Lentiviral Gene Therapy for Artemis-Deficient SCID.

BACKGROUND: The DNA-repair enzyme Artemis is essential for rearrangement of T- and B-cell receptors. Mutations in DCLRE1C, which encodes Artemis, cause Artemis-deficient severe combined immunodeficiency (ART-SCID), which is poorly responsive to allogeneic hematopoietic-cell transplantation. METHODS: We carried out a phase 1-2 clinical study of the transfusion of autologous CD34+ cells, transfected with a lentiviral vector containing DCLRE1C, in 10 infants with newly diagnosed ART-SCID. We followed them for a median of 31.2 months. RESULTS: Marrow harvest, busulfan conditioning, and lentiviral-transduced CD34+ cell infusion produced the expected grade 3 or 4 adverse events. All the procedures met prespecified criteria for feasibility at 42 days after infusion. Gene-marked T cells were detected at 6 to 16 weeks after infusion in all the patients. Five of 6 patients who were followed for at least 24 months had T-cell immune reconstitution at a median of 12 months. The diversity of T-cell receptor ß chains normalized by 6 to 12 months. Four patients who were followed for at least 24 months had sufficient B-cell numbers, IgM concentration, or IgM isohemagglutinin titers to permit discontinuation of IgG infusions. Three of these 4 patients had normal immunization responses, and the fourth has started immunizations. Vector insertion sites showed no evidence of clonal expansion. One patient who presented with cytomegalovirus infection received a second infusion of gene-corrected cells to achieve T-cell immunity sufficient for viral clearance. Autoimmune hemolytic anemia developed in 4 patients 4 to 11 months after infusion; this condition resolved after reconstitution of T-cell immunity. All 10 patients were healthy at the time of this report. CONCLUSIONS: Infusion of lentiviral gene-corrected autologous CD34+ cells, preceded by pharmacologically targeted low-exposure busulfan, in infants with newly diagnosed ART-SCID resulted in genetically corrected and functional T and B cells. (Funded by the California Institute for Regenerative Medicine and the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT03538899.).

Perinatal Caffeine Administration Improves Outcomes in an Ovine Model of Neonatal Hypoxia-Ischemia.

BACKGROUND: Neonatal hypoxic-ischemic encephalopathy disproportionately affects low- and middle-income countries, where ≈96% of affected infants reside. The current standard of care, therapeutic hypothermia, is frequently ineffective in this setting, likely because injury may be occurring earlier during labor. Here, we studied the pharmacokinetics, safety, and efficacy of perinatal caffeine administration in near-term lambs following global ischemic injury to support the development of earlier treatment strategies targeting the fetus in utero as well as the infant postnatally. METHODS: Ewes were randomly assigned to receive either 1 g IV caffeine citrate or placebo before delivery and placental transport assessed. Near-term lambs (141-143 days) of both sexes were subjected to severe global hypoxia-ischemia utilizing an acute umbilical cord occlusion model. Lambs that received caffeine in utero also received 20 mg/kg IV caffeine citrate following resuscitation and 10 mg/(kg·d) IV for 2 days. An additional cohort received 60 mg/kg followed by 30 mg/(kg·d) (low dose versus high dose) postnatally. Biochemical, histological, and neurological outcome measures in lambs were assessed over a 6-day period. RESULTS: Perinatal caffeine administration demonstrated excellent placental transport kinetics and was well tolerated with lamb plasma levels comparable to those targeted in neonates with apnea of prematurity. Caffeine administration resulted in a systemic immunomodulatory effect, evidenced by significant reductions in proinflammatory IP-10 levels. Treated lambs demonstrated improved neurodevelopmental outcomes, while histological analysis revealed that caffeine reduced gray matter injury and attenuated inflammation in the cingulate and parasagittal cortex. This neuroprotective effect was greater and via a different mode of action than we previously reported for azithromycin. A higher caffeine dosing regimen demonstrated significant toxicity. CONCLUSIONS: Perinatal caffeine administration is well tolerated, attenuates systemic and brain inflammation, and contributes to improvements in histological and neurological outcomes in an ovine model of neonatal hypoxic-ischemic encephalopathy.

Maximum a posteriori Bayesian methods out-perform non-compartmental analysis for busulfan precision dosing.

