Microdosing Studies Research Articles

Less is more? A review of psilocybin microdosing.

The applications of psilocybin, derived from 'magic mushrooms,' are vast, including a burgeoning practice known as microdosing, which refers to the administration of sub-hallucinogenic doses of psychedelic substances to obtain benefits without experiencing significant cognitive and perceptual distortion. However, current research is fairly new with several limitations and gaps that hinder adequate conclusions on its efficacy. This semi-structured review aimed to identify and highlight research gaps in the field of psilocybin microdosing for future research. A Preferred Reporting Items for Systematic Reviews and Meta-Analyses based strategy was employed, utilizing a chain of keywords and key phrases across multiple databases, augmented by a cross-sectional Google search for relevant grey literature in the form of the top 10 search results. A total of 40 studies and 8 unique websites were identified, summarized and tabulated into four distinct categories, namely non-clinical, clinical, observational and anecdotal evidence. The majority of available evidence originates from observational studies, while non-clinical and clinical study findings remain comparatively sparse and inconsistent. Web-based findings were consistent with current research findings. Key research gaps were highlighted: the imperative for more randomized placebo-controlled trials, exploration of dose-response ranges, psychological and personality testing of participants, utilization of active placebos, greater diversity in study populations, an increase in psilocybin-exclusive microdosing studies and the refinement of animal models. Definitive conclusions regarding the efficacy of psilocybin microdosing remain elusive, emphasizing the need for further study. Numerous research gaps necessitate consideration for future investigations.

Is microdosing a placebo? A rapid review of low-dose LSD and psilocybin research.

Some recent research and commentary have suggested that most or all the effects reported by people who microdose psychedelics may be explained by expectations or placebo effects. In this rapid review, we aimed to evaluate the strength of evidence for a placebo explanation of the reported effects of microdosing. We conducted a PubMed search for all studies investigating psychedelic microdosing with controlled doses and a placebo comparator. We identified 19 placebo-controlled microdosing studies and summarised all positive and null findings across this literature. Risk of bias was assessed using the Cochrane risk-of-bias tool for randomised trials. The reviewed papers indicated that microdosing with LSD and psilocybin leads to changes in neurobiology, physiology, subjective experience, affect, and cognition relative to placebo. We evaluate methodological gaps and challenges in microdosing research and suggest eight reasons why current claims that microdosing is predominately a placebo are premature and possibly wrong: (1) there have been only a small number of controlled studies; (2) studies have had small sample sizes; (3) there is evidence of dose-dependent effects; (4) studies have only investigated the effects of a small number of doses; (5) the doses investigated may have been too small; (6) studies have looked only at non-clinical populations; (7) studies so far have been susceptible to selection bias; and (8) the measured impact of expectancy is small. Considering the available evidence, we conclude that it is not yet possible to determine whether microdosing is a placebo.

Development of a sensitive LC-MS/MS assay to support human microdose study for an oral agonist of the GLP-1 receptor.

Aim: The purpose of this work was to determine the feasibility of supporting a clinical microdose study for PF-06882961 (danuglipron), an oral small molecule agonist of the GLP-1 receptor, by LC-MS/MS. Methodology: Statistical instrument parameteroptimization using response surface methodologywas employed to develop a LC-MS/MS method for the analyte, PF-06882961. Results: An LC-MS/MS method was developed and validated to support a proof of concept microdose pharmacokinetics preclinical study in monkeys, administered PF-06882961 (0.005mg total, average dose=0.0007mg/kg) via intravenous bolus injection. Conclusion: The present study demonstrated the feasibility of analyzing human microdose plasma samples for PF-06882961 by LC-MS/MS, instead of accelerator mass spectrometry, thereby reducing cost and eliminating synthesis and exposure to 14C labeled material.

The application of Phase 0 and microtracer approaches in early clinical development: past, present, and future.

Phase 0 microdosing studies were introduced to the drug development community approximately 20years ago. A microdose is defined as less than 1/100th of the dose calculated based on animal data to yield a pharmacological effect in humans, with a maximum of 100μg, or 30nmoles for protein products. In our experience, Phase 0microdose studies have not been fully embraced by the pharmaceutical industry. This notion is based on the number of Phase 0 studies that we have been involved in. Thus, we conducted at least 17 Phase 0 microdose studies in the Zero's (on average, two per year), but in the years beyond this, it was only 15 studies (1.4 per year); in these latter years, we did conduct a total of 23 studies which employed an intravenous (i.v.) microdose for absolute bioavailability (ABA) assessments (two per year on average), which are the most used and potentially informative type of clinical study using a microdose, albeit they are formally not microdose studies. In the current review, we summarize the past use of and experience with Phase 0 microdose designs in early clinical development, including intravenous 14C microdose ABA studies, and assess what is needed to increase the adoption of useful applications of Phase 0/microdose studies in the near future.

