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Purpose: Telomeres are specific DNA structure at the ends of chromosomes to protect chromosomes from fusion, recombination, and degradation. Telomere length changes are implicated in cell senescence, aging, tumorigenesis, and DNA repair. The standard method for measuring telomere length is Southern blot analysis. This method has several disadvantages, i.e., loss of DNA during membrane blotting, high background due to nonspecific binding of telomere probe to membrane, and loss of telomeric signal due to extensive washing. These limitations resulted in a low signal-to-noise ratio and, therefore, reduced sensitivity and reproducibility. The multi-step Southern blot is also highly labor-intensive. The present study was to develop a more quantitative assay of telomeric amount and length (TALA)., Methods: TALA was based on solution hybridization and did not require blotting, prehybridization, and washing. The major steps were (a) DNA preparation and digestion with restriction endonucleases, (b) hybridization between DNA and telomeric probe, (c) agarose gel electrophoresis, and (d) autoradiography and data analysis., Results: The telomere amount measured by TALA was linearly correlated with the amount of DNA analyzed (r2 = 0.985, P < 0.01). The telomere length measured by TALA also correlated with the telomere length determined by fluorescence in situ hybridization (r2 = 0.99, P < 0.01). Compared to the Southern blot analysis, TALA showed a 4-fold greater sensitivity, 4.6-fold higher signal-to-noise ratio, >2 fold-higher reproducibility, and 4-fold less time requirement., Conclusion: We report here a rapid, sensitive, and quantitative assay for measuring telomere length and amount.

Purpose: We previously reported that in patient tumors the expression of the mdrl p-glycoprotein (Pgp) resulted in a lower paclitaxel-induced inhibition of DNA precursor incorporation, but a higher apoptosis (Clin. Cancer Res. 4:2949-2955, 1998). The present study was to evaluate these findings in an experimental system where the Pgp effect can be studied without confounding factors such as the intra- and inter-tumor heterogeneity associated with patient tumors., Methods: To separate the effect of Pgp on intracellular paclitaxel accumulation from its effects on drug sensitivity, we compared the drug activity at various extracellular and intracellular drug concentrations using the human breast MCF7 tumor cells and its mdr1-transfected variant BC19 cells., Results: Compared to MCF7 cells, BC19 cells showed a 9-fold higher Pgp level and >13-fold higher mdrl expression. Intracellular paclitaxel accumulation was 80-130% lower in BC19 cells when the extracellular concentrations were < or = 100 nM, but the difference was reduced to <15% differences at higher extracellular concentrations of > or = 1,000 nM. For the G2/M block effect MCF7 cells were 43-fold more sensitive than BC19 cells at equal extracellular concentration, and 3.5-fold more sensitive at comparable intracellular concentrations. On the contrary. BC19 cells were more sensitive to the apoptotic effect: BC19 cells showed equal or higher apoptosis compared to MCF7 cells at extracellular concentrations above 100 nM, and a 30-100% higher apoptosis at comparable intracellular concentrations., Conclusions: These results confirm our previous observations in patient tumors and indicate that enhanced Pgp expression is associated with enhanced sensitivity to the apoptotic effect of paclitaxel and reduced sensitivity to its G2/M block effect, via yet-unknown mechanisms that are unrelated to the effect of Pgp on intracellular drug accumulation.

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.

