Factors Influencing Discontinuation of Clazosentan Therapy in Elderly Patients with Aneurysmal Subarachnoid Hemorrhage: A Retrospective Study from a Japanese Single Center.

BACKGROUND Clazosentan is an endothelin receptor antagonist approved in Japan for preventing cerebral vasospasm and vasospasm-associated cerebral ischemia and infarction. This study included elderly patients aged ≥75 years with aneurysmal subarachnoid hemorrhage (SAH) and aimed to evaluate the factors associated with discontinuing anti-vasospasm therapy with clazosentan. MATERIAL AND METHODS In this single-center retrospective observational study, we extracted diagnostic and therapeutic work-up data of consecutive 40 patients with SAH treated with clazosentan infusion (10 mg/h) as first-line anti-vasospasm therapy between May 2022 and August 2023. Patient data were compared between the discontinued and completed groups, and related factors for the discontinuation were further analyzed. RESULTS Clazosentan was discontinued in 22% (n=9) of patients due to intolerable dyspnea accompanied by hypoxemia at 5±3 days after therapy initiation, in which 44% (n=4) were elderly (≥75 years). Patients who discontinued clazosentan therapy showed significantly lower urine volumes compared with those who completed the therapy (P<0.05). Multivariate regression analysis revealed that day-to-day urine volume variance and older age were independent risk factors for drug cessation (P<0.05). The cut-off value for predicting clazosentan discontinuation was -0.7 mL/kg/h with sensitivity of 86% and specificity of 75% (area under the curve: 0.76±0.10; 95% confidence interval: 0.56-0.96; P=0.035). CONCLUSIONS Our results suggest that approximately 20% of SAH patients suffered from intolerable respiratory symptoms attributable to hypoxemia. We found that both reduced day-to-day urine volume variation and older age are independent risk factors for drug discontinuation.

Anti-tumor and cardiotoxic effects of microtubule polymerization inhibitors: The mechanisms and management strategies.

Microtubule polymerization inhibitors (MPIs) have long been used as anticancer agents because they inhibit mitosis. Microtubules are thought to play an important role in the migration of tumor cells and the formation of tumor blood vessels, and new MPIs are being developed. Many clinical trials of novel MPIs have been conducted in humans, while some clinical studies in dogs have also been reported. More attempts to apply MPIs not only in humans but also in the veterinary field are expected to be made in the future. Meanwhile, MPIs have a risk of cardiotoxicity. In this paper, we review findings on the pharmacological effects and cardiotoxicity of MPIs, as well as the mechanisms of their cardiotoxicity. Cardiotoxicity of MPIs involves not only the direct effects of MPIs on cardiomyocytes but also their effects on vascular function. For example, hypertension induced by impaired vascular function also contributes to the exacerbation of myocardial damage, and blood pressure control may be useful in reducing cardiotoxicity. By combined administration of MPIs and other anticancer agents, MPI efficacy may be enhanced, thereby potentially allowing to keep MPI dosage low. Measurement of myocardial injury markers in blood and echocardiography may be useful for monitoring cardiotoxicity. In particular, two-dimensional speckle tracking may have high sensitivity for the early detection of MPI-induced cardiac dysfunction. The exploration of the potential of new MPIs while understanding their toxicity and how to deal with them will lead to the further development of cancer chemotherapy.

Cardiopulmonary Events of the Elderly (≥75 Years) during Clazosentan Therapy after Subarachnoid Hemorrhage: A Retrospective Study from a Tertiary Stroke Center in Japan.

