Coronavirus disease 2019 (COVID-19) has become a serious public health problem worldwide. In general, healthcare workers are considered to be at higher risk of COVID-19 infection. However, the prevalence of COVID-19 among healthcare workers in Japan is not well characterized. In this study, we aimed to examine the seroprevalence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antibodies among 2160 healthcare workers in hospitals and clinics that are not designated to treat COVID-19 patients in Japan. The prevalence of SARS-CoV-2 immunoglobulin G was 1.2% in August and October 2020 (during and after the second wave of the pandemic in Japan), which is relatively higher than that in the general population in Japan (0.03-0.91%). Because of the higher risk of COVID-19 infection, healthcare workers should be the top priority for further social support and vaccination against SARS-CoV-2.

Body temperature is important for diagnosing illnesses. However, its assessment is often a difficult task, considering the large individual differences. Although 37 °C has been the gold standard of body temperature for over a century, the temperature of modern people is reportedly decreasing year by year. However, a mean axillary temperature of 36.89 ± 0.34 °C reported in 1957 is still cited in Japan. To assess the measured axillary temperature appropriately, understanding its distribution in modern people is important. This study retrospectively analyzed 2454 axillary temperature measurement data of healthy Japanese adults in 2019 (age range, 20–79 years; 2258 males). Their mean temperature was 36.47 ± 0.28 °C (36.48 ± 0.27 °C in males and 36.35 ± 0.31 °C in females). Approximately 5% of the 20–39-year-old males had body temperature ≥37 °C, whereas 8% had a temperature ≥ 37 °C in the afternoon. However, none of the subjects aged ≥50 years reported body temperature ≥37 °C. In multivariable regression analysis, age, blood pressure, pulse rate, and measurement time of the day were associated with axillary temperature. Our data showed that the body temperature of modern Japanese adults was lower than that reported previously. When assessing body temperature, the age, blood pressure, pulse rate, and measurement time of the day should be considered.

The concentration of cerebrospinal fluid total protein (CSF-TP) is important for the diagnosis of neurological emergencies. Recently, some Western studies have shown that the current upper reference limit of CSF-TP is quite low for older patients. However, little is reported about the concentration of CSF-TP in the older Asian population. In this study, we retrospectively analyzed the CSF-TP concentrations in healthy older Japanese volunteers. CSF samples in 69 healthy Japanese volunteers (age range: 55–73 years) were collected by lumbar puncture, and the data of CSF were retrospectively analyzed. The mean (standard deviation) CSF-TP was 41.7 (12.3) mg/dL. The older group (≥65 years old) had higher CSF-TP concentration than the younger group (55–64 years old). The 2.5th percentile and 97.5th percentile of CSF-TP were estimated as 22.5 and 73.2 mg/dL, respectively, which were higher than the current reference range in Japan (10–40 mg/dL). Conclusions: The reference interval of CSF-TP in the older population should be reconsidered for the precise diagnosis of neurological emergencies.

Nitrous oxide (N2O)-induced antinociception in mice is dependent on the neuromodulator nitric oxide (NO). In contrast to C57BL/6J (B6) mice, DBA/2J (D2) mice fail to respond to N2O with a robust antinociceptive response or with an increase in brain nitric oxide synthase (NOS) enzyme activity, suggesting that failure of D2 mice to respond to N2O might result from a deficit of NO function. Therefore, it was of interest to determine whether increasing the availability of NO might increase sensitivity of D2 mice to N2O. Male D2 mice were pretreated with sub-antinociceptive intracerebroventricular doses of the NO donor 3-morpholinosydnoimine or the NO precursor l-arginine then assessed for responsiveness to N2O-induced antinociception using the acetic acid abdominal constriction test. Both pretreatments increased the antinociceptive responsiveness of D2 mice to N2O. These results indicate that the NOS enzyme in D2 mice is functional and that the deficit in NO function that obstructs sensitivity to N2O-induced antinociception may lie in availability or utilization of l-arginine.

