Measurements of target activation in cells or tissues are key indicators of efficacy during drug development. In contrast to established methods that require reagents and multiple preprocessing steps, reagent-free in situ analysis of engaged drug targets or target-proximal pharmacodynamic signatures in solid tumors remains challenging. Here, we demonstrate that label-free quantification of histone acetylation-specific mass shifts by matrix-assisted laser desorption ionization (MALDI) mass spectrometry biotyping can be used for measurement of cellular potency of histone deacetylase inhibitors in intact cells. Furthermore, we employ MALDI mass spectrometry imaging of these mass shifts to visualize the spatiotemporal distribution of acetylated histones and thus the tumor-selective pharmacodynamic responses in a mouse model of gastrointestinal cancer. Taken together, our study suggests that the monitoring of drug-induced mass shifts in protein ion intensity fingerprints or images may be a powerful analytical tool in pharmacology and drug discovery.

A new method for the quantification of celecoxib in human plasma based on reversed-phase high-performance liquid chromatography (HPLC) coupled to atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) after liquid-liquid extraction is presented. The method is rapid, sensitive and highly selective. The retention time of celecoxib was 2.3 min. The limit of quantification was 5 microg/L. Rofecoxib was used as internal standard. After validation, the method was used to study the pharmacokinetic profile of celecoxib following administration of a single oral dose (200 mg) in 12 healthy volunteers. Since celecoxib should be metabolized primarily by cytochrome 2C9 (CYP2C9), a poor metabolizer (PM) for this cytochrome P450 enzyme was included in the study. Pharmacokinetic characteristics (mean +/- SD) of extensive metabolizers (EM) were t(max) 2.9+/-1.2h, c(max) 842+/-280 microg/L, AUC(infinity) 6246+/-2147 microg h/L and t(1/2) 7.8+/-2.7h. The area under the curve (AUC(infinity)) for the PM was 12561 microg h/L. However, we found no noticeable increase in half life in the PM (11.5 h) after a single dose of celecoxib.

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Background: Amatoxin-based antibody-drug conjugates (so called ATACs) are a new class of ADCs that use the RNA polymerase II inhibitor amanitin as toxic payload. HDP-101 is the first ATAC in the clinic, currently being tested in a phase I/II clinical trial in multiple myeloma patients. HDP-101 is given to patients intravenously (i.v.) as this is the most common administration route for ADCs. I.v. is often selected as route of administration in a clinical setting since it results in a bioavailability of 100%. However, i.v. dosing has disadvantages such as very high Cmax levels which can trigger toxicity, requires hospitalization of the patient for administration, and bears the risk of vessel damage. In this study, we demonstrate that subcutaneous (s.c.) dosing is a promising alternative route of administration for ATACs which is well tolerated and able to improve the therapeutic index (TI) of ATACs. Material and Methods: ATACs: ADCs based on cysteine-reactive and site-specific amatoxin-linker constructs synthesized at Heidelberg Pharma. Monkey Studies: Cynomolgus monkeys, escalating doses of ATACs i.v or s.c. q21d Mouse Studies: CB17 Scid mice, single dose of ATACs i.v. or s.c.; if applicable bleeding at 8 - 12 time points between 5 min and 14 days after dosing; tumor studies in s.c. and i.v. tumor models Results: The impact of the dosing route on organ distribution of ATACs was tested in a PK study after a single i.v. or s.c. ATAC dose. S.c. dosing resulted in increased serum half-life of ATACs and reduced Cmax values as compared to i.v. dosing, while the AUC was comparable. Toxicity of ATACs is most probably driven by its Cmax level in serum, whereas the efficacy is mainly driven by AUC. Thus, we hypothesized that s.c. dosing might improve the TI of ATACs. The maximal tolerated dose (MTD) of ATACs in mice was compared between s.c. and i.v. dosing. S.c. dosing resulted in higher MTDs as compared to i.v. dosing independent of the antibody and amatoxin payload variant used. These findings were also confirmed in cynomolgus monkeys, where s.c. dosing of the ATAC HDP-103 resulted in a reduced Cmax, a comparable AUC, and an improved HNSTD of HDP-103. The impact of s.c. dosing on anti-tumor efficacy of ATACs was investigated in several tumor models in mice. ATACs with different antibodies and amatoxin payloads were applied as single i.v. or s.c. dose. In all models tested, the anti-tumor efficacy of ATACs was similar between groups receiving s.c. or i.v. dosing. Conclusions: The present study demonstrates that s.c. dosing is a promising route of administration for ATACs as it not only refines the pharmacokinetic distribution of ATACs but may improve the TI in patients. By reducing Cmax, s.c. dosing improves the tolerability of ATACs in mice and cynomolgus monkeys. At the same time the anti-tumor efficacy of ATACs is maintained as the AUC is not negatively impacted. Thus, s.c. dosing improves the TI of ATACs and may represent a promising route of administration also in humans. Citation Format: Kristin Decker, Marija Vranic, Marisa Schmitt, Irina Dranova, Anikó Pálfi, Torsten Hechler, Andreas Pahl, Michael Kulke. Subcutaneous dosing of amatoxin-based ADCs increases the therapeutic index [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1523.