Clazosentan has been shown to prevent vasospasm and reduce mortality in patients after aneurysmal subarachnoid hemorrhage (SAH) and has been approved for clinical use in Japan; however, its systemic events in the elderly (aged ≥ 75 years) have not been well-documented. Here, we report serious/intolerable cardiopulmonary complications requiring discontinuation of drug therapy in elderly SAH patients. In this single-center case series study, medical records of consecutive SAH patients treated postoperatively with clazosentan (10 mg/h) between June 2022 and May 2023 were reviewed retrospectively. Thirty-three patients received clazosentan therapy, of whom six were elderly with a mean age of 80.3 ± 5.2 (range 75-89) years. Among them, despite no obvious medical history of systemic abnormalities, clazosentan was discontinued in three (50%) patients due to pleural effusion and hypoxemia with or without hypotension at 5 ± 3 days after therapy initiation, which was higher than the incidence for younger patients (15%). The elderly patients had significantly lower urine output (1935 ± 265 vs. 1123 ± 371 mL/day, p = 0.03) and greater weight gain (2.1 ± 1.1 vs. 4.2 ± 1.9 kg from baseline, p = 0.04) than patients who completed the therapy. One 89-year-old female developed congestive heart failure and hydrostatic pulmonary edema associated with increased intravascular and lung volumes even after therapy was discontinued, while the remaining two cases recovered within 2 days after drug cessation. These results suggest that elderly patients are more vulnerable to fluid retention and have a higher risk of cardiopulmonary complications during clazosentan therapy than younger patients. Careful monitoring of urine volume and weight gain and caution regarding age- and therapy-related hemodynamic insufficiencies are required.

Plasticity occurs in a specific phenotype of neurons in the nucleus tractus solitarius of dystrophin gene-mutated rats.

Duchenne muscular dystrophy (DMD) is a severe progressive neuromuscular disorder that causes cardiac and respiratory failure. Patients with DMD have tachycardia and autonomic nervous dysfunction at a young age, which can potentially worsen cardiorespiratory function. Therefore, we hypothesised that plasticity occurs in neurons of the cardiorespiratory brainstem nucleus (nucleus tractus solitarius [NTS]) due to DMD, thus affecting neuronal regulation because afferent information from cardiorespiratory organs changes with disease progression. Patch-clamp experiments were performed on second-order NTS neurons from Dmd-mutated (Dm) rats that showed no functional dystrophin protein expression, as confirmed by immunohistochemistry. NTS neurons are classified into two electrophysiological phenotypes: one showing a delayed onset of spiking from hyperpolarised membrane potentials, namely, delayed-onset spiking (DS)-type neurons, and the other showing a rapid onset, namely, rapid-onset spiking-type neurons. Neuroplasticity mainly occurs in DS-type neurons in Dm rats and is characterised by blunted neuronal excitability accompanied by reduced outward currents and a facilitatory effect on synaptic transmission, that is, an increased frequency of spontaneous and miniature excitatory postsynaptic currents (EPSCs) without changes in the amplitude and an increased amplitude of tractus solitarius-evoked EPSCs without changes in the paired-pulse ratio. Although we cannot rule out the possibility that the neuroplastic changes observed in Dm rats were caused by dystrophin deficiency in the neurons themselves, the plasticity could be caused by cardiorespiratory deterioration and/or adaptation in DMD patients.

Pretreatment with tadalafil attenuates cardiotoxicity induced by combretastatin A4 disodium phosphate in rats.

Combretastatin A4 disodium phosphate (CA4DP) is a prodrug of combretastatin A4 (CA4), a microtubule-disassembling agent that exhibits antitumor effects by inhibiting tumor cell proliferation and inducing morphological changes and apoptosis in vascular endothelial cells in tumors. However, cardiotoxicity induced by ischemia and hypertension is a severe adverse event. In this study, we focused on the fact that phosphodiesterase (PDE) 5 inhibitors dilate the heart and peripheral blood vessels and aimed to investigate whether co-administration of tadalafil, a PDE5 inhibitor, can attenuate cardiotoxicity without altering the antitumor effect of CA4DP. To investigate cardiotoxicity, CA4DP and/or tadalafil were administered to rats, and blood pressure, echocardiography, histopathology, and cGMP concentration in the myocardium were examined. Administration of CA4DP increased systolic blood pressure, decreased cardiac function, lowered cGMP levels in the myocardium, and led to necrosis of myocardial cells. Co-administration of tadalafil attenuated these CA4DP-induced changes. To investigate the antitumor effect, canine mammary carcinoma cell lines (CHMp-13a) and human umbilical vein endothelial cells were cultured with CA4 and/or tadalafil, and cell proliferation and endothelial vascular tube disruption were examined. CHMp-13a cells were transplanted into nude mice and treated with CA4DP and/or tadalafil. CA4-induced inhibition of cell proliferation and disruption of the endothelial vascular tube were not affected by co-treatment with tadalafil, and the antitumor effects of CA4DP in xenograft mice were not reduced by co-administration of tadalafil. These results revealed that myocardial damage induced by CA4DP was attenuated by co-administration of tadalafil while maintaining antitumor efficacy.

