Hennie M.M. Schlüper

New highly lipophilic camptothecin BNP1350 is an effective drug in experimental human cancer.

BNP1350, 7‐[(2‐trimethylsilyl)ethyl]‐20(S)‐camptothecin, is a novel semi‐synthetic, highly lipophilic, silicon‐containing camptothecin and an inhibitor of topoisomerase I. It has been supercomputer engineered for superior oral bioavailability, superior lactone stability, broad anti‐tumor activity, increased potency and insensitivity to Pgp/MRP/LRP drug resistance. We determined the efficacy of BNP1350 in experimental human colon cancer and compared its anti‐tumor effects with those of CPT‐11/SN‐38. We also determined a possible influence of Pgp, MRP and LRP on the efficacy of BNP1350. The in vitro anti‐proliferative capacity of the compounds using various exposure times was assessed in five colon cancer cell lines and indicated that BNP1350 was similarly effective or slightly more potent than SN‐38. Four cell lines of other origin with sublines expressing Pgp, MRP and/or LRP showed that BNP1350 was significantly more effective than SN‐38 (p < 0.05) and that the activity of BNP1350 was not reduced in multidrug‐resistant cells. For in vivo experiments, BNP1350 was given 1.0 mg/kg i.p. or 1.5 mg/kg p.o. daily × 5 and CPT‐11 20 mg/kg i.p. daily × 5 being equitoxic schedules in nude mice bearing s.c. human tumor xenografts. The schedules were studied in colon cancer xenografts COLO320, COLO205 or WiDr as well as in two Pgp‐positive xenografts 2780AD and BRO/mdr1.1 and the parental Pgp‐negative A2780 ovarian cancer xenografts and BRO melanoma xenografts. Growth inhibition of >50% was obtained for BNP1350 given i.p. in six out of the seven xenografts studied. BNP1350 was similarly effective when given i.p. or p.o. CPT‐11 was as effective as BNP1350, except in BRO and BRO/mdr1.1 xenografts. Pgp expression in xenografts in vivo confirmed that there was no negative influence on the efficacy of BNP1350. In conclusion, BNP1350 shows a broad spectrum of activity in experimental human tumors and is a suitable candidate for oral treatment of cancer. Int. J. Cancer 88:260–266, 2000.

Vascular endothelial growth factor-165 overexpression stimulates angiogenesis and induces cyst formation and macrophage infiltration in human ovarian cancer xenografts

Vascular endothelial growth factor (VEGF) is suggested to be an important regulator of angiogenesis in ovarian cancer. We have evaluated the effects of VEGF overexpression on the histology and growth rate of human ovarian cancer xenografts. OVCAR-3 human ovarian cancer cells were stably transfected with an expression vector encoding the 165-amino acid isoform of VEGF. As subcutaneous xenografts, moderately and highly VEGF(165)-overexpressing OVCAR-3 cells formed tumors with large cysts. Immunohistochemistry demonstrated an increase in the number of CD31-positive microvessels, some of which were larger in diameter than those in the parental tumors, as well as extensive vascular rimming around the cysts. Weakly VEGF(165)-overexpressing tumors also contained an increased number of CD31-positive microvessels and occasional vascular rimming, but cysts were not present. Immunohistochemistry further revealed the presence of monocytes and macrophages in both parental and VEGF(165)-overexpressing xenografts. Interestingly, the number of monocytes/macrophages was greatly increased in moderately and highly VEGF(165)-overexpressing xenografts and large areas populated with monocytes/macrophages were detected within the tumor stroma. Although the higher number of CD31-positive cells would suggest a better vascularization pattern in VEGF(165)-overexpressing xenografts, tumor growth rates were not increased when compared with that of parental xenografts. These data provide functional evidence for a role of VEGF(165) in cyst formation and monocyte/macrophage infiltration.

Novel camptothecin derivative BNP1350 in experimental human ovarian cancer: determination of efficacy and possible mechanisms of resistance.

