Ethnic variation and structure-function analysis of tauopathy-associated PERK alleles.

EIF2AK3, also known as PERK, plays a pivotal role in cellular proteostasis, orchestrating the Unfolded Protein Response (UPR) and Integrated Stress Response (ISR) pathways. In addition to its central position in intracellular stress regulation, human GWAS identify EIF2AK3 as a risk factor in tauopathies, neurodegenerative diseases caused by aberrant tau protein accumulation. Guided by these genomic indicators, our investigation systematically analyzed human PERK variants, focusing on those with potential tauopathy linkages. We assembled a comprehensive data set of human PERK variants associated with Wolcott Rallison Syndrome (WRS), tauopathies, and bioinformatically predicted loss-of-function, referencing the gnomAD, Ensembl, and NCBI databases. We found extensive racial/ethnic variation in the prevalence of common PERK polymorphisms linked to tauopathies. Using SWISS-MODEL, we identified structural perturbations in the ER stress-sensing luminal domain dimers/oligomers of tauopathy-associated PERK variants, Haplotypes A and B, in combination with another tauopathy-linked R240H mutation. Recombinant expression of disease-associated variants in vitro revealed altered PERK signal transduction kinetics in response to ER stress compared to the predominant non-disease variant. In summary, our data further substantiates that human PERK variants identified in tauopathy genetic studies negatively impact PERK structure, function, and downstream signaling with significant variations in prevalence among different racial and ethnic groups.

Effects of Enteric-Coated Formulation of Sodium Bicarbonate on Bicarbonate Absorption and Gastrointestinal Discomfort.

Sodium bicarbonate is used as an ergogenic supplement to enhance people's performances in various exercises. This study aimed to evaluate the effects of intestinal delivery of sodium bicarbonate on bicarbonate absorption and associated side effects in an experimental human trial. After preparing and assessing enteric-coated and uncoated sodium bicarbonate tablet formulations, pharmacokinetic analysis and gastrointestinal symptom tests were performed after oral administration in the human body. The dose required to increase blood bicarbonate concentration over 5 mmol∙L-1 for the purpose of improving performance during high-intensity exercise was also determined. Enteric-coated tablet formulation protects sodium bicarbonate under acidic conditions and releases bicarbonate in the intestine. Enteric-coated tablet formulation also reduced the oral dose required to achieve a blood bicarbonate concentration over 5 mmol∙L-1 from 300 mg∙kg-1 of uncoated tablet formulation to 225 mg∙kg-1. Gastrointestinal discomfort was significantly decreased for the group given 225 mg∙kg-1 enteric-coated tablets compared to that given 300 mg∙kg-1 uncoated tablets. These results suggest that enteric-coated tablet formulation could reduce the oral dose required in order to achieve a blood bicarbonate concentration over 5 mmol∙L-1 by 25%, from 300 mg∙kg-1 to 225 mg∙kg-1, along with its ability to reduce gastrointestinal discomfort associated with the dosage.

Tumor-homing peptide iRGD-conjugate enhances tumor accumulation of camptothecin for colon cancer therapy.

Poor intracellular uptake of therapeutics in the tumor parenchyma is a key issue in cancer therapy. We describe a novel approach to enhance tumor targeting and achieve targeted delivery of camptothecin (CPT) based on a tumor-homing internalizing RGD peptide (iRGD). We synthesized an iRGD-camptothecin conjugate (iRGD-CPT) covalently coupled by a heterobifunctional linker and evaluated its in vitro and in vivo activity in human colon cancer cells. In vitro studies revealed that iRGD-CPT penetrated cells efficiently and reduced colon cancer cell viability to a significantly greater extent at micromolar concentrations than did the parent drug. Furthermore, iRGD-CPT showed high distribution toward tumor tissue, effectively suppressed tumor progression, and showed enhanced antitumor effects relative to the parent drug in a mouse model, demonstrating that iRGD-CPT is effective in vivo cancer treatment. These results suggest that intracellular delivery of CPT via the iRGD peptide is a promising drug delivery strategy that will facilitate the development of CPT derivatives and prodrugs with improved efficacy.

Neurodegeneration risk factor, EIF2AK3 (PERK), influences tau protein aggregation.

