Estimation of minoxidil in human plasma using UHPLC-MS/MS and its application in pharmacokinetic study.

Measurement of drug concentration in biological matrices (such as serum, plasma, blood, urine, and saliva) is important to determine Bioavailability (BA) and/or Bioequivalence (BE) of a drug product which are required during the drug product development and approval process to support applications for new active substances (INDs, NDAs) and generic (ANDAs) drug products to make critical decisions on safety and efficacy. Because of their vital role, bioanalytical methods should be well-characterized, fully validated and documented to yield reliable results. In present work, a simple, specific, high throughput, accurate and sensitive UHPLC-MS/MS method has been developed and validated for quantification of Minoxidil in human plasma. The analyte and the internal standard were extracted from plasma by Liquid-Liquid Extraction using ethyl acetate. The chromatographic separation was achieved on Thermo Hypersil Gold column (4.6x50mm, 5µm) using acetonitrile-0.1% formic acid in water (60:40, v/v) at a flow rate of 0.400 ml/min. Detection by turbospray positive ionization mass spectrometry in the multiple reaction monitoring mode with a mass transition ion-pair of m/z 210.152 → 163.965 (Minoxidil) and m/z 220.267 → 169.089 (Internal Standard-Minoxidil D10) was found to be linear over the concentration range of 1.280 to 151.075 ng/ml. The method was fully validated as per USFDA guidelines and the results were within regulatory limits. The inter and intra-day precision ranged from 5.42 to 9.27% and 2.55-9.42% respectively. The inter and intra-day accuracy ranged from 89.2 to 98.9% and 102-105% respectively. The method was successfully applied to a BE study involving human volunteers.

Drug Delivery System Based On Minoxidil Nanoparticles Promotes Hair Growth In C57BL/6 Mice.

PURPOSE: We designed formulations based on minoxidil (MXD) nanoparticles (N-MXD) and examined whether N-MXD can increase drug delivery into the follicles. In addition, we investigated the effect of N-MXD on hair growth in C57BL/6 mice. METHODS: N-MXD (1%) was prepared as follows: methylcellulose, p-hydroxyalkylbenzoates, mannitol, and MXD were dispersed in purified water and milled using zirconia beads under refrigeration (5500 rpm, 30 s×15 times, intermittent milling). C57BL/6 mice were used to evaluate hair-growth effects. The expression levels of mRNA and protein for vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) were determined by real-time PCR and ELISA methods, respectively. RESULTS: The ratio of solid-MXD was approximately 60% in N-MXD, and the MXD nanoparticles (90-300 nm) were oblong in shape. For the design of nanomedicines, usability is important. Therefore, we measured the stability and toxicity after N-MXD treatment. No agglutination of MXD nanoparticles was detected for 2 weeks, and no redness or MXD powder residue was observed in the skin after repetitive applications of N-MXD. Next, we evaluated hair-growth effects by N-MXD treatment. MXD contents in the skin tissue from N-MXD were lower than for commercially available MXD formulations (CA-MXD). Conversely, MXD contents in the hair bulbs were higher for N-MXD than for CA-MXD, and the drug efficacy of N-MXD was also higher than that of CA-MXD. In addition, the mRNA and protein levels of IGF-1 and VEGF were enhanced by the repetitive application of N-MXD and CA-MXD, and the enhanced IGF-1 and VEGF levels were significantly higher for N-MXD than for CA-MXD. CONCLUSION: We designed a novel nanomedicine based on MXD nanoparticles and showed that N-MXD can deliver MXD into hair bulbs via hair follicles and that the therapeutic efficiency for hair growth is higher than for CA-MXD (solution type).

Evaluation of glucuronide metabolite stability in dried blood spots.

