Simultaneous analysis of mono-, di-, and tri-ethanolamine in cosmetic products using liquid chromatography coupled tandem mass spectrometry

Alkanolamines such as monoethanolamine (MEA), diethanolamine (DEA), and triethanolamine (TEA) are used as wetting agents in shampoos, lotions, creams, and other cosmetics. DEA is widely used to provide lather in shampoos and maintain a favorable consistency in lotions and creams. Although DEA is not harmful, it may react with other ingredients in the cosmetic formula after extended storage periods to form an extremely potent carcinogen called nitrosodiethanolamine (NDEA), which is readily absorbed through the skin and has been linked to the development of stomach, esophagus, liver, and bladder cancers. The purpose of this study was to develop a simultaneous quantification method for measurement of MEA, DEA, and TEA in cosmetic products. Liquid chromatography coupled tandem mass spectrometry (LC–MS/MS) was performed using a hydrophilic interaction liquid chromatography (HILIC) column with isocratic elution containing acetonitrile and 5 mM ammonium formate in water (88:12, v/v). Identification and quantification of alkanolamines were performed using MS/MS monitoring to assess the transition from precursor to product ion of MEA (m/z, 61.1 → 44.0), DEA (m/z, 106.1 → 88.0), TEA (m/z, 150.1 → 130.0), and the internal standard triethylamine (m/z, 102.2 → 58.0). Alkanolamines extractions were simplified using a single extraction with acetonitrile in the cosmetic matrix. Performance of the method was evaluated with quality parameters such as specificity, carry-over, linearity and calibration, correlation of determination (R²), detection limit, precision, accuracy, and recovery. Calibration curves of MEA (2.9–1000 ppb), DEA (1–1000 ppb), and TEA (1–1000 ppb) were constructed by plotting concentration versus peak-area ratio (analyte/internal standard with a correlation coefficient greater than 0.99). The intra- and inter-assay accuracy ranged from 92.92 to 101.15 % for all analytes. The intra- and inter-assay precision for MEA, DEA, and TEA showed all coefficients of variance were less than 9.38 % for QC samples. Limits of detection and limits of quantification were 2.00 and 15.63 ppb for MEA, 0.49 and 1.96 ppb for DEA, and 0.49 and 1.96 ppb for TEA, respectively. This novel quantification method simplified sample preparation and allowed accurate and reproducible quantification of alkanolamines in the ng/g cosmetic weight (ppb) range for several cosmetic products.     

Risk assessment of N-nitrosodiethylamine (NDEA) and N-nitrosodiethanolamine (NDELA) in cosmetics

N-nitrosamines and their precursors found in cosmetics may be carcinogenic in humans. Thus the aim of this study was to carry out risk assessment for N-nitrosamines (N-nitrosodiethanolamine [NDELA], N-nitrosodiethylamine [NDEA]) and amines (triethanolamine [TEA], diethanolamine [DEA]) levels in cosmetics determined using validated liquid chromatography–tandem mass spectrometry (LC–MS/MS) procedures. NDELA and NDEA concentrations were present at levels of “not detected” (N.D.) to 596.5 μg/kg and N.D. to 40.9 μg/kg, respectively. TEA and DEA concentrations ranged from N.D. to 860 μg/kg and N.D. to 26.22 μg/kg, respectively. The nitrite concentration (3–2250 mg/l), number of nitrosating agents to a maximum 5, and pH (3.93–10.09) were also assessed. The impact of N-nitrosamine formation on the levels of TEA, DEA, nitrite, and other nitrosating agents was also examined. N-nitrosamine concentrations correlated with the number of nitrosating agents and nitrite concentrations. Data demonstrated that higher nitrite concentrations and a greater number of nitrosating agents increased NDELA and NDEA yields. Further, the presence of TEA and DEA exerted a significant influence on N-nitrosamine formation. Risk assessments, including the margin of exposure (MOE) and lifetime cancer risk (LCR) for N-nitrosamines and margin of safety (MOS) for amines, were calculated using product type, use pattern, and concentrations. Exposure to maximum amounts of NDELA and NDEA resulted in MOE > 10,000 (based upon the benchmark dose lower confidence limit 10%) and LCR <1 × 10^?5, respectively. In addition, TEA and DEA concentrations in cosmetic samples resulted in MOS values >100. Therefore, no apparent safety concerns were associated with cosmetic products containing NDELA, NDEA, TEA, and DEA in this study. However, since amines and nitrosating agents produce carcinogenic nitrosamines, their use in cosmetics needs to be minimized to levels as low as technically feasible.     

