Posttraumatic administration of a sub-anesthetic dose of ketamine exerts neuroprotection via attenuating inflammation and autophagy.

As a complex disease, traumatic brain injury (TBI) can result in long-term psychiatric changes and sensorimotor and cognitive impairments. The TBI-induced loss of memory and long-term cognitive dysfunction are related to mechanistic factors including an increased inflammatory response, autophagy, edema, and ischemia. Many published studies have offered evidence for the neuroprotective effects and anti-inflammatory properties of ketamine for TBI patients. Nonetheless, there is a limited understanding of the accurate mechanism that underlies the potential neuroprotective effects of ketamine. Herein, it can be shown that posttraumatic administration of ketamine at a sub-anesthetic dose (10mg/kg ketamine, every 24h up to 7days) can prevent the TBI-induced production of IL-6 and TNF-α, attenuate deficits of dendrites and spines and exert beneficial effects on memory and behavior. Moreover, studies show that ketamine may activate the mTOR signaling pathway by p-mTOR induction to down-regulate the expression of crucial autophagic proteins such as LC3 and Beclin-1. According to these findings, ameliorating secondary brain injury and anti-inflammatory properties is closely related to the neuroprotection of ketamine, which supports the use of ketamine as a potential therapy for patients with TBI to alleviate functional deficits.

Structure of a retro-binding peptide inhibitor complexed with human alpha-thrombin.

The crystallographic structure of the ternary complex between human alpha-thrombin, hirugen and the peptidyl inhibitor Phe-alloThr-Phe-O-CH3, which is acylated at its N terminus with 4-guanidino butanoic acid (BMS-183507), has been determined at 2.6 A resolution. The structure reveals a unique "retro-binding" mode for this tripeptide active site inhibitor. The inhibitor binds with its alkyl-guanidine moiety in the primary specificity pocket and its two phenyl rings occupying the hydrophobic proximal and distal pockets of the thrombin active site. In this arrangement the backbone of the tripeptide forms a parallel beta-strand to the thrombin main-chain at the binding site. This is opposite to the orientation of the natural substrate, fibrinogen, and all the small active site-directed thrombin inhibitors whose bound structures have been previously reported. BMS-183507 is the first synthetic inhibitor proved to bind in a retro-binding fashion to thrombin, in a fashion similar to that of the N-terminal residues of the natural inhibitor hirudin. Furthermore, this new potent thrombin inhibitor (Ki = 17.2 nM) is selective for thrombin over other serine proteases tested and may be a template to be considered in designing hirudin-based thrombin inhibitors with interactions at the specificity pocket.

An efficient method for the rapid establishment of Epstein-Barr virus immortalization of human B lymphocytes.

Several methods have been developed for the immortalization of B lymphocytes by Epstein-Barr virus (EBV). We developed an efficient method which reduces the time from culture initiation to immortalization and cryopreservation. Two infections of EBV to lymphocytes, and the use of phorbol ester-induced EBV stock significantly improved immortalization efficiency and reduced the time between initiation and immortalization and cryopreservation. The resulting cell bank was used to produce DNA for genetic studies focusing on the genes involved in immune and autistic disorders.

Proximal extracranial vertebral artery disease in the New England Medical Center Posterior Circulation Registry.

OBJECTIVE: To describe the clinical features of patients with occlusive disease of the proximal (V1) segment of the vertebral artery. DESIGN AND PATIENTS: Patients with either occlusion or high-grade stenosis involving the V1 segment were chosen for study from the New England Medical Center Posterior Circulation Registry. The registry is a consecutive series of patients with signs and symptoms of posterior circulation ischemia seen at the New England Medical Center, Boston, Mass, during a 10-year period. Clinical features, radiographic findings, and patient outcome were reviewed. RESULTS: Of the 407 patients in the registry, 80 (20%) had V1 segment lesions. Patients could be classified into 5 groups: (1) V1 disease and coexistent severe intracranial occlusive disease of the posterior circulation (n=22); (2) V1 disease with evidence of artery-to-artery embolism (n=19); (3) suspected V1 disease with artery-to-artery embolism, but with other potential causes of stroke or less certain vascular diagnosis (n=20); (4) V1 disease associated with hemodynamic transient ischemic attacks (n=13); and (5) proximal vertebral arterial dissection (n=6). Hypertension, cigarette smoking, and coronary artery disease were common risk factors. Clinical features, location of infarct, and outcome differed between groups and reflected the presumed mechanisms of stroke. CONCLUSIONS: Occlusive disease involving the V1 segment of the vertebral artery is common in patients with posterior circulation ischemia, but is often associated with other potential mechanisms of stroke. However, in a series of patients seen at a tertiary referral center, occlusive disease of the V1 segment was the primary mechanism of ischemia in 9% of patients.

