Minoxidil Sulfate Research Articles

Mapping the Interaction Landscape of Adenosine and Minoxidil Sulfate Using an Independent Gradient Model Based on Hirshfeld Partition and Interaction Region Indicator.

Using theoretical techniques, the interactions between adenosine (AD) and minoxidil sulfate (MS) were studied using the DFT/M06-2X/aug-cc-pVDZ level of theory. The formation of the complex was thermodynamically favorable, so we carried out further studies. Frontier molecular orbital analysis shows that the HOMO-LUMO- gap is high (6.81 eV), which means that the complex is kinetically stable. The global reactivity parameters were also calculated to get insight about the chemical reactivity of the complex. Molecular electrostatic potential and charge analyses were carried out for the optimized complex. To study the charge transfer and inter- and intramolecular interactions present in the complex molecule, we employed natural bond orbital analysis. Noncovalent interaction (NCI) analysis along with IGMH and IRI analyses further helped us find the weak interactions. The presence of hydrogen bonds between the N atom (N27) of MS and H atom (H66) of AD and O atom (O32) of MS and H atom (H46) of AD was the main finding of these analyses. QTAIM investigation also supports this finding. EDA analysis was carried out to further study the intermolecular interactions present within the complex. Thus, the present investigation unveils the existence of weak NCIs in the complex.

Efficacy, Tolerability, and Superiority of Propylene Glycol-Free, North American Witch-Hazel (Hamamelis virginiana)-Based Solution of 5% Minoxidil Sulfate for the Treatment of Female Androgenetic Alopecia.

Androgenetic alopecia leads to progressive hair loss in susceptible individuals if left untreated. Topical minoxidil represents an evidence-based treatment for female androgenetic alopecia, though with variable success. Treatment of minoxidil non-responders remains challenging, as does treatment of patients with propylene glycol sensitivity or irritable scalp syndrome. Single-center, retrospective cohort of 50 female patients with androgenetic alopecia failing to respond to a minimum of 6 months of standard 5% topical minoxidil solution either once daily or b.i.d. depending on the severity of the alopecia. Patients were switched to propylene glycol-free, North American Witch Hazel (Hamamelis virginiana)-based solution of 5% minoxidil sulfate (5% minoxidil sensitive solution). Efficacy and safety of treatment were evaluated, including stereotactic global photography and epiluminiscence microscopy with digital imaging taken at baseline, at 3, and at 6 months of treatment. 70% of patients showed observable clinical improvement with combined global photographic and epiluminiscence microscopic assessment with digital imaging, and 22% epiluminiscence microscopic-only improvement as evidence of treatment efficacy. The treatment was well tolerated, particularly in patients with propylene glycol sensitivity and patients with irritable scalp syndrome. These results suggest that propylene glycol-free, North American witch hazel (Hamamelis virginiana)-based solution of 5% minoxidil is effective and safe for treatment of female androgenetic alopecia, specifically in minoxidil non-responders and patients with propylene glycol sensitivity or irritable scalp syndrome.

Comprehensive transcriptome data to identify downstream genes of testosterone signalling in dermal papilla cells

Testosterone-related steroid hormones are associated with various types of diseases, including prostate cancer and androgenetic alopecia (AGA). The testosterone or dihydroxy testosterone (DHT) circulates through the blood, binds to the androgen receptor (AR) in the cytoplasm, and finally enters the nucleus to activate downstream target genes. We previously found that immortalized dermal papilla cells (DPCs) lost AR expression, which may be explained by the repeated cell passages of DPCs. To compensate for the AR expression, DPCs that express AR exogenously were established. In this study, we performed an RNA-Seq analysis of the AR-expressing and non-AR-expressing DPCs in the presence or absence of DHT to identify the downstream target genes regulated by AR signalling. Furthermore, we treated DPCs with minoxidil sulphate, which has the potential to treat AGA. This is the first comprehensive analysis to identify the downstream genes involved in testosterone signalling in DPCs. Our manuscript provides high-priority data for the discovery of molecular targets for prostate cancer and AGA.

SULT1A1 (Minoxidil Sulfotransferase) enzyme booster significantly improves response to topical minoxidil for hair regrowth.

