Unbound Brain-to-Plasma Partition Coefficient, Kp,uu,brain—a Game Changing Parameter for CNS Drug Discovery and Development

Purpose More than 15 years have passed since the first description of the unbound brain-to-plasma partition coefficient (Kp,uu,brain) by Prof. Margareta Hammarlund-Udenaes, which was enabled by advancements in experimental methodologies including cerebral microdialysis. Since then, growing knowledge and data continue to support the notion that the unbound (free) concentration of a drug at the site of action, such as the brain, is the driving force for pharmacological responses. Towards this end, Kp,uu,brain is the key parameter to obtain unbound brain concentrations from unbound plasma concentrations. Methods To understand the importance and impact of the Kp,uu,brain concept in contemporary drug discovery and development, a survey has been conducted amongst major pharmaceutical companies based in Europe and the USA. Here, we present the results from this survey which consisted of 47 questions addressing: 1) Background information of the companies, 2) Implementation, 3) Application areas, 4) Methodology, 5) Impact and 6) Future perspectives. Results and conclusions From the responses, it is clear that the majority of the companies (93%) has established a common understanding across disciplines of the concept and utility of Kp,uu,brain as compared to other parameters related to brain exposure. Adoption of the Kp,uu,brain concept has been mainly driven by individual scientists advocating its application in the various companies rather than by a top-down approach. Remarkably, 79% of all responders describe the portfolio impact of Kp,uu,brain implementation in their companies as ‘game-changing’. Although most companies (74%) consider the current toolbox for Kp,uu,brain assessment and its validation satisfactory for drug discovery and early development, areas of improvement and future research to better understand human brain pharmacokinetics/pharmacodynamics translation have been identified. Supplementary Information The online version contains supplementary material available at 10.1007/s11095-022-03246-6.

The survey will be open on 22nd of September and end on 3rd of October.
The summary of the survey generated automatically by the KURT system will be circulated in PDF and Excel formats by 4th of October. After discussion, the results will be used in the preparation of the perspective paper for an honorary special issue of Pharmaceutical Research (PharmRes R ), dedicated to Professor Emerita Margareta Hammarlund-Udenaes. Other, please specify: Vaccine, gene therapy, Gene therapies (if not considered a part of "nucleotide therapeutics") 6. When did the first project teams or key scientists start advocating for applying the concepts of the unbound brain-to-plasma concentration ratio (Kp,uu,brain)? 8. What level of implementation and integration of Kp,uu,brain would you estimate in your company, as a % of project teams applying concepts and methodology as appropriate.
Vetenskapsområdet för medicin och farmaci, Uppsala universitet Sida 2 av 17 Survey on the use/implementation of Kp,uu,brain concept by pharma companies Less than 20 % 1 Between 20 and 80 % 5 More than 80 % 8 9. When was the Kp,uu,brain concept fully embedded in the project teams? I.e. ca 80% of the projects applying concepts and methodology as appropriate. Other, please specify: Not yet implemented, Application of free drug hypothesis also for targets within the CNS, both on-and off-targets 11. What was the key mechanism by which Kp,uu,brain was implemented in your organisation? 3 1 Yes, for every nominated drug candidate (e.g. prior to investment decision in GLP-tox studies) 2 Yes, for every nominated drug candidate, but only for CNS projects (e.g. prior to investment decision in GLP-tox studies) 3 Yes, Kp,uu,brain determination is part of our generic (company-wide) screening cascade and/or considered a prerequisite to conduct in vivo profiling (PK and/or PD) 4 No, Kp,uu,brain is not included in any defined company-wide processes however most project teams will have developed their own process and criteria for conducting Kp,uu,brain determination 5 There is little such process 6 Other, please specify Other, please specify: Answer c), but only for CNS projects . ?Only for CNS projects? applies also to question 8., Informal process for projects that are requiring brain exposure utilizing CNS strategy, Yes, for projects where CNS penetration and unbound brain exposure is a critical parameter e.g. for on-target efficacy, for on-target central side-effects, for offtarget central side-effects 13. Which department is primarily accountable for generating experimental data on Kp,uu,brain?
Note: Other functions may be responsible for conducting elements of the work.

