长效胰岛素类似物

综述几类能有效调控血糖的长效胰岛素类似物，如甘精胰岛素、脂肪酸酰化胰岛素、胰岛素前体类似物、PEG化胰岛素、胰岛素类似物聚合体等的性质、作用机制、免疫原性及组织选择性。新型长效胰岛素类似物的发现，克服了传统的长效胰岛素制剂存在的血药浓度峰谷明显、变异性高、夜间容易发生低血糖等缺点，为糖尿病的治疗开辟了新思路。     

Biochemical and Physiological Properties of a Novel Series of Long-Acting Insulin Analogs Obtained by Acylation with Cholic Acid Derivatives

Purpose This study was conducted to assess the suitability of insulin analogs acylated by various cholic acid derivatives for use as basal insulin, and to test the most promising of these, Lys^B29(N^ɛ-lithocholyl-γ-Glu) des(B30) human insulin (NN344) in pigs. Methods Circular dichroism spectroscopy and size-exclusion chromatography were used to explore the physicochemical properties of the analogs, and affinities for albumin and insulin receptors were determined. After subcutaneous injection in pigs, disappearance half-times were measured, and the plasma profile and glucose-lowering effect in a euglycemic clamp were assessed for NN344. Results NN344 showed glucose-lowering activity lasting more than 24 h. Glucose infusion rate was essentially constant from 5 to 19 h after injection. NN344 seemed to be a dodecamer in the presence of zinc ions and phenol. Without phenol, the apparent molecular mass was >5000 kDa. Formation of such a self-assembly at the site of s.c. injection and its subsequent slow decomposition might explain the long duration of action of NN344. A measurable affinity for albumin of the lithocholic acid ligand may also contribute to the prolonged action. Conclusions NN344 is a candidate for a neutral soluble basal insulin that might offer people with diabetes a prolonged duration, smooth, and predictable basal insulin supplement.     

Insulin Nanoparticles: A Novel Formulation Approach for Poorly Water Soluble Zn-Insulin

PURPOSE: To determine the feasibility of using wet milling technology to formulate poorly water soluble zinc-insulin as a stable, biologically active, nanoparticulate dispersion. METHODS: The feasibility of formulating zinc-insulin as a nanoparticulate dispersion using wet milling technology was studied. An insulin nanoparticulate formulation was reproducibly obtained after milling zinc-insulin in the presence of F68, sodium deoxycholate and water at neutral pH. The physical and chemical properties of these peptide particles were studied using electron microscopy, laser light scattering, HPLC and SDS-PAGE. To verify efficacy, hyperglycemic rats were dosed subcutaneously and intraduodenally with nanoparticles or solubilized insulin. Glucose and insulin levels were monitored on blood samples collected throughout the study. RESULTS: Zn-insulin (mean size = 16.162 microm) was processed using milling technology to form an aqueous-based nanoparticle dispersion with a mean particle size of less than 0.150 microm. The formulation was homogeneous and exhibited a unimodal particle size distribution profile using laser light diffraction techniques. Insulin, processed as a peptide-particle dispersion, was shown to be comparable to unprocessed powder using HPLC and SDS-PAGE. In addition, HPLC analyses performed on samples, heat-treated at 70 degrees C for 100 minutes, demonstrate that under conditions which effect the solubilized peptide, formulated as a peptide-particle dispersion, insulin was chemically stable. Also, when stored refrigerated, the insulin dispersion was chemically and physically stable. Finally, peptide particles of insulin, dosed subcutaneously and intraduodenally, were effective at lowering blood glucose levels of hyperglycemic rats. CONCLUSION: Water insoluble Zn-insulin can be formulated as a stable, biologically active nanometer-sized peptide particle dispersion using wet media milling technology.     

