Background and ObjectiveWhen eye diseases are treated by topical administration, the success of treatment lies in the effective drug concentration in the target tissue. This is why the drug’s pharmacokinetic, in the different substructures of the eye, needs to be explored more accurately during drug development. The aim of the present analysis was to describe by rabbit model, the distribution of a drug after ocular instillation in the selected eye tissues and fluids.MethodsBy a top-down population approach, we developed and validated a population pharmacokinetics (PopPK) model, using tissue concentrations (tear, naso-lacrymal duct, cornea and aqueous humor) of a new src tyrosine kinase inhibitor (FV-60165) in each anterior segment’s tissue and fluid of the rabbit eye. Inter-individual variability was estimated and the impact of the formulation (solution or nanosuspension) was evaluated.ResultsThe model structure selected for the eye is a 4-compartment model with the formulation as a significant covariate on the first-order rate constant between tears and the naso-lacrymal duct. The model showed a good predictive performance and may be used to estimate the concentration–time profiles after single or repeated administration, in each substructure of the eye for each animal included in the analysis.ConclusionsThis analysis allowed describing the distribution of a drug in the different selected tissues and fluids in the rabbit’s eyes after instillation of the prodrug as a solution or nanosuspension.

Recent advances in multiple myeloma (MM), have seen the advent of several therapeutic monoclonal antibodies (mAb) targeting antigens on malignant plasma cells such as CD38 and SLAMF7. Evaluation of depth of response through quantification of M-Protein, by Serum Protein Electrophoresis (SPEP) and Immuno-Fixation Electrophoresis (IFE) presents a particular challenge for clinical laboratories when therapeutic mAbs are used. Specifically, therapeutic mAbs can confound SPEP and IFE measurements when they overlap with patient serum M-protein. Such interference could lead to an inaccurate determination of depth of response according to International Myeloma Working Group (IMWG) criteria Isatuximab (Isa), an IgG-kappa, anti-CD38 mAb, binds a specific epitope on CD38, which is widely expressed on MM cells. ICARIA-MM was a phase 3, prospective, randomized, open-label, active-controlled, multicenter study comparing isatuximab in combination with pomalidomide plus dexamethasone (Isa-Pd) to pomalidomide plus dexamethasone (Pd) in patients with relapsed/refractory multiple myeloma (RRMM). ICARIA-MM enrolled patients who had received ≥2 prior lines of therapy, including lenalidomide (len) and a proteasome inhibitor (PI). ICARIA-MM demonstrated that Isa-Pd significantly improved PFS, overall response and depth of response with a trend in overall survival benefit and a manageable toxicity profile in these patients. To overcome the potential interference of isatuximab with M-Protein in patients enrolled in ICARIA-MM, we developed and validated a hybrid assay using Immuno-Capture and Liquid Chromatography coupled to High Resolution Mass Spectrometry (IC-LC-HRMS). This method enriches for serum immunoglobulins by immuno-capture. Heavy chains (HC) and light chains (LC) are then dissociated and sorted using Liquid Chromatography. M-Protein and isatuximab LC are analyzed using HRMS which allows to differentiate their monoisotopic intact mass. A small number of patients (24/154; 15.6% in the Isa-Pd arm and 5/153; 3.3% in the Pd arm) fulfilled all criteria for CR (100% M-protein reduction on SPEP, bone marrow <5% plasma cells), but remained IFE positive as per IRC (near-CR category). Serial serum samples from 22 of 24 patients in the Isa-Pd arm were available for MS analysis. Separation of the M-protein and isatuximab signals was performed by IC-LC-HRMS. Using a semi-quantitative approach and applying a sensitivity threshold of 0.25g/dL for IFE-positivity (sensitivity threshold of IFE test per Covance central lab), we identified 11 of 22 patients as having M-protein levels below the threshold for IFE positivity, indicating that the true CR-rate was underestimated in the ICARIA-MM study due to interference. Of the 11 patients with MS M-protein values below the IFE sensitivity threshold, 10 were IgG (8 IgGκ, 2 IgGλ) and one was lambda LC disease with undetectable baseline HC. Baseline myeloma isotype distribution was different in patients with MS M-protein values above IFE sensitivity threshold (4 IgA, 6 IgG, 1 kappa light chain). This confirms that M-protein interference mediated by isatuximab, an IgG kappa mAb, is predominantly seen in patients with IgG isotype (mainly IgGκ), and in LC patients in whom a detectable IgG heavy chain can now be identified. At primary analysis, 4 of 11 patients with MS M-protein values above IFE sensitivity level, versus 2 of 11 patients with MS M-protein values below IFE sensitivity level had progressed. Median PFS was 13.9mo in patients with MS M-protein values above IFE sensitivity level, versus 15.21mo in patients with MS M-protein values below IFE sensitivity level. In conclusion, MS can be successfully used to separate endogenous M-protein from therapeutic isatuximab serum levels. In this subpopulation with excellent response to Isa-Pd, while most patients are still progression-free, a trend can be observed towards longer PFS in patients who would be considered IF negative after MS versus patients remaining IF positive. Disclosures Finn: Sanofi Oncology: Employment. Macé:Sanofi Oncology: Employment. Campana:Sanofi: Employment. Le-Guennec:Sanofi: Employment. Muccio:Sanofi Oncology: Employment. Tavernier:Sanofi Oncology: Employment. Rouchon:Sanofi Oncology: Employment. Roccon:Sanofi Oncology: Employment. Dai:Sanofi Oncology: Employment. Boutet:Sanofi Oncology: Employment. Mouret:Sanofi Oncology: Employment. Pradeilles:Sanofi Oncology: Employment. Hugla:Sanofi Oncology: Employment. Engelvin:Sanofi Oncology: Employment. DiNoto:Sanofi Oncology: Employment. van de Velde:Sanofi: Employment. Fedeli:Sanofi Oncology: Employment. OffLabel Disclosure: Isatuximab is an investigational agent and has not yet been approved by the US Food and Drug administration or any other regulatory agency.