BACKGROUND: Stabilization of phase II metabolites is an important consideration during bioanalytical method development, method validation and sample analysis. Generic approaches to stabilize these metabolites during storage in liquid-based matrices include pH adjustment of samples prior to storage and/or temperature control; although a variety of other compound-specific stabilization techniques exist. Dried blood spot (DBS) technology is becoming a popular alternative to liquid matrix sampling in many preclinical and clinical applications. However, concerns remain regarding the stability of metabolites stored under ambient conditions using DBS. RESULTS: Experimental data have shown that, under ambient storage conditions, the stability of the glucuronides investigated herein stored as DBS is equivalent to that of liquid samples stored at -80°C. CONCLUSION: The decision to employ DBS technology for a given study needs to be considered on a case-by-case basis with an understanding of compound-specific metabolism characteristics and clinical study design.

Follicular and percutaneous penetration pathways of topically applied minoxidil foam.

In the past, it was assumed that the intercellular route was the only relevant penetration pathway for topically applied substances. Recent results on follicular penetration emphasize that the hair follicles represent a highly relevant and efficient penetration pathway and reservoir for topically applied substances. This study investigates a selective closure technique of hair follicle orifices in vivo assessing interfollicular and follicular absorption rates of topical minoxidil foam in humans. In delimited skin area, single hair orifices or interfollicular skin were blocked with a microdrop of special varnish-wax-mixture in vivo. Minoxidil foam (5%) was topically applied, and transcutaneous absorption was measured by a new surface ionization mass spectrometry technique in serum. Different settings (open, closed or none of both) enabled to clearly distinguish between interfollicular and follicular penetration of the topically applied minoxidil foam. Five minutes after topical application, minoxidil was detected in blood samples when follicles remained open, whereas with closed follicles 30 min were needed. Highest levels were found first when both pathways were open, followed by open follicles and subsequently by closed follicles. These results demonstrate the high importance of the follicular penetration pathway. Hair follicles are surrounded by a dense network of blood capillaries and dendritic cells and have stem cells in their immediate vicinity, making them ideal targets for drug delivery.

Cardiovascular toxicity of minoxidil in the marmoset.

The aim of the experiments was to assess the toxicity of minoxidil, a potent vasodilator, in marmosets. The animals were treated either at escalating doses from 2 to 40 mg/kg, escalating doses from 40 to 200 mg/kg or single doses of 150 mg/kg or 200 mg/kg. ECG recording and echocardiographic examination were conducted before and 1h after treatment. Necropsy and histopathology were performed 24h after the last dose. The treatment with minoxidil induced myocardial necrosis, coronary arteriopathy and degeneration of renal tubules in animals treated with 150 mg/kg or 200 mg/kg. Myocardial necrosis associated with fibrosis in some animals was located mainly in the left and right ventricles (including papillary muscles), but also in the right atrium, left atrium and/or interventricular septum. Arteriopathy was observed in small coronary arteries of the right or left atrium. ECG and echocardiographic examinations showed that in animals treated with 150 mg/kg or 200 mg/kg, there were positive chronotropic and inotropic effects that compensated for the hypotensive effect of the drug and were considered to have played a key role in the pathogenesis of the cardiovascular lesions. The cardiotoxicity of minoxidil in marmosets was similar to that described in dogs, but occurred at much higher doses. In conclusion minoxidil produced cardiovascular toxicity in the marmoset, which was probably due to the marked changes in the cardiac function associated with exaggerated pharmacological effects of the compound. The marmosets were found to be less sensitive than dogs to the cardiotoxicity of minoxidil.

Simultaneous effects of tocopheryl polyethylene glycol succinate (TPGS) on local hair growth promotion and systemic absorption of topically applied minoxidil in a mouse model.