Tauopathies are neurodegenerative diseases caused by pathologic misfolded tau protein aggregation in the nervous system. Population studies implicate EIF2AK3 (eukaryotic translation initiation factor 2 alpha kinase 3), better known as PERK (protein kinase R-like endoplasmic reticulum kinase), as a genetic risk factor in several tauopathies. PERK is a key regulator of intracellular proteostatic mechanisms-unfolded protein response and integrated stress response. Previous studies found that tauopathy-associated PERK variants encoded functional hypomorphs with reduced signaling in vitro. But, it remained unclear how altered PERK activity led to tauopathy. Here, we chemically or genetically modulated PERK signaling in cell culture models of tau aggregation and found that PERK pathway activation prevented tau aggregation, whereas inhibition exacerbated tau aggregation. In primary tauopathy patient brain tissues, we found that reduced PERK signaling correlated with increased tau neuropathology. We found that tauopathy-associated PERK variants targeted the endoplasmic reticulum luminal domain; and two of these variants damaged hydrogen bond formation. Our studies support that PERK activity protects against tau aggregation and pathology. This may explain why people carrying hypomorphic PERK variants have increased risk for developing tauopathies. Finally, our studies identify small-molecule augmentation of PERK signaling as an attractive therapeutic strategy to treat tauopathies by preventing tau pathology.

Daphne odora Exerts Depigmenting Effects via Inhibiting CREB/MITF and Activating AKT/ERK-Signaling Pathways.

Daphne odora, a blooming shrub, has been traditionally used for various medicinal purposes. However, information on its anti-melanogenic activity and dermal application is limited. In this study, the Daphne odora extract (DOE), with constituents including daphnetin, was used to investigate depigmenting activity and the underlying mechanism of Daphne odora. DOE inhibited in vitro and cellular tyrosinase activity in a dose-dependent manner, and reduced the α-MSH-induced melanin biosynthesis to a control level. The protein expressions of melanin synthesis-related enzymes were also significantly reduced by DOE. Moreover, DOE decreased the phosphorylation of cAMP-response element binding proteins (CREBs) induced by α-MSH in B16F10 cells, while it activated phosphorylated extra-cellular signal-regulated kinases (ERKs) and protein kinase B (AKT) expression. These results suggest that DOE might inhibit the melanogenesis signaling pathways by activating ERK- and AKT-signaling pathways to regulate the expression of CREB and MITF and its downstream pathways. Therefore, DOE could potentially be developed as a depigmenting agent.

Two-megahertz impedance index prediction equation for appendicular lean mass in Korean older people.

BACKGROUND: Whole-body bioelectrical impedance analysis (BIA) has been accepted as an indirect method to estimate appendicular lean mass (ALM) comparable to dual-energy X-ray absorptiometry (DXA). However, single or limited frequencies currently used for these estimates may over or under-estimate ALM. Accordingly, there is a need to measure the impedance parameter with appendicular lean-specific across multiple frequencies to more accurately estimate ALM. We aimed to validate muscle-specific frequency BIA equation for ALM using multifrequency BIA (MF-BIA) with DXA as the reference. METHODS: 195 community-dwelling Korean older people (94 men and 101 women) aged 70 ~ 92y participated in this study. ALM was measured by DXA and bioimpedance measures at frequencies of 5 kHz ~ 3 MHz were assessed for independent predictive variables. Regression analyses were used to find limb-specific frequencies of bioimpedance, to develop the ALM equations and to conduct the internal cross-validation. The six published equations and the final equation of MF-BIA were externally cross-validated. RESULTS: 195 participants completed the measurements of MF-BIA and DXA. Using bivariate regression analysis, the 2 MHz impedance index explained R2 = 91.5% of variability (P < 0.001) in ALM and predictive accuracy of standard error of estimate (SEE) was 1.0822 kg ALM (P < 0.001). Multiple stepwise regression analysis obtained in the development group had an adjusted R2 of 9.28% (P < 0.001) and a SEE of 0.97 kg ALM. The cross-validation group had no significant difference between the measured ALM and the predicted ALM (17.8 ± 3.9 kg vs. 17.7 ± 3.8 kg, P = .486) with 93.1% of R2 (P < 0.001) and 1.00 kg ALM of total error. The final regression equation was as follows: ALM = 0.247ZI@2 MHz + 1.254SEXM1F0 + 0.067Xc@5 kHz + 1.739 with 93% of R2 (P < 0.001), 0.97 kg ALM of SEE (Subjective Rating as "excellent" for men and "very good" for women). In the analysis of the diagnostic level for sarcopenia of the final regression, the overall agreement was 94.9% (k = 0.779, P < 0.001) with 71.4% of sensitivity, 98.8% of specificity, 91.3 of positive prediction value and 95.3% of negative prediction value. CONCLUSION: The newly developed appendicular lean-specific high-frequency BIA prediction equation has a high predictive accuracy, sensitivity, specificity, and agreement for both individual and group measurements. Thus, the high-frequency BIA prediction equation is suitable not only for epidemiological studies, but also for the diagnosis of sarcopenia in clinical settings.