The aims of the present study were (1) to determine the maximum tolerated doses of quetiapine and pramipexole when given to healthy Japanese male subjects using gradually increasing single doses; (2) to evaluate the feasibility of this exploratory method for further bioequivalence trials; and (3) to conduct bioequivalence trials using doses determined based on prior tolerability trials. For quetiapine, 18 participants received 25 mg in the first stage. In the second stage, participants were divided into three groups of six subjects each and allocated to receive 50 mg, 75 mg or 100 mg depending on the severity of adverse events in the first stage. For pramipexole, 18 participants received 0.125 mg in the first stage, and then received 0.25 mg, 0.375 mg, or 0.5 mg in the second stage in the same manner as quetiapine. In the group receiving 75 mg of quetiapine, three mild adverse events and seven moderate adverse events (including nightmare and syncope) were reported from all six subjects. In the group receiving 0.5 mg of pramipexole, three mild and five moderate adverse events were reported from five subjects. Therefore, we judged that doses equal to or greater than 75 mg of quetiapine and 0.5 mg of pramipexole are not well tolerated by healthy subjects. Based on these results, we conducted two-way crossover bioequivalence clinical trials with brand-name and generic formulations of 25 mg of quetiapine (25 mg tablets or 50% fine granules) and 0.125 mg of pramipexole, in subjects who did not participate in the tolerability studies. By calculating 90% confidence intervals of logarithmic transformed values of Cmax and AUCt, we found that the brand-name and generic formulations were bioequivalent.

Abstract FSK0808 is a recombinant human granulocyte colony-stimulating factor developed by Fuji Pharma Co., Ltd and Mochida Pharmaceutical Co., Ltd. as a biosimilar product of Gran®. We verified the pharmacokinetic/pharmacodynamic equivalence of FSK0808 and commercially available Gran® by a randomized crossover study of single intravenous dose (200 µg/m2) and single subcutaneous dose (400 µg/m2) in healthy Japanese adult male subjects. According to the bioequivalence guidelines, the area under the blood concentration – time curve by 48 hours after administration (AUC0–48) in a single intravenous drip (IVD) study, and AUC0–48 and maximum blood concentration (Cmax) in a single subcutaneous (SC) dose study were used as primary endpoints, and the pharmacodynamic parameters including absolute neutrophil count (ANC) or number of CD34 positive cells (CD34+ cells) as secondary endpoints. The safety was evaluated based on the characteristics and incidence of adverse reactions. As a result, the 90% confidence interval (CI) of the difference in mean value for AUC0–48 among drugs ranged from log(0.8) to log(1.25), in the IVD study, and those for Cmax and AUC0–48 were within the range of log(0.8)–log(1.25) in the SC study. Those for secondary endpoints were all within the range of log(0.8)–log(1.25). Thus, the pharmacokinetics/pharmacodynamics of both drugs were considered equivalent for all routes of administration, and the profiles of adverse reactions were also very similar.

C57BL/6 (C57) inbred mice respond to N2O with an increase in brain nitric oxide synthase (NOS) activity and a robust antinociceptive effect; DBA/2 (DBA) fail to exhibit these responses (Ishikawa and Quock, Brain Res. 976:262–263, 2003a). This study was conducted to determine whether increasing the availability of nitric oxide (NO) by administration of L‐arginine might increase responsiveness of DBA mice to N2O and ascertain the effect of N2O on brain levels of L‐arginine. Sensitivity to N2O was assessed using the acetic acid‐induced abdominal constriction test. Whole brain levels of L‐arginine were quantified by HPLC. Intracerebroventricular preloading of L‐arginine in subthreshold doses enhanced the N2O‐induced antinociceptive effects in both C57 and DBA mice. A 60‐min exposure to 70% N2O produced a 12‐fold increase in brain L‐arginine levels of C57 mice, compared to room air exposure. Similar treatment of DBA mice resulted in a 5‐fold increase in brain L‐arginine levels. While the cause of the differential responsiveness of inbred mice to N2O remains to be determined, it is apparent that N2O increases brain L‐arginine levels to produce its antinociceptive effect. (Supported in part by NIH Grant GM‐77153 and the Allen I. White Distinguished Professorship.)

