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It has long been known that regular physical exercise induces short and long term benefits reducing the risk of cardiovascular disease, diabetes, osteoporosis, cancer and improves sleep quality, cognitive level, mobility, autonomy in enderly. More recent is the evidence on the endocrine role of the contracting skeletal muscle. Exercise triggers the release of miokines, which act in autocrine, paracrine and endocrine ways controlling the activity of muscles but also of other tissues and organs such as adipose tissue, liver, pancreas, bones, and brain. The mechanism of release is still unclear. Neuromuscular electrical stimulation reproduces the beneficial effects of physical activity producing physiological metabolic, cardiovascular, aerobic responses consistent with those induced by exercise. In vitro, Electrical Pulse Stimulations (EPS) of muscle cells elicit cell contraction and mimic miokine release in the external medium. Here we show that, in cultured mouse myotubes, EPS induce contractile activity and the release of the myokine IL-6. Gadolinium highly reduces EPS-induced IL-6 release, suggesting the involvement of mechanical activated ion channels. The chemical activation of mechanosensitive Piezo1 channels with the specific agonist Yoda1 stimulates IL-6 release similarly to EPS, suggesting the involvement of Piezo1 channels in the control of the myokine release. The expression of Piezo1 protein in myotubes was confirmed by the Western blot analysis. To the best of our knowledge, this is the first evidence of a Piezo1-mediated effect in myokine release and suggests a potential translational use of specific Piezo1 agonists for innovative therapeutic treatments reproducing/enhancing the benefits of exercise mediated by myokines.

Aim: Mechanosensitive Piezo1 ion channels emerged recently as important contributors to various vital functions including modulation of the blood supply to skeletal muscles. The specific Piezo1 channel agonist Yoda1 was shown to regulate the tone of blood vessels similarly to physical exercise. However, the direct role of Piezo1 channels in muscle function has been little studied so far. We therefore investigated the action of Yoda1 on the functional state of skeletal muscle precursors (satellite cells and myotubes) as well as on adult muscle fibers. Methods: Immunostaining, electrophysiological intracellular recordings and Ca2+ imaging experiments were performed to localize and assess the effect of the chemical activation of Piezo1 channels with Yoda1, on myogenic precursors, adult myofibers and at the adult neuromuscular junction. Results: Piezo1 channels were detected by immunostaining in satellite cells and myotubes as well as in adult myofibers. In the skeletal muscle precursors, Yoda1 treatment stimulated the differentiation and cell fusion rather than the proliferation of satellite cells. Moreover, in myotubes, Yoda1 induced significant [Ca2+ ]i transients, without detectable [Ca2+ ]i response in adult myofibers. Furthermore, although expression of Piezo1 channels was detected around the muscle endplate region, Yoda1 application did not alter either the nerve-evoked or spontaneous synaptic activity or muscle contractions in adult myofibers. Conclusion: Our data indicate that the chemical activation of Piezo1 channels specifically enhances the differentiation of skeletal muscle precursors, suggesting a possible new strategy to promote muscle regeneration.

Mouse and human pluripotent stem cells have been widely used to study the development of the hematopoietic and immune systems. While not all cells can be derived with the same efficiency, immune cells such as natural killer (NK) cells and macrophages can be easily produced from pluripotent stem cells to enable development of new cell-based therapies. NK cells and macrophages are part of the innate immune system, the first line of defense against malignancies and infectious disease. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs)-derived NK cells can be produced at a clinical scale suitable for translation into clinical trials. Additionally, pluripotent stem cells can be genetically modified to produce hESC and iPSC-derived human NK cells with enhanced anti-tumor activity. These engineered NK cells can express a stabilized version of the high-affinity Fc receptor CD16, chimeric antigen receptors (CARs), or other strategies to enable more potent and targeted cellular immunotherapies. Moreover, macrophages can also be routinely and efficiently produced from hESCs and iPSCs as a tool to expand our knowledge of the basic biology of these cells. hESC/iPSC-derived macrophages can also be employed as a novel approach for cancer immunotherapy, as well as a strategy to repair or regenerate diseased/damaged tissues and organs.

Natural killer (NK) cells are known to mediate killing of various cancer types, but tumor cells can develop resistance mechanisms to escape NK cell-mediated killing. Here, we use a “two cell type” whole genome CRISPR-Cas9 screening system to discover key regulators of tumor sensitivity and resistance to NK cell-mediated cytotoxicity in human glioblastoma stem cells (GSC). We identify CHMP2A as a regulator of GSC resistance to NK cell-mediated cytotoxicity and we confirm these findings in a head and neck squamous cells carcinoma (HNSCC) model. We show that deletion of CHMP2A activates NF-κB in tumor cells to mediate increased chemokine secretion that promotes NK cell migration towards tumor cells. In the HNSCC model we demonstrate that CHMP2A mediates tumor resistance to NK cells via secretion of extracellular vesicles (EVs) that express MICA/B and TRAIL. These secreted ligands induce apoptosis of NK cells to inhibit their antitumor activity. To confirm these in vitro studies, we demonstrate that deletion of CHMP2A in CAL27 HNSCC cells leads to increased NK cell-mediated killing in a xenograft immunodeficient mouse model. These findings illustrate a mechanism of tumor immune escape through EVs secretion and identify inhibition of CHMP2A and related targets as opportunities to improve NK cell-mediated immunotherapy.

The so-called amphibole asbestos fibers are enriched with mineral iron ions, able to stimulate ROS production. We recently reported that crocidolite asbestos was able to interact with the cell membranes of Xenopus laevis oocytes, to alter their electrical membrane properties. Here, we found that applied iron ions (Fe3+) or H2O2 (for ROS generation) mimicked these effects, suggesting that at least one effect of iron-containing asbestos fiber exposure was mediated by ROS production. Furthermore, combined Fe3+ and H2O2 acted synergistically, producing a membrane effect stronger than that induced by these factors alone. Similar to crocidolite, these changes peaked within 30 minutes of incubation and vanished almost completely after 120 min. However, in the presence of cytochalasin D, which inhibits membrane actin repair mechanisms, crocidolite or applied Fe3+/H2O2 invariably produced oocyte cell death. While the electrophysiological modifications induced by crocidolite suggested a modification of an intrinsic chloride ion channel, the morphological appearance of the treated oocytes also indicated the formation of membrane “pores”; the effects of asbestos exposure may therefore consist of multiple (not necessarily exclusive) underlying mechanisms. In conclusion, using Xenopus oocytes allowed us for the first time, to focus on a specific membrane effect of crocidolite asbestos exposure, which deserves to be tested also on human lung cell lines. Much available evidence suggests that asbestos fibers damage cells through the production of ROS. Our present data confirm that crocidolite fibers can indeed trigger ROS-mediated damaging effects in the oocyte cell membrane, provided iron ions and H2O2 are available for ROS production.

