Atomic resolution structure of full-length human insulin fibrils.

Patients with type 1 diabetes mellitus who are dependent on an external supply of insulin develop insulin-derived amyloidosis at the sites of insulin injection. A major component of these plaques is identified as full-length insulin consisting of the two chains A and B. While there have been several reports that characterize insulin misfolding and the biophysical properties of the fibrils, atomic-level information on the insulin fibril architecture remains elusive. We present here an atomic resolution structure of a monomorphic insulin amyloid fibril that has been determined using magic angle spinning solid-state NMR spectroscopy. The structure of the insulin monomer yields a U-shaped fold in which the two chains A and B are arranged in parallel to each other and are oriented perpendicular to the fibril axis. Each chain contains two ß-strands. We identify two hydrophobic clusters that together with the three preserved disulfide bridges define the amyloid core structure. The surface of the monomeric amyloid unit cell is hydrophobic implicating a potential dimerization and oligomerization interface for the assembly of several protofilaments in the mature fibril. The structure provides a starting point for the development of drugs that bind to the fibril surface and disrupt secondary nucleation as well as for other therapeutic approaches to attenuate insulin aggregation.

Small-angle x-ray scattering investigation of the integration of free fatty acids in polysorbate 20 micelles.

A critical quality attribute for liquid formulations is the absence of visible particles. Such particles may form upon polysorbate hydrolysis resulting in release of free fatty acids into solution followed by precipitation. Strategies to avoid this effect are of major interest for the pharmaceutical industry. In this context, we investigated the structural organization of polysorbate micelles alone and upon addition of the fatty acid myristic acid (MA) by small-angle x-ray scattering. Two complementary approaches using a model of polydisperse core-shell ellipsoidal micelles and an ensemble of quasiatomistic micelle structures gave consistent results well describing the experimental data. The small-angle x-ray scattering data reveal polydisperse mixtures of ellipsoidal micelles containing about 22-35 molecules per micelle. The addition of MA at concentrations up to 100 µg/mL reveals only marginal effects on the scattering data. At the same time, addition of high amounts of MA (>500 µg/mL) increases the average sizes of the micelles indicating that MA penetrates into the surfactant micelles. These results together with molecular modeling shed light on the polysorbate contribution to fatty acid solubilization preventing or delaying fatty acid particle formation.

Self-Assembly of Exendin-4-Derived Dual Peptide Agonists is Mediated by Acylation and Correlated to the Length of Conjugated Fatty Acyl Chains.

Dual glucagon-like peptide-1/glucagon receptor agonists have emerged as promising candidates for the treatment of diabetes and obesity. Issues of degradation sensitivity and rapid renal clearance are addressed, for example, by the conjugation of peptides to fatty acid chains, promoting reversible albumin binding. We use combined dynamic and static light scattering to directly measure the self-assembly of a set of dual peptide agonists based on the exendin-4 structure with varying fatty acid chain lengths in terms of apparent molecular mass and hydrodynamic radius (RS). We use NMR spectroscopy to gain an insight into the molecular architecture of the assembly. We investigate conformational changes of the monomeric subunits resulting from peptide self-assembly and assembly stability as a function of the fatty acid chain length using circular dichroism and fluorescence spectroscopy. Our results demonstrate that self-assembly of the exendin-4-derived dual agonist peptides is essentially driven by hydrophobic interactions involving the conjugated acyl chains. The fatty acid chain length affects assembly equilibria and the assembly stability, although the peptide subunits in the assembly retain a dynamic secondary structure. The assembly architecture is characterized by juxtaposition of the fatty acyl side chains and a hydrophobic cluster of the peptide moiety. This cluster experiences local conformational changes in the assembly compared to the monomeric unit leading to a reduction in solvent exposure. The N-terminal half of the peptide and a C-terminal loop are not in contact with neighboring peptide subunits in the assemblies. Altogether, our study contributes to a thorough understanding of the association characteristics and the tendency toward self-assembly in response to lipidation. This is important not only to achieve the desired bioavailability but also with respect to the physical stability of peptide solutions.

