Polystyrene-b-poly (acrylic acid) nanovesicles coated by modified chitosans for encapsulation of minoxidil

Abstract In this work, polystyrene-b-poly (acrylic acid) (PS-b-PAA) nanovesicles were coated by modified chitosans aiming at studying its physicochemical parameters. The chitosan (CS) was chemically modified to add hydrophilic and/or hydrophobic groups, obtaining three modified chitosans. The PS-b-PAA nanovesicles were obtained by organic (1,4-dioxane) cosolvent method in water, resulting in nanovesicles with less than 150 nm of diameter (polydispersibility index - PDI at 90° = 0.106), measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM), and negative zeta potential (-37.5 ± 3.2 mV), allowing the coating of its surface with oppositely charged polysaccharides, such as the CS and the modified chitosans. The coating process was made by mixing the colloidal suspensions with the CS and the modified chitosans at specific ENT#091;CS-xENT#093;/ENT#091;PS-b-PAAENT#093; ratios (0.001 to 1.0 wt %) and measuring the change in size and surface charge by DLS and zeta potential. Upon reaching maximum adsorption, the zeta potential parameter was positively stabilized (+26.7 ± 4.1 mV) with a hydrodynamic diameter slightly longer (< 200 nm of diameter). The encapsulation efficiency (EE) of minoxidil, quantified by capillary electrophoresis, was 50.7%, confirming their potential as drug delivery carriers and the coating process showed the possibility of controlling the surface charge nature of these nanovesicles

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.

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.

Adenosine 5'-triphosphate-sensitive potassium channel activator induces the up-regulation of caveolin-1 expression in a rat brain tumor model.

This study was performed to determine whether minoxidil sulfate (MS), a selective Adenosine 5'-triphosphate-sensitive potassium channel (K (ATP) channel) activator, has an effect on the expression of caveolin-1 in the rat's brain tumor tissue. Using a rat brain glioma (C6) model, we found that the expression of caveolin-1 protein at tumor sites was greatly increased after intracarotid infusion of MS at a dose of 30 µg/kg/min for 15, 30, and 60 min via Western blot analysis. And the peak value of the caveolin-1 expression was observed in rats with glioma after 15 min of MS perfusion, which was significantly attenuated by reactive oxygen species (ROS) scavenger (N-2-mercaptopropionyl glycine, MPG). In addition, MPG also significantly inhibited the increase of blood-brain tumor barrier (BTB) permeability which was induced by MS. This led to the conclusion that the MS-induced BTB permeability increase may be related to the accelerated formation of caveolin-1 protein, and could be mediated by ROS.

Activation of KATP channels increases anticancer drug delivery to brain tumors and survival.

Several anticancer drugs are ineffective against brain tumor and do not impact patient survival because they fail to cross the blood-brain tumor barrier (BTB) effective levels. One such agent temozolomide is commonly used in brain tumor patients, which works better when combined with radiation or other anticancer agents. Likewise, trastuzumab (Herceptin, Her-2 inhibitor), which might be effective against Her2/neu over expressing gliomas may work well when combined with temozolomide. Nonetheless, both drugs do not cross the BTB to significantly impact patient survival. Beforehand we showed that potassium channel agonists when intracarotidly administered increased carboplatin and Her-2 antibody delivery in animal glioma models by triggering formation of brain vascular endothelial transcytotic vesicles. In this study, we investigated whether, intravenously administered, ATP-sensitive potassium channel (K(ATP)) activator (minoxidil sulfate; MS) increases temozolomide and Herceptin delivery to brain tumors to induce anti-tumor activity and increase survival in nude mice with Glioblastoma multiforme (GBM) cells. The results clearly demonstrate that when given intravenously temozolomide crosses BTB at a relatively low amount while Herceptin failed to cross the BTB. However, MS co-infusion with [(14)C]-temozolomide or fluorescently labeled-Herceptin resulted in improved and selective drug delivery to brain tumor. We also showed that combination treatment with temozolomide and Herceptin has enhanced anti-tumor effect which was more prominent than that of either treatment alone in increasing the survival in mice with GBM when co-infused with MS. Therefore, brain tumor patients may be benefited when anti-neoplastic agent delivery is increased selectively to the brain tumors using KATP channel agonists.

Attenuation of receptor-dependent and -independent vasoconstriction in the human radial artery.

