Suppression of Methotrexate-Induced Elevations in the Serum Alanine Aminotransferase Level of Patients with Rheumatoid Arthritis Who Had Prior Hepatitis B Infection

Background: Hepatitis after the reactivation of hepatitis B virus (HBV) has been recognized serious in the patients with rheumatoid arthritis (RA) treated with biologics. Objectives: The objective of the present study was to search some common background which might be relevant to the host factors that provoke such a serious hepatitis. Methods: We retrospectively collected and analyzed all data of serum alanine aminotransferase (ALT) levels in selected patients with RA at random. Results: A significant association (P < 0.001) between methotrexate (MTX) therapy and elevated serum ALT level was found only in the anti-HBcAb-negative RA group. The mean serum ALT level was significantly higher (P < 0.001) in patients with than without MTX therapy in the anti-HBcAb-negative RA group, although no significant difference was found (P = 0.73) in the anti-HBcAb-positive RA group. In addition, the anti-HBcAb-positive RA patients showed significantly lower mean serum level of ALT (P < 0.01) than anti-HBcAb-negatives provided they are naïve with biologics. But no significant difference between anti-HBcAb-positives and -negatives in the mean serum level of ALT was found among patients experienced with biologics (P < 0.8). Conclusions: The anti-HBcAb-positive RA group showed the suppression of MTX-induced elevations in serum ALT level. However, this suppression was not found in patients experienced in the treatment with biologics, although it was preserved in those who had not experienced biologics. Failure of this suppressive mechanism of ALT in anti-HBcAb-positive RA patients treated with biologics could be possibly assoHow to cite this paper: Noguchi, O., Nakamura, Y. and Kato, H. (2018) Suppression of Methotrexate-Induced Elevations in the Serum Alanine Aminotransferase Level of Patients with Rheumatoid Arthritis Who Had Prior Hepatitis B Infection. Health, 10, 531-541. https://doi.org/10.4236/health.2018.105042 Received: March 21, 2018 Accepted: May 12, 2018 Published: May 15, 2018 Copyright © 2018 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access


Introduction
Success in antiviral and remission-induction therapies for patients with rheumatoid arthritis (RA) who had prior hepatitis virus infection is an important clinical challenge to be addressed in clinical practice.Adequate methods for viral elimination and a sustained virological response have been established for the hepatitis C virus (HCV) [1].When remission-induction therapy of RA is initiated, however, special caution should be paid to patients with RA who are hepatitis B virus (HBV) carriers or who have prior HBV infection.Serious acute hepatitis after the reactivation of HBV [2] [3] has been reported after treatment with biologics in patients with RA who are both positive for the anti-hepatitis B core antibody (anti-HBcAb) and negative for the hepatitis B surface antigen (HBsAg) and even positive for the hepatitis B surface antibody (anti-HBsAb) [4] [5].
Hepatitis viruses and host factors are mentioned as the factors that are involved in the development of de novo hepatitis in patients with prior HBV infection.In the present study, we used medical records to randomly extract patients who were positive for anti-HBcAb and retrospectively examined changes in serum alanine transferase (ALT) levels after the first medical examination at our clinic.

Subjects
The medical records of 107 patients with RA who had been tested for HBsAg (Lumipulse Presto® HBsAg; Fujirebio, Tokyo, Japan), anti-HBcAb (Architect® Anti-HBc II; ABBOTT Japan, Chiba, Japan), and anti-HCV-Ab (Lumipulse Presto Ortho HCV; Fujirebio) between July 1997 and December 2011 except the alcohol abusers and patients with nonalcoholic steatohepatitis, were examined, although a large number of patients were selected at random and underwent the examination of HBV-related markers in addition to periodical routine laboratory tests happened to be done from September till December 2011 according to the recommendation of Japan Rheumatism Assoc that the time to monitor them should be every four weeks to three months.We then retrospectively assessed 103 patients after excluding 4 patients along with anti-HCV-Ab-positivity.A total of 103 patients with RA (22 men and 81 women; mean age ± SD [range]: 67.9 ± 11.8 years -negative RA groups, respectively (Table 1).Sociodemographic and clinical characteristics of patients with and without anti-HBcAb are shown on Table 2.
In all these 103 patients, an ALT kit (L-type ALT.J2; Wako Pure Chemical Industries, Osaka, Japan) was used to measure serum ALT levels.Serum ALT levels in their medical records at our institution were investigated to specify the maximum, minimum, and mean levels of serum ALT during individual clinical course within our Clinic.Subsequently, we extracted patients according to the history of presence or absence of treatment with biologics and to the presence or absence of this treatment at the time of reaching the maximum or minimum serum ALT level, and thus we attempted to compare biologic treatment-naive patients with patients who underwent the treatment when reaching the maximum or minimum serum ALT level.
A hepatitis episode was considered to have occurred when a serum ALT level greater than 40 IU/L persisted for at least 2 weeks in the anti-HBcAb-positive RA group and the anti-HBcAb-negative RA group.
An elevation in serum ALT level was considered attributable to self-limited transient hepatitis regarded as the mild drug-induced one in all patients.Although the dose reduction of methotrexate (MTX) and the dose increment of folic acid were conducted occasionally, no patients required special treatment apart from ursodeoxycholic acid therapy.Real-time polymerase chain reaction, however, was not conducted for the HBV DNA.
Treatment with biologics was conducted according to the established protocols.Blood samples were obtained immediately prior to their administration.
This study was approved by the ethics committee at Showa Inan General Hospital.Informed consent was not required for this study, because all patients were examined retrospectively based on unidentifiable personal information.the φ coefficient, significance level of which was examined according chi-square test.A two-tailed value of P < 0.05 was considered statistically significant.

