Low-energy defibrillation using a base-apex epicardial electrode.

BACKGROUND: Current implantable cardioverter defibrillators (ICDs) require electric conduction with high voltage and high energy, which can impair cardiac function and induce another malignant arrhythmia. As a result, there has been a demand for an ICD that can effectively operate with lower energy to mitigate the risks of a strong electric shock. METHODS: A pair of sheet-shaped electrodes covering the heart were analyzed in three configurations (top-bottom, left-right, and front-back) using a heart simulator. We also varied the distance between the two electrodes (clearance) to identify the electrode shape with the lowest defibrillation threshold (DFT). We also investigated the ICD shock waveform, shock direction, and the effect of the backside insulator of the electrode. RESULTS: The DFT was high when the clearance was too small and the DFT was high even when the clearance was too large, suggesting that an optimal value clearance. The top-bottom electrodes with optimal clearance showed the lowest DFT when the biphasic shocks set the top electrode to a high potential first and then the bottom electrode was set to a high potential. An interval between a first shock waveform and a second shock waveform should be provided for low-energy defibrillation. Because the insulator prevents unnecessary current flow to the backside, the DFT of the electrodes with insulators is less than those without insulators. CONCLUSION: Painless defibrillation using sheet-shaped electrodes on the epicardium is predicated on the basis of results using a heart simulator.

FAM83G-based Peptide Induces Apoptosis on Cultured Liver Cancer Cell.

BACKGROUND: Previously, AF-956, which contains S356 of FAM83G and an N-terminal antenna peptide for entry into colon cancer cells, is markedly antiproliferative compared to a control peptide (AF-859), which lacks the N-terminal antenna peptide, by inducing apoptosis via the inhibition of HSP27 phosphorylation at residues S15 and S82. OBJECTIVE: Because FAM83G-derived peptides are promising lead compounds for colon cancer treatment, we reanalyzed the effect of AG-066, which contains S356 of FAM83G and an N-terminal antenna peptide for entry into the liver cancer cells. METHODS: HepG2 liver cancer cells were incubated with either AF-859 or AG-066 at a concentration of 54 µM at 37 °C for 24, 48, and 72 h. The effects of AF-859 and AG-066 on the cultured HepG2 cells were estimated using an inverted light microscope. Furthermore, the DNA ladder method and the dead cell assay were performed by applying Live/Dead Cell Staining Kit II. Erk phosphorylation was estimated by western blotting. RESULTS: Treatment with AG-066 markedly reduced HepG2 viable cell counts compared to the AF- 859-treated HepG2 cells, as evident from the significantly increased number of dead cells in the culture medium. Additionally, AG-066 treatment increased cellular DNA laddering. We found no difference in Erk phosphorylation status between the AG-066- and AF-859-treated groups. CONCLUSION: This study illustrated that the peptide with a structure based on FAM83G functions as a spontaneous apoptosis inducer for liver cancer cells. Hence, it is a promising lead compound for the treatment of liver cancer.

UT-Heart: A Finite Element Model Designed for the Multiscale and Multiphysics Integration of our Knowledge on the Human Heart.

To fully understand the health and pathology of the heart, it is necessary to integrate knowledge accumulated at molecular, cellular, tissue, and organ levels. However, it is difficult to comprehend the complex interactions occurring among the building blocks of biological systems across these scales. Recent advances in computational science supported by innovative high-performance computer hardware make it possible to develop a multiscale multiphysics model simulating the heart, in which the behavior of each cell model is controlled by molecular mechanisms and the cell models themselves are arranged to reproduce elaborate tissue structures. Such a simulator could be used as a tool not only in basic science but also in clinical settings. Here, we describe a multiscale multiphysics heart simulator, UT-Heart, which uses unique technologies to realize the abovementioned features. As examples of its applications, models for cardiac resynchronization therapy and surgery for congenital heart disease will be also shown.

TBC1D8B, a GTPase-activating protein, is a novel apoptosis inducer.

