Liquid chromatography/electrospray ionization tandem mass spectrometry study of repaglinide and its forced degradation products.

RATIONALE: Stress stability studies of drugs have been recognized as an essential part of the drug development process. These studies are used to investigate the intrinsic stability of the drugs and for the development of a selective stability indicating assay method (SIAM). Stress testing is also useful for the formulation and packaging development, shelf-life determination and designing of manufacturing processes. As per regulatory guidelines, stress degradation studies and structural characterization should be carried out to establish degradation pathways of the drug, which is essential from both the efficacy and safety point of view. As the stress stability studies of repaglinide have not been reported in the literature, the present study has been undertaken. METHODS: Repaglinide (RP), an oral anti-diabetic drug, was subjected to hydrolysis (acidic, alkaline and neutral), oxidation, photolysis and thermal stress conditions as per International Conference on Harmonization (ICH) guidelines Q1A (R2). The chromatographic separation of the drug and its degradation products (DPs) was achieved on an Agilent XDB C-18 column using the gradient elution method with a mobile phase consisting of 20 mM ammonium acetate and acetonitrile at flow rate of 1.0 mL min-1 . The DPs were characterized using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with accurate mass measurements. RESULTS: The drug degraded under hydrolytic and oxidative stress, while it was stable under thermal and photolytic stress conditions. In total, six DPs were formed and the LC/MS method described here can resolve all DPs from the parent as well as from each other under various stress conditions. To elucidate the structures of DPs, fragmentation of the [M + H]+ ions of RP and its DPs was studied by using LC/ESI-MS/MS combined with accurate mass measurements. CONCLUSIONS: The forced degradation of RP carried out as per ICH guidelines results in the formation of six degradation products which have been characterized using LC/MS/MS in combination with accurate mass measurements.

Electrospray ionization tandem mass spectrometric study of protonated and alkali- cationized α/ε-hybrid peptides: differentiation of a pair of dipeptide positional isomers.

A new class of Boc-N-protected hybrid peptides derived from L- Ala and ε6-Caa (L-Ala = L-Alanine, Caa = C-linked carboamino acid derived from D-xylose) have been studied by positive ion electrospray ionization (ESI) ion-trap tandem mass spectrometry (MS/MS). MSn spectra of protonated and alkali-cationized hybrid peptides produce characteristic fragmentation involving the peptide backbone, the tert-butyloxycarbonyl (Boc) group, and the side chain. The dipeptide positional isomers are differentiated by the collision-induced dissociation (CID) of the protonated and alkali-cationized peptides. The CID of [M + H]+ ion of Boc-NH-L-Ala-ε-Caa- OCH3 (1) shows a prominent [M + H - C4H8]+ ion, which is totally absent for its positional isomer Boc-NH-ε-Caa-L-Ala-OCH3 (6), which instead shows significant loss of t-butanol. The formation of the [M + Cat - C4H8]+ ion is totally absent and [M + Cat - Boc + H]+ is prominent in the CID of the [M + Cat]+ ion of Boc-NH-L-Ala-ε-Caa- OCH3 (1), whereas the former is highly abundant and the latter is of low abundance for its positional isomer Boc-NH-ε-Caa-L-Ala-OCH3 (6). It is observed that 'b' ions are abundant when oxazolone structures are formed through a five-membered cyclic transition state in tetra-, penta-, and hexapeptides and the cyclization process for larger 'b' ions led to an insignificant abundance. However, the significant 'b' ion is formed in ε,α-dipeptide, which may have a seven-membered substituted 2-oxoazepanium ion structure. The MSn spectra of [M + Cat - Boc + H]+ ions of these peptides are found to be significantly different to those of [M + H - Boc + H]+ ions. The CID spectra of [M + Cat - Boc + H]+ ions of peptide acids containing L-Ala at the C-terminus show an abundant N-terminal rearrangement ion, [bn + 17 + Cat]+, which is absent for the peptide acids containing ε-Caa at the C-terminus. Thus, the results of these hybrid peptides provide sequencing information, the structure of the cyclic intermediate involved in the formation of the rearrangement ion, and distinguish a pair of dipeptide positional isomers.

Nutcracker syndrome in a young female with solitary functional left ovary: a surgical challenge.

Nutcracker syndrome (NCS), a rare clinical entity, when refractory to medical management warrants surgical intervention. In the following discussion, we present a case of NCS which was managed successfully by left renal vein transposition using a decompression shunt.

