(M1330-10-64) Evaluation of In Vitro Protein Binding of STK-001, an Antisense Oligonucleotide, in Plasma and Cerebrospinal Fluid

Purpose: STK-001 is currently evaluating in Phase 1/2a and Open Label Extension studies in the US and the UK for the treatment of Dravet Syndrome (DS) in children of age 2 to 18 years. STK-001 is an antisense oligonucleotide (ASO) containing 2’-O-methoxyethyl (MOE) and phosphorothioate modifications and is administered via intrathecal injection (IT) to circumvent the blood brain barrier. Protein binding affects drugs’ bioavailability and distribution, therefore, ICH M3 (R2) guidance recommends thatin vitro plasma protein binding data for animals and humans generally should be assessed before initiating human clinical trials [1]. Since the target of STK-001 is expressed in brain, we evaluated protein binding properties of STK-001 in both cynomolgus monkey and human, plasma and cerebrospinal fluid (CSF). There are pros and cons of various methods that have been established in the literature for assessment of protein binding including ultracentrifugation, gel electrophoresis using radiolabeled drug, and molecular weight cut-off (MWCO) ultrafiltration method followed by liquid chromatography-mass spectrometry (LC-MS) [2, 3]. We utilized MWCO/LC-MS method and adapted it for in vitro evaluation of protein binding of STK-001 [4]. This method allowed us to accurately determine the unbound fraction of STK-001 in plasma and CSF in cynomolgus monkey and human.

Methods: We selected the Amicon Ultra 50 kDa MWCO cellulose filter unit for the ultrafiltration method because of its minimal non-specific binding. Prior to use, the filter units were washed with PBS and then treated with PBS containing 0.5% Tween-80 to further reduce non-specific binding. To prepare the samples, selected concentrations of STK-001 were added to PBS, plasma, or CSF, followed by an incubation at 37 ℃ for 60 minutes. The test concentration range was selected based on the combination of observed exposures in monkey and anticipated exposures in clinic. Centrifugation was carried out at 25 ℃. The filtrates obtained were subjected to analysis using LC-MS to quantify the unbound or free portion of STK-001. Recovery of Amicon® Ultra -0.5 centrifugal filter (50 kDa MWCO) was evaluated using the test concentrations of STK-001 incubated in PBS. The spiked levels were adjusted to account for their respective recoveries. The percentage of bound fractions was calculated by subtracting the free fraction (that were adjusted for recoveries) from one hundred.

Results: Table 1 presents the results of the protein binding evaluation of STK-001 in human and monkey plasma. The results indicate that STK-001 exhibited high protein binding ( >98%) in both monkey and human plasma at all tested concentrations of STK-001 (1.00, 5.00, and 10.0 μg/mL). Table 2 presents the results of the protein binding evaluation of STK-001 in CSF from monkey and human at three different concentrations: 5.00, 50.0, and 100 μg/mL. In monkey CSF, the mean observed protein binding of STK-001 was 29.7%, 13.3%, and 7.25% at STK-001 concentrations of 5.00, 50.0, and 100 µg/mL, respectively. In human CSF, the mean observed protein binding was 36.8%, 15.5%, and 3.79% at STK-001 concentrations of 5.00, 50.0, and 100 µg/mL, respectively. The data indicates a decrease in protein bound fraction of STK-001 as the STK-001 concentration increased, both in monkey CSF (from 29.7% to 7.25%) and human CSF (from 36.8% to 3.79%).

Conclusion: A reliable MWCO/LC-MS based method with a recovery rate >90% was developed to assess the in vitro protein binding of STK-001 in monkey and human plasma and CSF at non-clinical and clinically relevant concentrations. Our results showed high protein binding ( >98%) in plasma in both species, at STK-001 concentration ranging from 1.00 µg/mL to 10.0 µg/mL. This result is consistent with observations made by others with ASOs of similar chemistry. Moderate to low protein binding of STK-001 was observed in CSF from both monkey and human. The observed relatively low protein binding in CSF compared to plasma can be attributed to the significantly lower total protein concentration in CSF (approximately 50-100 times lower) compared to plasma. These results indicate that a large percentage of unbound fraction of STK-001 is available to be distributed to central nervous system from CSF following IT administration.

References: 1. ICH guideline M3(R2) on non-clinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals. (2010).
2. Srikanth, C. H., et al. (2013). Correlation of in vitro and in vivo plasma protein binding using ultracentrifugation and UPLC-tandem mass spectrometry. The Analyst, 138(20), 6106-6116. https://doi.org/10.1039/c3an01116j
3. Rocca, C., et al. (2019). Evaluation of electrophoretic mobility shift assay as a method to determine plasma protein binding of siRNA. Bioanalysis, 11(21), 1927–1939. https://doi.org/10.4155/bio-2019-0151
4. Humphreys, S. C., et al. (2019). Plasma and Liver Protein Binding of N-Acetylgalactosamine–Conjugated Small Interfering RNA. Drug Metabolism and Disposition, 47(10), 1174–1182. https://doi.org/10.1124/dmd.119.086967

Table 1: Summary of Protein Binding of STK-001 in Monkey and Human Plasma

Table 2: Summary of Protein Binding of STK-001 in Monkey and Human CSF