Minoxidil is a widely used over-the-counter topical treatment for hair loss. The response rate for topical minoxidil is relatively low. Minoxidil is a pro-drug, converted to its active form, minoxidil sulfate, by SULT1A1 enzymes located in the scalp. Recently, a novel topical formula that increases the activity of SULT1A1 in hair follicles was reported. To evaluate any benefit of applying the SULT1A1 enzyme booster prior to daily 5% minoxidil treatment. Male androgenic alopecia patients were recruited to a randomized blinded placebo-controlled study. Patients were randomized to receive 5% topical minoxidil plus the novel formula or minoxidil plus a sham adjuvant. Patient's hair growth was monitored using global photography over 60days. Twenty-four males with androgenic alopecia (Norwood scale average 4.4, range 2-6) were randomized and completed the trial: 12 in the active arm and 12 in placebo. 75% of the subjects who used the SULT1A1 adjuvant with their daily minoxidil treatments for 60days regrew hair versus 33% of those using the placebo adjuvant (p=0.023). In a small cohort of androgenetic alopecia men, adding the SULT1A1 adjuvant to their daily minoxidil treatment regimen improved hair regrowth.

KATP Channel Openers Inhibit Lymphatic Contractions and Lymph Flow as a Possible Mechanism of Peripheral Edema.

Pharmacological openers of ATP-sensitive potassium (KATP) channels are effective antihypertensive agents, but off-target effects, including severe peripheral edema, limit their clinical usefulness. It is presumed that the arterial dilation induced by KATP channel openers (KCOs) increases capillary pressure to promote filtration edema. However, KATP channels also are expressed by lymphatic muscle cells (LMCs), raising the possibility that KCOs also attenuate lymph flow to increase interstitial fluid. The present study explored the effect of KCOs on lymphatic contractile function and lymph flow. In isolated rat mesenteric lymph vessels (LVs), the prototypic KATP channel opener cromakalim (0.01–3 µmol/l) progressively inhibited rhythmic contractions and calculated intraluminal flow. Minoxidil sulfate and diazoxide (0.01–100 µmol/l) had similar effects at clinically relevant plasma concentrations. High-speed in vivo imaging of the rat mesenteric lymphatic circulation revealed that superfusion of LVs with cromakalim and minoxidil sulfate (0.01–10 µmol/l) maximally decreased lymph flow in vivo by 38.4% and 27.4%, respectively. Real-time polymerase chain reaction and flow cytometry identified the abundant KATP channel subunits in LMCs as the pore-forming Kir6.1/6.2 and regulatory sulfonylurea receptor 2 subunits. Patch-clamp studies detected cromakalim-elicited unitary K+ currents in cell-attached patches of LMCs with a single-channel conductance of 46.4 pS, which is a property consistent with Kir6.1/6.2 tetrameric channels. Addition of minoxidil sulfate and diazoxide elicited unitary currents of similar amplitude. Collectively, our findings indicate that KCOs attenuate lymph flow at clinically relevant plasma concentrations as a potential contributing mechanism to peripheral edema.SIGNIFICANCE STATEMENTATP-sensitive potassium (KATP) channel openers (KCOs) are potent antihypertensive medications, but off-target effects, including severe peripheral edema, limit their clinical use. Here, we demonstrate that KCOs impair the rhythmic contractions of lymph vessels and attenuate lymph flow, which may promote edema formation. Our finding that the KATP channels in lymphatic muscle cells may be unique from their counterparts in arterial muscle implies that designing arterial-selective KCOs may avoid activation of lymphatic KATP channels and peripheral edema.

Novel topical booster enhances follicular sulfotransferase activity in patients with androgenetic alopecia: a new strategy to improve minoxidil response

Topical minoxidil has been used as a topical treatment for androgenetic alopecia (AGA) for more than 30 years. Approximately 60 to 70% of patients do not achieve hair growth. Minoxidil is a pro-drug. In order to exert biological activity, minoxidil requires conversion to minoxidil sulfate by sulfotransferase enzymes (SULT1A1). We have reported extensively that SULT1A1 activity in the outer root sheath (ORS) of the hair follicle correlates directly with topical minoxidil response. We have demonstrated the clinical utility and validity of a colorimetric test to measure the follicular SULT1A1 activity in plucked hair as a method to predict minoxidil responders. This same test also predicts clinical response to oral minoxidil.

