Development and validation of ultra-high-performance liquid chromatography-mass spectrometry method for the determination of raloxifene and its phase II metabolites in plasma: Application to pharmacokinetic studies in rats.

The aim of this study is to establish a reliable liquid chromatography-mass spectrometry method to simultaneously quantitate raloxifene, and its major metabolites, raloxifene-6-glucuronide, raloxifene-4'-glucuronide, and raloxifene-6-sulfate in rat plasma samples for pharmacokinetic studies. The separation of the analytes was achieved on a Waters BEH C18 column. Water (0.1% formic acid) and acetonitrile were used as the mobile phases for elution. A one-step protein precipitation using a mixture solvent was applied for plasma sample preparation. The method was validated following the FDA guidance. The results showed that the linear range were 1.95-1000 nM for raloxifene-6-glucuronide, and raloxifene-4'-glucuronide, 0.195-100 nM for raloxifene-6-sulfate, and 0.195-200 nM for raloxifene, respectively. The lower limit of quantification was 1.95, 1.95, 0.195, and 0.195 nM for raloxifene-6-glucuronide, raloxifene-4'-glucuronide, raloxifene-6-sulfate, and raloxifene, respectively. Only 20 µl of plasma sample was required since the method is sensitive. The intra- and interday variance is <15% and the accuracy is within 85-115%. The variance of matrix effect and recovery were <15%. The method was successfully applied in a pharmacokinetic study in rats with oral administration of raloxifene.

Sonochemical synthesis and fabrication of neodymium sesquioxide entrapped with graphene oxide based hierarchical nanocomposite for highly sensitive electrochemical sensor of anti-cancer (raloxifene) drug.

The ultrasound-assisted synthesis of a novel neodymium sesquioxide nanoparticles (Nd2O5 NPs) decorated graphene oxide (GO) nanocomposite under ultrasonic probe (Ultrasonic processor model-PR 1000; frequency-30 kHz; power of 100 W/cm2) has been reported. After then, SEM, TEM, XRD, EDX and electrochemical impedance spectroscopy characterized was analyzed using Nd2O5 NPs@GO nanomaterial. Furthermore, the nanomaterial modified GCE (glassy carbon electrode) shows excellent electrochemical sensing performance towards anti-cancer drug. Raloxifene is one of the important anti-cancer drug. Moreover, the fabricated electrochemical sensor has showed a wide linear range for raloxifene between 0.03 and 472.5 µM and nanomolar detection limit (18.43 nM). In addition, the Nd2O5 NPs@GO modified sensor has been applied to the determination of raloxifene in human blood and urine samples.

Design of a transdermal formulation containing raloxifene nanoparticles for osteoporosis treatment.

PURPOSE: In the clinical setting, raloxifene, a second-generation selective estrogen receptor modulator, is administered orally; however, the bioavailability (BA) is only 2% because of its poor solubility in aqueous fluids and its extensive first-pass metabolism. Therefore, it is expected that the development of a transdermally delivered formulation may reduce the necessary dose without compromising its therapeutic efficacy. In this study, we designed transdermal formulations containing raloxifene nanoparticles and evaluated their usefulness for osteoporosis therapy. METHODS: Raloxifene was crushed with methylcellulose by the bead mill method, and the milled raloxifene was gelled with or without menthol (a permeation enhancer) by Carbopol® 934 (without menthol, Ral-NPs; with menthol, mRal-NPs). The drug release and transdermal penetration were measured using a Franz diffusion cell, and the therapeutic evaluation of osteoporosis was determined in an ovariectomized rat model. RESULTS: The mean particle size of raloxifene in the transdermal formulation (Ral-NPs) was 173.7 nm. Although the raloxifene released from Ral-NPs remained in the nanoparticle state, the skin penetration of raloxifene nanoparticles was prevented by the stratum corneum in rat. On the other hand, inclusion of menthol in the formulation attenuated the barrier function of the stratum corneum and permitted the penetration of raloxifene nanoparticles through the skin. Moreover, macropinocytosis relates to the skin penetration of the formulation including menthol (mRal-NPs), since penetration was inhibited by treatment with 2 µM rottlerin, a macropinocytosis inhibitor. In addition, the application of 0.3% mRal-NPs (once a day) attenuated the decreases in calcium level and stiffness of the bones of ovariectomized rat. CONCLUSION: We prepared raloxifene solid nanoparticles by a bead mill method and designed a novel transdermal formulation containing nanoparticles and permeation enhancers. These trans-dermal formulations overcome the barrier properties of the skin and show high drug penetration through the transdermal route (BA 8.5%). In addition, we found that raloxifene transdermal formulations are useful for the treatment of osteoporosis in ovariectomized rat.