In this study, topical minoxidil solutions supplemented with TPGS in cosolvent systems of various compositions consisting of water, alcohol, and polyethylene glycol 400 were designed to evaluate the efficacy of promoting hair growth after topical application and the safety in terms of the amount of minoxidil absorbed through the skin into the circulation using C57BL/6J mice as a model. The commercial product of 2% Regaine) was used as the positive control. The role, which sulfotransferase activity plays in hair growth with treatment using minoxidil, was determined as well. The results revealed that the addition of 0.5% TPGS was able to enhance the proliferation of hair, but an increase in the amount of TPGS to 2% led to deterioration in the enhancement of hair growth. At the higher added amount (2.0%) of TPGS, the promotion of hair growth was slightly reduced for both cosolvent formulations F1 (100% water) and F3 (100% PEG 400), whereas it was reduced to a greater extent for the cosolvent formulations F8-F10. In comparison, the influences of cosolvent compositions with TPGS amounts of 0.0 and 2.0% on the promotion of hair growth were similar. On the contrary, variability in the promotion of hair growth by different solvent formulations was minimal when the added amount of TPGS was 0.5%. In general, a relationship between hair growth and sulfotransferase activities after topical application of 2% Regaine and minoxidil formulations containing various amounts of TPGS was not demonstrated. Plasma concentrations of minoxidil with 2% Regaine were found to be greater than those of 2% minoxidil in those cosolvent formulations containing various amounts of TPGS, while showing insignificant differences among those 10 cosolvent formulations with a fixed amount of TPGS. A tendency for the plasma concentration of minoxidil to increase after the topical administration of minoxidil formulations containing the higher amount of TPGS (2%) was noted.

Rapid determination of minoxidil in human plasma using ion-pair HPLC.

A rapid, simple and sensitive ion-pair high-performance liquid chromatography (HPLC) method has been developed for quantification of minoxidil in plasma. The assay enables the measurement of minoxidil for therapeutic drug monitoring with a minimum detectable limit of 0.5 ng ml(-1). The method involves simple, one-step extraction procedure and analytical recovery was complete. The separation was performed on an analytical 150 x 4.6 mm i.d. microbondapak C18 column. The wavelength was set at 281 nm. The mobile phase was a mixture of 0.01 M sodium dihydrogen phosphate buffer and acetonitrile (60:40, v/v) containing 2.5 mM sodium dodecyl sulphate adjusted to pH 3.5 at a flow rate of 1 ml/min. The column temperature was set at 50 degrees C. The calibration curve was linear over the concentration range 2-100 ng ml(-1). The coefficients of variation for inter-day and intra-day assay were found to be less than 8%.

Application of solid-phase extraction in the method development for determination of SEPA, an acronym for soft enhancement of percutaneous absorption, in human, rat, and rabbit serum using GC-FID method.

A new nonaqueous topical minoxidil formulation containing SEPA (2-n-nonyl-1,3-dioxolane) for enhancement of percutaneous absorption was under evaluation. SEPA does not have chromophore for either ultraviolet or fluorescence detection using liquid chromatography and has no functional groups for derivatization. Therefore, a direct gas-chromatographic method with flame-ionization detection (GC-FID) was developed. Owing to the limited detection response of the FID detection, it needs a selective and concentrated extract for GC-FID analysis to improve the assay sensitivity to meet the requirement for pharmacokinetic evaluation after topical application. In addition, SEPA is a very volatile compound. Any extraction procedures involving evaporation will result in a poor recovery. The application of solid-phase extraction (SPE) makes it possible to achieve a selective and a 10-fold concentrated extract with an absolute extraction recovery of approximately 90%, which greatly improved the assay sensitivity. This method involved the extraction of SEPA and the internal standard (2-n-heptyl-1,3-dioxolane) from serum (0.1-1 ml) with 100 microliter of hexane-chloroform (1:1, v:v) using a 50 mg 1.0 ml-1 phenyl SPE column (Varian, Harbor City, CA, USA), followed by direct GC-FID analysis on a fused-silica column chemically bonded with cross-linked methyl silicone gum phase (Hewlett Packard Ultra-1, 12 m x 0.2 mm x 0.33 micron, Avondale, PA, USA). The assay demonstrated a lower limit of quantitation of 2.5 ng ml-1 and a linear range of 2.5 to 250 ng ml-1 with intra- and inter-assay precision and accuracy of < or = 10%.