Preliminary study of heart rate variability in Criollo horses for the elucidation of their neurophysiological characteristics of autonomic nerve function.

The Criollo is an Argentine horse breed with a calm temperament. Although its temperament is considered to be related to its neurophysiological characteristics, the details of this are unknown. Therefore, we analyzed the heart rate variability in Criollos as a preliminary study to deepen the neurophysiological understanding of their autonomic function. Electrocardiograms were recorded from Criollos and Thoroughbreds, and the power spectrum of heart rate variability was analyzed. Compared with Thoroughbreds, Criollos showed (i) a significantly higher high-frequency component, which is an index of parasympathetic nerve activity, and (ii) tendency toward a lower ratio of low- to high-frequency power, which is an index of the autonomic balance. These results revealed that parasympathetic nerves might be more active in Criollos compared with Thoroughbreds.

Cardiac lesions in Duchenne muscular dystrophy model rats with out-of-frame Dmd gene mutation mediated by CRISPR/Cas9 system.

Duchenne muscular dystrophy (DMD) is a progressive muscular disorder caused by X-chromosomal DMD gene mutations. Recently, a new CRISPR/Cas9-mediated DMD rat model (cDMDR) was established and is expected to show cardiac lesions similar to those in humans. We therefore investigated the pathological and pathophysiological features of the cardiac lesions and their progression in cDMDR. For our cDMDR, Dmd-mutated rats (W-Dmd em1Kykn ) were obtained. Dmd heterozygous-deficient females and wild-type (WT) males were mated, and male offspring including WT as controls were used. (1) Hearts were collected at 3, 5, and 10 months of age, and HE- and Masson's trichrome-stained specimens were observed. (2) Electrocardiogram (ECG) recordings were made and analyzed at 3, 5, and 8 months of age. (3) Echocardiography was performed at 9 months of age. In cDMDR rats, (1) degeneration/necrosis of cardiomyocytes and myocardial fibrosis prominent in the right ventricular wall and the outer layer of the left ventricular wall were observed. Fibrosis became more prominent with aging. (2) Lower P wave amplitudes and greater R wave amplitudes were detected. PR intervals tended to be shorter. QT intervals were longer at 3 months but tended to be shorter at 8 months. Sinus irregularity and premature ventricular contraction were observed at 8 months. (3) Echocardiography indicated myocardial sclerosis and a tendency of systolic dysfunction. Pathological and pathophysiological changes occurred in cDMDR rat hearts and progressed with aging, which is, to some extent, similar to what occurs in humans. Thus, cDMDR could be a valuable model for studying cardiology of human DMD.

Histopathological and functional changes in a single-dose model of combretastatin A4 disodium phosphate-induced myocardial damage in rats.

