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J Huwyler

Publications and source records attributed to J Huwyler.

At least 19 recordsLinked to original sources
PubMed2003

Evaluation of the NR2B-selective NMDA receptor antagonist Ro 63-1908 on rodent behaviour: evidence for an involvement of NR2B NMDA receptors in response inhibition.

We have characterised the effects of the recently described NMDA NR2B subtype selective antagonist, Ro 63-1908, on spontaneous behaviour and in tasks sensitive to non-selective NMDA antagonists. In both rats and wild type mice, Ro 63-1908 (1-30mg/kg sc) produced a mild increase in motor activity of lesser magnitude than that elicited by dizocilpine. No signs of overt PCP-like stereotypy were seen in either species at equivalent doses. PPI was also unaffected. However, in mice lacking the NR2A subunit, Ro 63-1908 (3-30mg/kg) produced a profound hyperactivity of similar magnitude to dizocilpine but few other 'PCP-like' behaviours. In rats, Ro 63-1908 (1-10mg/kg) did not affect Morris water maze or delayed matching performance. In a 5-choice serial reaction time task, requiring rats to respond to a visual stimulus presented after a fixed time interval, Ro 63-1908 (0.3-3mg/kg) produced a dramatic increase in premature responses - accuracy was relatively unaffected. Finally in a DRL24 task, Ro 63-1908 (0.3-3mg/kg) reduced inter-response time, increased response rate, and consequently reduced efficiency. We conclude that the improved profile of Ro 63-1908 compared to NMDA channel blockers is due to both its selectivity for the NR2B vs. NR2A subunit containing receptors and its activity-dependent mechanism of action. However, in the 5-CSRT and DRL24 tasks, Ro 63-1908 produced behaviours suggestive of impaired response inhibition, implicating a critical role of NMDA NR2B transmission in this process.

Analysis of Variance↗
PubMed2002

Uptake of cationzied albumin coupled liposomes by cultured porcine brain microvessel endothelial cells and intact brain capillaries.

The suitability of protein-coupled liposomes as drug carriers for brain specific targeting was investigated using albumin (BSA) and cationized albumin (CBSA), respectively, as model proteins. Liposomes coated with polyethylene glycol (sterically stabilized, PEG-liposomes) were prepared from phosphatidylcholine, cholesterol, and a PEG-derivatized phospholipid and covalently coupled to thiolated BSA or CBSA. Liposomes were loaded with carboxy-fluorescein and rhodamine-labeled dipalmitoyl-phosphatidylethanolamine as hydrophilic and lipophilic marker compounds, respectively. The interaction of these constructs with monolayers of porcine brain capillary endothelial cells (BCEC) and freshly isolated porcine brain capillaries was studied by means of fluorescence assays and confocal laser scanning fluorescence microscopy (CLSFM). In contrast to BSA, CBSA was rapidly taken up by cultured BCECs. BSA-coupled liposomes did not interact with endothelial cells, whereas CBSA-coupled liposomes bound to cellular surfaces and exhibited time dependently a high intracellular accumulation. CBSA-conjugated liposomes were also taken up by intact brain capillaries. Cellular uptake could be inhibited by free cationized albumin, phenylarsineoxide, nocodazole, and filipin, but not by dansylcadaverine, suggesting a caveolae-mediated incorporation process. Immunostaining demonstrated a high expression of caveolin in the capillary endothelium. In conclusion, liposomes coupled to CBSA are taken up into brain endothelium via an endocytotic pathway and may therefore be a suitable carrier for drug delivery to the brain.

Animals↗
PubMed2000

Endocytosis and transcytosis of an immunoliposome-based brain drug delivery system.

