CCR1-specific non-peptide antagonist: efficacy in a rabbit allograft rejection model.

The classic signs of acute cellular rejection during organ transplantation include the infiltration of mononuclear cells into the interstitium. This recruitment of leukocytes into the transplanted tissue is promoted by chemokines like RANTES. Since RANTES is a potent agonist for the CC chemokine receptor CCR1, we examined whether the CCR1 antagonist BX 471 was efficacious in a rabbit kidney transplant rejection model. BX 471 was able to compete with high affinity with the CCR1 ligands MIP-1alpha and RANTES for binding to HEK 293 cells expressing rabbit CCR1. BX 471 was a competitive antagonist of rabbit CCR1 in Ca(2+) flux studies. Two separate studies in which animals were subcutaneously implanted with slow release pellets of BX 471 demonstrated that animals implanted with BX 471 had increased survival compared with untreated controls or animals implanted with placebo. The mean survival time for the placebo group was 12.33+/-1.7 days. The animals in the BX 471 treated group had mean survival times of 16.9+/-2.1 and 16.0+/-1.7 days, respectively, for the two studies. Analysis of the combined data by Student t-test gave a P value of 0.03 that is significant at the 0.05 level. In addition, there was a marked reduction in the urea and creatinine levels in the BX 471 treated animals compared with the control and placebo groups in both studies. Finally, pathologic analysis of the kidneys in the rabbit renal transplantation model from animals in the different groups showed that BX 471 was similar to cyclosporin in its ability to prevent extensive infarction of transplanted kidneys. Based on the data from these studies, BX 471 shows clear efficacy at the single dose tested compared with animals treated with placebo.

A non-peptide functional antagonist of the CCR1 chemokine receptor is effective in rat heart transplant rejection.

Chemokines like RANTES appear to play a role in organ transplant rejection. Because RANTES is a potent agonist for the chemokine receptor CCR1, we examined whether the CCR1 receptor antagonist BX471 is efficacious in a rat heterotopic heart transplant rejection model. Treatment of animals with BX471 and a subtherapeutic dose of cyclosporin (2.5 mg/kg), which is by itself ineffective in prolonging transplant rejection, is much more efficacious in prolonging transplantation rejection than animals treated with either cyclosporin or BX471 alone. We have examined the mechanism of action of the CCR1 antagonist in in vitro flow assays over microvascular endothelium and have discovered that the antagonist blocks the firm adhesion of monocytes triggered by RANTES on inflamed endothelium. Together, these data demonstrate a significant role for CCR1 in allograft rejection.

Identification and characterization of a potent, selective, and orally active antagonist of the CC chemokine receptor-1.

The CC chemokine receptor-1 (CCR1) is a prime therapeutic target for treating autoimmune diseases. Through high capacity screening followed by chemical optimization, we identified a novel non-peptide CCR1 antagonist, R-N-[5-chloro-2-[2-[4-[(4-fluorophenyl)methyl]-2-methyl-1-piperazinyl ]-2-oxoethoxy]phenyl]urea hydrochloric acid salt (BX 471). Competition binding studies revealed that BX 471 was able to displace the CCR1 ligands macrophage inflammatory protein-1alpha (MIP-1alpha), RANTES, and monocyte chemotactic protein-3 (MCP-3) with high affinity (K(i) ranged from 1 nm to 5.5 nm). BX 471 was a potent functional antagonist based on its ability to inhibit a number of CCR1-mediated effects including Ca(2+) mobilization, increase in extracellular acidification rate, CD11b expression, and leukocyte migration. BX 471 demonstrated a greater than 10,000-fold selectivity for CCR1 compared with 28 G-protein-coupled receptors. Pharmacokinetic studies demonstrated that BX 471 was orally active with a bioavailability of 60% in dogs. Furthermore, BX 471 effectively reduces disease in a rat experimental allergic encephalomyelitis model of multiple sclerosis. This study is the first to demonstrate that a non-peptide chemokine receptor antagonist is efficacious in an animal model of an autoimmune disease. In summary, we have identified a potent, selective, and orally available CCR1 antagonist that may be useful in the treatment of chronic inflammatory diseases.