Cardiotoxicity is a concern in the development of microtubule-disassembling agents (MDAs) as vascular-disrupting agents of tumors. This study investigated cardiotoxicity in rats induced by a single-dose of combretastatin A4 disodium phosphate (CA4DP), an MDA and discussed the use of this rat model in nonclinical studies of MDAs. First, CA4DP (120 mg/kg) was administered to rats intravenously, and cardiac histopathology and blood biomarkers were examined after 0.5, 24, and 72 h. Next, CA4DP (120 mg/kg) was administered to rats intravenously, and the electrocardiography and echocardiography results were analyzed. The results showed that at 0.5 h after dosing, plasma creatine kinase (CK), CK-muscle/brain (CK-MB), and fatty acid binding protein 3 levels increased. At 24 h, lactate dehydrogenase (LDH)-1, CK, and CK-MB levels increased, and multifocal vacuolar degeneration of myocardial cells was observed in the apical inner layer. At 72 h, LDH-1 levels were increased, and multifocal myocardial necrosis was observed in the interventricular septum and inner layer of the apex of left ventricular wall. Furthermore, at 0.5 h, heart rate (HR), ejection fraction (EF), and cardiac output (CO) decreased. At 24 h, CO decreased. Finally, at 72 h, HR, EF, and CO decreased, and depression of the T-wave amplitude was observed. In conclusion, myocardial injury, bradycardia, and depressed cardiac function were induced in rats by a single-dose of CA4DP. The lesion distribution and electrocardiographic features suggested that myocardial injury was induced by ischemia. These findings are similar to MDA-induced cardiotoxicity in humans, and this rat model will prove useful in studies of the cardiotoxicity in humans.

Combretastatin A4 disodium phosphate-induced myocardial injury.

Histopathological and electrocardiographic features of myocardial lesions induced by combretastatin A4 disodium phosphate (CA4DP) were evaluated, and the relation between myocardial lesions and vascular changes and the direct toxic effect of CA4DP on cardiomyocytes were discussed. We induced myocardial lesions by administration of CA4DP to rats and evaluated myocardial damage by histopathologic examination and electrocardiography. We evaluated blood pressure (BP) of CA4DP-treated rats and effects of CA4DP on cellular impedance-based contractility of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs). The results revealed multifocal myocardial necrosis with a predilection for the interventricular septum and subendocardial regions of the apex of the left ventricular wall, injury of capillaries, morphological change of the ST junction, and QT interval prolongation. The histopathological profile of myocardial lesions suggested that CA4DP induced a lack of myocardial blood flow. CA4DP increased the diastolic BP and showed direct effects on hiPS-CMs. These results suggest that CA4DP induces dysfunction of small arteries and capillaries and has direct toxicity in cardiomyocytes. Therefore, it is thought that CA4DP induced capillary and myocardial injury due to collapse of the microcirculation in the myocardium. Moreover, the direct toxic effect of CA4DP on cardiomyocytes induced myocardial lesions in a coordinated manner.

Cardiotoxic changes of colchicine intoxication in rats: electrocardiographic, histopathological and blood chemical analysis.

The microtubule inhibitor colchicine is cardiotoxic and is suggested to impair impulse formation and conduction. However, little is known about the electrocardiographic (ECG) changes induced by colchicine in experimental animals and the detailed pathogenesis of its cardiotoxicity. Therefore, we analyzed cardiotoxicity in colchicine-treated rats using electrocardiographic, histopathological and blood-chemistry approaches. A telemetry device for transmitting ECG data was implanted into male Crl:CD(SD) rats, and ECG tracings were obtained. At 6 weeks of age, 1.25 mg/kg colchicine was injected intravenously once daily for 2 consecutive days, and ECG waveforms and heart rate variability were analyzed. Furthermore, 1.25 mg/kg colchicine or vehicle was injected for 1 or 2 consecutive days in other rats at 6 weeks of age. One day after the final dosing, heart and blood samples were taken for histopathological and bloodchemical examination. ECG analysis revealed a prolonged RR interval, QRS duration, PR interval and QT interval. Heart rate variability analysis showed an increase in high frequency (HF) components as an index of parasympathetic nervous activity. In blood chemical examinations, colchicine induced high levels of parameters of cardiac injury and low levels and/or variations in Ca, inorganic phosphorus, potassium and chloride. Histopathologically, colchicine-treated rats showed eosinophilic granular degeneration and cytoplasmic vacuolation of ventricular myocardial cells but no remarkable change in the atrioventricular node. Not only blood chemical and histopathological changes but also ECG changes were induced in colchicine-treated rats, which indicated a decrease in myocardium excitability and conductivity, and these changes might be related to increased parasympathetic nervous activity and low blood Ca levels.