Interactions between tetraethylammonium and methohexitone at the chick neuromuscular junction: Experiments with the moving fluid electrode technique

The effect of methohexitone on the depolarization and contracture responses produced by tetraethylammonium (TEA), acetylcholine (ACh) and repetitive indirect stimulation were investigated, using the moving fluid electrode technique, in the chick biventer cervicis (BVC) nerve muscle preparation. TEA (4.8 × 10^?4?4.8 × 10^?2 M) produced contracture and depolarization responses which were concn-dependent. These responses were potentiated by methohexitone (8.8 × 10^?5 M). The mean ED₅₀S for the contracture responses in the control Krebs solution and with methohexitone were (mean ± S.E.M.) 6.5 ± 0.03 × 10^?3 M and 1.3 ± 0.04 × 10^?3 M (N = 6) respectively. The mean ED₅₀S for the depolarizations were (mean ± S.E.M.) 5.9 ± 0.1 × 10^?3 and 1.5 ± 0.06 × 10^?3 M (N = 6) respectively. ACh (5.5 × 10^?6?1.1 × 10^?2 M) produced contracture and depolarization responses which were concn-dependent. These responses were reduced by methohexitone (8.8 × 10^?5 M). The mean ED₅₀S for the contracture responses in the control Krebs solution and with methohexitone were (mean ± S.E.M. 2.4 ± 0.21 × 10^?4 and 2.3 ± 0.1 × 10^?3 M (N = 6) respectively. The mean (± S.E.M.) ED₅₀S for the depolarizations were 8.4 ± 0.33 × 10^?4 and 3.7 ± 0.14 × 10^?3 M (N = 6), respectively. Repetitive indirect stimulation, at 1–20 Hz, produced contraction and depolarization responses which were frequency-dependent. These responses were slightly potentiated by methohexitone (8.8 × 10^?5 M). The mean (± S.E.M.) frequency₅₀S for the contractions produced in the control Krebs solution and with methohexitone were 9.2 ± 0.1 and 8.5 ± 0.2 Hz (N = 6) respectively. The mean frequency₅₀S for the depolarizations were (mean ± S.E.M.) 7.2 ± 0.1 and 5.8 ± 0.19 Hz (N = 6) respectively. It is concluded that TEA may have a direct post-synaptic action, in addition to releasing ACh from the presynaptic nerve terminals. TEA produces more contracture tension than does ACh for a given level of membrane depolarization. Methohexitone, non-competitively, reduces the responses produced by applied ACh whereas it potentiates those produced by TEA and repetitive nerve stimulation.     

DERMAL AND SYSTEMIC TOXICITY AFTER APPLICATION OF SEMISYNTHETIC METAL-WORKING FLUIDS IN B6C3F1 MICE

About 10 million industrial workers of both sexes are exposed to metal-working fluids (MWFs) via inhalation, skin or both. Our preliminary results, following dermal application of 200 µl of 50% unused (neat) semisynthetic MWF (pH 7 or pH 9.7) to the unshaved backs of 6-wk-old B6C3F1 mice, twice a week for 6 wk, produced significant increase in weights of the liver of both sexes. The purpose of the present study was to determine if this weight change was related to oxidative stress subsequent to MWF exposure and also to determine whether ethanol intake influences this effect. Therefore, 6-mo-old mice of both sexes were exposed to MWFs following the protocol just described, except that the topical application was with 5% MWFs (pH 7 and 9.7, 5 d/wk) with or without adding 5% ethanol to their drinking water (7 d/wk) for 13 wk. The skin histamine levels and mast-cell numbers were significantly increased in the female group treated with 5% MWF (pH 7). The ascorbic acid levels in the liver (both sexes) (all groups except 5% MWF pH 9.7 males) and testes were reduced significantly. Malondialdehyde levels in the male liver were significantly increased with topical MWF exposure. Glutathione levels were reduced significantly in both male and female liver after 5% MWF (pH 7). Alcohol dehydrogenase activity of the male liver increased significantly after MWF (pH 7). These results suggest that MWFs are absorbed through the skin and produce toxicity in the liver of both sexes and in the male gonads. This may represent an important health risk to MWF-exposed industrial workers, and ethanol may exacerbate this risk.     

Absorption mechanism of oxymatrine in cultured Madin–Darby canine kidney cell monolayers

Context Oxymatrine (OMT) is beneficial to human health by exerting various biological effects. Objective To investigate the absorption mechanism of OMT and discover absorption enhancers using Madin–Darby canine kidney (MDCK) cell monolayers.

