3. Chapter. 2: Can a P-gp modulator assist in the
3.5. Discussion
In Chapter 1, the author administered MTX intravenously or intrathecally to rats with or without CysA, which was also intravenously or intrathecally injected. After 6 hr, the brain and CSF were sampled, and their MTX concentrations were compared.
CysA did not significantly affect MTX concentrations in the brain or CSF when MTX was intravenously injected. In contrast, when MTX was intrathecally administered, intravenously administered CysA was found to have a more prominent effect on MTX concentrations in the brain than in the CSF (87).
In the present study, a specific P-gp substrate, Rho123, increased brain MTX concentrations when MTX was intrathecally administered, which is consistent with my previous findings. This result suggests that this P-gp modulator competitively inhibited the excretion of MTX from the brain because both CysA and Rho123 are P-gp substrate and not RFC and PCFT substrate; folate analog (126).
Another possibility is that Rho123 potentiated the distribution of MTX from the blood into the brain. However, this is unlikely, because Rho123 did not significantly increase brain MTX concentrations when MTX was intravenously administered.
MTX may easily diffuse from CSF into the brain through supraependymal cells because significantly higher MTX concentrations were observed in the brain 6 hr after its intrathecal injection, compared with intravenous injection. In addition, MTX may be easily excreted from CSF and the brain into the blood, because its concentrations were markedly lower 12 hr than 6 hr after drug administration. Pacchionian granulation, the BBB or CSF-brain barrier have been suggested as excretion routes.
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In order to compare the MTX decrease in CSF and brain, the MTX concentration ratio 12 hr to 6 hr after drug administration was calculated (Table 3.3). While there was no significant difference in the CSF, Mit+Rit indicated significantly higher rate than Mit and Mit+Riv in the brain. This suggested that of these, the BBB may be inhibited the most by the Rho123 treatment, which is supported by the rapid decrease observed in MTX concentrations in CSF at 12 hr, even when Rho123 was co-administered and the slower decrease in MTX concentrations in the brain at 12 hr when Rho123 was co-administered.
However, the effects of Rho123 on MTX concentrations in the brain were weaker when it was intravenously rather than intrathecally injected. In contrast, CysA
potentiated the distribution of MTX in the brain after it was intravenously rather than intrathecally injected. Thus, Cys A and Rho123 may have the different affinities to the brain or different permeabilities through the BBB. As shown in Fig. 3.5, Rho123 concentrations in the brain and CSF were significantly higher in Mit+Rit than in Mit+Riv 6 and 12 hr after drug administration. It is speculated that although
intravenously administered Rho123 poorly penetrates into brain, its affinity to brain tissues is high. This affinity may owe to its lipophilicity or transporters involved in brain tissue, such as glial cells.
Although MTX does not appear to be a substrate for P-gp because of its negative charge, the results of the present study suggest that it is a P-gp substrate, even under in vivo conditions. Therefore, the co-administration of P-gp modulators with MTX may be effective, even for MTX-resistant tumors, because MTX resistant tumors have RFC functional disorders (66, 89). As such, combined cancer chemotherapy involving MTX with P-gp modulators may be effective for many CNS tumors.
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Since P-gp acts as a transporter not only in the brain, but also in other tissues, including the kidney, liver and intestine, P-gp modulators may alter the
pharmacokinetics of co-medicated drugs that are P-gp substrates, such as doxorubicin and etoposide (9, 24, 31, 40, 78-80, 119). However, in the present study, the plasma concentration-time profiles of MTX were not affected by the treatment with Rho123 at the dose, as shown in Fig. 3.2 and Table 3.2. This result suggests that the adverse effects associated with MTX chemotherapy are not potentiated by treatments with P-gp modulators. In addition, an i.t. injection of MTX may result in markedly higher MTX concentrations in the brain and markedly lower concentrations in other tissues than those after an i.v. injection because the i.t. dose is markedly lower than the i.v. dose.
This may result in less adverse effects. Therefore, an i.t. injection of MTX combined with an i.v. or i.t. injection of P-gp modulators, such as CysA and Rho123, may be an effective therapy for CNS tumors.
