Physical factors affecting chloroquine binding to melanin.

Chloroquine is an antimalarial drug but is also prescribed for conditions such as rheumatoid arthritis. Long-term users risk toxic side effects, including retinopathy, thought to be caused by chloroquine accumulation on ocular melanin. Although the binding potential of chloroquine to melanin has been investigated previously, our study is the first to demonstrate clear links between chloroquine adsorption by melanin and system factors including temperature, pH, melanin type, and particle size. In the current work, two Sepia melanins were compared with bovine eye as a representative mammalian melanin. Increasing the surface anionic character due to a pH change from 4.7 to 7.4 increased each melanin's affinity for chloroquine. Although the chloroquine isotherms exhibited an apparently strong interaction with each melanin, isosteric heat analysis indicated a competitive interaction. Buffer solution cations competed effectively at low surface coverage; chloroquine adsorption occurs via buffer cation displacement and is promoted by temperature-influenced secondary structure swelling.

Chloroquine and hydroxychloroquine binding to melanin: Some possible consequences for pathologies.

For many years chloroquine was used as a prophylactic agent against malaria, and more recently as a mild immunosuppressive. However, due to lengthy treatment periods, adverse effects have become apparent, which included retinopathy. The structurally related hydroxychloroquine is less toxic, thought to be owing to a lower tissue accumulation in melanin rich areas. This study primarily focused on quantifying melanin binding between chloroquine and hydroxychloroquine at physiological pH to investigate the potential link between binding and reported toxicity. In addition, for the first time this study quantified the actual extent of adsorption of chloroquine and hydroxychloroquine to melanin and examined the desorption profile of both drugs from melanin to demonstrate the affinity between the pigment and the solutes. The results suggest that there is a difference between the adsorption affinities of chloroquine and hydroxychloroquine, potentially explaining the differences in bioaccumulation in retinal tissue. In addition, both solutes displayed a strong physical attraction to the absorbent.

Butenolide endothelin antagonists with improved aqueous solubility.

Continued development around our ETA-selective endothelin (ET) antagonist 1 (CI-1020) has led to the synthesis of analogues with improved aqueous solubility profiles. Poor solubility characteristics displayed by 1 required a complex buffered formulation in order to conduct iv studies. To overcome the use of specific iv formulations for preclinical studies on additional drug candidates, analogues with improved aqueous solubility were desired. Several analogues were synthesized with substitution patterns that allowed for the formation of either acid or base addition salts. These derivatives had dramatically improved aqueous solubility. In addition, these analogues retained equivalent or improved ETA receptor selectivity and antagonist potency, versus 1, both in vitro and in vivo. Compound 29, which contains as a substituent the sodium salt of a sulfonic acid, has an ETA IC50 = 0.38 nM, ETA selectivity of 4200-fold, and ETA functional activity of KB = 7.8, all of which are similar or superior to those of 1. Compound 29 also has vastly superior aqueous solubility and solubility duration, compared to 1. Furthermore, 29 after iv infusion displays improved activity to 1 in preventing acute hypoxia-induced pulmonary hypertension in rats with an ED50 = 0.3 microg/kg/h.

PD 142893, SB 209670, and BQ 788 selectively antagonize vascular endothelial versus vascular smooth muscle ET(B)-receptor activity in the rat.

The purpose of this study was to determine whether vascular endothelial and vascular smooth-muscle endothelin ET(B) receptors could be quantitatively differentiated by PD 142893 (PD), SB 209670 (SB), and BQ 788 (BQ) in the same species by using closely matched experimental conditions. The isolated perfused rat kidney (vascular smooth muscle) and isolated perfused rat mesentery (vascular endothelium) were challenged with increasing bolus doses of sarafotoxin S6c in the absence and presence of antagonist. PD, SB, and BQ produced parallel concentration-dependent rightward shifts in the S6c dose-response curve in the kidney. PD and SB also produced parallel concentration-dependent rightward shifts in the S6c dose-response curve in the mesentery. In contrast, BQ produced an insurmountable antagonism. Schild-derived pA2 values for PD and SB were significantly greater for inhibiting endothelial versus smooth-muscle ET(B) receptors. Furthermore, PD and SB differed in their relative potency between the two assays. Because BQ produced an insurmountable antagonism in the mesentery, it was not possible quantitatively to compare the antagonist activity in the two assays. These results indicate that PD, SB, and BQ selectively antagonize endothelial ET(B)-receptor activity over smooth-muscle ET(B)-receptor activity.