Purpose: We have shown that the three human prostate xenograft tumors, i.e. the androgen-dependent CWR22 tumor, and the androgen-resistant CWR22R and CWR91 tumors, are comparable to patient tumors in their expression of prostate specific antigen, multidrug resistance p-glycoprotein, p53 and Bcl-2 and in their sensitivity to doxorubicin and paclitaxel. The present study used histocultures of these xenograft tumors to evaluate the antiproliferative and cytotoxic effects of several drugs (geldanamycin, cytochalasin E and thiacetazone), which have diverse action mechanisms and have shown activity against primary cultures of human prostate cancer cells. Suramin, a clinically active compound was included for comparison. Methods. The antiproliferative effect of 96 h drug treatment was measured by inhibition of DNA precursor incorporation, and the cytotoxic or cell kill effect was measured by in situ DNA end labeling of apoptotic and necrotic cells and by reduction of live cell density., Results: The rank order of molar potency was geldanamycin > cytochalasin E > suramin > or = thiacetazone. Thiacetazone produced antiproliferation only in CWR22 tumor and had no cytotoxicity, whereas the other three drugs produced both antiproliferation and cytotoxicity in all three tumors. Geldanamycin, but not cytochalasin E and suramin, showed greater antiproliferation and cytotoxicity in tumor cells compared to normal stromal cells. The two androgen-resistant tumors were 4 to >40-fold less sensitive than the androgen-dependent tumor to drug-induced antiproliferation but were about equally or 4 to >20-fold more sensitive to drug-induced cytotoxicity. The ratios of drug concentrations that produced 50% antiproliferation to the concentrations that produced 50% cytotoxicity ranged from <0.04 to 0.3 in CWR22 tumor, but ranged from 0.3 to 2.7 in CWR22R and CWR91 tumors, indicating a shift from antiproliferation as the predominant drug effect in the androgen-dependent tumor to cytotoxicity in the androgen-resistant tumors., Conclusions: Our results indicate (a) differential drug effects in human prostate xenograft tumors with antiproliferation and cytotoxicity as the predominant drug effect in the androgen-dependent and androgen-resistant tumors, respectively, (b) that progression of tumors from androgen-dependent state to androgen-resistant state appears to be associated with a lower sensitivity to drug-induced antiproliferation and an equal or greater sensitivity to drug-induced cytotoxicity, and (c) that geldanamycin but not thiacetazone warrants further development.

Purpose: The present study compared proliferative indices, i.e. incorporation of DNA precursor (i.e. thymidine or TdR, and bromodeoxyuridine or BrdU) and expression of proliferating cell nuclear antigen (PCNA), as molecular pharmacodynamic endpoints in evaluation of anticancer drug effect in human solid tumors., Methods: Tumor specimens obtained from patients were grown as histocultures. After treatment with doxorubicin, mitomycin C, and/or paclitaxel, cells labeled by [3H]TdR were identified using autoradiography, and cells labeled by BrdU and PCNA were identified using immunohistochemical techniques. Drug effect was measured as reduction of DNA precursor-labeled cells or PCNA-expressing cells., Results: The results indicate that (a) the two DNA precursors, TdR and BrdU, labeled the same cells and resulted in identical pharmacodynamics, (b) the pharmacodynamics established using inhibition of DNA precursor incorporation were qualitatively and quantitatively different from the pharmacodynamics established using inhibition of PCNA expression, (c) the inhibition of PCNA expression was erratic in some tumors, and (d) the differences in pharmacodynamics established using the two end points are drug-specific, with greater differences for paclitaxel than for mitomycin C., Conclusions: The erratic results measured by the PCNA labeling method suggest that this method may be less reliable than the conventional DNA precursor labeling method. The finding of identical pharmacodynamics of doxorubicin and paclitaxel established using BrdU and [3H]TdR indicates that the two precursors are interchangeable. Because the methodology for detecting BrdU incorporation requires less time and does not require the use of radioactivity, we conclude that inhibition of BrdU incorporation represents a useful endpoint for evaluating the antiproliferative activity of anticancer drugs in human solid tumors.

