Cancer Research The Future of Cancer Research: Science and Patient Impact Tumor Immunology: New Perspectives
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
[Cancer Research 59, 1054-1060, March 1, 1999]
© 1999 American Association for Cancer Research
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fuse, E.
Right arrow Articles by Sugiyama, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fuse, E.
Right arrow Articles by Sugiyama, Y.
[Cancer Research 59, 1054-1060, March 1, 1999]
© 1999 American Association for Cancer Research

Experimental Therapeutics

Altered Pharmacokinetics of a Novel Anticancer Drug, UCN-01, Caused by Specific High Affinity Binding to {alpha}1-Acid Glycoprotein in Humans

Eiichi Fuse, Hiromi Tanii, Katsumi Takai, Kazuki Asanome, Noriaki Kurata, Hiroyuki Kobayashi, Takashi Kuwabara1, Satoshi Kobayashi and Yuichi Sugiyama

Drug Development Research Laboratories, Pharmaceutical Research Institute, Kyowa Hakko Kogyo Co., Ltd., Shizuoka 411-8731, Japan [E. F., H. T., K. T., K. A., N. K., H. K., T.K., S. K.], and Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1, Hongo, Bunkyo-Ku, Tokyo 113, Japan [Y. S.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The large species difference in the pharmacokinetics/pharmacodynamics of 7-hydroxystaurosporine (UCN-01) can be partially explained by the high affinity binding of UCN-01 to human {alpha}1-acid glycoprotein (AGP) (Fuse et al., Cancer Res., 58: 3248–3253, 1998). To confirm whether its binding to human AGP actually changes the in vivo pharmacokinetics, we have studied the alteration in its pharmacokinetics after simultaneous administration of human AGP to rats: (a) the protein binding of UCN-01 was evaluated by chasing its dissociation from proteins using dextran-coated charcoal. The UCN-01 remaining 0.1 h after adding dextran-coated charcoal to human plasma or AGP was ~80%, although the values for other specimens, except monkey plasma (~20%), were <1%, indicating that the dissociation from human AGP was specifically slower than from other proteins; and (b) the pharmacokinetics of UCN-01 simultaneously administered with human AGP has been determined. The plasma concentrations after i.v. administration of UCN-01 with equimolar human AGP were much higher than those after administration of UCN-01 alone. The steady-state distribution volume and the systemic clearance were reduced to about 1/100 and 1/200, respectively. Human AGP thus reduced the distribution and elimination of UCN-01 substantially. On the other hand, dog AGP, which has a low binding affinity for UCN-01, did not change the pharmacokinetics of UCN-01 so much. Furthermore, human AGP markedly reduced the hepatic extraction ratio of UCN-01 from 0.510 to 0.0326. Also, human AGP (10 µM) completely inhibited the initial uptake of UCN-01 (1 µM) into isolated rat hepatocytes, whereas the uptake of UCN-01 was unchanged in the presence of human serum albumin (10 µM). In conclusion, the high degree of binding of UCN-01 to human AGP causes a reduction in the distribution and clearance, resulting in high plasma concentrations in humans.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
UCN-012 (Fig. 1)Citation is a protein kinase inhibitor, a new type of anticancer drug, which is presently under clinical investigation. It inhibits the growth of various cultured human cancer cell lines in vitro such as epidermoid carcinoma A431, fibrosarcoma HT1080, and non-small cell lung carcinoma A549 and exhibits potent antitumor activity in vivo against some of these cell lines (1, 2, 3, 4) . Also, the drug induces G1-phase accumulation in A431 and other cell lines (3 , 5) , and this G1-phase accumulation might be mediated through the direct inhibition of cyclin-dependent kinase 2 and indirect inhibition mediated by induction of p21/Cip1/WAF1 and p27/Kip1 (6) . In addition, UCN-01 synergistically enhances the antitumor activity of several standard agents, such as mitomycin C, in cultured human cancer cells both in vitro and in vivo (2 , 7 , 8) .



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 1. Chemical structures of [3H]UCN-01 and staurosporine (I.S.). I.S., internal standard; *, labeled position.

 
The plasma concentrations of UCN-01 in Phase I studies were much higher than expected. In fact, the CLtot (systemic clearance), Vdss (distribution volume at steady-state), and t1/2 (elimination half-lives) after i.v. infusion to cancer patients in the United States at 5.4 mg/m2 for 72 h were 0.0996–0.252 ml/h/kg, 88.6–113 ml/kg, and 253–947 h, and those after i.v. infusion to cancer patients in Japan at 0.65 and 1.3 mg/m2 for 3 h were 0.0407–0.102 ml/h/kg, 79.6–158 ml/kg, and 855–1660 h (9) . These pharmacokinetic properties of UCN-01 were very different from these seen in experimental animals, characterized by rapid clearance with a large distribution volume. The CLtot, Vdss, and t1/2 were 600–4000 ml/h/kg, 6000–17000 ml/kg, and 3–12 h, respectively (9 , 10) .

This dramatic species difference in the pharmacokinetics of UCN-01 could at least in part be explained by specific high affinity binding to human AGP (9) because the association constant (Ka) of UCN-01 for human AGP was 8 x 108 (M)-1, indicating very high affinity (9) . On the other hand, the Ka for dog AGP was 1/60 that, and UCN-01 exhibited only weak and nonspecific binding to rat AGP or HSA (9) . Thus, there is a large species difference in the binding to AGP.

Binding to plasma proteins is well known to affect drug disposition (11, 12, 13) and changes in the disposition of several drugs, e.g., imipramine (14 , 15) and propranolol (16) due to AGP have been reported. However, there are no reports describing the relationship between the species difference or affinity constants for AGP and changes in drug disposition. To confirm whether the high affinity binding of UCN-01 to human AGP actually changes the in vivo pharmacokinetics, we studied the effects of human AGP on the pharmacokinetics of UCN-01 including hepatic extraction and uptake into hepatocytes in rats. In addition, the protein binding was evaluated in terms of dynamic dissociation, not equilibrium binding. The dissociation of UCN-01 from plasma of various animals, including human or purified human plasma proteins, was measured by the DCC method (17 , 18) .


