A series of thiomorpholine sulfonamide hydroxamate TACE inhibitors, all bearing propargylic ether P1' groups, was explored. In particular, compound 5h has excellent in vitro potency against isolated TACE enzyme and in cells, oral activity in a model of TNF-alpha production and a collagen-induced arthritis model, was selected as a clinical candidate for the treatment of RA.

Potent and selective TACE and MMP inhibitors utilizing the diazepine and thiazepine ring systems were synthesized and evaluated for biological activity in in vitro and in vivo models of TNF-α release. Oral activity in the mouse LPS model of TNF-α release was seen. Efficacy in the mouse collagen induced arthritis model was achieved with diazepine 20. © 2005 Elsevier Ltd. All rights reserved.

Tumor necrosis factor (TNF)-alpha is a well validated therapeutic target for the treatment of rheumatoid arthritis. TNF-alpha is initially synthesized as a 26-kDa membrane-bound form (pro-TNF) that is cleaved by a Zn-metalloprotease named TNF-alpha-converting enzyme (TACE) to generate the 17-kDa, soluble, mature TNF-alpha. TACE inhibitors that prevent the secretion of soluble TNF-alpha may be effective in treating rheumatoid arthritis ( RA) patients. Using a structure-based design approach, we have identified a novel dual TACE/matrix metalloprotease (MMP) inhibitor 4-[[ 4( 2-butynyloxy) phenyl] sulfonyl]-N-hydroxy-2,2-dimethyl-(3S)thiomorpholinecarboxamide( TMI-1). This molecule inhibits TACE and several MMPs with nanomolar IC50 values in vitro. In cell-based assays such as monocyte cell lines, human primary monocytes, and human whole blood, it inhibits lipopolysaccharide LPS)-induced TNF-alpha secretion at submicromolar concentrations, whereas there is no effect on the TNF-alpha mRNA level as judged by RNase protection assay. The inhibition of LPS-induced TNF-alpha secretion is selective because TMI-1 has no effect on the secretion of other proinflammatory cytokines such as interleukin (IL)-1beta, IL-6, and IL-8. Importantly, TMI-1 potently inhibits TNF-alpha secretion by human synovium tissue explants of RA patients. In vivo, TMI-1 is highly effective in reducing clinical severity scores in mouse prophylactic collagen-induced arthritis ( CIA) at 5, 10, and 20 mg/kg p.o. b.i.d. and therapeutic CIA model at 100 mg/kg p.o. b.i.d. In summary, TMI-1, a dual TACE/MMP inhibitor, represents a unique class of orally bioavailable small molecule TNF inhibitors that may be effective and beneficial for treating RA.

A series of benzodiazepine MMP/TACE inhibitors bearing polar moieties has been synthesized in an effort to optimize inhibitory activity against LPS-stimulated TNF production in human monocytes and oral activity in a murine LPS model.

The SAR of a series of potent sulfonamide hydroxamate TACE inhibitors, all bearing a butynyloxy P1' group, was explored. In particular, compound 5j has excellent in vitro potency against isolated TACE enzyme and in cells, good selectivity over MMP-1 and MMP-9, and oral activity in an in vivo model of TNF-alpha production and a collagen-induced arthritis model.

Potent and selective bicyclic heteroaryl hydroxamic acid MMP and TACE inhibitors were synthesized by a novel convergent route. Selectivity and efficacy versus MMPs and TACE could be controlled by appropriate substitution on the scaffolds and by variation of the P1' group. Select compounds were found to be effective in in vivo models of arthritis.

The SAR of a series of potent sulfonamide hydroxamate TACE inhibitors bearing novel acetylenic P1' groups was explored. In particular, compound 4t bearing a butynyloxy P1' moiety has excellent in vitro potency against isolated TACE enzyme and in cells, good selectivity over MMP-1 and oral activity in an in vivo model of TNF-alpha production.

Plants are one of several novel hosts that can be used for the production of recombinant biopharmaceuticals such as cytokines, hormones, monoclonal antibodies, enzymes and vaccines. The novelty of this technology and its wide range of potential applications require an assessment of possible regulatory concerns in the clinical development of plant-derived biopharmaceuticals. General principles extrapolated from experience gained with biotechnology products from other sources can serve as a foundation to develop scientifically sound strategies for the large-scale production and clinical development of safe and effective biopharmaceuticals in plant hosts.

