The in vitro pharmacological profile of KR31080, a nonpeptide AT1 receptor antagonist

Summary— KR31080 (2-butyl-5-methyl-6-(1-oxopyridin-2-yl)-3-[[2'-(1H-tetrazol-5-yl) biphenyl-4-yl]methyl]-3H-imidazo[4,5-b] pyridine) is a potent inhibitor of angiotensin type 1 (AT₁) receptors in rabbit aorta and human recombinant AT₁ receptors. In the isolated rabbit thoracic aorta, KR31080 caused a nonparallel shift to the right of the concentration-response curves to angiotensin II (All) with decreased maximal response (pD'₂ = 10.1 ± 0.1), but had no effect on the contractile response induced by norepinephrine. KR31080 inhibited specific [¹²⁵I]AII binding to rabbit aortic membranes (AT, receptors) and [¹²⁵I][Sar¹, Ile⁸]AII binding to human recombinant AT₁ receptors in a concentration-dependent manner with IC₅₀ values of 0.84 ± 0.08 nM and 1.92 ± 0.15 nM, respectively, but did not inhibit specific [¹²⁵I)AII binding to bovine cerebellum membranes (ÀT₂ receptors). In the Scatchard analysis, KR31080 interacted with rabbit aortic AT₁ receptors in a competitive manner, similar to losartan. These results demonstrate that KR31080 is a potent and AT₁ selective angiotensin receptor antagonist which exerts a competitive antagonism in the [¹²⁵I]AII binding assay and insurmountable AT₁ receptor antagonism in the functional study.     

Detection of undiagnosed coagulopathies using routine rapid heparin neutralization.

OBJECTIVE: To determine the most frequent clinical causes of a prolonged activated partial thromboplastin time (APTT) result, and to determine whether a new heparin-removal device (the Hepchek, Pall Biomedical, Glen Cove, NY 11542) is capable of efficiently detecting the causes of these values. DESIGN: A combination of chart review and laboratory testing comparing the criterion standard--the heparin chromogenic substrate assay--with the Hepchek. Laboratory investigations were blinded and controlled. SETTING: Inpatient, acute-care hospital. PATIENTS: A total of 1,000 hospital patients with a variety of hemostatic disorders. MAIN OUTCOME MEASURE: The extent to which the Hepchek accurately identified the etiology of a prolonged APTT result. RESULTS: The APTT was prolonged in 25.2% of samples. The presence of heparin in the sample was confirmed by chromogenic assay or by using the Hepchek heparin-removal filter. The presence of heparin was confirmed in 12.8% of all samples and in more than 50% of all abnormal samples. The cause of the abnormal APTT was often unappreciated by the clinician. Bayesian analysis of the Hepchek's ability to diagnose heparin correctly as the cause of the abnormal APTT showed a sensitivity of 100% and specificity of 99.9%. CONCLUSION: Use of the Hepchek in the routine clinical laboratory is an efficient and rapid method of detecting heparin as a cause of isolated prolonged APTT results, and should reduce demands for unwarranted coagulation analyses and inappropriate treatment with blood products.     

Molecular cloning of agonistic and antagonistic monoclonal antibodies against human 4-1BB.

Previously, we prepared two different monoclonal antibodies (mAbs) against human 4-1BB (CD137): an agonistic mAb BBK-1 and an antagonistic mAb BBK-2. In this paper, we describe the molecular cloning of these two mAbs and present comparisons of their amino acid sequences. cDNAs encoding the heavy (H) and light (L) chains of the two mAbs were cloned by screening of cDNA libraries constructed from hybridomas secreting these mAbs. Comparisons of amino acid sequences of the two mAbs showed that, while the constant regions of the H and L chains were identical between the two mAbs, the variable region showed 45% identity in H chains and 48% identity in L chains. This suggests that these two mAbs recognize different epitopes of 4-1BB and may have different effects on the activity of 4-1BB.     

Dopamine differentially regulates dynorphin, substance P, and enkephalin expression in striatal neurons: in situ hybridization histochemical analysis.

Dopamine regulation of the levels of dynorphin, enkephalin, and substance P messenger RNAs in rat striatal neurons was analyzed with in situ hybridization histochemistry (ISHH). Relative levels of peptide mRNA expression in the patch and matrix compartments of the dorsolateral striatum were compared among control rats, rats treated for 10 d with apomorphine, rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal dopaminergic system, and rats with nigrostriatal dopaminergic lesions followed 2 weeks later by 10 d of apomorphine treatment. Image analysis of ISHH labeling demonstrated that the number of neurons expressing each peptide mRNA remained constant, whereas the relative level of peptide mRNA per neuron changed significantly, depending on the experimental treatment. Dynorphin mRNA expression increased following chronic apomorphine treatment: striatal patch neurons increased to an average of 100% above control values, whereas striatal matrix neurons showed only a 25% increase. Dynorphin mRNA expression decreased following 6-OHDA lesions: patch neurons showed an average 75% reduction in expression, whereas matrix neurons showed no significant change. In animals with 6-OHDA lesions followed by apomorphine treatment, both patch and matrix neurons showed an average increase in dynorphin expression of 300% above control levels. Changes in dynorphin mRNA levels with these treatments were matched by qualitative changes in dynorphin immunoreactivity both in the striatum and in striatonigral terminals in the substantia nigra. Neither substance P nor enkephalin mRNA levels showed a significant difference between the striatal patch and matrix compartments in any experimental condition (in the dorsolateral striatum). Substance P mRNA expression was increased an average of 50% after 10 d of apomorphine treatment and showed an average decrease of 75% following 6-OHDA lesions of the mesostriatal system. There was no significant change in the expression of substance P mRNA in striatal neurons compared to control values in rats with combined 6-OHDA lesion and apomorphine treatment. Enkephalin mRNA expression was not significantly altered by chronic apomorphine treatment but showed an average increase per cell of some 130% above control levels following 6-OHDA-induced lesions of the mesostriatal system. In animals with a 6-OHDA lesion and apomorphine treatment, enkephalin mRNA was also elevated but not significantly above the levels produced by the lesions alone. These data show that the expression of dynorphin, enkephalin, and substance P is differentially regulated by the mesostriatal dopaminergic system and, further, suggests that the mechanisms by which this regulation occurs may be different for the 3 peptide families.     

