Development and in vivo evaluation of intranasal formulations of parathyroid hormone (1-34)

For efficient intranasal transport of parathyroid hormone (1-34) [PTH(1-34)], there is a great medical need to investigate permeation enhancers for intranasal formulations. In this study, the development of PTH(1-34) intranasal formulations was conducted. Based on conformation and chemical stability studies, the most preferable aqueous environment was determined to be 0.008 M acetate buffer solution (ABS). Subsequently, citric acid and Kolliphor^® HS·15 were compared as permeation enhancers. The mechanisms of action of citric acid and Kolliphor^® HS·15 were investigated using an in vitro model of nasal mucosa, and Kolliphor^® HS·15 led to higher permeability of fluorescein isothiocyanate-labeled PTH(1-34) (FITC-PTH) by enhancing both the transcellular and paracellular routes. Moreover, citric acid showed severe mucosal toxicity resulting in cilia shedding, while Kolliphor^® HS·15 did not cause obvious mucosa damage. Finally, Kolliphor^® HS·15 was studied as a permeation enhancer using a liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The results showed that 5% and 10% Kolliphor^® HS·15 increased the bioavailability of PTH(1-34) to 14.76% and 30.87%, respectively. In conclusion, an effective and biosafe PTH(1-34) intranasal formulation was developed by using 10% Kolliphor^® HS·15 as a permeation enhancer. Intranasal formulations with higher concentrations of Kolliphor^® HS·15 for higher bioavailability of PTH(1-34) could be further researched.     

Purification and conformation of ribosomal protein L25 from E. coli ribosome

Ribosomal protein L25 from the large subunit of E. coli ribosomes has been purified using a new procedure involving a 2 m LiCl extraction followed by phosphocellulose chromatography in 6 m urea elution buffer. The conformation of purified L25 was studied employing circular dichroism and ultraviolet absorption spectroscopy in reconstitution buffer. The analysis of the far u.v. circular dichroism spectrum of L25 indicates L25 contains ～ 16%α-helix and ～ 19%β-structure. The conformation of L25 was also studied using the predictive methods of Chou & Fasman and Maxfield & Scheraga. Both of these methods predict approximately three times the percent α-helix present in L25 as compared with that determined from the analysis of the circular dichroism spectrum. A structure for L25 is predicted which contains two positively charged binding domains and is consistent with published binding data on the interaction of 5S RNA and L25. The large difference in the %α-helix as determined from the analysis of the circular dichroism spectrum and the predictive techniques is suggested to result from the denaturing effects of 6 m urea used in the preparation of ribosomal proteins.     

Comparative Signature Diagrams to Evaluate Biophysical Data for Differences in Protein Structure Across Various Formulations

A solution to the problem of being able to show statistically significant differences in the measurements of various levels of higher‐order protein structure has been an elusive one. We propose the use of comparative signature diagrams (CSDs) to this end. CSDs compare datasets from different biophysical techniques that fingerprint the secondary, tertiary, and quaternary structures of a protein molecule and display statistically significant differences in these datasets. In this paper, we explore the differences in the structures of two proteins (Granulocyte Colony Stimulating Factor [GCSF] and a monoclonal antibody [mAb]) in various formulations. These proteins were chosen based on the extent of differences in structure observed in the formulations. As an initial test, we utilize data from circular dichroism, 8‐anilino‐1‐naphthalene‐sulfonate and intrinsic fluorescence spectroscopy, and static light scattering measurements to fingerprint protein structure in the different formulations. Several layers of statistics were explored to visualize the regions of significant differences in the protein spectra. This approach provides a rapid, high‐resolution methodology to compare various structural levels of proteins using standard biophysical instrumentation.     