Quantitative analysis quantitation of 2-n-nonyl-1,3-dioxolane by stable-isotope dilution gas chromatography-mass spectrometry.

We report here a quantitative methodology developed for determination of SEPA (2-n-nonyl-1,3-dioxolane) in human serum. The method employed solid-phase extraction of SEPA and internal standard, [13C2]SEPA, from serum followed by gas chromatography-mass spectrometry analysis using EI monitoring m/z 73 and 75. We have investigated the utility of stable isotope dilution gas chromatography-mass spectrometry (GC-MS) for the determination of SEPA concentrations in serum using chemical ionization (positive ion, CI) or electron ionization (EI). The comparison of the specificity and sensitivity between EI and CI indicated that monitoring the m/z 73 ion in EI was superior to monitoring either MH+ or m/z 73 using CI. The method was simple, sensitive and accurate, demonstrating a lower limit of quantitation (LLOQ) of 0.25 ng/ml and intra- and inter-assay accuracy and precision of < or = 7.5%.

Relationship between serum concentrations, hemodynamic effects, and cardiovascular lesions in dogs treated with minoxidil.

The threshold hemodynamic changes associated with the cardiovascular (CV) toxicity of minoxidil (MNX) in the dog, characterized by subendocardial necrosis, right atrial hemorrhagic lesions, and coronary vascular medial hemorrhage and necrosis, have not been defined. To determine the relationship between serum concentration, hemodynamic effects [heart rate (HR) and mean arterial pressure (MAP)] and CV toxicity, groups of female Beagle dogs were treated with a continuous iv infusion of dextrose (control) or 0.05, 0.14, 0.43, 1.44, or 4.32 mg/kg/day of MNX for 3 days. Serum concentration of free MNX increased in a dose-related manner and reached steady state within 4 hr after the initiation of infusion. There was a time-dependent, apparently dose-related increase in HR at all doses. MAP was decreased at > or = 0.14 mg/kg/day in a time- and dose-related manner. The doses or steady-state serum concentrations of MNX that showed no significant hemodynamic effects and CV toxicity were approximately 0.05 mg/kg or 3.0 +/- 0.6 ng/ml and 0.14 mg/kg or 7.3 +/- 2.0 ng/ ml, respectively. CV toxicity occurred at a serum concentration of 16.6 +/- 1.9 ng/ml where HR was increased by 65 +/- 11 beats/min and MAP was decreased by 34 +/- 2 mm Hg. A serum concentration of 7.3 +/- 2 ng/ml of MNX that increased HR by 47 +/- 14 beats/min and decreased MAP by 17 +/- 8 mm Hg was not associated with CV toxicity. This study suggests that the threshold hemodynamic effects associated with the CV toxicity of MNX in the dog are a function of an increase in HR by at least 55 beats/min and a decrease in MAP by at least 30 mm Hg. In conclusion, the safety margin of drugs like MNX, where the mechanisms of toxicity are known to be related to their pharmacologic effects, should be based on the ratio of the pharmacokinetically and metabolically adjusted dose/serum concentration of the drug that evokes comparable pharmacologic effects in the animal model and humans rather than on the ratio of the nontoxic dose/serum concentration in animals to the efficacious dose in humans.

Absorption of minoxidil after topical application: effect of frequency and site of application.

The effect of application site and frequency on the systemic absorption of topical minoxidil was studied in 52 normal men. Subjects received 1 ml 3% minoxidil solution applied four, six, or eight times daily to the scalp or two, four, six, or eight times daily to the chest for 14 days. Serum and urine were collected and analyzed for minoxidil. No systemic minoxidil accumulation occurred from increasing application frequency to the scalp. Trends in the chest data suggest that absorption may have been lower with the twice-daily regimen. Absorption through the scalp and chest were similar for the lower-frequency regimens; however, trends in the eight-times-a-day regimens suggest that absorption may have been somewhat greater from application to the scalp. Systemic minoxidil accumulation resulting from frequent application is unlikely. The initial dose probably saturates the skin for a period of time longer than the dosing intervals examined.

