A human tumor necrosis factor p75 receptor agonist stimulates in vitro T cell proliferation but does not produce inflammation or shock in the baboon.

Tumor necrosis factor (TNF) is a potentially useful adjunct to anticancer therapies. However, the clinical utility of TNF has been limited by generalized toxicity and hypotension. Recently, studies have begun to dissect the individual proinflammatory and immunologic responses that result from TNF binding to its two cellular receptors, p55 and p75, in an attempt to develop TNF receptor agonists with reduced systemic toxicity. To evaluate a p75 receptor selective TNF mutant (p75TNF), TNF and p75TNF were administered to healthy anesthetized baboons. Intravenous infusion of the p75TNF produced none of the hemodynamic changes seen after the infusion of TNF. Infusion of p75TNF also failed to induce the plasma appearance of interleukins 6 and 8. However, p75TNF enhanced in vitro baboon thymocyte proliferation to concanavalin A, and infusion of p75TNF resulted in increased soluble p55 and p75 receptor plasma concentrations. Local skin necrosis and tissue neutrophil infiltration were seen after subcutaneous injections of TNF and p55TNF. Subcutaneous injection of p75TNF did not result in skin necrosis but did result in a modest dermal infiltration of lymphocytes and macrophages. The findings suggest that p75TNF may stimulate T cell proliferation without the systemic and local toxicity seen with TNF.

Peptidyl alpha-ketoheterocyclic inhibitors of human neutrophil elastase. 3. In vitro and in vivo potency of a series of peptidyl alpha-ketobenzoxazoles.

A series of peptidyl alpha-ketobenzoxazoles were synthesized and evaluated for their in vitro and in vivo inhibition of human neutrophil elastase (HNE). These compounds inhibit HNE by forming both a covalent bond between the ketone carbonyl carbon atom and the hydroxyl group of Ser-195 and a hydrogen bond between the benzoxazole nitrogen atom and His-57. Appending to the parent benzoxazole ring a variety of substituents which spanned a range of physicochemical properties had only a modest effect on in vitro potency (Ki = 3-0.4 nM). This apparent lack of a significant effect is believed to result from the fact that any increased ketone carbonyl activation by the ring substituent is counter balanced by a corresponding decrease in the hydrogen-bonding ability of the benzoxazole nitrogen atom. In contrast to the results in vitro, maximizing in vivo activity was critically dependent upon the choice of the benzoxazole ring substituent. Several substituted peptidyl alpha-ketobenzoxazoles effectively inhibited HNE-induced lung injury when administered intratracheally 24 h prior to the enzyme.

Nonpeptidic inhibitors of human neutrophil elastase. 7. Design, synthesis, and in vitro activity of a series of pyridopyrimidine trifluoromethyl ketones.

Using molecular modeling and the information derived from X-ray crystal structures of human neutrophil elastase (HNE) and porcine pancreatic elastase (PPE) complexed to peptidic ligands, we have developed a new series of nonpeptidic inhibitors of HNE, the pyridopyrimidine trifluoromethyl ketones (TFMKs). These bicyclic inhibitors were designed to extend the concept of the related pyridone trifluoromethyl ketones by incorporating a rigidly positioned carbonyl group to participate in a hydrogen bonding interaction with the backbone NH groups of Gly-218 and Gly-219 of the enzyme. In addition, the pyrimidine ring serves as a scaffold to vector substituents toward the S5-S4 subsites of the enzyme's extended binding pocket. Furthermore, the heteroatoms of the pyrimidine ring generally increase the aqueous solubility of the pyridopyrimidines relative to pyridone TFMKs. Pyridopyrimidine TFMKs containing a 6-phenyl substituent afforded potent inhibitors of elastase, and several inhibitors from this class of compounds possessed aqueous solubilities of > 0.1 mg/mL and Ki values of < or = 10 nM.

Peptidyl alpha-ketoheterocyclic inhibitors of human neutrophil elastase. 2. Effect of varying the heterocyclic ring on in vitro potency.

A series of peptidyl alpha-ketoheterocycles were synthesized and evaluated for their in vitro inhibition of human neutrophil elastase (HNE). Several heterocycles, including oxazoline and benzoxazole, afforded extremely potent inhibitors of HNE (1p-r) with nanomolar to subnanomolar Ki values. The structure-activity relationships revealed that for compounds with a Ki < 1000 nM potency tends to be positively correlated with the sigma I value of the heterocycle. Furthermore, the results in this study support the hypothesis that, in the covalent enzyme-inhibitor adduct, the azole nitrogen atom of the inhibitor heterocycle participates in a hydrogen-bonding interaction with the active-site His-57.