Transdermal delivery of raloxifene HCl via ethosomal system: Formulation, advanced characterizations and pharmacokinetic evaluation.

Raloxifene HCl belongs to a class of selective estrogen receptor modulators (SERMs) which is used for the management of breast cancer. The major problem reported with raloxifene is its poor bioavailability which is only up to 2%. The main objective of the present work was to formulate raloxifene loaded ethosomal preparation for transdermal application and compare it with an oral formulation of the drug. Five ethosomal formulations with different concentrations of ethanol and a conventional liposomes formulation were prepared by rotary evaporation method. The prepared systems were characterised by high resolution transmission electron microscopy (HRTEM), force emission electron microscopy (FESEM), atomic force microscopy (AFM), X-ray diffraction (XRD) and 31P NMR study. All these advanced characterization study established that the ethosome formulation was well defined by its size, shape and its bilayer formation. Transdermal flux of the optimized ethosome formulation was 22.14 ±â€¯0.83 µg/ml/cm2 which was 21 times higher when compared to the conventional liposomes. Confocal microscopy study revealed an enhanced permeation of coumarin-6 dye loaded ethosomes to much deeper layers of skin when compared with conventional liposomes. The gel was found to be pseudoplastic with elastic behaviour. In-vivo studies on rats showed a higher bioavailability of RXL (157% times) for ethosomal formulation when compared with the oral formulation. In conclusion, RXL loaded ethosomal formulation via transdermal route showed superior drug delivery properties as compared to oral formulation.

Poly(methyl vinyl ether-co-maleic acid) for enhancement of solubility, oral bioavailability and anti-osteoporotic effects of raloxifene hydrochloride.

Raloxifene HCl (RH) has poor water solubility and due to its extensive first pass metabolism; its bioavailability is only 2%. The purpose of the present study was to enhance the aqueous solubility, oral bioavailability and anti-osteoporotic effects of RH by electro-sprayed nanoparticles (NPs) in ovariectomized rats. NPs containing RH and different ratio of poly(methyl vinyl ether-co-maleic acid) (PMVEMA) were electrosprayed. The voltage, distance of needle to the collector, flow rate of the solution and polymeric percentage were optimized according to the size of NPs and drug solubility. The optimized formulation was characterized by SEM, XRD, DSC, and FTIR. The pharmacokinetic parameters were studies by oral administration of a single dose of 15mg/kg in Wistar rats. The anti-osteoporotic effects were studied in female ovariectomized rats. Animals were treated with 6mg/kg/day for 2months then serum calcium, phosphorous and alkaline phosphatase levels were measured. RH loaded electrosprayed NPs showed 10-fold enhanced solubility compared to the free drug. Moreover, the XRD and SEM tests displayed an amorphous state of drug in the NPs. FTIR and DSC tests revealed no interaction between the polymer and the drug. Serum calcium, phosphorous and alkaline phosphatase levels were significantly decreased in ovariectomized rats receiving oral RH NPs (P<0.05). No significant difference was detected between RH NPs and estradiol groups (P>0.05). Oral bioavailability of NPs showed 7.5-fold increase compared to the pure drug. The electrosprayed PMVEMA nanoparticles can enhance solubility, bioavailability and antiosteoporotic effects of RH.

Stearoyl-CoA desaturase-1, a novel target of omega-3 fatty acids for reducing breast cancer risk in obese postmenopausal women.