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Other, please add a comme... 6 1 1 Neuropharmacokinetic screening and profiling (from an efficacy standpoint). For instance using an estimate of Kp,uu,brain as gating criteria for entry into in vivo pharmacology studies. 2 By successfully correlating measured Kp,uu,brain with in vitro assays (such assays efflux ratios), it has become possible to use this in vitro methodology (efficiently and reliably) to screen and identify and prioritize molecules with desired Kp,uu,brain. 3 Definition of PK/PD relationship for CNS effects and/or prediction of therapeutic dose 4 CNS off-target safety assessment 5 CNS on-target safety assessment (e.g. the drug acts on a peripheral target which is also expressed in the brain, or a CNS target has additional functions which are sought to be avoided) 6 Other, please add a comment Other, please add a comment: In addition to the absolute Kp,uu value, project teams apply the unbound brain concentrations derived from a Kp,uu experiment to assess theoretical target coverage (e.g. Cu,br (nM) vs in vitro target IC50 (nM)). Hence a compound with a sub-optimal Kp,uu (e.g. 0.2) may prove progressable if sufficient unbound brain concentrations can be achieved to engage target (permitting peripheral side effect/TI profile) 18. Beyond rodents, in which other species is Kp,uu,brain determined (in vivo)? Select all that apply. Other, please specify: non-rodents only at late discovery stage (close to candidate selection) to strengthen human translation, There may occasionally be PET data used to calculate Kpuu in NHP and humans, NHP PET receptor occupancy data along with other data is used to infer NHP Kp,uu,brain, but Kp,uu,brain has not been measured directly from NHP.
19. What would be the drivers for inclusion of higher species (dog, NHP and human) in the assessment of Kp,uu,brain? Select all that apply. Reducing of uncertainty related to potential species differences for CNS targets 2 Mitigating risk of CNS side-effects which are not easily monitorable in early clinical trials 3 Better translation of human dose-exposure-CNS biomarker response relationships 4 Higher species (dog, NHP) are part of non-clinical safety/efficacy assessment 5 Does not apply as Kp,uu,brain is only determined in rodents 6 Other, please specify and include reference to species Other, please specify and include reference to species: Mitigation of species dependent transporter mediated efflux (eg. BCRP in monkey), Kp,uu,brain in other species is being rationally approached rather than experimentally determined, typically applying all relevant contextual data, NHP PET receptor occupancy data along with other data is used to infer NHP Kp,uu,brain, but Kp,uu,brain has not been measured directly from NHP. 20. How does the numerical value of Kp,uu,brain (any species) feed into predictions of therapeutic dose (typically underpinning MABEL, starting dose, tox margins, or Phase 2 dose ranges etc)? Other, please add comment: Kp,uu have been determined in specific pharmacological animal models that may influence BBB permeation (e.g. drug induced seizure animal model or trangsgenic mice models for neurological targets e.g. alzheimers) Yes, please add examples: Yes, we did it once for a target from which we know that the expression and the final pharmacological effect is age dependent in mice. The compound was a moderate P-gp substrate but there was no difference in Kp,uu,brain on age in mice. We never tested it for a strong P-gp substrate.
22. Do you investigate drug-drug interactions on the level of BBB using Kp,uu,brain? No, we typically consider the brain interstitium (Kp,uu,brain) to adequately represent exposure in brain cells 2 Yes. Please specify rationale 3 Other, please specify Other, please specify: Answer a) for Research, answer b) for Development, here we use calculated Kp,uu,cell to correct Fubrain from brain homogenate for lysosomal uptake and pH differences to derive a better estimation of Kp,uu,brain., Kp,uu,cell is assessed for in vitro cell models (tumor cell lines, hepatocyetes) and other tissues (liver) but not for brain.
24. Which in vivo methods are used at your company to determine Kp,uu,brain? Select all that apply.
Vetenskapsområdet för medicin och farmaci, Uppsala universitet Sida 7 av 17 Survey on the use/implementation of Kp,uu,brain concept by pharma companies Other, please specify... 5 2 1 Brain/blood sampling over time with assessment of AUCs in preclinical animals followed by binding correction in respective tissue 2 Brain/blood sampling at infusion steady-state in preclinical animals followed by binding correction in respective tissue 3 Brain/blood sampling at a single non steady-state time-point in preclinical animals followed by binding correction in respective tissue 4 Brain/blood microdialysis 5 Other, please specify Other, please specify: Plans to do PET imaging for compounds in Development, e.g. for compounds targeting brain tumors., Not yet broadly implemented 25. Do you employ cassette dosing as means to increase throughput and reduce animal usage in the determination of Kp,uu,brain. Select which option that best describes your situation. Other method, please specify: Muscle to brain ratio to estimate efflux at the BBB (Pharmaceutics, 11, 595 (2019), doi 10.3390/pharmaceutics11110595)., 96-well filter membrane impregnated with brain homogenate. The assay is called LIMBA (Lipid membrane binding assay). This assay is alos used to optimize against a high non-specific binding in the development of PET tracers. doi: 10.1016/j.ejps.2015.08.014, Imaging, Ultracentrifugation, ultrafiltration 27. Is brain free fraction using the brain homogenate method always measured in the same species as the in vivo study?