Previous research has found that hyperbaric oxygen (HBO2) produces an acute antinociceptive effect that is dependent on nitric oxide (NO). The present study was undertaken to determine whether HBO2-induced acute antinociception might involve a NO–cyclic GMP–protein kinase G–ATP-sensitive potassium (KATP) channel pathway. Male NIH Swiss mice were subjected to a 5-min HBO2 treatment (100% oxygen at 3.5 absolute atmospheres) and antinociception was assessed over the next 6 min still under HBO2 using the acetic acid abdominal constriction test. Pretreatment with 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide (carboxy-PTIO, an NO scavenger), 1H-[1,2,4]-oxadiazolo-[4,3-a]quinoxalin-1-one) (a soluble guanylyl cyclase-inhibitor, Rp-8-(4-chlorophenylthio)-guanosine-3′,5′-cyclic monophosphorothioate (a protein kinase G-inhibitor) or glibenclamide (an ATP-sensitive potassium channel-inhibitor) all led to antagonism of the HBO2-induced acute antinociception in a dose-dependent manner. These findings suggest that HBO2-induced acute antinociception might be due to activation of a NO–cyclic GMP–protein kinase G–KATP channel pathway.

The antinociceptive effect of nitrous oxide (N2O) is dependent on nitric oxide (NO); however, the next step in the pathway activated by NO is undetermined. The present study was conducted to test the hypothesis that a N2O action involves sequential activation of NO synthase, soluble guanylyl cyclase and protein kinase G to induce an antinociceptive effect in mice. The antinociceptive responsiveness of male NIH Swiss mice to N2O was assessed using the acetic acid abdominal constriction test. Different groups of mice were pretreated with either saline, the NO scavenger 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide (carboxy-PTIO), the guanylyl cyclase-inhibitor 1H-[1,2,4]-oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), the protein kinase G-inhibitor Rp-isomer of 8-(4-chlorophenylthio)-guanosine-3′,5′-cyclic monophosphorothioate (Rp-8-pCPT-cGMPS) or the selective phosphodiesterase V-inhibitor 1,2-dihydro-2-[(2-methyl-4-pyridinyl)methyl]-1-oxo-8-(2-pyrimidinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-2,7-naphthyridine-3-carboxylic acid methyl ester hydrochloride (T 0156). Vehicle (saline)-pretreated mice responded to N2O in a concentration-dependent manner. This antinociceptive effect was antagonized by systemic pretreatment with carboxy-PTIO and ODQ and central pretreatment with Rp-8-pCPT-cGMPS. In each case, the dose–response curve for N2O was progressively shifted to the right by increasing the dose of each pretreatment drug. On the other hand, N2O-induced antinociception was enhanced by systemic pretreatment with T 0156; the dose–response curve for N2O was shifted to the left. The ATP-sensitive potassium channel blocker glibenclamide was without influence on the antinociceptive effect of N2O. These results support the hypothesis that N2O-induced antinociception in mice is mediated by a NO–cyclic GMP–PKG pathway.

Previous studies have implicated nitric oxide (NO) in the antinociceptive response to the anesthetic gas nitrous oxide (N2O). The present study was conducted to confirm this NO involvement using pharmacological and gene knockdown and knockout strategies to inhibit the supraspinal and spinal production of NO. Antinociceptive responsiveness to 70% N2O was assessed using the acetic acid (0.6%) abdominal constriction test in NIH Swiss mice following intracerebroventricular (i.c.v.) or intrathecal (i.t.) pretreatment with the NOS-inhibitor l-NG-nitro arginine methyl ester (L-NAME) or an antisense oligodeoxynucleotide (AS-ODN) directed against neuronal NOS (nNOS). Experiments were also conducted in mice homozygous for a defective nNOS gene (nNOS−/−). Mice that were pretreated i.c.v. or i.t. with L-NAME (1.0 µg) both exhibited 80–90% reduction in the magnitude of the N2O-induced antinociceptive response. Mice that were pretreated i.c.v. or i.t. with nNOS AS-ODN (3 × 25 µg) exhibited a 60–80% antagonism of the antinociceptive response. Compared to wild-type mice, nNOS knockout mice showed a 60% reduction in N2O-induced antinociception. These findings consistently demonstrate that transient or developmental suppression of nNOS expression significantly reduces antinociceptive responsiveness to N2O. NO of both supraspinal and spinal origin, therefore, plays an important role in the antinociceptive response to N2O.

