Lorena Rodríguez-Páez

α-Asarone inhibits HMG-CoA reductase, lowers serum LDL-cholesterol levels and reduces biliary CSI in hypercholesterolemic rats

Our results showed that alpha-asarone was an inhibitor of hepatic HMG-CoA reductase and that the administration of alpha-asarone at 80 mg/kg body wt. for 8 days decreased serum cholesterol by 38% (p < 0.001) in hypercholesterolemic rats. This alpha-asarone treatment affected mainly the serum LDL-cholesterol levels, leaving serum HDL-cholesterol lipoproteins unaffected, with a consequent decrease of 74% in the LDL/HDL ratio. In addition, alpha-asarone especially stimulated bile flow in hypercholesterolemic rats (60%), increasing the secretion of bile salts, phospholipids and bile cholesterol. The drug also reduced the cholesterol levels of gallbladder bile, whereas the concentration of phospholipids and bile salts increased only slightly, leading to a decrease in the cholesterol saturation index (CSI) of bile in the hypercholesterolemic rats. This CSI decrease and the increase in bile flow induced by alpha-asarone may account for the cholelitholytic effect of alpha-asarone. It seems that alpha-asarone induced clearance of cholesterol from the bloodstream and that the excess of hepatic cholesterol provided by LDL-cholesterol is diverted to bile sterol secretion via a bile choleresis process. The inhibition of HMG-CoA reductase and the increase in bile flow induced by alpha-asarone, as well as the decrease in the CSI, could then explain the hypocholesterolemic and cholelitholytic effects of alpha-asarone.

SELECTIVE INHIBITION OF THE SPERM-SPECIFIC LACTATE DEHYDROGENASE ISOZYME-C4 BY N-ISOPROPYL OXAMATE

In the present study, we demonstrated that the attachment of the nonpolar isopropylic carbon chain in the nitrogen of oxamate, converted this competitive inhibitor of LDH isozymes into a powerful selective inhibitor of mouse LDH-C4. The comparative study of the inhibitory effect of oxamate and N-isopropyl oxamate on mouse LDH isozymes pointed out that the isopropylic carbon chain conferred upon N-isopropyl oxamate a high affinity for LDH-C4 and a marked decrease in the affinity for the other isozymes since oxamate showed more inhibitory effect on LDH-1 (Ki = 0.06 mM) and LDH-5 (Ki = 0.08 mM), and less inhibitory effect on LDH-C4 (Ki = 0.25 mM). On the other hand, N-isopropyl oxamate showed the highest inhibitory effect on LDH-C4 (Ki = 0.014 mM) and poor inhibitory effect on LDH-1 (Ki = 0.4 mM) and LDH-5 (Ki = 0.8 mM). Apparently, the enzymatic inactivation proceeded through a reversible binding of N-isopropyl oxamate, facilitated by nonpolar interactions with a hydrophobic region present only in the active site of mouse LDH-C4, resulting in a selective inhibition of this isozyme in comparison with the other LDH isozymes. N-isopropyl oxamate was also a powerful competitive inhibitor of LDH-C4 (Ki = 0.015 mM) compared with oxamate (Ki = 0.35 mM), using alpha-ketoisocaproate as a substrate.

Trypanocidal activity of N-isopropyl oxamate on cultured epimastigotes and murine trypanosomiasis using different Trypanosoma cruzi strains

The trypanocidal activity of N-isopropyl oxamate (NIPOx) and the ethyl ester of N-isopropyl oxamate (Et-NIPOx) were tested on cultured epimastigotes (in vitro) and on murine trypanosomiasis (in vivo) using five different T. cruzi strains. When benznidazole and nifurtimox, used for comparison, were tested we found that only three of these T. cruzi strains were affected, whereas the other two strains, Miguz and Compostela, were resistant to the in vitro and the in vivo trypanocidal activity of these substances. In addition, when NIPOx was tested on cultured epimastigotes and on mice parasitaemia, trypanocidal activity was not obtained on either of these T. cruzi strains. Our experiments strongly suggest that NIPOx does not penetrate intact epimastigotes due to the polarity of its carboxylate whereas Et-NIPOx, acting as a prodrug, exhibited in vitro and in vivo trypanocidal activity in the five tested T. cruzi strains.

