Protease Domain Exosites Regulate Plasma Recovery and Extravascular Distribution of Factor IX(a) Variants in Hemophilic Mice

Introduction: Among the coagulation protease, factor IX (FIX) is unique in that a substantial pool of extravascular factor exists that contributes to in vivo hemostasis. Extravascular distribution of FIX contributes to poor plasma recovery of infused FIX in hemophilia B (HB). The effect of mutations in the heparin (K126A/K132A) and antithrombin (R150A) binding exosites of FIX and FIXa on the distribution of these variants between the plasma and extravascular binding sites was examined in HB and hemophilia A (HA) mice, respectively. Methods: Recombinant human FIX wild type (18 U/kg) or equimolar FIX K126A/K132A was administered to HB mice (n=8) via tail vein injection with sacrifice 5 min post-injection following retro-orbital blood collection. Equimolar doses of human FIXa wild type (WT), K126A/K132A and R150A were similarly injected into HA mice (n=4-8), with blood collection and sacrifice 5 min post-injection. Plasma was isolated by centrifugation, organs (liver, spleen, kidneys, heart, lung, and brain) harvested, rinsed with PBS, weighed, with a portion made into tissue lysates and remainder frozen. FIX(a) content of tissue lysates was determined by a species-specific human FIX(a) ELISA with variant-specific standard curves. Tissue concentration was determined by total FIX(a) present in lysate (ng) divided by respective tissue weight (mg). Organ distribution was determined by extrapolating FIX(a) tissue concentration to total organ weight. The FIX(a) present in a specific organ was divided by the administered FIX(a) dose to determine the % organ distribution. Results: PK studies in HB mice demonstrated that FIX WT and R150A demonstrated a similar pattern and time course of elimination. In contrast, FIX K126A/K132A demonstrated ~2.4 fold higher plasma recovery relative to FIX WT. Based on plasma concentrations at 5 min post-injection (plasma volume 40 ml/kg), ~12.9% of FIX WT and 30.5% of FIX K126A/K132A was localized to the plasma compartment. Tissue lysates from the liver, spleen, kidney and brain demonstrated that liver had the highest FIX content by far, with ~41% of the FIX WT dose at 5 min post-injection. Other tissues demonstrated markedly less FIX WT content, including kidney (1%), spleen (0.1%) and brain (undetectable). FIX tissue concentration was significantly higher in the liver (0.69 ng/mg tissue) compared to other tissues, followed by kidney (0.07 ng/mg), spleen (0.02 ng/mg) and brain (undetectable). Comparison of FIX K126A/K132A tissue content to WT demonstrated reduced liver localization (25% dose) with concomitant reduction in tissue concentration (0.45 ng/mg). In contrast, FIX K126A/K132A increased localization to the kidney (1.7%) and spleen (0.37%) relative to WT. In protease PK studies, FIXa K126A/K132A and R150A both enhanced plasma recovery (2.2-2.5 fold) in HA mice compared to FIXa WT. Based on plasma concentrations at 5 min post-injection, ~16.1% of FIXa WT and 41.1% of FIXa K126A/K132A dose localized in the plasma compartment. Similar to zymogen, tissue lysates demonstrated that liver had the highest FIXa WT content (28%). Other tissues contained markedly less FIXa, including kidney (2.4%), spleen (0.2%), heart (0.5%) and brain (undetectable). Similarly, FIXa tissue concentration was 4-fold higher in the liver (0.47 ng/mg) compared to other tissues, followed by kidney (0.12 ng/mg), spleen and heart (0.06 ng/mg) and brain (undetectable). Localization of FIXa K126A/K132A to the liver was reduced by nearly half compared to FIXa WT (16%) with reduction in liver tissue concentration (0.29 ng/mg). FIXa K126A/K132A had increased localization to the kidney (3.3%) relative to WT. In contrast, FIXa R150A (not shown) had similar liver distribution (31.9% dose) and tissue concentration (0.53 ng/mg) to FIXa WT, despite increased localization to the plasma (36.2%). Conclusions: Liver is the predominant organ for extravascular FIX binding and the heparin binding exosite contributes to this localization, similar to the collagen IV binding site in the Gla domain. Heparan sulfate and collagen IV co-localize in the basement membrane suggesting synergistic roles. FIXa also localizes to extravascular sites via the heparin-binding exosite, although that binding may be modestly limited by endogenous FIX in the HA mouse. Disruption of the antithrombin binding exosite re-distributes FIXa to the plasma compartment by an independent mechanism, as liver content is unchanged. Disclosures Sheehan: Bayer: Consultancy, Research Funding; Roche: Consultancy, Research Funding; BioMarin: Consultancy, Research Funding.