9,11-Epoxy-9-homo-14-oxaprosta-5-enoic acid derivatives. Novel inhibitors of fatty acid cyclooxygenase.

A novel bicyclic prostaglandin analogue, [1R-[l alpha,2 beta (5Z),3 beta,4 alpha]]-7-[3-[(hexyloxy)methyl]- 7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (1), and cogeners were found to be potent inhibitors of fatty acid cyclooxygenase. Compound 1 was the only stereoisomer out of eight possible structures that was active. Ether 1 was 20 times more potent than indomethacin (IND) in inhibiting arachidonic acid (AA) induced aggregation of human platelet-rich plasma. Compound 1 was also more potent than IND in several in vivo assays, AA-induced sudden death in the conscious mouse (2 times) and AA-induced bronchoconstriction in the anesthetized guinea pig (16-45 times).

9,11-Epoxy-9-homo-14-thiaprost-5-enoic acid derivatives: potent thromboxane A2 antagonists.

A novel bicyclic prostaglandin analogue, (1S)-[1 alpha,2 alpha(Z),3 alpha,4 alpha]-7-[3-[(hexylthio)methyl]-7- oxabicyclo [2.2.1]hept-2-yl]-5-heptenoic acid ((-)-10), and its cogeners were found to be potent antagonists at the TxA2 receptor. Compound (-)-10 was the only stereoisomer out of eight possible structures that was active. Thioether (-)-10 was 30-40-fold more potent than another TxA2 antagonist, BM 13.177, in inhibiting arachidonic acid (AA) induced aggregation of human platelet-rich plasma. Compound (-)-10 was effective (I50 = 0.5 +/- 0.4 microM) in inhibiting 9,11-azo-PGH2-induced (0.1 microgram/mL) contraction of guinea pig tracheal spirals. The bronchoconstriction in anesthetized guinea pigs induced by AA was also effectively antagonized by (-)-10 (1 mg/kg, iv); however, in this assay (-)-10 exhibited some direct agonist activity. Radioligand binding studies in washed (human) platelets revealed that (-)-10 is one of the most potent ligands for the PGH2/TxA2 receptor yet described (Kd = 1.6 +/- 0.4 nM).

Interphenylene 7-oxabicyclo[2.2.1]heptane thromboxane A2 antagonists. Semicarbazone omega-chains.

A series of chiral interphenylene 7-oxabicyclo[2.2.1]heptane semicarbazones 19-26 were prepared and evaluated for their in vitro thromboxane (TxA2) antagonistic activity and in vivo duration of action. The potency of 19-26 was found to highly dependent on the substitution pattern of the interphenylene ring and decreased in the order ortho greater than meta much greater than para. SQ 35,091 (25), [1S-(1 alpha,2 alpha,3 alpha,4 alpha)]-2-[[3-[[[(phenylamino) carbonyl]hydrazono]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]methyl] benzenepropanoic acid, was identified as a potent and long-acting TxA2 antagonist. In human platelet rich plasma SQ 35,091 inhibited arachidonic acid (800 microM) and U-46,619 (10 microM) induced aggregation with I50 values of 3 and 12 nM, respectively. In contrast, no inhibition of ADP (20 microM) induced aggregation was observed at greater than 1000 microM. Receptor binding studies with [3H]-SQ 29,548 showed SQ 35,091 was a competitive antagonist with a Kd value of 1.0 +/- 0.1 nM in human platelet membranes. In vivo SQ 35,091 (0.2 mg/kg po) showed extended protection (T50 = 16 h) from U-46,619 (2 mg/kg iv) induced death in mice. These compounds have for the first time demonstrated that a metabolically stable interphenylene alpha-sidechain can be introduced into a prostanoid-like series of TxA2 antagonists with the maintainance of potent antagonistic activity.

