3-Aryl-1,2-diacetamidopropane derivatives as novel and potent NK-1 receptor antagonists.

Early structure-activity studies on racemic tryptophan ester and amide NK-1 antagonists 5-7 led to the discovery that the potency of the series could be markedly increased by moving the carbonyl function in these molecules to an off-chain position as in the 3-aryl-1,2-diacetamidopropane 9. Further medicinal chemistry incorporating this change resulted in the discovery of a novel series of highly potent aryl amino acid derived NK-1 antagonists of the R stereoisomeric series (IC50's = 100 pM to > 5 microM). Compounds in this series were shown to be competitive antagonists using an in vitro NK-1 smooth muscle assay, and this data correlated well with observed human NK-1 binding affinities. Two of these agents, (R)-25 and (R)-32, blocked intrathecal NK-1 agonist-driven [Ac-[Arg6,Sar9,Met(O2)11]- substance P 6-11 (Ac-Sar9)] nociceptive behavior in mice. Both compounds potently blocked the neurogenic dural inflammation following trigeminal ganglion stimulation in the guinea pig after intravenous administration. Further, upon oral administration in this model, (R)-32 was observed to be very potent (ID50 = 91 ng/kg) and have a long duration of action (> 8 h at 1 micrograms/kg). Compound (R)-32, designated LY303870, is currently under clinical development as an NK-1 antagonist with a long duration of action.

Pharmacological characterization of LY303870: a novel, potent and selective nonpeptide substance P (neurokinin-1) receptor antagonist.

LY303870 [(R)-1-[N-(2-methoxybenzyl)acetylamino]-3-(1H-indol-3-yl)-2-[N-(2-(4- (piperidin-1-yl)piperidin-1-yl)acetyl)amino]propane] is a new, potent and selective nonpeptide neurokinin-1 (NK-1) receptor antagonist. LY303870 bound selectively and with high affinity to human peripheral (Ki = 0.15 nM) and central (Ki = 0.10 nM) NK-1 receptors. LY303870 inhibited [125I]substance P (SP) binding to guinea pig brain homogenates with similar affinity; however, it had approximately 50-fold lesser affinity for rat NK-1 sites. The less active enantiomer, LY306155 [(S)-1-[N-(2-methoxybenzyl)acetylamino]-3-(1H-indol-3-yl)-2-[N-(2-(4- (piperidin-1-yl)piperidin-1-yl)acetyl)amino]-propane], was 1,000- to 15,000-fold less potent in all the species examined. LY303870 antagonized in vitro NK-1 receptor effects as demonstrated by blockade of SP-stimulated phosphoinositide turnover in UC-11 MG human astrocytoma cells (Ki = 1.5 nM) and interleukin-6 secretion from U-373 MG human astrocytoma cells (Ki = 5 nM). In addition, LY303870 inhibited SP-induced rabbit vena cava contractions (pA2 = 9.4) with high (50,000-fold) selectivity vs. NK-2 or NK-3 receptor-mediated responses. In vivo, LY303870 inhibited [Sar9,Met(O2)11]-SP induced guinea pig bronchoconstriction (ED50 = 75 micrograms/kg i.v.) and pulmonary microvascular leakage in the bronchi (ED50 = 12.8 micrograms/kg i.v.) and trachea (ED50 = 18.5 micrograms/kg i.v.). Therefore, LY303870 is a potent and selective NK-1 receptor antagonist in vitro and in vivo. The use of LY303870 will facilitate a better understanding of NK-1 receptors in physiological processes.

Cyclosporin A is a substance P (tachykinin NK1) receptor antagonist.

