Pharmacokinetics and pharmacodynamics of a single dose Nilotinib in individuals with Parkinson's disease

Abstract Nilotinib is a broad‐based tyrosine kinase inhibitor with the highest affinity to inhibit Abelson (c‐Abl) and discoidin domain receptors (DDR1/2). Preclinical evidence indicates that Nilotinib reduces the level of brain alpha‐synuclein and attenuates inflammation in models of Parkinson's disease (PD). We previously showed that Nilotinib penetrates the blood‐brain barrier (BBB) and potentially improves clinical outcomes in individuals with PD and dementia with Lewy bodies (DLB). We performed a physiologically based population pharmacokinetic/pharmacodynamic (popPK/PD) study to determine the effects of Nilotinib in a cohort of 75 PD participants. Participants were randomized (1:1:1:1:1) into five groups (n = 15) and received open‐label random single dose (RSD) 150:200:300:400 mg Nilotinib vs placebo. Plasma and cerebrospinal fluid (CSF) were collected at 1, 2, 3, and 4 hours after Nilotinib administration. The results show that Nilotinib enters the brain in a dose‐independent manner and 200 mg Nilotinib increases the level of 3,4‐Dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), suggesting alteration to dopamine metabolism. Nilotinib significantly reduces plasma total alpha‐synuclein and appears to reduce CSF oligomeric: total alpha‐synuclein ratio. Furthermore, Nilotinib significantly increases the CSF level of triggering receptors on myeloid cells (TREM)‐2, suggesting an anti‐inflammatory effect. Taken together, 200 mg Nilotinib appears to be an optimal single dose that concurrently reduces inflammation and engages surrogate disease biomarkers, including dopamine metabolism and alpha‐synuclein.