Oxamic acid analogues as LDH-C4-specific competitive inhibitors

We performed kinetic studies to determine whether oxamate analogues are selective inhibitors of LDH-C4, owing to their potential usefulness in fertility control and treatment of some cancers. These substances were shown to be competitive inhibitors of LDH isozymes and are able to discriminate among subtle differences that differentiate the active sites of LDH-A4, LDH-B4 and LDH-C4. N-Ethyl oxamate was the most potent inhibitor showing the highest affinity for LDH-C4. However, N-propyl oxamate was the most selective inhibitor showing a high degree of selectivity towards LDH-C4. Non-polar four carbon atoms chains, linear or branched, dramatically diminished the affinity and selectivity towards LDH-C4. N-Propyl oxamate significantly reduced ATP levels, capacitation and mouse sperm motility, in line with results shown by others, suggesting that LDH-C4 plays an essential role in mouse fertility.

In vitro and in vivo trypanocidal activity of the ethyl esters of N-allyl and N-propyl oxamates using different Trypanosoma cruzi strains

The trypanocidal activity of N-allyl (NAOx) and N-propyl (NPOx) oxamates and that of the ethyl esters of N-allyl (Et-NAOx) and N-propyl (Et-NPOx) oxamates were tested on cultured epimastigotes (in vitro) and murine trypanosomiasis (in vivo) using five different T. cruzi strains. NAOx and NPOx did not penetrate intact epimastigotes and therefore we were not able to detect any trypanocidal effect with these oxamates. Whereas the ethyl esters (Et-NAOx and Et-NPOx), acting as prodrugs, exhibited in vitro and in vivo trypanocidal activity on the five tested T. cruzi strains. On the contrary, when Nifurtimox and Benznidazole used as reference drugs were tested, we found that only three of the five tested T. cruzi strains were affected, whereas the other two strains, Miguz and Compostela, were resistant to the in vitro and in vivo trypanocidal activity of these compounds.

Inhibition of Trypanosoma cruzi α-Hydroxyacid Dehydrogenase-isozyme II by N-Isopropyl Oxamate and its Effect on Intact Epimastigotes

The effect of N-isopropyl oxamate on the activity of α-hydroxyacid dehydrogenase-isozyme II (HADH-isozyme II) from Trypanosoma cruzi was investigated. The kinetic studies showed that this substance was a competitive inhibitor of this isozyme. The attachment of the nonpolar isopropylic branched chain to the nitrogen of oxamate increased 12-fold the affinity of N-isopropyl oxamate for the active site of T. cruzi HADH-isozyme II. N-isopropyl oxamate was a selective inhibitor of HADH-isozyme II, since other T. cruzi dehydrogenases were not inhibited by this substance. Since HADH-isozyme II participates in the energy metabolism of T. cruzi, a trypanocidal effect can be expected with inhibitors of this isozyme. However, although it was not possible to detect any trypanocidal activity with N-isopropyl oxamate when the ethyl ester was tested as a possible trypanocidal prodrug, the expected trypanocidal effect was obtained, comparable to that obtained with nifurtimox and benznidazole.

2,4,5-trimethoxycinnamic acid: the major metabolite of α-asarone, retains most of the pharmacological properties of α-asarone

2,4,5-trimethoxycinnamic acid (TMC), the major and non toxic metabolite of α-asarone (2,4,5-trimethoxy-1-propenyl benzene), retains most of the pharmacological properties of α-asarone, since both substances, administered to hypercholesterolemic rats at 80 mg/kg body wt, decreased total serum cholesterol, lowered LDL-cholesterol levels and kept unaffected HDL-cholesterol levels. In addition, both substances increased bile flow, especially in hypercholesterolemic rats, by rising the secretion of bile salts, phospholipids and bile cholesterol. These drugs also reduced cholesterol levels of gallbladder bile, whereas phospholipids and bile salts concentrations were increased, decreasing the cholesterol saturation index (CSI). We also found that α-asarone was 20 times better inhibitor of HMG-CoA reductase than TMC. This effect on HMG-CoA reductase was the only property highly reduced in TMC in comparison with α-asarone, while the other pharmacological properties of α-asarone were retained by TMC. These experiments strongly suggest that TMC can be further studied as a possible hypocholesterolemic and cholelitholytic agent.

