Metabolic pathways in immune senescence and inflammaging: Novel therapeutic strategy for chronic inflammatory lung diseases. An EAACI position paper from the Task Force for Immunopharmacology.

The accumulation of senescent cells drives inflammaging and increases morbidity of chronic inflammatory lung diseases. Immune responses are built upon dynamic changes in cell metabolism that supply energy and substrates for cell proliferation, differentiation, and activation. Metabolic changes imposed by environmental stress and inflammation on immune cells and tissue microenvironment are thus chiefly involved in the pathophysiology of allergic and other immune-driven diseases. Altered cell metabolism is also a hallmark of cell senescence, a condition characterized by loss of proliferative activity in cells that remain metabolically active. Accelerated senescence can be triggered by acute or chronic stress and inflammatory responses. In contrast, replicative senescence occurs as part of the physiological aging process and has protective roles in cancer surveillance and wound healing. Importantly, cell senescence can also change or hamper response to diverse therapeutic treatments. Understanding the metabolic pathways of senescence in immune and structural cells is therefore critical to detect, prevent, or revert detrimental aspects of senescence-related immunopathology, by developing specific diagnostics and targeted therapies. In this paper, we review the main changes and metabolic alterations occurring in senescent immune cells (macrophages, B cells, T cells). Subsequently, we present the metabolic footprints described in translational studies in patients with chronic asthma and chronic obstructive pulmonary disease (COPD), and review the ongoing preclinical studies and clinical trials of therapeutic approaches aiming at targeting metabolic pathways to antagonize pathological senescence. Because this is a recently emerging field in allergy and clinical immunology, a better understanding of the metabolic profile of the complex landscape of cell senescence is needed. The progress achieved so far is already providing opportunities for new therapies, as well as for strategies aimed at disease prevention and supporting healthy aging.

Respiratory syncytial virus infection influences tight junction integrity.

Respiratory syncytial virus (RSV) is an important risk factor of asthma development and is responsible for severe respiratory tract infections. However, the influence of RSV infection on barrier function of bronchial epithelial cells in vitro and in vivo is still unclear. The aim of this study was to analyse the role of RSV in tight junction (TJ) regulation and to compare epithelial integrity between asthmatic and healthy individuals upon RSV infection. Healthy and asthmatic human bronchial epithelial cells (HBECs) were differentiated at air-liquid interface (ALI) and infected with RSV and ultraviolet (UV)-irradiated RSV. TJ expression and their integrity were analysed by quantitative polymerase chain reaction (qPCR), transepithelial resistance (TER) and paracellular flux. To determine the effect in vivo, BALB/c mice were infected intranasally with RSV or UV-irradiated RSV A2. Bronchoalveolar lavage and TJ integrity were analysed on days 1, 2, 4 and 6 post-infection by qPCR, bioplex and confocal microscopy. RSV increased barrier integrity in ALI cultures of HBEC from healthy subjects, but no effect was found in HBECs from asthmatics. This was not associated with an increase in TJ mRNA expression. In vivo, RSV induced lung inflammation in mice and down-regulated claudin-1 and occludin mRNA expression in whole lungs. Surprisingly, RSV infection was not observed in bronchial epithelial cells, but was found in the lung parenchyma. Decreased expression of occludin upon RSV infection was visible in mouse bronchial epithelial cells in confocal microscopy. However, there was no regulation of claudin-1 and claudin-7 at protein level.

Allergen immunotherapy for allergic asthma: A systematic review and meta-analysis.

BACKGROUND: To inform the development of the European Academy of Allergy and Clinical Immunology's (EAACI) Guidelines on Allergen Immunotherapy (AIT) for allergic asthma, we assessed the evidence on the effectiveness, cost-effectiveness and safety of AIT. METHODS: We performed a systematic review, which involved searching nine databases. Studies were screened against predefined eligibility criteria and critically appraised using established instruments. Data were synthesized using random-effects meta-analyses. RESULTS: 98 studies satisfied the inclusion criteria. Short-term symptom scores were reduced with a standardized mean difference (SMD) of -1.11 (95% CI -1.66, -0.56). This was robust to a prespecified sensitivity analyses, but there was evidence suggestive of publication bias. Short-term medication scores were reduced SMD -1.21 (95% CI -1.87, -0.54), again with evidence of potential publication bias. There was no reduction in short-term combined medication and symptom scores SMD 0.17 (95% CI -0.23, 0.58), but one study showed a beneficial long-term effect. For secondary outcomes, subcutaneous immunotherapy (SCIT) improved quality of life and decreased allergen-specific airway hyperreactivity (AHR), but this was not the case for sublingual immunotherapy (SLIT). There were no consistent effects on asthma control, exacerbations, lung function, and nonspecific AHR. AIT resulted in a modest increased risk of adverse events (AEs). Although relatively uncommon, systemic AEs were more frequent with SCIT; however no fatalities were reported. The limited evidence on cost-effectiveness was mainly available for sublingual immunotherapy (SLIT) and this suggested that SLIT is likely to be cost-effective. CONCLUSIONS: AIT can achieve substantial reductions in short-term symptom and medication scores in allergic asthma. It was however associated with a modest increased risk of systemic and local AEs. More data are needed in relation to secondary outcomes, longer-term effectiveness and cost-effectiveness.