Simple procedure for assessing diffuse subarachnoid hemorrhage successfully created using filament perforation method in mice.

The murine model of subarachnoid hemorrhage (SAH) is a valuable experimental tool for investigating molecular and cellular mechanisms, and the endovascular filament perforation technique can be used to simulate prominent pathophysiological features observed after human SAH; however, current validation methods for assessing an appropriate SAH model are limited. Here, we introduce a simple procedure for selecting a mouse model of diffuse SAH. SAH was induced in 24 mice using a standard filament perforation method. After confirming survival at 24 h, SAH was scored 0-1 based on T2*-weighted images on whole-brain magnetic resonance imaging (MRI) and visual surveillance of the cisterna magna (CM) through the dura mater. The CM-based SAH grading correlated well with a reference parameter defined by extracted brain (r2 = 0.53, p < 0.0001). The receiver operating characteristic curve revealed a sensitivity of 85% and a specificity of 91% for detecting diffuse SAH, with a similar area under the curve (0.89 ± 0.06 [standard error of the mean]) as the MRI-based grading (0.72 ± 0.10, p = 0.12). Our data suggest that confirming an SAH clot in the CM is a valuable way to select a clinically relevant diffuse SAH model that can be used in future experimental studies.

Protective role of protease-activated receptor-2 in anaphylaxis model mice.

Anaphylaxis is a severe life-threatening hypersensitivity reaction induced by mast cell degranulation. Among the various mediators of mast cells, little is known about the role of tryptase. Therefore, we aimed to elucidate the role of protease-activating receptor-2 (PAR-2), a receptor activated by tryptase, in murine anaphylactic models using PAR-2-deficient mice and newly generated tryptase-deficient mice. Anaphylaxis was induced by IgE-dependent and IgE-independent mast cell degranulation in mice. PAR-2 deficiency exacerbated the decrease in body temperature and hypotension during anaphylaxis; however, the number of skin mast cells, degree of mast cell degranulation, and systemic and local vascular hyperpermeability were comparable in PAR-2 knockout and wild-type mice. Nitric oxide, which is produced by endothelial nitric oxide synthase (eNOS), is an indispensable vasodilator in anaphylaxis. In the lungs of anaphylactic mice, PAR-2 deficiency promoted eNOS expression and phosphorylation, suggesting a protective effect of PAR-2 against anaphylaxis by downregulating eNOS activation and expression. Based on the hypothesis that the ligand for PAR-2 in anaphylaxis is mast cell tryptase, tryptase-deficient mice were generated using CRISPR-Cas9. In wild-type mice, the PAR-2 antagonist exacerbated the body temperature drop due to anaphylaxis; however, the effect of the PAR-2 antagonist was abolished in tryptase-deficient mice. These results suggest that tryptase is a possible ligand of PAR-2 in anaphylaxis and that the tryptase/PAR-2 pathway attenuates the anaphylactic response in mice.

Diffusion MRI Fiber Tractography and Benzodiazepine SPECT Imaging for Assessing Neural Damage to the Language Centers in an Elderly Patient after Successful Reperfusion Therapy.