Dose personalization improves patient outcomes for many drugs with a narrow therapeutic index and high inter-individuality variability, including busulfan. Non-compartmental analysis (NCA) and model-based methods like maximum a posteriori Bayesian (MAP) approaches are two methods routinely used for dose optimization. These approaches vary in how they estimate patient-specific pharmacokinetic parameters to inform a dose and the impact of these differences is not well-understood. Using busulfan as an example application and area under the concentration-time curve (AUC) as a target exposure metric, these estimation methods were compared using retrospective patient data (N = 246) and simulated precision dosing treatment courses. NCA was performed with or without peak extension, and MAP Bayesian estimation was performed using either the one-compartment Shukla model or the two-compartment McCune model. All methods showed good agreement on real-world data (correlation coefficients of 0.945-0.998) as assessed by Bland-Altman plots, although agreement between NCA and MAP methods was higher during the first dosing interval (0.982-0.994) compared to subsequent dosing intervals (0.918-0.938). In dose adjustment simulations, both NCA and MAP estimated high target attainment (> 98%) although true simulated target attainment was lower for NCA (63-66%) versus MAP (91-93%). The largest differences in AUC estimation were due to different assumptions for the shape of the concentration curve during the infusion phase, followed by how the methods considered time-dependent clearance and concentration-time points collected in earlier intervals. In conclusion, although AUC estimates between the two methods showed good correlation, in a simulated study, MAP lead to higher target attainment. When changing from one method to another, or changing infusion duration and other factors, optimum estimated exposure targets may require adjusting to maintain a consistent exposure.

Perinatal Azithromycin Provides Limited Neuroprotection in an Ovine Model of Neonatal Hypoxic-Ischemic Encephalopathy.

BACKGROUND: Hypoxic-ischemic brain injury/encephalopathy affects about 1.15 million neonates per year, 96% of whom are born in low- and middle-income countries. Therapeutic hypothermia is not effective in this setting, possibly because injury occurs significantly before birth. Here, we studied the pharmacokinetics, safety, and efficacy of perinatal azithromycin administration in near-term lambs following global ischemic injury to support earlier treatment approaches. METHODS: Ewes and their lambs of both sexes (n=34, 141-143 days) were randomly assigned to receive azithromycin or placebo before delivery as well as postnatally. Lambs were subjected to severe global hypoxia-ischemia utilizing an acute umbilical cord occlusion model. Outcomes were assessed over a 6-day period. RESULTS: While maternal azithromycin exhibited relatively low placental transfer, azithromycin-treated lambs recovered spontaneous circulation faster following the initiation of cardiopulmonary resuscitation and were extubated sooner. Additionally, peri- and postnatal azithromycin administration was well tolerated, demonstrating a 77-hour plasma elimination half-life, as well as significant accumulation in the brain and other tissues. Azithromycin administration resulted in a systemic immunomodulatory effect, demonstrated by reductions in proinflammatory IL-6 (interleukin-6) levels. Treated lambs exhibited a trend toward improved neurodevelopmental outcomes while histological analysis revealed that azithromycin supported white matter preservation and attenuated inflammation in the cingulate and parasagittal cortex. CONCLUSIONS: Perinatal azithromycin administration enhances neonatal resuscitation, attenuates neuroinflammation, and supports limited improvement of select histological outcomes in an ovine model of hypoxic-ischemic brain injury/encephalopathy.

Lentiviral Gene Therapy Combined with Low-Dose Busulfan in Infants with SCID-X1.

BACKGROUND: Allogeneic hematopoietic stem-cell transplantation for X-linked severe combined immunodeficiency (SCID-X1) often fails to reconstitute immunity associated with T cells, B cells, and natural killer (NK) cells when matched sibling donors are unavailable unless high-dose chemotherapy is given. In previous studies, autologous gene therapy with γ-retroviral vectors failed to reconstitute B-cell and NK-cell immunity and was complicated by vector-related leukemia. METHODS: We performed a dual-center, phase 1-2 safety and efficacy study of a lentiviral vector to transfer IL2RG complementary DNA to bone marrow stem cells after low-exposure, targeted busulfan conditioning in eight infants with newly diagnosed SCID-X1. RESULTS: Eight infants with SCID-X1 were followed for a median of 16.4 months. Bone marrow harvest, busulfan conditioning, and cell infusion had no unexpected side effects. In seven infants, the numbers of CD3+, CD4+, and naive CD4+ T cells and NK cells normalized by 3 to 4 months after infusion and were accompanied by vector marking in T cells, B cells, NK cells, myeloid cells, and bone marrow progenitors. The eighth infant had an insufficient T-cell count initially, but T cells developed in this infant after a boost of gene-corrected cells without busulfan conditioning. Previous infections cleared in all infants, and all continued to grow normally. IgM levels normalized in seven of the eight infants, of whom four discontinued intravenous immune globulin supplementation; three of these four infants had a response to vaccines. Vector insertion-site analysis was performed in seven infants and showed polyclonal patterns without clonal dominance in all seven. CONCLUSIONS: Lentiviral vector gene therapy combined with low-exposure, targeted busulfan conditioning in infants with newly diagnosed SCID-X1 had low-grade acute toxic effects and resulted in multilineage engraftment of transduced cells, reconstitution of functional T cells and B cells, and normalization of NK-cell counts during a median follow-up of 16 months. (Funded by the American Lebanese Syrian Associated Charities and others; LVXSCID-ND ClinicalTrials.gov number, NCT01512888.).

