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Endothelium-Mediated Action of Analogues of the Endogenous Neuropeptide Kyotorphin (Tyrosil-Arginine): Mechanistic Insights from Permeation and Effects on Microcirculation
Abstract
Kyotorphin (KTP) is an endogenous peptide with analgesic properties when administered into the central nervous system (CNS). Its amidated form (L-Tyr-L-Arg-NH2; KTP-NH2) has improved analgesic efficacy after systemic administration, suggesting blood-brain barrier (BBB) crossing. KTP-NH2 also has anti-inflammatory action impacting on microcirculation. In this work, selected derivatives of KTP-NH2 were synthesized to improve lipophilicity and resistance to enzymatic degradation while introducing only minor changes in the chemical structure: N-terminal methylation and/or use of D amino acid residues. Intravital microscopy data show that KTP-NH2 having a D-Tyr residue, KTP-NH2-DL, efficiently decreases the number of leukocyte rolling in a murine model of inflammation induced by bacterial lipopolysaccharide (LPS): down to 46% after 30 min with 96 mM KTP-NH2-DL. The same molecule has lower ability to permeate membranes (relative permeability of 0.38) and no significant activity in a behavioral test which evaluates thermal nociception (hot-plate test). On the contrary, methylated isomers at 96 mM increase leukocyte rolling up to nearly 5-fold after 30 min suggesting a pro-inflammatory activity. They have maximal ability to permeate membranes (relative permeability of 0.8) and induce long-lasting antinociception.
... The kyotorphins D-Tyr-L-Arg-NH 2 (KTP-NH 2 -DL), N-methyl-L-Tyr-D-Arg (Me-KTP-NH 2 -LD), N-methyl-L-Tyr-L-Arg-NH 2 (Me-KTP-NH 2 ), L-Tyr-L-Arg-NH 2 (KTP-NH 2 ), ibuprofen-Tyr-Arg (IbKTP), and ibuprofen-Tyr-Arg-NH 2 (IbKTP-NH 2 ) were produced on a small scale through standard solid-phase synthesis using 9-fluorenylmethoxycarbonyl (Fmoc)/tertbutyl (t-Bu) strategy, as described by Perazzo et al. [40] and Ribeiro et al. [28,31]. All peptides were purified by RP-HPLC, and purity (97-99%) was assessed by mass spectrometry and proton nuclear magnetic resonance ( 1 H NMR) (Supplementary material). ...
... More recently, D-Tyr-L-Arg-NH 2 (KTP-NH 2 -DL, 96 µM) also decreased the number of rolling leukocytes in a murine model of inflammation induced by LPS, but did not reveal a significant analgesic activity in the hot plate test (Perazzo et al., 2016). This KTP analog and others analyzed in the same study seem to have an action on the endothelium. ...
The endogenous peptide kyotorphin (KTP) has been extensively studied since it was discovered in 1979. The dipeptide is distributed unevenly over the brain but the majority is concentrated in the cerebral cortex. The putative KTP receptor has not been identified yet. As many other neuropeptides, KTP clearance is mediated by extracellular peptidases and peptide transporters. From the wide spectrum of biological activity of KTP, analgesia was by far the most studied. The mechanism of action is still unclear, but researchers agree that KTP induces Met-enkephalins release. More recently, KTP was proposed as biomarker of Alzheimer disease. Despite all that, KTP limited pharmacological value prompted researchers to develop derivatives more lipophilic and therefore more prone to cross the blood–brain barrier (BBB), and also more resistant to enzymatic degradation. Conjugation of KTP with functional molecules, such as ibuprofen, generated a new class of compounds with additional biological properties. Moreover, the safety profile of these derivatives compared to opioids and their efficacy as neuroprotective agents greatly increases their pharmacological value.
Kyotorphin (KTP, l-tyrosyl-l-arginine) is an endogenous analgesic neuropeptide first isolated from bovine brain in 1979. Previous studies have shown that kyotorphins possess anti-inflammatory and antimicrobial activity. Six kyotorphins—KTP-NH2, KTP–NH2–DL, ibuprofen-conjugated KTP (IbKTP), IbKTP-NH2, N-methyl-D-Tyr-L-Arg, and N-methyl-L-Tyr-D-Arg—were designed and synthesized to improve lipophilicity and resistance to enzymatic degradation. This study assessed the antimicrobial and antibiofilm activity of these peptides. The antifungal activity of kyotorphins was determined in representative strains of Candida species, including Candida albicans ATCC 10231, Candida krusei ATCC 6258, and six clinical isolates—Candida dubliniensis 19-S, Candida glabrata 217-S, Candida lusitaniae 14-S, Candida novergensis 51-S, Candida parapsilosis 63, and Candida tropicalis 140-S—obtained from the oral cavity of HIV-positive patients. The peptides were synthesized by standard solution or solid-phase synthesis, purified by RP-HPLC (purity >95 %), and characterized by nuclear magnetic resonance. The results of the broth microdilution assay and scanning electron microscopy showed that IbKTP-NH2 presented significant antifungal activity against Candida strains and antibiofilm activity against the clinical isolates. The absence of toxic activity and survival after infection was assessed after injecting the peptide in larvae of Galleria mellonella as experimental infection model. Furthermore, IbKTP-NH2 had strong antimicrobial activity against multidrug-resistant bacteria and fungi and was not toxic to G. mellonella larvae up to a concentration of 500 mM. These results suggest that IbKTP-NH2, in addition to its known effect on cell membranes, can elicit a cellular immune response and, therefore, is promising for biomedical application.
Kyotorphin is a unique biologically active neuropeptide (L-tyrosine-L-arginine), which is reported to have opioid-like analgesic actions through a release of Met-enkephalin from the brain slices. N-methyl-L-tyrosine-L-arginine (NMYR), an enzymatically stable mimetic of kyotorphin, successfully caused potent analgesic effects in thermal and mechanical nociception tests in mice when it was given through systemic routes. NMYR analgesia was abolished in μ-opioid receptor-deficient (MOP-KO) mice, and by intracerebroventricular (i.c.v.) injection of naloxone and of N-methyl L-leucine-L-arginine (NMLR), a kyotorphin receptor antagonist. In the Ca2+-mobilization assay using CHO cells expressing Gαqi5 and hMOPr or hDOPr, however, the addition of kyotorphin neither activated MOPr-mechanisms, nor affected the concentration-dependent activation of DAMGO- or Met-Enkephalin-induced MOPr activation, and Met-enkephalin-induced DOPr activation. NMYR-analgesia was significantly attenuated in preproenkephalin (PENK)- or proopioimelanocortin (POMC)-KO mice. The systemic administration of arginine, which is reported to elevate the level of endogenous kyotorphin selectively in midbrain and medulla oblongata, pain-related brain regions, caused significant analgesia, and the analgesia was reversed by i.c.v. injection of NMLR or naloxone. In addition, PENK- and POMC-KO mice also attenuated the arginine-induced analgesia. All these findings suggest that NMYR and arginine activate brain kyotorphin receptor in direct and indirect manner, respectively and both compounds indirectly cause the opioid-like analgesia through the action of endogenous opioid peptides.