Materials and methods Concentration effects on the transport of OMT were measured in the range of 1.0?×?10^?5–1.0?×?10^?3 M in 2?h. Then, the effect of time, direction, temperature and pH on the transport of OMT at 10^?4 M was studied. Moreover, P_app of OMT was determined in the absence/presence of cyclosporine and surfactants at 100?μM to further confirm the relative transport mechanism.

Results The P_{app AP→BL} ranged from (3.040?±?0.23)?×?10^?6 to (3.697?±?0.19)?×?10^?6?cm/s as the concentration varied from 10^?5 to 10^?3 M. OMT showed similar P_app at 4 and 37?°C (p?>?0.05). Increasing the apical pH 7.4 and 8.0 resulted in P_app versus pH 5.0 (p?<?0.01). Furthermore, in the presence of cyclosporine and surfactants including sodium citrate, sodium dodecyl sulphate (SDS) and deoxysodium cholate, P_app was (0.318?±?0.033)?×?10^?5, (0.464?±?0.048)?×?10^?5, (0.897?±?0.115)?×?10^?5 and (1.341?±?0.122)?×?10^?5?cm/s, respectively. In the presence of surfactants, P_app significantly increased up to 1.5–4.3-fold (p?<?0.05).

Discussion and conclusion OMT transport across MDCK cell monolayers was by passive diffusion. Sodium citrate, SDS and deoxysodium cholate serve as excellent absorption enhancers which are useful for the related research improving the oral bioavailability of OMT.     

Subcutaneous Absorption Kinetics and Local Tissue Distribution of Interferon and Other Solutes

Abstract— The subcutaneous absorption and consequent tissue distribution of interferon g was studied in an anaesthetized rat model. Interferon g showed a biphasic disappearance profile from a solution in a subcutaneous absorption cell. Both the initial rapid distribution phase and slower removal phase followed first order kinetics. The steady-state clearance of interferon g from the cell was 1·41 ± 0·38 × 10^?3 mL min^?1, and the absorption half-life (t½) was 3·8 ± 1·1 h (n = 4). Noradrenaline did not significantly alter either the clearance or absorption of interferon g (1·18 ± 0·44 × 10^?3 mL min^?1 P > 0·05, absorption t½ 4·96 ± 1·9 h, P > 0·05). Given that the clearance of smaller solutes, such as tritiated water, is significantly reduced when noradrenaline is coadministered, it is suggested that interferon g is removed via the lymphatic system rather than by the local blood supply. The amount of interferon g recovered in the plasma, urine and muscle is minimal relative to other solutes where the recovery is almost complete.     

Dermal and systemic toxicity after application of semisynthetic metal-working fluids in B6C3F1 mice

About 10 million industrial workers of both sexes are exposed to metal-working fluids (MWFs) via inhalation, skin or both. Our preliminary results, following dermal application of 200 microl of 50% unused (neat) semisynthetic MWF (pH 7 or pH 9.7) to the unshaved backs of 6-wk-old B6C3F1 mice, twice a week for 6 wk, produced significant increase in weights of the liver of both sexes. The purpose of the present study was to determine if this weight change was related to oxidative stress subsequent to MWF exposure and also to determine whether ethanol intake influences this effect. Therefore, 6-mo-old mice of both sexes were exposed to MWFs following the protocol just described, except that the topical application was with 5% MWFs (pH 7 and 9.7, 5 d/wk) with or without adding 5% ethanol to their drinking water (7 d/wk) for 13 wk. The skin histamine levels and mast-cell numbers were significantly increased in the female group treated with 55% MWF (pH 7). The ascorbic acid levels in the liver (both sexes) (all groups except 5%, MWF pH 9.7 males) and testes were reduced significantly. Malondialdehyde levels in the male liver were significantly increased with topical MWF exposure. Glutathione levels were reduced significantly in both male and female liver after 5% MWF (pH 7). Alcohol dehydrogenase activity of the male liver increased significantly after MWF (pH 7). These results suggest that MWFs are absorbed through the skin and produce toxicity in the liver of both sexes and in the male gonads. This may represent an important health risk to MWF-exposed industrial workers, and ethanol may exacerbate this risk.     

