Microdialysis as a Tool for Drug Quantification in the Bronchioles of Anaesthetized Pigs

Target site drug determinations are crucial for optimizing treatment of infectious diseases. There is limited knowledge of antibiotic drug penetration into the pulmonary epithelial lining fluid (PELF) and a lack of easily performed methods for continuous drug sampling hereof. The aim of this study was to develop a readily accessible microdialysis (MD) method for antibiotic drug quantification in PELF of pigs. The fluoroquinolone danofloxacin was administered to anaesthetized pigs allocated to three groups: intravenous injection, intravenous infusion and intramuscular injection. MD probes were guided through a tracheostomy into the distal bronchioles using an insertion tube. Intravenously administered inulin served as a marker of extracellular fluid contamination of PELF. Concentrations of free drug in MD fractions were compared to total and non‐protein‐bound drug concentrations in plasma. Rising and declining danofloxacin plasma concentrations were rapidly reflected in PELF, suggesting an efficient drug transport across the blood bronchial barrier. The AUC _{FREE DRUG PELF}/AUC _{FREE DRUG PLASMA} ratio was 1.8 (S.D. 0.4, 95% CL 1.4–2.3). Although the probes were placed without fiberscopic or other special equipment, the danofloxacin concentrations in PELF were consistent within the different administration groups. The described MD method for drug quantification in PELF is easily accessible and provides repeatable results. However, trace amount of inulin was detected in the MD fractions, suggesting a local tissue reaction induced by the MD membrane. The significance of this finding needs to be clarified in future studies.     

Penetration of minocycline into lung tissues.

The penetration of minocycline into different lung tissues and bronchial mucus was studied in 17 patients undergoing pulmonary surgery for cancer. The patients received oral minocycline 100 mg at night for 3 days preceding surgery. Minocycline concentrations were measured in plasma samples collected before the operation and in tissues and mucus taken from in and around the part of the lung that was surgically removed. Mean tissue or mucus concentration to plasma concentration ratios were 3.78 +/- 1.10 for lung parenchyma, 4.04 +/- 1.31 for bronchial walls, 3.37 +/- 1.00 for pulmonary arterial walls, 1.99 +/- 1.80 for intraluminal mucus collected from bronchi located in healthy tissue proximal to the tumour, 5.16 +/- 3.26 for intraluminal mucus collected in a bronchus distal to the tumour, and 3.06 +/- 1.99 for catheter-collected mucus from the trachea and principal bronchi. These results indicate that minocycline is found in high concentration in all types of lung tissue and mucus in man.     

REGIONAL DIFFERENCES IN THE MODULATORY ROLE OF THE EPITHELIUM IN SHEEP AIRWAY

• 1 The airway epithelium may modulate smooth muscle responsiveness via the release of biologically active substances, such as nitric oxide (NO) and prostaglandins. Based on regional differences in structure and function described for the airway epithelium, we performed a comparative study on the responsiveness of sheep isolated, epithelium‐intact or ‐denuded, first‐ to fourth‐order bronchi to acetylcholine (ACh).
• 2 We performed contractility studies using KCl or cholinergic stimuli in the presence or absence of NO or prostaglandin‐related drugs in epithelium‐intact and epithelium‐denuded bronchial strips obtained from all four airway regions. We also studied the expression of NO synthase (NOS), using the NADPH‐diaphorase staining technique, and the effect of airway epithelium removal on the synthesis of NO metabolites in the different bronchi orders.
• 3 There was no difference in the response of first‐ to fourth‐order epithelium‐intact bronchi to ACh (1 nmol/L–100 mmol/L) or KCl (5–100 mmol/L). Removal of the epithelium had no effect on ACh‐induced contractions of first‐ and second‐order bronchi, but increased responses of third‐ and fourth‐order bronchi to ACh. The NO synthase inhibitor N^G‐nitro‐l‐ arginine methyl ester (100 µmol/L) increased ACh‐induced contractions of fourth‐order epithelium‐intact bronchi only. The NO donor sodium nitroprusside (1 nmol/L–1 mmol/L) equally relaxed 1 µmol/L carbachol‐precontracted epithelium‐denuded first‐ and fourth‐order bronchi.
• 4 Although NAPDH–diaphorase staining demonstrated no regional differences in NOS expression, basal levels of NO metabolites were 4.5‐fold greater in fourth‐ compared with second‐order epithelium‐intact bronchi.
• 5 The cyclo‐oxygenase inhibitor indomethacin (10 µmol/L) had no effect on ACh‐induced contractions of first‐ to fourth‐order epithelium‐intact bronchi, but decreased responses of fourth‐order epithelium‐denuded bronchi to ACh. The contractile effect of the thromboxane A₂ mimetic U‐46619 (1 nmol/L–10 µmol/L) was greater in fourth‐ compared with first‐order epithelium‐denuded bronchi.
• 6 In conclusion, the sheep airway epithelium exhibits regional differences in its modulatory role and this is particularly apparent in small bronchi.