Incidence of postangiographic abnormalities revealed by diffusion-weighted MR imaging.

BACKGROUND AND PURPOSE: Occasionally we have observed anecdotal cases of asymptomatic hyperintensities on diffusion-weighted MR (DW-MR) examinations of the brain of patients who previously underwent routine cerebral angiography. These observations, as well as MR imaging and transcranial Doppler data in the literature suggesting a high rate of procedure-associated emboli, raise concern regarding the underdiagnosis of asymptomatic focal infarction associated with cerebral angiography. In order to determine whether asymptomatic diffusion abnormalities are frequently associated with procedures, we prospectively obtained DW-MR images before and after routine cerebral angiography. METHODS: Twenty consecutive patients, who met protocol criteria and received a routine three- or four-vessel diagnostic cerebral angiogram at our institution, were evaluated. Using a Bayesian estimate to establish an upper bound for the incidence of an event with zero occurrences in a study sample, the study group size was selected to exclude a 10% incidence of abnormalities revealed by DW-MR imaging of patients who underwent previous cerebral angiography. Two neuroradiologists evaluated imaging studies. RESULTS: Neither clinical signs nor abnormalities on DW-MR images were found, which suggested no infarction after angiography in our patient sample. Based on this data, an upper bound of 9% (95% confidence) is predicted for the appearance of abnormalities revealed by DW-MR imaging after cerebral angiography. CONCLUSION: Cerebral angiography is associated with an incidence of asymptomatic cerebral infarction of no more than 9%. It well may be substantially lower than this estimate; a more accurate evaluation of the true incidence would require a significantly larger study population. This test provides a convenient noninvasive means of assessing procedure-related cerebral infarction, such as that which occurs after carotid endarterectomy or vascular angioplasty and stenting.

Species selectivity of a small molecule antagonist for the CCR1 chemokine receptor.

The species specificity of a small molecule antagonist for the human CCR1 chemokine receptor, 2-2-diphenyl-5-(4-chlorophenyl)piperidin-1-yl)valeronitrile (CCR1 antagonist 1), has been examined using cloned CCR1 receptors from various species. The compound was able to bind to rabbit, marmoset, and human CCR1, and was able to block the functional activation of these receptors. However, it failed to significantly displace radiolabeled macrophage inflammatory protein-1alpha (MIP-1alpha) binding to mouse CCR1 at concentrations up to 10 microM. These data suggested that the antagonist binding site is well-conserved in rabbit, marmoset and human CCR1, but not in mouse CCR1. The functional selectivity and mechanism of action for CCR1 antagonist 1 were further characterized. CCR1 antagonist 1 blocked the increase in intracellular Ca(2+) stimulated by CCR1 agonists, but had no effect on N-formyl-Met-Leu-Phe (FMLP), monocyte chemotactic protein-1 (MCP-1) and stromal-derived factor 1alpha (SDF1alpha)-induced Ca(2+) mobilization, demonstrating functional selectivity for CCR1. Since CCR1 antagonist 1 is a functional antagonist of marmoset and rabbit CCR1 receptors, it should be possible to test its efficacy in animal models of disease.

Discovery of novel non-peptide CCR1 receptor antagonists.

Ligands for the CCR1 receptor (MIP-1alpha and RANTES) have been implicated in a number of chronic inflammatory diseases, most notably multiple sclerosis and rheumatoid arthritis. Because these ligands share a common receptor, CCR1, we sought to discover antagonists for this receptor as an approach to treating these disorders. A novel series of 4-hydroxypiperidines has been discovered by high throughput screening (HTS) which potently inhibits the binding of MIP-1alpha and RANTES to the recombinant human CCR1 chemokine receptor. The structure-activity relationships of various segments of this template are described as the initial HTS lead 1 was optimized synthetically to the highly potent receptor antagonist 6s. This compound has been shown to have at least 200-fold selectivity for inhibition of CCR1 over other human 7-TM receptors, including other chemokine receptors. In addition, data obtained from in vitro functional assays demonstrate the functional antagonism of compound 6s and structurally related analogues against the CCR1 receptor in a concentration dependent manner. The discovery and optimization of potent and selective CCR1 receptor antagonists represented by compound 6s potentially represent a novel approach to the treatment of chronic inflammatory diseases.

