IDO (IC50 = 4.52 nM)

PCC-0208009 functions as an indirect IDO1 inhibitor because it inhibits IDO1 activity in HeLa cells with an IC50 value of 4.52 nM while having no effect on enzyme activity in vitro[1]. IFN-γ-induced IDO protein and mRNA expression are dose-dependently suppressed by PCC0208009 (0-200 nM; 48 hours) [3].

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Effect of PCC-0208009/PCC on the viability and proliferation of HeLa cells [3]
The viability and proliferation of HeLa cells were examined after cells were treated with IFN-γ and PCC-0208009/PCC alone, or PCC plus IFN-γ. The results of the viability assay are shown in Figure 2(a); the percentages of living cells in the PCC and IFN-γ alone groups or in the combination treated groups had no significant differences compared with the vehicle group (P > 0.05). The results of the proliferation test are shown in Figure 2(b); the IRs in PCC and IFN-γ alone or in combination treated groups also showed no significant differences compared with the vehicle group (P > 0.05), with IRs less than 8%. These results indicated that 25–200 nM PCC or/and 100 ng/mL IFN-γ had no obvious effects on the viability and proliferation of HeLa cells.

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Effect of PCC-0208009/PCC on IDO activity and expression in HeLa cells [3]
IDO is highly expressed in HeLa cells induced by IFN-γ, which is widely used for activity screening of IDO inhibitors. After 10–20 h induction with 100 ng/mL IFN-γ, IDO was observed to be highly expressed in HeLa cells by Kyn/Trp determination. The IDO inhibition effects of PCC-0208009/PCC in HeLa cells were detected after 24 h induction with IFN-γ in this study. As shown in Figure 2(c), IDO activity (Kyn/Trp) was maintained at a high level in the IFN-γ group, and was highly expressed 10 h later. In the PCC group, the activity of IDO was effectively inhibited to the level of the vehicle group, from the drug addition to at least 72 h.
HeLa cells were treated with IFN-γ or IFN-γ plus PCC-0208009/PCC for 48 h, and the expressions of IDO at protein and mRNA levels were detected. The results from Western blot analysis are shown in Figure 2(d) and (e); the expression levels of IDO protein were almost undetectable in the vehicle group, but were significantly increased by IFN-γ treatment. The IDO protein expression induced by IFN-γ was dose-dependently suppressed by PCC, which showed significant differences at 100 and 200 nM (P < 0.05). The results from qRT-PCR are shown in Figure 2(f); IDO mRNA was expressed at low levels in the vehicle group and was significantly increased by IFN-γ treatment compared with the vehicle group (P < 0.05). The IDO mRNA expression induced by IFN-γ was dose-dependently suppressed by PCC, which showed significant differences at all doses compared with the IFN-γ group (P < 0.05).

In adult male Sprague Dawley rats weighing 180–200 grams, PCC-0208009 (single oral gavage; 50 mg/kg) was found in plasma and brain samples 60, 120, and 240 minutes after the drug was administered. The maximum concentrations of PCC0208009 were found in the plasma and brain 60 minutes after the drug was administered. The Kyn/Trp ratio also declines concurrently at 60, 120, and 240 minutes after dosing, peaking at 60 minutes in the brain and plasma[1]. In adult male Sprague Dawley rats (180 g–200 g), PCC0208009 (oral gavage; once; 12–50 mg/kg) is detected 30, 60, and 90 minutes after administration to assess the antinociceptive effects of PCC0208009 on neuropathic pain[1].

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In rats receiving spinal nerve ligation (SNL), IDO1 expression level was increased in ACC and amygdala. PCC-0208009 attenuated pain-related behaviors in the formalin test and SNL model and increased cognition and anxiogenic behaviors in SNL rats at doses that did not affect locomotor activity and sleeping. PCC0208009 inhibited IDO1 expression in ACC and amygdala by inhibiting the IL-6-JAK2/STAT3-IDO1-GCN2-IL-6 pathway. In addition, PCC0208009 reversed synaptic plasticity at the functional and structural levels by suppressing NMDA2B receptor and CDK5/MAP2 or CDK5/Tau pathway in ACC and amygdala.[1]
Conclusion and implications: These results support the role of IDO1-mediated molecular mechanisms in neuropathic pain and suggest that the IDO1 inhibitor PCC0208009 demonstrates selective pain suppression and could be a useful pharmacological therapy for neuropathic pain.[1]

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In the mouse glioma GL261 heterotopic model, the effects of PCC-0208009 on l-kynurenine/tryptophan (Kyn/Trp), tumor growth, flow cytometry for T cells within tumors, and immunohistochemistry for IDO and Ki67 were examined. In the rat glioma C6 orthotopic model, animal survival, flow cytometry for T cells within tumors, and immunohistochemistry for proliferating cell nuclear antigen (PCNA) and IDO were examined. The results show that PCC0208009 is a highly effective IDO inhibitor, not only directly inhibiting IDO activity but also participating in the gene regulation of IDO expression at the transcription and translation levels. PCC0208009 significantly enhanced the anti-tumor effects of temozolomide in GL261 and C6 models, by increasing the percentages of CD3+, CD4+, and CD8+ T cells within tumors and suppressing tumor proliferation. These findings indicate that PCC0208009 can potentiate the anti-tumor efficacy of temozolomide and suggest that combination of IDO inhibitor-based immunotherapy with chemotherapy is a potential strategy for brain tumor treatment.[3]

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Effects of PCC-0208009 in formalin test[1]
Intraplantar injection of formalin (2.5%, 20 μL) induced a typical biphasic nocifensive response including flinching and licking. The second-phase (15 min–45 min) was significantly inhibited by PCC0208009 in a dose-dependent manner (25, 50, 100 mg·kg−1, Fig. 2; F(5,78) = 18.10, p < 0.001). In addition, no significant difference was observed between 60 mg·kg−1 pregabalin and 50 or 100 mg·kg−1 PCC0208009 groups (p > 0.05).