Overexpressed TBC1D8B, a GTPase-activating protein, significantly reduced cultured HCT116 human colon cancer cell number. We tested N-terminal TBC1D8B, which is identical to wild type TBC1D8B from amino acid positions 1 to 427 and possesses a modified sequence from position 428 to 435 (ECGGLFLL) because of the introduction of a premature stop codon at position 436 to narrow down the minimum requirement element. The N-terminal TBC1D8B contains two GRAM domains but not the TBC domain essential for Rab-GTPase activity. The N-terminal TBC1D8B overexpression significantly reduced the cultured HCT116 cell number. When we tested C-terminal TBC1D8B, containing the portion of TBC1D8B absent in the N-terminal TBC1D8B, the cell number reduction was not observed. The N-terminal TBC1D8B overexpression significantly increased the coronin 1B expression and reduced the phosphorylation of serine 51 in eIF2α, respective markers of apoptosis and cell death/survival. Also, caspase 3 and poly ADP-ribose polymerase increased cleavage in suspended cells overexpressing the N-terminal TBC1D8B. Taken together, it is not the TBC domain for Rab-GTPase activity, but amino acids 1 to 435, including the two GRAM domains, that is enough for TBC1D8B to cause spontaneous apoptosis. TBC1D8B could be a potential anticancer therapeutic molecule.

An application of a patient-specific cardiac simulator for the prediction of outcomes after mitral valve replacement: a pilot study.

Despite advancements in preoperative prediction of patient outcomes, determination of the most appropriate surgical treatments for patients with severely impaired cardiac function remains a challenge. "UT-Heart" is a multi-scale, multi-physics heart simulator, which can be used to assess the effects of treatment without imposing any burden on the patients. This retrospective study aimed to assess whether UT-Heart can function as a tool that aids decision making for performing mitral valve replacements (MVR) in patients with severe mitral regurgitation (MR) and impaired left ventricular (LV) function. We used preoperative clinical data to create a patient-specific heart model using UT-Heart for a patient who had dilated cardiomyopathy with severe MR. After confirming that this heart model reproduced the preoperative state of the patient, we performed an in silico MVR operation without changing any parameters, such as the end-diastolic volume of the left ventricle, systemic vascular resistance, and the number of myocardiocytes. Among the functional changes introduced by in silico surgery, we found two indices, forward flow and the mechanical efficiency of the work done to the systemic circulation, which may relate positively to the favorable outcome observed in the real world. Thus, multi-scale, multi-physics heart simulators can reproduce the pathophysiology of MR with impaired LV function. By performing in silico MVR and examining the resultant functional changes, we identified two indices, whose usefulness should be tested in future studies.

Ionic mechanisms of ST segment elevation in electrocardiogram during acute myocardial infarction.

ST elevation on an electrocardiogram is a hallmark of acute transmural ischemia. However, the underlying mechanism remains unclear. We hypothesized that high ischemic sensitivities of epicardial adenosine triphosphate-sensitive potassium (IKATP) and sodium (INa) currents play key roles in the genesis of ST elevation. Using a multi-scale heart simulation under moderately ischemic conditions, transmural heterogeneities of IKATP and INa created a transmural gradient, opposite to that observed in subendocardial injury, leading to ST elevation. These heterogeneities also contributed to the genesis of hyper-acute T waves under mildly ischemic conditions. By contrast, under severely ischemic conditions, although action potentials were suppressed transmurally, the potential gradient at the boundary between the ischemic and normal regions caused ST elevation without a contribution from transmural heterogeneity. Thus, transmural heterogeneities of ion channel properties may contribute to the genesis of ST-T changes during mild or moderate transmural ischemia, while ST elevation may be induced without the contribution of heterogeneity under severe ischemic conditions.

FAM83G Is a Novel Inducer of Apoptosis.

The family with sequence similarity 83 (FAM83) protein family G (FAM83G) possesses a predicted consensus phosphorylation motif for serine/threonine-protein kinase D1/protein kinase C mu (PKD1/PKCµ) at serine residue 356 (S356). In this study, overexpressed wild-type FAM83G coimmunoprecipitated with PKD1/PKCµ in Chinese hamster ovary (CHO) cells inhibited heat shock protein 27 (HSP27) phosphorylation at S82 and reduced the living cell number. The expression of a FAM83G phosphorylation-resistant mutant (S356A-FAM83G) had no effect on the living cell number or the induction of spontaneous apoptosis. By contrast, the introduction of a synthetic peptide encompassing FAM83G S356 into HCT116 and HepG2 cells decreased HSP27 S15 and S82 phosphorylation and induced spontaneous apoptosis. On the other hand, the introduction of FAM83G phosphorylation-resistant mutant synthesized peptides (S356A-AF-956 and S356A-AG-066) did not reduce the living cell number or induce spontaneous apoptosis. The endogenous expression of HSP27 and FAM83G was apparently greater in HCT116 and HepG2 cells compared with in CHO cells. In various types of lung cancer cell lines, the FAM83G messenger RNA (mRNA) level in non-small lung cancer cells was at a similar level to that in non-cancerous cells. However, the FAM83G mRNA level in the small cell lung cancer cell lines was variable, and the HSP27 mRNA level in FAM83G mRNA-rich types was greater than that in FAM83G mRNA-normal range types. Taken together, these data demonstrate that FAM83G S356 phosphorylation modulates HSP27 phosphorylation and apoptosis regulation and that HSP27 is a counterpart of FAM83G.

