Biochemical assay reagent/probe

Response correlation with drug efflux [2]
We evaluated the efflux of 3, 3'-diethyloxacarbocyanine iodide/DiOC2 from pretreatment leukemic blast cells as a measure of functional drug resistance. Figure 3 shows the relationship between measured leukemic blast cell drug efflux levels and saturation of CD33 sites with response. Elimination of leukemia appeared to be correlated with a low capacity by leukemic blast cells to extrude DiOC2. For example, of the 30 patients evaluated with the assay in whom doses of CMA-676 saturated >75% of available CD33 sites on peripheral blood blast cells, 8 of 17 patients with leukemic blast cells that showed ≤40 channel numbers of DiOC2 efflux had <5% blasts in the bone marrow after treatment. In contrast, none of the 13 patients with leukemic blast cells expressing >40 channel numbers of DiOC2 efflux entered remission.

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Fluorescence studies of a carbocyanine dye 3, 3'-diethyloxacarbocyanine iodide (DOCI) in a series of monohydroxy alcohols reveal a monotonic increasing trend for both the fluorescence quantum yield and fluorescence lifetime with solvent viscosity. The results were interpreted in terms of retardation of the photoisomerization rate of DOCI with increasing solvent viscosity, and a definite inverse relationship between the two parameters was established. This relationship was utilized to estimate the microviscosity experienced by DOCI in supramolecular assemblies like micelles and microemulsions of several well-known surfactants and amphiphilic block copolymer. In most of the assemblies, the local environment around DOCI was characterized by rather high microviscosity, comparable to those of high alcohols between butanol and decanol. Our observations suggest that the rod-like DOCI molecule is largely embedded among the hydrophobic tails of the surfactant molecules, which exert a viscous drag strong enough to hinder its photoisomerization. The fluorescence properties of DOCI were also utilized to determine the critical micellization concentration of several surfactants in water. Interestingly, the microviscosity reported by DOCI in water-free AOT reverse micelles is significantly high, and can be explained by considering enhanced electrostatic attraction between the DOC+ cation and the anionic AOT head-groups [1].

Laboratory investigations [2]
Total hP67.6 antibody concentrations in plasma samples were determined using an enzyme-linked immunosorbent assay (ELISA). Formation of antigen-antibody CMA-676 bound to peripheral blood mononuclear cells was detected by flow microfluorimetry. Cells were incubated with biotinylated goat monoclonal anti-human IgG4, followed by avidin-fluorescein isothiocyanate. Cells incubated with avidin-fluorescein isothiocyanate alone comprised the negative control. The saturation percentage was defined as 100 times the ratio of the fluorescence intensity of patient mononuclear cells (minus the negative control) over the maximum fluorescence intensity (minus the negative control). Maximum achievable saturation was determined by incubating patient mononuclear cells from the same time point with saturating amounts of CMA-676 in vitro before the addition of the anti-human IgG4 antibody. The efflux of 3, 3'-diethyloxacarbocyanine iodide (DiOC2) from CD33-positive blast cells was measured as an indication of functional drug efflux.11 Serum samples obtained from each patient before CMA-676 administration, on day 7 after initial doses, and on days 7, 14, 21, and 28 after administration of the final dose of CMA-676 were analyzed for anti-hP67.6 (humanized mouse antibody) or anti-calicheamicin/linker immune response by ELISA. [2]

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Absorption and fluorescence spectra of the 3, 3'-diethyloxacarbocyanine iodide/DOCI solutions were measured in a Shimadzu UVPC-3200 spectrophotometer and a PerkinElmer LS55 fluorimeter, respectively. Picosecond fluorescence dynamics studies of the solutions were conducted with a time-correlated single photon counting (TCSPC) setup employing a picosecond diode laser (IBH NanoLED-07) operating at a λex = 440 nm and a repetition rate of 0.9 MHz as excitation source. The overall temporal and spectral resolution of the setup were ∼70 ps and ∼10 nm, respectively. All spectroscopic measurements were performed at 22 °C [1].

