Decreased bone mineral density in ovariectomized mice is ameliorated after subsequent repeated intermittent administration of (R)‐ketamine, but not (S)‐ketamine

Abstract Aim Depression is a common symptom in people with osteoporosis. (R)‐ketamine produced greater potency and longer‐lasting antidepressant‐like actions than (S)‐ketamine in rodents. Here, we examined the effects of two ketamine enantiomers on the reduced bone mineral density (BMD) in the ovariectomized (OVX) mice which is an animal model of postmenopausal osteoporosis. Methods Female ddY mice were OVX or sham‐operated. Subsequently, saline (10 mL/kg/d, twice weekly), (R)‐ketamine (10 mg/kg/d, twice weekly), or (S)‐ketamine (10 mg/kg/d, twice weekly) was administered intraperitoneally into OVX or sham mice for the 6 weeks. The femur from all mice was collected 3 days after the final injection, and BMD in the femur was measured. Results The reduction of cortical BMD and total BMD in the OVX mice was significantly ameliorated after subsequent repeated intermittent administration of (R)‐ketamine, but not (S)‐ketamine. Conclusion The study shows that (R)‐ketamine can ameliorate the reduced cortical BMD and total BMD in OVX mice. Therefore, (R)‐ketamine would be a novel therapeutic drug for women with osteoporosis.


| INTRODUC TI ON
Osteoporosis is the common chronic disease characterized by low bone mass and structural deterioration of bone tissue, leading to fragility of the bone. Depression is common in people with osteoporosis. A meta-analysis demonstrated that bone mineral density (BMD) in patients with depression is reduced compared to subjects without depression, 1 indicating depression as a risk factor for low BMD. [2][3][4] Furthermore, the use of antidepressants is associated with osteoporotic fracture in elderly peoples. 5,6 Importantly, the use of selective serotonin reuptake inhibitors (SSRIs) is associated with decreased BMD and increased fracture risk in humans. 7 Therefore, the development of the novel antidepressants which also have beneficial effects for osteoporosis is an unmet medical need.
In this study, we investigated the effects of (R)-ketamine and (S)ketamine on the reduced BMD in OVX mice.
Mice were housed under controlled conditions for temperature (23 ± 1°C) and humidity (55 ± 5%) with a 12-h light-dark cycle (lights on from 7:00 to 19:00). Mice were allowed free access to food (CE-2; CLEA Japan, Inc, Tokyo, Japan) and water. The study was approved by the Chiba University Institutional Animal Care and Use Committee (permission number: 1-364).
Other reagents were purchased commercially.

| OVX model and repeated administration of ketamine enantiomers
The schedule of surgery and treatment is shown in Figure 1 or sham surgery (n = 7) under isoflurane anesthesia. Subsequently, saline (10 mL/kg/d, twice weekly), (R)-ketamine (10 mg/kg/d, twice weekly), or (S)-ketamine (10 mg/kg/d, twice weekly) was administered to mice for 6 weeks. The femur from all mice was collected 3 days after the final injection ( Figure 1A).

| Determination of BMD
We quantified BMD of the femur using an experimental animal CT system (Latheta LCT-200; Hitachi Ltd.), as reported previously. 23,25 We performed the calibration using a phantom before were scanned together. We determined cortical BMD, cancellous BMD, total BMD, and plane BMD using the Latheta software (version 3.40). 23,25

| Statistical analysis
The data were shown as the mean ± standard error of the mean (SEM). The data of body weight were analyzed using the repeatedmeasures one-way analysis of variance (ANOVA). The data of BMD were analyzed using the one-way ANOVA, followed by post hoc Tukey's HSD test. The P-values of <.05 were considered statistically significant.

