Standardized kinetic microassay to quantify differential chemosensitivity on the basis of proliferative activity

Conventionally in vitro cytotoxicity assays are performed as single-end-point determinations. To compensate for the diversity of growth rates among different cell lines in this report we describe a computerized kinetic chemosensitivity assay based on quantification of biomass by staining cells with crystal violet. As a prerequisite four human breast cancer cell lines (MDA-MB-231, MCF-7, T-47-D and ZR-75-1) were characterized with regard to oestrogen and progesterone receptor content, modal chromosome number and proliferation kinetics depending on the number of passages in culture. With prolonged time in culture for ZR-75-1 exposed to various concentrations of cisplatinum a dose-related increase in drug effect was observed. Owing to a correction of the T/C values for the initial cell mass (at the time when drug is added) a sharp distinction between cytostatic and cytocidal drug effects becomes obvious in plots of corrected T/C values versus time of incubation. The influence of the untreated control on the corrected T/C values and possible time courses of theoretical inhibition profiles (reflecting cytostatic, transient cytotoxic or cytocidal drug effects as well as development of resistance) and their relationship to the corresponding growth curves of drug-treated cells are discussed. Chemosensitivity assays with diethylstilbestrol dipropionate, tamoxifen, melphalan, cisplatinum, vinblastine, Adriamycin and 5-fluorouracil prove the theoretical considerations to be true for MDA-MB-231, MCF-7, T-47-D and ZR-75-1 human breast cancer cell lines in practice

Computerized determination of growth kinetic curves and doubling times from cells in microculture

In this paper we describe the microcomputer-aided determination of cell proliferation kinetics and doubling times utilizing a crystal violet assay and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay in microtitration plates. The analysis of spectrophotometric data provides the doubling times at any time of incubation. Plots of doubling time versus time of incubation give reproducible information on the exact duration of the logarithmic growth phase. This method is applicable to anchorage-dependent as well as anchorage-independent cells when colorimetric or fluorometric data are accessible

Subcutaneous versus intravenous administration of rituximab: pharmacokinetics, CD20 target coverage and B-cell depletion in cynomolgus monkeys.

The CD20-specific monoclonal antibody rituximab (MabThera(®), Rituxan(®)) is widely used as the backbone of treatment for patients with hematologic disorders. Intravenous administration of rituximab is associated with infusion times of 4-6 hours, and can be associated with infusion-related reactions. Subcutaneous administration of rituximab may reduce this and facilitate administration without infusion-related reactions. We sought to determine the feasibility of achieving equivalent efficacy (measured by endogenous B-cell depletion) and long-term durability of CD20 target coverage for subcutaneously administered rituximab compared with intravenous dosing. In these preclinical studies, male cynomolgus monkeys were treated with either intravenous rituximab or novel subcutaneous formulation of rituximab containing human recombinant DNA-derived hyaluronidase enzyme. Peripheral blood samples were analyzed for serum rituximab concentrations, peripheral B-cell depletion, and CD20 target coverage, including subset analysis according to CD21+ status. Distal lymph node B-cell depletion and CD20 target coverage were also measured. Initial peak serum concentrations of rituximab were significantly higher following intravenous administration than subcutaneous. However, the mean serum rituximab trough concentrations were comparable at 2 and 7 days post-first dose and 9 and 14 days post-second dose. Efficacy of B-cell depletion in both peripheral blood and distal lymph nodes was comparable for both methods. In lymph nodes, 9 days after the second dose with subcutaneous and intravenous rituximab, B-cell levels were decreased by 57% and 42% respectively. Similarly, levels of peripheral blood B cells were depleted by >94% for both subcutaneous and intravenous dosing at all time points. Long-term recovery of free unbound surface CD20 levels was similar, and the duration of B-cell depletion was equally sustained over 2 months for both methods. These results demonstrate that, despite initial peak serum drug level differences, subcutaneous rituximab has similar durability, pharmacodynamics, and efficacy compared with intravenous rituximab

Depletion of Peripheral Blood CD21+ and CD21- B-cell subsets.

<p>Short and long-term analysis of PBMC B-cell depletion of (<b>A</b>) CD21+ and (<b>B</b>) CD21- B-cell subsets (as identified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080533#pone-0080533-g002" target="_blank">Figure 2C</a>). Individuals animal samples with mean shown for baseline, day 2, day 9 and day 14 post second dose (top) and percent remaining as normalized to PBS vehicle treated group (bottom) (group mean ± SD (n=4 rituximab treated groups, n=3 PBS vehicle treated groups).</p

Pharmacokinetics of serum rituximab in cynomolgus monkeys.

<p>PK analysis of serum rituximab levels after (<b>A</b>) a single SC dose of rituximab preformulated in rHuPH20 administered at 20 mg/kg (Individual animal samples with mean score shown (n=3)), and (<b>B</b>) 2 × 10 mg/kg doses of SC (preformulated in rHuPH20) or standard IV rituximab, given 7 days apart (Individuals animal samples with mean shown (n=4)). </p

CD20 target coverage and B-cell depletion in lymph nodes.

<p>Lymph node analysis of (<b>A</b>) CD20 target coverage as determined by flow cytometric staining for free surface CD20 levels on (B cells identified as in Figure 2A) and (<b>B</b>) depletion of those lymph node B-cellsat baseline and 9 days after second dose of subcutaneous or intravenous rituximab as determined by a ratio to CD4+/CD3+ T cells (Individuals animal samples with mean shown (n=4 rituximab treated groups, n=3 PBS vehicle treated groups)). </p

CD20 target coverage in B-cells and according to CD21+ status.

<p>Paradigms for flow cytometry staining: (<b>A</b>) CD20-independent identification of B cells using fsc/scc lymphocyte gating followed by CD4/CD3 negative gating and CD40 positive CD16 negative gating shows a progressively increasing specific B cell population; (<b>B</b>) Free surface CD20 levels on B cells (as identified in A) with and without rituximab treatment to show target coverage of CD20 compared to CD4+/CD3+ T cells; (<b>C</b>) Identification of CD21+ and CD21- peripheral blood B-cell subsets (within the CD4-/CD3-/CD16-/CD40+ gated B cells as identified in A) showing CD21, CD40 and CD20 levels of the subsets.</p

CD20 target coverage and B-cell depletion in Peripheral Blood.

<p>PBMC analysis of (<b>A</b>) CD20 target coverage as determined by flow cytometric staining for free surface CD20 levels on (B cells identified as in Figure 2A) and (<b>B</b>) depletion of those PBMC B-cells at baseline and 2, 9, and 14 days after second dose of subcutaneous or intravenous rituximab as determined by a ratio to CD4+/CD3+ T cells (Individuals animal samples with mean shown (n=4 rituximab treated groups, n=3 PBS vehicle treated groups)); and long-term PBMC analysis of (<b>C</b>) percent free surface CD20 target coverage and (<b>D</b>) percent remaining B-cells out to 63 days; normalized to PBS vehicle (group mean ± SD (n=4 rituximab treated groups, n=3 PBS vehicle treated groups)) with ex-vivo rituximab treatment of PBS vehicle sample to show maximal target coverage.</p