**4. The association between IFN treatment and changes in the TRP-KYN pathway on depression as a side effect in humans and mice**

### **4.1. Changes in the levels of serum TRP and its metabolites in HCV patients with IFN-α therapy**

In order to further clarify the relationship between the IDO1-induced KYN pathway and the development of depressive symptoms during IFN-α therapy, we conducted a study in which we measured TRP metabolites of the KYN pathway in the serum of HCV patients undergoing IFN-α therapy.

A total of 49 patients (32 males and 17 females; mean age 54.0 ± 2.3 years) suffering from CHC were recruited. **Table 1** shows the clinical characteristics of patients with HCV. In this study, most of patients were treated with recombinant (r) IFN-α 2b or pegylated (PEG)-IFN-α 2b (21 patients (42.9%) received each medicine, respectively). Five patients (10.2%) were treated with natural (n) IFN-α, and others received PEG-IFN-α 2a (2.0%) and rIFN-α 2a (2.0%), individually. All interferons have almost the same efficiency and induce about the same activation of the KYN pathway [36]. No patient had a past record of psychiatric treatment, and all were off from depressive symptoms prior to IFN-α treatment. They did not take any antidepressant medications during the study period. At an average of 104.2 ± 15.8 days after the IFN-α administration, some patients presented with apathy, social isolation tendencies, melancholy, depressed mood, and an intention to stop IFN administration. Patients who felt depressed mood were referred for psychiatric

**3. Side effect: IFN-induced depression and tryptophan metabolism**

42 Pharmacokinetics and Adverse Effects of Drugs - Mechanisms and Risks Factors

5-HT pathway to the KYN pathway, resulting in a decrease in 5-HT levels.

IFN-α has been shown to develop depression in many diseases, not only CHC, but also in melanoma, chronic myelogenous leukemia, and renal cell carcinoma [26]. However, CHC patients may be more susceptible to developing IFN-induced depression than patients with other disorders, possibly due to a baseline 5-HT system dysfunction. Depression in CHC patients may result from changes in platelet 5-HT function, with decreased 5-HT concentrations during CHC infection compensated for by a decrease in reuptake and metabolism [1]. Immune activation, particularly by IFN-γ, affects the catabolism of TRP, a precursor of 5-HT, by inducing expression of IDO1. IDO1 is the first and rate-limiting enzyme that converts TRP to N-formyl-L-kynurenine, which is further metabolized to QUIN (**Figure 2**). IFN treatment of CHC patients results in a decrease in plasma TRP and an increase in plasma KYN [8]. Another clinical study with cancer patients has shown that immunotherapy with IFN-α significantly increases the severity of depressive symptoms, which is related to a depletion of serum 5-HT and induction of the catabolism of TRP to KYN [27]. Thus, TRP catabolism switches from the

IDO1 is induced by several pro-inflammatory cytokines including IFNs (IFN-α/β, γ), TNF-α, and interleukin 6 (IL-6). It is also widely accepted that IFNs, especially IFN-γ, are essential factors for IDO1 induction since two ISREs and IFN-γ-activated site (GAS) element sequences are found in the 5′-flanking region of the IDO1 gene [28]. Recent preclinical studies in mice have demonstrated that pharmacological inhibition of IDO1 enzymatic activity or genetic deletion of IDO1 abrogates acute and chronic inflammation-dependent behavioral changes induced by peripheral or central administration of lipopolysaccharide (LPS) [29–33]. Additionally, it has been reported that peripheral administration of KYN alone can induce depression-like behavior in rats [34]. In a clinical study, patients receiving IFN-α therapy showed increases in the total Montgomery-Asberg Depression Rating Score (MADRS), an index of depressive symptoms similar to the KYN/TRP ratio; this indicates IDO1 activity and the KYN/KA ratio, which reflects a neurotoxic challenge [35]. These findings suggest that only TRP depletion itself may not be required for the induction of behavioral changes as a result of IDO1 activation; and that KYN and its neuroactive metabolites are more related to cytokine-induced depression-like behaviors than TRP depletion. However, it is still unclear whether direct activation of IDO1 and KYN metabolites plays a definitive role in the induction of depressive symptoms by IFN-α treatment.

**4. The association between IFN treatment and changes in the TRP-KYN** 

**4.1. Changes in the levels of serum TRP and its metabolites in HCV patients with IFN-α** 

In order to further clarify the relationship between the IDO1-induced KYN pathway and the development of depressive symptoms during IFN-α therapy, we conducted a study in which we measured TRP metabolites of the KYN pathway in the serum of HCV patients undergoing

**pathway on depression as a side effect in humans and mice**

**therapy**

IFN-α therapy.