     

Epithelium-derived inhibitory factor in human bronchus

The potencies of histamine and methacholine were significantly increased by approximately 2- and 5-fold respectively in human non-diseased isolated bronchi on removal of the epithelium. In contrast, no increases in spasmogen potency were observed following epithelium removal in bronchi obtained from a sample of asthmatic human lung. The failure of epithelium removal to increase asthmatic bronchial sensitivity to histamine may have been due to a reduction in the release of an epithelium-derived inhibitory factor (EpDIF) resulting from disease-induced epithelial damage. A co-axial bioassay system in which endothelium-denuded rat aorta was used as the assay tissue was used to detect the release of a vasorelaxant EpDIF from human bronchial tissue. Histamine (100 microM) and methacholine (25 microM), in the presence of indomethacin (5 microM), reduced phenylephrine-induced tone in endothelium-denuded rat aorta in co-axial assemblies by 75 +/- 11 and 67 +/- 9% respectively. Removal of the bronchial epithelium abolished these responses, indicating that they were mediated by an EpDIF. It is possible that human airway smooth muscle is sensitive to this vasorelaxant EpDIF and that the absence of the source of this factor following epithelium removal caused the increases in sensitivity to spasmogens. Alternatively, the human bronchial epithelium may also release an EpDIF selective for airway smooth muscle.     

HISTAMINE AND CARBACHOL CONTRACTILE RESPONSES IN PROXIMAL AND DISTAL AIRWAYS OF THE RABBIT

Airway preparations from the trachea, main bronchi, subsegmental bronchi and the parenchyma of rabbits were studied. The proximal airways, the trachea and main bronchi, were less sensitive to histamine than the distal airways of the subsegmental bronchi and lung parenchymal strip. The proximal airways were more sensitive to carbachol than the distal airways. These results may assist in the interpretation of airway responses in vivo.     

Evaluation of ofloxacin penetration into the skin after a single oral dose assessed by cutaneous microdialysis

An antibacterial drug can exert its therapeutic action if it is present in target tissue at proper concentration. Cutaneous microdialysis is a relatively new technique, which allows to determine drug concentration in the skin. The aim of the study was to evaluate the ofloxacin concentrations in plasma and skin following a single oral dose of 0.4 g. Drug concentration in the skin was assessed by applying cutaneous microdialysis. The penetration of the studied agent into dermal microdialysate was compared with its penetration into theoretical peripheral compartment. Maximum ofloxacin concentration in plasma was 9.26 micromol/l on average and was achieved after about 1.7 h. Mean peak concentrations in cutaneous microdialysate and in theoretical peripheral compartment were comparable (4.16 versus 4.50 micromol/l), but time to peak concentration in theoretical peripheral compartment was significantly longer than in microdialysates (5.8 and 2.0 h, respectively). Degree of penetration into cutaneous microdialysate was about 0.54. Cutaneous microdialysis seems to be a valuable technique to evaluate drug penetration into the skin.     