Purpose: The present study evaluated the cytostatic and apoptotic effects of a 24-hr paclitaxel treatment in ovarian tumors., Methods: Three-dimensional histocultures of surgical specimens from patients (n = 17) were used. The cytostatic effect was measured by inhibition of 96-hr cumulative DNA precursor incorporation and induction of apoptosis was determined by morphological changes., Results: Paclitaxel produced partial inhibition of DNA precursor incorporation in about 40% of tumors (maximum inhibition of approximately 30%) and induced apoptosis in about 90% of tumors (maximum apoptotic index of approximately 15%). In responsive tumors, maximum cytostatic and apoptotic effects were achieved at < or = 1 microM with no further enhancement by increasing the drug concentration to 10 microM. In individual tumors, the apoptotic effect inversely correlated with cytostatic effect (r2 = 0.27, p = 0.031), and the maximal apoptotic index correlated with the LI for the untreated controls (r2 = 0.38, p < 0.01). More than 95% of apoptotic cells after paclitaxel treatment were labeled with DNA precursor. The incomplete cytostatic and apoptotic effects of paclitaxel and the link between DNA synthesis and apoptosis in ovarian tumors are similar to our previous findings in other human solid tumors., Conclusions: These findings suggest that (a) apoptosis is the major paclitaxel effect in advanced ovarian tumors, (b) tumor sensitivity to drug-induced cytostatic effect is opposite to sensitivity to apoptotic effect, (c) paclitaxel-induced apoptosis increases with increased cell proliferation and is completed after DNA synthesis, and (d) further increasing the dose to elevate plasma concentration beyond 1 microM may not improve treatment outcome.

Purpose: The goal of this study was to develop physiologically based pharmacokinetic (PBPK) models for 2',3'-dideoxyinosine (ddI) in rats when the drug was administered alone (ddI model) and with pentamidine (ddI + pentamidine model), and to use these models to evaluate the effect of our previously reported pentamidine-ddI interaction on tissue ddI exposure in humans., Methods: The PBPK models consisted of pharmacologically relevant tissues (blood, brain, gut, spleen, pancreas, liver, kidney, lymph nodes, muscle) and used the assumptions of perfusion-rate limited tissue distribution and linear tissue binding of ddI. The required physiologic model parameters were obtained from the literature, whereas the pharmacokinetic parameters and the tissue-to-plasma partition coefficients were calculated using plasma and tissue data., Results: The ddI model in rats yielded model-predicted concentration-time profiles that were in close agreement with the experimentally determined profiles after an intravenous ddI dose (5% deviation in plasma and 20% deviation in tissues). The ddI + pentamidine model incorporated the pentamidine-induced increases of ddI partition in pancreas and muscle. The two PBPK models were scaled-up to humans using human physiologic and pharmacokinetic parameters. A comparison of the model-predicted plasma concentration-time profiles with the observed profiles in AIDS patients who often received ddI with pentamidine showed that the ddI model underestimated the terminal half-life (t1/2, beta) by 39% whereas the ddI + pentamidine model yielded identical t1/2, beta and area-under-the-curve as the observed values (< 1% deviation). Simulations of ddI concentration-time profiles in human tissues using the two models showed that pancreas and lymph nodes received about 2- to 30-fold higher ddI concentration than spleen and brain, and that coadministration of pentamidine increased the AUC of ddI in the pancreas by 20%., Conclusions: Data of the present study indicate that the plasma ddI concentration-time profile in patients were better described by the ddI + pentamidine model than by the ddI model, suggesting that the pentamidine-induced changes in tissue distribution of ddI observed in rats may also occur in humans.