BACKGROUND: Intravenous thrombolysis and mechanical thrombectomy are the first-line reperfusion therapies for acute ischemic stroke. Here, we describe the utility of diffusion magnetic resonance imaging (MRI) fiber tractography and 123I-iomazenil benzodiazepine receptor single-photon emission computed tomography to estimate the prognosis of post-stroke aphasia after successful reperfusion therapy. CASE REPORT: An 81-year-old man was admitted to the hospital approximately 3.5 h after the onset of symptoms, including decreased consciousness, right hemiparesis, and aphasia. An MRI revealed acute cerebral infarction due to M1 segment occlusion. Intravenous alteplase thrombolysis followed by endovascular thrombectomy resulted in recanalization of the left middle cerebral artery territory. A subsequent MRI showed no new ischemic or hemorrhagic lesions. Although the patient's motor hemiparesis gradually recovered, motor aphasia persisted. Diffusion MRI fiber tractography performed 2 weeks after admission revealed partial injury to the left arcuate fasciculus, indicated by lower fractional anisotropy values than on the contralateral side. A decreased benzodiazepine receptor density was also detected in the left perisylvian and temporoparietal cortices. The patient showed no clear signs of further improvement in the chronic stage post-stroke and was discharged to a nursing home after 3 months. CONCLUSIONS: The application of functional neuroimaging techniques to assess neuronal damage to the primary brain regions 2 weeks after reperfusion therapy for large-vessel occlusion may allow for an accurate prognosis of post-stroke aphasia. This may have a direct clinical implication for navigating subacute-to-chronic phases of rehabilitative care.

Persistence and accumulation of micronucleated hepatocytes in liver of rats after repeated administration of diethylnitrosamine.

A repeat-dose micronucleus assay in adult rat liver was recently developed [Mutat. Res. 747 (2012) 234-239]. This assay demonstrated a high detectability of hepatocarcinogens at relatively low doses, as indicated by dose-dependent micronucleus induction. Because the adult rat liver is known to have a long life-span, this desirable property of the assay will be an advantage in detecting micronucleated hepatocytes (MNHEPs) that have persisted for long periods in the liver following repeated dosing. However, no data directly supporting the underlying mechanisms have been published to date. In the present study, we verified the mechanisms by means of pulse-labeling of micronucleated hepatocytes with the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU). The rodent hepatocarcinogen diethylnitrosamine (DEN) was repeatedly administered orally to male Crl:CD (SD) rats (6 weeks old) for up to 2 weeks, and EdU was injected intraperitoneally on days 1, 7, or 14. Hepatocytes were isolated by use of a non-perfusion technique at 24h, 1 week, or 2 weeks after EdU injection and analyzed for EdU incorporation and micronucleus formation. The results of our study confirmed that MNHEPs labeled with EdU on the first day of DEN administration persisted until 2 weeks post-administration in the rat livers. However, the frequency of MHNEPs among EdU-labeled hepatocytes decreased over time. In addition, the number of terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL)-positive cells in the liver tissue increased, suggesting selective removal of micronucleated cells. Theoretical calculation of the cumulative MNHEP frequency on each of the days on which DEN was administered, taking into account the rate of loss, came out closer to the actual value observed in the liver micronucleus test. Taken together, these results indicate that although micronucleated cells induced in rat livers by administration of the genotoxic hepatocarcinogen DEN undergo selective removal, they persist for a long time in a certain proportion, and repeated administration results in their accumulation and increased frequency.

Polyoxazoline-Conjugated l-Asparaginase: An Antibody-Production-Free Therapeutic Agent for Acute Lymphoblastic Leukemia.

l-asparaginase (ASNase), an enzyme that catalyzes the hydrolysis of l-asparagine into l-aspartic acid, is frequently used as a medication for acute lymphoblastic leukemia (ALL). However, when derived from bacterial sources, this enzyme can elicit side effects, including allergic or hypersensitivity reactions, owing to immune responses. Here, we describe the synthesis of polyoxazoline-conjugated ASNase (POx-ASNase) and investigate its enzyme activity, anticancer efficacy, immunogenicity, and retention in the bloodstream. The water-soluble POx was coupled with surface lysine residues of ASNase using a bifunctional cross-linker. The average number of polymers bound to each enzyme was determined as 10. Although the enzymatic activity of POx-ASNase decreased to 56% of that of native ASNase, its temperature and pH dependencies remained unaltered. Remarkably, the lyophilized powder form of POx-ASNase retained its catalytic ability for 24 months. POx-ASNase demonstrated nearly identical anticancer efficacy compared to naked ASNase against leukemia and lymphoma cells (MOLT-4, CLBL-1, and K562) while displaying no cytotoxicity toward normal cells. Animal experiments conducted using rats revealed that the POx decoration suppressed the generation of anti-ASNase IgM and IgG antibodies with no detection of anti-POx antibodies. The half-life within the bloodstream extended to 34 h, representing a 17-fold increase compared to unmodified ASNase. These findings suggest that POx-ASNase serves as an anticancer therapeutic agent, characterized by the absence of antibody production and notably extended circulation persistence.