Synthesis and calcium channel modulation effects of isopropyl 1,4‐dihydro‐2,6‐dimethyl‐3‐nitro‐4‐phenylpyridine‐5‐carboxylates possessing ortho‐, meta‐, and para‐CH2S(O)nMe and –S(O)nMe (n = 0–2) phenyl substituents

A group of isopropyl 1,4‐dihydro‐2,6‐dimethyl‐3‐nitro‐4‐phenylpyridine‐5‐carboxylates ( 13–15 ) possessing ortho‐, meta‐, and para‐CH₂S(O)nMe and –S(O)nMe (n = 0–2) phenyl substituents were synthesized using a modified Hantzsch reaction. Calcium channel (CC) modulating activities were determined using guinea pig ileum longitudinal smooth muscle (GPILSM) and guinea pig left atrium (GPLA) in vitro assays. This class of –CH₂S(O)nMe and –S(O)nMe (n = 0–2) compounds ( 13–15a–f ) exhibited weaker CC antagonist activity on GPILSM (IC₅₀ = > 1.1 × 10^–5 to 4.1 × 10^–6 M range) than the reference drug nifedipine (IC₅₀ = 1.4 × 10^–8 M). The oxidation state of the sulfur atom was a determinant of smooth muscle CC antagonist activity where the relative activity profile was generally thio ( 13 , ‐CH₂SMe, ‐SMe) and sulfonyl ( 15 , ‐CH₂SO₂Me, ‐SO₂Me) > sulfinyl ( 14 , ‐CH₂SOMe, ‐SOMe). The point of attachment of the phenyl substituent was a determinant of activity for the –CH₂SMe ( 13a–c ), ‐CH₂SOMe ( 14a–c ) and SOMe ( 14d–f ) isomers where the relative potency order was meta and para > ortho. Compounds in this group ( 13–15 ), unlike Bay K 8644 (EC₅₀ = 2.3 × 10^–7 M on GPILSM), did not exhibit an agonist effect on GPILSM. The meta‐CH₂SMe ( 13b ), ortho‐CH₂SMe ( 13c ), meta‐SMe ( 13e ), and ortho‐CH₂SO₂Me ( 15c ) C‐4 phenyl derivatives exhibited respectable in vitro cardiac positive inotropic activities (EC₅₀ = 1.00 × 10^–6 to 7.57 × 10^–6 M range) relative to the reference drug Bay K 8644 (EC₅₀ = 7.70 × 10^–7 M) in the GPLA assay. In contrast to Bay K 8644, which acts as an undesirable calcium channel agonist on smooth muscle (GPILSM), compounds 13b (IC₅₀ = 4.11 × 10^–6 M), 13c (IC₅₀ = 2.29 × 10^–5 M), 13e (IC₅₀ = > 1.20 × 10^–5 M) and 15c (IC₅₀ = 6.22 × 10^–6 M) exhibited a desirable simultaneous calcium channel antagonist effect on smooth muscle at a similar ( 13b , 15c ), or lower ( 13c , 13e ), concentration relative to its cardiac agonist EC₅₀ value. Model compounds such as 13b , 13c , 13e , and 15c , that exhibit dual cardioselective agonist / smooth muscle selective antagonist activities, represent a novel type of 1,4‐dihydropyridine CC modulators that offer a potential approach to drug discovery targeted toward the treatment of congestive heart failure and for use as probes to study the structure–function relationship of calcium channels. Drug Dev. Res. 51:177–186, 2000. © 2001 Wiley‐Liss, Inc.     

Biological evaluation of selected 3,4‐dihydro‐2(1H)‐quinoxalinones and 3,4‐dihydro‐1,4‐benzoxazin‐2‐ones: Molecular docking study

In order to investigate new potential therapeutically active agents, we investigated the biological properties of two small libraries of quinoxalinones and 1,4‐benzoxazin‐2‐ones. The results obtained showed that compounds 5 , 9–11 have good cytotoxic activity against HeLa cells where the lowest IC₅₀ value (10.46 ± 0.82 μM/mL) was measured for compound 10 . Additionally, the most active compounds ( 5 , 9 – 11 ) showed much better selectivity for MRC‐5 cells (up to 17.4) compared to cisplatin. In vitro evaluation of the inhibition of the enzyme α‐glucosidase showed that compounds 10 and 11 exert significant inhibition of the enzyme at 52.54 ± 0.09 and 40.09 ± 0.49 μM, respectively. Competitive experiments with ethidium bromide (EB) indicated that all tested compounds have affinity to displace EB from the EB‐DNA complex through intercalation, suggesting good competition with EB (K_sv = (3.1 ± 0.2), (5.1 ± 0.1), (5.6 ± 0.2), and (6.3 ± 0.2) × 10³ M^?1). A molecular docking study was also performed to better understand the binding modes and to conclude the structure–activity relationships of the synthesized compounds.