Immunoliposomes conjugated with the OX26 monoclonal antibody to the rat transferrin receptor can be used for brain delivery of small molecules. In the present study the uptake of OX26-immunoliposomes by target cells as well as their transcytosis across the blood-brain barrier was investigated. Microscopy of RG2 rat glioma cells incubated with fluorescence labeled OX26-immunoliposomes revealed intracellular co-localization of liposomal cargo, the liposomal membrane bilayer and the OX26 monoclonal antibody. The distinct particulate staining pattern was indicative for accumulation of OX26-immunoliposomes within endosomal or lysosomal compartments. Prolonged incubations demonstrated endosomal release of the liposomal cargo propidium iodide to the cytoplasm. A maximum of 50% of propidium iodide was released from the endosomal compartment after 24 hours of incubation. Transcytosis was studied using an in vitro model of the blood-brain barrier consisting of immortalized RBE4 rat brain endothelial cells. OX26-immunoliposomes did permeate across the RBE4 cell monolayer and showed a permeability coefficient of P(app) = 1.6 x 10(-5) ml/s. Transport was inhibited at low temperature, by competition with free OX26 or by exchanging the OX26 monoclonal antibody for an unspecific isotype antibody. Transcytosis of OX26-immunolipsomes was confirmed in vivo by the brain perfusion and capillary depletion technique. OX26-immunoliposomes were detected within the post-vascular compartment of brain parenchyma (PS product = 2.4 microl/g/min.) and were not associated with the brain microvasculature.

Animals↗
PubMed2000

Evidence that nicotinic alpha(7) receptors are not involved in the hyperlocomotor and rewarding effects of nicotine.

Neuronal nicotinic receptors are comprised of combinations of alpha(2-9) and beta(2-4) subunits arranged to form a pentameric receptor. Currently, the principal central nervous system (CNS) subtypes are believed to be alpha(4)beta(2) and a homomeric alpha(7) receptor, although other combinations almost certainly exist. The identity of the nicotinic receptor subtype(s) involved in the rewarding effects of nicotine are unknown. In the present study, using some recently described subtype selective nicotinic agonists and antagonists, we investigated the role of the alpha(7) nicotinic receptor in the mediation of nicotine-induced hyperactivity and self-administration in rats. The alpha(7) receptor agonists AR-R 17779 and DMAC failed to stimulate locomotor activity in both nicotine-nontolerant and -sensitized rats. In contrast, nicotine and the putative alpha(4)beta(2) subtype selective agonist SIB1765F increased activity in both experimental conditions. In nicotine-sensitized rats, the high affinity (including the alpha(4)beta(2) subtype) nicotinic antagonist dihydro-beta-erythroidine (DHbetaE), but not the selective alpha(7) antagonist methyllycaconitine (MLA), antagonized a nicotine-induced hyperactivity. Similarly, DHbetaE, but not MLA, pretreatment reduced nicotine self-administration. Electrophysiology experiments using Xenopus oocytes expressing the human alpha(7) receptor confirmed AR-R 17779 and DMAC to be potent agonists at this site, and further studies demonstrated the ability of systemically administered AR-R 17779 to penetrate into the CNS. Taken together, these results indicate a negligible role of alpha(7) receptors in nicotine-induced hyperlocomotion and reward in the rat, and support the view for an involvement of a member from the high-affinity nicotinic receptor subclass, possibly alpha(4)beta(2). Issues such as drug potency, CNS penetration, and desensitization of the alpha(7) receptor are discussed.

Animals↗
PubMed1999

HIV protease inhibitor ritonavir: a more potent inhibitor of P-glycoprotein than the cyclosporine analog SDZ PSC 833.

The effect of P-glycoprotein inhibition on the uptake of the HIV type 1 protease inhibitor saquinavir into brain capillary endothelial cells was studied using porcine primary brain capillary endothelial cell monolayers as an in vitro test system. As confirmed by polymerase chain reaction and Western blot analysis, this system functionally expressed class I P-glycoprotein (pgp1A). P-Glycoprotein isoforms pgp1B or pgp1D could not be detected. The uptake of saquinavir into endothelial cells could be described as the result of a diffusional term of uptake and an oppositely directed saturable extrusion process. Net uptake of saquinavir into cultured brain endothelial cells could be increased significantly up to 2-fold by SDZ PSC 833 in a dose-dependent manner, with an IC(50) of 1.13 microM. In addition, the HIV protease inhibitor ritonavir inhibited p-glycoprotein-mediated extrusion of saquinavir with an IC(50) of 0.2 microM, indicating a high affinity of ritonavir for p-glycoprotein. In conclusion, we showed that the HIV protease inhibitor ritonavir is a more potent inhibitor of P-glycoprotein than the multidrug resistance (MDR)-reversing agent SDZ PSC 833. The inclusion of this drug in combination regimens may greatly facilitate brain uptake of HIV protease inhibitors, which is especially important in patients suffering from AIDS dementia complex.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Modulation of multidrug resistance protein expression in porcine brain capillary endothelial cells in vitro.