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and Reagents.
UCN-01 and staurosporine (Fig. 1)Citation , as the internal standard for HPLC analysis, were produced by a fermentation technique in our laboratories as described previously (4) . [3H]UCN-01 (Fig. 1)Citation synthesized at Amersham International (Buckinghamshire, United Kingdom) was purified immediately before use. The radiochemical purity determined by HPLC was almost 100%, and the specific radioactivity was 666 kBq/nmol. UCN-01 dissolved in DMSO was diluted with 66 mM phosphate buffer containing 50 mM sodium chloride (pH 7.4, PBS) or plasma from various animal species, including humans, before use. The final concentration of DMSO was <1%. Activated charcoal (column chromatography, 60–150 mesh) and Dextran T70 were purchased from Nacalai Tesque (Tokyo, Japan) and Pharmacia Biotech AB (Uppsala, Sweden), respectively. Essential fatty acid and {gamma}-globulin-free lyophilized HSA prepared from fraction V (lot 93H9345), human AGP (Cohn Fraction VI, lot 113H9308 or lot 90H9317), and human {gamma}-globulin (Cohn Fraction II, III, lot 37F9315) were purchased from Sigma Chemical (St. Louis, MO). BSA (Fraction V Protease Free) was obtained from Seikagaku Kogyo (Tokyo, Japan). Collagenase (Clostridium histolyticum) and liquid paraffin were obtained from Wako Pure Chemical (Osaka, Japan). Anti-human AGP IgG fraction from goat (Incstar, Stillwater, MN) and HRP-conjugated anti-human AGP antibody from goat (EY Laboratories, San Mateo, CA) were used. All other chemicals and solvents were of analytical grade. Vessels and the tips of dispensers for protein solutions were siliconized with Sigmacote (Sigma) to avoid nonspecific adsorption of proteins and UCN-01.

Plasma and Animals.
Blood collected from a healthy volunteer with written informed consent was centrifuged at 2100 x g for 10 min at 4°C to provide control plasma. Animal plasma was obtained from male ddY strain mice (7 weeks of age; Nihon SLC, Hamamatsu, Japan), male SD strain rats (7 weeks of age; Nihon SLC), male LRE strain beagle dogs (2 years of age; HRP, Covance Research Products, Kalamazoo, MI), and male cynomolgus monkeys (4–5 years of age; C.V. Universal Fauna, Indonesia). In the experiments on the effects of AGP on plasma concentration-time profiles, hepatic extraction, and uptake into hepatocytes, male SD strain rats of 6–8 weeks of age (Nihon SLC; 210–310 g at the start of the experiments) were housed for about 1 week under controlled conditions (23 ± 1°C and 55 ± 5% humidity) and lighting (12-h light/dark cycle) with free access to food and water. All experiments using laboratory animals were approved by the Welfare Committee for Experimental Animals in our institute.

DCC Methods.
DCC, to which proteins exhibited little nonspecific binding, was prepared by the method of Sablonnière et al. (18) with minor modification. Charcoal (2 g) was suspended in distilled water (~50 ml), stirred for 10 s, and centrifuged at 1200 x g for 10 min. The supernatant was discarded by decantation. This washing procedure was repeated three times to remove light particles. The charcoal pellet resuspended in 25 ml of PBS containing dextran (0.2 g) was defined as DCC. DCC was added to an equal volume of human plasma or solutions of HSA (600 µM), human AGP (20 µM), and {gamma}-globulin (67 µM) in the presence of 10, 20, and 40 µM (final concentrations, 5, 10, and 20 µM) UCN-01 at 37°C. Then, the suspension was agitated by mixer. At 0.1 and 2 h after mixing, an aliquot was centrifuged at 4°C, ~20,000 x g for 3 min, and the supernatant was used to measure the UCN-01 concentration.

In the same way as the protein solutions, an aliquot of supernatant was collected to determine the concentration of UCN-01 at 0.1 and 2 h after the addition of DCC to an equal volume of human, monkey, dog, rat, or mouse plasma at 10 µM (final concentration, 5 µM) UCN-01 at 37°C. UCN-01 was stable in these matrices without DCC at 37°C (data not shown).

Plasma Concentrations of UCN-01 after i.v. Administration of UCN-01 with or without Human/Dog AGP.
For dosing (19) , UCN-01 was dissolved at 0.175 mg/ml in citric acid·H2O (1.2 mg/ml), Na2HPO4·12H2O (5.6 mg/ml), and NaCl (6 mg/ml) with protection from light and stirring overnight at 4°C. In the experiment involving simultaneous administration of UCN-01 and human or dog AGP, each AGP sample was also added to the solution about 1 h before dosing at 15.25 mg/ml, almost equimolar to UCN-01. The doses of UCN-01 and each AGP were 0.35 and 30.5 mg/kg (0.725 and 0.726 µmol/kg; Mr 482.5 and 42,000), respectively. The dosing solution of UCN-01 or the mixture of UCN-01 and AGP was rapidly administered to rats at a volume of 2 ml/kg via the femoral vein. For the analysis of the plasma concentrations of UCN-01, ~0.25 ml blood was withdrawn from the tail vein and transferred to heparinized capillary tubes at 0.05, 0.1, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 32, and 48 h after dosing. During the simultaneous dosing of UCN-01 and human AGP, samples were also collected at 72 h after dosing. Each plasma sample was separated and stored at -70°C until analysis.

Effects of Human AGP on Hepatic Extraction of UCN-01 in Rats.
As described in the above section, UCN-01 was dissolved in citric acid·H2O (1.2 mg/ml), and Na2HPO4·12H2O and NaCl were added. Hepatic extraction was measured by the method reported previously (19) . Under light ether anesthesia, cannulation of the femoral artery and vein and the hepatic vein was carried out by the usual method, using PE-50 polyethylene tubing. After recovery from the anesthesia, the drug was infused via the femoral venous cannula using an infusion pump (model 975; Harvard Apparatus, South Natick, MA). The infusion rate was set at 0.0652 ml/min. The infusion was started after bolus administration of 3 mg/kg as a loading dose. The infusion dose rate of UCN-01 was 25 µg/kg/min. For simultaneous infusion with human AGP, the solutions for the bolus and infusion dosing contained 0.1 g/100 ml (24 µM) human AGP, the physiological concentration in human plasma. The molar ratio of UCN-01 and human AGP was 10:1 in the dosing solution. Femoral arterial and hepatic venous blood samples (~0.25 ml) were collected at 20, 30, and 40 min after infusion in heparinized capillary tubes. Plasma was separated and stored at -70°C until the day of assay.

The hepatic extraction ratio (EH) was calculated from the hepatic venous plasma concentrations divided by the femoral arterial plasma concentrations at 40 min after the start of the UCN-01 infusion.

Effects of Serum Proteins on UCN-01 Uptake into Rat Hepatocytes.
Hepatocytes were isolated from rats by a two-step collagenase perfusion technique (20) . The isolated hepatocytes were washed twice with Hank’s buffer and twice with albumin-free Krebs-Henseleit buffer. The cell viability (>85%) was checked by the trypan blue (Flow Laboratories, Irvine, Scotland) exclusion test.