| INTRODUC TI ON
Nilotinib (Tasigna ® , AMN107, Novartis, Switzerland) is known as a break point cluster (BCR)-Abl (Abelson) tyrosine kinase inhibitor approved by the U.S. FDA for adults with chronic myeloid leukemia (CML) at oral doses of 600-800 mg daily. [1][2][3] Nilotinib is also a potent inhibitor of discoidin domain receptors (DDR) 1 and 2. 4, 5 We previously demonstrated that a low dose of Nilotinib (1-10 mg/ kg daily) penetrates the blood-brain barrier (BBB), reduces inflammation and degrades misfolded alpha-synuclein in several animal models of neurodegenerative diseases. [6][7][8][9] Nilotinib also increases dopamine (DA) levels and improves motor and cognitive outcomes in Parkinson's disease (PD) and Alzheimer's disease (AD) models. 6,[9][10][11][12][13][14][15] In a small, 12-patient pilot study, Nilotinib appeared to be potentially effective in treating the motor and nonmotor symptoms in patients with PD and dementia with Lewy bodies (DLB) and seemed to affect surrogate disease biomarkers, including DA metabolism and alpha-synuclein. 16 PD is the second most common neurodegenerative disorder that causes motor and nonmotor symptoms. PD is characterized by loss of DA-producing neurons in the substantia nigra (SN) pars compacta and formation of intracellular inclusions known as Lewy bodies (LBs) that primarily contain aggregated alpha-synuclein. Cerebrospinal fluid (CSF) levels of alpha-synuclein oligomers longitudinally increase in PD compared to aged-matched controls. [17][18][19] Additionally, the ratio of oligomeric to total alpha-synuclein also increases in the CSF of PD patients when compared to control and this increased ratio has been associated with motor decline. 20,21 Homovanillic acid (HVA) and 3,4-Dihydroxyphenylacetic Acid (DOPAC) are two primary metabolites of DA and can be used as a CSF marker of DA metabolism.
Decreased CSF levels of DOPAC have been shown to be an early marker for PD, 22 and similarly HVA has been shown to be decreased in the CSF of PD patients compared to controls. 23 Measuring CSF HVA and DOPAC as well as alpha-synuclein levels may provide an important pharmacodynamic effect of Nilotinib treatment in PD.
The R47H and other variants of triggering receptors on myeloid cells (TREM)-2, which result in loss of TREM2 function, are strong risk factors for PD. [24][25][26] Activated microglia in the SN proliferate and produce reactive oxygen species and pro-inflammatory cytokines, resulting in progressive degeneration of DA neurons in PD. 27,28 TREM2 may regulate microglial response and phagocytosis. TREM2 inhibits inflammatory responses in microglia via suppression of NF-kB pathways and activation of innate immunity, 29 while TREM2 loss of function results in reduced microglial phagocytosis. [30][31][32] Therefore, measuring TREM2 levels in the CSF may provide another important pharmacodynamic effect indicating neuroinflammation and the phagocytic activity of microglia to potentially reduce alphasynuclein levels after treatment of PD patients with Nilotinib.
To determine the pharmacokinetics and pharmacodynamics of Nilotinib in individuals with PD, we designed a physiologically based population pharmacokinetic/pharmacodynamic (popPK/PD) study and measured plasma and CSF concentration of Nilotinib as well as DOPAC, HVA, total and oligomeric alpha-synuclein and TREM2 levels.
Seventy-five participants were randomized into five groups (n = 15) of an open-label random single dose (RSD) study that included placebo, 150 mg, 200 mg, 300 mg, and 400 mg Nilotinib. This RSD study in a homogenous cohort of participants with PD provides a valuable insight into the potential mechanisms of action of Nilotinib and its effects on neuroinflammation and potential CSF biomarkers of disease. Baseline visits were scheduled 2-4 weeks after screening and results from all screening procedures were reviewed and all inclusion/ exclusion criteria were met prior to baseline assessments. After recruitment, an open-label RSD study was performed and participants (N = 75) were randomized into five groups (n = 15; 1:1:1:1:1) and received a single daily dose of placebo, 150 mg, 200 mg, 300 mg or 400 mg Nilotinib ( Figure 1). Because we previously demonstrated that Nilotinib is detectable up to 4 hours after drug administration in PD and DLB patients, 16 blood collection and lumbar puncture (LP) were staggered as shown in Figure 1 between 1 and 4 hours and blood was drawn half an hour prior to LP. All LPs were performed roughly within 2 hours from the last levodopa dose. Nilotinib was also taken on an empty stomach at least 2 hours after a meal. Following the 1 week wash-out period, all participants were randomized into three groups of placebo, 150 mg and 300 mg doses for 12 months (NCT02954978). This double-blinded, placebo-controlled study is currently underway and LP and blood will be collected at the end of 1 year treatment to obtain physiological population pharmacokinetics and pharmacodynamics. We performed this seamless, openlabel, RSD experiment as part of a larger long-term study with two active arms (150 mg and 300 mg) and placebo.

| Plasma and CSF collection
We previously showed that Nilotinib was detected in the human CSF 4 hours after administration. 16 Blood draw (15 mL) and LP (∼15 mL CSF) were performed on all patients roughly 2 hours after they received the last levodopa dose and at 1, 2, 3, or 4 hours after oral administration of Nilotinib. Plasma was isolated immediately after blood draw and aliquoted and stored at −80°C. CSF was aliquoted and stored at −80°C. Freeze/thaw cycles were avoided. To avoid CSF contamination with blood, the first 1 mL of CSF collection was discarded and all samples were centrifuged at 1000 g for 15 minutes.
Samples that contained >25 ng/mL hemoglobin were eliminated and were not tested for biomarkers. The hemoglobin levels in CSF samples were measured human hemoglobin ELISA Quantitation Set (Cat # E80-136) Kit (Bethyl Lab Inc, Montgomery, TX) according to the manufacturer's instructions. F I G U R E 1 Schematic diagram of a phase II study to determine the pharmacokinetics and pharmacodynamics of Nilotinib in a cohort of 75 participants with Parkinson's disease. The Random Single Dose (RSD) study was performed as part of a randomized double-blind, placebo controlled study for 1 year. A total of 15 participants were enrolled in each group (placebo:150 mg:200 mg:300 mg:400 mg) in the RSD study and lumbar puncture (LP) was performed in each group in a random fashion at 1, 2, 3 and 4 hours. CSF and plasma were analyzed to determine Nilotinib concentration and biomarkers of disease within each treatment group (dose study) and at each time point (exploratory time-dependent study)