Identification and characterization of small molecule functional antagonists of the CCR1 chemokine receptor.

The CC chemokines macrophage inflammatory protein-1alpha (MIP-1alpha) and RANTES (regulated on activation normal T cell expressed) have been implicated in rheumatoid arthritis and multiple sclerosis. Since their effects are mediated through the CCR1 chemokine receptor, we set up a small molecule CCR1 antagonist program to search for inhibitors. Through high capacity screening we discovered a number of 4-hydroxypiperidine compounds with CCR1 antagonist activity and report their synthesis and in vitro pharmacology here. Scatchard analysis of the competition binding data revealed that the compounds had Ki values ranging from 40 to 4000 nM. The pharmacological profile of the most potent member of this series, compound 1 (2-2-diphenyl-5-(4-chlorophenyl)piperidin-lyl)valeronitri te), was further evaluated. Compound 1 showed concentration-dependent inhibition of MIP-1alpha-induced extracellular acidification and Ca2+ mobilization demonstrating functional antagonism. When given alone, the compound did not elicit any responses, indicating the absence of intrinsic agonist activity. Compound 1 inhibited MIP-1alpha- and RANTES-induced migration in peripheral blood mononuclear cells in a dose-responsive manner. Selectivity testing against a panel of seven transmembrane domain receptors indicated that compound 1 is inactive on a number of receptors at concentrations up to 10 microM. This is the first description of CCR1 receptor antagonists that may be useful in the treatment of chronic inflammatory diseases involving MIP-1alpha, RANTES, and CCR1.

Responding to health care reform: preparing tomorrow's practitioners.

At a time when all of healthcare is undergoing change and evolution, the healthcare education community has so far escaped intense public scrutiny. To remain valid, we must address the education and the reeducation of healthcare professionals. Baccalaureate health administration programs, in particular, can step in and respond to the changing needs of the industry, the market, and of healthcare professionals by creating articulation formats that provide flexible verti for associate degreed healthcare professionals. Such programs enable a diverse constellation of healthcare professionals to obtain the managerial and organizational skills that are cal and horizontal access key to career mobility in today's turbulent healthcare arena.

Similar rates of phosphatidylinositol hydrolysis following activation of wild-type and truncated rat neurokinin-1 receptors.

The substance P (neurokinin-1) receptor belongs to the family of seven putative transmembrane domain receptors that are coupled via G proteins to phospholipase C activation. Homologous desensitization of substance P-stimulated responses has been described in various systems. The rat neurokinin-1 receptor and a truncated mutant lacking the carboxyl-terminal region were expressed in Chinese hamster ovary cells to examine the mechanisms of substance P-induced desensitization. Wild-type and truncated receptor-bearing cells were indistinguishable in agonist binding affinity and EC50 of substance P-induced accumulation of 3H-inositol phosphates. Substance P-induced responses continued for 30-45 min in cells expressing wild-type and truncated receptors as well as in rat LRM-55 and human U373 cells, which express endogenous neurokinin-1 receptors. In transfected cells expressing the wild-type receptor, CP-96,345 added 15 min after substance P blocked further responses, demonstrating the continuing presence of responsive receptors. The rates of accumulation of 3H-inositol phosphates were four times greater in the initial 15 s of stimulation than for the next 20 min for both wild-type and truncated receptor types. This decrease in rate of substance P-stimulated phosphatidylinositol hydrolysis is therefore not dependent on the carboxyl-terminal region of the rat neurokinin-1 receptor, which contains 26 serine and threonine residues. These results are discussed in relation to current ideas regarding neurokinin-1 receptor desensitization.

