Development of Sustained Release System Based on High Water-Absorbable Gel Formation Using Croscarmellose Sodium, Alkaline Excipients and HPMC (ACSH SR System); Novel Application of Croscarmellose Sodium as a Gel Former.

PURPOSE: Croscarmellose sodium, generally used as a superdisintegrant in pharmaceutical formulations, is hydrolyzed to form the gel structure under basic pH conditions. Utilizing this property of croscarmellose sodium, we developed a novel sustained release (SR) system. METHODS: Immediate release (IR) and SR tablets containing croscarmellose sodium, alkaline excipients and/or hydroxypropyl methylcellulose (HPMC) were prepared and examined for wet strength and in vitro drug release behavior. In vivo oral drug absorption was evaluated for IR tablets, HPMC tablets and our novel SR tablets in fasted Beagle dogs. RESULTS: To form the gel structure even under the physiological condition, alkaline excipients were added into the formulation containing croscarmellose sodium. Furthermore, HPMC was used to make the gel structure strong enough against mechanical destructive forces. The novel alkalized croscarmellose sodium-HPMC (ACSH) SR tablet, consisting of croscarmellose sodium, alkaline excipients, and HPMC, successfully sustained the release of acetaminophen, ibuprofen, or nicardipine hydrochloride, compared with the IR tablets. The ACSH SR system provided a better release of acetaminophen than the HPMC tablet without croscarmellose sodium in the release study using a small volume of liquid, suggesting that substantial release and subsequent absorption would be expected in the distal intestinal segments after oral dosing. The in vivo oral absorption study revealed that the ACSH SR system successfully suppressed and prolonged the plasma concentrations of acetaminophen. CONCLUSION: This novel ACSH SR system prepared with croscarmellose sodium, alkaline excipients, and HPMC, would be a promising SR formulation for enabling substantial drug absorption in the distal intestinal segments.

Highly Potent and Oral Macrocyclic Peptides as a HIV-1 Protease Inhibitor: mRNA Display-Derived Hit-to-Lead Optimization.

Human immunodeficiency virus type-1 (HIV-1) protease is essential for viral propagation, and its inhibitors are key anti-HIV-1 drug candidates. In this study, we discovered a novel HIV-1 protease inhibitor (compound 16) with potent antiviral activity and oral bioavailability using a structure-based drug design approach via X-ray crystal structure analysis and improved metabolic stability, starting from hit macrocyclic peptides identified by mRNA display against HIV-1 protease. We found that the improvement of the proteolytic stability of macrocyclic peptides by introducing a methyl group to the α-position of amino acid is crucial to exhibit strong antiviral activity. In addition, macrocyclic peptides, which have moderate metabolic stability and solubility in solutions containing taurocholic acid, exhibited desirable plasma total clearance and oral bioavailability. These approaches may contribute to the successful discovery and development of orally bioavailable peptide drugs.

Optimization of Cyclic Peptide Property Using Chromatographic Capacity Factor on Permeability of Passive Cell Membrane and Human Induced Pluripotent Stem Cell-Derived Intestinal Membrane.

Cyclic peptides have attracted increasing attention as a privileged class of molecules addressing undruggable targets. Cell permeability of cyclic peptides has remained a challenging issue owing to their molecular properties. Various efficiency metrics have emerged to assess this issue. Among them, the lipophilic permeability efficiency (LPE) metric is the difference between an experimental 1,9-decadiene-water partition coefficient at pH 7.4 (log Ddec/w) and calculated octanol/water partition coefficients (ALogP). This metric provides insight into how structural changes affect permeability. Here, we demonstrate the chromatographic capacity factor (log k') of cyclic peptides using reversed-phase liquid chromatography as an alternative to log Ddec/w, which enables efficient and reliable experimental lipophilicity for the adoption of LPE in early drug discovery. The log k' indicates the passive membrane permeability of cyclic peptides and can be used to optimize passive membrane permeability in combination with other parameters. In addition, intestinal membrane permeability of cyclic peptides on human induced pluripotent stem cell-derived intestinal epithelial cells was achieved with log k' and high passive membrane permeability, although cyclic peptides are P-glycoprotein substrates. These approaches could facilitate optimization of properties of cyclic peptides for oral administration and contribute to the successful discovery and development of cyclic peptides.

