Quantitation of crystalline and amorphous forms of anhydrous neotame using 13C CPMAS NMR spectroscopy

Although most drugs are formulated in the crystalline state, amorphous or other crystalline forms are often generated during the formulation process. The presence of other forms can dramatically affect the physical and chemical stability of the drug. The identification and quantitation of different forms of a drug is a significant analytical challenge, especially in a formulated product. The ability of solid-state 13C NMR spectroscopy with cross polarization (CP) and magic-angle spinning (MAS) to quantify the amounts of three of the multiple crystalline and amorphous forms of the artificial sweetener neotame is described. It was possible to quantify, in a mixture of two anhydrous polymorphic forms of neotame, the amount of each polymorph within 1-2%. In mixtures of amorphous and crystalline forms of neotame, the amorphous content could be determined within 5%. It was found that the crystalline standards that were used to prepare the mixtures were not pure crystalline forms, but rather a mixture of crystalline and amorphous forms. The effect of amorphous content in the crystalline standards on the overall quantitation of the two crystalline polymorphic forms is discussed. The importance of differences in relaxation parameters and CP efficiencies on quantifying mixtures of different forms using solid-state NMR spectroscopy is also addressed.     

Conformational flexibility models for the receptor in structure based drug design

The problem of incorporating receptor flexibility in routine in silico screening of databases of small chemical compounds for the purposes of structure based drug design is still an unsolved problem. The main reason behind this difficulty is the large number of degrees of freedom that have to be considered to represent receptor flexibility. In this paper we review protein flexibility models that have been developed to limit the number of additional search parameters. These models can be roughly divided into five different categories. These are a) use of soft receptors which relax energetic penalties due to steric clashes, b) selection of a few critical degrees of freedom in the receptor binding site, c) use of multiple receptor structures either individually or by combining them using an averaging scheme, d) use of modified molecular simulation methods, and e) use of collective degrees of freedom as a new basis of representation for protein flexibility. All these flexible receptor models strive to balance an improvement in the accuracy of the binding predictions with an increase in computational cost. In addition, other challenges such as the development of accurate solvation models and scoring functions make the receptor flexibility problem even harder.     

市售阿奇霉素固体制剂的溶出行为

目的考察不同制药公司生产的阿奇霉素制剂(片剂、颗粒剂、干混悬剂)的体外溶出度。方法以硫酸溶液(φ=73.5%)为显色剂,用分光光度法测定阿奇霉素溶出度。结果不同制药公司及同一公司生产的不同批号的片剂、干混悬剂的溶出度基本上能达到《中华人民共和国药典》规定标准(即45 min溶出度分别≥80%)。各公司生产的颗粒剂,其溶出度差异显著。结论建议对颗粒剂制定溶出度标准。     

Conformational similarities and dissimilarities between the stereoisomeric forms of endomorphin-2

In this study, taking into account both the l-d and cis-trans isomerisms, a comprehensive structural characterization and a comparative conformational analysis were performed on the 32 stereoisomeric forms of opioid tetrapeptide, endomorphin-2. For all stereoisomers, the Φ-Ψ and χ conformational spaces were explored, in the course of which the conformational distributions, as well as the rotamer states of aromatic side chains were characterized in detail. Furthermore, the typical β- and γ-turn structures, as well as the characteristic intramolecular interactions (i.e., H-bonds, aromatic-aromatic and proline-aromatic interplays) were determined. The afore-mentioned structural and conformational features identified for each stereoisomeric form were compared with one another, considering all 32 stereoisomers. The results obtained from this comparative study indicated that both similarities and dissimilarities could be observed between the stereoisomeric forms, with regard to their structural and conformational properties. This theoretical work supplied several valuable observations concerning the effects of both l-d and cis-trans isomerisms on the three-dimensional structure of parent peptide and its stereoisomers. Nevertheless, in the course of this structural investigation, it was clarified how the structural and conformational features of stereoisomeric forms differed from one another.     

Conformational flexibility of beta-secretase: molecular dynamics simulation and essential dynamics analysis.

AIM: Based on the structural analysis to reveal the mechanism of ligand binding to beta-secretase and the specificity of each binding sub-site. METHODS: Molecular dynamics was used to simulate on the ligand free beta-secretase and ligand bound beta-secretase. The trajectories were analyzed using the essential dynamics, and the significant conformational change was illustrated employing the DynDom program. RESULTS: The essential dynamics and DynDom analyses clearly showed that the beta-secretase experienced a large conformational change upon the substrate or inhibitor binding. The flap structure adopted a swing motion, gradually covering the active site to facilitate the ligand binding process. Residues Ser86 and Ile87 served as the hinge point. Inhibitor-enzyme interaction analysis revealed that residues at P2, P1, and P1' positions of the inhibitor were very important for the binding, and residues at P2' and P3' positions may be modified to improve the binding specificity. S3 subsite of the enzyme still had space to modify the inhibitors in increasing the binding affinity. CONCLUSION: The information presented here is valuable and could be used to identify small molecular inhibitors of beta-secretase.     