Nonpeptidic inhibitors of human leukocyte elastase. 2. Design, synthesis, and in vitro activity of a series of 3-amino-6-arylopyridin-2-one trifluoromethyl ketones.

A series of potent nonpeptidic inhibitors of the enzyme human leukocyte elastase (HLE) is reported. These inhibitors contain a 3-amino-2-pyridone ring as a central template in which the pyridone carbonyl and 3-position NH group are thought to form important hydrogen bonding interactions with the Val-216 residue of HLE. Substitution of the 6-position of the pyridone ring by various alkyl and aryl groups was found to afford increases in the in vitro potency of these inhibitors. A 6-position phenyl group, compound 10f, was found to result in a large increase in binding affinity, which was not obtained when the phenyl group was placed in either the 4- or 5-position of the molecule. Compound 10f was found to have good selectivity for HLE over other proteolytic enzymes, with the exception of bovine pancreatic chymotrypsin (BPC). Substitution of the 6-phenyl group in these molecules was found to decrease binding affinity for BPC without adversely affecting affinity for HLE.

Nonpeptidic inhibitors of human leukocyte elastase. 3. Design, synthesis, X-ray crystallographic analysis, and structure-activity relationships for a series of orally active 3-amino-6-phenylpyridin-2-one trifluoromethyl ketones.

A series of nonpeptidic inhibitors of human leukocyte elastase (HLE) is reported. These trifluoromethyl ketone-based inhibitors contain a 3-amino-6-phenylpyridone group as a central template. The effect of varying the N-3 substituent in these inhibitors on in vitro potency, physical properties, and oral activity in a hamster based, HLE-induced lung damage model is described. The variety of substituents at this position that have little effect on in vitro potency supports the idea that this region of the molecule does not interact strongly with the enzyme. One exception to this generality is 13k, which is substituted with a (4-acetamidophenyl)sulfonyl group. This compound has a K(i) of 0.7 nM and is, in vitro, the most potent inhibitor in the series. In contrast, variation of the N-3 substituent was found to have a dramatic effect on activity after oral administration. Several analogs, including the parent amine, 7, formamide, 2u, benzyl sulfamide, 13e, and benzyl sulfonamide, 13f, show significant activity when administered at an oral dose of 2.5 mg/kg. Support for the modeling-based design concepts was obtained through in vitro SAR results and X-ray crystallographic analysis of the complex between 13d and porcine pancreatic elastase (PPE), a closely related enzyme.

Discovery and biological activity of orally active peptidyl trifluoromethyl ketone inhibitors of human neutrophil elastase.

Previously we had shown that tripeptidyl trifluoromethyl ketones (TFMKs) possessing an N-terminal diarylacylsulfonamide, such as ICI 200,880 and ICI 200,355, displayed unparalleled protection against the lung damage induced by human neutrophil elastase (HNE) when the inhibitors were administered intratracheally. Since the diarylacylsulfonamides were designed specifically to afford a long residence time in the lung, it was not unexpected that inhibitors from this class of TFMKs were not active when administered orally. Upon evaluating a large number of peptidyl TFMKs possessing a variety of N-terminal groups, several compounds were identified which demonstrated oral activity. Compounds were evaluated for their oral activity by measuring their ability to inhibit the increase in lung weight relative to body weight (Lw/Bw), the increase in red blood cells, and the increase in white blood cells induced by intratracheally administered HNE (100 micrograms/hamster). A number of tripeptidyl trifluoromethyl ketones containing neutral N-terminal groups displayed good oral activity, while those containing basic, acidic, or polar groups did not. Compound 50, possessing an N-terminal 4-(CH3O)C6H4CO group, was particularly effective, reducing Lw/Bw by 77%, red cells by 89%, and white cells by 91% when dosed at 37.5 mg/kg orally. Thus, by modifying the N-terminal group of tripeptidyl TFMKs, inhibitors can be designed which are effective in vivo when administered either orally or intratracheally.

Orally active trifluoromethyl ketone inhibitors of human leukocyte elastase.

This paper describes the development a series of peptidyl trifluoromethyl ketone inhibitors of human leukocyte elastase which are found to have excellent pharmacological profiles. Methods have been developed that allow for the synthesis of these inhibitors in stereochemically pure form. Two of these compounds, 1k and 1l, have high levels of oral bioavailability in several species. Compound 1l has entered development as ZD8321 and is presently undergoing clinical evaluation. These compounds demonstrate that peptidyl trifluoromethyl ketone inhibitors can achieve high levels of oral activity and bioavailability, and therefore they may prove useful as therapeutic agents in the treatment of diseases in which elastase is implicated.