Mastication stimuli regulate the heartbeat rate through rhythmic regulation by the hypothalamic-autonomic system; molecular and telemetric studies in weaning-stage rats.

Mastication stimuli have been demonstrated to affect memory function and autonomic nerve activity; however, this process has not been well studied during weaning compared to old age. Previously, we conducted molecular analyses of the thalamus and hippocampus to elucidate the mechanisms underlying this memory-enhancing effect in weaning-stage rats. In this study, we aimed to evaluate the effect of masticatory stimuli on the regulation of heartbeat rate (HR) through the hypothalamic-autonomic system. Three-week-old male rats were administered a powdered diet (P group) or chow-diet (C group) for 10 days. Thereafter, transcriptome analysis was performed. Vasopressin, cocaine-amphetamine-regulated transcript prepropeptide, corticotropin-releasing hormone, and thyrotropin-releasing hormone, which are involved in sympathetic activation of heart rate, were downregulated in the C group. Electrocardiograms were recorded continuously for 12 days under the same condition. Interestingly, rats in the C group had a significantly lower HR than those in the P group on day 11. We checked several parameters representing the autonomic regulation of HR. The C group had higher values for the high-frequency band integration of the HR power spectrum (parasympathetic marker) and root mean square successive difference of R-wave intervals (parasympathetic marker) relative to the P group. Such findings provide a molecular and physiological basis for understanding the regulation of cardiovascular function in response to masticatory stimuli in the autonomic nervous system.

Poly(2-ethyl-2-oxazoline)-Conjugated Hemoglobins as a Red Blood Cell Substitute.

Hemoglobin wrapped covalently with poly(2-ethyl-2-oxazoline)s (POx-Hb) is characterized physicochemically and physiologically as an artificial O2 carrier for use as a red blood cell (RBC) substitute. The POx-Hb is generated by linkage of porcine Hb surface-lysines to a sulfhydryl terminus of the POx derivative, with the average binding number of the polymers ascertained as 6. The POx-Hb shows moderately higher colloid osmotic activity and O2 affinity than the naked Hb. Human adult HbA conjugated with POx also possesses equivalent features and O2 binding properties. The POx-Hb solution exhibits good hemocompatibility, with no influence on the functions of platelets, granulocytes, and monocytes. Its circulation half-life in rats is 14 times longer than that of naked Hb. Hemorrhagic shock in rats is relieved sufficiently by infusion of the POx-Hb solution, as revealed by improvements of circulatory parameters. Serum biochemistry tests and histopathological observations indicate no acute toxicity or abnormality in the related organs. All results indicate that POx-Hb represents an attractive alternative for RBCs and a useful O2 therapeutic reagent in transfusion medicine.

Ivabradine ameliorates cardiomyopathy progression in a Duchenne muscular dystrophy model rat.

