Specific Distribution of TOP-53 to the Lung and Lung-Localized Tumor Is Determined by Its Interaction with Phospholipids

4396 Vol. 6, 4396–4401, November 2000 Clinical Cancer Research

Specific Distribution of TOP-53 to the Lung and Lung-Localized Tumor Is Determined by its Interaction with Phospholipids

Masahiko Yoshida, Takashi Kobunai, (Fig. 1; Ref. 1). As a result, VP-162 and VM-26 have been Kumio Aoyagi, Hitoshi Saito, Teruhiro Utsugi, developed and introduced for the treatment of cancer patients. 1 However, their mechanism of action appeared to differ from that Konstanty Wierzba, Yuji Yamada of podophyllotoxin, though both were found to induce DNA Cancer Research Laboratory, Hanno Research Center, Taiho fragmentation that later was found to be the result of the inhi- Pharmaceutical Co. Ltd., Hanno-City, Saitama 357 [M. Y., T. K., K. A., T. U., K. W., Y. Y.], and Antimicrobials Research Laboratory, bition of DNA topoisomerase II (2). Taiho Pharmaceutical Co. Ltd., Tokushima 771-01 [H. S.], Japan Recently, VP-16 has been widely used in cancer chemotherapy of small cell lung cancer and malignant lymphoma (3–5), but its efficacy against a NSCLC is low because of ABSTRACT insufficient inhibition of DNA topoisomerase II and, among We have investigated the mechanism of TOP-53 dis- other factors, a poor tissue distribution, especially to the lung, tribution to the lung and lung-localized tumor. In contrast which may also be considered a disadvantage. To exert a desired to etoposide (VP-16), TOP-53 contains a basic aminoalkyl pharmacological effect, a sufficient amount of an active com- group that may predispose it to interact specifically with pound must be present in the “effector” tissue(s), which in the phospholipids, consequently leading to an increase of case of cancer disease involves an organ invaded by a primary drug accumulation in the tissues. Therefore, we have tumor and the other tissues being targeted during the process of studied its interaction with phospholipids in vitro using an its metastasizing. organic solvent-water partition system. TOP-53 appeared During the search for new and potent topoisomerase II- ؋ ؊2 inhibitors active against NSCLCs, we have focused on the ؍ to have the most potent binding affinity (Ka 563 10 ␮M) to phosphatidylserine (PhS), whereas VP-16 showed development of new chemical entities related to the structure of no interaction with any phospholipid tested. PhS content podophyllotoxin (6–8). Keeping in mind the penetration of a determined after HPLC separation varied among tested drug in the effector organ, the glucopyranoside-like function tissues; however, large quantities were found in normal was omitted in the drug design process and replaced with a basic lung and lung cancer tissues far exceeding those present aminoalkyl function to fortify an interaction of the compounds in the liver and kidney. The predicted tissue-to-plasma with the intracellular molecules, consequently leading to an increase of intracellular drug accumulation. partition coefficient values, estimated based on PhS con- As a result of such an approach, we have found a novel tent and its binding affinity, resembled those experimen- podophyllotoxin derivative, TOP-53, that is a non-glycoside tally determined. We concluded that tissue distribution of derivative with an aminoalkyl residue directly connected to TOP-53 is determined by PhS content in the tissues and position 4-␤ of podophyllotoxin through the C-C bond instead by binding affinity. As a result of specific accumulation in of the ether (O-C) bond. TOP-53 exerted significant antitumor the lung, TOP-53 appeared to show a strong antitumor activity against murine solid tumors and human cancer xe- against cancer (%171 ؍ activity (increase of life span nografts; its antitumor activity was especially well manifested in metastasizing to the lung, whereas VP-16 was less effec- lung metastatic tumors, supposedly as a result of its specific ؍ tive (increase of life span 78%). These results suggest accumulation in the lung tissue (9). On the basis of these results, that TOP-53 may have an advantage over VP-16 in the Phase I clinical study of TOP-53 has been initiated in Japan. treatment of lung cancers in patients. Recent trends in the development of clinically active compounds indicate the necessity of using experimental systems INTRODUCTION closely reflecting clinical conditions. For instance, the evaluation of an anticancer agent against pancreatic cancer requires a Several attempts have been made to develop anticancer model of a tumor localized in the pancreas. Furthermore, the agents by chemical modification of podophyllotoxin, a com- accumulation of a drug in the organ in question will determine pound known to interact with the microtubular system of the cell to some extent its final pharmacological effect. Very good