     

Cardiovascular effects elicited by milonine, a new 8,14-dihydromorphinandienone alkaloid

Abstract: The mechanisms underlying the cardiovascular responses evoked by milonine (i.v.), an alkaloid, were investigated in rats. In normotensive rats, milonine injections produced hypotension and tachycardia, which were attenuated after N^w‐nitro‐l ‐arginine methyl esther (l ‐NAME; 20 mg/kg, i.v.). In phenylephrine (10 μM), pre‐contracted mesenteric artery rings, milonine (10^?10 M to 3 × 10^?4 M) caused a concentration‐dependent relaxation (EC₅₀ = 1.1 × 10^?6 M, E_max = 100 ± 0.0%) and this effect was rightward shifted after either removal of the vascular endothelium (EC₅₀ = 1.6 × 10^?5, p < 0.001), or after l ‐NAME 100 μM (EC₅₀ = 6.2 × 10^?5, p < 0.001), hydroxocobalamin 30 μM (EC₅₀ = 1.1 × 10^?4, p < 0.001) or ODQ 10 μM (EC₅₀ = 1.9 × 10^?4p < 0.001). In addition, in rabbit aortic endothelial cells, milonine increased NO₃^? levels. The relaxant effect induced by milonine was attenuated in the presence of KCl (20 mM), a modulator efflux K⁺ (EC₅₀ = 1.2 × 10^?5, p < 0.001), or different potassium channel blockers such as glibenclamide (10 μM) (EC₅₀ = 6.3 × 10^?5, p < 0.001), TEA (1 mM) (EC₅₀ = 2.3 × 10^?5 M, n = 6) or Charybdotoxin (0.2 μM) plus apamin (0.2 μM) (EC₅₀ = 3.9 × 10^?4 M, n = 7). In addition, pre‐contraction with high extracellular potassium concentration prevented milonine‐induced vasorelaxation (EC₅₀ = 1.0 × 10^?4, p < 0.001). Milonine also reduced CaCl₂‐induced contraction in Ca²⁺‐free solution containing KCl (60 mM). In conclusion, using combined functional and biochemical approaches, we demonstrated that the hypotensive and vasorelaxant effects produced by milonine are, at least in part, mediated by the endothelium, likely via nitric oxide release, activation of nitric oxide‐cGMP pathway and opening of K⁺ channels.     

Mechanistic study of absorption and first‐pass metabolism of GL‐V9, a derivative of wogonin

GL‐V9, a derivative of wogonin, has potent anti‐cancer activity. The absorption and metabolism of this compound have not been investigated systematically. This study aims to illustrate the pharmacokinetic characters of GL‐V9 by exploring its metabolic status under different administration routes. To further clarify the absorption mechanism of GL‐V9, an in situ single‐pass perfusion model and a Caco‐2 cell monolayer model were used. Meanwhile, a microsomal incubation system was used to evaluate the enzyme kinetic parameters. In vivo, the obtained gastrointestinal availability (F_a × F_g) was 21.28 ± 5.38%. The unmetabolized fraction in the gut wall (F_{gut wall}) was 98.59 ± 9.74%, while the hepatic bioavailability (F_h) was 29.11 ± 5.22%. These results indicated that poor absorption and extensive metabolism may contribute greatly to the low bioavailability of GL‐V9. The effective permeability (P_eff) in the duodenum and jejunum was 1.34 ± 0.50 × 10^?4 and 0.90 ± 0.27 × 10^?4 cm/s, respectively. The high permeability of GL‐V9 indicated that other unknown factors (such as metabolism) may account for its systemic exposure problem. Studies in rat liver microsomal (RLMs) confirmed this hypothesis, and the Cl_{int, CYP450s and UGT} of GL‐V9 was 0.20 ml/min/mg protein. In conclusion, these results suggest that GL‐V9 possesses higher permeability than wogonin and the metabolism of GL‐V9 is related to its disposition in rat intestine and liver.     

角质层及其类脂对5-氟脲嘧啶经皮渗透的作用

采用离体皮肤扩散等技术研究了5-Fu在人皮肤各层次中的渗透性质和1,8-CN对药物渗透及皮肤热转变的影响。实验表明5-Fu在皮肤各层次中有相近分配系数,但扩散性质不同。角质层、全皮层、脱脂角质层和去角质层全皮扩散系数依次为1.32×10^-7,1.01×10^-7,1.37×10^-6和54.09×10^-6cm²/h。用1,8-CN处理上述皮肤样品12h后,5-Fu在各组织的分配均减少,角质层和全皮的通透性显著增加(P<0.05),对去脂角质层和去角质层全皮的通透性无明显影响。结合DSC分析证明,角质层是5-Fu经皮渗透的重要屏障,类脂对5-Fu的扩散和1,8-CN的增效具重要意义。     

Single and mixed chemically modified carbon paste ion‐selective electrodes for determination of ketotifen fumarate