ACD856, a novel positive allosteric modulator of Trk receptors, single ascending doses in healthy subjects: Safety and pharmacokinetics

PurposeAlzeCure Pharma AB is developing novel positive allosteric modulators of Trk-receptors for treatment of Alzheimer’s disease, depression, other psychiatric conditions and other disorders where cognition is impaired. The preceding candidate drug ACD855 was shown to have a too long half-life in humans to allow further development. To de-risk the development of the follow-up compound ACD856, the oral single ascending dose study of ACD856 in humans was preceded by an intravenous microdose study, assessing the elimination half-life in plasma.MethodsA phase 0 study with a microdose of ACD856 (0.100 mg), was conducted in six healthy male subjects all receiving ACD856. Sequentially, a randomized, placebo-controlled, double-blind Phase I single ascending oral dose study (1 – 150 mg) was conducted, including 56 healthy subjects. Both studies assessed the safety and tolerability, as well as the PK properties of ACD856 after single dose intravenous and oral administration.ResultsACD856 was well tolerated with no treatment emergent, or dose related adverse events or other safety assessments. In the microdose study, ACD856 exhibited a bi-exponential plasma decline, low distribution volume, low plasma clearance with a half-life of approximately 20 hours. Orally, ACD856 exhibited rapid absorption, an almost complete bioavailability and a dose proportional increase in exposure. While the Cmax was lowered and delayed by food intake, the effect on plasma half-life and the overall bioavailability was low. No renal elimination of ACD856 was detected. ConclusionThe prediction proved accurate demonstrating the value of conducting a microdose study prior to ascending dose studies.Trial registrationNCT05783830 March 24, 2023 (microdose study, retrospectively registered) and NCT05077631 October 14, 2021 (single ascending dose study).

Abstract C024: Trial in progress: Phase 1 study of BAL0891 as monotherapy and in combination with chemotherapy in patients with advanced solid tumors

Abstract Background: BAL0891 is a first-in-class small-molecule mitotic checkpoint inhibitor (MCI) that targets threonine tyrosine kinase (TTK) and polo-like kinase 1 (PLK1) to disrupt spindle assembly checkpoint (SAC) regulation and induce tumor cell death. It has a more potent anti-tumor activity as compared to TTK-specific inhibitors due to its combined prolonged effect on TTK and transient effect on PLK1. In preclinical studies, BAL0891 demonstrated potent anti-tumor activity as a monotherapy and in combination with paclitaxel and carboplatin. In the first-in-human microdose study of TTK-CS-001 in healthy subjects, key BAL0891 pharmacokinetic (PK) parameters were investigated (i.e., clearance, half-life, exposure), and no safety signals were detected. Trial design: This clinical trial is a multiple-cohort, open-label phase 1 study with 3 dose-escalation substudies, investigating intravenous (IV) BAL0891 as monotherapy, and in combination with carboplatin or paclitaxel, in patients with advanced solid tumors. Primary objectives: 1) To evaluate safety/tolerability 2) To estimate the maximum tolerated dose (MTD) and/or recommended Phase 2 dose (RP2D) of BAL0891 monotherapy and in combination with carboplatin/paclitaxel. Eligibility: Patients ages ≥18 years with advanced/metastatic solid tumors that are refractory to, or intolerant of existing therapy may participate in this study. Patients enrolled in dose level ≥4 of Substudy 1 and all patients in Substudies 2 and 3 must have measurable disease. Substudy 1: Monotherapy BAL0891: IV doses of BAL0891 on Days 1 (D1) and 8 (D8) every 3 weeks (Q3W). A Bayesian logistic regression model (BLRM) will guide dose escalations, with 8 nominal DLs of 5/10/20/40/80/160/320/480 mg. Substudy 2: Phase 1 BAL0891 D1, D8 + carboplatin AUC5-6 D1 Q3W. Substudy 3: Phase 1 BAL0891 D1, D15 + paclitaxel 70-80 mg/m2 D1, D8, D15 Q4W. Substudies 2 and 3 may begin once the maximum tolerated dose (MTD) of BAL0891 monotherapy has been established or at the first signs of expected target toxicity and/or efficacy with monotherapy. Enrollment began in February 2023 and is ongoing in the United States and South Korea. Clinical trial information; NCT05768932 Citation Format: Shivaani Kummar, Minal Barve, Eric Feldman, Seunghyun Ma, Jaime Merchan, Seock-Ah Im, Sun Young Rha. Trial in progress: Phase 1 study of BAL0891 as monotherapy and in combination with chemotherapy in patients with advanced solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C024.

Dried blood spots for erythropoietin analysis: Detection of micro-doses, EPO c.577del variant and comparison with in-competition matching urine samples.