7-Oxabicyclo[2.2.1]heptyl carboxylic acids as thromboxane A2 antagonists: aza omega-chain analogues.

A novel bicyclic prostaglandin analogue, [1S-[1 alpha, 2 alpha (Z), 3 alpha, 4 alpha]]-7-[3-[[[[(1- Oxoheptyl)amino]acetyl]amino]-methyl]-7-oxabicyclo[2.2.1]hept-2- yl]-5-heptenoic acid [-)-7) was found to be a potent and selective thromboxane A2 (TxA2) receptor antagonist. Unlike the related series of omega-chain allylic alcohols, amide 7 and its congeners were uniformly free of direct contractile activity in vitro (bovine coronary) and in vivo (anesthetized guinea pig). Amide 7 was effective in the inhibition of (a) arachidonic acid induced platelet aggregation of human platelet-rich plasma (I50 = 0.18 +/- 0.006 microM), (b) 11,9-epoxymethano-PGH2 induced platelet aggregation of human platelet-rich plasma (I50 = 0.24 microM), (c) 11,9-epoxymethano-PGH2 induced contraction of guinea pig trachea (Kb = 3.0 +/- 0.3 nM) or rat aorta (Kb = 8.8 +/- 1.1 nM), and (d) arachidonic acid induced bronchoconstriction in the anesthetized guinea pig (0.1-1.0 mg/kg iv). Amide 7 inhibited the binding of [5,6-3H2]-[1S- (1 alpha, 2 alpha (Z), 3 alpha, 4 alpha)]-7-[3-[[2-[(Phenyl- amino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5- heptenoic acid to human platelet membranes in a specific and saturable manner with a Kd = 49.6 +/- 1.4 nM.

Interphenylene 7-oxabicyclo[2.2.1]heptane oxazoles. Highly potent, selective, and long-acting thromboxane A2 receptor antagonists.

A series of interphenylene 7-oxabicyclo[2.2.1]heptane oxazoles (2) were prepared and evaluated for their thromboxane (TxA2) antagonistic activity in vitro and duration of action in vivo. Examination of the carboxyl side chain indicated that the interphenylene ring substitution pattern and, to a lesser extent, chain length were important factors in determining TxA2 antagonistic potency. For the carboxyl side chain, ortho substitution, a single methylene spacer between the interphenylene and oxabicycloheptane rings, and a propionic acid side-chain length were determined to be optimal. With respect to the oxazole side chain a wide range of amide substituents with diverse structures and lipophilicities were compatible with potent antagonistic activity. Finally, an acidic functional group on the alpha-chain and a hydrogen bond acceptor on the 4-position of the oxazole ring were critical for potent activity. From the analogs prepared 42 (BMS-180,291: [(+)-1S-(1 alpha, 2 alpha, 3 alpha, 4 alpha)-2-[[3-[4-[(n- pentylamino)carbonyl]-2-oxazolyl]-7-oxabicyclo[2.2.1]hept-2- yl]methyl]benzenepropanoic acid) was found to be a potent, selective, and orally-active TxA2 antagonist with a long duration of action and has been selected as a candidate for clinical development. In human platelet-rich plasma, 42 inhibited arachidonic acid (800 microM) and U-46,-619 (10 microM) induced aggregation with I50 values of 7 and 21 nM, respectively. Radioligand binding studies of 42 with [3H]-SQ 29,548 showed a Kd value of 4.0 +/- 1.0 nM in human platelet membranes. Both in vitro and in vivo studies indicated 42 was devoid of direct agonistic activity. In vivo 42 (0.2 mg/kg, po) showed extended protection (T50 = 14.4 h) from U-46,619 (2 mg/kg, iv) induced death in mice, and a single oral dose of 42 (3 mg/kg) abolished U46,619-induced platelet aggregation ex vivo in African green monkeys for > 24 h.

