Anti-babesial activity of a series of 6,7-dimethoxyquinazoline-2,4-diamines (DMQDAs).

Diminazene aceturate (DA), imidocarb dipropionate (ID), atovaquone (ATO), azithromycin (AZI), clindamycin, and quinine have been used to treat animal and human babesiosis for many years, despite their negative effects and rising indications of resistance. Thus, finding anti-babesial compounds that can either treat the infection or lower the dose of drugs given has been a primary objective. Quinazolines are one of the most important nitrogen heterocycles, with a wide range of pharmacological activities including analgesic, anti-inflammatory, sedative-hypnotic, anti-histaminic, anti-cancer, and anti-protozoan properties. The present study investigated the anti-babesial activities of twenty 6,7-dimethoxyquinazoline-2,4-diamines on Babesia spp. One candidate, 6,7-dimethoxy-N4-ethylisopropyl-N2-ethyl(pyridin-4-yl)quinazoline-2,4-diamine (SHG02), showed potent inhibition on Babesia gibsoni in vitro, as well as on B. microti and B. rodhaini in mice. Our findings indicate that the candidate compound SHG02 is promising for further development of anti-babesial drugs and provides a new structure to be explored for developing anti-Babesia therapeutics.

Quest for a potent antimalarial drug lead: Synthesis and evaluation of 6,7-dimethoxyquinazoline-2,4-diamines.

Quinazolines have long been known to exert varied pharmacologic activities that make them suitable for use in treating hypertension, viral infections, tumors, and malaria. Since 2014, we have synthesized approximately 150 different 6,7-dimethoxyquinazoline-2,4-diamines and evaluated their antimalarial activity via structure-activity relationship studies. Here, we summarize the results and report the discovery of 6,7-dimethoxy-N4-(1-phenylethyl)-2-(pyrrolidin-1-yl)quinazolin-4-amine (20, SSJ-717), which exhibits high antimalarial activity as a promising antimalarial drug lead.

In vitro and in vivo characterization of anti-malarial acylphenoxazine derivatives prepared from basic blue 3.

BACKGROUND: Basic blue 3 is a promising anti-malarial lead compound based on the π-delocalized lipophilic cation hypothesis. Its derivatives with nitrogen atoms bonded to carbon atoms at the 3- and 7-positions on the phenoxazine ring were previously shown to exert potent antiprotozoal activity against Plasmodium falciparum, Trypanosoma cruzi, Trypanosoma brucei rhodesiense, and Leishmania donovani parasites in vitro. However, compounds with nitrogen modification at the 10-position on the phenoxazine ring were not evaluated. METHODS: Six acylphenoxazine derivatives (ITT-001 to 006) with nitrogen modification at the 10-position on the phenoxazine ring, which were synthesized from basic blue 3, were characterized and evaluated for anti-malarial activity in vitro with an automated haematology analyzer (XN-30) and light microscopy. Intensity of self-fluorescence was measured using a fluorometer. Localization of basic blue 3 was observed by fluorescence microscopy. Cytotoxicity was evaluated using human cell lines, HEK293T and HepG2 cells. Finally, anti-malarial activity was evaluated in a rodent malaria model. RESULTS: All the six derivatives showed anti-malarial efficacy even against chloroquine-, pyrimethamine-, and artemisinin-resistant field isolates similar to the sensitive strains and isolates in vitro. The efficacy of basic blue 3 was the strongest, followed by that of ITT-001 to 004 and 006, while that of ITT-005 was the weakest. Basic blue 3 showed strong self-fluorescence, whereas ITT derivatives had five- to tenfold lower intensity than that of basic blue 3, which was shown by fluorescence microscopy to be selectively accumulated in the plasmodial cytoplasm. In contrast, ITT-003, 004, and 006 exhibited the lowest cytotoxicity in HEK293T and HepG2 cells in vitro and the highest selectivity between anti-malarial activity and cytotoxicity. The in vivo anti-malarial assay indicated that oral administration of ITT-004 was the most effective against the rodent malaria parasite, Plasmodium berghei NK65 strain. CONCLUSIONS: The six ITT derivatives were effective against chloroquine- and pyrimethamine-resistant strains and artemisinin-resistant field isolates as well as the sensitive ones. Among them, ITT-004, which had high anti-malarial activity and low cytotoxicity in vitro and in vivo, is a promising anti-malarial lead compound.