Altered fatty acid metabolism and reduced stearoyl-coenzyme a desaturase activity in asthma.

BACKGROUND: Fatty acids and lipid mediator signaling play an important role in the pathogenesis of asthma, yet this area remains largely underexplored. The aims of this study were (i) to examine fatty acid levels and their metabolism in obese and nonobese asthma patients and (ii) to determine the functional effects of altered fatty acid metabolism in experimental models. METHODS: Medium- and long-chain fatty acid levels were quantified in serum from 161 human volunteers by LC/MS. Changes in stearoyl-coenzyme A desaturase (SCD) expression and activity were evaluated in the ovalbumin (OVA) and house dust mite (HDM) murine models. Primary human bronchial epithelial cells from asthma patients and controls were evaluated for SCD expression and activity. RESULTS: The serum desaturation index (an indirect measure of SCD) was significantly reduced in nonobese asthma patients and in the OVA murine model. SCD1 gene expression was significantly reduced within the lungs following OVA or HDM challenge. Inhibition of SCD in mice promoted airway hyper-responsiveness. SCD1 expression was suppressed in bronchial epithelial cells from asthma patients. IL-4 and IL-13 reduced epithelial cell SCD1 expression. Inhibition of SCD reduced surfactant protein C expression and suppressed rhinovirus-induced IP-10 secretion, which was associated with increased viral titers. CONCLUSIONS: This is the first study to demonstrate decreased fatty acid desaturase activity in humans with asthma. Experimental models in mice and human epithelial cells suggest that inhibition of desaturase activity leads to airway hyper-responsiveness and reduced antiviral defense. SCD may represent a new target for therapeutic intervention in asthma patients.

High-dose bee venom exposure induces similar tolerogenic B-cell responses in allergic patients and healthy beekeepers.

BACKGROUND: The involvement of B cells in allergen tolerance induction remains largely unexplored. This study investigates the role of B cells in this process, by comparing B-cell responses in allergic patients before and during allergen immunotherapy (AIT) and naturally exposed healthy beekeepers before and during the beekeeping season. METHODS: Circulating B cells were characterized by flow cytometry. Phospholipase A2 (PLA)-specific B cells were identified using dual-color staining with fluorescently labeled PLA. Expression of regulatory B-cell-associated surface markers, interleukin-10, chemokine receptors, and immunoglobulin heavy-chain isotypes, was measured. Specific and total IgG1, IgG4, IgA, and IgE from plasma as well as culture supernatants of PLA-specific cells were measured by ELISA. RESULTS: Strikingly, similar responses were observed in allergic patients and beekeepers after venom exposure. Both groups showed increased frequencies of plasmablasts, PLA-specific memory B cells, and IL-10-secreting CD73- CD25+ CD71+ BR 1 cells. Phospholipase A2-specific IgG4-switched memory B cells expanded after bee venom exposure. Interestingly, PLA-specific B cells showed increased CCR5 expression after high-dose allergen exposure while CXCR4, CXCR5, CCR6, and CCR7 expression remained unaffected. CONCLUSIONS: This study provides the first detailed characterization of allergen-specific B cells before and after bee venom tolerance induction. The observed B-cell responses in both venom immunotherapy-treated patients and naturally exposed beekeepers suggest a similar functional immunoregulatory role for B cells in allergen tolerance in both groups. These findings can be investigated in other AIT models to determine their potential as biomarkers of early and successful AIT responses.

Correlations between selected parameters of nasal cavity in neonates and young infants - computed tomography study.