In conclusion, P-gp appears to play an important role in the excretion of MTX from the brain. The i.t. administration of MTX with a P-gp modulator may maintain a sufficient MTX concentration in the brain for a longer period of time without increasing systemic body exposure. If P-gp modulators appropriately control MTX concentrations in the brain, this combined chemotherapy may lead to promising outcomes for patients with CNS tumors.
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3.5. Acknowledgments
This chapter is based on an article described by Ogushi et al. under the title of “CAN a P-gp modulator assist in the control of methotrexate concentrations in the rat brain? −inhibitory effects of rhodamine 123, a specific substrate for P-gp, on methotrexate excretion from the rat brain and its optimal route of administration.” in J. Vet. Med Sci. 79(2): 320-327, 2017.
- 59 - Table 3.1 Definition of administration groups
Group MTX Rho123
Miv i.v.
Mit i.t.
Miv+Riv i.v. i.v.
Mit+Riv i.t. i.v.
Mit+Rit i.t. i.t.
This table shows 5 administration groups defined by the combinations of the drugs and their administration routes as follows. Miv: MTX (i.v.)+ saline (i.t.), Mit: MTX (i.t.), Miv+Riv: MTX (i.v.) +Rho123 (i.v.)+ saline (i.t.), Mit+Riv: MTX (i.t.) +Rho123 (i.v.), Mit+Rit: MTX (i.t.)+Rho123 (i.t.).
Each value is the mean ± S.D. (n=5). The doses of MTX and Rho123 administered were 2 and 0.2 mg/body, respectively.
Table 3.2 Pharmacokinetic parameters of MTX after administration of with or without Rho123 to rats.
Group C0
(μM) kel
(1/hr)
AUC
(μM䞉hr) Vd
(l/body) Cltot
(l/hr) t1/2 (min)
Miv 1.3 ±
0.28
1.2 ± 0.17
1.25 ±
0.27 10 ± 2.2 12 ± 3.1 35 ± 3.2 Mit 0.51 ±
0.19
0.49 ± 0.22
1.02 ±
0.28 84 ± 20
Miv+Riv 1.3 ± 0.20
0.96 ± 0.10
1.41 ±
0.23 12 ± 1.4 12 ± 2.2 43 ± 2.9 Mit+Riv 0.58 ±
0.12
0.72 ± 0.26
0.86 ±
0.052 58 ± 9.0
Mit+Rit 1.3 ± 0.53
0.97 ± 0.32
1.45 ±
0.24 43 ± 8.3
Abbreviations stand for as follows: C0; initial concentration, kel; elimination rate constant, AUC;
area under the plasma concentration-time curves, Vd; volume of distribution, Cltot; total body clearance, t1/2; elimination half-life, Miv: MTX (i.v.)+ saline (i.t.), Mit: MTX (i.t.), Miv+Riv:
MTX (i.v.) +Rho123 (i.v.)+ saline (i.t.), Mit+Riv: MTX (i.t.) +Rho123 (i.v.), Mit+Rit: MTX (i.t.)+Rho123 (i.t.). Each value is a mean ± S.D. (n=5). Doses of MTX and Rho123 were 2 and 0.2 mg/body, respectively. Each parameter was not significantly different among these groups.
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Fig. 3.1. A chemical structure of Rhodamine123 (Rho123)
C21H17ClN2O3 = 380.82. The cationic dye, Rho123 is known as a specific P-gp substrate, and the P-gp function in MDR cells is assessed by the Rho123 efflux from the MDR cells (64). Rho123 localizes specifically in mitochondria and is selectively toxic to carcinoma cells in vitro (12, 72, 73) and in vivo (11, 73).
However, accumulation and consequent cytotoxicity of Rho123 in sensitive cells are not increased by one of the P-gp modulator, verapamil (71). In female Sprague-Dawley rats, Rho123 level in plasma eliminated in a triphasic manner (t1/2Ș: 15min., t1/2ș: 1hr, t1/2Ț: 4.7hr) (107).