Personalized Perioperative Multi-scale, Multi-physics Heart Simulation of Double Outlet Right Ventricle.

For treatment of complex congenital heart disease, computer simulation using a three-dimensional heart model may help to improve outcomes by enabling detailed preoperative evaluations. However, no highly integrated model that accurately reproduces a patient's pathophysiology, which is required for this simulation has been reported. We modelled a case of complex congenital heart disease, double outlet right ventricle with ventricular septal defect and atrial septal defect. From preoperative computed tomography images, finite element meshes of the heart and torso were created, and cell model of cardiac electrophysiology and sarcomere dynamics was implemented. The parameter values of the heart model were adjusted to reproduce the patient's electrocardiogram and haemodynamics recorded preoperatively. Two options of in silico surgery were performed using this heart model, and the resulting changes in performance were examined. Preoperative and postoperative simulations showed good agreement with clinical records including haemodynamics and measured oxyhaemoglobin saturations. The use of a detailed sarcomere model also enabled comparison of energetic efficiency between the two surgical options. A novel in silico model of congenital heart disease that integrates molecular models of cardiac function successfully reproduces the observed pathophysiology. The simulation of postoperative state by in silico surgeries can help guide clinical decision-making.

Dapagliflozin Inhibits Cell Adhesion to Collagen I and IV and Increases Ectodomain Proteolytic Cleavage of DDR1 by Increasing ADAM10 Activity.

Dapagliflozin, empagliflozin, tofogliflozin, selective inhibitors of sodium-glucose cotransporter 2 (SGLT2), is used clinically to reduce circulation glucose levels in patients with type 2 diabetes mellitus by blocking the reabsorption of glucose by the kidneys. Dapagliflozin is metabolized and inactivated by UGT1A9. Empagliflozin is metabolized and inactivated by UGT1A9 and by other related isoforms UGT2B7, UGT1A3, and UGT1A8. Tofogliflozin is metabolized and inactivated by five different enzymes CYP2C18, CYP3A4, CYP3A5, CYP4A11, and CYP4F3. Dapagliflozin treatment of HCT116 cells, which express SGLT2 but not UGT1A9, results in the loss of cell adhesion, whereas HepG2 cells, which express both SGLT2 and UGT1A9, are resistant to the adhesion-related effects of dapagliflozin. PANC-1 and H1792 cells, which do not express either SGLT2 or UGT1A9, are also resistant to adhesion related effects of dapagliflozin. On the other hand, either empagliflozin or tofogliflozin treatment of HCT116, HepG2, PANC-1, and H1792 cells are resistant to the adhesion-related effects as observed in dapagliflozin treated HCT116 cells. Knockdown of UGT1A9 by shRNA in HepG2 cells increased dapagliflozin sensitivity, whereas the overexpression of UGT1A9 in HCT116 cells protected against dapagliflozin-dependent loos of cell adhesion. Dapagliflozin treatment had no effect on cellular interactions with fibronectin, vitronectin, or laminin, but it induced a loss of interaction with collagen I and IV. In parallel, dapagliflozin treatment reduced protein levels of the full-length discoidin domain receptor I (DDR1), concomitant with appearance of DDR1 cleavage products and ectodomain shedding of DDR1. In line with these observations, unmetabolized dapagliflozin increased ADAM10 activity. Dapagliflozin treatment also significantly reduced Y792 tyrosine phosphorylation of DDR1 leading to decrement of DDR1 function and detachment of cancer cells. Concomitant with these lines of results, we experienced that CEA in patients with colon cancer, which express SGLT2 but not UGT1A9, and type 2 diabetes mellitus treated by dapagliflozin in addition to chemotherapy was decreased (case 1). CEA in patients with colon cancer, which express SGLT2 but not UGT1A9, and type 2 diabetes mellitus was treated by dapagliflozin alone after radiation therapy was decreased but started to rise after cessation of dapagliflozin (case 2). CA19-9 in two of patients with pancreatic cancer and type 2 diabetes mellitus was resistant to the combination therapy of dapagliflozin and chemotherapy (case 3 and 4 respectively). PIVKAII in patients with liver cancer and type 2 diabetes mellitus, and CYFRA in patients with squamous lung cancer and type 2 diabetes mellitus was also resistant the combination therapy of dapagliflozin and chemotherapy (case 5 and 6 respectively). Taken together, these data suggest a potential role for dapagliflozin anticancer therapy against colon cancer cells that express SGLT2, but not UGT1A9.

