Evidence that pioglitazone, metformin and pentoxifylline are inhibitors of glycation

Enhanced formation and accumulation of advanced glycation end products (AGEs) have been proposed to play a major role in the pathogenesis of diabetic complications, and atherosclerosis, leading to the development of a range of diabetic complications including nephropathy, retinopathy and neuropathy. Several potential drug candidates as AGE inhibitors have been reported recently. Aminoguanidine is the first drug extensively studied. However, there are no currently available medications known to block AGE formation. We have previously reported a number of novel and structurally diverse compounds as potent inhibitors of glycation and AGE formation. We have now studied several of the existing drugs, which are in therapeutic practice for lowering blood sugar or the treatment of peripheral vascular disease in diabetic patients, for possible inhibitory effects on glycation. We show that that three compounds; pioglitazone, metformin and pentoxifylline are also inhibitors of glycation.     

Effect of pentoxifylline on Wrb antigen

Pentoxifylline, a hemorrheologic agent that lowers whole blood viscosity by increasing red cell membrane deformability, recently was approved by the Food and Drug Administration for the treatment of intermittent claudication. The effect of this drug on the phenotypic expression of red cell blood group antigens was studied with cells collected from six patients with intermittent claudication. After in vivo treatment with pentoxifylline, the serologic expression of the Wrb antigen increased. Comparative studies, using hemagglutination titration techniques, with red cells collected before treatment and 1 month after treatment, showed an increase in titer of at least two tubes and an increase in score of greater than 10 in all six patient samples drawn after treatment. No in vivo serologic changes were observed in any of the other antigens studied (A, B, D, C, E, c, e, M, N, S, s, U, P1, Leb, K, k, Fya, Fyb, Jka, Jkb, Yta). Protein analysis (sodium-dodecylsulfate polyacrylamide gel electrophoresis, silver stain) of red cell membranes prepared from blood collected before treatment and 1 month after treatment showed an increase in band density in the 24,000 and 14,000 dalton regions in the samples drawn after treatment. In vitro treatment of red cells with pentoxifylline and one of its major metabolites did not affect the phenotypic expression of any of the antigens studied, including Wrb.     

Anti-Tcb, an antibody that defines a red cell antigen antithetical to Tca

A patient's serum, used as a source of anti-Goa typing serum, was found to contain a second antibody that detected a 'new' red cell antigen present in approximately 5 percent of blacks, but not yet found in Caucasians. Among blacks, the 'new' antigen shows an antithetical relationship to the high frequency antigen Tca, as evidenced by tests on eight unrelated Tc(a-) black women and the families of two of these propositi. The antigen, designated Tcb, has not been found on the red cells of Tc(a-) persons whose racial origins differ from the propositi of this report, which suggests that more than one genetic mechanism can produce the Tc(a-) phenotype. The presence of both anti-Goa and anti-Tcb in the same serum appears to be a coincidental and unrelated finding.     

Evidence that proteases are involved in superoxide production by human polymorphonuclear leukocytes and monocytes.

The possible participation of proteases in superoxide (O2-) production by human polymorphonuclear leukocytes (PMN) and monocytes was explores using various protease inhibitors and substrates. Protease inhibitors of serine proteases and synthetic inhibitors that modify the active site of serine proteases. Substrates used were synthetic substrates of the chymotrypsin type as well as trypsin type of protease. All these inhibitors and substrates inhibited O2- oroduction by human PMN and monocytes induced by cytochalasin E and concanavalin A, though PMN were more sensitive to these inhibitors and substrates than monocytes. Inhibition appeared rapidly even when the inhibitors were added at the same time as the stimulants, during the "induction time of O2-production" or at the time of maximum O2- production, whereas much greater inhibition was observed when the cells were preincubated with the inhibitors. These observations suggest that enzymatically active serine proteases are essential for these phagocytic cells to initiate and maintain the O2- production in response to the stimuli. The inhibitory effect of the inhibitor and substrate for chymotrypsin type protease was greater than that of those substances for trypsin-type protease. Macromolecular inhibitors also inhibited the O2- production. These findings suggest that the serine proteases involved in the O2- production by human PMN and monocytes are similar to chymotrypsin rather than trypsin, and are possibly located at the cell surface membrane.     

