Effects of gastric inhibitory polypeptide,glucagon‐like peptide‐1 and glucagon‐like peptide‐1 receptor agonists on Bone Cell Metabolism

The relationship between gut and skeleton is increasingly recognized as part of the integrated physiology of the whole organism. The incretin hormones gastric inhibitory polypeptide (GIP) and glucagon‐like peptide‐1 (GLP‐1) are secreted from the intestine in response to nutrient intake and exhibit several physiological functions including regulation of islet hormone secretion and glucose levels. A number of GLP‐1 receptor agonists (GLP‐1RAs) are currently used in treatment of type 2 diabetes and obesity. However, GIP and GLP‐1 cognate receptors are widely expressed suggesting that incretin hormones mediate effects beyond control of glucose homeostasis, and reports on associations between incretin hormones and bone metabolism have emerged. The aim of this MiniReview was to provide an overview of current knowledge regarding the in vivo and in vitro effects of GIP and GLP‐1 on bone metabolism. We identified a total of 30 pre‐clinical and clinical investigations of the effects of GIP, GLP‐1 and GLP‐1RAs on bone turnover markers, bone mineral density (BMD), bone microarchitecture and fracture risk. Studies conducted in cell cultures and rodents demonstrated that GIP and GLP‐1 play a role in regulating skeletal homeostasis, with pre‐clinical data suggesting that GIP inhibits bone resorption whereas GLP‐1 may promote bone formation and enhance bone material properties. These effects are not corroborated by clinical studies. While there is evidence of effects of GIP and GLP‐1 on bone metabolism in pre‐clinical investigations, clinical trials are needed to clarify whether similar effects are present and clinically relevant in humans.     

Physiological mechanisms for the increase in renal solute‐free water clearance by a glucagon‐like peptide‐1 mimetic

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The glucagon‐like peptide‐1 receptor agonist Exendin‐4, ameliorates contrast‐induced nephropathy through suppression of oxidative stress,vascular dysfunction and apoptosis independent of glycaemia

Contrast‐induced nephropathy (CIN) is a leading cause of hospital‐acquired acute kidney injury, particularly in diabetic patients. Previous studies have shown renoprotective effects of glucagon‐like peptide‐1 (GLP‐1) signalling; however, its role in CIN remains unexplored. This study investigates the prophylactic effect of exendin‐4, a GLP‐1R agonist, against CIN in a rat model mimicking both healthy and diabetic conditions. Animals were randomly divided into 7 groups: a control sham group (n = 8), and 2 identical sets of 3 disease groups, one received exendin‐4 before exposure to contrast medium (CM), while the other served as untreated control. The 3 disease groups represented diabetes (n = 8), CIN (n = 8), or diabetes and CIN combined (n = 8). Untreated groups showed deteriorating renal function as indicated by significantly higher levels of serum creatinine and blood urea nitrogen, malondialdehyde, and endothelin‐1 and caspase‐3 expression compared to the sham control group. This was accompanied by a significant decrease in tissue reserves of reduced glutathione, superoxide dismutase, nitrate and endothelin nitric oxide synthase as well as deteriorating renal histology. The CM‐induced changes in diabetic rats indicate impaired renal function, oxidative stress, vascular dysfunction, and apoptosis, and were significance higher in intensity compared to non‐diabetic rats. Pretreatment with exendin‐4 ameliorated all the aforementioned CM‐induced nephropathic effects independent of the glycemic state. To our knowledge, this is the first study describing the prophylactic renoprotective effects of exendin‐4 against CIN. With the current pharmaceutical use of exendin‐4 as a hypoglycaemic agent, the GLP‐1R agonist becomes an interesting candidate for human clinical trials on CIN prevention.     