New modified carbon paste electrodes for determination of ketotifen fumarate in its pure and pharmaceutical preparations were constructed. The used modifiers are ketotifen phosphotungestate (Keto₃PT), and ketotifen tetraphenylborate (Keto‐TPB). Single and mixed ion‐associate electrodes were prepared. Both Keto‐TPB and mixed (Keto‐TPB and Keto₃PT) electrodes have a linearity range of 1.00 × 10^?5‐1.00 × 10^?2 mol L^?1. The slopes were 58.30 and 54.20 mV/decade for Keto‐TPB and mixed chemically modified carbon paste electrodes (CMCPE), respectively. The limits of detection were 1.42 × 10^?6 and 1.00 × 10^?5 mol L^?1 for Keto‐TPB and mixed CMCPEs, respectively. The potential variation due to pH change is considered acceptable in the pH ranges 4.44–9.11 and 2.50–9.00 for Keto‐TPB and mixed ion‐exchanger CMCPE, respectively. The response time was ≤10 s for both electrodes. Selectivity coefficients values towards different inorganic cations, sugars, and amino acids reflect high selectivity of the prepared electrodes. Potentiometric titrations and standard addition methods were applied for the determination of ketotifen ion in its pure samples and pharmaceutical formulations (Zaditen tablet and syrup) using proposed electrodes. The electrodes were also tested in flow injection analysis (FIA). The results obtained from both methods were statistically treated by F‐ and t‐tests. The carbon paste electrodes have the advantages of being more easily prepared and longer life span compared to the plastic membrane electrodes previously reported. Copyright © 2011 John Wiley & Sons, Ltd.     

NKH477: A novel bronchodilator produces potentiation and tachyphylaxis reversal to salbutamol in isolated guinea pig trachea

The relaxant responses of the β₂‐adrenoceptor agonist salbutamol and NKH477, an activator of adenylate cyclase, were compared and the possible potentiating effect of NKH477 on salbutamol‐induced bronchodilatation was measured together with the effectiveness of NKH477 in reversing tachyphylaxis development to salbutamol. The in vitro bronchodilator effect of salbutamol and NKH477 (10^–9 – 10^–5 M) was measured on isolated guinea pig tracheal ring segments precontracted with carbachol (10^–6 M). Both salbutamol and NKH477 produced a concentration‐dependent relaxation. EC₅₀ values were determined from cumulative concentration–response curves. Salbutamol was more potent than NKH477 in relaxing the tracheal preparations (7.1 ± 0.1 compared to 6.1 ± 0.2, respectively). NKH477 produced a significant increase in the salbutamol‐induced bronchodilator effect. The potency values recorded for salbutamol were 7.1 ± 0.1, 7.4 ± 0.2, 7.6 ± 0.1, and 8.6 ± 0.4 in the absence and presence of NKH477 (3 × 10^–8 M, 10^–7 M, and 3 × 10^–7 M, respectively). Reproducible relaxant responses could be elicited to salbutamol and NKH477 after 24 h incubation in Krebs' solution. Tachyphylaxis to the relaxant effects of salbutamol was experimentally induced by 24‐h incubation of the preparations in Krebs' solution containing salbutamol (10^–6, 3 × 10^–6 or 10^–5 M). The potency of salbutamol was reduced to 6.9 ± 0.2, 6.8 ± 0.2, and 6.0 ± 0.2 after 24 h incubation with salbutamol 10^–6 M, 3 × 10^–6 M or 10^–5 M, respectively. NKH477 (3 × 10^–7 M) produced a complete reversal of tachyphylaxis to salbutamol‐induced relaxation in salbutamol pretreated tissues. The potency of salbutamol was increased to 7.4 ± 0.2, 7.1 ± 0.1, and 7.3 ± 0.1 after the addition of NKH477 (3 × 10^–7 M) to the preparations preincubated (24 h) with salbutamol 10^–6, 3 × 10^–6, or 10^–5 M, respectively. NKH477 shares with salbutamol the ability to relax airway smooth muscle and produces an apparent increase of the bronchodilator effects of salbutamol and reverses tachyphylaxis. Drug Dev. Res. 48:154–159, 1999. © 1999 Wiley‐Liss, Inc.     