Electrospray ionization tandem mass spectrometry of protonated and alkali-cationized Boc-N-protected hybrid peptides containing repeats of D-Ala-APyC and APyC-D-Ala: formation of [b(n-1) + OCH3 + Na]+ and [b(n-1) + OH + Na]+ ions.

RATIONALE: Differentiation and structural characterization of positional isomers of non-natural amino acid hybrid peptides by using electrospray ionization tandem mass spectrometry (ESI-MS(n) ) is desirable because of their fundamental importance from the view point of peptide mass spectrometry and also of their increasing importance in the area of research towards biomedical and material applications; hence, the present study is undertaken. METHODS: Electrospray ionization ion-trap tandem mass spectrometry (ESI-MS(n)) was used to characterize and differentiate three pairs of positional isomers of Boc-N-protected hybrid peptides containing repeats of D-Ala-APyC and APyC-D-Ala (D-Ala = D-alanine and APyC = trans-3-aminopyran-2-carboxylic acid). RESULTS: ESI-MS(n) spectra of protonated and alkali-cationized positional isomeric peptides display characteristic fragmentation involving the peptide backbone, the Boc group, and the side chain. It is observed that abundant rearrangement ions [b(n-1) + OCH(3) + Na](+) or [b(n-1) + OH + Na](+) are formed when D-Ala is present at C-terminus and the presence of APyC at the C-terminus inhibits the formation of rearrangement ions. In addition, abundant b(n-1)(+) ions are formed, presumably with stable oxazolone structures, when the C-terminus of b(n-1) (+) ions possessed D-Ala. CONCLUSIONS: The present study demonstrates that ESI tandem mass spectrometry is very useful for differentiating positional isomers of hybrid peptides containing D-Ala and APyC amino acids. While the protonated peptides give rise to characteristic sequencing ions, the cationized peptides produce additional rearrangement ions ([b(n-1) + OCH(3) + Na](+) and [b(n-1) + OH + Na](+)) which helps distinguish between the presence of D-Ala and APyC amino acids at the C-terminus.

SEROLOGICAL DIAGNOSIS OF TOXOPLASMA GONDII INFECTION IN VARIOUS PATIENT POPULATION IN THE ARMED FORCES.

The study included 200 healthy age and sex matched controls (120 healthy volunteers and 80 healthy antenatal cases without any bad obstetric history) and 300 cases comprising patients with bad obstetric history (BOH) 80 cases, seizure or encephalitis 50 cases, cervical lymphadenopathy 30 cases, congenital deformity 30 cases, HIV infected 80 cases and patients on long term immunosuppression 30 cases. Anti-toxoplasma IgG & IgM were measured by ELISA and individuals found positive for anti Toxoplasma gondii IgM or with high titre of IgG were subjected to repeat testing after 3 weeks for rise in titre. True sero-prevalence of T gondii infection was found to be 19% and it increased with increasing age in the population. 2.33% patients were found to have acute toxoplasma infection. Statistical significance was seen only in patients with BOH and children born with congenital abnormality.

An investigation into the effect and mechanisms of action of nicotine in inflammatory bowel disease.

OBJECTIVE AND DESIGN: To determine the effect of nicotine on colonic inflammation in the trinitrobenzenesulphonic acid (TNBS) model of inflammatory bowel disease in comparison with sulphasalazine. MATERIALS: Male Wistar rats were used for the in-vivo and ex-vivo studies. In-vitro studies were performed using human leukemia peripheral blood monocyte cells (THP-1 cells) grown in continuous culture. TREATMENT: Rats were given access to either nicotine (5 or 100 microg/mL) or sulphasalazine (375 microg/mL) in their drinking water for 10 or 2 days respectively before and 3 days after TNBS administration. THP-1 cells were treated with nicotine (10(-14) to 10(-11) M) for 2 h before and after stimulation with 3 microg/mL lipopolysaccharide (LPS). METHODS: Inflammation in the TNBS model was assessed by measuring the tissue myeloperoxidase activity, leukotriene B4 concentration, inducible nitric oxide protein expression, the ex-vivo production of tumour necrosis factor alpha (TNFalpha), macroscopic damage score, plasma corticosterone levels and by a qualitative histological evolution. The effect of nicotine on TNFalpha production in LPS stimulated THP-1 monocyte cells in-vitro was also determined. Statistical comparisons were made using the Mann-Whitney U-test for the macroscopic damage score and an ANOVA for all other parameters. RESULTS: TNBS treated rats given access to 100 microg/mL nicotine in their drinking water had a marked reduction in several of the markers of inflammation compared to control TNBS treated rats, but a greater reduction was found at 5 microg/mL nicotine or 375 microg/mL sulphasalazine, the latter producing comparable reductions in inflammation to the low dose nicotine. Nicotine also caused a significant reduction in TNFalpha release from THP-1 cells. CONCLUSIONS: Nicotine reduced inflammation in the TNBS model of colonic damage confirming the use of nicotine in IBD although the choice of dose requires further investigation. The mechanism of action of nicotine does not involve increased corticosterone levels, but may be a consequence of a reduction in TNFalpha or leukotriene B4 production.