"Depression (−)": HCV patients without depression, "Depression (+)": HCV patients with depression following IFN-α therapy [47]. HCV, hepatitis C virus; AST, aspartate aminotransferase; ALT, alanine aminotransferase.


For all HCV patients, blood was collected before the onset of IFN-α therapy, as well as 2 and 4 weeks after the onset of therapy, and after the end or cessation of therapy. See **Figure 3a** for a detailed blood sampling schedule.\* *p*<0.05 *versus* Depression (−) [47].

**Table 1.** Clinical information for HCV patients undergoing IFN-α therapy.

evaluation and identified as major depressive disorder (MDD) by a psychiatrist. Nineteen of the HCV patients were diagnosed with depressive symptoms [depression (+)], while 30 of them did not present depressive symptoms [depression (−)]. The diagnosis to verify the incidence of depressive symptoms associated to MDD was made according to the DSM-IV (Diagnostic and Statistical Manual of Mental Disorders fourth edition) and ICD-10 (International Statistical Classification of Disease and Related Health problems-10) base on clinical interviews.

For all HCV patients, blood was collected before the onset of IFN-α therapy as well as 2 and 4 weeks after initiation of treatment. There was a no significant time difference for blood sampling between depression (−) and (+) patients (**Table 1b** and **Figure 3**).

Previous studies suggested that IDO1-mediated TRP metabolism could be implicated in the development of depression, as a side effect of IFN-α therapy in HCV patients. We also found that HCV patients showed decreased TRP and increased KYN concentrations without any changes in KA, AA, and 3-HAA concentrations during IFN-α therapy (**Figure 3b** and **Table 2a**). Furthermore, depression (+) patients presented a higher increase in 3-HK concentration compared to depression (−) patients during treatment (**Table 2a**). Ogawa et al. recently showed that plasma TRP concentration was significantly decreased in MDD patients compared to healthy controls [37]. Teraishi et al. also demonstrated increased KYN metabolites along the TRP-KYN-QUIN pathway, but not the KYN-KA pathway, in MDD patients [38]. Our results showed that the level of 3-HK in the serum significantly increased in depression (+) patients are consistent with these findings. We also investigated the ratios of 3-HK/KA (reflecting neurotoxic indices) [39, 40] and KYN/TRP (reflecting IDO1 activity) in depression (−) and depression (+) HCV patients during IFN-α treatment (**Figure 3c** and **Table 2b**). The ratios of KYN/TRP and 3-HK/KA in both groups increased during treatment. However, in depression (+) patients, the ratios of KYN/TRP and 3-HK/KA increased much larger in depression (−) patients during treatment (**Table 2b**). In these patients, the serum KYN/TRP and 3-HK/KA ratios increased more at the diagnosis of depression, but at 70.3 ± 9.1 days post therapy, they returned to the same levels as before onset of the therapy (data not shown). The severity of depression was not assessed during treatment, using neither the MADRS nor Hamilton Depressing Rating Scale. Therefore, we could not clearly show the direct association between the aggravation of depressive symptoms and changes in TRP metabolites. However, our results suggest that HCV patients with a high sensitivity for IDO1 activation by IFNs are highly susceptible to the depression-related side effects of IFN-α treatment.

### **4.2. The effects of chronic** *Ifn-γ* **gene expression on depression-like behavior in mice**

We hypothesized that the high induction of IDO1 and the imbalance of TRP metabolites induced by IFNs in humans may be related to psychiatric side effects, such as depression. Previous studies have shown that all three IFNs (IFN-α, -β, and -γ) induce strong IDO1 activity in human peripheral blood mononuclear cells [41, 42]. In contrast, in mouse, IDO1 is induced more markedly by IFN-γ than IFN-α, which has only a weak direct IDO1-stimulatory effect. Therefore, we investigated whether IDO1 activity induced by *Ifn-γ* gene transfer impaired behavior in mice.

