Combination of valspodar with cytostatic drugs in vitro . 106

First, the efficacy of valspodar co-administered to certain cytostatic drugs was investigated in vitro. MDR resistant Kb-V1 cells (Kb-V1/VBL, see chapter 4.4) were incubated with paclitaxel or vinblastine in combination with various concentrations of valspodar. The cytostatic drugs as well as the modulator valspodar were given at concentrations that did not affect the growth of the Kb-V1/VBL cells when the compounds were administered as single drugs. The results of the chemosensitivity assay are illustrated in Fig. 5.9.

Figure 5.9: Chemosensitivity of p-gp expressing MDR resistant Kb-V1/VBL cells against vin-blastine (a) and paclitaxel (b) in combination with different concentrations of val-spodar. The chemosensitivity of the cells is increased by valval-spodar.

The co-incubation of paclitaxel or vinblastine with valspodar led to a transient

inhi-5.3 Results 107 bition of the Kb-V1/VBL cell proliferation due to the blockade of the p-gp activity in a valspodar concentration dependent manner. A valspodar concentration of 1 µM even fully restored the paclitaxel sensitivity of the Kb-V1/VBL cells as indicated by the com-plete growth inhibition. Using vinblastine as cytostatic drug the cell proliferation was at least transiently inhibited. The co-application of valspodar led to enhanced toxicity of the co-administered cytostatic drug. This proves the ability of valspodar to inhibit p-gp activity.

5.3.2.2 Co-application of valspodar with vinblastine for the treatment of NCI-H460 lung cancer in the brains of nude mice

NCI-H460 human lung cancer cells were intracerebrally implanted into nude mice. This procedure resulted in aggressively growing intracerebral neoplasms (see chapter 3 and 5.3.1.3). Every five days nude mice received 0.8 mg/kg of vinblastine intraperitoneally with (group 1) or without (group 2) 50 mg/kg valspodar. Valspodar was given orally 4 h before the vinblastine injection. In the literature, valspodar is described as a cytotoxic agent with an anticancer effect independent from the modulatory action on MDR (Kreis et al. 2001). Therefore, it was given to another group of mice that served as a control (group 3). The valspodar vehicle was also administered to mice as a control (group 4) because cremophor RH40, a derivative of cremophor EL, is an ingredient of the vehicle.

50 mg/kg valspodar,

Figure 5.10: Application scheme for the treatment of NCI-H460 brain metastases in nude mice with vinblastine co-administered with valspodar. Each group consists of 6 to 8 nude mice.

108 In vivo models of human lung cancer brain metastases For cremophor EL a modulating effect on p-gp is described in literature (Chervinsky et al.

1993, Woodcock et al. 1990). In addition, mice received 0.9 % sodium chloride solution, the vehicle of vinblastine, to represent the "untreated" control (group 5). The survival plot was determined according to Kaplan-Meyer.

Survival did not significantly differ within the five groups (Fig. 5.11). Until day 15 the combination treatment seemed to exhibit an advantage compared to the other groups because no mouse died in group 1 until this day. In contrast, in the other groups at least 2 mice had died by day 15 indicated by a survival rate of 80% at best. On day 20 all living animals were very weak and apathetic, and the experiment was aborted. In group 1 where mice received the combination of vinblastine and valspodar, 2 of 8 mice (25 %) were still living. In the control groups 3 and 4, all animals had died. However, 1 animal remained alive in the vinblastine group 2 (16.7 %) as well as in the untreated group 5

Figure 5.11: Kaplan-Meyer survival plot for the treatment of intracerebrally grown NCI-H460 lung cancer in nude mice with vinblastine and valspodar. The animals (n = 6 - 8) were treated every 5 days starting on day 1. Group 1 received vinblastine (0.8 mg/kg, i.p.) and valspodar (50 mg/kg, p.o., 4 h before vinblastine), group 2 vinblastine alone and the groups 3 to 5 served as control groups and received either valspodar, valspodar vehicle or sodium chloride solution, respectively. No significant differences within the five groups were observed.

5.3 Results 109 (16.7 %). In this model no significant increase in survival time was achieved by systemic treatment with vinblastine and even the co-application of valspodar had no significant effect on the life span of the nude mice.

Up to day 5 the mice put on weight slightly (data not shown). Two to 3 days after the 2^nd treatment a clear decrease in body weight was observed in the treated groups 1 and 2 and also in the control groups 3 to 5 on day 10 after the 3^rd treatment. There are no differences between the vinblastine/valspodar group and mice, which received vinblastine alone. There are at least three reasons for the obvious body weight reduction. The first one is the increased toxicity of vinblastine caused by the co-administration of valspodar;

this is well known from clinical studies (Bates et al. 2001). Another reason for the de-crease in body weight in the vinblastine treated groups is the narrow therapeutic index of vinblastine and the corresponding systemic toxicity. The third reason is the aggressive and rapid growth of the NCI-H460 cell line (see chapter 3).