Accuracy of Estimated Bioimpedance Parameters with Octapolar Segmental Bioimpedance Analysis.

The validity of the impedance parameters of the five body segments estimated using octapolar segmental bioelectrical impedance analysis (OS-BIA) has not been confirmed. This study aimed to verify the accuracy of the resistance (R), reactance (Xc), and phase angle of each five-body segment. The accuracy of the OS-BIA at 50 kHz was measured based on the direct tetrapolar segmental BIA. The differences in the estimated impedance parameters of the five body segments were compared to those measured from the OS-BIA in elderly men (N = 73) and women (N = 63). The estimated 50 kHz-R (Ω) was significantly higher than the measured 50 kHz-R in the right and left arms, and lower than the measured 50 kHz-R of the trunk, right leg, and left leg (all, p < 0.05). The estimated 50 kHz-phase angles in all the five body segments were significantly lower than the measured ones (all, p < 0.05). The findings suggest that the estimated impedance parameters, R, Xc, and phase angle of the trunk, were remarkedly underestimated, limiting the assessment of the physiological state of the organs in the body. Therefore, further intensive research is needed in the field of estimated segmental BIA in the future.

Melatonin-stimulated exosomes enhance the regenerative potential of chronic kidney disease-derived mesenchymal stem/stromal cells via cellular prion proteins.

Chronic kidney disease (CKD) is caused by dysfunctional kidneys, which result in complications like cardiovascular diseases. Chronic kidney disease-induced pathophysiological conditions decrease efficacy of autologous mesenchymal stem/stromal cell (MSC)-based therapy by reducing MSC functionality. To enhance therapeutic potential in patients with CKD, we isolated exosomes derived from melatonin-treated healthy MSCs (MT exosomes) and assessed the biological functions of MT exosome-treated MSCs isolated from patients with CKD (CKD-MSCs). Treatment with melatonin increased the expression of cellular prion protein (PrPC ) in exosomes isolated from MSCs through the upregulation of miR-4516. Treatment with MT exosomes protected mitochondrial function, cellular senescence, and proliferative potential of CKD-MSCs. MT exosomes significantly increased the level of angiogenesis-associated proteins in CKD-MSCs. In a murine hindlimb ischemia model with CKD, MT exosome-treated CKD-MSCs improved functional recovery and vessel repair. These findings elucidate the regenerative potential of MT exosome-treated CKD-MSCs via the miR-4516-PrPC signaling axis. This study suggests that the treatment of CKD-MSCs with MT exosomes might be a powerful strategy for developing autologous MSC-based therapeutics for patients with CKD. Furthermore, miR-4516 and PrPC could be key molecules for enhancing the regenerative potential of MSCs in ischemic diseases.

Melatonin suppresses senescence-derived mitochondrial dysfunction in mesenchymal stem cells via the HSPA1L-mitophagy pathway.