An in vitro system of electrical stimulation was used to explore whether an innovative “noisy” stimulation protocol derived from human electromyographic recordings (EMGstim) could promote muscle regeneration. EMGstim was delivered to cultured mouse myofibers isolated from Flexor Digitorum Brevis, preserving their satellite cells. In response to EMGstim, immunostaining for the myogenic regulatory factor myogenin, revealed an increased percentage of elongated myogenin-positive cells surrounding the myofibers. Conditioned medium collected from EMGstim-treated cell cultures, promoted satellite cells differentiation in unstimulated myofiber cell cultures, suggesting that extracellular soluble factors could mediate the process. Interestingly, the myogenic effect of EMGstim was mimicked by exogenously applied ATP (0.1 μM), reduced by the ATP diphosphohydrolase apyrase and prevented by blocking endogenous ATP release with carbenoxolone. In conclusion, our results show that “noisy” electrical stimulations favor muscle progenitor cell differentiation most likely via the release of endogenous ATP from contracting myofibres. Our data also suggest that “noisy” stimulation protocols could be potentially more efficient than regular stimulations to promote in vivo muscle regeneration after traumatic injury or in neuropathological diseases.

Adenosine is a powerful modulator of skeletal neuromuscular transmission, operating via inhibitory or facilitatory purinergic-type P1 receptors. To date, studies have been focused mainly on the effect of adenosine on presynaptic P1 receptors controlling transmitter release. In this study, using two-microelectrode voltage-clamp and single-channel patch-clamp recording techniques, we have explored potential postsynaptic targets of adenosine and their modulatory effect on nicotinic acetylcholine receptor (nAChR)-mediated synaptic responses in adult mouse skeletal muscle fibers in vitro. In the whole-mount neuromuscular junction (NMJ) preparation, adenosine (100 μM) significantly reduced the frequency of the miniature endplate currents (MEPCs) and slowed their rising and decay time. Consistent with a postsynaptic site of action, adenosine and the potent P1 receptor agonist NECA significantly increased the open probability, the frequency and the open time of single nAChR channels, recorded at the endplate region. Using specific ligands for the P1 receptor subtypes, we found that the low-affinity P1 receptor subtype A2B was responsible for mediating the effects of adenosine on the nAChR channel openings. Our data suggest that at the adult mammalian NMJ, adenosine acts not only presynaptically to modulate acetylcholine transmitter release, but also at the postsynaptic level, to enhance the activity of nAChRs. Our findings open a new scenario in understanding of purinergic regulation of nAChR activity at the mammalian endplate region.

Efficient targeting in tumor therapies is still an open issue: systemic biodistribution and poor specific accumulation of drugs weaken efficacy of treatments. Engineered nanoparticles are expected to bring benefits by allowing specific delivery of drug to the tumor or acting themselves as localized therapeutic agents. In this study we have targeted epithelial ovarian cancer with inorganic nanoparticles conjugated to a human antibody fragment against the folate receptor over-expressed on cancer cells. The conjugation approach is generally applicable. Indeed several types of nanoparticles (either magnetic or fluorescent) were engineered with the fragment, and their biological activity was preserved as demonstrated by biochemical methods in vitro. In vivo studies with mice bearing orthotopic and subcutaneous tumors were performed. Elemental and histological analyses showed that the conjugated magnetic nanoparticles accumulated specifically and were retained at tumor sites longer than the non-conjugated nanoparticles.

Loss of MeCP2 (Methyl CpG binding protein 2) in Rett syndrome (RTT) causes brain weight decrease, shrinkage of the cortex with reduced dendritic arborization, behavioral abnormalities, seizures and cardio-respiratory complications. The observed monoamine neurotransmitters reduction in RTT suggested antidepressants as a possible therapy. We treated MeCP2-null mice from postnatal-day 28 for two weeks with desipramine, already tested in RTT, or mirtazapine, an antidepressant with limited side-effects, known to promote GABA release. Mirtazapine was more effective than desipramine in restoring somatosensory cortex thickness by fully rescuing pyramidal neurons dendritic arborization and spine density. Functionally, mirtazapine treatment normalized heart rate, breath rate, anxiety levels and eliminated the hopping behavior observed in MeCP2-null mice, leading to improved phenotypic score. These morphological and functional effects of mirtazapine were accompanied by reestablishment of the GABAergic and glutamatergic receptor activity recorded in cortex and brainstem tissues. Thus, mirtazapine can represent a new potential pharmacological treatment for the Rett syndrome.

The mode of interaction of asbestos fibres with cell membranes is still debatable. One reason is the lack of a suitable and convenient cellular model to investigate the causes of asbestos toxicity. We studied the interaction of asbestos fibres with Xenopus laevis oocytes, using electrophysiological and morphological methods. Oocytes are large single cells, with a limited ability to endocytose molecular ligands; we therefore considered these cells to be a good model for investigating the nature of asbestos/membrane interactions. Electrophysiological recordings were performed to compare the passive electrical membrane properties, and those induced by applying positive or negative voltage steps, in untreated oocytes and those exposed to asbestos fibre suspensions. Ultrastructural analysis visualized in detail, any morphological changes of the surface membrane caused by the fibre treatment. Our results demonstrate that Amosite and Crocidolite-type asbestos fibres significantly modify the properties of the membrane, starting soon after exposure. Cells were routinely depolarized, their input resistance decreased, and the slow outward currents evoked by step depolarizations were dramatically enhanced. Reducing the availability of surface iron contained in the structure of the fibres with cation chelators, abolished these effects. Ultrastructural analysis of the fibre-exposed oocytes showed no evidence of phagocytic events. Our results demonstrate that asbestos fibres modify the oocyte membrane, and we propose that these cells represent a viable model for studying the asbestos/cell membrane interaction. Our findings also open the possibly for finding specific competitors capable of hindering the asbestos-cell membrane interaction as a means of tackling the long-standing asbestos toxicity problem.

The endothelial vasodilation mechanism(s) has been investigated in aortic rings of hypophysectomized male rats as well as hypophysectomized rats treated for 7 days with growth hormone (GH, 400 microg/kg, s.c.) or hexarelin (80 microg/kg, s.c.). Tissue preparations from intact animals were taken as controls. The results obtained indicate that the release of 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha) from aortic rings of hypophysectomized rats was markedly reduced (51%; p

Paclitaxel poliglumex (CT-2103; XYOTAX) is an innovative macromolecular taxane designed to increase the therapeutic index of paclitaxel. This large macromolecule conjugate of paclitaxel and poly-L-glutamic acid accumulates in tumor tissues by taking advantage of the enhanced permeability of tumor vasculature and lack of lymphatic drainage. Paclitaxel poliglumex prolongs exposure to active drug and minimizes systemic exposure. Preclinical studies in animal tumor models demonstrate enhanced safety and efficacy relative to paclitaxel when administered as a single agent or in conjunction with radiation. Clinical pilot studies with paclitaxel poliglumex showed improved outcomes compared to standard taxanes and allowed a more convenient administration schedule. Human pharmacokinetic data are consistent with prolonged tumor exposure to active drug and a limited systemic exposure. Based on these results, three ongoing randomized phase III trials were initiated to test the efficacy of paclitaxel poliglumex in patients with advanced non-small cell lung carcinoma.