Rapid-Acting and Human Insulins: Hexamer Dissociation Kinetics upon Dilution of the Pharmaceutical Formulation.

PURPOSE: Comparison of the dissociation kinetics of rapid-acting insulins lispro, aspart, glulisine and human insulin under physiologically relevant conditions. METHODS: Dissociation kinetics after dilution were monitored directly in terms of the average molecular mass using combined static and dynamic light scattering. Changes in tertiary structure were detected by near-UV circular dichroism. RESULTS: Glulisine forms compact hexamers in formulation even in the absence of Zn2+. Upon severe dilution, these rapidly dissociate into monomers in less than 10 s. In contrast, in formulations of lispro and aspart, the presence of Zn2+ and phenolic compounds is essential for formation of compact R6 hexamers. These slowly dissociate in times ranging from seconds to one hour depending on the concentration of phenolic additives. The disadvantage of the long dissociation times of lispro and aspart can be diminished by a rapid depletion of the concentration of phenolic additives independent of the insulin dilution. This is especially important in conditions similar to those after subcutaneous injection, where only minor dilution of the insulins occurs. CONCLUSION: Knowledge of the diverging dissociation mechanisms of lispro and aspart compared to glulisine will be helpful for optimizing formulation conditions of rapid-acting insulins.

Readmissions of patients with diabetes mellitus and foot ulcers after infra-popliteal bypass surgery - attacking the problem by an integrated case management model.

BACKGROUND: Patients with neuroischemic diabetic foot syndrome (DFS) may need arterial revascularization, minor amputations, débridements as well as meticulous wound care. Unfortunately, postoperative outpatient care is frequently inadequate. This is especially true for Germany, where the in- and outpatient sectors are funded and managed separately, with poor communication between the two. Thus, many patients may be readmitted to the hospital following successful treatment and discharge. In an attempt to overcome these problems, we looked at whether an integrated case management (CM) system for outpatient care according to in-hospital standards might improve patients care and avoid readmissions. In addition we analyzed the length of hospital stay (LOS) as well as hospital costs. PATIENTS AND METHODS: In this retrospective cohort study patients with DFS, bypass surgery and foot surgery after implementation of the CM (study group; n = 376) were compared with a matched historic control group (HCG; n = 190) including the flat rate revenues (G-DRG K01B). Following a standardized assessment, integrated trans-sectoral CM care was offered to 116 patients (CMP). RESULTS: The proportion of patients who were readmitted to hospital was reduced in CMP compared to HCG (8.8 vs. 16.4 %; p < 0.01), with consequent reduction of case consolidations (9.7 % versus 17.8 %, p < 0.001). Although initially, the mean LOS was higher in the CMP patients, the reduction in readmissions meant that this integrated CM program improved the hospital's economic situation. CONCLUSIONS: A hospital-based integrated CM system significantly reduces the hospital readmissions in patients with neuroischemic DFS following bypass surgery, with lower hospital costs.

In-situ investigation of solid phase evolution during lyophilization of mannitol-based antibody formulations using an XRPD climate chamber.

Crystalline mannitol is commonly used as bulking agent in antibody formulations to provide structure to the lyophilized cake and prevent collapse. Depending on the lyophilization process conditions mannitol can either crystallize as α-, ß-, δ-mannitol, mannitol-hemihydrate, or transition to its amorphous state. While crystalline mannitol helps to create a firmer cake structure this is not true for amorphous mannitol. The hemihydrate is also an undesired physical form as it may reduce the drug product stability by releasing bound water molecules into the cake. Our aim was to simulate lyophilization processes in an X-ray powder diffraction (XRPD) climate chamber. In the climate chamber, the process can be carried out fast with low sample quantities to determine optimal process conditions. Insights on the emergence of desired anhydrous mannitol forms helps to adjust the process parameters in larger scale freeze-dryers. In our study we have identified the critical process steps for our formulations and then varied relevant process parameters, which were the annealing temperature, annealing time and temperature ramp rate of the freeze-drying process. Furthermore, the effect of the presence of antibodies on excipient crystallization was investigated by performing the studies on placebo solutions versus two respective antibody formulations. A comparison of the products obtained in a freeze-dryer and the simulated process in the climate chamber showed good accordance demonstrating the method as suitable tool to identify ideal process conditions on a laboratory scale.