Mesenchymal stem cells (MSCs) are a popular cell source for stem cell-based therapy. However, continuous ex vivo expansion to acquire large amounts of MSCs for clinical study induces replicative senescence, causing decreased therapeutic efficacy in MSCs. To address this issue, we investigated the effect of melatonin on replicative senescence in MSCs. In senescent MSCs (late passage), replicative senescence decreased mitophagy by inhibiting mitofission, resulting in the augmentation of mitochondrial dysfunction. Treatment with melatonin rescued replicative senescence by enhancing mitophagy and mitochondrial function through upregulation of heat shock 70 kDa protein 1L (HSPA1L). More specifically, we found that melatonin-induced HSPA1L binds to cellular prion protein (PrPC ), resulting in the recruitment of PrPC into the mitochondria. The HSPA1L-PrPC complex then binds to COX4IA, which is a mitochondrial complex IV protein, leading to an increase in mitochondrial membrane potential and anti-oxidant enzyme activity. These protective effects were blocked by knockdown of HSPA1L. In a murine hindlimb ischemia model, melatonin-treated senescent MSCs enhanced functional recovery by increasing blood flow perfusion, limb salvage, and neovascularization. This study, for the first time, suggests that melatonin protects MSCs against replicative senescence during ex vivo expansion for clinical application via mitochondrial quality control.

Comparison of tadalafil pharmacokinetics after administration of a new orodispersible film versus a film-coated tablet.

BACKGROUND: An orodispersible film (ODF) of tadalafil may provide increased convenience for erectile dysfunction (ED) patients as compared to conventional tablet formulations. In this study, we aimed to compare the pharmacokinetic, safety, and tolerability profiles of a newly developed ODF formulation of tadalafil to those of a film-coated tablet (FCT) of tadalafil. MATERIALS AND METHODS: This study was conducted in healthy male subjects using an open-label, randomized sequence, two-period, two-formulation, single-dose, crossover design. The subjects were randomly assigned to one of two sequences of the two formulations: both the test drug (ODF) and the reference drug (FCT) contained 20 mg of tadalafil. Blood samples were collected up to 72 h after administration. Plasma concentrations of tadalafil were analyzed using liquid chromatography-tandem mass spectrometry. Geometric mean ratios (GMRs) of the ODF to FCT formulations and their 90% CIs for the pharmacokinetic parameters were estimated. Safety and tolerability were assessed throughout the study. RESULTS: Forty healthy male subjects were enrolled, and 36 of these completed the study. The GMRs (90% CIs) of the maximum plasma concentration and the area under the plasma concentration-time curve from time zero to the time of the last quantifiable concentration for tadalafil were 0.927 (0.882-0.974) and 0.972 (0.918-1.029), respectively. Both ODF and FCT formulations were well tolerated, and no clinically significant changes from the baseline were observed after dosing. CONCLUSION: The pharmacokinetics of the tadalafil ODF formulation did not differ significantly from those of the FCT formulation. Furthermore, the safety and tolerability profiles of the ODF formulation were comparable to those of the FCT formulation. Therefore, this tadalafil ODF formulation offers a convenient treatment option for patients with erectile dysfunction.

Paracellular permeation-enhancing effect of AT1002 C-terminal amidation in nasal delivery.

BACKGROUND: The identification of permeation enhancers has gained interest in the development of drug delivery systems. A six-mer peptide, H-FCIGRL-OH (AT1002), is a tight junction modulator with promising permeation-enhancing activity. AT1002 enhances the transport of molecular weight markers or agents with low bioavailability with no cytotoxicity. However, AT1002 is not stable in neutral pH or after incubation under physiological conditions, which is necessary to fully uncover its permeation-enhancing effect. Thus, we increased the stability or mitigated the instability of AT1002 by modifying its terminal amino acids and evaluated its subsequent biological activity. METHODS: C-terminal-amidated (FCIGRL-NH2, Pep1) and N-terminal-acetylated (Ac-FCIGRL, Pep2) peptides were analyzed by liquid chromatography-mass spectrometry. We further assessed cytotoxicity on cell monolayers, as well as the permeation-enhancing activity following nasal administration of the paracellular marker mannitol. RESULTS: Pep1 was nontoxic to cell monolayers and showed a relatively low decrease in peak area compared to AT1002. In addition, administration of mannitol with Pep1 resulted in significant increases in the area under the plasma concentration-time curve and peak plasma concentration at 3.63-fold and 2.68-fold, respectively, compared to mannitol alone. In contrast, no increase in mannitol concentration was shown with mannitol/AT1002 or mannitol/Pep2 compared to the control. Thus, Pep1 increased the stability or possibly reduced the instability of AT1002, which resulted in an increased permeation-enhancing effect of AT1002. CONCLUSION: These results suggest the potential usefulness of C-terminal-amidated AT1002 in enhancing nasal drug delivery, which may lead to the development of a practical drug delivery technology for drugs with low bioavailability.