BACKGROUND: Vasodilator strategies used to treat bypass grafts in the operating theatre, such as nitrates, phosphodiesterase inhibitors and calcium channel antagonists have a broad but short-lived effect against a variety of vasoconstrictor stimuli. Treatments that react irreversibly with proteins modulating vasoconstriction have the advantage that their effects can last well into the postoperative period. In addition systemic effects are avoided as the treatment is localised to the treated graft. This study investigated the use of two clinically applied drugs; fluphenazine (SKF7171A, HCl), an irreversible calmodulin antagonist and minoxidil sulphate, an irreversible potassium channel opener. Treatments were tested against receptor and non-receptor-mediated contraction in the human radial artery. METHOD: Isometric tension was measured in response to angiotensin II, KCl and vasopressin in 108 radial artery rings (taken from 31 patients undergoing coronary artery bypass grafting). Control responses were compared with rings pretreated with fluphenazine or minoxidil sulphate. Vasopressin responses were also compared in the presence of glyceryl trinitrate or the reversible Rho kinase inhibitor Y27632. RESULTS: Fluphenazine pretreatment significantly suppressed vasoconstriction to all agonists tested. Maximal responses to angiotensin II, vasopressin and KCl were reduced by 42+/-19%, 35+/-8% and 48+/-15% respectively, without any measurable effect on the EC(50). Minoxidil sulphate showed no discernable effect. Vasopressin-induced contraction was also reduced by high levels of glyceryl trinitrate (220 microM; 50 microg/ml) or 10 microM Y27632. CONCLUSIONS: The irreversible calmodulin antagonist fluphenazine has potential to be developed as an inhibitor of contraction in arterial graft vessels. The involvement of Rho kinase indicates that other vasoconstrictors and surgical stress can sensitize radial artery to vasopressin-induced contraction. Strategies targeting this pathway also have future potential.

Different effects of KCa and KATP agonists on brain tumor permeability between syngeneic and allogeneic rat models.

The blood-brain tumor barrier (BTB) significantly limits delivery of effective concentrations of chemotherapeutic drugs to brain tumors. Previous studies suggest that BTB permeability may be modulated via alteration in the activity of potassium channels. In this study, we studied the relationship of BTB permeability increase mediated by potassium channel agonists to channel expression in two rat brain tumor models. Intravenous infusion of KCO912 (K(ATP) agonist), minoxidil sulfate (K(ATP) agonist) or NS1619 (K(Ca) agonist) increased tumor permeability more in the 9L allogeneic brain tumor model than in the syngeneic brain tumor model. Consistently, expression of both K(ATP) and K(Ca) channels in 9L tumors was increased to a significantly greater extent in Wistar rats (allogeneic) as compared to Fischer rats (syngeneic). Furthermore, as a preliminary effort to understand clinical implication of potassium channels in brain tumor treatment, we determined the expression of K(ATP) in surgical specimens. K(ATP) mRNA was detected in glioblastoma multiforme (GBM) from nineteen patients examined, with a wide range of expression levels. Interestingly, in paired GBM tissues from seven patients before and after vaccination therapy, increased levels of K(ATP) were detected in five patients after vaccination that had positive response to chemotherapy after vaccination. The present study indicates that the effects of potassium channel agonists on BTB permeability are different between syngeneic and allogeneic models which have different expression levels of potassium channels. The expression of potassium channels in brain tumors is variable, which may be associated with different tumor permeability to therapeutic agents among patients.

Adenosine 5'-triphosphate-sensitive potassium channel-mediated blood-brain tumor barrier permeability increase in a rat brain tumor model.

Brain tumor microvessels/capillaries limit drug delivery to tumors by forming a blood-brain tumor barrier (BTB). The BTB overexpresses ATP-sensitive potassium (K(ATP)) channels that are barely detectable in normal brain capillaries, and which were targeted for BTB permeability modulation. In a rat brain tumor model, we infused minoxidil sulfate (MS), a selective K(ATP) channel activator, to obtain sustained, enhanced, and selective drug delivery, including various sized molecules, across the BTB to brain tumors. Glibenclamide, a selective K(ATP) channel inhibitor, significantly attenuated the MS-induced BTB permeability increase. Immunocytochemistry and glibenclamide binding studies showed increased K(ATP) channel density distribution on tumor cells and tumor capillary endothelium, which was confirmed by K(ATP) channel potentiometric assay in tumor cells and brain endothelial cells cocultured with brain tumor cells. MS infusion in rats with brain tumors significantly increased transport vesicle density in tumor capillary endothelial and tumor cells. MS facilitated increased delivery of macromolecules, including Her-2 antibody, adenoviral-green fluorescent protein, and carboplatin, to brain tumors, with carboplatin significantly increasing survival in brain tumor-bearing rats. K(ATP) channel-mediated BTB permeability increase was also demonstrated in a human, brain tumor xenograft model. We conclude that K(ATP) channels are a potential target for biochemical modulation of BTB permeability to increase antineoplastic drug delivery selectively to brain tumors.