BACKGROUND: Correlations between selected metric parameters of the nasal cavity and nasopharynx in children without atresia may be useful for anticipating probable dimensions of this region in living subjects, in terms of changes with age. MATERIALS AND METHODS: One hundred and eighty children, age range 0-3 years, were divided into five age groups, and measurements of 18 distances between structures of nasal cavity and nosopharynx were performed on computed tomography scans. Correlation coefficients and relations between parameters have been determined. RESULTS: Our study confirmed the existence of statistically significant correlations between linear dimensions within nasal cavity in children. The analysis demon-strated that for the values of following indexes: nasal septum length/piriform aperture width, and maximum length of the nasal septum/posterior nares width no statistically significant differences have been noted between age groups of children. All correlations have been positive. No statistically significant differences have been noted between the maximum width of the vomer and osseous parameters measured, both in the anterior and posterior part of the nasal cavity, and nasal septum length. CONCLUSIONS: The size of posterior nares changed with age in children by a constant value. So far, no such an analysis has been carried out assessing potential correlations between linear dimensions for the entire nasal cavity, nasopharynx, length of the nasal septum in children, as well as proportions of individual linear dimensions of the anatomical structures analysed, in various age groups.

Adiponectin and leptin receptors expression in Barrett's esophagus and normal squamous epithelium in relation to central obesity status.

UNLABELLED: Esophageal adenocarcinoma incidence is rapidly increasing which may be due to the growing incidence of Barrett's esophagus (BE) and obesity. The mechanisms linking obesity and progression of Barrett's carcinogenesis is poorly understood. The aim of the study was to evaluate the expression of adipokines receptors in BE and in normal squamous epithelium in the same patients in correlation with obesity parameters. METHODS: Expression of adiponectin receptors 1 and 2 protein (AdipoR1, AdipoR2) as well as leptin receptor protein (ObR) in biopsies from 27 BE and normal squamous epithelium (N) in the same patients as well as in obese and normal controls were assessed with Western-blot analysis. These correlations were confirmed with the quantitative RT-PCR (qRT-PCR). AdipoR1 and ObR protein levels were similar in BE mucosa and squamous epithelium in the same patients in Western-blot analysis (2303 vs. 2448 OB units; 106927 vs. 103390, respectively; p>0.05). RT-PCR analysis confirmed this observation for AdipoR1, R2 and ObR genes expression (0.11±0.08 vs. 0.19±0.24, p=0.78; 0.24±0.36 vs. 0.33±0.49, p=0.5375; 0.71±0.8 vs. 1.33±2.95, p=1.0; respectively). Using linear correlation analysis we found the positive correlation between AdipoR1 expression in Barrett's epithelium compared to squamous epithelium in the same patients (N) (r=0.5; p=0.008) and between ObR expression in BE and N (r=0.8; p<0.001). The AdipoR1 and ObR protein levels were significantly higher in BE patients compared to controls and obese controls (2303 vs. 895 vs. 1674 and OD units, p<0.05). CONCLUSIONS: in opposite to the prior hypothesis adiponectin and leptin receptors activation in BE may be not caused by obesity.

Hydrogen peroxide and nitrite reduction in exhaled breath condensate of COPD patients.

Chronic obstructive pulmonary disease (COPD) is predominantly the result of years of cigarette smoking. Increased oxidative stress in COPD derives from the increased burden of inhaled oxidants (cigarette smoke), air pollution and the increase in reactive oxygen and nitrogen species (ROS and RNS), generated by some inflammatory, immune, and structural airways cells. In view of the lack of therapy that might inhibit the progress of the disease, there is an urgent need for a successful therapeutic approach. Apocynin is a molecule inhibiting activation of NADPH oxidase - enzyme generating ROS and RNS precursor. Thus, our aim was to analyze apocynin influence on hydrogen peroxide and nitrite concentrations in EBC of COPD patients. Apocynin reduced concentration of H(2)O(2) in COPD patients 60 and 120 min after apocynin inhalation, in comparison to placebo (0.43 µM vs. 0.59 µM, and 0.4 µM vs. 0.59 µM respectively, p < 0.05). Moreover, apocynin decreased NO(2)(-) ions concentration in airways of COPD patients after apocynin nebulization (3.97 µM vs. 4.48 µM after 30 min, 3.82 µM vs. 4.48 µM after 60 min, and 3.76 µM vs. 4.48 µM after 30 min respectively, p < 0.05). No adverse effects have been observed. The results suggest that apocynin might be considered as anti-inflammatory agent, and, possibly used in therapy of COPD.

Apocynin reduces reactive oxygen species concentrations in exhaled breath condensate in asthmatics.

