Mechanisms of nitrate tolerance

Summary There is now little dispute that clinical tolerance of organic nitrates occurs, particularly when these drugs are used by themselves to treat patients with stable angina pectoris and congestive heart failure. Classical hypotheses of nitrate tolerance suggest the phenomenon to result from vascular depletion of critical sulfhydryl groups, which are necessary to bring about vasorelaxation from nitrates. While this mechanism of nitrate tolerance probably operates when isolated blood vessels are exposed to high concentrations of nitrate in vitro, there is little evidence to suggest that it contributes to clinical nitrate tolerance. Instead, emerging data suggest that nitrates can cause significant shifts in fluid distribution and secretion of neurohormonal factors that can modulate their vasorelaxant effects. use of angiotensin converting enzyme inhibitors and diuretics in conjunction with nitrates may alleviate the development of tolerance, but the experience has not been universally favorable. Other receptor-effector systems that affect cardiovascular function, such as the adrenergic system, may also be affected by nitrate tolerance. The mechanisms of nitrate tolerance are therefore likely to be multifactorial, involving vascular biochemical changes, physiologic compensation, and possibly receptor regulation.     

A Modified Product Inhibition Model Describes the Nonlinear Pharmacokinetics of Nicorandil in Rats

Nicorandil, a vasodilator which acts through both cyclic GMP accumulation and K⁺ channel opening, has been used in the treatment of various cardiovascular diseases. We have examined the pharmacokinetics of nicorandil in the rat as a function of dose, as both i.v. boluses (9 doses, 0.75 – 12 mg, n = 1-4 per dose), and as a 5-hr infusion followed by a 5-hr washout (6 doses, 10-500 µg/kg/min, n = 3 per dose). Plasma nicorandil concentrations were determined by HPLC. Nicorandil plasma concentrations increased disproportionately with dose, but nicorandil elimination obeyed apparent monoexponential kinetics, and the apparent half-life (t_1/2) increased with dose. In addition, the approach to apparent steady-state during the infusion phase was not overtly sensitive to the drastic changes in t_1/2observed. Pharmacokinetic modelling with several nonlinear models, viz: Michaelis-Menten with parallel first-order, cosubstrate depletion and competitive product inhibition, were carried out. Addition of the sulfhydryl donor, N-acetyl-L-cysteine, did not change the pharmacokinetics of nicorandil, providing experimental indication that a cosubstrate depletion model might not be applicable. To describe the unique pharmacokinetics, a modified product inhibition model was developed. This new model includes the classic competitive product inhibition equation, describing both parent and product kinetics, and it incorporates, in addition, separate first-order elimination rate constants for both nicorandil and the inhibiting metabolite. Experimental evidence showed that N-(2-hydroxyethyl) nico-tinamide, the major metabolite of nicorandil in rats, and nicotinamide (niacinamide) itself, indeed inhibited nicorandil elimination.     

Use of an in Vitro Model for the Assessment of Muscle Damage from Intramuscular Injections: in Vitro-in Vivo Correlation and Predictability with Mixed Solvent Systems

The potential of binary mixtures of propylene glycol–water, ethanol–water, and polyethylene glycol 400–water to cause skeletal muscle damage (myotoxicity) following intramuscular injection was examined with an in vitro model using the isolated rat muscle. At moderate concentrations (20–40%, v/v) of the organic cosolvent, the order of myotoxicity was propylene glycol > ethanol polyethylene glycol 400. The in vitro results were then compared with in vivo toxicity in rabbits after injection of normal saline, 40% (v/v) polyethylene glycol 400, 40% (v/v) propylene glycol, indocyanine green in normal saline, and indocyanine green in 40% (v/v) propylene glycol. Employing the area under the creatine kinase activity curve from 0 to 72 hr as the index of skeletal muscle damage, an excellent in vitro–in vivo correlation was observed. The basic myotoxicity relationships obtained from the binary cosolvent systems were then used to examine the myotoxicity of ternary organic cosolvent mixtures. Several mixed solvent systems with the same theoretical molar solubilization power for a model compound, diazepam, were selected to determine (1) if myotoxicity can be reduced by changing the composition of the ternary mixtures and (2) if myotoxicity of the individual components is additive. For the solvent systems containing propylene glycol, ethanol, and water, the total myotoxicity equaled the sum of the individual myotoxicity of each component. In contrast, for the solvent systems containing polyethylene glycol 400, the total myotoxicity was only half of the sum of individual toxicities. These results suggest that polyethylene glycol 400 in mixed cosolvent systems might have a protective effect on the myotoxicity generated by intramuscular injections.     