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Effects of PCC-0208009 on the ongoing pain in SNL rats[1]
Ongoing pain is the cardinal symptom of neuropathic pain. Here, we tested whether PCC-0208009 could reduce the ongoing pain using CPP and open-field test assays, two widely used assays to detect ongoing pain. During the pre-conditioning phase, all rats preferred the chamber with black wall and smooth floor and there was no significant difference among the groups. 12.5, 25, and 50 mg·kg−1 PCC0208009 significantly increased the time the rats spent in drug-paired chamber as compared to the vehicle group (Fig. 3A; F(5,66) = 17.43, p < 0.001). In addition, 25 and 50 mg·kg−1 PCC0208009 demonstrated similar effects with 30 mg·kg−1 pregabalin in inducing CPP (p > 0.05).

Because the presence of pain reduces the general locomotion, we used the open-field test to directly measure whether PCC-0208009 could restore pain-suppressed locomotion in the SNL rats. As can be seen in Fig. 3, vehicle-treated SNL rats had lower frequency of locomotion and rearing as compared to the sham rats. Both pregabalin and 50 mg·kg−1 PCC0208009 significantly reversed pain-induced hypolocomotion (Fig. 3B-C; locomotion, F(5,66) = 2.48, p < 0.05; rearing, F(5,66) = 2.34, p = 0.051), while all the three doses of PCC0208009 demonstrated similar magnitude with pregabalin (30 mg·kg−1) in increasing the locomotor activity and rearing behavior (p > 0.05).

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Effects of PCC-0208009 on SNL-induced mechanical and thermal hypersensitivity [1]
Rats were tested for mechanical allodynia after a single PCC-0208009 dose. The average PWT decreased to 2.83 g ± 0.76 g within 10 d after the surgery in the SNL group, which was significantly different from the sham rats (12.09 g ± 0.30 g). 12.5, 25, and 50 mg·kg−1 PCC-0208009 dose-dependently increased the PWT at 30, 60 and 90 min post-single dose administration, with the peak anti-allodynia effect observed 60 min after drug administration (Fig. 3D; F(7, 88) = 143.53, p < 0.001; pregabalin, F(7, 88) = 78.77, p < 0.001; 12.5 mg·kg−1 PCC0208009, F(7, 88) = 12.00, p < 0.01; 25 mg·kg−1 PCC0208009, F(7, 88) = 21.53, p < 0.001; 50 mg·kg−1 PCC0208009, F(7, 88) = 42.76, p < 0.001).

We further tested the antinociceptive effect of PCC-0208009 at 60 min post-dosing in rats that received repeated drug treatment. PCC0208009 (12.5, 25, 50 mg·kg−1) significantly increased the PWT in a dose-dependent manner on POD17 and POD24 (Fig. 3F; F(5, 66) = 109.59, p < 0.001; pregabalin, F(5, 66) = 81.36, p < 0.001; 12.5 mg·kg−1 PCC0208009, F(5, 66) = 12.04, p < 0.01; 25 mg·kg−1 PCC0208009, F(5, 66) = 34.96, p < 0.001; 50 mg·kg−1 PCC0208009, F(5, 66) = 53.69, p < 0.001). Furthermore, the high dose of PCC0208009 (50 mg·kg−1) produced similar effect with 30 mg·kg−1 pregabalin on POD17 and POD24 (p > 0.05).

Thermal allodynia was tested after a single dose of PCC0208009 on POD11. PWL of the SNL-vehicle rats markedly decreased from 10.76 s ± 0.56 s to 6.40 s ± 0.45 s. PCC0208009 at the doses of 12.5, 25, and 50 mg·kg−1 increased the PWL at 30, 60 and 90 min post administration in a dose-dependent manner (Fig. 3E; F(7, 88) = 39.91, p < 0.001; pregabalin, F(7, 88) = 35.88, p < 0.001; 25 mg·kg−1 PCC0208009, F(7, 88) = 23.13, p < 0.05; 50 mg·kg−1 PCC0208009, F(7, 88) = 29.37, p < 0.001).

Repeated treatment with PCC-0208009 on thermal allodynia was also examined on POD18 and POD25. Both 25 and 50 mg·kg−1 PCC0208009 significantly increased the PWL as compared to the vehicle group (Fig. 3G; F(5, 66) = 45.05, p < 0.001; pregabalin, F(5, 66) = 51.82, p < 0.001; 12.5 mg·kg−1 PCC0208009, F(5, 66) = 14.63, p < 0.01; 25 mg·kg−1 PCC0208009, F(5, 66) = 34.24, p < 0.001; 50 mg·kg−1 PCC0208009, F(5, 66) = 33.08, p < 0.001). PCC0208009 (50 mg·kg−1) demonstrated better although statistically insignificant antinociceptive effect than 30 mg·kg−1 pregabalin (p > 0.05).

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Effects of PCC-0208009 on SNL-induced cognitive deficit and anxiogenic behaviors[1]
Cognitive impairment and emotional disorders like anxiogenic effects were important comorbid manifestations of chronic neuropathic pain in the clinic although this is rarely evaluated in preclinical pain studies along with pain sensitivity. Rats were treated daily with PCC0208009 for 28 days in order to examine its effect on cognition-altering and anxiogenic effects induced by neuropathic pain.