Adding a New Dimension to the Amorphous Solid Dispersion Phase Diagram: Studying Dissolution Kinetics of Crystalline Drugs in a Polymer Matrix Using Temperature Dependent XRPD and DSC.

Hot-melt-extrusion (HME) is an enabling technology used for poorly soluble active pharmaceutical ingredients (APIs) to increase the bioavailability by embedding the drug in a water soluble and often amorphous carrier such as a polymer. Knowledge of the most critical factors impacting the dissolution rate of crystalline API in the polymer during manufacturing will provide useful insight for process improvement. In this study, crystalline APIs (Acetaminophen, APAP and Indomethacin, IMC) were analyzed in a polymeric matrix (Copovidone, PVP-VA64) via X-Ray Powder Diffraction (XRPD) and Differential Scanning Calorimetry (DSC) to follow the dissolution process under various conditions in a down-scaled static laboratory system. The combination of in-situ XRPD measurements and a kinetic model based on DSC data proved to be a suitable tool to investigate the dissolution process and can be applied to various APIs and polymers to avoid residual crystallinity and thermal degradation. Thus, the temperature-composition phase diagram in a thermodynamic equilibrium is augmented by the kinetic component as new dimension. The obtained findings set the foundation for investigating the dissolution kinetics and enable the transition from a static to a dynamic system.

An example of how to establish the thermodynamic stability relationship between two polymorphs of a compound highly prone to solvate formation.

Upon transition from research to development, a new chemical entity, which acts upon the Kv1.5-potassium channel and blocks potassium flow in the atrium of the human heart, has been subjected to a crystallization screen. The sodium salt of an anthranilic acid amide with a heteroarylsulfonyl side chain forms solvates from all tested organic solvents. Solvent-free crystalline phases can only be obtained by drying certain solvates under suitable conditions. Two well crystalline solvent-free phases can be obtained this way. Three different methods were applied to determine their thermodynamic stability relationship from melting, solution and eutectic melting data. The different approaches are discussed and compared with respect to their accuracy and limitations.

The quaternary structure of insulin glargine and glulisine under formulation conditions.

The quaternary structures of insulin glargine and glulisine under formulation conditions and upon dilution using placebo or water were investigated using synchrotron small-angle X-ray scattering. Our results revealed that insulin glulisine in Apidra® is predominantly hexameric in solution with significant fractions of dodecamers and monomers. Upon dilution with placebo, this equilibrium shifts towards monomers. Insulin glargine in Lantus® and Toujeo® is present in a stable hexamer/dimer equilibrium, which is hardly affected by dilution with water down to 1 mg/ml insulin concentration. The results provide exclusive insight into the quaternary structure and thus the association/dissociation properties of the two insulin analogues in marketed formulations.

An example of how to handle amorphous fractions in API during early pharmaceutical development: SAR114137--a successful approach.