Purpose: Previous studies by other investigators have shown an enhancement of mitomycin C (MMC) activity at acidic extracellular pH (pHe) in monolayer cultures of human cells. The goal of the present study was to determine if the efficacy of intravesical MMC therapy in patients treated for superficial bladder cancer can be enhanced by using acidified dosing solutions. We evaluated (a) the effect of pHe on MMC activity in patient bladder tumors in vitro, and (b) the pH dependency of MMC activity in 2-dimensional monolayer and 3-dimensional multilayer cultures of human bladder RT4 tumor cells., Methods: Patient bladder tumors were maintained as 3-dimensional histocultures. RT4 cells were harvested and maintained as monolayer cultures or as 3-dimensional cell pellets on a collagen gel matrix. The cell pellets were 300-450 cell layers and 4,000-5,000 microns in diameter. Tumors or cells were incubated for 2 hr with MMC-containing media at pHe of 5, 6, and 7.4. The drug effect was measured by the inhibition of DNA precursor (thymidine) incorporation. The stability of MMC as a function of pHe was determined. About 24% of MMC was degraded following 2 hr exposure at pHe 5 and < or = 2% at pHe 6 and 7.4., Results: The drug concentrations required to inhibit thymidine incorporation by 50% (IC50) were corrected for the degraded MMC at acidic pHe. The results showed no pH-dependent MMC activity in human patient bladder tumors nor in RT4 multilayer cultures; the IC50 values were about 10 micrograms/ml at all three pHe. In contrast, the monolayer RT4 cultures showed a pH-dependent MMC cytotoxicity; the IC50 were 0.1, 0.8 and 1.2 micrograms/ ml at pHe 5, 6 and 7.4, respectively (p < 0.05). Pre-incubation of multilayered RT4 cultures in acidic pH medium for 8 hr enhanced the MMC activity; the IC50 was reduced by about 5 fold at pHe about 3 fold at pHe 6. Similar pH-dependent MMC activity was found when multilayers were pre-treated for 1 hr with 0.5 microgram/ml nigericin, a proton ionophore known to cause the intracellular pH (pHi) to equilibrate with pHe., Conclusions: These data suggest that the difference in the pH dependency of MMC activity in the monolayer and multilayer systems was due to the different experimental conditions. The time lag for pHi to equilibrate with pHe in the multilayer systems and the instability of MMC at low pHe imply that the efficacy of intravesical MMC therapy is unlikely to be enhanced by using acidic dosing solution.

Purpose: This study compared the sensitivity of rat prostate MAT-LyLu primary and lymph node metastatic tumors to taxol., Methods: Tumors were established by subcutaneous implantation of tumor cells in a hind leg (primary site) of male Copenhagen rats. Lymph node metastases were used for serial transplantation. Eleven pairs of primary and metastatic tumors between the sixth and twentieth generations were harvested and maintained as 3-dimensional histocultures. The effects of taxol (24 hr treatment at 1 nM to 10 microM) were measured by the appearance of apoptotic cells, and by the inhibition of DNA precursor (thymidine) incorporation. To determine the basis of differential sensitivity of primary and metastatic tumors to the DNA inhibition, we examined the expression of multidrug resistance pglycoprotein (Pgp) and the accumulation of 3H-taxol after 24 hr exposure and the retention after a 48 hr washout period., Results: The fraction of apoptotic cells increased linearly with the logarithm of taxol concentration to a maximal value of 25%; the concentration-response curves for primary and metastatic tumors were superimposable. Taxol produced a sigmoidal, concentration-dependent inhibition of thymidine incorporation; the maximal inhibition of approximately 40% was reached at 0.1 and 1 microM for primary and metastatic tumors, respectively. Within the primary or metastatic subgroups, the IC30 (drug concentration that produced a 30% inhibition of DNA synthesis) among consecutive generations varied by < 5 fold, but the primary tumor consistently showed a lower IC30 than the daughter or the parent metastatic tumor (mean, 20-fold; median, 15-fold; range, 6- to 56-fold). The finding that the lower drug sensitivity in metastatic tumors was not exhibited in its daughter primary tumor but was regained in its daughter metastatic tumors suggests that the chemoresistant phenotype is maintained only in lymph nodes and not in the primary site. There were no differences in the Pgp status (neither tumor expressed Pgp), accumulation and retention of taxol in primary and metastatic tumors., Conclusions: Taxol induced apoptosis and inhibited DNA synthesis in the rat MAT-LyLu primary and lymph node metastatic tumors. The apoptotic effect was not different among the two tumors, whereas the primary tumor was more sensitive to the inhibition of DNA synthesis. The differential sensitivity of the two tumors to the DNA effect is not associated with a difference in Pgp expression, drug accumulation nor drug retention, and appears to be associated with changes that are linked to lymph node metastasis.