In the Morris water maze test, SNL rats spent more time to search the platform than sham rats and 50 mg·kg−1 PCC-0208009 prevented this effect (Fig. 4C; F(23,216) = 9.03, p < 0.001). During the probe test (POD25), SNL rats spent less time in the target quadrant than sham rats and 12.5, 25 and 50 mg·kg−1 PCC0208009 groups prevented this impairment (Fig. 4D; F(5,54) = 5.82, p < 0.001).
In the EOM test, SNL rats spent less time in the open area (Fig. 4E; F(5,66) = 4.37, p < 0.01) and were less active (Fig. 4F; F(5,66) = 5.57, p < 0.001) than the sham rats, suggesting an anxiety-like phenotype. 25 and 50 mg·kg−1 PCC0208009 significantly increased the time the rats spent in the open area without altering the locomotor activity in the SNL rats.

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Effects of PCC-0208009 on the locomotion, sedation and loss of righting reflex[1]
In the rota-rod assay, SNL rats demonstrated significantly reduced time spent on the rotor drum and the speed to fall off the drum. 50 mg·kg−1 PCC0208009 significantly increased the duration and maximal speed before falling off (Fig. 5A; F(5, 66) = 4.29, p < 0.01; Fig. 5B; F(5, 66) = 4.18, p < 0.01), whereas 30 mg·kg−1 pregabalin failed to achieve statistical significance (p > 0.05).

In the open-field test after a single dose treated, 12.5, 25, or 50 mg·kg−1 PCC-0208009 did not significantly reduce the overall locomotor activity as compared to control rats but 30 mg·kg−1 pregabalin significantly reduced the locomotor activity in rats (p < 0.001).

In the pentobarbital-induced loss of righting reflex experiment, PCC-0208009 at the doses up to 100 mg·kg−1 did not enhance a sub-threshold dose of pentobarbital-induced loss of righting reflex such that the mice showed similar sleep latency (Fig. 5E; F(5, 66) = 7.02, p < 0.001) and sleep duration (Fig. 5F; F(4, 55) = 27.19, p < 0.001) as compared to normal control mice, as well as the number of mice entering sleep (Fig. 5D; Mann-Whitney test, p < 0.01). For comparison, 60 mg·kg−1 pregabalin significantly increased the loss of righting reflex in the mice (p < 0.001).

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Effects of PCC-0208009 on the IDO1 enzyme activity in ACC and amygdala in SNL rats As compared with sham group, the Kyn/Trp ratio in the ACC and amygdala in SNL rats was significantly increased, which was also observed in the spinal cord dorsal horn and hippocampus, suggesting increased IDO1 enzyme activity (Fig. 6A; spinal cord dorsal horn, F(2,15) = 4.84, p < 0.05; hippocampus, F(2,15) = 8.21, p < 0.01; ACC, F(2,15) = 11.41, p < 0.001; amygdala, F(2,15) = 5.05, p < 0.05). The Ido1 mRNA and IDO1 protein expression were also elevated in SNL rats as compared to the sham rats. PCC0208009 at a dose that significantly reduced the pain-related behaviors in SNL rats (50 mg·kg−1) significantly decreased the Kyn/Trp ratio in the ACC, amygdala, hippocampus and the spinal cord dorsal horn, and significantly reduced the Ido1 mRNA (Fig. 6B; ACC, F(2,14) = 8.41, p < 0.005; amygdala, F(2,14) = 23.73, p < 0.001) and protein expression (Fig. 6C-D; ACC, F(2,14) = 46.64; amygdala, F(2,14) = 16.45, all p < 0.001) in ACC and amygdala.

In the NOR test, the rats that received SNL surgery spent much less time to explore the novel object as compared with the sham group (Fig. 4A; F(5,66) = 3.40, p < 0.01 for familiar object; F(5,66) = 27.22, p < 0.001 for novel object), and the recognition index was significantly increased by repeated treatment with 25 or 50 mg·kg−1 PCC-0208009 (Fig. 4B; F(5,66) = 15.42, p < 0.001).

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Effects of PCC-0208009 on the IL-6-JAK2/STAT3-IDO1-GCN2 pathway in the ACC and amygdala in SNL rats[1]
Because IL-6 activation increases IDO1 expression through JAK2/STAT3 pathway [8], we examined the effect of the indirect IDO1 inhibitor PCC0208009 on this pathway in SNL rats. SNL surgery upregulated the co-expression of IDO1 and GFAP in astrocytes of the ACC and amygdala (Fig. 6G), along with the increased GFAP protein expression (Fig. 6E-F; ACC, F(2,12) = 69.83; amygdala, F(2,15) = 23.21; all p < 0.001). SNL surgery also significantly increased the production of pro-inflammatory cytokines (IL-6, IL-1β) and the expression of its associated receptors (IL-6Rα, IL-1R1) (Fig. 7A; ACC, F(2,15) = 20.33; amygdala, F(2,15) = 10.61; all p < 0.001) (Fig. 7A; ACC, F(2,15) = 40.12; amygdala, F(2,15) = 26.42; all p < 0.001) (Fig. 7B-C; ACC, F(2,15) = 36.89; amygdala, F(2,15) = 28.67; all p < 0.001) (Fig. 7D-E; ACC, F(2,12) = 27.24; amygdala, F(2,15) = 24.21; all p < 0.001), the phosphorylation of GCN2 at Thr667 (Fig. 7F-G; ACC, F(2,15) = 31.99; amygdala, F(2,15) = 55.71; all p < 0.001), JAK2 at Y1008 (Fig. 7H-I; ACC, F(2,15) = 26.61; amygdala, F(2,15) = 10.48; all p < 0.001) and STAT3 at Tyr705 (Fig. 7J-K; ACC, F(2,15) = 23.91; amygdala, F(2,15) = 38.39; all p < 0.001) in the ACC and amygdala. These changes were significantly reduced by 50 mg·kg−1 PCC0208009 treatment.