The abuse of recombinant erythropoietin (rEPO) and other erythropoietin (EPO) receptor agonists (ERAs) in sports prompted the need for sensitive detection methods of these substances. Dried blood spot (DBS) samples offer an easy solution for simultaneous collection of blood and urine during a doping control, but sensitivity issues are often presented as a challenge for routine EPO analysis from DBS. Its potential use for detecting rEPO micro-doses and the EPO gene c.577del variant thus needed further demonstration. Here, capillary blood collected from the arm skin of 111 athletes with Tasso-M20 (17.5μL/spot), collected during professional triathlon competitions, were analysed. Also, venous blood samples from healthy volunteers were used to prepare several spots of 20 μL on Mitra VAMS (from an rEPO micro-dose study) and Whatman filter paper (from an EPO gene variant study). Immunopurification of 2 spots with MAIIA EPO Purification Gel Kit and analysis with sodium N-lauroylsarcosinate polyacrylamide gel electrophoresis (SAR-PAGE)/Western blot resulted in sensitive detection of (1) micro-doses of rEPO from Mitra VAMS, (2) endogenous EPO from Tasso-M20 in all in-competition subjects, and (3) the EPO c.577del variant from Whatman filter paper. Additionally, in-competition endogenous EPO was detected in DBS even when matching urine samples had undetectable EPO. In conclusion, this work demonstrated that DBS can be a useful complementary matrix to urine samples for EPO detection.

Abstract CT246: An open-label, single-center, phase 0, microdose study to demonstrate delivery of TTX-MC138-NODAGA-Cu64 to radiographically confirmed metastases in subjects with advanced solid tumors

Abstract Purpose: Cu-64 labeled TTX-MC138, a microRNA-10b (miR-10b) inhibitor, will be evaluated in a Phase 0 clinical study conducted under an Exploratory IND to evaluate delivery of the molecule to metastatic lesions in subjects with advanced solid tumors by using positron emission tomography-magnetic resonance imaging (PET-MRI). Background: miR10b is a master regulator of the viability of metastatic tumor cells and promotes capacity of tumor cells to migrate and invade surrounding tissue. These findings prompted design of a miR10b-targeted therapeutic consisting of an anti-miR10b antagomir conjugated to ultrasmall iron oxide nanoparticles (MN), termed TTX-MC138. In mouse models of cancer, TTX-MC138 caused durable regressions of established metastases with no systemic toxicity. Clinical development will investigate if TTX-MC138 would accumulate in clinical metastases. TTX-MC138 is radiolabeled with isotope Cu-64 to generate the radiopharmaceutical TTX-MC138-NODAGA-Cu64. PET-MRI will assess pharmacokinetics (PK) of the radiolabeled product in humans and determine uptake in metastatic lesions. Methods: This open-label, single-center, single-arm, phase 0, microdose study in subjects with advanced solid tumors and radiographically confirmed metastases will evaluate delivery of TTX-MC138-NODAGA-Cu64 by using PET-MRI; establish PK of TTX-MC138-NODAGA-Cu64; and assess safety of TTX-MC138-NODAGA-Cu64. Approximately 12 subjects age ≥ 18 years are expected to be enrolled in the study. Eligible subjects will have confirmed diagnosis of at least 1 metastatic solid tumor (that is not amenable to curative therapy unless participation in the study would delay standard therapy), Eastern Cooperative Oncology Group (ECOG) performance status of 0 to 1, and at least 1 target lesion per Response Evaluation Criteria in Solid Tumors version 1.1 (at least 10 mm per MRI from fludeoxyglucose [FDG] PET-MRI). Study duration is up to 46 days and includes a screening visit, dosing period (2 visits), and a follow-up visit. On Day 1, subjects receive 1 microdose of TTX-MC138-NODAGA-Cu64 by intravenous bolus. Days 1 and 2 include whole body PET-MRI imaging, ECG and blood collection. Safety data are collected at all visits. A Data Review Committee may review specific study data, including safety data. The primary endpoint will measure percent injected dose per cubic centimeter (%ID/cc) tissue of TTX-MC138-NODAGA-Cu64 delivered to metastatic lesions. Secondary endpoints may include evaluation of PK of key components of TTX-MC138-NODAGA-Cu64, metabolite analysis, and target engagement. Safety data, including incidence of adverse events, clinical lab data, ECGs, vital signs and concomitant medications, will be summarized. Enrollment is expected to start in 2023. Citation Format: Andreas Varkaris, Peter Caravan, Neil Robertson, Subrata Ghosh, Michael Warren, Zdravka Medarova, Susan Duggan. An open-label, single-center, phase 0, microdose study to demonstrate delivery of TTX-MC138-NODAGA-Cu64 to radiographically confirmed metastases in subjects with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT246.

A naïve pooled data approach for extrapolation of Phase 0 microdose trials to therapeutic dosing regimens.

