Noncytotoxic suramin as a chemosensitizer in patients with advanced non-small-cell lung cancer: a phase II study.

BACKGROUND: The purpose of this study was to evaluate the potential of noncytotoxic doses of suramin to reverse chemotherapy resistance in advanced chemonaive and chemoresistant non-small-cell lung cancer patients. PATIENTS AND METHODS: Patients received paclitaxel (Taxol) (200 mg/m(2)) and carboplatin (area under the concentration-time curve 6 mg/ml/min) every 3 weeks. The total suramin per cycle dose was calculated using a nomogram derived from the preceding phase I trial to obtain the desirable plasma concentration range of 10-50 microM. RESULTS: Thirty-nine response-assessable chemonaive patients (arm A) received 213 cycles. Thirty-eight cycles were administered to 15 patients with demonstrated resistance to paclitaxel and carboplatin (arm B). The pattern/frequency of toxic effects was similar to those expected for paclitaxel/carboplatin, and pharmacokinetic analyses (199 cycles) showed suramin plasma concentrations maintained between 10 and 50 microM in 94% of cycles. In arm A, response evaluation criteria in solid tumors (RECIST) response rate was 36% (95% confidence interval 22% to 54%; two complete, 12 partial); 15 patients (38%) had disease stabilization for > or =4 months; median progression-free survival (intention to treat) was 6.4 months; median overall survival (OS) 10.4 months and 1-year survival rate 38%. In arm B, no RECIST responses occurred; four patients had disease stabilization for > or =4 months; median OS was 132 days and 1-year survival rate 7%. Plasma basic fibroblast growth factor levels were higher in chemopretreated/refractory patients compared with chemonaive patients (P = 0.05). Sequence analysis of the EGFR tyrosine kinase domain in a long-term disease-free survivor revealed an ATP-binding pocket mutation (T790M). CONCLUSIONS: Noncytotoxic suramin did not increase paclitaxel/carboplatin's toxicity and the suramin dose was predicted from clinical parameters. No clinically significant reversal of primary resistance was documented, but a modulatory effect in chemotherapy-naive patients cannot be excluded. Controlled randomization is planned for further evaluation of this treatment strategy.

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 microm), 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.

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.

Nontoxic doses of suramin enhance activity of doxorubicin in prostate tumors.

We recently reported that acidic and basic fibroblast growth factors (aFGF and bFGF) confer a broad-spectrum chemoresistance in solid tumors, and that inhibitors of these proteins enhanced the antitumor activity of several anticancer drugs. The present study investigated the effect of FGF inhibitors on doxorubicin activity in human prostate PC3 tumors. In in vitro studies, conditioned medium (CM) obtained from histocultures of rat MAT-LyLu lung metastases and different combinations of recombinant FGF induced a 7- to 15-fold doxorubicin resistance. Suramin had no effect on the doxorubicin activity in the absence of CM or FGF, but reversed the CM- and FGF-induced resistance by > or =90% at concentrations that had no cytotoxicity (i.e., 1-17 microM suramin). In the in vivo study, immunodeficient mice bearing well established, subcutaneous PC3 tumors (approximately 100 mg in size) were treated intravenously with doxorubicin (5 mg/kg) and suramin (10 mg/kg), administered twice weekly for 3 weeks. The suramin dose, selected to yield plasma concentration of below 50 microM, had neither antitumor activity nor toxicity. Doxorubicin alone reduced tumor growth rate by approximately 60%, reduced the density of nonapoptotic tumor cells by approximately 60%, enhanced the apoptotic cell fraction by 4-fold, and reduced the body weight by approximately 15% (p < 0.05 compared with control). Addition of suramin to doxorubicin therapy did not increase weight loss but significantly enhanced the antitumor effect, resulting in complete inhibition of tumor growth, an additional 3-fold reduction in the density of nonapoptotic tumor cells, and an additional 2-fold enhancement of the apoptotic tumor cell fraction (p < 0.05 compared with all other groups). These data indicate significant enhancement of the effectiveness of doxorubicin in prostate tumors by nontoxic and subtherapeutic doses of suramin.

Nontoxic doses of suramin enhance activity of paclitaxel against lung metastases.