Effects of Insulin Concentration and Self-Association on the Partitioning of Its A-21 Cyclic Anhydride Intermediate to Desamido Insulin and Covalent Dimer

Purpose. In the pH range 2–5, human insulin degrades via deamidation at the A-21 asn and covalent dimerization. Both products form via a common cyclic anhydride intermediate, a product of intramolecular nucleophilic attack by the A-21 carboxyl terminus. This study examines the influence of [insulin] and self-association on the partitioning of the intermediate to products. Methods. Insulin self-association was characterized (pH 2–4) by concentration difference spectroscopy. Deamidation rates (pH 2–4) and concurrent rates of covalent dimer formation (pH 4) were determined versus [insulin] at 35°C by initial rates. A mathematical model was developed to account for the overall rate and product composition profile versus pH and [insulin]. Results. Between pH 2–4, insulin self-associates to form non-covalent dimers with a pH independent association constant of 1.8 × 10⁴ M ^–1. The overall rate of degradation is governed by intermediate formation, while product distribution is determined by competition between water and the phe B-l amino group of insulin for the anhydride. In dilute solutions, deamidation is first-order in [insulin] while covalent dimerization is second-order. Thus, deamidation predominates in dilute solutions but the fraction of covalent dimer formed increases with [insulin]. At high [insulin], self-association inhibits covalent dimer formation, preventing exclusive degradation via this pathway. The model accurately predicts a maximum in covalent dimer formation near pH 4. Conclusions. A mechanism is described which accounts for the complex dependence of insulin's degradation rate and product distribution profile on pH (between 2–5) and [insulin]. If these results can be generalized, they suggest that covalent aggregation in proteins may be inhibited by self-association.     

Formulating Insulin for Oral Administration: Preparation of Hyaluronan-Insulin Complex

PURPOSE: To investigate the behaviour of peptides and hyaluronan in strong acid solutions containing electrolytes in the preparation of a new formulation of insulin, hyaluronan-insulin complex, and to evaluate the in vivo oral activity of the formulation. METHODS: Individual processing parameters in the preparation of the insulin complex were first refined, and two formulations were subsequently investigated. The chemical structure, particle size and hydrophilic/hydrophobic properties of the insulin complex in these formulations were studied using light scattering techniques, amino acid analysis, atomic force microscopy and cryo-transmission electron microscopy. The in vivo activity of oral hyaluronan-insulin complex was then evaluated by measuring the decrease in blood glucose concentrations in streptozotocin diabetic rats. RESULTS: Five of seven batches of the two insulin complex formulations fit the baseline criteria for approval of the new formulation. The formulation consists of a transparent aqua sol containing a solid hydrophobic phase as precipitate. Glucose-lowering activity was demonstrated after oral administration of the insulin complex'to diabetic rats. CONCLUSION: A new insulin formulation, a hyaluronan-insulin complex, has been developed and oral activity has been demonstrated.     

2011～2013年南京地区胰岛素及胰岛素类似物应用情况分析

目的:评价南京地区2011~2013年胰岛素及胰岛素类似物的临床应用情况和变化趋势。方法:对南京地区43家医院2011~2013年胰岛素及胰岛素类似物的品种、销售金额、用药频度(DDDs)等进行统计和分析。结果:胰岛素及胰岛素类似物的使用总金额逐年增长,平均年增长率16%。甘精胰岛素、精蛋白生物合成人胰岛素(预混30R)、门冬胰岛素30、生物合成人胰岛素、门冬胰岛素等5个品种3年均排在金额排序的前5位,且所占比例均超过80%。而精蛋白生物合成人胰岛素(预混30R)、门冬胰岛素30、甘精胰岛素、生物合成人胰岛素、普通胰岛素等5个品种3年均排在DDDs值排序的前5位。结论:南京地区胰岛素及胰岛素类似物临床应用广泛,使用量和金额均呈快速增长趋势。     

Pharmacokinetic and Pharmacodynamic Properties of Insulin Aspart and Human Insulin

