Bone morphogenetic protein 2/SMAD signalling in human ligamentocytes of degenerated and aged anterior cruciate ligaments.

OBJECTIVE: Anterior cruciate ligament (ACL) degeneration leads to knee instability and favors osteoarthritis (OA) progression. During ageing the growth factor sensitivity of ligaments changes but nothing is known about BMP2-signalling and -sensitivity in degenerated ACLs. This study addressed the question whether a dysregulated BMP2 signalling might contribute to age- and OA-dependent ACL degeneration. METHOD: ACL samples from patients with/without OA of different ages (<60 and ≥60 years, males, females) were graded histopathologically (n = 45). After stimulation of cultured ACL fibroblasts with 5 nM BMP2 for different time points, phosphorylation of SMAD1/5/8 and gene expression of crucial BMP2 signalling proteins, ligamentogenic and chondrogenic transcription factors, scleraxis (SCX) and SOX9, were analyzed. RESULTS: ACL samples displayed different grades of degeneration, often associated with synovitis and calcium deposits. Degeneration correlated significantly with synovitis. ACL fibroblasts expressed BMP type I receptors ALK3 and ALK6 and the BMP type II receptor BMPRII. Donors could be divided into "responders" and "non responders" since their BMP2 mediated SMAD1/5/8 phosphorylation level differed. Basal ID1 expression was lower in cells derived from OA compared with non-OA patients and BMP2 led to an ID1 induction in both. Irrespective of BMP2 stimulation, the donor age significantly influenced the expression profile of BMP6 and SCX but not BMP signalling. The BMP2-mediated SMAD6 expression differed between OA and healthy ACL fibroblasts. CONCLUSION: Our data indicate that the expression level of BMP2/SMAD target genes such as ID1 and SMAD6 was reduced in ACL fibroblasts derived from OA compared with non OA patients.

Regulation of osteoarthritis-associated key mediators by TNFα and IL-10: effects of IL-10 overexpression in human synovial fibroblasts and a synovial cell line.

Synovial fibroblasts (SF) contribute to the pathogenesis of osteoarthritis (OA), but the effects of intra-articular cytokines on SF are not completely understood. The aim of this study was to characterize the interplay between tumor necrosis factor (TNF)α and the anti-inflammatory interleukin (IL)-10. Non-immortalized human SF and SF of the human cell line K4IM were stimulated with recombinant TNFα, IL-10, or TNFα + IL-10 (10 ng/ml each) for 24 h or transduced with an adenoviral vector overexpressing human IL-10 (hIL-10) and subsequently treated with 10 ng/ml TNFα for 24 h. Effects on the gene expression and protein synthesis of IL-6, IL-10, matrix metalloproteinases (MMP)-1, -3, type I collagen, ß1-integrin, and CD44 were investigated via real-time detection polymerase chain reaction, immunofluorescence labeling, flow cytometry, and Western blotting. IL-10 release by transduced SF was confirmed with enzyme-linked immunosorbent assay. Both cell populations were activated by TNFα and by TNFα + IL-10, increasing their gene expression and protein synthesis of IL-6, IL-10, MMP-1, and MMP-3 and altering the synthesis of type I collagen, ß1-integrin, and CD44. hIL-10 overexpression greatly elevated the gene expression and protein synthesis of IL-10. However, transduction did not significantly affect the gene expression of IL-6, MMP-1, and MMP-3 in SF. The increased expression of pro-inflammatory and catabolic mediators in TNFα-activated SF indicates their role in OA pathogenesis, suggesting they are a potential therapeutic target. Although the vigorousness of the responses of non-immortalized SF and K4IM clearly differ, the K4IM cell line seems to be a suitable model for non-immortalized human SF.

Elevated plasma clara cell secretory protein concentration is associated with high-grade primary graft dysfunction.