Structural characterization of recombinant therapeutic proteins by circular dichroism

Most of the protein therapeutics are now produced by recombinant DNA technology. The advantages of recombinant proteins are related to their higher specificity and to their safety as exposure to animal or human diseases. However, several problems are still present in development of recombinant proteins as therapeutics, such as low bioavailability, short serum half-life, and immune response. Their successful application hinges on the protein stereochemical stability, and on the folding and the tendency to aggregate induced by purification steps and storage. All these aspects determine the failure of many potential protein therapies, and limitations in the development of the formulation. The application of multiple analytical techniques is important in order to obtain a detailed product profile and to understand how manufacturing can influence product structure and activity. Surely the protein conformation is a key aspect to be assessed, because a specific conformation is often essential for the biological function of the protein. Thus, there is a growing need to perform structural studies under the conditions in which the proteins operate, and to monitor the structural changes of the protein. Circular dichroism has been increasingly recognised as a valuable and reliable technique to get this information. In particular, examples will be here reported on the use of circular dichroism spectroscopy in the structural characterization of free and formulated recombinant proteins, looking at the prediction of the secondary structure, propensity to conformational changes, stability, and tendency to aggregate.     

氯化镍对人红细胞膜损伤机理的波谱学研究

为探讨镍对红细胞膜损伤作用的机理,采用傅立叶变换红外吸收技术和园二色光谱技术,分别研究了氯化镍对人红细胞膜蛋白和重组于卵磷脂脂质体上ｂａｎｄ３蛋白的二级结构影响,同时采用５－脱氧不饱和硬脂酸标记的电子自旋共振技术,研究了膜脂质流动性的变化．结果表明：在１－１００μｍｏｌ·Ｌ－１浓度范围内,镍离子可使红细胞膜蛋白发生变性和不可逆聚合,使膜浅层脂质流动性增加;０．０５,０．１０,０．５０ｍｍｏｌ·Ｌ－１的镍可使ｂａｎｄ３的α－螺旋和无规卷曲含量分别有减少和增加的趋势．     

Long-Term and High-Temperature Storage of Supercritically-Processed Microparticulate Protein Powders

Purpose. The long-term and high-temperature storage of dry, micron-sized particles of lysozyme, trypsin, and insulin was investigated. Subsequent to using supercritical carbon dioxide as an antisolvent to induce their precipitation from a dimethylsulfoxide solution, protein microparticles were stored in sealed containers at -25, -15, 0, 3, 20, 22, and 60°C. The purpose of this study was to investigate the suitability of supercritical antisolvent precipitation as a finishing step in protein processing. Methods. Karl Fisher titrations were used to determine the residual moisture content of commercial and supercritically-processed protein powders. The secondary structure of the dry protein particles was determined periodically during storage using Raman spectroscopy. The proteins were also redissolved periodically in aqueous buffers and assayed spectrophotometrically for biological activity and by circular dichroism for structural conformation in solution. Results. Amide I band Raman spectra indicate that the secondary structure of the protein particles, while perturbed from that of the solution state, remained constant in time, regardless of the storage temperature. The recoverable biological activity upon reconstitution for the supercritically-processed lysozyme and trypsin microparticles was also preserved and found to be independent of storage temperature. Far UV circular dichroism spectra support the bioactivity assays and further suggest that adverse structural changes, with potential to hinder renaturation upon redissolution, do not take place during storage. Conclusions. The present study suggests that protein precipitation using supercritical fluids may yield particles suitable for long-term storage at ambient conditions.     

Circular dichroism conformational study of the chemotactic peptide formyl-L-methionyl-L-leucyl-L-phenylalanine

The solution circular dichroism of the chemotactic agent formyl-L-methionyl-L-leucyl-L-phenylalanine was found to be highly solvent dependent. In relatively nonpolar solvents (fluoroalcohols) the circular dichroism is that characteristic of a folded conformation. In H₂SO₄ the tripeptide remains intact and displays a circular dichroism typical of highly solvated and totally disordered peptides. The order-to-disorder transition can be followed by addition of water to trifluoroethanol.     

