MEN4901/T-0128, a New Camptothecin Derivative–Carboxymethyldextran Conjugate, Has Potent Antitumor Activities in a Panel of Human Tumor Xenografts in Nude Mice Free

Camptothecin (CPT) is a potent, antitumor agent acting mainly through inhibition of topoisomerase I during the S-phase of the cell cycle (1). Although CPT shows potent antitumor activity in vitro and in vivo, its low water solubility and untoward toxicity have severely restricted its clinical applications (2). The design of tailor-made synthetic polymer–based and polysaccharide-based CPT conjugates provides a synthetic approach that can overcome some of the problems (3–8). Linking CPT to a high molecular weight anionic polymer enhances solubility and improves distribution to the tumor through enhanced permeability and retention (9) and the constitution of the peptide spacer has a major impact on sustained release of the active drug and biological activity (4–8, 10, 11). MEN4901/T-0128 is composed of the novel CPT analogue 10-(3′-aminopropyloxy)-7-ethyl-(20S)-CPT (T-2513) and carboxymethyldextran, which are linked via a triglycine spacer (Fig. 1) and shows enhanced antitumor activity relative to T-2513 in our previous studies (5). MEN4901/T-0128 was highly resistant to hydrolysis both in plasma and in buffer at physiologic pH, whereas cysteine proteases were able to cleave MEN4901/T-0128 to a mixture of T-2513 and monoglycyl-T-2513 (Gly-T-2513) in a time-dependent manner in vitro (12).

In this paper, we present the results of the evaluation of the in vivo antitumor activity of MEN4901/T-0128 against a panel of seven human tumors xenografted in nude mice and the pharmacokinetics of MEN4901/T-0128 in tumor-bearing mice in comparison with T-2513 and irinotecan (CPT-11; ref. 13).

Chemicals. MEN4901/T-0128, T-2513, Gly-T-2513, SN-38 (7-ethyl-10-hydroxy-CPT), and 7-ethyl-10-aminopentyloxy-CPT were synthesized at Medicinal Chemistry Research Laboratories, Tanabe Seiyaku Co., Ltd. (Saitama, Japan). A detailed characterization of MEN4901/T-0128 has been published previously (5). CPT-11 was purchased from Daiichi Pharmaceutical Co., Ltd. (Tokyo, Japan). All other chemicals and reagents used in this study were of analytic grades. 7-Ethyl-10-aminopentyloxy-CPT was used as the internal standard for the high-performance liquid chromatography analysis; its stock solution was prepared in DMSO at a concentration of 500 μg/mL and diluted with a 1:4 mixture of acetonitrile and 35 mmol/L ammonium formate buffer (pH 3.0) to yield the internal standard solution at a final concentration of 50 ng/mL.

Dose Formulation and Administration. MEN4901/T-0128 was formulated in physiologic saline at concentrations of 8/3, 8/9, and 8/27 mg/mL (expressed as T-2513) immediately before use and administered i.v. weekly for 4 weeks at its maximum tolerated dose (MTD) of 80 mg/kg (expressed as T-2513), 1/3 MTD, 1/9 MTD, or 1/27 MTD at a dose volume of 0.3 or 0.1 mL per 10 g body weight. The MTD was defined as the maximum dose that produced animal body weight loss of <20% without causing drug-related lethality. T-2513 and CPT-11 were formulated in physiologic saline immediately before use and administered i.v. weekly for 4 weeks at their MTD of 60 mg/kg or 1/3 MTD.

For pharmacokinetic studies in tumor-bearing mice, each drug was formulated in physiologic saline and administered as a single i.v. injection at a dose of 10 or 40 mg/kg (expressed as T-2513) for MEN4901/T-0128, 10 or 40 mg/kg for T-2513, and 40 mg/kg for CPT-11.