Purpose: This study examined the pharmacokinetic interaction between 2',3'-dideoxyinosine (ddI) and pentamidine., Background: ddI and pentamidine are often coadministered to patients with acquired immunodeficiency syndrome, and are both associated with pancreatic toxicity. Information on potential interaction would be useful to assess the need for dose modification and the basis of the higher incidence of pancreatic toxicity associated with coadministration of the two drugs., Methods: ddI (200 mg/kg) and pentamidine (10 mg/kg) were administered by continuous infusion to rats over 3 hr, either alone or concomitantly. Drug analysis was by high pressure liquid chromatography with UV or fluorescence detection, or by radioimmunoassay., Results: Pentamidine coadministration significantly increased the apparent volume of distribution at steady state of ddI from 1.4 to 3.4 l/kg (p = 0.004), and increased the mean residence time from 36.3 to 50.0 min (p = 0.015). Pentamidine enhanced the distribution of ddI from plasma into pancreas (p = 0.001) and muscle (p = 0.026). ddI distribution into spleen and liver was also increased, with differences approaching statistical significance (p = 0.08 and 0.06, respectively). In contrast, ddI coadministration did not affect the total body clearance but increased the urinary excretion and the renal clearance of pentamidine by about 5-fold (p = 0.0003)., Conclusions: These data indicate that pentamidine increased the distribution of ddI into pancreas and muscle, whereas ddI increased the renal elimination of pentamidine.

Purpose: To evaluate the intratracheal route of administration as an alternative to oral administration for 2',3'-dideoxyinosine (ddI)., Methods: A ddI dose (40 mg/kg/300 microliters or 6.5 mg/kg/50 microliters) was instilled into the trachea in female Fisher rats and an intravenous tracer dose (9 micrograms/kg) of 3H-ddI was administered concomitantly to determine the drug clearance. Plasma concentrations were analyzed for the rate and extent of absorption., Results: ddI was rapidly absorbed from the lungs, with a bioavailability of 63% at 40 mg/kg and 101% at 6.5 mg/kg. By comparison, our previous data showed an oral bioavailability of about 15% (Pharm Res., 9:822, 1992). The distribution of a dye solution instilled intratracheally showed that a fraction of the 300 microliters dose spilled over to the gastrointestinal tract, where the entire 50 microliters dose was retained in the lungs. The different distribution of the two doses volumes likely contributed to the different bioavailability, with a fraction of the higher dose/volume degraded in the gastrointestinal tract after the spillover. Absorption of ddI from the airspace of the lung was biexponential, suggesting two absorption processes., Conclusions: These data indicate significantly higher and less variable bioavailability of ddI by the intratracheal route of delivery compared to the oral route. Furthermore, the complete bioavailability at the lower dose/volume indicates no significant pulmonary first pass elimination for ddI.

Purpose: To use the drug kinetics in dermis to predict the in vivo blood concentration after topical administration., Methods: A two-step pharmacokinetic model was established. The first step was to calculate the drug input rate or flux from the skin to the systemic circulation using the drug kinetic parameters in dermis. These parameters include (a) distance over which the drug concentration declines by 50%, (b) drug concentration at the epidermal-dermal junction, and (c) minimal plateauing drug concentration in the muscle layer. These parameters were experimentally determined from the drug concentration-tissue depth profiles in the dermis, after the application of a topical dose of ddI (200 mg/kg) to rats. The second step was to use the drug input rate together with the systemic disposition pharmacokinetics of ddI in rats to predict the plasma concentration-time profiles. The model-predicted plasma concentration-time profiles were compared with the observed profiles, to determine the validity of the proposed pharmacokinetic model., Results: The observed steady state concentration (CSS) in individual animals (n = 6) deviated from the predicted values by 3 to 55% with 3 of 6 rats showing a < 15% deviation. The mean observed CSS of all animals deviated from the mean predicted values by less than 15%., Conclusions: The close agreement between the observed and the model-predicted drug concentrations indicates that the systemic drug input can be calculated from the drug kinetics in the dermis.