We recently reported that acidic (aFGF) and basic (bFGF) fibroblast growth factors confer a broad spectrum chemoresistance in solid tumors, and that suramin, an inhibitor of multiple growth factors including aFGF and bFGF, enhanced the in vitro antitumor activity of several anticancer drugs including paclitaxel (Song, S., et al., Proc. Natl. Acad. Sci. USA, 97: 8658-8663, 2000). The present study investigated in vitro and in vivo interaction between paclitaxel and suramin, using human PC3-LN cells which, upon i.v. injection into immunodeficient mice, yielded lung metastases in 100% of animals. In in vitro studies, conditioned medium (CM) obtained from histocultures of rat lung metastases induced a 3-fold resistance. The addition of suramin had no effect in the absence of CM but reversed the CM-induced resistance; calculations based on the IC(50) values indicate a complete reversal in the presence of <20 microM suramin. Analysis by the combination index method indicates a synergistic interaction between paclitaxel and suramin. In in vivo studies, animals with well-established lung metastases (at least five nodules of 1 mm in diameter) were treated i.v. with paclitaxel (15 mg/kg) and suramin (10 mg/kg) administered twice weekly for 3 weeks. Single-drug therapy with paclitaxel or suramin did not reduce body weight. Suramin alone had no antitumor activity. Paclitaxel alone reduced the tumor size by approximately 75%, reduced the density of nonapoptotic cells by approximately 70% in residual tumors, and enhanced the fraction of apoptotic cells by approximately 3-fold. The addition of suramin to paclitaxel therapy enhanced the antitumor effect, resulting in an additional 5-fold reduction of tumor size, an additional 9-fold reduction of the density of nonapoptotic cells, and an additional 30% increase in the apoptotic cell fraction. These data indicate significant enhancement of the efficacy of paclitaxel by suramin and support the use of nontoxic doses of suramin with paclitaxel in the treatment of lung cancer.

Determinants of drug delivery and transport to solid tumors.

This presentation addresses the barriers and determinants and the importance of drug-induced apoptosis in drug transport and delivery to organs and solid tumors. In particular, we examined the roles of interstitial space, drug removal by capillaries, tissue structure and tissue composition on drug distribution. Drug transport in bladder tissues is described by the distributed model which combined monodimensional Fickian diffusion and first order removal of drug by the perfusing blood. Microscopic evaluation of the spatial drug distribution in bladder, prostate and tongue indicates heterogeneous drug distribution with large and erratic concentration gradient. In general, drug distribution favors interstitial space and vasculature, with little penetration in muscles. Drug penetration into 3-dimensional solid tumors is typically 5- to 10-fold slower than in monolayer cultures. 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. Accordingly, the delivery of a highly protein-bound drug to cells in a solid tumor is affected by its apoptotic effects and is therefore determined by the drug concentration and the treatment duration, i.e. treatment schedule. Under in vitro and in vivo conditions, the delivery of highly protein-bound drugs to tumor can be enhanced by using a pretreatment that induces apoptosis and reduction in cell density, and by using treatment schedules designed to take advantage of these drug-induced changes in tumor tissue composition. In conclusion, in addition to the usual processes involved in drug transport such as distribution through vascular space, transport across microvessel walls, and diffusion through interstitial space in tumor tissue, other factors including tissue structure and composition and alteration by drug-induced apoptosis are important determinants of drug distribution in organs and solid tumors.

Kinetics of P-glycoprotein-mediated efflux of paclitaxel.

Paclitaxel is a substrate of the mdr1 P-glycoprotein (Pgp). The objective of the present study was to determine the kinetics of the Pgp-mediated efflux and its contribution to the overall efflux of paclitaxel at the clinically achievable concentration range of 1 to 1500 nM. Human breast carcinoma BC19 cells that were derived from MCF7 cells by mdr1 transfection and show a >10-fold higher level of the Pgp protein were used to measure the uptake and efflux of [(3)H]paclitaxel. A computational model of intracellular paclitaxel pharmacokinetics was developed to analyze for the Pgp efflux parameters. The results show a saturable Pgp-mediated efflux in BC19 cells; the dissociation constant was 14 nM, and the maximal efflux rate was 2.8 x 10(-4) pmol/h/cell. The contribution of Pgp-mediated efflux to the total efflux decreased with increasing extracellular drug concentrations; the Pgp efflux accounted for 86 and 34% of total efflux at 1 and 1500 nM, respectively. The validity of the model was confirmed by the close agreement between the model-predicted data and the experimentally obtained data (approximately 6% deviation) describing the effect of cell density and intracellular-to-extracellular concentration gradient on the kinetics of drug accumulation and efflux. In conclusion, our results indicate that the Pgp-mediated efflux represents a major efflux mechanism of paclitaxel at the low end of the clinically observed drug concentration range, but accounts for only a minor part of the efflux at higher concentrations in BC19 cells.