Primary graft dysfunction (PGD) is the leading cause of early posttransplant morbidity and mortality after lung transplantation. Clara cell secretory protein (CC16) is produced by the nonciliated lung epithelium and may serve as a plasma marker of epithelial cell injury. We hypothesized that elevated levels of CC16 would be associated with increased odds of PGD. We performed a prospective cohort study of 104 lung transplant recipients. Median plasma CC16 levels were determined at three time points: pretransplant and 6 and 24 h posttransplant. The primary outcome was the development of grade 3 PGD within the first 72 h after transplantation. Multivariable logistic regression was performed to evaluate for confounding by donor and recipient demographics and surgical characteristics. Twenty-nine patients (28%) developed grade 3 PGD within the first 72 h. The median CC16 level 6 h after transplant was significantly higher in patients with PGD [13.8 ng/mL (IQR 7.9, 30.4 ng/mL)] than in patients without PGD [8.2 ng/mL (IQR 4.5, 19.1 ng/mL)], p = 0.02. Elevated CC16 levels were associated with increased odds of PGD after lung transplantation. Damage to airway epithelium or altered alveolar permeability as a result of lung ischemia and reperfusion may explain this association.

Elevated plasma long pentraxin-3 levels and primary graft dysfunction after lung transplantation for idiopathic pulmonary fibrosis.

Primary graft dysfunction (PGD) after lung transplantation may result from ischemia reperfusion injury (IRI). The innate immune response to IRI may be mediated by Toll-like receptor and IL-1-induced long pentraxin-3 (PTX3) release. We hypothesized that elevated PTX3 levels were associated with PGD. We performed a nested case control study of lung transplant recipients with idiopathic pulmonary fibrosis (IPF) or chronic obstructive pulmonary disease (COPD) from the Lung Transplant Outcomes Group cohort. PTX3 levels were measured pretransplant, and 6 and 24 h postreperfusion. Cases were subjects with grade 3 PGD within 72 h of transplantation and controls were those without grade 3 PGD. Generalized estimating equations and multivariable logistic regression were used for analysis. We selected 40 PGD cases and 79 non-PGD controls. Plasma PTX3 level was associated with PGD in IPF but not COPD recipients (p for interaction < 0.03). Among patients with IPF, PTX3 levels at 6 and 24 h were associated with PGD (OR = 1.6, p = 0.02 at 6 h; OR = 1.4, p = 0.008 at 24 h). Elevated PTX3 levels were associated with the development of PGD after lung transplantation in IPF patients. Future studies evaluating the role of innate immune activation in IPF and PGD are warranted.

In vitro and in vivo neo-cartilage formation by heterotopic chondrocytes seeded on PGA scaffolds.

Implantation of tissue-engineered heterotopic cartilage into joint cartilage defects might be an alternative approach to improve articular cartilage repair. Hence, the aim of this study was to characterize and compare the quality of tissue-engineered cartilage produced with heterotopic (auricular, nasoseptal and articular) chondrocytes seeded on polyglycolic acid (PGA) scaffolds in vitro and in vivo using the nude mice xenograft model. PGA scaffolds were seeded with porcine articular, auricular and nasoseptal chondrocytes using a dynamic culturing procedure. Constructs were pre-cultured 3 weeks in vitro before being implanted subcutaneously in nude mice for 1, 6 or 12 weeks, non-seeded scaffolds were implanted as controls. Heterotopic neo-cartilage quality was assessed using vitality assays, macroscopical and histological scoring systems. Neo-cartilage formation could be observed in vitro in all PGA associated heterotopic chondrocytes cultures and extracellular cartilage matrix (ECM) deposition increased in vivo. The 6 weeks in vivo incubation time point leads to more consistent results for all cartilage species, since at 12 weeks in vivo construct size reductions were higher compared with 6 weeks except for auricular chondrocytes PGA cultures. Some regressive histological changes could be observed in all constructs seeded with all chondrocytes subspecies such as cell-free ECM areas. Particularly, but not exclusively in nasoseptal chondrocytes PGA cultures, ossificated ECM areas appeared. Elastic fibers could not be detected within any neo-cartilage. The neo-cartilage quality did not significantly differ between articular and non-articular chondrocytes constructs. Whether tissue-engineered heterotopic neo-cartilage undergoes sufficient transformation, when implanted into joint cartilage defects requires further investigation.