Neuropeptide Kyotorphin (KTP) is a potent analgesic if administered directly into the brain. In contrast, KTP-amide (KTP-NH2) is analgesic, neuroprotective and anti-inflammatory following systemic administration, albeit its mechanism of action is unknown. The aim of this study was to shed light on the mechanism of action of KTP-NH2 at the molecular level. KTP-NH2 does not inhibit the enkephalinases angiotensin-converting-enzyme and dipeptidyl-peptidase 3. Intravital microscopy showed that KTP-NH2 decreased the number of rolling leukocytes in a mouse model of inflammation induced by lipopolysaccharide (LPS). Pre-treatment with metyrapone abrogated the action of KTP-NH2. Interestingly, stimulating rolling leukocytes using CXCL-1 is also counteracted by the KTP-NH2, but this effect is not abrogated by metyrapone. We conclude that KTP-NH2 has dual action: a glucocorticoid-mediated action, which is dominant in the full-fledged LPS-induced inflammation model, and a glucocorticoid-independent mechanism, which is predominant in models in which leukocyte rolling is stimulated but inflammation is not totally developed.
Chronic brain ischemia is a prominent risk factor for neurological dysfunction and progression for dementias, including Alzheimer’s disease (AD). In rats, permanent bilateral common carotid artery occlusion (2VO) causes a progressive neurodegeneration in the hippocampus, learning deficits and memory loss as it occurs in AD. Kyotorphin (KTP) is an endogenous antinociceptive dipeptide whose role as neuromodulator/neuroprotector has been suggested. Recently, we designed two analgesic KTP-derivatives, KTP-amide (KTP-NH2) and KTP-NH2 linked to ibuprofen (IbKTP-NH2) to improve KTP brain targeting. This study investigated the effects of KTP-derivatives on cognitive/behavioral functions (motor/spatial memory/nociception) and hippocampal pathology of female rats in chronic cerebral hypoperfusion (2VO-rat model). 2VO-animals were treated with KTP-NH2 or IbKTP-NH2 for 7 days at weeks 2 and 5 post-surgery. After behavioral testing (week 6), coronal sections of hippocampus were H&E-stained or immunolabeled for the cellular markers GFAP (astrocytes) and NFL (neurons). Our findings show that KTP-derivatives, mainly IbKTP-NH2, enhanced cognitive impairment of 2VO-animals and prevented neuronal damage in hippocampal CA1 subfield, suggesting their potential usefulness for the treatment of dementia.
This paper addresses the mechanisms behind selective endothelial permeability and their regulations. The singular properties of each of the seven blood-tissues barriers. Then, it further revisits the physical, quantitative meaning of permeability, and the way it should be measured based on sound physical chemistry reasoning and methodologies. Despite the relevance of permeability studies one often comes across inaccurate determinations, mostly from oversimplified data analyses. To worsen matters, the exact meaning of permeability is being lost along with this loss of accuracy. The importance of proper permeability calculation is illustrated with a family of derivatives of kyotorphin, an analgesic dipeptide.
In Alzheimer's disease (AD), besides the characteristic deterioration of memory, studies also point to a higher pain tolerance in spite of sensibility preservation. A change in the normal tau protein phosphorylation is also characteristic of AD, which contributes to the pathogenesis of the disease and is useful in early diagnosis. Kyotorphin (KTP) is an endogenous analgesic dipeptide (Tyr-Arg) for which there is evidence of eventual neuroprotective and neuromodulatory properties. The objective of this work was to study the possible correlation between KTP and phosphorylated tau protein (p-tau) levels in cerebro-spinal fluid (CSF) samples of AD patients. CSF samples were collected from 25 AD patients and 13 age-matched controls (N), where p-tau and KTP levels were measured. We found a statistically significant difference between p-tau/KTP values in AD and N groups with an inverse correlation between p-tau and KTP values in AD samples. These results suggest that in the future KTP may be a candidate biomarker for neurodegeneration and may be a lead compound to be used pharmacologically for neuroprotection.
Time-lapse fluorescence microscopy is one of the main tools used to image subcellular structures in living cells. Yet for decades it has been applied primarily to in vitro model systems. Thanks to the most recent advancements in intravital microscopy, this approach has finally been extended to live rodents. This represents a major breakthrough that will provide unprecedented new opportunities to study mammalian cell biology in vivo and has already provided new insight in the fields of neurobiology, immunology, and cancer biology.
The adverse side-effects associated with opioid administration restrain their use as analgesic drugs and call for new solutions to treat pain. Two kyotorphin derivatives, kyotorphin-amide (KTP-NH2) and ibuprofen-KTP-NH2 (IbKTP-NH2) are promising alternatives to opioids: they trigger analgesia via an indirect opioid mechanism and are highly effective in several pain models following systemic delivery. In vivo side-effects of KTP-NH2 and IbKTP-NH2 are, however, unknown and were evaluated in the present study using male adult Wistar rats. For comparison purposes, morphine and tramadol, two clinically relevant opioids, were also studied. Results showed that KTP-derivatives do not cause constipation after systemic administration, in contrast to morphine. Also, no alterations were observed in blood pressure or in food and water intake, which were only affected by tramadol. A reduction in micturition was detected after KTP-NH2 or tramadol administrations. A moderate locomotion decline was detected after IbKTP-NH2-treatment. The side-effect profile of KTP-NH2 and IbKTP-NH2 support the existence of opioid-based mechanisms in their analgesic actions. The conjugation of a strong analgesic activity with the absence of the major side-effects associated to opioids highlights the potential of both KTP-NH2 and IbKTP-NH2 as advantageous alternatives over current opioids.
Mammalian cell membranes regulate homeostasis, protein activity, and cell signaling. The charge at the membrane surface has been correlated with these key events. Although mammalian cells are known to be slightly anionic, quantitative information on the membrane charge and the importance of electrostatic interactions in pharmacokinetics and pharmacodynamics remain elusive. Recently, we reported for the first time that brain endothelial cells (EC) are more negatively charged than human umbilical cord cells, using zeta-potential measurements by dynamic light scattering. Here, we hypothesize that anionicity is a key feature of the blood-brain barrier (BBB) and contributes to select which compounds cross into the brain. For the sake of comparison, we also studied the membrane surface charge of blood components-red blood cells (RBC), platelets, and peripheral blood mononuclear cells (PBMC). To further quantitatively correlate the negative zeta-potential values with membrane charge density, model membranes with different percentages of anionic lipids were also evaluated. From all the cells tested, brain cell membranes are the most anionic and those having their lipids mostly exposed, which explains why lipophilic cationic compounds are more prone to cross the blood-brain barrier.
Intravital microscopy is an extremely powerful tool that enables imaging several biological processes in live animals. Recently, the ability to image subcellular structures in several organs combined with the development of sophisticated genetic tools has made possible extending this approach to investigate several aspects of cell biology. Here we provide a general overview of intravital microscopy with the goal of highlighting its potential and challenges. Specifically, this review is geared toward researchers that are new to intravital microscopy and focuses on practical aspects of carrying out imaging in live animals. Here we share the know-how that comes from first-hand experience, including topics such as choosing the right imaging platform and modality, surgery and stabilization techniques, anesthesia and temperature control. Moreover, we highlight some of the approaches that facilitate subcellular imaging in live animals by providing numerous examples of imaging selected organelles and the actin cytoskeleton in multiple organs.