| Mass spectrometry to evaluate Nilotinib pharmacokinetics
The samples were resolved on an Acquity UPLC BEH C18 1.7 μm, 2.1 × 50 mm column online with a triple quadrupole mass spectrometer (Xevo-TQ-S, Waters Corporation) operating in the multiple reaction monitoring (MRM) mode. The instrument parameters were optimized to gain maximum specificity and sensitivity of ionization for the parent (m/z = 530.27 Nilotinib) and daughter ions (m/z = 289.01 Nilotinib) using the "IntelliStart" feature of MassLynx software (Waters Corporation). The metabolite ratios were calculated by normalizing the peak area of endogenous metabolites within tissue samples normalized to the internal standard Nilotinib_13C_2H3.

| Quantification of DA metabolites DOPAC and HVA by LC-MS/MS
Concentrations of DOPAC and HVA in the CSF samples were measured by ultrahigh performance liquid chromatography with electrospray tandem mass spectrometry (UHPLC-MS/MS) following derivatization with benzoyl chloride as previously described. 33 Briefly, the UHPLC-MS/MS system included a PAL autosampler

| Total alpha-synuclein ELISA measurement
Solid phase alpha-synuclein sandwich ELISA (Cat#SIG38974, Biolegend) was performed on CSF and plasma. To avoid freezethaw cycles, immediately after LP and blood draws, 15 ml CSF and 5 ml plasma were aliquoted on ice into 0.5 ml tubes and stored at −80°C. Fresh aliquots were used to perform ELISA. All samples were analyzed side by side using the same reagents. Total alpha-

| Oligomeric alpha-synuclein ELISA measurement
Solid phase human alpha-synuclein oligomer sandwich ELISA (Cat# MBS730762, Mybiosource) was performed on CSF. To avoid freezethaw cycles, immediately after LP and blood draws, 15 ml of CSF and 10 mL of plasma were aliquoted on ice into 0.5 ml tubes and stored at −80°C. Fresh aliquots were used to perform ELISA. All samples were analyzed side by side using the same reagents. Fifty microliters of standards or CSF samples was added to the appropriate wells.

| Multiplex Xmap TREM2 ELISA
Xmap technology uses magnetic microspheres that are internally coded with two fluorescent dyes. Through precise combinations of these two dyes, multiple proteins are simultaneously measured within a sample. Each of these spheres is coated with a specific capture antibody. The capture antibody binds to the detection antibody and a reporter molecule, completing the reaction on the surface of the bead.
All samples were analyzed in parallel using the same reagents. Twentyfive microliters of human CSF or plasma was incubated overnight at  The plate was immediately read at 405 nm and 50 μL of 3N NaOH was added to each well to stop the reaction.

| Experimental design and data analysis
An open-label RSD study was performed in which participants (N = 75) were randomized into five groups (n = 15; 1:1:1:1:1) and received a single daily dose of placebo, 150 mg, 200 mg, 300 mg, or 400 mg Nilotinib ( Figure 1). As Nilotinib is detectable up to 4 hours after drug administration in PD and DLB patients, 16

| Pharmacokinetics of Nilotinib in plasma and CSF
Nilotinib was not detected in the plasma in the placebo group and the concentration of Nilotinib detected in the plasma (Figure 2A) was similar across all different dose groups of Nilotinib. The 400 mg Nilotinib group had a single patient as an outlier. Similarly, the level of Nilotinib in the CSF ( Figure 2B) was similar across all groups compared with the placebo group, with more outliers appearing as the dose increases. These data suggest that Nilotinib enters the brain in a dose-independent manner. The ratio of unbound CSF: plasma Nilotinib ( Figure 2C) appears to be the same in all dose groups and an average of 0.5-1% Nilotinib is detected in the CSF, suggesting that Nilotinib crosses the BBB and is detected as free or unbound in the CSF at low concentrations.