Defining Longer-Term Outcomes in an Ovine Model of Moderate Perinatal Hypoxia-Ischemia.

Hypoxic-ischemic encephalopathy (HIE) is the leading cause of neonatal morbidity and mortality worldwide. Approximately 1 million infants born with HIE each year survive with cerebral palsy and/or serious cognitive disabilities. While infants born with mild and severe HIE frequently result in predictable outcomes, infants born with moderate HIE exhibit variable outcomes that are highly unpredictable. Here, we describe an umbilical cord occlusion (UCO) model of moderate HIE with a 6-day follow-up. Near-term lambs (n = 27) were resuscitated after the induction of 5 min of asystole. Following recovery, lambs were assessed to define neurodevelopmental outcomes. At the end of this period, lambs were euthanized, and brains were harvested for histological analysis. Compared with prior models that typically follow lambs for 3 days, the observation of neurobehavioral outcomes for 6 days enabled identification of animals that recover significant neurological function. Approximately 35% of lambs exhibited severe motor deficits throughout the entirety of the 6-day course and, in the most severely affected lambs, developed spastic diparesis similar to that observed in infants who survive severe neonatal HIE (severe, UCOs). Importantly, and similar to outcomes in human neonates, while initially developing significant acidosis and encephalopathy, the remainder of the lambs in this model recovered normal motor activity and exhibited normal neurodevelopmental outcomes by 6 days of life (improved, UCOi). The UCOs group exhibited gliosis and inflammation in both white and gray matters, oligodendrocyte loss, neuronal loss, and cellular death in the hippocampus and cingulate cortex. While the UCOi group exhibited more cellular death and gliosis in the parasagittal cortex, they demonstrated more preserved white matter markers, along with reduced markers of inflammation and lower cellular death and neuronal loss in Ca3 of the hippocampus compared with UCOs lambs. Our large animal model of moderate HIE with prolonged follow-up will help further define pathophysiologic drivers of brain injury while enabling identification of predictive biomarkers that correlate with disease outcomes and ultimately help support development of therapeutic approaches to this challenging clinical scenario.

Correction: Preservation of myocardial contractility during acute hypoxia with OMX-CV, a novel oxygen delivery biotherapeutic.

[This corrects the article DOI: 10.1371/journal.pbio.2005924.].

Preservation of myocardial contractility during acute hypoxia with OMX-CV, a novel oxygen delivery biotherapeutic.

The heart exhibits the highest basal oxygen (O2) consumption per tissue mass of any organ in the body and is uniquely dependent on aerobic metabolism to sustain contractile function. During acute hypoxic states, the body responds with a compensatory increase in cardiac output that further increases myocardial O2 demand, predisposing the heart to ischemic stress and myocardial dysfunction. Here, we test the utility of a novel engineered protein derived from the heme-based nitric oxide (NO)/oxygen (H-NOX) family of bacterial proteins as an O2 delivery biotherapeutic (Omniox-cardiovascular [OMX-CV]) for the hypoxic myocardium. Because of their unique binding characteristics, H-NOX-based variants effectively deliver O2 to hypoxic tissues, but not those at physiologic O2 tension. Additionally, H-NOX-based variants exhibit tunable binding that is specific for O2 with subphysiologic reactivity towards NO, circumventing a significant toxicity exhibited by hemoglobin (Hb)-based O2 carriers (HBOCs). Juvenile lambs were sedated, mechanically ventilated, and instrumented to measure cardiovascular parameters. Biventricular admittance catheters were inserted to perform pressure-volume (PV) analyses. Systemic hypoxia was induced by ventilation with 10% O2. Following 15 minutes of hypoxia, the lambs were treated with OMX-CV (200 mg/kg IV) or vehicle. Acute hypoxia induced significant increases in heart rate (HR), pulmonary blood flow (PBF), and pulmonary vascular resistance (PVR) (p < 0.05). At 1 hour, vehicle-treated lambs exhibited severe hypoxia and a significant decrease in biventricular contractile function. However, in OMX-CV-treated animals, myocardial oxygenation was improved without negatively impacting systemic or PVR, and both right ventricle (RV) and left ventricle (LV) contractile function were maintained at pre-hypoxic baseline levels. These data suggest that OMX-CV is a promising and safe O2 delivery biotherapeutic for the preservation of myocardial contractility in the setting of acute hypoxia.

Reduced Toxicity Conditioning for Nonmalignant Hematopoietic Cell Transplants.

