Parkinson's disease therapy with Istradefylline and blood biomarkers of epigenetics

Istradefylline (IST), an adenosine A2A receptor antagonist, has been used since 2013 in Japan as an adjunctive therapy to levodopa (L‐DOPA) for patients with Parkinson's disease (PD). The long‐term use of IST as an adjunct to L‐DOPA (IST‐LD) was herein investigated to clarify the cooperative potential to keep motor functions, and an epigenetic modification for disease‐specific up‐regulated A2AR signals.


| INTRODUC TI ON
Treatments with L-DOPA restore LTP by early genes expression change; however, continued treatment is expected to enhance D 1 R/MSN trafficking at the cell surface with phosphorylation by GCPR kinase and linked activation of the GNAL/Gα-olf, those promote higher signal transduction and synaptic transmission, prone to develop LID. 6-10 L-DOPA also activates c-JNK signaling with downstream ERK activation, which plays relevant role to maintain LID. 6,11,12 A 2A R forms a functional complex with D 2 R/MSNs and antagonizes its functions reciprocally and independently. The adenosine A 2A R antagonist/KW6002/IST suppresses the presynaptic terminals of the globus pallidus by reducing the excessive output of the basal ganglia. 1,2,13 Increased A 2A R expressions were observed in the drug-naïve PD putamen and blood lymphocytes using Western blot assays or radioligand binding assays with A 2A R antagonist/[ 3 H] ZM241385, those were regulated by post-transcriptional epigenetic activation of ADORA2A gene. [14][15][16][17] The treatment of PD with IST has been covered by the national health insurance system in Japan since 2013 as non-dopaminergic adjunctive therapy to L-DOPA, and this treatment has been shown to exert robust effects on motor dysfunctions in both the off and on periods. 18 for up-regulated A 2A R expression, those have not yet been evaluated in any clinical trials. 20,21 In the present study, we examined the effects of the long-term use of IST as an adjunct to L-DOPA (IST-LD) in clinical practice and assessed blood biomarkers of epigenetics, including the A 2A R-p, A 2A R-m, and DNA methylation of the ADORA2A gene in peripheral blood lymphocytes (PBL).

| Subjects
The The present study was approved by the local Ethics Committee of our hospital, and informed consent were obtained from recruited patients and HC subjects.

| Statistical analysis
The Wilcoxon signed-rank test and a binomial logistic regression analysis were used. All statistical analyses were conducted using IBM SPSS statistics version 12.0. P values of <.05 were considered to be significant.

| Subjects
The demographics and clinical characteristics of 62 PD patients at baseline and 36 months after the IST-LD treatment are shown in dose were decreased compared with those of the baseline, but no significant differences were observed within the CGI-I categories.
Regarding biomarkers in blood samples, A2AR-m/GAPDH expressions had a significant influence on effectiveness (P < .05).

| Clinical global effectiveness of IST-LD with CGI-I
CGI-I after 10 days showed 11% MI, 45% SI, 32% NC, and 11% AW ( Figure 1A). After 36 months of treatment, up to 58% of patients (MI + SI) maintained their motor functions and well-being of daily life ( Figure 1B). Discontinuation of IST occurred in 11% of participants due to other incidental comorbidities, such as pneumonia and cerebral infarction.

| Locomotive function and off time
Locomotive function scores improved by 4.2 points over baseline in MI patients (P < .05) (Figure 2A). AW patients showed an increase of 3.6 points from the baseline (P < .05), which was evaluated at 10 days as aggravated/discontinued (Figure 2A). Off-time scores improved by 2 points over the baseline with 4 hours of daily off time in MI patients (P < .05), while AW patients showed aggravation from the baseline, which was significant (P < .05) ( Figure 2B). Expression levels were compared between HC and PD, and among the patient groups before and after the IST treatment