A quantitative assay of telomerase activity.

PURPOSE: Telomerase is a ribonucleoprotein that extends telomeres at the ends of chromosome. Increased telomerase activity is associated with cellular immortality. The currently available assay for telomerase, i.e., telomeric repeat amplification protocol (TRAP), consists of 2 steps: (a) telomerase-mediated extension of an oligonucleotide primer by the enzyme-containing extracts of cells and tissues, and (b) amplification of the telomerase-extended primer products by polymerase chain reaction (PCR) and detection of the PCR products. It is generally accepted that the current TRAP assay lacks quantitative precision. The present study was to develop a quantitative telomerase assay with greater precision and sensitivity. METHODS: This new method used the primer extension method as in TRAP, plus the following modifications: (a) used a lysis buffer that yielded complete lysis of nuclei; (b) removal of PCR inhibitors by phenol/chloroform extraction after primer extension; and (c) used primers for the internal standard that were designed to reduce their competition with the telomerase products for PCR. RESULTS: The modified method showed a good correlation (r2 = 0.99, P < 0.001) between telomerase amount (expressed as total protein in cell lysate) and its activity (expressed as telomerase products). Compared to the conventional TRAP, the new method (a) was more sensitive (average of 5.5-fold in cultured cancer cells and >5.9-fold in patient tumors), (b) had a lower inter- and intra-day variability (>3fold), and (c) showed a 2 to 4-fold broader range of linearity in the standard curve. The higher assay sensitivity further enabled the use of a nonradioactive method, i.e., ethidium bromide staining of DNA, to detect the TRAP products, as opposed to the use of radioactive nucleotide and the more labor-intensive autoradiography mandated by the conventional TRAP. CONCLUSION: We report here a quantitative assay for telomerase activity in cultured human cancer cells and patient tumors.

Determinants of paclitaxel uptake, accumulation and retention in solid tumors.

This report addresses the determinants of the rate and extent of paclitaxel accumulation in tumors. In a 2-dimensional system such as monolayers where the drug is directly in contact with tumor cells, drug accumulation is determined by the extracellular-to-intracellular concentration gradient, the drug binding to extracellular and intracellular macromolecules, the presence of the mdrl p-glycoprotein (Pgp). and the time-dependent and drug concentration-dependent changes in tubulins and cell density. Intracellular pharmacokinetic models were developed to depict the effects of these parameters. Computer simulation results indicate that at the clinically relevant concentration range of 1 to 1,000 nM, (a) the binding affinity and the number of intracellular saturable drug binding sites are important for drug accumulation at low and high extracellular concentrations, respectively, (b) saturation in the drug binding to the high affinity intracellular binding sites (e.g., tubulin/microtubule) occurs at extracellular drug concentration above 100 nM, (c) treatment with 1,000 nM paclitaxel for >4 hr results in increased levels of tubulin/microtubule and consequently increased intracellular drug accumulation, whereas the continued cell proliferation after treatment with low drug concentrations results in reduced intracellular accumulation, and (d) saturation of Pgp in mdr1-transfected cells occurs at the high end of the clinically relevant concentration range. In a 3-dimensional system such as the solid tumor histocultures, which contain tumor cells as well as stromal cells, the drug accumulation into the inner cell layers is determined by the unique properties of solid tumors, including tumor cell density and spatial arrangement of tumor and stromal tissues. Most interestingly, drug penetration is modulated by the drug-induced apoptosis; the reduced cell density due to apoptosis results in an enhancement of the rate of drug penetration into the inner cell layers of solid tumors. In conclusion, the uptake, accumulation, and retention of paclitaxel in solid tumors are determined by (a) factors that are independent of biological changes in tumor cells induced by paclitaxel, i.e., ratio of extracellular and intracellular concentrations, and drug binding to extracellular and intracellular macromolecules, and (b) factors that are dependent on the time- and drug concentration-dependent biological changes induced by paclitaxel, i.e., induction of apoptosis, enhancement of tubulin/microtubule production, and induction of Pgp expression.

Expression of DT-diaphorase and cytochrome P450 reductase correlates with mitomycin C activity in human bladder tumors.