The role of pro-inflammatory and immunoregulatory cytokines in tendon healing and rupture: new insights.

Owing to limited self-healing capacity, tendon ruptures and healing remain major orthopedic challenges. Increasing evidence suggests that post-traumatic inflammatory responses, and hence, cytokines are involved in both cases, and also in tendon exercise and homeostasis. This review summarizes interrelations known between the cytokines interleukin (IL)-1ß, tumor necrosis factor (TNF)α, IL-6 and vascular endothelial growth factor (VEGF) in tendon to assess their role in tendon damage and healing. Exogenic cytokine sources are blood-derived leukocytes that immigrate in damaged tendon. Endogenous expression of IL-1ß, TNFα, IL-6, IL-10 and VEGF was demonstrated in tendon-derived cells. As tendon is a highly mechanosensitive tissue, cytokine homeostasis and cell survival underlie an intimate balance between adequate biomechanical stimuli and disturbance through load deprivation and overload. Multiple interrelations between cytokines and tendon extracellular matrix (ECM) synthesis, catabolic mediators e.g. matrix-degrading enzymes, inflammatory and angiogenic factors (COX-2, PGE2, VEGF, NO) and cytoskeleton assembly are evident. Pro-inflammatory cytokines affect ECM homeostasis, accelerate remodeling, amplify biomechanical adaptiveness and promote tenocyte apoptosis. This multifaceted interplay might both contribute to and interfere with healing. Much work must be undertaken to understand the particular interrelation of these inflammatory and regulatory mediators in ruptured tendon and healing, which has relevance for the development of novel immunoregulatory therapeutic strategies.

Differing in vitro biology of equine, ovine, porcine and human articular chondrocytes derived from the knee joint: an immunomorphological study.

For lack of sufficient human cartilage donors, chondrocytes isolated from various animal species are used for cartilage tissue engineering. The present study was undertaken to compare key features of cultured large animal and human articular chondrocytes of the knee joint. Primary chondrocytes were isolated from human, porcine, ovine and equine full thickness knee joint cartilage and investigated flow cytometrically for their proliferation rate. Synthesis of extracellular matrix proteins collagen type II, cartilage proteoglycans, collagen type I, fibronectin and cytoskeletal organization were studied in freshly isolated or passaged chondrocytes using immunohistochemistry and western blotting. Chondrocytes morphology, proliferation, extracellular matrix synthesis and cytoskeleton assembly differed substantially between these species. Proliferation was higher in animal derived compared with human chondrocytes. All chondrocytes expressed a cartilage-specific extracellular matrix. However, after monolayer expansion, cartilage proteoglycan expression was barely detectable in equine chondrocytes whereby fibronectin and collagen type I deposition increased compared with porcine and human chondrocytes. Animal-derived chondrocytes developed more F-actin fibers during culturing than human chondrocytes. With respect to proliferation and extracellular matrix synthesis, human chondrocytes shared more similarity with porcine than with ovine or equine chondrocytes. These interspecies differences in chondrocytes in vitro biology should be considered when using animal models.

IL-10 overexpression differentially affects cartilage matrix gene expression in response to TNF-alpha in human articular chondrocytes in vitro.