Allogeneic hematopoietic cell transplantation (HCT) for children with nonmalignant disorders is challenged by potential drug-related toxicities and poor engraftment. This retrospective analysis expands on our single pediatric medical center experience with targeted busulfan, fludarabine, and intravenous (IV) alemtuzumab as a low-toxicity regimen to achieve sustained donor engraftment. Sixty-two patients received this regimen for their first HCT for a nonmalignant disorder between 2004 and 2018. Donors were matched sibling in 27%, 8/8 HLA allele-matched unrelated in 50%, and 7/8 HLA allele-mismatched in 23% (some of whom received additional immunoablation with thiotepa or clofarabine). Five patients experienced graft failure for a cumulative incidence of 8.4% (95% CI, 1 to 16%). In engrafted patients, the median donor chimerism in whole blood and CD3, CD14/15, and CD19 subsets at 1-year were 96%, 90%, 99%, and 99%, respectively. Only one patient received donor lymphocyte infusions (DLIs) for poor chimerism. Two patients died following disease progression despite 100% donor chimerism. The 3-year cumulative incidence of treatment-related mortality was 10% (95% CI, 2 to 17%). Overall survival and event-free-survival at 3-years were 87% (95% CI, 78 to 95%) and 80% (95% CI, 70 to 90%), respectively. The 6-month cumulative incidence of grade II to IV acute graft-versus-host disease (GVHD) was 7% (95% CI, 3 to 13%), while the 3-year cumulative incidence of chronic GVHD was 5% (95% CI, 0 to 11%). These results suggest that use of targeted busulfan, fludarabine and IV alemtuzumab offers a well-tolerated option for children with nonmalignant disorders to achieve sustained engraftment with a low incidence of GVHD.

Lower Exposure to Busulfan Allows for Stable Engraftment of Donor Hematopoietic Stem Cells in Children with Mucopolysaccharidosis Type I: A Case Report of Four Patients.

Busulfan is an alkylating agent routinely used in conditioning regimens prior to allogeneic hematopoietic cell transplantation (HCT) for various nonmalignant disorders, including inborn errors of metabolism. The combination of model-based dosing and therapeutic drug monitoring (TDM) of busulfan pharmacokinetics (PK) to a lower exposure target has the potential to reduce the regimen-related toxicity while opening marrow niches sufficient for engraftment in diseases such as mucopolysaccharidosis type I (MPS I). We present four cases of the severe form of MPS I or Hurler syndrome, demonstrating successful and stable CD14/15 donor chimerism following the prospective application of model-based dosing and TDM aimed to achieve lower busulfan exposure. All patients received a busulfan-based conditioning regimen with a median cumulative area-under-the-curve (cAUC) target of 63.7 mg h/L (range, 62.4 to 65.0) in protocol-specific combination of chemotherapeutic regimen. The donor source was unrelated umbilical cord blood for three patients and matched sibling donor bone marrow for one patient. The observed median busulfan cAUC was 66.1 mg h/L (range, 65.2 to 70.6) and was within 10% of the intended target. Stable, full donor myeloid chimerism was achieved for three patients, while one patient achieved a stable mixed chimerism (76% donor CD14/15 at 53 months) without a recurring need for enzyme replacement. The normalization of α-L-iduronidase enzyme levels followed the attainment of successful donor myeloid chimerism in all patients. Regimen-related toxicity remained low with no evidence of acute graft-versus-host disease (GVHD) grades II to IV and chronic GVHD.

Population Pharmacokinetics of Clofarabine as Part of Pretransplantation Conditioning in Pediatric Subjects before Hematopoietic Cell Transplantation.

The primary objective of this work was to characterize the pharmacokinetics (PK) of systemic clofarabine (clo-fara) in pediatric allogeneic hematopoietic cell transplantation (HCT) recipients receiving either nucleoside monotherapy or a dual nucleoside analog preparative regimen. Fifty-one children (median age, 4.9 years; range, .25 to 14.9 years) undergoing allogeneic HCT for a variety of malignant and nonmalignant disorders underwent PK assessment. Plasma samples were collected over the 4 to 5 days of clo-fara treatment and quantified for clo-fara, using a validated liquid chromatography/tandem mass spectrometry assay. Nonlinear mixed-effects modeling was used to develop the population PK model, including identification of covariates that influenced drug disposition. In agreement with previously published models, a 2-compartment PK model with first-order elimination best described the PK of clo-fara. Final parameter estimates for clo-fara were consistent with previous reports and were as follows: clearance (CL), 23 L/h/15 kg; volume of the central compartment, 42 L/15 kg; volume of peripheral compartment, 47 L/15 kg; and intercompartmental CL, 9.8 L/h/15 kg. Unexplained variability was acceptable at 33%, and the additive residual error (reflective of the assay) was estimated to be 0.36 ng/mL. Patient-specific factors significantly impacting clo-fara CL included actual body weight and age. The covariate model was able to estimate clo-fara CL with good precision in children spanning a wide age range from infancy to early adulthood and demonstrates the need for variable dosing in children of different ages. For example, the dose required for a 6-month and 1-year old was approximately 43% and 17% lower, respectively, than the typical 40 mg/m2dose to achieve the median AUC0-24of 1.04 mg·h/L in the study population. Despite the known renal elimination of clo-fara, no significant clinical parameters for renal function were retained in the final model (P> .05). Coadministration of fludarabine with clo-fara did not alter the CL of clo-fara (P> .05). These results will help inform individualized dosing strategies for clo-fara to improve clinical outcomes and limit drug-related adverse events in children undergoing HCT.