Asthma is an inflammatory airway disease, and oxidative stress was proven to be involved in its pathogenesis. Apocynin effectively inhibits the main source of reactive oxygen species (ROS)-nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-by blocking its activation. The aim of this study was to investigate the effect of inhaled apocynin on ROS and RNS (reactive nitrogen species) concentration in 14 nonsmoking mild asthmatics. Effects of nebulized apocynin (0.5 mg/mL) were assessed in exhaled breath condensate (EBC) after 30, 60, and 120 minutes, and safety parameters have been analyzed. Apocynin significantly decreased H2O2 concentration in EBC in comparison with placebo after 60 and 120 minutes. Moreover, apocynin significantly reduced NO(-2) concentration 30 and 60 minutes after nebulization and caused a significant decrease of NO(-3) concentration in EBC 60 and 120 minutes after administration, comparing with placebo. No adverse events have been observed throughout the study. This research confirmed anti-inflammatory properties of nebulized apocynin, which might be an effective and safe drug in bronchial asthma.

Apocynin decreases hydrogen peroxide and nitrate concentrations in exhaled breath in healthy subjects.

The imbalance between reactive oxygen species (ROS) synthesis and antioxidants might be involved in the pathogenesis of many inflammatory diseases. NADPH oxidase, an enzyme responsible for ROS production, may represent an attractive therapeutic target to inhibit, for the treatment of these diseases. Apocynin is an inhibitor of activation of NADPH oxidase complex present in the inflammatory cells. In double blind, placebo-controlled, cross-over study, we investigated the effect of nebulized apocynin on ROS synthesis in 10 nonsmoking healthy volunteers. Apocynin (6ml of 0.5mg/ml) was administered by nebulization and its effects on H(2)O(2), NO(2)(-) and NO(3)(-) generation were assessed after 30, 60 and 120min by collecting exhaled breath condensate (EBC) samples using an EcoScreen analyzer. Additionally, respiratory parameters have been evaluated, utilizing spirometry and DLCO. We also analyzed peripheral blood differential counts and NO(2)(-) serum level, cough scale control and blood pressure as safety parameters. Apocynin caused reduction of H(2)O(2) concentration in EBC as compared to placebo, after 60min. of inhalation (0.18microM vs. 0.31microM, p<0.05) as well as after 120min. (0.2microM vs. 0.31microM, p<0.05). Similarly, apocynin significantly decreased concentration of NO(3)(-) as compared to placebo, after 60 and 120min. (6.8microM vs. 14.4microM and 6.5microM vs. 14.9microM respectively, p<0.05). Apocynin was well tolerated and no adverse events have been observed throughout the study. Thus, as apocynin significantly influence ROS concentration, it might have also antiinflammatory properties. As it is safe, it may have a potential to become a drug in airway inflammatory diseases treatment.

Tumour necrosis factor (TNF-alpha) alpha converting enzyme and soluble TNF-alpha receptor type 1 in psoriasis patients in relation to the chronic alcohol consumption.

BACKGROUND: Tumour necrosis factor alpha converting enzyme (TACE) is a major sheddase of TNF-alpha and its receptors, essential for the generation of soluble, mature molecules. The regulation of the TACE activity by ethanol in vitro has been suggested recently. The alcohol abuse is a frequent problem among psoriasis patients. The aim of the study was to analyse the relationship between long-term alcohol consumption and the concentration of TACE in peripheral blood mononuclear cells (PBMC) and its substrate--soluble TNF-alpha receptor type 1 (sTNF-R1) in plasma in psoriasis patients. METHODS: The study has been conducted among 44 patients (aged 30-59 years) with early-onset, plaque-type psoriasis. Thirty-eight patients (aged 29-61 years) with other than psoriasis chronic dermatologic disorders were controls. The data on alcohol consumption during previous 10 years were obtained with a structured questionnaire. The severity of the disease was assessed using Psoriasis Area and Severity Index (PASI), and concentrations of TACE in PBMC lysate and sTNF-R1 in plasma was assessed with a quantitative sandwich enzyme immunoassay technique. RESULTS: The TACE concentration correlated to that of sTNF-R1 (R = 0.52 in psoriasis patients and R = 0.56 in controls, P < 0.05). The concentrations of TACE were 2.62 +/- 0.32 ng/mL in patients and 1.29 +/- 0.25 ng/mL in controls (P < 0.05), and corresponding sTNF-R1 concentrations were 2.54 +/- 0.27 ng/mL and 1.79 +/- 0.14 ng/mL (P < 0.05), respectively. The concentrations of TACE and sTNF-R1 in patients correlated to the intensity of alcohol consumption (R = 0.56, and R = 0.52, P < 0.05, respectively) and were the highest in excessive drinking psoriasis patients (2.94 +/- 0.34 and 2.67 +/- 0.13 ng/mL). CONCLUSION: The alcohol abuse may contribute to the increase of TACE expression in PBMC and also to the elevated plasma sTNF-R1 concentration in psoriasis patients.

