P-gp/P-glycoprotein

Up to 0.75 μg/mL, valspodar (PSC 833) exhibits no cytotoxic effects. When valspodar (0.25, 0.5, and 0.75 μg/mL) is given in addition to DOX-L, the MDR cell type's cell-kill effectiveness increases noticeably. The most lethal combination of DOX and valspodar (0.5 and 0.75 μg/mL) kills almost 70% of the resistant cells[1]. When PSC833 is pretreated, the IC50 value of NSC 279836 in MDA-MB-435mdr cells is reduced to 0.4±0.02 μM in MDR cells, virtually reversing the resistance of MDR cells to NSC 279836[3].

Valspodar (10 mg/kg, op) has a low mean blood-to-plasma ratio of roughly 0.52 which indicates that it shows negligible blood-cell partitioning. Valspodar exhibits a significant volume of distribution and delayed clearance. Comparable to its structural homolog CsA, valspodar exhibits broad distribution and low hepatic extraction[2]. When Valspodar/PSC833 is preadministered to mice, the NSC 279836 fluorescence intensity in MDR tumors is increased to 94% of that in tumors of the wild type[3].

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Valspodar is a P-glycoprotein inhibitor widely used in preclinical and clinical studies for overcoming multidrug resistance. Despite this, the pharmacokinetics of valspodar in rat, a commonly used animal model, have not been reported. Here, we report on the pharmacokinetics of valspodar in Sprague–Dawley rats following intravenous and oral administration of its Cremophor EL formulation, which has been used for humans in clinical trials. After intravenous doses, valspodar displayed properties of slow clearance and a large volume of distribution. Its plasma unbound fraction was around 15% in the Cremophor EL formulation used in the study. After 10 mg kg−1 orally it was rapidly absorbed with an average maximal plasma concentration of 1.48 mg l−1 within approximately 2 h. The mean bioavailability of valspodar was 42.8%. In rat, Valspodar showed properties of low hepatic extraction and wide distribution, similar to that of its structural analogue cyclosporine A [2].

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Mitoxantrone Accumulation and Effects of Pgp Inhibitors in Xenograft Tumors in Living Mice. Confocal tumor images were used to evaluate mitoxantrone accumulation and the effect of Valspodar/PSC833 on the accumulation in MDA-MB-435 xenograft tumors in vivo. Images of the MDA-MB-435wt xenograft tumors demonstrated similar intracellular mitoxantrone localization in vivo as that observed in vitro and showed mitoxantrone localized in both nuclei and cytoplasm of the cells. However, images of MDR xenograft tumors in vivo showed that mitoxantrone mainly localized in the nuclei area (Fig. 3). Tumor image analysis demonstrated significant differences of mitoxantrone fluorescent intensity in the MDR and wild-type tumors (Table 2) (p < 0.05). Mitoxantrone fluorescent intensity in the MDR tumors was only 61% of that in the wild-type tumors. Preadministration of Valspodar/PSC833 to mice increased mitoxantrone fluorescent intensity in MDR tumor to 94% of that in the wild-type tumors. A similar intracellular localization pattern of mitoxantrone was observed in MDR and the wild-type tumors in the pretreated mice. Mitoxantrone accumulation in MDR tumors in the pretreated mice increased 36% compared with that in the nonpretreated animals.

In vitro cytotoxicity of various formulations against T47D/TAMR-6 cells is investigated by MTT assay. A 104 T47D/TAMR-6 cells are cultured in 96-well plate containing RPMI medium and incubated overnight to allow cell attachment. After 48 hours incubation, fresh medium containing serial concentration of various drug formulations, including free DOX, DOX-L, mixture of DOX-L and free Valspodar (PSC 833), mixture of DOX-L and PSC-L and DOX/PSC-L are added. The plates are then incubated for an additional 48 hours before washing with normal saline followed by adding MTT solution (0.5 mg/mL) to each well, and incubated for 4 h at 37°C. Then, the medium is removed, and DMSO is added to dissolve the formazan crystals. The plates are mildly shaken for 10 min to ensure the dissolution of formazan. The formazan dye is measured spectrophotometrically using microplate reader at 570 nm with reference standard of 690 nm as described before.