Aza-tricyclic substance P antagonists.

The synthesis and structure-activity relationships of a series of aza-tricyclic analogs of the quinuclidine substance P (SP) antagonist 1 are described. The SP receptor affinity of these compounds was found to vary according to the size of the new ring fused to the quinuclidine and the mode of fusion. Correlations between receptor affinity and (1) the steric bulk of the newly introduced ring fusion and (2) the dihedral angle between the benzhydryl and benzylamino substituents of these aza-tricyclic compounds were explored.

Substance P increases neutrophil adhesion to bronchial epithelial cells.

Polymorphonuclear neutrophils (PMNs) accumulate within the airways during acute and chronic bronchitis and can adhere to bronchial epithelium. Substance P (SP), a neuropeptide released from the primary afferent nerve endings, has been shown to have a proinflammatory effect on PMNs. To test the hypothesis that SP could modulate PMN bronchial epithelial cell adherence, bovine bronchial epithelial cells (BBECs) were isolated and cultured, and the capacity of SP to modulate PMN-BBEC adherence was evaluated. SP interacted with BBECs to induce an increase in PMN adhesion (14.7 +/- 1.2% vs 5.3 +/- 0.7% adherence, p < 0.01). The effect of SP was both time- and dose-dependent with maximal responses at 6 h and 10(-10) M. The effect was reproduced by the carboxyl-terminal sequence of the molecule (SP 6-11). Importantly, pretreatment of the BBECs with the tachykinin SP receptor (NK1) antagonist, CP-96,345, significantly reduced the increase in adhesion induced by SP (p < 0.01). Furthermore, treatment of the BBECs with antibodies against CD11a (LFA-1), CD11b (MAC-1), or ICAM-1 significantly decreased SP-induced adherence (p < 0.01 all comparisons). Conversely, SP stimulation of PMN induced a dose-dependent, rapid (within 5 min) increase in adherence. This effect was also mediated by the carboxy end of the molecule and was decreased by CP-96,345, again suggesting that this effect was NK1 mediated. These data demonstrate the SP has the capacity for modulating PMN-BBEC interactions and suggest an important role for SP in modulating airway inflammation.

CP-96,345, a substance P antagonist, inhibits rat intestinal responses to Clostridium difficile toxin A but not cholera toxin.

Toxin A from Clostridium difficile mediates acute inflammatory enterocolitis in experimental animals, while cholera toxin causes noninflammatory secretory diarrhea. The purpose of this study was to investigate whether an antagonist to the peptide substance P, a constituent of primary sensory neurons known to participate in inflammatory responses, would inhibit toxin A-mediated enteritis in the rat ileum. Pretreatment of rats with CP-96,345 (2.5 mg per kg of body weight), a substance P antagonist, dramatically inhibited fluid secretion (P < 0.01) and mannitol permeability (P < 0.01) in ileal loops exposed to toxin A. The protective effects, which were dose dependent, caused a significant reduction of inflammation in the lamina propria, reduction of the necrosis of intestinal epithelial cells, and complete inhibition of toxin A-mediated release of rat mast cell protease II, a specific product of rat mucosal mast cells. An inactive enantiomer of the substance P antagonist, CP-96,344, had no effect. In contrast, pretreatment with CP-96,345 had no inhibitory effect on the intestinal effects caused by administration of cholera toxin into the ileal loops. From these data, we conclude that the peptide substance P is involved in the secretory and inflammatory effects of toxin A but not of cholera toxin.

Selective inhibition of the carotid body sensory response to hypoxia by the substance P receptor antagonist CP-96,345.