Introduction: Before the nerve contacts the skeletal muscle, the nicotinic acetylcholine receptors (nAChRs) form aggregates known as prepatterned clusters. We investigated their role in the occurrence of Ca2+ spikes and twitching during myogenesis. Methods: Cultured mouse myotubes were used as cell models. Cells were subjected to a combination of immunostaining, Ca2+ imaging and electrophysiological analysis. Results: A single prepatterned nAChR cluster per myotube was generally detected. A correlation between formation of the prepatterned clusters and occurrence of Ca2+ spikes and twitching was observed. Increase in size of the prepatterned clusters raised the frequency of Ca2+ spikes and twitching. Blockade of the electrical activity triggered by the autocrine activation of prepatterned nAChR induced over-numbered nAChR clusters. Conclusions: Prepatterned nAChR aggregation is required for Ca2+ spikes and twitching of developing myotubes. Moreover, prepatterned nAChR-driven electrical activity preserves the distribution of nAChRs, mimicking the effect of synaptic activity before innervation. Muscle Nerve 46: 112–121, 2012

Cell membranes isolated from nervous tissue can be easily injected into Xenopus oocytes, thereby effectively "microtransplanting" functional neurotransmitter receptors. This technique therefore allows a direct functional characterization of the original membrane receptor/ion channel proteins and the associated molecules while still embedded in their natural lipid environment. Cell membranes will contain components from different types of cells, i.e. neurons and glial cells, expressing their own receptors, with possibly different properties. To study the receptor properties of a single cell type, we injected oocytes with membranes isolated only from glia (gliosomes) of adult mouse neocortex and we focused our work on GABA(A) receptors incorporated in the oocyte cell membrane. We found that GABA(A)-activated currents allowed a good biophysical and pharmacological characterization of glial GABA(A) receptors. Therefore, the microtransplantation of gliosomes into oocytes can represent a good model to study the electrical and pharmacological properties of adult glial cells under different physiological and pathological conditions. Moreover, since gliosomes can be isolated from frozen tissues, this approach can be extended to post-mortem human tissues.

It is known that Alzheimer's disease (AD) is a synaptic disease that involves various neurotransmitter systems, particularly those where synaptic transmission is mediated by acetylcholine or glutamate (Glu). Nevertheless, very little is known about the properties of neurotransmitter receptors of the AD human brain. We have shown previously that cell membranes, carrying neurotransmitter receptors from the human postmortem brain, can be transplanted to frog oocytes, and their receptors will still be functional. Taking advantage of this fact, we have now studied the properties of Glu receptors (GluRs) from the cerebral cortices of AD and non-AD brains and found that oocytes injected with AD membranes acquired GluRs that have essentially the same functional properties as those of oocytes injected with membranes from non-AD brains. However, the amplitudes of the currents elicited by Glu were always smaller in the oocytes injected with membranes from AD brains. Western blot analyses of the same membrane preparations used for the electrophysiological studies showed that AD membranes contained significantly fewer GluR2/3 subunit proteins. Furthermore, the corresponding mRNAs were also diminished in the AD brain. Therefore, the smaller amplitude of membrane currents elicited by Glu in oocytes injected with membranes from an AD brain is a consequence of a reduced number of GluRs in cell membranes transplanted from the AD brain. Thus, using the comparatively simple method of microtransplantation of receptors, it is now possible to determine the properties of neurotransmitter receptors of normal and diseased human brains. That knowledge may help to decipher the etiology of the diseases and also to develop new treatments.

The ability of hexarelin, a recently synthesized hexapeptide with a strong growth hormone (GH)-releasing activity, or of GH itself to display a protectant activity against postischemic ventricular dysfunction in senescent hearts was studied in 24-month-old male rats. Heart preparations from control (saline-treated) senescent rats, subjected to moderate ischemia, showed at reperfusion: (a) a low recovery of postischemic left ventricular developed pressure (LVDP; 37% of the preischemic values; from 90 +/- 5.7 to 33.5 +/- 3.8 mm Hg; p0.01; n = 10) coupled to a substantial increase in coronary perfusion pressure (CPP; 71% over baseline; from 68.3 +/- 5.2 to 116.8 +/- 4.6 mm Hg; p0.01; n = 10); (b) a marked increase of creatine kinase (CK) released in the perfusates (6.6-fold increase over preischemic values; from 45 +/- 4 to 298 +/- 25 mU/min/g wet tissue; p0.001; n = 10). In vivo administration of hexarelin (80 microg/kg, b.i.d., s.c.) for 21 days resulted in a striking heart protection against reperfusion stunning. In fact, the recovery of LVDP at reperfusion was almost complete (90% of the preischemic values; from 93 +/- 5.8 to 83.7 +/- 5.9 mm Hg; p0.05; n = 9), and the increase in coronary resistance was minimal (from 67 +/- 5.8 to 79.7 +/- 6.9 mm Hg; p0.05; n = 9). Furthermore, the concentration of CK in the perfusates was increased only twofold (from 45.8 +/- 5.5 to 90 +/- 7.2 mU/min/g wet tissue; p0.05; n = 9), with a gradual return toward basal values at the end of reperfusion. The protectant activity of hexarelin was divorced from any detectable alteration of the somatotropic function, as assessed by pituitary GH messenger RNA (mRNA) and plasma insulin-like growth factor I levels. In vivo administration of GH (400 microg/kg b.i.d., s.c.) for the same time lapse resulted in only a partial protectant activity: 55% of LVDP recovery (from 91.5 +/- 6.2 to 50 +/- 3.5 mm Hg; p0.01; n = 6); 65% increase of coronary resistance (from 68 +/- 4.3 to 112.2 +/- 5.2 mm Hg; p0.01; n = 6); 5.3-fold increase of CK concentrations in heart perfusates on reperfusion (from 43.8 +/- 3.8 to 232 +/- 16 mU/min/g wet tissue; p0.001; n = 6). Evaluation of the rate of release of 6-keto-prostaglandin F1alpha (PGF1alpha), the stable metabolite of prostacyclin, in heart perfusates, and assessment of the vasopressor activity of angiotensin II on the coronary vasculature, did not show any change in these parameters among the three experimental groups. Collectively these data indicate that hexarelin displays a strong heart-protectant activity against myocardial stunning in senescent rats. The protection afforded by the peptide is likely due to a direct cardiotropic action and is far greater than that of GH. Neither compound appears able to interfere with the endothelium-dependent relaxant mechanism.

The ability of hexarelin, an effective growth hormone (GH)-releasing hexapeptide, to reverse the worsening of cardiac dysfunction in GH-deficient animals was studied in young male rats passively immunized by administration of an anti-GH-releasing hormone (GHRH) serum. Heart preparations from anti-GHRH serum-treated rats, undergoing low-flow ischemia and reperfusion, showed: (1) a progressive increase of left ventricular end-diastolic pressure during the ischemic period and a poor recovery of contractility at reperfusion with a consistent decrease of the left ventricular-developed pressure; (2) a decreased rate of formation of 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha), a stable metabolite of prostacyclin, in perfusates from preischemic and reperfusion periods; (3) an increased vasopressor activity of angiotensin II. Hexarelin (80 microg/kg, bid, s.c.), administered for 15 days to anti-GHRH serum-treated rats, restored to normal the impaired somatotropic function and counteracted the ischemic damage, improving postischemic left ventricular developed pressure to values higher than those of controls. Furthermore, both the generation of 6-keto-PGF1alpha and the vasopressor activity of angiotensin II reverted to those of control preparations. Administration of hexarelin to control rats induced a considerable improvement of postischemic ventricular function of the perfused hearts which was similar to that present in preparations from anti-GHRH serum-treated rats given hexarelin. This protective activity was divorced from any further stimulation of somatotropic function. Collectively, these data indicate that, in GH-deficient rats, hexarelin is capable of restoring somatotropic function and has a beneficial effect in myocardial ischemia and reperfusion damage. In addition, the increased responsiveness of the coronary vasculature to angiotensin II and the decreased generation of prostacyclin in hearts from GH-deficient rats would indicate that for prevention of injury and dysfunction of the vascular endothelium a normal somatotropic function is mandatory.