The experiment was repeated and this time the brains were supposed to be

ana-Figure 5.12: Tolerance experiment for the vinblastine/valspodar combination. Mice received 50 mg/kg valspodar p.o. 4 h before i.p. injection of 0.8 mg/kg vinblastine.

Decrease in body weight is an indicator of systemic toxicity. The combination of vinblastine and valspodar was only tolerated by 2 mice (a,f).

110 In vivo models of human lung cancer brain metastases lyzed by a morphometrical method described at Fellner (2001). Unfortunately, the mice treated with vinblastine and valspodar already died 2 days after the 1^st treatment. There-fore, the experiment was not repeated. The intraperitoneal administration of vinblastine 0.8 mg/kg, every 5 days, to 5 tumor-free female nude mice was well tolerated. The appli-cation of the vinblastine/valspodar combination was tested in healthy mice as well and in contrast to the result described above most of the mice did not withstand the treatment.

Fig. 5.12 shows the body weights of 8 male nude mice, which received 50 mg/kg valspodar orally, and 4 h later 0.8 mg/kg vinblastine intraperitoneally.

Two days after the 1^sttreatment 2 mice died (b,g), another 2 mice (c,d) died 4 days after the 1^st treatment. Again 3 days after the 2^nd treatment 2 mice (e,h) died. Only 2 mice (a,f) sustained the combination treatment with vinblastine and valspodar. Subsequently performed HPLC investigations of the used vinblastine solutions proved that incorrect preparation of the vinblastine solutions can be excluded as a possible cause of the fatalities.

5.3.2.3 Co-application of valspodar with paclitaxel for the treatment of DMS 114 lung cancer in the brain of nude mice

In a second study nude mice with intracerebrally grown DMS 114 tumors were treated with a combination of paclitaxel and valspodar. The animals were treated with 3 mg/kg of paclitaxel intravenously on day 8 after tumor cell implantation and with 2 mg/kg of

50 mg/kg valspodar,

Figure 5.13: Application scheme for the treatment of DMS 114 brain metastases with paclitaxel co-administered with valspodar. Each group consists of 6 to 8 nude mice.

5.3 Results 111 paclitaxel on day 15, with (group 1) and without (group 2) orally administered 50 mg/kg of valspodar 4 h before each paclitaxel dose. The control groups received either 50 mg/kg of valspodar (group 3), the valspodar vehicle (group 4) or 0.9 % sodium chloride solution (group 5), the vehicle of paclitaxel (cp. chapter 5.3.2.2). The mice were killed 20 days after implantation and the tumor volume in each brain was determined morphometrically.

In each group 4 brains were used for the morphometric analysis. After the HE-staining of the brains of group 1, a tumor of enormous size was detected in one brain. Paclitaxel and valspodar had been administered to mice in group 1. In theory, the brains of group 1 should possess small or no tumors due to a therapeutical benefit of the valspodar pretreat-ment. Thereupon, only the brains of the mice assigned to the control group 4 (valspodar vehicle) were examined as the morphometric examination is very time consuming. As-suming a globular or ellipsoidal tumor growth, the tumor volumes were estimated from the HE-stained sections.

Figure 5.14: Mean estimated tumor volumes of treated group 1 (50 mg/kg valspodar p.o., 4 h before 3 and 2 mg/kg paclitaxel i.v., respectively) and untreated group 4 (valspodar vehicle). There is no significant difference within the two groups.

Paclitaxel administration even in combination with valspodar, did not have a tumor reducing effect on intracerebrally grown DMS 114 tumors.

Fig. 5.14 presents the estimated tumor area for the paclitaxel/valspodar treated group 1 and the control group 4. In this study, there were no statistically significant differences between the mean tumor volumes within the two examined groups. Only 3 of the 4

112 In vivo models of human lung cancer brain metastases examined brains per group could be used for the volume estimation. In group 4, one brain was incorrectly prepared, so only part of the brain which did not contain any tumor was available for evaluation. In group 1 no tumor was detected in one brain. It cannot be excluded that the injection of the tumor cells had failed in this animal as the tumor volume of another DMS 114 brain tumor was assessed to 39 mm³. Hence, no therapeutic benefit could be achieved in this tumor model by systemic treatment with paclitaxel even in combination with valspodar.

Figure 5.15: Decrease in body weight is an indicator of systemic toxicity. The arrows indicate drug administration. The animals (n = 6 - 8) were treated on day 8 (3 mg/kg paclitaxel i.v.) and on day 15 (2 mg/kg paclitaxel i.v.). Group 1 received pa-clitaxel and valspodar (50 mg/kg, p.o., 4 h before papa-clitaxel), group 2 papa-clitaxel alone and the groups 3 to 5 served as control groups and received either valspodar, valspodar vehicle or sodium chloride solution, respectively.