The isolated hepatocytes were diluted to 7 x 105 to 2 x 106 cells/ml and kept on ice. Uptake of [3H]UCN-01 at 0°C or 37°C was initiated by adding 22.5 µl of the ligand solution to the cell suspension (2 ml of 7 x 105 to 2 x 106 cells/ml) immediately or after preincubation (37°C for 10 min) at final concentrations of 0.5% DMSO, 0.5% methanol, 0.64 kBq/ml, and 1 µM UCN-01. After 10 s, the reaction was terminated by separating the cells from the medium using centrifugal filtration (21) . In the experiment on the uptake-time course, uptake was performed for 10, 20, 35, 50, and 70 s. In the experiment on the UCN-01 concentration dependence of uptake, the final concentrations of UCN-01 were 0.2, 1, 5, 20, 50, and 100 µM. An aliquot of the hepatocyte suspension was placed into a 0.4-ml centrifuge tube (Sarstedt, Nümbrecht, Germany) containing 100 µl of a mixture of silicone and liquid paraffin (4:1, density 1.015) atop 50 µl of 3 M potassium hydroxide, followed by centrifugation at 10,000 x g for 1 min at room temperature. After centrifugation, the sample tube was cut midway through the silicone-oil interface. The hepatocytes pelleted through the oil layer into the potassium hydroxide fraction, and the supernatants containing the medium were placed in individual scintillation vials and left overnight at room temperature to dissolve the cells. Fifty µl of 3 M hydrochloric acid were added to neutralize the potassium hydroxide. Then, Ultima Gold (Packard Instrument, Meriden, CT) was added to the vial, and the radioactivity in the cells and medium was determined in a liquid scintillation counter (LS-6500; Beckman, Fullerton, CA). The amount adhering to the cells at time 0 was extrapolated by linear regression of points (10–70 s) at 0°C. In general, the initial uptake velocity was calculated using linear regression of two or more points during the early period. In this experiment on UCN-01, the uptake velocity calculated from the first and second time points (about 10 and 20 s) was lower than that from the first time point (10 s). In view of this, the initial uptake velocity (Vo) was determined by dividing the difference between the amount taken up for 10 s at 37°C and the amount adhering by the incubation period. The cellular uptake was corrected by the reported values of the adhering fluid volume and the intracellular space (21) . The Vo and the substrate concentration (S) were plotted as a Vo-Vo/S plot (Eadie-Hofstee plot), and the inverse of the slope of the regression line by the least-squares method was taken as the Michaelis constant (Km). The maximum uptake velocity (Vmax) was calculated by multiplying the intercept of the regression line by Km.

The effects of various plasma proteins on the uptake velocity were studied by adding human/dog AGPs at 0.5 or 10 µM or HSA at 10 µM to the hepatocyte suspension. The percentage inhibition of the Vo by each plasma protein was calculated. The relationship between the percentage inhibition of Vo at 0.1, 0.5, 0.75, 1, 1.25, 1.5, and 10 µM human AGP and the specific binding ratio of UCN-01 to human AGP was also evaluated.

Determination of UCN-01 Concentrations.
The UCN-01 concentrations were determined as reported previously using a highly sensitive HPLC method with fluorescence detection (22) . The range for quantitation was 0.2–100 ng/ml. The accuracy and precision of the intra- and interday assays were <15%. For the experiment involving DCC treatment, the residual percentage was calculated by the relative internal standard ratio of the peak height of DCC-treated to DCC-untreated samples.

Determination of Human AGP Concentrations.
The plasma concentrations of human AGP were measured by ELISA involving goat anti-serum (IgG fraction) against human AGP as a coating antibody and goat HRP-conjugated anti-human AGP antibody as enzyme-labeled antibody. After addition of sodium citrate/phosphate buffer (pH 4.0) containing 0.01% H2O2 and 0.04 g/100 ml 2,2-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid), the absorbance at 412 nm was measured by microplate reader (THERMOmax; Molecular Devices, Menlo Park, CA) and analyzed by SOFTmax (version 2.32; Molecular Devices) software. Human AGP samples diluted with control rat plasma at concentrations of 100-5000 ng/ml were used as standards. The accuracy and precision for intra- and interday assays were within ±20%. There was no cross-reactivity against rat AGP, and UCN-01 had no effects on this assay. Human AGP has multiplicity, e.g., the genetic variants and carbohydrate modification (13) . The difference of reactivity against them was not determined.

Pharmacokinetic Analysis.
The plasma concentration-time profile of UCN-01 in individual rats was analyzed using model-independent approaches (9 , 23) . The plot of the logarithm of the plasma concentration versus time showed triphasic elimination. The slope of the third phase (elimination rate constant of the ß phase: ß) was determined by log-linear regression analysis, and those of the second and first phases (elimination rate constants at {alpha} and {pi} phases: {alpha} and {pi}) were determined by the method of residuals. The half-lives of the respective phases (t1/2{pi}, t1/2{alpha}, and t1/2ß) were calculated as 0.693/{pi}, 0.693/{alpha}, and 0.693/ß. The area under the plasma concentration-time curve (AUC0–{infty}) and the area under the moment curve were determined by the trapezoidal rule using the extrapolated value (Co) at time zero, calculated by the residual method and the observed data with extrapolation to infinity using ß. The systemic clearance (CLtot), the apparent distribution volume of the central compartment (V1), the mean residence time (MRT), and the apparent distribution volume at steady-state (Vdss) were calculated as Dose/AUC0–{infty}, Dose/Co, the area under the moment curve/AUC0–{infty}, and CLtot x MRT, respectively.