Application of gene therapy to acute inflammatory diseases.

The application of gene therapy to acute inflammation has not received as much research attention as has the treatment of genetically-based diseases, cancer, and viral infections. However, gene therapy as a drug delivery system offers several theoretical and practical advantages over current protein delivery systems. These include the ability to target therapies to individual tissues or cell types, to locally produce proteins that can act intracellularly or in an autocrine, juxtacrine, or paracrine fashion, and to sustain new protein synthesis for periods up to several weeks after a single administration. Although retrovirus, herpes simplex, and adeno-associated virus have been proposed for gene therapy in cancer and in genetic diseases, nonviral and adenovirus approaches appear most applicable as drug delivery systems due to their rapid onset and short duration of transgene expression. The relative modest transduction efficiencies obtained at present with nonviral approaches, and the inherent inflammatory properties of first-generation adenovirus constructs, however, have limited their usefulness to date. The present review discusses the theoretical and practical benefits of specific gene therapy approaches for the treatment of acute inflammatory diseases, as well as our experiences with liposome:plasmid DNA and adenovirus-based approaches. Although a number of technical and theoretical hurdles remain before it can be evaluated in humans with acute inflammation, gene therapy offers a novel approach for the treatment of acute inflammation, and will likely enter the armamentarium of critical care physicians in the near future.

Visceral ischemia-reperfusion injury promotes tumor necrosis factor (TNF) and interleukin-1 (IL-1) dependent organ injury in the mouse.

Acute visceral ischemia and subsequent reperfusion injury, which accompanies the surgical repair of a thoracoabdominal aorta aneurysm, is associated with high rates of morbidity and mortality. The purpose of the present study was to determine whether endogenous tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) production contributes to organ dysfunction in animals subjected to visceral ischemia secondary to 30 min of supraceliac aortic occlusion. C57BL6/j mice were treated with either a TNF binding protein (TNF-bp-10 mg/kg) or an anti-IL-1 receptor type 1 antibody (150 micrograms) 2 h prior to 30 min of supraceliac aortic occlusion. An additional group of mice received 30 min of infrarenal aortic occlusion to determine the contribution of lower torso ischemia-reperfusion injury to the changes seen following supraceliac aortic occlusion. Visceral organ ischemia for 30 min produced by supraceliac aortic occlusion followed by 2 h of reperfusion produced measurable TNF-alpha in 38% of untreated mice, but TNF-alpha was undetectable in both sham-operated mice and following infrarenal aortic occlusion. After 2 h of reperfusion, lung myeloperoxidase levels were significantly elevated in the mice experiencing visceral ischemia-reperfusion compared with either a sham operation or infrarenal ischemia-reperfusion (11.6 +/- 1.3 U/g vs. 3.4 +/- .2 U/g and 3.7 +/- 1.0 U/g, respectively, p < .05). Pretreatment with TNF-bp and anti-IL-1 antibody decreased lung neutrophil recruitment (7.2 +/- 1.2 U/g and 4.6 +/- 1.1 U/g) and capillary membrane permeability changes in mice following visceral ischemia-reperfusion. The present study demonstrates that brief (30 min) clinically relevant visceral ischemia produces TNF-alpha and IL-1 dependent lung injury.

Pharmacokinetics, immunogenicity, and efficacy of dimeric TNFR binding proteins in healthy and bacteremic baboon.

Immunogenicity, pharmacokinetics, and therapeutic efficacy of three novel dimeric soluble tumor necrosis factor (TNF)-receptor I constructs [TNF-binding protein (bp)] were evaluated in 28 baboons, 12 of which were healthy and 16 were challenged with a lethal Escherichia coli bacteremia. The three constructs differed only in the number of extracellular domains of the TNF receptor I and were dimerized with polyethylene glycol. Although all three constructs had generally similar pharmacokinetics when administered to a naive animal, they differed quantitatively in their immunogenicity. Antibodies were detected more frequently, and titers were significantly higher (P < 0.05) in both healthy and septic baboons that received the 4.0-domain TNF-bp construct, compared with animals receiving the 2.6-domain construct. When the TNF-bp constructs were administered a second time (21 days later), the half-lives of the three constructs were significantly shorter in animals that had an antibody response after the first injection. In contrast, all three TNF-bp constructs were equally effective at improving outcome, blocking a systemic TNF-alpha response, and attenuating the cytokine responses when administered at a dose of 1.0 mg/kg body wt 1 h before a lethal E. coli infusion. The findings suggest that immunogenicity of TNF-bp constructs can be altered by changing the number of functional domains, without affecting their capacity to neutralize TNF-alpha and to abrogate TNF-mediated pathology.