BACKGROUND/OBJECTIVES: Conversion of saturated fatty acids to monounsaturated fatty acids by the enzyme stearoyl-Co-A-desaturase (SCD-1) is emerging as a major factor in promoting carcinogenesis including breast cancer. The aim of our study was to explore the regulation of SCD-1 by Raloxifene and omega-3 fatty acids in women at increased risk of breast cancer based on high breast density. SUBJECTS/METHODS: As a reflection of SCD-1 activity, we measured the ratios of palmitoleic acid (C16:1n7) to palmitic acid (C16:0) (SCD-16) and oleic acid (C18:1n9) to steric acid (C18:0) (SCD-18) in plasma samples of postmenopausal women enrolled in our clinical trial (NCT00723398) designed to test the effects of the antiestrogen, Raloxifene and/or the omega-3 preparation Lovaza, on breast density, a validated biomarker of breast cancer risk. RESULTS: We report that Lovaza but not Raloxifene-reduced SCD-16 and SCD-18 for the 2-year duration of the trial. Importantly, decreasing levels of SCD-16 and SCD-18 were associated with a progressive reduction in breast density but only in obese women (body mass index ⩾30). CONCLUSIONS: Body mass index-related factors play an important role in the reduction of breast density and hence breast cancer risk by omega-3 fatty acids. SCD-1 may be a useful biomarker in future clinical trials testing the benefit of nutritional interventions in reducing obesity-associated breast cancer risk.

Effects of raloxifene against letrozole-induced bone loss in chemically-induced model of menopause in mice.

INTRODUCTION: The deleterious effects of letrozole, an aromatase inhibitor, used in the adjuvant treatment of breast cancer in postmenopausal women, on bone are well-documented and represent a major drawback to its clinical use. Raloxifene, a selective estrogen receptor modulator and a clinically approved anti-osteoporotic drug, has been recently demonstrated to be efficacious in women with breast cancer. The present study evaluated the effects of preventive and curative treatment with raloxifene on letrozole-induced alterations of bone microarchitecture and turnover markers in a chemically-induced menopause model in mice. METHOD: Swiss strain albino female mice were made menopausal by inducing ovotoxicity using vinyl cyclohexene di epoxide (VCD, 160 mg/kg for 15 days followed by 30 days drug-free period) confirmed by ovarian histology and serum estradiol levels. Effects on femoral and lumbar bones were evaluated by micro CT determination of bone volume, trabecular number, separation, thickness, connective density and trabecular pattern factor and bone turnover markers including ALP, TRAP5b, hydroxyproline and RANKL. In addition to these, markers of Wnt signaling (sclerostin and dickkopf-1) were also evaluated. To rule out the involvement of pharmacokinetic interaction, plasma levels of letrozole and raloxifene were measured following drugs alone and in combination. RESULTS: Though bone loss was observed in VCD treated mice (as indicated by micro CT measurements), it was further enhanced with letrozole administration (1 mg/kg) for one month particularly in epiphysis of femoral bones. Raloxifene (15 mg/kg), whether administered concurrently or post-letrozole was able to revert the structural alterations and changes in turnover markers caused by letrozole to varying degrees (p < 0.01 or p < 0.001). Further, estrogen deficiency following letrozole treatment in ovotoxic mice was associated with significant increase in sclerostin and dickkopf-1 in both lumbar and femur bones (p < 0.001) which was attenuated with preventive and curative treatment with raloxifene (p < 0.05). The plasma levels of letrozole remained unaffected by raloxifene administration and vice versa. CONCLUSIONS: Our study indicates the potential of raloxifene in preventing and attenuating letrozole-induced bone loss. Further, these effects were found to be independent of a pharmacokinetic interaction between the two drugs.

Formulation and optimization of raloxifene-loaded solid lipid nanoparticles to enhance oral bioavailability.

The main aim of this study was to improve the oral bioavailability of raloxifene (RXF), a selective estrogen receptor modulator, by incorporation into solid lipid nanoparticles (SLN). RXF-loaded SLN was prepared by homogenization-sonication technique and characterized through physicochemical, pharmacokinetic, and cytotoxicity studies. The optimized SLN formulation exhibited a spherical shape with average size around 140 nm, easing its transport across the lymphatic system. Augmentation in the profiles of C(max) (308%) and AUC (270%) indicated a significant enhancement in the rate and extent of bioavailability by SLN formulations compared to free drug. In vitro cytotoxicity study performed in NIH-3T3 cells revealed that RXF-SLN was cytocompatible, and SLN remained unchanged during the freeze-drying process. Furthermore, the optimized formulation was quite stable at room temperature for more than two months, exemplifying its superior performance. In conclusion, SLN provides a promising platform for the pronounced enhancement of RXF bioavailability.