Evaluation of protein stability and in vitro permeation of lyophilized polysaccharides-based microparticles for intranasal protein delivery

Biocompatible microparticles prepared by lyophilization were developed for intranasal protein delivery. To test for the feasibility of this formulation, stability of the incorporated protein and enhancement of in vitro permeation across the nasal epithelium were evaluated. Lyophilization was processed with hydroxypropylmethylcellulose (HPMC) or water soluble chitosan (WCS) as biocompatible polymers, hydroxypropyl-β-cyclodextrin (HP-β-CD) and d-alpha-tocopheryl poly(ethylene glycol 1000) succinate (TPGS 1000) as permeation enhancers, sugars as cryoprotectants and lysozyme as the model protein. As a result, microparticles ranging from 6 to 12 μm were developed where the maintenance of the protein conformation was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism and fluorescence intensity detection. Moreover, in vitro bioassay showed that the lysozyme activity was preserved during the preparation process while exhibiting less cytotoxicity in primary human nasal epithelial (HNE) cells. Results of the in vitro release study revealed slower release rate in these microparticles compared to that of the lysozyme itself. On the other hand, the in vitro permeation study exhibited a 9-fold increase in absorption of lysozyme when prepared in lyophilized microparticles with HPMC, HP-β-CD and TPGS 1000 (F4-2). These microparticles could serve as efficient intranasal delivery systems for therapeutic proteins.     

Effect of Device Design and Formulation on the In Vitro Comparability for Multi-Unit Dose Dry Powder Inhalers

The focus of this investigation was to understand the design space to achieve comparable in vitro performance of two multi-unit dose dry powder inhalers (DPIs)—Flixotide® Accuhaler® (reference product) and MultiHaler® (test product). Flow field, pressure drop and particle trajectories within the test and reference DPI devices were modelled via computational fluid dynamics (CFD). Micronized fluticasone propionate (FP) was characterized to determine particle size distribution (PSD), specific surface area (SSA) and surface interfacial properties using cohesive-adhesive balance (CAB). CFD simulations suggested that the pressure drop and airflow velocity in the MultiHaler® were greater than Accuhaler®. Two modified test devices (MOD MH 1 and MOD MH 2) were manufactured with the introduction of by-pass channels in the airflow path, which achieved comparable specific resistance and airflow path between the test and reference devices. Assessment of reference product formulation in modified test devices suggested that MOD MH 2 achieved comparable in vitro performance to the reference product. CAB analysis suggested that adhesion of all FP batches to lactose was different, with batch D showing greatest and batch A least adhesion to lactose. Test DPI formulations were manufactured using four different batches of FP with milled or sieved lactose, and showed that batch A FP formulated with sieved lactose in MOD MH 2 device demonstrated the highest degree of similarity to the Accuhaler® in vitro deposition. Application of CFD modelling and material characterization of formulation raw materials enabled the modification of device and formulation critical material attributes to create an in vitro comparable device/formulation system to the reference product.

Electronic supplementary material

The online version of this article (doi:10.1208/s12248-015-9775-z) contains supplementary material, which is available to authorized users.Key words: aerosolization, computational fluid dynamics, device design, dry powder inhaler, in vitro comparability, in vitro performance     

Synthesis of a metal binding protein designed on the α/β scaffold of charybdotoxin*

The α/β scaffold of the scorpion toxin charybdotoxin has been used for the engineering of a metal binding site. Nine substitutions, including three histidines as metal ligands, have been introduced into the original toxin sequence. The newly designed sequence, 37 amino acids long, has been assembled by solid-phase synthesis and HBTU (2-(1H-benzotriazol-l-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) coupling of Fmoc-protected amino acids. Formation of the three disulfide bonds occurred efficiently and rapidly in the presence of glutathione, and this post-synthesis modification has facilitated the purification task enormously. The process of synthesis and purification was performed in less than a week with an overall 10.2°, yield. Circular dichroism analysis showed that the newly designed protein is folded in a α/β structure, similarly to the parent toxin. Electronic absorption spectroscopy, circular dichroism and gel filtration experiments have been used to show that Cu²⁺ and Zn²⁺ ions bind with high affinity to the newly engineered protein. These results demonstrate that the α/β fold, common to all scorpion toxins, is a very versatile basic structure, tolerant for substitutions and able to present new sequences in a predetermined conformation. The chemical approach is shown to be effective, rapid and practical for the production of novel designed small proteins. © Munksgaard 1995.     