85-kDa cytosolic phospholipase A2 group IValpha gene promoter polymorphisms in patients with severe asthma: a gene expression and case-control study.

Cytosolic phospholipase A(2) (cPLA(2)) group IValpha is a critical enzyme involved in the liberation of arachidonic acid from cellular membranes. cPLA(2)(-/-) mice have reduced allergen-induced bronchoconstriction and bronchial hyperresponsiveness. The goal of this study was to investigate polymorphisms of the (CA)(n) and (T)(n) microsatellites and surrounding regions in the cPLA(2)alpha gene promoter. We analysed the cPLA(2) promoter regions containing (CA)(n) and (T)(n) repeats in 87 patients with severe asthma and in 48 control subjects by bidirectional sequencing. Functional studies were performed utilizing reporter genes derived from subjects with varying numbers of these repeats, and on constructs with a series of deletions. We found that the (CA)(n) and (T)(n) regions are polymorphic and that constructs with CA or T repeats or CA and T repeats deleted revealed, respectively, a 41.8 +/- 7%, 22.3 +/- 5% and 100 +/- 20% increase in reporter gene activity. A lower number of CA or T repeats caused higher cPLA(2) promoter luciferase activity. The group of shorter alleles of the (CA)(n) microsatellite region (n = 12-18) (P(cor) = 0.00006), and the group of shorter alleles of (T)(n) repeats region (n = 17-38) (P(cor) = 0.0039) occurred significantly more often in patients with severe asthma. We also found novel SNPs in positions -292 C > G, -185 A > C, -180 T > C and -165 A > C. Two of them were associated with the severe asthma phenotype: -180T allele (P(cor) = 0.03996) and -185 A allele (P(cor) = 0.03966). These results demonstrate that (CA)(n) and (T)(n) repeats may have an influence on cPLA(2) transcription which might play a role in severe asthma pathogenesis.

Restriction endonucleases that resemble a component of the bacterial DNA repair machinery.

It has long been known that most Type II restriction endonucleases share a conserved core fold and similar active-sites. The same core folding motif is also present in the MutH protein, a component of the bacterial DNA mismatch repair machinery. In contrast to most Type II restriction endonucleases, which assemble into functional dimers and catalyze double-strand breaks, MutH is a monomer and nicks hemimethylated DNA. Recent biochemical and crystallographic studies demonstrate that the restriction enzymes BcnI and MvaI share many additional features with MutH-like proteins, but not with most other restriction endonucleases. The structurally similar monomers all recognize approximately symmetric target sequences asymmetrically. Differential sensitivities to slight substrate asymmetries, which could be altered by protein engineering, determine whether the enzymes catalyze only single-strand nicks or double-strand breaks.

Synthesis and properties of N-methyl and N-phenyl derivatives of 1,4-dioxo-1,2,3,4-tetrahydropyrido[3,4-d]pyridazines.

2-Methoxy- and 2-ethoxy-6-methyl-3,4-pyridinedicarboximides (XI, XII) reacted with N-methylhydrazine giving 2- and 3-methyl derivatives of the appropriate 1,4-dioxo-1,2,3,4-tetrahydropyrido[3,4-d]pyridazines (XV, XIII and XVI, XIV). In both cases 3-methyl isomer (XIII, XIV) was formed in higher yield than 2-methyl derivative (XV, XVI). Reaction of the imide XII with N-phenylhydrazine gave the salts of the suitable N-phenyldihydropyrido[3,4d]pyridazines with NH2-NHC6H5 (XXI and XXII) which transformed into N-phenylaminoimide (XXIII) during the boiling in 80% CH3COOH. Imide XXIII isomerized to the appropriate 2-phenyl and 3-phenylpyrido[3,4-d]pyridazines (XXIV - main reaction product and XXV) under the influence of heating in ethanolic solution of C2H5ONa. Some of N-phenylpiperazinylhydroxyalkyl(alkyl) derivatives of compound XXIV (XXVII, XXVIII) were pharmacologically active.

Synthesis and properties of tetra(hexa)hydropyrazolo [1,2-a]pyrido[3,4-d]pyridazine derivatives.