Duchenne muscular dystrophy (DMD) is an X-linked recessive myopathy caused by dystrophin mutations. Inevitable progressive cardiomyopathy is a current leading cause of premature death although respiratory management has improved the prognosis of patients with DMD. Recent evidence shows that reducing the heart rate is expected as one of the promising strategies for heart failure treatment, but administering a sufficient dose of ß-blocker for patients with DMD with tachycardia is difficult because of their low blood pressure (BP). Thus, this study aimed to clarify the role of ivabradine, which suppresses cardiac sinus node pacemakers without decreasing BP, in ameliorating cardiomyopathy progression in a rat model with DMD. A trans-oral single ivabradine administration demonstrated a declined dose-dependent heart rate without any significant BP reduction. Trans-gastric repeated administrations of 5 mg/kg of ivabradine twice a day for 3 months showed ameliorated cardiomyopathy in DMD rats based on echocardiography and histopathological observations (left ventricular dysfunction, right ventricular dysfunction, and myocardial fibrosis) as compared with vehicle administration. Our finding indicates that ivabradine is expected as another treatment choice for patients with DMD having tachycardia.

Co-administration of JQ1, a bromodomain-containing protein 4 inhibitor, enhances the antitumor effect of combretastatin A4, a microtubule inhibitor, while attenuating its cardiotoxicity.

Combretastatin A4 (CA4) inhibits microtubule polymerization, and clinical trials of the prodrug, CA4 disodium phosphate (CA4DP), as an anti-cancer agent have been conducted. However, CA4DP has not been marketed to date because the margin between the effective dose and the cardiotoxic dose is insufficient. Meanwhile, bromodomain-containing protein 4 (BRD4) has been reported to be required for recovery from mitotic arrests induced by anti-microtubule drugs. BRD4 has also been reported to be involved in the progression of heart failure. Therefore, we hypothesized that the combined use of CA4DP with BRD4 inhibitors can enhance the antitumor effect and attenuate CA4DP-induced cardiotoxicity. In this study, the antitumor effect and cardiotoxicity caused by the co-administration of CA4DP with JQ1, a BRD4 inhibitor, were evaluated. CA4 or JQ1 alone reduced the viability of cultured canine mammary tumor cells (CHMp-13a). Viability was further reduced by co-administration, through the suppression of c-Myc. BRD4 positivity in CHMp-13a cytoplasm showed a significant increase when treated with CA4 alone, while the increase was not significant following co-administration. In CHMp-13a xenograft-transplanted mice, co-administration of CA4DP and JQ1 suppressed tumor growth significantly. In CA4DP-induced cardiac injury model rats, echocardiography showed a CA4DP-induced decrease in cardiac function and histopathology showed cardiomyocyte necrosis. Meanwhile, these cardiac changes tended to be milder following the co-administration of CA4DP and JQ1. These results suggest that CA4DP-JQ1 co-administration enhances the antitumor effect of CA4DP while attenuating its cardiotoxicity and therefore potentially open the doors to the development of a novel cancer chemotherapy with reduced cardiotoxicity risks.

Polyoxazoline-conjugated porcine serum albumin as an artificial plasma expander for dogs.

Veterinary medicine has made tremendous progress for domestic dogs, which are irreplaceable family members enriching human life. Nevertheless, no adequate supply system exists for their blood products. This study examined the synthesis, structure, safety, and efficacy of poly(2-ethyl-2-oxazoline)-conjugated porcine serum albumin (POx-PSA) as an artificial plasma expander for dogs. The aqueous POx-PSA solution showed moderately high colloid osmotic pressure and good blood cell compatibility. Actually, lyophilized powder stored for 1 year can regenerate into a homogeneous solution. The circulation half-life of POx-PSA in rats was 2.1-fold longer than that of naked PSA. Rats produced neither anti-PSA IgG antibody nor anti-POx IgG antibody, which suggests excellent immunological stealth properties of POx-PSA. Complete resuscitation of hemorrhagic shock in rats was achieved soon after injection of POx-PSA solution. Serum biochemistry tests and histopathological observations indicated no abnormality in the related organs. When POx-PSA was administered to dogs intravenously, (i) no serum biochemical or hematological alteration was observed, also (ii) no overt deterioration of animal health was observed. These results indicate that POx-PSA has potential as an artificial plasma expander for dogs.