The preferred approach to determine the pharmacokinetic (PK) and pharmacodynamic (PD) properties of insulin analogues is the euglycemic glucose clamp. Currently, non-compartmental data analytical approaches are used to analyze data. The purpose of the present study is to propose a novel compartmental-model for analysis of data from glucose clamp studies. Data used in this trial only involved 18 of the 20 originally treated subjects. Data was obtained from a crossover trial where 18 healthy subjects each received a single subcutaneous (sc) dose of 1.2 nmol/kg (body weight) insulin aspart (IAsp) or 1.2 nmol/kg human insulin (HI) during a euglycemic glucose clamp after overnight fast. Serum insulin and glucose concentrations were measured and the glucose infusion rate (GIR) was adjusted after dosing, to maintain blood glucose near basal levels. Individual model parameters were estimated for IAsp, HI, and the corresponding glucose and GIR data. We found statistically significant differences between most of the HI and IAsp pharmacokinetic parameters, including the sigmoidicity of the time course of absorption (1.5 for HI vs. 2.1 for IAsp (unit less), P=0.0005, Wilcoxon Signed-rank test), elimination rate constant (0.010 min^–1 for HI vs. 0.016 min^–1 for IAsp (P=0.002)). The PD model parameters were mostly not different, except for the rate of insulin action (0.012 min^–1 for HI vs. 0.017 min^–1 for IAsp (P=0.03)). The model may provide a framework to account for different PK properties when estimating the PD properties of insulin and insulin analogues in glucose clamp experiments.     

Solution Stability of Poloxamer 188 Under Stress Conditions

Poloxamer 188 (P188) is a triblock copolymer of the form polyethylene oxide–polypropylene oxide–polyethylene oxide (PEO–PPO–PEO). The center PPO block is hydrophobic, and the side PEO blocks are hydrophilic, resulting in surface-active properties. P188 has been used in the pharmaceutical industry as an excipient in various formulations and drug delivery systems. Although the chemical stability of P188 in the solid state has been reported, there are very few reports detailing the solution state stability. In this study, we report the solution state stability of P188 conducted to evaluate the effects of P188 concentration, temperature, pH and buffer type, and trace metals on chemical stability. The degradation chemistry of P188 and identification of degradation products was studied using various analytical techniques (ultraviolet, gas chromatography–mass spectrometry, and liquid chromatography-mass spectrometry). The degradation of P188 in solution was found to be strongly dependent on temperature, P188 concentration, and buffer type. For the first time, we report that in histidine buffer, oxidation of both P188 and histidine may occur at pharmaceutically relevant conditions. We observed degradation of both histidine and P188 as well as species formed from the mutual interactions of the degradation products from the 2 types of molecules.     

Analogs of oxytocin containing a pseudopeptide Leu-Gly bond of cis and trans configuration*

Analogs of deamino-oxytocin wherein the Leu-Gly peptide bond has been replaced by a tetrazole moiety or by a double bond of trans configuration were synthesized and their biological activities evaluated. Trans double bond was found to be the most appropriate substitution for the amide bond (uterotonic activity 24% of the deamino-oxytocin). In the case of all three analogs low but prolonged galactogogic activity was found and the ratio of uterotonic in vitro and in vivo activity was surprisingly high (ranging from 4.5 to 20).     

Characterization of a Solid State Reaction Product from a Lyophilized Formulation of a Cyclic Heptapeptide. A Novel Example of an Excipient-Induced Oxidation

Purpose. To elucidate the structure of a degradation product arising from a lyophilized formulation of a cyclic heptapeptide, and to provide a mechanism to account for its formation. Methods. Preparative HPLC was used to isolate the degradate in quantities sufficient for structural studies. A structure assignment was made on the basis of the compounds spectroscopic properties (UV, MS, NMR) and the results of amino acid analysis. Results. The degradate was identified as a benzaldehyde derivative arising from the oxidative deamination of an aminomethyl phenylalanine moiety. The extent of formation of this product is influenced by the amount of mannitol used as an excipient in the formulation. A mechanism is proposed whereby reducing sugar impurities in mannitol act as an oxidizing agent via the intermediacy of Schiff base adducts which subsequently undergo tautomerization and hydrolysis. Conclusions. Reducing sugar impurities in mannitol are responsible for the oxidative degradation of the peptide via a mechanism that involves Schiff base intermediates. This mechanism may be a potential route of degradation of other arylmethyl amines in mannitol-based formulations.     