Vetenskapsområdet för medicin och farmaci, Uppsala universitet Sida 8 av 17 Survey on the use/implementation of Kp,uu,brain concept by pharma companies

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No, the company typically... 1 9 Other, please specify... 2 0 1 No, the company typically uses a single species and assumes species-independency of drug brain tissue binding properties. Please specify which species is used. 2 Other, please specify No, the company typically uses a single species and assumes species-independency of drug brain tissue binding properties. Please specify which species is used.: Rat, We use porcine brain polar lipids. Correlates very well with brain homogenates from rat, mouse, monkey, human brain., rat, rat, rat brain homogenate, Rat 28. What elements of Kp,uu,brain methodology has been internally validated in your company?
Select all that apply. Other, please specify... 6 1 1 Kp,uu,brain assessment using brain microdialysis 2 Brain tissue uptake and binding assessment using brain homogenate vs brain slice assays 3 Kp,uu,brain versus ratio of Kp,brain in e.g. mdr1a/b double knockout and wild type mice 4 Validation against literature values of Kp,uu,brain 5 Validation against literature values of fu,brain 6 Other, please specify Other, please specify: validation in characterizing cross compound and cross series relationship between unbound brain concentration and pharmacodynamic responses measured preclinically.
29. Do you use BBB cell culture models to predict Kp,uu,brain? Select all that apply. Physiologically-based pharmacokinetic models for the brain tissue with parameters derived from chemical properties and/or in vitro assays 3 No, we do not use any in silico approach 4 Other, please specify Other, please specify: We've developed individual in-house in silico models for fu,brain, fu,plasma, MDCK P-gp efflux ratio, and passive permeability although these separate models have not been combined together and validated for ability to predicted Kp,uu,brain 31. Do you use CSF as a surrogate fluid to assess the free brain concentration? Other, please specify : CSF only used for large molecule (mAbs) brain exposure assessment, Answer b), but only for compounds which are no transporter substrates.
32. Do project teams (continue to) ask to measure (total) brain exposure in addition to plasma concentrations in standard PK or PD studies?
Yes, this is a common pra... 1

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No, brain tissue samples... 3 7 1 Yes, this is a common practice in at least some projects 2 Yes, but only if concomitant biomarker responses are measured in the same tissue 3 No, brain tissue samples are typically only taken in the studies dedicated to estimate Kp,uu,brain 33. For the purpose of determining Kp,uu,brain in pharmacology studies, do project teams typically conduct brain sampling of the animals in the study (including any satellite groups).
