Haptoglobin 1-1 Isoform Predicts Higher Serum Haptoglobin Concentration and Lower Multiorgan Failure Risk in Sickle Cell Disease

Haptoglobin (HP) is the major scavenger of cell-free hemoglobin in circulation. When haptoglobin is depleted, cell-free hemoglobin can lead to organ damage through direct oxidative injury, upregulation of inflammatory pathways, and depletion of nitric oxide. A common polymorphism in HP gives rise to structurally and functionally distinct phenotypes: HP 1-1, HP 2-1, and HP 2-2. In people of African descent, the HP 1-1 and 2-1 phenotypes have been associated with higher serum HP concentrations compared to the HP 2-2 phenotype (PMID 10807979). In hemolytic diseases, such as sickle cell disease (SCD), the consequence of the HP polymorphism is unclear but could influence cell-free hemoglobin processing and susceptibility to organ damage. We first investigated whether the HP isoforms are associated with HP concentrations at steady-state. Between 10/2009 and 6/2018, we recruited 431 SCD patients into a longitudinal registry at our institution during a routine clinic visit, which we defined as steady-state. Baseline clinical and laboratory data were collected at the time of enrolment. The median age of the cohort was 32 (interquartile range [IQR], 24 - 43) years, 43% were male, 76% were SCD SS or Sβ 0-thalassemia genotype, and 46% were on hydroxyurea at the time of enrolment. Genotyping of the HP polymorphisms by PCR demonstrated the HP 1-1 isoform in 30% (n = 129), the HP 2-1 isoform in 47% (n = 203), and the HP 2-2 isoform in 23% (n = 99) of the SCD cohort. Serum samples were available in 244 of the 431 SCD patients at steady-state. Serum concentrations of HP, measured by ELISA, were higher in those with the HP 1-1 isoform compared to the HP 2-1 or 2-2 isoform (Figure). In a linear regression model, independent variables associated with steady-state serum HP concentration included SCD SS or Sβ 0-thalassemia genotype (β -1.8; 95% CI: -2.5 to -1.0; P < 0.001), LDH (natural log β -0.9; 95% CI: -0.2 to -1.6; P = 0.018), hydroxyurea use (β 0.6; 95% CI: 0.1 to 1.1; P = 0.02), and HP 1-1 isoform (β 0.5, 95% CI: 0 to 1.1; P = 0.049), adjusting for age and sex. Between 9/2020 and 5/2021, we collected an additional 38 serum samples from SCD patients during a hospitalization for vaso-occlusive crisis (VOC) and serum samples from 7 SCD patients during a hospitalization for acute chest syndrome. The HP 1-1 isoform was associated with higher serum HP concentrations during VOC and during acute chest syndrome compared to the HP 2-1 or 2-2 isoforms (Figure). Interestingly, the serum HP trended higher from steady-state to VOC and to acute chest syndrome in SCD patients with the HP 1-1 isoform (β +1.1, P = 0.085) but trended lower in those with the HP 2-1 or 2-2 isoform (β -0.5, P = 0.099). Next we tested whether the HP 1-1 isoform is protective against multi-organ failure during hospitalizations for acute chest syndrome. For the longitudinal analysis, we focused on SCD patients with > 6 months of follow up at our institution (n = 391). A multiorgan failure event was defined as organ dysfunction involving at least 2 of the following organ systems: lung, kidney, liver, or central nervous system (PMID 8109600). With a median follow up of 6.8 (IQR, 3.5 - 8.9) years, we observed an acute chest syndrome episode complicated by multiorgan failure in 14% (53/391) of SCD patients. A significantly lower proportion of SCD patients with the HP 1-1 isoform developed a multiorgan failure event versus those with the 2-1 or 2-2 isoforms (5.7% vs. 17.2%, respectively; P < 0.001). On logistic regression analysis, acute chest syndrome with multiorgan failure was associated with the HP 1-1 isoform (OR 0.3, 95% CI: 0.1 to 0.6; P = 0.002) and baseline LDH (natural log OR 2.7, 95% CI: 1.2 - 6.3; P = 0.02), after adjusting for age, sex, SCD genotype and hydroxyurea use. In conclusion, we demonstrate that the HP 1-1 isoform is independently associated with higher serum HP concentrations at steady-state as well as during hospitalizations for VOC or acute chest syndrome compared to the HP 2-1 or 2-2 isoforms. Furthermore, the HP 1-1 isoform is protective against developing the devastating multiorgan failure syndrome during acute chest syndrome episodes. Our findings highlight the clinical significance of the HP isoforms and cell-free hemoglobin scavenging in SCD. Future studies assessing HP induction and scavenging function based on HP isoform may help elucidate high-risk patients for aggressive measures, such as rapid initiation of exchange transfusion therapy or HP replacement therapy. Figure 1 Figure 1. Disclosures Gordeuk: Modus Therapeutics: Consultancy; Novartis: Research Funding; Incyte: Research Funding; Emmaus: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; CSL Behring: Consultancy. Saraf: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Steady-State Serum IgG Trough Levels Are Adequate for Pharmacokinetic Assessment in Patients with Immunodeficiencies Receiving Subcutaneous Immune Globulin