Animals. Male and female BALB/cA Jcl nude mice were obtained from CLEA Japan, Inc. (Tokyo, Japan) and bred under specific pathogen-free conditions in small vinyl isolators at constant temperature (23 ± 2°C) and humidity (50 ± 5%) using autoclaved water, bedding and cages, and γ-ray-irradiated food (CL-2; CLEA Japan). Male BALB/c nude mice, 7 weeks of age, were purchased from Nippon SLC (Hamamatsu, Japan) and acclimated for a period of at least 1 week before tumor implantation. The Animal Ethics Committee of Tanabe Seiyaku Co., Ltd. reviewed and approved all aspects of the protocol concerning animal welfare.

Tumor Lines. The human tumor lines used for in vivo evaluation were gastric cancer H-81 (squamous cell carcinoma, poorly differentiated), colon cancer H-110 (adenocarcinoma, well differentiated), lung cancer Mqnu-1 (squamous cell carcinoma, poorly differentiated), lung cancer H-74 (papillary adenocarcinoma), esophageal cancer H-204 (squamous cell carcinoma, moderately differentiated), liver cancer H-181 (hepatocellular carcinoma), and pancreatic cancer H-48 (tubular adenocarcinoma, well differentiated). These tumor lines have been characterized previously (14–16). Each tumor was maintained by serial s.c. passages in nude mice of the same sex as the donor patient; five lines (H-81, Mqnu-1, H-204, H-181, and H-48) were transplanted into male mice and the remaining two into female mice. In addition, human gastric carcinoma St-4, used in the pharmacokinetic studies, was kindly provided by Dr. T. Kubota (Department of Surgery, School of Medicine, Keio University, Tokyo, Japan; ref. 17) and has been maintained by serial s.c. passages in male nude mice. Its chemosensitivity to MEN4901/T-0128, T-2513, and CPT-11 has been reported in detail (5).

Assay of In vivo Antitumor Activity of MEN4901/T-0128. The methods of chemotherapy and data analysis are similar to those reported previously (14). Briefly, tumor tissues harvested from s.c. growth in nude mice were inspected and any capsular or necrotic parts were removed. Tumor tissues were then cut into cubic fragments as uniformly as possible. Cubes of tumor fragments, 2 to 3 mm in diameter, were transplanted with a trocar under the dorsal skin of 4- to 6-week-old BALB/c AJcl nude mice on day 0, and allowed to grow for 8 to 18 days. Each tumor was measured twice a week with a sliding caliper in three dimensions, maximum diameter (L), diameter at right angles to the length (W), and thickness (D) at the 0.5 mm accuracy level. The tumor volume was estimated by the following formula: V = (L × W × D) / 2. Animals with estimated mean tumor volumes of 80 to 200 mm³ were allocated to treatment and control groups of seven animals each with almost equal mean tumor sizes and standard errors. Animals in the treatment groups received i.v. administration of drug weekly for 4 weeks and untreated control animals did not receive any treatment. The tumor volume and body weight of each animal were recorded twice a week, and all animals were sacrificed 4 weeks after the initiation of treatment. The tumor was removed from each mouse and weighed. The tumor growth inhibition rate (IR) was calculated using the formula: IR (%) = (1 − T / C) × 100, where T and C are the mean tumor weight of treatment and control groups, respectively. The drug was assessed as “effective” (18, 19) and “markedly effective” (19) when the IR was no less than 58% and 80%, respectively. “Shrinking effect” refers to the mean estimated tumor volume at the end of the experiment being smaller than that at the initiation of treatment.

Statistical Analysis. Data are expressed as the means ± SE. Tumor weights of the treatment and control groups were evaluated by two-tailed Student's t test. The difference was considered statistically “significant” when P < 0.05, whereas difference levels with P < 0.01 and P < 0.001 were considered “highly significant” and “very highly significant,” respectively.

Histologic Assessment. The tumor samples collected as described above were fixed in 10% buffered neutral formalin solution, embedded in paraffin, and cut to 5-μm-thick sections. Sections were stained with H&E, examined microscopically, and assessed according to the criteria proposed by Shimosato et al. (20).