De-agglomeration Effect of the US Pharmacopeia and Alberta Throats on Carrier-Based Powders in Commercial Inhalation Products

The US pharmacopeia (USP) and Alberta throats were recently reported to cause further de-agglomeration of carrier-free powders emitted from some dry powder inhalers (DPIs). This study assessed if they have similar influences on commercially available carrier-based DPIs. A straight tube, a USP throat, and an Alberta throat (non-coated and coated) were used for cascade impaction testing. Aerosol fine particle fraction (FPF ≤ 5 μm) was computed to evaluate throat-induced de-agglomeration. Computational fluid dynamics are employed to simulate airflow patterns and particle trajectories inside the USP and Alberta throats. For all tested products, no significant differences in the in vitro aerosol performance were observed between the USP throat and the straight tube. Using fine lactose carriers (<10 μm), Symbicort^® and Oxis^™ showed minimal impaction inside the Alberta throat and resulted in similar FPF among all induction ports. For products using coarse lactose carriers (>10 μm), impaction frequency and energy inside the Alberta throat were significant. Further de-agglomeration was noted inside the non-coated Alberta throat for Seretide^® and Spiriva^®, but agglomerates emitted from Relenza^®, Ventolin^®, and Foradil^® did not further break up into smaller fractions. The coated Alberta throat considerably reduced the FPF values of these products due to the high throat retention, but they generally agreed better with the in vivo data. In conclusion, depending on the powder formulation (including carrier particle size), the inhaler, and the induction port, further de-agglomeration could happen ex-inhaler and create differences in the in vitro measurements.

Electronic supplementary material

The online version of this article (doi:10.1208/s12248-015-9802-0) contains supplementary material, which is available to authorized users.KEY WORDS: dry powder inhaler, idealized mouth-throat geometry, lactose carrier, powder aerosols, USP throat     

圆二色谱测定技术在小分子化合物与DNA相互作用研究中的应用

圆二色谱(CD)是研究DNA构象随环境条件(如温度、离子强度和pH)的改变而变化的检测技术,也是研究DNA与配基(包括小分子和蛋白质等大分子)相互作用的有力工具。DNA的圆二色谱是由其骨架结构中的不对称糖分子和由这些糖分子的构型决定的螺旋结构产生的。根据配基对原有的DNA圆二色谱信号的影响,以及诱导产生的圆二色谱新信号(ICD)的不同特点,不仅可以得知配基与DNA具有相互作用,还可以推断配基与DNA结合的不同模式。研究表明这些ICD信号是由配基的电子跃迁偶极矩与DNA的碱基电子跃迁偶极矩在DNA不对称环境中发生偶合产生的。如果使用不同序列的短链DNA进行深入的研究,还可获知配基与DNA相互作用中的序列特异性。实验已经证明,圆二色谱法虽然灵敏度较低,但它具有快速、简便、样品用量少、对构象变化敏感及不受分子大小的限制等特点,可以作为核磁共振(NMR)和X-光单晶衍射技术的补充。本文将介绍圆二色谱技术在研究DNA与小分子化合物相互作用中的应用以及本实验室利用圆二色谱技术研究抗肿瘤新药的初步结果。由于圆二色谱可作为新药研究中发现先导化合物辅助筛选的手段,因此可用于发现更多以DNA为靶点的药物,如抗肿瘤药、抗菌药、抗病...     