Pharmacokinetic-pharmacodynamic modeling for hepatic delivery and efficacy of antisense oligonucleotides with lipophilic ligands in mice.

Conjugation with lipophilic ligands such as cholesterol and α-tocopherol dramatically improves the delivery and efficacy of antisense oligonucleotides (ASOs) in the liver. To estimate the hepatic ASO concentration and the efficacy of ASOs conjugated with lipophilic ligands in mice, we constructed a pharmacokinetic-pharmacodynamic (PK-PD) model that consisted of a two-linear compartment model for the plasma and the hepatic ASO concentration, and two indirect response models for the hepatic apolipoprotein B (Apo-B) mRNA and plasma total cholesterol. The model provided a good fit of the hepatic ASO concentration although it showed an overprediction of Apo-B mRNA and an underprediction of the plasma total cholesterol within 2-fold at a later time after single intravenous administration of ASOs conjugated with lipophilic ligands. In addition, the model simulations indicated that the efficacy at a dose regimen of ASOs conjugated with lipophilic ligands (0.2 mg/kg, once a week) in mice was comparable to that at an effective dose of unchanged ASO (2.5 mg/kg, once a week). Although further studies are required to refine the parameters of the PK-PD model, this approach could be used to guide dose-ranging pharmacological studies for ASOs conjugated with lipophilic ligands in mice.

In-vitro-in-silico investigation of the negative food effect of zolpidem when administered as immediate-release tablets.

OBJECTIVES: The main objective of the present work was to combine in-vitro and in-silico tools to better understand the in-vivo behavior of the immediate release (IR) formulation of zolpidem in the fasted and fed states. METHODS: The dissolution of zolpidem was evaluated using biorelevant media simulating the gastric and intestinal environment in the fasted and fed states. Additionally, the influence of high viscosity and high fat content on the release of zolpidem under fed state conditions was investigated. The in-vitro results were combined with a physiologically based pharmacokinetic (PBPK) model constructed with Simcyp® to simulate the zolpidem pharmacokinetic profile in both prandial states. KEY FINDINGS: In vitro biorelevant dissolution experiments representing the fasted and fed states, combinedwith PBPKmodelling, were able to simulate the plasma profiles from the clinical food effect studies well. Experiments reflecting the pH and fat content of themeal led to a good prediction of the zolpidem plasma profile in the fed state, whereas increasing the viscosity of the gastricmedia led to an under-prediction. CONCLUSIONS: This work demonstrates that the combination of biorelevant dissolution testing and PBPK modelling is very useful for understanding the in-vivo behavior of zolpidem in the fasted and fed states. This approach could be implemented in the development of other drugs exhibiting negative food effects, saving resources and bringing new drug products to the market faster.

Discovery of a potent orally bioavailable retinoic acid receptor-related orphan receptor-gamma-t (RORγt) inhibitor, S18-000003.

The retinoic acid receptor-related orphan receptor-gamma-t (RORγt) is the master transcription factor responsible for regulating the development and function of T-helper 17 (Th17) cells, which are related to the pathology of several autoimmune disorders. Therefore, RORγt is an attractive drug target for such Th17-mediated autoimmune diseases. A structure-activity relationship (SAR) study of lead compound 1 yielded a novel series of RORγt inhibitors, represented by compound 6. Detailed SAR optimization, informed by X-ray cocrystal structure analysis, led to the discovery of a potent orally bioavailable RORγt inhibitor 25, which inhibited IL-17 production in the skin of IL-23-treated mice by oral administration.

Improved Human Pharmacokinetic Prediction of Hepatically Metabolized Drugs With Species-Specific Systemic Clearance.