Cough accompanied by an increased sensitivity of the cough reflex is the most common symptom of inflammatory airway disease. This symptom is also frequently reported in patients receiving angiotensin-converting enzyme (ACE) inhibitors as therapy for heart failure or hypertension, although the underlying mechanism is unknown. We have investigated the possibility that the inflammatory peptide bradykinin, normally degraded by ACE, causes sensitization of airway sensory nerves and an enhancement of the cough reflex in conscious guinea pigs. Treatment of guinea pigs for two weeks with captopril led to an increased cough response to inhaled citric acid, which was prevented by concomitant treatment with the bradykinin receptor antagonist icatibant. A similar icatibant-sensitive enhancement of citric acid-evoked cough was seen in untreated animals after prior inhalation of bradykinin, although cough evoked by hypertonic saline was unaffected. In electrophysiological studies performed in vitro, responses of single vagal C fibers to capsaicin, applied to receptive fields of single-fiber units in the trachea, were also markedly increased after perfusion with bradykinin, whereas A delta fiber responses to hypertonic saline were unaffected. These results indicate that bradykinin-evoked sensitization of airway sensory nerves may underlie the pathogenesis of ACE-inhibitor cough. Bradykinin receptor antagonists may be of benefit in treating chronic cough seen with this and other inflammatory conditions.

Implantation of myoblasts is a strategy used to enhance the regeneration of skeletal muscle tissue in vivo. In mouse models, myogenic cell lines and primary cells have been employed with different yields of adult muscle tissue formed. The present work is a study of some developmental features of expanded primary mouse myoblasts (i28), which have been shown to form muscle tissue. i28 myoblasts were differentiated in vitro and the expression of acetylcholine receptor channels and maturation of the excitation–contraction coupling mechanism were investigated using patch clamp and videoimaging techniques. In all the developing cells the embryonic isoform of the acetylcholine receptors was present. Skeletal muscle-type excitation–contraction coupling (i.e., a mechanical link between voltage-dependent calcium channels and ryanodine receptor channels) was detected in about 75% of differentiating i28 myotubes. Only these cells showed spontaneous changes in cytosolic free calcium concentration associated with twitches. Our findings are the first description of the physiological properties of expanded primary myoblasts which are used for implantation and confirm that they are a heterogeneous cell population. In comparison to permanent cell lines, the Ca2+ signaling is more similar to that described in mature nonexpanded muscle fibers. This suggests that cultured primary cells are, so far, the most suitable cell type for muscle regeneration.

After incubation of equimolar amounts of cisplatin (CDDP) and glutathione (GSH) in phosphate buffer pH 7.4 at 37 degrees C, we detected two CDDP-GSH adducts whose structures, characterized by LC-MS, corresponded to cis-[Pt(NH3)2Cl(SG)] and cis-([Pt(NH3)2Cl]2(mu-SG))+. The latter is a new CDDP-GSH adduct, which was postulated but never structurally characterized so far. Rats and patients were given a 15-min intravenous infusion of CDDP (10 mg/kg to rats and 25 mg/m2 to patients) preceded by a GSH intravenous administration (200 mg/kg to rats as a bolus and 1.5 g/m2 to patients as a 15-min infusion). After the administrations, CDDP-GSH adducts were absent in rat and human plasma ultrafiltrates. The discrepancy between in vitro and in vivo findings can be explained based on pharmacokinetic considerations.

Acute myocardial infarction was induced in New Zealand male albino rabbits. 3 days after surgery, the mortality rate and plasma CPK activity were reduced by 64% and 66% respectively by intravenous infusion of Oligotide (32 mg/kg/h for 6h), a single-stranded oligodeoxyribonucleotide of mammalian origin. Perfused hearts, obtained from Oligotide-treated rabbits 3 days after surgery, showed a strength of contraction in the range of that of hearts of sham-operated animals and an ameliorated postsynaptic β-adrenergic and cholinergic receptor function. In addition, in these hearts, “ex vivo” treatment with Oligotide resulted in almost complete preservation of both adrenergic and cholinergic responses to their specific agonists. These data suggest that Oligotide by protecting the hearts from the ischemic damage and possibly by restricting left ventricular tissue necrosis may have normalized the biological responses of this organ to isoproterenol and preserved the integrity of coronary endothelial-dependent relaxant function.

The effects of hexarelin, a growth hormone (GH) secretagogue, and human GH on the mechanical and metabolic changes measured in isolated rat hearts submitted to 5 min of Ca2+ deprivation followed by reperfusion with Ca2+-containing medium, the so-called calcium paradox phenomenon, were studied. Hexarelin (80 microg/kg bid, subcutaneously) administered for 7 d to male rats effectively antagonized the sudden increase in resting tension measured in vitro on Ca2+ repletion. Moreover, during Ca2+ repletion the release of creatine kinase activity (an index of cell damage) in the perfusate of these hearts was reduced up to 40% compared with controls. By contrast, administration of hexarelin for 3 d or GH (400 microg/kg bid, subcutaneously) for 7 d did not affect the mechanical and metabolic alterations induced by the calcium paradox. To assess its direct and acute cardiac effects, hexarelin (8 microg/mL) was perfused in vitro in recirculating conditions for 60 min through the hearts of normal rats. In this case, hexarelin did not stimulate heart contractility and failed to prevent ventricular contracture upon Ca2+ readmission, whereas diltiazem, a Ca2+channel blocker, effectively antagonized the calcium paradox phenomenon. We conclude that short-term in vivo exposure to hexarelin, but not GH, enables cardiac myocyites to prevent cytoplasmatic electrolytic unbalance and to control intracellular Ca2+ gain, two functions largely impaired during the calcium paradox phenomenon. Moreover, because the effect of hexarelin is not acute but dependent on the length of in vivo treatment, we suggest that it requires modifications of myocardiocyte physiology.

Cell membranes, carrying neurotransmitter receptors and ion channels, can be ‘microtransplanted’ into frog oocytes. This technique allows a direct functional characterization of the original membrane proteins, together with any associated molecules they may have, still embedded in their natural lipid environment. This approach has been previously demonstrated to be very useful to study neurotransmitter receptors and ion channels contained in cell membranes isolated from human brains. Here, we examined the possibility of using the microtransplantation method to study acetylcholine receptors from normal and denervated rat skeletal muscles. We found that the muscle membranes, carrying their fetal or adult acetylcholine receptor isoforms, could be efficiently microtransplanted to the oocyte membrane, making the oocytes become sensitive to acetylcholine. These results show that oocytes injected with skeletal muscle membranes efficiently incorporate functional acetylcholine receptors, thus making the microtransplantation approach a valuable tool to further investigate receptors and ion channels of human muscle diseases.