Pharmacokinetic and Pharmacological Profiles of Free and Liposomal Recombinant Human Erythropoietin After Intravenous and Subcutaneous Administrations in Rats

Purpose. Recombinant human erythropoietin (Epo) is used frequently through intravenous (i.v.) and subcutaneous (s.c.) administration for the clinical treatment of the last stage of renal anemia. We encapsulated Epo in liposomes to develop an alternative administration route. The purpose of our study was to evaluate the pharmacokinetics and the pharmacological effects of liposomal Epo in comparison with the Epo after i.v. and s.c. administration to rats. Methods. Epo was encapsulated in liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and soybean-derived sterol mixture (SS) prepared by the reversed-phase evaporation vesicle method. After filtration through a 0.1 m polycarbonate membrane, liposomes were gel filtered (Epo/liposomes). Results. Epo/liposomes showed higher pharmacological activity than Epo/liposomes before gel filtration after i.v. administration to rats. Non-encapsulated Epo lost its activity, whereas encapsulated Epo in liposomes retained it. The pharmacological effects of Epo/liposomes were greater than those of Epo after i.v. administration. Epo/liposomes afforded 3–9 times higher AUC, lower clearance and lower steady-state volume of distribution than Epo after both i.v. and s.c. administrations. Epo/liposomes had an improved pharmacokinetic profile compared with Epo. S.c. administration of Epo/liposomes at 7 h may penetrate primarily (40% of dose) through the blood as a liposome and partly (7% of dose) in lymph. Conclusions. Epo/liposomes may reduce the frequency of injections required for a certain reticulocyte effect in comparison to Epo. The lower clearance of Epo/liposomes may increase the plasma concentrations of Epo, which increases the efficacy.     

Protective effects of melatonin and glucagon‐like peptide‐1 receptor agonist (liraglutide) on gastric ischaemia–reperfusion injury in high‐fat/sucrose‐fed rats

Ischaemia–reperfusion (I–R) injury is a serious pathology that is often encountered with thrombotic events, during surgery when blood vessels are cross‐clamped, and in organs for transplantation. Increased oxidative stress is the main pathology in I–R injury, as assessed in studies on the heart, kidney, and brain with little data available on gastric I–R (GI–R). Liraglutide is a GLP‐1 receptor agonist that has insulinotropic and weight reducing actions, and melatonin that has been much studied as a chronotropic hormone; have also studied as being anti‐oxidative stress agents. Herein, we aimed to explore the effects of liraglutide and melatonin on GI–R injury with high‐fat/sucrose diet. Rats were divided into six groups; two diet‐control, two melatonin‐ and two liraglutide‐pretreated groups. All rats were subjected to 30 minutes of gastric ischaemia followed by 1 hour of reperfusion. Gastric tissues were assessed for the percentage of DNA fragmentation, myeloperoxidase activity, total oxidant status, total antioxidant capacity, oxidative stress index, BMI and histopathological examination. We showed that high‐fat feeding for four weeks prior to GI–R significantly increased BMI, oxidative stress indices and decreased total antioxidant capacity, with a neutral effect on apoptosis compared to controls. Pretreatment with either melatonin (10 mg/kg per day orally) or liraglutide (25 μg/kg per day ip) reverses these effects. Furthermore, both drugs reduced weight only in HFS‐fed rats. Both liraglutide and melatonin have nearly similar protective effects on gastric I–R injury through decreasing the oxidative stress and apoptosis.     

Possible interaction of quinolone antibiotics with peptide transporter 1 in oral absorption of peptide‐mimetic drugs