The changes in body weight are depicted in Fig. 5.15. Particularly in mice treated with the paclitaxel/valspodar combination (group 1) the body weight was reduced after each treatment. The body weight of mice assigned to the control groups 3 to 5 remained relatively constant over a long period of time and decreased at the end of the experiment due to the tumor burden. The body weight reduction of animals which received

pacli-5.3 Results 113 taxel alone, was lower than in case of the paclitaxel/valspodar treated mice. Thus, the co-application of valspodar increases the toxicity of paclitaxel. This finding is in good agreement with results of clinical trials (Advani et al. 2001).

5.3.3 Discussion

In both studies no therapeutic effect of valspodar co-administration on the growth of brain tumors was observed. The dosage of valspodar (50 mg/kg) was sufficient to inhibit p-gp activity in the mouse. Lemaire et al. (1996) investigated the brain-to-blood partition coefficient of valspodar in rats 2 h after the intravenous administration of radioactively labelled valspodar in doses of 0.1 to 30 mg/kg. A valspodar dosage of 10 mg/kg achieved a saturation effect of the p-gp inhibition at the BBB. As the oral bioavailability of valspodar reached 53 % (Covelli 1998), a concentration of 20 mg/kg should already be sufficient for a complete p-gp inhibition at the BBB.

The ineffectiveness of the co-application of vinblastine and valspodar is discussed in the literature. Lyubimov et al. (1996) and Drion et al. (1996) described that the increase in vinblastine concentration achieved by co-administration of valspodar, was lower in the brain than in liver and kidney. Similarly, in our study the vinblastine concentrations in the brain were too low to achieve an antitumor effect. The intolerance of the combination treatment in mice could be explained by the narrow therapeutic index of vinblastine.

Alterations of the pharmacokinetic parameters of vinblastine caused by the combination with valspodar have profound consequences on the safety of the treatment. In other stu-dies, the co-application was only tolerated with acceptable toxicities in case of a vinblastine dose reduction (van Asperen et al. 1996, Bates et al. 2004). But with the optimized dosage only a slight benefit was observed in both studies, because the vinblastine concentration was too low after the dose reduction to have an effect on the tumor growth.

In the literature, studies with the combination of paclitaxel and valspodar are described as well. By co-application of the p-gp modulator increased paclitaxel brain levels were achieved (Fellner et al. 2002, Kemper et al. 2003) and toxicity remained acceptable due to paclitaxel dose reduction (Fracasso et al. 2000). Moreover, Fellner (2001) determined a paclitaxel concentration of 146 nmol/g in the nude mouse brain 24 h after the

intra-114 In vivo models of human lung cancer brain metastases venous injection of 4 mg/kg of paclitaxel when the animals were pretreated with 50 mg/kg valspodar p.o. (4 h before paclitaxel injection). The in vitro chemosensitivity of DMS 114 against paclitaxel was tested within the scope this thesis (see chapter 5.3.1.2). A paclitaxel concentration of 5 nM (equivalent to 5 nmol/g) had a cytocidal effect on the in vitro growth of DMS 114 cells. Hence, the paclitaxel brain levels detected by Fell-ner should be intrinsically high enough to achieve a therapeutic effect on the DMS 114 brain tumors in nude mice. However, under the conditions described by Fellner et al. the treatment of DMS 114 brain metastases failed. It is conceivable that the chemosensitivity of the selected DMS 114 tumor against paclitaxel in vivo is reduced compared to the chemosensitivity in vitro.

Furthermore, the injected number of tumor cells plays a decisive role for the success of a treatment experiment. In preliminary tests with different cell numbers (30,000 to 300,000 DMS 114 cells) tumors of sufficient size grew after an injection of 30,000 and 100,000 tumor cells, respectively. As in the treatment experiment the investigated tumors were partly too big in size for a morphometric analysis, the preliminary tests were repeated with 10,000 and 30,000 cells, respectively (4 mice per group). After 22 days the brains were collected and the prepared sections were analyzed. Only in 5 of 8 brains tumors were detectable. Since the mice received no treatment, probably the injection of the tumor cells was incorrect. In one brain, no injection channel was detected and in another case, the tumor had grown between the skull and the dura mater. The detected tumors differed in size independent of the injected tumor cell number. Thus, the procedure of the tumor cell injection is complicated and it is difficult to produce tumors of a sufficient size (10 -15 mm³) especially in case of the DMS 114 cell line.

In case of the NCI-H460 cell line, the cell injection is important as well. Additionally, the tumor growth is aggressive and rapid both in vitro and in vivo. Since the cells possess the mRNA encoding for p-gp (see chapter 5.3.1.1), it is possible that the cells became chemoresistant during therapy due to the continuous contact with the p-gp inducer vinblastine. Thus, this cell line is unsuited for the brain metastases model studying effects of p-gp inhibition at the BBB, because of its rapid growth, the high chemoresistance and the existence of p-gp encoding mRNA.

For further treatment studies with the DMS 114 tumor, the model should be improved.

5.4 Summary 115