flow cytometer.
4.7 Conclusions and summary
The calcein-AM efflux assay represents a convenient method to characterize the MDR1 phenotype with respect to the activity of expressed p-gp in cells. The method is useful for the investigation of p-gp modulators. However, the assay is not able to distinguish between transported substrates of p-gp and compounds with inhibitory properties. The prediction of the p-gp interaction is important for the screening of drug candidates. Furthermore, the characterization of the kinetics of the p-gp transport process has to be investigated for the development of potent p-gp inhibitors. This is especially true for agents with CNS activity. Further efforts have to be made to elucidate p-gp binding sites and the complex allosteric interactions (Martin et al. 2000) as well as to develop new assays for differentiation between inhibitors, transported substrates and non-transported substances.
Today it is difficult to perform this classification with only one assay (Schwab et al. 2003) and the characteristics of substances in different assays must be considered to obtain information about the site of interaction for the tested compound (Polli et al. 2001).
Computer based models are also used as computational filters to select drugs lacking the interaction with p-gp (Penzotti et al. 2002). However, the calcein-AM efflux assay is most suitable for primary screening of drug candidates and should be confirmed by transport assays and in vivo experiments.
92 Establishment and application of a calcein-AM efflux assay
In vivo models of human lung cancer brain metastases 93
Chapter 5
In vivo models of human lung cancer brain metastases
5.1 Introduction
Multidrug resistance (MDR) is one of the major obstacles to successful tumor therapy.
The overexpression of P-glycoprotein 170 (p-gp) is the best characterized phenotype of MDR, although this is not the only mechanism involved (Gottesman and Pastan 1993).
P-gp is not only expressed in chemoresistant tumor cells but also within normal cells of the human body, particularly in liver, kidney and the blood-brain-barrier (BBB). The localization of p-gp at the BBB is one of the drawbacks in the chemotherapy of primary and secondary brain tumors because of the protective function of p-gp that results in efflux transport of chemotherapeutic agents (Schinkel et al. 1995). A further problem in the treatment of brain metastases is the primary chemoresistance of tumors preferably metastasizing to the brain (Sawaya and Bindal 1995). However, in case of secondary brain tumors chemotherapy is preferred to locally acting methods such as radiation therapy and surgery because the whole brain is treated. In addition, the cytostatic drugs can act on the primary tumor as well as on metastases in organs other than the brain.
Secondary brain tumors represent approximately 40 % of intracranial neoplasms. In the United States annually approximately 170,000 cancer patients develop brain metastases.
In adults, primary tumors that often metastasize to brain are lung cancer (50 %), breast
94 In vivo models of human lung cancer brain metastases cancer (15 - 20 %), unknown primary neoplasms (10 - 15 %), melanoma (10 %), renal and colon cancer (both 5 %) (Kaye and Laws 1995). Brain metastases from unknown primary tumors are most likely from lung cancer (72 %). Furthermore, breast, renal and colon cancer produce single metastases, whereas metastases from lung cancer and melanoma are normally multiple (Wen and Loeffler 1999, Khosla 2004). Brain metastases contribute to cancer morbidity and mortality, and are responsible for the neurologic breakdown in cancer. As mentioned before the majority of secondary brain tumors originate from lung cancer which is characterized by a high fatality rate due to the advanced stage of disease at time of diagnosis and its aggressive biologic nature (Tuveson and Jacks 1999). Hence, need for new approaches to the treatment of lung tumor brain metastases is obvious.
Possible new treatment strategies, e.g. the combination of anticancer drugs with ABC efflux transporter modulators, have to be examined first regarding their effectiveness in preclinical studies. For that purpose animal models are required that simulate the situation in the human body. At present, various mouse models are established including mice, which develop lung tumors spontaneously or after chemical induction, as well as transgenic mice, which express viral and cellular oncogenes (Tuveson and Jacks 1999).
However, none of these models account for the development of brain metastases.
At our department an orthotopic tumor model, using nude mice bearing intracerebral human gliobalstoma, was developed to study the therapy of primary brain tumors (Fellner et al. 2002). For that purpose, human glioblastoma cells were injected intracerebrally into nude mice according to Altenschöpfer (1998). The anticancer agent paclitaxel was co-administered intravenously with valspodar, an inhibitor of the efflux transporter p-gp, to increase the paclitaxel concentration in the brain. The paclitaxel dose had to be reduced from 3 to 2 mg paclitaxel per kg body weight within the experiment due to the body weight reduction that is an indication of the toxicity of the combination with valspodar.
In this study a significant increase in the brain paclitaxel concentration and a reduction of the tumor volume by 90 % was achieved with the combination treatment. Since the glioblastoma cells expressed no p-gp, the observed effect could only be explained by the p-gp inhibition at the BBB.
The aforementioned model was very useful to demonstrate that the combination stra-tegy is a promising approach to the treatment of primary brain tumors. In principle, this
5.2 Material and methods 95