The novel camptothecin derivative BNP1350 (7‐[2‐trimethylsilyl)ethyl]‐20(S)‐camptothecin), also known as Karenitecin, has been developed for superior oral bioavailability and increased lactone stability. In our study, we describe the antiproliferative effects of BNP1350, SN‐38 and topotecan in 4 human ovarian cancer cell lines. BNP1350 was found to be slightly more potent than SN‐38 (p<0.01) and was considerably more potent than topotecan (p<0.01). We extended these studies to well‐established human ovarian cancer xenografts in which we compared the growth inhibition induced by BNP1350 with that of topotecan given in equitoxic schedules. The growth inhibition in all 3 xenografts induced by BNP1350 was ≥75%, which was significantly better than that resulting from topotecan (p<0.05). We then selected BNP1350‐resistant variants of the A2780 human ovarian cancer cell line, 2780K4 (resistance factor: 41) and 2780K32 (resistance factor: 90), to analyze possible resistance mechanisms. These variants exhibited cross‐resistance against all camptothecins tested. In comparison with 2780K4 cells, 2780K32 cells were relatively more resistant against SN‐38, topotecan, DX‐8951f and BNP1350. In addition, 2780K32 cells were highly cross‐resistant against mitoxantrone. In both 2780K4 and 2780K32, the amount of topoisomerase I was not changed but the catalytic activity was reduced. Furthermore, 2780K32 cells clearly overexpressed the breast cancer resistance protein (BCRP), as demonstrated for both the gene and the protein. In contrast to topotecan, BNP1350 proved not to be a good substrate for BCRP. Overall, we conclude that BNP1350 offers advantages over topotecan expressed by high efficacy in experimental human ovarian cancer and poor affinity for BCRP.

Comparison of the monoclonal antibodies 17-1A and 323/A3: the influence of the affinity on tumour uptake and efficacy of radioimmunotherapy in human ovarian cancer xenografts

The low-affinity monoclonal antibody (MAb) chimeric 17-1A(c-17-1A) and the high-affinity MAb mouse 323/A3 (m-323/A3) were used to study the effect of the MAb affinity on the tumour uptake and efficacy of radioimmunotherapy in nude mice bearing subcutaneously the human ovarian cancer xenografts FMa, OVCAR-3 and Ov.Pe. Both MAbs are directed against the same pancarcinoma glycoprotein. In vitro, the number of binding sites on tumour cells at 4 degrees C was similar for both MAbs, but m-323/A3 had an approximately 5-fold higher affinity (1.3-3.0x10(9) M-1) than c-17-1A (3.0-5.4x10(8) M-1). This difference in affinity was more extreme at 37 degrees C, when no binding of c-17-1A could be observed. MAb m-323/A3 completely blocked binding of c-17-1A to tumour cells, whereas the reverse was not observed. Immunohistochemistry showed a similar but more intense staining pattern of m-323/A3 in human ovarian cancer xenografts than of c-17-1A. In vivo, the blood clearance in non-tumour-bearing nude mice was similar for both MAbs with terminal half-lives of 71.4 h for m-323/A3 and 62.7 h for c-17-1A. MAb m-323/A3 targeted better to tumour tissue, but was more heterogeneously distributed than c-17-1A. The cumulative absorbed radiation dose delivered by m-323/A3 to tumour tissue was 2.5- to 4.7-fold higher than that delivered by c-17-1A. When mice were treated with equivalent radiation doses of 131(I)m-323/A3 and 131(I)c-17-1A, based on a correction for the immunoreactivity of the radiolabelled MAbs, m-323/A3 induced a better growth inhibition in two of the three xenografts. When the radiation doses were adjusted to obtain a similar amount of radiation in the tumour c-17-1A was more effective in tumour growth inhibition in all three xenografts.

Induction of breast cancer resistance protein by the camptothecin derivative DX-8951f is associated with minor reduction of antitumour activity

DX-8951f (exatecan mesylate), a new water-soluble derivative of camptothecin, is currently being evaluated in phase II clinical trials. Resistance may be acquired when treating cancer patients with DX-8951f. Therefore, we selected a subline of the human ovarian cancer cell line A2780 for resistance against DX-8951f to investigate possible mechanisms of resistance. This DX-8951f-resistant subline, designated 2780DX8 (resistance factor=9.3), displayed a typical cross-resistance pattern including compounds, such as topotecan (resistance factor =34), SN-38 (resistance factor =47), mitoxantrone (resistance factor =59) and doxorubicin (resistance factor =2.9), which have previously been associated with the expression of breast cancer resistance protein. 2780DX8 cells did not show changes in the topoisomerase I gene, in topoisomerase I protein levels or catalytic activity. Overexpression of breast cancer resistance protein could be detected, both at the mRNA and protein level, while staining for Pgp, MRP1, or LRP was negative. GF120918, an inhibitor of breast cancer resistance protein, was able to reverse the DX-8951f-induced resistance in 2780DX8 cells. In vivo experiments in well-established 2780DX8 human tumour xenografts demonstrated that the growth inhibition induced by CPT-11 was more affected by breast cancer resistance protein expression than that of DX-8951f. These data indicate for the first time that DX-8951f is able to induce breast cancer resistance protein as a mechanism of resistance. Breast cancer resistance protein, however, results in only minor reduction of antitumour activity of DX-8951f which is an advantage over topotecan and CPT-11/SN-38.