Kyotorphin (KTP; L-Tyr-L-Arg), an endogenous neuropeptide, is potently analgesic when delivered directly to the central nervous system. Its weak analgesic effects after systemic administration have been explained by inability to cross the blood-brain barrier (BBB) and detract from the possible clinical use of KTP as an analgesic. In this study, we aimed to increase the lipophilicity of KTP by amidation and to evaluate the analgesic efficacy of a new KTP derivative (KTP-amide - KTP-NH(2) ).
We synthesized KTP-NH(2) . This peptide was given systemically to assess its ability to cross the BBB. A wide range of pain models, including acute, sustained and chronic inflammatory and neuropathic pain, were used to characterize analgesic efficacies of KTP-NH(2) . Binding to opioid receptors and toxicity were also measured.
KTP-NH(2) , unlike its precursor KTP, was lipophilic and highly analgesic following systemic administration in several acute and chronic pain models, without inducing toxic effects or affecting motor responses and blood pressure. Binding to opioid receptors was minimal. KTP-NH(2) inhibited nociceptive responses of spinal neurons. Its analgesic effects were prevented by intrathecal or i.p. administration of naloxone.
Amidation allowed KTP to show good analgesic ability after systemic delivery in acute and chronic pain models. The indirect opioid-mediated actions of KTP-NH(2) may explain why this compound retained its analgesic effects although the usual side effects of opioids were absent, which is a desired feature in next-generation pain medications.
The ketogenic diet is a high-fat, low-carbohydrate regimen that forces ketone-based rather than glucose-based cellular metabolism. Clinically, maintenance on a ketogenic diet has been proven effective in treating pediatric epilepsy and type II diabetes, and recent basic research provides evidence that ketogenic strategies offer promise in reducing brain injury. Cellular mechanisms hypothesized to be mobilized by ketone metabolism and underlying the success of ketogenic diet therapy, such as reduced reactive oxygen species and increased central adenosine, suggest that the ketolytic metabolism induced by the diet could reduce pain and inflammation. To test the effects of a ketone-based metabolism on pain and inflammation directly, we fed juvenile and adult rats a control diet (standard rodent chow) or ketogenic diet (79% fat) ad libitum for 3-4 weeks. We then quantified hindpaw thermal nociception as a pain measure and complete Freund's adjuvant-induced local hindpaw swelling and plasma extravasation (fluid movement from the vasculature) as inflammation measures. Independent of age, maintenance on a ketogenic diet reduced the peripheral inflammatory response significantly as measured by paw swelling and plasma extravasation. The ketogenic diet also induced significant thermal hypoalgesia independent of age, shown by increased hindpaw withdrawal latency in the hotplate nociception test. Anti-inflammatory and hypoalgesic diet effects were generally more robust in juveniles. The ketogenic diet elevated plasma ketones similarly in both age groups, but caused slowed body growth only in juveniles. These data suggest that applying a ketogenic diet or exploiting cellular mechanisms associated with ketone-based metabolism offers new therapeutic opportunities for controlling pain and peripheral inflammation, and that such a metabolic strategy may offer significant benefits for children and adults.
The contribution of behavioral endpoint to results obtained in the 55 degrees C rat hot plate procedure was assessed. Specifically, the use of a hind paw lick-only endpoint was compared to that of a hind paw lick-or-jump endpoint. Effects of prototypical analgesic and non-analgesic compounds on response latency increases were determined under each condition. Whereas the effects of morphine, oxycodone and codeine were similar under each condition, effects of a number of non-analgesic agents differed markedly depending upon the endpoint used. Clozapine, chlorpromazine, thioridazine, atropine, scopolamine, benactyzine, yohimbine, idazoxan and cyproheptadine produced dose-dependent increases in response latency under the hind paw lick-only condition but did not increase latencies when the hind paw lick-or-jump endpoint was used. Haloperidol, sulpiride, benztropine, methyl atropine, phentolamine, prazosin, methiothepin, methysergide, diphenhydramine, pargyline and diazepam failed to increase response latencies under the hind paw lick-only condition. Moreover, whereas diazepam, chlorpromazine, pentobarbital, dantrolene and ethanol produced dose-dependent increases in the height required for successful aerial righting, increases in hind paw lick-or-jump latencies occurred only following near-anesthetic doses of pentobarbital and ethanol. These data indicate that the hind paw lick endpoint is susceptible to perturbation by extraneous pharmacologic activities. Drugs exerting muscarinic cholinergic and alpha 2-adrenergic antagonist effects are particularly able to disrupt this behavior. Disruption is not associated specifically with any other pharmacologic action, although other activities may interfere with the response. In contrast, the hind paw lick-or-jump endpoint fails to detect skeletal muscle relaxant activity and only detects gross motor impairment when near-anesthetic doses of drugs are used. The present data suggest that detection of non-analgesic drug activities by rat hot plate can be minimized by use of a hind paw lick-or-jump endpoint.
We attempted to identify the kyotorphin receptor and the post receptor mechanisms mediated by GTP-binding proteins (G-proteins), using reconstitution techniques. The specific binding of [3H]kyotorphin in rat brain membranes was composed of high affinity (Kd = 0.34 nM) and low affinity (Kd = 9.07 nM) binding. As the high affinity binding disappeared in the presence of guanosine 5'-O-(3-thiotriphosphate) and MgCl2, we investigated the kyotorphin receptor-mediated changes in membrane G-protein activity by measuring low Km GTPase activity. Kyotorphin produced a stimulation of low Km GTPase, and this stimulation was antagonized by Leu-Arg, a synthetic dipeptide which showed a potent displacement of [3H]kyotorphin binding, yet in itself had no effect on the low Km GTPase. The kyotorphin stimulation of low Km GTPase was abolished by pretreating membranes with islet-activating protein, pertussis toxin, and was recovered by reconstitution with purified G-protein, Gi, but not with Go. Similar evidence of selective coupling of kyotorphin receptor to Gi was obtained with the phospholipase C assay. Kyotorphin-induced stimulation of phospholipase C was also abolished by islet-activating protein-treatment and recovered by reconstitution with Gi but not with Go. These findings indicate that specific high and low affinity kyotorphin receptors exist in the rat brain and that the kyotorphin receptor is functionally coupled to stimulation of phospholipase C, through Gi. This study provides the first evidence of a selective involvement of Gi in the receptor-mediated activation of phospholipase C.