CTNI-16. TROTABRESIB (CC-90010, BMS-986378), A REVERSIBLE, POTENT ORAL BROMODOMAIN AND EXTRATERMINAL INHIBITOR (BETi) IN PATIENTS WITH HIGH-GRADE GLIOMAS: A PHASE 1 OPEN-LABEL ‘WINDOW OF OPPORTUNITY’ STUDY

Trotabresib is a potent, reversible oral BETi with antitumor activity in patients with advanced malignancies (Moreno et al. ESMO 2020. Abstract 5270). The CC-90010-GBM-001 study (NCT04047303) enrolled patients with progressive or recurrent astrocytoma or recurrent glioblastoma scheduled for salvage resection. Patients were treated with trotabresib 30 mg daily for 4 days before surgery, then trotabresib 45 mg daily 4 days on/24 days off after recovery. Primary objectives were trotabresib tumor tissue concentration and plasma pharmacokinetics (PK). Secondary and exploratory objectives included safety, antitumor activity, cerebrospinal fluid concentration, and pharmacodynamics (PD). Twenty patients were enrolled; blood PK, blood PD, and tumor PD data were available for 14, 12, and 11 patients, respectively. Geometric mean peak trotabresib plasma concentration on day 4 was 1.92 μM; median time to peak concentration was 1.5 hours. At the time of resection, geometric mean trotabresib concentrations in plasma and brain tumor tissue were 1.01 and 0.68 μM, respectively. Blood CCR1 mRNA was reduced ≥ 50% from baseline after dose 4. Blood HEXIM1 mRNA increased at 72–96 hours following first dose, and at the time of surgery the percentage increase was related to plasma trotabresib concentration. Tumor HEXIM1 RNA increased in 10 of 11 patients. Eighteen patients (90%) had ≥ 1 treatment-related adverse event (TRAE). Nine patients (45%) had grade 3/4 TRAEs, most frequently thrombocytopenia (5 patients [25%]). Only 1 patient had serious TRAEs (hemiparesis and lethargy). Two patients died of intracranial hemorrhage unrelated to study drug. Of 16 patients evaluable for antitumor response, 7 had stable disease per RANO criteria, with 3 ongoing beyond data cutoff at cycles 4–11. Median progression-free survival was 1.9 months (95% CI, 1.4–3.3). Overall, trotabresib showed good tumor tissue penetration, with PD signals of response, and was well tolerated. A study of trotabresib + temozolomide in first-line glioblastoma is ongoing (NCT04324840).

Kidney and Liver Tissue Tacrolimus Concentrations in Adult Transplant Recipients—The Influence of the Whole Blood and Tissue Concentrations on Efficiency of Treatment during Immunosuppressive Therapy

Tacrolimus (TAC) has a narrow therapeutic index and highly variable pharmacokinetic characteristics. Close monitoring of the TAC concentrations is required in order to avoid the risk of acute rejection or adverse drug reaction. The results in some studies indicate that inter-tissue TAC concentrations can be a better predictor with regards to acute rejection episode than TAC concentration in whole blood. Therefore, the aim of the study was to assess the correlation between dosage, blood, hepatic and kidney tissue concentration of TAC measured by a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) and clinical outcomes in a larger cohort of 100 liver and renal adult transplant recipients. Dried biopsies were weighed, mechanically homogenized and then the samples were treated with a mixture of zinc sulfate—acetonitrile to perform protein precipitation. After centrifugation, the extraction with tert-butyl methyl ether was performed. The analytical range was proven for TAC tissue concentrations of 10–400 pg/mg. The accuracy and precision fell within the acceptance criteria for intraday as well as interday assay. There was no correlation between dosage, blood (C0) and tissue TAC concentrations. TAC concentrations determined in liver and kidney biopsies ranged from 8.5 pg/mg up to 160.0 pg/mg and from 7.1 pg/mg up to 215.7 pg/mg, respectively. To the best of our knowledge, this is the first LC-MS/MS method for kidney and liver tissue TAC monitoring using Tac13C,D2 as the internal standard, which permits measuring tissue TAC concentrations as low as 10 pg/mg.