Hyaluronan Production by Renomedullary Interstitial Cells: Influence of Endothelin, Angiotensin II and Vasopressin.

The content of hyaluronan (HA) in the interstitium of the renal medulla changes in relation to body hydration status. We investigated if hormones of central importance for body fluid homeostasis affect HA production by renomedullary interstitial cells in culture (RMICs). Simultaneous treatment with vasopressin and angiotensin II (Ang II) reduced HA by 69%. No change occurred in the mRNA expressions of hyaluronan synthase 2 (HAS2) or hyaluronidases (Hyals), while Hyal activity in the supernatant increased by 67% and CD44 expression reduced by 42%. The autocoid endothelin (ET-1) at low concentrations (10-10 and 10-8 M) increased HA 3-fold. On the contrary, at a high concentration (10-6 M) ET-1 reduced HA by 47%. The ET-A receptor antagonist BQ123 not only reversed the reducing effect of high ET-1 on HA, but elevated it to the same level as low concentration ET-1, suggesting separate regulating roles for ET-A and ET-B receptors. This was corroborated by the addition of ET-B receptor antagonist BQ788 to low concentration ET-1, which abolished the HA increase. HAS2 and Hyal2 mRNA did not alter, while Hyal1 mRNA was increased at all ET-1 concentrations tested. Hyal activity was elevated the most by high ET-1 concentration, and blockade of ET-A receptors by BQ123 prevented about 30% of this response. The present study demonstrates an important regulatory influence of hormones involved in body fluid balance on HA handling by RMICs, thereby supporting the concept of a dynamic involvement of interstitial HA in renal fluid handling.

Inhibition of mTOR activity in diabetes mellitus reduces proteinuria but not renal accumulation of hyaluronan.

OBJECTIVES: Accumulation of extracellular matrix (ECM) components is an early sign of diabetic nephropathy. Also the glycosaminoglycan hyaluronan (HA) is elevated in the renal interstitium during experimental diabetes. The mammalian target of rapamycin (mTOR) pathway participates in the signaling of hyperglycemia-induced ECM accumulation in the kidney, but this has not yet been investigated for HA. We hypothesized that interstitial HA accumulation during diabetes may involve mTOR activation. METHODS: Diabetic rats (6 weeks post-streptozotocin (STZ)) were treated with rapamycin to inhibit mTOR or vehicle for 2 additional weeks. Kidney function (glomerular filtration rate, renal blood flow, urine output) and regional renal HA content were thereafter analyzed. The ability of the animals to respond to desmopressin was also tested. RESULTS: Diabetic animals displayed hyperglycemia, proteinuria, hyperfiltration, renal hypertrophy, increased diuresis with reduced urine osmolality, and reduced weight gain. Cortical and outer medullary HA was elevated in diabetic rats. Urine hyaluronidase activity was almost doubled in diabetic rats compared with controls. The ability to respond to desmopressin was absent in diabetic rats. Renal blood flow and arterial blood pressure were unaffected by the diabetic state. In diabetic rats treated with rapamycin the proteinuria was reduced by 32%, while all other parameters were unaffected. CONCLUSION: Regional renal accumulation of the ECM component HA is not sensitive to mTOR inhibition by rapamycin, while proteinuria is reduced in established STZ-induced diabetes. Whether the diabetes-induced renal accumulation of HA occurs through different pathways than other ECM components, or is irreversible after being established, remains to be shown.

Supersaturated state of diazepam injection following dilution with infusion fluid.

