Pharmacokinetic study on the paclitaxel distribution in nude mice . 161

In this study, the influence of the p-gp modulators elacridar and tariquidar on the pa-clitaxel distribution in brain, liver, kidney and plasma of nude mice was investigated.

The analyzed paclitaxel concentrations after elacridar or tariquidar administration were compared to the results obtained in the valspodar study carried out by S. Fellner. The paclitaxel distribution in the different tissues is summarized in Figs. 7.6 to 7.9.

In liver (Fig. 7.6), paclitaxel levels were very high (> 15 nmol/g) after 1.5 hours. The values decreased to similar paclitaxel levels in all four groups. No statistically significant differences were determined between the different p-gp modulator groups and the control group.

162 Effect of P-gp modulators on the paclitaxel distribution in nude mice

Figure 7.6: Effect of p-gp modulator co-administration (50 mg/kg, p.o.) on paclitaxel levels in liver (mean values ± SEM). Paclitaxel was given intravenously at a dosage of 8 mg/kg. Valspodar (¥), elacridar (N) and tariquidar (H) were used as p-gp modulators; untreated mice served as a control (◦).

Figure 7.7: Effect of p-gp modulator co-administration (50 mg/kg, p.o.) on paclitaxel levels in kidney (mean values ± SEM). Paclitaxel was given intravenously at a dosage of 8 mg/kg. Valspodar (¥), elacridar (N) and tariquidar (H) were used as p-gp modulators; untreated mice served as a control (◦).

7.3 Results 163 In contrast to the drug levels determined in liver, in the kidney the highest concen-tration of paclitaxel was found in mice that received valspodar (Fig. 7.7). In fact, 1.5 hours after injection the paclitaxel levels were high in all examined groups with values up to 15.5 nmol paclitaxel per g of tissue. Then in elacridar and tariquidar treated mice, the paclitaxel content in kidney decreased to achieve concentrations similar to those in the untreated control group. In contrast, co-administration of valspodar led to a slight increase in the paclitaxel concentration after 3 hours compared to the values obtained after 1.5 hours. Paclitaxel levels were 7-fold higher after 4.5 hours than after 24 hours.

However, this paclitaxel content was still significantly different from the concentrations in the other groups. The paclitaxel concentrations determined in kidney were slightly lower in comparison to the levels measured in liver.

Figure 7.8: Effect of p-gp modulator co-administration (50 mg/kg, p.o.) on paclitaxel levels in plasma (mean values ± SEM). Paclitaxel was given intravenously at a dosage of 8 mg/kg. Valspodar (¥), elacridar (N) and tariquidar (H) were used as p-gp modulators; untreated mice served as a control (◦).

The paclitaxel concentration time profile in plasma (Fig. 7.8) followed a similar pattern as observed in kidney. Relative to the control group the paclitaxel level in plasma of mice that received valspodar increased by 2.5-fold after 1.5 hours and 10-fold after 3 hours. In mice pretreated with elacridar or tariquidar the paclitaxel concentrations were similar to

164 Effect of P-gp modulators on the paclitaxel distribution in nude mice those in plasma of the control mice and decreased during the experiment to values lower than 0.2 nmol/g after 24 hours. By contrast, in the plasma of valspodar treated mice, no decrease in the paclitaxel concentration was observed.

Figure 7.9: Effect of p-gp modulator co-administration (50 mg/kg, p.o.) on paclitaxel levels in brain (mean values ± SEM). Paclitaxel was given intravenously at a dosage of 8 mg/kg. Valspodar (¥), elacridar (N) and tariquidar (H) were used as p-gp modulators; untreated mice served as a control (◦).

The concentration of paclitaxel in the brains of nude mice was also measured (Fig. 7.9).

The administration of paclitaxel alone resulted in low levels of the drug in the brain. The concentrations were near the limit of quantitation (0.1 nmol/g). Brain levels were up to 57 times lower (1.5 and 3 hours after injection) compared to those in liver and kidney.

Co-administration of valspodar substantially increased the paclitaxel concentration in the brain. Although the levels were 5- to 8.5-fold higher than in the control group the new p-gp modulators elacridar and tariquidar were not able to induce the same increase in the concentration of paclitaxel in the brain as that achieved by valspodar administration.

Co-application of elacridar and tariquidar led only to an increase by 4.9 to 7 fold and 4.7 to 6.6 fold, respectively. Despite this slight increase, brain paclitaxel levels observed after pretreatment with elacridar and tariquidar remained significantly different from the control levels. Thus, the brain was the only examined tissue, where administration of

7.3 Results 165 elacridar and tariquidar resulted in different paclitaxel concentrations compared to the control levels. Furthermore, in contrast to liver, kidney and plasma, where paclitaxel concentrations strongly declined after 24 hours, the paclitaxel levels in the brain remained relatively constant within 24 hours.