Low Exposure Busulfan Conditioning to Achieve Sufficient Multilineage Chimerism in Patients with Severe Combined Immunodeficiency.

After allogeneic hematopoietic cell transplantation (HCT), the minimal myeloid chimerism required for full T and B cell reconstitution in patients with severe combined immunodeficiency (SCID) is unknown. We retrospectively reviewed our experience with low-exposure busulfan (cumulative area under the curve, 30 mg·hr/L) in 10 SCID patients undergoing either first or repeat HCT from unrelated or haploidentical donors. The median busulfan dose required to achieve this exposure was 5.9 mg/kg (range, 4.8 to 9.1). With a median follow-up of 4.5 years all patients survived, with 1 requiring an additional HCT. Donor myeloid chimerism was generally >90% at 1 month post-HCT, but in most patients it fell during the next 3 months, such that 1-year median myeloid chimerism was 14% (range, 2% to 100%). Six of 10 patients had full T and B cell reconstitution, despite myeloid chimerism as low as 3%. Three patients have not recovered B cell function at over 2 years post-HCT, 2 of them in the setting of treatment with rituximab for post-HCT autoimmunity. Low-exposure busulfan was well tolerated and achieved sufficient myeloid chimerism for full immune reconstitution in over 50% of patients. However, other factors beyond busulfan exposure may also play critical roles in determining long-term myeloid chimerism and full T and B cell reconstitution.

Pharmacokinetics and Model-Based Dosing to Optimize Fludarabine Therapy in Pediatric Hematopoietic Cell Transplant Recipients.

A prospective multicenter study was conducted to characterize the pharmacokinetics (PK) and pharmacodynamics (PD) of fludarabine plasma (f-ara-a) and intracellular triphosphate (f-ara-ATP) in children undergoing hematopoietic cell transplantation (HCT) and receiving fludarabine with conditioning. Plasma and peripheral blood mononuclear cells (PBMCs) were collected over the course of therapy for quantitation of f-ara-a and f-ara-ATP. Nonlinear mixed-effects modeling was used to develop the PK model, including identification of covariates impacting drug disposition. Data from a total of 133 children (median age, 5 years; range, .2 to 17.9) undergoing HCT for a variety of malignant and nonmalignant disorders were available for PK-PD modeling. The implementation of allometric scaling of PK parameters alone was insufficient to describe drug clearance, particularly in very young children. Renal impairment was predicted to increase drug exposure across all ages. The rate of f-ara-a entry into PBMCs (expressed in pmoles per million cells) decreased over the course of therapy, resulting in 78% lower f-ara-ATP after the fourth dose (1.7 pmoles/million cells [range, .2 to 7.2]) compared with first dose (7.9 pmoles/million cells [range, .7 to 18.2]). The overall incidence of treatment-related mortality (TRM) was low at 3% and 8% at days 60 and 360, respectively, and no association with f-ara-a exposure and TRM was found. In the setting of malignancy, disease-free survival was highest at 1 year after HCT in subjects achieving a systemic f-ara-a cumulative area under the curve (cAUC) greater than 15 mg*hour/L compared to patients with a cAUC less than 15 mg*hour/L (82.6% versus 52.8% P = .04). These results suggest that individualized model-based dosing of fludarabine in infants and young children may reduce morbidity and mortality through improved rates of disease-free survival and limiting drug-related toxicity. ClinicalTrials.gov Identifier: NCT01316549.

Randomised placebo-controlled safety and tolerability trial of FK506 (tacrolimus) for pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a devastating disease characterised by occlusive pulmonary vasculopathy. Activation of bone morphogenetic protein receptor 2 (BMPR2) signalling by FK506 (tacrolimus) reverses occlusive vasculopathy in rodent PAH models. Here, we determined the safety and tolerability of low-level FK506 therapy in stable PAH patients.We performed a randomised, double-blind, placebo-controlled, 16-week, single-centre, phase IIa trial in PAH patients with New York Heart Association functional class II/III symptoms using three FK506 target levels (<2, 2-3 and 3-5 ng·mL-1). 23 patients were randomised and 20 patients completed the trial.FK506 was generally well tolerated, with nausea/diarrhoea being the most commonly reported adverse event and no observation of line infections in patients on intravenous prostacyclin therapy. PAH patients had significantly lower BMPR2 expression in peripheral blood mononuclear cells versus healthy controls (n=13; p=0.005), which improved after FK506 treatment. While we observed that some patients responded with a pronounced increase in BMPR2 expression as well as improvement in 6-min walk distance, and serological and echocardiographic parameters of heart failure, these changes were not significant.Low-level FK506 is well tolerated and increases BMPR2 in subsets of PAH patients. These results support the study of FK506 in a phase IIb efficacy trial.