Results
To study the association between elevated serum ALT level and MTX therapy, we first investigated whether patients were on MTX therapy when the maximum or minimum serum level of ALT was reached.Eighty patients (85.1%) were on MTX therapy when serum ALT levels reached the maximum, while 47 patients (50%) were on MTX therapy when ALT reached the minimum.These results suggest a significant association between elevated serum ALT level and MTX therapy (P < 0.001) (Table 3).This association was found not in the anti-HBcAb-positive RA group (P < 0.8) (Table 4) but in the anti-HBcAb-negative RA group (P < 0.001) (Table 5).Namely, changes in methotrexate-induced elevation in serum ALT level were found only in RA patients without prior HBV infection.Next, we examined the maximum, minimum, and mean serum levels of ALT, regardless of treatment with biologics or MTX.Consequently, no significant differences were found in these levels between the anti-HBcAb-positive RA group and the anti-HBcAb-negative RA group (Table 6), although the minimum    serum ALT levels tended to be higher in the former group (P < 0.2).In addition, the hepatitis episodes tended to be less frequent in patients with RA who had prior HBV infection (P < 0.2) (Table 7).We conjectured that elevated serum ALT level was associated with MTX therapy in RA patients and that MTX-associated elevations in serum ALT level were suppressed in the anti-HBcAb-positive RA group.Therefore, clinicians need to give heed to the presence or absence of MTX therapy and to prior HBV infection that can be indicated by anti-HBc and/or -anti-HBs antibody along with anamnesis when interpreting changes in serum ALT levels in patients with RA.
Accordingly, to further evaluate changes in serum ALT levels, patients were examined based on the positivity or negativity for anti-HBcAb and on the presence or absence of MTX therapy with regard to the maximum, minimum, and mean serum levels of ALT (Figure 1).
Both the anti-HBcAb-positive and -negative RA groups showed significantly higher (P < 0.05 and P < 0.01, respectively) maximum ALT levels in the presence of MTX therapy against the absence of MTX therapy.Of note was the fact that the level of significance was greater in the anti-HBcAb-negative RA group.The anti-HBcAb-positive RA group showed no significant difference in the mean ALT level between presence and absence of MTX-administration (P = 0.73).
However, only the anti-HBcAb-negative RA group showed a significant increase (P < 0.001) in the mean ALT level in the presence of MTX therapy, as compared with absence of MTX therapy.Namely, the suppression of the MTX-induced increase in serum ALT levels was confirmed in the anti-HBcAb-positive RA group, which was suggested from the fact that the increased serum ALT level found in the anti-HBcAb-negative RA group was lost to find in the anti-HBcAb-positive RA group in the presence of MTX therapy (Table 4 and Table 5).
On the other hand, neither the anti-HBcAb-positive group nor the anti-HBcAb-negative RA group showed a significant difference in the minimum ALT level between the presence and absence of MTX therapy.However, of note was the fact that the anti-HBcAb-positive RA group tended to show the high minimum ALT level (P < 0.1) in the presence of MTX therapy against the anti-HBcAb-negative counterpart (Figure 1).
To examine the effects of treatment with biologics on serum ALT levels, subsequently, patients with RA were categorized to two groups according to the presence or absence of history of treatment with biologics.Furthermore, only patients with RA, who had been on treatment with biologics when serum ALT levels reached the maximum or minimum, were extracted and compared with those who were neither on the treatment nor the history thereof (Figure 2).Consequently, no significant difference was found in serum ALT level between the anti-HBcAb-positive and -negative RA groups in the presence of treatment with biologics (Figure 2 †).In the absence of treatment with biologics (Figure 2 ‡), however, the mean serum ALT level was significantly lower (P < 0.01) in the anti-HBcAb-positive group than in the anti-HBcAb-negative group.This result indicates the possibility that a significant difference (P < 0.01) in serum ALT level between the anti-HBcAb-positive and -negative RA groups was lost in the presence of treatment with biologics.Furthermore, no significant difference was found between the anti-HBcAb-positive and -negative RA groups with respect to background on the characteristics of MTX therapy (Table 8).