Simultaneous determination of minoxidil and minoxidil sulfate by high-performance liquid chromatography with UV-detection and its applications

AimsMinoxidil is a hair growth drug for treating androgenetic alopecia. Although minoxidil is generally administered as a topical formulation, this prodrug must be converted to its active form (minoxidil sulfate) by sulfotransferase in hair follicle tissue. Therefore, its effect may be affected by the sulfureting of minoxidil and de-sulfureting of minoxidil sulfate in hair follicles. To investigate the biotransformation of minoxidil sulfate to minoxidil in hair follicle component cells, a method for simultaneous determination of minoxidil and minoxidil sulfate by high-performance liquid chromatography with UV-detection was established. Main methodsMinoxidil and minoxidil sulfate were separated by a reversed-phase chromatography. Minoxidil sulfate was incubated with human hair follicle keratinocyte- and dermal papilla cell-derived arylsulfatases as well as snail-derived sulfatase. The enzyme mixture was applied on HPLC system directly. Key findingsMinoxidil and minoxidil sulfate were separated simultaneously. The limit of detection of minoxidil and minoxidil sulfate was 25 and 100 pg inj−1, respectively. Snail-derived sulfatase as well as human hair follicle keratinocyte- and dermal papilla cell-derived arylsulfatases hydrolyzed minoxidil sulfate to minoxidil. Arylsulfatase in hair follicle metabolized the active form of minoxidil to the inactive form. Inactivation of active minoxidil (sulfate) was inhibited by the natural substrate of arylsulfatase A, ascorbate-2-sulfate. SignificanceArylsulfatase inhibitors may sustain the effect of minoxidil sulfate in AGA therapy. The developed technique was effective for studies of minoxidil metabolism and bioavailability.

LC–MS bioanalytical method for simultaneous determination of latanoprost and minoxidil in the skin

The association of minoxidil sulphate and latanoprost is currently emerging as a promising strategy for the treatment of androgenic alopecia, which is the most common type of scalp hair loss. In order to support the development of new pharmaceutical products containing such drugs combination, this study proposes a simple and efficient LC–MS bioanalytical method to simultaneously quantify minoxidil sulphate and latanoprost in different skin layers. Compounds separation was performed by liquid chromatography using a C18 column with gradient elution of a mobile phase composed of 0.1 % formic acid in acetonitrile and water at a flow rate of 0.5 mL min−1. Determinations were executed using mass spectrometry equipped with an ESI interface operating in a positive ionization mode. Quantification was performed using selective ion mode monitoring of m/z 210.1 for minoxidil sulphate and 433.3 for latanoprost. The matrix effect was very pronounced in samples containing some skin layers or electrolyte solution. Accordingly, a calibration curve for each contaminant group was built, leading to correlation coefficient values higher than 0.99. Additionally, lower limits of detection and quantification were obtained, and precision (repeatability and intermediate precision) achieved results with a coefficient of variation less than 15 %. Drug recovery from skin samples was higher than 70 %, fulfilling the recommendations. Also, the bioanalytical method was successfully tested in in vitro skin penetration studies proving its effectiveness in the development of topical formulations containing both drugs.

Colored Polymeric Nanofiber Loaded with Minoxidil Sulphate as Beauty Coverage and Restoring Hair Loss

Polymeric nanofibers fabricated by electrospinning either blank (PVA) or loaded with minoxidil sulphate have yielded optimum fibers with an average diameter 273 nm, and 511 nm, respectively. Thermal analysis of nanofibers indicated no chemical interaction. The NMR spectrum confirmed stability of nanofiber as there were no interactions between functional groups. Prepared nanofibers showed a 47.4% encapsulation efficiency and 73% yield. In vitro drug release of minoxidil sulphate from nanofiber exhibited an initial burst release followed by a slower release pattern. Stability studies revealed that minoxidil nanofiber was stable if stored at room temperature and protected from light with only loss of 9.6% of its nominal concentration within 6 months. As a result, the prepared solid/colored formula serves as an ideal formulation for such instable drug in liquid formula taking the advantage of the attractiveness of beauty colored coverage, and the simple, and non-tousled application.

Effect of Minoxidil on Trabecular Outflow via the Paracellular Pathway

PurposeTo investigate the pathway and effects of minoxidil on trabecular outflow in cultured human trabecular meshwork (TM) cells.MethodsAfter exposing primarily cultured TM cells to 0, 10, 50, or 100 µM minoxidil sulfate (MS), trabecular outflow was assessed by measuring TM cell monolayer permeability to carboxyfluorescein and transepithelial electrical resistance. To assess the pathway of permeability changes, caveolin-1, occludin, and claudin-5 levels were measured via western blot. Generation of reactive oxygen species (ROS) was measured using the dichlorofluorescein diacetate assay. To assess the involvement of nitric oxide (NO) in minoxidil-induced permeability increase, the degrees of endothelial nitric oxide synthase mRNA expression and NO production were measured with reverse transcription polymerase chain reaction and Griess assays, respectively. Permeability was also measured with co-exposure to 50 µM N-acetyl cysteine.ResultsMS significantly increased TM cell monolayer permeability (p < 0.05) and decreased transepithelial electrical resistance (p < 0.05). MS decreased the degree of endothelial nitric oxide synthase mRNA expression but did not affect NO production. MS decreased occludin and claudin-5 levels but did not affect caveolin-1 level. MS at 100 µM increased the generation of ROS, and MS-induced permeability increase was attenuated after co-exposure to 50 µM N-acetyl cysteine.ConclusionsMinoxidil may preferentially increase trabecular permeability via a paracellular pathway by downregulation of tight junction proteins. This minoxidil-induced permeability through the TM may be mediated by generation of ROS.