The immunosuppressive cyclic undecapeptide, cyclosporin A, inhibited the binding of [125I]substance P to tachykinin NK1 receptors expressed by human IM-9 lymphoblastoid cells, U-373 MG human astrocytoma cells and guinea pig lung parenchyma with IC50 values of 425 +/- 58, 783 +/- 180, and 784 +/- 163 nM respectively. The dihydro derivative of cyclosporin A (dihydro-cyclosporin A) was an equally effective inhibitor, but the O-acetylated derivative (cyclosporin A-OAc) was 3-4 fold less potent. The cyclosporin compounds also inhibited [125I]neurokinin A binding to human NK2 receptors with potencies slightly less than at NK1 sites. In contrast, they were 8-20-fold less effective inhibitors of [125I]MePhe7-neurokinin B binding to guinea pig NK3 receptors (p < 0.001). Thus, the cyclosporin A compounds showed selectivity for NK1 and NK2 receptors. The structure-activity pattern for the effects of cyclosporin A compounds at tachykinin receptors differs from the pattern previously described for their immunosuppressive activity. All three compounds inhibited substance P induced interleukin-6 (IL-6) secretion from U-373 MG astrocytoma cells with potencies similar to their NK1 receptor binding affinities. In addition, cyclosporin A blocked substance P induced phosphatidylinositol (PI) turnover in U-373 MG cells without blocking the corresponding response to histamine. This novel pharmacological profile of the cyclosporin A compounds as NK1 receptor antagonists does not appear to correlate with other known in vitro cyclosporin A functions.

Interleukin-6 secretion from human astrocytoma cells induced by substance P.

Functional NK-1 (substance P) receptors have been demonstrated previously on astrocytes from primary newborn rat brain cultures and human astrocytoma cells lines by specific [125I]-Bolton Hunter substance P (SP) binding and by SP-induced phosphoinositol turnover. In addition, these cells have been shown to release cytokines upon stimulation with interleukin-1 (IL-1) and lipopolysaccharide (LPS). Since SP has also been shown to induce cytokine release from rat glial cells, this neuropeptide may contribute to the pathophysiology of neuronal inflammation in humans by stimulating cytokine production in the brain. We, therefore, explored whether SP could induce U-373 MG human astrocytoma cells, via specific NK-1 receptor activation, to secrete interleukin-6 (IL-6), a cytokine implicated as a key mediator of immune and inflammatory responses. SP stimulated IL-6 production in a concentration-dependent manner with an MC50 (concentration inducing 50% of the maximum response) of 45 nM. IL-6 was detected in the cell culture supernatant fluids 2 h post stimulation and secretion peaked at 12 h. SP induced IL-6 secretion was not mediated by IL-1 since neutralizing anti-IL-1 (alpha and beta) antibody treatment had no effect on the SP response. The selective NK-1 receptor agonist, [Sar9, Met(O2)11]-SP, was comparably effective to SP in stimulating IL-6 secretion; however, selective NK-2 and NK-3 receptor agonists were 250-500-fold less effective. In addition, the non-peptide NK-1 receptor antagonist, (+/-)CP-96,345, inhibited SP (Ki = 4 nM), but not IL-1-induced IL-6 release. These selectivity and specificity studies confirmed the presence of functional NK-1 type receptors linked to IL-6 release. The results of this study support a role for SP as a modulator of immune and/or inflammatory processes in the human CNS.

beta-Amyloid peptide in vitro toxicity: lot-to-lot variability.

beta A4 peptide (beta AP) accumulates in amyloid plaques of Alzheimer's disease and may contribute to neuronal degeneration. Conflicting observations have been reported regarding the direct in vitro and in vivo neurotoxicity of beta AP. We have assessed in vitro beta AP toxicity in high density primary rat hippocampal cultures and found marked lot-to-lot differences in the neurotoxic properties of beta AP. One lot of beta AP from a commercial supplier resulted in significant direct neurotoxicity at 10 microM, while 2 other lots from the same supplier were essentially nontoxic. Three additional lots of beta AP from unrelated sources were also nontoxic at 10 microM. Initial biochemical characterization has not yet revealed any marked differences among the various lots of beta AP. Low levels of endotoxin (ca., 1 EU/ml) were detected in several beta AP preparations but did not correlate with neurotoxicity. Our observation that lot-to-lot variability of beta AP occurred even under identical in vitro culture conditions may account for part of the present controversy in this area.

Selectivity and specificity of new, non-peptide, quinuclidine antagonists of substance P.

Two members of a new class of non-peptide antagonists of substance P, (+-)-cis-3-(2-methoxybenzylamino)-2-benzhydrylquinuclidine [(+/-)-CP-96,345; I] and (+-)-cis-3-(2-chlorobenzylamino)-2-benzhydrylquinuclidine [II], were tested for their ability to antagonize neurokinin-induced contractions of the rabbit cava and jugular veins (NK-1), the rabbit pulmonary artery (NK-2) and the rat portal vein (NK-3 system). Compound 1 is the most potent NK-1 receptor antagonist identified until now; its apparent affinity (pA2 = 9.52) is at least two log units higher than those of other NK-1 antagonists. Compound II is less active. Both compounds have been found to be almost inactive as NK-2 and NK-3 antagonists and should, therefore, be considered as selective for the NK-1 receptor. The new compounds have no direct myotropic effects and are specific for neurokinin (NK-1) receptors since they do not affect the myotropic effects of angiotensin, noradrenaline and bradykinin in the rabbit cava and jugular veins.