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Pgp Expression, Resistance to Mitoxantrone, and the Resistance Reversal by Valspodar/PSC833. [3]
Pgp expression was determined by Western blot assay. Cell lysates used for the analysis were prepared from crude cell membranes as described previously (Shen et al., 2008). Protein concentration in the cell lysates was determined with the Bradford assay (Bradford, 1976), and equal amounts of proteins were loaded on gels. Cell lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The blot was then probed with the primary antibody C219 (dilution 1:1000), followed by reaction with horseradish peroxidase-conjugated secondary antibody. The signal was detected using enhanced chemiluminescence and exposure of X-ray film.

Colony formation assays were used to evaluate the resistance of MDA-MB-435 cells to mitoxantrone and the reversal of the resistance by Valspodar/PSC833 (Shen et al., 2008). Cells were seeded in flasks and incubated under standard conditions overnight. Each group of three flasks of cells was treated with a different dose of mitoxantrone for 1 h. To assess the reversal of MDR by PSC833, the cells were pretreated with PSC833 30 min before mitoxantrone exposure. The cells were then washed, harvested, counted, and seeded into triplicate culture dishes. Colonies were fixed and visually counted after 14 days.

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Mitoxantrone Intracellular Accumulation and Effects of Valspodar/PSC833. [3]
MDA-MB-435 cells were seeded on coverslips in 12-well plates and allowed to grow overnight. On the following day, cells were washed with phosphate-buffered saline, incubated with or without 3 mg/ml PSC833 for 30 min before being treated with 5 μM mitoxantrone for 2 h, and then examined using confocal microscopy.

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Dynamic Assessment of Mitoxantrone Net Uptake, Efflux, and Effects of Valspodar/PSC833 in MDA-MB-435 Cells. [3]
MDA-MB-435 cells were seeded on coverslips overnight. The coverslips were mounted in microscope chambers. They were placed on the microscope stage and perfused sequentially with mitoxantrone-free medium for 6 min, medium with 5 μM mitoxantrone for 2 h (uptake perfusion), and then mitoxantrone-free medium again for 1 h (efflux perfusion). Serial images at 2-min intervals were collected and analyzed. To study the effect of PSC833 on the time course of mitoxantrone accumulation and efflux, MDA-MB-435 cells grown on coverslips were pre-exposed to 3 mg/ml Valspodar/PSC833 for 30 min before being mounted in microscope chambers for perfusion as described above.

Male Sprague–Dawley rats (250-350 g) wer housed in temperature-controlled rooms with 12 h of light per day. The animals had free access to food and water prior to experimentation. Rats are divided into two groups: one group (n=6) receives intravenous dose (5 mg/kg) of Valspodar and the other group administered valspodar orally (10 mg/kg). Stereoselective pharmacokinetics of desbutylhalofantrine, a metabolite of halofantrine, in the rat after administration of the racemic metabolite or parent drug. After surgery, the rats are transferred to their regular holding cages and allowed free access to water, but food is withheld overnight. The next morning, rats are transferred to the metabolic cages and dosed with valspodar.

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Animals [2]
Male Sprague–Dawley rats (250–350 g) were housed in temperature-controlled rooms with 12 h of light per day. The animals had free access to food and water prior to experimentation. Rats were divided into two groups: one group (n = 6) received intravenous dose (5 mg kg−1) of valspodar and the other group (n = 5) administered Valspodar orally (10 mg kg−1). The right jugular vein of all rats was cannulated with Silastic® Laboratory Tubing under isofluorane anaesthesia as previously described. After surgery, the rats were transferred to their regular holding cages and allowed free access to water, but food was withheld overnight. The next morning, rats were transferred to the metabolic cages and dosed with valspodar.

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Drug administration and sample collection [2]
Standard Valspodar formulation (Valspodar 50 mg and Cremophor EL 600 mg ml−1 in ethanol) was diluted in saline to a final concentration of 5 mg ml−1 and used for both intravenous and oral administration (CitationWatanabe et al. 1996). The intravenous dose was injected over 2 min via the jugular vein cannula, immediately followed by injection of normal saline solution. At the time of first sample withdrawal, the first 0.2 ml volume of blood were discarded. For oral dosing, the rats received the desired dose by oral gavage. For both routes of administration, food was provided to animals 4 h after the dose administration. Serial blood samples (0.15–0.25 ml) were collected at 0.08, 0.25, 0.75, 1, 2, 4, 6, 9, and 12 h post-dose for intravenous dosing and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 24, and 48 h post-dose for oral dosing into polypropylene microcentrifuge tubes. Heparin in normal saline was used to flush the cannula after each collection of blood. Blood samples were immediately centrifuged for 3 min; plasma was separated and stored at −20°C until analysis. The plasma concentrations of valspodar were analysed by a liquid chromatography-mass spectrometry (LC-MS) method and the plasma concentration versus time curve was profiled.