Quality evaluation of minoxidil topical solutions obtained from magistral pharmacies

The objective of this study was to evaluate quality parameters of magistral topical solutions containing minoxidil (A, B and C), comparing the results with the ones obtained for the industrial formulation. Organoleptic tests, evaluation of the pH and density, centrifuge test, drug content determination, comparison of indicated dosages and in vitro follicular penetration of minoxidil were performed. Regarding the organoleptic properties, differences in color and viscosity were observed between the magistral (composed of minoxidil sulfate) and the industrial formulations (composed of minoxidil base). For pH values, the magistral solutions presented considerably more acidic pH, compared to the industrial sample. For the density test, the samples with the highest ethanol percentages (B and C) presented lower density. In the centrifuge test, none of the samples showed changes. Considering the drug content test, only the industrial sample and the magistral sample C showed drug percentage within the expected (90-110%), indicating lack of correction factor determination by the magistral pharmacies. Furthermore, it was observed that the dosage indicated by the magistral pharmacies do not correspond to the dose indicated by the industry, being significantly lower. All topical solutions tested presented hair follicle penetration of minoxildil, without statistical difference. The results indicate that there is a failure in the magistral pharmacies regarding the production and the indication of dosage of minoxidil topical solutions.

Evaluation of Hair Growth Promoting Activity of Maharanga bicolor and Hibiscus rosa sinensis

Alopecia is common and most frequently complaint dermatological disorder since the time immemorial. Various herbal and allopathic formulations are reported to have hair growth promoting activity. Minoxidil and Finasteride are only US FDA approved drugs for alopecia and hair loss. Maharanga bicolor and Hibiscus rosa sinensis are two ethnomedicinal herbs commonly used for hair growth promotion. Present study was conducted to determine hair growth promoting activity of petroleum ether extract of M. bicolor and H. rosa sinensis in Aloe vera gel. Minoxidil Sulphate and Different formulations of petroleum ether extract of M. bicolor and H. rosa sinensis was formulated in Aloe vera gel and applied to denuated area of rats for 30 days. Hair growth pattern, hair growth initiation, hair growth completion and length of hair was measured. M. bicolor (2%) formulation shows shortest hair growth initiation time on 6th day compared to 11th day in blank sample. Formulation containing 2% of each extract have shortest hair growth completion time on 19th day of sample application and showed potent hair growth pattern observed by blind investigation. The result suggests that petroleum ether extract of M. bicolor and H. rosa sinensis possess good hair growth promoting activity.&#x0D; Key words: Maharanga bicolor, Hibiscus rosa sinensis, Hair growth promotion, Activity

Tretinoin enhances minoxidil response in androgenetic alopecia patients by upregulating follicular sulfotransferase enzymes.

Minoxidil sulfate is the active metabolite required to exert the vasodilatory and hair growing effects of minoxidil. For hair growth, sulfotransferase enzymes expressed in outer root sheath of the hair follicle sulfonate minoxidil. The large intra-subject variability in follicular sulfotransferase was found to predict minoxidil response and thus explain the low response rate to topical minoxidil in the treatment of androgenetic alopecia. A method to increase minoxidil response would be of significant clinical utility. Retinoids have been reported to increase minoxidil response. The purported mechanism of action was retinoid modulation of skin permeation to minoxidil; however, evidence to the contrary supports retinoids increase dermal thickness. In order to elucidate the effect of topical retinoids on minoxidil response, we studied the effect of topical tretinoin on follicular sulfotransferase. In this study, we demonstrate that topical tretinoin application influences the expression of follicular sulfotransferase. Of clinical significance, in our cohort, 43% of subjects initially predicted to be nonresponders to minoxidil were converted to responders following 5 days of topical tretinoin application. To the best of our knowledge, this is the first study to elucidate the interaction mechanism between topical minoxidil and retinoids and thus provides a pathway for the development of future androgenetic alopecia treatments.