Statistical Analysis.
All data are presented as the mean ± SD or the mean of three animals or experiments. Statistical analysis was carried out using Windows-SAS System Release 6.12 (SAS Institute Japan, Tokyo, Japan). Any significant difference in the UCN-01 concentrations in the ratios of UCN-01 remaining in human plasma or purified protein solutions due to the DCC treatment was estimated by the Tukey test with equal variance and any significant difference identified by Bartlett’s test and a one-way ANOVA. In the case of a significant difference in variance, the analysis was discontinued. A significant difference in the ratios of UCN-01 remaining due to the DCC treatment between human plasma and each purified protein solution or each experimental animal plasma sample was estimated by the unpaired t test. The effect of human or dog AGP on the pharmacokinetic parameters of UCN-01 was estimated by Student’s t test with equal variance recognized by the F test or the Aspin-Welch test with unequal variance by the F test. The effects of various proteins on the uptake velocity of UCN-01 into rat hepatocytes were estimated by a one-way ANOVA, followed by the Dunnett test. For all tests, a probability of P < 0.05 was considered significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Evaluation of the Dissociation of UCN-01 from Proteins after Adding DCC to Various Purified Human Protein Solutions and Plasma of Animal Species Including Humans.
The ratios of UCN-01 remaining in the supernatant were determined 0.1 and 2 h after adding DCC to human plasma or purified proteins at final concentrations of 5, 10, and 20 µM UCN-01 at 37°C (Table 1)Citation . The ratios of UCN-01 remaining 0.1 h after adding DCC to human plasma were relatively high and decreased clearly with the increase in the concentration of UCN-01. Those in human AGP were almost equal to those in human plasma (79.3 ± 6.6% for human AGP versus 85.9 ± 6.2% for human plasma), whereas those in HSA (0.397%) and {gamma}-globulin (0.293%) were much lower than in human plasma. The ratios of UCN-01 remaining 2 h after adding DCC to human plasma and AGP were also much higher than those in HSA and {gamma}-globulin. No clear difference in the ratios of UCN-01 remaining between human AGP and plasma was found except that at 5 µM UCN-01.


View this table:
[in this window]
[in a new window]
  		Table 1 Ratios of UCN-01 remaining in human plasma and proteins 0.1 or 2 h after adding DCC (final concentration, 40 mg charcoal/ml suspension) at 37°C

 
The ratios of UCN-01 remaining in the supernatant were determined 0.1 and 2 h after adding DCC to plasma samples of various animals at a final concentration of 5 µM UCN-01 at 37°C (Table 2)Citation . The ratio of UCN-01 remaining 0.1 h after adding DCC to human plasma was ~80%. On the other hand, although the ratio remaining in monkey plasma was relatively high, that in plasma samples of mouse, rat, and dog was <0.6% and much lower than that in human plasma. The ratio of UCN-01 remaining 2 h after adding DCC to human plasma was clearly higher than that for plasma samples of other experimental animals. These results suggest that the dissociation of UCN-01 from AGP was slow and specific for humans.


View this table:
[in this window]
[in a new window]
  		Table 2 Ratios of UCN-01 remaining in plasma of various animals including humans 0.1 or 2 h after adding DCC (final concentration, 40 mg charcoal/ml suspension) at 37°C

 
Altered Pharmacokinetics of UCN-01 after Simultaneous Administration of Human or Dog AGP.
The plasma concentration-time profiles of UCN-01 and human AGP after bolus i.v. administration of UCN-01 alone or with equimolar human or dog AGP (0.73 µmol/kg) to male rats are shown in Fig. 2Citation , and the pharmacokinetic parameters of UCN-01 are shown in Table 3Citation . The profile of UCN-01 after giving UCN-01 alone was almost identical to the reported results (19) . The plasma concentrations of UCN-01 after i.v. administration of UCN-01 with human AGP were much higher than those after administration of UCN-01 alone. The value 3 min after dosing, the first sampling point, was 92 times higher than after giving UCN-01 alone. The pharmacokinetic parameters of UCN-01 after giving UCN-01 alone and simultaneous administration of UCN-01 with human AGP were compared. The V1 for coadministration was close to the plasma volume (12 , 24) and about 1/50 that after administration of UCN-01 alone (Table 3)Citation . The Vdss and CLtot decreased to about 1/100 and 1/200 those after administration of UCN-01 without human AGP (Table 3)Citation . Human AGP limited the distribution and elimination of UCN-01 in rats after simultaneous dosing. The elimination half-life after administration of UCN-01 with human AGP was about 1.7-fold longer than that after administration of UCN-01 alone. The plasma concentrations of human AGP decreased more slowly than those of UCN-01, and the CLtot and elimination half-life of human AGP were 0.37- and 2.1-fold that of UCN-01, respectively. However, the difference in the above pharmacokinetic parameters of UCN-01 after simultaneous administration with dog AGP compared with those after giving UCN-01 alone was about 2-fold.



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 2. Plasma concentration-time profiles of UCN-01 ({circ}, •, {blacktriangleup}) and human AGP ({diamond}) after bolus i.v. administration of UCN-01 alone ({circ}) or with human AGP (•) or dog AGP ({blacktriangleup}) to male rats. The doses of UCN-01, human AGP, and dog AGP were 0.35, 30.5, and 30.5 mg/kg, respectively. Values are means of three rats; bars, SD.

 

View this table:
[in this window]
[in a new window]
  		Table 3 Pharmacokinetic parameters of UCN-01 and human AGP after bolus i.v. administration of UCN-01 alone or with human AGP or dog AGP to male rats

 
Effects of Human AGP on Hepatic Extraction Ratio in Rats.
The plasma concentrations of UCN-01 during infusion of UCN-01 with or without human AGP in rats are shown in Fig. 3Citation . The plasma concentrations of UCN-01 tended to increase even at 40 min after simultaneous administration of UCN-01 with human AGP, unlike after giving UCN-01 alone (19) . The arterial plasma concentration was ~5 µM and was ~11-fold that for UCN-01 alone. The apparent EH of UCN-01 40 min after infusion of UCN-01 with human AGP was 0.0326 ± 0.0184 and dramatically reduced, in comparison with that after infusion of UCN-01 alone (0.510; Ref. 19 ).



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 3. Plasma concentration-time profiles of UCN-01 during i.v. infusion of UCN-01 alone (({square}, {blacksquare}; Ref. 19 ) or with human AGP ({circ}, •) to male rats. The open and closed symbols show hepatic venous and femoral arterial plasma concentrations. The infusion of UCN-01 started after a loading dose of 6200 nmol/kg. The infusion rate of UCN-01 was 52 nmol/min/kg. The concentration of human AGP in the loading and infusion solutions was 0.1 g/100 ml. Values are means of three rats; bars, SD.