The Role of Intramolecular Nucleophilic Catalysis and the Effects of Self-Association on the Deamidation of Human Insulin at Low pH

The influence of intramolecular catalysis and self-association on the kinetics of deamidation at the A-21 Asn residue of human insulin was explored at low pH and 35°C. Observed rate constants of overall insulin degradation were determined as a function of pH over a pH range of 2.0–5.0 and as a function of total insulin concentration between pH 2.0–4.0. The pH-rate behavior of both monomeric and associated insulin degradation from pH 2.0 to 5.0 indicated intramolecular catalysis by the unionized carboxyl terminus of the A chain. Anhydride trapping with aniline at pH 3.0 provided evidence supporting the formation of a cyclic anhydride intermediate in the rate limiting step indicative of intramolecular nucleophilic catalysis. Insulin in the presence of aniline at low pH formed two anilide products, A-21 N²-phenyl asparagine and N²-phenyl aspartic acid human insulin, at the expense of desamido A-21 formation, consistent with the partitioning of a common intermediate. Self-associated insulin degraded at a rate approximately 2.5 times greater than that of the monomer at pH 2.0 and pH 3. However, self-association had a negligible or slight stabilizing effect on insulin decomposition at pH 4.0. An apparent downward shift in the pK_a of the carboxyl terminus of approximately 0.75 units upon self-association and a catalytic rate constant which increases with -COOH acidity are proposed to account for these observations.In partial fulfillment for a Ph.D. degree in the     

Synthesis of Palmitoyl Derivatives of Insulin and Their Biological Activities

In order to improve the lipophilicity of peptides, bovine insulin was chosen for the chemical modification using palmitic acid. The N-hydroxysuccinimide ester of palmitic acid was used for attachment to terminal amino groups, and p-methoxybenzoxycarbonyl azide was used for protection of the glycine-Al amino terminus. We obtained two purified derivatives of insulin, Bl-monopalmitoyl- and Bl,B29-dipalmitoyl-insulin, which were confirmed to be more lipophilic than the parent insulin on high-performance liquid chromatography (HPLC). The hypoglycemic effects of both products were measured in rats after intravenous and intramuscular injections. The mono derivative was more active than the di derivative and produced a longer effect duration than the native insulin after intravenous injection. The derivatives were also shown to be less immunoreactive as judged by an enzyme immunoassay.     

Stability of Acyl Derivatives of Insulin in the Small Intestine: Relative Importance of Insulin Association Characteristics in Aqueous Solution

The stability of insulin and its acyl derivatives in the small intestine was examined in vitro. When these compounds were incubated in small intestinal fluid at 37°C, proteolysis of monoacyl insulins was reduced by increasing the carbon number of the fatty acid attached to Phe-B₁ of the insulin molecule. In contrast, Phe-B₁ and Lys-B₂₉ diacylated insulins were more susceptible to hydrolysis than native insulin. Similar results were obtained using homogenates of the small intestinal mucosa, although the extent of the contribution of acylation to insulin degradation differed. The mechanism of the accelerated insulin proteolysis by diacylation was studied by circular dichroism (CD). The negative maxima at 270 nm in the CD spectra were attenuated for the diacyl derivatives, indicating that insulin association was inhibited by diacylation. Therefore, the increased proportion of monomers available for insulin proteolysis represents a main factor that makes diacyl derivatives unstable.     

重组人胰岛素V8酶肽图谱的毛细管区带电泳分析

目的：建立重组人胰岛素Ｖ８酶肽图谱的毛细管电泳分析方法。方法：利用毛细管区带电泳分离模式,采用熔凝石英毛细管柱,检测了重组人胰岛素经Ｖ８蛋白酶解所产生的５个肽片段;比较了重组与天然人胰岛素的Ｖ８蛋白酶肽图谱;鉴定了毛细管区带电泳和反相高效液相色谱的肽图谱中各峰的对应关系。结果：重组人胰岛素与天然人胰岛素的分子结构具有同一性;毛细管区带电泳和反相高效液叮色谱２种方法具有不同的分离机理。结论：毛细管区     

A Quantitative Assessment of the Significance of Molecular Mobility as a Determinant for the Stability of Lyophilized Insulin Formulations