Magnetic resonance imaging confirmation of resolution of periodontoid pannus formation following C1/C2 posterior transarticular screw fixation.

Chronic odontoid fractures are considered unstable spinal lesions. Chronic instability in this region leads to the development of an inflammatory pannus, which can progress resulting in spinal cord compression radiographically and a myelopathy syndrome clinically. In this report we document three cases of reversal of pannus after C1/C2 transarticular screw fixation of an unstable odontoid fracture. Three patients were identified with chronic odontoid fractures and spinal cord compression due to periodontoid pannus formation. All patients presented with a progressive myelopathy syndrome. All patients underwent preoperative and postoperative magnetic resonance imaging (MRI) of the craniovertebral junction. C1/C2 transarticular screw fixation was performed for stabilization of C1/C2. Postoperatively there were no complications. Postoperative MRI at 6 months demonstrated resolution of the ventral pannus. Moreover, all patients exhibited improvement of preoperative neurological deficits. MRI is the imaging technique of choice for diagnosis and follow-up of patients with chronic odontoid fractures and ventral pannus. C1/C2 transarticular screw fixation provides a viable method for spinal stabilization in this region. In addition, stabilization can result in resolution of inflammatory pannus formation secondary to instability of the C1/C2 articulation.     

Spontaneous neuronal hyperactivity in the medial and intralaminar thalamic nuclei of patients with deafferentation pain

Electrical activity was recorded from single cells in the thalamus of 10 patients with chronic pain associated with deafferentation. Under local anesthesia, these patients underwent either electrode implantation or thalamotomy for treatment of their pain. In eight of the 10 patients, single units were identified as discharging spontaneously in high-frequency, often rhythmic, bursts. The discharges were of two types: short bursts comprised of two to six spikes with a burst frequency of one to four per second; and long trains of 30 to 80 spikes of similar frequency. Reconstruction of electrode trajectories indicated that recordings were made from the region corresponding to the lateral aspect of the mediodorsal thalamic nucleus, the central lateral nucleus, a small part of the central median nucleus, and the parafascicular nucleus. In the eight patients in whom spontaneous neuronal burst activity was exhibited, it was impossible to study activity evoked by natural cutaneous stimulation due to the continuous spontaneous neuronal discharges. Both animal and human studies have suggested that pain related to deafferentation is accompanied by spontaneous hyperactivity in the dorsal horn of the spinal cord and in the ventral posterior thalamic nuclei. The authors present evidence of spontaneous neuronal hyperactivity in the intralaminar thalamic nuclei of patients with pain related to deafferentation. The findings suggest that spontaneous neuronal discharge in patients with pain related to deafferentation is more widespread in the central nervous system than has been previously appreciated. The results have important implications for the surgical treatment of chronic pain.     

Changes in outcome with sphincter preserving surgery for rectal cancer in Korea, 1991-2000.

AIM: To report the clinical and oncological data of patients operated on for rectal cancers 3-5 cm from the AV over a 10 year period, including the Sphincter preservation (SP) rate. METHODS: We reviewed medical records of 304 patients with rectal cancers 3-5 cm from the AV who underwent surgical resection from January 1991 through December 2000. The 10 years were divided into three periods based on the introduction of new surgical techniques, specifically, ultralow anterior resection (ULAR) with double stapling in March 1994 and ULAR with coloanal anastomosis in April 1997. The rates of SP, complications and patient survival during these periods were compared. RESULTS: The SP rate increased significantly over the 10 years, from 16.4% in period I (January 1991-February 1994), to 53.0% in period II (March 1994-March 1997), to 86.5% in period III (April 1997-December 2000) (p<0.001). Over time, the age of the patients increased (p=0.004), the length of the distal resection margin became shorter (p=0.005), and the rate of lymph node metastasis increased (p=0.016). The factors significantly influencing SP were the period (p<0.001) and the distance from the AV (p<0.001). Over time, morbidity did not increase, and overall and disease free survival rates did not decrease. In contrast, the overall survival of N2 cases significantly increased over time (p=0.0492). CONCLUSION: Over 10 years, the SP rate in rectal cancers 3-5 cm from the AV was significantly increased by the introduction of the double stapling and coloanal anastomosis techniques. These surgical methods, however, had no effect on morbidity, disease free survival and overall survival rates.     

Regulation of NGF gene expression in CNS glia by cell-cell contact.

Nerve growth factor (NGF) gene expression in central nervous system (CNS) glia appears to be associated with active glial growth. To study the underlying molecular mechanisms, we examined the effects of a number of growth-related factors on NGF mRNA expression in glial cultures. Our results suggest that glial membrane interaction, as a consequence of growth, actively inhibits NGF gene expression in CNS glia.