Evidence that cytotoxic T cells are part of the host's response to influenza pneumonia

Cytotoxic T cells were detected in the cervical lymph nodes, lungs, spleen, and peripheral blood of mice with influenza. Lymphocytes decreased in the peripheral circulation and increased in the lung during the period of acute inflammation and pneumonia. Peak cytotoxic T- cell activity was present at the time of marked pulmonary infiltration, and it decreased with resolution of the pneumonia. The cytotoxic T cells in the lung were shown to be H-2 restricted and specific for the hemagglutinin of the infecting virus. The results indicate that hemagglutinin specific cytotoxic T cells are (a) induced during influenza infection; (b) they circulate in the blood; (c) they are present in greatest number; and (d) they have their peak cytotoxic effect when pneumonia is most marked. We interpret the results to indicate that specific cytotoxic T cells in the infected target organ are part of the immunological and pathological response to virus infection.     

Evidence that the clinical effects of cholinesterase inhibitors are related to potency and targeting of action

Since degenerative alterations associated with cholinergic changes in the brains of demented patients occur in specific regions, optimal efficacy may be achieved by targeting the actions of potent cholinesterase inhibitors in relevant regions. When evaluating the activities of these agents, only cerebrospinal fluid (CSF)-based studies in demented patients provide reliable data. Preclinical or healthy volunteer studies of cholinesterase inhibitory activity using plasma or erythrocytes as an enzyme source are inconclusive due to differences between enzymes, their relative activities, and the profiles of their isoforms from different sources, with additional changes during disease progression. Tacrine and rivastigmine inhibit both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in the CSF of Alzheimer's disease patients. Both enzymes are involved in the breakdown of acetylcholine (ACh) in the brain and dual inhibition may lead to greater, broader efficacy, as well as a greater potential for disease modification. However, potent and rapid elevation in levels of ACh may also induce more acute tolerability problems, such as nausea and vomiting. Only rivastigmine appears to show brain region-selectivity, particularly for regions involved in attention and behaviour and that are known to degenerate during the progression of various dementia types. This selectivity is due to preferential inhibition of the G1 form of AChE and, probably, also BuChE. Cholinesterase inhibitors that lack preferential selectivity for particular isoforms may provide less targeted actions. This may explain the relatively higher incidences of certain peripheral side effects observed during maintenance treatment with some of these drugs. All cholinesterase inhibitors interact via ACh, additionally available due to enzyme inhibition, with nicotinic and muscarinic receptors (nAChRs and mAChRs). Allosteric modulation of a presynaptic nAChR has been shown in vitro with many of these agents, and it has been proposed, but not demonstrated, that this may result in an increased release and potentiation of ACh in the brain. The clinical relevance of this mechanism is unknown. The rapidly reversible actions of donepezil, tacrine and galantamine may lead to tolerance due to their ability to upregulate target enzyme activities; however upregulation is not seen with the slowly reversible (pseudo-irreversible) inhibitor rivastigmine. Available clinical data support the hypothesis that potent, slowly reversible inhibitors of AChE and BuChE targeted to the G1 isoforms may lead to greater, broader and more sustained benefits. However, further investigation of the cholinesterase inhibitors to elucidate more definitely the clinical consequences of their differing pharmacological properties is required.     

Incidence of Wra antigen and anti-Wra in a Spanish population

BACKGROUND: This study reports the incidence of Wr(a) antigen and anti-Wr(a) in Valencia, Spain. STUDY DESIGN AND METHODS: The incidence of the Wr(a) antigen in 110,000 healthy blood donors was estimated. Likewise, the incidence of anti-Wr(a) was analyzed in a population consisting of 730 healthy blood donors, 356 pregnant women, and 581 patients who received transfusions from the area of Valencia, Spain. RESULTS: The incidence of Wr(a) antigen was 1 in 785. Overall, anti-Wr(a) was found in 59 samples: 20 healthy blood donors (1/37), 18 pregnant women (1/20), and 21 patients who received transfusions (1/28). The most frequent immunoglobulin class of anti-Wr(a) in healthy blood donors was immunoglobulin M, either alone (8 cases) or plus immunoglobulin G (IgG; 8 cases); the IgG1 and IgG3 were the IgG subclasses most frequently detected in pregnant women (12 cases) and in patients who received transfusions (12 cases). Only 51 percent of the anti-Wr(a) appeared to have the potential to be clinically significant. CONCLUSION: These data show that the incidence of Wr(a) antigen and anti-Wr(a) among the population from Valencia is similar to that reported in other European areas and suggest that the development of anti-Wr(a) is facilitated by the presence of a hyperactive immune system. The clinical relevance of anti-Wr(a) is limited, however.     