Multidrug resistance-associated protein (MRP) is a transport system that is involved in the elimination of xenobiotics and biologically active endogenous substrates. Recently, the presence of MRP has been demonstrated in cultured brain capillary endothelial cells (BCECs). The time-dependent, functional expression of MRP in porcine BCECs was investigated to assess the value of this cell culture model for drug transport at the blood-brain barrier. Western blot analysis was used to investigate MRP expression in freshly isolated porcine BCECs and compared to MRP expression at days 8 and 10 in culture. Subcellular localization of MRP was investigated by immunocytochemistry with an MRP-specific monoclonal antibody, MRPr1. Functional activity of MRP was assessed by efflux studies with the fluorescent MRP substrate glutathione-methylfluorescein (GS-MF). No significant MRP expression was detected in freshly isolated endothelial cells. However, MRP expression is up-regulated in cell culture in a time-dependent manner. Immunostaining revealed predominantly perinuclear and, to a lesser degree, plasma membrane localization of MRP. At 10 degrees C GS-MF efflux was significantly decreased, indicating the involvement of an energy-dependent transport system. Efflux of GS-MF was apparently inhibited by MK571, a specific inhibitor for MRP. Porcine BCECs demonstrate up-regulation of functional MRP expression during culture, as observed in human tissue, and therefore might serve as a useful in vitro system for studying MRP-mediated blood-brain barrier transport.

ATP-Binding Cassette Transporters↗
PubMed1998

Examination of blood-brain barrier transferrin receptor by confocal fluorescent microscopy of unfixed isolated rat brain capillaries.

Isolated rat brain capillaries were analyzed by confocal microscopy. Fluorescent immunoliposomes bearing the OX26 anti-transferrin receptor monoclonal antibody were synthesized and incubated with freshly isolated unfixed microvessels to visualize binding to luminal and abluminal membranes of the endothelium. Intactness of the endothelial structure was demonstrated by computer-aided reconstruction of a series of consecutive optical sections. These results indicate that analysis of unfixed brain capillaries by confocal microscopy offers the possibility of assigning the presence of membrane receptors to either the luminal or the basolateral plasma membrane domain.

Animals↗
PubMed1998

Transport of the beta-lactam antibiotic benzylpenicillin and the dipeptide glycylsarcosine by brain capillary endothelial cells in vitro.

Peripherally administered beta-lactam antibiotics, which are structural analogs of tripeptides, may cause neurotoxic reactions or induce seizures. Previous in vivo studies provided evidence for brain uptake of these antibiotics. In the present work, we studied the extent and mechanism of the uptake of benzylpenicillin and glycylsarcosine by brain microvessel endothelial cells in vitro, using freshly isolated and cultured porcine brain capillary endothelial cells. Characterization of the cell culture model demonstrated the functional expression of the system transporting the neutral amino acids leucine and phenylalanine. The initial rate of uptake of benzylpenicillin was >3-fold greater than the rate of uptake of the extracellular marker sucrose (ratio, 3.29 +/- 0.37), whereas uptake of glycylsarcosine did not differ from that of sucrose. The differences in cellular uptake correlated with the octanol/buffer partition coefficients for glycylsarcosine and benzylpenicillin (1.16 x 10(-3) for glycylsarcosine and 6.83 x 10(-2) for benzylpenicillin). The concentration-dependent uptake of benzylpenicillin (1-2000 microM) was not saturable and was not sensitive to shifts in pH or temperature. The permeability-surface area product for the uptake of benzylpenicillin at pH 7.4 was determined from these experiments and was found to be 8.1 x 10(-5) ml/sec/g of brain. This value was very close to the value determined in in vivo studies. Uptake of benzylpenicillin and glycylsarcosine was not reduced in the presence of 1 mM ceftibuten or 100 microM probenecid. The findings with cultured cell monolayers were confirmed using freshly isolated endothelial cells. These in vitro data are compatible with benzylpenicillin, but not glycylsarcosine, being able to penetrate endothelial cells. Uptake of benzylpenicillin by brain capillary endothelial cells occurs by a slow nonsaturable process, with no evidence for carrier-mediated transport.