The ACTN3 R577X variant in sprint and strength performance.

PURPOSE: The aim of this study is to examine the association between the distribution of ACTN3 genotypes and alleles in power, speed, and strength-oriented athletics. METHODS: ACTN3 genotyping was carried out for a total of 975 Korean participants: top-level sprinters (n = 58), top-level strength athletes (n = 63), and healthy controls (n = 854). RESULTS: Genetic associations were evaluated by chi-squire test or Fisher's exact test. In the power-oriented group composed of sprinters and strength athletes, the frequency of the XX genotype was significantly underrepresented (11.6%) in comparison to its representation in the control group (11.6% versus 19.1%, P < 0.05). When the power-oriented group was divided into strength-oriented and speed-oriented groups, no significant difference in the ACTN3 XX genotype was found between the strength-oriented athletes and the controls (15.9% versus 19.1%, P < 0.262). Only the speed-oriented athletes showed significant differences in the frequency distributions of the ACTN3 XX genotype (6.9% versus 19.1%, P < 0.05) from that of the controls. CONCLUSION: The ACTN3 genotype seems to mainly affect sports performance and especially speed.

The impact of AT1002 on the delivery of ritonavir in the presence of bioadhesive polymer, carrageenan.

New insights into the modification of the tight junctions theoretically offer the opportunity to regulate the diffusion barrier and then make it possible to investigate a permeation enhancer of low-bioavailability therapeutic agents. AT1002, a minimum biologically active fragment of zonula occludens toxin which reversibly opens intercellular tight junctions after binding to the Zonulin receptor, increased the transport of various molecular weight markers or low-bioavailability agents. The objective of this study was continuously to evaluate the permeation-enhancing ability of AT1002 in the presence of the bioadhesive agent, carrageenan after intranasal administration of the antiretroviral drug, ritonavir, and the permeation enhancement ratio compared with the previous results. The permeation-enhancing effect of AT1002 was significantly promoted by the bioadhesive agent, carrageenan. The administration of ritonavir with AT1002 and carrageenan resulted in a 2.55-fold increase in AUC(0-240min) and a 2.48-fold increase in C(max) compared with the control group. However, AT1002 in the absence of carrageenan did not produce a statistic enhancement in the absorption of ritonavir. Hence, AT1002 together with the addition of carrageenan may open a new approach of research in the tight junction modulated permeation enhancer, and allow the development of the mucosal drug delivery of therapeutic agents.

The influence of stabilizer and bioadhesive polymer on the permeation-enhancing effect of AT1002 in the nasal delivery of a paracellular marker.

Permeation enhancers are of major interest to improve the low bioavailability of therapeutic agents due to poor membrane permeation. AT1002, a six-amino acid fragment of Zonula occludens toxin, was reported to possess permeation-enhancing effects. However, further studies were suggested to focus on the peptide nature of AT1002 like stability and membrane clearance to accurately reflect its permeation-enhancing potential. Thus, this paper focused on the susceptibility of AT1002 for identifying additives to minimize the instability of AT1002, and the permeation-enhancing effect of AT1002 when co-administered with a bioadhesive polymer. The stability study showed that AT1002 were unstable in neutral to basic pH conditions and with increasing incubation time, and 5% dextrose and the 1% mixture of amino acids (arginine, cysteine, glycine) significantly minimized the instability of AT1002 at pH 7.4 for at least 6 hours, respectively. In the intranasal study of a paracellular marker, the administration of mannitol with AT1002 in 5% dextrose solution led to statistically significant 3.14- and 2.17-fold increases in C(max) and AUC(0-360min) in the presence of carrageenan over the control. Thus, the addition of carrageenan as a bioadhesive polymer and dextrose as a stabilizer together with AT1002 may allow the development of the mucosal drug delivery of low-bioavailability therapeutic agents.

The influence of AT1002 on the nasal absorption of molecular weight markers and therapeutic agents when co-administered with bioadhesive polymers and an AT1002 antagonist, AT1001.