Reduction of potassium currents and phosphatidylinositol 3-kinase-dependent AKT phosphorylation by tumor necrosis factor-(alpha) rescues axotomized retinal ganglion cells from retrograde cell death in vivo.

Tumor-necrosis-factor-alpha (TNF-alpha) prevented secondary death of retinal ganglion cells (RGCs) after axotomy of the optic nerve in vivo. This RGC rescue was confirmed in vitro in a mixed retinal culture model. In accordance with our previous findings, TNF-alpha decreased outward potassium currents in RGCs. Antagonism of the TNF-alpha-induced decrease in outward potassium currents with the potassium channel opener minoxidilsulfate (as verified by electrophysiology) abolished neuroprotection. Western blot analysis revealed an upregulation of phospho-Akt as a consequence of TNF-alpha-induced potassium current reduction. Inhibition of the phosphatidylinositol 3-kinase-Akt pathway with wortmannin decreased TNF-alpha-promoted RGC survival. These data point to a functionally relevant cytokine-dependent neuroprotective signaling cascade in adult CNS neurons.

New windows on the mechanism of action of K(ATP) channel openers.

K(ATP) channel openers are a diverse group of drugs with a wide range of potential therapeutic uses. Their molecular targets, the K(ATP) channels, exhibit tissue-specific responses because they possess different types of regulatory sulfonylurea receptor subunits. It is well recognized that complex interactions occur between K(ATP) channel openers and nucleotides, but the cloning of the K(ATP) channel has introduced a new dimension to the study of these events and has furthered our understanding of the molecular basis of the action of K(ATP) channel openers.

The effect of minoxidil analogues and metabolites on the contraction of collagen lattices by human skin fibroblasts.

Minoxidil, in addition to its effect on hypertension and hair growth, has been proposed as a potential antifibrotic agent. Minoxidil inhibits the contraction of collagen lattices by human fibroblasts in vitro. However, the mechanism of inhibition is unknown. As minoxidil is metabolised in the body to minoxidil glucuronide and minoxidil sulphate, we investigated the potencies of these metabolites to inhibit collagen lattice contraction. We also studied selected analogues of minoxidil to assess the influence of certain functional groups in the inhibition. The major metabolite, minoxidil glucuronide, proved to be inactive, whereas minoxidil sulphate was considerably more active than minoxidil. In terms of the structural analogues, the substitution of one amino group by a methyl group resulted in loss of the inhibitory activity; removal of the nitroxide oxygen led to stronger inhibition than with minoxidil. Further studies are planned to learn more about a possible role for minoxidil in wound contraction.

Stimulation of Cl conductance by minoxidil sulfate and K conductance by minoxidil in eccrine clear cells.

Minoxidil sulfate (MXS), an antihypertensive agent and hair growth promoter, has been reported to stimulate K channels in vascular smooth muscle cells. We now report that MXS stimulates whole cell Cl currents, whereas minoxidil (MX) stimulates K currents in dissociated eccrine clear cells. Using whole cell clamp techniques we observed that: 1) 1 mM MXS stimulated sweat secretion in vitro; 2) MXS depolarized the membrane potential by as much as 40 mV; 3) MXS stimulated membrane conductance, increased inward current and shifted the reversal potential to the right when physiological electrolyte solutions were used; 4) in symmetrical Cl (Cl/Cl) solutions without permeable cations, MXS induced outwardly rectifying current-voltage (I-V) relationships; 5) in the Cl/Cl solutions, the MXS-induced current responses to imposed voltage pulses showed time-dependent activation, especially at the depolarizing potentials; 6) the reversal potential of the MXS-stimulated I-V curves in the Cl/Cl solutions shifted to the right by 55 mV when [Cl] in the bath was decreased from 157 to 7 mM; 7) MXS did not elevate cytosolic Ca or cAMP, although prolonged exposure to a Ca-free solution abolished the effect of MXS and 8) MXS-stimulated conductance was partially inhibited by diphenylamine-2-carboxylic acid, a blocker of Cl channels. The data suggest that MXS stimulates Cl channels, most likely depolarization-activated, outwardly rectifying channels. In contrast, the parent compound MX hyperpolarized the membrane potential and stimulated outward current without elevating cytosolic Ca and was independent of extracellular Ca, suggesting that MX stimulates Ca-insensitive K currents.