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Effects of PCC-0208009 on the synaptic plasticity in SNL rats [1]
Golgi staining was used to examine the morphological changes on the dendrites and dendritic spines in the ACC and amygdala (Fig. 8A). The total dendritic spine density (Fig. 8B; ACC, F(2,72) = 55.76; amygdala, F(2,72) = 37.72; all p < 0.001), number of mushroom-like (Fig. 8B; ACC, F(2,72) = 28.13; amygdala, F(2,72) = 31.29; all p < 0.001) and thin-like (Fig. 8B; ACC, F(2,72) = 9.52; amygdala, F(2,72) = 21.76; all p < 0.001) spine density were significantly increased in the rats that received SNL surgery as compared to sham rats, and 50 mg·kg−1 PCC0208009 treatment prevented these morphological changes. PCC/PCC-0208009 on Kyn/Trp in GL261 subcutaneous mouse model [3]
To investigate the biochemical mechanism of PCC in vivo, mouse model bearing GL261 were generated. The levels of the pharmacodynamic biomarkers Kyn/Trp and PCC in the plasma and tumors were determined at different time points after mice were i.g. administered a single dose of PCC at 100 mg/kg. As shown in Figure 3(a), PCC was highly distributed in the tumors and plasma. Compared to pre-dose (0 h), the ratios of Kyn/Trp in the tumor and plasma samples were all significantly decreased from 2 to 8 h after drug administration (P < 0.05) (Figure 3(b)).

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Combinatorial treatment with PCC/PCC-0208009 and TMZ on GL261 subcutaneous mouse model [3]
To explore the anti-tumor effects and mechanisms of PCC in combination with TMZ on tumor growth in vivo, mouse GL261 subcutaneous model was prepared. Mice were treated with Vehicle, PCC, TMZ, or PCC plus TMZ. The body weights of animals and the volumes and weights of tumors were measured. Tumor samples were analyzed by flow cytometry for T cell populations and were immunohistochemically examined for IDO and Ki67 expression.

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Body weights of tumor-bearing mice [3]
As shown in Figure 3(c) and Table 1, compared with the vehicle group, no significant effects on the body weight of animals were observed in the PCC-0208009/PCC group during the study (P > 0.05). From day 12 and day 16 to the end of this study, mouse body weights in the TMZ and PCC plus TMZ groups were noted to have significant decreases compared with the vehicle group (P < 0.05). The body weight decreased by 12.56% and 12.66% in the TMZ group and PCC plus TMZ group, respectively, compared to that at the beginning of the study (P < 0.05). However, no decrease in body weight was observed between the PCC plus TMZ group and the TMZ group (P > 0.05), indicating that PCC did not increase the side effects of TMZ.

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Tumor volume and tumor weight [3]
The suppression of tumor growth was observed in the PCC/PCC-0208009, TMZ and PCC plus TMZ groups, and PCC combined with TMZ showed stronger suppression of tumor growth than PCC or TMZ alone (Figure 3(d)). At the end of the study, the mean tumor weights in PCC, TMZ, and combinatorial treatment groups were obviously smaller than those in the vehicle group (P < 0.05), and the tumor IRs were 42.59%, 53.01%, and 70.83%, respectively (Figure 3(e) and Table 1). No significant difference in tumor weight was observed between the PCC group and TMZ group (P > 0.05). The mean tumor weights in the PCC plus TMZ group were less than those in the PCC and TMZ groups (P < 0.05).

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T cell subtypes in GL261 tumor [3]
To understand the immunological changes in animals treated with PCC/PCC-0208009 and TMZ, T cell populations within tumors were analyzed. As shown in Figure 4(a) and (b), compared with the vehicle group, the percentages of CD3+, CD4+, and CD8+ T cells in the PCC group were noted to have slight increases (P > 0.05) while there were significant decreases in the TMZ group (P < 0.05). In the PCC plus TMZ group, the percentage of CD3+ cells was significantly higher than those in the vehicle and TMZ groups (P < 0.05), and the percentages of CD4+ and CD8+ were higher than those in the vehicle group (P > 0.05) and significantly higher than those in the TMZ group (P < 0.05).

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IDO and Ki67 expression in GL261 tumor [3]
Immunohistochemical detection was carried out to observe the changes in IDO and Ki67 expression in GL261 tumors. The Ki67 protein is a cellular marker for proliferation. As shown in Figure 4(c) and (d), Ki67 expression was noted at a high level in the vehicle group. Considerable decreases in Ki67 expression were observed in the PCC-0208009/PCC and TMZ groups (P > 0.05), and a significant decrease was observed in the PCC plus TMZ group compared to the vehicle group (P < 0.05). There was no significant difference in Ki67 expression between the TMZ and PCC groups (P > 0.05). The expression of IDO is shown in Figure 4(e) and (f). The high expression level of IDO was observed in GL261 tumors in the vehicle group, while IDO expression in the PCC group was significantly decreased (P < 0.05).

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Combinatorial treatment with PCC-0208009/PCC and TMZ on C6 orthotopic rat model [3]
The syngeneic intracranial orthotopic model of rat glioma C6 were prepared by implanting tumor cells into the caudate nucleus of SD rats. The day of tumor inoculation was designated day 1.

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Animal survival and body weights [3]
The animal survival curve is shown in Figure 5(a). No significant prolongation in animal survival was observed in the PCC-0208009/PCC or TMZ groups compared with the vehicle group (P > 0.05). Animal survival in the PCC plus TMZ group was significantly prolonged than compared with the vehicle group (P < 0.05) and compared with the PCC and TMZ groups (P < 0.05).

As shown in Figure 5(b), compared with the vehicle group, no significant effects on the body weight of rats were observed in the PCC-0208009/PCC group during the study (P > 0.05); from day 8 to the end of this study and from day 8 to day 21, rat body weights in the TMZ group and PCC plus TMZ group were noted to show significant decreases (P < 0.05). However, no additional decrease in body weight was observed between the PCC plus TMZ group and the TMZ group (P > 0.05), indicating that PCC did not increase the side effects of TMZ in this model.