Pharmacological characterization of potent, long-acting thromboxane receptor antagonists, SQ 33,261 and SQ 33,552.

SQ 33,261 ([1S-[1 alpha,2 alpha(Z),3 alpha,4 alpha]]-6-[3-[[2- [(phenylamino)carbonyl]hydrazono]methyl]-7-oxabicyclo[2.2.1]hept-2 - yl]-4-hexenoic acid) and SQ 33,552 ([1S-[1 alpha,2 alpha(Z),3 alpha,4 alpha]]-6-[3-[[[[(4- chlorophenyl)amino]carbonyl]hydrazono]methyl]-7- oxabicyclo[2.2.1]hept-2-yl]-4-hexenoic acid) are potent thromboxane (Tx) A2 receptor antagonists. They inhibited platelet aggregation in platelet-rich plasma induced by the TxA2 mimetic, U-46,619 (10 microM), with IC50 values of 200 and 70 nM, respectively. Neither compound inhibited ADP (20 microM)-induced platelet aggregation (IC50 greater than 1000 microM). SQ 33,261 and SQ 33,552 competitively antagonized U-46,619-induced contraction of rat aortic strips with respective pA2 values of 9.0 and 10.1 and KB values of 1.2 and 0.1 nM. They also competitively antagonized U-46,619-induced contraction of guinea pig tracheal strips with pA2 values of 8.9 and 9.9 and KB values of 1.9 and 0.4 nM, respectively. SQ 33,261 and SQ 33,552 (p.o.) were potent inhibitors of U-46,619 (2 mg/kg i.v.)-induced death in mice with ID50 values of 8 and 1 micrograms/kg, respectively. SQ 33,261 and SQ 33,552 (0.2 mg/kg p.o.), also had long duration of action in this assay with 50% survival times of 7 and 15 hr, respectively. SQ 33,261 at 0.01 and 1.0 mg/kg i.v., inhibited arachidonic acid-induced bronchoconstriction and reversed arachidonic acid-induced hypertension to a hypotensive response. SQ 33,552 inhibited TxA2 synthase at high concentrations (IC50 = 307 microM), whereas SQ 33,261 was inactive. Neither compound inhibited cyclooxygenase or caused an elevation of platelet cyclic AMP levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Retro-binding thrombin active site inhibitors: identification of an orally active inhibitor of thrombin catalytic activity.

A series of retro-binding inhibitors of human alpha-thrombin was prepared to elucidate structure-activity relationships (SAR) and optimize in vivo performance. Compounds 9 and 11, orally active inhibitors of thrombin catalytic activity, were identified to be efficacious in a thrombin-induced lethality model in mice.

Oxidative activation of acylguanidine prodrugs: intestinal presystemic activation in rats limits absorption and can be inhibited by co-administration of ketoconazole.

1. The disposition of acyl prodrugs was studied to improve the delivery of a guanidine-containing parent compound with poor membrane permeability and poor absorption. 2. The prodrugs were evaluated in vitro and in vivo for conversion to drug. Prodrugs were evaluated for hydrolytic or oxidative bioactivation in intestinal homogenate and rat liver S9 or microsomes. The disposition of the prodrugs in vivo was monitored in bile duct-cannulated rats. 3. Compounds with n-alkylacyl groups were efficiently bioactivated, but were hydrolysed before absorption. 4. Hydrolytic bioactivation could be blocked in vitro by branching in the alkyl chain. These compounds showed modest improvements in absorption, despite favourable permeability. Experiments with liver microsomes demonstrated efficient NADPH-dependent oxidative bioactivation, which was proposed to occur through a CYP-mediated side chain oxidation followed by cyclization and release of parent compound. Ketoconazole co-administration yielded approximately a twofold increase in absorption. 5. The hydrolytically stable prodrugs were successful in increasing absorption of parent drug and were efficiently bioactivated, but they did not yield increased systemic levels of drug.