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3639 Development of Thioredoxin Monothiol Derivatives for Mitigating Radiation Induced Hematopoietic Injury

Program: Oral and Poster Abstracts
Session: 802. Chemical Biology and Experimental Therapeutics: Poster II
Hematology Disease Topics & Pathways:
Research, Acquired Marrow Failure Syndromes, Translational Research, Bone Marrow Failure Syndromes, drug development, hematopoiesis, Diseases, Therapies, Biological Processes
Sunday, December 10, 2023, 6:00 PM-8:00 PM

Yubin Kang, MD1, Jian Wu, MD, PhD1*, Xiaobei Wang, PhD1*, Parker Mathews2*, Shaima Jabbar, PhD1*, George William Schaaf, DVM3*, John Olson, MA3*, Joel Ross, PhD2*, Nelson J. Chao, MD4, Mark Cline, DVM, PhD3* and Peter B. Heifetz, PhD5*

1Division of Hematologic Malignancies and Cellular Therapy, Duke University, Durham, NC
2Duke University, Durham, NC
3Wake Forest School of Medicine, Winston-Salem, NC
4Division of Hematologic Malignancies and Cellular Therapy, Department of Medicine, Duke University, Durham, NC
5OrPro Therapeutics, Inc, San Diego, CA

Hematopoietic stem cells (HSCs) are among the most sensitive cells to radiation injury and contribute to many of the manifestations of acute radiation syndrome. There is an unmet need for effective agents that can be used to rescue radiation injury and enhance HSC recovery especially when administered post-irradiation. We previously identified the stress-protective human thioredoxin-1 protein (TRX) as a novel molecule for enhancing HSC recovery in mouse models of radiation injury. Treatment of lethally irradiated mice with recombinant TRX 24 hours after irradiation increased survival by 45%. However, native or recombinant TRX has a very short half-life (0.5–1 hr), which limits its clinical application, and the ability of exogenous TRX to internalize to the cytosol and support cellular proliferation is a potential concern.

To overcome these limitations, an engineered monocysteinic, chemically pre-reduced TRX variant in which the second active-site Cys at position 35 has been replaced by Ser was developed (ORP100S). This modification stabilizes the mixed-disulfide transition state intermediate formed during docking and reduction of TRX-specific target protein Cys disulfide bonds. The resulting ORP100S Cys32–target Cys linkage shifts the thiol-disulfide equilibrium and confers a longer duration and more potent activity compared to native TRX. Compared to TRX, ORP100S does not increase multiplication of EML hematopoietic cells in vitro, consistent with enhanced extracellular regulatory/stress-protective activity and attenuated intracellular proliferative activity.

We first tested the effectiveness of ORP100S in mitigating lethal total body irradiation (TBI) in C57Bl/6 mice. Animals were irradiated (9.5 Gy) and 24 hours later were administered (IV, tail-vein injection) either PBS control buffer or ORP100S in PBS at 32 μg/mouse, every other day for a total of five doses. While all lethally-irradiated mice receiving PBS alone died within 2 weeks, half of the mice receiving ORP100S survived through termination of the study at 40 days. We further performed a dose-ranging efficacy study. At 24 hrs after TBI (8.45Gy), C57Bl/6 mice were given PBS or ORP100S in PBS (32 μg, 64 μg, 128 μg, 320 μg IV; 128μg SQ) every other day for a total of five doses. Nine of ten 128 μg SQ ORP100S-treated mice and all treatment groups given ORP100S at greater than 64 μg IV survived to the end of study on Day 45.

Because subcutaneous injection is a preferred route of administration in the event of radiation exposure incidents where large populations may require treatment in the field, we performed a pharmacokinetic (PK) study to establish optimal SQ dosing of ORP100S, the timing of initial administration (24 hr or 48 hr after radiation), and the duration of treatment (one injection or five injections). PK and pharmacodynamic (PD) studies were also performed to compare IV and SQ administration of 64 μg or 128 μg ORP100S. Plasma samples were collected before injection and at 15 min, 30 min, 60 min, 2 hr, 4 hr, 8 hr and 16 hr. ORP100S levels in plasma were analyzed using a custom hybrid immunocapture LC/MS-MS assay with selective quantification of the ORP100S tryptic peptide SMPTFQFFK following immunoprecipitation. We found that at two hr post injection, SQ administration of ORP100S achieved similar plasma level of ORP100S compared to IV injection.

We also performed a repeated-dose toxicology study of ORP100S in C57Bl/6 mice. Animals were injected with 0, 128 μg, 1,280 μg, 2,560 μg, 5,120 μg IV or SQ in a volume of 200 μl once daily for five days. The mice were weighed daily and monitored for any changes in activities and behaviors. Terminal blood samples were collected at euthanasia the day following the final dose. Blood cell counts, chemistry panels, and biomarkers relevant to radiation effect were measured, and organs (brain, lung, heart, kidney, liver, spleen, bone marrow) were harvested for histological examination. A confirmatory study utilizing non-human primates (cynomolgous macaques) is in progress utilizing a sub-lethal 4 Gy radiation dose.

In summary, we demonstrate that ORP100S, a monocysteinic, thioredoxin derivative, is a potent mitigator of radiation-induced hematopoietic injury even when delivered many hours post-exposure. Our preclinical study results support safety and efficacy with clear potential for this novel agent to advance to clinical studies for the treatment of acute radiation syndrome.

Disclosures: Kang: OrPro Therapeutics: Other: Patent application. Heifetz: OrPro Therapeutics, Inc: Current Employment, Other: President and CEO.

*signifies non-member of ASH