We previously reported that induction of selective GH deficiency in the rat exacerbates cardiac dysfunction induced by experimental ischemia and reperfusion performed on the explanted heart. In the same model, short-term treatment with hexarelin, a GH-releasing peptide, reverted this effect, as did GH. To ascertain whether hexarelin had non-GH-mediated protective effects on the heart, we compared hexarelin and GH treatment in hypophysectomized rats. Hexarelin (80 microg/kg sc), given for 7 days, prevented exacerbation of the ischemia-reperfusion damage induced by hypophysectomy. We also demonstrate that hexarelin prevents increases in left ventricular end diastolic pressure, coronary perfusion pressure, reactivity of the coronary vasculature to angiotensin II, and release of creatine kinase in the heart perfusate. Moreover, hexarelin prevents the fall in prostacyclin release and enhances recovery of contractility. Treatment with GH (400 microg/kg sc) produced similar results, whereas administration of EP 51389 (80 microg/kg sc), another GH-releasing peptide that does not bind to the heart, was ineffective. In conclusion, we demonstrate that hexarelin prevents cardiac damage after ischemia-reperfusion, and that its action is not mediated by GH but likely occurs through activation of specific cardiac receptors.

A physiologic pharmacokinetic model of cyclosporin has been developed in the rat aimed at predicting the time course of drug concentrations in blood, organs, and tissues. The model assumes that tissue distribution is perfusion-rate limited and that each tissue acts as a well-stirred compartment. The unbound equilibrium distribution ratios as well as the values of the fraction unbound and the distributon isotherm of cyclosporin between erythrocytes and plasma are included in the rate equations describing the time course of the drug concentration in each tissue. Parameter values for the rat were obtained experimentally from a continuous infusion study, in which 2.7 and I3.9mg/kg per day doses of cyclosporin were administered subcutaneously to each of two groups of rats by osmotic pumps for 6 days. Steady-state cyclosporin concentrations in blood, CSF, and 18 different organs and tissues, were determined by a monoclonal antibody RIA. Differences in values of the unbound equilibrium distribution ratios in some tissues and unbound clearance indicated that both the processes of distribution and elimination may have elements of nonlinearity over the range of dosing rales tested. The model was evaluated in the rat with a kinetic experiment in which a 6-mg/kg dose of cyclosporin was infused intravenously over 15 min, with measurements of blood concentrations until 56 hr. Good agreement was obtained for the volume of distribution at steady state (blood), V xs between the perfusion model and that calculated from the kinetic experiment. Also, the model prediction of the blood concentration temporal profile agreed closely with that observed except in the early moments, when distribution out of blood occurred considerably slower than predicted. On scaling the model up to humans, good agreement was found between the predicted plasma concentration-time profile and V ss ,and experimental data from the literature. Both rat and human data suggest that partition into adipose tissue plays an important role in the pharmacokinetics of cyclosporin.

The ubiquitin-proteasome pathway plays a central role in regulation of the production and destruction of cellular proteins. These pathways mediate proliferation and cell survival, particularly in malignant cells. The successful development of the 20S human proteasome inhibitor bortezomib for the treatment of relapsed and refractory multiple myeloma has established this targeted intervention as an effective therapeutic strategy. Herein, the potent, selective, and orally bioavailable threonine-derived 20S human proteasome inhibitor that has been advanced to preclinical development, [(1R)-1-[[(2 S,3 R)-3-hydroxy-2-[(6-phenylpyridine-2-carbonyl)amino]-1-oxobutyl]amino]-3-methylbutyl]boronic acid 20 (CEP-18770), is disclosed.

Modulating protein ubiquitination via proteasome inhibition represents a promising target for cancer therapy, because of the higher sensitivity of cancer cells to the cytotoxic effects of proteasome inhibition. Here we show that CEP-18770 is a novel orally-active inhibitor of the chymotrypsin-like activity of the proteasome that down-modulates the nuclear factor-κB (NF-κB) activity and the expression of several NF-κB downstream effectors. CEP-18770 induces apoptotic cell death in multiple myeloma (MM) cell lines and in primary purified CD138-positive explant cultures from untreated and bortezomib-treated MM patients. In vitro, CEP-18770 has a strong antiangiogenic activity and potently represses RANKL–induced osteoclastogenesis. Importantly, CEP-18770 exhibits a favorable cytotoxicity profile toward normal human epithelial cells, bone marrow progenitors, and bone marrow–derived stromal cells. Intravenous and oral administration of CEP-18770 resulted in a more sustained pharmacodynamic inhibition of proteasome activity in tumors relative to normal tissues, complete tumor regression of MM xenografts and improved overall median survival in a systemic model of human MM. Collectively, these findings provide evidence for the utility of CEP-18770 as a novel orally active proteasome inhibitor with a favorable tumor selectivity profile for the treatment of MM and other malignancies responsive to proteasome inhibition.

NCX 4016, a nitro-ester of aspirin endowed with antithrombotic activity, appears to have clinical potential in treating cardiac complications related to coronary insufficiency. This compound has been shown to improve postischemic ventricular dysfunction and to reduce myocardial infarct size in the rabbit. The cardioprotection conferred by NCX 4016 (10, 30, and 100 mg/kg) and aspirin (ASA, 54 mg/kg) was evaluated in anesthetized rats subjected to 30 min of myocardial ischemia followed by 120 min of reperfusion (MI/R). Drugs were given orally for 5 consecutive days. NCX 4016 displayed remarkable cardioprotection in rats subjected to MI/R as was evident in the reduction of ventricular premature beats and in the incidence of ventricular tachycardia and fibrillation; they were reduced dose dependently and correlated with survival of all rats treated with the higher dose of NCX 4016. In these animals, infarct size was restricted proportionally to the dose of NCX 4016 associated with diminution of both plasma creatine phosphokinase and cardiac myeloperoxidase activities. ASA showed only a minor degree of protection against MI/R damage. Rats treated with N(G)-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg) demonstrated aggravated myocardial damage in terms of arrhythmias, mortality, and infarct size. Supplementation of nitric oxide (NO) with NCX 4016 (100 mg/kg) greatly reduced the worsening effect caused by L-NAME. The beneficial effects of NCX 4016 appear to derive in large part from the NO moiety, which modulates a number of cellular events leading to inflammation, obstruction of the coronary microcirculation, arrhythmias, and myocardial necrosis.

