Real-World Clinical Profile of Patients Prescribed Evolocumab in Japan.

BACKGROUND: Real-world utilization data for evolocumab, the first proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor to be introduced in Japan in 2016, to date are limited. This study aimed to clarify the current real-world patient user profiles of evolocumab based on large-scale health claims data. METHODS AND RESULTS: This retrospective database study examined patients from a health administrative database (MDV database) who initiated evolocumab between April 2016 (baseline) and November 2021. Characteristics and clinical profiles of this patient population are described. In all, 4,022 patients were included in the final analysis. Most evolocumab prescriptions occurred in the outpatient setting (3,170; 78.82%), and 940 patients (23.37%) had a recent diagnosis of familial hypercholesterolemia. Common recent atherosclerotic cardiovascular disease events at baseline included myocardial infarction (1,633; 40.60%), unstable angina (561; 13.95%), and ischemic stroke (408; 10.14%). Comorbidity diseases included hypertension (2,504; 62.26%), heart failure (1,750; 43.51%), diabetes (1,199; 29.81%), and chronic kidney disease (297; 7.38%). Among the lipid-lowering regimens concomitant with evolocumab, ezetimibe+statin was used most frequently (1,281; 31.85%), followed by no concomitant lipid-lowering regimen (1,190; 29.59%), statin (950; 23.62%), and ezetimibe (601; 14.94%). The median evolocumab treatment duration for all patients was 260 days (interquartile range 57-575 days). CONCLUSIONS: This study provides real-world insights into evolocumab utilization in Japan for optimizing patient care and adherence to guideline-based therapies to better address hypercholesterolemia in Japan.

Characterization of Motor Neuron Prostaglandin E2 EP3 Receptor Isoform in a Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a motor neuron disease with adult onset, characterized by progressive loss of motor neurons. Prostaglandin E2 (PGE2), a lipid mediator, exerts its biological functions by binding to four subtypes of E-prostanoid (EP1-4). Among them, EP3 has been shown to have multiple isoforms, EP3α, EP3ß, and EP3γ, produced by alternative splicing. Since PGE2 has been shown to have important pathophysiological roles in ALS, experiments were performed to identify EP3 receptor isoform(s) in spinal motor neurons of wild-type (WT) and ALS model (G93A) mice. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of adult mice demonstrated expression of EP3α and EP3γ mRNAs in the lumbar spinal cord, whereas EP3ß mRNA was barely detectable. Laser capture microdissection was used to dissect out motor neurons from frozen samples of lumbar spinal cord in these mice for analysis by real-time PCR. We found that expression of EP3γ mRNA was predominant in these neurons, whereas EP3α and EP3ß mRNAs were undetectable. At the early symptomatic stage, the mRNA expression profiles of these splice isoforms in G93A motor neurons were comparable to those in neurons from WT mice. These results suggest that the PGE2-to-EP3 signaling pathway is mediated mainly by the EP3γ isoform in the motor neurons of mice, and that modulation of the EP3γ isoform in motor neurons may be a promising new therapeutic approach for ALS.

Mithramycin, an agent for developing new therapeutic drugs for neurodegenerative diseases.

Mithramycin A (MTM) has been shown to inhibit cancer growth by blocking the binding of Sp-family transcription factors to gene regulatory elements and is used for the treatment of leukemia and testicular cancer in the United States. In contrast, MTM has also been shown to exert neuroprotective effects in normal cells. An earlier study showed that MTM protected primary cortical neurons against oxidative stress-induced cell death. Recently, we demonstrated that MTM suppressed endoplasmic reticulum (ER) stress-induced neuronal death in organotypic hippocampal slice cultures and cultured hippocampal cells through attenuation of ER stress-associated signal proteins. We also found that MTM decreased neuronal death in area CA1 of the hippocampus after transient global ischemia/reperfusion in mice and restored the ischemia/reperfusion-induced impairment of long-term potentiation in this area. MTM has been shown to prolong the survival of Huntington's disease model mice and to attenuate dopaminergic neurotoxicity in mice after repeated administration of methamphetamine. In this review, we provide an up to date overview of neuroprotective effects of MTM and less toxic MTM analogs, MTM SK and MTM SDK, on some of the neurodegenerative diseases and discuss the promise of MTM as an agent for developing new therapeutic drugs for such diseases.