| Effects of ketamine enantiomers on the reduced BMD of OVX mice
The time course of body weight was statistically significant, indicating that body weight of all groups was increased from day 2 to day 43. However, there were no changes among the four groups at each time point ( Figure 1B). Figure 2A shows the representative CT images of the femur from the four groups. One-way ANOVA of all BMD data revealed statistical significances among the four groups. Both cortical BMD and total BMD were significantly higher in the (R)ketamine-treated OVX group than those of saline-treated OVX group ( Figure 2B,D). In contrast, (S)-ketamine did not ameliorate the reduction of cortical BMD and total BMD in the OVX mice ( Figure 2B,D). Cancellous BMD and plane BMD were significantly lower in the saline-treated OVX group than those of saline-treated sham group. However, both ketamine enantiomers did not ameliorate the reduction of cancellous BMD and plane BMD in the OVX mice ( Figure 2C,E).

| D ISCUSS I ON
In the present study, we showed that the reduction of cortical BMD and total BMD in the OVX mice was significantly ameliorated after subsequent repeated intermittent administration of (R)-ketamine, but not (S)-ketamine. Greater beneficial effects of (R)-ketamine compared to (S)-ketamine in the OVX mice were consistent with the greater potency of antidepressant-like effects of (R)-ketamine compared to (S)-ketamine in rodent models of depression. [12][13][14][15][16] Considering comorbidity of depression in patients with osteoporosis, it is possible that (R)-ketamine could produce beneficial effects in female patients with osteoporosis.
Fukumoto et al 14 reported that two ketamine enantiomers share similar pharmacokinetic profiles in rodents, indicating that the differential effects between two ketamine enantiomers for the reduced BMD in the OVX mice are not due to differences in their pharmacokinetic profiles. Therefore, it is unlikely that NMDAR might play a major role in the beneficial actions of (R)ketamine in OVX mice. Nonetheless, further studies investigating the molecular and cellular mechanisms underlying the beneficial effects of (R)-ketamine in animal models of osteoporosis are needed.
As aforementioned in the introduction, the use of SSRIs is associated with hip fracture risk in the general elderly population. A retrospective cohort study of a 10-year period showed that veterans with SSRI usage were associated with 56.7% more likely to suffer hip fracture and 34.6% more likely to develop osteoporosis. 26 Collectively, it seems that SSRIs may not be suitable for elderly MDD patients with a risk for osteoporosis. Furthermore, the use of SSRI was associated with increased hip fracture risk in patients with hemodialysis (adjusted odd ratio = 1.25). 27 Considering the risk of SSRI use for hip fracture, (R)-ketamine could be a potential alternative drug for elderly patients with osteoporosis.
In 2019, (S)-ketamine nasal spray of Johnson & Johnson was approved for treatment-resistant patients with MDD although several concerns were addressed. 28,29 In contrast, (R)-ketamine did not cause psychotomimetic and dissociative side effects in healthy control subjects, whereas the same dose of (S)-ketamine produced these side effects in the healthy subjects. 18 In addition, a pilot study showed low incidence of dissociation in treatment-resistant MDD patients after a single administration of (R)-ketamine. 19 Thus, it is recognized that detrimental side effects (ie, psychotomimetic effects and dissociation) of (R,S)-ketamine are associated with (S)ketamine. 30 Taken together, it is likely that (R)-ketamine is a potential therapeutic drug for osteoporosis.
In conclusion, this study suggests that (R)-ketamine, but not (S)ketamine, significantly ameliorated the reduced BMD in OVX mice. Therefore, it is likely that (R)-ketamine would be a potential therapeutic drug for patients with osteoporosis.

ACK N OWLED G M ENTS
This study was supported by AMED (to KH, JP20dm0107119).

CO N FLI C T O F I NTE R E S T
Dr Hashimoto is the inventor of filed patent applications on "The use of R-ketamine in the treatment of psychiatric diseases" and "Preventive or therapeutic agent and pharmaceutical composition for inflammatory diseases or bone diseases" by the Chiba University.
Dr Hashimoto also declares that he has received research support and consultant fees from Dainippon Sumitomo, Otsuka, and Taisho.
The other author declares no conflict of interest.

AUTH O R CO NTR I B UTI O N S
KH is responsible for the design of the research and experiment and supervised the experimental analyses. KH wrote the paper. YF performed the experiments. YF analyzed the data. All authors read and approved this paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available in Appendix S1 of this article.