The different impact of valspodar, elacridar and tariquidar co-administration on the paclitaxel levels in the brain compared to plasma is presented as concentration ratios determined at different points in time in Fig. 7.10. Irrespective of the administered modulator, more paclitaxel was found in plasma than in brain 1.5 hours after paclitaxel injection. However, additional 1.5 hours later, the brain/plasma ratio had changed in case of elacridar or tariquidar co-application: 2.5 and 1.5 times higher paclitaxel levels were detected in brain than in plasma. This effect lasted for 4.5 hours, and was more pronounced with tariquidar (ratio 4.5) than with elacridar (ratio 1.2). The brain/plasma ratios of paclitaxel after valspodar co-administration were 2- to 15-fold lower than in the presence of the new p-gp modulators, indicating that p-gp inhibition by valspodar does

Figure 7.10: Brain-plasma ratio of paclitaxel after co-administration of valspodar (black bar;

not determined at 4.5 h), elacridar (light grey bar) and tariquidar (dark grey bar).

Three hours after paclitaxel injection more paclitaxel is found in brain than in plasma if elacridar or tariquidar were co-administered.

166 Effect of P-gp modulators on the paclitaxel distribution in nude mice not lead to a specific accumulation of paclitaxel in the brain of nude mice.

As the measured elacridar concentrations in the brain were at least 50 times higher than the IC₅₀value (ca. 20 nM) determined for p-gp inhibition in vitro, in another experiment the dosage of the modulator was reduced to 5 mg/kg. This dosage should be sufficient by far to achieve the elacridar concentration required for p-gp inhibition. The effect of 5 mg/kg elacridar on the distribution of paclitaxel in different tissues and plasma of nude mice compared to untreated mice is summarized in Fig. 7.11.

In liver, kidney and plasma of mice treated with elacridar at reduced dosage, the paclitaxel concentrations were not significantly different from the control values. As ex-pected, the paclitaxel levels in liver, kidney and plasma obtained after co-administration

Figure 7.11: Effect of elacridar co-administration (5 mg/kg, p.o.) on paclitaxel levels in brain (a), liver (b), kidney (c) and plasma (d) of nude mice (black bar; mean values

± SEM). Paclitaxel was intravenously given at a dosage of 8 mg/kg. Untreated mice served as a control (light grey bar).

7.4 Discussion 167 of 5 mg/kg of elacridar were comparable to those after a dosage of 50 mg/kg. However, the reduced dose of elacridar did not result in an increase of the paclitaxel concentration in brain. All measured paclitaxel levels in the brain were near the limit of quantitation. In all tissue and plasma samples a decrease in the paclitaxel concentration was determined during the 4.5 hour study period. Only little information is available on the absorption of the lipophilic elacridar and its distribution into adipose tissue. Due to the lack of in-creased paclitaxel levels in the brain after p.o. administration of 5 mg/kg elacridar, it may be speculated, that the modulator is only partly absorbed and distributed preferentially to adipose tissue. This would explain why a higher dosage is required to inhibit the p-gp at the BBB.

7.4 Discussion

The 3^rd generation p-gp modulators elacridar and tariquidar proved to be highly potent p-gp inhibitors in vitro (see chapter 6). Now, their efficacy was investigated in vivo by quantification of the paclitaxel levels in different tissues of nude mice depending on the co-application of the MDR modulators. Fortunately, the two modulators were detectable in the HPLC chromatograms which were recorded for paclitaxel analysis. The recovery of elacridar and tariquidar was determined afterwards, the calculated modulator distribu-tion in the different tissues could be estimated although sample preparadistribu-tion and HPLC method were developed for the detection and quantification of paclitaxel. The high SEM values in the determination of accuracy and precision were explainable by the following facts. Interfering peaks that were observed particularly in liver and kidney and could only be estimated in control samples might influence the results especially at low modulator concentrations. Since the tariquidar peak was detected close to the peak of the internal standard the separation of both peaks was impaired at higher modulator concentrations.

The peak area that is attributed to interfering peaks is negligible at modulator concentra-tions greater than 2.5µM. At high elacridar concentrations the pronounced tailing of the peak hampered the exact quantification of elacridar. The asymmetry factor of elacridar peaks at high modulator concentrations was close to the acceptable value of 2.5. Factors higher than 3 result in unprecisely defined peak areas because the point at which the

168 Effect of P-gp modulators on the paclitaxel distribution in nude mice peak reaches the base line is not well identifiable (Meyer 1992). To determine the con-centrations of elacridar and tariquidar correctly, a new method has to be developed. Due to the low recovery, particularly from plasma, the procedure of the sample preparation should be adapted to the modulators. The preparation of modulator samples according to a different method failed due to the small tissue homogenate volume especially in case of brain samples.