The synthesis of tetra(hexa)hydropyrazolo[1,2-a]pyrido[3,4-d]pyridazine derivatives (14-21) and the results of pharmacological screening are described in this paper. All compounds tested were non-toxic and showed a significant analgesic action. The analgesic effects were associated with the suppression of the spontaneous locomotor activity. Furthermore most of the synthesized compounds displayed a weak antimycobacterial action in the preliminary screening.

Effect of acute administration of 1,2,3,4-tetrahydroisoquinoline on the levels of glutathione and reactive oxygen species, and on the enzymatic activity of gamma-glutamyl transpeptidase in dopaminergic structures of rat brain.

The effect of acute administration of 1,2,3,4-tetrahydroisoquinoline, an endogenous substance suspected of producing Parkinsonism in humans, on the levels of glutathione and reactive oxygen species and on the enzymatic activity of gamma-glutamyl transpeptidase was investigated in the substantia nigra, striatum and cortex of rat brain. Four hours after a single dose of 1,2,3,4-tetrahydroisoquinoline (100 mg/kg i.p.), a significant increase in tissue glutathione level was found in the dopaminergic structures studied. The most pronounced effect was observed in the substantia nigra and cortex, and the weakest in the striatum. At the same time, significant inhibition of gamma-glutamyl transpeptidase was observed in the substantia nigra, cortex and striatum whose extent strictly corresponded to the increase in glutathione levels in those structures. Moreover, in 1,2,3,4-tetrahydroisoquinoline-treated rats, the production of reactive oxygen species was significantly reduced in the substantia nigra, whereas it was markedly enhanced in the striatum.Our results suggest that the increase in tissue glutathione level in the dopaminergic structures studied results from inhibition of gamma-glutamyl transpeptidase and refers to the extracellular pool of this peptide. Moreover, it is likely that both the 1,2,3,4-tetrahydroisoquinoline-induced alterations in glutathione level and the enhanced production of reactive oxygen species in the striatum may have implications for the pathogenesis of Parkinson's disease.

[Why does nitroglycerin tolerance appear?]. / Dlaczego pojawia sie tolerancja na nitrogliceryne?

Nitroglycerin (NTG) and other organic nitrates are beneficial drugs for the treatment of acute forms of coronary artery disease. The major limitation of organic nitrates use in clinical practice is the rapid development of hemodynamic tolerance. In general mechanisms of tolerance can be categorized into two different groups. One group of mechanisms is related to changes in the vascular smooth muscle bioactivation of organic nitrates. The other mechanisms are related to physiological response to increase nitric oxide (NO) production.

Gravimetric and spectrophotometric determination of some phenothiazine and imidazole derivatives in coated tablets and tablets.

Two drugs in the form of coated tablets: Promazin (promazine hydrochloride) (1) and Thioridazin (thioridazine hydrochloride) (2), and tablets Clotrimazolum (clotrimazole) (3) were assayed gravimetrically and spectrophotometrically in the same process using complexes with ammonium molybdate. Stoichiometry of these complexes was established by elemental analysis and analysis of the incineration residue (MoO3). The complexes were subsequently characterized using their IR and UV spectra and melting points. The active substances of the complexes were also determined spectrophotometrically. Using this method Beers Law was found to hold for the concentration ranges of 5-40 microg/ml (complex of 1), 5-60 (microg/ml (complex of 2) and 2-10 microg/ml (complex of 3). The method was validated in terms of precision, linearity, detection limit and quantification limit. The two methods of drug determination, used in a single analytical process verify each other.

Gravimetric and spectrophotometric determination of some drugs--tertiary amine hydrochlorides in coated tablets.

Four drugs in the form of coated tablets: Amitryptylinum (Amitryptyline) (1), Imipramin (Imipramine) (2), Chloropyribenzaminum (Chloropyramine) (3), and Phenazolinum (Antazoline) (4) were determined gravimetrically and spectrophotometrically in the same process by using complexes with ammonium molybdate. Stoichiometry of the complexes was established by elemental analysis and analysis of the incineration residue (MoO3). The complexes were characterized by IR and UV spectra and melting points. Contents of the drugs in the complexes were also determined spectrophotometrically. In this method Beer's law was found to obey over the concentration ranges 10-80 micrograms/ml (complex of 1), 10-100 micrograms/ml (complexes of 2 and 3), and 10-60 micrograms/ml (complex of 4). This method was validated in terms of precision, linearity, limit of detection and limit of quantitation. The two methods of the drug determination used in a single process of analysis, verify each other.