A new family of nitroderivatives of conventional non-steroidal anti-inflammatory drugs capable of releasing nitric oxide has been synthesized. Among these compounds, a nitroderivative of aspirin (NCX 4016), which displays antiplatelet and vasodilating activities, appears to have clinical potential in cardiac pathology related to coronary insufficiency.In this study the beneficial effects of NCX 4016 and aspirin were evaluated in vitro in a model of myocardial ischemia-reperfusion of the rabbit and in vivo in a model of acute myocardial infarction of the same animal species.The NCX 4016 (from 1 x 10(-5) M to 3 x 10(-4) M) caused dose-dependent cardiac protection in isolated rabbit hearts subjected to low flow ischemia-reperfusion. Inhibition of 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha) generation and proportional reduction of creatine kinase (CK) activity at reperfusion was observed. Aspirin (1 x 10(-4)M) markedly worsened the post-ischemic ventricular dysfunction and this event was paralleled by a 63% increase in CK activity and abolition of 6-keto-PGF1alpha formation. Perfusion of the hearts with NG-monomethyl-L-arginine (1 x 10(-5) M) worsened the ischemia-reperfusion damage in perfused hearts. This event was prevented by prior treatment with NCX 4016 (1 x 10(-4) M) but not with aspirin (1 x 10(-4) M). Ligation of the first antero-lateral branch of the left coronary artery in rabbits resulted in acute myocardial infarction with a mortality rate of 60% at 24 hours. NCX 4016 (0.5 mg/kg/min for 2 hours) significantly reduced the mortality rate by 10%, protected the rabbits against electrocardiogram derangement and almost abolished CK activity in plasma and myeloperoxidase activity in cardiac tissue. Aspirin was devoid of any protective activity.In the rabbit NCX 4016 appears to exert a relevant cardioprotection likely mediated by nitric oxide donation. These results suggest that this nitroderivative of aspirin may lead to innovative therapy in myocardial ischemia and infarction.

This study investigates the microvascular permeability changes in tracheal tissue of rats exposed to hyperbaric oxygen (HBO). Rats, following exposure to HBO or ambient air (control animals) for 1.5, 3 and 6 h, were prepared for recording of nitric oxide exhaled (FENO) in air using a chemiluminescence analyser. The level of FENO was not statistically different in the two groups. Plasma exudation, evaluated by measuring the leakage of Evans blue (EB) dye into the tracheal tissue, was significantly elevated (48, 86 and 105% at 1.5, 3 and 6 h, respectively) in HBO-treated rats. Plasma exudation in the trachea of control rats was significantly increased (42%, P0.05) by NG-nitro-L-arginine methyl ester (L-NAME), whereas it was significantly reduced (31%, P0.05) in rats exposed to HBO for 3 h. N-acetylcysteine (NAC) and flunisolide significantly prevented the increase in plasma leakage in HBO-treated rats. In contrast, indomethacin was devoid of anti-exudative activity in these experiments. Western immunoblot showed a significant increase in the level of inducible nitric oxide synthase (iNOS) protein in the tracheal homogenates of HBO-treated rats, as compared to basal levels. These results indicate that nitric oxide (NO) is involved in the maintenance of microvascular permeability in tracheal tissue of rats. The protective effect observed with the steroid seems to support this hypothesis. Furthermore, the beneficial action of NAC underlines that reactive oxygen species participate in the microvascular permeability changes observed in tracheal tissue of rats exposed to HBO.

The aim of the present study was to evaluate the nitric oxide (NO) concentrations present in end-expired gas (FENO) of different animal species under basal and stimulated conditions using a clinical chemiluminescence analyser, which has been developed for measurement of single exhalations in humans. Anaesthetised, tracheotomised and artificially ventilated guinea pigs, rats and rabbits were prepared for recording systemic blood pressure and FENO. Stable levels of FENO were detected in expired air over a 1-h observation period in the three animal species tested. Rabbits exhibited the highest concentrations and output (FENO 12.9+/-1.0 ppb, VNO 9.0+/-0.7 nl min-1), followed by guinea pigs (FENO 6.2+/-0.70 ppb, VNO 1.7+/-0.19 nl min-1) and rats (FENO 0.9+/-0.01 ppb, VNO 0.25+/-0.00 nl min-1). L-arginine (1 g kg-1 i.v.) evoked significant increments in VNO in guinea pigs and rabbits but was ineffective in rats. However, L-arginine showed a direct effect on blood pressure in all the animal species tested, causing a rapid fall in the mean arterial blood pressure (MABP; 38, 48 and 50% decrease in rabbits, guinea pigs and rats, respectively; P0.05). An inhibitor of endogenous NO synthesis, NG-nitro-L-arginine methyl ester (L-NAME, 20 mg kg-1 i.v.), decreased both basal and L-arginine-induced VNO in guinea pigs and rabbits, but was ineffective in rats. L-NAME increased MABP in all the animal species tested (58% in guinea pigs, 43% in rats and 18% in rabbits; P0.05). The results indicate that it is possible to detect NO in the exhaled air of different animal species using a clinical chemiluminescence analyser and that different species exhibit striking differences in the levels of basal and stimulated NO output.

BBR 3464 is a novel trinuclear platinum anticancer agent designed on the hypothesis that new clinically useful platinum based anticancer agents should have novel structures unrelated to those of agents currently used in the clinic. BBR 3464 shows outstanding cytotoxicity both against sensitive (L1210) and cisplatin (CDDP) resistant (L1210/CDDP) murine leukemia cell lines. In fact, BBR 3464 is 30 times more cytotoxic than CDDP against the L1210 cell line and also shows a complete lack of cross-resistance in L1210/CDDP (resistance index 0.8). BBR 3464 and CDDP cellular uptake in L1210 and L1210/CDDP eells and binding to nuclear DNA were studied after incubation with 3.34 microM BBR 3464 and 66.7 microM CDDP, respectively, for up to 4 hours with 133.4 and 266.8 microM of CDDP and 6.68, 13.36 microM of BBR 3464 for 2 hours to determine concentration-cellular uptake and concentration-DNA binding relationships. CDDP and BBR 3464 cellular uptake and their extent of binding to DNA increased as function of time and in a concentration dependent manner in both cell lines. CDDP uptake and binding to DNA were higher in L1210 eells than in L1210/CDDP cells whereas BBR 3464 uptake and binding to DNA were similar in both cell lines. In L1210/CDDP murine leukemia cells BBR 3464 seems to overcome the CDDP cross-resistance related to impaired accumulation and reduction of binding to DNA.

The ability of hexarelin, a recently synthesized hexapeptide with a remarkable growth hormone (GH)-releasing activity, to reverse signs of cardiovascular dysfunction in GH-deficient animals was studied in young male rats made GH deficient by the administration of an anti-GH-releasing hormone serum (GHRH-Ab) for 20 days. Heart preparations from GHRH-Ab treated rats, subjected to low-flow ischemia and reperfusion, showed: a progressive increase of left ventricular end-diastolic pressure during the ischemic period and a poor recovery of contractility at reperfusion as compared to control hearts; a decreased rate of formation of 6-keto-PGF1 alpha, the stable metabolite of prostacyclin, in perfusates of both preischemic and reperfusion period; an increased vasopressor activity of angiotensin II on the coronary vasculature. The endothelium-dependent relaxing function in GH-deficient rats was also evaluated in aortic ring preparations, which showed: a decreased rate of formation of 6-keto-PGF1 alpha, an hyperreactivity to endothelin-1, a markedly reduced vasopressor response to NG-monomethyl-L-arginine (the nitric oxide synthase inhibitor) and a decreased vasodilator response to acetylcholine of precontracted aortic tissue. Hexarelin (80 micrograms/kg, s.c. twice daily), administered for 15 days to GHRH-Ab-treated rats, fully restored the somatotropic function and reversed all the signs of cardiac and endothelial dysfunction. In fact, in heart preparations from rats treated with hexarelin the trend of the ischemic damage was similar to that observed in control rats and both the rate of formation of 6-keto-PGF1 alpha and the vasopressor activity of angiotensin II were reverted to control levels. Furthermore, all the parameters of endothelial function were in the normal range. These results indicate that GH deficiency in rats is responsible for an impairment of cardiac function that is associated with a damage of the endothelium-dependent relaxing function not limited to coronary vessels but widespread in the circulation. These alterations are fully reverted by an in vivo treatment with hexarelin.

