The median-effect equation derived from the mass-action law principle at equilibrium-steady state via mathematical induction and deduction for different reaction sequences and mechanisms and different types of inhibition has been shown to be the unified theory for the Michaelis-Menten equation, Hill equation, Henderson-Hasselbalch equation, and Scatchard equation. It is shown that dose and effect are interchangeable via defined parameters. This general equation for the single drug effect has been extended to the multiple drug effect equation for n drugs. These equations provide the theoretical basis for the combination index (CI)-isobologram equation that allows quantitative determination of drug interactions, where CI 1 indicate synergism, additive effect, and antagonism, respectively. Based on these algorithms, computer software has been developed to allow automated simulation of synergism and antagonism at all dose or effect levels. It displays the dose-effect curve, median-effect plot, combination index plot, isobologram, dose-reduction index plot, and polygonogram for in vitro or in vivo studies. This theoretical development, experimental design, and computerized data analysis have facilitated dose-effect analysis for single drug evaluation or carcinogen and radiation risk assessment, as well as for drug or other entity combinations in a vast field of disciplines of biomedical sciences. In this review, selected examples of applications are given, and step-by-step examples of experimental designs and real data analysis are also illustrated. The merging of the mass-action law principle with mathematical induction-deduction has been proven to be a unique and effective scientific method for general theory development. The median-effect principle and its mass-action law based computer software are gaining increased applications in biomedical sciences, from how to effectively evaluate a single compound or entity to how to beneficially use multiple drugs or modalities in combination therapies.

http://www.protocol-online.org/forums/uploads/monthly_09_2010/post-8165-008503000%201284677034.ipb

Theoretical Basis, Experimental Design, and Computerized Simulation of Synergism and Antagonism in Drug Combination Studies

Congeners of nitrogen monoxide (NO) are neuroprotective and neurodestructive. To address this apparent paradox, we considered the effects on neurons of compounds characterized by alternative redox states of NO: nitric oxide (NO.) and nitrosonium ion (NO+). Nitric oxide, generated from NO. donors or synthesized endogenously after NMDA (N-methyl-D-aspartate) receptor activation, can lead to neurotoxicity. Here, we report that NO.- mediated neurotoxicity is engendered, at least in part, by reaction with superoxide anion (O2.-), apparently leading to formation of peroxynitrite (ONOO-), and not by NO. alone. In contrast, the neuroprotective effects of NO result from downregulation of NMDA-receptor activity by reaction with thiol group(s) of the receptor's redox modulatory site. This reaction is not mediated by NO. itself, but occurs under conditions supporting S-nitrosylation of NMDA receptor thiol (reaction or transfer of NO+). Moreover, the redox versatility of NO allows for its interconversion from neuroprotective to neurotoxic species by a change in the ambient redox milieu. The details of this complex redox chemistry of NO may provide a mechanism for harnessing neuroprotective effects and avoiding neurotoxicity in the central nervous system.

A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds

The heme oxygenase (HO) system consists of two forms identified to date: the oxidative stress-inducible protein HO-1 (HSP32) and the constitutive isozyme HO-2. These proteins, which are different gene products, have little in common in primary structure, regulation, or tissue distribution. Both, however, catalyze oxidation of heme to biologically active molecules: iron, a gene regulator; biliverdin, an antioxidant; and carbon monoxide, a heme ligand. Finding the impressive heme-degrading activity of brain led to the suggestion that "HO in brain has functions aside from heme degradation" and to subsequent exploration of carbon monoxide as a promising and potentially significant messenger molecule. There is much parallelism between the biological actions and functions of the CO- and NO-generating systems; and their regulation is intimately linked. This review highlights the current information on molecular and biochemical properties of HO-1 and HO-2 and addresses the possible mechanisms for mutual regulatory interactions between the CO- and NO-generating systems.

THE HEME OXYGENASE SYSTEM:A Regulator of Second Messenger Gases

http://academic.oup.com/milmed/article-pdf/146/7/506/24126715/milmed-146-7-506.pdf

Goodman and Gilman's The Pharmacological Basis of Therapeutics

The heme oxygenases, which consist of constitutive and inducible isozymes (HO-1, HO-2), catalyze the rate-limiting step in the metabolic conversion of heme to the bile pigments (i.e., biliverdin and bilirubin) and thus constitute a major intracellular source of iron and carbon monoxide (CO). In recent years, endogenously produced CO has been shown to possess intriguing signaling properties affecting numerous critical cellular functions including but not limited to inflammation, cellular proliferation, and apoptotic cell death. The era of gaseous molecules in biomedical research and human diseases initiated with the discovery that the endothelial cell-derived relaxing factor was identical to the gaseous molecule nitric oxide (NO). The discovery that endogenously produced gaseous molecules such as NO and now CO can impart potent physiological and biological effector functions truly represented a paradigm shift and unraveled new avenues of intense investigations. This review covers the molecular and biochemical characterization of HOs, with a discussion on the mechanisms of signal transduction and gene regulation that mediate the induction of HO-1 by environmental stress. Furthermore, the current understanding of the functional significance of HO shall be discussed from the perspective of each of the metabolic by-products, with a special emphasis on CO. Finally, this presentation aspires to lay a foundation for potential future clinical applications of these systems.

/pdf/heme-oxygenase-1-carbon-monoxide-from-basic-science-to-2rhjm6458g.pdf

Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications.