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T cell subtypes in C6 tumor [3]
To understand the immunological mechanisms of underlying the anti-tumor effects of PCC-0208009/PCC and combinatorial treatment of PCC and TMZ, T cell populations within tumors were analyzed. As shown in Figure 5(c) and (d), compared with the vehicle group, the percentages of CD3+, CD4+, and CD8+ T cells in the PCC group were increased, ranging from 40% to 70%, and were slightly decreased in the TMZ group. In the PCC plus TMZ group, the percentages of CD3+, CD8+, and CD4+ T cells were significantly increased compared with the vehicle and TMZ groups by approximately about twofold.

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PCNA and IDO expression in C6 tumor by immunohistochemistry [3]
The results from immunohistochemical staining are shown in Figure 6. PCNA expression is a useful prognostic and diagnostic biomarker for gliomas.19 The expression of PCNA was very low in the brain tissue and highly expressed in the tumor of the vehicle group (Figure 6(a)). Compared with the vehicle group, PCNA in the PCC-0208009/PCC, TMZ, and PCC plus TMZ groups was significantly decreased (P < 0.05). The PCNA in the PCC plus TMZ group was significantly decreased compared to the PCC and TMZ groups (P < 0.05). IDO expression in the PCC group was significantly decreased in the C6 tumor compared with that in the vehicle group (P < 0.05) (Figure 6(b)).

IDO1 activity assay [1]
The spinal cord and brain Trp and Kyn concentrations were analyzed by LC-MS/MS system. Rats (n = 6 per group) were euthanized and the L3-L4 spinal dorsal horns, hippocampus, ACC and amygdala tissues were dissected and harvested after drug administrations for IDO1 activity determination. All tissues were homogenized by deionized water (10 mg of tissue in 40 μL), centrifuged and supernatants collected. The ratio of Kyn/Trp in each tissue was determined using the method describe in the PK study.

Western Blot Analysis[3]
Cell Types: HeLa cells
Tested Concentrations: 0, 50, 100, 200 nM
Incubation Duration: 48 hrs (hours)
Experimental Results: The IDO protein expression induced by IFN-γ was dose-dependently suppressed by PCC, which demonstrated significant differences at 100 and 200 nM (P < 0.05).

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RT-PCR [3]
Cell Types: HeLa cells
Tested Concentrations: 0, 50, 100, 200 nM
Incubation Duration: 48 hrs (hours)
Experimental Results: The IDO mRNA expression by IFN-γ was dose-dependently suppressed by PCC, which demonstrated significant differences at all doses compared with the IFN-γ group.

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Effects of PCC/PCC-0208009 on the viability and proliferation of HeLa cells [3]
HeLa cells were seeded into 96-well plates at 6 × 103 cells/well. After culture for 10–12 h, the culture medium was replaced with fresh medium with 100 ng/mL interferon gamma (IFN-γ), or PCC-0208009 at 25, 50, 100, or 200 nM with or without 100 ng/mL IFN-γ, and medium containing 0.1% DMSO was used as the vehicle treatment. At 72 h after the addition of drugs, cell viability and proliferation were observed. In the viability assay, adherent cells were washed, trypsinized, and counted using a CountStar IC1000 Automated Cell Counter, and viability of the counted cells was confirmed by 0.1% trypan blue exclusion, which was indicated as a percentage of the living cells. In the proliferation test, cells were assayed using the 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide (MTT) method. The cell proliferation rate of control cells was represented as 100%, and the relative cell proliferation rates of the other groups were calculated.

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Inhibition effects of PCC/PCC-0208009 on IDO activity in HeLa cells [3]
HeLa cells were seeded in 96-well plates at 6 × 103 cells/well. After culture for 10–12 h, the medium was replaced with fresh medium only or medium containing 100 ng/mL IFN-γ. Except for the vehicle group, the other groups were all induced by IFN-γ for 24 h. Then, the medium was replaced with fresh medium only, medium containing 100 ng/mL IFN-γ, or containing 100 nM PCC-0208009. At 0.5, 1, 2, 5, 10, 24, 48, and 72 h after the addition of drugs, the cell supernatants were harvested for determination of l-kynurenine (Kyn) and tryptophan (Trp) by liquid chromatography tandem-mass spectrometry (LC-MS/MS).

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Effects of PCC/PCC-0208009 on IDO expression in HeLa cells [3]
HeLa cells were seeded in 6-well plates at 2 × 105 cells/well. After culture for 10–12 h, the medium was replaced with fresh medium, 100 ng/mL IFN-γ, or 100 ng/mL IFN-γ plus PCC-0208009 at 50, 100, and 200 nM, respectively. After incubation for 48 h, cells were harvested. The expression of IDO at the protein and mRNA levels was detected by Western blot and quantitative real-time polymerase chain reaction (qRT-PCR), respectively.

Animal/Disease Models: Adult male Sprague Dawley rats (180 g-200 g) [1]
Doses: 50 mg/kg
Route of Administration: Single po (oral gavage)
Experimental Results: The highest concentrations of PCC-0208009 in plasma and brain were observed at 60 min after administration.

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Animal/Disease Models: Adult male SD (Sprague-Dawley) rats bearing spinal nerve ligation (SNL)[1]
Doses: 12.5 mg/kg, 25 mg/kg, 50 mg/kg
Route of Administration: po (oral gavage); once
Experimental Results: demonstrated the behavioral tests and the timelines.

