Quantitation of drug content in a low dosage formulation by transmission near infrared spectroscopy

A transmission near infrared (NIR) spectroscopic method has been developed for the nondestructive determination of drug content in tablets with less than 1% weight of active ingredient per weight of formulation (m/m) drug content. Tablets were manufactured with drug concentrations of ∼0.5%, 0.7%, and 1.0% (m/m) and ranging in drug content from 0.71 to 2.51 mg per tablet. Transmission NIR spectra were obtained for 110 tablets that constituted the training set for the calibration model developed with partial least squares regression. The reference method for the calibration model was a validated UV spectrophotometric method. Several data preprocessing methods were used to reduce the effect of scattering on the NIR spectra and base the calibration model on spectral changes related to the drug concentration changes. The final calibration model included the spectral range from 11 216 to 8662 cm⁻¹ the standard normal variate (SNV), and first derivative spectral pretreatments. This model was used to predict an independent set of 48 tablets with a root mean standard error of prediction (RMSEP) of 0.14 mg, and a bias of only −0.05 mg per tablet. The study showed that transmission NIR spectroscopy is a viable alternative for nondestructive testing of low drug content tablets, available for the analysis of large numbers of tablets during process development and as a tool to detect drug agglomeration and evaluate process improvement efforts. Published: March 24, 2006     

Tris is a non-innocent buffer during intein-mediated protein cleavage

Fusion protein purification systems based on self-cleavable protein splicing elements are well established nowadays and have the advantage of producing recombinant proteins with their native amino acid composition while abolishing the need of an additional proteolytic cleavage step for removal of a purification tag. However, a potential disadvantage is the concomitant generation of reactive thioester intermediates during the protein self-splicing process, which are prone to undergo side reactions yielding undesired adducts. We followed the formation of these adducts as well as ways to avoid them with electrospray ionization mass spectrometry using one of our target proteins, Triticum aestivum (wheat) E(c)-1, a plant metallothionein with the ability to bind a total of six zinc or cadmium ions in the form of metal-thiolate clusters. Our investigations show that one of the most commonly used buffer substances, tris(hydroxymethyl)aminomethane (Tris), has to be applied with caution in combination with the described purification system, as it can itself react with the thioester intermediate forming a yet unreported stable adduct. This makes Tris a so called non-innocent buffer during the protein isolation procedure. Additionally, the results presented open up an interesting possibility to directly couple the one-step purification strategy with selective carboxy-terminal protein or peptide modification, e.g. the addition of fluorophors or PEGylation of peptides. Unrelated to the purification system used, we further observed a high amount of N-formylmethionine in the mass spectra when the protein of interest was expressed in cadmium-supplemented growth media.     

The βE-Domain of Wheat Ec-1 Metallothionein: A Metal-Binding Domain with a Distinctive Structure

Metallothioneins (MTs) are ubiquitous cysteine-rich proteins with a high affinity for divalent metal ions such as Zn^II, Cu^I, and Cd^II that are involved in metal ion homeostasis and detoxification, as well as protection against reactive oxygen species. Here we show the NMR solution structure of the β_E-domain of the early cysteine-labeled protein (E_c-1) from wheat (β_E-E_c-1), which represents the first three-dimensional structure of a plant MT. The β_E-domain comprises the 51 C-terminal residues of E_c-1 and exhibits a distinctive unprecedented structure with two separate metal-binding centers, a mononuclear Zn^II binding site constituted by two cysteine and two highly conserved histidine residues as found in certain zinc-finger motifs, and a cluster formed by three Zn^II ions coordinated by nine Cys residues that resembles the cluster in the β-domain of vertebrate MTs. Cys-metal ion connectivities were determined by exhaustive structure calculations for all 7560 possible configurations of the three-metal cluster. Backbone dynamics investigated by ¹⁵N relaxation experiments support the results of the structure determination in that β_E-E_c-1 is a rigidly folded polypeptide. To further investigate the influence of metal ion binding on the stability of the structure, we replaced Zn^II with Cd^II ions and examined the effects of metal ion release on incubation with a metal ion chelator.     

Reversal of haloperidol-induced orofacial dyskinesia and neuroinflammation by isoflavones

Molecular Biology Reports - Schizophrenia is a mental illness and its pharmacological treatment consists in the administration of antipsychotics like haloperidol. However, haloperidol often causes...     

Alteration of Cytokines Levels in the Striatum of Rats: Possible Participation in Vacuous Chewing Movements Induced by Antipsycotics

Antipsychotic drugs have been used in the treatment of schizophrenia and their long-term use can cause movement disorders, such as tardive dyskinesia (TD) in humans mainly typical ones such as haloperidol. Neuroinflammation has been implicated to the use of antipsychotics besides its participation in TD remains unclear. Thus, the aim of this study was to investigate the relation of cytokines with vacuous chewing movements (VCMs) in rats comparing typical and atypical antipsychotics. Rats were treated with haloperidol or risperidone for 28 days. On day 29, rats were subjected to behavioral analysis (quantification of crossing and rearing numbers and VCMs) with subsequent measurement of cytokines levels in the striatum. Haloperidol, but not risperidone treatment significantly decreased the number of crossing and rearing and increased the VCMs when compared with control group. Both antipsychotics were able to increase the levels of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α and IFN-γ) and decrease the anti-inflammatory cytokine (IL-10) in striatum of rats. However, IL-1β and IFN-γ levels were higher in animals treated with haloperidol than risperidone. Furthermore, positive correlations were observed between the cytokines (IL-1β and IFN-γ) and VCM numbers. Thus, the results suggest a role of inflammatory markers in the development of movement disorders, especially IL-1β and IFN-γ.