Novel selective quinazoline inhibitors of endothelin converting enzyme-1.

PD 069185 is a highly selective and structurally novel inhibitor of endothelin converting enzyme-1 (ECE-1). PD 069185 is a trisubstituted quinazoline with an IC50 value of 0.9 +/- 0.1 microns for inhibition of human ECE-1 from the solubilized membrane fraction of CHO cells stably transfected with human ECE-1 cDNA. Kinetic analysis revealed that PD 069185 is best fit with a competitive inhibition model with a Ki value of 1.1 +/- 0.1 microns and binds in a reversible manner. The closely related enzyme, ECE-2, is not inhibited at up to 100 microns PD 069185. In addition, PD 069185 at 200-300 microns has little effect on other metalloproteases, such as neutral endopeptidase 24.11, stromelysin, gelatinase A, and collagenase, showing a high ECE-1 specificity. Data are also presented to show that this series of inhibitors are effective in inhibiting ECE-1 in intact cells and in attenuating the increase in perfusion pressure induced by big ET-1 in isolated rat mesentery. These non-peptidic ECE-1 inhibitors should serve as a valuable tool to study the pathophysiological role of endothelin and the therapeutic potential of ECE-1 inhibitors.

Pharmacological characterization of PD 156707, an orally active ETA receptor antagonist.

We describe the pharmacological characteristics of PD 156707 (sodium 2-benzo[1,3]dioxol-5-yl-4-(4-methoxy-phenyl)-4-oxo-3-(3,4,5- trimethoxy-benzyl)-but-2-enoate), a potent, orally active, nonpeptide antagonist of the endothelin A (ETA) receptor subtype. PD 156707 was designed on the basis of a compound identified by screening the Parke-Davis chemical library. PD 156707 is highly selective for the ETA receptor (ETAR) and inhibits the binding of [125I]-ET-1 to cloned human ETAR and ETBR with Ki values of 0.17 and 133.8 nM, respectively. PD 156707 antagonizes ET-1-stimulated phosphoinositide hydrolysis in Ltk- cells expressing cloned human ETAR with an IC50 value of 2.4 nM. PD 156707 inhibits vasoconstriction in isolated blood vessels mediated by ETAR (rabbit femoral artery) and ETBR (rabbit pulmonary artery) with pA2 values of 7.5 and 4.7, respectively. PD 156707 administered orally to rats blocked subsequent ETAR-mediated pressor responses in vivo but had no effect on ETBR-mediated dilator responses. As a potent and orally active ETA-selective antagonist, PD 156707 will be useful in defining the physiological and pathological roles of ETAR.

Cardiac hypertrophy in rats after intravenous administration of CI-959, a novel antiinflammatory compound: morphologic features and pharmacokinetic and pharmacodynamic mechanisms.

CI-959 is an antiallergic/antiinflammatory agent currently in development. In rats, daily bolus intravenous administration of CI-959 at doses > or = 10 mg/kg was associated with development of cardiac hypertrophy. There was no morphologic or biochemical evidence of myocyte injury, and cardiac hypertrophy rapidly reversed after treatment was discontinued. Cardiac hypertrophy was not evident when CI-959 was given orally or by continuous intravenous infusion with ALZA osmotic pumps. Maximum plasma drug concentrations (Cmax) were significantly higher when CI-959 was given by bolus intravenous injection, suggesting that cardiac effects were dependent on high Cmax concentrations. When neonatal rat cardiomyocytes were exposed to CI-959 in vitro, there was no evidence of myocyte enlargement or increased protein content. Cardiac hypertrophy was prevented by pretreatment with nonselective beta- and beta 1-selective adrenoceptor blockers as well as with central sympatholytics. beta 2- and alpha-adrenoceptor blockers were ineffective in preventing cardiac hypertrophy. Bolus intravenous CI-959 administration resulted in prolonged hypotension and associated increase in plasma catecholamine levels, with apparent inhibition of reflex tachycardia. We conclude that CI-959-associated cardiac hypertrophy in rats was not a direct drug effect but instead was probably mediated by endogenous catecholaminergic stimulation of cardiac beta 1-adrenoceptors.

Methylnaloxonium diffuses out of the rat brain more slowly than naloxone after direct intracerebral injection.