BACKGROUND: Significant precipitation produced by the dilution of diazepam (DZP) injection with an infusion fluid is a great concern for the clinical practice. In this study, the precipitation behavior under different conditions was investigated. METHOD: For the sample preparation, DZP injections (Horizon injection and Cercine injection) were diluted with various infusion fluids (Saline, 5% glucose infusion fluid and Soldem 3A) at designated dilution ratios ranging from 1× to 40× (5 mg/mL to 0.125 mg/mL). In addition, to measure the solubility of DZP in the samples, the saturated solutions of DZP were prepared. The DZP concentrations in the samples were measured by high-performance liquid chromatography (HPLC). This study also investigated the precipitate using various analytical methods: infrared microscopy, (1)H-NMR, differential scanning calorimetry, and powder X-ray deflection. RESULTS: First, the compatibility of injection with infusion fluids was investigated. Significant precipitation occurred at dilution ratios ranging from 2× to 20×. No significant effects of formulations and infusion fluids on the compatibility were observed. The solubility of DZP was then further investigated. The concentration of DZP dissolved in the admixtures was higher than the solubility. This indicated that DZP existed in a supersaturated state in the infusion fluid admixtures. In the next phase of this study, the precipitate was investigated using various analytical methods. Results showed that the precipitate in infusion fluid admixtures was mostly composed of DZP, but also contained small amounts of the ingredients of DZP injection, such as benzoic acid and benzyl alcohol. CONCLUSIONS: This study clarified details of the precipitation occurring after dilution of DZP injection with infusion fluids. It is worth noting that DZP in an infusion admixture existed in a supersaturated state. These findings offer important insight into the clinical practice of DZP injection.

Contribution of glucose to crystallization of phenytoin in injectable dosage form by dilution with infusion fluids.

The crystallization of phenytoin occurring after its dilution with infusion fluid is a major concern in the clinical use of injectable phenytoin. To gain further understanding of the crystallization, this study assessed details of the involvement of glucose in this action. For sample preparation, phenytoin crystals were created by diluting the injectable phenytoin with infusion fluids with different glucose concentrations at different temperature, and then the characteristics of the crystallization (e.g., crystal size in the long direction, accumulated amount over 24 h, and crystallization rate constant) were measured. Results of the analysis of variance indicated that the glucose concentration and temperature had significant impacts on the crystallization. The mode of action of the glucose concentration was suggested to be different from that of the incubation temperature. This study also examined the molecular mobility of components (i.e., glucose, propylene glycol, phenytoin) in the admixtures using diffusion NMR techniques. The findings will provide valuable information for the clinical use of injectable phenytoin.

Proteomics of the rodent malaria parasite using matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight tandem mass spectrometry.

Plasmodium berghei strain NK65 is a rodent malaria parasite species widely used as a model of the human-infectious malaria parasite. Because a rodent malaria parasite model allows issues to be addressed which would not be possible with human-infectious species, e.g., mode of action and in vivo screening, a convenient method to analyze the malaria parasite proteome is required. The proteins of P. berghei separated using two-dimensional polyacrylamide gel electrophoresis were analyzed using matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight tandem mass spectrometry.

A 5% glucose infusion fluid provokes significant precipitation of phenytoin sodium injection via interruption of the cosolvent effect of propylene glycol.

The precipitation of phenytoin sodium injection provoked by mixing with infusion fluids renders its use in clinical practice difficult, as rapid intravenous (i.v.) push and i.v. infusion are supposed to be avoided. As some of its aspects remain unclear, this study tried to elucidate this precipitation mechanism. In particular, this study focused on the significant precipitation induced by glucose infusion fluid. The precipitation provoked by 5% glucose infusion fluid was obviously different from the precipitation that accompanied simple pH reduction, in terms of the growth mode and morphology of crystals. In addition, the effect of glucose was partially unrelated to pH reduction. NMR measurements including a two-dimensional nuclear Overhauser effect spectroscopy (2D-NOESY) spectrum indicated the specific interaction between glucose and propylene glycol, which is incorporated into phenytoin sodium injection as a solubilizing agent. These results led to the conclusion that this interaction was crucial for the precipitation of phenytoin, as it diminished the solubilizing effect of propylene glycol, resulting in the enhancement of the crystallization of phenytoin. The determination of phenytoin solubility in aqueous solutions at different pH values revealed that phenytoin incorporated in the admixture could be dissolved completely, as long as the injection was diluted with saline or water. These findings offer a profound insight into the formulation design of phenytoin sodium injection and its use in clinical practice.