The compound 8-epi-prostaglandin F2 alpha (8-epi-PGF2 alpha), a F2-isoprostane formed mainly via a non-cyclooxygenase pathway, has been shown to constrict both human and guinea pig airway smooth muscle in vitro. We investigated whether this compound has any activity on bronchial resistance and plasma exudation in tracheal tissue of anaesthetised guinea pigs. 8-Epi-PGF2 alpha (12.5, 25 and 50 micrograms kg-1 i.v.) elicited a dose-dependent increase in intratracheal pressure. Diphenhydramine (2 mg kg-1 i.v.), indomethacin (1 mg kg-1 i.v.) and NG-nitro-L-arginine methyl ester (L-NAME, 10 mg kg-1 i.v.) did not affect the 8-epi-PGF2 alpha (25 micrograms kg-1 i.v.)-induced bronchoconstriction. On the contrary, while atropine (0.5 mg kg-1 i.v.) injected 10 min prior to 8-epi-PGF2 alpha (25 micrograms kg-1 i.v.) significantly reduced to 55% (P < 0.05) the increase in intratracheal pressure induced by the isoprostane, the selective thromboxane A2 receptor antagonist, ICI-192,605 (0.5 mg kg-1 i.v.) abolished it (95%, P < 0.001). Furthermore, 8-epi-PGF2 alpha (50, 100 and 200 micrograms kg-1 i.v.) increased plasma leakage, measured according to the Evans Blue dye technique, in tracheal tissue. This effect was particularly evident when 8-epi-PGF2 alpha was injected at the dose of 200 micrograms kg-1 i.v. (105% increase; P < 0.01) and it was markedly reduced by ICI-192,605 (0.5 mg kg-1 i.v.) (36%; P < 0.01) but not by diphenhydramine (2 mg kg-1 i.v.). In isolated perfused lungs, 8-epi-PGF2 alpha (2.5 micrograms ml-1 min-1 perfused for 2 min) increased the thromboxane A2 formation which was significantly reduced by ICI-192,605 (16 micrograms ml-1 min-1 perfused for 5 min) and abolished by indomethacin (1 microgram ml-1 min-1 perfused for 15 min). These data indicate that 8-epi-PGF2 alpha induces bronchoconstriction in guinea pig in vivo and that this effect, which seems to be related to activation of thromboxane A2 receptor, is associated to an augment of vascular permeability with plasma leakage in airway smooth muscle.

Rats were exposed to hyperbaric oxygen (HBO = 100% oxygen; 2.5 atmospheres absolute pressure) for 6 h. Isovolumic left heart preparations from these animals were subjected to global low flow-ischemia (perfusion rate from 12 ml/min to 2 ml/min for 40 min) and reperfusion. Hearts from rats not exposed to HBO underwent the same ischemic-reperfusion procedure (controls). As compared to control, HBO treatment caused in ex vivo hearts a significant aggravation of cardiac ischemic picture as indicated by a marked increase in left ventricular end diastolic pressure (LVEDP) and reduced post ischemic left ventricular developed pressure (LVDP). At the end of the ischemic and reperfusion periods LVEDP values were 6.8 (p0.001) and 8 (p0.001) times higher than the corresponding control values. Moreover, LVDP and coronary perfusion pressure (CPP) values were decreased (2.8 times; p0.001) and increased (56%; p0.001), respectively, as compared to control preparations. These events were also associated with a considerable impairment of the cardiac tissue to generate 6-keto-PGF1 alpha. Treatments of rats with different doses of acetylcysteine (N-acetylcysteine, CAS 616-91-1, NAC; 0.25-0.5-1 g/kg p.o.) before HBO displayed a clear-cut and dose-related protective activity in hearts subjected to ischemia-reperfusion. Also the generating capacity of 6-keto-PGF1 alpha from these hearts were restored according to the dose of NAC employed. When aortic rings from rats exposed to HBO were considered, they showed a reduced capacity to release 6-keto-PGF1 alpha and an increased sensitivity to endothelin-1. At the same time, the relaxant activity of acetylcholine in these tissues was almost lost. Again, NAC treatment of the animals before HBO restored in a dose-dependent way the capacity of the aortic rings to generate 6-keto-PGF1 alpha. This event was paralleled by normalized responses of the preparations to endothelin-1 and acetylcholine. Taken together these results clearly indicate that acute HBO treatment of the rats markedly aggravates the ischemic-reperfusion damage in ex vivo hearts. This event is coupled with a compromised integrity of cardiac and extracardiac endothelial cell functions. The protective activity of NAC observed in this study once more emphasises its therapeutic role in increasing antioxidant defence mechanisms.

In the rat, plasma leakage in various vascular beds, including the whole lung, occurs after administration of lipopolysaccharide (LPS). LPS-induced microvascular plasma leakage in many organs is associated with an enhanced formation of nitric oxide (NO) after the induction of nitric oxide synthase (iNOS). However, there is limited information concerning the relationship between NO and plasma leakage into the airways. LPS (10 mg/kg, intravenously) caused a significant leakage of Evans blue dye, a marker of microvascular permeability, at 240 min in the trachea which was inhibited by the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME; 10 mg/kg, intravenously), or dexamethasone (1 mg/kg, intravenously). This effect was paralleled by an increase in calcium-independent iNOS activity, assessed by measuring the conversion of radiolabeled L-arginine to L-citrulline, in LPS-treated animals. In contrast, L-NAME significantly increased plasma leakage in the trachea of vehicle-treated rats and this effect was inhibited by indomethacin. These results suggest that under "physiological" conditions endogenous NO suppresses plasma leakage but when iNOS is expressed the increased production of NO enhances plasma leakage. These findings may implicate a role for NO in the maintenance of airway function and in the inflammatory process occurring in diseases such as asthma, where iNOS is known to be expressed.

The two enantiomers of cispentacin, an antifungal antibiotic, are determined by reversed phase HPLC, after derivatization with Marfey's reagent, in 24 h urine samples collected from rats treated sc and iv with 20 mg/kg cispentacin racemate obtained by synthesis. The application range of the method is 25-250 mg/L for each enantiomer with a precision of 4.0-9.0%. Comparison with an authentic sample of natural origin (-)-cispentacin indicated that (-) enantiomer is excreted unchanged in smaller percentage than (+) enantiomer, which is almost completely eliminated as such.

The rate and extent of passive pharmacokinetic processes, such as passive gastrointestinal absorption, tissue distribution and glomerular filtration, depend on the physical-chemical properties of a drug. In the case of chiral drugs, since enantiomers have the same properties, in general we would not expect passive processes to be stereoselective. On the contrary, enantioselectivity would occur for processes involving a drug interaction with a chiral macromolecule (e.g. receptors, enzymes, transport proteins), like the carrier-mediated absorption, protein binding, active renal and biliary secretion, metabolism. However, a few exceptions aside, the degree of stereoselectivity of these processes is relatively modest. In this paper we reviewed several examples of stereoselective kinetic processes and discussed the limits of the interpretation of pharmacokinetic data resulting from the application of non-stereospecific analytical methods.