Cartilage-specific extracellular matrix synthesis is the prerequisite for chondrocyte survival and cartilage function, but is affected by the pro-inflammatory cytokine TNF-alpha in arthritis. The aim of the present study was to characterize whether the immunoregulatory cytokine IL-10 might modulate cartilage matrix and cytokine expression in response to TNF-alpha. Primary human articular chondrocytes were treated with either recombinant IL-10, TNF-alpha or a combination of both (at 10ng/mL each) or transduced with an adenoviral vector overexpressing human IL-10 and subsequently stimulated with 10ng/ml TNF-alpha for 6 or 24h. The effects of IL-10 on the cartilage-specific matrix proteins collagen type II, aggrecan, matrix-metalloproteinases (MMP)-3, -13 and pro-inflammatory cytokines were evaluated by real-time RT-PCR and immunohistochemistry. Transduced chondrocytes overexpressed high levels of IL-10 which significantly up-regulated collagen type II expression. TNF-alpha suppressed collagen type II and aggrecan, but increased MMP and cytokine expression in chondrocytes compared to the non-stimulated controls. The TNF-alpha mediated down-regulation of aggrecan expression was significantly antagonized by IL-10 overexpression, whereas the suppression of collagen type II was barely affected. The MMP-13 and IL-1beta expression by TNF-alpha was slightly reduced by IL-10. These results suggest that IL-10 overexpression modulates some catabolic features of TNF-alpha in chondrocytes.

Hyperhomocysteinemia enhances vascular inflammation and accelerates atherosclerosis in a murine model.

Although hyperhomocysteinemia (HHcy) is a well-known risk factor for the development of cardiovascular disease, the underlying molecular mechanisms are not fully elucidated. Here we show that induction of HHcy in apoE-null mice by a diet enriched in methionine but depleted in folate and vitamins B6 and B12 increased atherosclerotic lesion area and complexity, and enhanced expression of receptor for advanced glycation end products (RAGE), VCAM-1, tissue factor, and MMP-9 in the vasculature. These homocysteine-mediated (HC-mediated) effects were significantly suppressed, in parallel with decreased levels of plasma HC, upon dietary supplementation with folate and vitamins B6/B12. These findings implicate HHcy in atherosclerotic plaque progression and stability, and they suggest that dietary enrichment in vitamins essential for the metabolism of HC may impart protective effects in the vasculature.

Interleukin-10 modulates pro-apoptotic effects of TNF-alpha in human articular chondrocytes in vitro.

The aim of this study is to determine if there is an antagonistic effect between tumour necrosis factor (TNF)-alpha and the immunoregulatory interleukin (IL)-10 on chondrocytes survival. Serum-starved primary human articular chondrocytes were stimulated with either 10 ng/ml recombinant TNF-alpha, IL-10 or a combination of both (at 10 ng/ml each). Chondrocyte apoptosis was determined by measuring caspase-3/7, -8 and -9 activities using caspase assays. Mitochondrial apoptotic inducer bax, and the suppressor bcl-2 were evaluated using western blotting at 48 h. Results indicated that TNF-alpha increased caspase activities and resulted in a significant (p = 0.001) increase in bax/bcl-2 ratio. Stimulation with IL-10 did not alter caspase activities, while co-treatment with IL-10 and TNF-alpha inhibited TNF-alpha induced caspase activities and significantly (p > 0.004) impaired bax/bcl-2 ratio. At 24 h, mRNA levels for collagen type II, TNF-alpha and IL-10 were determined using real-time RT-PCR. Stimulation with TNF-alpha or TNF-alpha and IL-10 significantly inhibited collagen type II and increased IL-10 and TNF-alpha mRNA expression. IL-10 modulated the pro-apoptotic capacity of TNF-alpha in chondrocytes as shown by the decrease in caspase activities and bax/bcl-2 ratio compared to TNF-alpha stimulated chondrocytes, suggesting a mostly antagonistic interplay of IL-10 and TNF-alpha on mitochondrial apoptotic pathways.

Hyperhomocyst(e)inemia and endothelial dysfunction in IDDM.