Selective accumulation of a novel antimalarial rhodacyanine derivative, SSJ-127, in an organelle of Plasmodium berghei.

SSJ-127, a novel antimalarial rhodacyanine derivative, has shown potent antimalarial activity against chloroquine-resistant Plasmodium strains in vitro and subcutaneous administration of SSJ-127 results in a complete cure of a mouse malaria model. SSJ-127 was detected by fluorescence microscopy in the mouse malaria parasites Plasmodium berghei after exposure of infected red blood cells to the compound in vitro and in vivo. Selective accumulation of SSJ-127 in an organelle is observed in all blood stages of live malaria parasites. The organelle is clearly different from the mitochondrion and the nucleus in terms of morphology. The shape of the organelle changed during the asexual blood stages of the parasite. There was always a close association between the organelle and the mitochondrion. These results raised the possibility that SSJ-127 accumulates in an apicoplast of the malaria parasite and affects protozoan parasite-specific pathways.

Increased oxidized protein hydrolase activity in serum and urine of diabetic rat models.

Oxidative stress is implicated in the pathogenesis of diabetes mellitus. We investigated whether or not oxidized protein hydrolase (OPH), which preferentially degrades oxidized and glycated proteins, has a preventive or delaying role in diabetes mellitus. Using streptozotocin (STZ)-induced diabetic Wistar and Goto-Kakizaki (GK) rats as models of types 1 and 2 diabetes, respectively, we traced the changes in serum and urinary OPH activity with the pathological progress. Serum OPH activity increased nearly in parallel with increases in the blood glucose level in the two rat models, clarifying first in this study that serum OPH activity increases from a very early stage of diabetes. These findings suggest that increased serum OPH has at least a delaying action against the progression of diabetes through the removal of damaged proteins. Urinary OPH activity increased only in STZ-induced diabetic rats with microalbuminuria, but not in GK rats still without marked renal disorder, indicating the leakage of circulating OPH; the increase in urinary OPH activity may be a useful marker for diabetic nephropathy. OPH may provide a new therapeutic strategy against diabetes and its complications.

Enhanced activity of serum and urinary hyaluronidases in streptozotocin-induced diabetic Wistar and GK rats.

Using streptozotocin-induced diabetic Wistar and GK rats as models of type 1 and type 2 diabetes, respectively, we investigated the changes in serum and urinary hyaluronidase activity with the pathological progress. The serum hyaluronidase levels of streptozotocin-induced rats started to increase on the third day after injection and thereafter maintained approximately threefold higher levels compared with control rats; those of GK rats were already higher ( approximately twofold) from the beginning of the experiment. The increases of serum hyaluronidase activity in both diabetic rats were similar to those of blood glucose level, indicating that diabetes mellitus was accompanied by enhanced activity of circulating hyaluronidase from the early phase of its development. In zymography, every serum from diabetic and control rats gave two hyaluronidase isomers, a major 73-kDa band (Hyal-1 type) and a minor 132-kDa band, suggesting that the increases in serum hyaluronidase activity were not due to the appearance of novel isomers. The hyaluronidase activity in 24-h urine of streptozotocin-induced rats was 3-, 7-, and 11-fold higher at the 8th, 15th, and 18th week than that of control rats, respectively, and the urinary hyaluronidase activity of GK rats was not significantly different from controls. There was a good correlation between the urinary hyaluronidase activity and the albumin excretion. Thus the increase in urinary hyaluronidase activity may reflect enhanced glomerular permeability in streptozotocin-induced diabetic rats and may be a useful marker for diabetic nephropathy. Relative resistance to SDS-denaturation in zymography of rat serum and urinary hyaluronidases compared with human serum hyaluronidase are also shown.