Another problem was the poor solubility of elacridar and particularly tariquidar in the vehicle if dosed at 50 mg/kg. Different solvents mentioned in literature such as hydroxypropylmethyl cellulose (HMPC) and tween 80 solutions (Mistry et al. 2001) were tested, but proved to be unsuited, and therefore the modulators were administered in suspension. Thus, the exact dose of soluble modulator that was given to each mouse remained unknown. By improving the solubility of these compounds, the ideal modulator concentration could be determined.

Despite the problems associated with the exact determination of the modulator con-centrations, high elacridar and tariquidar levels were analyzed particularly in liver and kidney. Even in the nude mouse brain, the modulator levels were at least 10 to 50 times higher than those required for p-gp inhibition in vitro (see IC50 values in the calcein-AM efflux assay). Elacridar and tariquidar were able to significantly increase the paclitaxel concentrations up to 0.3 nmol/g after 24 hours in the brain compared to the control.

However, the increase ín brain paclitaxel levels caused by the new modulators was not as high as in case of valspodar administration (about 0.5 nmol/g after 24 hours), indi-cating an incomplete inhibition of p-gp. This observation should not necessarily lead to the conclusion that elacridar and tariquidar are weaker p-gp modulators in vivo. For the evaluation of the efficacy of a p-gp inhibitor with respect to the increase in brain concen-trations of a co-administered drug it is also necessary to take into account the influence of the inhibitor on the drug plasma level. Kemper et al. (2003) examined to what extent increased plasma levels contribute to higher brain levels. They determined the paclitaxel concentrations in the brains of p-gp knockout mice after administration of valspodar. The paclitaxel brain levels were even higher as in untreated knockout mice, suggesting that increased plasma levels due to reduced excretion also account for higher brain levels in wild type mice receiving valspodar. Since the plasma levels of paclitaxel measured by

7.4 Discussion 169 S. Fellner were markedly increased after valspodar administration compared to the con-trol, the increased levels in the brain might have partly resulted from the high plasma levels in the valspodar group. In case of the new modulators, no marked elevations of the paclitaxel levels in plasma were observed compared to the control. Thus, the elevated paclitaxel concentrations in brain of elacridar and tariquidar treated mice only resulted from the inhibition of p-gp at the BBB. The potency of elacridar found in this study is in line with previous reports on its effects on the brain penetration of docetaxel (Kemper et al. 2004c) and morphine (Letrent et al. 1998).

Another important conclusion can be drawn from the experiment with reduced elacridar dosage. In this case there was no increase in paclitaxel levels at all compared to the control samples. Thus, a relatively high modulator dose is required to show marked elevated paclitaxel levels in the brain. In case of valspodar a further increase in dosage is not reasonable. In experiments done by Kemper et al. (2003) a single dose of 25 mg/kg was found to be sufficient to achieve maximum inhibition of p-gp and other enzymes involved in metabolic elimination of paclitaxel. In addition, higher valspodar doses did not lead to enhanced paclitaxel levels, but could result in toxic side effects if valspodar plasma levels were higher than 3.5µM. In contrast, elacridar can be administered at high concentrations up to 1 g orally without any toxic side effects to patients (Kruijtzer et al. 2002).

Paclitaxel was specifically accumulated in brain in case of elacridar and tariquidar co-administration. The favorable brain/plasma concentration ratios indicate a preferential modulation of p-gp at the BBB and lower systemic toxicity for the combination of pacli-taxel with the new modulators. Several clinical studies in which paclipacli-taxel was combined with a presumed selective and potent p-gp modulator, have shown that dose reductions of the cytostatic were not necessary (Boniface et al. 2002). The fact that no increase in the paclitaxel levels in liver and kidney was measured after co-administration of elacridar and tariquidar, confirmed the suitability of these substances for the combination treatment with cytostatic drugs. By contrast, valspodar caused considerably elevated paclitaxel concentrations in plasma due to substantial reduction of paclitaxel clearance (p-gp inhi-bition in kidneys). The increased systemic exposure to paclitaxel led to enhanced toxic effects such as bone marrow suppression and neurotoxicity. Hence, it can be concluded that 3^rd generation p-gp modulators are not more efficient in inhibiting p-gp in vivo, but

170 Effect of P-gp modulators on the paclitaxel distribution in nude mice these substances are much more suitable for a well tolerated combination chemotherapy compared to 2^nd generation modulators such as valspodar.