Thilo Spruß

Transport of paclitaxel (Taxol) across the blood-brain barrier in vitro and in vivo.

Paclitaxel concentrations in the brain are very low after intravenous injection. Since paclitaxel is excluded from some tumors by p-glycoprotein (p-gp), the same mechanism may prevent entry into the brain. In vitro, paclitaxel transport was examined in capillaries from rat brains by confocal microscopy using BODIPY Fl-paclitaxel. Western blots and immunostaining demonstrated apical expression of p-gp in isolated endothelial cells, vessels, and tissue. Secretion of BODIPY Fl-paclitaxel into capillary lumens was specific and energy-dependent. Steady state luminal fluorescence significantly exceeded cellular fluorescence and was reduced by NaCN, paclitaxel, and SDZ PSC-833 (valspodar), a p-gp blocker. Leukotriene C(4) (LTC(4)), an Mrp2-substrate, had no effect. Luminal accumulation of NBDL-cyclosporin, a p-gp substrate, was inhibited by paclitaxel. In vivo, paclitaxel levels in the brain, liver, kidney, and plasma of nude mice were determined after intravenous injection. Co-administration of valspodar led to increased paclitaxel levels in brains compared to monotherapy. Therapeutic relevance was proven for nude mice with implanted intracerebral human U-118 MG glioblastoma. Whereas paclitaxel did not affect tumor volume, co-administration of paclitaxel (intravenous) and PSC833 (peroral) reduced tumor volume by 90%. Thus, p-gp is an important obstacle preventing paclitaxel entry into the brain, and inhibition of this transporter allows the drug to reach sensitive tumors within the CNS.

Computerized Determination of Growth Kinetic Curves and Doubling Times from Cells in Microculture

In this paper we describe the microcomputer-aided determination of cell proliferation kinetics and doubling times utilizing a crystal violet assay and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay in microtitration plates. The analysis of spectrophotometric data provides the doubling times at any time of incubation. Plots of doubling time versus time of incubation give reproducible information on the exact duration of the logarithmic growth phase. This method is applicable to anchorage-dependent as well as anchorage-independent cells when colorimetric or fluorometric data are accessible.

Hyaluronidase enhances the activity of Adriamycin in breast cancer models in vitro and in vivo

SummaryThe effect of hyaluronidase and a combination of hyaluronidase with Adriamycin was investigated on several breast cancer models in vitro and in vivo. In vitro enzyme treatment (using concentrations up to 80 000 IU/l) of murine (MXT−, MXT±, and MXT+) and human (MCF-7, ZR-75-1 and T-47-D) breast cancer cell lines did not inhibit tumour cell proliferation (measured by a kinetic crystal violet assay) in either case. Although highdose hyaluronidase (1.2×106 IU/kg) was ineffective, when administered peritumourally to the MXT M3.2 mammary carcinoma of the B6D2F1 mouse, it is remarkable that five “megadoses” were excellently tolerated. However, the antineoplastic activity of Adriamycin against the oestrogen-receptor-positive variant of the MXT tumour was significantly enhanced by combination with concentrations of hyaluronidase that were inactive per se, both in vitro and in vivo. Interestingly, the enhancement of the in vivo antitumour activity was not compromised by toxic side-effects.

Establishment and characterization of the follicular thyroid carcinoma cell line ML-1

Abstract.The present study focuses on the establishment and characterization of a new follicular thyroid carcinoma cell line. The human cell line ML-1 was derived from a dedifferentiated follicular thyroid carcinoma relapse, which progressed despite preceding surgery followed by two radioiodine therapies. More than 90% of the cells of this line express thyroglobulin, chondroitin sulfate, and vimentin antigens, but only about 70% show cytokeratin filaments and a negative surface charge density such as human erythrocytes. More importantly, cells of this line are able to take up iodine and/or glucose both in vitro and in vivo and to secrete thyroglobulin, chondroitin sulfate, and fibronectin into the interstitial space. In addition, triiodothyronine is released constitutively into culture supernatants. Moreover, it is also suitable for xenotransplantation studies because it is tumorigenic in NMRI nude mice in vivo. The cell line forms tumors with follicular structures when transplanted to nude mice. Due to these unique features the ML-1 cell line can be considered as a very suitable test model for pharmacological and cell biological studies. Since chemicals may interfere with the production of thyroid hormones, this cell line represents also a tool for toxicological investigations.