RESPONSIVENESS AND SENSITIVITY TO CHOLINERGIC AGONISTS AND ANTAGONISTS IN BOVINE ISOLATED BRONCHIAL MUSCLE

1. Airways derived from different levels of the lung exhibit a difference in the reactivity and sensitivity to agonists. We have evaluated the effect of acetylcholine and cholinergic selective (pirenzepine, gallamine and 4-dipherylacetoxymethyl piperidine [4-DAMP]) and non-selective (atropine) antagonists on bovine proximal and distal smooth muscle preparations. 2. The distal preparations are more sensitive to acetylcholine than proximal bronchi. The relaxant effect of three selective antagonists on the distal and proximal tissues was the same when the results for each drug were compared. 3. Atropine and 4-DAMP were more potent than pirenzepine and gallamine in relaxing both proximal and distal bovine smooth muscle preparations. 4. These data suggest that the muscarinic sites on the smooth muscle of bovine airways are of the M3 subtype.     

Does Bosentan Protect Diabetic Brain Alterations in Rats? The Role of Endothelin‐1 in the Diabetic Brain

Diabetes mellitus (DM) is a major problem all over the world, affecting more people in recent years. Individuals with diabetes are more prone to disease than non‐diabetics, especially vascular complications. The aim of this study was to examine the roles of the endothelin (ET)‐1 in brain damage formed in a streptozocin (STZ)‐induced diabetes model, and the effect of bosentan, which is the non‐specific ET1 receptor blocker in the prevention of the diabetes‐induced brain damage. To examine the effects of bosentan (50 mg/kg and 100 mg/kg) in this study, the rats were given the drug for 3 months. The rats were divided into four groups: the sham group (n = 10), the diabetic control group (n = 10), the group of diabetic rats given bosentan 50 mg/kg (n = 10) and the group of diabetic rats given bosentan 100 mg/kg (n = 10). Diabetes was induced in the rats by STZ (60 mg/kg i.p.). On day 91, all rats were killed. Brain tissues of the rats were measured by molecular, biochemical and histopathological methods. Antioxidant levels in the therapy groups were observed as quite near to the values in the healthy group. In this study, while the brain eNOS levels in the diabetic groups decreased, the ET1 and iNOS levels were found to be increased. However, in the diabetes group, hippocampus and cerebellum, pericellular oedema and a number of neuronal cytoretraction were increased in neuropiles, whereas these results were decreased in the therapy group. Based on all of these results, ET1 will not be ignored in diabetes‐induced cerebral complications.     

Diffusion Studies of Nanometer Polymersomes Across Tissue Engineered Human Oral Mucosa

Purpose  To measure the diffusion of nanometer polymersomes through tissue engineered human oral mucosa. Methods   In vitro models of full thickness tissue engineered oral mucosa (TEOM) were used to assess the penetration properties of two chemically different polymersomes comprising two of block copolymers, PMPC-PDPA and PEO-PDPA. These copolymers self-assemble into membrane-enclosed vesicular structures. Polymersomes were conjugated with fluorescent rhodamine in order to track polymersome diffusion. Imaging and quantification of the diffusion properties were assessed by confocal laser scanning microscopy (CLSM). Results  TEOM is morphologically similar to natural oral mucosa. Using CLSM, both formulations were detectable in the TEOM within 6 h and after 48 h both penetrated up to 80 μm into the TEOM. Diffusion of PMPC-PDPA polymersomes was widespread across the epithelium with intra-epithelial uptake, while PEO-PDPA polymersomes also diffused into the epithelium. Conclusions  CLSM was found to be an effective and versatile method for analysing the level of diffusion of polymersomes into TEOM. The penetration and retention of PMPC-PDPA and PEO-PDPA polymersomes means they may have potential for intra-epithelial drug delivery and/or trans-epithelial delivery of therapeutic agents.     