Microdosing is a strategy to obtain knowledge of human pharmacokinetics prior to Phase I clinical trials. The most frequently used method to extrapolate microdose (≤100 μg) pharmacokinetics to therapeutic doses is based on linear extrapolation from a noncompartmental analysis (NCA) with a two-fold acceptance criterion between pharmacokinetic metrics of the extrapolated microdose and the therapeutic dose. The major disadvantage of NCA is the assumption of linear extrapolation of NCA metrics. In this study, we used a naïve pooled data (NPD) modeling approach to extrapolate microdose pharmacokinetics to therapeutic pharmacokinetics. Gemcitabine and anastrozole were used as examples of intravenous and oral drugs, respectively. Data from microdose studies were used to build a parent-metabolite model for gemcitabine and its metabolite 2',2'-difluorodeoxyuridine (dFdU) and a model for anastrozole. The pharmacokinetic microdose models were extrapolated to therapeutic doses. Extrapolation of the microdose showed differences in pharmacokinetic shape for gemcitabine and dFdU between the simulated and observed therapeutic concentrations, whereas the observed therapeutic concentrations for anastrozole were captured by the extrapolation. This study demonstrated the possible use and feasibility of an NPD modeling approach for the evaluation and application of microdose studies in early drug development. Last, physiologically-based pharmacokinetic modeling might be an alternative for microdose extrapolation of drugs with complex pharmacokinetics such as gemcitabine.

PET as a Translational Tool in Drug Development for Neuroscience Compounds.

In central nervous system drug discovery programs, early development of new chemical entities (NCEs) requires a multidisciplinary strategy and a translational approach to obtain proof of distribution, proof of occupancy, and proof of function in specific brain circuits. Positron emission tomography (PET) provides a way to assess in vivo the brain distribution of NCEs and their binding to the target of interest, provided that radiolabeling of the NCE is possible or that a suitable radioligand is available. PET is therefore a key tool for early phases of drug discovery programs. This review will summarize the main applications of PET in early drug development and discuss the usefulness of PET microdosing studies performed with direct labelling of the NCE and PET occupancy studies. The purpose of this review is also to propose an alignment of the nomenclatures used by drug metabolism and pharmacokinetic scientists and PET imaging scientists to indicate key pharmacokinetic parameters and to provide guidance in the performance and interpretation of PET studies.

Microdosing as a Potential Tool to Enhance Clinical Development of Novel Antibiotics: A Tissue and Plasma PK Feasibility Study with Ciprofloxacin.

Background and ObjectiveIn microdose studies, drug pharmacokinetics is measured in humans after administration of subtherapeutic doses. While previous microdose studies focused primarily on plasma pharmacokinetics, we set out to evaluate the feasibility of microdosing for a pharmacokinetic assessment in subcutaneous tissue and epithelial lining fluid.MethodsHealthy subjects received a single intravenous bolus injection of a microdose of [14C]ciprofloxacin (1.1 µg, 7 kBq) with (cohort A, n = 9) or without (cohort B, n = 9) a prior intravenous infusion of a therapeutic dose of unlabeled ciprofloxacin (400 mg). Microdialysis and bronchoalveolar lavage were applied for determination of subcutaneous and intrapulmonary drug concentrations. Microdose [14C]ciprofloxacin was quantified by accelerator mass spectrometry and therapeutic-dose ciprofloxacin by liquid chromatography–tandem mass spectrometry.ResultsThe pharmacokinetics of therapeutic-dose ciprofloxacin (cohort A) in plasma, subcutaneous tissue, and epithelial lining fluid was in accordance with previous data. In plasma and subcutaneous tissue, the dose-adjusted area under the concentration–time curve of microdose ciprofloxacin was similar in cohorts A and B and within an 0.8-fold to 1.1-fold range of the area under the concentration–time curve of therapeutic-dose ciprofloxacin. Penetration of microdose ciprofloxacin into subcutaneous tissue was similar in cohorts A and B and comparable to that of therapeutic-dose ciprofloxacin with subcutaneous tissue-to-plasma area under the concentration–time curve ratios of 0.44, 0.44, and 0.38, respectively. Penetration of microdose ciprofloxacin into epithelial lining fluid was highly variable and failed to predict the epithelial lining fluid penetration of therapeutic-dose ciprofloxacin.ConclusionsOur study confirms the feasibility of microdosing for pharmacokinetic measurements in plasma and subcutaneous tissue. Microdosing combined with microdialysis is a potentially useful tool in clinical antimicrobial drug development, but its applicability for the assessment of pulmonary pharmacokinetics with bronchoalveolar lavage requires further studies.Clinical Trial RegistrationClinicalTrials.gov NCT03177720 (registered 6 June, 2017).Supplementary InformationThe online version contains supplementary material available at 10.1007/s40262-021-01091-1.