**Figure 3.** Changes in the levels of serum TRP and its metabolites in HCV patients receiving IFN-α therapy. Original data from Ref. [47]. (a) Schematic depiction of the collection schedule for blood sampling from depression (−) and depression (+) HCV patients. The range of time points and average collection time point (a−d) per group are listed in **Table 1b**. (b) Serum TRP, KYN, KA, and 3-HK concentrations in HCV patients at 2 and 4 weeks after the onset of therapy, expressed as a percentage of the concentration before IFN-α therapy. (c) Serum KYN/TRP and 3-HK/KA ratios in HCV patients are shown as a percentage of values before IFN-α therapy. Rectangles indicate non-depressive HCV patients [Depression (−)] and circles indicate HCV patients with depressive symptoms [Depression (+)]. Each data point represents the mean ± SEM of values

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*p*<0.05, ##*p*<0.01 *versus* depression (−) patients. Detailed statistical analyses are shown in **Table 2** [47].

*p*<0.05, \*\*\**p*<0.001 *versus* before the onset of

obtained from n = 30 depression (−) patients and n = 19 depression (+) patients. \*

IFN-α therapy, #

evaluation and identified as major depressive disorder (MDD) by a psychiatrist. Nineteen of the HCV patients were diagnosed with depressive symptoms [depression (+)], while 30 of them did not present depressive symptoms [depression (−)]. The diagnosis to verify the incidence of depressive symptoms associated to MDD was made according to the DSM-IV (Diagnostic and Statistical Manual of Mental Disorders fourth edition) and ICD-10 (International Statistical Classification of Disease and Related Health problems-10)

For all HCV patients, blood was collected before the onset of IFN-α therapy as well as 2 and 4 weeks after initiation of treatment. There was a no significant time difference for blood sam-

Previous studies suggested that IDO1-mediated TRP metabolism could be implicated in the development of depression, as a side effect of IFN-α therapy in HCV patients. We also found that HCV patients showed decreased TRP and increased KYN concentrations without any changes in KA, AA, and 3-HAA concentrations during IFN-α therapy (**Figure 3b** and **Table 2a**). Furthermore, depression (+) patients presented a higher increase in 3-HK concentration compared to depression (−) patients during treatment (**Table 2a**). Ogawa et al. recently showed that plasma TRP concentration was significantly decreased in MDD patients compared to healthy controls [37]. Teraishi et al. also demonstrated increased KYN metabolites along the TRP-KYN-QUIN pathway, but not the KYN-KA pathway, in MDD patients [38]. Our results showed that the level of 3-HK in the serum significantly increased in depression (+) patients are consistent with these findings. We also investigated the ratios of 3-HK/KA (reflecting neurotoxic indices) [39, 40] and KYN/TRP (reflecting IDO1 activity) in depression (−) and depression (+) HCV patients during IFN-α treatment (**Figure 3c** and **Table 2b**). The ratios of KYN/TRP and 3-HK/KA in both groups increased during treatment. However, in depression (+) patients, the ratios of KYN/TRP and 3-HK/KA increased much larger in depression (−) patients during treatment (**Table 2b**). In these patients, the serum KYN/TRP and 3-HK/KA ratios increased more at the diagnosis of depression, but at 70.3 ± 9.1 days post therapy, they returned to the same levels as before onset of the therapy (data not shown). The severity of depression was not assessed during treatment, using neither the MADRS nor Hamilton Depressing Rating Scale. Therefore, we could not clearly show the direct association between the aggravation of depressive symptoms and changes in TRP metabolites. However, our results suggest that HCV patients with a high sensitivity for IDO1 activation by IFNs are highly susceptible to the depression-related side effects of

**4.2. The effects of chronic** *Ifn-γ* **gene expression on depression-like behavior in mice**

We hypothesized that the high induction of IDO1 and the imbalance of TRP metabolites induced by IFNs in humans may be related to psychiatric side effects, such as depression. Previous studies have shown that all three IFNs (IFN-α, -β, and -γ) induce strong IDO1 activity in human peripheral blood mononuclear cells [41, 42]. In contrast, in mouse, IDO1 is induced more markedly by IFN-γ than IFN-α, which has only a weak direct IDO1-stimulatory effect. Therefore, we investigated whether IDO1 activity induced by *Ifn-γ* gene transfer impaired

pling between depression (−) and (+) patients (**Table 1b** and **Figure 3**).

44 Pharmacokinetics and Adverse Effects of Drugs - Mechanisms and Risks Factors

base on clinical interviews.

IFN-α treatment.

behavior in mice.