Mitomycin C (MMC) is activated by DT-diaphorase (DTD) and cytochrome P450 reductase (P450R). In cancer cell lines, MMC cytotoxicity is correlated with DTD and P450R expression levels. The present study investigated the relationship between enzyme expression/activity and MMC cytotoxicity in patient bladder tumors. DTD and P450R expression was detected by competitive reverse transcription-PCR and their activity was measured by bioreductive assays. The expression of DTD and P450R in patient tumors (n = 29), as ratios to beta-actin levels, varied from 0 to 90% and 0 to 29%, respectively. The DTD expression was significantly correlated with P450R expression (r(2), 0.32; P < 0.01), whereas the average DTD level was 2-fold higher than that of P450R (P < 0.01). Among the 29 tumors, 21 provided sufficient materials to evaluate tumor sensitivity to MMC. The concentration of MMC required to produce 50% inhibition (IC(50)) of DNA precursor incorporation for a 2-h treatment ranged from 0.17 to 18.1 microg/ml, indicating a 110-fold intertumor variation, with the high-grade and more invasive tumors being less chemosensitive compared with the low-grade and less invasive tumors. Tumor sensitivity to MMC, as indicated by the inverse of IC(50) values, was positively correlated with the expression of DTD (r(2), 0.28; P < 0.05) and P450R (r(2), 0.26; P < 0.05). Multivariate analysis indicates DTD expression and P450R expression as better determinants of MMC activity compared with other pathobiological factors (e.g., tumor grade, stage, and labeling index) that have been shown to significantly correlate with MMC activity. Eleven tumors were studied for the relationship between gene expression level and enzyme activity of DTD and P450R. The DTD activity was significantly correlated with the gene expression level (r(2), 0.84; P < 0.001). For P450R, there is a trend of a correlation between enzyme activity and its mRNA level, but the correlation was not statistically significant (r(2), 0.28; P = 0.09). These data indicate that the sensitivity of patient bladder tumors to MMC is correlated with the expression of DTD and P450R in tumors and suggest that the lower expression of these enzymes in the high-grade and more invasive tumors is a cause of the lower efficacy of intravesical MMC in these tumors.

Methods to improve efficacy of intravesical mitomycin C: results of a randomized phase III trial.

BACKGROUND: Intravesical chemotherapy (i.e., placement of the drug directly in the bladder) with mitomycin C is beneficial for patients with superficial bladder cancer who are at high risk of recurrence, but standard therapy is empirically based and patient response rates have been variable, in part because of inadequate drug delivery. We carried out a prospective, two-arm, randomized, multi-institutional phase III trial to test whether enhancing the drug's concentration in urine would improve its efficacy. METHODS: Patients with histologically proven transitional cell carcinoma and at high risk for recurrence were eligible for the trial. Patients in the optimized-treatment arm (n = 119) received a 40-mg dose of mitomycin C, pharmacokinetic manipulations to increase drug concentration by decreasing urine volume, and urine alkalinization to stabilize the drug. Patients in the standard-treatment arm (n = 111) received a 20-mg dose without pharmacokinetic manipulations or urine alkalinization. Both treatments were given weekly for 6 weeks. Primary endpoints were recurrence and time to recurrence. Treatment outcome was examined by use of Kaplan-Meier analysis with log-rank tests. Statistical tests were two-sided. RESULTS: Patients in the two arms did not differ in demographics or history of intravesical therapy. Dysuria occurred more frequently in the optimized arm but did not lead to more frequent treatment termination. In an intent-to-treat analysis, patients in the optimized arm showed a longer median time to recurrence (29.1 months; 95% confidence interval [CI] = 14.0 to 44.2 months) and a greater recurrence-free fraction (41.0%; 95% CI = 30.9% to 51.1%) at 5 years than patients in the standard arm (11.8 months; 95% CI = 7.2 to 16.4 months) and 24.6% (95% CI = 14.9% to 34.3%) (P =.005, log-rank test for time to recurrence). Improvements were found in all risk groups defined by tumor stage, grade, focality, and recurrence. CONCLUSIONS: This study identified a pharmacologically optimized intravesical mitomycin C treatment with statistically significantly enhanced efficacy.

Enhancement of paclitaxel delivery to solid tumors by apoptosis-inducing pretreatment: effect of treatment schedule.