Carotid bodies are sensory organs for monitoring arterial oxygen and CO2. Previous studies have shown that chemoreceptor tissue contains substance P (SP) and exogenously administered SP augments chemosensory discharge. In the present study, we examined the physiological importance of SP in carotid body chemoreception by using a selective nonpeptide SP [neurokinin (NK) 1] receptor antagonist CP-96,345. In experiments performed on anesthetized cats, sensory discharge was recorded from the carotid body in situ. To control for alterations in blood flow, additional studies were conducted on the carotid body in vitro. In in vivo studies, close carotid body (intraarterial) administration of CP-96,345 attenuated the sensory response to hypoxia in a dose-dependent manner with 73% of the response abolished at doses of 0.3-0.6 mg/kg. Comparable doses of the (2R,3R)-enantiomer had no effect on hypoxia-induced excitation, indicating that the effect of CP-96,345 was not due to nonspecific action. In contrast, the carotid body response to high CO2 was not affected by CP-96,345, implying that only the hypoxic response is mediated by NK-1 receptor and confirming that the effect of the SP antagonist was not due to nonspecific actions. Marked attenuation of the sensory response to hypoxia was also obtained in the carotid body in vitro, suggesting that the effects of the NK-1 antagonist were not secondary to cardiovascular changes. These results demonstrate that CP-96,345 attenuates or abolishes the chemosensory response to hypoxia but not to CO2 and suggest that SP mediates the hypoxia-induced sensory excitation in the cat carotid body via NK-1 receptor activation.

Pharmacology of CP-99,994; a nonpeptide antagonist of the tachykinin neurokinin-1 receptor.

(+)-(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine (CP-99,994) binds selectively and with high affinity (Ki = 0.25 nM) to neurokinin (NK)-1 tachykinin receptors in a human cell line and in guinea pigs where it acts as an antagonist as evidenced by its blockade of substance P-induced excitation of locus coeruleus neurons in vitro. Subcutaneously administered CP-99,994 antagonized locomotor activity in guinea pigs induced by intraventricular infusion of [Sar9,Met(O2)11]-substance P (50 micrograms) with an ID50 = 0.59 mg/kg, indicating that CP-99,994 penetrates into the central nervous system. Orally administered CP-99,994 potently blocked (ID50 = 4 mg/kg) the leakage of Evans blue dye into trachea and bronchi elicited by exposure of guinea pigs to aerosol capsaicin (1 mM). CP-99,994 has reduced affinity (IC50 = 3 microM) for the L-type calcium channel in contrast to CP-96,345 (IC50 = 27 nM) an earlier nonpeptide antagonist. Thus, CP-99,994 represents an important pharmacological tool for investigating the physiological role of substance P and a potentially novel therapeutic agent for treating a variety of diseases.

NK1 receptors mediate tachykinin-induced increase in microvascular clearance in hamster cheek pouch.

The purpose of this study was to determine the receptor subtype(s) that mediates tachykinin-induced neurogenic plasma extravasation in the hamster cheek pouch. Changes in microvascular clearance were quantified by counting the number of leaky sites and calculating the clearance of fluorescein isothiocyanate-dextran [mol wt 70,000 (Dextran 70)] during suffusion of the cheek pouch with substance P, neurokinin A, neurokinin B, and capsaicin. Suffusion of substance P, capsaicin, and neurokinin A, but not neurokinin B, was associated with a significant concentration-dependent increase in leaky site formation and clearance of fluorescein isothiocyanate-Dextran 70 (P < 0.05). However, the responses to substance P and capsaicin were significantly greater than those to neurokinin A. Pretreatment with the selective, nonpeptide NK1 receptor antagonist, CP-96,345, significantly attenuated substance P- and capsaicin-induced but not neurokinin A-induced responses (P < 0.05). These effects were specific, since the 2R,3R enantiomer, CP-96,344, was inactive, and CP-96,345 had no significant effect on adenosine-induced responses. We conclude that, in the hamster cheek pouch, NK1 receptors are the predominant receptors that mediate neurogenic plasma extravasation.

Two nonpeptide tachykinin antagonists act through epitopes on corresponding segments of the NK1 and NK2 receptors.