In vitro permeation of diclofenac sodium from novel microemulsion formulations through rabbit skin

In order to increase topical penetration of the nonsteroidal anti‐inflammatory drug, diclofenac sodium, new microemulsion formulations were prepared to increase drug solubility and in vitro penetration of the drug. The influence of dimethyl sulfoxide and propylene glycol were also investigated as enhancers on the in vitro penetration of diclofenac sodium using Franz diffusion cells using excised dorsal rabbit skin. Factorial randomized design was performed to analyze the results of in vitro permeation studies. Microemulsions prepared with isopropyl alcohol were superior to those prepared with propanol. Enhancers had different effects depending on the formulation. Propylene glycol was superior to dimethyl sulfoxide when incorporated into isopropyl alcohol microemulsion, whereas dimethyl sulfoxide was superior to propylene glycol in propanol microemulsions. There were no observable histopathological differences between the skin of the control group and the treated groups at the light microscope level due to swelling of the skin tissue. The present study shows that microemulsion formulations containing isopropyl alcohol as co‐surfactant and propylene glycol as enhancer represent a promising approach for a topical vehicle for diclofenac sodium. Drug Dev. Res. 65:17–25, 2005. © 2005 Wiley‐Liss, Inc.     

Enhancement of ocular drug penetration

Although new drugs have recently been developed within the field of ophthalmology, the eye's various defense mechanisms make it difficult to achieve an effective concentration of these drugs within the eye. Drugs administered systemically have poor access to the inside of the eye because of the blood-aqueous and blood-retinal barriers. And although topical instillation of drugs is very popular in ophthalmology, topically applied drugs are rapidly eliminated from the precorneal area. In addition, the cornea, considered a major pathway for ocular penetration of topically applied drugs, is an effective barrier to drug penetration, since the corneal epithelium has annular tight junctions (zonula occludens), which completely surround and effectively seal the superficial epithelial cells. Various drug-delivery systems have been developed to increase the topical bioavailability of ophthalmic drugs by enhancement of the ocular drug penetration. The first approach is to modify the physicochemical property of drugs by chemical and pharmaceutical means. An optimum promoiety can be covalently bound to a drug molecule to obtain a prodrug that can chemically or enzymatically be converted to the active parent drug, either within the cornea or after the corneal penetration. Along these same lines, the transient formation of a lipophilic ion pair by ionic bonding is also useful for improving ocular drug penetration. The second approach is to modify the integrity of the corneal epithelium transiently by coadministration of an amphiphilic substance or by chelating agents that act as drug-penetration enhancers. The third approach modifies the integrity of the corneal epithelium transiently by physical techniques including iontophoresis and phonophoresis. This paper reviews the absorption behavior and ocular membranes penetration of topically applied drugs, and the various approaches for enhancement of ocular drug penetration in the eye.     

64排螺旋CT评价中央型肺癌气管、支气管及肺门血管侵犯的应用研究

目的 探讨64排螺旋CT血管成像诊断中央型肺癌（CLC）气管、支气管及肺门血管侵犯的准确性.方法 收集43例临床确诊的CLC患者64排螺旋CT检查结果,并以病理诊断结果为参照标准判断其准确性.结果 43例患者气管、支气管受侵,CT诊断气管、主支气管、叶支气管、近端段支气管的准确性分别为100%、97.67%、96.74%、88.37%.38例患者肺门血管受侵,CT诊断肺门血管受侵和可疑受侵的准确性分别为88.23%、75.00%.结论 64排螺旋CT能比较准确地显示CLC气管、支气管、肺门气管侵犯的情况.     