A fluorimetric Morgan-Elson assay method for hyaluronidase activity.

Despite their physiological importance, hyaluronidases (HAases) have long been "neglected enzymes," due, presumably, in part to the lack of rapid, sensitive assays. Currently, the colorimetric Morgan-Elson assay method, which is based upon the generation of a new reducing N-acetyl-D-glucosamine terminus with each cleavage reaction, is most widely employed but is yet insensitive. We, therefore, reinvestigated the colorimetric method and established the fluorimetric Morgan-Elson assay for HAase activity, with the optimized tetraborate reagent. The fluorimetric assay, requiring neither specialized reagents nor a long time to perform, provided high sensitivity, nearly comparable to that of enzyme-linked immunosorbent assay (ELISA)-like assays, with a detection limit of 5 x 10(-3)NFU/ml of bovine testicular HAase after 1-h incubation. The increased sensitivity permitted rapid measurement of low HAase activity in biological samples such as human and rabbit serum HAases, the latter of which has not been detected either by an ELISA-like assay or by zymography. Human serum HAase was easily characterized it along with its optimum pH and kinetic parameters.

Increased hyaluronidase activity in the kidney of streptozotocin-induced diabetic rats.

We investigated changes in renal hyaluronidase activity in streptozotocin (STZ)-induced diabetic rats during the progression of diabetes. Prior to the study, we characterized rat renal hyaluronidase activity to find that its optimum pH is 3.5 and that it consists of two isomers of 73 and 63 kDa, as detected by zymography. Hyaluronidase activity was traced in one whole kidney and in the cortex and medulla of the other kidney up to the 18th week after STZ injection. Whole kidney hyaluronidase activity started to increase on day 3 and reached a maximum level 2.4 times that of the controls in the 3rd week. Cortical hyaluronidase showed a similar tendency to that of whole kidney hyaluronidase, while medullary hyaluronidase activity continued to increase until the 8th week, suggesting their different involvements in the progression of diabetic nephropathy. In zymography, the intensities of the two isomer bands increased with the progression of diabetes, but the intensity ratio did not change significantly and no new isomer band appeared. Renal HAase activity increased only in STZ-induced diabetic rats, but not in spontaneously diabetic Goto-Kakizaki rats still without remarkable renal disorder. Based on these findings, increased renal HAase activity may serve as a useful marker for diabetic nephropathy.

Microanalysis of hyaluronan oligosaccharides by polyacrylamide gel electrophoresis and its application to assay of hyaluronidase activity.

We established a rapid, sensitive polyacrylamide gel electrophoresis (PAGE) method for the analysis of hyaluronan (HA) oligosaccharides. Using mini-slab gels, but not large-slab gels so far reported, HA oligosaccharides of 5 to more than 50 repeating disaccharide units could be separated into discrete ladder-like bands in a short electrophoresis time of 45 min. Using a combined Alcian blue and silver staining protocol, the detection limit was less than 1 ng per band for 11 repeating disaccharide units, indicating 50 times higher sensitivity than that of an earlier-described sensitivity-enhanced PAGE method. Our PAGE method was applicable to the assay of hyaluronidase activity. When a total of multiple band intensities for 18-24 repeating disaccharide units was used as a measure of activity, as little as 3 x 10(-4) NFU of bovine testicular hyaluronidase was detectable on a 1-h incubation. This sensitivity permitted rapid measurements of human and rabbit serum hyaluronidases, the latter of which having never been detected even by a sensitive enzyme-linked immunosorbent assay (ELISA). Since this PAGE assay does not require specialized reagents and instruments and since it provides information on both the activity and the enzymatic HA degradation pattern, there may be many potential applications.