| Effects of Nilotinib on DA metabolism
To determine the effects of Nilotinib on the metabolism of DA in the

| Effects of Nilotinib on CSF alpha-synuclein
The concentration of total alpha-synuclein in the CSF was unchanged in all Nilotinib-treated groups (n = 15) compared with placebo ( Figure 4A) and the concentration of oligomeric alphasynuclein in the CSF was unchanged in all Nilotinib-treated groups (n = 15) compared with placebo. No difference was observed in oligomeric: total alpha-synuclein ratio in CSF ( Figure 4C) as well as plasma ( Figure 4D). Further exploratory evaluation of CSF levels of oligomeric alpha-synuclein over time shows that oligomeric alphasynuclein is unaltered at 1 hour ( Figure 4E The ratio of oligomeric: total CSF alpha-synuclein was unaltered at 1 hour ( Figure 4M), 2 hours ( Figure 4N), and 4 hours ( Figure 4P) after Nilotinib dosing but a significant reduction in oligomeric: total alpha-synuclein ratio was observed in the 200 mg ( Figure 4O) and 400 mg ( Figure 4O) groups compared with placebo.

| Effects of Nilotinib on plasma alpha-synuclein
The plasma concentration of total alpha-synuclein was unchanged in all Nilotinib-treated groups ( Figure 5A Oligomeric alpha-synuclein was not detected in the plasma.

| Effects of Nilotinib on soluble TREM2 in the CSF
The CSF level of soluble TREM2 shows a slight (10%) increase in the 150 mg Nilotinib group ( Figure 5E, n = 15) compared with placebo, The plasma levels of TREM2 were not changed in Nilotinib-treated groups compared with placebo ( Figure 5F), suggesting that Nilotinib at the dose used in this study affects soluble CSF TREM2, independent of its plasma levels.
As expected, after a single dose of Nilotinib, the level of p-NF-H was unchanged in all groups in the current study (data not shown).
To demonstrate the effects of Nilotinib on markers of cell death, we analyzed p-HF-H levels in CSF samples collected from a previous study after treatment with 150 mg (n = 5) and 300 mg (n = 7) Nilotinib for 6 months. 16 Previous results show that Nilotinib may significantly reduce CSF neuron-specific enolase (NSE) and S100B, which are markers of neuronal and glial cell death, respectively. 16 Analysis of CSF samples demonstrates that the level of cell death biomarker p-NF-H was significantly reduced between baseline and 6-month treatment with 150 mg ( Figure 5G) and 300 mg ( Figure 5H) Nilotinib, suggesting Nilotinib treatment may lead to neuroprotective effects.