Hyaluronidase significantly enhances the efficacy of regional vinblastine chemotherapy of malignant melanoma

The regional chemotherapy of the human malignant melanomas (SK-MEL-2, -3, -5, -24) implanted in NMRInu/nu mice with a combination of the hyaluronic-acid-cleaving enzyme hyaluronidase (HYase) and vinblastine is a very effective therapeutic procedure. In three out of four melanoma models (SK-MEL-2,-3, -5) the weekly peritumoral administration of high-dose HYase (100 000 IU/kg) 4 h prior to the injection of 0.3 mg/kg vinblastine in the vicinity of the tumor (seven weekly therapeutic cycles) caused marked antitumor effects, while HYase and vinblastine were inactive when given alone. The pretreatment with HYase, which is well tolerated by the test animals, prevented local inflammation reactions commonly seen after subcutaneous vinblastine administration. Tumor growth and metastatic behavior of the melanomas used were neither increased nor reduced by HYase after peritumoral administration without subsequent vinblastine injection. The curative activity of the regional chemotherapy with HYase/vinblastine could be demonstrated on the SK-Mel-3 melanoma. After an observation time of 18 weeks tumor cells could no longer be detected in the subcutaneous region of the former lesion. Only macrophages, which had abundantly incorporated melanin, gave evidence of previously growing tumors. In contrast to the controls, no metastases could be observed in the axillary lymph nodes of the test animals.

Efficacy of BCNU and paclitaxel loaded subcutaneous implants in the interstitial chemotherapy of U-87 MG human glioblastoma xenografts.

Nude mice were challenged with human U-87 MG glioblastoma tumors to assess the efficacy of different cytostatics and different application protocols. While the intraperitoneal application of BCNU solutions (3 times 20 mg BCNU/kg) had no effect on tumor growth, the application of polymer matrices made of a physical mixture of poly(1,3-bis[carboxyphenoxpropane]-co-sebacic acid) 20:80 with poly(D,L-lactic-co-glycolic acid) loaded with 0.25 mg BCNU, slowed down the growth of tumors significantly. When the animals were treated with implants carrying 0.25 mg BCNU they responded to the treatment whether the tumor had been inoculated recently (9 days ago) or whether it was fully established (after 20 days). After its sensitivity was proven, the xenograft model was used to further investigate the efficacy of anticancer drugs and some treatment regimens using polymer implants. Thus the tumor model allowed to discriminate between the efficacy of different doses of BCNU. Only implants loaded with 0.75 or 1 mg of BCNU led to a substantial suppression of tumor growth over approximately 2 months. While BCNU was only able to suppress the growth of the tumor, the combination of BCNU with paclitaxel led to a complete remission in some animals. These preliminary results suggest that combinations of cytostatics might improve local chemotherapy of malignant glioma substantially. Based on our data it will be worthwhile to investigate implants that release drugs such as BCNU and paclitaxel closer. Amongst other factors we will try to elucidate the effect of repetitive doses of drugs using programmable implants.