OBJECTIVE: While elevated blood levels of homocyst(e)ine represent an independent risk factor for macrovascular disease, we assessed the link between hyperhomocyst(e)inemia and diabetic microvascular diseases. RESEARCH DESIGN AND METHODS: Plasma levels of homocyst(e)ine and thrombomodulin (TM), markers of endothelial cell damage, were measured before and 3 h after oral methionine loading in 75 patients with IDDM and 40 healthy control subjects matched for sex and age. Exclusion criteria were hyperlipidemia, hypertension, smoking, or positive family history for cardiovascular disease. RESULTS: IDDM patients had higher pre- and postload plasma levels of homocyst(e)ine than did healthy control subjects (12.0 vs. 7.7 mumol/l and 27.6 vs. 16.0 mumol/l; P < 0.001). Of 75 IDDM patients, 26 had plasma homocyst(e)ine levels above the normal range (means +/- 2 SD of values obtained in the control group). These IDDM patients with hyperhomocyst(e)inemia had higher plasma TM levels (62.2 vs. 38.2 ng/ml, P < 0.001), higher albumin excretion rates (485 vs. 115 mg/l, P < 0.005), and a higher prevalence of late diabetic complications (nephropathy, 76 vs. 33%; retinopathy, 69 vs. 51%; neuropathy, 57 vs. 41%; and macroangiopathy, 57 vs. 33%) compared with IDDM patients with normal plasma homocyst(e)ine. In vitro experiments with human umbilical vein cells showed an increased release of TM into the culture supernatant only when endothelial cells were pretreated with advanced glycation end product (AGE)-albumin before L-homocystine was added. A synergistic action of homocyst(e)ine and AGEs might contribute to vascular complications in patients with diabetes. CONCLUSIONS: Hyperhomocyst(e)inemia is common in nephropathic diabetic patients and may contribute to the enhanced morbidity and mortality from cardiovascular diseases characteristically observed in IDDM patients with diabetic nephropathy.

Hyperhomocyst(e)inemia and endothelial dysfunction in IDDM.

OBJECTIVE: Considering that elevated blood levels of homocyst(e)ine represent a known independent risk factor for macrovascular disease, we assessed the link between hyperhomocyst(e)inemia and diabetic microvascular complications. RESEARCH DESIGN AND METHODS: Homocyst(e)ine and thrombomodulin plasma levels, a marker of endothelial cell damage, were measured before and 3 h after oral methionine loading in 75 patients with stable, well-controlled IDDM and 40 healthy control subjects matched for sex and age. Exclusion criteria were hyperlipidemia, hypertension, smoking, or positive family history for cardiovascular disease. RESULTS: IDDM patients had higher pre- and postload homocyst(e)ine plasma levels than did healthy control subjects (12.0 vs. 7.7 mumol/l and 27.6 vs. 16.0 mumol/l; P < 0.001). Of 75 IDDM patients, 26 had homocyst(e)ine plasma levels above the normal range (defined as mean +2 SD of values obtained in the control group). The IDDM patients with hyperhomocyst(e)inemia had higher thrombomodulin plasma levels (62.2 vs. 38.2 ng/ml; P < 0.001), higher albumin excretion rates (485 vs. 115 mg/l; P < 0.005), and a higher prevalence of late diabetic complications (nephropathy, 76 vs. 33%; retinopathy, 69 vs. 51%; neuropathy, 57 vs. 41%; macroangiopathy, 57 vs. 33%) compared with IDDM patients with normal plasma homocyst(e)ine. In vitro experiments with human umbilical vein cells show an increased release of thrombomodulin into the culture supernatant only when endothelial cells were pretreated with advanced glycation end product (AGE)-albumin before L-homocystine was added. A synergistic action of homocyst(e)ine and AGEs might contribute to vascular complications of patients with diabetes. CONCLUSIONS: Hyperhomocyst(e)inemia is common in nephropathic diabetic patients and may contribute to the enhanced morbidity and mortality from cardiovascular diseases characteristically observed in IDDM patients with diabetic nephropathy.

The glycine site on the NMDA receptor: structure-activity relationships and possible therapeutic applications.