Received 1/14/00; revised 6/15/00; accepted 8/18/00. 2 The abbreviations used are: VP-16, etoposide; PhS, phosphatidyl ser- The costs of publication of this article were defrayed in part by the ine; PhG, phosphatidyl glycerol dipalmitol; PhI, phosphatidyl inositol; payment of page charges. This article must therefore be hereby marked PhC, phospatidyl choline dipalmitoyl; PhA, phosphatidic acid; PhE,

advertisement in accordance with 18 U.S.C. Section 1734 solely to phosphatidyl ethanolamine dipalmitoyl; Ka, association constant; Kp, indicate this fact. tissue-to-plasma partition coefficient; ILS, increase of life span; HPLC, 1 To whom requests for reprints should be addressed, at Cancer Re- high-performance liquid chromatography; AUC, area under the (con- search Laboratory, Taiho Pharmaceutical Co., Ltd., Cancer Research centration-time) curve; NSCLC, non-small cell lung cancer; LLC, Lewis Laboratory, 1-27 Misugidai, Hanno-City, Saitama 357-8527, Japan. lung carcinoma.

Experimental Metastasis of LLC. LLC cells, passaged

in C57BL/6 mice, were injected i.v. into male BDF1 mice as a cell suspension prepared in saline (3.0 ϫ 105 cells/0.2 ml/ mouse). The survival of drug-treated and control animals was observed, and the antitumor activity was evaluated as the percentage of ILS relative to control animals. The observation was terminated on day 60 and the live mice were considered long- term survivors in this experimental model. Control animals were able to survive approximately 21 days, and their death was caused by the extensive metastatic foci developed in the lung. Measurement of Tissue Distribution of TOP-53 and

Fig. 1 Chemical structures of TOP-53 and VP-16. VP-16. TOP-53 or VP-16 were injected i.v. into BDF1 mice bearing experimental lung metastasis at equal doses of 12 mg/ kg. The main tissues, such as normal lung tissue, lung metastatic nodules, liver, kidney, spleen, heart, and blood, were collected response of cancers localized in the lung to TOP-53 treatment raised the question of the mechanism(s) involved in its tissue at 0-min, 5-min, 15-min, 1-h, 2-h, 4-h, 8-h, 12-h, 24-h, and 48-h distribution, particularly in the lung. intervals after i.v. injection. Four animals were used per one There are few reports indicating the presence of drug- experimental point. The blood samples were centrifuged at 3000 ϳ macromolecule interactions determining tissue distribution of a rpm for 15 min to obtain plasma samples. An aliquot of 100– drug. Specific binding of DNA intercalators determines their 500 mg of a tissue sample was homogenized with 4 volumes of tissue affinity (10). The intercalators prefer DNA-rich tissues. acetonitrile, and then the organic phase was separated by cen- On the other hand, Vinca alkaloids having a high binding trifugation at 3000 rpm for 15 min. The resulting supernatant affinity to tubulin tend to accumulate in the tissues rich in was transferred to a conical test tube and evaporated to dryness tubulin (11, 12). Many basic drugs, such as imipramine, quini- under a stream of nitrogen. The dried residue was dissolved in ␮ dine, and propranolol, were shown to preferentially accumulate 200 l of a mobile phase (20 mM KH2PO4/methanol, 60:40) and in the lung because of specific interaction with phosphatidyl analyzed by the HPLC. The analysis was performed on octa- serine, a phospholipid widely distributed in that organ. decyl silane II column (4.6 ϫ 150 mm, 5 ␮m; GL Science, Inc., In this study, we tried to identify a mechanism of specific Tokyo Japan), with a mobile phase consisting of 20 mM