| D ISCUSS I ON
The data show that Nilotinib enters the brain and alters surrogate biomarkers of PD in a dose-independent manner. An equal concentration of unbound free Nilotinib was detected in the CSF of all dose groups, suggesting that perhaps the drug target (c-Abl, DDRs, etc.) may provide a sink for Nilotinib in the brain. We previously showed that Nilotinib was detected in the human CSF 4 hours after administration but inhibition of CSF Abl extended up to 6 hours, 16 suggesting that free CSF Nilotinib may not be the final or total concentration of drug that enters the brain. Nilotinib exhibits strong and irreversible binding to its target and the level of CSF Nilotinib may reflect the dynamic pharmacological properties of tyrosine kinase inhibitors via interaction with the ATP-binding cassette efflux transporters, Furthermore, our mouse studies showed that Nilotinib concentration in the brain increased in an underproportional manner with dose. In humans, plasma concentrations were similar for all doses between 150 and 400 mg. CSF levels and CSF/plasma ratios were also independent of the dose. This is a logical consequence of the constant plasma levels across doses, since the distribution of Nilotinib into the brain depends on plasma levels.
The apparent dose-independent effect of Nilotinib on potential disease biomarkers in the CSF may be due to the effect of the drug at multiple targets, especially at higher (400 mg) concentrations, suggesting that multi-target engagement abrogates Nilotinib action and may result in more side-and off-target effects in the CNS. It is possible that a balance between drug level in the CNS and target engagement is best achieved at lower concentrations that lead to more specific binding of Nilotinib to either c-Abl and/or DDR1/2. We have shown in several experimental models that knockdown of either Abl or DDRs alone results in reduction of alpha-synuclein, and protection of DA neurons, reminiscent of Nilotinib effects in this study.
Nilotinib displays a high binding affinity and potently inhibit Abl (IC50 < 30 nmol/L) and DDRs (IC50 at 3-6 nmol/L) and it is highly F I G U R E 4 A, The concentration of CSF total alpha-synuclein was unchanged in all Nilotinib-treated groups (n = 15) compared to placebo and B, CSF concentration of oligomeric alpha-synuclein was unchanged in all Nilotinib-treated groups (n = 15) compared to placebo. C, No difference was observed in oligomeric: total alpha-synuclein levels in (C) CSF as well as plasma (D). An exploratory evaluation of CSF levels of oligomeric alpha-synuclein over time shows that oligomeric alpha-synuclein is not altered at (E) 1 hour (F) 2 hours and (H) 4 hours after Nilotinib administration, but a significant reduction in oligomeric alpha-synuclein was observed in the 400 mg group after 3 hours (G) of dosing (* indicates P < 0.05). No significant changes in total CSF alpha synuclein were observed in all groups and at all time-points (I-L). The ratio of oligomeric: total CSF alpha-synuclein was unaltered at (M) 1 hour and (N) 2 hours and (P) 4 hours after Nilotinib dosing but (O) a significant reduction in oligomeric: total alpha-synuclein was observed in the 200 mg and 400 mg groups compared to placebo (** indicates P < 0.01). For total alpha-synuclein and oligomeric alpha-synuclein time points n = 4 (except at 4 hours n = 3), at 1-hourr 200 mg group n = 3, at 2 hours n = 4 except 400 mg, at 3 hours n = 4 except 300 mg group n = 3 likely that dual inhibition of these receptors facilitates the effects of Nilotinib action. A wider concentration range (50- We previously demonstrated that Nilotinib treatment for 6 months results in a significant increase in CSF HVA levels in PD and DLB patients from baseline to 2 and 6 months. 16 The present data show that a single dose of 200 mg Nilotinib results in a significant increase in DOPAC, suggesting that Nilotinib alters DA metabolism. Previous work showed that CSF HVA levels may be unreliable biomarkers for PD (1995). 37 However, our results show that not only HVA but also DOPAC is altered when participants were off any MOA-B inhibitors (selegiline and rasagiline) for at least 6 weeks before screening. Additionally, it was recently reported that de NOVO PD patients show a peak of CSF HVA levels around 1.5-2 hours after administration of 200 mg levodopa and the level of CSF HVA remains constant for a few (up to 4) hours. 38 In the current study, LPs were performed within 2 hours (on-state) after last levodopa administration and were staggered for up to 4 hours. The results show that the levels of HVA and DOPAC in the placebo group are unchanged, supporting previous findings 38 and suggesting a plateau of CNS HVA and DOPAC in the present cohort for at least 4 hours.
In the previous study HVA was measured at baseline and 2 months, when MOA-B inhibitors were eliminated and levodopa treatment was reduced in most patients. However, the level of HVA continued to rise between 2 and 6 months. The single dose effect of Nilotinib on HVA and DOPAC strongly supports alteration of DA metabolism that maybe a long-term disease modifying effect of Nilotinib in PD.