     

Resveratrol Protects Against Vacuous Chewing Movements Induced by Chronic Treatment with Fluphenazine

Typical antipsychotics, which are commonly used to treat schizophrenia, cause motor disorders such as tardive dyskinesia (TD) in humans and orofacial dyskinesia (OD) in rodents. The disease mechanisms as well as treatment effectiveness are still unknown. In this study, we investigated the effect of resveratrol, a polyphenol with neuroprotective properties, on behavioral changes induced by chronic treatment with fluphenazine in rats and the possible relationship between monoamine oxidase (MAO) activity and vacuous chewing movements (VCMs). Rats were treated for 18 weeks with fluphenazine enantate [25 mg/kg, intramuscularly (i.m.), every 21 days] and/or resveratrol (20 mg/kg, offered daily in drinking water). Next, body weight gain, behavioral parameters (VCMs and open field tests—locomotor and rearing activity), and MAO activity were evaluated. Fluphenazine treatment reduced body weight gain, number of crossings and rearings, and the co-treatment with resveratrol did not affect these alterations. Fluphenazine increased the prevalence and intensity of VCMs and the co-treatment with resveratrol reduced the VCMs. Furthermore, a negative correlation was found between the number of VCMs and MAO-B activity in the striatum of rats. Our data suggest that resveratrol could be promissory to decrease OD. Moreover, MAO-B activity in the striatum seems to be related to VCMs intensity.     

A genetically encoded Förster resonance energy transfer sensor for monitoring in vivo trehalose-6-phosphate dynamics

Trehalose-6-phosphate is a pivotal regulator of sugar metabolism, growth, and osmotic equilibrium in bacteria, yeasts, and plants. To directly visualize the intracellular levels of intracellular trehalose-6-phosphate, we developed a series of specific Förster resonance energy transfer (FRET) sensors for in vivo microscopy. We demonstrated real-time monitoring of regulation in the trehalose pathway of Escherichia coli. In Saccharomyces cerevisiae, we could show that the concentration of free trehalose-6-phosphate during growth on glucose is in a range sufficient for inhibition of hexokinase. These findings support the hypothesis of trehalose-6-phosphate as the effector of a negative feedback system, similar to the inhibition of hexokinase by glucose-6-phosphate in mammalian cells and controlling glycolytic flux.     

Harpagophytum procumbens Prevents Oxidative Stress and Loss of Cell Viability In Vitro

Harpagophytum procumbens, popularly known as devil’s claw, is a plant commonly used in the treatment of diseases of inflammatory origin. The anti-inflammatory effects of H. procumbens have been studied; however, the mechanism of action is not elucidated. It is known that excess of reactive oxygen and nitrogen species may contribute to increasing tissue damage due to inflammation. In the present study, we examined the effects of H. procumbens infusion, crude extract and fractions on lipid peroxidation (brain homogenates) induced by different pro-oxidants (Fe²⁺ or sodium nitroprusside) and the effects of ethyl acetate fraction (rich in phenolic compounds) on antioxidant defenses (catalase activity and thiol levels) and cell damage (brain cortical slices) induced by different pro-oxidants. All tested extracts of H. procumbens inhibited lipid peroxidation in a concentration-dependent manner. Furthermore, the ethyl acetate fraction had the highest antioxidant effects either by decreasing lipid peroxidation and cellular damage or restoring thiols levels and catalase activity. Taken together, our results showed that H. procumbens acts either by preventing oxidative stress or loss of cell viability. Thus, the previously reported anti-inflammatory effect of H. procumbens could also be attributed to its antioxidant activity.     

Harpagophytum Procumbens Ethyl Acetate Fraction Reduces Fluphenazine-Induced Vacuous Chewing Movements and Oxidative Stress in Rat Brain

Neurochemical Research - Long-term treatment with fluphenazine is associated with manifestation of extrapyramidal side effects, such as tardive dyskinesia. The molecular mechanisms related to the...     

Metal ion binding properties of <Emphasis Type="Italic">Tricium aestivum</Emphasis> E<Subscript>c</Subscript>-1 metallothionein: evidence supporting two separate metal thiolate clusters

Metallothioneins are ubiquitous low molecular mass, cysteine-rich proteins with an extraordinary high metal ion content. In contrast to the situation for the vertebrate forms, information regarding the properties of members of the plant metallothionein family is still scarce. We present the first spectroscopic investigation aiming to elucidate the metal ion binding properties and metal thiolate cluster formation of the Tricium aestivum (common wheat) early cysteine-labeled plant metallothionein (E_c-1). For this, the protein was overexpressed recombinantly in Escherichia coli. Recombinant E_c-1 is able to bind a total of six divalent d ¹⁰ metal ions in a metal thiolate cluster arrangement. The pH stability of the zinc and cadmium clusters investigated is comparable to stabilities found for mammalian metallothioneins. Using cobalt(II) as a paramagnetic probe, we were able to show the onset of cluster formation taking place with the addition of a fourth metal ion equivalent to the apo protein. Limited proteolytic digestion experiments complemented with mass spectrometry and amino acid analysis provide clear evidence for the presence of two separate metal thiolate clusters. One cluster consists of four metal ions and is made up by a part of the protein containing 11 cysteine residues, comparable to the situation found in the mammalian counterparts. The second cluster features two metal ions coordinated by six cysteine residues. The occurrence of the latter cluster is unprecedented in the metallothionein superfamily so far. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This article is dedicated to Prof. Bernhard Lippert on the occasion of his 60th birthday.