The design, synthesis, and pharmacological evaluation of brain-targeted chemical delivery systems (CDS) for a kyotorphin analogue (Tyr-Lys) are described. The brain-targeted compound contains the active peptide in a packaged, disguised form, flanked between the lipophilic cholesteryl ester on the C-terminus and the 1, 4-dihydrotrigonellyl redox targetor, attached to the N-terminus through strategically selected L-amino acid(s) spacer. It was found that for successful brain targeting, the epsilon-amine of Lys needs to be also converted to a lipophilic function. Through sequential enzymatic bioactivation, the Tyr-Lys dipeptide is released in a sustained manner, producing significant and prolonged analgesic activity as demonstrated by the rat tail latency test. An alternate strategy was also employed. Lys was replaced by a redox amino acid pair, Nys+ left and right arrow Nys, the nicotinamide left and right arrow 1,4-dihydronicotinamide analogues of Lys (Nys+ is 2-amino-6-(3-carbamoyl-1-pyridiniumyl)hexanoic acid). The Nys form is lipophilic and facilitates delivery in addition to the C- and N-terminal lipophilic functions. Enzymatic oxidation to Nys+ provides the lock-in, followed by removal of the lipophilic groups, releasing Tyr-Nys+ from the brain-targeted analogue (BTRA). Nys+ was shown to be an effective substitution for Arg or Lys. The activities of the CDS and BTRA, respectively, were antagonized by naloxone, supporting the designed brain-targeted processes. The most potent compound is the two-proline spacer containing CDS (CDS-PP), followed by the BTRA.
The study of pain in awake animals raises ethical, philosophical, and technical problems. We review the ethical standards for studying pain in animals and emphasize that there are scientific as well as moral reasons for keeping to them. Philosophically, there is the problem that pain cannot be monitored directly in animals but can only be estimated by examining their responses to nociceptive stimuli; however, such responses do not necessarily mean that there is a concomitant sensation. The types of nociceptive stimuli (electrical, thermal, mechanical, or chemical) that have been used in different pain models are reviewed with the conclusion that none is ideal, although chemical stimuli probably most closely mimic acute clinical pain. The monitored reactions are almost always motor responses ranging from spinal reflexes to complex behaviors. Most have the weakness that they may be associated with, or modulated by, other physiological functions. The main tests are critically reviewed in terms of their sensitivity, specificity, and predictiveness. Weaknesses are highlighted, including 1) that in most tests responses are monitored around a nociceptive threshold, whereas clinical pain is almost always more severe; 2) differences in the fashion whereby responses are evoked from healthy and inflamed tissues; and 3) problems in assessing threshold responses to stimuli, which continue to increase in intensity. It is concluded that although the neural basis of the most used tests is poorly understood, their use will be more profitable if pain is considered within, rather than apart from, the body's homeostatic mechanisms.
The role of spinal cord mu-opioid receptor (MOR)-expressing dorsal horn neurons in nociception and morphine analgesia is incompletely understood. Using intrathecal dermorphin-saporin (Derm-sap) to selectively destroy MOR-expressing dorsal horn neurons, we sought to determine the role of these neurons in (1) normal baseline reflex nocifensive responses to noxious thermal stimulation (hotplate, tail flick) and to persistent noxious chemical stimulation (formalin) and (2) the antinociceptive activity of intrathecal and systemic morphine in the same tests. Lumbar intrathecal Derm-sap (500 ng) produced (1) partial loss of lamina II MOR-expressing dorsal horn neurons, (2) no effect on MOR-expressing dorsal root ganglion neurons, and (3) no change in baseline tail-flick and hotplate reflex nocifensive responses. Derm-sap treatment attenuated the antinociceptive action of both intrathecal and systemic morphine on hotplate responses. Derm-sap treatment had two effects in the formalin test: (1) increased baseline nocifensive responding and (2) reduced antinociceptive action of systemic morphine. We conclude that MOR-expressing dorsal horn neurons (1) are not essential for determining nocifensive reflex responsiveness to noxious thermal stimuli, (2) are necessary for full antinociceptive action of morphine (intrathecal or systemic) in these tests, and (3) play a significant role in the endogenous modulation of reflex nocifensive responses to persistent pain in the formalin test. Thus, one would predict that altering the activity of MOR-expressing dorsal horn neurons would be antinociceptive and of interest in the search for new approaches to management of chronic pain.
Recently, a designed class of efficient analgesic drugs derived from an endogenous neuropeptide, kyotorphin (KTP, Tyr-Arg) combining C-terminal amidation (KTP-NH2) and N-terminal conjugation to ibuprofen (Ib), IbKTP-NH2, was developed. The Ib moiety is an enhancer of KTP-NH2 analgesic action. In the present study, we have tested the hypothesis that KTP-NH2 is an enhancer of the Ib anti-inflammatory action. Moreover, the impact of the IbKTP-NH2 conjugation on microcirculation was also evaluated by a unified approach based on intravital microscopy in the murine cremasteric muscle. Our data show that KTP-NH2 and conjugates do not cause damage on microcirculatory environment and efficiently decrease the number of leukocyte rolling induced by lipopolysaccharide (LPS). Isothermal titration calorimetry showed that the drugs bind to LPS directly thus contributing to LPS aggregation and subsequent elimination. In a parallel study, molecular dynamics simulations and NMR data showed that the IbKTP-NH2 tandem adopts a preferential "stretched" conformation in lipid bilayers and micelles, with the simulations indicating that the Ib moiety is anchored in the hydrophobic core, which explains the improved partition of IbKTP-NH2 to membranes and the permeability of lipid bilayers to this conjugate relative to KTP-NH2. The ability to bind glycolipids concomitant to the anchoring in the lipid membranes through the Ib residue explains the analgesic potency of IbKTP-NH2 given the enriched glycocalyx of the blood-brain barrier cells. Accumulation of IbKTP-NH2 in the membrane favors both direct permeation and local interaction with putative receptors as the location of the KTP-NH2 residue of IbKTP-NH2 and free KTP-NH2 in lipid membranes is the same.
Amidated kyotorphin (L-Tyr-L-Arg-NH2; KTP-NH2) causes analgesia when systemically administered. The lipophilic ibuprofen-conjugated derivative of KTP-NH2 has improved analgesic efficacy. However, fast degradation by peptidases impacts negatively in the pharmacodynamics of these drugs. In this work, selected derivatives of KTP and KTP-NH2 were synthesized to combine lipophilicity and resistance to enzymatic degradation. Eight novel structural modifications were tested for the potential to transverse lipid membranes and to evaluate their efficacy in vivo. The rationale behind the design of the pool of the eight selected molecules consisted in the addition of individual group at the N-terminus, namely the tert-butyloxycarbonyl (Boc), γ-aminobutyric acid (GABA), acetyl, butanoyl, and propanoyl or in the substitution of the tyrosine residue by an indole moiety and in the replacement of the peptidic bond by a urea-like bond in some cases. All the drugs used in the study are intrinsically fluorescent, which enables the use of spectrofluorimetry to sample the drugs in the permeation assays. The results show that the BOC and indolyl derivatives of KTP-NH2 have maximal ability to permeate membranes with concomitant maximal analgesic power. Overall, the results demonstrate that membrane permeation is correlated with analgesic efficacy. However, this is not the only factor accounting for analgesia. KTP-NH2 for instance has low passive permeation but is known to have central action. In this case, hypothetical transcytosis over the blood-brain barrier seems to depend on dipeptide transporters.