TREM2 is a receptor expressed on microglia 39 and plays an important role in neurodegeneration. 40,41 The R47H and other variants of TREM2, which result in loss of function, are reported to be risk factors for PD. [24][25][26] The role of TREM2 in neurodegeneration remains controversial 41,42 as partial or incomplete loss of TREM2 leads to differential effects on microglia and neurodegenerative pathology. 43 Microglia activation in the SN of the PD brain may produce neurotoxic molecules, resulting in progressive degeneration of DA neurons. 27,28 TREM2 is implicated in the regulation of a regulatory microglial response. 44 TREM2 loss of function results in reduced microglial phagocytosis, 30-32 while TREM2 function inhibits inflammatory responses in microglia via suppression of NF-kB pathways and is strongly implicated in microglia innate immunity. 29 Overexpression of TREM2 reduces 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neuropathology including DA neuron degeneration and neuroinflammation by negatively regulating NF-κB signaling pathways in mice. 45 Our results show that a single dose of 200 mg Nilotinib significantly increases soluble CSF TREM2 levels and this increase is concurrent with improved DA metabolism and a potential reduction of oligomeric alpha-synuclein, in agreement with recent findings. 45 We previously found that both TREM2 and its adaptor protein DNAX-activating protein of 12 kDa (DAP12) 46 are reduced after DDRs knockdown, suggesting that alteration of this tyrosine F I G U R E 5 A, The plasma concentration of total alpha-synuclein was unchanged in all Nilotinib-treated groups compared to placebo at 1hour post Nilotinib dosing. Plasma total alpha-synuclein (B) was significantly reduced in the 150 mg group (and trended toward a decrease in the 200 mg group) compared to placebo (* indicates P < 0.05). C, No difference was observed in total alpha-synuclein levels at (C) 3 hours (D) and 4 hours compared to placebo. For all plasma alpha-synuclein time points n = 4 (except at 4 hours n = 3). E, Graph shows TREM2 levels in the CSF (n = 15) and (F) shows TREM2 levels in the plasma (n = 15) after treatment with single dose Nilotinib. G, Graph shows the CSF levels of phosphorylated neurofilaments between baseline and after 6-months treatment with 150 mg Nilotinib (n = 5) and (H) shows the CSF levels of phosphorylated neurofilaments between baseline and after 6-months treatment with 300 mg Nilotinib (n = 7) kinase receptor, which is potently targeted by Nilotinib, 4,5 may result in alteration of TREM2 signaling. DDRs levels are increased in postmortem PD brains and Nilotinib enhances neurotoxic protein clearance, attenuates cell death and reduces the number of TREM2 + microglia, 47 suggesting regulation of myeloid-derived microglia and protection against neurotoxic proteins.
Oligomeric alpha-synuclein was not detected in the plasma, but we detected a significant effect on total alpha-synuclein levels with lower single doses (150 and 200 mg) of Nilotinib. Nilotinib significantly reduced the concentration of alpha-synuclein that is elevated in the blood of PD patients. [48][49][50][51] These results are consistent with our previous findings that demonstrated Nilotinib effects on lowering blood alpha-synuclein in mice. 7 There is evidence that autophagy is disregulated in the blood of PD patients, [52][53][54][55] and the effects of Nilotinib on autophagy may underlie its effects on plasma alphasynuclein. However, a single dose Nilotinib had no effect on total CSF alpha-synuclein level using the same assay and conditions used in the measurement of plasma alpha-synuclein. The levels of CSF oligomeric alpha-synuclein appeared to change particularly with lower concentrations at 3 hours after Nilotinib administration. These data are consistent with increased CSF levels of TREM2, which enhances the phagocytosis function of microglia and suppresses inflammation, 56 suggesting that reduction in oligomeric alphasynuclein may be the result of increased phagocytosis. We previously demonstrated that Nilotinib promotes autophagic clearance of neurotoxic proteins and improves motor and cognitive symptoms in several models of neurodegeneration without evidence of increased inflammation. 6,7,9,[11][12][13][57][58][59] Measurement of CSF alpha-synuclein with other PD-related biomarkers like TREM2 and DA metabolism may validate the utility of CSF and/or plasma alpha-synuclein as a biomarker, not for the diagnosis or progression of PD, but for specific drug effects like Nilotinib. We observed some variability in oligomeric CSF alpha-synuclein but the combination with TREM2 and DA metabolism as well as plasma alpha-synuclein suggest that with longer drug exposure oligomeric alpha-synuclein that longitudinally increases in the CSF of PD patients 17,19-21 may be a reliable measure of Nilotinib effects on changes of PD-related biomarkers. 60,61 In conclusion, this study demonstrates that Nilotinib penetrates the BBB and is detected in the CSF in a concentration-independent manner. An optimal dose of 200 mg Nilotinib potentially induces a concurrent change in DA metabolism, reduction in oligomeric alphasynuclein, and elevation of TREM2 levels in the CSF.

ACK N OWLED G EM ENTS
The authors acknowledge Jan Kehr and Staffan Schmidt at Pronexus