Does carbon monoxide have a physiological function

Studies on the physiological role of heme oxygenase (HO) require an inhibitor that will selectively inhibit HO activity without inhibiting the activity of either nitric oxide synthase (NOS) or soluble guanylyl cyclase (sGC). The objective of this study was to test a series of metalloporphyrins that have previously been shown to inhibit HO activity, for their ability to inhibit HO without inhibiting NOS or sGC activities. Measurement of activity of HO in rat brain microsomes and NOS in rat brain cytosol was made for samples incubated with metalloporphyrins (0.15–50 μM), including zinc protoporphyrin IX, zinc deuteroporphyrin IX 2,4-bis-ethylene glycol (ZnBG), chromium mesoporphyrin IX (CrMP), tin protoporphyrin IX, and zinc N -methylprotoporphyrin IX. CrMP and ZnBG were found to be the most selective inhibitors of HO activity (i.e., caused the greatest inhibition of HO activity, 89 and 80%, respectively, without inhibition of NOS activity). Based on these results, sGC activity in rat lung cytosol incubated with CrMP or ZnBG (0.15–15 μM) was measured. ZnBG did not affect basal sGC activity but did potentiate S -nitroso- N -acetylpenicillamine (SNAP)-induced sGC activity. CrMP did not affect either basal or SNAP-induced activity. It was concluded that of the five metalloporphyrins studied, CrMP, at a concentration of 5 μM, was a selective inhibitor of HO activity and was the most useful metalloporphyrin for the conditions tested. Thus, CrMP would appear to be a valuable chemical probe in elucidating the physiological role of HO.

Selective inhibition of heme oxygenase, without inhibition of nitric oxide synthase or soluble guanylyl cyclase, by metalloporphyrins at low concentrations.

SUMMARYThe expanding role of intravenous nitroglycerin (GTN) in the management of critically ill hospitalized patients demands a clear knowledge of its pharmacodynamics and kinetics in both normal and diseased states. Accordingly, we studied 16 patients with congestive cardiac failure to establish the relationship between blood levels of GTN and its physiologic effects during and after an i.v. infusion. The end point of this study was either a greater than 25% fall in pulmonary capillary wedge presure or more than a tenfold increment over the initial GTN infusion rate. Infusion rate of GTN and blood concentration correlated well (r = 0.75, p < 0.001). Patients were divided into two groups based on their blood GTN concentration. Group I patients (n = 8) achieved blood GTN concentrations of 1.2-11.1 ng/ml and all reached the hemodynamic end point. The minimum effective blood GTN concentration was 1.2 ng/ml at an infusion rate of 15 μg/min. Group 2 patients (n = 8) had blood levels greater than 11.1 ng/ml and only three achieved the hemodynamic end point. Group 2 had greater systemic venous congestion than group 1 (right atrial pressure 19 ± 4 mm Hg (SD) vs 10 ± 4 mm Hg [p < 0.0011). In addition, group 2 had lower total body clearance of GTN (3.6 ± 1.8 1/min) than group 1 (13.8 ± 5.8 1/min) (p < 0.005). The low clearance of GTN in group 2 patients may be explained in part by impaired hepatic metabolism secondary to severe systemic venous congestion. Complete blood GTN data were available on five patients after cessation of the GTN infusion and revealed a short half-life of 1.9 minutes. Some patients failed to reach the hemodynamic end point with high infusion rates of GTN (220-4 μg/min), and blood levels of 42.2-481.3 ng/ml. There was no evidence of toxicity despite these high GTN blood levels.

Pharmacokinetic-hemodynamic studies of intravenous nitroglycerin in congestive cardiac failure.

Pharmacokinetic analysis of nitroglycerin (GTN) has been hampered by the lack of a sensitive and specific method for measuring GTN in blood. Therefore, we examined the appearance of GTN in blood after administering 0.6 mg sublingually in 10 studies of normal volunteers. We used a gas-liquid chromatographic method with electron-capture detection and isosorbide dinitrate as the internal standard. GTN appeared in blood at 0.5 minutes, reached a peak of 2.3 +/- 0.36 ng/ml at 2 minutes, fell to 50% of peak value at 7.5 minutes and was barely detectable at 20 minutes. These blood levels paralleled the changes in heart rate and systolic blood pressure. These data show rapid appearance and disappearance of GTN from blood after sublingual administration, a large volume of distribution, and a rapid rate of total body clearance that precludes the liver from being the sole elimination site. This method for analysis of GTN and isosorbide dinitrate should be helpful in defining the role of chronic nitrate therapy.

Blood levels after sublingual nitroglycerin.

(1985). Exposure to Toxic Agents: The Heme Biosynthetic Pathway and Hemoproteins as Indicator. CRC Critical Reviews in Toxicology: Vol. 15, No. 2, pp. 151-180.

Gerald S. Marks

Papers

Does carbon monoxide have a physiological function

Selective inhibition of heme oxygenase, without inhibition of nitric oxide synthase or soluble guanylyl cyclase, by metalloporphyrins at low concentrations.

Pharmacokinetic-hemodynamic studies of intravenous nitroglycerin in congestive cardiac failure.

Blood levels after sublingual nitroglycerin.

Exposure to toxic agents: the heme biosynthetic pathway and hemoproteins as indicator.