Highly sensitive quantification of pemetrexed in human plasma using UPLC-MS/MS to support microdosing studies.

Pemetrexed is an antifolate drug approved for the treatment of non‐small‐cell lung cancer and mesothelioma. Assessing pemetrexed pharmacokinetics after administration of a microdose (100 μg) may facilitate drug–drug interaction and dose individualization studies with cytotoxic drugs, without causing harm to patients. Therefore, a highly sensitive bioanalytical assay is required.A reversed‐phase ultra‐high performance liquid chromatography method was developed to determine pemetrexed concentrations in human ethylenediaminetetraacetic acid–plasma after microdosing. [13C5]‐Pemetrexed was used as the internal standard. The sample preparation involved solid‐phase extraction from plasma. Detection was performed using MS/MS in a total run time of 9.5 min.The assay was validated over the concentration range of 0.0250–25.0 μg/L pemetrexed. The average accuracies for the assay in plasma were 96.5 and 96.5%, and the within‐day and between‐day precision in coefficients of variations was <8.8%. Extraction recovery was 59 ± 1 and 55 ± 5% for pemetrexed and its internal standard. Processed plasma samples were stable for 2 days in a cooled autosampler at 10°C.The assay was successfully applied in a pharmacokinetic curve, which was obtained as a part of an ongoing clinical microdosing study.

Abstract 1311: PET-MRI microdosing can determine the delivery of the experimental cancer therapeutic, MN-anti-miR10b, to metastatic lesions in a murine model of breast cancer

Abstract Conventional therapies, which are effective against primary cancer, oftentimes are not successful at treating metastatic cancer. In our earlier work, we identified miRNA-10b as a master regulator of the viability of metastatic tumor cells. This knowledge allowed us to design a miR-10b inhibitor, termed MN-anti-miR10b, capable of delivery to metastatic sites via intravenous injection. In mouse models of breast cancer, we demonstrated that MN-anti-miR10b, when combined with a low-dose cytostatic, caused lifelong complete regressions of established metastases with no evidence of systemic toxicity. As a first step towards translating MN-anti-miR10b for the treatment of metastatic breast cancer, we needed to determine if MN-anti-miR10b, which is so effective in mice, will also accumulate in human metastases. Towards that goal, we developed a method to radiolabel MN-anti-miR10b with Cu-64, inject a microdose of the radiolabeled MN-anti-miR10b into murine models of metastatic breast cancer, and determine the uptake of the therapeutic by the metastatic lesions using positron emission tomography-magnetic resonance imaging (PET-MRI). Specifically, we injected a microdose of 64Cu-MN-anti-miR10b into mice bearing luciferase-expressing metastatic breast adenocarcinoma and performed in vivo bioluminescence (BLI) and PET-MRI to assess uptake by the metastatic lesions. The lesions were identified by BLI, based on their luciferase expression. PET-MRI clearly demonstrated uptake of 64Cu-MN-anti-miR10b, measurable at a microdose. Ex vivo PET-MRI confirmed that the activity was associated with the metastatic lesions, as identified at necropsy. Uptake by metastatic organs was also demonstrated by scintigraphy and shown to be significantly higher than in the same organs devoid of BLI-detectable metastatic lesions. Finally, 64Cu-MN-anti-miR10b injected as a non-carrier added microdose had comparable biodistribution to that injected at a standard therapeutic dose. The value of the current study is in setting the stage for clinical PET-MRI microdosing, which would be able to answer a key question on the path to drug development without the associated cost to do a full clinical trial – the question of delivery to the target organ sites. Since the PET technique is sensitive enough to determine the concentration of radiolabeled drug with sensitivity approaching the sub-picomolar range, as little as a microgram of the radiolabeled drug is sufficient to perform a PET-MRI study in humans. In addition to proving delivery, the microdosing studies will reveal the pharmacokinetic behavior of MN-anti-miR10b which will allow one to establish dosing during therapy. Finally, in clinical trials, one can use microdosing PET-MRI to select patients for treatment, based on which patients' metastases accumulate the therapeutic. Citation Format: Marianne Le Fur, Byunghee Yoo, Nicholas Rotile, Pamela Pantazopoulos, Alana Ross, Iris Zhou, Peter Caravan, Zdravka Medarova. PET-MRI microdosing can determine the delivery of the experimental cancer therapeutic, MN-anti-miR10b, to metastatic lesions in a murine model of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1311.

Dose-linearity of the pharmacokinetics of an intravenous [14 C]midazolam microdose in children.