Evidence for a new segregant series of B cell antigens that are encoded in the HLA-D region and that stimulate secondary allogenic proliferative and cytotoxic responses

Five new histocompatibility antigens, designated secondary B cell or (SB) antigens, have been identified by secondary allogeneic proliferative and cytotoxic responses. The reagents used to define the SB antigents are lymphocytes primed between donors matched for all known HLA antigens. The SB antigens stimulate weak primary allogeneic proliferative responses (a mean relative response of 8%) but strong secondary proliferative responses. Strong secondary cell-mediated cytotoxicity is generated against target antigens that are distinguishable from the SB antigens defined by proliferation. Studies by direct lysis and by cold-target inhibition indicate that these target antigens are preferentially expressed on B cells relative to T cells. The SB antigens segregate with HLA, and the gene(s) encoding the SB1, 3, and 4 antigens maps centromeric to HLA-B. The SB antigens are major histocompatibility antigens not only because they are encoded by major histocompatibility complex (MHC) genes, but also by the functional criteria that the proliferative and cytotoxic responses to SB antigens are not restricted by HLA-DR or HLA-A,-B. Parallel studies of the SB antigens and the DR antigens with respect to: (a) their preferential expression on B cells, (b) their function in secondary allogeneic proliferative and cytotoxic respones, and (c) the location of their structural gene within the MHC. However, the SB antigens and the DR antigens are clearly distinct antigens, because population studies indicate that they can occur independently, and family studies indicate that specific SB antigens segregate with HLA haplotypes having different D and DR specificities. Our data are consistent with the hypotheses that the SB antigens are a new segregant series of B cell alloantigens, and that the SB gene and the DR gene derive from a duplicated ancestral gene.     

Evidence that nicotinic alpha(7) receptors are not involved in the hyperlocomotor and rewarding effects of nicotine

Neuronal nicotinic receptors are comprised of combinations of alpha(2-9) and beta(2-4) subunits arranged to form a pentameric receptor. Currently, the principal central nervous system (CNS) subtypes are believed to be alpha(4)beta(2) and a homomeric alpha(7) receptor, although other combinations almost certainly exist. The identity of the nicotinic receptor subtype(s) involved in the rewarding effects of nicotine are unknown. In the present study, using some recently described subtype selective nicotinic agonists and antagonists, we investigated the role of the alpha(7) nicotinic receptor in the mediation of nicotine-induced hyperactivity and self-administration in rats. The alpha(7) receptor agonists AR-R 17779 and DMAC failed to stimulate locomotor activity in both nicotine-nontolerant and -sensitized rats. In contrast, nicotine and the putative alpha(4)beta(2) subtype selective agonist SIB1765F increased activity in both experimental conditions. In nicotine-sensitized rats, the high affinity (including the alpha(4)beta(2) subtype) nicotinic antagonist dihydro-beta-erythroidine (DHbetaE), but not the selective alpha(7) antagonist methyllycaconitine (MLA), antagonized a nicotine-induced hyperactivity. Similarly, DHbetaE, but not MLA, pretreatment reduced nicotine self-administration. Electrophysiology experiments using Xenopus oocytes expressing the human alpha(7) receptor confirmed AR-R 17779 and DMAC to be potent agonists at this site, and further studies demonstrated the ability of systemically administered AR-R 17779 to penetrate into the CNS. Taken together, these results indicate a negligible role of alpha(7) receptors in nicotine-induced hyperlocomotion and reward in the rat, and support the view for an involvement of a member from the high-affinity nicotinic receptor subclass, possibly alpha(4)beta(2). Issues such as drug potency, CNS penetration, and desensitization of the alpha(7) receptor are discussed.     

Ryanodine: antithetical calcium channel effects in skeletal muscle sarcoplasmic reticulum

The effects of ryanodine, a neutral alkaloid, on the Ca++ uptake and Ca++ release properties of a skeletal muscle isolated sarcoplasmic reticulum (SR) preparation were evaluated. Ryanodine had no effect on the rate of oxalate-facilitated Ca++ uptake in this SR. Ruthenium red, which reportedly blocks Ca++ channels, increased Ca++ uptake by 2-fold in the SR. Although no effect of ryanodine on Ca++ transport by SR was observed, notable effects on Ca++-induced Ca++ release pathways were discovered. Ryanodine acts on the same Ca++ channels that are affected by ruthenium red. When these Ca++ channels were activated by Ca++ to an open state in the presence of ryanodine, then ryanodine maintained the channel in an activated, open state. However, once Ca++ was taken up by the SR, ryanodine tended to lock the channel in a closed state, producing a condition refractory to Ca++-induced Ca++ release. Thus, dual, opposing effects of ryanodine were demonstrated. In a fast-reaction kinetics measurement of Ca++-induced Ca++ release, both rate and amount of Ca++ release were reduced by ryanodine, and Ca++ appeared to act in a noncompetitive, antagonistic mode. These unusual, antithetical effects of ryanodine on the Ca++ efflux pathway are not mechanistically defined by this study, but they reveal the potential value of ryanodine as a probe for exploring Ca++ channel function.