The limited penetration of paclitaxel into solid tumors may limit its therapeutic efficacy. We recently showed a correlation between an increase in interstitial space and an enhancement of drug delivery in solid tumors. The present study evaluated whether this observation can be used to develop a treatment strategy, where an apoptosis-inducing pretreatment with paclitaxel is used to enhance its own delivery to solid tumors. In histocultures of human pharynx FaDu xenograft tumors, pretreatment with 1 microM nonradiolabeled paclitaxel, which resulted in approximately 25% apoptosis and a 25% reduction in cell density, enhanced the penetration rate of [(3)H]paclitaxel. Likewise, dividing a total drug exposure to two treatments, separated by an interval to allow apoptosis to occur, resulted in higher drug penetration rate and accumulation compared with giving the same drug exposure continuously. Similar results were obtained in rats bearing subcutaneously implanted prostate MAT-LyLu tumors; fractionation of the dose, to include 1) a pretreatment that yielded sufficient and clinically relevant plasma concentration to induce apoptosis and 2) a second dose given at an interval selected to allow apoptosis and reduction in tumor cell density to occur, resulted in higher tumor concentration compared with other treatments using the same total dose but either did not include an apoptosis-inducing pretreatment or did not allow for apoptosis to occur. We conclude that the pharmacological effect of paclitaxel affects its own delivery to solid tumors and that modifications of the paclitaxel treatment schedule can enhance drug delivery in solid tumors.

Fibroblast growth factors: an epigenetic mechanism of broad spectrum resistance to anticancer drugs.

Based on the observation that removal of tumors from metastatic organs reversed their chemoresistance, we hypothesized that chemoresistance is induced by extracellular factors in tumor-bearing organs. By comparing chemosensitivity and proteins in different tumors (primary vs. metastases) and different culture systems (tumor fragment histocultures vs. monolayer cultures derived from the same tumor), we found elevated levels of acidic (aFGF) and basic (bFGF) fibroblast growth factors in the conditioned medium (CM) of solid and metastatic tumors. These CM induced broad spectrum resistance to drugs with diverse structures and action mechanisms (paclitaxel, doxorubicin, 5-fluorouracil). Inhibition of bFGF by mAb and its removal by immunoprecipitation resulted in complete reversal of the CM-induced chemoresistance, whereas inhibition/removal of aFGF resulted in partial reversal. Using CM that had been depleted of aFGF and/or bFGF and subsequently reconstituted with respective human recombinant proteins, we found that bFGF but not aFGF induced chemoresistance whereas aFGF amplified the bFGF effect. aFGF and bFGF fully accounted for the CM effect, indicating these proteins as the underlying mechanism of the chemoresistance. The FGF-induced resistance was not due to reduced intracellular drug accumulation or altered cell proliferation. We further showed that an inhibitor of aFGF/bFGF (suramin) enhanced the in vitro and in vivo activity of chemotherapy, resulting in shrinkage and eradication of well established human lung metastases in mice without enhancing toxicity. These results indicate elevated levels of extracellular aFGF/bFGF as an epigenetic mechanism by which cancer cells elude cytotoxic insult by chemotherapy, and provide a basis for designing new treatment strategies.

Computational model of intracellular pharmacokinetics of paclitaxel.

The intracellular pharmacokinetics of paclitaxel is closely related to its pharmacodynamics. Although drug transport across the cell membrane and extracellular and intracellular drug binding have been shown to affect intracellular drug accumulation, their quantitative relationship is unknown. This study was designed to establish a mathematical model for computing the intracellular paclitaxel pharmacokinetics. As a starting point, the model assumes drug transport into and out of cells via passive diffusion. Experimental data on the intracellular pharmacokinetics of [(3)H]paclitaxel were obtained using monolayer cultures of human breast MCF7 tumor cells, which have negligible expression of the mdr1 P-glycoprotein. The results indicate that, in addition to drug binding and microtubule concentration, changes in cell number due to cell growth and drug effects also affected intracellular drug accumulation. A kinetic model was developed to describe several concomitant processes: 1) saturable drug binding to extracellular proteins, 2) saturable and nonsaturable drug binding to intracellular components, 3) time- and concentration-dependent drug depletion from culture medium, 4) cell density-dependent drug accumulation, and 5) time- and drug concentration-dependent enhancement of tubulin concentration. The model was validated by the close prediction (<7% deviation) of the effects of extracellular-to-intracellular concentration gradient and cell density on the kinetics of drug accumulation and efflux. This model was used to predict the effects of changing several parameters (number and binding affinity of intracellular binding sites, free fraction, and concentration of drug in extracellular fluid) on intracellular drug accumulation. In conclusion, the computational model of intracellular paclitaxel pharmacokinetics provides the means to predict drug concentration in cells.