The pharmacokinetics of 2.5- to 10-mg oral doses of minoxidil in healthy volunteers.

The dose proportionality of minoxidil was investigated by studying its pharmacokinetics after administration of single, oral doses of 2.5, 5.0, and 10.0 mg. The study, which was a Latin square cross-over design, was performed in 30 young, nonobese, normal subjects. Treatments were separated by a 4-day washout period. Serum and urine levels of minoxidil were determined by high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA). Supine blood pressure and pulse were monitored during each study phase. Minoxidil concentrations determined by RIA were highly correlated with concentrations determined by HPLC; only the HPLC data was used in the pharmacokinetic analyses. No significant effects were observed for dose normalized Cmax, tmax, volume of distribution, minoxidil renal clearance, or the percentage of the dose excreted as either minoxidil or minoxidil glucuronide. Significant differences in apparent oral clearance, dose normalized AUC, and terminal elimination rate constant (beta) were observed between the 2.5-mg dose and the higher doses, but no differences in these parameters between the 5.0- and 10.0-mg doses were apparent. Thus, the available data support dose-independent pharmacokinetics for minoxidil over this range of doses. Repeated measures analysis of variance detected significant time and treatment effects on supine blood pressure and pulse rate, but the effects were generally small and of little clinical significance. The results support the hypothesis that minoxidil has little effect on blood pressure in normotensive subjects.

Topical minoxidil in extensive alopecia areata, including 3-year follow-up.

Perhaps the most intriguing aspect of topical minoxidil is the fact that this drug can promote hair growth in two unrelated conditions: alopecia areata (AA) and androgenetic alopecia. The two conditions have quite different underlying mechanisms. In AA, hair follicles respond to some signal or cell injury by entering a state of aborted cyclical activity; this state can reverse itself spontaneously, or it can be temporarily circumvented with nonspecific immunomodulating agents. In androgenetic alopecia, genetically marked hair follicles undergo progressive, androgen-mediated miniaturization; antiandrogens have been conventionally sought to intercept this process. It is not known how minoxidil promotes hair growth except that living follicles capable of stimulation and hypertrophy are required. It may be that minoxidil influences some fundamental signal to the follicular apparatus, irrespective of the pathophysiology involved. We have used topical minoxidil solution in 90 patients, aged 7-63 years, with extensive AA affecting 25-100% of the scalp. One study was double-blind, and placebo-controlled for an entire year. Minoxidil-treated patients responded better than placebo-treated patients. Both 3 and 5% topical minoxidil solutions have been used, and treatment with the 3% solution has continued for up to 3 years. The results of these studies will be discussed. While topical minoxidil is not very effective for those with 100% scalp hair loss, it is an effective, easy and safe treatment for those with AA affecting 25-99% of the scalp.

The effect of once-daily minoxidil on blood pressure and plasma lipids.

Once-daily minoxidil administration was added to the treatment regimen of 11 patients with hypertension that was inadequately controlled by nadolol 160 mg and chlorthalidone 50 mg given once daily. Additional diuretic therapy was needed by five patients. During treatment without minoxidil and at three and six months of maintenance minoxidil therapy, respectively, 24-hour postdose supine blood pressure fell significantly (P less than .01) from 142 +/- 19/96 +/- 6 mm Hg to 132 +/- 16/87 +/- 4 and 131 +/- 12/87 +/- 4 mm Hg, and home recordings showed that minoxidil induced a mean increase of about 5 beats/min in heart rate. Resting plasma renin activity was not significantly altered. High-density lipoprotein (HDL) cholesterol increased from 31.6 +/- 9.9 to 35.2 +/- 10.9 mg/dL (P less than .05) and to 34.4 +/- 11.3 mg/dL (NS), and low-density lipoprotein (LDL) cholesterol decreased from 146 +/- 36 to 136 +/- 32 mg/dL (P less than .05) and to 126 +/- 35 mg/dL (P less than .01) over the same periods. At six months, approximately 20% changes in ratios of HDL cholesterol to either LDL or total cholesterol were seen. These changes occurred despite a three-pound mean increase in body weight (P less than .05); these alterations are potentially beneficial in terms of reducing the estimated risk of coronary artery disease.