Cocultivation of SIPC-2 myeloma cells with clone A31, BALB/3T3 cells results in the continuous production of a C-type virus by the A31 cells that has been designated S/A-1. S/A-1 virus possessed the essential properties of a murine retrovirus, including a buoyant density of 1.16 g/cm3, C-type morphology, high molecular weight (70 S) RNA, and an RNA-dependent DNA polymerase. After cloning by three endpoint dilution passages in A31, and SIRC cells, the virus was shown to possess ecotropic as well as xenotropic activity. For example, the S/A-1 virus producibility infected mouse cells, including BALB/3T3, NIH/3T3, SC-1, 3T3 FL, and F1 cells as well as non-mouse cells, including normal rat kidney, rabbit corneal (SIRC), Chinese hamster peritoneal, mink lung, Pekin duck embryo, human rhabdomyosarcoma, and guinea pig embryo cells. The S/A-1 virus infected A31, cells do not induce XC-syncytia and the virus does not induce foci in mink lung cells. Thus, the data suggest that it is an amphotropic virus whose ecotropism is different (i.e., NB-tropic) from the previously reported amphotropic viruses (i.e., N-tropic). In addition, S/A-1 virus is different from the mink cell focus-inducing (MCF) viruses (J. W. Hartley, N. K. Wolford, L. J. Old, and W. P. Rowe 1977, Proc. Nat. Acad. Sci. USA74, 789–792) in terms of its lack of focus-inducing activity on mink lung cells, B-tropism, and ability to replicate in human rhabdomyosarcoma and F1 mouse cells. The structural proteins of cloned S/A-1 virus were compared to those of 1504-A virus, a previously described amphotropic virus isolated from feral mice (J. W. Hartley and W. P. Rowe 1976, J. Virol.19 19–26; S. Rasheed, M. B. Gardner, and E. Chan 1976, J. Virol.19, 13–18), by electrophoresis on a linear 7–20% acrylamide slab gel and staining with Coomassie blue. S/A-1 differed from 1504-A in terms of (a) the mobility of a protein with a molecular weight of approximately 12,000 daltons (pl5) and (b) the fact that it has a “double” p30.

The antithrombotic drug Defibrotide (DFT) (a polydeoxyribonucleotide with a mean MW of 20,000 Daltons) reduces the number of leukocytes and platelets in thrombi. Because leukocytes and platelets are of importance in the genesis of endothelial lesions leading to atherosclerosis, DFT was given to challenge leukocytosis in rabbits with diet-induced atherosclerosis (0.25% cholesterol for 16 weeks). After 9 weeks of cholesterol feeding and at the end of experiment, oral DFT (60 mg/Kg per day) had decreased the leukocyte count raised by the cholesterol diet. Leukocyte stickiness and leukocyte differential counts were not modified by either oral cholesterol or by oral cholesterol plus oral DFT. At the end of experiment, oral DFT had normalized the platelet count increased by cholesterol diet. The red blood cell count decreased by oral cholesterol at 9 weeks and at the end of experiment was normalized by DFT. The % of aortae endothelial surface involved in the atherosclerotic process was decreased by oral DFT. The frequencies of intimal thickening in blood vessels of kidneys and hearts and in cardiac valves were reduced by oral DFT by 47%, 29% and 17%, although these reductions were not statistically significant. It is suggested that DFT, by preventing the increase in the number of leukocytes and platelets and deactivating them, as demonstrated in papers already published, was able to counteract against the atherosclerotic process.

The binding of single-stranded polydeoxyribonucleotides to adenosine A1 and A2 receptors was investigated. Defibrotide, a natural substance with established anti-thrombotic and anti-ischaemic effects, displaced [3H]CHA (N6-cyclohexyl-adenosine) and [3H]NECA (5'-N-ethylcarboxamido-adenosine) concentration dependently, completely and competitively. Ki values of 371 +/- 68 and 688 +/- 115 micrograms/ml (mean +/- S.E.M. of 4-5 replications) were computed for adenosine A1 and A2 sites, respectively. Higher and lower molecular weight polydeoxyribonucleotides displayed comparable affinity, whereas a double-stranded polydeoxyribonucleotide and a polyanion with a negative charge comparable to that of defibrotide were inactive. Defibrotide did not affect the total number of binding sites in radioligand saturation experiments. Defibrotide relaxed the K(+)-contracted guinea-pig trachealis muscle (IC50 = 4001 micrograms/ml) about one-third as potently as the CHA-contracted preparation and as potently as the resting preparation. NECA, a mixed adenosine A1/A2 receptor agonist, behaved similarly. The effects were abolished by the adenosine A1/A2 receptor blocker 8-phenyltheophylline, but not by the selective A1 blocker, 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine. These results demonstrate that defibrotide binds to adenosine receptors and triggers pharmacological responses comparable to those of a known agonist.