AimsDrug disposition in children may vary from adults due to age‐related variation in drug metabolism. Microdose studies present an innovation to study pharmacokinetics (PK) in paediatrics; however, they should be used only when the PK is dose linear. We aimed to assess dose linearity of a [14C]midazolam microdose, by comparing the PK of an intravenous (IV) microtracer (a microdose given simultaneously with a therapeutic midazolam dose), with the PK of a single isolated microdose.MethodsPreterm to 2‐year‐old infants admitted to the intensive care unit received [14C]midazolam IV as a microtracer or microdose, followed by dense blood sampling up to 36 hours. Plasma concentrations of [14C]midazolam and [14C]1‐hydroxy‐midazolam were determined by accelerator mass spectrometry. Noncompartmental PK analysis was performed and a population PK model was developed.ResultsOf 15 infants (median gestational age 39.4 [range 23.9–41.4] weeks, postnatal age 11.4 [0.6–49.1] weeks), 6 received a microtracer and 9 a microdose of [14C]midazolam (111 Bq kg−1; 37.6 ng kg−1). In a 2‐compartment PK model, bodyweight was the most significant covariate for volume of distribution. There was no statistically significant difference in any PK parameter between the microdose and microtracer, nor in the area under curve ratio [14C]1‐OH‐midazolam/[14C]midazolam, showing the PK of midazolam to be linear within the range of the therapeutic and microdoses.ConclusionOur data support the dose linearity of the PK of an IV [14C]midazolam microdose in children. Hence, a [14C]midazolam microdosing approach may be used as an alternative to a therapeutic dose of midazolam to study developmental changes in hepatic CYP3A activity in young children.

Abstract 2155: High-plex spatial profiling analysis of multidrug CIVO microdose studies in cancer patients

Abstract Background: CIVO microdosing studies performed in patient tumors in situ allow drug developers to assess localized tumor and microenvironment responses to multiple agents without having to expose patients to high systemic drug levels. By concentrating microdoses of multiple different drugs within a living tumor in situ, it is possible to compare tumor and immune responses in spatially resolved regions of the same tumor. This type of early phase (Phase 0) clinical study represents a new path for drug developers to gain insight into drug efficacy, tumor associated immune cell modulation, biomarker discovery and validation, and microenvironment interactions for new drugs earlier in the drug development process. Traditionally, analysis of FFPE samples from these microdosing studies involve routine immunohistochemistry, immunofluorescence, and in situ assays that reveal changes in protein and RNA expression. These assays offer tissue wide protein and gene expression information but have limited multiplexing capabilities and dynamic range, as well as rapidly consume precious trial samples. Novel technologies such as NanoString’s GeoMxTM Digital Spatial Profiler (DSP) enable high-plex spatially resolved analysis of proteins and RNA transcripts in single FFPE tissue sections. In this proof of principle study, we utilized GeoMxTM Digital Spatial Profiler for protein and RNA expression on single FFPE sections from patient sarcoma tumors that were microdosed with multiple FDA approved drugs. Methods: Single FFPE sections from microdosed patient tumor samples were IF stained for DNA and CD3 and whole slide imaged. 600-micron and 100-micron diameter regions of interests (ROIs) were selected for DSP analysis within drug and control microinjection sites. Imaging and barcode counts were performed using GeoMxTM DSP and nCounter systems. Results: DSP protein analysis highlighted phospho-S6 and phospho-ERK upregulation in response to doxorubicin compared to vehicle site. Additionally, ROIs sampled along the doxorubicin exposure gradient showed a dose-dependent reduction of phosphorylation of both S6 and ERK proteins. DSP RNA analysis revealed drug specific transcript regulation of multiple genes in microdosed tumors, including upregulation of chemokines CXCL9 and CXCL10 at sites of doxorubicin and aldesleukin injection but not to other chemotherapy agents. Conclusions: Early phase CIVO microdosing studies combined with high-plex DSP opens the door to generating multi-omics data for multiple microdosed drugs within small patient studies. Analysis of protein and RNA expression using DSP enabled collection of targeted region of interest high density data from single FFPE sections, conserving precious patient biopsy samples. Through continued expansion of the GeoMxTM DSP analyte panels, collecting an ever-increasing depth of protein and gene expression data is possible in Phase 0 CIVO microdosing studies. Citation Format: Gary B. Deutsch, Seth M. Pollack, Matthew J. Thompson, Kenneth R. Gundle, Jessica A. Bertout, Jason P. Frazier, Emily Beirne, Marc O. Grenley, JingJing Gong, Yan Liang, Joseph M. Beechem, Richard A. Klinghoffer, Robert G. Maki. High-plex spatial profiling analysis of multidrug CIVO microdose studies in cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2155.