Probing the binding of the coumarin drugs using peptide fragments of DNA gyrase B protein

Abstract: Bacterial DNA gyrase, has been identified as the target of several antibacterial agents, including the coumarin drugs. The coumarins inhibit the gyrase action by competitive binding to the ATP‐binding site of DNA gyrase B (GyrB) protein. The high in vitro inhibitory potency of coumarins against DNA gyrase reactions has raised interest in studies on coumarin–gyrase interactions. In this context, a series of low‐molecular weight peptides, including the coumarin resistance‐determining region of subunit B of Escherichia coli gyrase, has been designed and synthesized. The first peptide model was built using the natural fragment 131–146 of GyrB and was able to bind to novobiocin (K_a = 1.8 ± 0.2 × 10⁵/m ) and ATP (K_a = 1.9 ± 0.4 × 10³/m ). To build the other sequences, changes in the Arg¹³⁶ residue were introduced so that the binding to the drug was progressively reduced with the hydrophobicity of this residue (K_a = 1.3 ± 0.1 × 10⁵/m and 1.0 ± 0.2 × 10⁵/m for Ser and His, respectively). No binding was observed for the change Arg¹³⁶ to Leu. In contrast, the binding to ATP was not altered, independently of the changes promoted. On the contrary, for peptide–coumarin and peptide–ATP complexes, Mg²⁺ appears to modulate the binding process. Our results demonstrate the crucial role of Arg¹³⁶ residue for the stability of coumarin–gyrase complex as well as suggest a different binding site for ATP and in both cases the interactions are mediated by magnesium ions.     

The flavonolignan‐silymarin protects enzymatic,hematological, and immune system against γ‐radiation‐induced toxicity

The main focus of this study is evaluation of radioprotective efficacy of silymarin, a flavonolignan, against γ‐radiation‐induced damage to hematological, vital organs (liver and intestine), and immune system. Survival studies revealed that silymarin (administered orally for 3 days) provided maximum protection (67%) at 70 mg/kg body weight (b.wt.) against lethal 9 Gy γ‐irradiation (dose reduction factor = 1.27). The study revealed significant (p < 0.05) changes in levels of catalase (12.57 ± 2.58 to 30.24 ± 4.89 units), glutathione peroxidase (6.23 ± 2.95 to 13.26 ± 1.36 µg of reduced glutathione consumed/min/mg protein), glutathione reductase (0.25 ± 5.6 to 11.65 ± 2.83 pM NADPH consumed/min/mg protein), and superoxide dismutase (11.74 ± 0.2 to 16.09 ± 3.47 SOD U/mg of protein) activity at 30th day. Silymarin pretreated irradiated group exhibited increased proliferation in erythrocyte count (1.76 ± 0.41 × 10⁶ to 9.25 ± 0.24 × 10⁶), hemoglobin (2.15 ± 0.48g/dL to 14.77 ± 0.25g/dL), hematocrit (4.55 ± 0.24% to 37.22 ± 0.21%), and total leucocyte count (1.4 ± 0.15 × 10⁶ to 8.31 ± 0.47 × 10⁶) as compared with radiation control group on 15th day. An increase in CD4:CD8 ratio was witnessed (0.2–1%) at 30th day time interval using flow cytometry. Silymarin also countered radiation‐induced decrease (p < 0.05) in regulatory T‐cells (T_regs) (11.23% in radiation group at 7th day versus 0.1% in pretreated silymarin irradiated group at 15th day). The results of this study indicate that flavonolignan‐silymarin protects enzymatic, hematological, and immune system against γ‐radiation‐induced toxicity and might prove useful in management of nuclear and radiological emergencies. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 641–654, 2016.     

Biopharmaceutics classification and intestinal absorption of chikusetsusaponin IVa

The objective of this study was to investigate the absorption behavior of chikusetsusaponin IVa (CHS‐IVa) in the rat intestine using single‐pass intestinal perfusion (SPIP) and to classify CHS‐IVa into the biopharmaceutics classification system (BCS). The equilibrium solubility of CHS‐IVa was determined by the shaker method. The absorption mechanism of CHS‐IVa in the intestine was studied by comparing the P_eff of different concentrations of CHS‐IVa. The intestinal site dependence of CHS‐IVa absorption was studied by comparing the P_eff of the same concentration of CHS‐IVa in different intestinal segments. The relationship between CHS‐IVa and intestinal efflux protein was studied by perfusion with an efflux protein inhibitor. The permeability of CHS‐IVa was investigated by comparing the P_eff of CHS‐IVa and the reported value. The solubility of CHS‐IVa over the pH range 1.0–7.5 was 14.4 ± 0.29 to 16.9 ± 0.34 mg/ml. The P_eff of CHS‐IVa in the duodenum was 1.76 × 10^?3 to 2.00 × 10^?3 cm/min. The P_eff of CHS‐IVa in the jejunum was 1.26 × 10^?3 to 1.39 × 10^?3 cm/min. The P_eff of CHS‐IVa in the ileum was 1.25 × 10^?3 to 1.31 × 10^?3 cm/min. The P_eff of CHS‐IVa in the colon was 1.02 × 10^?3 to 1.08 × 10^?3 cm/min. There was no statistical difference of the P_eff in the four segments at different CHS‐IVa concentrations. The P_eff of CHS‐IVa (0.07, 0.7 and 7.0 mg/ml) were all notably smaller than the reported P_eff (3.00 × 10^?3 cm/min) in the jejunum. The P_eff of CHS‐IVa was not influenced by verapamil (P‐gp inhibitor), indomethacin (MRP inhibitor) and pantoprazole (BCRP inhibitor). CHS‐IVa was classified as high solubility, low permeability and BCS III. The main absorptive tracts were the upper intestinal tracts and the rank order of intestinal permeability was duodenum > jejunum ≈ ileum > colon. The transport mechanism of CHS‐IVa in all intestinal segments might be primarily passive transport. CHS‐IVa was not a substrate of P‐gp, MRP and BCRP.     