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Mitoxantrone Accumulation and Effects of Valspodar/PSC833 in Xenograft Tumors in Living Mice. [3]
Living nude mice bearing subcutaneous MDA-MB-435wt and MDA-MB-435mdr tumor xenografts in opposite flanks were injected with or without 50 mg/kg PSC833 intraperitoneally 1 h before receiving intravenous injection of 12.5 mg/kg mitoxantrone. Confocal tumor images were taken 2 h after the mice had received mitoxantrone. To obtain the images, the mice were anesthetized and a small skin incision was made to expose the tumor xenografts. The mice were placed on the microscope stage connected to a water circulator set to 37°C.

Metabolism / Metabolites
Valspodar's known human metabolites include 9-(3-hydroxy-2-methylpropyl)-1,4,7,10,12,15,19,25,28-nonylmethyl-33-[(E)-2-methylhex-4-enoyl]-6,18,24-tris(2-methylpropyl)-3,21,30-tris(propane-2-yl)-1,4,7,10,13,16,19,22,25,28,31-undecazatecyclotritane-2,5,8,11,14,17,20,23,26,29,32-undecane.

[1]. Co-delivery of NSC 123127 and PSC 833 (Valspodar) by stealth nanoliposomes for efficient overcoming of multidrug resistance. J Pharm Pharm Sci. 2012 Sep;15(4):568-82.

[2]. Pharmacokinetics of PSC 833 (valspodar) in its Cremophor EL formulation in rat.2010,40(1):55-61.

[3]. Dynamic Assessment of NSC 279836 Resistance and Modulation of Multidrug Resistance by Valspodar (PSC833) in Multidrug Resistance Human Cancer Cells. J Pharmacol Exp Ther. 2009 Aug;330(2):423-9.

SDZ PSC 833 is a homologous cyclic peptide. Valspodar has been used in therapeutic trials for various cancers, including cancer, sarcoma, leukemia, lymphoma, and breast cancer. Valspodar is an analog of cyclosporine A. Valspodar inhibits P-glycoprotein (a multidrug resistance efflux pump), thereby restoring the retention and activity of certain drugs in certain drug-resistant tumor cells. This drug can also induce caspase-mediated apoptosis. (NCI04) Valspodar is a protein drug currently in Phase III clinical trials (covering all indications) and has 18 investigational indications. Objective: This study aims to develop stealthy nanoliposomes co-encapsulated with the highly effective multidrug resistance (MDR) modulator PSC 833 and doxorubicin (DOX) to improve the efficacy of anticancer drugs and reduce their adverse reactions. Methods: To improve drug encapsulation efficiency, we tested different liposome preparation methods and investigated the effects of various parameters, including the molar ratio of drug to lipid, the molar percentage of cholesterol, and lipid composition. The final product was prepared using a thin-film hydration method, with a lipid composition of EPC:DSPE-PEG2000:Chol (60:5:30 mol). After preparing empty liposomes, doxorubicin (DOX) and sodium polystyrene sulfonate (PSC) 833 were loaded into the liposomes using the ammonium sulfate gradient method and the remote thin-film method, respectively. The physical properties of the optimized liposomes (DOX/PSC-L) were evaluated, including particle size, zeta potential, encapsulation efficiency, in vitro drug release, and stability. Furthermore, the in vitro cytotoxicity of different liposome formulations and drug solutions to the drug-resistant human breast cancer cell line T47D/TAMR-6 was evaluated using the MTT assay.
Results: The optimal formulation had a narrow particle size distribution, with an average particle size of 91.3 ± 0.2 nm and a zeta potential of -6 ± 1.2 V. The encapsulation efficiency of DOX and PSC 833 exceeded 95% and 65.5%, respectively. In doxorubicin (DOX) resistant T47D/TAMR-6 cells, the dual-drug stealth liposomes showed significantly higher cytotoxicity than treatment with free doxorubicin and liposomal doxorubicin combined with free PSC 833 (P < 0.05).
Conclusion: Co-encapsulation of doxorubicin and PSC 833 is a promising anticancer agent that can effectively reverse drug resistance and should be further investigated in animal tumor xenograft models. [1]