Accurate prediction of human pharmacokinetics (PK) is important for the choice of promising compounds in humans. As the predictability of human PK by an empirical approach is low for drugs with species-specific PK, the utility of a physiologically based pharmacokinetic (PBPK) model was verified using 16 hepatically metabolized reference drugs. After the prediction method for total clearance (CLtot) and distribution volume at steady state (Vdss) in the conventional PBPK model had been optimized, plasma concentrations following a single oral administration of each reference drug to healthy volunteers were simulated, and the prediction accuracy for human PK was compared between empirical approaches and the optimized PBPK model. In the drugs with low species-specific CLtot, there was little difference in predictability for maximum concentration (Cmax), time to maximum plasma concentration (Tmax), and area under the curve (AUC) (absolute average fold error: 1.3-2.4). In contrast, the optimized PBPK model predicted Cmax and AUC of the drugs with high species-specific CLtot with lower absolute average fold error (Cmax and AUC: 2.8 and 3.2, respectively) than those of the empirical approach (Cmax and AUC: 2.6-4.9 and 3.9-10.7, respectively). Therefore, the optimized PBPK model is useful for human PK prediction of drugs with species-specific CLtot.

Gene Silencing Activity and Hepatic Accumulation of Antisense Oligonucleotides Bearing Cholesterol-Conjugated Thiono Triester at the Gap Region.

Cholesterol (Chol) conjugation to the 5' or 3' end of antisense oligonucleotide (ASO) enables delivery to the liver, and Chol conjugation at the gap region can also be expected to improve delivery to the liver. In this study, we synthesized ASOs bearing the Chol-conjugated thiono triester and evaluated their activity and hepatic accumulation. We found that Chol conjugations at the gap region improved in vitro activity and hepatic accumulation when compared to unconjugated ASOs. However, Chol conjugation with phosphorothioate linkage did not improve in vivo activity in the liver, suggesting the importance of cleaving the phosphodiester between ASO and Chol. These results offer useful information for tuning the oligonucleotide structure to improve pharmaceutical properties and designing ASOs for multiple ligand conjugations and combinations with end modification.

Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides.

Gapmer antisense oligonucleotides cleave target RNA effectively in vivo, and is considered as promising therapeutics. Especially, gapmers modified with locked nucleic acid (LNA) shows potent knockdown activity; however, they also cause hepatotoxic side effects. For developing safe and effective gapmer drugs, a deeper understanding of the mechanisms of hepatotoxicity is required. Here, we investigated the cause of hepatotoxicity derived from LNA-modified gapmers. Chemical modification of gapmer's gap region completely suppressed both knockdown activity and hepatotoxicity, indicating that the root cause of hepatotoxicity is related to intracellular gapmer activity. Gene silencing of hepatic ribonuclease H1 (RNaseH1), which catalyses gapmer-mediated RNA knockdown, strongly supressed hepatotoxic effects. Small interfering RNA (siRNA)-mediated knockdown of a target mRNA did not result in any hepatotoxic effects, while the gapmer targeting the same position on mRNA as does the siRNA showed acute toxicity. Microarray analysis revealed that several pre-mRNAs containing a sequence similar to the gapmer target were also knocked down. These results suggest that hepatotoxicity of LNA gapmer is caused by RNAseH1 activity, presumably because of off-target cleavage of RNAs inside nuclei.

In vivo use of the CYP inhibitor 1-aminobenzotriazole to increase long-term exposure in mice.

1-Aminobenzotriazole (ABT) is a well-known in vivo nonspecific inhibitor of cytochrome P450 (CYP) enzymes. An effective dosing regimen of ABT for a multiple-administration study is needed to conduct pharmacological studies for proof-of-concept, although it has been established for single-administration study, to characterize the pharmacokinetics of drug candidates. This study demonstrated a suitable dosing vehicle of ABT for continuous administration and increased exposure to antipyrine, which is a nonspecific probe of CYP, using ABT for a long period in mice. The dosing vehicle of ABT was 0.5% (w/v) hydroxypropyl methylcellulose and 0.5% (v/v) Tween 80 in N,N-dimethylacetamide/20% hydroxypropyl-ß-cyclodextrin aqueous solution (2:8, v/v) based on the duration of apparent solubility. After implantation of an ALZET osmotic pump with ABT, the plasma concentrations of ABT were maintained at more than 4.1 µg/ml over 336 h. Compared with the vehicle group, the CLtot of antipyrine with ABT decreased to approximately one-fourth, and the BA of antipyrine with ABT increased up to 3-fold. In addition, the enhancement of exposure of antipyrine by ABT was maintained over the 336 h. The body weight, food consumption and hematological parameters of mice did not change with ABT administration for 16 days. These findings demonstrated that pretreatment of ABT can increase long-term exposure using continuous administration with the ALZET osmotic pump in mice with no overt toxicity. It is concluded that the in vivo use of 1-aminobenzotriazole can be applied to pharmacological studies for proof-of-concept, thus contributing to the selection of drug candidates at an early drug discovery stage. Copyright © 2016 John Wiley & Sons, Ltd.