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Formalin-induced inflammatory pain model [1]
Formalin test was carried out in mice according to a modified protocol in a transparent test chamber (23 cm × 13 cm × 14 cm; length × width × height). Sham (0.5% CMC-Na, p.o.), vehicle (0.5% CMC-Na, p.o.), pregabalin (60 mg·kg−1, p.o.) or PCC-0208009 (25, 50, 100 mg·kg−1, p.o.) were administered 60 min before formalin injection in a volume of 10 mL·kg−1 body weight (n = 14 mice per group) and then mice were acclimatized in the test cage for 10 min prior to formalin injection. 20 μL of 2.5% formalin (36.5–38% formaldehyde solution diluted in 0.9% saline) was injected to the right hind paw subcutaneously (s.c.) (sham mice were injected with 20 μL 0.9% saline) and pain-like nocifensive behaviors were monitored as 5 min bins for a total duration of 60 min. A mirror placed behind the observation chamber allowed the unhindered observation of the formalin-injected paw. The cumulative licking, shaking and lifting time (s) of the injected paw were counted as described. Behavioral response during 0–5 min post-formalin was considered Phase I and that during 15 min-45 min post-formalin injection was considered Phase II.

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Animal model of neuropathic pain [1]
A model of neuropathic pain was induced by the ligation of L5 spinal nerve as described previously. Briefly, rats were anesthetized with sodium pentobarbital (50 mg·kg−1, i.p.). Thereafter, a 2 cm long incision was made at the level of the posterior iliac crest to access the right lumbar spinal nerves under the cold-light. L5 spinal nerve was identified and carefully dissected free from the adjacent L4 spinal nerve and then tightly ligated distal to the dorsal root ganglia using 6–0 silk suture. The SNL operation was completed by closing the muscles and skin with a 3–0 and 2–0 silk suture in layers. Daily intramuscular injection of 0.4 mL penicillin was administered post-surgically for three days to prevent infection. In the sham surgery, the L5 nerve was not ligated. Rats were allowed to recover for 7 days before behavioral testing.

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Experiment design in SNL rats [1]
The timelines and experimental designs of the behavioral tests were shown in Fig. 1E. The experiments from 1 to 4 were all for SNL rats, and each group in experiments was 12 rats but were not the same animals. Experiment 1: to evaluate the antinociceptive effects of an acute dose of PCC-0208009 on neuropathic pain, six groups of rats (n = 12 per group) with the following treatments were used: sham (0.5% CMC-Na, p.o.), vehicle (0.5% CMC-Na, p.o.), pregabalin (30 mg·kg−1, p.o.) and PCC0208009 (12.5, 25 and 50 mg·kg−1, p.o.). Mechanical allodynia and thermal hyperalgesia were measured before and 30, 60 and 90 min after the drug administration on day 10 post-SNL surgery (POD10) and POD 11, respectively.

Experiment 2: to measure the antinociceptive effects of PCC-0208009 on spontaneous pain, conditioned place preference (CPP) was performed during POD10 to POD17 and six groups of rats (n = 12 per group) with the following treatments were used: sham (0.5% CMC-Na, p.o.), vehicle (0.5% CMC-Na, p.o.), pregabalin (30 mg·kg−1, p.o.) and PCC0208009 (12.5, 25 and 50 mg·kg−1, p.o.).

Experiment 3: to measure the effects of repeated administration of PCC-0208009 on the neuropathic pain and motor function, six groups of rats (n = 12 per group) with the following treatments were used: sham (0.5% CMC-Na, p.o.), vehicle (0.5% CMC-Na, p.o.), pregabalin (30 mg·kg−1, p.o.) and PCC0208009 (12.5, 25 and 50 mg·kg−1, p.o.). Drugs were administered p.o. twice daily for 21 consecutive days. Mechanical allodynia was measured 60 min after drug administration on POD17 and POD24, and thermal hyperalgesia was measured 60 min after drug administration on POD18 and POD25. Rota-rod and open field tests were performed 60 min after drug administration on POD26-POD28 and POD29, respectively. Besides, the ACC and amygdala tissues for immunofluorescence assay were collected on POD14. The spinal cord dorsal horn, hippocampus, ACC and amygdala tissues for IDO1 activity assay as well as the ACC and amygdala tissues for IL-6, IL-1β enzyme linked immunosorbent assay (ELISA), Golgi-cox stain, western blotting and reverse transcription-polymerase chain reaction (RT-PCR) were collected on POD30 (sham n = 6; vehicle, n = 6; PCC0208009 50 mg·kg−1, n = 6).

Experiment 4: to measure the effects of repeated administration of PCC-0208009 on anxiety-like and cognition-related behaviors, seven groups of rats (n = 12 per group) with the following treatments were used: sham (0.5% CMC-Na, p.o.), vehicle (0.5% CMC-Na, p.o.), diazepam (3 mg·kg−1), risperidone (1 mg·kg−1) and PCC0208009 (12.5, 25 and 50 mg·kg−1). Drugs were administered p.o. twice daily for 19 consecutive days. Novel object recognition (NOR) test was evaluated 60 min postdose on POD14 and POD15, Morris water maze (MWM) test was performed 60 min postdose on POD21 and POD25, and elevated zero maze test (EOM) was performed 60 min postdose on POD28.

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Pentobarbital-induced loss of righting reflex in normal mice [1]
Mice (n = 12 per group) were acclimated to the environment in a transparent test chamber (23 cm × 13 cm × 14 cm; length × width × height) for at least 2 h and randomly assigned into six groups: vehicle (0.5% CMC-Na, p.o.), diazepam (3 mg·kg−1, p.o.), pregabalin (60 mg·kg−1, p.o.) and PCC-0208009 (25, 50, 100 mg·kg−1, p.o.) before testing. Forty-five min before pentobarbital sodium (28 mg·kg−1, i.p.) injection, mice received the respective drug administrations. Immediately after pentobarbital administration, the loss of righting reflex was tested for 30 min. The number of mice with the loss of righting reflex for at least 1 min within the 30 min test period was recorded. Sleep latency was defined as the time between pentobarbital injection and the time that loss of righting reflex was observed, and sleep duration was defined as the time between the onset of loss of righting reflex and the time that mice regained righting reflex.