Table 3.3 MTX concentration ratio of 12hr to 6hr after administration
12h / 6h Mit Mit+Riv Mit+Rit
CSF 1.2 ± 0.43 (%) 0.79 ± 0.38 (%) 2.35 ± 1.27 (%) Brain 4.2 ± 0.73 (%) 0.66 ± 0.30 (%) * 18.54 ± 6.87 (%)
Abbreviations stand for as follows: Mit: MTX (i.t.), Miv+Riv: MTX (i.v.) +Rho123 (i.v.)+
saline (i.t.), Mit+Riv: MTX (i.t.) +Rho123 (i.v.), Mit+Rit: MTX (i.t.)+Rho123 (i.t.). Each value is a mean ± S.D. (n=5). *Brains of Mit+Riv indicated significantly larger ratio than the others.
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Fig. 3.2 MTX concentration in plasma after administrations of MTX with or without Rho123 to rats.
●; MTX (i.v.)+ saline (i.t.), ■; MTX (i.t.), ▲; MTX (i.v.) +Rho123 (i.v.)+ saline (i.t.), □;
MTX (i.t.) +Rho123 (i.v.), ○; MTX (i.t.)+Rho123 (i.t.). Each value is a mean±S.D. (n=5).
Each value is a mean±S.D. (n=5). Doses of MTX and Rho123 were 2 and 0.2 mg/body, respectively. There was no significant difference in AUC.
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Fig. 3.3 Total MTX concentrations in brain and CSF at 6 (A) and 12hr (B) after administrations of MTX with or without Rho123 to rats.
Abbreviations stand for as follows: Miv: MTX (i.v.)+ saline (i.t.), Mit: MTX (i.t.), Miv+Riv:
MTX (i.v.) +Rho123 (i.v.)+ saline (i.t.), Mit+Riv: MTX (i.t.) +Rho123 (i.v.), Mit+Rit: MTX (i.t.)+Rho123 (i.t.). Black and white columns represent brain and CSF concentrations, respectively. Each value is a mean±S.D. (n=5). Doses of MTX and Rho123 were 2 and 0.2 mg/body, respectively.
* The Mit+Rit group revealed significantly higher MTX levels in the brain than those of the other 3 groups at 12hr post administration (P<0.05, Scheffe’s multiple comparison test).
- 63 -
Fig. 3.4 Ratios of MTX concentration in brain to plasma AUC (BBR) and in CSF to plasma AUC (CBR) at 6 (A) and 12hr (B) after administrations of MTX with or without Rho123 to rats.
Abbreviations stand for as follows: Miv: MTX (i.v.)+ saline (i.t.), Mit: MTX (i.t.), Miv+Riv:
MTX (i.v.) +Rho123 (i.v.)+ saline (i.t.), Mit+Riv: MTX (i.t.) +Rho123 (i.v.), Mit+Rit: MTX (i.t.)+Rho123 (i.t.). Black and white columns represent BBR and CBR, respectively. Each value is a mean±S.D. (n=5). Doses of MTX and Rho123 were 2 and 0.2 mg/body, respectively. # CBR was significantly higher than BBR in Mit (p<0.05).
* The Mit+Rit group revealed significantly higher MTX levels in the brain than those of the other 3 groups at 12hr post administration (P<0.05, Scheffe’s multiple comparison test).
ݡݡ
- 64 -
Fig. 3.5 Rho123 concentrations in the brain and CSF at 6 (A) and 12 hr (B) after the administration of MTX with Rho123 to rats.
This figure is composed of charts A and B, which show Rho123 concentrations at 6 and 12 hr post administration respectively. The abbreviations used are as follows: Miv+Riv;
MTX (i.v.) +Rho123 (i.v.)+ saline (i.t.), Mit+Riv; MTX (i.t.) +Rho123 (i.v.), Mit+Rit;
MTX (i.t.)+Rho123 (i.t.). The doses of MTX and Rho123 were 2 and 0.2 mg/body, respectively. Black and white columns represent brain and CSF concentrations, respectively. Each value is the mean ± S.D. (n=5).
*Mit+Rit revealed significantly higher concentration in both CSF and brain, compared with the other groups (P<0.05, Scheffe’s multiple comparison test).