Arginine-enhanced enteral nutrition augments the growth of a nitric oxide-producing tumor.

BACKGROUND: Arginine-enhanced diets have been shown to be beneficial in tumor-bearing hosts, but no data exist regarding their effects in hosts bearing nitric oxide (NO)-producting tumors. OBJECTIVE: To examine the effect of arginine supplementation on the growth of a NO-producing murine breast cancer cell line. METHODS: EMT-6 cells were grown in various concentrations of arginine in the presence or absence of the inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine (1 mmol/L). Forty-eight hours later, nitrite accumulation and viable cell number were assessed. BALB/c mice were then pair-fed basal purified diets (n = 10), 4% casein diets (isonitrogenous control, n = 5), or 4% arginine-enhanced diets (n = 10). One week later, 10(5) EMT-6 cells were implanted subcutaneously into the dorsal flank. After tumor implantation, five mice fed basal purified diets and five mice fed arginine-enhanced diets also received aminoguanidine (100 mg/kg subcutaneously twice daily). Two weeks after tumor cell implantation, tumor size (mean diameter), animal weight, serum and tumor nitrite and nitrate levels were measured. RESULTS: There was minimal nitrite accumulation in arginine-free media, while increasing the arginine concentration increased nitrite levels. Viable cell number did not increase in arginine-free media, but increased nearly twofold in 100 and 1000 mumol/L arginine. In 5000 and 10,000 mumol/L arginine, the difference in viable cell number was not statistically different than that seen in arginine-free media, whereas the addition of aminoguanidine blocked nitrite accumulation and increased viable cell number at these arginine concentrations. Arginine-enhanced diets stimulated tumor growth in vivo more than twofold over tumor growth in mice fed isonitrogenous control or basal purified enteral diets. Mice fed arginine-enhanced diets also had increased serum nitrite and nitrate levels over mice fed basal purified enteral diets, whereas tumors from mice fed arginine-enhanced diets had nitrite and nitrate levels similar to mice fed basal purified enteral diets. Aminoguanidine blocked the increase in serum nitrite and nitrate, but failed to block the increased tumor growth in mice receiving the arginine-supplemented diets. CONCLUSIONS: Arginine concentration influences the growth of EMT-6 tumor cells in vitro and dietary arginine supplementation augments tumor growth in vivo. The mechanism of the growth modulation in vitro is NO-dependent whereas the enhanced tumor growth in vivo is NO-independent.

CT measurement of main pulmonary artery diameter.

The aim of this study was to determine the upper limit of the normal main pulmonary artery diameter using a modern CT system. This was measured at the level of the pulmonary artery bifurcation in 100 normal subjects using unenhanced contiguous 10 mm CT slices viewed at fixed mediastinal window settings (400/20). These normal subjects were then compared with similar unenhanced 10 mm images from 12 patients with proven pulmonary arterial hypertension (mean pulmonary artery pressure > 20 mmHg). The main pulmonary artery diameter in normal subjects was 2.72 cm (SD = 0.3). Main pulmonary artery diameter in patients with pulmonary arterial hypertension was significantly greater (p < 0.01) at 3.47 cm (SD = 0.33). A pulmonary artery diameter of 3.32 cm (main pulmonary artery diameter + 2 SD) had a 58% sensitivity and 95% specificity for the presence of pulmonary arterial hypertension. It is concluded that, using unenhanced axial 10 mm CT sections, the upper limit of normal main pulmonary artery diameter is 3.32 cm. Pulmonary arterial hypertension should be considered in patients with values above this level.

Spiral CT optimization for measurement of bronchial lumen diameter using an experimental model.