Determination of Pharmacokinetics of MEN4901/T-0128 in Nude Mice. Pharmacokinetic evaluations were done in male BALB/c nude mice bearing human gastric cancer St-4 in two experiments. In the first experiment, one group (24 mice) received MEN4901/T-0128 at a dose equivalent to 10 mg of T-2513/kg and the other (15 mice) received T-2513 at a dose of 10 mg/kg. The average tumor burden per mouse on the day of drug administration was 250 ± 75 mm³ (n = 39). In the second experiment, one group (30 mice) received MEN4901/T-0128 at a dose equivalent to 40 mg of T-2513/kg and two groups (15 mice per group) received T-2513 or CPT-11 at a dose of 40 mg/kg. The average tumor burden per mouse on the day of drug administration was 248 ± 67 mm³ (n = 60).

The blood samples were collected into a heparinized syringe by exsanguination via abdominal aorta under deep diethyl ether anesthesia at predetermined times after administration (three animals per time point) and immediately centrifuged (10,000 rpm) at 4°C for 10 minutes. The resulting plasma samples (50 μL) were diluted with 150 μL 35 mmol/L ammonium formate buffer (pH 3.0), vortexed with 200 μL of the internal standard solution, centrifuged (5,000 × g) at 4°C for 5 minutes, and the supernatant was filtered through a membrane filter (M_r 5,000, Ultrafree-MC, UFC3 LCC 00, Nihon Millipore K.K., Tokyo, Japan). The tumor, liver, spleen, lung, and kidney were removed quickly (stored at −20°C until high-performance liquid chromatography analysis), homogenized in 4 volumes of 1/15 mol/L phosphate buffer (pH 7.4) in an ice bath, and centrifuged (3,500 × g) at 4°C. The supernatants (200 μL) were vortexed with 200 μL of the internal standard solution for 10 seconds and centrifuged (5,000 × g) at 4°C for 5 minutes.

Aliquots (100 μL) of plasma samples or tissue homogenates were hydrolyzed in 400 μL HCl, 6 mol/L, at 100°C for 4 hours and the resulting CPT analogues were assayed using a procedure similar to that described previously (5, 12). Briefly, aliquots (20 μL) of the plasma or tumor/tissue samples described above were chromatographed on a C18 reversed-phase column (Inertsil ODS-2; ID 4.6 × 150 mm; GL Sciences, Tokyo, Japan) using a gradient elution consisted of acetonitrile and 35 mmol/L ammonium formate buffer (pH 3.0). The high-performance liquid chromatography apparatus (LC-6A; Shimadzu Seisakusho, Kyoto, Japan) was linked to a fluorescence detector (RF-535; Shimadzu Seisakusho), which was set at an excitation wavelength of 360 nm and an emission wavelength of 420 nm for T-2513, Gly-T-2513, and CPT-11, and at 380 nm and 540 nm for SN-38, respectively. The area under the concentration-time curve (AUC) values were calculated from the mean plasma and tumor/tissue concentrations using the i.v. one-compartment model and linear trapezoidal rule, respectively.

Antitumor Effect. The antitumor activity of MEN4901/T-0128 was evaluated against a panel of seven human tumors xenografted in nude mice including gastric (H-81), colon (H-110), lung (Mqnu-1, H-74), esophageal (H-204), liver (H-181), and pancreatic (H-48) cancer lines, in comparison with T-2513 and CPT-11 using a q7d × 4 schedule. As shown in Table 1, MEN4901/T-0128 was “markedly effective” in each of these tumor models and the tumor growth was significantly suppressed in animals receiving MEN4901/T-0128 treatment. “Shrinking effects” were observed in the models of Mqnu-1 and H-48 (Fig. 2A and C, respectively) at the doses as low as 1/9 of its MTD (80 mg/kg, q7d × 4). “Shrinking effects” were also seen in the models of H-81, H-110, and H-74 at 1/3 of its MTD, and in the models of H-204 and H-181 at its MTD. The IRs of MEN4901/T-0128 treatment at 1/3 of its MTD were 97.5%, 98.5%, 99.7%, 90.7%, 78.8%, 81.2%, and 98.8% in the models of H-81, H-110, Mqnu-1, H-74, H-204, H-181, and H-48, respectively. On the other hand, treatment at MTD of T-2513 (60 mg/kg, q7d × 4) and CPT-11 (60 mg/kg, q7d × 4) were “inactive” in these models except for Mqnu-1 (IR of 79.7% and 85.9%, respectively).