The value of intracerebral injections as a means of relating brain structure and function is dependent on the degree of site specificity of the injection. The purpose of this study was to compare the distribution over time of naloxone and its quaternary derivative, methylnaloxonium, after intracerebral microinjection. One microliter of tritiated naloxone (NAL) or methylnaloxonium (MN 10.0 ng, 12.8 nCi for both drugs) was infused directly into the n. raphe pontis. Each animal was then decapitated at a specific time (2.5, 5.0, 15, 30, or 60 min), the brain was removed and dissected into hindbrain, cerebellum, midbrain and cortex. Tritium beta emissions of brain homogenates were measured 1 day later, MN remained better localized to the injection site than did the same volume of the more lipophilic NAL. Within 15 minutes, less than 5% of the NAL remained in the hindbrain compared with nearly 40% of the MN. These results that MN may be a better probe than NAL for investigating the relationship of opioid receptor anatomy and function, particularly for dependent variables requiring sustained time courses.

Lack of effect of metoclopramide on cisplatin-induced nephrotoxicity in rats.

Metoclopramide has gained acceptance as an effective drug for the control of nausea and vomiting in cancer patients receiving cisplatin chemotherapy. In addition to its emetogenic side effects, cisplatin is known to be nephrotoxic. This animal study was undertaken to determine if the severity of cisplatin-induced nephrotoxicity is altered by the coadministration of metoclopramide. Female F-344 rats received subcutaneous saline or metoclopramide 30 min before and 120 min after intravenous administration of saline or cisplatin. Animals were sacrificed on the 5th day following treatment. Decreases in body weight, elevations in serum urea nitrogen and histopathological changes were of similar magnitude in all cisplatin treated groups regardless of concomitant metoclopramide administration. Saline dosed control animals did not exhibit these changes. It is concluded that the coadministration of metoclopramide did not significantly alter the nephrotoxic effects of cisplatin in this experiment.

Myocardial effects of mitoxantrone and doxorubicin in the mouse and guinea pig.

Mitoxantrone (DHAQ) was compared to doxorubicin for myocardial effects in the mouse and the guinea pig. Histologically, DHAQ induced a high incidence of focal myocardial damage in mice, similar to that observed with doxorubicin. Functionally, like doxorubicin, DHAQ significantly reduced the rate of contraction and histamine responsiveness of guinea pig right atria in vitro. Additionally, long-term ip administration of either DHAQ or doxorubicin reduced the atrial response to histamine in vitro. These data suggest that DHAQ may have a spectrum of myocardial activity similar to that of doxorubicin.

Effect of ICRF-187 on doxorubicin-induced myocardial effects in the mouse and guinea pig.

ICRF-187 was tested for cardioprotective activity in doxorubicin-treated mice and guinea pigs. Pretreatment with i.p. ICRF-187 caused a significant decrease in the indicence of i.v. doxorubicin-induced myocardial histological damage in the mouse. I.p. ICRF-187 did not, however, reduce the effect of i.p. doxorubicin on a functional myocardial effect of this antitumour drug, a reduced histamine responsiveness of right atria in vitro. These data suggest that ICRF-187 may not be specific for all the cardiac effects of doxorubicin.

Development and Localization of Carboxypeptidase Activity in Embryo-less Barley Half-kernels.

Distal half-kernels of barley (Hordeum vulgare L.), after imbibition for 1 day, produced a carboxypeptidase that was active on N-carbobenzoxy-l-phenylalanyl-l-alanine and on N-carbobenzoxy-l-phenylalanyl-l-phenylalanine. For the ensuing 2 days, activity increased linearly and thereafter increased at reduced rates. Electrofocusing of an imbibed half-kernel homogenate produced coincident peaks of activity on both substrates. Experiments with dissected imbibed (3 days) half-kernels showed that the enzyme arose in the aleurone layer. Enzyme production was inhibited by 6-methylpurine, cordycepin, cycloheximide, and p-fluorophenylalanine, and activity was inhibited by phenylmethylsulfonylfluoride. The enzyme did not hydrolyze endopeptidase substrates over a range of pH.Gibberellic acid accelerated the rate of release from the aleurone, but was not essential for release and did not appreciably affect the ultimate amount of carboxypeptidase produced. In these respects, the carboxypeptidase appears to be unique among the known hydrolases produced by barley aleurone tissue.