Effect of counter-ions and penetration enhancers on the skin permeation of flurbiprofen

The aim of this work was to investigate the percutaneous absorption of flurbiprofen (FP) using counter-ions as enhancers as well as to compare their enhancing activity with penetration enhancers in vitro. The in vitro permeation studies of FP were performed in isopropyl myristate (IPM) solution by two-chamber diffusion cells, using rat abdominal skin as a model. Among the penetration enhancers examined, including the cosolvents of propylene glycol and ethanol (EtOH), oleic acid, menthol, laurocapram, sorbitan monooleate, and N-methyl-2-pyrrolidone (NMP), 10% (w/w) EtOH and NMP exhibited the most potent solubilization and enhancing effects of FP from IPM, with a flux of (372.60 ± 45.12) µg/cm²/h and (474.21 ± 46.64) µg/cm²/h, respectively. Ten percent (w/w) EtOH/IPM binary system was used to investigate the effect of the counter-ions, namely diethylamine (DEA), triethylamine (TEA), ethanolamine (EtA), diethanolamine (DEtA), triethanolamine (TEtA), and N-(2′-hydroxyethanol)-piperdine (HEPP). The cumulative amounts were markedly increased in the presence of the counter-ions, and the highest flux of (1297.53 ± 121.81) µg/cm²/h was obtained by DEA. This was related to the decreased lipophilicity and different physicochemical properties of the ion-pairs. In particular, we proved the formation of an FP/amine ion-pair in solution by ¹H-NMR. The results suggest that the counter-ions are more efficient than penetration enhancers. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 1826–1837, 2010     

Pharmacokinetics of 14C‐ortho‐phenylphenol following intravenous administration in pigs

Workers in the USA are exposed to industrial formulations, which may be toxic. These formulations often contain preservatives or biocides such as ortho‐phenylphenol (OPP). There are limited data describing OPP following intravenous administration to assess truly the clearance of this chemical in humans and other species. In vivo experiments were conducted in pigs to determine related pharmacokinetic parameters. ¹⁴C‐OPP was administered as an intravenous bolus dose. Blood, feces, urine and tissue samples were collected for analysis by liquid scintillation. Data were analyzed using non‐compartmental and compartmental pharmacokinetic model approaches. These data fitted a three‐compartment model and showed that the half‐life of ¹⁴C‐OPP following the intravenous bolus in pigs was 46.26 ± 10.01 h. The kidneys play a crucial role in clearance of ¹⁴C‐OPP with a large percentage of the dose being found in the urine (70.3 ± 6.9% dose). Comparisons with other species suggest that ¹⁴C‐OPP clearance in pigs (2.48 ml h^–1 kg^–1) is less than that in humans (18.87 ml h^–1 kg^–1) and rats (35.51 ml h^–1 kg^–1). Copyright © 2016 John Wiley & Sons, Ltd.     

新类型酸性色素定量胺类化合物的研究:专利蓝Ⅴ测定胺类药物

前报认为此新类型蓝色酸性色素的基本构造为二氨基三苯甲烷磺酸,据此,找出了另一种同类型的酸性色素——专利蓝Ⅴ(PBV)。对它进行了配合物的吸收峰、试剂空白的降低、试剂浓度的选定、最佳pH值的选择、检出限、检量线、色素与两种胺的分子配合比、PBV测定法的精密度、五种胺配合物的吸收系数比较等研究,证明它与二甲苯蓝、羊毛罂粟蓝有类似的性质以及同样的灵敏度。进一步说明所认定的此类型的基本构造是对的。