Continuous or Transient High Level of Glucose Exposure Differentially Increases Coronary Artery Endothelial Cell Proliferation and Human Colon Cancer Cell Proliferation.

We studied effect of high glucose levels on coronary artery endothelial cell proliferation and human colon cancer cell proliferation. To examine the long-term effect of glucose exposure on cell growth, cells were cultured for 14 days in the absence or presence of 183 mg/dL D-glucose addition in the culture medium. Short effect of elevated glucose levels was examined by addition of 183 mg/dL D-glucose addition in the culture medium for just one hour per day followed by changing the culture to standard medium (5.5 mM D-glucose) during the next 23-hours period. Cell proliferation was estimated by 2,3-Bis (2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carbox-anilide (XTT) assay and phosphor-Erk western blot analysis. We found that coronary artery endothelial cell proliferation was significantly increased in the culture medium with the acute one-hour addition of 183 mg/dL D-glucose compared to the absence or chronic presence of 183 mg/dL D-glucose addition in the culture medium. In contrast, colon cancer cell proliferation was significantly increased in the continuous presence of 183 mg/dL D-glucose addition in the culture medium compared to the acute one-hour addition of glucose. The extent of Erk2 phosphorylation paralleled with the relative changes in cellular proliferation in both cell types. Taken together, these results suggested that continuous or transient high level of glucose exposure differentially effects coronary artery endothelial and human colon cancer cell proliferation.

Effect of dapagliflozin on colon cancer cell [Rapid Communication].

Dapagliflozin is a SGLT2 (Sodium/Glucose cotransporter 2) inhibitor that reduces circulating glucose levels in type 2 diabetic patients by blocking the SGLT2-dependent reabsorption of glucose in the kidney. Dapagliflozin is metabolized by UGT1A9 (UDP Glucuronosyltransferase 1 family, Polypeptidase A9), suppressing its SGLT2 inhibitor activity. However little information is available on whether dapagliflozin acts in the absence of dapagliflozin metabolism. Treatment with 0.5µM dapagliflozin significantly reduced the number of HCT116 cells, which express SGLT2 but not UGT1A9. This was independent of SGLT2 inhibition, as the SGLT2 inhibitor phlorizin had no effect. Dapagliflozin also enhanced Erk phosphorylation but without changing levels of uncleaved and cleaved PPAR and uncleaved caspase-3, suggesting that the cause of the decrease in HCT116 cell number was apoptosis independent cell death. Taken together, these data indicate a new potential role for dapagliflozin as an anticancer reagent in tumor cell populations that do not express UGT1A9.

Synip phosphorylation is required for insulin-stimulated Glut4 translocation and glucose uptake in podocyte.

Previously we reported that the phosphorylation of Synip on serine 99 is required for Synip dissociation from Syntaxin4 and insulin-stimulated Glut4 translocation in cultured 3T3-L1 adipocytes. We also reported that the dissociated Synip remains anchored to the plasma membrane by binding to Phosphatidylinositol (3,4,5)-triphosphate. Recently Synip was reported to arrest SNARE-dependent membrane fusion as a selective t-SNARE binding inhibitor. In this study, we have found that Synip is expressed in podocytes although at a somewhat lower level than in adipocytes. To determine whether phosphorylation of Synip on serine 99 is required for insulin-stimulated Glut4 translocation and glucose uptake in podocytes we expressed a phosphorylation deficient Synip mutant (S99A-Synip) that inhibited insulin-stimulated Glut4 translocation and 2-deoxyglucose uptake in adipocytes. We conclude that serine 99 phosphorylation of Synip is required for Glut4 translocation and glucose uptake in both adipocytes and podocytes, suggesting that defects in Synip phosphorylation may underlie insulin resistance and associated diabetic nephropathy.

