Dynamic Mass Balance Modeling for Chemical Distribution Over Time in In Vitro Systems With Repeated Dosing.

As toxicologists and risk assessors move away from animal testing and more toward using in vitro models and biological modeling, it is necessary to produce tools to quantify the chemical distribution within the in vitro environment prior to extrapolating in vitro concentrations to human equivalent doses. Although models predicting chemical distribution in vitro have been developed, very little has been done for repeated dosing scenarios, which are common in prolonged experiments where the medium needs to be refreshed. Failure to account for repeated dosing may lead to inaccurate estimations of exposure and introduce bias into subsequent in vitro to in vivo extrapolations. Our objectives were to develop a dynamic mass balance model for repeated dosing in in vitro systems; to evaluate model accuracy against experimental data; and to perform illustrative simulations to assess the impact of repeated doses on predicted cellular concentrations. A novel dynamic in vitro partitioning mass balance model (IV-MBM DP v1.0) was created based on the well-established fugacity approach. We parameterized and applied the dynamic mass balance model to single dose and repeat dosing scenarios, and evaluated the predicted medium and cellular concentrations against available empirical data. We also simulated repeated dosing scenarios for organic chemicals with a range of partitioning properties and compared the in vitro distributions over time. In single dose scenarios, for which only medium concentrations were available, simulated concentrations predicted measured concentrations with coefficients of determination (R 2) of 0.85-0.89, mean absolute error within a factor of two and model bias of nearly one. Repeat dose scenario simulations displayed model bias <2 within the cell lysate, and ∼1.5-3 in the medium. The concordance between simulated and available experimental data supports the predictive capacity of the IV-MBM DP v1.0 tool, but further evaluation as empirical data becomes available is warranted, especially for cellular concentrations.

Short-Term Steroid Treatment of Rhesus Macaque Increases Transduction.

Repeat dosing poses a major hurdle for the development of an adeno-associated virus (AAV)-based gene therapy for cystic fibrosis, in part because of the potential for development of an immune reaction to the AAV1 capsid proteins. Here, to dampen the immune response to AAV1, we treated Rhesus monkeys with methylprednisolone before and after the instillation of two doses of AAV1Δ27-264-CFTR into their airways at 0 and 30 days, followed by a single dose of AAV1-GFP on day 60. Animals were euthanized on day 90, except for one monkey that was sacrificed at 1 year. No adverse events occurred, indicating that the two AAV1 vectors are safe. rAAV1-CFTR and AAV1-GFP vector genomes and mRNA transcripts were detectable in all lung sections and in the liver and pancreas at day 90 and after 1 year at levels comparable with animals necropsied at 90 days. The numbers of vector genomes for cystic fibrosis transmembrane regulator (CFTR) and green fluorescent protein (GFP) detected here were higher than those found in the monkeys infected without methylprednisolone treatment that we tested previously.1 Also, lung surface and keratin 5-positive basal cells showed higher CFTR and GFP staining than did the cells from the uninfected monkey control. Positive immunostaining, also detected in the liver and pancreas, remained stable for at least a year. All animals seroconverted for anticapsid antibodies by 90 days post-treatment. The neutralizing antibody titer declined in the animal necropsied at 1 year. Conclusion: AAV1 safely and effectively transduces monkey airway and basal cells. Both the presence of vector genomes and transduction from AAV1-CFTR and AAV1-GFP virus seen in the monkeys 4 months to 1 year after the first instillation suggest that repeat dosing with AAV1-based vectors is achievable, particularly after methylprednisolone treatment.

Sequential dosing of convalescent COVID-19 plasma with significant temporal clinical improvements in a persistently SARS-COV-2 positive patient.

The current global pandemic, SARS-CoV-2 infection, is still extending across the world affecting millions of lives to the date. While new successful vaccines are available with promising outcomes to minimize the spread and to reduce the severity of the disease, optimal therapeutic options still remain elusive. COVID-19 convalescent plasma (CCP) is an investigational treatment option which studies suggesting signals of efficacy and favorable outcomes only for patients treated very early in course of the disease. Benefits of the use of CCP later in the disease remain highly debated and therefore are not common practice. We hereby report a case of severe SARS-CoV-2 infection in a young male patient with prolonged COVID-19 positivity who received repeat doses of CCP treatments later in the disease with temporal clinical improvement. This patient's case highlights the need of further studies evaluating efficacy of repeated dosing of CCP. This also suggests a potential of successful use of CCP later in the disease in selected COVID-19 patients.

Repeat Dosing of AAV2.5T to Ferret Lungs Elicits an Antibody Response That Diminishes Transduction in an Age-Dependent Manner.

Readministration of recombinant adeno-associated virus (rAAV) may be necessary to treat cystic fibrosis (CF) lung disease using gene therapy. However, little is known about rAAV-mediated immune responses in the lung. Here, we demonstrate the suitability of the ferret for testing AAV2.5T-mediated CFTR delivery to the lung and characterization of neutralizing-antibody (NAb) responses. AAV2.5T-SP183-hCFTRΔR efficiently transduced both human and ferret airway epithelial cultures and complemented CFTR Cl- currents in CF airway cultures. Delivery of AAV2.5T-hCFTRΔR to neonatal and juvenile ferret lungs produced hCFTR mRNA at 200%-300% greater levels than endogenous fCFTR. Single-dose (AAV2.5T-SP183-gLuc) or repeat dosing (AAV2.5T-SP183-fCFTRΔR followed by AAV2.5T-SP183-gLuc) of AAV2.5T was performed in neonatal and juvenile ferrets. Repeat dosing significantly reduced transgene expression (11-fold) and increased bronchoalveolar lavage fluid (BALF) NAbs only in juvenile, but not neonatal, ferrets, despite near-equivalent plasma NAb responses in both age groups. Notably, both age groups demonstrated a reduction in BALF anti-capsid binding immunoglobulin (Ig) G, IgM, and IgA antibodies after repeat dosing. Unique to juvenile ferrets was a suppression of plasma anti-capsid-binding IgM after the second vector administration. Thus, age-dependent immune system maturation and isotype switching may affect the development of high-affinity lung NAbs after repeat dosing of AAV2.5T and may provide a path to blunt AAV-neutralizing responses in the lung.