Changing prevalence and knowledge of urinary incontinence among Hong Kong Chinese women

A territory-wide telephone survey was conducted in Hong Kong to assess the prevalence, knowledge, and treatment-seeking behaviour of Chinese women with urinary incontinence, using validated Chinese version of Urogenital Distress Inventory (UDI-6) and Incontinence Impact Questionnaire (IIQ-7). Women, 540, aged between 17 to 77 years were interviewed. Of the respondents, 40.8% reported stress urinary incontinence, 20.4% had urge incontinence and 15.9% had mixed incontinence. Among these, 16.0% reported quality of life impairment; 9.3% felt frustrated with low morale, and 15.2% had nervous and anxiety problems. However, as many as 78.3% of the respondents did not know that stress urinary incontinence is a disease entity, and 60.6% thought that leakage of urine was a normal aging process. For those respondents having stress urinary incontinence, the first treatment of choice was physiotherapy. The second choice was medication, and surgical treatment was the last option. Respondents with stress urinary incontinence showed higher education level.     

Pharmacodynamic modeling of thein vitro vasodilating effects of organic mononitrates

The in vitrodose-relaxation curves of four isomeric organic mononitrates: L-isoidide mononitrate (L-IIMN), isosorbide-2-mononitrate (IS-2-MN), isomannide mononitrate (IMMN), and isosorbide-5-mononitrate (IS-5-MN), were determined with rat aorta rings. These mononitrates relaxed vascular tissue in a concentration-dependent manner. Based upon the EC ₅₀ obtained from the Hill equation, the relative potency of L-IIMN: IS-2-MN: IMMN: IS-5-MN was 43.2:12.2:2.3:1. The Hill exponential coefficients were identical (value of about 1.5) in these four isomeric mononitrates, suggesting that they are likely to have a common mechanism of action. For all four isomers, relaxation was fairly immediate after addition of the tested compound into the tissue bath, with a 2- to 3- min-delay to reach steady-state effect. The rates of relaxation were then used to construct a pharmacodynamic model that described the time course of relaxation for these compounds. This theoretical analysis suggested that in vitronitrate action is mediated by a catenary process, consistent with published biochemical evidence that suggests a series of reactions involving metabolic activation to nitric oxide, production of cyclic GMP, and myosin light-chain phosphorylation to produce relaxation. Via this pharmacodynamic model, the half-lives of nitric oxide and cGMP in the smooth muscle cells were estimated to be 15.2 and 23.1 sec, respectively, consistent with literature reports. Results from the present study indicated the potential use of in vitropharmacodynamic modeling in confirming mechanism(s) of drug action obtained through biochemical or other methods.Supported in part by National Institutes of Health grants HL22273 and GM42850.     

The effect of dose on the disposition of lead in rats after intravenous and oral administration

Single doses of lead acetate were administered to 250- to 350-g rats by both iv (0.5–15 mg Pb/kg) and po (1–100 mg Pb/kg) routes, and blood lead concentrations were measured up to 25 days following dosing. The area under the blood lead concentration vs time curve (AUC) after iv dosing increased in proportion to increases in the dose. Total blood lead clearance and renal lead clearance were not related to the magnitude of the injected dose.After oral dosing, blood lead concentrations (and AUC) did not increase proportionately with dose. After a 1 mg/kg po lead dose, the extent of absorption was estimated at 42%; this decreased to 2% when the dose was increased to 100 mg/kg. Lead concentrations in the blood, kidneys, liver, and brain of both adult and suckling rats recovered 24 hr after various single po doses also indicated that the extent of lead absorption decreased substantially with increasing dose. Blood and kidney lead concentrations in adult rats exposed for 14 days to lead via drinking water also were not proportional to the apparent amount of lead ingested. The results are consistent with published in vitro data which suggested that the mechanism for gastrointestinal absorption of lead is largely capacity-limited in adolescent and adult rats. Because blood lead concentrations were not a linear function of the oral dose in the rat, the relationship between oral dose and toxic effects of lead may not be a simple one. This factor should be considered when safe lead exposure levels in man are to be established via extrapolation of data from high levels of exposure.     

Evaluation of an LC-MS/MS assay for 15N-nitrite for cellular studies of L-arginine action

The utility of an LC-MS/MS assay for nitrite determination in studying l-arginine (ARG) cellular action was examined in vitro. EA.hy926 human endothelial cells or cellular fractions (membrane and cytosol) were exposed to 0-500 μM of ¹⁵N₄-ARG for 2 h. ¹⁴N-nitrite and ¹⁵N-nitrite in the cell lysate and in the incubation medium were derivatized with 2,3-diaminonaphthalene (DAN) to form ¹⁴N- and ¹⁵N-naphthotriazole (i.e., ¹⁴N-NAT and ¹⁵N-NAT). Peak responses of ¹⁴N-NAT and ¹⁵N-NAT were analyzed by LC-MS/MS with 1H-naphth[2,3-d]imidazole as an internal standard. The calibration curves of DAN-derivatized ¹⁴N-NAT and ¹⁵N-NAT from ¹⁴N-nitrite and ¹⁵N-nitrite were linear. Intra- and inter-day variability of the quantification was below 14.2% in quality control samples. Following incubation of EA.hy926 cells with ¹⁵N₄-ARG, saturable increases of ¹⁵N-nitrite accumulation with increasing ¹⁵N₄-ARG exposure were observed clearly. This increase however could not be detected by the classical fluorescence method, nor were changes in ¹⁴N-nitrite level observed. When cellular fractions were exposed to ¹⁵N₄-ARG, ¹⁵N-nitrite formation was only observed in the membrane fragments. The sensitive and selective LC-MS/MS method reported here can be applied to quantify accumulated nitrite levels in human endothelial cells. The selectivity of this stable-isotope labeled LC-MS/MS method offers an advantage over other traditional methods for elucidating cellular ARG action when its stable isotope is employed as a substrate.     