 
Effects of Various Serum Proteins including Human AGP on Uptake of UCN-01 into Rat Hepatocytes.
The amount of [3H]UCN-01 taken up into rat hepatocytes versus time at 37°C and 0°C are shown in Fig. 4ACitation . At 37°C, >60% of the added [3H]UCN-01 radioactivity was taken up by hepatocytes over 10 s and >80% over 35 s (Fig. 4A)Citation . The uptake of UCN-01 was rapid and marked. The concentration ratio of UCN-01 in hepatocytes to that in the medium was ~500. The uptake of UCN-01 into rat hepatocytes at 0°C was lower than that at 37°C and was linear with time for 70 s. The amount at time 0 extrapolated by linear regression of the plot of the uptake of [3H]UCN-01 versus time at 0°C, estimated as the amount adhering on the cell surface (25) , was 21.6% of the added [3H]UCN-01. The initial uptake velocity (Vo) at 0.2–100 µM UCN-01 calculated from the uptake for 10 s with subtraction of the amount adhering was analyzed by an Eadie-Hofstee plot. The kinetic parameters were: Vmax 201 nmol/min/7 x 105 cells and Km 90.9 µM(Fig. 4B)Citation . Metabolic inhibitors (rotenone and carbonylcyanide p-trifluoromethoxyphenyl hydrazone) did not alter the uptake velocity of UCN-01 (data not shown). The effects of several plasma proteins on the Vo of UCN-01 were evaluated (Fig. 5)Citation . The Vo of UCN-01 at a concentration of 1 µM was not altered by adding HSA, which showed only nonspecific binding to UCN-01 (9) , at a concentration of 10 µM in the medium. On the other hand, human AGP inhibited the uptake of UCN-01 by ~50% and 100% at concentrations of 0.5 and 10 µM human AGP. Although dog AGP with an association constant of 1.3 x 107M-1 (9) did not have any effect on the uptake of UCN-01 at 0.5 µM dog AGP, it inhibited the Vo by 72.1% at 10 µM dog AGP. The inhibitory effect of each AGP clearly corresponded to the binding affinity for UCN-01 (9) . Furthermore, the percentage inhibition of Vo by human AGP over the wider concentration range was determined (Fig. 6)Citation . In the conditions, the unbound, specific bound (i.e., high affinity binding), and nonspecific bound fractions were calculated using the reported binding parameters of UCN-01 to human AGP (9) . There was best correlation between the percentage inhibition of Vo by human AGP and the specific binding ratios of UCN-01 to human AGP (r2 = 0.989; Fig. 6Citation ).



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 4. A, time courses of UCN-01 uptake into isolated rat hepatocytes at 37°C ({circ}) and 0°C (•). UCN-01 concentration, 1 µM; hepatocytes, 2 x 106 cells/ml. ——, regression line obtained by linear least-squares regression. B, Eadie-Hofstee plot of the rate of UCN-01 uptake into isolated rat hepatocytes. S, UCN-01 concentration; hepatocytes, 7 x 105 cells/ml. ——, regression line obtained by linear least-squares regression. Each point represents the mean of triplicate measurements.

 


View larger version (39K):
[in this window]
[in a new window]
  		Fig. 5. Effects of various serum proteins on the initial velocity of UCN-01 uptake into isolated rat hepatocytes at 37°C. n.d., not detectable; UCN-01 concentration, 1 µM. **, significant difference from control (P < 0.001, Dunnett test). Data represent the means (n = 3); bars, SD.

 


View larger version (12K):
[in this window]
[in a new window]
  		Fig. 6. A, the effect of human AGP concentration on the initial uptake velocity of UCN-01 (1 µM) into isolated rat hepatocytes at 37°C with and without human AGP (0.1, 0.5, 0.75, 1.0, 1.25, 1.5, and 10 µM). B, relationship between the calculated specific-binding fraction of UCN-01 and inhibition ratio of the initial uptake velocity (A) of the control (without human AGP). ——, regression line (y = 0.935x + 0.351, r2 = 0.989). Data represent the means (n = 3).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
There was a clear relationship between the pharmacokinetic parameters of UCN-01 and the binding affinity for AGP, i.e., the small distribution volume and low clearance in patients could be explained by the high degree of binding to human AGP (9) . However, there was no direct evidence that the high affinity of human AGP for UCN-01 actually limits the distribution and elimination of UCN-01. We have measured the affinity constant of UCN-01 with AGP at equilibrium (9) . In this report, we also evaluate the binding in a more dynamic way. The ratios of UCN-01 remaining after adding charcoal to plasma from different species, including human or plasma proteins, were evaluated using the DCC method (17 , 18) . Provided that the amount of charcoal to proteins in the matrix is sufficient and rebinding of the ligand to proteins can be neglected, the ratios of the ligand remaining in the supernatant shortly after DCC treatment reflect the rate of dissociation of the ligand from the proteins. The residual UCN-01 in human AGP after DCC treatment was much higher than that in other plasma proteins or animal plasmas, and the specificity of human AGP was very marked. In addition, the ratio of UCN-01 remaining in human AGP 2 h after DCC treatment did not fall to one-half the value at 0.1 h, and the dissociation half-life of UCN-01 from human AGP was estimated to be longer than 2 h. The half-lives of UCN-01 dissociation from the other matrices were estimated as ~0.01–0.05 h.

In our study, the pharmacokinetics of UCN-01 after simultaneous administration with human AGP to rats have been studied to confirm whether the high affinity binding of UCN-01 to human AGP actually changes the in vivo pharmacokinetics. The pharmacokinetics of UCN-01 after its administration with equimolar dog AGP, which had 1/60 the binding affinity of human AGP, was also studied. The linear pharmacokinetics of UCN-01 in rats was confirmed over the dose range 0.35–3.5 mg/kg (19) . In this study, the dose was set at 0.35 mg/kg (0.725 µmol/kg). In the administered solution, 86.4% of the UCN-01 was calculated to be bound to human AGP using the binding parameters (9) . The effects of human AGP on the pharmacokinetics of UCN-01 were seen immediately after administration, and the V1, 46.7 ml/kg, was almost equal to the plasma volume (Table 3)Citation . The CLtot, 15.0 ml/h/kg, fell to about 1/200 after administration of UCN-01 alone (2950 ml/h/kg; Table 3Citation ). These results suggested that the high degree of binding of UCN-01 to human AGP limited distribution into tissues and inhibited elimination of UCN-01. On the other hand, the CLtot (1770 ml/h/kg), V1 (1340 ml/kg), and Vdss (5500 ml/kg) after administration of UCN-01 with dog AGP fell by only 40–60% after giving UCN-01 alone.

If the binding of UCN-01 to human AGP in the administered solution is constant in plasma after administration to rats, the initial plasma concentration of UCN-01 should be comparable with the maximum binding capacity, nP, the product of the number of binding sites per molecule (n), and the protein concentration (P). The nPs for human and dog AGPs calculated using the n of human and dog AGPs (9) , multiplied by the AGP concentrations at time 0, were 11.8 and 5.61 µM. Although the concentration of UCN-01 3 min after giving UCN-01 with human AGP was comparable with the nP for human AGP, the value after giving it with dog AGP was less than 1/10 the nP for dog AGP. These results indicate that UCN-01 dissociated from dog AGP rapidly due to the low affinity, although human AGP bound to UCN-01 tightly and confined it to plasma. Actually, the binding constant of UCN-01 for dog AGP was ~1/60 that for human AGP (9) . Also, the ratio of UCN-01 remaining 0.1 h after adding DCC to dog plasma was substantially lower than that in human plasma, suggesting the rapid dissociation of UCN-01 from dog AGP (Table 2)Citation . Although many researchers have reported that variations in AGP affected the pharmacokinetics of several drugs such as imipramine (14 , 15) and propranolol (16) , the change in the pharmacokinetics of UCN-01 was more drastic than for these drugs.