Investigating the Central Nervous System Disposition of Actinomycin D: Implementation and Evaluation of Cerebral Microdialysis and Brain Tissue Measurements Supported by UPLC-MS/MS Quantification

Actinomycin D is a potent cytotoxic drug against pediatric (and other) tumors that is thought to barely cross the blood–brain barrier. To evaluate its potential applicability for the treatment of patients with central nervous system (CNS) tumors, we established a cerebral microdialysis model in freely moving mice and investigated its CNS disposition by quantifying actinomycin D in cerebral microdialysate, brain tissue homogenate, and plasma. For this purpose, we developed and validated an ultraperformance liquid chromatography–tandem mass spectrometry assay suitable for ultra-sensitive quantification of actinomycin D in the pertinent biological matrices in micro-samples of only 20 µL, with a lower limit of quantification of 0.05 ng/mL. In parallel, we confirmed actinomycin D as a substrate of P-glycoprotein (P-gp) in in vitro experiments. Two hours after intravenous administration of 0.5 mg/kg, actinomycin D reached total brain tissue concentrations of 4.1 ± 0.7 ng/g corresponding to a brain-to-plasma ratio of 0.18 ± 0.03, while it was not detectable in intracerebral microdialysate. This tissue concentration exceeds the concentrations of actinomycin D that have been shown to be effective in in vitro experiments. Elimination of the drug from brain tissue was substantially slower than from plasma, as shown in a brain-to-plasma ratio of approximately 0.53 after 22 h. Because actinomycin D reached potentially effective concentrations in brain tissue in our experiments, the drug should be further investigated as a therapeutic agent in potentially susceptible CNS malignancies, such as ependymoma.

Intestinal Region-Specific and Layer-Dependent Induction of TNFα in Rats with Streptozotocin-Induced Diabetes and after Insulin Replacement

Tumour necrosis factor alpha (TNFα) is essential in neuroinflammatory modulation. Therefore, the goal of this study is to reveal the effects of chronic hyperglycaemia and insulin treatment on TNFα expression in different gut segments and intestinal wall layers. TNFα expression was mapped by fluorescent immunohistochemistry and quantitative immunogold electron microscopy in myenteric ganglia of duodenum, ileum and colon. Tissue TNFα levels were measured by enzyme-linked immunosorbent assays in muscle/myenteric plexus-containing (MUSCLE-MP) and mucosa/submucosa/submucous plexus-containing (MUC-SUBMUC-SP) homogenates. Increasing density of TNFα-labelling gold particles is observed in myenteric ganglia from proximal to distal segments and TNFα tissue levels are much more elevated in MUSCLE-MP homogenates than in MUC-SUBMUC-SP samples in healthy controls. In the diabetics, the number of TNFα gold labels is significantly increased in the duodenum, decreased in the colon and remained unchanged in the ileal ganglia, while insulin does not prevent these diabetes-related TNFα changes. TNFα tissue concentration is also increased in MUSCLE-MP homogenates of diabetic duodenum, while decreased in MUC-SUBMUC-SP samples of diabetic ileum and colon. These findings support that type 1 diabetes has region-specific and intestinal layer-dependent effects on TNFα expression, contributing to the regional damage of myenteric neurons and their intestinal milieu.

Mycorrhizal Symbioses Enhance Competitive Weed Growth in Biochar and Nutrient-Amended Soils