The aim of the study was to determine optimal parameters for demonstrating sublobar bronchi on spiral CT. Measurements were obtained from five parallel polyethylene tubes embedded in foam matrix with similar radiographic characteristics to segmental and subsegmental bronchi and to lung parenchyma, respectively. Collimation widths of 1.5, 2, 3 and 4 mm were used, with a pitch of 1 or 1.5 and a reconstruction interval of 1 mm or 2 mm. Various slice planes were used. Images acquired orthogonally were viewed normally. Images acquired in planes oblique or parallel to the long axes of the tubes were reformatted into a plane orthogonal to the long axes of the tubes to be comparable with the directly acquired orthogonal images. Tube diameters were measured at lung window settings (L, -400; W, 1300) and compared with known true inner and outer tube diameters. Measurements from images acquired orthogonal to the tube long axes were accurate regardless of slice thickness. Images acquired obliquely or parallel only produced accurate measurements at the lowest slice thickness (1.5 mm). Pitch and reconstruction interval had no effect on measurement error in any scan plane. It is concluded that a slice thickness of 1.5 mm or less, with a pitch of 1.5, should be used when acquiring images at angles other than orthogonal to the long axes of experimental tubes equivalent to the segmental and subsegmental bronchi. It is suggested that similar parameters should be used in vivo and that the examination should be targeted to the area of the bronchial tree in question to reduce patient dose and length of breath-hold.

CT dimensions of the normal pericardium.

Previous studies have suggested that the upper limit of the thinnest portion of the pericardium is 3-4 mm using 10 mm CT slices. However, these studies suffered from small sample sizes, long data acquisition times and unconventional viewing parameters. We have measured the width of the thinnest portion of the normal pericardium using 10 mm (100 patients) and 1 mm (100 patients) high resolution CT (HRCT) slices with modern CT equipment and fixed mediastinal window settings (400/20). The pericardium was identified in all patients and was best seen anterior to the heart. The pericardium is exceptionally well seen using 1 mm HRCT slices and this may be the optimal technique for visualization of the pericardium. The upper limit of the thinnest portion of the normal pericardium (mean value + 2 SD) was 1.2 mm (10 mm CT slices) and 0.7 mm (1 mm HRCT slices). These values are substantially lower than those previously reported and in line with anatomical findings.

Inhibitors of bacterial signal peptidase: a series of 6-(substituted oxyethyl)penems.

A series of 6-(substituted oxyethyl)penem esters having the (5S) stereochemistry which are potent inhibitors of Escherichia coli leader peptidase is described. Structure-activity relationships are discussed.

6-(substituted methylene)penems, potent broad spectrum inhibitors of bacterial beta-lactamase. IV. Kidney stability, serum binding and additional biological evaluation of racemic derivatives.

Sodium (5RS)-Z-6-(substituted methylene)penem-3-carboxylates (3) are extremely potent inhibitors of bacterial beta-lactamases, but some members of this group of compounds are highly bound to human serum, while others are readily degraded by renal dehydropeptidase I enzyme. Consequently, the stability of a variety of 6-(substituted methylene)penems (3) to human kidney homogenate, their binding to human serum and their activity in a mouse infection model was investigated at an early stage, and were instrumental in the selection of the 1,2,3-triazolylmethylene derivatives (e.g. 3k) as a class of compounds worthy of further evaluation.

A new non-invasive test for the detection of compartment syndromes.

Chronic exertional compartment syndrome (CECS) is currently diagnosed using invasive pressure measurements. We report the use of 99Tcm-methoxyisobutyl isonitrile (99Tcm-MIBI) scintigraphy as a new non-invasive method of diagnosis. Forty-six patients with suspected chronic compartment syndrome underwent graded treadmill exercise to reproduce the presenting symptoms. At peak exercise, 300 MBq of 99Tcm-MIBI were injected intravenously. Subsequent cross-sectional imaging provided by emission tomography demonstrated regional abnormalities in muscle perfusion in the calf. A repeat study was performed at rest the following day. All patients in whom there was a strong clinical suspicion of CECS were considered for invasive pressure measurements. Statistical analysis of the results for investigation of CECS using 99Tcm-MIBI versus pressure studies gave P = 0.06. A comparison of 99Tcm-MIBI versus outcome gave P < 0.0001. The sensitivity was 80% and the specificity 97% for 99Tcm-MIBI studies based on outcome. The positive predictive value was 89% and the negative predictive value 94%. Thus 99Tcm-MIBI can detect compartment syndromes with good positive and negative predictive values. It is relatively simple, cheap and less invasive than pressure measurements. This technique shows promise in the diagnosis of CECS.

Finite element analysis of a total knee replacement by using Gauss point contact constraints.

Finite element methods have been applied extensively and with much success in the analysis of orthopaedic hip and knee implants. Very recently a burgeoning interest has developed, in the finite element community, in how numerical models can be constructed for the solution of problems in contact mechanics. New developments in this area are of paramount importance in the design of implants for orthopaedic surgery. Modern techniques are described for finite element contact analysis and applied to two problems of stress analysis in a plastic tibial component. In the former, results are compared with a previous finite element analysis and with Hertzian solutions. In the latter, an estimate of the extent of convergence of the finite element solutions is provided.