The elimination half-life of UCN-01 after coadministration with human AGP increased only by ~70% and was apparently shorter than that in clinical trials (9) . The difference may be due to the experimental conditions, such as the shorter period of plasma sampling in this study than in the Phase I trials. That is, a longer elimination phase may be missed because of being short of observing time. In addition, although AGP levels are generally constant in humans, the human AGP administered to rats was eliminated from the body (Fig. 2Citation ; Table 3Citation ). The pharmacokinetic parameters of human AGP obtained in this study (Table 3)Citation approximated the values in the literature (26 , 27) . The elimination half-life of UCN-01 in Phase I trials (9) was longer than that after i.v. administration of 125I- or 131I-labeled human AGP in humans, 2–7 days (28 , 29) , and UCN-01 may retard the elimination of human AGP itself. However, no clear effects of UCN-01 on the elimination of human AGP after simultaneous administration with UCN-01 were found in our study. In addition, human AGP is eliminated more slowly than UCN-01. These facts suggest that UCN-01 is eliminated in the unbound form after dissociating from human AGP but not in the bound form with human AGP. In our in vitro study, we actually found dissociation of UCN-01 from human AGP (Table 1)Citation .

The effects of human AGP on the extraction of UCN-01 in liver, the main eliminating organ, were investigated. As predicted, the addition of human AGP to the administered solution dramatically reduced the EH of UCN-01, and little elimination of UCN-01 occurred in the liver (Fig. 3)Citation . The effects of plasma proteins on the uptake into isolated rat hepatocytes were further evaluated. The hepatocytes:medium concentration ratio of UCN-01 was about 500 at steady-state, and enrichment of UCN-01 by uptake into hepatocytes was shown. The uptake clearance in liver was very high and was calculated as 906,000 ml/h/kg using the Vmax/Km (Fig. 4B)Citation . Metabolic inhibitors did not alter the uptake of UCN-01, and no ATP-dependent active transport was observed (data not shown). The effect of several plasma proteins on the uptake of UCN-01 into rat hepatocytes was evaluated (Fig. 5)Citation . HSA, which showed only nonspecific binding to UCN-01 (9) , had no effect on the uptake of UCN-01, and the degrees of inhibition by human and dog AGPs almost reflected the difference in the affinity for UCN-01 between these AGPs. The percentage inhibition by these AGPs was comparable with the specific binding ratios but not the sum of specific and nonspecific binding ratios (Fig. 6)Citation . This indicated that only specific binding inhibited the uptake of UCN-01 into rat hepatocytes.

In this report, we have focused on the effects of human AGP on the pharmacology of UCN-01 in rats. It is predicted that the high degree of binding of UCN-01 to human AGP and the altered pharmacokinetics produced by human AGP modify the antiproliferative activity of UCN-01. From this standpoint, studies of the effects of human AGP on the uptake and antiproliferative activity of UCN-01 in human cancer cell lines are on-going.

The interpatient variation in AGP levels is relatively large, and intrapatient variation due to certain disease, including cancer, is known (13) . This indicates differences in efficacy and/or toxicity as well as pharmacokinetics among patients treated with UCN-01. Although the plasma concentrations of UCN-01 were very high, most drug molecules in plasma were predicted to be bound to human AGP so that the "free" UCN-01 as well as "total" concentrations in plasma should be monitored in each patient. In addition, the genetic variants (F1, S, and A) of human AGP are known and could potentially affect drug-binding properties (30) . This may cause differences in pharmacokinetic behavior among patients. It will be important to determine whether UCN-01 can bind to every variant.

If bound UCN-01 was not released from human AGP, the plasma concentration of UCN-01 would need to be above the AGP levels to exhibit the desired pharmacological effects in patients. However, dissociation of UCN-01 from human AGP occurred, although this was much slower than in experimental animals (Tables 2Citation and 3Citation ; Fig. 2Citation ). This implies that most of the administered UCN-01 was in the AGP-bound form and was gradually released as "free" into the plasma compartment. In addition, biologically, it is necessary that several cell types have relatively prolonged exposure to show the antiproliferative activity of UCN-01 (3 , 31) . The implication of the pharmacological features of UCN-01, i.e., low free concentrations and long exposure in patients, should be further evaluated in clinical and nonclinical studies.

The conclusions of our study are as follows: (a) dissociation of UCN-01 from protein was very slow in human AGP and plasma; (b) distribution and elimination of UCN-01 was dramatically reduced after administration of equimolar human AGP; (c) hepatic extraction of UCN-01 and uptake into hepatocytes was completely inhibited by human AGP. These results clearly suggest that the unusually high plasma concentrations of UCN-01 in humans are due to the high affinity of UCN-01 for human AGP.


   ACKNOWLEDGMENTS
 
We thank Drs. Edward A. Sausville and William D. Figg, National Cancer Institute, for many constructive discussions. We also thank Akitoshi Hashimoto, Natsuko Sato, Rika Ito, and Yuko Takahashi for skillful technical assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, Drug Development Research Laboratories, Pharmaceutical Research Institute, Kyowa Hakko Kogyo Co., Ltd., 1188, Shimotogari, Nagaizumi-Cho, Sunto-Gun, Shizuoka 411-8731, Japan. Phone: 81-559-89-2021; Fax: 81-559-86-7430; E-mail; takashi.kuwabara{at}kyowa.co.jp Back

2 The abbreviations used are: UCN-01, 7-hydroxystaurosporine; AGP, {alpha}1-acid glycoprotein; HSA, human serum albumin; DCC, dextran-coated charcoal; HRP, horseradish peroxidase. Back

