Development of orally active oxytocin antagonists: studies on 1-(1-[4-[1-(2-methyl-1-oxidopyridin-3-ylmethyl)piperidin-4-yloxy]-2- methoxybenzoyl]piperidin-4-yl)-1,4-dihydrobenz[d][1,3]oxazin-2-one (L-372,662) and related pyridines

The previously reported oxytocin antagonist L-371,257 (2) has been modified at its acetylpiperidine terminus to incorporate various pyridine N-oxide groups. This modification has led to the identification of compounds with improved pharmacokinetics and excellent oral bioavailability. The pyridine N-oxide series is exemplified by L-372,662 (30), which possessed good potency in vitro (Ki = 4.1 nM, cloned human oxytocin receptor) and in vivo (intravenous AD50 = 0.71 mg/kg in the rat), excellent oral bioavailability (90% in the rat, 96% in the dog), good aqueous solubility (>8.5 mg/mL at pH 5.2) which should facilitate formulation for iv administration, and excellent selectivity against the human arginine vasopressin receptors. Incorporation of a 5-fluoro substituent on the central benzoyl ring of this class of oxytocin antagonists enhanced in vitro and in vivo potency but was detrimental to the pharmacokinetic profiles of these compounds. Although lipophilic substitution around the pyridine ring of compound 30 gave higher affinity in vitro, such substituents were a metabolic liability and caused shortfalls in vivo. Two approaches to prevent this metabolism, addition of a cyclic constraint and incorporation of trifluoromethyl groups, were examined. The former approach was ineffective because of metabolic hydroxylation on the constrained ring system, whereas the latter showed improvement in plasma pharmacokinetics in some cases.     

Conformal proton therapy for prostate carcinoma

The suppression of apoptosis may contribute to the carcinogenicity of the peroxisome proliferators (PPs), a class of non-genotoxic rodent hepatocarcinogens. Our previous work demonstrated that the PP nafenopin suppressed both spontaneous and transforming growth factor beta1 (TGFbeta1)-induced hepatocyte apoptosis both in vivo and in vitro. Here, we extend these observations by demonstrating the ability of nafenopin to suppress apoptosis induced by other major candidates for the signalling of cell death in the liver. Treatment of rat or mouse hepatocyte monolayers with TGFbeta1 or the DNA damaging drugs etoposide or hydroxyurea induced high levels of apoptosis. Western blot analysis did not support a role for either p53 or p21waf1 in etoposide-induced apoptosis in rat hepatocytes. Treatment of mouse hepatocytes with an agonistic anti-Fas antibody also resulted in an induction of high levels of apoptosis. Pre-addition and continued exposure to nafenopin suppressed apoptosis induced by all three stimuli. Overall, our studies demonstrate that the ability of nafenopin to protect hepatocytes from apoptosis is not restricted to species or apoptotic stimulus. It is possible, therefore, that the PPs may suppress apoptosis by acting on diverse signalling pathways. However, it seems more likely that nafenopin suppresses hepatocyte apoptosis elicited by each death stimulus by impinging on a core apoptotic mechanism.     

Production and characterisation of monoclonal antibodies specific for bovine interleukin-4

This study assessed physical activity patterns in a sample of urban African Americans, whose participation in physical activity has not previously been well-described. From questions administered by interviewers during health fair screenings in 19 churches in East Baltimore, information regarding participation in regular, leisure-time activity (defined as 30 minutes of activity, 5 days per week), time spent walking on the job, and distance walked to and from work was assessed from 365 adults (69% women). Regular, leisure-time activity participation was 18% for men and 16% for women. When the definition of physical activity participation was broadened to include: (1) spending over half the day walking at work; (2) walking at least 10 blocks to and from work; as well as (3) regular, leisure-time activity, 41% of men and 38% of women were active. These data suggest that, while a small percentage of African Americans participate in regular physical activity, a substantial percentage are regularly active when non-leisure-time activity is assessed. To accurately characterize overall participation, physical activity derived from a variety of sources, including transportation and work-related activity, should be assessed.     