Comparative Characterization of Hepatic Distribution and mRNA Reduction of Antisense Oligonucleotides Conjugated with Triantennary N-Acetyl Galactosamine and Lipophilic Ligands Targeting Apolipoprotein B.

TriantennaryN-acetyl galactosamine (GalNAc, GN3) and lipophilic ligands such as cholesterol andα-tocopherol conjugations dramatically improve the distribution and efficacy of second-generation antisense oligonucleotides (ASOs) in the whole liver. To characterize ligands for delivery to liver cells based on pharmacokinetics and efficacy, we used a locked nucleic acid gapmer of ASO targeting apolipoprotein B as a model compound and evaluated the amount of ASO and apolipoprotein B mRNA in the whole liver, hepatocytes, and nonparenchymal (NP) cells as well as plasma total cholesterol after administration of ASO conjugated with these ligands to mice. Compared with unconjugated ASO, GN3 conjugation increased the amount (7-fold) and efficacy (more than 10-fold) of ASO in hepatocytes only and showed higher efficacy than the increased rate of the amount of ASO. On the other hand, lipophilic ligand conjugations led to increased delivery (3- to 5-fold) and efficacy (5-fold) of ASO to both hepatocytes and NP cells. GN3 and lipophilic ligand conjugations increased the area under the curve of ASOs and the pharmacodynamic duration but did not change the half-life in hepatocytes and NP cells compared with unconjugated ASO. In the liver, the phosphodiester bond between ASO and these ligands was promptly cleaved to liberate unconjugated ASO. These ligand conjugations reduced plasma total cholesterol compared with unconjugated ASO, although these ASOs were well tolerated with no elevation in plasma transaminases. These findings could facilitate ligand selection tailored to liver cells expressed in disease-related genes and could contribute to the discovery and development of RNA interference-based therapy.

Surface Plasmon Resonance Assay of Binding Properties of Antisense Oligonucleotides to Serum Albumins and Lipoproteins.

In the present study, we developed an assay to evaluate the kinetic binding properties of the unconjugated antisense oligonucleotide (ASO) and lipophilic and hydrophilic ligands conjugated ASOs to mouse and human serum albumin, and lipoproteins using surface plasmon resonance (SPR). The lipophilic ligands conjugated ASOs showed clear affinity to the albumins and lipoproteins, while the unconjugated and hydrophilic ligand conjugated ASOs showed no interaction. The SPR method showed reproducible immobilization of albumins and lipoproteins as ligands on the sensor chip, and reproducible affinity kinetic parameters of interaction of ASOs conjugated with the ligands could be obtained. The kinetic binding data of these ASOs to albumin and lipoproteins by SPR were related with the distributions in the whole liver in mice after administration of these conjugated ASOs. The results demonstrated that our SPR method could be a valuable tool for predicting the mechanism of the properties of delivery of conjugated ASOs to the organs.

Irbesartan attenuates atherosclerosis in Watanabe heritable hyperlipidemic rabbits: noninvasive imaging of inflammation by 18F-fluorodeoxyglucose positron emission tomography.

The purpose of this study was to assess the usefulness of 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET) in evaluating the antiatherogenic effects of irbesartan, an angiotensin II type 1 receptor blocker. Watanabe heritable hyperlipidemic rabbits were divided into the irbesartan-treated group (75 mg/kg/d; n  =  14) and the control group (n  =  14). After a 9-month treatment, rabbits underwent 18F-FDG PET. Using the aortic lesions, autoradiography and histologic examinations were performed. PET imaging clearly visualized the thoracic lesions of control rabbits and showed a significant decrease in the 18F-FDG uptake level of irbesartan-treated rabbits (78.8% of controls; p < .05). Irbesartan treatment significantly reduced the plaque size (43.1% of controls) and intraplaque macrophage infiltration level (48.1% of controls). The 18F-FDG uptake level in plaques positively correlated with the plaque size (r  =  .65, p < .05) and macrophage infiltration level (r  =  .57, p < .05). Noninvasive imaging by 18F-FDG PET is useful for evaluating the therapeutic effects of irbesartan and reflects inflammation, a key factor involved in the therapeutic effects.