Pharmacology and structure-activity relationships for KATP modulators: tissue-selective KATP openers.

KATP openers are recognized as having a therapeutic potential for the treatment of various cardiovascular and noncardiovascular diseases. However, the first-generation agents open KATP in a variety of tissues that limit their potential clinical utility. This review describes our studies aimed at the identification of a common pharmacophore among structurally diverse KATP openers and the discovery of tissue-selective agents that may offer advantages over the first-generation agents for the treatment of cardiovascular diseases. Compounds (e.g., BMS-182264) combining the structural features of KATP openers cromakalim and pinacidil, show smooth-muscle relaxing and antihypertensive activities comparable to their predecessors, indicating cromakalim and pinacidil may express their biologic effects through similar structural requirements. The hypothesis about common features is further supported by radioligand-binding studies showing cromakalim, pinacidil, and the combination compound BMS-182264 bind to a similar receptor site in rat aortic smooth-muscle cells. Regardless of having a pharmacologic profile similar to cromakalim and pinacidil in vascular smooth muscle, BMS-182264 had no effect on action potential duration in guinea pig papillary muscle, indicating smooth-muscle selectivity for BMS-182264. We demonstrate that no correlation exists between anti-ischemic and smooth-muscle relaxing potencies for a variety of structurally different KATP openers. Efforts to find KATP openers selective for the ischemic myocardium led to the identification of BMS-180448 which, despite having similar anti-ischemic potency to cromakalim, was significantly less active as a smooth-muscle relaxant. This compound was shown to be efficacious as an anti-ischemic agent in vivo without affecting hemodynamic variables, a potential liability of first-generation compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

Synergistic effects of glyburide and U-37883A, two structurally different vascular ATP-sensitive potassium channel antagonists.

Glyburide, a sulfonylurea, and U-37883A, a guanidine (4-Morpholinecarboximidine-N-1-Adamantyl-N' cyclohexylhydrochloride), have been previously characterized as antagonists of the vascular ATP-sensitive K+ channels (KATP). In this report, the in vitro interaction between these two chemically distinct KATP antagonists was investigated using isolated rabbit mesenteric artery. The KATP antagonism was functionally studied as the inhibition of vasodilation produced by various KATP openers as follows: pinacidil (1 microM), minoxidil sulfate (5 microM), cromakalim (0.5 microM) and RP-49356 (1 microM). Glyburide alone produced inhibition in the concentration range of 50 to 500 nM with the glyburide IC50 ranging from 72 to 148 nM. U-37883A alone produced inhibition in the concentration range of 0.5 to 5 microM, with the U-37883A IC50 ranging from 0.78 to 1.4 microM. In the presence of a threshold U-37883A concentration of 0.5 microM, the glyburide inhibition dose-response curve against pinacidil was significantly shifted to the left and the glyburide IC50 was lowered from 72 to 3.9 nM, representing an 18-fold decrease. Similarly, in the presence of a threshold glyburide concentration of 50 nM, the U-37883A inhibition dose-response curve against pinacidil was significantly shifted to the left and the U-37883A IC50 was lowered from 780 to 96 nM, representing an eightfold decrease. Thus, glyburide and U-37883A potentiated each other's effects as KATP blockers. This synergistic interaction between glyburide and U-37883A was observed independently of the pinacidil, cromakalim or minoxidil sulfate used to produce vasodilation.(ABSTRACT TRUNCATED AT 250 WORDS)

4-morpholinecarboximidine-N-1-adamantyl-N'-cyclohexylhydrochloride (U-37883A): pharmacological characterization of a novel antagonist of vascular ATP-sensitive K+ channel openers.