Purpose The purpose was to explore a method for quantitatively assessing the contribution of molecular mobility to the chemical reactivity of amorphous solids. Degradation of insulin in lyophilized formulations containing trehalose and poly(vinylpyrrolidone)(PVP) was chosen as a model system, and the temperature- and glass transition temperature (T_g)-dependence of the degradation rate was analyzed to obtain the relative contributions of molecular mobility and that of the chemical activational barrier reflected in the energy of activation.Methods Insulin degradation and dimerization in lyophilized trehalose and PVP formulations were monitored at various relative humidities (6–60% RH) and temperatures (10–60°C) by reverse-phase high-performance liquid chromatography (HPLC) and high-performance size-exclusion chromatography (HP-SEC), respectively. The T_g and fragility parameter of the lyophilized insulin formulations were determined by differential scanning calorimetry (DSC).Results Insulin degradation in the initial stage was describable with first-order kinetics for both of the trehalose and PVP formulations. The temperature- and T_g-dependence of the degradation rate indicated that the reactivity of insulin in the trehalose formulation is affected by molecular mobility at low humidity (12% RH), such that the ratio of the observed rate constant (k′) to the rate constant governed only by the activational barrier (k) was 0.051 at the T_g. At higher humidities, in contrast, the value of k′/k was much higher (0.914, 0.978, and 0.994 for 23% RH, 33% RH, and 43% RH, respectively), indicating that insulin degradation rate is determined predominantly by the activational barrier. For insulin degradation in the PVP formulation at temperatures below T_g, the contribution of molecular mobility to the degradation rate appeared to be negligible, as the extrapolated value of t₉₀ at the T_g exhibited a large difference between the formulations with differing T_g values (because of differing water contents).Conclusions The reactivity of insulin in the trehalose and PVP formulations can be described by an equation including factors reflecting the activational barrier (activation energy and frequency coefficient) and factors reflecting the molecular mobility (T_g, fragility parameter and a constant representing the relationship between the molecular mobility and the reaction rate). Thus, analysis of temperature dependence based on the proposed equation allows quantitative assessment of the significance of molecular mobility as a factor affecting chemical reactivity.     

TOPOCHEMICALLY RELATED HORMONE STRUCTURES: Synthesis of Partial Retro-Inverso Analogs of LH-RH

The syntheses of five partial retro-inverso luteinizing hormone-releasing hormone (LH-RH)analogs, [g-Tyr⁵, m-Gly⁶]LH-RH, [g-Tyr⁵-r-Gly⁶, R,S-m-Leu⁷]LH-RH, [g-p-Glu¹, m-His²]LH-RH, [g-p-Glu¹ -r-D-His-R,S-m-Trp³]LH-RH, and [g-Pro⁹- propionyl-des-Gly¹⁰]LH-RH, have been accomplished by solution methods. The choice of sequence to be reversed was based on suggested biodegradation mechanisms of LH-RH. A (gem)-diamino alkylidene residue, which was produced via Curtius rearrangement of a peptide segment, and a 2-substituted malonyl residue mark the initiating and terminating site, respectively, of the reversed sequence.     

Novel Side‐Chain Glucosylated and Adamantylated [Asp2/Glu2]Enkephalin Analogs: Synthesis and In Vitro Growth Inhibition of Human Tumor Cells

A series of new backbone‐modified Leu‐ and Met‐enkephalin analogs ( 13 – 20 a and b ) were synthesized. Backbone manipulations involved the replacement of the Gly² residue in Tyr‐Gly‐Gly‐Phe‐Leu/Met with side‐chain glucosylated or adamantylated D /L ‐aspartic or ‐glutamic acids. The in vitro antiproliferative activity of these compounds was evaluated for several cell lines and the results were compared with the effect of Met‐enkephalin, the native opioid growth factor. The tested compounds modestly inhibited the growth of the tumor cells (20–50% inhibition at millimolar concentrations). Among the tested compounds, Tyr‐D ‐Glu(AdNH)‐Gly‐Phe‐Met ( 20b ) showed significant antiproliferative activity, somewhat more pronounced on MCF‐7 (breast carcinoma) and MOLT‐4 (lymphoblastic leukemia) cells.     