L-glutamate is the most important fast excitatory neurotransmitter in the mammalian central nervous system. Glutamate receptors are classified into two main categories: ionotropic and metabotropic. The N-methyl-D-aspartate (NMDA) receptor, which is associated with an ion channel, seems to play an important role in glutamate excitotoxicity, a process thought to be involved in a number of neurodegenerative disorders such as focal cerebral ischaemia (stroke), Parkinsonís, Huntingtonís, Alzheimerís disease, schizophrenia and epilepsy. The unique glycine site on the NMDA receptor, discovered by Johnson and Ascher in 1987, represents an interesting target for the development of neuroprotective compounds. Glycine antagonists may offer advantages over other NMDA antagonists in terms of their side-effect profile, especially in the long-term treatment of chronical neurodegenerative disorders but also in the treatment of serious medical emergencies with a significant morbidity and mortality like status epilepticus or stroke. So far it is not clear whether NMDA receptor antagonists including glycine antagonists would be suitable for chronic administration because of their effects on cognition, learning and motor function. High-affinity, in vivo potent, glycine antagonists of great structural diversity (i. e. pyrido[2,3-b]pyrazine-N-oxides, indole-2-carboxylates, 4-substituted-3-phenylquinoline-2(1H)-ones and alkyl-substituted 1,4-dihydro-quinoxaline-2,3-diones) are now available and their suitability for long-term treatment of chronical neurodegenerative disorders has to be investigated in clinical trials.

The NMDA receptor complex: a promising target for novel antiepileptic strategies.

Antiepileptic drugs (AEDs) cover a broad spectrum of pathological conditions ranging from seizures following congenital or acquired brain disorders to behavioural and psychiatric disorders and recently neuropathic pain. The need for novel antiepileptics raises from the expanding field of indications as well as from the fact, that special seizure types are refractory to common AEDs. In addition, many of the conventional antiepileptic drugs exhibit an unfavourable side-effect profile. Since there is growing evidence, that NMDA receptor activation might play a crucial role in epilepsy, NMDA receptor antagonists have become compounds of interest in preventing and treating seizures. This review focuses on NMDA receptor antagonistic compounds, which are already in use for the treatment of epileptic seizures (i. e. MgSO4, felbamate) and compounds in clinical trials (i. e. remacemide, ADCI). Further interest is put on NMDA antagonists in preclinical and biological testing (memantine, dizocilpine, conantokins, Co101244/PD174494, ifenprodil, arcaine, L-701,324, eliprodil, CGP40116, LY235959, LY233053, MRZ2/576, LU73068, 4-Cl-KYN). Some of the latter compounds are predominantely of academic interest (i. e. 4-Cl-KYN), others (i. e. dizocilpine, LY235959, LY233053) might be of therapeutical value when combined with conventional AEDs. In order to reduce adverse effects in antiepileptic medication using NMDA antagonists, special interest will be focused on subtype selective compounds. In this respect, Co101244, a novel potent and selective NR1/2B NMDA receptor antagonist might be a lead for therapeutically promising compounds.

Glucocorticoids decrease the production of parathyroid hormone-related protein in vitro but not in vivo in the Walker carcinosarcoma 256 rat model.

In 50-90% of cases, humoral hypercalcemia of malignancy (HHM) is due to tumor secretion of parathyroid hormone-related protein (PTHrP). Glucocorticoids are sometimes used as calcium lowering agents and there are in vitro results showing that glucocorticoids diminish PTHrP production. In this study we tested whether the serum-calcium-lowering effect of glucocorticoids is due to decreased PTHrP production by the tumor. As an animal and cell culture model we used the Walker carcinosarcoma (WCS) 256, a rat mammary carcinoma cell line producing PTHrP. In vitro, dexamethasone caused a dose-dependent inhibition of PTHrP production, whereby already 1-5 nmol/L revealed a significant decrease by WCS 256 cells. In contrast to these in vitro results, in WCS 256 tumor-bearing rats, dexamethasone (4 mg/kg body weight on day 4, and 1 mg/kg body weight from day 5 until day 7 after WCS transplantation; circulating dexamethasone levels > 20 nmol/L) did not decrease PTHrP production, PTHrP secretion, serum calcium, or tumor weight in vivo. We conclude that, in this PTHrP-mediated model of humoral hypercalcemia of malignancy, glucocorticoids do not decrease PTHrP production and secretion in vivo and do not show a calcium-lowering effect.