OBJECTIVES: The purpose of this study was to demonstrate the effects of the tight junction permeation enhancer, AT1002, on the nasal absorption of molecular weight markers and low bioavailable therapeutic agents co-administered with bioadhesive polymers or zonulin antagonist. METHODS: The bioadhesive polymers, carrageenan and Na-CMC, were prepared with AT1002 to examine the permeation-enhancing effect of AT1002 on the nasal absorption of inulin, calcitonin and saquinavir after nasal administration to Sprague-Dawley rats. Blood samples were collected over a 6-hour period from a jugular cannula. In addition, we determined whether AT1002 exerts a permeation-enhancing effect via activation of PAR-2 specific binding to a putative receptor of zonulin. To examine this zonulin antagonist, AT1001, was administered 30 min prior to dosing with an AT1002/inulin solution and blood samples were collected over a 6-hour period. KEY FINDINGS: The bioadhesive polymers did not directly increase the absorption of inulin, calcitonin and saquinavir, but promoted the permeation-enhancing effect of AT1002 when delivered nasally, thereby significantly increasing the absorption of each drug. Pre-treatment with AT1001 antagonized the zonulin receptor and significantly minimized the permeation-enhancing effect of AT1002. CONCLUSION: These findings will assist in understanding the permeation-enhancing capability of and the receptor binding of AT1002. Further, combining AT1002 with carrageenan supports the development of the mucosal delivery of therapeutic agents that have low bioavailability even with bioadhesive agents.

Enhanced nasal absorption of hydrophilic markers after dosing with AT1002, a tight junction modulator.

AT1002 is a six-mer synthetic peptide, H-FCIGRL-OH, that retains the delta G and Zot biological activity of reversibly opening tight junctions and increases the paracellular transport of drugs. The objective of this study was to evaluate the possible use of AT1002 in enhancing the nasal availability of macromolecules using large paracellular markers as model agents. Male Sprague-Dawley rats cannulated in the jugular vein were randomly assigned to receive radiolabelled paracellular markers, [14C]PEG4000 or [14C]inulin, with/without AT1002, for each intranasal study. The plasma concentration of PEG4000 with AT1002 (10mg/kg) was significantly higher than that from PEG4000 control over 360 min following intranasal administration. The AUC0-360 min and Cmax from the PEG4000/AT1002 (10mg/kg) treatment were statistically (p<0.05) increased to 235% and 357%, of control, respectively. When inulin was administered with AT1002 (10 mg/kg), the plasma concentration was significantly higher (p<0.05) than control over 360 min, and increases (p<0.05) of 292% and 315% for AUC0-360 min and Cmax over control were observed, respectively. AT1002 significantly increased the nasal absorption of molecular weight markers, PEG4000 and inulin. This study suggests that AT1002 may be used to enhance the systemic availability of macromolecules when administered concurrently.

Effect of the six-mer synthetic peptide (AT1002) fragment of zonula occludens toxin on the intestinal absorption of cyclosporin A.

Zonula occludens toxin (Zot) and its biologically active fragment, delta G, have been shown to reversibly open tight junctions (TJ) in endothelial and epithelial cells. Recently, a six-mer synthetic peptide H-FCIGRL-OH (AT1002) was identified and synthesized that retains the Zot permeating effect on intercellular TJ. The objective of this study was to evaluate the biological activity of AT1002 on enhancing the oral administration of cyclosporin A (CsA). The intestinal permeability enhancing effect of AT1002 on the transport of CsA across Caco-2 cell monolayers was examined after the following treatments, i.e., CsA, CsA/protease inhibitors (PI), CsA/PI/benzalkonium chloride (BC), CsA/AT1002, CsA/PI/AT1002, and CsA/PI/BC/AT1002 (CsA 0.5 microCi/ml, PI (bestatin 15 mM and E-64 5mM), BC 0.005 w/v%, and AT1002 5mM, respectively). Apparent permeability coefficients (P app) were calculated for each treatment. In addition, four treatments, i.e., CsA, CsA/PI/BC, CsA/AT1002, and CsA/PI/BC/AT1002 (CsA 120 microCi/kg, PI (bestatin 30 mg/kg and E-64 10mg/kg), BC 0.1 w/v%, and AT1002 doses of 5, 10 or 40 mg/kg, respectively) were prepared and administered intraduodenally to male Sprague-Dawley rats (230-280 g, n=4-5). Blood samples were collected at 0, 20, 60, and 120 min post-dosing and CsA plasma concentrations were determined subsequently using a Beckman Liquid Scintillation Counter. No significant increases in CsA transport were observed in the Caco-2 cell culture experiments following pre-treatment with AT1002 (5mM). Even though, AT1002 appeared to increase the P app of CsA in each treatment (CsA/AT1002, 1.54+/-0.13 x 10(-6)cm/s and CsA/PI/AT1002, 1.76+/-0.05 x 10(-6)cm/s) compared to each control (CsA and CsA/PI), respectively. The plasma concentration of CsA was significantly increased over a range of 1.55-2.50 at 10 and 40 mg/kg dose of AT1002. Also, AUC 0-120 min of CsA over a range of 1.64-2.14 and the Cmax of CsA over a range of 1.77-2.56 was statistically and significantly increased at 10 and 40 mg/kg of AT1002 after the intraduodenal administration of CsA/PI/BC/AT1002 to Sprague-Dawley rats. AT1002 significantly increased the in vivo oral absorption of CsA in the presence of PI. This study suggests that AT1002-mediated tight junction modulation, combined with metabolic protection and stabilization, may be used to enhance the low oral bioavailability of certain drugs when administered concurrently.