Discussion
Serum ALT level is a well-known marker of hepatocyte injury [8] that is frequently examined in routine laboratory testing.The reference value at our institution ranges between 6 and 40 IU/L, and serum ALT levels can vary in the same Anti-HBcAb

Anti-HBcAb
Anti-HBcAb Anti-HBcAb Anti-HBcAb Anti-HBcAb Since no significant differences were found in serum ALT levels between RA patients who had prior HBV infection and patients who did not as a whole, they were subcategorized according to the presence or absence of MTX therapy (Figure 1).Consequently, significant elevations in the maximum (P < 0.01) and mean (P < 0.001) serum ALT levels were found in the anti-HBcAb-negative RA group in the presence of MTX therapy against the absence of MTX therapy.On the contrary, the anti-HBcAb-positive RA group in the presence of MTX therapy showed no significant elevations in the minimum(P < 0. Hepatocyte apoptosis has been reported as a cardinal pathophysiological mechanism in MTX-induced hepatotoxicity from both histological [10] and biochemical viewpoints [11].Accordingly, a suppressive mechanism on elevations in serum ALT level or a protection mechanism of hepatocytes, which was observed with RA patients who had HBV infection in the present study, possibly relies on the suppression of hepatocyte apoptosis.One of the reported suppressive mechanisms of apoptosis is the modulation of the Nrf2-NF-κB pathways [11] and might be involved in patients whose hepatocytes are infected with HBV and who do not present de novo hepatitis.Furthermore, HBV needs to prevent the apoptosis of host cells for its survival and spread in the liver [12].We consider that our data provide clinical evidence to support the presence of the suggested suppressive mechanism on hepatocyte apoptosis in our patients.
On the other hand, the tendency for higher minimum serum ALT levels in RA patients who had prior HBV infection remains a mystery.One plausible mechanism is that the permeability of aged hepatocyte membranes increases along with the fact the expected viability of hepatocytes is extended by a suppressive mechanism on hepatocyte apoptosis, resulting in the persistence of the higher minimum serum ALT levels.

Conclusion
In conclusion, the suppression of MTX-induced increases in serum ALT levels was considered attributable to a suppressive mechanism on the apoptosis of HBV-infected hepatocytes.On the other hand, the suppression of an increase in mean serum ALT level was not found in anti-HBcAb-positive RA patients during treatment with biologics, which might suggest the failure (so to speak, "biologic-induced failure") of the suppressive mechanism on the apoptosis of HBV-infected hepatocytes.If this is true, the relevant failure would be one of the explanations about serious hepatitis that may develop subsequent to the reactivation of HBV due to treatment with biologics.Therefore, the elucidation of this mechanism by immuno-virological approaches potentially leads to the development of new categories of drugs (e.g., inhibitors of hepatocyte apoptosis) and therapeutic modalities (e.g., immunomodulation) for patients with hepatitis.Indeed, our outcomes are not conclusive because we don't have immuno-virological evidence relevant to this clinical phenomenon, and the patient population is just small.Therefore, a detailed immuno-virological study and a large-sized cohort study in other medical institutions are required to confirm our speculation.

Figure 1 .
Figure 1.The maximum, minimum, and mean serum levels of ALT in patients with RA according to the positivity or negativity for anti-HBcAb and to the presence or absence of MTX therapy.*: Student's t-test; **: Welch's t-test; ***: The Mann-Whitney U-test.The bars represent standard deviations.ALT, alanine aminotransferase; anti-HBcAb, hepatitis B core antibody; MTX, methotrexate; RA, rheumatoid arthritis.

Figure 2 .
Figure 2. The maximum, minimum, and mean serum levels of ALT in patients with RA according to the positivity or negativity for anti-HBcAb and to the presence or absence of the history of treatment with biologics.*: Student's t-test; **: Welch's t-test; ***: The Mann-Whitney U-test.†: Presence of history of treatment with biologics; ‡: Absence of history of treatment with biologics.The bars represent standard deviations.RA, rheumatoid arthritis; ALT, alanine aminotransferase; anti-HBcAb, hepatitis B core antibody; MTX, methotrexate; RA, rheumatoid arthritis.

7 )
and mean(P = 0.73) serum ALT level, although exhibited significantly less elevations (P < 0.05) in the maximum serum ALT level.Taken together, these findings based on the measured values of serum ALT levels suggest the presence of a suppressive mechanism on MTX-induced increases in serum ALT level in patients who had prior HB infection.

Table 1 .
Characteristics of patients with rheumatoid arthritis.

Table 2 .
Comparison of the sociodemographic and clinical characteristics of the patients with and without HBcAb.

Table 3 .
Associations between serum ALT levels and MTX therapy in patients with rheumatoid arthritis.

Table 4 .
Associations between serum ALT levels and MTX therapy in the RA patients with anti-HBcAb-positivity.

Table 5 .
Associations between serum ALT levels and MTX therapy in the RA patients with anti-HBcAb-negativity.

Table 6 .
Serum ALT levels in anti-HBcAb-positive and -negative patients with rheumatoid arthritis.

Table 7 .
Relationships between the hepatitis episode and the immunity status of anti-HBcAb in patients with rheumatoid arthritis.

Table 8 .
Background on the characteristics of MTX therapy classified by immunity status of anti-HBcAb in patients with RA who have never been treated with biologics.