**Figure 3.** Changes in the levels of serum TRP and its metabolites in HCV patients receiving IFN-α therapy. Original data from Ref. [47]. (a) Schematic depiction of the collection schedule for blood sampling from depression (−) and depression (+) HCV patients. The range of time points and average collection time point (a−d) per group are listed in **Table 1b**. (b) Serum TRP, KYN, KA, and 3-HK concentrations in HCV patients at 2 and 4 weeks after the onset of therapy, expressed as a percentage of the concentration before IFN-α therapy. (c) Serum KYN/TRP and 3-HK/KA ratios in HCV patients are shown as a percentage of values before IFN-α therapy. Rectangles indicate non-depressive HCV patients [Depression (−)] and circles indicate HCV patients with depressive symptoms [Depression (+)]. Each data point represents the mean ± SEM of values obtained from n = 30 depression (−) patients and n = 19 depression (+) patients. \* *p*<0.05, \*\*\**p*<0.001 *versus* before the onset of IFN-α therapy, # *p*<0.05, ##*p*<0.01 *versus* depression (−) patients. Detailed statistical analyses are shown in **Table 2** [47].


To conduct this experiment, for murine *Ifn-γ* gene transfer, the plasmid pCpG-Muγ was constructed by inserting a BglII/NheI murine *Ifn-γ* cDNA fragment into the BglII/NheI site of the pCpG-mcs vector (**Figure 4a**). The prepared plasmid pCpG-Muγ was dissolved in normal saline and injected into the tail veins of the mice for over 5 s on day 0. The injection volume was approximately 9% (v/w) of body weight. To eliminate the possibility of tissue damage or inflammation by the hydrodynamic injection, a control plasmid, which was the empty vector without the *Ifn-γ* gene (pCpG-mcs), was injected (0.05 pmol/mouse; IFN-γ transfected (−) mice). A previous study demonstrated that sustained IFN-γ concentrations were observed in mice receiving pCpG-Muγ at a dose of 0.2 pmol/mouse and more than 1000 pg/mL of IFN-γ was detected in the serum from 6 to 31 days after injection of pCpG-Muγ [43]. We also confirmed that the injected plasmid, pCpG-Muγ (IFN-γ transfected (+) mice) significantly increased IDO1 activity in the frontal cortex over a dose of 0.05 pmol/mouse compared to IFN-γ transfected (−) mice (**Figure 4c**). Therefore, the plasmid dose was fixed at 0.05 pmol/mouse for subsequent experiments, which corresponded to

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**Figure 4.** *Ifn-γ* gene transfer. Original data from Ref. [47]. (a) Schematic depiction of the pCpG-Muγ plasmid construct (InvivoGen, San Diego, CA). (b) Schematic depiction of the time schedule for animal experiments. (c) Increase of IDO1 activity in the frontal cortex of mice 28 days after *Ifn-γ* gene transfer [47]. βGlo MAR, β-globin matrix attachment region; mCMV enh, mouse cytomegalovirus enhancer; hEF1 prom, human elongation factor1 promoter; I140, synthetic 5′UTR containing an intron 140; MCS, multi cloning site; SV40 pAn, Simianvirus 40 polyadenylation; IFN-β S/MAR, interferon β gene scaffold/matrix attachment region; EM2K, CpG-free version of the bacterial EM7 promoter; Zeo, Zeocin; R6K ori,

0.10–0.12 μg of DNA/mouse.

R6K origin.

Percent value of serum TRP, KYN, 3-HK, KA, AA, and 3-HAA concentrations in HCV patients at 2 and 4 weeks after the onset of therapy, compared to the concentration (100%) before IFN-α therapy. In the clinical samples, some metabolites were difficult to separate clearly by HPLC. Therefore, the degree of freedom (df) values differ by the measured molecules. "Depression (−)": HCV patients without depression, "Depression (+)": HCV patients with depression.\* *p*<0.05,

\*\*\**p*<0.001 *versus* before the therapy;

##*p*<0.01 *versus* Depression (−) [47].


Serum KYN/TRP reflects IDO1 activity, and 3-HK/KA reflects neurotoxic indices. Both ratios in HCV patients were shown as % of value compared to the value (100%) before IFN-α therapy, at 2 and 4 weeks after the onset of therapy.\* *p*<0.05,

\*\*\**p*<0.001 *versus* before the therapy; #

*p*<0.01 *versus* Depression (−) [47].