Development of raloxifene-solid dispersion with improved oral bioavailability via spray-drying technique.

The purpose of this study was to develop a raloxifene-loaded solid dispersion with enhanced dissolution rate and bioavailability via spray-drying technique. Solid dispersions of raloxifene (RXF) were prepared with PVP K30 at weight ratios of 1:4, 1:6 and 1:8 using a spray-drying method, and characterized by differential scanning calorimetry, X-ray powder diffraction, scanning electron microscopy, and solubility and dissolution tests. The bioavailability of the solid dispersion in rats was also evaluated compared to those of RXF powder and commercial product. Results showed that the RXF-loaded solid dispersion was in amorphous form with increased solubility and dissolution rate. The absorption of RXF from solid dispersion resulted in approximately 2.6-fold enhanced bioavailability compared to pure drug. Moreover, RXF-loaded solid dispersion gave similar AUC, C(max) and T(max) values to the commercial product, suggesting that it was bioequivalent to the commercial product in rats. These findings suggest that an amorphous solid dispersion of RXF could be a viable option for enhancing the oral bioavailability of RXF.

Preparation and evaluation of raloxifene-loaded solid dispersion nanoparticle by spray-drying technique without an organic solvent.

The aim of this study was to improve the physicochemical properties and bioavailability of a poorly water-soluble drug, raloxifene by solid dispersion (SD) nanoparticles using the spray-drying technique. These spray-dried SD nanoparticles were prepared with raloxifene (RXF), polyvinylpyrrolidone (PVP) and Tween 20 in water. Reconstitution of optimized RXF-loaded SD nanoparticles in pH 1.2 medium showed a mean particle size of approximately 180 nm. X-ray diffraction and differential scanning calorimetry indicated that RXF existed in an amorphous form within spray-dried nanoparticles. The optimized formulation showed an enhanced dissolution rate of RXF at pH 1.2, 4.0, 6.8 and distilled water as compared to pure RXF powder. The improved dissolution of raloxifene from spray-dried SD nanoparticles appeared to be well correlated with enhanced oral bioavailability of raloxifene in rats. Furthermore, the pharmacokinetic parameters of the spray-dried SD nanoparticles showed increased AUC(0-∞) and C(max) of RXF by approximately 3.3-fold and 2.3-fold, respectively. These results suggest that the preparation of RXF-SD nanoparticles using the spray drying technique without organic solvents might be a promising approach for improving the oral bioavailability of RXF.

Electrochemistry of raloxifene on glassy carbon electrode and its determination in pharmaceutical formulations and human plasma.

The electrochemical behavior of raloxifene (RLX) on the surface of a glassy carbon electrode (GCE) has been studied by cyclic voltammetry (CV). The CV studies were performed in various supporting electrolytes, wide range of potential scan rates, and pHs. The results showed an adsorption-controlled and quasi-reversible process for the electrochemical reaction of RLX, and a probable redox mechanism was suggested. Under the optimum conditions, differential pulse voltammetry (DPV) was applied for quantitative determination of the RLX in pharmaceutical formulations. The DPV measurements showed that the anodic peak current of the RLX was linear to its concentration in the range of 0.2-50.0µM with a detection limit of 0.0750µM, relative standard deviation (RSD %) below 3.0%, and a good sensitivity. The proposed method was successfully applied for determination of the RLX in pharmaceutical and human plasma samples with a good selectivity and suitable recovery.

Increased antitumor potential of the raloxifene prodrug, raloxifene diphosphate.

Raloxifene (RAL) significantly reduced the incidence of breast cancer in women at high risk of developing the disease. Unlike tamoxifen (TAM), an increased incidence of endometrial cancer was not observed in women treated with RAL. However, RAL, having two hydroxyl moieties, can be conjugated rapidly through phase II metabolism and excreted, making it difficult to achieve adequate bioavailability by oral administration in humans. As a result, higher doses must be administered to obtain an efficacy equivalent to that achieved with TAM. To improve oral bioavailability and antitumor potential, RAL diphosphate was prepared as a prodrug. RAL diphosphate showed several orders of magnitude lower binding potential to both ER alpha and ER beta and weak antiproliferative potency on cultured human MCF-7 and ZR-75-1 breast cancer cells, as compared to RAL. However, RAL diphosphate has a much higher bioavailability than RAL, endowing it with higher antitumor potential than RAL against both 7,12-dimethylbenz(a)anthracene-induced mammary carcinoma in rats and human MCF-7 breast cancer implanted in athymic nude mice. The RAL prodrug may provide greater clinical benefit for breast cancer therapy and prevention.