A trifluoroacetic acid (TFA) labile protecting group for the guanidine side chain function of arginine has been developed. NG-(2,2,5,7,8-Pentamethylchroman-6-sulphonyl-L-arginine is cleaved rapidly in TFA or 50% TFA in dichloromethane at room temperature. The preparations of Fmoc.Arg(Pmc).OH and Bnpeoc.Arg(Pmc).OH are described.Graphical abstract2,2,5,7,8 Pentamethylchroman-6-sulphonyl chloride (10) affords effective TFA-labile NG-protected derivatives for Arg in solution and solid phase peptide synthesis
The acid-labile 5-(4-(9-fluorenylmethyloxycarbonyl)aminomethyl-3,5-dimethoxyphenoxy)valeric acid (PAL) handle 1 is described for the solid-phase synthesis of C-terminal peptide amides. The pure para isomer of 1 was prepared by each of two efficient five-step routes, in overall yields from 52% to 74%. The handle 1 was coupled onto a variety of amino group-containing supports to provide a general starting point for stepwise assembly of peptide chains according to a wide range of chemistries. In particular, protocols based on the base-labile Nα-9-fluorenylmethyloxycarbonyl (Fmoc) group worked well with PAL handle 1. For small model peptides, final cleavage of tert-butyl side-chain protecting groups and of the anchoring linkage proceeded smoothly in trifluoroacetic acid-dichloromethane-dimethyl sulfide (14:5:1) (reagent A) at 25°C for 2 h. For cleavage of complex peptides that contain several sensitive side-chain functionalities, or that include arginine residues blocked with the 4-methoxy-2,3,6-trimethylphenylsulfonyl (Mtr) or 2,2,5,7,8-pentamethylchroman-6-ylsulfonyl (Pmc) groups, a mixture of trifluoroacetic acid-thioanisole-1,2-ethanedithiol-anisole (90:5:3:2) (reagent R), applied for 2-8 h at 25°C, was preferred. A side reaction involving alkylation at tryptophan was elucidated, and conditions were developed to minimize its occurrence. The methodology was demonstrated by syntheses of over a hundred peptides, among which acyl carrier protein (65-74) amide (natural and retro sequences), luteinizing hormone-releasing hormone, adipokinetic hormone, PHI porcine fragment (18-27), and human gastrin-I are highlighted in this report. In comparative studies, the yields and purities of peptide amides prepared with PAL were shown to be equivalent or superior to those found for products prepared by alternative procedures from the recent literature.
[Ala8]-dynorphin A, a potent kappa-selective opioid heptadecapeptide with numerous sensitive side-chain residues, has been prepared by solid-phase synthesis using base-labile N(alpha)-9-fluorenylmethyloxycarbonyl (Fmoc) protection and side-chain anchoring to a tris(alkoxy)benzylamide ''PAL''-resin. Final cleavage and deprotection was carried out with reagent R, trifluoroacetic acid-thioanisole-1,2-ethanedithiol-anisole (90:5:3:2), reagent K, trifluoroacetic acid-phenol-water-thioanisole-1,2-ethanedithiol (82.5:5:5:5:2.5), and reagent B, trifluoroacetic acid-phenol-water-triisopropylsilane (88:5:5:2); optimal reaction and workup conditions are described. Crude peptide products were evaluated by analytical high-performance liquid chromatography (HPLC), capillary zone electrophoresis (CZE), and direct fast atom bombardment and ion electrospray mass spectrometry (FABMS and ESMS). Tryptophan alkylation side reactions involving a 2,4,6-trimethoxybenzyl (Tmob) group from asparagine or 2,2,5,7,8-pentamethylchroman-6-ylsulfonyl (Pmc) groups from arginines occur under insufficiently long cleavage times and/or when certain scavenger components are omitted from the cleavage cocktails, but can be minimized under the best conditions. Conditions with reagent B, 1 h, 25-degrees-C, and extractive workup, were followed up preparatively to provide [Ala8]-dynorphin A in excellent purity (>99%) and 58% overall isolated yield based on the C-terminal amino acid anchored on the resin.
Proteases have been shown to signal to cells through the activation of a novel class of receptors coupled to G proteins: the protease-activated receptors (PARs). Those receptors are expressed in a wide range of cells, which ultimately are all involved in mechanisms of inflammation and pain. Numerous studies have considered the role of PARs in cells, organ systems or in vivo, highlighting the fact that PAR activation results in signs of inflammation. A growing body of evidences discussed here suggests that these receptors, and the proteases that activate them, interfere with inflammation and pain processes. Whether a role for PARs has been clearly defined in inflammatory and pain pathologies is discussed in this review. Further, the pros and cons for considering PARs as targets for the development of therapeutic options for the treatment of inflammation and pain are discussed.
The characteristics of kyotorphin (Tyr-Arg)-induced release of Met-enkephalin from the striatum and the spinal cord of guinea pig were determined by superfusing the slices in vitro and then carrying out radioimmunoassays. Depolarization by 50 mM K+ induced a marked release of Met-enkephalin-like immunoreactivity. The potassium-induced release of Met-enkephalin was calcium-dependent. In preparations from the striatum, the addition of kyotorphin to the superfusion medium produced a concentration-dependent increase in Met-enkephalin. The kyotorphin-induced release of Met-enkephalin was calcium-dependent and was abolished by tetrodotoxin. Similar effects of kyotorphin were seen in the spinal cord preparations. Electrical field stimulation of the striatal slices at a frequency of 10 Hz also evoked significant and calcium-dependent increases in the release of Met-enkephalin and markedly enhanced the kyotorphin-induced release of Met-enkephalin, as compared to the controls not given field stimulation. These results suggest that kyotorphin depolarizes the so-called enkephalinergic neurons and releases Met-enkephalin from the nerve terminals. This effect of kyotorphin may be a possible mechanism related to the manifestation of analgesia.
The pharmaceutical potential of natural analgesic peptides is mainly hampered by their inability to cross the blood-brain barrier, BBB. Increasing peptide-cell membrane affinity through drug design is a promising strategy to overcome this limitation. To address this challenge, we grafted ibuprofen (IBP), a nonsteroidal anti-inflammatory drug, to kyotorphin (l-Tyr-l-Arg, KTP), an analgesic neuropeptide unable to cross BBB. Two new KTP derivatives, IBP-KTP (IbKTP-OH) and IBP-KTP-amide (IbKTP-NH(2)), were synthesized and characterized for membrane interaction, analgesic activity and mechanism of action. Ibuprofen enhanced peptide-membrane interaction, endowing a specificity for anionic fluid bilayers. A direct correlation between anionic lipid affinity and analgesic effect was established, IbKTP-NH(2) being the most potent analgesic (from 25 μmol · kg(-1)). In vitro, IbKTP-NH(2) caused the biggest shift in the membrane surface charge of BBB endothelial cells, as quantified using zeta-potential dynamic light scattering. Our results suggest that IbKTP-NH(2) crosses the BBB and acts by activating both opioid dependent and independent pathways.