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In multiple myeloma (MM) disease, malignant plasma cells produce excessive quantities of a monoclonal immunoglobulin (Ig), known as M-protein. M-protein levels are measured in the serum of patients with MM using electrophoresis techniques to determine the response to treatment. However, therapeutic monoclonal antibodies, such as isatuximab, may confound signals using electrophoresis assays. We developed a robust assay based on immunocapture and liquid chromatography coupled to high-resolution mass spectrometry (IC-HPLC-HRMS) in order to eliminate this interference. Following immunocapture of Ig and free light chains (LC) in serum, heavy chains (HC) and LC were dissociated using dithiothreitol, sorted by liquid chromatography and analyzed using HRMS (Q-Orbitrap). This method allowed the M-proteins to be characterized and the signals from isatuximab and M-proteins to be discriminated. As Mprotein is specific to each patient, no standards were available for absolute quantification. We therefore used alemtuzumab (an IgG kappa mAb) as a surrogate analyte for the semiquantification of M-protein in serum. This assay was successfully validated in terms of selectivity/specificity, accuracy/precision, robustness, dilution linearity, and matrix variability from 10.0 to 200 μg/mL in human serum. This method was used for clinical assessment of samples and eliminated potential interference due to isatuximab when monitoring patients with MM.

Pyoverdines and pyochelin are siderophore compounds secreted by Pseudomonas aeruginosa present in cystic fibrosis affected patients. The available literature on the quantification of pyoverdines and pyochelin in sputa of cystic fibrosis patients is based on detecting the fluorescence properties of the targets detecting pyoverdines and pyochelin without differentiating the types by chromagraphic separation. Within these studies pyochelin was always far less detected than pyoverdines. This is in contrast to gene expression data and suggets the pyochelin fluorescence detection method lacks sensitivity which results in the molecule being non-detectable. In this report, we describe a novel and sensitive LC/MS/MS method for quantitation of pyochelin in human sputum from cystic fibrosis affected patients with Pseudomonas aeruginosa (culture positive) infection. Using a validated bioanalytical method, 26 sputum samples from cystic fibrosis patients from a biobank were analyzed. Data showed the LC/MS/MS method met bioanalytical validation acceptance criteria and results of pyochelin were consistent with reported results of Pseudomonas aeruginosa concentration in cystic fibrosis patients. This new approach for quantitation of pyochelin in human sputum is more sensitive, reproducible and easier to reliably measure the biomarker pyochelin in cystic fibrosis patients. Monitoring pyoverdines and pyochelin in the sputum samples from cystic fibrosis patients may provide better analytical tools for cystic fibrosis new drug development.