The potential influence of CO2, as an agent for euthanasia, on the pharmacokinetics of basic compounds in rodents.

Rodent tissue distribution and pharmacokinetic studies were performed on basic compounds Org A and Org B in support of central nervous system drug discovery programs. A consistent observation from these studies was that drug concentrations in plasma obtained by cardiac puncture after CO(2) euthanasia were markedly higher compared with those from other sampling methods (serial sampling, isoflurane anesthesia, or cervical dislocation). Further investigations demonstrated that CO(2) euthanasia led to a reduction in blood pH in both rats and mice, which was not observed with the other sampling methods. The use of CO(2) euthanasia resulted in a decrease in the brain/plasma ratio of Org B, largely as a result of increased plasma concentrations. The pharmacokinetics of a basic drug, raloxifene, in rat were also influenced by sampling technique. CO(2) euthanasia before sampling, resulted in a 2- to 3-fold increase in the area under the drug concentration-time curve, a decrease in plasma clearance, and a decrease in the steady-state volume of distribution compared with isoflurane anesthesia. It is proposed that a decrease in the pH of blood relative to that of other tissues, as a consequence of CO(2) exposure, results in a redistribution of basic compounds out of the tissues, leading to higher concentrations in plasma.

Pharmacokinetics of raloxifene in male Wistar-Hannover rats: influence of complexation with hydroxybutenyl-beta-cyclodextrin.

Raloxifene is a highly insoluble, highly metabolized serum estrogen receptor modulator approved for use in the treatment of osteoporosis. Hydroxybutenyl-beta-cyclodextrin (HBenBCD) is a novel solubility enhancer previously demonstrated to increase the oral bioavailability of tamoxifen, letrozole, and itraconazole. The current study evaluated the pharmacokinetics of raloxifene in oral and intravenous formulations with HBenBCD in male Wistar-Hannover rats. Analytical methodology to measure raloxifene and its metabolites was developed by measuring raloxifene metabolism in vitro. Formulation with HBenBCD significantly increased raloxifene oral bioavailability. Mean+/-S.D. oral bioavailabilities were 2.6+/-0.4% for raloxifene formulated with microcrystalline cellulose, 7.7+/-2.1% for a solid capsule formulation of raloxifene:HBenBCD complex, and 5.7+/-1.3% for a liquid-filled capsule formulation containing raloxifene:HBenBCD/PEG400/H(2)O. Relative to raloxifene/microcrystalline filled capsules, the presence of HBenBCD in the solid capsule formulation afforded: (i) a decrease in raloxifene T(max) (2.5+/-0.5h versus 4.0+/-0.5h); (ii) a two-fold increase in raloxifene C(max) and a three-fold increase in raloxifene AUC; and (iii) a 12-fold increase in raloxifene glucuronide C(max) and a 6.5-fold increase in raloxifene glucuronide AUC. Hence, these studies demonstrate that raloxifene formulations containing HBenBCD significantly increased the oral bioavailability in rats relative to formulations that did not contain HBenBCD.

Development and validation of a liquid chromatography-tandem mass spectrometry assay for determination of raloxifene and its metabolites in human plasma.

This paper describes the development and validation of a method for the detection of raloxifene (Ral) and its two glucuronide metabolites, raloxifene-6-glucuronide (M1) and raloxifene-4'-glucuronide (M2), in human plasma samples. Both glucuronides were synthesized enzymatically, purified and used as authentic standards. The assay involves a simple solid phase extraction (SPE) procedure of 0.5 mL of human plasma and subsequent analysis by LC-MS-MS. The recoveries were higher than 71% and chromatographic separation of all the analytes was accomplished in less than 7 min. Linear ranges (r(2)>0.99) were found from 0.200 to 340 microg/L, from 1.600 to 2720 microg/L and from 0.088 to 60.00 microg/L, for M1, M2 and Ral, respectively. The limits of detection achieved were 8, 11 and 6 ng/L for M1, M2 and Ral, respectively. The method presented was successfully applied to a genetic polymorphism study of 47 plasma samples from women taking Evista (raloxifene hydrochloride).