Mitochondrial colocalization with Ca2+ release sites is crucial to cardiac metabolism.

In cardiomyocyte subcellular structures, colocalization of mitochondria with Ca2+ release sites is implicated in regulation of cardiac energetics by facilitating Ca2+ influx into mitochondria to modulate the tricarboxylic acid (TCA) cycle. However, current experimental techniques limit detailed examination of this regulatory mechanism. Earlier, we developed a three-dimensional (3D) finite-element cardiomyocyte model featuring a subcellular structure that integrates excitation-contraction coupling and energy metabolism. Here, using this model, we examined the influence of distance between mitochondria and Ca2+ release sites by comparing a normal (50-nm) distance model and a large (200-nm) distance model (LD). The influence of distance was minimal under a low pacing rate (0.25 Hz), but under a higher pacing rate (2 Hz), lower levels of mitochondrial Ca2+ and NADH, elevated phosphate, and suppressed force generation became apparent in the LD model. Such differences became greater when functional impairments (reduced TCA cycle activity, uncoupling effect, and failing excitation-contraction coupling) were additionally imposed. We concluded that juxtaposition of the mitochondria and the Ca2+ release sites is crucial for rapid signal transmission to maintain cardiac-energy balance. The idealized 3D model of cardiac excitation-contraction and metabolism is a powerful tool to study cardiac energetics.

Effects of an hERG activator, ICA-105574, on electrophysiological properties of canine hearts.

In short QT syndrome, inherited gain-of-function mutations in the human ether a-gogo-related gene (hERG) K(+) channel have been associated with development of fatal arrhythmias. This implies that drugs that activate hERG as a side effect may likewise pose significant arrhythmia risk. hERG activators have been found to have diverse mechanisms of activation, which may reflect their distinct binding sites. Recently, the new hERG activator ICA-105574 was introduced, which disables inactivation of the hERG channel with very high potency. We explored characteristics of this new drug in several experimental models. Patch clamp experiments were used to verify activation of hERG channels by ICA-105574 in human embryonic kidney cells stably-expressing hERG channels. ICA-105574 significantly shortened QT and QTc intervals and monophasic action potential duration (MAP(90)) in Langendorff-perfused guinea-pig hearts. We also administered ICA-105574 to anesthetized dogs while recording ECG and drug plasma concentrations. ICA-105574 (10 mg/kg) significantly shortened QT and QTc intervals, with a free plasma concentration of approximately 1.7 µM at the point of maximal effect. Our data showed that unbound ICA-105574 caused QT shortening in dogs at concentrations comparable to the half maximal effective concentration (EC(50), 0.42 µM) of hERG activation in the patch clamp studies.

A three-dimensional simulation model of cardiomyocyte integrating excitation-contraction coupling and metabolism.

Recent studies have revealed that Ca(2+) not only regulates the contraction of cardiomyocytes, but can also function as a signaling agent to stimulate ATP production by the mitochondria. However, the spatiotemporal resolution of current experimental techniques limits our investigative capacity to understand this phenomenon. Here, we created a detailed three-dimensional (3D) cardiomyocyte model to study the subcellular regulatory mechanisms of myocardial energetics. The 3D cardiomyocyte model was based on the finite-element method, with detailed subcellular structures reproduced, and it included all elementary processes involved in cardiomyocyte electrophysiology, contraction, and ATP metabolism localized to specific loci. The simulation results were found to be reproducible and consistent with experimental data regarding the spatiotemporal pattern of cytosolic, intrasarcoplasmic-reticulum, and mitochondrial changes in Ca(2+); as well as changes in metabolite levels. Detailed analysis suggested that although the observed large cytosolic Ca(2+) gradient facilitated uptake by the mitochondrial Ca(2+) uniporter to produce cyclic changes in mitochondrial Ca(2+) near the Z-line region, the average mitochondrial Ca(2+) changes slowly. We also confirmed the importance of the creatine phosphate shuttle in cardiac energy regulation. In summary, our 3D model provides a powerful tool for the study of cardiac function by overcoming some of the spatiotemporal limitations of current experimental approaches.

PEGylation of osteoprotegerin/osteoclastogenesis inhibitory factor (OPG/OCIF) results in decreased uptake into rats and human liver.