Velvetleaf (Abutilon theophrasti) is a highly competitive weed in agroecosystems that is well-studied for its efficient nitrogen (N) acquisition, yet research on its phosphorus (P) uptake is lacking. One pathway may be through symbioses with arbuscular mycorrhizal fungi (AMF) which increase nutrient acquisition. These AMF benefits can be further enhanced by soil amendment with biochar, although effects may vary with different biochar production characteristics. We implemented a fully factorial nutrient and biochar addition experiment in a greenhouse for six months to determine how AMF nutrient uptake impacts plant growth and how these effects vary between two biochar types. We measured total above- and belowground biomass, plant tissue concentration (N and P), AMF colonization and activity rates, and soil media N and P availability. Overall, we observed few statistically significant results, however AMF N uptake may have been more beneficial to velvetleaf than AMF P uptake as evidenced by increased biomass and tissue N concentrations in treatments where N was only accessible by AMF. Additionally, by maintaining root to shoot ratios biochar may have provided plants with N and P (through sorption of nutrients to surfaces or its inherent properties) when nutrients were more difficult to access. We also found variable plant responses across the two biochar types used. Understanding how nutrient and biochar additions can influence weed competition is important for anticipating potential undesirable consequences of novel soil amendments such as biochar.

Brain-Targeted Polysorbate 80-Emulsified Donepezil Drug-Loaded Nanoparticles for Neuroprotection

Most Alzheimer’s disease drugs do not work efficiently because of the blood–brain barrier. Therefore, we designed a new nanopreparation (PS-DZP-CHP): cholesterol-modified pullulan (CHP) nanoparticle with polysorbate 80(PS) surface coverage, as donepezil (DZP) carrier to realize brain tissue delivery. By size analysis and isothermal titration calorimetry, we chose the optimal dosing ratio of the drug with nanomaterials (1:5) and designed a series of experiments to verify the efficacy of the nanoparticles. The results of in vitro release experiments showed that the nanoparticles can achieve continuous drug release within 72 h. The results of fluorescence observation in mice showed a good brain targeting of PS-DZP-CHP nanoparticles. Furthermore, the nanoparticle can enhance the drug in the brain tissue concentration in mice. DZP-CHP nanoparticles were used to pretreat nerve cells with Aβ protein damage. The concentration of lactate dehydrogenase was determined by MTT, rhodamine 123 and AO-EB staining, which proved that DZP-CHP nanoparticles had a protective effect on the neurotoxicity induced by Aβ25–35 and were superior to free donepezil. Microthermal perpetual motion meter test showed that PS-DZP-CHP nanoparticles have an affinity with apolipoprotein E, which may be vital for this nanoparticle targeting to brain tissue.

Influence of pre-analytical sample preparation on drug concentration measurements in peritoneal tissue: an ex-vivo study

Objectives Biopsy morphology (surface/depth ratio) and sample processing might affect pharmacological measurements in peritoneal tissue. Methods This is an ex-vivo study on inverted bovine urinary bladders (IBUB). We compared cisplatin (CIS) and doxorubicin (DOX) concentration in 81 standardized transmural punch biopsies of different diameters (6 and 12 mm). Then, we assessed the effect of dabbing the peritoneal surface before analysis. After automatized tissue homogenization with ceramic beads followed by lyophilisation, DOX concentration was quantified by high-performance liquid chromatography (HPLC), CIS concentration by atomic absorption spectroscopy. Experiments were performed in triplicate; the analysis was blinded to the sample origin. Comparisons were performed using non-parametric tests. Results Concentrations are given in mean (CI 5–95%). Results were reproducible between experiments (for CIS p=0.783, for DOX p=0.235) and between different localizations within the IBUB (for CIS p=0.032, for DOX p=0.663). Biopsy diameter had an influence on CIS tissue concentration (6 mm biopsies: 23.2 (20.3–26.1), vs. 12 mm biopsies: 8.1 (7.2–9.2) ng/mg, p<0.001) but not on DOX: (0.46, 0.29–0.62) vs. 0.43 (0.33–0.54) ng/mg respectively, p=0.248). Dabbing the peritoneal surface reduced DOX tissue concentration (dry biopsies: 0.28 (0.12–0.43) vs. wet biopsies: 0.64 (0.35–0.93) ng/mg, p=0.025) but not CIS (23.5 (19.0–28.0) vs. 22.9 (18.9–26.9) ng/mg, respectively, p=0.735). Conclusions Measurements of drug concentration in peritoneal tissue can be influenced by the biopsy’s surface/depth ratio and after drying the biopsy’s surface. This influence can reach a factor three, depending on the drug tested. The biopsy technique and the pre-analytical sample preparation should be standardized to ensure reliable pharmacological measurements in peritoneal tissue.