Raloxifene pharmacokinetics in males with normal and impaired renal function.

AIMS: To compare raloxifene pharmacokinetics between renally impaired and healthy subjects. METHODS: Raloxifene 120 mg was administered to 10 males with renal impairment (creatinine 2-4 mg dl(-1)) and to 10 healthy males. Data were analysed by two noncompartmental and one compartmental nonlinear regression methods. RESULTS: The medians (95% confidence interval) of the area under the curves (AUC) were 35.1 (25.8, 74) and 20.5 (16.8, 28.0) h ng ml(-1) per mg kg(-1), P < 0.01, and of the clearances (CL/F) were 28.5 (13.5, 38.8) and 48.8 (35.8, 59.4) l h(-1) kg(-1), P < 0.01, in renally impaired and healthy subjects, respectively. 95% Confidence intervals on the differences for AUC and CL/F were 6.5-44.1 and -35.1 to -7.9, respectively. CONCLUSION: Exposure to raloxifene was twice as high in males with renal impairment compared with healthy subjects.

Divergent effects of raloxifene HCI on the pharmacokinetics and pharmacodynamics of warfarin.

PURPOSE: Evista (raloxifene HCl) is a nonsteroidal selective estrogen receptor modulator that displays estrogen agonist effects on bone and lipid metabolism but estrogen antagonist effects on the breast and endometrium. The potential for drug-drug interaction between raloxifene and warfarin was assessed in 15 healthy postmenopausal women. METHODS: Single doses of warfarin (20 mg) were administered prior to and during 2 weeks of dosing with raloxifene 120 mg/day. Each warfarin dose was followed by pharmacokinetic sampling and prothrombin time measurements. RESULTS: Raloxifene administration resulted in 7.1% and 14.1% decreases in the clearance (CLp/F) and 7.4% and 9.8% decreases in the volume of distribution (Vss/F) of R- and S-warfarin, respectively (all p < or = 0.05). In contrast to the slightly higher plasma concentrations of R- and S-warfarin, raloxifene reduced the maximum prothrombin time (PTmax) by 10% and the area under the PT versus time curve from 0-120 h (AUCPT) by 8% (p < 0.01). CONCLUSIONS: Raloxifene administration may result in a small increase in systemic warfarin exposure that is associated with a diminution, not augmentation, of the pharmacodynamic effect. Due to the small magnitude of this effect, concomitant administration of raloxifene and warfarin is not likely to result in clinically significant drug-drug interaction.

Raloxifene reduces atherosclerosis: studies of optimized raloxifene doses in ovariectomized, cholesterol-fed rabbits.

OBJECTIVE: We have previously shown that raloxifene, a selective oestrogen receptor modulator, 35 mg/day inhibits atherosclerosis in ovariectomized, cholesterol-fed rabbits. This effect was only partial as compared to 17beta-oestradiol 4 mg/day; however, plasma raloxifene concentrations were low relative to those obtained in raloxifene-treated women. We therefore investigate the effects of raloxifene at higher doses. DESIGN: The study on atherosclerosis in ovariectomized, cholesterol-fed rabbits (n = 80) compared raloxifene 70 mg/day and 210 mg/day to 17beta-oestradiol 4 mg/day and placebo. RESULTS: After 48 weeks of therapy, the aortic cholesterol content in the 70 mg/day and 210 mg/day raloxifene treatment groups were 471 +/- 56 nmol/mg protein and 456 +/- 56 nmol/mg protein, respectively. This was significantly less than in the placebo group (654 +/- 69 nmol/mg protein; P < 0.05). In the oestrogen-treated group, the aortic cholesterol content was 357 +/- 62 nmol/mg protein (P < 0.01 as compared to placebo). Differences in serum lipids between the treatment groups could only partly explain the effect on aortic cholesterol content, indicating that additional anti-atherogenic mechanisms may contribute to the decrease in aortic atherosclerosis. This anti-atherosclerotic activity of raloxifene was observed at plasma concentrations comparable to those in postmenopausal women during raloxifene treatment. CONCLUSIONS: We conclude that clinically relevant raloxifene treatment inhibits aortic atherosclerosis in ovariectomized, cholesterol-fed rabbits.