Regulated LC-MS/MS bioanalysis technology for therapeutic antibodies and Fc-fusion proteins using structure-indicated approach

In recent studies, the development of bioanalysis technologies using liquid chromatography-tandem mass spectrometry (LC-MS/MS) has attracted attention. Our developed nano-surface and molecular-orientation limited (nSMOL) proteolysis enables Fab-specific proteolysis and is optimal for LC-MS/MS analysis of antibody drugs and Fc-fusion proteins in biological samples. In this nSMOL method, antibodies and Fc-fusion proteins are held in pores of the particle and the subsequent proteolysis is carried out with protease-immobilized nanoparticles. The Fab of antibodies or fused region of Fc-fusion protein can be held to orient toward the reaction solution. The access of the immobilized protease is limited to a part in the structure of protein substrate on the particle surface. Thus, nSMOL proteolysis reacts selectively at the Fab complementarity-determining region of antibodies or N-terminal specific domain of Fc-fusion proteins and can be applied to both types of drugs. We have already evaluated drug concentrations in biological samples pretreated with nSMOL proteolysis using LC-MS/MS for more than twenty drugs, of which ten drugs have been fully validated and published. In this review, we discuss the development and application of LC-MS/MS bioanalysis, which enables the bioanalysis of therapeutic antibodies and Fc-fusion proteins by focusing on a structure-based approach.     

Influence of decamethonium and suxamethonium on the conformation of tryptophan side chain chromophores of membrane bound extrajunctional acetylcholine receptors

The influence of decamethonium and suxamethonium on the conformation of skeletal plasma membrane proteins was investigated by means of circular dichroism measurements. The CD-spectra of native membranes from both innervated and denervated rat diaphragm resembled the characteristics of proteins containing substantial quantities of α-helix. The development of extrajunctional receptors after chronic denervation induced a change in the protein pattern of the plasma membrane; however, no difference in the overall conformation of the proteins could be detected compared to those of the innervated membrane. It is, therefore, suggested that the newly formed membrane bound acetylcholine receptor protein contains a particularly high content of ordered secondary structure in the same order of magnitude as the other proteins present in the membrane. Decamethonium and suxamethonium gave rise to a distinct change of the CD-spectra of plasmalemmal microsomes derived from denervated rat diaphragm: the occurrence of three additionally ellipticity bands at 235, 215 and near 192 nm. Position and sign of the bands coincided with those known for trytophanyl-N-acetylamide. The presence of this side chain chromophore was confirmed by measurements of the trytophan fluorescence of the membrane. The tryptophan fluorescence increased considerably with the time of denervation reflecting a rise in the tryptophan content of the denervated membrane by 55 per cent. Both the increase in tryptophan content and the effect of the neuromuscular agents on the circular dichroism spectra were related to the presence of extrajunctional acetylcholine receptors. No change in protein conformation of the innervated membranes could be detected. Furthermore, this effect could be exerted only when the inside of the microsomes were exposed to the neuromuscular agent during sonication, thus manifesting the inside out nature of the microsomes, i.e. both agents induced a conformational alteration only if they had access to the former extracellular surface of the membrane now facing inwards. Experiments on the concentration dependency of the effect underlined the specifity of the conformational alteration. Suxamethonium induced a conformational alteration in the same concentration range (3 × 10^?6?3 × 10^?3 M) as acting upon the intact chronically denervated rat diaphragm. It is suggested that a trytophan side chain chromophore, which is in close vicinity to or part of the acetylcholine receptor, is held in an asymmetric environment, if decamethonium or suxamethonium is bound to the membrane.     

EFFECT OF TEMPERATURE UPON THE CIRCULAR DICHROISM OF BRADYKININ

Analysis of the effect of temperature on the circular dichroism spectrum of bradykinin has led to a more precise understanding of the solution conformation of the peptide. Circular dichroism and ¹³C n.m.r. have been used in a complementary fashion to support the picture that bradykinin spends a maximum of about 20% of its time in a partially ordered conformation featuring a γ-turn with Pro⁷ as the second residue. Since the γ-turn probability is insensitive to temperature, some other conformational effect dominated by the structure of water presumably produces the pronounced change in the circular dichroism spectrum with increasing temperature.     