Lipopolysaccharide and silica-stimulated mononuclear cell prostaglandin production in ulcerative colitis.

Basal, lipopolysaccharide (LPS) and silica-stimulated prostaglandin (PG) production were compared between peripheral blood mononuclear cells (PBMNC) from UC patients and healthy subjects (HS). Basal and LPS-stimulated PBMNC PGI2, but not PGE2, production was greater in UC. LPS stimulated both PGE2 and PGI2 by PBMNC from HS and UC patients. Silica stimulated production of both PGs by cells from HS but only PGE2 by cells from UC patients. The differences in responses to silica and LPS may result from differences in activation of NFkappaB or, alternatively, prior sensitisation to one of these agents. That PBMNC PGE2 production is not increased in UC, as it is in Crohn's disease, suggests that there are differences in PBMNC behaviour between these two diseases.

The effect of an inhibitor of matrix metalloproteinases on colonic inflammation in a trinitrobenzenesulphonic acid rat model of inflammatory bowel disease.

BACKGROUND: Recent publications have reported that matrix metalloproteinases (MMPs) are expressed in colonic tissue taken from ulcerative colitis and Crohn's disease patients. AIM: To evaluate the effects of a matrix metalloproteinase inhibitor, marimastat, on colonic inflammation in experimental colitis induced by trinitrobenzenesulphonic acid (TNBS)-ethanol in the rat. METHODS: Rats were dosed (by mouth) for 7 days (b.d.) with either sulphasalazine (50 mg/kg), marimastat (40 mg/kg) or vehicle. TNBS-ethanol was administered rectally on the 4th day of dosing. On the last day of dosing, colons were removed and assessed for inflammation using myeloperoxidase activity, production of soluble TNFalpha (tumour necrosis factor alpha), clinical score and histological assessment. In addition, the bioavailability and effect of marimastat on a range of MMPs were assessed in-vitro. RESULTS: In this study we have confirmed that marimastat is a broad spectrum MMPI with a bioavailability of 5%. TNBS rats dosed with sulphasalazine had a significantly lower (P < 0.05) myeloperoxidase activity, TNFalpha production and a markedly lower clinical score. Similarly, rats dosed with marimastat had a significantly lower (P < 0.05) myeloperoxidase activity and clinical score, but the TNFalpha production was not significantly reduced. CONCLUSIONS: Dosing rats with TNBS-induced colitis using sulphasalazine or marimastat produced a significant reduction in tissue injury and inflammation.

Examination of the metabolic status of rat air pouch inflammatory exudate by high field proton NMR spectroscopy.

High field proton (1H) NMR spectroscopy was employed to investigate the metabolic status of rat air pouch inflammatory exudates obtained subsequent to the induction of inflammation with carrageenan, and the 1H NMR profiles of these fluids were compared and contrasted with those of inflammatory human synovial fluid, rat plasma and human serum. The characteristic biochemical features obtained from 1H NMR analysis of these exudates consisted of (1) substantially elevated levels of lactate (11.40+/-1.46x10-3 mol dm-3 for samples collected at a time point of 24 h post induction) with little or no NMR-detectable glucose, data consistent with a hypoxic environment and consequent anaerobic metabolism in the inflamed air pouch, and (2) high levels of the ketone body 3-d-hydroxybutyrate, providing evidence for an increased utilization of fats for energy by lymphocytes, the predominant leucocytes present in this environment. These phenomena represent a pathological extreme of the abnormal metabolic status of inflammatory human synovial fluids.

Colony stimulating factors regulate nitric oxide and prostaglandin E2 production in rat cartilage chondrocytes.