Angiotensin II enhances insulin sensitivity in healthy volunteers under euglycemic conditions.

OBJECTIVE: It has been postulated that vasoconstrictors cause insulin resistance. This effect has been documented for epinephrine but not for angiotensin II (Ang II). The aim of this study was to investigate the effect of the latter on insulin sensitivity. DESIGN: In order to study the influence of subpressor doses of Ang II on insulin-mediated glucose uptake under euglycemic conditions, eight healthy volunteers were allocated in random order to sham infusion or infusion of Ang II (first 0.75 ng/kg per min and subsequently 1.5 ng/kg per min). In addition, in seven of the subjects Ang II was infused after 3 days of indomethacin pretreatment (150 mg/day). METHODS: Insulin-mediated glucose uptake (expressed as M value) was measured with the euglycemic clamp technique. Insulin levels were measured enzymatically, plasma renin activity, Ang II, aldosterone and C-peptide levels by radioimmunoassay, blood pressure by Dinamap and muscle blood flow by plethysmography. RESULTS: The M value after sham infusion was 7.81 +/- 1.52 mg/kg per min and after 1.5 ng/kg Ang II per min was 9.76 +/- 1.26 mg/kg per min (P < 0.001). Indomethacin pretreatment did not abolish the Ang II-induced rise in the M value. Mean arterial blood pressure during the euglycemic clamp was unchanged with sham infusion and the low dose of Ang II. It increased slightly with the higher dose of Ang II. Inferior limb muscle perfusion was higher after infusion of Ang II than after sham infusion; this effect was not obliterated by indomethacin pretreatment. CONCLUSIONS: Ang II increases insulin-mediated glucose uptake: that is, it enhances insulin sensitivity by mechanisms independent of prostaglandins. The observations are of potential relevance to the changes in insulin sensitivity in some forms of hypertension.

From peptide to non-peptide. 3. Atropisomeric GPIIbIIIa antagonists containing the 3,4-dihydro-1H-1,4-benzodiazepine-2,5-dione nucleus.

The benzodiazepinedione class of non-peptidal GPIIbIIIa antagonists has been modified to allow the isolation of noninterconverting rotational isomers, or atropisomers, with the aim of examining their structure-activity relationships as compared to active RGD-containing peptides and other non-peptidal antagonists. Resolution of these antagonists was accomplished by the introduction of a tert-butyl group at N1 and a chlorine at C9 on the 3,4-dihydro-1H-1,4-benzodiazepine-2,5-dione nucleus and enantiospecific substitution on the beta-alanine side chain attached to N4. The relative configuration was determined by single-crystal X-ray analysis. Further, conformational analyses using ab initio calculations were performed to assess the conformational preferences about the beta-alanine side chain. The data support a good topographical correlation between the benzodiazepinedione class of antagonists and the "cupped" presentation of the RGD tripeptide sequence found in the cyclic peptide G4120. The relationship between these compounds with other peptidal and non-peptidal antagonists is discussed.

(H+,K+)-ATPase inhibiting 2-[(2-pyridylmethyl)sulfinyl]benzimidazoles. 4. A novel series of dimethoxypyridyl-substituted inhibitors with enhanced selectivity. The selection of pantoprazole as a clinical candidate.

[(Pyridylmethyl)sulfinyl]benzimidazoles 1 (PSBs) are a class of highly potent antisecretory (H+,K+)-ATPase inhibitors which need to be activated by acid to form their active principle, the cyclic sulfenamide 4. Selective inhibitors of the (H+,K+)-ATPase in vivo give rise to the nonselective thiophile 4 solely at low pH, thus avoiding interaction with other thiol groups in the body. The propensity to undergo the acid-catalyzed transformation is dependent on the nucleophilic/electrophilic properties of the functional groups involved in the formation of 2 since this step is both rate-determining and pH-dependent. The aim of this study was to identify compounds with high (H+,K+)-ATPase inhibitory activity in stimulated gastric glands possessing acidic pH, but low reactivity (high chemical stability) at neutral pH as reflected by in vitro (Na+,K+)-ATPase inhibitory activity. The critical influence of substituents flanking the pyridine 4-methoxy substituent present in all derivatives was carefully studied. The introduction of a 3-methoxy group gave inhibitors possessing a combination of high potency, similar to omeprazole and lansoprazole, but increased stability. As a result of these studies, compound 1a (INN pantoprazole) was selected as a candidate drug and is currently undergoing phase III clinical studies.