Background: Amatoxin-based antibody-drug conjugates (ATACs) are a new class of ADCs using amanitin, a specific inhibitor of RNA polymerase II, as toxic payload. A first ATAC targeting B-cell maturation antigen (BCMA) is currently being tested in phase I/II clinical trials in multiple myeloma patients. During the clinical trials, the effect of repeated dosing on safety as well as anti-tumor efficacy of the ATAC will be assessed. In preclinical studies a reduction of toxicity was observed after repeated dosing with ATACs regardless of the target or antibody. In the present study, we demonstrate the effect of ATAC pre-treatment on tolerability in pre-clinical models. Material and Methods: Antibody: Engineered monoclonal antibodies produced at Heidelberg Pharma Toxic warhead: Cysteine reactive amanitin-linker constructs were synthesized at Heidelberg Pharma and conjugated site-specifically to the antibody. Animal models: Tolerability in CB17 Scid mice and Cynomolgus monkeys; Efficacy in a subcutaneous LNCaP prostate cancer model; Treatment: ADC (ATAC): escalating dosing i.v. q21d (tolerability) or repeated dosing i.v. q7dx4 (efficacy). Results: The impact of repeated dosing on the tolerability of a non-targeting ATAC was tested in CB17 Scid mice. The mice were initially treated with the maximal tolerated dose (MTD) of an ATAC, or PBS followed by a second higher dose 21 days after the first dose. Pre-treatment improved the tolerability of a second, higher dose of the same non-targeting ATAC from 12.5% to 80% survival. As ATAC toxicity is mainly caused by liver toxicity, the liver damage markers AST, ALT and LDH are used as toxicity markers in non-human primate studies. The induction of liver damage markers observed after repeated or escalated dosing was significantly lower than the induction after single or first dose treatment. Taken together, these data indicate that ATAC treatment can induce tolerability in animal models. Furthermore, in a subcutaneous LNCaP prostate cancer model, multiple ATAC dosing did improve the anti-tumor efficacy as compared to single dose treatment, indicating that the induced tolerability is not associated with reduced anti-tumor efficacy. Conclusions: ATAC pre-treatment induces tolerability in preclinical animal models without a loss in efficacy. Since this effect of ATAC pre-treatment was observed across different animal species, it likely translates also into humans. Consequently, if reduced liver toxicity and less adverse effects after repeated dosing with ATACs are also observed in the upcoming clinical trials, it might have a significant impact on treatment regimen and clinical success of ATACs. Citation Format: Kristin Decker, Marisa Schmitt, Can Araman, Franziska Ebeling, Irina Dranova, Torsten Hechler, Anikó Pálfí, Andreas Pahl, Michael Kulke. Treatment with antibody-targeted amanitin conjugates induces tolerability in preclinical models without triggering tolerance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1761.