**Table 2.** Changes in TRP-KYN pathway in HCV patients undergoing IFN-α therapy.

To conduct this experiment, for murine *Ifn-γ* gene transfer, the plasmid pCpG-Muγ was constructed by inserting a BglII/NheI murine *Ifn-γ* cDNA fragment into the BglII/NheI site of the pCpG-mcs vector (**Figure 4a**). The prepared plasmid pCpG-Muγ was dissolved in normal saline and injected into the tail veins of the mice for over 5 s on day 0. The injection volume was approximately 9% (v/w) of body weight. To eliminate the possibility of tissue damage or inflammation by the hydrodynamic injection, a control plasmid, which was the empty vector without the *Ifn-γ* gene (pCpG-mcs), was injected (0.05 pmol/mouse; IFN-γ transfected (−) mice). A previous study demonstrated that sustained IFN-γ concentrations were observed in mice receiving pCpG-Muγ at a dose of 0.2 pmol/mouse and more than 1000 pg/mL of IFN-γ was detected in the serum from 6 to 31 days after injection of pCpG-Muγ [43]. We also confirmed that the injected plasmid, pCpG-Muγ (IFN-γ transfected (+) mice) significantly increased IDO1 activity in the frontal cortex over a dose of 0.05 pmol/mouse compared to IFN-γ transfected (−) mice (**Figure 4c**). Therefore, the plasmid dose was fixed at 0.05 pmol/mouse for subsequent experiments, which corresponded to 0.10–0.12 μg of DNA/mouse.

**(a) Changes in the levels of serum TRP and its metabolites**

2 w after onset of therapy

4 w after onset of therapy

\*\*\**p*<0.001 *versus* before the therapy; ##*p*<0.01 *versus* Depression (−) [47].

2 w after onset of therapy

4 w after onset of therapy

#

\*\*\**p*<0.001 *versus* before the therapy;

*p*<0.01 *versus* Depression (−) [47].

**(b) Changes in serum KYN/TRP and 3-HK/KA ratios**

KYN/TRP 115.7 ± 5.69 138.3±8.84\*

3-HK/KA 129.6 ± 8.67 171.1 ± 18.6\*\*\*,

**Depression (−) Depression (+)**

46 Pharmacokinetics and Adverse Effects of Drugs - Mechanisms and Risks Factors

**% of value before IFN-α therapy** *t* **df** *p* **value**

TRP 95.4 ± 2.93 100.5 ± 3.98 0.965 40 0.340 KYN 108.6 ± 4.77 118.1 ± 4.24\* 1.200 39 0.237 3-HK 117.0 ± 7.13 152.6 ± 10.4\*\*\*, ## 2.886 38 0.006 KA 97.4 ± 5.51 95.9 ± 7.13 0.136 38 0.892 AA 119.9 ± 7.42 115.5 ± 7.11 0.381 41 0.706 3-HAA 102.9 ± 6.53 121.8 ± 12.6 1.452 37 0.155

TRP 92.0 ± 2.55 93.3 ± 6.49 0.213 39 0.833 KYN 104.8 ± 4.38 114.4 ± 6.38 1.204 39 0.236 3-HK 123.0 ± 9.01 155.0 ± 11.5\*\*\* 2.005 36 0.053 KA 91.9 ± 5.12 88.8 ± 6.98 0.341 40 0.735 AA 107.5 ± 5.32 103.6 ± 11.3 0.361 40 0.720 3-HAA 101.9 ± 6.52 104.5 ± 14.8 0.182 36 0.857

"Depression (−)": HCV patients without depression, "Depression (+)": HCV patients with depression.\*

KYN/TRP 115.6 ± 4.55 114.1 ± 5.95 0.198 42 0.844 3-HK/KA 129.1 ± 9.52 144.0±9.06\* 1.036 39 0.308

,

Serum KYN/TRP reflects IDO1 activity, and 3-HK/KA reflects neurotoxic indices. Both ratios in HCV patients were shown as % of value compared to the value (100%) before IFN-α therapy, at 2 and 4 weeks after the onset of therapy.\*

**Depression (−) Depression (+)**

**Table 2.** Changes in TRP-KYN pathway in HCV patients undergoing IFN-α therapy.

Percent value of serum TRP, KYN, 3-HK, KA, AA, and 3-HAA concentrations in HCV patients at 2 and 4 weeks after the onset of therapy, compared to the concentration (100%) before IFN-α therapy. In the clinical samples, some metabolites were difficult to separate clearly by HPLC. Therefore, the degree of freedom (df) values differ by the measured molecules.

**% of value before IFN-α therapy** *t* **df** *p* **value**