Abstract 4136: Direct intratumoral microdosing via the CIVO® platform reveals anti-tumor immune responses induced by the SUMO inhibitor TAK-981

Abstract The complex interplay between a drug, the tumor and the surrounding microenvironment is a critical determinant in how a patient will respond to their selected cancer therapy. Yet to date, this complex response has been difficult to evaluate prior to late stage clinical investigation. To enable a means for testing investigational agents earlier in the development process but still directly in human patients, in a way that limits the risk of adverse effects and provides an indication of efficacy, Presage Biosciences developed a technology called CIVO. This platform allows for simultaneous assessment of multiple drugs or drug combinations directly in a single solid tumor, in the context where the drugs are ultimately intended to be used, directly in the patient. Here we demonstrate the potential for using the CIVO platform in Phase 0 microdosing studies to detect complex responses to investigational agents. In this study, we used the CIVO platform to assess the impact of TAK-981 on the native tumor immune microenvironment of animal models. TAK-981 is a novel and selective small molecule inhibitor of the SUMOylation enzymatic cascade currently in Phase I clinical trials. SUMOylation is a reversible post-translational modification that regulates protein function by covalent attachment of a small ubiquitin-like modifier (SUMO) protein to protein substrates. TAK-981 was microinjected into tumors from a syngeneic mouse model of B cell lymphoma and responses assessed via immunohistochemistry and in situ hybridization following tumor resection. An early inflammatory response was evident by 24 hours, including the accumulation of neutrophils, inflammatory macrophages and a Type I interferon response. The chemokine IP10 was secreted around TAK-981 injection sites and was accompanied by the accumulation of cytotoxic T lymphocytes, likely recruited from the local tumor microenvironment. A localized cell death response was observed proximal to TAK-981 injection sites by 72 hours and was likely induced by the granzyme B-bearing cytotoxic T cells enriched at TAK-981 sites. Abscopal studies demonstrated that the local immune modulation induced by TAK-981 translated into an adaptive immune response. The results from this study were consistent with findings from systemically dosed in vivomouse efficacy studies carried out at Takeda, demonstrating that the local responses to agents microdosed intratumorally via CIVO are predictive of responses induced by systemic drug exposure. These studies highlight the unique capability of TAK-981 to promote antitumor immunity, which may be further evaluated using the CIVO platform in a Phase 0 trial in human solid tumor patients. Citation Format: Beryl A. Hatton, Marc Grenley, James Garnsey, Vaishali Shinde, Dennis Huszar, Connor Burns, Sally Ditzler, Angela Merrell, Joyoti Dey, Emily Beirne, Richard A. Klinghoffer. Direct intratumoral microdosing via the CIVO® platform reveals anti-tumor immune responses induced by the SUMO inhibitor TAK-981 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4136.

1187-P: The Novel Biguanide NT1195 Is an Oral Antidiabetes Drug for Patients with Kidney Disease

Metformin, the most prescribed drug for T2D, has significant deficiencies in potency, pharmacokinetics (PK) and safety, especially in patients with kidney disease. Despite these deficiencies no improvements have been made to the biguanide (BIG) drug class. Novel BIGs with greater efficacy and without the risk of lactic acidosis are needed for T2D in patients with impaired renal function. A library of novel BIGs was screened for functional activity against a panel of organic cation transporters (OCTs). This OCT activity profile has allowed us to select BIGs with increased potency, improved PK properties and reduced elimination in urine. NT1195 was identified from this library and examined in more detail. This BIG is 5-10x more potent in mouse OGTT than metformin and is potent in the db:db mouse model, lowering plasma glucose after 8 days of dosing when metformin did not. NT1195 is more effective at elevating GLP-1 levels and inhibited gastric emptying in mice, where metformin did not. Improved potency alone is not sufficient to create the next generation of BIG therapeutics; it is also important to improve drug safety and to reduce the risk of lactic acidosis. Detailed PK studies in mice show that NT1195 has a 28.5h half-life (metformin = 1.97h), high levels of exposure (6x &amp;gt;metformin) and renal clearance that is 50x less than metformin. This latter finding suggests an increase in the safety of NT1195 in patients with impaired renal function, since we anticipate that NT1195 will be cleared by non-renal processes and that drug levels will not be affected by kidney disease. PK studies in cynomolgus monkeys provide similar findings for NT1195 with good exposure and a long half-life after oral administration. Also, NT1195 was found to increases glucose disposal in an IVGTT in insulin resistant monkeys. Safety studies are underway that will prepare NT1195 for a micro-dose study in T2D patients with and without kidney disease. Disclosure K.W. Batchelor: None. J.N. Livingston: Consultant; Self; CohBar, Inc. Employee; Self; NovoTarg Therapeutics. K.K. Brown: None. K. Kavanagh: Research Support; Self; Takeda Development Centre Europe Ltd. J.E. Cobb: Consultant; Self; NovaTarg Therapeutics. Funding National Institute of Diabetes and Digestive and Kidney Diseases

Exploring the effect of microdosing psychedelics on creativity in an open-label natural setting