Nitroglycerin-inhibited whole blood aggregation is partially mediated by calcitonin gene-related peptide–a neurogenic mechanism

1. The role of the vasculature and calcitonin gene-related peptide (CGRP) in nitroglycerin (NTG)-mediated platelet inhibition was studied.
2. In vitro incubations of CGRP in whole blood induced a dose-dependent inhibition of platelet aggregation with an IC₅₀ of 62.1 nM.
3. The platelet inhibition induced by CGRP was blocked by co-incubation of 0.53 μM CGRP_8-37, as well as 30 μM N^G-nitro-monomethyl-L-arginine (L-NMMA).
4. In a separate group of experiments, 100 nM NTG in rat whole blood (WB) induced platelet inhibition of 6.0±1.3% (mean±s.d.), which was enhanced to 77.6±3.5% by the addition of rat aortic tissue (AT) (P<0.001). The inclusion of CGRP_8-37 with NTG and AT in WB reduced platelet inhibition to 31.6±6.8% (P<0.01). Incubation of WB and AT with 30 μM L-NMMA reduced NTG-induced inhibition of platelet aggregation to 26.4±4.2% (P<0.001).
5. It is concluded that vascular tissue contributes to the antiplatelet mechanism of action of NTG. Furthermore, NTG apparently evokes the release of CGRP from vascular tissue and this neuropeptide contributes to the antiplatelet actions of NTG.
6. The antiplatelet activity of CGRP in whole blood is mediated primarily through the activation of nitric oxide synthase.

     

Application of Pharmacodynamic Modeling for Designing Time-Variant Dosing Regimens to Overcome Nitroglycerin Tolerance in Experimental Heart Failure

Purpose. Prolonged continuous administration of nitroglycerin (NTG) leads to hemodynamic tolerance. We used a previously developed pharmacokinetic-pharmacodynamic (PK/PD) model of NTG tolerance in experimental heart failure to test whether dosage regimens, designed from this model, may allow avoidance of tolerance development upon continuous NTG inftision. Methods. Simulation experiments (using ADAPT II) were performed to evolve a time-variant infusion regimen that would theoretically provide sustained hemodynamic effect (30% reduction in left ventricular end-diastolic pressure, LVEDP) throughout 10 hours of drug dosing. A computer controlled infusion pump was utilized to deliver this time-variant input. Infusion experiments were then conducted in CHF rats to challenge the predictability of the applied PK/PD model. Results. Simulations showed that exponentially increasing input functions provided more sustained LVEDP effects when compared to linear or hyperbolic input functions delivering the same total NTG dose. A computer-selected infusion regimen of 6.56e^{0.00156×minutes} g/min was anticipated to provide the desired hemodynamic profile in our animal model. Experiments conducted in rats with congestive heart failure (n = 4) confirmed the prediction of sustained hemodynamic effect without tolerance (28 ± 4% reduction in LVEDP at 10 hrs). Conclusions. These findings support the utility of our PK/PD model of NTG tolerance in predicting NTG action, and serve as an example of therapeutic optimization through PK/PD considerations.     

Complexation of Nifedipine with Substituted Phenolic Ligands

The bioavailability of nifedipine in man is highly variable. This may be partly due to its poor aqueous solubility (5–6 µg/ml over pH 2.2–10.0, as determined in this laboratory). We initiated this study to examine the enhancement of aqueous nifedipine solubility via complexation. A series of substituted aromatic ligands was studied to identify those structural features important for complexation with nifedipine. The studies were performed at 25°C employing the solubility technique, using pH 2.2 or 7.0 buffers at an ionic strength of 0.25 M. The apparent equilibrium complexation constants for the 1:1 and/or 1:2 complexes were determined, where appropriate. A linear free-energy approach was used to relate K _1:1 with Hammett's sigma () and fractional partition coefficient () parameters. The following correlation was obtained: log (K _l:l/K _o = 0.31 + 0.l0 + 0.36 (r ² = 0.86, P < 0.003, N = 9), where K _o is the complexation constant for phenol. Statistical analyses showed that was more important than in affecting nifedipine complexation. The exact location of this interaction on the nifedipine molecule is undefined at present.