Using Improved Serial Blood Sampling Method of Mice to Study Pharmacokinetics and Drug-Drug Interaction.

In pharmacokinetic evaluation of mice, using serial sampling methods rather than a terminal blood sampling method could reduce the number of animals needed and lead to more reliable data by excluding individual differences. In addition, using serial sampling methods can be valuable for evaluation of the drug-drug interaction (DDI) potential of drug candidates. In this study, we established an improved method for serially sampling the blood from one mouse by only one incision of the lateral tail vein, and investigated whether our method could be adapted to pharmacokinetic and DDI studies. After intravenous and oral administration of ibuprofen and fexofenadine (BCS class II and III), the plasma concentration and pharmacokinetic parameters were evaluated by our method and a terminal blood sampling method, with the result that both methods gave comparable results (ibuprofen: 63.8±4.0% and 64.4%, fexofenadine: 6.5±0.7% and 7.9%, respectively, in bioavailability). In addition, our method could be adapted to DDI study for cytochrome P450 and organic anion transporting polypeptide inhibition. These results demonstrate that our method can be useful for pharmacokinetic evaluation from the perspective of reliable data acquisition as well as easy handling and low stress to mice and improve the quality of pharmacokinetic and DDI studies.

Using improved serial blood sampling method of mice to study pharmacokinetics and drug-drug interaction.

In pharmacokinetic evaluation of mice, using serial sampling methods rather than a terminal blood sampling method could reduce the number of animals needed and lead to more reliable data by excluding individual differences. In addition, using serial sampling methods can be valuable for evaluation of the drug-drug interaction (DDI) potential of drug candidates. In this study, we established an improved method for serially sampling the blood from one mouse by only one incision of the lateral tail vein, and investigated whether our method could be adapted to pharmacokinetic and DDI studies. After intravenous and oral administration of ibuprofen and fexofenadine (BCS class II and III), the plasma concentration and pharmacokinetic parameters were evaluated by our method and a terminal blood sampling method, with the result that both methods gave comparable results (ibuprofen: 63.8 ± 4.0% and 64.4%, fexofenadine: 6.5 ± 0.7% and 7.9%, respectively, in bioavailability). In addition, our method could be adapted to DDI study for cytochrome P450 and organic anion transporting polypeptide inhibition. These results demonstrate that our method can be useful for pharmacokinetic evaluation from the perspective of reliable data acquisition as well as easy handling and low stress to mice and improve the quality of pharmacokinetic and DDI studies.

Pharmacokinetic-pharmacodynamic modeling for reduction of hepatic apolipoprotein B mRNA and plasma total cholesterol after administration of antisense oligonucleotide in mice.

Second-generation antisense oligonucleotides (ASOs) demonstrate excellent biological stability and in vitro/in vivo potency, and thus are considered to be attractive candidates for drugs to treat various diseases. A pharmacokinetic-pharmacodynamic (PK-PD) model of ASOs is desired for the design of appropriate PK and pharmacological studies. The objective of this study was to develop a PK-PD model to accurately simulate hepatic ASO concentration and its efficacy from plasma ASO concentration. After single subcutaneous administration of an ASO targeting hepatic apolipoprotein B (Apo-B) mRNA to mice, the ASO was absorbed rapidly and showed biphasic decline with time from the plasma and liver (t1/2: 1-3 and 81-183 h, Tmax: 0.25-0.50 and 4-8 h). After administration, hepatic Apo-B mRNA and plasma total cholesterol began decreasing at 4-8 and 8-24 h, and their Tmax values were observed at 24-72 and 72 h. To develop the PK-PD model based on the mechanisms of ASOs, we described the plasma and hepatic ASO concentration with linear two-compartment models. In addition, we inserted two indirect response models for mRNA and plasma total cholesterol. Model predictions from plasma ASO concentration gave excellent fits to the observed values of hepatic ASO concentration, Apo-B mRNA and plasma total cholesterol after single or multiple subcutaneous administrations. Our PK-PD model could accurately predict hepatic ASO concentrations and their efficacies from plasma ASO concentrations. This PK-PD model could be a useful tool for suggesting PK and pharmacological study protocols for various liver-targeted second-generation ASOs.