Animals↗
PubMed1997

High-performance liquid chromatographic method for an automated determination of local anaesthetics in human plasma.

A method is described that allows the rapid and precise determination of the local anaesthetics bupivacaine and etidocaine from biological fluids. This method uses a fully automated system with solid-phase extraction in combination with a column-switching technique. Both sample extraction on a LiChrocart pre-column and elution onto the analytical LiChrospher column, were performed automatically and concomitantly using conventional HPLC equipment in conjunction with an OSP-2 on-line sample preparator from Merck combined with UV detection. Recoveries were found to be 96.7 and 96.4% for 2 micrograms/ml bupivacaine and etidocaine, respectively. Lower limits of quantification were found to be 0.05 microgram/ml plasma for both of the compounds.

Anesthetics, Local↗
PubMed1997

Transport of clonidine across cultured brain microvessel endothelial cells.

Brain penetration of clonidine, an alpha-2 adrenoceptor agonist, was studied using an in vitro cell culture system consisting of primary cultures of porcine brain capillary endothelial cells. Uptake of clonidine was measured as a function of its concentration in the incubation mixture. Saturation of uptake was apparent and could be described by Michaelis-Menten-type kinetics (K(M) = 1.34 mM; Vmax = 0.099 nmol/min/cm2). Saturation was not observed at a low temperature (4 degrees C). Transendothelial transport experiments revealed that translocation of clonidine cannot be attributed solely to paracellular leakage. Uptake was reduced at low extracellular pH or by using an incubation buffer that contained the K+ ionophore valinomycin. Time-dependent uptake of clonidine and transendothelial transport were slower than expected considering the high octanol-to-buffer partition coefficient of this compound. On the basis of transendothelial transport experiments, we concluded that the carrier system responsible for active transport of clonidine is located at both the apical and the basolateral membrane domain.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Receptor mediated delivery of daunomycin using immunoliposomes: pharmacokinetics and tissue distribution in the rat.

Pharmacokinetics and tissue distribution of daunomycin and different liposomal formulations of daunomycin were determined. Special emphasis was thereby given to immunoliposome-mediated drug delivery. Three different types of 85 nm liposomes were used for this study: 1) conventional liposomes, 2) liposomes sterically stabilized with 2000 Dalton polyethylene glycol and 3) immunoliposomes prepared by coupling a control IgG2a or monoclonal antibody to the distal end of the polyethylene glycol spacer. The antibody used was the OX26 monoclonal antibody to the rat transferrin receptor. Daunomycin and liposomes were administered by i.v. injection to the rat. Daunomycin and daunomycin in conventional liposomes were rapidly cleared from the plasma compartment. When compared to the free drug, daunomycin in conventional liposomes did accumulate to higher levels in liver and spleen and to lower levels in heart, lung and liver. In contrast, daunomycin in liposomes sterically stabilized with polyethylene glycol could not be detected in heart, lung, kidney, liver and spleen. Using nonspecific IgG2a isotype immunoliposomes, tissue concentrations of immunoliposomes were reduced by at least a factor of two. Attachment of more than 29 OX26 monoclonal antibodies per liposome did not increase tissue levels in heart, kidney or lung. Tissue levels of OX26 immunoliposomes were reduced in all organs by coinjection of unbound OX26. In vitro, endocytosis of fluorescent immunoliposomes by RG2 rat glioma cells was observed. These data indicate that receptor mediated drug delivery to different tissues can be achieved using OX26 conjugated immunoliposomes.

Animals↗
PubMed1996

Brain drug delivery of small molecules using immunoliposomes.