Dilated intercellular spaces and acid reflux at the distal and proximal oesophagus in patients with non-erosive gastro-oesophageal reflux disease

BACKGROUND: Acid exposure of proximal oesophagus and dilated intercellular space diameters of oesophageal epithelium are relevant in the perception of gastro-oesophageal reflux. AIM: To explain the relationship between gastro-oesophageal reflux disease symptoms, acid exposure and intercellular space diameter along the oesophageal epithelium and to assess time-related variability of intercellular space diameter. METHODS: Thirty-three non-erosive reflux disease (NERD), six erosive oesophagitis patients and 12 asymptomatic controls underwent oesophageal manometry and 24-h dual-channel oesophageal pH-monitoring following endoscopy. Biopsies were taken 5 cm above the LES and 10 cm below the UES, at comparable levels, as pH sensors. A total of 100 intercellular space diameters per patient/control were measured blindly at transmission electron microscopy. In 15 patients, the investigation was repeated after 1 year. RESULTS: In all NERD patients, acid exposure was higher at mid-proximal oesophagus (P < 0.01) and mean intercellular space diameters, at distal and mid-proximal oesophagus, was three- and twofold higher (1.5 and 0.82 micro m, respectively) compared with controls. Intra-patient intercellular space diameter values were stable over time, not overlapping with those of controls. CONCLUSIONS: Dilation of intercellular space diameter occurs along the distal and proximal oesophageal epithelium in NERD patients and could be responsible for the enhanced perception of proximal acid reflux. This finding appears to be time-reproducible and to represent a sensitive, histopathological marker of NERD.     

Pharmacokinetic interactions between clozapine and sertraline in smokers and non‐smokers

Clozapine is an effective antipsychotic drug for treatment‐resistant schizophrenia. Sertraline is a widely prescribed antidepressant and often concomitantly applied to address negative symptoms or depression. However, data on interactions between clozapine and sertraline are inconsistent. The aim of our study was to evaluate pharmacokinetic interactions between clozapine and sertraline analysing a therapeutic drug monitoring database of 1644 clozapine‐medicated patients. We compared four groups: non‐smokers (n = 250) and smokers (n = 326) with co‐medication without known effects on cytochrome P450 and without sertraline, and non‐smokers (n = 18) and smokers (n = 17) with sertraline co‐medication. Measured and dose‐corrected concentrations (C/D) of clozapine were compared between the groups using non‐parametrical tests with a significance level of 0.05. Post hoc analyses included pairwise comparisons to account for smoking status. Although we detected significant differences for clozapine levels and C/D values between study groups (P < .001 for Kruskal‐Wallis test in both cases), post hoc analyses revealed no differences for concentrations and C/D values of clozapine (P > .05 for Mann‐Whitney U test in both cases). A negative correlation between the sertraline dose and the clozapine concentration was found in non‐smokers (Spearman's rank correlation, rs = ?0.535, P = .048). A potential pharmacokinetic interaction between clozapine and a standard therapeutic sertraline dose seems to be of minor clinical importance.     

Evaluation of the potentiating effects of antidepressants on the contractile response to noradrenaline in guinea pig urethra smooth muscles

We investigated the potential augmenting effects of 19 clinically available antidepressants on noradrenaline (NA)‐induced contractions in guinea pig urethra smooth muscle (USM). Concentration‐response curves for NA‐induced contractions in guinea pig USM strips were obtained in the absence or presence of selected antidepressants. Desipramine, an active metabolite of imipramine, produced a contraction and potentiated NA‐induced contraction at the distal urethra without affecting the proximal urethra. Further, nortriptyline and amoxapine, tricyclic antidepressants, produced a contraction and potentiated NA‐induced contraction at the distal urethra. NA‐induced contraction was unaffected or reduced by imipramine, clomipramine, trimipramine, and amitriptyline at the proximal and distal urethra. Maprotiline, a tetracyclic antidepressant, potentiated NA‐induced contraction at the distal urethra. NA‐induced contraction was unaffected by mianserin at the proximal and distal urethra. Paroxetine, a selective serotonin reuptake inhibitor (SSRI), potentiated NA‐induced contraction at the distal urethra, while NA‐induced contraction was unaffected by fluvoxamine, sertraline, and escitalopram at the proximal and distal urethra. Milnacipran, a serotonin‐noradrenaline reuptake inhibitor (SNRI), potentiated NA‐induced contraction at the proximal and distal urethra, whereas duloxetine potentiated it at the distal urethra. Mirtazapine slightly inhibited NA‐induced contraction at the distal urethra. Aripiprazole and sulpiride did not affect NA‐induced contractions at the proximal nor distal urethra. Trazodone inhibited NA‐induced contraction at both urethras. Desipramine, nortriptyline, amoxapine, maprotiline, paroxetine, milnacipran, and duloxetine likely induce urinary disturbance by increasing urethral resistance and augmenting NA‐induced contraction, which should be carefully considered when delivering guidance for drug administration to patients.     