Prostaglandin E2-induced cell death is mediated by activation of EP2 receptors in motor neuron-like NSC-34 cells.

Prostaglandin E2 (PGE2) was shown to induce neuronal death in the CNS. To characterize the neurotoxicity of PGE2 and E-prostanoid receptors (EP) in motor neurons, we investigated PGE2-induced cell death and the type(s) of EP responsible for mediating it in NSC-34, a motor neuron-like cell line. Immunoblotting studies showed that EP2 and EP3 were dominantly expressed in NSC-34 cells and motor neurons in mice. Exposure to PGE2 and butaprost, an EP2 agonist, but not sulprostone, an EP1/3 agonist, resulted in decreased viability of these cells. These results suggest that PGE2 induces cell death by activation of EP2 in NSC-34 cells.

Relevance of the hippocampal endoplasmic reticulum stress response in a mouse model of chronic kidney disease.

It has been shown that the incidence of cognitive impairment increases with the severity of chronic kidney disease (CKD). A previous study has demonstrated that hippocampal oxidative stress contributes to cognitive dysfunction in CKD model mice. Endoplasmic reticulum (ER) stress is thought to contribute significantly to neuronal dysfunction, but its role in the hippocampal dysfunction seen in CKD still remains unclear. The present study examined whether the ER stress response as well as oxidative stress was activated in the hippocampus of CKD model mice. Western blotting revealed that the expression level of 4-hydroxy-2-nonenal (HNE)-protein adducts, a marker of oxidative stress, was increased in the hippocampus 8 weeks after 5/6 nephrectomy. In these mice, the expression level of glucose-regulated protein 78 (GRP78), a typical ER stress marker, also showed a pronounced increase in the hippocampus. Correlation analyses showed that the levels of these two marker proteins in the hippocampus are positively correlated with the serum concentrations of BUN and creatinine. These results suggest that ER stress as well as oxidative stress are induced in the hippocampus of CKD mice and that the levels of these stress markers in the hippocampus are correlated with the renal impairment caused by CKD.

N-acetylcysteine selectively protects cerebellar granule cells from 4-hydroxynonenal-induced cell death.

4-hydroxynonenal (HNE), an aldehydic product of membrane lipid peroxidation, has been shown to induce neurotoxicity accompanied by multiple events. To clarify mechanisms of neuroprotective compounds on HNE-induced toxicity, the protective effects of N-acetylcysteine (NAC), alpha-tocopherol (TOC), ebselen and S-allyl-L-cysteine (SAC) were compared in cerebellar granule neurons. The decrease in MTT reduction induced by HNE was significantly suppressed by pretreatment of the neurons with 1000 microM NAC or 10 and 100 microM TOC; however, lactate dehydrogenase (LDH) release and propidium iodide (PI) fluorescence studies revealed that neuronal death was suppressed by NAC but not by TOC. Treatment of these neurons with HNE resulted in a drastic reduction of mitochondrial membrane potential, and this reduction was also prevented by NAC but not by TOC. Ebselen and SAC, a garlic compound, were unable to protect these neurons against HNE-induced toxicity. Pretreatment with NAC also prevented HNE-induced depletion of intracellular glutathione (GSH) levels in these neurons. These results suggest that NAC, but not other antioxidants such as TOC, SAC and ebselen, exerts significant protective effects against HNE-induced neuronal death in cerebellar granule neurons, and that this neuroprotective effect is due, at least in part, to preservation of mitochondrial membrane potential and intracellular GSH levels.

Protective action of mithramycin against neurodegeneration and impairment of synaptic plasticity in the hippocampal CA1 area after transient global ischemia.