Nitrous oxide (N(2)O)-induced antinociception is thought to result from nitric oxide (NO)-dependent neuronal release of endogenous opioid peptides in the central nervous system. The present study employed microdialysis to determine whether exposure to N(2)O stimulates proopiomelanocortin (POMC) neurons to release β-endorphin in the arcuate nucleus (ARC) of the hypothalamus and the periaqueductal gray (PAG) of the midbrain. Male Sprague-Dawley rats were stereotaxically implanted with microdialysis probes in the ARC or PAG. Exposure to 70% N(2)O significantly increased dialysate levels of oxidation products of NO as well as β-endorphin, compared to levels in fractions collected under room air. These increases in the ARC and PAG were abolished by systemic pretreatment with L-N(G)-nitro arginine methyl ester (L-NAME). These findings suggest an association between increased NO activity and the stimulated release of β-endorphin during exposure of rats to N(2)O.

BACKGROUND:Artemisinin selectively kills cancer cells which have more intracellular free iron than do normal cells. Hyperbaric oxygen (HBO(2)) may be beneficial in the treatment of cancer. The hypothesis of this study was that HBO(2) enhances anticancer activity of artemisinin.MATERIALS AND METHODS:After pretreatment with 12 μM holotransferrin, Molt-4 human leukemia cells were cultured in 10 μM artemisinin and exposed for 90 min to one of three different conditions: control, room air control, and HBO(2). Cell growth was determined for 48 h after exposure.RESULTS:Differences in growth were noted after 6 h of incubation. After 48 h of incubation, growth of cells treated with artemisinin alone or HBO(2) alone was 85% of that of cells grown under artemisinin-free control conditions. Combined artemisinin and HBO(2) treatment resulted in an additional 22% decrease in growth.CONCLUSION:Combined HBO(2) and artemisinin exposure may be an effective anticancer chemotherapeutic strategy.

ART reacts with free iron to form cytotoxic free radicals. It has been shown to be selective in killing cancer cells compared to its effect on normal cells because cancer cells contain more intracellular free iron. Furthermore, there is evidence in the literature that HBO2‐100% oxygen administered at elevated atmospheric pressure (ATA)‐may be beneficial in the treatment of cancer. The hypothesis of the present study is that the anticancer activity of ART can be enhanced by exposure to HBO2. In our studies, after pretreatment with 12 µM human holotransferrin, 10 µM ART was added to the culture and Molt‐4 cells (3.2 × 105 cells/mL) were exposed for 90 min to one of three different conditions: control (37ºC, 5% CO2 / 95% RA, normal ATA); room air [room temperature (RT), RA, normal ATA]; and HBO2 (RT, 100 % O2, 2.5 ATA). Cell growth and viability were determined at 1, 2, 4, 6, 12, 24 and 48 hrs. Differences in growth among treatments were noted after 6 hr of incubation. After 48 hr of incubation, growth of cells exposed to ART alone or HBO2 alone was 85.3% of that of cells grown under ART‐free control conditions. Combined exposure to ART and HBO2 resulted in an additional 22.1% decrease in growth. These findings suggest the possibility that combined HBO2 and ART exposure can be developed into an effective chemotherapeutic strategy for cancer treatment. (Supported by the Washington State University College of Pharmacy.)

Hyperbaric oxygen (HBO2) therapy induces analgesia in various conditions of pain in humans. In mice, HBO2 treatment evokes an acute antinociceptive response in the abdominal constriction test. To demonstrate the dependence of HBO2-induced antinociception on nitric oxide (NO), antinociceptive responsiveness to HBO2 was assessed after three different approaches that interfered with NO production. HBO2-induced antinociception was significantly attenuated by intracerebroventricular and intrathecal pretreatment with an inhibitor of NO synthase (NOS) enzyme and also by an antisense oligodeoxynucleotide directed against neuronal NOS. The antinociceptive effect was also significantly reduced in mice homozygous for a defective neuronal NOS gene. On the basis of these results, we conclude that neuronal NO is critical in the expression of the acute antinociceptive effect of HBO2.