Influence of preparation method on polynucleotide conformation and pharmacological activity of lipoplex

Conformations of polyinosinic acid [poly(I)] and polycytidylic acid [poly(C)] in liposomes (lipoplex) were investigated by both circular dichroism (CD) spectroscopy and fluorescence resonance energy transfer (FRET) measurements, and compared with those in aqueous solution. The results indicate that poly(I) and poly(C) take double-stranded structure in aqueous solution at pH 6.5-7.5 in the presence of NaCl at higher concentration than 50mM. Although lipoplex was prepared without NaCl to avoid aggregation of lipoplex particles, poly(I) and poly(C) were double-stranded in pre-mixed poly(I)/poly(C) lipoplex (pre-mixed LIC), prepared by adding a mixed solution of poly(I) and poly(C) to the cationic liposomes. However, poly(I) and poly(C) did not take double-stranded structure in separately mixed LIC, prepared by separate addition of poly(I) solution and poly(C) solution to the cationic liposomes. The physicochemical properties (particle diameter and zeta potential) of pre-mixed LIC and separately mixed LIC were not different, but the anti-proliferative effect of pre-mixed LIC on human epidermoid carcinoma A431 cells was about eight times greater than that of separately mixed LIC. Our results indicate that polynucleotide conformation in lipoplex is markedly influenced by the preparation method, and the polynucleotide conformation in lipoplex has a substantial effect on pharmacological activity.     

Choice of LC-MS Methods for the Absolute Quantification of Drug-Metabolizing Enzymes and Transporters in Human Tissue: a Comparative Cost Analysis

The quantification of drug-metabolizing enzymes and transporters is important for in vitro-in vivo extrapolation (IVIVE) of xenobiotic clearance, which has become an integral part of drug development. There are different mass spectrometry-based techniques used for quantitative proteomics, and as more laboratories are opting for the use of these methods, selecting the most appropriate tool is becoming a concern. For the first time, we attempt to determine the significance of cost of different LC-MS methods of quantitative analysis of these proteins and to present a framework to objectively assess the choice of the techniques. Based on our analysis, quantification using labeled internal standards is more expensive per sample but provides higher quality data than label-free quantification. Quantification using absolute quantification synthetic peptides is the approach of choice for analyzing less than nine proteins, whereas when quantifying a defined set of proteins (10–50), such as enzymes, in a reasonably large number of samples (20–100), the quantification concatemer technique is more economical, followed by label-free quantification. When analyzing proteomes or sub-proteomes (≥500 proteins), label-free quantification is more cost-effective than the use of labeled internal standards. A cost-benefit approach is described to assess the choice of the most appropriate mass spectrometry-based approach for the quantification of proteins relevant to IVIVE.

Electronic supplementary material

The online version of this article (doi:10.1208/s12248-014-9712-6) contains supplementary material, which is available to authorized users.KEY WORDS: cost, drug-metabolizing enzymes, LC-MS, performance, transporters     

Development and Advanced Validation of an Optimized Method for the Quantitation of Aβ42 in Human Cerebrospinal Fluid

Cerebrospinal fluid (CSF) biomarkers have been extensively utilized in the diagnosis of Alzheimer’s disease (AD) and characterization of progression. One important CSF biomarker is the amyloid beta 42 (Aβ₄₂) peptide, a key player in AD pathogenesis. The INNOTEST® Aβ₄₂ ELISA kit has been widely used but an advanced level of method development and validation has not been reported. To support a clinical trial in AD, we successfully completed a Good Laboratory Practices (GLP)-level validation of the method to establish the parameters of precision, accuracy, parallelism, selectivity, specificity, and linearity of dilution of the assay in CSF matrix, as well as CSF storage stability. Several modifications were required to optimize the assay and ensure consistent results in a clinical-trial setting. These included the use of additional calibrators, an adjusted standard curve range, a minimum required dilution (MRD) of CSF by 6-fold to avoid matrix interference and mitigation of analyte adsorption to labware by the addition of Tween-20. The optimized method displayed a quantitative range of 375–4,500 pg/mL. The inter-assay precision was ≤12.1 % CV and the inter-assay relative accuracy was ≤10.9 % absolute bias, bringing the total error of the assay to ≤23 %. The intra-assay precision of the assay at the high validation standard and below was ≤5.5 % CV; this enables sensitive detection of biomarker changes across a therapeutic regime. The INNOTEST® Aβ₄₂ ELISA kit, modified as reported here, may be appropriate for many applications, including regulatory agency acceptable clinical diagnosis and pharmacodynamic assessment.