The so-called pharmaceutical solid chain, which encompasses drug substance micronisation to the final tablet production, at pilot plant scale is presented as a case study for a novel, highly potent, pharmaceutical compound: SAR114137. Various solid-state analytical methods, such as solid-state Nuclear Magnetic Resonance (ssNMR), Differential Scanning Calorimetry (DSC), Dynamic Water Vapour Sorption Gravimetry (DWVSG), hot-stage Raman spectroscopy and X-ray Powder Diffraction (XRPD) were applied and evaluated to characterise and quantify amorphous content during the course of the physical treatment of crystalline active pharmaceutical ingredient (API). DSC was successfully used to monitor the changes in amorphous content during micronisation of the API, as well as during stability studies. (19)F solid-state NMR was found to be the method of choice for the detection and quantification of low levels of amorphous API, even in the final drug product (DP), since compaction during tablet manufacture was identified as a further source for the formation of amorphous API. The application of different jet milling techniques was a critical factor with respect to amorphous content formation. In the present case, the change from spiral jet milling to loop jet milling led to a decrease in amorphous API content from 20-30 w/w% to nearly 0 w/w% respectively. The use of loop jet milling also improved the processability of the API. Stability investigations on both the milled API and the DP showed a marked tendency for recrystallisation of the amorphous API content on exposure to elevated levels of relative humidity. No significant impact of amorphous API on either the chemical stability or the dissolution rate of the API in drug formulation was observed. Therefore, the presence of amorphous content in the oral formulation was of no consequence for the clinical trial phases I and II.

Enantiomeric characterization and structure elucidation of Otamixaban.

Otamixaban is a potent (Ki=0.5 nM) fXa inhibitor currently in late-stage clinical development at Sanofi for the management of acute coronary syndrome. Being unproductive in obtaining a suitable crystal of Otamixaban, the required enantiomeric characterization has been accomplished using vibrational circular dichroism (VCD) spectroscopy. Selected by a spectrum similarity index, the calculated spectra of several higher energy conformers were found to match well with the observed spectra. The characteristic IR bands of these conformers were also identified and attributed to the solvation effect. Combined with both the single crystal x-ray diffraction results for an intermediate and the proton NMR study, the absolute configuration of Otamixaban is unambiguously determined to be (R,R).

From laboratory to pilot plant: the solid-state process development of a highly potent cathepsin S/K inhibitor.

The solid-state development for the low dose drug molecule SAR114137, a selective and reversible inhibitor of cysteine cathepsin S/K, is reported. Six polymorphic forms as well as various solvate phases were discovered by an extensive polymorphism screening. The solid phase characterizations revealed that phase 1, from which a single crystal structure could be obtained, is the thermodynamically most stable form and therefore it was chosen for pharmaceutical development. The successful scale-up from development laboratory into pilot plant for the crystallization and drying processes is presented. Testing of different drying techniques, like agitated drying in conical or filter dryers as well as spray drying, proved them to be very promising alternatives to the conventional tray drying process and might be used during the industrialization phase of the project. The use of online analytical tools (e.g., Raman spectroscopy) for a better process understanding and as tools for process optimization is shown. Furthermore, wet milling by ultrasound was performed on laboratory scale and discussed as potential option to reach the desired particle size distribution necessary for a good content uniformity of the API in an oral formulation.

Challenges in the development of hydrate phases as active pharmaceutical ingredients--an example.

The challenges during pilot plant scale-up of the SAR474832 API (active pharmaceutical ingredient) production in view of crystallization, isolation, drying and micronization are reported. A variety of different solid-state analytical and spectroscopic techniques (also coupled methods) were applied in order to understand the complex phase transition behaviour of the crystallographic phase (form 1) chosen for development: a partially non-stoichiometric channel-hydrate (x (1+1.25) H(2)O) crystallizing from pure water in the crystal habit of fine needles, which tend to agglomerate upon isolation and drying. Processes have been developed for drying, sieving and micronization by jetmilling to avoid non-desired phase transitions (overdrying effects) into other hydrate forms. Special methods have been established to minimize, monitor and control the formation of amorphous content during the particle size reduction steps. By optimizing all production parameters it was possible to produce API batches in 10 kg scale with physical quality suitable for oral formulations (e.g. particle size d 90 value<20 µm, water content and crystallographic phase corresponding to desired form 1 of SAR474832).