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     

Regional differences in awareness and attitudes regarding genetic testing for disease risk and ancestry

Little is known about the lay public’s awareness and attitudes concerning genetic testing and what factors influence their perspectives. The existing literature focuses mainly on ethnic and socioeconomic differences; however, here we focus on how awareness and attitudes regarding genetic testing differ by geographical regions in the US. We compared awareness and attitudes concerning genetic testing for disease risk and ancestry among 452 adults (41% Black and 67% female) in four major US cities, Norman, OK; Cincinnati, OH; Harlem, NY; and Washington, DC; prior to their participation in genetic ancestry testing. The OK participants reported more detail about their personal ancestries (p = 0.02) and valued ancestry testing over disease testing more than all other sites (p < 0.01). The NY participants were more likely than other sites to seek genetic testing for disease (p = 0.01) and to see benefit in finding out more about one’s ancestry (p = 0.02), while the DC participants reported reading and hearing more about genetic testing for African ancestry than all other sites (p < 0.01). These site differences were not better accounted for by sex, age, education, self-reported ethnicity, religion, or previous experience with genetic testing/counseling. Regional differences in awareness and attitudes transcend traditional demographic predictors, such as ethnicity, age and education. Local sociocultural factors, more than ethnicity and socioeconomic status, may influence the public’s awareness and belief systems, particularly with respect to genetics.     

Inhibition of Mitochondrial Respiratory Chain in the Brain of Adult Rats After Acute and Chronic Administration of Methylphenidate

Methylphenidate (MPH) is frequently prescribed for the treatment of attention deficit/hyperactivity disorder. It was previously demonstrated that MPH altered brain metabolic activity. Most cell energy is obtained through oxidative phosphorylation, in the mitochondrial respiratory chain. However, there are still few studies about MPH effects on the brain of adult rats. Thus, in the present study we evaluated the effect of acute or chronic administration of MPH on the activities of mitochondrial respiratory chain complexes I–IV in the brain of adult rats. For acute administration, a single injection of MPH was given to 60-day-old rats. For chronic administration, MPH injections were given to 60-day-old rats once daily for 28 days. Our results showed that complexes I, II, III and IV were inhibited after acute or chronic MPH administration in the hippocampus, prefrontal cortex, striatum and cerebral cortex. On the other hand, cerebellum was not affected.     

Pteridine–sulfonamide conjugates as dual inhibitors of carbonic anhydrases and dihydrofolate reductase with potential antitumor activity

Recent evidences suggest that cancer treatment based on combination of cytostatic and conventional chemostatic therapeutics, which are usually cytotoxic, can provide an improved curative option. On the sequence of our previous work on methotrexate (MTX) derivatives, we have developed and evaluated novel MTX analogues, containing a pteridine moiety conjugated with benzenesulfonamide derivatives, thus endowed with the potential capacity for dual inhibition of dihydrofolate reductase (DHFR) and carbonic anhydrases (CA). These enzymes are often overexpressed in tumors and are involved in two unrelated cellular pathways, important for tumor survival and progression. Their simultaneous inhibition may turn beneficial in terms of enhanced antitumor activity.Herein we report the design and synthesis of several diaminopteridine–benzenesulfonamide and -benzenesulfonate conjugates, differing in the nature and size of the spacer group between the two key moieties. The inhibition studies performed on a set of CAs and DHFR, revealed the activities in the low nanomolar and low micromolar ranges of concentration, respectively. Some inhibitors showed selectivity for the tumor-related CA (isozyme IX). Cell proliferation assays using two tumor cell lines (the non-small cell lung carcinoma, A549, and prostate carcinoma, PC-3) showed activities only in the millimolar range. Nevertheless, this fact points out the need of improving the cell intake properties of these new compounds, since the general inhibitory profiles revealed their potential as anticancer agents.     

Influence of the Use of Statin on the Stability of Erythrocyte Membranes in Multiple Sclerosis

Multiple sclerosis (MS) probably occurs by oxidative, inflammatory and autoimmune mechanisms. This study investigated the influence of statin on the stability of erythrocyte membranes in MS patients. The population was composed of one group with simvastatin therapy (20 mg/day), another group without statin therapy and a healthy control group. The stability of erythrocytes was evaluated by the half-transition points, H₅₀ and D₅₀, obtained from the curves of hemolysis induced by hypotonic shock and ethanol action, respectively. Erythrocytes of MS patients were less stable against lysis by both chaotropes. This behavior may be merely a consequence of the lifestyle of MS patients or it may be intrinsically associated with the conjunct of factors responsible for the development of the disease. The use of statin by MS patients was associated with lower levels of LDL and total cholesterol, as expected, and with higher stability of erythrocytes against ethanol compared to the values of untreated MS patients.     