| Changes in blood biomarkers
The expression level of the A 2A R-p in PBL appeared to be higher in PD patients with IST-naïve than the healthy controls (HC) ( Figure 3A).
Since all samples of PBL from PD patients and HC showed the 559bp band corresponding to A 2A R-m by RT-PCR (data not shown), we examined A 2A R-m expression levels by semi-quantitative real-time PCR and found that they were significantly higher in PD than HC F I G U R E 4 Changes in DNA demethylation in PD. A, DNA demethylation differences were compared across the samples of juvenile monozygotic twin pair of the IST-naïve early stage of PD and NC. B, DNA demethylation differences were compared across the samples of IST-LD at 12 mo in monozygotic twin pair. Surrogate global DNA methylation sequencing (LINE-1 kit) was used. Dsemethylation difference (%) of the promoter, exon, intron, and CpG islands and shores are shown. Ch, chromosomes; and encoding genes located on chromosomes are shown. GBA1, glucocerebrosidase 1; SNCA, α-synuclein; BDNF, brain-derived neurotrophic factor; MAPT, microtubule-associated protein tau; GNAL, G protein subunit alpha L; ADORA2A, adenosine 2A receptor; DRD1, Dopamine D1 receptor. C, DNA demethylation average was compared across the samples of IST-LD at 12 mo in 5 PD patients. Human Jurkat genomic DNA was compared as standards. #, large demethylation differences (>10%) 33,34 ( Figure 3B). After 12 months of the IST treatment, A 2A R-m expression levels appeared to be decreased in samples obtained from MI or SI responsiveness except for AW, suggesting positive test may be associated to CGI-I effectiveness.

| DNA demethylation for the whole genome in the monozygotic twin with PD
Using surrogate global DNA methylation sequencing, DNA demethylation differences were compared across the samples of juvenile monozygotic twin pair of the naïve early stage of PD and NC; since they have the same genome, demethylation differences showed epigenetic modifications ( Figure 4A). After 12 months of the IST-LD treatment, the difference was restored to small (Decrement > 5%) in the promotor and CpG islands of the several genomic sites including ADORA2A ( Figure 4B).

| Chronological changes in the DNA demethylation of ADORA2A and DRD1 after IST-LD
Five PD patients compared across the samples of 12 months of IST-LD treatment, using human Jurkat genomic DNA as normal controls. MI or SI responsiveness (PD2-5) showed demethylation were restored to small at the ADORA2A (Decrement > 10%). DNA demethylation of DRD1 did not change, whereas MI with severe LID (PD5) altered both ADORA2A and DRD1 ( Figure 4C).

| D ISCUSS I ON
In the present study, the long-term treatment with IST-LD partially reversed locomotive dysfunction and motor off time, and indicated the potential to preserve global daily activities improvement, expected to decline with disease progression and age-related comorbidities. Although our cohort was small and did not include a placebo arm, it is important to note that another long-term natural history study on Rasagiline (ADAGIO follow-up study) achieved similar outcomes with a slower rate of progression. 23 The overall rate of discontinuation was 22% (aggravation/withdrew 11% and comorbidities 11%), which was similar to the findings of the previous long-term and larger prospective studies. 24,25 The present study revealed that IST-LD spared the L-DOPA dose (LEDD at baseline and prospective, prodromal PD stages, both in the substantia nigra and with blood lymphocytes. [14][15][16][17]31,32 Our monozygotic twin pair with juvenile PD or PD with IST-naïve patients showed the intrinsic demethylation of ADORA2A those were chronologically restored after the IST treatment. Alternatively, the patient with severe LID at baseline was diminished after the long-term IST treatment that may be associated with reduced demethylation levels of DRD1.
Taken together, IST-LD has the potential to influence biological off targets of the disease process by restoring intrinsic ADORA2A biomarker of PD. 35 Regenerative medicine will play an essential role in the treatment of PD in the near future. 36 However, pharmacotherapy will still be crucial for preventing disease-specific pathological evolution and accelerated neurodegeneration.

ACK N OWLED G M ENTS
We thank Akihisa Mori, PhD (Research fellow of Kyowa Kirin Co., Ltd.), for his helpful discussions.