Mithramycin A (MTM) is an antibiotic used for the treatment of hypercalcemia and several types of cancer. We have reported previously that MTM protects against endoplasmic reticulum (ER) stress-induced neuronal death in organotypic hippocampal slice cultures. In the present study, the neuroprotective effect of MTM against ischemia/reperfusion-induced neuronal injury was evaluated in the hippocampus in mice. Neuronal damage was apparent in area CA1 of the hippocampus after transient global ischemia/reperfusion. The expression of C/EBP homologous protein (CHOP), a key transcription factor for ER stress-induced neuronal death, showed a pronounced increase in area CA1 in these mice. Treatment of the mice with MTM significantly decreased both the number of neurons stained with Fluoro-Jade B and the level of CHOP expression in the hippocampus. MTM did not affect the increase of 78-kDa glucose-regulated protein induced by ischemia/reperfusion. MTM also restored the ischemia/reperfusion-induced impairment of long-term potentiation in the hippocampus, without any change in paired pulse facilitation. These results suggest that administration of MTM protects hippocampal neurons against injury induced by transient global ischemia/reperfusion through attenuation of ER stress-associated signals, and ameliorates neuronal injury induced by ischemia/reperfusion in the hippocampus.

Characterization of neuronal and astroglial responses to ER stress in the hippocampal CA1 area in mice following transient forebrain ischemia.

Transient forebrain ischemia has been shown to cause neuronal injury in the CA1 area of the hippocampus in mice. In addition to neuronal injury, astrocytes in area CA1 undergo apoptosis under ischemic conditions. Although failure of impaired astrocytes to take up glutamate is thought to contribute to the pathogenesis of cerebral ischemia, the molecular mechanism underlying this phenomenon remains unexplored. In the present study, we investigated neuronal and astroglial responses to endoplasmic reticulum (ER) stress, which is an important sequela of transient forebrain ischemia in the hippocampus of mice. Cellular injury was observed in area CA1 of the hippocampus 72h after reperfusion, and ssDNA positivity was detectable in some glial cells as well as neurons in this area. An increase of 78-kDa glucose-regulated protein (GRP78), an indicator of ER stress, was detected in pyramidal neurons and astrocytes in this area after the insult. Immunohistochemical analysis showed that caspase-12 was increased in pyramidal neurons and astrocytes located in the extrapyramidal cell layer. Immunoreactivity for C/EBP homologous protein (CHOP) was increased significantly in pyramidal cells but not in astrocytes. These results suggest that astrocytes as well as pyramidal neurons in area CA1 undergo apoptosis through an ER stress-dependent mechanism after ischemia. Unlike the situation in neuronal apoptosis, CHOP does not play a role in the cell death of astrocytes.

Apolipoprotein E-deficient mice are more vulnerable to ER stress after transient forebrain ischemia.

Apolipoprotein E-deficient (apoE(-/-)) mice have been shown to have increased vulnerability to neuronal damage induced by cerebral ischemia; however, the mechanism of this increased vulnerability remains unclear. In order to define the role of the apoE protein against ischemia-induced ER stress and cell death, experiments were performed to compare ER stress-associated chaperones and signal proteins in the hippocampus of apoE(-/-) mice to those of WT mice after being subjected to forebrain ischemia and reperfusion. Although neuronal loss in area CA1-CA3 of the hippocampus was observed 3 days after ischemia in both types of mice, the damage in apoE(-/-) mice was more severe. In apoE(-/-) mice, a more extensive increase in 78-kDa glucose-regulated protein (GRP78) was observed after the insult, whereas the level of GRP94 was not changed. The expression of both C/EBP homologous protein (CHOP) and caspase-12 was increased in the hippocampus in both WT and apoE(-/-) mice after ischemia. The increased levels of CHOP in apoE(-/-) mice were significantly higher than those in WT mice, whereas the levels of caspase-12 in the two were comparable. Furthermore, whereas the levels of c-Jun N-terminal kinase (JNK), p-JNK1 and p-JNK2 in WT mice were unchanged after ischemia, they were significantly increased in apoE(-/-) mice 24h and 48h after ischemia. These results suggest that increased vulnerability of the hippocampus to forebrain ischemia and reperfusion in apoE(-/-) mice is at least partly attributable to perturbed induction of an ER chaperone, GRP 94, and enhancement of the CHOP- and JNK-dependent apoptotic pathway in the hippocampus.