Thermococcus kodakarensis Mutants Deficient in Di-myo-Inositol Phosphate Use Aspartate To Cope with Heat Stress

Many of the marine microorganisms which are adapted to grow at temperatures above 80°C accumulate di-myo-inositol phosphate (DIP) in response to heat stress. This led to the hypothesis that the solute plays a role in thermoprotection, but there is a lack of definitive experimental evidence. Mutant strains of Thermococcus kodakarensis (formerly Thermococcus kodakaraensis), manipulated in their ability to synthesize DIP, were constructed and used to investigate the involvement of DIP in thermoadaptation of this archaeon. The solute pool of the parental strain comprised DIP, aspartate, and α-glutamate. Under heat stress the level of DIP increased 20-fold compared to optimal conditions, whereas the pool of aspartate increased 4.3-fold in response to osmotic stress. Deleting the gene encoding the key enzyme in DIP synthesis, CTP:inositol-1-phosphate cytidylyltransferase/CDP-inositol:inositol-1-phosphate transferase, abolished DIP synthesis. Conversely, overexpression of the same gene resulted in a mutant with restored ability to synthesize DIP. Despite the absence of DIP in the deletion mutant, this strain exhibited growth parameters similar to those of the parental strain, both at optimal (85°C) and supraoptimal (93.7°C) temperatures for growth. Analysis of the respective solute pools showed that DIP was replaced by aspartate. We conclude that DIP is part of the strategy used by T. kodakarensis to cope with heat stress, and aspartate can be used as an alternative solute of similar efficacy. This is the first study using mutants to demonstrate the involvement of compatible solutes in the thermoadaptation of (hyper)thermophilic organisms.Hyperthermophilic bacteria and archaea isolated from saline environments accumulate unusual organic solutes in response to osmotic as well as heat stress. Mannosylglycerate, mannosylglyceramide, di-myo-inositol phosphate, mannosyl-di-myo-inositol phosphate (DIP), diglycerol phosphate, and glycero-phospho-myo-inositol are examples of compatible solutes highly restricted to thermophiles and hyperthermophiles (27, 31). Our team has, over several years, examined the compatible solute composition in a large number of hyperthermophiles and their accumulation under stressful conditions. The data reveal a trend toward specialization of roles in thermoadaptation and osmoadaptation. Indeed, mannosylglycerate and diglycerol phosphate typically accumulate in response to increased NaCl concentration in the growth medium, whereas the levels of DIP and derivatives consistently increase at supraoptimal growth temperatures (11, 16, 17, 27, 31).DIP is widespread among extreme archaeal hyperthermophiles, such as Methanotorris igneus, Aeropyrum pernix, Stetteria hydrogenophila, Pyrodictium occultum, Pyrolobus fumarii, Archaeoglobus spp., and all the members of the Thermococcales examined thus far, except Palaeococcus ferrophilus (5, 7, 11, 13, 16, 18, 31). This organic solute has also been found in representatives of the two hyperthermophilic bacterial genera, Aquifex and Thermotoga (14, 17, 22).The specific chemical nature of solutes encountered in hyperthermophiles, together with their accumulation in response to elevated temperatures, led to the hypothesis that they play a role in thermoprotection of cellular components in vivo. However, there is a lack of convincing experimental evidence, such as that obtained with suitable mutants. Progress toward understanding the physiological functions of these solutes critically depends on two conditions: the availability of genetic tools to manipulate hyperthermophilic organisms and knowledge about the genes and enzymes implicated in the synthesis of these unusual solutes.Thermococcus kodakarensis (formerly Thermococcus kodakaraensis) is a member of the order Thermococcales with an optimal growth temperature of 85°C and is able to grow at temperatures up to 94°C in batch cultures. The NaCl concentration for optimal growth matches that of seawater (1). T. kodakarensis is the only marine hyperthermophile for which a number of genetic tools have been developed, including Escherichia coli-T. kodakarensis shuttle vectors and a reliable gene disruption system (19, 29, 32, 34). The genome of T. kodakarensis possesses a gene encoding CTP:inositol-1-phosphate cytidylyltransferase/CDP-inositol:inositol-1-phosphate transferase (IPCT/DIPPS), a key enzyme in DIP synthesis (2, 25, 26). This enzyme catalyzes the synthesis of CDP-inositol from CTP and inositol-1-phosphate as well as the transfer of the inositol group from CDP-inositol to a second molecule of inositol-1-phosphate to yield a phosphorylated form of DIP (2). Therefore, we set out to investigate whether DIP was involved in thermoadaptation of T. kodakarensis. A DIP-deficient mutant was constructed by deleting the IPCT/DIPPS gene; subsequently, this strain was complemented in this activity by inserting the gene under the control of a constitutive promoter, resulting in a construct with restored ability to synthesize DIP. The effects of heat and osmotic stress on the pattern of solute accumulation and on the growth profiles of the two mutants provided evidence for the involvement of DIP in thermoprotection.     