Vetenskapsområdet för medicin och farmaci, Uppsala universitet Sida 10 av 17 Survey on the use/implementation of Kp,uu,brain concept by pharma companies No, the estimate of Kp,uu,brain is obtained separately in a dedicated study and applied to measurement of plasma concentration to derive CNS exposure 2 Yes, it is generally routine to determine (total) brain concentrations across PD studies for future use 3 Other, please specify Other, please specify: Sometimes; depends on PD model/# animals, stagegate of compound(s), etc., Not yet broadly implemented, Sometimes estimate of Kp,uu,brain comes from separate dedicated "PK" study and applied to measurements of plasma concentration to derive CNS exposure, other times brain concentration is determined from PD studies for KPuu,brain determination and PK/PD analysis 34. In determining the temporal aspect of CNS exposure in pharmacology (e.g. unbound brain concentration at defined time-points such as pre-dose trough levels in repeated dosing studies) which of the of the following statements best reflects the way your company works: Brain exposure at specific time points is usually not considered for any major applications (including PK/PD and prediction of therapeutic dosage). 2 It happens that there is a need or interest in assessing brain exposure at specific time points. In such instances we conduct sampling of brain at those timepoints to enable calculation of exposure. 3 It happens that there is a need or interest in assessing brain exposure at specific time points. In such instances exposure is estimated from the plasma concentration and Kp,uu,brain determined based on AUC or at steady state (from the same study or separate dedicated study Other, please specify... 6 6 1 Endothelial permeability (e.g. in situ brain perfusion) 2 CSF concentrations (e.g. Kp,uu,CSF) 3 Total brain concentrations (including total brain-to-plasma ratio) 4 fu,brain (as a standalone parameter) 5 fu,plasma (as a standalone parameter) 6 Other, please specify Other, please specify: Efflux ratio in transfected cells of MDR1 and BCRP, Total brain exposure is always used together with fu,brain, Muscle to brain ratio (Pharmaceutics, 11, 595 (2019) Yes, we have such values established and use them for compound prioritization. Please specify specific Kp,uu,brain value the projects/organization would consider for a compound being ?progressable?.: generally 0.3, but can be project dependent., Our aspirational cut-off for a candidate molecule to move into the clinic is >0.5. For early leads deemed progressable, a cut-off of 0.3 is typically applied. There may be specific projects where the pursued target give rise adjusted cut-offs (e.g. high risk of peripheral side effects)., case by case, depends on the team (either >0.3 or >0.5), typically 0.5-2, but where PD response is observed, it may go outside this range (e.g. lower), >0.3 is good CNS exposure, >0.3, Cutoff values are specific to a given project, whether ?good? brain penetration is warranted (or not warranted) and expected impact on therapeutic index and/or dosing regimen. Any value of Kp,uu,brain that allows an acceptable therapeutic index and dosing regimen is considered an acceptable Kp,uu,brain. Generally, cut-off value of >0.3 defines ?good? brain penetration and value Other, please specify: Values are defined for "likely equilibrium", but all other values "depend" on the specific situation/molecule. 37. Do you use transgenic animal lines for clarification of the certain BBB mechanism and verify the Kp,uu,brain?
Vetenskapsområdet för medicin och farmaci, Uppsala universitet Sida 12 av 17 Survey on the use/implementation of Kp,uu,brain concept by pharma companies Yes, please provide a short summary: Kp,uu determined in 3 rats -standard deviation is assessed and should fall within limits for data to be aggregated and reported as a mean in the database, Aceptance criteria are based on quality of the in vivo and in vitro experimental data and from the context of all that is known in the project 39. How do you interpret and report development data for submission to regulatory authorities that are based on total brain concentrations, such as Quantitative whole body autoradiography (QWBA) data? Please describe as applicable.
• QWBA is qualitative and signals maybe be contaminated by metabolites. If not intensity in the brain, suggesting poor brain penetration.
• Reported as total concentrations for regulatory purposes • We do not use QWBA data for Kpuu assessesment because data represent total drug related radioactivity (parent plus potential metabolites), i.e. not only parent drug.
• We don't use QWBA results to draw any conclusion about the brain penetration of a compound • As qualitative yes or no CNS penetration, since metabolite and parent are not separated in QWBA study • Yes -we report a Kp and fu,b typically, sometimes Kp,uu. QWBA data are reported as such -no specific call-out to brain.
• Primarily imaging data has been used and reported semi-quantitatively • not applicable • typically only when negative, where there is a lack of compound in the brain • These data are used for prediction of committed effective dose in human • Images shared • Report QWBA • Such data are only interpreted at the level of total exposures • Quantitative whole body autoradiography (QWBA) data and in vitro efflux data Measurement of Kp,uu,brain requires additional animals and has therefore increased the use of experimental animals. 2 By enabling better selection of molecules to enter in vivo testing we have been able to reduce the use of animals. 3 There is no clear impact on the number of animals used for each project in isolation. However by enabling better selection of compounds it has increased the probability of success and can therefore be seen as a reduction in animal use.
44. How do you evaluate the adequacy of the current toolbox for Kp,uu,brain assessment and its validation? Select all that apply. 1 Satisfying for early drug development 2 In principle satisfying for early drug development, but throughput is still limiting 3 The implementation of the concept for late drug development requires additional validation 4 Larger cross-validation and reproducibility studies are needed 5 Not satisfying, please specify Not satisfying, please specify: -We need a better understand of unbound intracellular concentrations for the cells types of interest (both for efficacy and for safety); -We need tools to explain/verify mechanistically unexpected Kp,uu values 45. What are the required, yet missing, aspects for successful translation of Kp,uu,brain concept from preclinical animals to patients? Select all that apply.