Airborne fine particulate matter causes murine bronchial hyperreactivity via MAPK pathway‐mediated M3 muscarinic receptor upregulation

Regarding the human health effects, airborne fine particulate matter 2.5 (PM_2.5) is an important environmental risk factor. However, the underlying molecular mechanisms are largely unknown. The present study examined the hypothesis that PM_2.5 causes bronchial hyperreactivity by upregulated muscarinic receptors via the mitogen‐activated protein kinase (MAPK) pathway. The isolated rat bronchi segments were cultured with different concentration of PM_2.5 for different time. The contractile response of the bronchi segments were recorded by a sensitive myograph. The mRNA and protein expression levels of M₃ muscarinic receptors were studied by quantitative real‐time PCR and immunohistochemistry, respectively. The muscarinic receptors agonist, carbachol induced a remarkable contractile response on fresh and DMSO cultured bronchial segments. Compared with the fresh or DMSO culture groups, 1.0 µg/mL of PM_2.5 cultured for 24 h significantly enhanced muscarinic receptor‐mediated contractile responses in bronchi with a markedly increased maximal contraction. In addition, the expression levels of mRNA and protein for M₃ muscarinic receptors in bronchi of PM_2.5 group were higher than that of fresh or DMSO culture groups. SB203580 (p38 inhibitor) and U0126 (MEK1/2 inhibitor) significantly inhibited the PM_2.5‐induced enhanced contraction and increased mRNA and protein expression of muscarinic receptors. However, JNK inhibitor SP600125 had no effect on PM_2.5‐induced muscarinic receptor upregulation and bronchial hyperreactivity. In conclusion, airborne PM_2.5 upregulates muscarinic receptors, which causes subsequently bronchial hyperreactivity shown as enhanced contractility in bronchi. This process may be mediated by p38 and MEK1/2 MAPK pathways. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 371–381, 2017.     

EPITHELIAL DISRUPTION BY PROTEASES AUGMENTS THE RESPONSIVENESS OF PORCINE BRONCHIAL SEGMENTS

1. The effect of disruption to the epithelium of intact porcine bronchi was examined by comparing the responsiveness to agonists applied to the adventitial and luminal surfaces. The development of smooth muscle tone was measured as an increase in pressure in an isovolumic bronchial segment of approximately 2 mm i.d. The reactivity and sensitivity to acetylcholine (ACh) introduced intraluminally was greatly attenuated when compared with adventitial addition. 2. Luminal exposure to K+ and vanadate (VO3-) had little effect compared with strong responses obtained by adventitial application. 3. Intraluminal exposure to trypsin, chymotrypsin and elastase (1 mg/mL) selectively augmented both the sensitivity and the reactivity to the luminal addition of ACh, K+ and carbachol. 4. Mechanical removal of the epithelium produced a 716-fold increase in sensitivity to ACh introduced luminally but had no effect on ACh applied adventitially. 5. The inhibitory effects of luminally introduced isoprenaline on electrical field stimulation responses were also significantly potentiated in segments stripped of epithelium. 6. The evidence presented here indicates that the responsiveness of in vitro airways segments is highly influenced by the epithelial layer, which acts most prominently as a barrier inhibiting the penetration of luminally introduced agonists.