[1]. 3,3′-Diethyloxacarbocyanine iodide: A new microviscosity probe for micelles and microemulsions. Colloids and Surfaces A: Physicochemical and Engineering Aspects.
[2]. Selective ablation of acute myeloid leukemia using antibody-targeted chemotherapy: a phase I study of an anti-CD33 calicheamicin immunoconjugate. Blood. 1999 Jun 1;93(11):3678-84.

C3-oxacyanine is a C3 cyanine dye with 3-ethyl-1,3-benzoxazole-2(3H)-yl units at both ends. It is a fluorescent dye. It is an organic iodide salt, belonging to the cyanine dye class and also a 1,3-benzoxazole compound. It contains a C3-oxacyanine cation. Our results indicate that in monohydroxy alcohols, the photoisomerization rate constant of 3,3'-diethyloxacyanine iodide/DOCI follows the relationship kiso ∝ 1/ηa, (a < 1), over a wide viscosity range. We also found that the kiso values in most micelles and microemulsions are in the range of 1–3 × 10⁹ s⁻¹, comparable to those in higher alcohols between butanol and decanol. Clearly, the effective local viscosity experienced by DOCI in these surfactant aggregates is similar to that in higher alcohols and can be calculated using the relationship between kiso and η in monohydroxy alcohols. From a physical point of view, this similarity can be explained by assuming that the rod-shaped DOCI molecules (including cyanine bonds) are embedded between the hydrophobic tails of the surfactant molecules. Thus, photoisomerization around the bonds is inhibited by the friction of these tails, which are structurally similar to the long hydrocarbon chains of higher alcohols. However, in AOT reverse micelles, the viscosity of DOCI is unusually high compared to all other media. Here, the absence of water as a dielectric results in a strong electrostatic attraction between the DOC+ cation and the anionic head group of AOT, which further restricts the movement of DOCI molecules. [1]

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Most patients with acute myeloid leukemia (AML) have leukemia blast cells that express the CD33 antigen, but hematopoietic stem cells do not. We conducted a study to determine whether selectively eliminating cells that express the CD33 antigen could restore normal hematopoietic function in AML patients. In a dose-escalation study, 40 patients with relapsed or refractory CD33(+) AML received an immunoconjugate (CMA-676) consisting of a humanized anti-CD33 antibody linked to the potent antitumor antibiotic calicimycin. We assessed pre-treatment functional resistance using the ability of leukemia cells to efflux 3,3'-diethyloxacarcyanine iodide (DiOC2). Leukemia cells were cleared from the blood and bone marrow in 8 of the 40 patients (20%); blood cell counts returned to normal in 3 patients (8%). A higher clinical remission rate was observed in leukemia with low in vitro dye efflux. CMA-676 infusions were generally well tolerated, with fever and chills being the most common toxicities following infusion. Two patients receiving the highest dose (9 mg/m2) developed neutropenia more than 5 weeks after their last CMA-676 administration. These results indicate that CD33-targeting immunoconjugates can selectively deplete malignant hematopoietic function in some patients with acute myeloid leukemia (AML). [2]

C21H21IN2O2

460.31

460.065

C, 54.80; H, 4.60; I, 27.57; N, 6.09; O, 6.95

905-96-4

6538326

Purplish red to red solid powder

278ºC (dec.)(lit.)

1.086

0

4

4

26

474

0

[I-].O1C(=CC=CC2=[N+](CC)C3C(=CC=CC=3)O2)N(CC)C2C1=CC=CC=2

FIZZUEJIOKEFFZ-UHFFFAOYSA-M

InChI=1S/C21H21N2O2.HI/c1-3-22-16-10-5-7-12-18(16)24-20(22)14-9-15-21-23(4-2)17-11-6-8-13-19(17)25-21;/h5-15H,3-4H2,1-2H3;1H/q+1;/p-1

(2Z)-3-ethyl-2-[(E)-3-(3-ethyl-1,3-benzoxazol-3-ium-2-yl)prop-2-enylidene]-1,3-benzoxazole;iodide

3,3'-Diethyloxacarbocyanine iodide; 905-96-4; C3-oxacyanine; 57441-62-0; 3,3'-Diethyloxacarbocyanine (iodide); DOCI dye; Oxacarbocyanine (C3); DiOC2(3) [3,3'-Diethyloxacarbocyanine Iodide];

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 20.83 mg/mL (45.25 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.52 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)