Lectin staining of peritoneal mesothelial cells in vitro.

A survey of lectin-binding specificities present on rodent and human mesothelial cells propagated and maintained in tissue culture was made using fluorescein isothiocynate conjugated (FITC) lectins. Rodent and human cells exhibited cell-associated fluorescence following exposure to the FITC-lectins from C. ensiformis, T. vulgaris, A. hypogaea, E. cristagalli and B. simplicifolia, but not with lectins from G. max and D. biflorus. Rodent cells were also positive for FITC-M. pomifera lectin binding. Human, but not rodent, cells were positive for FITC-T. purpureas lectin binding. Exposure of rabbit mesothelial cells in vitro to FITC-lectins that bound to the cell surface resulted in the appearance of discrete loci of putatively intracellular fluorescence. Exposure of cells to ferritin-labelled T. vulgaris lectin at 37 degrees C for as little as 7.5 minutes resulted in the appearance of ferritin-size particles in intracellular vesicles. These results demonstrate 1. the presence of lectin-binding sites in and on peritoneal mesothelial cells from rodents and humans and 2. a possible role of such sites in mediating the entry of lectin-like endogenous molecules into the vacuolar apparatus of these cells.

In vivo and in vitro characterization of mesothelial lipid inclusions.

The nature of intracytoplasmic lipid inclusions found in cultured rabbit and rat peritoneal mesothelial cells was examined by ultrastructural and biochemical techniques. Transmission electron microscopy also demonstrated extracellular release of these lipid bodies. Differential fixation with tannic acid revealed 2 types of inclusions, lamellated (lamellar bodies) and nonlamellated (homogeneous). The lamellar bodies were found near or in the Golgi apparatus and on the cell surface where occasionally they were observed in exocytotic pouches. The homogeneous inclusions were the predominant species being found primarily intracellularly. Lipid bodies obtained from the culture media over the cells displayed on electron microscopy the same morphological characteristics as those seen intracellularly. Exposure of confluent cultures of mesothelial cells to the vital lipid stain Nile Red caused the appearance of intensely fluorescent droplets in or on the cells at wave lengths consistent with staining for phosphatidylcholine-rich vesicles. Incubation of the cells with (14C)-choline and subsequent analysis of phospholipid formation revealed high rates of (14C)-phosphatidylcholine addition to both intra- and extracellular lipid pools. Taken together, mesothelial cells exhibit lipid bodies similar in ultrastructure to the surfactant containing organelles of Type II pneumocytes.

Autosomal recessive polycystic kidney disease: characterization of human peritoneal and cystic kidney cells in vitro.

Renal cystic epithelia and peritoneal mesothelia from two humans with autosomal recessive polycystic kidney disease (ARPKD) were grown in culture. Cystic epithelial and mesothelial cells formed continuous monolayers in vitro. By electron microscopy, cystic renal cells exhibited a single apical cilium and numerous short, stubby microvilli, both in situ and in vitro. Mesothelial cells exhibited intra- and extracellular membrane-limited, lipid-filled vesicles and surface microvilli. Cystic kidney cells in vitro stained positive for lectins from Cancanavalia ensiformis (concanavalin A), Triticum vulgaris, Erythrina cristagalli, Ulex europeaus, and Arachis hypogaea. Immunocytochemical and lectin staining revealed the renal and peritoneal cells to be of collecting tubule and mesothelial origin, respectively. Both cell types showed large depositions of glycogen granules in the cytoplasm during propagation in certain culture media; in kidney cells, dibutyryl cyclic adenosine monophosphate (cAMP) abolished glycogen depositions. Glycogen deposition also was observed in liver tissue obtained by needle biopsy from one patient. No bacteria were cultured from nor endotoxin detected in the renal cyst fluid. Relative to serum, the cyst fluids contained low sodium, potassium, and chloride levels. Thus, cultured ARPKD cells demonstrate a number of characteristics that are different from cells derived from the autosomal dominant form of renal cystic disease (ADPKD).