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Locomotor activity in normal rats [1]
Rats (n = 12 per group) were transferred to the test room at least 2 h before the test and were randomly assigned into five groups: vehicle (0.5% CMC-Na, p.o.), pregabalin (30 mg·kg−1, p.o.) and PCC-0208009 (12.5, 25, 50 mg·kg−1, p.o.). Fifty-five minutes after the respective drug administrations, rats were placed into test boxes (50 cm × 50 cm × 50 cm; length × width × height) and the locomotor activity was recorded and analyzed for 10 min by TopScan monitoring system.

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Effects on the pharmacodynamic biomarker Kyn/Trp [3]
When the tumor volumes reached approximately 300–400 mm3, the mice were intragastrically (i.g.) administered a single dose of PCC-0208009/PCC at 100 mg/kg. At 0, 2, 4, and 8 h after administration, plasma and tumor were collected for the detection of Trp, Kyn, and PCC, with five animals per time point. Trp, Kyn, and PCC were all measured by LC-MS/MS.

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Anti-tumor effects of PCC-0208009/PCC in combination with TMZ [3]
When the tumors reached approximately 100 mm3, the mice were randomized into four groups: Vehicle, PCC-0208009/PCC, TMZ, and PCC plus TMZ; each group contained 10 mice. PCC was i.g. administered at 100 mg/kg twice daily, TMZ was i.g. administered at 100 mg/kg once every 2 days, and the vehicle group was i.g. administered 1% SCMC twice daily. The dosing volume was 0.1 mL/10 g. During the study, the body weight of animals and tumor volumes were measured once every 3 or 5 days. At the end of the study, tumors were collected and quickly weighed. The mean tumor weight (MTW) in each group was obtained. The inhibition rate (IR in %) was calculated by the following formula: IR (%) = [(MTWvehicle − MTWtreatment)/MTWvehicle] × 100. Five tumors were randomly selected for flow cytometry analysis, and three tumors were randomly selected for immunohistochemical detection.

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Animal survival study [3]
According to the body weight, animals were randomly divided into four groups: Vehicle, PCC-0208009/PCC, TMZ, and PCC plus TMZ. Each group contained 10 animals. PCC was i.g. administered at 50 mg/kg twice daily, TMZ was i.g. administered at 50 mg/kg once every 2 days, and the vehicle group was i.g. administered with 1% SCMC twice daily, from day 5 to day 35. The dosing volume was 0.2 mL/100 g. During the study, the body weight was measured twice every week and the survival times of rats were recorded and analyzed. Animals were kept in the study until the rats were dead or dying.

The PK/PD profile of PCC-0208009 after a single oral administration in rats [1]
In order to assess the mean plasma or brain concentration-time profiles and the inhibition of IDO1 enzyme activity to produce Kyn from Trp in rats, PK/PD testing was conducted in rats. PCC0208009 was detected at 60, 120 and 240 min after drug administration in plasma and brain samples, and the highest concentrations of PCC0208009 in plasma (Fig. 1B) and brain (Fig. 1C) were observed at 60 min after administration. Concomitantly, the Kyn/Trp ratio decreased at 60, 120 and 240 min postdose, with the minimum level in the plasma and the brain seen at 60 min post-dose (Fig. 1D).

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Distribution of PCC-0208009/PCC in rat brain [3]
At 15 days after tumor inoculation, the rats were i.g. administered a single dose of PCC-0208009/PCC at 50 mg/kg, and the cerebrum and cerebellum were harvested for the detection of PCC content. The PCC contents in the cerebrum and cerebellum were 122.3 and 113.7 nM/g at 0.5 h, 70.6 and 63.5 nM/g at 2.5 h, 32.8 and 34.3 nM/g at 6.5 h, respectively. These results indicated that PCC could cross the blood-brain barrier and distribute into the brain.

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Pharmacokinetic (PK) and pharmacodynamic (PD) studies [1]
The PCC-0208009 concentration and the Kyn/Trp ratio in the plasma and brain of rats were measured. Healthy Sprague-Dawley rats were given a single oral dose of PCC-0208009 at 50 mg·kg−1. Plasma and brain samples were collected at 60, 120 and 240 min post-drug administration. Brain samples were homogenized in purified water (weight to volume ratio of 4:1). The concentrations of PCC0208009 in the plasma and brain were determined using a validated high-performance LC-MS/MS method, analyzed by an Agilent 1100 series HPLC system and a TSQ Quantum Access tandem mass spectrometer equipped with electrospray ionization (ESI) source. Samples (5 μL) were pretreated by a liquid–liquid extraction with 3 mL of tert-butyl methyl ether-dichloromethane (3/2, v/v). The mobile phase used acetonitrile-water (80:20, v/v) containing 0.02 mM ammonium acetate and was kept at a flow rate of 0.2 mL·min−1. A symmetry C18 column (150 mm × 2.1 mm i.d., 3.5 μm, Waters, USA, SN: 0124362291265) was used for the HPLC system. The following parameters were applied: spray voltage of 4 kV; sheath gas and auxiliary gas pressures of 30 and 5 psi, respectively; capillary temperature of 350℃; and argon gas pressure of 1.5 milli-Torr. The collision induced dissociation voltage was 18 V for all samples. The transitions (precursor to product) monitored were m/z 436.1 → 253.9. The quantities of Kyn and Trp were determined according to methods previously established in the laboratory

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Distribution of PCC-0208009/PCC in the rat brain [3]
After tumor inoculation for 15 days, rats were i.g. administered a single dose of PCC at 50 mg/kg. At 0.5, 2.5, and 6.5 h after dosing, the cerebrum and cerebellum were harvested for detection of PCC content using LC-MS/MS.