The study investigated whether quinolone antibiotics inhibit the PEPT1‐mediated uptake of its substrates. Among the quinolones examined, lomefloxacin, moxifloxacin (MFLX) and purlifloxacin significantly inhibited the uptake of PEPT1 substrate phenylalanine‐Ψ(CN‐S)‐alanine (Phe‐Ψ‐Ala) in HeLa/PEPT1 cells to 31.6 ± 1.3%, 27.6 ± 2.9%, 36.8 ± 2.2% and 32.6 ± 1.4%, respectively. Further examination showed that MFLX was an uncompetitive inhibitor, with an IC₅₀ value of 4.29 ± 1.29 mm . In addition, MFLX significantly decreased the cephalexin and valacyclovir uptake in HeLa/PEPT1 cells. In an in vivo study in rats, the maximum plasma concentration (C_max) of orally administered Phe‐Ψ‐Ala was significantly decreased in the presence of MFLX (171 ± 1 ng/ml) compared with that in its absence (244 ± 9 ng/ml). The area under the concentration–time curve (AUC) of orally administered Phe‐Ψ‐Ala in the presence of MFLX (338 ± 50 ng/ml · h) tended to decrease compared with that in its absence (399 ± 75 ng/ml · h). The oral bioavailability of Phe‐Ψ‐Ala in the presence and absence of MFLX was 41.7 ± 6.2% and 49.2 ± 9.2%, respectively. The results indicate that administration of quinolone antibiotics concomitantly with PEPT1 substrate drugs may potentially result in drug–drug interaction. Copyright © 2016 John Wiley & Sons, Ltd.     

Synthesis and conformational analysis of the insulin‐like 4 gene product

Abstract: Insulin‐like 4 (INSL‐4) is a protein expressed in the early placenta. Its primary structure is insulin‐like with reference to the distribution of cysteine residues and the single chain pro‐form. Insulin‐like 4 was generated by solid‐phase peptide synthesis of the two chains followed by the sequential synthesis of the three disulfide bonds. Two disulfide isomers were produced, one with an insulin‐like disulfide bonding pattern and the other with a reversed chain orientation. The CD spectra of the two disulfide isomers were indistinguishable without any features produced by periodic structures. In addition, the hydrodynamic properties of the two isomers were identical which implied a very open structure of the disulfide‐bonded two‐chain molecules. It appears that insulin‐likeness cannot be defined solely on the basis of the primary structure of cDNA.     

Novel furan‐containing peptide‐based inhibitors of protein arginine deiminase type IV (PAD4)

Protein arginine deiminase type IV (PAD4) is responsible for the posttranslational conversion of peptidylarginine to peptidylcitrulline. Citrullinated protein is the autoantigen in rheumatoid arthritis, and therefore, PAD4 is currently a promising therapeutic target for the disease. Recently, we reported the importance of the furan ring in the structure of PAD4 inhibitors. In this study, the furan ring was incorporated into peptides to act as the “warhead” of the inhibitors for PAD4. IC₅₀ studies showed that the furan‐containing peptide‐based inhibitors were able to inhibit PAD4 to a better extent than the furan‐containing small molecules that were previously reported. The best peptide‐based inhibitor inhibited PAD4 reversibly and competitively with an IC₅₀ value of 243.2 ± 2.4 μm . NMR spectroscopy and NMR‐restrained molecular dynamic simulations revealed that the peptide‐based inhibitor had a random structure. Molecular docking studies showed that the peptide‐based inhibitor entered the binding site and interacted with the essential amino acids involved in the catalytic activity. The peptide‐based inhibitor could be further developed into a therapeutic drug for rheumatoid arthritis.     

Pharmacokinetics of pyrrole‐imidazole polyamides after intravenous administration in rat