The present study evaluated the kinetics of drug penetration in the dermis. A rat was given a dermal dose of 2',3'-dideoxyinosine (ddI). At 6 hr, the skin tissue was excised, immediately frozen and sectioned, and the decline of drug concentration as a function of tissue depth was determined. The tissue concentration-depth profile showed a semilogarithmic decline, as would be expected in a distributed tissue kinetic model which incorporates diffusion and capillary membrane transport. The goodness of fit of the profiles by the simple diffusion and the distributed models were compared using four statistical criteria, i.e., coefficient of determination. Akaike Information criterion, Schwartz criterion and Imbimbo criterion. These analyses showed that the decline of tissue concentration versus tissue depth in the dermis was better described by the distributed model than by the diffusion model in all 7 animals. To examine the effect of blood perfusion on the tissue concentration-depth profiles, some of the tissues were frozen after 1 and 2 hr storage at room temperature. In contrast to the adjacent tissues frozen immediately, the concentration-depth profiles in tissues frozen after a 1-2 hr delay were described equally well by distributed and diffusion models. A comparison of the concentration-depth profiles in the tissues processed immediately or after a delay showed a 7 fold more shallow slope and a 60% lower concentration at the epidermis-dermis interface after storage. However, storage did not alter the total amount of drug in the entire dermis. Drug degradation during storage was further ruled out by the insignificant ddI degradation in 10% skin homogenate (a half-life of approximately 70 hr).(ABSTRACT TRUNCATED AT 250 WORDS)

This study explored the topical route for administering of 2',3'-dideoxyinosine (ddI), a nucleoside analog used for treating patients with acquired immunodeficiency syndrome. A dose of ddI (approximately 180 mg/kg) dispersed in approximately 1 g ointment base was applied, with or without occlusion, to the back of high follicular density (HFD) and low follicular density (LFD) rats. The systemic ddI clearance was determined using a concomitant administration of an intravenous tracer dose of [3H]ddI. At 24 hr, the experiment was terminated and skin sections at the application site were removed. After topical application, average plateau plasma levels of about 0.6 microgram/ml were achieved within 1 to 2 hr and maintained for 24 hr. Occlusion gave a more uniform plasma profile but did not increase the bioavailability. The systemic bioavailability in HFD and LFD rats was about the same at 33%. In addition, a depot of about 16% of the dose was recovered by rinsing the application area and extracting the drug from the excised application site. These data indicate that about 50% of the dermal dose penetrated the skin barrier in 24 hr. The similar bioavailability in the HFD and LFD rats further suggests an unimportant role for the transfollicular absorption route for ddI. The effect of a mixture of penetration enhancers, Azone and propylene glycol (5:95), was studied in HFD rats. Coadministration of ddI with the enhancers did not increase the ddI bioavailability. However pretreatment and coadministration with the enhancers significantly increased the bioavailability to 62%, which is a conservative estimate because the plasma drug level was still at a plateau when the experiment was terminated at 24 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

This investigation was to establish a clinically relevant experimental model to evaluate the pharmacodynamics of drugs used for head and neck cancers. A total of 83 surgical samples of primary and lymph nodal metastatic tumors was obtained from 66 patients. Fragments of these tumors were cultured on a collagen gel matrix. The tumor cell labeling index (LI) was determined by [3H]thymidine incorporation and autoradiography. Seventeen tumors (20%) were contaminated. About 80% of the remaining 65 tumors were successfully cultured for at least 2 weeks. The cultured tumor fragments retained the morphology and architecture of the freshly removed specimens; both tumor and stromal cells were present. The tumor cell LI after 2-3 weeks in culture, determined from the most proliferative area of the tissue, averaged 77 +/- 12% for primary tumors and 78 +/- 12% for nodal metastases. The activity of three clinically active agents, 5-fluorouracil (FU), cisplatin (DDP), and mitomycin C (MMC), was evaluated in 47 tumors. All three drugs inhibited the tumor LI. The concentrations needed to produce a 50% inhibition of the tumor LI (IC50) were within the clinically achievable concentration range. The intertumor variation in the IC50 for FU (60-fold) was considerably greater than that for DDP and MMC (7- to 8-fold). The nodal metastatic tumors appeared to be less sensitive to FU than the primary tumors, while there were no apparent differences for DDP or MMC.(ABSTRACT TRUNCATED AT 250 WORDS)