Received 10/30/98. Accepted 1/ 4/99.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Akinaga S., Gomi K., Morimoto M., Tamaoki T., Okabe M. Antitumor activity of UCN-01, a selective inhibitor of protein kinase C, in murine and human tumor models. Cancer Res., 51: 4888-4892, 1991.[Abstract/Free Full Text]
  2. Akinaga S., Nomura K., Gomi K., Okabe M. Enhancement of antitumor activity of mitomycin C in vitro and in vivo by UCN-01, a selective inhibitor of protein kinase C. Cancer Chemother. Pharmacol., 32: 183-189, 1993.[Medline]
  3. Seynaeve C. M., Stetler-Stevenson M., Sebers S., Kaur G., Sausville E. A., Worland P. J. Cell cycle arrest and inhibition by the protein kinase antagonist UCN-01 in human breast carcinoma cells. Cancer Res., 53: 2081-2086, 1993.[Abstract/Free Full Text]
  4. Takahashi I., Kobayashi E., Asano K., Yoshida M., Nakano H. UCN-01, a selective inhibitor of protein kinase C from Streptomyces. J. Antibiot., 40: 1782-1784, 1987.[Medline]
  5. Kawakami K., Futami H., Takahara J., Yamaguchi K. UCN-01, 7-hydroxyl-staurosporine, inhibits kinase activity of cyclin-dependent kinase and reduces the phosphorylation of the retinoblastoma susceptibility gene product in an A549 human lung carcinoma cell line. Biochem. Biophys. Res. Commun., 219: 778-783, 1996.[Medline]
  6. Akiyama T., Yoshida T., Tsujita T., Shimizu M., Mizukami T., Okabe M., Akinaga S. G1 phase accumulation induced by UCN-01 is associated with dephosphorylation of Rb and CDK2 proteins as well as induction of CDK inhibitor p21/Cip1/WAF1/Sdil in p53-mutated human epidermoid carcinoma A431 cells. Cancer Res., 57: 1495-1501, 1997.[Abstract/Free Full Text]
  7. Pollack I. F., Kawecki S., Lazo J. S. Blocking of glioma proliferation in vitro and in vivo and potentiating the effects of BCNU and cisplatin: UCN-01, a selective protein kinase inhibitor. J. Neurosurg., 84: 1024-1032, 1996.[Medline]
  8. Bunch R. T., Eastman A. Enhancement of cisplatin-induced cytotoxicity by 7-hydroxystaurosporine (UCN-01), a new G2-checkpoint inhibitor. Clin. Cancer Res., 2: 791-797, 1996.[Abstract]
  9. Fuse E., Tanii H., Kurata N., Kobayashi H., Shimada Y., Tamura T., Sasaki Y., Tanigawara Y., Lush R. D., Headlee D., Figg W. D., Arbuck S. G., Senderowicz M., Sausville E. A., Akinaga S., Kuwabara T., Kobayashi S. Unpredicted clinical pharmacology of UCN-01 caused by specific binding to human {alpha}1-acid glycoprotein. Cancer Res., 58: 3248-3253, 1998.[Abstract/Free Full Text]
  10. Fuse, E., Tanii, H., Kurata, N., Kuwabara, T., Kobayashi, S. Alteration of pharmacokinetics of a novel anticancer drug, UCN-01, in rats by human {alpha}1-acid glycoprotein. Proceedings of the 10th NCI-EORTC symposium on new drugs in cancer therapy, Annals of Oncology, Suppl 2, in press.
  11. Wagner J. G. Protein binding Wagner J. G. eds. . Pharmacokinetics for the Pharmaceutical Scientist, : 243-258, Technomic Publishing Lancaster 1993.
  12. Wilkinson G. R. Plasma and tissue binding considerations in drug disposition. Drug Metab. Rev., 14: 427-465, 1983.[Medline]
  13. Kremer J. M. H., Wilting J., Janssen L. H. M. Drug binding to human alpha-1-acid glycoprotein in health and disease. Pharmacol. Rev., 40: 1-47, 1988.[Medline]
  14. Holladay J. W., Dewey M. J., Yoo S. D. Steady-state kinetics of imipramine in transgenic mice with elevated serum AAG levels. Pharm. Res. (NY), 13: 1313-1316, 1996.[Medline]
  15. Yoo S. D., Holladay J. W., Fincher T. K., Baumann H., Dewey M. J. Altered disposition and antidepressant activity of imipramine in transgenic mice with elevated alpha-1-acid glycoprotein. J. Pharmacol. Exp. Ther., 276: 918-922, 1996.[Abstract/Free Full Text]
  16. Yasuhara M., Fujiwara J., Kitade S., Katayama H., Okumura K., Hori R. Effect of altered plasma protein binding on pharmacokinetics and pharmaco-dynamics of propranolol in rats after surgery: role of alpha-1-acid glycoprotein. J. Pharmacol. Exp. Ther., 235: 513-520, 1985.[Abstract/Free Full Text]
  17. Yuan J., Yang D. C., Birkmeier J., Stolzenbach J. Determination of protein binding by in vitro charcoal adsorption. J. Pharmacokinet. Biopharm., 23: 41-55, 1995.[Medline]
  18. Sablonnière B., Dallery N., Grillier I., Formstecher P., Dautrevaux M. Physicochemical parameters affecting the charcoal adsorption assay for quantitative retinoid-binding measurement. Anal. Biochem., 217: 110-118, 1994.[Medline]
  19. Kurata, N., Kuwabara, T., Tanii, H., Fuse, E., Akiyama, T., Akinaga, S., Kobayashi, H., Yamaguchi, K., and Kobayashi, S. Pharmacokinetics and pharmacodynamics of a novel protein kinase inhibitor, UCN-01. Cancer Chemother. Pharmacol., in press, 1999.
  20. Seglen P. O. Preparation of rat liver cells. III. Enzymatic requirement for tissue dispersion. Exp. Cell Res., 82: 391-398, 1973.[Medline]
  21. Yamazaki M., Suzuki H., Hanano M., Tokui T., Komai T., Sugiyama Y. Na+-independent multispecific anion transporter mediates active transport of pravastatin into rat liver. Am. J. Physiol., 264: G36-G44, 1993.[Abstract/Free Full Text]
  22. Kurata N., Kuramitsu T., Tanii H., Fuse E., Kuwabara T., Kobayashi H., Kobayashi S. Development of a highly sensitive high-performance liquid chromatographic method for measuring an anticancer drug, UCN-01 in human plasma or urine. J. Chromatogr. B, 708: 223-227, 1998.
  23. Gibaldi M., Perrier D. Noncompartment analysis based on statistical moment theory Gibaldi M. Perrier D. eds. . Pharmacokinetics, : 409-417, Marcel Dekker New York 1982.
  24. Davies B., Morris T. Physiological parameters in laboratory animals and humans. Pharm. Res., 10: 1093-1095, 1993.[Medline]
  25. Hassen A. M., Lam D., Chiba M., Tan E., Geng W., Pang K. S. Uptake of sulfate conjugates by isolated rat hepatocytes. Drug Metab. Dispos., 24: 792-797, 1996.[Abstract]
  26. Keyler D. E., Pentel P. R., Haughy D. B. Pharmacokinetics and toxicity of high-dose human {alpha}1-acid glycoprotein infusion in the rat. J. Pharm. Sci., 76: 101-104, 1987.[Medline]
  27. Parivar K., Tolentino L., Taylor G., Oie S. Elimination of non-reactive and weakly reactive human {alpha}1-acid glycoprotein after induction of the acute phase response in rats. J. Pharm. Pharmacol., 44: 447-450, 1992.[Medline]
  28. Brée F., Houin G., Barré J., Moretti J. L., Wirquin V., Tillement J. P. Pharmacokinetics of intravenously administered 125I-labelled human {alpha}1-acid glycoprotein. Clin. Pharmacokinet., 11: 336-342, 1986.[Medline]
  29. Weisman S., Goldsmith B., Winzler R., Lepper M. H. Turnover of plasma orosomucoid in man. J. Lab. Clin. Med., 57: 7-15, 1961.
  30. Hervé F., Duché J., d’Athis P., Marché C., Barré J., Tillement J. Binding of disopyramide, methadone, dipyridamole, chlorpromazine, lignocaine and progesterone to the two main genetic variants of human alpha-1-acid glycoprotein: evidence for drug-binding differences between the variants and for the presence of two separate drug-binding sites on alpha-1-acid glycoprotein. Pharmacogenetics, 6: 403-415, 1996.[Medline]
  31. Fuse E., Kobayashi T., Inaba M., Sugiyama Y. Prediction of the maximal tolerated dose (MTD) and therapeutic effect of anticancer drugs in humans: integration of pharmacokinetics with pharmacodynamics and toxicodynamics. Cancer Treat. Rev., 21: 133-157, 1995.[Medline]