Population pharmacokinetics of busulfan in pediatric and young adult patients undergoing hematopoietic cell transplant: a model-based dosing algorithm for personalized therapy and implementation into routine clinical use.

BACKGROUND: Population pharmacokinetic (PK) studies of busulfan in children have shown that individualized model-based algorithms provide improved targeted busulfan therapy when compared with conventional dose guidelines. The adoption of population PK models into routine clinical practice has been hampered by the tendency of pharmacologists to develop complex models too impractical for clinicians to use. The authors aimed to develop a population PK model for busulfan in children that can reliably achieve therapeutic exposure (concentration at steady state) and implement a simple model-based tool for the initial dosing of busulfan in children undergoing hematopoietic cell transplantation. PATIENTS AND METHODS: Model development was conducted using retrospective data available in 90 pediatric and young adult patients who had undergone hematopoietic cell transplantation with busulfan conditioning. Busulfan drug levels and potential covariates influencing drug exposure were analyzed using the nonlinear mixed effects modeling software, NONMEM. The final population PK model was implemented into a clinician-friendly Microsoft Excel-based tool and used to recommend initial doses of busulfan in a group of 21 pediatric patients prospectively dosed based on the population PK model. RESULTS: Modeling of busulfan time-concentration data indicates that busulfan clearance displays nonlinearity in children, decreasing up to approximately 20% between the concentrations of 250-2000 ng/mL. Important patient-specific covariates found to significantly impact busulfan clearance were actual body weight and age. The percentage of individuals achieving a therapeutic concentration at steady state was significantly higher in subjects receiving initial doses based on the population PK model (81%) than in historical controls dosed on conventional guidelines (52%) (P = 0.02). CONCLUSIONS: When compared with the conventional dosing guidelines, the model-based algorithm demonstrates significant improvement for providing targeted busulfan therapy in children and young adults.

Primary Immune Deficiency Treatment Consortium (PIDTC) report.

The Primary Immune Deficiency Treatment Consortium (PIDTC) is a network of 33 centers in North America that study the treatment of rare and severe primary immunodeficiency diseases. Current protocols address the natural history of patients treated for severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, and chronic granulomatous disease through retrospective, prospective, and cross-sectional studies. The PIDTC additionally seeks to encourage training of junior investigators, establish partnerships with European and other International colleagues, work with patient advocacy groups to promote community awareness, and conduct pilot demonstration projects. Future goals include the conduct of prospective treatment studies to determine optimal therapies for primary immunodeficiency diseases. To date, the PIDTC has funded 2 pilot projects: newborn screening for SCID in Navajo Native Americans and B-cell reconstitution in patients with SCID after hematopoietic stem cell transplantation. Ten junior investigators have received grant awards. The PIDTC Annual Scientific Workshop has brought together consortium members, outside speakers, patient advocacy groups, and young investigators and trainees to report progress of the protocols and discuss common interests and goals, including new scientific developments and future directions of clinical research. Here we report the progress of the PIDTC to date, highlights of the first 2 PIDTC workshops, and consideration of future consortium objectives.

Development and validation of a liquid chromatography-tandem mass spectrometry assay to quantify plasma busulfan.

BACKGROUND: Busulfan is an anti-leukemic, DNA alkylating agent that is used in conditioning regimens for patients undergoing hematopoietic stem cell transplantation. Because of the large intraindividual and interindividual variations seen in busulfan pharmacokinetics, therapeutic drug monitoring is necessary. Currently at the authors' institution, plasma busulfan in adults is measured by gas chromatography-mass spectrometry (GC-MS) at a reference laboratory, whereas pediatric specimens are sent to a different reference laboratory also for GC-MS analysis. As the result turnaround time is not optimal and this practice is of significant cost, a liquid chromatography-tandem mass spectrometry assay to quantify plasma busulfan was developed. METHODS: Protein precipitation with D8-busulfan (deuterated internal standard) in acetonitrile was carried out on 50 µL of heparinized plasma. Gradient elution with ammonium acetate, formic acid, water, and methanol at 0.6 mL/min had a 4-minute run time. Multiple reaction monitoring was employed using Q1/Q3 transitions of 264/151 and 264/55 for busulfan and 272/159 and 272/62 for D8-busulfan. RESULTS: Sample preparation took ∼30 minutes for 6 patient samples. Six calibrators were used (0-2000 ng/mL) with 3 quality controls (means of 12, 356, and 1535 ng/mL). The limits of detection and quantitation were 1 and 6 ng/mL, respectively. Extraction recovery was ∼77% and ion suppression ∼5%. Within-run and between-run precision studies yielded <15% coefficient of variation at the limit of quantitation and <6% coefficient of variation through the rest of the linear range. Method comparisons between this assay and 2 GC-MS assays revealed mean biases of 7% and 1%. CONCLUSIONS: An accurate, rapid, and sensitive liquid chromatography-tandem mass spectrometry assay for quantification of plasma busulfan was developed. This assay reduces current specimen volume requirements, reduces result turnaround time for patients and clinicians, and additionally saves institutional funds.