Hyaluronidase pretreatment produces selective melphalan enrichment in malignant melanoma implanted in nude mice

Abstract Preclinical and clinical observations suggest that the administration of hyaluronidase (Hyase) shortly before that of chemotherapy increases the access and, thus, the effectiveness of aniticancer drugs in tumors. To examine this hypothesis as well as the selectivity of such a therapeutic approach potentially beneficial in isolated limb perfusion, the Hyase-induced distribution of melphalan was measured in tumor-bearing nude mice with respect to the mode of drug administration using RP-18 ion-pair high-performance liquid chromatography (HPLC) with fluorimetric detection. Melphalan alone (50 μmol/kg) or a combination of melphalan (50 μmol/kg) and Hyase (100,000 IU/kg) was injected either i.p. or s.c. in the vicinity of the tumors. The s.c. melphalan injection caused a 4-fold rise in melphalan concentration (59 μM) in the tumors as compared with i.p. application (15 μM). Only minor effects were observed with respect to the route of melphalan application on its distribution in other tissues (ca. 13 μM in plasma, 15 μM in muscle, 30 μM in the liver, 26 μM in the kidney, and 21 μM in the testicle). Irrespective of the route of Hyase coadministration, the enzyme increased the concentration of i.p. injected melphalan in all tissues to ca. 20 μM in the tumor, 15 μM in plasma, 27 μM in muscle, 40 μM in the liver, 29 μM in the kidney, and 28 μM in the testicle. In contrast, s.c. injected melphalan was selectively accumulated by the tumors after both s.c. and i.p. Hyase administration (462 and 388 μM, respectively). Melphalan enrichment in the tumors was higher (16- to 32-fold higher than in the other tissues) after i.p. administration of Hyase since, in contrast to s.c. injection of the enzyme, its i.p. administration caused a decrease in the concentration of the cytostatic in all other tissues as compared with the s.c. administration of melphalan alone.

Programmable biodegradable implants

Pulsatile release implants were developed that release substances up to 58 days post implantation. With a cylindrical size of 2 mm diameter and 1.8 mm height the matrices can carry as much as 1 mg of drug and allow even for intracranial implantation into a rodent model. The matrices are made of materials that have been used for parenteral applications in humans before such as surface eroding polyanhydrides and bulk eroding poly(D,L-lactic acid) or poly(D,L-lactic acid-co-glycolic acid). The onset of drug release is controlled by the degradation of bulk eroding polymers which are known to exhibit a certain stability over a defined period of time and which start eroding after they reach a critical degree of degradation. The time of drug release onset was found to depend on the molecular weight and the chemical state of the carboxylic acid end of the polymer chain. For testing the onset of release in vivo a nude mouse model was developed where the release of Evans blue could be observed visually after subcutaneous application. By combining individual matrices with different release onset, a therapeutic system can be composed that releases drugs after implantation at predetermined time points in a preprogrammed way. Potential applications for such matrices is vaccination and local tumor therapy.

Tumor-inhibiting [1,2-bis(fluorophenyl)ethylenediamine]platinum(II) complexes. V. Synthesis and evaluation of enantiomeric [1,2-bis-(4-fluorophenyl)ethylenediamine]dichloroplatinum(II) complexes

SummaryThe enantiomeric [1,2-bis(4-fluorophenyl)ethylenediamine]dichloroplatinum(II) complexes were synthesized and tested on the hormone-sensitive human MCF7 breast cancer cell line and on the P388 leukemia of the mouse. They showed a strong and comparable activity on both tumor models.

Dunning R3327-G prostate carcinoma of the rat: an appropriate model for drug evaluation

SummaryModels for testing new drugs for the treatment of hormone-dependent prostate cancer are restricted to a few in vivo rat tumour lines; most of them originating from the Dunning R3327 adenocarcinoma. The original tumour and the R3327-H line grow rather slowly leading to a long duration of therapeutic experiments. The R3327-G subline can be transplanted as a cell suspension or tumour fragments, which give rise to fast and rather homogeneously growing androgen-dependent tumours. Their growth is strongly inhibited by castration or administration of diethylstilbestrol. Experiments were terminated 5 weeks after transplantation. Best results were obtained when treatment was started 1 day after transplantation. Histological sections showed therapy-dependent changes in the microarchitecture of these prostate tumours. The direct inhibitory effect of antiandrogens on prostate tumours was demonstrated when castrated, testosterone-propionate-supplemented animals were used. The short duration of experiments and reproducible responses to standard therapies are the advantages of this tumour model.