This study describes the in vitro and in vivo characteristics of a guanidine 4-morpholinecarboximidine-N-1-adamantyl-N'-cyclohexyl-hydroc hloride (U-37883A), as an antagonist of vascular ATP-sensitive K+ channels (KATP). In isolated rabbit mesenteric artery, the antagonistic effects of U-37883A (0.5-5 microM) were studied against vasorelaxation produced by cromakalim (0.5 microM), minoxidil sulfate (5 microM), pinacidil (1 microM) and RP-49356 (1 microM). The dose-response curves for U-37883A against all four potassium channel openers were similar with U-37883A, IC50S ranging from 0.78 to 1.4 microM, suggesting that U-37883A is producing inhibition by acting at a step that is common to all four potassium channel openers during their activation of the vascular KATP. In contrast, U-37883A at 10 microM did not inhibit relaxation dose-response curves by nitroglycerine, forskolin or D600. U-37883A (1 or 10 microM) effectively inhibited as well as reversed 42K efflux-stimulated by cromakalim (1 microM) or minoxidil sulfate (5 microM). Finally, U-37883A (3 mg/kg i.v.) was found to inhibit significantly as well as reverse hypotension produced by minoxidil (1 mg/kg i.v. or 3 mg/kg p.o.), cromakalim (0.5 mg/kg p.o.) and pinacidil (0.3 mg/kg i.v.) in rats, cats and dogs. In contrast, the in vivo responses to phenylephrine, nitroglycerine, sodium nitroprusside or isoproterenol were not altered. U-37883A thus appears to be the first nonsulfonylurea shown to block consistently and selectively the in vitro as well as the in vivo pharmacological responses to various potassium channel openers. This structurally novel KATP antagonist therefore would be useful for further characterizing the mechanisms of pharmacological modulation of the KATP in a variety of cell systems.

Minoxidil sulfate is the active metabolite that stimulates hair follicles.

An important step in understanding minoxidil's mechanism of action on hair follicles was to determine the drug's active form. We used organ-cultured vibrissa follicles to test whether it is minoxidil or its sulfated metabolite, minoxidil sulfate, that stimulates hair growth. Follicles from neonatal mice were cultured with or without drugs and effects were assessed by measuring incorporation of radiolabeled cysteine in hair shafts of the treated follicles. Assays of minoxidil sulfotransferase activity indicated that vibrissae follicles metabolize minoxidil to minoxidil sulfate. Dose-response studies showed that minoxidil sulfate is 14 times more potent than minoxidil in stimulating cysteine incorporation in cultured follicles. Three drugs that block production of intrafollicular minoxidil sulfate were tested for their effects on drug-induced hair growth. Diethylcarbamazine proved to be a noncompetitive inhibitor of sulfotransferase and prevented hair growth stimulation by minoxidil but not by minoxidil sulfate. Inhibiting the formation of intracellular PAPS with chlorate also blocked the action of minoxidil but not of minoxidil sulfate. Acetaminophen, a potent sulfate scavenger blocked cysteine incorporation by minoxidil. It also blocked follicular stimulation by minoxidil sulfate apparently by directly removing the sulfate from the drug. Experiments with U-51,607, a potent minoxidil analog that also forms a sulfated metabolite, showed that its activity was inhibited by both chlorate and diethylcarbamazine. These studies show that sulfation is a critical step for hair-growth effects of minoxidil and that it is the sulfated metabolite that directly affects hair follicles.

Glyburide blocks the relaxation response to BRL 34915 (cromakalim), minoxidil sulfate and diazoxide in vascular smooth muscle.

BRL 34915 [6-cyano-3,4-dihydro-2,2-dimethyl-trans-4-(2-oxo-1-pyrrolidyl) 2H-benzo(b) pyran-3-ol], minoxidil sulfate and diazoxide may relax vascular smooth muscle via hyperpolarization due to an opening of membrane potassium channels. We therefore examined the effects of several potassium channel antagonists on the relaxation response to these vasodilators in isolated rat portal venous strips which were mounted in vitro for detecting changes in isometric force. BRL 34915 (IC50 = 4.7 X 10(-8) M), minoxidil sulfate (IC50 = 1.4 X 10(-7) M) and diazoxide (IC50 = 5 X 10(-6) M) elicited concentration-dependent relaxations of the spontaneous, myogenic contractions in venous strips. The relatively nonselective potassium channel antagonists tetraethylammonium ion (0.3-10 X 10(-3) M) and 4-aminopyridine (1-10 X 10(-3) M) caused concentration-dependent shifts (5- to 50-fold) in the relaxation responses to each vasodilator. Charybdotoxin (up to 10(-7) M) and apamin (up to 10(-7) M), known to be antagonists of high and low conductance calcium-activated potassium channels, respectively, had no inhibitory effect on the relaxation-response curves to BRL 34915, minoxidil sulfate or diazoxide. Glyburide (10(-7) to 3 X 10(-5) M), a sulfonylurea which has been shown to block the ATP-modulated potassium channel in insulin-secreting cells, caused concentration-dependent shifts to the right (up to 100-fold) of the IC50 value for BRL 34915 and diazoxide, and at 10(-6) M, abolished the relaxation response to minoxidil sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)