- 65 -
4. Chapter 3.
Effects of leucovorin (folinic
acid) in the methotrexate
-treated rat brain
- 66 -
4.1. Abstract
Folinic acid (FA) is generally administered to patients with CNS tumors in order to treat severe neurological disorders caused by MTX; therefore, the author herein examined the effects of the co-administration of FA on MTX concentrations in the rat brain and CSF as well as the pharmacokinetics of MTX. MTX was intravenously or intrathecally administered to rats with or without FA. MTX concentrations were assessed by HPLC.
No significant differences were observed in pharmacokinetic parameters, including kel, Vd, AUC, Cltot, and t1/2, between the FA-treated and non-treated groups.
MTX concentrations were not significantly different in the brain or CSF 6 hrs after the intrathecal administration of MTX. However, compare to intravenous administration of MTX, intravenous administration of both FA and MTX significantly decreased MTX concentrations in the brains and CSF.
These results suggest that FA inhibits the influx of MTX into the brain and CSF, possibly by competing with folate carriers, but has no effect on its efflux from these regions. Therefore, FA may be administered to CNS tumor patients receiving intrathecal MTX therapy in order to treat the adverse effects of MTX without affecting its
concentrations in the brain and CSF.
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4.2. Introduction
MTX is an anticancer drug that is used to treat CNS tumors. However, its influx into the CNS is strongly restricted because of its water solubility. The CNS is also protected from xenobiotics such as MTX by BBB and B-CSFB at the choroid plexus (47, 102, 116). Various methods have been developed to deliver MTX into the CNS at sufficient concentrations, such as its administration via an intravenous (i.v.) route at high doses and an intrathecal (i.t.) route in humans and animals (3, 15, 43, 48, 64, 65, 95).
Since MTX is a folate analog, its carrier-mediated transportation generally depends on folate carriers, such as reduced folate carriers (RFC), proton-coupled folate transporters (PCFT), and folate receptor-mediated endocytosis (FR). These carriers are also expressed at the BBB and B-CSFB. RFC is expressed at the apical membrane. RFC is a bidirectional transporter and since it is an organic anion antiporter driven by the organic phosphate gradient, it favors transport from CSF into the ependymal cells. In adult humans, PCFT, which transport folates from blood into the CSF, are expressed along the basolateral membrane of the choroid plexus, and FR, extracting folate from CSF, are abundantly expressed at the apical brush-border membrane within the CSF and, to a lesser extent, at the basolateral membrane (53, 81, 114, 125, 126).
However, multidrug transporters of the ATP-binding cassette family, such as P-gp, and MRPs may also play an important role in regulating the distribution and efflux of MTX in the brain and choroid plexus. As described Chapter 1 and Chapter 2, the potent P-gp and MRP1 modulator, CysA and specific P-gp modulator, Rho123 potentiated MTX concentrations in the rat brain when MTX was administered i.t.. However, neither
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modulator potentiated MTX concentrations when it was administered i.v. (87, 88).
Based on these findings, P-gp appears to be crucially involved in the efflux of MTX from the rat brain. However, it currently remains unclear whether folate carriers contribute to the efflux of MTX from the brain and CSF.
Folinic acid (FA) supplementation is essential in patients with CNS tumors for the treatment of severe neurological disorders caused by MTX (14). It may disturb the distribution of MTX in the brain because it is a folate derivative and, thus, may share folate carriers with MTX.
A previous study reported that a folate deficiency disrupted BBB function by targeting P-gp and tight junctions and also that FA supplementation restored BBB function to normal levels (114). MRP have been shown to transport not only MTX, but also folate derivatives, such as folic acid and FA (105, 123). These findings suggest that FA affects the retention of MTX in the brain when used to treat MTX-related adverse effects. Therefore, the author herein investigated the effects of co-medication with FA and i.v.- or i.t.-administered MTX on the retention of MTX in the rat brain.
- 69 -
4.3. Materials and methods
4.3.1. Animals
In the same manner as Chapter 1 and Chapter 2, male Sprague-Dawley rats (8 weeks old) were used (weighing between 251 and 310 g).
4.3.2. Chemicals
MTX and its polyglutamates were prepared as described in Chapter 1 and Chapter 2. FA solution (Leucovorin®) was purchased as a hydrochloride salt (Pfizer Inc., New York, U.S.A.).