Role of HIF-1alpha in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis

As a result of deprivation of oxygen (hypoxia) and nutrients, the growth and viability of cells is reduced. Hypoxia-inducible factor (HIF)-1alpha helps to restore oxygen homeostasis by inducing glycolysis, erythropoiesis and angiogenesis. Here we show that hypoxia and hypoglycaemia reduce proliferation and increase apoptosis in wild-type (HIF-1alpha+/+) embryonic stem (ES) cells, but not in ES cells with inactivated HIF-1alpha genes (HIF-1alpha-/-); however, a deficiency of HIF-1alpha does not affect apoptosis induced by cytokines. We find that hypoxia/hypoglycaemia-regulated genes involved in controlling the cell cycle are either HIF-1alpha-dependent (those encoding the proteins p53, p21, Bcl-2) or HIF-1alpha-independent (p27, GADD153), suggesting that there are at least two different adaptive responses to being deprived of oxygen and nutrients. Loss of HIF-1alpha reduces hypoxia-induced expression of vascular endothelial growth factor, prevents formation of large vessels in ES-derived tumours, and impairs vascular function, resulting in hypoxic microenvironments within the tumour mass. However, growth of HIF-1alpha tumours was not retarded but was accelerated, owing to decreased hypoxia-induced apoptosis and increased stress-induced proliferation. As hypoxic stress contributes to many (patho)biological disorders, this new role for HIF-1alpha in hypoxic control of cell growth and death may be of general pathophysiological importance.     

Differential induction of cytochrome P450 gene expression by 4n-alkyl-methylenedioxybenzenes in primary rat hepatocyte cultures

A well-characterized primary rat hepatocyte culture system was used to examine induction patterns of cytochrome 450 gene expression by a series of 4-n-alkyl-methylenedioxybenzene (MDBs) derivatives. Hepatocytes were treated for 24, 48, or 72 hours with 0-500 microM of the MDB compounds, and total cellular RNA and protein from each treatment was evaluated by hybridization and immunochemical techniques. Exposure to MDB congeners possessing increasing 4-n-alkyl side-chain length (C0-C8) resulted in dose- and structure-dependent activation of CYP2B1, 2B2, 3A1, 1A1, and 1A2 gene expression. At equivalent 100 microM concentrations, the C6 and C8 MDB congeners were more effective than the prototypical inducer phenobarbital (PB) with respect to induction potency of CYP2B1, CYP2B2, and CYP3A1 gene expression. In contrast to PB, longer side-chain-substituted MDBs effectively induced CYP1A1 and CYP1A2 gene expression, in addition to the CYP2B and CYP3A genes. At equivalent molar concentrations, the catechol derivative of C6-MDB was ineffective in its ability to induce CYP gene expression, indicating the importance of the intact methylenedioxy bridge in the induction mechanism. Levels of MDB-inducible CYP2B1 and CYP2B2 mRNA were highly correlated with CYP2B1/2 apoprotein levels, ascertained by immunoblot analysis of cultured hepatocyte S9 fractions. Compared with results from previous in vivo analysis (12), the current data indicate that pharmacodynamic factors may influence MDB induction profiles and that differences in MDB effects on CYP gene expression result depending on distinct structure-activity relationships.     

Morphologic comparison of atherosclerotic lesions in native coronary arteries and saphenous vein graphs with intracoronary angioscopy in patients with unstable angina

The purpose of this study was to investigate platelet effects on postischemic heart function in conjunction with adenosine effects on intracoronary platelet adhesion. Homologous platelets were infused into the coronaries of isolated guinea pig hearts, either during low-flow ischemia or during reperfusion, and external heart work (EHW) and intracoronary platelet adhesion were determined. In most experiments, thrombin was added to the perfusate. The influence of endogenous adenosine was studied by use of the uptake blocker dipyridamole and the unspecific adenosine-receptor blocker theophylline, the A1-receptor blocker 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), and the A2-receptor blocker 3,7-dimethyl-1-propargylxanthine (DMPX). The importance of nitric oxide and prostaglandin I2 (PGI2) was tested by using nitro-L-arginine (NOLAG) and indomethacin, respectively. When platelets were applied with thrombin during low-flow ischemia, EHW recovered to only 63 +/- 4% of the preischemic value, as compared with 89 +/- 3% without platelets (p < 0.05). Despite thrombin, platelets incurred no significant functional loss when applied in the first minute of reperfusion (but again in the fifth minute); however, when theophylline was also present, recovery of EHW amounted to only 42 +/- 12%. Intracoronary adhesion of platelets was negligible without thrombin, and highest during low-flow ischemia with thrombin (35 +/- 3% of the applied number). No adhesion occurred during the first minute of reperfusion, whereas in the fifth minute, adhesion was again 20.8 +/- 4%. Dipyridamole increased adenosine release and attenuated adhesion at this time. Theophylline increased adhesion in the first minute of reperfusion (33 +/- 6.4%), whereas NOLAG and indomethacin proved to be ineffective. DPCPX and DMPX each increased platelet retention during the first minute of reperfusion, their effects being additive. Intracoronary adhesion of platelets induced by thrombin in isolated hearts can reduce postischemic recovery of heart function. During reperfusion, but not during low-flow, endogenous adenosine can prevent platelet adhesion and loss of myocardial function, an action mediated both by A1- and A2-receptor-dependent mechanisms.     