The pharmacokinetics of pyrrole (Py)‐imidazole (Im) polyamides was studied in rats after the intravenous administration of these compounds. Py‐Im polyamide (A) was composed of Ac‐ImPyPy‐ImPyPy‐β‐Dp (β: β‐alanine, Dp: N,N‐dimethylaminopropylamide). Py‐Im polyamide (B) was composed of Ac‐PyIm‐β‐ImIm‐PyPy‐β‐PyPy‐β‐Dp. Py‐Im polyamide (C) was composed of Ac‐PyPy‐β‐PyImPy‐PyPyPy‐β‐ImPy‐β‐Dp. The molecular weight of Py‐Im polyamide (A) was 1035.12, that of Py‐Im polyamide (B) was 1422.51 and that of Py‐Im polyamide (C) was 1665.78. After the intravenous injection of Py‐Im polyamide (A) at 1.3, 2.0, 7.5 and 15.0 mg/kg, Py‐Im polyamides (B) and (C) at 1.0, 2.0, 3.0 and 5.0 mg/kg, the average systemic clearance and the volume of distribution at the steady state obtained by a non‐compartmental method were in the ranges of 4.6–6.4 ml/min/kg and 244–412 ml/kg, 8.9–10.3 ml/min/kg and 1990–4567 ml/kg, and 7.3–11.9 ml/min/kg and 407–667 ml/kg, respectively. Dose linearity of Py‐Im polyamides was observed. The plasma concentration‐time profiles after the intravenous administration of Py‐Im polyamides (A) and (B) were fitted well by a two‐compartment model. Py‐Im polyamide (C) was observed at high concentrations in the lungs. The plasma concentration‐time profiles after the intravenous administration of Py‐Im polyamide (C) were described using a catenary two‐compartment model. This model is useful for describing the time course after the administration of high‐molecular‐weight Py‐Im polyamides. Copyright © 2009 John Wiley & Sons, Ltd.     

Pharmacokinetic study of chloramphenicol in patients with liver disease

Summary The pharmacokinetics of intravenous chloramphenicol has been studied in 42 patients with liver disease and in 8 controls. The half-life of chloramphenicol (t_1/2) was increased in the various liver disorders, the metabolic clearance rate (MCR) and apparent volume of distribution (Vd) were decreased and the area under the time — concentration curve (AUC) showed an increase. The t_1/2 of chloramphenicol showed a significant correlation with serum albumin and prothrombin time index.     

A site‐specific PEGylated analog of exendin‐4 with improved pharmacokinetics and pharmacodynamics in vivo

Objectives Our aim was to improve the in vivo pharmacokinetics and pharmacodynamics of exendin‐4 by using site‐specific PEGylation. Methods We designed the PEGylated peptide based on its structure and activity relationship and prepared the conjugate by two steps of chromatographic purification. After obtained the conjugate we confirmed its glucose‐lowering activity in normal mice and determined its half‐life in SD rats. Then we evaluated its anti‐diabetic activity in a multiple low‐dose Streptozocin (STZ)‐induced diabetic mice model. Key findings With the process established in this study the product conjugate was obtained with a yield of over 60% and purity of above 99%. The conjugate maintained its original conformation after modification. In SD rats its half‐life was prolonged to 27.12 ± 5.75 h which was 17.61‐fold longer than that of the natural exendin‐4 for which the half‐life was only 1.54 ± 0.47 h. Its anti‐diabetic activity was significantly improved in the diabetic mice. Conclusions Compare with native exendin‐4, the C‐terminal site‐specific PEGylated analog of exendin‐4 obtained in this study has an improved pharmacokinetics and pharmacodynamics in vivo and could be regarded as a potential candidate for the future development of anti‐diabetic drugs.     

A solid‐phase synthetic strategy for the preparation of peptide‐based affinity labels: synthesis of dynorphin A analogs

Abstract: Solid‐phase synthetic methodology was developed for the preparation of peptide‐based affinity labels. The initial peptides synthesized were dynorphin A (Dyn A) analogs [Phe(p‐X)⁴,d ‐Pro¹⁰]Dyn A(1–11)NH₂ containing isothiocyanate (X = –N=C=S) and bromoacetamide (X = –NHCOCH₂Br) groups. The peptides were assembled on solid supports using Fmoc‐protected amino acids, and the side chain amine to be functionalized, Phe(p‐NH₂), was protected by the Alloc (allyloxycarbonyl) group. Following removal of the Alloc group by palladium(0), the reactive isothiocyanate and bromoacetamide functionalities were successfully introduced while the peptides were still attached to the resin. Synthesis of these peptides was carried out on polystyrene (PS) and polyethylene glycol–polystyrene (PEG–PS) resins containing the PAL [peptide amide linker, 5‐(4‐Fmoc‐aminomethyl‐3,5‐dimethoxyphenoxy)valeric acid] linker. Both the rate of Alloc deprotection and the purity of the crude affinity‐labeled peptides obtained were found to be dependent on the resin used for peptide assembly.     