Colony stimulating factors (CSFs) are now widely used in cancer treatment and immunological disease therapy. Both granulocyte CSF (G-CSF) and granulocyte-macrophage CSF (GM-CSF) are used to increase neutrophil counts in Felty syndrome. In the present study, the effects of macrophage CSF (M-CSF), G-CSF, GM-CSF and interleukin-3 (IL-3) (10 ng/ml) on the production of nitric oxide and prostaglandin E2 (PGE2) by cartilage explants were examined over 24 and 48 h. The effects of these CSFs were also measured in combination with IL-1 beta (10 ng/ml). M-CSF, GM-CSF and IL-3 had no effect on nitrite production. However, both IL-1 beta and G-CSF caused a significant increase (p < 0.05) in nitrite levels at 48 h. NG-L-arginine-methyl-ester was used to inhibit nitrite production induced by G-CSF and this implicated nitric oxide synthase activity. When G-CSF and IL-1 beta were used in a combined treatment, nitrite levels were significantly increased (p < 0.05) at both 24 and 48 h. Both IL-3 alone and in combination with IL-1 beta caused elevated PGE2 production in this model. PGE2 levels were also significantly increased by stimulation with GM-CSF and IL-3 combined with IL-1 beta. These findings demonstrate that GM-CSF, G-CSF and IL-3 may induce changes in the production of inflammatory mediators such nitric oxide and PGE2 in cartilage chondrocytes. Hence, CSFs may play a vital role in influencing cartilage metabolism in rheumatoid and osteoarthritis.

Evaluating the antioxidant potential of new treatments for inflammatory bowel disease using a rat model of colitis.

BACKGROUND: Reactive oxygen species may mediate tissue injury in inflammatory bowel disease. Aminosalicylates have antioxidant activity and the antioxidants, superoxide dismutase and allopurinol, are of reported benefit in inflammatory bowel disease. AIM: To develop a convenient technique for testing the antioxidant potential of standard and novel therapeutic agents for use in inflammatory bowel disease. METHODS: Amplified chemiluminescence was used to measure reactive oxygen species production by colonic biopsy specimens from rats with acetic acid induced colitis and to assess the in vitro effect of conventional antioxidants, standard therapies and proposed novel therapies for inflammatory bowel disease. RESULTS: The model was validated by demonstrating that the profile of effects on chemiluminescence of acetic acid induced colitis biopsy specimens given by conventional antioxidants (sodium azide, catalase, copper-zinc superoxide dismutase, dimethyl sulphoxide, N-acetylcysteine and ascorbate) and standard therapies (5-aminosalicylate and hydrocortisone) resembled that previously reported using biopsy specimens from ulcerative colitis. Human recombinant manganese superoxide dismutase did not alter chemiluminescence. Two novel compounds, LY231617 (10 mM) and amflutizole (20 mM), reduced chemiluminescence by 98% (n = 5, p = 0.009) and 88% (n = 5, p = 0.03), respectively. CONCLUSIONS: The similarity of the chemiluminescence responses of colonic biopsy specimens from acetic acid induced colitis and ulcerative colitis to a range of conventional antioxidants and standard treatments suggests that this model is a useful method for testing the antioxidant potential of new therapies for inflammatory bowel disease. The antioxidant actions of dimethyl sulphoxide, ascorbate, and the novel compounds, amflutizole and LY231617 in this model suggest that these agents merit further assessment in the treatment of inflammatory bowel disease.

Generation of lipid peroxidation products in culinary oils and fats during episodes of thermal stressing: a high field 1H NMR study.

The oxidative deterioration of glycerol-bound polyunsaturated fatty acids (PUFAs) in culinary oils and fats during episodes of heating associated with normal usage (30-90 min at 180 degrees C) has been monitored by high field 1H NMR spectroscopy. Thermal stressing of PUFA-rich culinary oils generated high levels of n-alkanals, trans-2-alkenals, alka-2,4-dienals and 4-hydroxy-trans-2-alkenals via decomposition of their conjugated hydroperoxydiene precursors, whereas only low concentrations of selected aldehydes were produced in oils with a low PUFA content, lard and dripping when subjected to the above heating episodes. Samples of repeatedly used, PUFA-rich culinary oils obtained from restaurants also contained high levels of each class of aldehyde. The dietary, physiological and toxicological ramifications of the results obtained are discussed.

The role of the contractile fibroblast in the capsules around tissue expanders and implants.

The occurrence, structure and contractility of myofibroblasts in the capsules around tissue expanders and static implants has been studied in the rat, pig and humans. The capsules showed a characteristic layered structure with myofibroblasts being the predominant cell type. Capsular strips contract in vitro in a manner characteristic of fibroblast contraction. The contractile ability decreased with the time since expander insertion; and increased with expander exposure, peri-expander infection and clinical evidence of adverse capsular contracture. An hypothesis is proposed that capsular contracture is analogous to wound contraction, and that intraimplant pressure usually inhibits capsular contraction. Evidence is shown from intraexpander pressure measurements to support this hypothesis. The clinical implications for tissue expansion and breast augmentation are discussed.