Immunoliposomes (antibody-directed liposomes) were used in the present study for delivery of the antineoplastic agent daunomycin to the rat brain. A coupling procedure was introduced, which allows conjugation of a thiolated antibody to maleimide-grafted 85-nm liposomes sterically stabilized with PEG. Antibody was thereby coupled to the terminal end of a PEG-conjugated linker lipid. No brain uptake of PEG-conjugated liposomes carrying [3H]daunomycin was observed. However, brain targeting of immunoliposomes carrying [3H]daunomycin was mediated by the OX26 monoclonal antibody to the rat transferrin receptor, which is selectively enriched at the brain microvascular endothelium that comprises the blood-brain barrier in vivo. Coupling of 30 OX26 antibodies per liposome resulted in optimal brain delivery. Saturation of delivery was observed at higher antibody densities. Determination of brain levels of immunoliposomes over 24 h revealed that immunoliposomes accumulate in brain tissue. Brain targeting of immunoliposomes was not observed in immunoliposomes conjugated with a mouse IgG2a isotype control. In addition, coinjection of free OX26 saturated plasma clearance of immunoliposomes. Since a single liposome may carry > or = 10,000 drug molecules, the use of PEG-conjugated immunoliposomes increases the drug carrying capacity of the monoclonal antibody by up to 4 logarithmic orders in magnitude. In summary, specific OX26-mediated targeting of daunomycin to the rat brain was achieved by the use of an immunoliposome-based drug delivery system.

Animals↗
PubMed1996

Relevance of p-glycoprotein for the enteral absorption of cyclosporin A: in vitro-in vivo correlation.

1. The interaction of cyclosporin A (CyA) with p-glycoprotein during intestinal uptake was investigated by a combination of in vitro experiments with human Caco-2 cells and an intubation study in healthy volunteers. 2. CyA uptake into the cells was not saturable and exhibited only a low temperature sensitivity, suggesting passive diffusion. When the permeation of CyA across Caco-2 monolayers from the apical to the basolateral side was determined, overall transport had an apparently saturable component up to a concentration of 1 microM. At higher concentrations permeation increased over-proportionally. Calculation of the kinetic parameters of apical to basolateral permeation suggested a diffusional process with a KD of 0.5 microliter min-1 per filter, which was overlayed by an active system in basolateral to apical direction with a KM of 3.8 microM and a Jmax of 6.5 picomol min-1 per filter. 3. CyA permeation was significantly higher when the drug was given from the basolateral side as compared to the permeation from the apical side. Apical to basolateral transport of CyA was increased in the presence of vinblastine, daunomycin and a non-immunosuppressive CyA-derivative. All compounds inhibit p-glycoprotein-mediated transport processes. Basolateral to apical permeation of CyA showed a dose-dependent decrease in the presence of vinblastine. Permeation of daunomycin across Caco-2 cell monolayers was also higher from the basolateral to the apical side than vice versa. Basolateral to apical permeation was decreased in the presence of SDZ PSC 833 and cyclosporin A. 4. Western blot analysis of Caco-2 cells with the monoclonal antibody C219 confirmed the presence of p-glycoprotein in the used cell system. 5. When the absorption of CyA in the gastrointestinal (GI)-tract of healthy volunteers was determined, a remarkable decrease of the plasma AUC could be observed dependent on the location of absorption in the rank order stomach > jejunum/ileum > colon. The decrease in absorption exhibited a marked correlation (r = 0.994) to the expression of mRNA for p-glycoprotein over the GI-tract (stomach < jejunum < colon). 6. All data provide evidence that CyA is a substrate of p-glycoprotein in the GI-tract, which might explain the local differences and the high variability in cyclosporin absorption found in vivo.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Evidence for P-glycoprotein-modulated penetration of morphine-6-glucuronide into brain capillary endothelium.

1. Morphine-6-glucuronide is one of the major metabolites of morphine. The potent analgesic action of this compound together with its potential lower apparent toxicity in man, when compared with morphine, indicated its clinical importance. 2. Primary cultures of porcine brain capillary endothelial cells were used to study brain penetration of morphine-6-glucuronide. Biochemical characterization of the cell cultures revealed a marked enrichment in enzymatic activity of alkaline phosphatase (56 fold) and angiotensin converting enzyme (230 fold) as compared to whole brain tissue. By immunostaining the presence of vimentin, factor VIII, the tight junction associated protein ZO-1, and P-glycoprotein was shown. Functional characterization revealed that the carrier system responsible for transport of neutral amino acids was intact. 3. Uptake and transport of morphine-6-glucuronide was marginal and in the range of the extracellular marker sucrose. However, uptake of morphine-6-glucuronide was enhanced significantly (P < 0.0001) in presence of the inhibitors of P-glycoprotein, verapamil or vincristine. The finding that morphine-6-glucuronide may serve as a substrate for P-glycoprotein was confirmed in multidrug-resistant P388 tumour cells. 4. We conclude that penetration of the blood-brain barrier by morphine-6-glucuronide may depend on the expression of the product of the multidrug-resistance (MDR) gene in brain capillary endothelial cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Rapid and highly automated determination of morphine and morphine glucuronides in plasma by on-line solid-phase extraction and column liquid chromatography.