Comparison of the biodistribution and the efficacy of monoclonal antibody 323 A3 labeled with either 131I or 186Re in human ovarian cancer xenografts

PURPOSE The radionuclide 186Re has favorable physical characteristics for use in radioimmunotherapy, including the emission of beta-particles of a high-energy and a low-abundance of gamma-emission. The gamma-emission, in particular, is ideal for tumor imaging and poses less hazards to the patient and the medical personnel when compared with the gamma-emission of the widely used radionuclide 131I. In the present study, we determined whether 186Re-labeled monoclonal antibody 323/A3 may be better suited for the treatment of ovarian cancer than 131I-323/A3. METHODS AND MATERIALS We compared the biodistribution and the efficacy of 186Re- and 131I-labeled 323/A3 in nude mice bearing s.c. the human ovarian cancer xenografts FMa, OVCAR-3 and Ov.Pe. 186Re was conjugated to 323/A3 with the use of the S-benzoylmercaptoacetyltriglycine (S-benzoyl-MAG3) chelate. RESULTS A molar ratio of Re-MAG3:323/A3 of 3:1 did not affect the integrity and the pharmacokinetic behaviour of the MAb. The tumor uptake and the retention of 186Re- and 131I-labeled 323/A3 were comparable, but the cumulative absorbed radiation dose in the tumor delivered by 186Re-323/A3 was 1.3-fold higher than that of 131I-323/A3. When mice were treated with equivalent radionuclide doses, the tumor growth inhibition induced by 186Re-323/A3 was similar or slightly better when compared with the efficacy of 131I-323/A3. When mice were treated with radionuclide doses that were adjusted to obtain equal cumulative absorbed radiation doses in the tumor for both conjugates, 131I-323/A3 was slightly more effective in the inhibition of the growth of FMa and OVCAR-3 xenografts. CONCLUSIONS The favorable physical characteristics of 186Re as well as its efficacy when conjugated to a MAb indicate 186Re as an attractive radionuclide in radioimmunotherapy of ovarian cancer patients.

Addition of cisplatin improves efficacy of 131I-labeled monoclonal antibody 323/A3 in experimental human ovarian cancer

UNLABELLED This study was conducted to determine whether the cytotoxic agent cisplatin (CDDP), also known as a radiosensitizer, can improve the efficacy of the 131I-labeled monoclonal antibody (MAb) 323/A3 in the treatment of experimental human ovarian cancer. METHODS AND MATERIALS Nude mice bearing well-established subcutaneous FMa, OVCAR-3, or Ov.Pe xenografts were injected twice with a 2-week interval either with a bolus of CDDP, 131I-323/A3, or with a combination of both modalities. CDDP was injected at various timepoints when combined with 131I-323/A3. The efficacy of the treatment was expressed as the specific growth delay (SGD). The growth inhibitory effect of the combination was characterized to detect additivity or synergism, using the mean relative tumor volumes at 2, 4, and 6 weeks after the last injection as endpoints. RESULTS The efficacy of 131I-323/A3 was superior to that of the maximum tolerated dose (MTD) of CDDP (6 mg/kg) in all three xenografts. The addition of CDDP to 131I-323/A3 could increase the growth inhibition in the CDDP-responsive FMa and OVCAR-3 xenografts, but not in Ov.Pe xenografts. Although this improved antitumor effect was additive rather than synergistic, the combination was more effective when compared with that of the MTD of each of the modalities alone. The time interval between the administration of a bolus injection of CDDP and 131I-323/A3 had no effect on the extent of growth inhibition in OVCAR-3 xenografts. CONCLUSION The addition of CDDP to 131I-323/A3 resulted in an additive inhibitory effect on the growth of CDDP-responsive xenografts. As the combination of radioimmunotherapy and CDDP was more effective in the inhibition of the tumor growth when compared with that of the MTD of each of the modalities alone, this treatment may therefore be considered of use in patients with ovarian cancer responsive to CDDP.