Two Alternative Pathways for the Synthesis of the Rare Compatible Solute Mannosylglucosylglycerate in Petrotoga mobilis

The compatible solute mannosylglucosylglycerate (MGG), recently identified in Petrotoga miotherma, also accumulates in Petrotoga mobilis in response to hyperosmotic conditions and supraoptimal growth temperatures. Two functionally connected genes encoding a glucosyl-3-phosphoglycerate synthase (GpgS) and an unknown glycosyltransferase (gene Pmob_1143), which we functionally characterized as a mannosylglucosyl-3-phosphoglycerate synthase and designated MggA, were identified in the genome of Ptg. mobilis. This enzyme used the product of GpgS, glucosyl-3-phosphoglycerate (GPG), as well as GDP-mannose to produce mannosylglucosyl-3-phosphoglycerate (MGPG), the phosphorylated precursor of MGG. The MGPG dephosphorylation was determined in cell extracts, and the native enzyme was partially purified and characterized. Surprisingly, a gene encoding a putative glucosylglycerate synthase (Ggs) was also identified in the genome of Ptg. mobilis, and an active Ggs capable of producing glucosylglycerate (GG) from ADP-glucose and d-glycerate was detected in cell extracts and the recombinant enzyme was characterized, as well. Since GG has never been identified in this organism nor was it a substrate for the MggA, we anticipated the existence of a nonphosphorylating pathway for MGG synthesis. We putatively identified the corresponding gene, whose product had some sequence homology with MggA, but it was not possible to recombinantly express a functional enzyme from Ptg. mobilis, which we named mannosylglucosylglycerate synthase (MggS). In turn, a homologous gene from Thermotoga maritima was successfully expressed, and the synthesis of MGG was confirmed from GDP-mannose and GG. Based on the measurements of the relevant enzyme activities in cell extracts and on the functional characterization of the key enzymes, we propose two alternative pathways for the synthesis of the rare compatible solute MGG in Ptg. mobilis.Thermophilic and hyperthermophilic organisms, like the vast majority of other microorganisms, accumulate compatible solutes in response to water stress imposed by salt. In fact, many of the (hyper)thermophiles known were isolated from geothermal areas venting seawater (36). However, the compatible solutes of thermophilic and hyperthermophilic prokaryotes are generally different from those of their mesophilic counterparts and some, namely, di-myo-inositol-phosphate (DIP), mannosyl-di-myo-inositol-phosphate (MDIP), diglycerol phosphate, and mannosylglyceramide, are confined to organisms that grow at extremely high temperatures (19, 22, 34, 38). Mannosylglycerate (2-α-d-mannosylglycerate; MG), for example, is a common compatible solute of thermophiles and hyperhermophiles (23, 27, 38) but has also been found in mesophilic organisms, such as red algae, where it was first identified (6). It should also be noted that there is a growing awareness that compatible solutes are involved in other types of stress; trehalose, for example, plays a role in osmotic stress, heat stress, desiccation, and freezing (9). Some compatible solutes of thermophilic organisms are extremely rare and have been encountered in only one or two, generally closely related, species. Among them are mannosylglyceramide in Rhodothermus marinus, diglycerol phosphate in Archaeoglobus fulgidus, and, more recently, mannosylglucosylglycerate (α-d-1→2-mannopyranosyl-α-d-1→2-glucopyranosylglycerate; MGG) identified in Petrotoga miotherma (16, 19, 38).The species of the genus Petrotoga represent slightly thermophilic members of the generally hyperthermophilic and deep-branching bacteria of the order Thermotogales (2, 3, 31). Organisms of this genus have all been isolated from hot oilfield water (21, 25), and have an optimum temperature for growth of 55 to 60°C in medium containing NaCl in the range of 0.5 to 10% (16). In Ptg. miotherma, the levels of MGG increased during low-level osmotic adaptation, whereas glutamate and proline were used for protection against hyperosmotic stress (16). The hyperthermophilic Thermotoga spp. accumulate primarily di-myo-inositol-phosphate and mannosyl-di-myo-inositol-phosphate during osmotic adjustment or during growth at temperatures above the optimum for growth (37).The novel compatible solute MGG is a derivative of glucosylglycerate (2-α-d-glucosylglycerate; GG) identified in the free form in Erwinia chrysanthemi, in the marine cyanobacteria Prochlorococcus marinus and Synechococcus sp. PCC7002, and in the thermophilic bacterium Persephonella marina, the latter of which possesses two alternative pathways for its synthesis (8, 13, 14, 18, 37). Glucosylglycerate has also been detected in trace amounts in Mycobacterium smegmatis, where it probably is the precursor of a polysaccharide involved in the regulation of fatty acid synthesis, as well as in the polar head group of a glycolipid from Nocardia otitidiscaviarum (17, 30).Two alternative pathways for the synthesis of GG have been identified and characterized. In the two-step reaction scheme, the synthesis of GG involves the condensation of nucleoside diphosphate (NDP)-glucose and d-3-phosphoglycerate (3-PGA) into glucosyl-3-phosphoglycerate (GPG), which in turn is dephosphorylated to yield GG. Yet, in a single-step pathway, the synthesis of GG occurs via the condensation of ADP-glucose with d-glycerate (13). Similar routes to those described above also lead to the synthesis of mannosylglycerate in Rhodothermus marinus (4).Two functionally connected genes encoding an “actinobacterial”-type glucosyl-3-phosphoglycerate synthase (GpgS) and an unknown glycosyltransferase were detected in the genome of Petrotoga mobilis (12). In this study, we examine the synthesis of MGG through a phosphorylating pathway (with a phosphorylated intermediate) from 3-phosphoglycerate and UDP-glucose to the final compatible solute, in cell extracts and by functional characterization of recombinant enzymes. We also examine a second nonphosphorylating pathway (no phosphorylated intermediates) that could represent an alternative route for the synthesis of MGG in Ptg. mobilis that could lead to the direct conversion of GG and GDP-mannose to MGG. Pathway multiplicity likely reflects a crucial role for MGG in the physiology of Ptg. mobilis during stress adaptation.     