Patients with primary immunodeficiency diseases often require lifelong immunoglobulin (IG) therapy. Most clinical trials investigating IG therapies characterize serum immunoglobulin G (IgG) pharmacokinetic (PK) profiles by serially assessing serum IgG levels. This retrospective analysis evaluated whether steady-state serum IgG trough level measurement alone is adequate for PK assessment. Based on individual patient serum IgG trough levels from two pivotal trials (phase 2/3 European [NCT01412385] and North American [NCT01218438]) of weekly 20% subcutaneous IG (SCIG; Cuvitru, Ig20Gly), trough level-predicted IgG AUC (AUCτ,tp) were calculated and compared with the reported AUC calculated from serum IgG concentration-time profiles (AUCτ). In both studies, mean AUCτ,tp values for Ig20Gly were essentially equivalent to AUCτ with point estimates of geometric mean ratio (GMR) of AUCτ,tp/AUCτ near 1.0 and 90% CIs within 0.80–1.25. In contrast, for IVIG, 10%, mean AUCτ,tp values were lower than AUCτ by >20%, (GMR [90% CI]: 0.74 [0.70–0.78] and 0.77 [0.73–0.81] for the two studies, respectively). Mean AUCτ,tp values calculated for 4 other SCIG products (based on mean IgG trough levels reported in the literature/labels) were also essentially equivalent to the reported AUCτ (differences <10% for all except HyQvia, a facilitated SCIG product), while differences for IVIG products were >20%. In conclusion, steady-state serum IgG levels following weekly SCIG remain stable, allowing for reliable prediction of AUC over the dosing interval using trough IgG levels. These findings indicate that measuring steady-state serum IgG trough levels alone may be adequate for PK assessment of weekly SCIG.

Effect of Genetic Polymorphisms of CYP2C19 on the Steady-state Serum Concentrations of Valproic Acid in Chinese Han Patients With Schizophrenia

Valproic acid is an anticonvulsant, which is also widely used for treating psychiatric disorders. Some clinical trials have demonstrated benefits of valproic acid augmentation therapy in schizophrenia. Interindividual variability in valproic acid dose and serum concentration may reflect functional consequences of genetic polymorphisms in genes encoding drug-metabolizing enzymes. The aim of this study was to determine the relationship between serum concentrations of valproic acid and single nucleotide polymorphisms of the cytochrome P450 (CYP) 2C19 gene in patients with schizophrenia. All patients had been receiving fixed dose of valproic acid for at least 2 weeks. The daily doses were 0.5–1.5 g. No other drugs except olanzapine were coadministered. Serum concentrations of valproic acid were measured using the ultra-high performance liquid chromatography method with mass-spectrometric detection. The CYP2C19 (CYP2C19*2 G681A and CYP2C19*3 G636A) genotypes were identified by real-time PCR analyses. The mean concentration/dose ratios of valproic acid was significantly higher in patients with 1 (P = 0.029) or 2 (P = 0.007) mutated alleles for CYP2C19 than in those without mutated alleles. The mean concentration/dose ratios of valproic acid was significantly higher in patients with CYP2C19 *1/*2 genotype (P = 0.029) or CYP2C19 *2/*3 genotype (P = 0.014) than in those with CYP2C12 *1/*1 genotype. The findings of this study suggest that CYP2C19 genotypes play an important role in controlling steady-state serum concentrations of valproic acid in Chinese Han population.

Noninvasive Cellular Oxygenation Measurement During Graded Hypoxia Using Visible–Near-Infrared Spectroscopy

In critically ill patients, direct knowledge of intracellular pO2 would allow for identification of cellular hypoxia, which when prolonged leads to organ failure. We have developed a visible–near-infrared optical system that noninvasively measures myoglobin saturation, which is directly related to intracellular pO2, from the surface of the skin. We used an animal model of graded hypoxia from low levels of inspired oxygen ( n = 5) and verified that low intracellular pO2 is correlated with high steady-state serum lactate values. In addition, the pO2 gradient between arterial blood and inside muscle cells was 83 mm Hg at 21% O2, but fell to 24 mm Hg at 8% O2. Continuous myoglobin saturation measurement in skeletal muscle displayed the same trends as cerebral oxygenation with no lag in changes over time, demonstrating its relevance as a measure of systemic oxygenation.

The Pharmacokinetics of Medetomidine Administered Subcutaneously during Isoflurane Anaesthesia in Sprague-Dawley Rats

Anaesthetic protocols involving the combined use of a sedative agent, medetomidine, and an anaesthetic agent, isoflurane, are increasingly being used in functional magnetic resonance imaging (fMRI) studies of the rodent brain. Despite the popularity of this combination, a standardised protocol for the combined use of medetomidine and isoflurane has not been established, resulting in inconsistencies in the reported use of these drugs. This study investigated the pharmacokinetic detail required to standardise the use of medetomidine and isoflurane in rat brain fMRI studies. Using mass spectrometry, serum concentrations of medetomidine were determined in Sprague-Dawley rats during medetomidine and isoflurane anaesthesia. The serum concentration of medetomidine for administration with 0.5% (vapouriser setting) isoflurane was found to be 14.4 ng/mL (±3.0 ng/mL). The data suggests that a steady state serum concentration of medetomidine when administered with 0.5% (vapouriser setting) isoflurane can be achieved with an initial subcutaneous (SC) dose of 0.12 mg/kg of medetomidine followed by a 0.08 mg/kg/h SC infusion of medetomidine. Consideration of these results for future studies will facilitate standardisation of medetomidine and isoflurane anaesthetic protocols during fMRI data acquisition.