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This study is the first to report the detailed pharmacokinetics of valspodar after intravenous and oral administration in rats. This study investigated the pharmacokinetics of valspodar in rats and compared it with the pharmacokinetics in humans and cyclosporine A (CyA) in rats. After intravenous administration, the plasma concentration-time curve of valspodar showed a multi-exponential decrease and a long elimination half-life. After oral administration, the drug showed a rapid absorption phase, reaching peak at about 2 hours after administration, with an average bioavailability of over 40%. Overall, the pharmacokinetic characteristics of valspodar in rats were similar to those of cyclosporine A (CyA), although there were significant differences in oral bioavailability and hematopoietic/protein binding. [2] P-glycoprotein (Pgp) is a member of the ATP-binding cassette transporter family and is one of the main causes of multidrug resistance (MDR). We used confocal microscopy to study the role of Pgp in mediating the efflux of the anticancer drug mitoxantrone and the effect of the specific Pgp inhibitor valspodar (PSC833) in reversing MDR. In this study, we quantitatively analyzed and compared the net uptake and efflux of mitoxantrone in human cancer cells MDA-MB-435 (MDR) expressing Pgp and their parental wild-type cells, as well as the effect of PSC833. MDR cells transfected with the human Pgp-encoding gene MDR1 construct were about 8 times more resistant to mitoxantrone than wild-type cells. The accumulation of mitoxantrone in MDR cells was 3 times lower than that in wild-type cells. The net uptake of mitoxantrone in the nucleus and cytoplasm of MDR cells was only 58% and 67% of that in the corresponding intracellular compartments of wild-type cells, respectively. Pretreatment with PSC833 increased the accumulation of mitoxantrone in MDR cells to 85% of that in wild-type cells. In live animals, the accumulation of mitoxantrone in MDA-MB-435mdr xenograft tumors was 61% of that in wild-type tumors. Pretreatment with PSC833 in animals before mitoxantrone treatment increased the accumulation of mitoxantrone in MDR tumors to 94% of that in wild-type tumors. These studies provide direct in vitro and in vivo visualization of how Pgp processes anticancer compounds and how Pgp inhibitors modulate MDR in drug-resistant cancer cells. [3]

C63H111N11O12

1214.62

1213.841

C, 62.30; H, 9.21; N, 12.68; O, 15.81

121584-18-7

121584-18-7

5281884

White to yellow solid powder

1.0±0.1 g/cm3

1290.1±65.0 °C at 760 mmHg

734.0±34.3 °C

0.0±0.3 mmHg at 25°C

1.467

4.1

4

12

15

86

2410

11

C/C=C/C[C@@H](C)C(=O)[C@H]1C(=O)N[C@H](C(=O)N(CC(=O)N([C@H](C(=O)N[C@H](C(=O)N([C@H](C(=O)N[C@H](C(=O)N[C@@H](C(=O)N([C@H](C(=O)N([C@H](C(=O)N([C@H](C(=O)N1C)C(C)C)C)CC(C)C)C)CC(C)C)C)C)C)CC(C)C)C)C(C)C)CC(C)C)C)C)C(C)C

YJDYDFNKCBANTM-QCWCSKBGSA-N

InChI=1S/C63H111N11O12/c1-26-27-28-41(16)53(76)52-57(80)67-49(38(10)11)61(84)68(19)33-48(75)69(20)44(29-34(2)3)56(79)66-50(39(12)13)62(85)70(21)45(30-35(4)5)55(78)64-42(17)54(77)65-43(18)58(81)71(22)46(31-36(6)7)59(82)72(23)47(32-37(8)9)60(83)73(24)51(40(14)15)63(86)74(52)25/h26-27,34-47,49-52H,28-33H2,1-25H3,(H,64,78)(H,65,77)(H,66,79)(H,67,80)/b27-26+/t41-,42+,43-,44+,45+,46+,47+,49+,50+,51+,52+/m1/s1

(3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-1,4,7,10,12,15,19,25,28-nonamethyl-33-[(E,2R)-2-methylhex-4-enoyl]-6,9,18,24-tetrakis(2-methylpropyl)-3,21,30-tri(propan-2-yl)-1,4,7,10,13,16,19,22,25,28,31-undecazacyclotritriacontane-2,5,8,11,14,17,20,23,26,29,32-undecone

Valspodar; 121584-18-7; Amdray; Sdz psc 833; PSC-833; Psc 833; Sdz-psc-833; PSC833;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~82.33 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.06 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)