This article has been cited by other articles:


Home page
 Cancer Res.Home page
M. McGahren-Murray, N. H.A. Terry, and K. Keyomarsi
The Differential Staurosporine-Mediated G1 Arrest in Normal versus Tumor Cells Is Dependent on the Retinoblastoma Protein
Cancer Res., October 1, 2006; 66(19): 9744 - 9753.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
P. N. Lara Jr, P. C. Mack, T. Synold, P. Frankel, J. Longmate, P. H. Gumerlock, J. H. Doroshow, and D. R. Gandara
The Cyclin-Dependent Kinase Inhibitor UCN-01 Plus Cisplatin in Advanced Solid Tumors: A California Cancer Consortium Phase I Pharmacokinetic and Molecular Correlative Trial
Clin. Cancer Res., June 15, 2005; 11(12): 4444 - 4450.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
D. Komander, G. Kular, M. Deak, D. R. Alessi, and D. M. F. van Aalten
Role of T-loop Phosphorylation in PDK1 Activation, Stability, and Substrate Binding
J. Biol. Chem., May 13, 2005; 280(19): 18797 - 18802.
[Abstract] [Full Text] [PDF]

Home page
 J Clin PharmacolHome page
E. Fuse, T. Kuwabara, A. Sparreboom, E. A. Sausville, and W. D. Figg
Review of UCN-01 Development: A Lesson in the Importance of Clinical Pharmacology
J. Clin. Pharmacol., April 1, 2005; 45(4): 394 - 403.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Katsuki, V. T. G. Chuang, K. Nishi, K. Kawahara, H. Nakayama, N. Yamaotsu, S. Hirono, and M. Otagiri
Use of Photoaffinity Labeling and Site-directed Mutagenesis for Identification of the Key Residue Responsible for Extraordinarily High Affinity Binding of UCN-01 in Human {alpha}1-Acid Glycoprotein
J. Biol. Chem., January 14, 2005; 280(2): 1384 - 1391.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
A. Sparreboom, H. Chen, M. R. Acharya, A. M. Senderowicz, R. A. Messmann, T. Kuwabara, D. J. Venzon, A. J. Murgo, D. Headlee, E. A. Sausville, et al.
Effects of {alpha}1-Acid Glycoprotein on the Clinical Pharmacokinetics of 7-Hydroxystaurosporine
Clin. Cancer Res., October 15, 2004; 10(20): 6840 - 6846.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
C. J. Strock, J.-I. Park, M. Rosen, C. Dionne, B. Ruggeri, S. Jones-Bolin, S. R. Denmeade, D. W. Ball, and B. D. Nelkin
CEP-701 and CEP-751 Inhibit Constitutively Activated RET Tyrosine Kinase Activity and Block Medullary Thyroid Carcinoma Cell Growth
Cancer Res., September 1, 2003; 63(17): 5559 - 5563.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
C. Yu, G. Krystal, L. Varticovksi, R. McKinstry, M. Rahmani, P. Dent, and S. Grant
Pharmacologic Mitogen-activated Protein/Extracellular Signal-regulated Kinase Kinase/Mitogen-activated Protein Kinase Inhibitors Interact Synergistically with STI571 to Induce Apoptosis in Bcr/Abl-expressing Human Leukemia Cells
Cancer Res., January 1, 2002; 62(1): 188 - 199.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
Y. Dai, C. Yu, V. Singh, L. Tang, Z. Wang, R. McInistry, P. Dent, and S. Grant
Pharmacological Inhibitors of the Mitogen-activated Protein Kinase (MAPK) Kinase/MAPK Cascade Interact Synergistically with UCN-01 to Induce Mitochondrial Dysfunction and Apoptosis in Human Leukemia Cells
Cancer Res., July 1, 2001; 61(13): 5106 - 5115.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
E. A. Kruger and W. D. Figg
Protein Binding Alters the Activity of Suramin, Carboxyamidotriazole, and UCN-01 in an ex Vivo Rat Aortic Ring Angiogenesis Assay
Clin. Cancer Res., July 1, 2001; 7(7): 1867 - 1872.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
Z. Shi, A. Azuma, D. Sampath, Y.-X. Li, P. Huang, and W. Plunkett
S-Phase Arrest by Nucleoside Analogues and Abrogation of Survival without Cell Cycle Progression by 7-Hydroxystaurosporine
Cancer Res., February 1, 2001; 61(3): 1065 - 1072.
[Abstract] [Full Text]

Home page
 Clin. Cancer Res.Home page
S. Harvey, R. Decker, Y. Dai, G. Schaefer, L. Tang, L. Kramer, P. Dent, and S. Grant
Interactions between 2-Fluoroadenine 9-{beta}-D-Arabinofuranoside and the Kinase Inhibitor UCN-01 in Human Leukemia and Lymphoma Cells
Clin. Cancer Res., February 1, 2001; 7(2): 320 - 330.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services