Characterization of Rhodamine-123 as a Tracer Dye for Use In In vitro Drug Transport Assays

Fluorescent tracer dyes represent an important class of sub-cellular probes and allow the examination of cellular processes in real-time with minimal impact upon these processes. Such tracer dyes are becoming increasingly used for the examination of membrane transport processes, as they are easy-to-use, cost effective probe substrates for a number of membrane protein transporters. Rhodamine 123, a member of the rhodamine family of flurone dyes, has been used to examine membrane transport by the ABCB1 gene product, MDR1. MDR1 is viewed as the archetypal drug transport protein, and is able to efflux a large number of clinically relevant drugs. In addition, ectopic activity of MDR1 has been associated with the development of multiple drug resistance phenotype, which results in a poor patient response to therapeutic intervention. It is thus important to be able to examine the potential for novel compounds to be MDR1 substrates. Given the increasing use rhodamine 123 as a tracer dye for MDR1, a full characterisation of its spectral properties in a range of in vitro assay-relevant media is warranted. Herein, we determine λmax for excitation and emission or rhodamine 123 and its metabolite rhodamine 110 in commonly used solvents and extraction buffers, demonstrating that fluorescence is highly dependent on the chemical environment: Optimal parameters are 1% (v/v) methanol in HBSS, with λex = 505 nm, λem = 525 nm. We characterise the uptake of rhodamine 123 into cells, via both passive and active processes, and demonstrate that this occurs primarily through OATP1A2-mediated facilitated transport at concentrations below 2 µM, and via micelle-mediated passive diffusion above this. Finally, we quantify the intracellular sequestration and metabolism of rhodamine 123, demonstrating that these are both cell line-dependent factors that may influence the interpretation of transport assays

Subcutaneous daratumumab plus standard treatment regimens in patients with multiple myeloma across lines of therapy (PLEIADES): an open-label Phase II study

© 2020 The Authors. Daratumumab is a CD38-targeting monoclonal antibody approved for intravenous (IV) infusion for multiple myeloma (MM). We describe the Phase II PLEIADES study of a subcutaneous formulation of daratumumab (DARA SC) in combination with standard-of-care regimens: DARA SC plus bortezomib/lenalidomide/dexamethasone (D-VRd) for transplant-eligible newly diagnosed MM (NDMM); DARA SC plus bortezomib/melphalan/prednisone (D-VMP) for transplant-ineligible NDMM; and DARA SC plus lenalidomide/dexamethasone (D-Rd) for relapsed/refractory MM. In total, 199 patients were treated (D-VRd, n = 67; D-VMP, n = 67; D-Rd, n = 65). The primary endpoints were met for all cohorts: the ≥very good partial response (VGPR) rate after four 21-day induction cycles for D-VRd was 71·6% [90% confidence interval (CI) 61·2–80·6%], and the overall response rates (ORRs) for D-VMP and D-Rd were 88·1% (90% CI 79·5–93·9%) and 90·8% (90% CI 82·6–95·9%). With longer median follow-up for D-VMP and D-Rd (14·3 and 14·7 months respectively), responses deepened (ORR: 89·6%, 93·8%; ≥VGPR: 77·6%, 78·5%), and minimal residual disease–negativity (10‒5) rates were 16·4% and 15·4%. Infusion-related reactions across all cohorts were infrequent (≤9·0%) and mild. The median DARA SC administration time was 5 min. DARA SC with standard-of-care regimens demonstrated comparable clinical activity to DARA IV–containing regimens, with low infusion-related reaction rates and reduced administration time

Phase II study results of a replacement therapy for hereditary angioedema with subcutaneous C1-inhibitor concentrate.

BackgroundHereditary angioedema (HAE) due to C1 inhibitor deficiency manifests as recurrent swelling attacks that can be disabling and sometimes fatal. Long-term prophylaxis with twice-weekly intravenous injections of plasma-derived C1-inhibitor (pdC1-INH) has been established as an effective treatment. Subcutaneous (SC) administration of pdC1-INH has not been studied in patients with HAE.MethodsThis open-label, dose-ranging, crossover study (COMPACT Phase II) was conducted in 18 patients with type I or II HAE who received two of twice-weekly 1500, 3000, or 6000 IU SC doses of highly concentrated volume-reduced CSL830 for 4 weeks each. The mean trough plasma levels of C1-INH functional activity, C1-INH and C4 antigen levels during Week 4, and overall safety and tolerability were evaluated. The primary outcome was model-derived steady-state trough C1-INH functional activity.ResultsAfter SC CSL830 administration, a dose-dependent increase in trough functional C1-INH activity was observed. C1-INH and C4 levels both increased. The two highest dose groups (3000 and 6000 IU) achieved constant C1-INH activity levels above 40% values, a threshold that was assumed to provide clinical protection against angioedema attacks. Compared with intravenous injection, pdC1-INH SC injection with CSL830 showed a lower peak-to-trough ratio and more consistent exposures. All doses were well tolerated. Mild-to-moderate local site reactions were noted with pain and swelling being the most common adverse event.ConclusionsSubcutaneous volume-reduced CSL830 was well tolerated and led to a dose-dependent increase in physiologically relevant functional C1-INH plasma levels. A clinical outcome study of SC CSL830 in patients with HAE warrants further investigation

Phase II study results of a replacement therapy for hereditary angioedema with subcutaneous C1-inhibitor concentrate

Background Hereditary angioedema (HAE) due to C1 inhibitor deficiency manifests as recurrent swelling attacks that can be disabling and sometimes fatal. Long-term prophylaxis with twice-weekly intravenous injections of plasma-derived C1-inhibitor (pdC1-INH) has been established as an effective treatment. Subcutaneous (SC) administration of pdC1-INH has not been studied in patients with HAE. Methods This open-label, dose-ranging, crossover study (COMPACT Phase II) was conducted in 18 patients with type I or II HAE who received two of twice-weekly 1500, 3000, or 6000 IU SC doses of highly concentrated volume-reduced CSL830 for 4 weeks each. The mean trough plasma levels of C1-INH functional activity, C1-INH and C4 antigen levels during Week 4, and overall safety and tolerability were evaluated. The primary outcome was model-derived steady-state trough C1-INH functional activity. Results After SC CSL830 administration, a dose-dependent increase in trough functional C1-INH activity was observed. C1-INH and C4 levels both increased. The two highest dose groups (3000 and 6000 IU) achieved constant C1-INH activity levels above 40% values, a threshold that was assumed to provide clinical protection against angioedema attacks. Compared with intravenous injection, pdC1-INH SC injection with CSL830 showed a lower peak-to-trough ratio and more consistent exposures. All doses were well tolerated. Mild-to-moderate local site reactions were noted with pain and swelling being the most common adverse event. Conclusions Subcutaneous volume-reduced CSL830 was well tolerated and led to a dose-dependent increase in physiologically relevant functional C1-INH plasma levels. A clinical outcome study of SC CSL830 in patients with HAE warrants further investigation