Analysis of fludrocortisone acetate and its solid dosage forms by high-performance liquid chromatography.

A newly developed reversed-phase high-performance liquid chromatographic assay and test method for determining content uniformity are described for fludrocortisone acetate. The method is stability indicating and separates most known degradation products and impurities. In addition, the method is simple, sensitive, accurate, and relatively free of interferences. The coefficient of variation for multiple weight assays is between 0.3 and 1.8%.     

Conformational flexibility of transmembrane helix VII of the human serotonin transporter impacts ion dependence and transport

The serotonin transporter (SERT) regulates the serotonin concentration in the synapse and is a target of several antidepressant and psychostimulant drugs. Previous work suggested that the middle transmembrane helices (TMHs) of the biogenic amine transporters (TMHs) play a role in substrate and ion recognition. We focused our present studies on exploring the role of TMH VII in transporter function and ion recognition. Residues divergent between human SERT and Drosophila SERT (hSERT and dSERT, respectively) were identified and mutated in hSERT to the corresponding identity in dSERT. hSERT mutants V366S, M370L, S375A, and T381S exhibited a decrease in transport capacity. To further explore the role of these residues in the transport process, we generated cysteine mutants at multiple positions. Pretreatment with [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET) caused a decrease in transport of [³H]5-HT in the V366C and M370C mutants. The hSERT V366S, M370L, and M370C mutations also altered the sodium and chloride dependence for substrate transport. Interpretation of our results in the context of a homology model of SERT based on the crystal structure of the Aquifex aeolicus leucine transporter suggests flexibility in the conformation of TMH VII that impacts ion dependence and substrate transport.     

Near infrared spectroscopy in the development of solid dosage forms

The use of near infrared (NIR) spectroscopy has rapidly grown partly due to demands of process analytical applications in the pharmaceutical industry. Furthermore, newest regulatory guidelines have advanced the increase of the use of NIR technologies. The non-destructive and non-invasive nature of measurements makes NIR a powerful tool in characterization of pharmaceutical solids. These benefits among others often make NIR advantageous over traditional analytical methods. However, in addition to NIR, a wide variety of other tools are naturally also available for analysis in pharmaceutical development and manufacturing, and those can often be more suitable for a given application. The versatility and rapidness of NIR will ensure its contribution to increased process understanding, better process control and improved quality of drug products. This review concentrates on the use of NIR spectroscopy from a process research perspective and highlights recent applications in the field.     

Vascular permeability enhancement in solid tumor: various factors,mechanisms involved and its implications

Most solid tumors are known to exhibit highly enhanced vascular permeability, similar to or more than the inflammatory tissues. Common denominators affecting both cancer and inflammatory lesions are now well known: bradykinin (BK), nitric oxide (NO), peroxynitrite (ONOO(-)), prostaglandins (PGs), collagenases or matrix metalloproteinases (MMPs) and others. Incidentally, enzymes involved in these mediator syntheses are upregulated or activated. Initially described vascular permeability factor (VPF) (proteinaceous) was later identified to be the same as vascular endothelial growth factor (VEGF), which promotes angiogenesis of cancer tissues as well. These mediators cross-talk or co-upregulate each other, such as BK-NO-PGs system. Therefore, vascular permeability observed in solid tumor may reflect the other side of the coin (angiogenesis). The vascular permeability and accumulation of plasma components in the interstitium described here is applicable for predominantly macromolecules (molecular weight, Mw>45 kDa), but not for low molecular compounds as most anticancer agents are. Macromolecular compounds (e.g., albumin, transferrin) or many biocompatible water-soluble polymers show this effect. Furthermore, they are not cleared rapidly from the sites of lesion (cancer/inflammatory tissue), thus, remain for prolonged time, usually for more than a few days. This phenomenon of "enhanced permeability and retention effect" observed in cancer tissue for macromolecules and lipids is coined "EPR effect", which is now widely accepted as a gold standard for anticancer drug designing to seek more cancer-selective targeting using macromolecular drugs. Consequently, drastic reduction of the systemic side effect is observed, while the macromolecular drugs will continuously exert antitumor activity. Other advantages of macromolecular drugs are also discussed.     

Studying the bioavailability of fenozan acid in various medicinal forms

The bioavailability of fenozan acid as an antioxidant from tablets and rectal suppositories was studied in vitro and in vivo (rabbits). The complete release of fenozan acid from tablets in vitro takes 45 min, while 75% of the drug content is released from tablets within 21 min. The absolute bioavailability of fenozan acid from tablets and rectal suppositories in vivo amounts to 46.8 and 51.4%, respectively. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 40, No. 1, pp. 3–5, January, 2006.     