4.3.3. Drug Administration and Sampling Protocol
The author considered two points to determine the FA and MTX dose.
Primarily, in order to avoid increases in intracranial pressure, i.t. injections should be performed after removing the same volume of CSF as the injection volume. Since the author could collect about 0.2 ml CSF from the rats, the author determined the FA and MTX injection volume as 0.1 ml respectively. Secondly, the author determined MTX and FA dose as following. When MTX is administered intrathecally, the clinical dose of MTX is 0.2-0.4 mg/kg in human. When MTX is administered intravenously, it is 5-10 mg/ body in human. However, since some of MTX is stored as polyglutamates, the author needed to analyze the polyglutamate concentrations. In addition, MTX
polyglutamates’ peaks were less than monoglutamate MTX. For that reason, the author administered MTX to rats at 2 mg/body (0.1 ml) to detect MTX and its polyglutamates in rat brains and CSF.
Next, FA is generally administered at 0.1-0.25 mg/kg to prevent the adverse
- 70 -
effects by MTX in human. However, since one of the author’s aims in this study was to examine if FA show competitive inhibition to MTX, the author defined FA dose as the maximum dose the author could administer intrathecally (0.3 mg/0.1ml).
In the same manner as Chapter 1 and Chapter 2, the administration of all drugs was performed. MTX (2 mg/body) and FA (0.3 mg/body) were simultaneously
injected into animals via an i.v. or i.t. route.
The author defined 5 groups as follows: group Miv: MTX (i.v.) + saline (i.t.), group Mit: MTX (i.t.), group Miv+Fiv: MTX (i.v.) + FA (i.v.) + saline (i.t.), group Mit+Fiv: MTX (i.t.) + FA (i.v.), and group Mit+Fit: MTX (i.t.) + FA (i.t.) (Table 4.1).
Blood (0.2 ml) was sampled from the caudal vein 1, 2, 3, 4, 5, and 6 hr after drug administration. In my preliminary study, the half-lives of i.v.- and i.t.-administered MTX were 35±3.2 and 84±20 min, respectively.
Rats in each group were euthanized 6 hr after drug administration following the sampling of CSF (0.1 ml) from the cisterna, and brains were collected (n=5,
respectively). In the same manner as Chapter 1 and Chapter 2, the brain samples were obtained and stored.
4.3.4. Sample Preparation
In the same manner as Chapter 1 and Chapter 2, the brain, CSF and plasma samples were prepared and subjected to a HPLC analysis for MTX.
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4.3.5. HPLC analysis
In the same manner as Chapter 1 and Chapter 2, MTX and its polyglutamates were analyzed using a HPLC system.
4.3.6. Pharmacokinetic Analysis
In the same manner as Chapter 2, a one-compartment open model was used to analyze the pharmacokinetics of MTX, and C0, kel, AUC, t1/2, Vd, and Clto) were calculated.
4.3.7. Statistical Analysis
In the same manner as Chapter 1 and Chapter 2, data are displayed as
meanssSD. Differences in mean values between groups were analyzed by Scheff«s multiple comparison test after a one-way ANOVA. Equal variances among the groups were confirmed by Bartlett’s test. Since the author examined if FA competitively inhibit MTX to penetrate into the brains and CSF, comparisons between Miv and Miv+Fiv were conducted using the one-sided Student’s t-test. Differences were considered to be significant at 3<0.05.
- 72 -
4.4. Results
As shown in Fig.4.1, no significant differences were observed in plasma concentration-time courses between the FA-treated and non-treated groups.
Furthermore, pharmacokinetic parameters, including kel, Vd, AUC, Cltot, and t1/2, were not significantly different between the FA-treated and non-treated groups (Table 4.2).
Fig. 4.2.A shows MTX concentrations in the brain and CSF 6 hrs after the administration of MTX. No significant differences were observed in MTX
concentrations in the brain or CSF among the i.t.-administered groups: Mit, Mit+Fiv, and Mit+Fit, suggesting that FA did not significantly affect MTX concentrations in the brain or CSF regardless of its i.v. or i.t. administration.