HBO2 is approved by the FDA for limited clinical indications but not for chronic pain. Earlier we showed that HBO2 treatment produces an antinociceptive effect mediated by nitric oxide (NO) and opioid mechanisms [Zylstra et al., FASEB J 22, 711.16 & 711.17, 2008]. In this study, we assessed the antinociceptive effect of multiple HBO2 treatments. Male NIH Swiss mice (20‐30 g) were exposed to HBO2 at 2.5 ATA for 4×60‐min then returned to their home cages for different time intervals before testing with the acetic acid‐induced abdominal constriction test. Results revealed a robust antinociception of at least 6 hr after the last HBO2 treatment. This response was followed a day later by an emerging and equally robust late‐phase antinociceptive effect that surprisingly lasted for two weeks following the last HBO2 treatment. The early phase was antagonized by pretreatment with naltrexone (NTX) and the late phase was antagonized by the selective 5‐HT1A antagonist WAY‐100635 but not NTX. Continuous treatment with NTX or the NO synthase‐inhibitor L‐NAME during the 4 days of HBO2 treatment also reduced the late‐phase response. Based on these findings, we suggest that the early‐phase antinociception involves an opioid pathway, while the longer‐acting late‐phase antinociception involves an NO‐opioid‐5‐HT1A pathway. (Supported in part by NIH Grant GM‐77153, the College of Pharmacy and the Chico Hyperbaric Center.)

Earlier we reported that a 60‐min exposure to HBO2 induced an antinociceptive response that lasted for up to 90 min following exposure [Zylstra et al., FASEB J 22:711.17, 2008]. Recent studies have reported a sustained antinociceptive effect of a selective, high‐efficacy 5‐HT1A receptor agonist in rodents [Deseure et al., Eur J Pharmacol 568:134, 2007]. To determine whether supraspinal or spinal 5‐HT1A receptors might be involved in HBO2‐ or N2O‐induced antinociception, male NIH Swiss mice (20‐30 g) were exposed to HBO2 (2.5 ATA, 60 min) then removed to room air for 90 min or N2O acutely (11 min). Mice were pretreated with intracerebroventricular (i.c.v.) or intrathecal (i.t.) selective 5HT1A antagonist WAY‐100635 prior to antinociceptive testing using the abdominal constriction test. Results show that both i.c.v. and i.t. pretreatments with WAY‐100635 produced comparable and dose‐related antagonism of the antinociceptive response to HBO2. On the other hand, i.c.v. and i.t. pretreatment with WAY‐100635 had no effect on N2O‐induced antinociception. These results suggest that both supraspinal and spinal 5HT1A receptors are involved in the 90‐min antinociceptive response to a 60‐min exposure to HBO2. However, there does not appear to be a role for 5HT1A receptors in the antinociceptive response to N2O. (Supported in part by NIH Grant GM‐77153, the College of Pharmacy and the Chico Hyperbaric Center.)

Hyperbaric oxygen (HBO(2)) therapy is reported to be beneficial in transient brain ischemia. The present study was conducted to determine the influence of HBO(2) on metabolites of nitric oxide (NO) in brain and spinal cord of rats. Rats were exposed to room air (RA), normobaric air (NBA), normobaric oxygen (NBO(2)), hyperbaric air (HBA) or HBO(2), the last two conditions at 2.5ATA (atmosphere absolute) for 60 min. The results demonstrate that, compared to the NBA control, oxygen alone generally reduced tissue levels of NO(x)(-) (nitrite plus nitrate). On the other hand, 2.5ATA alone tended to have a slight, if any, effect on tissue levels of NO(x)(-). The combination of oxygen and pressure (i.e., HBO(2)) generally led to an increase in tissue levels of NO(x)(-). Based on these findings, it is concluded that HBO(2) appears to markedly increase NO function most notably in the corpus striatum, brainstem, cerebellum and spinal cord.

N2O‐induced antinociception in mice is dependent on the neuromodulator NO. In contrast to C57BL/6 mice, DBA/2 mice fail to respond to N2O with a robust antinociceptive response or with an increase in brain nitric oxide synthase (NOS) enzyme activity, suggesting that failure of DBA/2 mice to respond to N2O might result from a deficit of NO function (Ishikawa and Quock, Brain Res. 976:262–263, 2003). Therefore, it was of interest to determine whether increasing the availability of NO might increased sensitivity of DBA mice to N2O. Male DBA/2 mice (20–25 g) were pretreated with sub‐antinociceptive intracerebroventricular doses of the NO donor 3‐morpholinosydnoimine (SIN‐1) or the NO precursor L‐arginine (L‐Arg) then assessed for responsiveness to N2O‐induced antinociception using the acetic acid abdominal constriction test. Both pretreatments increased the antinociceptive responsiveness of DBA/2 mice to N2O. These results show that the NOS enzyme in DBA/2 mice is functional and that the deficit in NO function that obstructs sensitivity to N2O‐induced antinociception may lie in some other component or function related to NO. (Supported in part by NIH Grant GM‐77153).