Pharmacokinetics of Briquilimab as a Conditioning Agent for Hematopoietic Stem Cell Transplantation in Patients With Severe Combined Immunodeficiency, Myelodysplastic Syndrome, or Acute Myeloid Leukemia.

For successful engraftment of donor hematopoietic stem cells (HSC), conditioning with chemotherapy and/or radiation prior to hematopoietic cell transplantation (HCT) has been required to open marrow niche space and minimize the risk of immune rejection. Briquilimab, a humanized IgG1 monoclonal antibody that blocks the interaction between the c-Kit receptor and stem cell factor on various C-Kit expressing tissues including HSC, is a potential nonmyeloablative conditioning agent in clinical development for patients with severe combined immunodeficiency (SCID), myelodysplastic syndromes (MDS), and acute myeloid leukemia (AML). This study aimed to characterize pharmacokinetics (PK) and develop a population PK model of briquilimab after single intravenous infusions of 4 different doses in patients with SCID, MDS, or AML receiving HCT. The PK data was collected from 2 different studies: JAS-BMT-CP-001 and JSP-CP-003. JAS-BMT-CP-001 is a phase 1/2 open-label study of briquilimab as a conditioning agent prior to allogenic HCT in SCID patients. The participants received single intravenous infusions of 0.1, 0.3, 0.6, or 1.0 mg/kg. JSP-CP-003 was a phase 1a/b open-label study of briquilimab in combination with a standard conditioning regimen of low dose total body irradiation and fludarabine in MDS or AML subjects undergoing HCT. The participants received a single intravenous dose of 0.6 mg/kg briquilimab. In both studies, briquilimab PK samples were obtained at pre-treatment, 5 minutes post-end of infusion, 4- and 24-hours post-start of infusion, any time between 2 days and 30 days postinfusion, and on the day of HCT prior to donor cell infusion.The population PK model was developed using the PK data from these 2 clinical studies, and the effect of participants' baseline characteristics on the briquilimab PK was evaluated. PK simulations were performed using the developed PK model to calculate the time to reach target concentrations for HCT. A total of 49 participants (21 SCID adult and pediatric participants with a median age of 12 yr and 28 MDS/AML adult participants with a median age of 70 yr) were included in the PK analysis. A 2-compartment model with combined linear and non-linear elimination best described the PK of briquilimab. Body weight was determined as the sole covariate of the PK parameters among the explored covariates. For a typical subject with a body weight 70 kg, the estimated parameters for clearance, maximum metabolic rate of Michaelis Menten elimination, Michaelis Menten constant, central volume, peripheral volume, and intercompartmental clearance were 17.6 mL/h, 51,434.8 ng/h, 71.5 ng/mL, 3444.0 mL, 1613.3 mL, and 21.2 mL/h, respectively. The median time to reach target concentrations of 500, 1000, and 2000 ng/mL after a single dose of 0.6 mg/kg was calculated as 12.3, 10.4, and 7.7 days, respectively. The PK of intravenous briquilimab was characterized in subjects with SCID, MDS, or AML receiving HCT, and a population PK model was developed to estimate briquilimab clearance to use as a guide to the timing of donor cell infusion post-briquilimab. Body weight was identified as a significant covariate on elimination and volume of distribution of briquilimab.

Diagnosing and Grading of Sinusoidal Obstructive Syndrome after Hematopoietic Stem Cell Transplant of Children, Adolescent and Young Adults treated in a Pediatric Institution with Pediatric Protocols.