The anti-ischemic action of ITF 296 was evaluated in isovolumic, electrically driven rabbit heart preparations subjected to temporary ischemia and reperfusion. Reduction of perfusion rate from 20 to 0.2 ml/min produced a sharp decrease of peak systolic pressure, left ventricular (LV)-developed pressure, and LV dP/dt, which culminated in complete ventricular arrest within 3-4 min. Thereafter, LV end-diastolic pressure (LVEDP) progressively increased, suggesting a severe ischemic episode. Reperfusion with 20 ml Krebs solution/min after 40 min of low-flow perfusion produced only minimal recovery from the rhythm disturbances associated with cardiac mechanical activity. During reperfusion, loss of myocardial elasticity was associated with a significant increase in coronary vascular resistance, as indicated by the augmentation of coronary perfusion pressure. Injection of ITF 296 (0.3-10 microM) into the perfusion system dose-dependently inhibited the increase in LVEDP that took place during ischemia and progressively improved the recovery of a regular rhythm in the isolated heart. Isosorbide dinitrate (ISDN) at a concentration of 10 microM exerted a protective action on the myocardium similar to that obtained with ITF 296 (3 microM). Treatment with ITF 296 (1 and 10 microM) resulted in a dose-dependent increase in the rate of 6-keto-PGF1 alpha biosynthesis during both the ischemia and the reperfusion period (+157% over basal values). Similar results were obtained when hearts were pretreated with ISDN (10 microM). Infusion of a buffer containing NG-monomethyl-L-arginine (L-NMMA; NO synthase inhibitor at 10 microM) just before reduction of flow, markedly exacerbated the effects of ischemia and reperfusion on the myocardium and increased coronary perfusion pressure. The effects of L-NMMA were clearly antagonized by ITF 296 (10 microM) or L-arginine (100 microM). Mechanical activity and sinus rhythm during reperfusion were rapidly and completely restored, and coronary resistance values were close to the preischemic values. As with ISDN, ITF 296 significantly increased the rate of synthesis of 6-keto-PGF1 alpha both under basal conditions and during reperfusion.

In anaesthetized ventilated guinea-pig, acetaldehyde (CAS 75-07-0) (40-80 mg/kg i.v.) elicits a dose-dependent increase in intratracheal pressure accompanied by an increase in circulating histamine. When acetaldehyde is injected repeatedly at 15 min intervals in capsaicin-desensitized animals, it already loses its activity at the second administration (50% reduction; p0.01); this does not happen in control animals. This phenomenon is even more marked when acetaldehyde is given at the dose of 80 mg/kg i.v., since at the third injection both the bronchoconstriction and the increase in blood histamine are almost completely reduced to baseline values. The increase in intratracheal pressure caused by acetaldehyde (20, 40, 80 mg/kg i.v.) is associated with a dose related increase in microvascular permeability and leakage of protein-bound Evans blue in lower tracheal tissue. This event and the bronchoconstrictor response caused by acetaldehyde (40 mg/kg i.v.) are 87% and 35% inhibited, respectively, (p0.01) in tachykinin-depleated animals. On the contrary, thiorphan (2 mg/kg i.v.) remarkably potentiates both the rise in intratracheal pressure (110%; p0.01) and Evans blue extravasation (215%; p0.01) induced by acetaldehyde (20 mg/kg i.v.) in normal guinea-pigs. Furthermore, treatment with CP-96,345, a selective tachykinin NK1-receptor antagonist, only prevents plasma extravasation in lower tracheal tissue (82% inhibition; p0.01) without affecting the bronchoconstriction caused by acetaldehyde (40 mg/kg i.v.).(ABSTRACT TRUNCATED AT 250 WORDS)

S-acetyl- and S-phenylacetyl-glutathione derivatives were synthesized by using a new procedure. The derivatives were incubated with rat plasma and red blood cells, and also with cytosol from rat liver, kidney and heart, or tissue slices from rat heart, kidney and liver. A limited hydrolysis of the compounds occurs in plasma, whereas hydrolysis occurs to a larger extent in tissue cytosols. Both purified and crude gamma-glutamyl-transpeptidase from different sources recognized the S-acetyl- and S-phenylacetyl derivatives as substrates. Intracellular glutathione increases after incubating the derivatives with red blood cells. A potential role of S-acetyl- and S-phenylacetyl-glutathione in replenishing cells with exogenous glutathione is envisaged.

Cyclosporin was administered (6 mg kg−1, i.v.) over 15 min, or (10 mg kg−1) by gavage, to two groups of 5 rats. Following i.v. infusion, cyclosporin exhibited triphasic behaviour with mean ± s.e.m. disposition half-lives of 9·0 ± 1·3 min, 4·0 ± 0·5 h and 16·0 ± 1·7 h. Following oral administration, peak blood concentration (Cmax) of 1290 ± 93 ng mL−1 was reached after 5 h, when cyclosporin absorption essentially ceased. The absolute bioavailability (F) of cyclosporin was 24·0%. Standard laboratory rat chow consisting of 2% corn oil did not appear to alter cyclosporin absorption kinetics.

After the i.v. administration of a single 10 mg/Kg dose of [14C] teicoplanin to rats, no substantial differences were found between males and females as regards the hematic profile, the excretion pattern and tissue distribution. About 74% of the administered radioactivity was recovered in the 0-120 hours interval and more than 90% of this amount was found in the first 24 hours. In the 0-120 hour interval, 7% of the dose was excreted with the faeces, while the total 14C excreted (including the cage washing) was 82% of the dose. A high affinity of [14C] teicoplanin for the kidneys and especially for the cortex area was displayed in the distribution study. Similar findings were observed when 7 intravenous doses (10 mg/Kg each) were given to male rats at 24 hour intervals. The percentage of the total dose found in urine seven days after the last administration remained close to 70% and also the excretion data observed in each collection interval after the last treatment were comparable with those obtained in the single dose study. The distribution in tissues was very similar to that of the single dose experiment. The high 14C concentration level found in the kidneys of animals killed at 5 min after the last treatment was cleared with a disappearance rate comparable with that observed in the single dose experiment.

The pharmacokinetic profile of [14C]teicoplanin was studied in male Sprague-Dawley rats given a single 10,000-U/kg intravenous dose. The disposition of the antimicrobial activity in the body was estimated by a three-compartment open model. Plasma concentration data were fitted to a three-exponent equation. The profile of total 14C in plasma was similar to that of the microbiological activity. The cumulative recovery of total 14C 5 days after drug administration averaged 76.3% of the administered dose in the urine and 8.7% in the feces. The residual dose remaining in the animal carcasses was 11.1%. Teicoplanin was widely distributed in the body. In almost all organs, the maximum concentration of [14C]teicoplanin was already reached at the first time of killing, which was 0.25 h after the administration of drug. The liver, kidneys, skin, and fat contained most of the residual dose found in the animal carcasses 120 h after administration and behaved as a deep compartment with the adrenal glands and spleen.