Breed group effects on milk production of Brazilian crossbred dairy cows

Lactation records (n = 2362) of 1402 crossbred cows in 22 cooperating dairy herds in southeastern Brazil were evaluated. Cows were mixtures of Zebu (Gir, Guzera, and unknown) and European breeding (mostly Holstein). Lactation milk yields were expressed as total, 3050-d, or deviated 305-d yields, either adjusted or unadjusted for days in milk (DIM). Mean DIM was 280. Arithmetic means unadjusted for DIM were 1942, 1666, and 5 kg per record. Milk yields of daughters from sires of 6/8 and 7/8 European breeds were higher than yields of daughters from sires of 5/8 European breeds when data were either adjusted or unadjusted for DIM. The differences associated with breed group of sire were only slightly reduced when records were adjusted for DIM. There was no evidence of a decline in milk yield as the fraction of European breeding of the sire increased from 6/8 to 7/8. For a given breed group of sire, whether the grandsire was purebred or crossbred had no detectable effect. These results should be useful in determining strategies for crossbreeding of dairy cows in tropical areas, particularly when crossbred sires are used.     

T lymphocyte responses to anti-neutrophil cytoplasmic autoantibody (ANCA) antigens are present in patients with ANCA-associated systemic vasculitis and persist during disease remission

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel has been identified in the cardiac muscle of a number of mammalian species, including humans. The goal of this study was to begin quantifying the structural requirements necessary for arylaminobenzoate block of the CFTR channel. The cardiac cAMP-dependent Cl- current (ICl) was measured using the whole-cell arrangement of the patch-clamp technique in guinea pig ventricular myocytes during stimulation of protein kinase A with forskolin. At drug concentrations below the IC50 value for channel block, reduction of ICl by the arylaminobenzoates occurred in a strongly voltage-dependent manner with preferential inhibition of the inward currents. At higher drug concentrations, block of both the inward and outward ICl was observed. Increasing the length of the carbon chain between the benzoate and phenyl rings of the arylaminobenzoates resulted in a marked increase in drug block of the channel, with IC50 values of 47, 17, and 4 microM for 2-benzylamino-5-nitro-benzoic acid, 5-nitro-2-(2-phenylethylamino)-benzoic acid, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), respectively. Increasing the carbon chain length further with the compound 5-nitro-2-(4-phenylbutylamino)-benzoic acid, caused no additional increase in the potency of drug block (IC50 = 4 microM). Inhibition of ICl by the arylaminobenzoates was modulated by the pH of the external solution; increasing the pH from 7.4 to 10.0 greatly weakened NPPB block, whereas decreasing the pH to 6.4 enhanced block. In addition, block of ICl was observed during intracellular dialysis of NPPB, and this action was not affected by raising the external pH.     

Functional dissociation between local and systemic immune response during anti-melanoma peptide vaccination

Peptide vaccination against tumor Ags can induce powerful systemic CTL responses. However, in the majority of patients, no tumor regression is noted. To study this discrepancy, we analyzed CTL reactivity in a melanoma patient (F001) vaccinated with g209-2M peptide, a single residue variant of gp100(209-217). G209/g209-2M-reactive CTL were identified in post- but not prevaccination PBL. Limiting dilution analysis identified one predominant CTL clone (C1-35), with TCR Vbeta6s2, recognizing g209/HLA-A*0201-expressing targets. Additionally, two autologous melanoma lines (F001TU-3 and -4) and 20 separate tumor-infiltrating lymphocyte cultures were generated from a fine needle aspirate of a metastatic lesion progressing after initial response to vaccination. Both F001TU did not express gp100 and were not recognized by C1-35. Loss of gp100 by F001TU correlated with a marked reduction of gp100 expression in the same metastatic lesion compared with prevaccination. Thus, ineffectiveness of C1-35 and tumor progression could be best explained by loss of target Ag expression. Interestingly, 12 of 20 tumor-infiltrating lymphocyte cultures recognized F001TU, but none demonstrated g209/g209-2M reactivity, suggesting a functional dissociation between systemic and local immune response. This study suggests that vaccination effects must be analyzed in the target tissue, rather than in the systemic circulation alone.