Low-dose daily aspirin reduces topical minoxidil efficacy in androgenetic alopecia patients.

Topical minoxidil is the only US FDA approved OTC drug for the treatment of androgenetic alopecia (AGA). Minoxidil is a pro-drug converted into its active form, minoxidil sulfate, by the sulfotransferase enzymes in the outer root sheath of hair follicles. Previously, we demonstrated that sulfotransferase activity in hair follicles predicts response to topical minoxidil in the treatment of AGA. In the human liver, sulfotransferase activity is significantly inhibited by salicylic acid. Low-dose OTC aspirin (75-81 mg), a derivative of salicylic acid, is used by millions of people daily for the prevention of coronary heart disease and cancer. It is not known whether oral aspirin inhibits sulfotransferase activity in hair follicles, potentially affecting minoxidil response in AGA patients. In the present study, we determined the follicular sulfotransferase enzymatic activity following 14 days of oral aspirin administration. In our cohort of 24 subjects, 50% were initially predicted to be responders to minoxidil. However, following 14 days of aspirin administration, only 27% of the subjects were predicted to respond to topical minoxidil. To the best of our knowledge, this is the first study to report the effect of low-dose daily aspirin use on the efficacy of topical minoxidil.

Induction of hair follicle dermal papilla cell properties in human induced pluripotent stem cell-derived multipotent LNGFR(+)THY-1(+) mesenchymal cells

The dermal papilla (DP) is a specialised mesenchymal component of the hair follicle (HF) that plays key roles in HF morphogenesis and regeneration. Current technical difficulties in preparing trichogenic human DP cells could be overcome by the use of highly proliferative and plastic human induced pluripotent stem cells (hiPSCs). In this study, hiPSCs were differentiated into induced mesenchymal cells (iMCs) with a bone marrow stromal cell phenotype. A highly proliferative and plastic LNGFR(+)THY-1(+) subset of iMCs was subsequently programmed using retinoic acid and DP cell activating culture medium to acquire DP properties. The resultant cells (induced DP-substituting cells [iDPSCs]) exhibited up-regulated DP markers, interacted with human keratinocytes to up-regulate HF related genes, and when co-grafted with human keratinocytes in vivo gave rise to fibre structures with a hair cuticle-like coat resembling the hair shaft, as confirmed by scanning electron microscope analysis. Furthermore, iDPSCs responded to the clinically used hair growth reagent, minoxidil sulfate, to up-regulate DP genes, further supporting that they were capable of, at least in part, reproducing DP properties. Thus, LNGFR(+)THY-1(+) iMCs may provide material for HF bioengineering and drug screening for hair diseases.

Solid effervescent formulations as new approach for topical minoxidil delivery

Currently marketed minoxidil formulations present inconveniences that range from a grease hard aspect they leave on the hair to more serious adverse reactions as scalp dryness and irritation. In this paper we propose a novel approach for minoxidil sulphate (MXS) delivery based on a solid effervescent formulation. The aim was to investigate whether the particle mechanical movement triggered by effervescence would lead to higher follicle accumulation. Preformulation studies using thermal, spectroscopic and morphological analysis demonstrated the compatibility between effervescent salts and the drug. The effervescent formulation demonstrated a 2.7-fold increase on MXS accumulation into hair follicles casts compared to the MXS solution (22.0±9.7μg/cm2 versus 8.3±4.0μg/cm2) and a significant drug increase (around 4-fold) in remaining skin (97.1±29.2μg/cm2) compared to the drug solution (23.5±6.1μg/cm2). The effervescent formulations demonstrated a prominent increase of drug permeation highly dependent on the effervescent mixture concentration in the formulation, confirming the hypothesis of effervescent reaction favoring drug penetration. Clinically, therapy effectiveness could be improved, increasing the administration interval, hence, patient compliance. More studies to investigate the follicular targeting potential and safety of new formulations are needed.

Iontophoresis of minoxidil sulphate loaded microparticles, a strategy for follicular drug targeting?