OBJECTIVES: Our aim was to investigate the effect of PEGylation on the uptake of osteoprotegerin/osteoclastogenesis inhibitory factor (OPG/OCIF) into rat liver, kidney and spleen, and human liver. METHODS: Copolymer of polyethyleneglycol allylmethylether and maleamic acid sodium salt with OCIF (poly(PEG)-OCIF) (0.5 mg/kg) was administered to rats and the concentrations of poly(PEG)-OCIF in the liver, kidney and spleen at 15 min after administration were measured by ELISA. For human liver uptake, the liver perfusion of OCIF and (3)H-labelled poly(PEG)-OCIF was conducted using fresh human liver block. KEY FINDINGS: The tissue uptake of poly(PEG)-OCIF in rats was significantly lower compared with that of OCIF. In fresh human liver perfusion, (3)H-poly(PEG)-OCIF was rarely taken up into the liver. On the other hand, more than 50% of the perfused OCIF was taken up. CONCLUSIONS: PEGylation of OCIF using poly(PEG) dramatically suppressed the uptake of OCIF into human liver as well as into rat liver and could be a promising approach for improving the pharmacokinetic and pharmacological effects of OCIF in the clinical setting.

Extranodal natural killer/T-cell lymphoma, nasal type: the significance of radiotherapeutic parameters.

BACKGROUND: The objective of this study was to investigate the correlation between local recurrence and radiotherapeutic parameters, including dose and RT radiotherapy (RT) field. METHODS: The current study included 35 patients who were diagnosed with immunohistochemically confirmed nasal natural killer (NK)/T-cell lymphoma between 1976 and 2004. There were 21 males and 14 females, and they ranged in age from 18 years to 76 years (median, 51 yrs). The primary tumor originated in the nasal cavity in 28 patients, and 32 patients had Stage I disease. Seventeen patients received treatment solely with RT, and the remaining 18 patients received a combination of chemotherapy and RT. The median tumor dose was 50 grays (Gy) (range, 22-60 Gy). Twenty-seven patients received RT to include all macroscopic lesions, all paranasal sinuses, the palate, and the nasopharynx. Eight patients received RT to all macroscopic lesions with generous margins. RESULTS: A complete remission (CR) or a CR/unconfirmed was achieved in 28 patients (80%). The 5-year overall survival (OAS) rate, disease-free survival (DFS) rate, and local control probability (LCP) were 47.3%, 42.9%, and 65.2%, respectively. Patients who received RT only to macroscopic lesions fared less well in terms of LCP (LCP 5 years, 71.9% vs. 41.7%; P=0.007). The difference in RT field also affected both the OAS rate and the DFS rate. Patients who received RT doses>or=50 Gy tended to achieve favorable local control. CONCLUSIONS: In the management of nasal NK/T-cell lymphoma, the RT field affected treatment outcomes. RT doses>or=50 Gy resulted in favorable local control.

Enhancement of skin permeation of ketotifen by supersaturation generated by amorphous form of the drug.

Pressure sensitive adhesive (PSA) matrices containing amorphous ketotifen were prepared and evaluated for enhanced skin permeability of the drug. A solvent casting method using silicone-typed PSA was employed, and n-hexane, an original solvent for the PSA and one more solvent, dichloromethane, tetrahydrofuran, acetone, ethyl acetate or toluene, were used for complete dissolution of ketotifen and high dispersion in an amorphous state of the drug. Presence of the amorphous form was judged based on the in vitro drug release rate from the matrix. As a result, dichloromethane and tetrahudrofuran were selected as appropriate dilution solvents. In vitro permeation experiments through excised hairless mouse skin revealed that the steady-state flux from the amorphous ketotifen-dispersed matrices was about five times greater than that of the crystalline ketotifen-dispersed matrices, and that the enhancement ratio was in good agreement with the solubility ratio of the amorphous to crystalline form of the drug. Comparison of the skin permeation profiles of amorphous ketotifen-dispersed matrices between two different drug contents suggested that the steady-state flux was not influenced by the drug content. In addition, at both drug contents, the period of the steady-state permeation coincided with the time until the amorphous drug was depleted from the matrix. These results suggest that the increase in skin permeation of ketotifen from PSA matrix was due to the supersaturation generated by amorphous form, and that the amorphous form was stable during the application period.