Abstract P432: Racial Differences In Endothelial Function And Insulin Sensitivity

Racial disparities in health outcomes continue to be a significant public health concern and African Americans (AA) are disproportionately burdened by several risk factors for cardiovascular disease. Endothelial dysfunction has been shown to be a predictor of CVD and metabolic factors, including insulin resistance, are associated with endothelial dysfunction. Compared to EA, AA have impaired endothelial function and are more insulin resistant (less insulin sensitive). However, it remains unclear how insulin resistance may contribute to endothelial dysfunction in AA and EA. The purpose of the present study was to evaluate the relationship between insulin sensitivity and endothelial function in AA and EA. It was hypothesized that insulin sensitivity would be associated with endothelial function in both AA and EA. Skeletal muscle insulin sensitivity was measured by hyperinsulinemic-euglycemic glucose clamp technique in 112 lean, overweight, and obese AA and EA adults without diabetes. Insulin was infused at 120 mU/m 2 /min for 3 hours and an infusion of 20% dextrose was adjusted to maintain blood glucose at the fasting level. Insulin sensitivity (10 -4 .dL.kg -1 .min -1 /(μU/mL)) was defined as M/(G x ΔI), where M is steady state glucose infusion rate, G is steady state serum glucose concentration, and ΔI is the difference between basal and steady state serum insulin concentrations. M was adjusted for total lean body mass which was measured by dual-energy X-ray absorptiometry (DXA). Endothelial function was assessed by percent change in flow-mediated brachial artery dilatation (FMD). Changes in brachial artery diameter during reactive hyperemia were measured using ultrasound. Increased blood flow was induced via blood pressure cuff around the forearm, with a 5-minute inflation at 50 mmHg above the subject’s systolic blood pressure. Brachial arterial flow was determined using a pulsed-Doppler signal at baseline and 10-15 seconds after cuff release. Arterial diameter was measured at end-diastolic phase from super-VHS recordings. For reactive hyperemia response, measurements with the 5 largest diameters were averaged and the percent increase from baseline was determined as FMD. A total of 55 AA and 57 EA were included in the analysis. Mean insulin sensitivity was 4.89 in AA and 7.97 in EA (p < .0001) and mean FMD was 10.69 in AA and 10.14 in EA (p = .595). Linear regression analysis indicated a significant relationship between insulin sensitivity and endothelial function in AA but not in EA (p= .005 and p= .5, respectively). These results suggest that insulin sensitivity may play a role in determining endothelial function in AA.

CTC enumeration and characterization as a pharmacodynamic marker in the phase I clinical study of APX3330, an APE1/Ref-1 inhibitor, in patients with advanced solid tumors.

e14531 Background: APE1/Ref-1 is a dual-function protein with a pleiotropic role in regulating transcription factors involved in cancer cell signaling via redox control, as well as responding to oxidative and base DNA damage. APX3330 is a highly selective inhibitor of the Ref-1 mediated redox function in tumors, while enhancing the neuronal protective function of APE1. APX3330 is undergoing clinical development as an anti-tumor agent that also protects and reverses oxidative damage to neurons. We now report on the CTC analysis conducted as part of the study (NCT0337508). Results of the clinical study are reported in a separate abstract. Methods: A total of 19 patients with 10 various solid tumors, including rectal, pancreatic, colon, endometrial, gall bladder, hepatocellular, prostate, melanoma, bladder, and ovarian cancers received APX3330 in escalating divided daily doses of 240, 360, 480, 600, and 720mg. Blood samples were collected from all patients prior to receiving APX3330 and after achieving steady-state serum concentrations of the drug. Samples were sent to Epic Sciences to analyze circulating tumor cells (CTCs) in peripheral blood via their CTC Platform. Results: Cumulatively, 37 samples from 19 patients were received. 35/37 (95%) samples passed technical quality control and were feasible for downstream analysis. 9/17 (53%) baseline samples (BL) had CTCs detected, while 9/18 (50%) on-treatment (OTx) draws had CTCs detected. 16 patients had BL and OTx samples that were further evaluated with the longitudinal analysis. Of these, 7/16 (44%), 6/16 (38%), and 3/16 (19%) patients showed a reduction trend, an increase trend, and no change in all CTC populations (delta greater than or equal to 0 CTC/ml), respectively. Patient follow-up is ongoing and the correlation of CTC biomarkers with clinical outcomes is pending. Conclusions: APX3330 is undergoing clinical evaluation as an anti-tumor agent that protects against and reverses CIPN. In this phase I study, 44% of evaluable blood samples showed a reduction in CTCs after initiation of treatment with APX3330. Additional studies are now being planned. Clinical trial information: NCT0337508.