The feasibility of targeting drugs to hair follicles by a combination of microencapsulation and iontophoresis has been evaluated. Minoxidil sulphate (MXS), which is used in the treatment of alopecia, was selected as a relevant drug with respect to follicular penetration. The skin permeation and disposition of MXS encapsulated in chitosan microparticles (MXS-MP) was evaluated in vitro after passive and iontophoretic delivery. Uptake of MXS was quantified at different exposure times in the stratum corneum (SC) and hair follicles. Microencapsulation resulted in increased (6-fold) drug accumulation in the hair follicles relative to delivery from a simple MXS solution. Application of iontophoresis enhanced follicular delivery for both the solution and the microparticle formulations. It appears, therefore, that microencapsulation and iontophoresis can act synergistically to enhance topical drug targeting to hair follicles.

Chitosan nanoparticles for targeting and sustaining minoxidil sulphate delivery to hair follicles

This work developed minoxidil sulphate-loaded chitosan nanoparticles (MXS-NP) for targeted delivery to hair follicles, which could sustain drug release and improve the topical treatment of alopecia. Chitosan nanoparticles were obtained using low-molecular weight chitosan and tripolyphosphate as crosslink agent. MXS-NP presented a monomodal distribution with hydrodynamic diameter of 235.5±99.9nm (PDI of 0.31±0.01) and positive zeta potential (+38.6±6.0mV). SEM analysis confirmed nanoparticles average size and spherical shape. A drug loading efficiency of 73.0±0.3% was obtained with polymer:drug ratio of 1:1 (w/w). Drug release through cellulose acetate membranes from MXS-NP was sustained in about 5 times in comparison to the diffusion rate of MXS from the solution (188.9±6.0μg/cm2/h and 35.4±1.8μg/cm2/h). Drug permeation studies through the skin in vitro, followed by selective recovery of MXS from the hair follicles, showed that MXS-NP application resulted in a two-fold MXS increase into hair follicles after 6h in comparison to the control solution (5.9±0.6μg/cm2 and 2.9±0.8μg/cm2). MXS-loading in nanoparticles appears as a promising and easy strategy to target and sustain drug delivery to hair follicles, which may improve the topical treatment of alopecia.

Novel enzymatic assay predicts minoxidil response in the treatment of androgenetic alopecia

Topical minoxidil is the most common drug used for the treatment of androgenetic alopecia (AGA) in men and women. Although topical minoxidil exhibits a good safety profile, the efficacy in the overall population remains relatively low at 30-40%. To observe significant improvement in hair growth, minoxidil is typically used daily for a period of at least 3-4 months. Due to the significant time commitment and low response rate, a biomarker for predicting patient response prior to therapy would be advantageous. Minoxidil is converted in the scalp to its active form, minoxidil sulfate, by the sulfotransferase enzyme SULT1A1. We hypothesized that SULT1A1 enzyme activity in the hair follicle correlates with minoxidil response for the treatment of AGA. Our preliminary retrospective study of a SULT1A1 activity assay demonstrates 95% sensitivity and 73% specificity in predicting minoxidil treatment response for AGA. A larger prospective study is now under way to further validate this novel assay.

Minoxidil sulfate induced the increase in blood–brain tumor barrier permeability through ROS/RhoA/PI3K/PKB signaling pathway

Adenosine 5′-triphosphate-sensitive potassium channel (KATP channel) activator, minoxidil sulfate (MS), can selectively increase the permeability of the blood–tumor barrier (BTB); however, the mechanism by which this occurs is still under investigation. Using a rat brain glioma (C6) model, we first examined the expression levels of occludin and claudin-5 at different time points after intracarotid infusion of MS (30 μg/kg/min) by western blotting. Compared to MS treatment for 0 min group, the protein expression levels of occludin and claudin-5 in brain tumor tissue of rats showed no changes within 1 h and began to decrease significantly after 2 h of MS infusion. Based on these findings, we then used an in vitro BTB model and selective inhibitors of diverse signaling pathways to investigate whether reactive oxygen species (ROS)/RhoA/PI3K/PKB pathway play a key role in the process of the increase of BTB permeability induced by MS. The inhibitor of ROS or RhoA or PI3K or PKB significantly attenuated the expression of tight junction (TJ) protein and the increase of the BTB permeability after 2 h of MS treatment. In addition, the significant increases in RhoA activity and PKB phosphorylation after MS administration were observed, which were partly inhibited by N-2-mercaptopropionyl glycine (MPG) or C3 exoenzyme or LY294002 pretreatment. The present study indicates that the activation of signaling cascades involving ROS/RhoA/PI3K/PKB in BTB was required for the increase of BTB permeability induced by MS. Taken together, all of these results suggested that MS might increase BTB permeability in a time-dependent manner by down-regulating TJ protein expression and this effect could be related to ROS/RhoA/PI3K/PKB signal pathway.