High resolution proton NMR investigations of rat blood plasma. Assignment of resonances for the molecularly mobile carbohydrate side-chains of 'acute-phase' glycoproteins.

An intense broad resonance at 2.14 ppm present in high field (400, 500 and 600 MHz) Hahn spin-echo 1H-NMR spectra of rat blood plasma, but absent from those of human blood plasma is attributable to the presence of terminal O-acetylsialate sugars in the molecularly mobile carbohydrate side-chains of 'acute-phase' glycoproteins (predominantly alpha 1-acid glycoprotein). The presence of such alternative acetylsugars in the carbohydrate side-chains of rat plasma glycoproteins are of much physiological and experimental significance in view of the regular use of these animals in model systems of human inflammatory conditions.

Assessment of a human recombinant manganese superoxide dismutase in models of inflammation.

We evaluated a novel human recombinant preparation of manganese superoxide dismutase (MnSOD) for anti-inflammatory and anti-oxidant activity compared with a copper zinc (CuZn) SOD preparation. The results showed that administration of MnSOD (50, 100 and 200 micrograms kg-1) in the Freund's Complete Adjuvant (FCA) mediated paw oedema model suppressed the inflammation at 4 hours by 43, 25 and 43% (P < 0.001, P < 0.01 and P < 0.001 at respective doses). However, 24 hours post-challenge, MnSOD (50 and 100 micrograms kg-1), suppressed the inflammation by 19% (P < 0.001). In contrast, Mn SOD at higher doses (400-800 micrograms kg-1; 2 mgkg-1) exacerbated the inflammatory response at 4 hours. This pro-inflammatory response declined progressively by 24 hours. Furthermore, CuZn SOD produced no significant effects on the inflammatory response. In the carrageenan-induced synovitis model, Mn SOD (25 and 50 micrograms; intra-articular administration) exacerbated the inflammation at 48 hours. In contrast, Mn SOD at 5 micrograms produced a significant suppression (44%, P < 0.05) in knee joint swelling at 24 hours. The CuZn SOD preparation produced marked pro-inflammatory effects in the joints whilst it lacked activity in the FCA-mediated paw oedema model. These findings support a therapeutic potential of MnSOD in inflammatory disorders, however the compound has a complex pharmaco-dynamic profile.

Effect of endothelin-1 on croton oil-induced granulation tissue in the rat. A pharmacologic and immunohistochemical study.

BACKGROUND: To date no attempts have been made to determine the role of the endothelial cell derived product, endothelin-1 (ET-1) in granulation tissue development. This study investigates the cellular immunolocalization of ET-1 and its pharmacologic effect on myofibroblast-mediated rat croton oil-induced granulation tissue contraction. EXPERIMENTAL DESIGN: The distribution, cellular localization and temporal production of ET-1 in the tissues was determined by immunohistochemistry at days 7, 14, 21, and 28. The contractile response of the granulation tissue to ET-1 was tested over the same time period, and it effects modified by use of calcium antagonists. The pharmacologic profile was correlated to the ultrastructural development of contractile fibroblast-like cells within the tissue. RESULTS: Endothelin-1 caused reversible concentration-dependent contraction of the granulation tissue. The 21-day granulation tissue was the most responsive, with a maximum increase in tension of 458.9 +/- 41.1 mg; this response could be inhibited by use of calcium antagonists. Of the calcium antagonists tested, verapamil (1 x 10(-4) M) was the most potent inhibitor, giving a 43% reduction in maximum amplitude of the response. It is suggested that entry of extracellular calcium via the L-type potential operated calcium channel, is involved in ET-1 induced responses in contractile fibroblast-like cells or myofibroblasts. Ultrastructural analysis showed a correlation between the pharmacologic sensitivity of the tissue and the development of contractile fibroblast-like cells. The number of cells expressing the phenotypic characteristics of a myofibroblast increased with time, and were first observed at day 7. Immunohistochemistry revealed the presence of increasing numbers of ET-1 labeled cells throughout the time course of study. The ET-1 positive cells were localized to the capillaries. Immunolabeling of serial sections with the rodent endothelial cell specific lectin, Bandeiraea simplicifolia isolectin B4 and factor VIII-related antigen, confirmed the specific localization of ET-1 to endothelial cells. CONCLUSIONS: We present evidence that ET-1 may be an endogenous modulator of myofibroblast-mediated granulation tissue contraction and that the use of calcium antagonists could afford a possible therapeutic control in the treatment of fibrocontractive diseases.