Minoxidil analysis in human plasma using high-performance liquid chromatography with electrochemical detection. Application to pharmacokinetic studies.

A method is described for determination of minoxidil in human plasma using high-performance liquid chromatography with electrochemical detection. The method is specific and sensitive (500 pg/ml), however, minoxidil and minoxidil sulfate cannot be differentiated due to rapid autohydrolysis of minoxidil sulfate to minoxidil. The extraction procedure employs a C18 preparatory column to remove endogenous plasma constituents which would interfere with the assays. The calibration curves are linear for concentrations from 500 pg to 10 ng/ml. Within-day and between-day reproducibility are satisfactory with coefficient of variation less than 5.7% for all concentrations. Sample recovery from extraction is consistent at 45 to 55% at low and high concentrations, respectively. A pharmacokinetic study in a hypertensive volunteer receiving two different oral doses of minoxidil (1.25 and 2.5 mg) on different occasions demonstrates the utility of the method.

Dose-response study of topical minoxidil in male pattern baldness.

Eighty-nine healthy men with male pattern baldness completed a 6-month double-blind, placebo-controlled study of 0.01%, 0.1%, 1%, and 2% topical minoxidil. Subjects on 2% topical minoxidil had a statistically significant increase in mean total target area hair count over baseline compared to the placebo, 0.01%, and 0.1% topical minoxidil groups (p = 0.04). Changes from baseline were more impressive with the 2% topical minoxidil group but not significantly different from the 1% topical minoxidil group in all parameters of objective response to treatment. The investigator, however, rated more subjects as having at least a moderate cosmetic response to treatment in the 2% versus 1% topical minoxidil treatment group. These results indicate that 1% topical minoxidil is the lowest effective concentration of topical minoxidil for male pattern baldness of those tested. Because of the more impressive changes in hair counts and the cosmetic preference for the 2% versus 1% topical minoxidil, 2% topical minoxidil may be the standard preferred treatment for male pattern baldness.

Topical minoxidil for male pattern baldness.

Significant hair growth occurred in 53% of the 81 patients completing a 1 year trial of topical minoxidil. The average reduction in the diameter of the balding crown was 3.48 cm for all patients. There were no minoxidil related changes in laboratory tests during the study period. Psychosocial studies of our patients indicate that 95% assessed the effectiveness of topical minoxidil as moderate or excellent. The majority of those participating in the study thought that their personal presentation of self was of equal or greater importance than their work performance. It was concluded that topical minoxidil has the potential to improve male pattern baldness significantly without apparent risk and to be a means by which individual presentation of self may be improved.

Efficacy of minoxidil in the treatment of severe hypertension in systemic disorders.

Minoxidil, an orally effective vasodilator, was successful in lowering blood pressure in 10 patients with systemic diseases complicated by hypertension refractory to medical management. Follow-up was for periods up to 58 months. In patients not requiring dialysis prior to therapy, renal function was preserved, and in patients on dialysis, bilateral nephrectomy was avoided. Although sodium retention and reflex tachycardia were common, they were managed by beta-adrenergic blockade with propranolol and diuresis with furosemide. Results did not differ qualitatively or quantitatively from those achieved in 31 refractory hypertensives without systemic illness. Pharmacologic studies performed on hemodialysis in a patient with polyarteritis revealed a dialysis clearance of 14C-labeled minoxidil of 48 ml/min. Venous minoxidil concentrations decreased by 32% across six single dialysis treatments. Nineteen percent of the administered dose was recovered in the feces, an increased fraction compared to normal subjects.