In contrast, as shown in Fig. 4.2.B, MTX concentrations in the brain and CSF were lower in the Miv+Fiv group than in the Miv group. MTX concentrations in the brain and CSF were significantly lower in the Miv+Fiv group than in the Miv group (P<0.05).
The brain to CSF MTX concentration ratio was compared among these groups, as shown in Fig. 4.3. The i.t.-administered groups: Mit+Fiv and Mit+Fit, had a slightly higher ratio than the Mit group. No significant differences were observed between the Miv and Miv+Fiv groups, suggesting that MTX concentrations were not shifted to the brains or the CSF.
- 73 -
4.5. Discussion
Since FA supplementation is essential for patients with CNS tumors to treat severe neurological disorders caused by MTX (14), the mechanisms by which it affects MTX in the brain and CSF need to be elucidated in more detail. Therefore, there were two aims in the present study.
(1) To clarify whether folate carriers (RFC, PCFT, and FR) are involved in effluxion of MTX from the CNS.
(2) To clarify whether folate carriers are involved in the influx of MTX into the CNS from blood.
Fig. 4.1 and Table 4.2 showed that FA did not have a significant pharmacokinetic interaction with MTX (2 mg/body) at the dose administered (0.3 mg/body), suggesting that FA did not affect the renal excretion of MTX.
FA did not significantly affect MTX concentrations in the brain or CSF when MTX was administered i.t., as shown in Fig. 4.2.A. MTX concentrations in the CSF were not significantly different between the Mit+FAiv and Mit+FAit groups, indicating that FA did not affect MTX transporters on the basolateral membrane or apical brush-border membrane at the B-CSFB. Therefore, folate transporters do not appear to contribute to the efflux of MTX from the brain and CSF.
Furthermore, as shown in Fig. 4.2 B, FA inhibited the influx of MTX into the brain and CSF. A significant difference was observed in MTX concentrations in the brain and CSF between the Miv and Miv+Fiv groups. In addition, the brain to CSF MTX concentration ratio was not significantly different between the Miv and Miv+Fiv
- 74 -
groups (Fig. 4.3), indicating that FA decreased MTX concentrations without shifting to the brains or the CSF. This indicates that FA did not only inhibit the influx of MTX into the CSF.
In other words, it is suggested that FA impaired both the BBB and B-CSFB functions in charge of PCFT and a part of FR to penetrate MTX into the brains and CSF (114, 125, 126). Probably, although FA competitively impaired all of the folate carriers, the physiological function to influx folates from blood into the CNS is superior to the function to efflux folates from the CNS, because FA did not significantly impaired to efflux intrathecally administered MTX in this study. Hence, it is suggested that FA competitively inhibit to influx MTX into the brain and CSF without significant pharmacokinetic interaction.
In conclusion, the results of the present study suggest that folate carriers play an important role in the influx of MTX into the brain and CSF, but not in its efflux from the CNS. Regarding the optimal dosage schedule, MTX is classified one of S-phase specific but self-limiting drugs. While the optimal schedule for the S-phase specific drugs was one which provided effective serum concentrations at intervals shorter than the median DNA synthesis time of cancers, increasing the concentration of S-phase specific but self-limiting drugs above some minimum level does not increase the effect to cancer cells but does increase host toxicity (104). Hence, it is important for a successful chemotherapy to keep MTX at minimum level for a longer time.
In addition, though FA did not affect plasma MTX concentration in this study, it is known that FA supplement is clinically available to decrease systemic host toxicity.
Therefore, even though FA were administered, the author might not have to change the
- 75 -
dosage schedule. Thus, FA may be administered to patients with CNS tumors receiving i.t. MTX therapy to treat the adverse effects of MTX without affecting its concentrations in the brain and CSF; however, FA may decrease these concentrations in patients
receiving i.v. MTX therapy at a high dose.
㻌
4.6 Acknowledgement
This chapter is based on an article described by Ogushi et al. under the title of “Effects of leucovorin (folinic acid) in the methotrexate-treated rat brain.”
in J. Vet. Med Sci.doi: 10.1292/jvms.17-0666, 2018.