Suppressive effects of irbesartan on inflammation and apoptosis in atherosclerotic plaques of apoE-/- mice: molecular imaging with 14C-FDG and 99mTc-annexin A5.

OBJECTIVES: To investigate the effects of irbesartan on inflammation and apoptosis in atherosclerotic plaques by histochemical examination and molecular imaging using (14)C-FDG and (99m)Tc-annexin A5. BACKGROUND: Irbesartan has a peroxisome proliferator-activated receptor gamma (PPARγ) activation property in addition to its ability to block the AT1 receptor. Accordingly, irbesartan may exert further anti-inflammatory and anti-apoptotic effects in atherosclerotic plaques. However, such effects of irbesartan have not been fully investigated. Molecular imaging using (18)F-FDG and (99m)Tc-annexin A5 is useful for evaluating inflammation and apoptosis in atherosclerotic plaques. METHODS: Female apoE(-/-) mice were treated with irbesartan-mixed (50 mg/kg/day) or irbesartan-free (control) diet for 12 weeks (n = 11/group). One week after the treatment, the mice were co-injected with (14)C-FDG and (99m)Tc-annexin A5, and cryostat sections of the aortic root were prepared. Histochemical examination with Movat's pentachrome (plaque size), Oil Red O (lipid deposition), Mac-2 (macrophage infiltration), and TUNEL (apoptosis) stainings were performed. Dual-tracer autoradiography was carried out to evaluate the levels of (14)C-FDG and (99m)Tc-annexin A5 in plaques (%ID×kg). In vitro experiments were performed to investigate the mechanism underlying the effects. RESULTS: Histological examination indicated that irbesartan treatment significantly reduced plaque size (to 56.4%±11.1% of control), intra-plaque lipid deposition (53.6%±20.2%) and macrophage infiltration (61.9%±20.8%) levels, and the number of apoptotic cells (14.5%±16.6%). (14)C-FDG (43.0%±18.6%) and (99m)Tc-annexin A5 levels (45.9%±16.8%) were also significantly reduced by irbesartan treatment. Irbesartan significantly suppressed MCP-1 mRNA expression in TNF-α stimulated THP-1 monocytes (64.8%±8.4% of un-treated cells). PPARγ activation was observed in cells treated with irbesartan (134%±36% at 3 µM to 3329%±218% at 81 µM) by a PPARγ reporter assay system. CONCLUSIONS: Remissions of inflammation and apoptosis as potential therapeutic effects of irbesartan on atherosclerosis were observed. The usefulness of molecular imaging using (18)F-FDG and (99m)Tc-annexin A5 for evaluating the therapeutic effects of irbesartan on atherosclerosis was also suggested.

Quantitative imaging of a therapeutic peptide in biological tissue sections by MALDI MS.

BACKGROUND: Therapeutic peptides and proteins are being increasingly explored as potential therapeutic agents for molecular-targeted therapy, and the requirement for quantitative bioanalytical tools for such molecules has been discussed. RESULTS: The distribution of octreotide, a synthetic octapeptide analog of somatostatin, in the liver and kidney of mice administered with the analog was clearly visualized by MALDI-Imaging MS (IMS). The developed MALDI-IMS analytical method successfully quantified the amount of octreotide on tissue sections (accuracy was 76-127%) and the 2,5-dihydroxybenzoic acid-based normalization method was effective. CONCLUSION: The results of this study suggest that MALDI-IMS enables the quantification of an administered therapeutic peptide on biological tissue sections, as well as visualization of the in vivo distribution of the peptide.

Quantitative determination of apoptosis of pancreatic ß-cells in a murine model of type 1 diabetes mellitus.