The affinity of a drug candidate for a biological membrane (its lipophilicity) is closely related to the pharmacologically crucial events of absorption, biodistribution, metabolization and excretion. The evolution of knowledge of biological membranes during the past two decades contrasts with the rudimentary parameter most commonly used to assess lipophilicity: P(o/w), the octanol-water partition coefficient. P(o/w) is especially unrealistic when testing molecules that are polar or partially charged. By contrast, lipid vesicle-based methods determine the extent of the actual partition of a drug to a membrane much more accurately, and have the additional advantage of enabling the choice of the lipid composition considered most suitable to answer a specific biological or pharmaceutical question. In addition, some of these methods are appropriate for high throughput screening, thus shifting determinations of membrane partition to a more preliminary stage of drug development. This streamlines research and development, by saving the time and money that would be spent on unpromising leads.
Herpes Simplex Virus type 1 (HSV-1) vectors are known to inhibit nociceptive transmission at the spinal cord after peripheral applications. Similar approaches may also be useful when applied at the supraspinal pain control system as the system includes pronociceptive (facilitatory) components. We performed a study aimed to analyse the migration of HSV-1 along with the inhibition of pronociception from the medullary dorsal reticular nucleus (DRt), a major facilitatory component of the supraspinal pain control system. To study the migration, a HSV-1 vector expressing lacZ under control of the human cytomegalovirus (hCMV) promoter was injected in the DRt and the expression of beta-galactosidase (beta-gal) was detected at 2, 4, 7, 10 and 14 days. Numerous beta-gal-immunoreactive neurons were observed at the injection site until day 4, and at some of the brain areas projecting to the DRt until day 7. To block the pronociceptive effects of the DRt, a HSV-1 vector expressing the preproenkephalin transgene, under the control of hCMV promoter, was injected into the DRt. Behavioural evaluation was performed at the time-points referred above, using the paw withdrawal latency test to evaluate thermal nociceptive responses. Anti-hyperalgesic effects persisted during 4 days, decreasing after that time-point. The present study demonstrates that selective migration of HSV-1 should be considered in gene therapy strategies based on HSV-1 injections into the brain. The study also shows that it is possible to decrease pain facilitation from the brain using opioidergic inhibition of pronociceptive supraspinal areas.
It is generally accepted that morphine exerts its analgesic effect by binding to specific opiate receptors in the brain and spinal cord. Since Hughes et al. isolated and identified two endogenous pentapeptides, Met- and Leu-enkephalin, from the brain and found that they acted as agonists at opiate receptors, alpha-, beta- and gamma-endorphins, larger peptides than enkephalins and having morphine-like activity, have been identified in either the brain or pituitary of various species. Several studies have demonstrated that enkephalins possess analgesic properties and that they are distributed in the pain-mediated pathways in the central nervous system. These findings suggest that enkephalins are important neurotransmitters or neuromodulators regulating pain transmission. We now report the isolation of a novel substance which has a Met-enkephalin releasing action. Our findings suggest the possibility of a regulating mechanism for the release of endogenous opioid peptides, especially Met-enkephalin.
One of the main problems still hampering solid-phase peptide synthesis using orthogonal protection strategies based on the 9-fluorenylmethoxycarbonyl amino protecting group is the difficult removal of currently used arginine arylsulphonyl guanidino protecting groups. Poor acid liability of 4-methoxy-2,3,6-trimethylbenzenesulphonyl-protected arginine has led to the popularity of the newer 2,2,5,7,8- pentamethylchroman-6-sulphonyl guanidino protecting group. This group was initially believed to have liability to trifluoroacetic acid, the reagent commonly used to simultaneously deprotect peptides and detach them from the synthesis resin, comparable to tert.-butyl and trityl type protecting groups used for the protection of other peptide side-chain functionalities. In a comparison of three established cleavage/deprotection mixtures we have shown that this is not always the case, particularly in multiple arginine peptides. We have found that only hard-acid deprotection with trimethylsilyl bromide reliably removed both arylsulphonyl guanidino protecting groups from a variety of arginine-containing peptides.
1. A new simple method is described for quantitating the adhesiveness of circulating polymorphonuclear leucocytes, or granulocytes, to the walls of blood vessels. The cheek pouch of anaesthetized hamsters or a small part of the mesentery of anaesthetized mice were prepared for continuous microscopic observation of selected venules. Those granulocytes which moved sufficiently slowly to be individually visible were counted for 1 or 2 min periods as they rolled past a selected point on one side of a vessel. The velocity distribution of these cells was determined by analysing films. Films were used also to measure mean blood flow velocity in the venules by observing embolizing platelet thrombi induced by the iontophoretic application of adenosine diphosphate. Emigration of granulocytes into the tissues was quantitated by enumerating them in standard areas of stained histological sections.
2. In control experiments with hamster cheek pouch venules, the rolling granulocyte count usually passed through a maximum shortly after the preparation was set up and then fell to a low constant value. In mouse mesentery venules the count remained at a low approximately constant value from the beginning for at least 3 hr.
3. The mean velocity of blood flow in the venules was between 900 and 200 μ/sec. All rolling granulocytes moved much more slowly; in hamster cheek pouch venules the mean velocity was about 20 μ/sec and in mouse mesentery venules about 10 μ/sec. Around these means the velocity distribution of individual cells was narrow.
4. Rolling of granulocytes was abolished by superfusing ethylenediamine tetra‐acetate (EDTA, 0·1 M ) suggesting that the phenomenon depends on calcium or magnesium ions.
5. Agents were applied locally to the observed venules. Human serum albumin, trypsin or histamine in high concentrations did not affect the rolling granulocyte count.
6. The rolling granulocyte count was increased during the application of Hammarsten casein or Escherichia coli culture filtrate which are chemotactic to granulocytes in vitro . These agents did not cause alterations in mean blood flow velocity in the observed venules which might have accounted for the effect on the rolling granulocyte counts. When E. coli culture filtrate was perfused through mouse intestine the increase in rolling granulocyte count in the draining venous blood was proportional to the logarithm of the concentration of filtrate.
7. The rolling granulocyte count was also increased by the local application of plasma globulin permeability factor or lymph node permeability factor.
8. Granulocyte counts in standard histological sections showed no significant increases in control preparations but considerable increases following the application of Hammarsten casein.
To examine the physiological role of the analgesic dipeptide, kyotorphin (Tyr-Arg), which was isolated from the mammalian brain, its subcellular localization was studied. Kyotorphin was determined using high-performance liquid chromatography (HPLC) with an electrochemical detector. This dipeptide was found to be concentrated in the crude mitochondrial (P2) fraction. Further subfractionation of the P2 fraction revealed that kyotorphin was exclusively localized in the synaptosomal fraction.This finding suggests the possibility that kyotorphin has a neurotransmitter/neuromodulator role in the brain.