葛根素对高胰岛素环境下大鼠肝细胞一氧化氮合成的影响

目的：观察葛根素对高胰岛素环境下大鼠肝细胞一氧化氮（NO）合成的影响。方法：体外培养BRL大鼠肝细胞株,用高胰岛素诱导其形成胰岛素抵抗细胞模型,观察葛根素对肝细胞一氧化氮合成的影响。结果：葛根素可调节高胰岛素环境下大鼠肝细胞一氧化氮合酶（NOS）的活性,增加肝细胞NO的合成。结论：葛根素可通过适度调节肝细胞NO的产生,从而促进肝细胞对葡萄糖转化,改善肝细胞胰岛素抵抗的作用。     

The Effect of Intestinal Bacteria Adherence on Drug Diffusion Through Solid Films Under Stationary Conditions

Purpose. To study the in vitro and in vivo the role of surface bacterial adhesion on the diffusion of model drugs at stationary conditions. Methods. Salicylic acid (SA) diffusion through ethyl cellulose (EC) films was measured in vitro in side-by-side diffusion cells with and without E. coli of intestinal origin. Insulin (I) release from paper strips coated or uncoated with pectin films, with or without antibiotic treatment, was measured in vivo in conscious rats after cecal implantation by comparing blood glucose levels at T_max of the pharmacodynamic effect. Results. During five hours of diffusion studies which were performed immediately following incubation of EC films with bacteria, the diffusion rate of S A throughout the films was 2.72-fold lower in the presence of bacteria compared with the diffusion rate in the control studies conducted without bacteria. The mean blood glucose levels dropped in the rat to 40.6 ± 21.6% of glucose basal levels within 2.4 ± 1.4 h when uncoated I solid carriers were used. Glucose levels did not change for pectin-coated dosage forms. After antibiotic treatment which prevented the formation of bacterial biofilm on the surface of the I solid dosage forms, blood glucose levels dropped to 22.0 ± 4.7% and 50.9 ± 20.5% of glucose basal levels within 7.4 ± 2.6 h and 1.8 ± 0.9 h for pectin uncoated or coated dosage forms, respectively. Maximum bacterial adherence occurred at stationary conditions (RPM = 0), while at maximum agitation (200 RPM), almost no adherence occurred. Conclusions. (a) Bacterial adherence slows down the diffusion rate of SA through EC films; (b) Under stationary conditions bacterial adherence may also interfere with drug release from biodegradable (pectin) films; (c) Successful functioning of biodegradable colon-specific delivery systems depends on agitation and surface friction in the lumen of the colon.     

Assembly and Dissociation of Human Insulin and LysB28ProB29-Insulin Hexamers: A Comparison Study

Purpose. Investigations into the kinetic assembly and dissociation of hexameric Lys^B28Pro^B29-human insulin (LysPro), a rapid-acting insulin analog produced by the sequence inversion of amino acids at positions B28 and B29, were designed to explain the impact that the sequence inversion has on the formulation and pharmacokinetics of the insulin analog. Methods. The kinetics of phenolic ligand binding to human insulin and LysPro were studied by stopped-flow spectroscopy. The kinetics of R₆ hexamer disruption were studied by extraction of Co(II) with EDTA. Results. Phenolic ligand binding to human insulin yielded rate constants for a fast and slow phase that increased with increasing ligand concentration and are attributed to the T₆ T₃R₃ and T₃R₃ R₆ transitions, respectively. However, the kinetics of phenolic ligand binding with LysPro was dominated by rates of hexamer assembly. The kinetic differences between the insulin species are attributed to alterations at the monomer-monomer interface in the dimer subunit of the LysPro analog. The extraction of Co(II) from both hexameric complexes by EDTA chelation is slow at pH 8.0 and highly dependent on ligand concentration. Cobalt extraction from LysPro was pH dependent. Of the various phenolic ligands tested, the relative affinities for binding to the human and LysPro hexamer are resorcinol > phenol > m-cresol. Conclusions. The extraction data support the formation of an R₆-type LysPro hexamer under formulation conditions, i.e., in the presence of divalent metal and phenolic ligand, that is similar in nature to that observed in insulin. However, the formation kinetics of LysPro identify a radically different monomeric assembly process that may help explain the more rapid pharmacokinetics observed with the hexameric formulation of LysPro insulin relative to human insulin.