This study explored the rectal route of administration for 2',3'-dideoxyinosine (ddI). Rats were given a rectal infusion of nonradiolabeled ddI (200 mg/kg in 0.7 mL saline) over 35 min along with an intravenous (iv) bolus injection of [8-(3)H]ddI (20 microCi, equivalent to 2.1 micrograms), which was used to calculate the absolute rectal bioavailability of ddI. Maximal plasma concentrations of rectally administered unlabeled ddI were 5.4 +/- 2.2 micrograms/mL and were reached at the end of the infusion. The rectal bioavailability averaged 15.6 +/- 4.4% (n = 9). The second aim of this study was to examine the kinetics of ddI absorption from the colorectal region. Analyses of the absorption rate-time profiles by the Loo-Riegelman and deconvolution methods showed biphasic absorption: a rapid phase during infusion and a slow phase postinfusion. These profiles were inconsistent with a mammillary model with absorption from a single site with one apparent rate constant. The model which gave the best fit for infusion and postinfusion data consisted of two different sites (colon and rectum) with different apparent absorption rate constants. The two sites were connected by a first-order transfer of drug solution from rectum to colon. The apparent absorption rate constant in the rectum was 39-fold higher than that in the colon. In conclusion, these results show absorption of ddI from the colorectal region and suggest the rectal route as an alternative to the oral route. The data further suggest different absorption sites in the colorectal region, with a more rapid absorption in the rectum than in the colon.

The pharmacokinetics of 2',3'-dideoxyinosine (ddI) were examined in rats given intravenous doses of 8, 40, or 200 mg/kg. The concentrations of ddI in whole blood and plasma were identical. The concentration decline was multiexponential, with mean half-lives of 2 and 20 min for the first and second phases, respectively. At the highest dose, a slower third phase with a half-life of 56 min was observed. The total-body clearances were 99, 77, and 37 ml/min-kg for the 8, 40, and 200 mg/kg doses. The steady-state volume of distribution showed a trend for a decrease with increasing doses, but the difference was not statistically significant. Twenty-four-hour urinary recovery of unchanged drug for the three doses was similar at about 20%, suggesting that a major fraction of the dose was metabolized. Urinary excretion of ddI metabolite, hypoxanthine, accounted for less than 5% of the dose. Renal and metabolic clearances decreased with increased doses. ddI was metabolized in blood; the addition of inorganic phosphate, a cosubstrate in phosphorylase-mediated nucleoside catabolism, enhanced the degradation by about fourfold. In summary, these data indicate equal distribution of ddI in the extracellular and intracellular spaces in blood, its enzymatic degradation in blood, and nonlinear elimination kinetics.

Determination of the depth of penetration of locally applied drug therapy and evaluation of possible mechanisms of drug transport require knowledge of drug concentration-versus-tissues depth profiles. A method to determine the drug concentration-depth profile is needed. We have devised such a method and used it to determine the penetration of mitomycin C (MMC) in the dog bladder wall after intravesical drug instillation. This method is based on sectioning of frozen tissue into 40-microns segments, followed by drug extraction and high-pressure liquid chromatography analysis. Tissue concentrations could be detected with a sensitivity of 1 ng/sample, or 20 ng/g for tissue samples of approximately 2 x 2 cm. This sensitivity was sufficient to describe the penetration of MMC in the bladder wall of dogs, using an identical instillation technique, dwell time, and MMC concentration as in human patients. Tissue concentrations were expressed relative to tissue weight or tissue protein contents. For MMC, standardization to tissue weight yielded a better mathematical fit of the concentration-versus-depth profiles than standardization to protein content. The time interval between tissue harvesting and freezing was critical. The MMC concentration at the urothelial side of dog bladders was 2- to 10-fold higher in samples processed immediately after harvesting, compared to samples processed after 1 hr or longer. This significant decrease was not due to drug metabolism in situ. In separate in vitro experiments, we found that the degradation of MMC in 8% tissue homogenate was relatively slow, with only a 30% decline in concentration over 24 hr.(ABSTRACT TRUNCATED AT 250 WORDS)