A pharmacodynamic analysis method to determine the relative importance of drug concentration and treatment time on effect.

PURPOSES: The pharmacodynamics of most drugs follow the empirical relationship, C(n) x T = h, where C is drug concentration, T is exposure time and h is drug exposure constant. The value of n indicates the relative importance of C and T in determining the effect. An n value greater than 1.0 indicates that for two infusions that produce the same C x T, a short infusion that delivers high concentrations over a short duration will produce a greater C(n) x T and therefore a greater effect, compared to a long infusion that delivers lower concentrations. The reverse is true for an n value less than 1.0 and would support the use of a slow infusion. Hence, it is important to determine the n values and whether the n value significantly differs from 1.0. This report describes a three-step method for this purpose. METHODS: First, we obtained experimental data on the relationship between drug concentration, treatment time and effect, and analyzed the data with a three-dimensional surface response method to obtain the pharmacodynamic model parameters and the magnitude of data variability. The experiments used mitomycin C and two human cancer cell lines, i.e. bladder RT4 and pharynx FaDu cells. The n values obtained from four experiments ranged from 1.04 to 1.16 for FaDu cells and from 1.14 to 1.46 for RT4 cells. The variability in the effect data decreased from 11.9% at 0% effect to 6.14% at 100% effect. Second, these results were used with Monte Carlo simulations to generate 100 concentration-time-effect data sets, which contained randomly and normally distributed data variability comparable to the experimentally observed variability, for each experimentally determined n value. This is analogous to performing 100 experiments under the same experimental conditions. Third, we analyzed the simulated data sets to obtain 100 estimated n values. The frequency with which these estimated n values fell above or below 1.0 indicated the probability that the experimentally determined n value used in the Monte Carlo simulations was truly different from 1.0. We defined this frequency for individual experiments as F(one), and calculated the overall probability for multiple experiments (F(multiple)). A probability of greater than 97.5% (i.e. P < 0.05 for a two-tailed test) was considered statistically significant. RESULTS: Analysis of the mitomycin C pharmacodynamic data yielded F(one) and F(multiple) of 99% to 100% for FaDu and RT4 cells, indicating that the n values for these cells were significantly higher than 1.0. A comparison of the statistical significance of the n value analyzed by the three-step pharmacodynamic analysis method, a conventional statistical method such as the Student's t-test and nonlinear regression analysis, indicated two advantages for the pharmacodynamic method: fewer experiments were required (theoretically only one experiment with three replicates would be sufficient) and a higher statistical significance of the n value was obtained. CONCLUSIONS: In summary, the three-step pharmacodynamic study design and analysis method can be used to define the relative importance of drug concentration and treatment time on drug effect.

Cremophor reduces paclitaxel penetration into bladder wall during intravesical treatment.

PURPOSE: We have previously shown that paclitaxel, when dissolved in water and instilled into the bladder, readily penetrates the urothelium. The FDA-approved formulation uses Cremophor and ethanol to dissolve paclitaxel. In the present study, the effects of this solvent system on the urine, bladder tissue, and plasma pharmacokinetics of intravesical paclitaxel were evaluated. METHODS: Plasma, urine, and tissue pharmacokinetics were determined in five dogs treated for 120 min with paclitaxel (500 microg per 20 ml of 0.22% w/v Cremophor and 0.21% v/v ethanol) by intravesical instillation. Equilibrium dialysis was used to determine the free fraction of paclitaxel and the presence of Cremophor micelles was verified using a fluorescent probe method. RESULTS: The average bladder tissue concentration was > 1600-fold higher than the plasma concentration. Comparison of the results for paclitaxel dissolved in Cremophor/ethanol with our previous results of paclitaxel dissolved in water (500 microg per 20 ml) indicates that Cremophor/ethanol decreased the paclitaxel partition across the urothelium and reduced the average bladder tissue concentration by 75%, but did not alter the rate of paclitaxel penetration across the bladder wall, the urine pharmacokinetics or the plasma pharmacokinetics of paclitaxel. For Cremophor, the urine concentrations during the 120-min treatment ranged from 0.12% to 0.22%, and the concentration in bladder tissue from 0.00004% to 0.0009%. The threshold Cremophor concentration for micelle formation was 0.008%. We found that ethanol at concentrations up to 1% and Cremophor at concentrations below 0.01% did not alter the free fraction of paclitaxel, whereas Cremophor at higher concentrations, i.e. 0.065% and 0.25%, significantly reduced the free fraction by two- to six-fold, respectively. These results indicate that during intravesical instillation of the FDA-approved paclitaxel formulation, the concentration of Cremophor in urine was sufficient to form micelles, resulting in sequestration of paclitaxel into micelles, reduction in the free fraction of paclitaxel and consequently a reduction in paclitaxel penetration across the urothelium. In contrast, the Cremophor concentrations in bladder tissue were inadequate to form micelles and thus did not alter the drug penetration through the bladder tissue. CONCLUSIONS: We conclude that intravesical paclitaxel treatment using the FDA-approved formulation provides a significant bladder tissue targeting advantage, although the advantage is lower than when paclitaxel is dissolved in water.