A novel opioid analgesic peptide termed “Kyotorphin” was isolated and identified from bovine brain, using an in vivo analgesia assay method. The analgesic activity was assessed by a combination of intracisternal injection and the tail pinch test in mice. The sequence of this peptide was determined as Tyr-Arg by amino acid analysis and the N-terminal determination by the dansylation method. The natural peptide was confirmed to be identical to the synthetic peptide on TLC, HVPE and HPLC. The median analgesic dose, ED50 of kyotorphin was 34.7 nmol/mouse with the tail pinch test and the potency was 4.2 times greater than that of met-enkephalin. With the hot plate test, the ED50 of kyotorphin was 15.7 nmol/mouse and this effect was completely reversed by the pretreatment with naloxone (0.1 mg/kg, s.c.).The present analgesic assay method can thus be effectively applied to endogenous opioid substances to establish analgesic actions.
Intracerebroventricular (i.c.v.) administration of l ‐arginine ( l ‐Arg), at 10–100 μg per mouse, produced antinociception in mice, as assessed by the tail flick test; this antinociception was reversed by pretreatment (s.c.) with naltrindole (NTI), a δ‐selective opioid antagonist, and by co‐administered l ‐leucyl‐ l ‐arginine (Leu‐Arg), a kyotorphin (endogenous Met‐enkephalin releaser) receptor antagonist.
l ‐N G ‐nitroarginine methyl ester ( l ‐NAME), a NO synthase inhibitor, but not d ‐N G ‐nitroarginine methyl ester, given i.c.v. at 3–10 μg per mouse, exhibited antinociceptive activity that was resistant to naloxone (s.c.), NTI (s.c.) and Leu‐Arg (i.c.v.).
The l ‐NAME (i.c.v.)‐induced antinociception was not reversed by l ‐Arg (i.c.v.), which was antinociceptive by itself, but was abolished by combined injection of l ‐Arg plus Leu‐Arg (i.c.v.) or by l ‐Arg (i.c.v.) after NTI (s.c.).
Methylene blue (MB), a soluble guanylate cyclase inhibitor, at 0.1–1 μg per mouse, produced antinociception by i.c.v. administration. The antinociception induced by MB (i.c.v.) or l ‐NAME (i.c.v.) was reversed by co‐administered dibutyryl cyclic GMP.
These findings suggest that l ‐Arg plays a dual role in nociceptive processing in the brain, being antinociceptive via the kyotorphin‐Met‐enkephalin pathway and nociceptive via the NO‐cyclic GMP pathway.
Bradykinin (BK) given into the plantar (i.pl.) of the mouse hind-limb produced a flexor response. The flexor responses were dependent on BK doses (0.02-20 pmol, i.pl.), and were completely abolished by Hoe140, a B2-type BK receptor antagonist. Kyotorphin, an analgesic neuropeptide which shows enkephalin release in brain slices, produced a dose-dependent reduction of the BK-induced nociceptive responses in ranges of 10 pmol to 1 nmol (i.pl.). Such analgesic effects of kyotorphin were reversed by leucine-arginine, a specific kyotorphin receptor antagonist, but not by naloxone. The kyotorphin-analgesia was also abolished by pertussis toxin (PTX) pretreatment. These results suggest that peripheral analgesic effects of kyotorphin are mediated through mechanisms of kyotorphin specific receptor and PTX-sensitive Gi/Go, and that the enkephalin release is not necessary for this analgesia.
The effects of kyotorphin (KTP), a dipeptide (L-Tyr-L-Arg), on the level of sensory attention to stimuli of different modalities in rats and the exploratory behavior in goldfish were investigated. In both cases KTP was found to suppress the exploratory activity. When 5-HTP, a precursor of serotonin synthesis, is activated the inhibitory effects of KTP increased. It is assumed that the regulatory effect of KTP on the exploratory behavior of animals is mediated by the monoaminergic (neurotransmitting) brain systems, as distinct from its analgetic effects, which are mediated by the opioid brain systems.
Evidence is fast accumulating which indicates that Alzheimer's disease is a vascular disorder with neurodegenerative consequences rather than a neurodegenerative disorder with vascular consequences. It is proposed that two factors need to be present for AD to develop: (1) advanced ageing, (2) presence of a condition that lowers cerebral perfusion, such as a vascular-risk factor. The first factor introduces a normal but potentially insidious process that lowers cerebral blood flow in inverse relation to increased ageing; the second factor adds a crucial burden which further lowers brain perfusion and places vulnerable neurons in a state of high energy compromise leading to a cascade of neuronal metabolic turmoil. Convergence of the two factors above will culminate in a critically attained threshold of cerebral hypoperfusion (CATCH). CATCH is a hemodynamic microcirculatory insufficiency that will destabilize neurons, synapses, neurotransmission and cognitive function, creating in its wake a neurodegenerative state characterized by the formation of senile plaques, neurofibrillary tangles, amyloid angiopathy and in some cases, Lewy bodies. Since any of a considerable number of vascular-related conditions must be present in the ageing individual for cognition to be disturbed, CATCH identifies an important aspect of the heterogeneic disease profile assumed to be present in the AD syndrome. It is proposed that CATCH initiates AD by distorting regional brain capillary structure involving endothelial cell shape changes and impairment of nitric oxide (NO) release which affect signaling between the immune, cardiovascular and nervous systems. Evidence is presented that in many tissues there is a basal level of NO being produced and that the actions of several signaling molecules may initiate increases in basal NO levels. Moreover, these temporary increases in basal NO levels exert inhibitory cellular actions, via cellular conformational changes. Findings indicate that (a) constitutive NO is responsible for a basal or 'tonal' level of NO; (b) this NO keeps particular types of cells in a state of inhibition and (c) activation of these cells occurs through disinhibition. Consequently, tissues not maintaining a basal NO level are more prone to excitatory, immune, vascular and neural influences. Under such circumstances, these tissues cannot be down-regulated to normal basal levels, thus prolonging their excitatory state. Thus, the clinical convergence of advanced ageing in the presence of a chronic, pre-morbid vascular risk factor, can, in time, contribute to an endotheliopathy involving basal NO deficit, to the degree where regional metabolic dysfunction leads to cognitive meltdown and to progressive neurodegeneration characteristic of Alzheimer's disease.
Accidents by Thalassophryne nattereri fish venom are characterised by severe local symptoms and signs including pain of fast onset, oedema and necrosis with impaired muscle regeneration. These effects have been related to alterations in hemostatic mechanisms and cytolytic effects rather than to conventional inflammatory pathways. In this work we evaluated the effects induced by the venom on microcirculatory vessels, platelets and blood coagulation. Effects evoked by topical application of venom on cremaster muscle were visualised through intravital microscopy. Stasis was observed, concomitantly with the presence of thrombi in venules and focal transient constrictions in arterioles, all of which impaired the blood flow. Significant alterations on vessel walls took place few minutes after venom application, characterised by increment in thickness, probably by deposition of fibrin. Increase in vascular permeability was also observed in venules. Additionally, the action of the venom was locally restricted since no alteration on systemic blood coagulation was observed. Venom lacked a direct pro-coagulant activity, but exerted a strong cytolytic effect on platelets and endothelial cells in vitro. These data suggest that venom action on endothelium may contribute to blood stasis and to the formation of platelet and fibrin thrombi, with the consequent ischemia, contributing to the local effects of the venom.
