Ifenprodil for the treatment of methamphetamine use disorder: An exploratory, randomized, double-blind, placebo-controlled trial.

AIM: No effective pharmacological interventions have been developed for patients with methamphetamine use disorder. Ifenprodil is a blocker of G protein-activated inwardly rectifying potassium channels, which play a key role in the mechanism of action of addictive substances. We conducted a randomized, double-blind, exploratory, dose-ranging, placebo-controlled trial to examine the clinical efficacy of ifenprodil for the treatment of methamphetamine use disorder. METHODS: Participants were assigned to three groups: placebo, 60 mg/d ifenprodil, or 120 mg/d ifenprodil. The drug administration period was 84 days. The primary outcome was the use or nonuse of methamphetamine during the drug administration period in the placebo group vs 120 mg/d ifenprodil group. We also assessed drug use status, relapse risk based on the Stimulant Relapse Risk Scale (SRRS), drug craving, and methamphetamine in urine as secondary outcomes. We further evaluated drug use status and SRRS subscale scores in patients who were not taking addiction medications during the study. RESULTS: Ifenprodil did not affect the primary or secondary outcomes. However, the additional analyses showed that the number of days of methamphetamine use during the follow-up period and scores on the emotionality problems subscale of the SRRS improved in the 120 mg/d ifenprodil group. The safety of ifenprodil was confirmed in patients with methamphetamine use disorder. CONCLUSION: The present findings did not confirm the efficacy of ifenprodil for methamphetamine use disorder treatment based on the primary or secondary outcomes, but we found evidence of its safety and efficacy in reducing emotionality problems. CLINICAL TRIAL REGISTRATION: The study was registered at the University Hospital Medical Information Network Clinical Trial Registry (no. UMIN000030849) and Japan Registry of Clinical Trials (no. jRCTs031180080). The main registration site is jRCT (https://jrct.niph.go.jp/).

Direct visualization of replication dynamics in early zebrafish embryos.

We analyzed DNA replication in early zebrafish embryos. The replicating DNA of whole embryos was labeled with the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU), and spatial regulation of replication sites was visualized in single embryo-derived cells. The results unveiled uncharacterized replication dynamics during zebrafish early embryogenesis.

Direct Visualization of DNA Replication Dynamics in Zebrafish Cells.

Spatiotemporal regulation of DNA replication in the S-phase nucleus has been extensively studied in mammalian cells because it is tightly coupled with the regulation of other nuclear processes such as transcription. However, little is known about the replication dynamics in nonmammalian cells. Here, we analyzed the DNA replication processes of zebrafish (Danio rerio) cells through the direct visualization of replicating DNA in the nucleus and on DNA fiber molecules isolated from the nucleus. We found that zebrafish chromosomal DNA at the nuclear interior was replicated first, followed by replication of DNA at the nuclear periphery, which is reminiscent of the spatiotemporal regulation of mammalian DNA replication. However, the relative duration of interior DNA replication in zebrafish cells was longer compared to mammalian cells, possibly reflecting zebrafish-specific genomic organization. The rate of replication fork progression and ori-to-ori distance measured by the DNA combing technique were ∼ 1.4 kb/min and 100 kb, respectively, which are comparable to those in mammalian cells. To our knowledge, this is a first report that measures replication dynamics in zebrafish cells.

Functional and heterologous expression of human protein O-linked mannose ß-1,2-N-acetylglucosaminyltransferase 1 in zebrafish.

Although membrane-associated proteins are related to many diseases and are important targets for drug discovery, their expression is often difficult in bacterial hosts such as Escherichia coli. To overcome this limitation, here, we focused on a novel host-vector system in zebrafish for the expression of human protein O-linked mannose ß-1,2-N-acetylglucosaminyltransferase 1 (hPOMGnT1) which is related to muscle-eye-brain disease. For the expression of hPOMGnT1, the vector pZex-EGFP-pXI-hPOMGnT1 was constructed and injected into fertilized eggs. Using this system, we demonstrated that recombinant hPOMGnT1 was successfully expressed in the whole bodies of zebrafish embryos.

Protein O-mannosylation is necessary for normal embryonic development in zebrafish.

Two distinct cDNAs corresponding to two zebrafish protein O-mannosyltransferase genes, zPOMT1 and zPOMT2, were cloned from early developmental embryos. Gene expression analysis revealed that zPOMT1 and zPOMT2 were expressed in similar patterns during early embryonic development and in all adult tissues. To study the regulation of zPOMT1 and zPOMT2 mRNA distribution during zebrafish embryogenesis, we injected enhanced green fluorescent protein (EGFP) mRNA fused to the 3'untranslated regions of each zPOMT gene. The distribution of EGFP resulting from the two constructs was similar. Injection of antisense morpholino oligonucleotides of zPOMT1 and zPOMT2 resulted in several severe phenotypes-including bended body, edematous pericaridium and abnormal eye pigmentation. Immunohistochemistry using anti-glycosylated alpha-dystroglycan antibody (IIH6) and morphological analysis revealed that the phenotypes of zPOMT2 knockdown were more severe than those of zPOMT1 knockdown, even though the IIH6 reactivity was lost in both zPOMT1 and zPOMT2 morphants. Finally, only when both zPOMT1 and zPOMT2 were expressed in human embryonic kidney 293T cells were high levels of protein O-mannosyltransferase activity detected, indicating that both zPOMT1 and zPOMT2 were required for full enzymatic activity. Moreover, either heterologous combination, zPOMT1 and human POMT2 (hPOMT2) or hPOMT1 and zPOMT2, resulted in enzymatic activity in cultured cells. These results indicate that the protein O-mannosyltransferase machinery in zebrafish and humans is conserved and suggest that zebrafish may be useful for functional studies of protein O-mannosylation.