Expression of von Willebrand factor in plasma and platelets of cats.

Immunochemical methods that are used to assess von Willebrand factor in human beings and dogs were used to assess von Willebrand factor in 3 cat species. Our findings indicated that the expression and multimeric composition of von Willebrand factor in plasma and platelets of cats were similar to those reported in human beings and dogs. We suggest that these methods may be used to evaluate von Willebrand disease in members of the cat family used in this study.

Isolation and propagation in vitro of peritoneal mesothelial cells.

Mesothelial cells lining the peritoneal cavity are the primary site of molecular exchange during peritoneal dialysis, a life support system for over 50,000 patients worldwide. In this study, techniques are described for the isolation and propagation in culture of peritoneal mesothelial cells from rats and rabbits. For comparison, mesothelial cells were also obtained from the serosal surface of human colonic tissue. By electron microscopy the cultured cells were found to exhibit microvilli, a well-developed endoplasmic reticulum and golgi apparatus, micropinocytotic vesicles, and lipid-filled intracellular vesicles. Immunochemical probes revealed the expression by these cells in vitro of cytokeratin, fibronectin, vimentin, and keratin, but not von Willebrand factor. Mesothelial cells from rat, rabbit, and human exhibited contact inhibition, but differences in growth rates and dependence on supplements to the growth media. This work provides a multispecies comparison of the behavior of mesothelial cells in vitro for the purpose of developing an experimental system for the study of mesothelial cell biology and the role of these cells in peritoneal dialysis.

Canine platelet von Willebrand factor: quantification and multimeric analysis.

Washed canine platelets were shown to express a significant level of von Willebrand factor (vWf). Canine platelet vWf differed from canine plasma vWf by the absence of satellite bands associated with each multimer when resolved by SDS-agarose gel electrophoresis. Expression and multimeric composition of canine platelet vWf was quite similar to that of human platelet vWf. Quantification in both lysed, washed canine platelets and in releasate of washed canine platelets yielded estimates of platelet vWf at approximately 2% of circulating vWf in this species, with approximately 15% of this being released into the fluid phase on activation. This contrasts with findings in humans, in which approximately 10%-25% of circulating vWf is compartmentalized in platelets. The difference in relative levels of canine and human platelet vWf could not be accounted for by differences in platelet ultrastructure. The decreased relative level may account for reports that canine platelets contain no vWf.

Metabolic studies in head injury patients: a preliminary report.

The caloric and nitrogen balances of 10 patients with head injury were studied. On the basis of caloric deficits, two groups of five patients each were identified. Both groups were similar except for the average daily caloric supply. Lower caloric deficits were associated with better neurologic outcomes. Large caloric deficits developed by the fourth or fifth day after injury when supplementation was inadequate.

Childhood cerebellopontine angle teratoma associated with congenital hydrocephalus.

A case of childhood teratoma in the cerebellopontine angle associated with shunted congenital hydrocephalus is presented. The need for detailed computed axial scanning with and without contrast in congenital hydrocephalus not associated with intraventricular hemorrhage or myelodysplasia is emphasized.

Effect of parenteral nutrition on cold-induced vasogenic edema in cats.

The effect of standard parenteral nutritional formulas on cold-induced vasogenic edema formation in cats was examined and compared to the effects of 5% dextrose, 0.9% saline, and 40.5% mannitol. The amount of vasogenic edema formed during a 3-hour period of fluid infusion following cold injury was quantified by a computerized graphics tablet determination of the volume of Evans blue-dyed white matter. Specific gravity measurements were taken as a measure of white matter water content. Serum osmolality, urine output, arterial blood gases, hematocrit, body temperature, and systolic blood pressure were measured periodically throughout the infusion period. Parenteral nutritional formulas and a 40.5% mannitol solution produced greater changes in serum osmolality than did 5% dextrose or 0.9% saline. Greater changes in serum osmolality were associated with larger calculated volumes of edema in the injured hemisphere and lower water contents in the uninjured hemisphere. The data indicate that hyperosmolar solutions may potentiate vasogenic edema formation when the blood-brain barrier is open.

Anterior cervical epidural abscess: the use of intraoperative spinal sonography.

A case of acute cervical epidural abscess is presented. The use of intraoperative spinal sonography is discussed as a valuable adjunct in the evaluation and treatment of these uncommon lesions.