Review article: the continuing development of proton pump inhibitors with particular reference to pantoprazole.

Inhibition of the gastric proton pump is gaining acceptance as the treatment of choice for severe gastrooesophageal reflux disease, and for treatment of duodenal and gastric ulceration. Three of these drugs are now available (omeprazole, lansoprazole and pantoprazole) and more are being developed. Proton pump inhibitors share the same core structure, but differ in terms of substituents on this core. The substitutions are able to modify some important chemical properties of the compounds. For example, pantoprazole is significantly more acid-stable than omeprazole or lansoprazole. E3810 is significantly less stable than the other compounds. We present an explantation for this finding that depends on the relative pK values for the pyridine and benzimidazole nitrogens, especially the former. Pantoprazole formulated in an enteric-coated tablet displays high bioavailability and linear pharmacokinetics whether on single or multiple dose regimens. Although all three proton pump inhibitors provide a similar chemical conversion to sulphenamides, which are highly reactive cysteine reagents, these reagents derivatize different cysteines in the extracytoplasmic or membrane domain of the pump and inhibit the pump at different rates. Whereas the differences in chemical reactivity can be explained by the solution chemistry of the compounds, selective derivatization of different cysteines on the protein argues for an involvement of pump structure in response to the presence of the proton pump inhibitor on its luminal surface. This suggests that the proton pump inhibitors, which were originally designed to take advantage of only the highly acidic space generated in the parietal cell by the production of the sulphenamide, are made even more selective by the protein they target. Pantoprazole is metabolized by a combination of phase I and phase II metabolism, and has also been shown to have a very low potential for drug interaction. Studies of acid secretion in man have shown this compound to be an effective and long lasting inhibitor of acid secretion. The pharmacodynamics explain the cumulative effect of repeated doses and maximal acid secretory capacity with a once daily dosage.

Serum lipid changes on low salt diet. Effects of alpha 1-adrenergic blockade.

An increase of some serum lipid fractions has been documented in normotensive healthy volunteers and patients with essential hypertension during acute drastic restriction of salt intake. To clarify the potential role of vasopressor systems, particularly the sympathetic system, in the lipid changes induced by salt restriction, we compared fasting serum lipids, glucose, insulin, and C-peptide levels in 16 normotensive healthy volunteers during 7 days of high (200 mmol/day) and 7 days of low (20 mmol/day) salt intake. The individuals were examined on either placebo or on the alpha 1-adrenergic blocker doxazosin (2 mg/day). The study was carried out using a single blind parallel group random order design with two arms of treatment. In the volunteers on placebo, total cholesterol (corrected for hemoconcentration) was significantly higher (P < .01) during low salt intake. The same was true for LDL-cholesterol, whereas HDL-cholesterol and triglycerides did not change with salt intake. The lipid changes, and, in parallel, the changes of hemoconcentration indicators, were more pronounced after 2 days than after 7 days of low salt intake. The rise of total and LDL-cholesterol on low salt was blunted after alpha 1-adrenergic blockade with doxazosin. Fasting glycemia was similar on low salt and high salt, respectively, but in placebo treated volunteers, C-peptide levels were significantly (P < .01) higher on low, rather than high, salt intake. alpha 1-Adrenergic blockade with doxazosin attenuated the rise of C-peptide levels on low salt. The results confirm previous findings that levels of total cholesterol and LDL-cholesterol change inversely with salt intake in normotensive healthy volunteers.(ABSTRACT TRUNCATED AT 250 WORDS)