Transduction of Surface and Basal Cells in Rhesus Macaque Lung Following Repeat Dosing with AAV1CFTR.

To test the effectiveness of repeat dosing, we sprayed two doses (1013 vg each) of AAV1Δ27-264-CFTR into airways of four rhesus monkeys at 0 and 30 days, followed by a single dose of 1013 vg of AAV1GFP on day 60. Monkeys were sacrificed on day 90. No adverse events occurred, indicating that AAV1 vectors are safe. An elevated anti-AAV1 neutralizing titer was established by the third dose. A positive ELISPOT to the adeno-associated virus (AAV) capsid but not to cystic fibrosis transmembrane conductance regulator (CFTR) occurred after the third dose in three monkeys. AAV1-CFTR and GFP vectors were detectable in all lung sections and in the heart, liver, and spleen. The CFTR protein was higher in treated monkeys than in an untreated monkey. GFP protein was detected in treated lungs. Lung surface and keratin 5-positive basal cells showed higher CFTR staining than in the uninfected monkey and were positive for GFP staining, indicating widespread gene transduction by AAV1CFTR and GFP. AAV1 safely and effectively transduces monkey airway and basal cells. Both the significant numbers of vector genomes and transduction from AAV1CFTR and GFP virus seen in the monkeys 3 months after the first instillation suggest that repeat dosing with AAV1-based vectors is achievable.

Probable pulmonary embolism with repeat administration of prothrombin complex concentrate in a factor Xa inhibitor patient.

WHAT IS KNOWN AND OBJECTIVE: Prothrombin complex concentrate (PCC) is a plasma-derived concentrate used to replenish clotting factors. There are limited recommendations for treating coagulopathy induced by direct oral anticoagulants (DOAC). Data are limited regarding both total dose and repeated dosing with this population. CASE SUMMARY: We describe a case of an adult patient anticoagulated with apixaban who received two 35 unit/kg doses of PCC resulting in suspected pulmonary embolism. WHAT IS NEW AND CONCLUSION: Treatment of DOAC-induced bleeding remains an "off-label" indication for PCC. Additional doses should be given with caution if given at all and patients monitored closely.

Nonadditive Effects of Repetitive Administration of Lipoplexes in Immunocompetent Mice.

Repetitive administration is routinely used to maintain therapeutic drug levels, but previous studies have documented an accelerated blood clearance of some lipid-based delivery systems under these conditions. To assess the effect of repetitive administration, non-PEGylated lipoplexes (+/-0.5) were administered 4 times via tail vein injection at 3-day intervals to immunocompetent BALB/c mice bearing 4T1 tumors. This study measured the effect of repeat administration of nontargeted lipoplexes on clearance, cytokine/chemokine response, plasmid distribution, reporter gene expression, and liver toxicity. We do not observe a refractory period or a statistically significant difference in blood clearance between the first administration and subsequent injections of this lipoplex formulation, consistent with the absence of a cytokine/chemokine response. However, we do see a significant effect on both plasmid accumulation and expression, an enhancement of 26-fold and 10-fold in tumor plasmid levels and expression, respectively, after 4 injections as compared to that after a single injection. In addition, in vivo imaging suggests that expression in other organs had diminished rapidly 72 h after each administration, in contrast to relatively constant expression in the tumor. Taken together, the findings indicate that gene delivery to tumors can be dramatically enhanced by employing repetitive administration.

Safety, tolerability and pharmacokinetics of repeat dosing of the antibiotic GSK1322322, a peptide deformylase inhibitor: a randomized placebo-controlled study.

OBJECTIVES: GSK1322322 is a potent inhibitor of peptide deformylase, an essential bacterial enzyme required for protein maturation. In this two-part, double-blind, randomized, placebo-controlled, Phase 1 study (study identifier: PDF112668), the safety, tolerability and pharmacokinetics of single and repeat oral-dose GSK1322322 (500-1500 mg) in healthy adult and elderly volunteers were evaluated. PATIENTS AND METHODS: Part A included GSK1322322 doses of 500, 750, 1000 and 1500 mg in healthy adults; Part B evaluated 1000 mg of GSK1322322 in healthy elderly volunteers. Volunteers received a single morning dose of a powder-in-bottle formulation of GSK1322322 or placebo on day 1, no dosing on day 2 and twice-daily dosing on days 3-12. RESULTS: Of 52 enrolled volunteers, 40 and 12 volunteers were treated with GSK1322322 and placebo, respectively. Mean plasma GSK1322322 trough concentration increased with increasing dose and reached steady-state after 2 days of repeat dosing. After single dosing of GSK1322322, maximum plasma concentration and exposure (AUC) were dose proportional from 500 to 1500 mg. However, after repeat dosing, AUC values at steady-state increased slightly more than proportionally, possibly because of a slightly longer terminal elimination t½ after repeat dosing (compared with single-dose t½) at higher doses (1000 and 1500 mg). There was no age effect or diurnal variation in the GSK1322322 pharmacokinetic profile. GSK1322322 was generally well tolerated-all adverse events were mild to moderate in intensity. CONCLUSIONS: Repeat oral GSK1322322 (500-1500 mg) for 10 days was well tolerated. These data warrant further clinical investigation of GSK1322322.