Conformational flexibility of the methyltetrazolethiomethyl side chain of beta-lactam antibiotics. A computer graphics study

Analysis of X-ray crystallographic data for cephalosporins and 1- oxacephalosporins shows that, although the 1-methyl-1H- tetrazol -5- ylthiomethyl side chain at position 3 of a bicyclic beta-lactam nucleus has certain conformational preferences, it also has considerable flexibility. Both the C4 = C3-C3'-S and C3-C3'-S-C5" torsional angles are frequently observed in the vicinity of +/- 90 degrees. The distance between the tetrazole ring carbon C5" and the 4-carboxyl carbon ranges from 4.07 to 5.65 A. Mean bond lengths and bond angles for the 3 and 4 side chains are tabulated.     

The cardiovascular effects of caffeine in various forms

We have investigated the influence of various procedures which alter the solubility of caffeine on its physiological effects. Pretreatment of caffeine prior to injection by a) addition of sodium salicylate or sodium benzoate, b) increase in temperature or c) preliminary dissolution in hydrochloric acid, did not change the cardiovascular effects when injected i.v. at equivalent doses in urethane-anaesthetized rats. Addition of chlorogenic acid, a molecule found in coffee and other plants and reported to modify, by complexation, the physiological effects of caffeine, had no significant effect on the depressor responses to caffeine but did diminish caffeine-induced tachycardia at high doses of caffeine (10 and 30 mg/kg i.v.).     

Imaging and behaviour of solid oral dosage forms in vivo

The kinetics of the hydrolysis of methyl, ethyl and n-propyl 4-hydroxybenzoate esters and a subsequent decarboxylation of 4-hydroxybenzoic acid have been studied at pH 1.26–10.59 and the sterilizing temperature of 130.5°C. Enthalpies (ΔH1) and entropies (ΔS1) of activation are reported for k_obs and the derived rate constants. The decarboxylation reaction showed a maximum rate near the mid-pH region where the esters were more susceptible to hydrolysis. The overall pathway for the degradation of the esters was accounted for by a consecutive reaction sequence:

where A represents ester, B, 4-hydroxybenzoic acid and C, phenol. Interference with the spectrophotometric assay by the subsequent oxidation of phenol, made necessary the exhaustive de-oxygenation of solutions for the kinetic runs where phenol was formed. The esters were found to be sufficiently stable to withstand a heat sterilization process within the pH range 3–6. The rate of phenol formation by decarboxylation of the acid was greater in the pH range where the esters are frequently used commercially, as preservatives.     

Interaction between polyethylene films and bromhexine HCl in solid dosage forms. I. Effects of moisture contents in the solid dosage forms on the sorption of the drug

The decrease of bromhexine HCl contents in granules and tablets was determined when the preparations were stored in polyethylene film package. Effects of temperature, contact area with film, excipients and moisture contents in the preparation on the remaining amount of bromhexin HCl were studied in order to investigate the interaction mechanism between bromhexine HCl and polyethylene film. It was observed that the decrease of bromhexine HCl was due to the sorption to the polyethylene film. The results indicated that the moisture contents of the dosage forms determined the rate of sorption predominantly, and that removal of adsorbed water from dosage forms was effective to prevent bromhexine HCl content decrease.     

Conformational analysis of opioid peptides in the solid states and the membrane environments by NMR spectroscopy

Determination of conformations and structures of opioid peptides in the membrane environments is an essential step to understand the action of the peptide to the specialized receptors. This information not only gains insight into the structure-function relationship of opioid peptide but also gives proper guidelines to design a new drug to have same neuroendocrine functions. This review provides the structural studies of three types of opioid peptide families such as enkephalin, beta-endorphin and dynorphin in the solid states and the membrane environments. The structures of enkephalins show that they take beta-bend, extended and double beta-bend structures in the crystals. Moreover, enkephalin molecules take a variety of structures in the crystals and are easily converted to the other structures with slightly different torsion angles. On the other hand, beta-bend structures are mostly seen in the membrane environments. Membrane bound structure of dynorphin shows that the N-terminus forms alpha-helical structure and is inserted into the membrane with the helical axis almost perpendicular to the membrane surface. It is discussed that the helical region of the extracellular loop II of the kappa-opioid receptor may interact with the helical region of dynorphin with a high affinity in the membrane environments. beta-endorphin takes alpha-helical structure at N-terminus and the central regions and the rest of regions take unordered structure when the bind to the membrane. Since the membrane bound structures of opioid peptides differ from those of the solution states, membrane association is an important process for exerting the affinity and the selectivity to the specific opioid receptors.     

Gel electrophoresis slab technique to separate the forms of acetylcholinesterase molecule

We have developed a vertical slab technique to separate the different forms of acetylcholinesterase molecule with the help of polyacrylamid gel electrophoresis, wich is based on the variation of concentration. With this method we got three double and one single paler band in the homogenates of rat cerebellum and medulla spinalis.