UNLABELLED: Type 1 diabetes mellitus is characterized by a significant deficit in pancreatic ß-cell mass, presumably caused by ß-cell apoptosis. We investigated the incidence of ß-cell apoptosis in streptozotocin-treated mice and nonobese diabetic (NOD) mice with (99m)Tc-annexin A5. METHODS: Vehicle-treated mice, streptozotocin-treated mice, and NOD mice at the ages of 5, 9, 16, and 20 wk (5-8 mice per group) were injected with (99m)Tc-annexin A5 and sacrificed 6 h later for autoradiography, and the regional (99m)Tc-annexin A5 level in the pancreas was evaluated. Pancreatic islets were identified by insulin immunohistochemical staining, and apoptotic cells were determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. The (99m)Tc-annexin A5 level in pancreatic islets was expressed as the percentage injected dose per area of pancreatic islets and normalized by animal body weight (%ID × 10(6)/mm(2)/kg). The level of apoptotic cells in pancreatic islets was expressed as the number of TUNEL-positive cells per area of pancreatic islets (cells/mm(2)). RESULTS: The (99m)Tc-annexin A5 accumulation level was significantly higher (2.5 ± 0.7 vs. 0.7 ± 0.1 %ID × 10(6)/mm(2)/kg, P < 0.05) and the number of TUNEL-positive cells was significantly higher (1,170 ± 535 vs. 5 ± 6 cells/mm(2), P < 0.05) in the pancreatic islets of the streptozotocin-treated mice than in those of the vehicle-treated mice. The (99m)Tc-annexin A5 accumulation level was significantly higher (1.1 ± 0.4 vs. 0.5 ± 0.1 %ID × 10(6)/mm(2)/kg, P < 0.05) and the number of TUNEL-positive cells was significantly higher (152 ± 82 vs. 4 ± 9 cells/mm(2), P < 0.05) in the pancreatic islets of 16-wk-old NOD mice than in those of 5-wk-old NOD mice. In addition, the level of (99m)Tc-annexin A5 correlated with the number of TUNEL-positive cells in the pancreatic islets of the streptozotocin-treated mice (r = 0.821, P < 0.001) and NOD mice (r = 0.721, P < 0.001). CONCLUSION: There is significant islet cell apoptosis with (99m)Tc-annexin A5 accumulation in the pancreas of both streptozotocin and NOD mice.

Effect of glycosylation on biodistribution of radiolabeled glucagon-like peptide 1.

OBJECTIVE: Glycosylation is generally applicable as a strategy for increasing the activity of bioactive proteins. In this study, we examined the effect of glycosylation on biodistribution of radiolabeled glucagon-like peptide 1 (GLP-1) as a bioactive peptide for type 2 diabetes. METHODS: Noninvasive imaging studies were performed using a gamma camera after the intravenous administration of (123)I-GLP-1 or (123)I-α2, 6-sialyl N-acetyllactosamine (glycosylated) GLP-1 in rats. In ex vivo biodistribution studies using (125)I-GLP-1 or (125)I-glycosylated GLP-1, organ samples were measured for radioactivity. Plasma samples were added to 15% trichloroacetic acid (TCA) to obtain TCA-insoluble and TCA-soluble fractions. The radioactivity in the TCA-insoluble and TCA-soluble fractions was measured. RESULTS: In the noninvasive imaging studies, a relatively high accumulation level of (123)I-GLP-1 was found in the liver, which is the major organ to eliminate exogenous GLP-1. The area under the time-activity curve (AUC) of (123)I-glycosylated GLP-1 in the liver was significantly lower (89%) than that of (123)I-GLP-1. These results were consistent with those of ex vivo biodistribution studies using (125)I-labeled peptides. The AUC of (125)I-glycosylated GLP-1 in the TCA-insoluble fraction was significantly higher (1.7-fold) than that of GLP-1. CONCLUSIONS: This study demonstrated that glycosylation significantly decreased the distribution of radiolabeled GLP-1 into the liver and increased the concentration of radiolabeled GLP-1 in plasma. These results suggested that glycosylation is a useful strategy for decreasing the distribution into the liver of bioactive peptides as desirable pharmaceuticals.