evaluated the growth of human prostate tumors in histoculture, an in vitro culture technique that maintains three-dimensional tissue structure and organization. Eighty-six percent of 50 tumor specimens from 50 patients were successfully cultured. The histocultures showed proliferation of epithelial tumor cells and stromal cells. Prostate specific antigen (PSA) and prostatic acid phosphatase (PAP) were detectable by immunohistochemistry for at least 8 weeks. Synthesis of PSA was further confirmed by its presence in medium. The mean thymidine labeling index (LI) of the neoplastic cells was 62%. There was no correlation between thymidine LI and tumor grade. The explants maintained their characteristics for at least 8 weeks as indicated by unchanged thymidine LI, PSA and PAP immunohistochemistry after 2 and 8 weeks in culture. A 24 to 48 hour delay in processing the tissues for culture did not reduce the thymidine LI. The thymidine LI and PSA and PAP staining in tumors cultured in a Minimal Essential Medium (MEM)-based or a PFMR-4-based culture medium were similar, suggesting an insignificant effect of the medium on cell proliferation. Migration of neoplastic cells from the tissue fragments into the collagen gel matrix was noted in 1 of 10 samples cultured in MEM-based medium versus 8 of 10 samples cultured in PFMR-4 medium (p < 0.01). Exposure of 13 patient tumors to suramin, doxorubicin and 5-fluorouracil at clinically relevant concentrations and duration showed tumor sensitivity in 23%, 31% and 15% of the specimens. These values approximate the historical clinical response rates. These data suggest that the histoculture system holds promise for short-term culture of human patient prostate tumor specimens for biologic and pharmacologic studies.

Inhibition of Intestinal Pyrimidine Nucleoside Phosphorylases

The activity of 5′-deoxy-5-fluorouridine (dFUR) depends on its activation to 5-fluorouracil (FU) by pyrimidine nucleoside phosphorylases. These enzymes are found in tumors and normal tissues, with the highest activity in the small intestines. The present study examined the inhibition of dFUR phosphorolysis in intestinal tissues. dFUR metabolism in intestinal homogenates was inhibited by uracil (U), uridine (UR), and thymidine (TdR), which are the normal substrates for the phosphorylases. Conversely dFUR reduced the metabolism of these inhibitors. A good agreement was found between the observed data and the computer-fitted data using the equations for competitive inhibition between dFUR and the inhibitors. In the absence of inhibitors, the Vmax of dFUR phosphorolysis was 47.1 ± 4.9 µM/min and the apparent Km was 910 ± 167 µM. The Vmax was unaltered by the inhibitors, while the Km was increased with increasing inhibitor concentrations. The maximal inhibition of dFUR metabolism by UR and TdR was about 80%. The Ki,s were 372 µM for U, 87.2 µM for UR, and 112 µM for TdR and are orders of magnitude higher than their reported endogenous serum concentrations. The rate of dFUR phosphorolysis to FU in the intact intestinal epithelial crypt cells, indicated by the ratio of FU to dFUR in the intracellular fluid, was reduced by UR in a concentration-dependent fashion. These data indicate that the naturally occurring pyrimidines inhibit competitively the dFUR metabolism by the intestinal phosphorylases, that this inhibition occurs at concentrations much higher than the circulating endogenous levels, and that phosphorolysis is the major route of dFUR metabolism.

Abstract 3485: Uncertainty envelope to evaluate drug interactivity with statistical analysis

Introduction. Development of new therapies for cancer and infections frequently involves combinations of two or more drugs. Existing methods to quantify drug interactivity are suitable for analyzing drugs with parallel concentration-effect curves and similar maximum drug effects. This study established a method for the remaining situations. Methods. Equations describing zero-interactivity effects of combination therapy were developed based on the principle that a drug cannot interact with itself (i.e., additivity only). These equations were used to convert the concentration-effect (C-E) curve of the combination to C-E curve for each of the two drugs in the combination as if they were used as single agents (single agent-equivalent curves). Confidence intervals (CI) of these curves were generated using the error propagation method. Effect data of drug combinations were analyzed by the new and existing methods. Results. The calculated single agent-equivalent curves plus 95% CI defined the boundaries of an envelope for additive drug effects (Uncertainty Envelope). The Envelope shape and size were determined by differences in the shapes of single agent C-E curves. The nature of drug interactivity is indicated by the location of experimental data points of the combination; data points located within the Envelope indicate additivity, whereas points above and below the Envelope respectively indicate statistically significant synergy and antagonism. In comparison with the existing methods, the Envelope method yielded more conservative results (additivity vs. synergy or antagonism). Conclusion. Uncertainty Envelope method enables the interactivity analysis between drugs with nonparallel C-E curves and unequal maximal effect, with statistical confidence. Citation Format: Liang Zhao, Jessie L.-s. Au, M. Guill Wientjes. Uncertainty envelope to evaluate drug interactivity with statistical analysis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3485. doi:10.1158/1538-7445.AM2015-3485