The molecular mechanism of action for two chemically distinct and highly selective, nonpeptide antagonists, CP-96,345 and SR-48,968, was studied by development of a series of chimeric constructs between their respective target receptors, the NK1 (substance P) and NK2 (neurokinin A) receptors. The binding affinities of the natural peptide ligands, substance P and neurokinin A, were not affected by exchanging almost the entire C-terminal half of the NK1 receptor with the corresponding segment of the NK2 receptor. In contrast, it was found that transfer from the NK2 to the NK1 receptor of a segment corresponding to transmembrane segment VI, the amino-terminal half of transmembrane segment VII, and the connecting extracellular loop 3 completely switched the susceptibility for the nonpeptide antagonists. This chimeric exchange, corresponding to 17 nonconserved residues, conveyed full susceptibility for the NK2-specific compound SR-48,968 to the previously unresponsive NK1 receptor--i.e., the Ki value for inhibition of binding of 125I-labeled substance P decreased from > 10,000 to 0.97 nM. At the same time the affinity for the NK1-selective compound CP-96,345 decreased > 30-fold. The actual binding site for SR-48,968 was localized to this region of the NK2 receptor by use of [3H]SR-48,968, which did not bind to the NK1 receptor but bound with similar high affinities to the wild-type NK2 receptor and to the chimeric NK1 receptor with the NK2 receptor segment incorporated around transmembrane segments VI and VII, Kd = 1.5 nM and 1.0 nM, respectively. Our data indicate that two chemically very different nonpeptide antagonists, CP-96,345 and SR-48,968, act through epitopes located around transmembrane segment VI on their respective target receptors and that at least the nonconserved residues in these epitopes are not important for the binding of the natural peptide ligands, substance P and neurokinin A.

A new NK1 receptor antagonist (CP-99,994) prevents the increase in tracheal vascular permeability produced by hypertonic saline.

The increase in tracheal vascular permeability evoked by hypertonic saline depends on capsaicin-sensitive sensory nerves, which contain substance P and other neuropeptides. The present study was performed to determine whether a novel, nonpeptide, selective antagonist of the NK1 tachykinin receptor CP-99,994, [(+)-(2S-3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine], can prevent the effect of substance P, capsaicin and hypertonic saline on tracheal vascular permeability. CP-99,994 was also tested against a nonpeptide inflammatory mediator, platelet-activating factor (PAF), to assess the selectivity of its action. Anesthetized F-344 rats were injected with either substance P (5 micrograms/kg i.v.), capsaicin (100 micrograms/kg i.v.) or PAF (10 micrograms/kg i.v.), or were exposed to ultrasonically nebulized 3.6% NaCl. In each group, some of the rats were pretreated with CP-99,994 (1 to 4 mg/kg i.v.), and some with its vehicle (0.9% NaCl). Groups of rats injected with substance P or exposed to hypertonic saline were pretreated with the (2R, 3R)-enantiomer CP-100,263, [(-)-(2R-3R)-3-(2-methoxybenzylamino)-2-phenylpiperidine] (2 or 4 mg/kg i.v.). The magnitude of the increase in tracheal vascular permeability was measured by quantifying the extravasation of Evans blue dye. CP-99,994 prevented the increase in tracheal vascular permeability produced by inhalation of hypertonic saline, by substance P and by capsaicin, but did not prevent the effect of PAF. CP-100,263 did not affect substance P- and hypertonic saline-induced increase in vascular permeability. These results indicate that the NK1 receptor antagonist CP-99,994 produces stereoselective inhibition of neurogenic plasma extravasation evoked by inhalation of hypertonic saline.

Discovery of CP-96,345 and its characterization in disease models involving substance P.

Studies with CP-96,345, a potent, selective, orally active, nonpeptide NK1 receptor antagonist, have provided considerable insight into SP pharmacology. Rather than being a primary neurotransmitter, SP prolongs the nociception produced by other neurotransmitters. By controlling endothelial permeability, SP plays a major role in inflammation and inflammatory aspects of asthma, possibly by regulating the access of neutrophils to an inflammatory site. These results indicate potential therapeutic applications for SP antagonists in the treatment of chronic pain, inflammation, and inflammatory aspects of asthma, and signal a new era in the clinical management of these important diseases.