Casein kinase 2 (CK2) is a widely expressed protein kinase. Over the last several years a long list of protein substrates has evolved, many of which have proven or hypothesized roles in nociceptive signal transmission. However, CK2 has not itself been demonstrated to participate in nociception prior to this time. We set out to test the hypothesis that spinal CK2 regulates nociception using several pain models. Our first studies focused on the ability of the selective CK2 inhibitors 4,5,6,7-tetrabromobenzotriazole (TBBT) and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) to reduce formalin-stimulated pain behaviors in mice. Both phases of the response to subcutaneous formalin were strongly inhibited by intrathecal administration of TBBT or DRB in dose-dependent fashion. Likewise, using the complete Freund's adjuvant (CFA) model of chronic inflammatory pain, TBBT was observed to strongly reduce mechanical allodynia. The inhibition of spinal CK2 with either inhibitor did not, however, alter withdrawal latencies in the hotplate thermal pain model while intrathecal morphine was very effective. Immunohistochemical studies demonstrated all three known CK2 subunits, alpha, alpha' and beta to be expressed in spinal cord tissue as did real-time PCR experiments. While mRNA levels for each of the subunits was transiently enhanced after formalin or CFA hindpaw injection, overall spinal cord protein levels were not elevated in a sustained fashion. Our results indicate that CK2 participates in inflammatory nociception both in the acute and chronic phases. Simple changes in the abundance of spinal CK2 subunits do not likely underlie these phenomena, however.
The aim of this study was to develop a novel predictive medium-throughput screening method for drug permeability, with use of a tight barrier of liposomes on a filter support. To our knowledge no one has succeeded in depositing membrane barriers without the use of an inert solvent such as hexadecane. The first part of the study involved development of a protocol for preparation of these barriers, which were made of liposomes from egg phosphatidylcholin in phosphate buffer pH 7.4 with 10 % (v/v) ethanol. The liposomes were deposited into the pores and onto the surface of a filter support (mixed cellulose ester) by use of centrifugation. Solvent evaporation and freeze-thaw cycling were then used to promote fusion of liposomes. A tight barrier could thus be obtained as shown with calcein permeability and electrical resistance. In the second part of the study the model was validated using 21 drug compounds, which cover a wide range of physicochemical properties and absorption (F(a)) in humans (13-100%). The drug permeation studies were carried out at room temperature with phosphate buffer (pH 7.4) in both acceptor and donor chambers. The apparent permeability coefficients obtained from the phospholipid vesicle based model correlated well with literature data on human absorption in vivo, which suggests that its performance is adequate and that the method is suitable for rapid screening of passive transport of new chemical entities. The results obtained from our model were compared with polar surface area (PSA) and experimental logD and with results obtained by established permeability screening methods such as immobilized liposome chromatography (ILC), the PAMPA models and the Caco-2 model. Our approach seems to model the in vivo absorption better than PSA, experimental logD, the ILC and PAMPA models, when similar conditions are used as in our assay, and equally well as the Caco-2 model and the Double Sink PAMPA (DS-PAMPA) model.
Leukocyte rolling is an important step for the successful recruitment of leukocytes from blood to tissues mediated by a specialized group of glycoproteins termed selectins. Because of the dynamic process of leukocyte rolling, binding of selectins to their respective counter-receptors (selectin ligands) needs to fulfill three major requirements: (1) rapid bond formation, (2) high tensile strength, and (3) fast dissociation rates. These criteria are perfectly met by selectins, which interact with specific carbohydrate determinants on selectin ligands. This chapter describes the theoretical background, technical requirements, and analytical tools needed to quantitatively assess leukocyte rolling in vivo and in vitro. For the in vivo setting, intravital microscopy allows the observation and recording of leukocyte rolling under different physiological and pathological conditions in almost every organ. Real-time and off-line analysis tools help to assess geometric, hemodynamic, and rolling parameters. Under in vitro conditions, flow chamber assays such as parallel plate flow chamber systems have been the mainstay to study interactions between leukocytes and adhesion molecules under flow. In this setting, adhesion molecules are immobilized on plastic, in a lipid monolayer, or presented on cultured endothelial cells on the chamber surface. Microflow chambers are available for studying leukocyte adhesion in the context of whole blood and without blood cell isolation. The microscopic observation of leukocyte rolling in different in vivo and in vitro settings has significantly contributed to our understanding of the molecular mechanisms responsible for the stepwise extravasation of leukocytes into inflamed tissues.
To clarify the activational role of ovarian hormones on pain and analgesia, the present study determined whether estradiol (E2) modulation of nociception and morphine antinociception in adult female rats depends on (1) the dose of E2 and (2) the interval between E2 treatment and nociceptive testing. Female rats were ovariectomized (OvX) and either oil vehicle (0), or E2 (0.25, 2.5 or 25 microg/0.1 ml vehicle) was injected s.c. two consecutive days of every four days for five cycles before testing. Either 4, 24, 48 or 96 h after the last injection, nociception was evaluated on the 50 degrees C hotplate and warm water tail withdrawal tests before and after escalating doses of s.c. morphine. Lordosis behavior and uterine weight were assessed in other rats at the same E2 doses and time points. E2 significantly lengthened latency to respond on the hotplate test at 24 h after the last injection, but had no significant effect on tail withdrawal latencies. The lower doses of E2 significantly increased morphine antinociceptive potency at 4-24 h on one or both tests, but the intermediate E2 dose significantly decreased morphine potency at 48 h on the hotplate test. Thus, E2 modulation of morphine antinociception in the adult female rat is bidirectional, and occurs at E2 doses producing cyclic changes in sexual behavior, uterine weight and vaginal cytology that are similar to those observed in gonadally intact, cycling females.
× 10 mL), and n-pentane (2 × 10 mL), and dried in vacuo. The resulting white solid was dissolved in water and lyophylized to afford the corresponding deprotected dipeptides
DOI: 10.1021/acschemneuro.6b00099
ACS Chem. Neurosci. 2016, 7, 1130−1140
mL), diethyl ether (2 × 10 mL), and n-pentane (2 × 10 mL), and
dried in vacuo. The resulting white solid was dissolved in water and
lyophylized to afford the corresponding deprotected dipeptides.
Synthesis of KTP-NH 2 -DL. By the general procedure,
tBu)−OH (4 equiv) provided H-Tyr-D-Arg- NH 2 (66 mg, 70%) as a colorless solid
- Fmoc-Tyr
equiv) and Fmoc-Tyr(tBu)−OH (4 equiv) provided H-Tyr-D-Arg-
NH 2 (66 mg, 70%) as a colorless solid. HPLC (water/acetonitrile,
56 mg, 58%) as a colorless solid. HPLC (water/ acetonitrile, 0.1% TFA, 2−100%, 17 min): retention time, 1
- Tyr-Arg
Tyr-Arg-NH 2 (56 mg, 58%) as a colorless solid. HPLC (water/
acetonitrile, 0.1% TFA, 2−100%, 17 min): retention time, 1.66 min;
86 g, 90%) as a colorless solid
- Arg-Nh
Arg-NH 2 (4.86 g, 90%) as a colorless solid. 1 H NMR (DMSO-d 6, 400