Effect of oxamic analogues on functional mice sperm parameters

Abstract The present study evaluates the effect of oxamate derivatives (N-ethyl, N-propyl, N-butyl oxamates) on functional murine sperm parameters, towards a new male non-hormonal contraceptive. These derivatives are selective inhibitors of lactate dehydrogenase-C4 (LDH-C4). LDH-C4 is a sperm-specific enzyme that plays an important role in ATP production for maintaining progressive motility as well as to induce capacitation and hyperactivation. The results demonstrate that all oxamate derivatives selectively inhibited LDH-C4 in mouse sperm extracts. The IC50 values for hexokinase and glyceraldehyde-3-phosphate dehydrogenase were at least an order of magnitude greater than LDH-C4 IC50 values. Prodrugs of oxamate derivatives assayed on sperm cells diminished normal sperm motility parameters, acrosome reaction, and cell viability in a concentration dependent manner. Also, we performed in vivo studies to determine the potential toxicity and possible contraceptive ability of these inhibitors. Mouse sperm were more sensitive to the N-butyl oxamate ethyl ester (NBOXet). Furthermore, results showed that NBOXet was of a low toxicity substance that diminished the total and progressive motility as well as the kinematic parameters of sperm cells. Data from in vitro and in vivo studies showed that N-butyl oxamate and its prodrug, are selective inhibitors of sperm LDH-C4, has low toxicity, and inhibits sperm progressive motility, offering some of the desirable characteristics of a male contraceptive: effect, low toxicity, and selectivity.

Trypanosoma cruzi: inhibition of alpha-hydroxyacid dehydrogenase isozyme II by N-allyl and N-propyl oxamates and their effects on intact epimastigotes

N-allyl (NAOx) and N-propyl (NPOx) oxamates were designed as inhibitors of alpha-hydroxyacid dehydrogenase (HADH) isozyme II from Trypanosoma cruzi. The kinetic studies showed that NAOx and NPOx were competitive inhibitors of HADH-isozyme II (Ki = 72 microM, IC50 = 0.33 mM and 70 microM, IC50 = 0.32 mM, respectively). The attachment of the allylic and propylic chains to nitrogen of the competitive inhibitor oxamate (Ki = 0.91 mM, IC50 = 4.25 mM), increased 12.6 and 13-folds respectively, the affinity for T. cruzi HADH-isozyme II. NAOx and NPOx were selective inhibitors of HADH-isozyme II, because other T. cruzi dehydrogenases were not inhibited by these substances. Since HADH-isozyme II participates in the energy metabolism of T. cruzi, a trypanocidal effect can be expected with these inhibitors. However, we were not able to detect any trypanocidal activity with these oxamates. When the corresponding ethyl esters of N-allyl (Et-NAOx) and N-propyl (Et-NPOx) oxamates were tested as a possible trypanocidal prodrugs, in comparison with nifurtimox and benznidazole, the expected trypanocidal effects were obtained.

Trypanocidal activity of the ethyl esters of N-propyl and N-isopropyl oxamates on intracellular amastigotes of Trypanosoma cruzi acute infected mice

In this investigation we studied the trypanocidal activity of the ethyl esters of N-propyl (Et-NPOX) and N-isopropyl (Et-NIPOX) oxamates on bloodstream trypomastigotes and on the clinically relevant intracellular amastigotes of Trypanosoma cruzi acute infected mice. In the infected and treated mice, the levels of parasitemia were drastically reduced between days 15 and 20 of treatment and almost to zero between days 35 and 40. We also found that Et-NPOX completely eliminated amastigote nests in the myocardium of mice infected with INC-5 or NINOA T. cruzi strain, and in skeletal muscle the reduction in the number of amastigote nests was between 60 and 80% in both strains. Also, Et-NIPOX reduced by 60–80% the number of amastigote nests in the myocardium and skeletal muscle of mice infected with these T. cruzi strains. In contrast, nifurtimox, used for comparison, produced a reduction of amastigote nests of only 20–40% in the studied tissues in both strains.