Electronic supplementary material

The online version of this article (doi:10.1208/s12248-012-9360-7) contains supplementary material, which is available to authorized users.Key words: AB42, Alzheimer, biomarker, enzyme-linked immunosorbent assay, method validation     

In Vitro,Pharmacokinetic, Pharmacodynamic,and Safety Comparisons of Single and Combined Administration of Tiotropium and Salmeterol in COPD Patients Using Different Dry Powder Inhalers

In vitro Andersen cascade impactor-sized mass (ISM) and aerodynamic fine particle mass (FPM) <5 μm for tiotropium and salmeterol combined in a novel inhalation powder formulation containing 7.5 μg tiotropium/25 μg salmeterol (TSHH) were similar (within ±15%) to reference products containing 18 μg of tiotropium (Spiriva® HandiHaler®) (TioHH) and 50 μg of salmeterol (Serevent® Diskus®) (SalD). The pharmacokinetics (PK), pharmacodynamics, safety, and tolerability of the novel fixed-dose TSHH formulation administered once daily was compared with the single-agent therapies TioHH (once daily [qd]) and SalD (twice daily [bid]) and with the jointly administered combination of TioHH (qd) plus SalD (bid) in a randomized, 22-week, open-label, four-way crossover study in 50 patients with chronic obstructive pulmonary disease (COPD). For tiotropium, TSHH and TioHH were bioequivalent based on mean steady-state plasma area under the plasma concentration–time curves (AUC), while the urinary excretion amount was higher for TSHH and not bioequivalent to TioHH. Tiotropium peak plasma concentrations at steady state (C_max,ss) were 40% higher with TSHH. For salmeterol, substantial differences were observed in plasma AUCs and C_max,ss. No significant differences in 8-h forced expiratory volume in 1 s or forced vital capacity were detected for the TSHH (qd) against the combination of TioHH (qd) with SalD (bid). Maintenance therapy with tiotropium plus salmeterol as TSHH or as the jointly administered reference products is superior to either agent alone, safe, and well tolerated in COPD patients. In vitro results were not predictive of clinical PK findings for both tiotropium and salmeterol for the TSHH dry powder inhaler product.

Electronic supplementary material

The online version of this article (doi:10.1208/s12248-015-9751-7) contains supplementary material, which is available to authorized users.     

(L-2-HYDROXY-3-MERCAPTOPROPIONIC-ACID)OXYTOCIN

The role of the α-amino group of oxytocin in affecting the conformation of oxytocin and its binding to neurophysin was studied by a comparison of the circular dichroism and binding properties of oxytocin with those of (L-2-hydroxy-3-mercaptopropionic acid)oxytocin which contains a hydroxyl in place of the oxytocin amino group. The circular dichroism properties of (L-2-hydroxy-3-mercaptopropionic acid)oxytocin were very similar to those of deamino-oxytocin (in which the amino group of oxytocin is replaced by a hydrogen) but differed significantly from those of oxytocin, particularly under conditions in which the oxytocin α-amino group is protonated. The protonated oxytocin α-amino group is known to participate in a salt-bridge with a neurophysin carboxyl at neutral pH, but oxytocin appears to bind to neurophysin without salt-bridge formation below pH 2. Nonetheless, (L-2-hydroxy-3-mercaptopropionic acid)oxytocin, like deamino-oxytocin, was found not to bind to the hormone-binding site of neurophysin with measurable affinity at either neutral pH or low pH. The results indicate that, in the binding of oxytocin to neurophysin, the protonated oxytocin α-amino group plays a role more complex than that of carboxylate charge neutralization and suggest that this role involves an effect on oxytocin conformation. However, highly specific bonding interactions between the α-amino group and neurophysin, additional to those of salt-bridge formation, are not precluded.