IntroductionTaking microdoses (a mere fraction of normal doses) of psychedelic substances, such as truffles, recently gained popularity, as it allegedly has multiple beneficial effects including creativity and problem-solving performance, potentially through targeting serotonergic 5-HT2A receptors and promoting cognitive flexibility, crucial to creative thinking. Nevertheless, enhancing effects of microdosing remain anecdotal, and in the absence of quantitative research on microdosing psychedelics, it is impossible to draw definitive conclusions on that matter. Here, our main aim was to quantitatively explore the cognitive-enhancing potential of microdosing psychedelics in healthy adults.MethodsDuring a microdosing event organized by the Dutch Psychedelic Society, we examined the effects of psychedelic truffles (which were later analyzed to quantify active psychedelic alkaloids) on two creativity-related problem-solving tasks: the Picture Concept Task assessing convergent thinking and the Alternative Uses Task assessing divergent thinking. A short version of the Ravens Progressive Matrices task assessed potential changes in fluid intelligence. We tested once before taking a microdose and once while the effects were expected to be manifested.ResultsWe found that both convergent and divergent thinking performance was improved after a non-blinded microdose, whereas fluid intelligence was unaffected.ConclusionWhile this study provides quantitative support for the cognitive-enhancing properties of microdosing psychedelics, future research has to confirm these preliminary findings in more rigorous placebo-controlled study designs. Based on these preliminary results, we speculate that psychedelics might affect cognitive metacontrol policies by optimizing the balance between cognitive persistence and flexibility. We hope this study will motivate future microdosing studies with more controlled designs to test this hypothesis.

O-33 Feasibility of a paediatric micro-dose study of [14c]midazolam to study the ontogeny of cyp3a-mediated drug metabolism

Background Microdose studies present an interesting innovation to study age-related changes in drug metab-olism in young children. To further delineate maturation of intestinal and hepatic CYP3A activity, in this pilot study we aimed to study the feasibility of an oral [14C]midazol-am (MDZ) microdosing study in children. Methods Children admitted to the paediatric intensive care unit were eligible to receive a single oral [14C]MDZ microdose when they received IV midazolam for thera-peutic reasons and had an arterial line in place enabling blood sampling. Blood samples were taken up to 24 hours after dose administration. Plasma concentrations of [14C] MDZ and the metabolite [14C]OH-MDZ were determined by accelator mass spectrometry (AMS). Pharmacokinetic (PK) parameters were estimated using non-compartmen-tal PK models with PKSolver software (Microsoft Excel). Results Of 139 eligible patients, 125 were excluded and informed consent was obtained from parents of nine chil-dren [median age 3.3 months (range 12 days – 4.2 years)] who received a midazolam microdose (19.3 [18.7–21.3] ng/kg; 58 [56-64] Bq/kg). [14C]MDZ and [14C]1-OH-MDZ were detectable at expected concentrations: plasma [14C]MDZ AUC0-∞ was 49.9 (4.0–107.7) ng/L*h, Cmax was 7.5 (1.5–22.2) ng/L, Tmax was 0.5 (0.3–3.1) hour, T0.5 was 4.6 (1.1–14.0) hour, CL/F was 0.4 (0.2–5.3) L/h/kg and Vss/F was 3.1 (1.7–10.7) L/kg. Plasma [14C]1-OH-MDZ AUC0-∞ was 7.8 (1.3–28.3) ng/L*h and CL/F was 2.4 (0.7–14.6) L/h/kg. Plasma Cmax of [14C]MDZ normalised to a dose of 0.1 mg/kg was 39.9 (7.0–114.9) ng/ml. Conclusion We demonstrate the feasibility of an oral [14C]MDZ microdose to study MDZ and 1-OHMDZ dis-position in young infants and children with AMS. This method can be used to study developmental changes in intestinal and hepatic CYP3A activity.

Discovery of Clinical Candidate 4-[2-(5-Amino-1H-pyrazol-4-yl)-4-chlorophenoxy]-5-chloro-2-fluoro-N-1,3-thiazol-4-ylbenzenesulfonamide (PF-05089771): Design and Optimization of Diaryl Ether Aryl Sulfonamides as Selective Inhibitors of NaV1.7.

A series of acidic diaryl ether heterocyclic sulfonamides that are potent and subtype selective NaV1.7 inhibitors is described. Optimization of early lead matter focused on removal of structural alerts, improving metabolic stability and reducing cytochrome P450 inhibition driven drug-drug interaction concerns to deliver the desired balance of preclinical in vitro properties. Concerns over nonmetabolic routes of clearance, variable clearance in preclinical species, and subsequent low confidence human pharmacokinetic predictions led to the decision to conduct a human microdose study to determine clinical pharmacokinetics. The design strategies and results from preclinical PK and clinical human microdose PK data are described leading to the discovery of the first subtype selective NaV1.7 inhibitor clinical candidate PF-05089771 (34) which binds to a site in the voltage sensing domain.