distribution of TOP-53 to the lung. Because TOP-53 interacts KH2PO4/methanol (60:40, v/v) at a flow rate of 1 ml/min, and with neither DNA nor with tubulin, but possesses a basic ami- the elution was monitored at the wavelength of 254 nm using a noalkyl chain, it prompted us: (a) to investigate its interaction SPD-6V UV-detector (Shimadzu, Kyoto, Japan). Tissue:drug with anionic phospholipids; (b) to identify the most specific concentrations were calculated by the external standard method. phospholipid; (c) to determine its content in the lung and other The plasma samples were extracted with 4 volumes of chloro- tissues; and (d) to correlate its tissue distribution in selected form, the organic phase being separated by centrifugation and organs and in tumor tissue. evaporated to dryness. The dried residue was dissolved in 200 ␮ l of mobile phase (20 mM KH2PO4/methanol, 60:40 containing MATERIALS AND METHODS 66 ␮M EDTA) and analyzed by reverse-phase HPLC as indi- Drugs and Reagents. TOP-53 was synthesized by Taiho cated above, except for detection, which was performed with an Pharmaceutical Co., Ltd. (Tokyo, Japan) as described previ- E-110 electrochemical detector (Eicom, Tokyo Japan). The ously (6). VP-16 was obtained from Nippon Kayaku (Tokyo, above extraction procedure was associated with a satisfactory Japan). Quinidine sulfate dihydrate of analytical grade was recovery rate of ϳ80–95%, depending on the type of tissue purchased from Wako (Osaka, Japan). The following standard used. phospholipids were obtained from Sigma (St. Louis, MO) and Pharmacokinetic Analysis. The blood plasma and tissue used without further purification: PhS (No. P-7769, bovine concentration time courses were subjected to a noncompartmen- brain), PhG (No. P-5650), PhI, (No. P-0639, soybean), saturated tal pharmacokinetic analysis using the WINNONLIN program, PhC, (No. P-6267), PhA (No. P-9511, egg yolk lecithin), and version 1.1 (Scientific Consulting, Inc.). PhE (No. P-3273). All other reagents were of an analytical Binding of TOP-53, VP-16, and Quinidine to Phospho- grade. For in vivo experiments, TOP-53 and VP-16 were dis- lipids. Binding of TOP-53, VP-16, and quinidine to phospho- solved in saline or in saline containing 6.5% Tween 80 and 3.5% lipids was determined by using an organic solvent-water parti- DMSO, respectively. tion system according to the slightly modified method of Animals. Specific pathogen-free male C57BL ϫ DBA/2 Nishiura et al. (13). Briefly, 2 ml of buffer solution [0.25 M

F1 (hereafter called BD2F1) mice were purchased from Japan sucrose, 0.05 M Tris-HCl buffer (pH 7.4)] containing a drug at SLC (Hamamatsu, Japan). Male BALB/c nu/nu mice were pur- concentrations ranging from 1–100 ␮M was vortexed with 2 ml chased from Clea (Tokyo, Japan). of the heptane solution containing an individual phospholipid (8 Tumor Cells. LLC was maintained as a serial passage in ␮g as an inorganic phosphorus/ml) in a screw-capped glass tube C57BL/6 mice, the inbred mouse strain of the tumor origin. at room temperature for 30 s. The mixture was then centrifuged Human lung cancer A549 was a gift from the Japanese Cancer at 2000 rpm for 10 min, and the separated aqueous phase was Research Resource Bank, Tokyo, Japan. analyzed by HPLC. The binding parameters of quinidine to an

individual phospholipid were calculated from Scatchard plots as described by Weber et al. (14) and by Sastrasinh et al. (15). Measurement of Tissue Phospholipids Concentrations in Mice. Phospholipids were extracted from tissue samples according to the method of Folch et al. (16). A tissue sample (0.15–0.5 g) was homogenized with 9 volumes of a chloroform/ methanol (2:1 v/v) mixture, and the homogenate was centrifuged at 2500 rpm for 10 min. The separated organic layer was washed out with 0.5 ml of saline. After centrifugation, the organic layer was transferred into a test tube and evaporated to dryness under a stream of nitrogen. The individual phospholipids (PhC, PhE, PhA, PhI, PhG, and PhS) were separated by the HPLC method. The HPLC fractionation was performed according to the method reported by Sasaki (17). The concentration of each individual phospholipid was determined after its conver- sion to inorganic phosphorus as described by Ames and Dubin (18), and the inorganic phosphorus was assayed by the procedure of Chen et al. (19). The final concentration of phospholipid was calculated by multiplying the concentration of inorganic phosphorus by 25, as reported in the literature (19, 20). Observed and Predicted Kp for TOP-53. The observed Kp values were calculated based on plasma and tissue concentrations of TOP-53 determined at 2 h after drug administration. The predicted Kp values were calculated based on the concentration of PhS in a tissue and the binding constant of TOP-53 to PhS. The calculations were performed according to Yata et al. (21), using the following formula: ϭ ϫ ͑ ͒ ϫ Ϫ Kp 14.3 log nK CPhS 8.09