[1]. PCC0208009, an indirect IDO1 inhibitor, alleviates neuropathic pain and co-morbidities by regulating synaptic plasticity of ACC and amygdala. Biochem Pharmacol. 2020 Jul;177:113926.

[2]. Development of a series of novel o-phenylenediamine-based indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors. Bioorg Med Chem Lett. 2018 Feb 15;28(4):732-736.

[3]. PCC0208009 enhances the anti-tumor effects of temozolomide through direct inhibition and transcriptional regulation of indoleamine 2,3-dioxygenase in glioma models. Int J Immunopathol Pharmacol. Jan-Dec 2018;32:2058738418787991.

Background and purpose: Indoleamine 2, 3-dioxygenase 1 (IDO1) has been linked to neuropathic pain and IDO1 inhibitors have been shown to reduce pain in animals. Some studies have indicated that IDO1 expression increased after neuropathic pain in hippocampus and spinal cord, whether these changes existing in anterior cingulate cortex (ACC) and amygdala remains obscure and how IDO1 inhibition leads to analgesia is largely unknown. Here, we evaluated the antinociceptive effect of PCC0208009, an indirect IDO1 inhibitor, on neuropathic pain and examined the related neurobiological mechanisms. Experimental approach: The effects of PCC0208009 on pain, cognition and anxiogenic behaviors were evaluated in a rat model of neuropathic pain. Motor disorder, sedation and somnolence were also assessed. Biochemical techniques were used to measure IDO1-mediated signaling changes in ACC and amygdala. Key results: In rats receiving spinal nerve ligation (SNL), IDO1 expression level was increased in ACC and amygdala. PCC0208009 attenuated pain-related behaviors in the formalin test and SNL model and increased cognition and anxiogenic behaviors in SNL rats at doses that did not affect locomotor activity and sleeping. PCC0208009 inhibited IDO1 expression in ACC and amygdala by inhibiting the IL-6-JAK2/STAT3-IDO1-GCN2-IL-6 pathway. In addition, PCC0208009 reversed synaptic plasticity at the functional and structural levels by suppressing NMDA2B receptor and CDK5/MAP2 or CDK5/Tau pathway in ACC and amygdala. Conclusion and implications: These results support the role of IDO1-mediated molecular mechanisms in neuropathic pain and suggest that the IDO1 inhibitor PCC0208009 demonstrates selective pain suppression and could be a useful pharmacological therapy for neuropathic pain. [1]

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A novel series of o-phenylenediamine-based inhibitors of indoleamine 2,3-dioxygenase (IDO) has been identified. IDO is a heme-containing enzyme, overexpressed in the tumor microenvironment of many cancers, which can contribute to the suppression of the host immune system. Synthetic modifications to a previously described diarylether series resulted in an additional degree of molecular diversity which was exploited to afford compounds that demonstrated significant potency in the HeLa human cervical cancer IDO1 assay. [2]

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Indoleamine 2,3-dioxygenase (IDO), which is highly expressed in human glioblastoma and involved in tumor immune escape and resistance to chemotherapy, is clinically correlated with tumor progression and poor clinical outcomes, and is a promising therapeutic target for glioblastoma. IDO inhibitors are marginally efficacious as single-agents; therefore, combination with other therapies holds promise for cancer therapy. The aim of this study was to investigate the anti-tumor effects and mechanisms of the IDO inhibitor PCC0208009 in combination with temozolomide. The effects of PCC0208009 on IDO activity inhibition, and mRNA and protein expression in HeLa cells were observed. In the mouse glioma GL261 heterotopic model, the effects of PCC0208009 on l-kynurenine/tryptophan (Kyn/Trp), tumor growth, flow cytometry for T cells within tumors, and immunohistochemistry for IDO and Ki67 were examined. In the rat glioma C6 orthotopic model, animal survival, flow cytometry for T cells within tumors, and immunohistochemistry for proliferating cell nuclear antigen (PCNA) and IDO were examined. The results show that PCC0208009 is a highly effective IDO inhibitor, not only directly inhibiting IDO activity but also participating in the gene regulation of IDO expression at the transcription and translation levels. PCC0208009 significantly enhanced the anti-tumor effects of temozolomide in GL261 and C6 models, by increasing the percentages of CD3+, CD4+, and CD8+ T cells within tumors and suppressing tumor proliferation. These findings indicate that PCC0208009 can potentiate the anti-tumor efficacy of temozolomide and suggest that combination of IDO inhibitor-based immunotherapy with chemotherapy is a potential strategy for brain tumor treatment. [3]

C29H35N7O

497.6345

497.29

C, 69.99; H, 7.09; N, 19.70; O, 3.21

1668565-74-9

90718185

White to off-white solid powder

6.5

3

5

9

37

686

0

O=C(N([H])C1C([H])=C([H])C(C([H])([H])[H])=C([H])C=1[H])N([H])C1C([H])=C(C2=C([H])C([H])=C([H])C([H])=C2C2N=NN([H])N=2)C([H])=C([H])C=1N(C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H])C([H])([H])C([H])(C([H])([H])[H])C([H])([H])[H]

CJNMMPAEIYFQIJ-UHFFFAOYSA-N

InChI=1S/C29H35N7O/c1-19(2)17-36(18-20(3)4)27-15-12-22(24-8-6-7-9-25(24)28-32-34-35-33-28)16-26(27)31-29(37)30-23-13-10-21(5)11-14-23/h6-16,19-20H,17-18H2,1-5H3,(H2,30,31,37)(H,32,33,34,35)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 2.5 mg/mL (5.02 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.02 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)