Regional heterogeneity and pharmacodynamics in human solid tumor histoculture.

PURPOSE: Human solid tumor histocultures represent a clinically relevant experimental system for pharmacodynamic study. The evaluation of the drug-induced antiproliferative effect in histocultures is usually performed by manual microscopic scoring of individual cells. This procedure, because of its labor intensive nature, is performed on a single microscopic field, i.e. the field with the highest proliferative activity. Because regional heterogeneity in a solid tumor may result in different drug sensitivities in different parts of a tumor, there is the question as to whether the pharmacodynamic data determined in the most proliferative field is representative of those in the whole tumor. This question was addressed in the present study. METHODS: A recently developed automated image analysis method was used to measure the labeling index of tumor cells. The drug-induced inhibition of DNA precursor incorporation into nuclei of cells in the region with the highest proliferative activity was compared to the inhibition in cells in the entire histoculture. This study was performed in human bladder tumor histocultures treated with several drugs (doxorubicin, mitomycin C, paclitaxel and 5-fluorouridine). A total of 724 pairs of data obtained from untreated and drug-treated histocultures (each data point representing the average of 1 to 6 tumor histocultures) were analyzed. RESULTS: The absolute value of the labeling index in the most proliferative region (LI(one)) was significantly higher than the absolute value of the labeling index in the whole tumor (LI(all)), in both untreated and drug treated samples (mean difference of 18%, range 1-27%). However, when the absolute LI values in drug-treated samples were normalized to the values in untreated controls and expressed as a percentage of control, and used to construct the concentration-response curves, the two curves obtained using LI(one) and LI(all) yielded comparable pharmacodynamic parameters, i.e. curve shape parameters and drug concentrations that produce 30, 50, and 70% inhibition. CONCLUSION: These results indicate comparable pharmacodynamics in the most proliferative region and the whole tumor, and confirm the validity of using the most proliferative field for evaluating chemosensitivity in solid tumor histocultures.

Determinants of paclitaxel penetration and accumulation in human solid tumor.

The present study examined the determinants of the penetration and accumulation of [(3)H]paclitaxel (12-12,000 nM) in three-dimensional histocultures of patient tumors and of a human xenograft tumor in mice. The results showed 1) significant and saturable drug accumulation in tumors, 2) extensive drug retention in tumors, and 3) a slower penetration but a more extensive accumulation in the xenograft tumor compared with patient tumors. Drug penetration was not rate-limited by drug diffusion from medium through the matrix supporting the histocultures. The difference in the expression of the mdr1 P-glycoprotein did not fully account for the difference in the drug accumulation in xenograft and patient tumors. Autoradiography and imaging were used to evaluate the spatial relationship between tumor architecture, tumor cell distribution, and drug distribution as a function of time and initial drug concentration in culture medium. The tumor cell density and the kinetics of drug-induced apoptosis were also evaluated. The results indicate that a high tumor cell density is a barrier to paclitaxel penetration and that the apoptotic effect of paclitaxel enhances its penetration in solid tumor. These factors are responsible for the time- and concentration-dependent drug penetration rate, with drug penetration confined to the periphery until apoptosis and reduction of epithelial cell density occurred at 24 h, after which time paclitaxel penetrated the inner parts of the tumor.

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.