where, nK is the binding capacity and CPhS is the tissue concentration of PhS. The observed Kp values of TOP-53 were initially correlated with PhS concentrations found in rat tissues, which were collected from the publications of Yata et al. (21) and Nishiura et al. (13). The correctness of using PhS concentration data derived from different species is justified by the fact that there is a small variation in lipid composition, particularly PhS concentration, among diverse animal species, as reported by Baxter et al. (22) Statistical Evaluation. Bonferroni t test was used for statistical evaluation. Fig. 2 Tissue distribution of TOP-53 (A) and VP-16 (B) in mice bearing LLC metastasizing to the lung. The experiment was performed ϫ 5 RESULTS 9 days after i.v. implantation of 3 10 LLC cells (fully developed tumor nodules in the lung). The animals were given TOP-53 or VP-16, Tissue Distribution of TOP-53 and VP-16 in Mice Bear- i.v., at the dose level of 12 mg/kg. ing LLC Lung Metastasis. TOP-53 rapidly disappeared from mouse plasma, and its concentration declined biphasically; however, its high concentrations were retained for a long period of time, especially in the lung and lung metastasis, spleen, and also VP-16 concentrations in the lung and lung metastasis were in the organs of elimination (kidney and liver; Fig. 2A). The much lower than those in the plasma (Fig. 2B). plasma and tissue concentrations of VP-16 rapidly declined with Binding of TOP-53, VP-16, and Quinidine to Phospho- time, and there was no significant retention in tested mouse lipids. TOP-53 showed the highest binding affinity to phos- ϭ tissues. The maximum plasma concentration of TOP-53 reached phatidylserine as manifested by the association constant of Ka Ϫ2 the level of 3.2 ␮g/ml, whereas those in the lung and lung 563 ϫ 10 ␮M, whereas the binding to other phospholipids was metastasis were 41.4 and 39.2 ␮g/g of tissue, respectively. The of low affinity. Also, quinidine showed a similar binding pat- respective AUCs were 2.9, 259.7, 292.4 ␮g/hr/ml or g of tissue. tern, with the highest affinity to PhS as expressed by the ϭ ϫ Ϫ2␮ On the other hand, the maximum concentration of VP-16 in association constant of Ka 242 10 M. The binding to plasma was 21.6 ␮g/ml, and those in the lung and lung metas- other phospholipids was of very low affinity. In contrast to tasis were 11.9 and 11.2 ␮g/g of tissue, respectively. The AUCs TOP-53, VP-16 exerted only a weak interaction with either were 11.4, 16.1, 27.8 ␮g/hr/ml or g of tissue, respectively. phospholipids tested (Table 1).

Fig. 3 The relationship between observed Kp values of TOP-53 in mice and tissue PhS concentrations reported by Yata et al. (21) and Nishiura et al. (13) in rats (A) and determined in the recent experiment in the main tissues of tumor-bearing mice (B).