Detection of human neutrophil elastase with peptide‐bound cross‐linked ethoxylate acrylate resin analogs

Abstract: An assessment of elastase‐substrate kinetics and adsorption at the solid–liquid interface of peptide‐bound resin was made in an approach to the solid‐phase detection of human neutrophil elastase (HNE), which is found in high concentration in chronic wound fluid. N‐succinyl‐alanine‐alanine‐proline‐valine‐p‐nitroanilide (suc‐Ala‐Ala‐Pro‐Val‐pNA), a chromogenic HNE substrate, was attached to glycine‐cross‐linked ethoxylate acrylate resins (Gly‐CLEAR) by a carbodiimide reaction. To assess the enzyme‐substrate reaction in a two‐phase system, the kinetic profile of resin‐bound peptide substrate hydrolysis by HNE was obtained. A glycine and di‐glycine spacer was placed between the resin polymer and substrate to assess the steric and spatial requirements of resin to substrate with enzyme hydrolysis. The enzymatic activities of suc‐Ala‐Ala‐Pro‐Val‐pNA and suc‐Ala‐Ala‐Pro‐Ala‐pNA on the solid‐phase resin were compared with similar analogs in solution. An increase in visible wavelength absorbance was observed with increasing amounts of substrate‐resin and enzyme concentration. Enzyme hydrolysis of the resin‐bound substrate was also demonstrated on a polypropylene surface, which was employed for visible absorbance of released chromophore. A soluble active substrate analog was released from the resin through saponification of the ethoxylate ester linkages in the resin polymer. The resin‐released conjugate of the HNE substrate demonstrated an increased dose response with increasing enzyme concentration. The synthesis and assay of elastase substrates bound to CLEAR resin gives an understanding of substrate‐elastase adsorption and activity at the resin's solid–liquid interface for HNE detection with a solid‐phase peptide.     

Prediction of Human Bioavailability from Human Oral Administration Data and Animal Pharmacokinetic Data Without Data from Intravenous Administration of Drugs in Humans

Purpose  To predict the absolute oral bioavailabilities (BAs) of drugs in humans without using pharmacokinetic data from intravenous administration in humans. Methods  The distribution volume of the terminal phase () in humans was predicted by three methods using animal pharmacokinetic data. Then, total body clearance (CL_tot) was calculated by multiplying the elimination rate constant and , and the BA was calculated as a ratio between CL_tot and oral clearance. The predicted and observed values were compared for 67 drugs for which pharmacokinetic data after intravenous administration in humans were available. Results  For , predicted values within twice the observed value were obtained for 72.1% of drugs by both methods Ia and Ib, respectively, in which only rat pharmacokinetic data were used. The corresponding percentage was 75.0% for method II, in which pharmacokinetic data from animals other than rats were used. For BA, predicted values within 1.3 times the observed values were obtained for 66.7% and 57.4% of drugs by methods Ia and Ib, respectively, and 75.0% by method II. Conclusions  Using the present methods, it is possible to predict BA from human oral administration data combined with animal pharmacokinetic data to a certain level without using intravenous injection data. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Synthesis and evaluation of a new series of peptide‐based endothelin receptor antagonists

Abstract: Novel peptide‐based endothelin (ET) receptor antagonists were designed and synthesized in our laboratory. BQ‐485, HIM‐CO‐Leu‐d ‐Trp‐d ‐Trp‐OH, was selected as the leading compound. The primary structures of these new tripeptides were ABO‐CO‐Leu‐d ‐Trp‐d ‐AA(X)‐OH. The introduction of unnatural aromatic amino acids into these tripeptides was useful in the structure–activity relationship studies. Among the 20 tripeptides, 16 of them showed high activities against the contraction of rat aortic smooth muscles induced by ET‐1.