Increased corticosterone levels in mice subjected to the rat exposure test

In recent years, there has been a notable interest in studying prey-predator relationships to develop rodent-based models for the neurobehavioral aspects of stress and emotion. However, despite the growing use of transgenic mice and results showing important differences in the behavioral responses of rats and mice, little research has been conducted regarding the responses of mice to predators. The rat exposure test (RET), a recently developed and behaviorally validated prey-predator (mouse-rat)-based model, has proven to be a useful tool in evaluating the defensive responses of mice facing rats. To further validate the RET, we investigated the endocrine and behavioral responses of mice exposed to this apparatus. We first constructed a plasma corticosterone secretion curve in mice exposed to a rat or to an empty cage (control). Rat-exposed mice showed a pronounced rise in corticosterone levels that peaked 15 min from the beginning of the predator exposure. The corticosterone levels and behavioral responses of mice exposed to a rat or to a toy in the RET apparatus were then measured. We observed high plasma corticosterone levels along with clear avoidance behaviors represented by decreases in tunnel and surface area exploration and increases in risk assessment behaviors and freezing. This strongly suggests that the test elicits a repertoire of behavioral responses compatible with an aversion state and indicates that it is a promising model for the evaluation of prey-predator interactions. However, more physiological, neurochemical, and pharmacological studies are needed to further validate the test.     

The role of deer as vehicles to move ticks, Ixodes ricinus, between contrasting habitats

In Europe the most important hosts maintaining Ixodes ricinus tick populations are deer. Therefore, excluding deer by fencing or culling are potential tick management tools. Here we test the hypothesis that deer act as vehicles for moving ticks between two distinct habitats: forest and open heather moorland. We utilised an ideal “natural experiment” whereby forests were either fenced or unfenced to prevent or allow deer to move between habitats. We aimed to test the hypothesis that deer cause a net movement of ticks from high tick density areas, i.e. forests, to low tick density areas, i.e. open moorland. We recorded I. ricinus and host abundance in 10 unfenced and seven fenced forests and their respective surrounding heather moorland. We found that fenced forests had fewer deer and fewer I. ricinus nymphs than unfenced forests. However, we found no evidence that fencing forests reduced I. ricinus abundance on adjacent heather moorland. Thus there was insufficient evidence for our hypothesis that deer cause a net movement of ticks from forest onto adjacent moorland. However, we found that deer abundance generally correlates with I. ricinus abundance. We conclude that fencing can be used as a tool to reduce ticks and disease risk in forests, but that fencing forests is unlikely to reduce ticks or disease risk on adjacent moorland. Instead, reducing deer numbers could be a potential tool to reduce tick abundance with implications for disease mitigation.