Table 1 Binding affinity of TOP-53, quinidine, and VP-14 to Table 2 Concentration of phospholipid components in each tissue phospholipids and tumor ␮ Ϫ1 Association constant (Ka, M ) of test compound Lung Lung tumor Liver Kidney Phospholipid TOP-53 Quinidine VP-16 PhS 2.97 Ϯ 0.30a 3.14 Ϯ 0.24 0.65 Ϯ 0.14 1.58 Ϯ 0.16 Ϯ Ϯ b Ϯ ϫ Ϫ2 ϫ Ϫ2 ϫ Ϫ2 PhG 0.88 0.19 1.06 0.15 ND 0.48 0.08 PhS 563 10 242 10 3.37 10 PhI 0.23 Ϯ 0.13 0.46 Ϯ 0.07 2.52 Ϯ 0.17 1.67 Ϯ 0.25 PhA 14.7 ϫ 10Ϫ2 10.4 ϫ 10Ϫ2 39.2 ϫ 10Ϫ2 Ϫ2 Ϫ2 Ϫ2 PhA ND ND ND ND PhG 11.2 ϫ 10 14.5 ϫ 10 15.8 ϫ 10 Ϯ Ϯ Ϯ Ϯ Ϫ2 Ϫ2 Ϫ2 PhE 4.96 0.46 4.87 0.34 6.44 0.62 7.57 0.93 PhI 7.95 ϫ 10 48.7 ϫ 10 10.0 ϫ 10 Ϯ Ϯ Ϯ Ϯ ϫ Ϫ2 ϫ Ϫ2 ϫ Ϫ2 PhC 6.86 0.49 7.03 0.60 10.55 0.47 7.86 1.05 PhE 10.7 10 2.47 10 23.1 10 Total 15.91 Ϯ 0.99 16.56 Ϯ 1.03 20.17 Ϯ 0.46 19.16 Ϯ 0.60 PhC 1.69 ϫ 10Ϫ2 0.24 ϫ 10Ϫ2 6.02 ϫ 10Ϫ2 a Each value represents the mean Ϯ SD of three mice. The value of phospholipid concentration in each tissue and tumor (mg/g of tissue). b ND, not detected. Tissue Phospholipid Concentration in Mice. Tissue composition of phospholipids and their respective concentrations are shown in Table 2. The liver and kidney contained number of mouse tissues (Fig. 3B). However, no correlation has higher concentrations of total phospholipids than did lung and been found between total tissue phospholipid concentrations and lung cancer tissue. However, there is a tissue-related distribution tissue distribution of TOP-53 (data not shown). Therefore, PhS pattern, namely, the lung tissue contains the increased amount of may be considered as a determinant of tissue distribution of PhS (2.97 Ϯ 0.30 mg/g of tissue) when compared with those of TOP-53. As a matter of fact, the additional line of evidence is liver and kidney (0.65 Ϯ 0.62 and 1.58 Ϯ 0.16 mg/g of tissue, derived from the binding properties of TOP-53 to PhS. On the respectively), whereas the content of PhI shows an opposite basis of the binding parameters of TOP-53 to PhS, the Kp value trend. The concentrations of PhS in the lung and lung cancer are was calculated (predicted Kp value) and then correlated with the comparable, indicating that both tissues contain a similar bind- observed Kp values in mice, resulting in significant correlation ing capacity for the drugs specifically interacting with PhS. (r ϭ 0.81) as shown in Fig. 4. Relationship between Kp Values and Tissue Phospho- Antitumor Activity of TOP-53 against Metastasizing lipids Concentration. On the basis of the assumption that the Lung Cancer. The increased survival time of tumor-bearing concentrations of phospholipids are similar in different animal animals after treatment either with TOP-53 or VP-16 is shown species at the initial stage, we have compared the Kp values of in Fig. 5. The treatment with TOP-53 at its optimum dosing TOP-53 with the reported PhS concentrations for rats (21, 13). schedule resulted in significant prolongation of the survival of As shown in Fig. 3A), there was significant correlation between tumor-bearing animals, their survival having been increased by the Kp values of TOP-53 observed in mice and PhS tissue 171%. On the other hand, the treatment with an optimal dose of concentrations derived from rats. Also, significant relationship VP-16 was associated with a significantly lower survival, only (r ϭ 0.81) was found between the observed Kp value of TOP-53 78%. Moreover, four of six mice treated with TOP-53 were able and the tissue concentration of PhS measured in a limited to survive due to the curative effect of TOP-53. No long-term

Fig. 4 The correlation between observed and predicted Kp values of TOP-53. The predicted Kp values were calculated based on the binding Fig. 5 Antitumor activity of TOP-53 and VP-16 against LLC metas- parameters of TOP-53 to PhS and its tissue content in tumor-bearing tasizing to the lung. TOP-53 and VP-16 were administered i.v. starting mice. from day 7 at the optimal schedules of q7dϫ3 and qdϫ5, respectively. The percentage of ILS (%ILS) was calculated as [(MST of the treated group Ϫ MST of the control group)/MST of the control group] ϫ 100, where MST ϭ mean survival time. #, P Ͻ 0.01; TOP-53- versus surviving animals were observed in the case of treatment with VP-16-treated group determined by Bonferroni t test. VP-16.