New analogues of camptothecins: Activity and resistance

Semisynthetic analogues of camptothecin have been developed with the aim of better efficacy and reduced toxicity when compared to topotecan and CPT-11. Among them are DX-8951f [(1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline10,13(9H,15H)-dione methanesulfonate dihydrate]1 and BNP1350 (7-[(2-trimethylsilyl)ethyl]-20(S)camptothecin),2 which are currently in phase I–II clinical trials. DX-8951f is a water-soluble derivative, while BNP1350 is a highly lipophilic compound. Preclinical activity data indicate that both drugs have the potential for a greater therapeutic index than camptothecins in clinical use. We have studied DX8951f and BNP1350 for their efficacy in a variety of human tumor xenografts. In addition, we analyzed possible mechanisms of resistance in drug-selected variants of the human ovarian cancer cell line A2780.

Doxorubicin compared with related compounds in a nude mouse model for human ovarian cancer

Eight human ovarian cancer lines grown in nude mice were used to compare the activity of doxorubicin, epirubicin, mitoxantrone and menogaril. The tumour lines were different in histological subtype, tumour doubling time and sensitivity to doxorubicin. The compounds were administered intravenously at the maximum tolerated dose twice with one week in between when tumours measured 50-150 mm3. Growth inhibition greater than 50% was obtained for doxorubicin in 8/8, for epirubicin in 4/8, for mitoxantrone in 5/8 and for menogaril in 2/8 tumour lines. In MRI-H-207, doxorubicin was the only drug able to induce complete remission. Compared with doxorubicin, mitoxantrone and menogaril were given in proportionally higher doses than those administered to patients, but did not result in superior antitumour activity.

Determination of tumor-related factors of influence on the uptake of the monoclonal antibody 323/A3 in experimental human ovarian cancer.

The epithelial glycoprotein 40 (EGP40) is an important target in the clinic for radioimmunolocalization and monoclonal antibody (MAb)‐mediated therapy of cancer. We determined which tumor‐related factors (including antigen distribution and density, vascularization and perfusion) were involved in the uptake of the anti‐EGP40 MAb 323/A3 in 4 different human ovarian cancer xenografts grown s.c. in nude mice. The reactivity pattern of 323/A3 in all xenografts in vitro was similar and showed a strong and homogeneous distribution of the EGP40 antigen. FMa xenografts, however, showed the highest uptake of 323/A3 in vivo, which was 5.5‐, 6.2‐ and 10.0‐fold higher than that in OVCAR‐3, Ov.Pe and Ov.Sh xenografts, respectively. FMa xenografts contained 2.1‐ to 3.5‐fold more antigen per gram protein when compared with the antigen content of the other xenografts. FMa and Ov.Sh xenografts demonstrated a better vascularization pattern, whereas Ov.Pe and OVCAR‐3 xenografts were moderately to poorly vascularized. FMa xenografts were also better perfused, as was shown by a 1.6‐ to 1.8‐fold higher uptake of the 99mTc‐labeled blood flow marker hexamethylpropyleneamine oxime (HMPAO). The tumor uptake of the non‐specific MAb E48 was 2.2‐ to 11.2‐fold lower when compared with that of 323/A3, but the sequence of uptake was similar (FMa > OVCAR‐3 = Ov.Pe > Ov.Sh), indicating the lowest extravasation of MAbs in Ov.Sh xenograft tissue. Since both the antigen content and the perfusion appeared to be important factors of influence on the tumor uptake of 323/A3, attempts were made to manipulate these determinants to improve the tumor uptake. Neither γ‐interferon nor 5‐fluorouracil were able to increase EGP40 expression in human ovarian cancer cells in vitro. Treatment of tumor‐bearing mice with the calcium‐antagonist flunarizine did not result in an improved perfusion, although a slight increase in the initial tumor uptake of 323/A3 was observed in Ov.Sh‐bearing mice. Our results illustrate the relative contribution of various tumorrelated factors that determine the usefulness of a MAb for imaging and therapy of cancer. Int. J. Cancer 71:237–245, 1997.