Body Weight Changes after MEN4901/T-0128 Treatment. No animals in any of the treatment groups died for a period of up to 4 weeks after initiation of treatment. Figure 2B and D shows body weight changes of tumor-bearing animals in the models of Mqnu-1 and H-48, respectively. In the Mqnu-1 model, a 17.9% reduction in body weight at nadir was observed in animals receiving MEN4901/T-0128 at its MTD 10 days after initiation of treatment and host recovery was not seen until 4 weeks after initiation of treatment. A 9.9% loss of body weight was seen in animals receiving MEN4901/T-0128 at 1/3 of its MTD during the early stage of the treatment (7 days after initiation of treatment) but weight gain occurred in association with tumor shrinkage, which resulted in good physical conditions 4 weeks after initiation of the treatment. As shown in Table 1, a >10% reduction in body weight at nadir was also observed in animals receiving MEN4901/T-0128 at MTD or 1/3 of its MTD in the H-81, H-110, H-204, and H-181 models; however, the loss of body weight in these models may have been partly due to cachexia induced by the tumors (the body weight losses of untreated controls ranged 7.3-10.8% at nadir).

Histologic Examination. The histologic effects of MEN4901/T-0128, T-2513, and CPT-11 on the tumor sections were evaluated; each sample was assessed using the classification of Shimosato et al. (20) and results are summarized in Table 1. Among the seven models, tumor tissues were almost completely destroyed in three models (H-110, Mqnu-1, and H-74), most of which reached grade IV when treated with MEN4901/T-0128 at its MTD. Mqnu-1 is more sensitive to chemotherapy and was cured histologically even at 1/9 of its MTD. At 1/27 of its MTD, grade IIB (severe destruction of tumor structure with few viable tumor cells) and grade IIA were observed; these results were favorably compared with those obtained in the tumors from animals treated with CPT-11 at doses of 60 mg/kg and much superior to those treated with CPT-11 at doses of 20 mg/kg. Representative tumors are shown in Fig. 3. In the H-48 model, at least grade IIB was observed in the tumors from animals treated with MEN4901/T-0128 at 1/9 of its MTD. Less damage was seen in three other models when treated with MEN4901/T-0128 at 1/3 of its MTD.

Pharmacokinetics of MEN4901/T-0128, T-2513, and CPT-11. Pharmacokinetic evaluations were done in nude mice bearing human gastric cancer St-4 and results are summarized in Table 2. I.v. administration of T-2513 resulted in a rapid clearance from the systemic circulation. A comparison of the plasma pharmacokinetics of the two groups receiving T-2513 or CPT-11 at a dose of 40 mg/kg did not disclose any significant difference in their parameters (Table 2). In contrast, MEN4901/T-0128 showed a significantly longer circulation time (Table 2). Following i.v. administration at doses equivalent to 40 and 10 mg of T-2513/kg, plasma levels of MEN4901/T-0128 reached C_max of 658 ± 28 and 144 ± 2.1 μg equivalent of T-2513 per milliliter 5 minutes after administration and decreased slowly with terminal t_1/2 of 30.2 and 14.5 hours and AUC values of 23,900 and 3,550 μg equivalent of T-2513 h/mL, respectively. The kinetics of MEN4901/T-0128 at the doses equivalent to 40 and 10 mg of T-2513/kg, respectively, is characterized by low plasma clearance (CL = 0.0017 and 0.0028 L/kg/h) and low volume of distribution (V_d = 0.073 and 0.059 L/kg), indicating that MEN4901/T-0128 was mainly present in the systemic circulation and slowly eliminated from the animal body. The plasma T-2513 levels remained very low at any time points with an AUC value of 10.6 μg·h/mL and plasma Gly-T-2513 levels were below the quantification limit of 0.005 μg/mL after administration of MEN4901/T-0128 at the dose equivalent to 40 mg of T-2513/kg.