DISCUSSION of other drugs (21, 24). On the basis of the binding affinity of TOP-53 was shown to exert excellent antitumor activity TOP-53 to PhS, and knowing the tissue concentration of PhS, against lung cancers, especially against NSCLC, exceeding the we were able to predict the Kp (21) that appeared to correlate antitumor activity of VP-16 (9). The cell-killing kinetics of well with the observed values. This finding indicated that TOP-53 were both concentration and time-dependent, therefore TOP-53 is predominantly bound to the intracellular PhS. the AUC determines its antitumor effect (23). TOP-53 was Taking into account tissue distribution of endogenous PhS, found to be active against lung-localized cancers because of its the lung tissue was shown to be rich in PhS (21, 22) in pronounced accumulation in that organ as revealed by the ex- comparison with other organs. This finding may explain a periments on its biodisposition. In this study, we have tried to significant accumulation of TOP-53 in the lung. On the other clarify the mechanism(s) of specific tissue distribution of TOP- hand, PhS concentration was also found to be high in the 53, assuming the presence of specific intracellular molecules tumor originating from the lung, indicating that TOP-53 can that have an ability to bind TOP-53 and to concentrate in it. penetrate well not only lung tissue but also tumor tissue Tissue distribution of several drugs depends on the pres- localized in the lung, which is important for antitumor activ- ence of specific binding molecules present in an organ; e.g., ity of TOP-53. In our experiments we have confirmed that DNA intercalating agents distribute well to the tissues rich in TOP-53 was specifically distributed to the lung tissue, a DNA content (10), whereas Vinca alkaloids tend to accumulate well-penetrated tumor mass, resulting in good antitumor ac- in the tissues rich in tubulin protein (11). The drugs containing tivity. In the other experiment (data not shown), TOP-53 basic amino groups, but not having the above-mentioned prop- showed a weak antitumor activity against cancers localized in erties, also can be well distributed even in tissues of a nontarget the liver, that organ containing lower concentration of PhS organ (13, 21, 24). Yata et al. (21) have reported that such drugs and thus showing a lower accumulation of TOP-53, indicat- can be specifically bound to PhS, a phospholipid present at ing that specific tissue distribution may determine the activ- different concentrations in the tissues that may serve as an ity of a compound. Also it is important to keep in mind that determinant of tissue distribution. Because TOP-53 also con- the liver is an eliminating organ, thus additionally lowering tains a basic side chain that may predispose it to a specific the distribution of a drug and its antitumor activity. However, interaction with the phospholipids, it was of great interest to one may also expect some tumors to contain a large amount investigate whether TOP-53 may specifically interact with phos- of PhS; therefore, they may accumulate TOP-53 specifically pholipids, and if so, what kind of phospholipid is responsible for despite its lower accumulation tendency in normal tissue of that specific interaction. the invaded organ. We did not examine the concentration of We have found that TOP-53 was able to interact with PhS in the tumors of other origin, but this point seems to be phospholipids, and the most specific interaction was observed important for additional consideration. with PhS, a phospholipid reported to determine the distribution The distribution pattern of VP-16 and, in consequence, its

biological properties, differed significantly from those of TOP- ses of 1-␤-alkyl-1-desoxypodophyllotoxin derivatives and biological 53. There was no interaction between phospholipids and VP-16, activity. Chem. Pharm. Bull. (Tokyo), 41: 907–912, 1993. therefore its tissue penetration, accumulation, and retention 9. Utsugi, T., Shibata, J., Sugimoto, Y., Aoyagi, K., Wierzba, K., Kobunai, were significantly lower than those of TOP-53. The lack of T., Terada, T., Oh-hara, T., Tsuruo, T., and Yamada, Y. Antitumor activity of a novel podophyllotoxin derivative (TOP-53) against lung cancer and specific intracellular binding sites for VP-16 determined its low lung metastatic cancer. Cancer Res., 56: 2809–2814, 1996. cellular uptake as revealed in the intracellular uptake studies 10. Terasaki, T., Iga, T., Sugiyama, Y., Sawada, Y., and Hanano, M. (data not shown). Tissue distribution of VP-16 is characterized Nuclear binding as a determinant of tissue distribution of adriamycin, by low Kp values, thus contrasting the properties of TOP-53. daunomycin, adriamycinol, daunorubicinol and actinomycin D. J. Phar- Moreover, the retention of VP-16 in mouse tissues is far shorter macobio-Dyn., 7: 269–277, 1984. than that of TOP-53. One may expect that these properties, to 11. Wierzba, K., Sugiyama, Y., Okudaira, K., Iga, T., and Haruno, M. some extent, are responsible for weaker antitumor activity of Tubulin as a major determinant of tissue distribution of vincristine. VP-16, especially against lung-localized cancers. J. Pharm. Sci., 76: 872–875, 1987. Because TOP-53 is an AUC-dependent drug, its activity 12. Wierzba, K., Sugiyama, Y., Iga, T., and Haruno, M. Kinetic study on the mechanism of tissue distribution of vinblastine. J. Pharmacobio- depends on both concentration and exposure time (25–28). 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Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2000 American Association for Cancer Research. Specific Distribution of TOP-53 to the Lung and Lung-Localized Tumor Is Determined by its Interaction with Phospholipids

Masahiko Yoshida, Takashi Kobunai, Kumio Aoyagi, et al.

Clin Cancer Res 2000;6:4396-4401.

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