A high-performance liquid chromatography (HPLC) method has been developed for the determination of morphine and its main metabolites, morphine-6-glucuronide (M-6-G) and morphine-3-glucuronide (M-3-G), in plasma or cerebrospinal fluid. Samples were extracted using on-line solid-phase extraction followed by reversed-phase HPLC with fluorescence detection. Recoveries of 20 ng morphine and morphine glucuronides in plasma were over 95%. The limit of detection using 400 microliters of a biological matrix was 0.85, 3.4 and 1.0 ng/ml of M-3-G, M-6-G and morphine, respectively. Inter- and intra-day assay precision was better than 10%. The main advantages of the present described method are increased recoveries (> 95%) and a high degree of automation allowing a high speed in routine analysis. The time required for the fully automated analysis of one sample was less than 26 min.

Automation↗
PubMed1994

Leukotriene B4 formation upon halothane-induced lipid peroxidation in liver membrane fractions under low O2 concentrations in vitro.

Lipid peroxidation was induced in rat liver membrane fractions in vitro upon NADPH-dependent metabolic activation of the anesthetic agent halothane at low O2 concentrations. Halothane-induced lipid peroxidation was dependent on time, concentration of halothane, and the calculated O2 concentrations present in the system. Lipid peroxidation was inducible at increasing O2 concentrations up to 12 microM, decreased at higher O2 concentrations up to 48 microM, and was not detectable at normoxic conditions. Leukotriene B4 (LTB4) was identified as a product arising upon lipid peroxidation by reverse-phase high-pressure liquid chromatography combined with a radioimmunoassay. LTB4 formation was maximal under conditions of maximal lipid peroxidation at a calculated O2 concentration of 12 microM. Even at high concentrations, the 5-lipoxygenase inhibitors MK886 (10 microM), ZD2138 (20 microM), and ZM230487 (20 microM) were not inhibitory in halothane-induced lipid peroxidation nor in the associated formation of LTB4. Synthetic LTB4 was transformed into its 20-hydroxy derivative by omega-oxidation in an O2-concentration-dependent manner, being considerably reduced at the low O2 concentrations that maximally promoted lipid peroxidation. The collective evidence of these data raises the possibility that exposure to halothane might lead to peroxidation-associated net synthesis of LTB4 through 5-lipoxygenase-independent escape routes in liver tissue under physiologically or pathophysiologically low O2 concentrations.

Aerobiosis↗
PubMed1993

Mechanisms of halothane toxicity: novel insights.

Exposure of individuals to halothane causes, in 20% of patients, a mild form of hepatotoxicity. In contrast, a very small subset of individuals only develops halothane hepatitis, which is thought to have an immunological basis. Sera of halothane hepatitis patients contain antibodies directed against some discrete liver trifluoroacetyl (TFA)-protein adducts, which arise upon oxidative biotransformation of halothane and include protein disulfide isomerase, microsomal carboxylesterase, calreticulin, ERp72, GRP 78 and ERp99. No immune response occurs in the majority of human individuals, although evidence suggests that TFA-protein adducts arise in all halothane-exposed individuals. The lack of immunological responsiveness of individuals might be due to tolerance, induced by a presumed repertoire of self-peptides that molecularly mimic TFA-protein adducts. Thus, constitutively expressed proteins of 52 and 64 kDa have been identified that confer molecular mimicry of TFA-protein adducts. The 64 kDa protein corresponds to the E2 subunit of the mitochondrial pyruvate dehydrogenase complex. Lipoic acid, the prosthetic group of the E2 subunit, is involved in the molecular mimicry process. A fraction of halothane hepatitis patients exhibit irregularities in the expression levels of the 52 kDa protein and the E2 subunit protein. Molecular mimicry of TFA-protein adducts by the 52 kDa protein and the E2 subunit protein might play a role in the susceptibility of individuals to development of halothane hepatitis.

Animals↗