Sinusoidal obstructive syndrome (SOS), or veno-occlusive disease, of the liver has been recognized as a complex, life-threatening complication in the posthematopoietic stem cell transplant (HSCT) setting. The diagnostic criteria for SOS have evolved over the last several decades with a greater understanding of the underlying pathophysiology, with 2 recent diagnostic criteria introduced in 2018 (European Society of Bone Marrow Transplant [EBMT] criteria) and 2020 (Cairo criteria). We sought out to evaluate the performance characteristics in diagnosing and grading SOS in pediatric patients of the 4 different diagnostic criteria (Baltimore, Modified Seattle, EBMT, and Cairo) and severity grading systems (defined by the EBMT and Cairo criteria). Retrospective chart review of children, adolescent, and young adults who underwent conditioned autologous and allogeneic HSCT between 2017 and 2021 at a single pediatric institution. A total of 250 consecutive patients underwent at least 1 HSCT at UCSF Benioff Children's Hospital San Francisco for a total of 307 HSCT. The day 100 cumulative incidence of SOS was 12.1%, 21.1%, 28.4%, and 28.4% per the Baltimore, Modified Seattle, EBMT, and Cairo criteria, respectively (P < .001). We found that patients diagnosed with grade ≥4 SOS per the Cairo criteria were more likely to be admitted to the Pediatric Intensive Care Unit (92% versus 58%, P = .035) and intubated (85% versus 32%, P = .002) than those diagnosed with grade ≥4 per EBMT criteria. Age <3 years-old (HR 1.76, 95% [1.04 to 2.98], P = .036), an abnormal body mass index (HR 1.69, 95% [1.06 to 2.68], P = .027), and high-risk patients per our institutional guidelines (HR 1.68, 95% [1.02 to 2.76], P = .041) were significantly associated with SOS per the Cairo criteria. We demonstrate that age <3 years, abnormal body mass index, and other high-risk criteria associate strongly with subsequent SOS development. Patients with moderate to severe SOS based on Cairo severity grading system may correlate better with clinical course based on ICU admissions and intubations when compared to the EBMT severity grading system.

Cyclophosphamide and Thiotepa Increases Risk of Transplant-Associated Thrombotic Microangiopathy.

Transplant associated thrombotic microangiopathy (TA-TMA) is a complication of hematopoietic cell transplant (HCT) associated with endothelial injury resulting in severe end organ damage, acute and long-term morbidity, and mortality. Myeloablative conditioning is a known risk factor, though specific causative agents have not been identified. We hypothesized that the combination of cyclophosphamide and thiotepa (CY + TT) is particularly toxic to the endothelium, placing patients at elevated risk for TA-TMA. We conducted a retrospective review of pediatric and young adult patients who received conditioned autologous and allogeneic HCT between 2012 and August 2023 at UCSF Benioff Children's Hospital, San Francisco. We excluded patients undergoing gene therapy or triple tandem transplants for brain tumors. Neuroblastoma tandem transplants were classified a single transplant occurrence. High dose N-acetylcysteine (NAC) prophylaxis was incorporated into the institutional standard of care from December 2016-May 2019 and May 2022-August 2023. Defibrotide was given prophylactically to patients deemed high-risk for sinusoidal obstruction syndrome (SOS) per institutional guidelines or on clinical trial NCT#02851407 for SOS prophylaxis or NCT#03384693 for TA-TMA prophylaxis. Kaplan-Meier analysis was used to estimate the 1-year cumulative incidence of TA-TMA. Univariate analysis was performed for each of the potential risk factors of interest using log-rank tests and bivariate analysis with Cox regression models using backward selection and hazard ratios were built using all covariates with a univariate P-value < .2 for allogeneic HCT. SPSS (v29) was used to estimate all summary statistics, cumulative incidences, and uni- and bi-variate analyses. A total of 558 transplants were performed with 43 patients developing TA-TMA, for a 1-year cumulative incidence of 8.6% (95% CI, 5.9-11.3) and 7.2% (95% CI, 2.9-11.5) in allogeneic and autologous HCTs, respectively (P = .62). In allogeneic recipients (n = 417), the 1-year cumulative incidence of TA-TMA with CY + TT as part of conditioning was 35.7% (95% CI, 15.7-55.7) compared to 11.7% (95% CI, 7.2-16.2) with either CY or TT alone, and 1.2% (95% CI, 0-2.8) if neither agent was included in the conditioning regimen (P < .001). Use of either CY or TT (HR = 10.14; P = .002) or CY + TT (HR = 35.93; P < .001), viral infections (HR = 4.3; P = .017) and fungal infections (HR = 2.98; P = 0.027) were significant factors resulting in increased risk for developing TA-TMA. In subjects undergoing autologous HCT (n = 141), the 1-year cumulative incidence of TA-TMA with CY + TT was 19.6% (95% CI, 8.8-30.6) while TA-TMA did not occur in patients receiving either CY or TT alone or when neither were included (P < .001). TA-TMA occurred only in patients with neuroblastoma receiving CY + TT as part of their conditioning. For autologous patients who received CY + TT, those who were CMV seronegative at the time of HCT had an incidence of TA-TMA of 6.7% (95% CI, 0.1-15.7) compared to 38.1% (95% CI, 35-41.2) for those CMV seropositive (P = .007). These data show that CY or TT alone or in combination as part of pre-transplant conditioning prior to HCT increase the incidence of TA-TMA. Alternative conditioning excluding the combination of CY + TT should be considered whenever possible to limit the development of TA-TMA.