As shown in Fig. 4 and Table 2, i.v. administration of MEN4901/T-0128 at a dose equivalent to 40 mg of T-2513/kg resulted in high and sustained levels of MEN4901/T-0128 and T-2513 in the tumor (Gly-T-2513 was not detected at any time points during the period of 14 days). The tumor MEN4901/T-0128 levels reached a C_max of 31.4 μg equivalent of T-2513/g tissue 6 hours after administration and decreased with a terminal t_1/2 of 49.0 hours and an AUC value of 3,030 μg equivalent of T-2513·h/g tissue, whereas the tumor T-2513 levels reached a C_max of 7.38 μg/g tissue 3 days after administration and decreased with a terminal t_1/2 of 37.3 hours and an AUC value of 848 μg·h/g tissue. This tumor T-2513 AUC value was ∼17 and 1,000 times greater than the tumor AUC values of T-2513 (49.2 μg·h/g tissue) and SN-38 (0.66 μg·h/g tissue) achieved in animals receiving T-2513 and CPT-11, respectively, at a dose of 40 mg/kg (Table 2).

Body distribution analysis showed a high MEN4901/T-0128 accumulation in the liver and spleen (Table 2), both of which have an active reticuloendothelial system (21), suggesting that the reticuloendothelial system may have a sizable impact on the distribution of MEN4901/T-0128.

The present study investigated the antitumor activity of MEN4901/T-0128 in a panel of seven human tumor xenografts. As summarized in Table 1, MEN4901/T-0128 given as a q7d × 4 schedule showed a marked antitumor activity in each of these tumor models, indicating that MEN4901/T-0128 produces tumor-shrinking effects against a panel of human tumor xenografts including those refractory to CPT-11 and T-2513. In addition, histologic analysis of the tumors taken from these animals indicates that MEN4901/T-0128 is superior to CPT-11 and T-2513, and that cure can be expected with MEN4901/T-0128 in four of seven models. The IRs for CPT-11 (60 mg/kg, q7d × 4) in the Mqnu-1 and H-181 models reported here are in agreement with those reported earlier for CPT-11 (60 mg/kg, q4d × 4; refs. 15, 16).

Body distribution analysis in tumor-bearing mice showed that MEN4901/T-0128 improves the pharmacokinetic profile of T-2513 (Table 2). The prolonged T-2513 accumulation in the liver and spleen not only suggests that the reticuloendothelial system may play a major role in the distribution of MEN4901/T-0128, but also supports the idea that lysosomal cathepsin B is responsible for the release of T-2513 from the macromolecular conjugate (12) because cathepsin B activity is reported to be very high in these tissues (22). The reason for the absence of Gly-T-2513 in these tissues remains unclear; one possible explanation is that Gly-T-2513 released from MEN4901/T-0128 may have been converted to T-2513 rapidly in vivo. Besides the liver and spleen, tumor tissues showed a greater T-2513 accumulation than other normal tissues, which is most likely responsible for the superior antitumor activity of MEN4901/T-0128 over T-2513 or CPT-11.

In summary, MEN4901/T-0128 consistently produced regression/suppression of human tumor xenografts that are highly refractory to CPT-11. Improved pharmacokinetic properties of MEN4901/T-0128 in terms of plasma half-life, tissue distribution, and tumor T-2513 accumulation may account for the effectiveness. Because the efficacies of some drugs in this human cancer-nude mouse panel correlated well with their clinical outcomes in patients with the same type of cancers (14, 16, 23, 24), the findings provide direct support that MEN4901/T-0128 is more efficacious than CPT-11 and is an excellent candidate for clinical trials for the treatment of solid tumors. Although MEN4901/T-0128 embodies many of the essential properties indicated for clinical success, it is not peerless as other polymer- and polysaccharide-based CPT conjugates have shown comparable pharmacokinetic properties (3, 4, 6–8, 10, 11).