Enhanced intestinal absorption of salmon calcitonin (sCT) from proliposomes containing bile salts.

PURPOSE: The feasibility of using proliposomes containing salmon calcitonin (sCT) and absorption enhancing agents, as an oral delivery system, to improve the intestinal absorption of sCT was explored using rats and Caco-2 cell systems. METHODS: Seventeen surfactants were examined for their effects with reference to accelerating the permeability of sCT (300 microg/ml) across Caco-2 cell monolayers, and damage to the intestinal epithelial cells, as measured by the change in transepithelial electrical resistance (TEER) across the cell monolayer. Proliposomes containing sCT (0.75%, w/w) and sodium taurodeoxycholate (NaTDC, 2.5%, w/w) (TDC proliposomes) were prepared according to the standard method using sorbitol and phosphatidylcholine as core and wall-forming materials, respectively, administered intra-duodenally to rats, and plasma concentrations of sCT were subsequently determined by LC-MS. RESULTS: Among the surfactants examined, some bile salts including NaTDC appeared to be the most advantageous when estimated based on the balance between the permeation enhancement (e.g., a 10.8-fold increase in the permeability of sCT for 0.1% NaTDC) and damage to the cells (e.g., a 3.55-fold decrease in the TEER value for 0.1% NaTDC). The administration of TDC proliposomes resulted in a 7.1-fold increase in the bioavailability (i.e., 0.49%) of sCT, when administered duodenally to rats. The size of the reconstituted liposomes in water was significantly smaller (e.g., 23.2 nm, number weighted diameter), and the entrapment efficiency (EE) of sCT in the reconstituted liposomes was 2.8-fold larger (54.9%), for TDC proliposomes, compared to proliposomes prepared without NaTDC (sCT proliposomes). CONCLUSION: A 7.1-fold increase in the bioavailability of sCT could be achieved from the TDC proliposomes. In addition to the intrinsic activity of the bile salt to fluidize the membrane, the simultaneous delivery of sCT and NaTDC to the site of absorption in the intestine via proliposomes and the subsequent formation of lipophilic ion-pair complexes between sCT and NaTDC at the site might have been contributing factors in this outstanding absorption enhancement.

Preparation and evaluation of proliposomes containing salmon calcitonin.

Salmon calcitonin (sCT)-containing proliposomes were prepared by penetrating a methanol-chloroformic solution of sCT and phosphatidylcholine (PC) into microporous sorbitol particles, followed by vacuum evaporation of the solvent. As a result, sCT proliposomes with free-flowing flowability were obtained. On contact with water, the proliposomes were rapidly converted into a liposomal dispersion, in which a certain amount of sCT was entrapped by the liposomes. The apparent permeability of sCT across Caco-2 cell monolayers was increased as the result of incorporating sCT into the proliposomes, suggesting that the pharmacokinetics of sCT would be modified through the administration of proliposomes. This is the first study that reports the successful loading of sCT, a protein drug, in proliposomes. The development of various dosage forms of sCT, especially solid dosage forms, appears be feasible using proliposomes.