Evans blue/ Albumin

Fig. 2 EPR effect and drug accumulation in solid tumor. (A) and (B) CT scan of human liver with hepatocellular carcinoma. When SMANCS/Lipiodol was infused arterially it was selectively taken up in the tumor. CT scan of (A) was obtained 2 days after the initial arterial infusion via a catheter, in which SMANCS/Lipiodol was selectively accumulated in the massive carcinoma seen as white area in the liver (due to high electron density of Lipiodol ). After 6 months with intermitted infusions of SMANCS two more times, tumor size (containing SMANCS) has remarkably regressed (B). (C) EPR effect in mouse sarcoma S-180 in mouse skin. Two dark blue circular areas are tumors marked by 0, where putative macro-molecular drug, Evans blue/albumin (MW 70 kDa), accumulated selectively. Note that the background of the normal skin shows no uptake of blue albumin. Mouse was killed at 6 h after the injection of Evans blue and it was quantified after extraction...

Fig. 6 NO-dependent vascular permeability of solid tumors (S-180) as quantified by uptake of Evans blue albumin complex in tumor of various sizes in mice. Mice were administered with NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-l-oxyl 3-oxide (PTIO) orally (dose PTIO, 125 mg/kg 1 4 in 8 h). Note that EPR effect is greatly suppressed by PTIO, which is a fraction of NO contributing to EPR effect. Further note that when the tumor size is larger than 0.25 g, the contribution of NO in EPR effect is less pronounced. This implicates the importance of NO at the early stages of tumor growth (see text) (from [42] after modification)...

Recently, we found that EPR effect is further enhanced by applying nitroglycerin ointment onto the skin of superficial tumors including breast cancer induced by chemical carcinogen, 7,12-dimethylbenz[a]anthracene (DMBA) in rats, and also transplanted mice tumors (Seki and Maeda, in preparation). The increase of Evans blue/albumin delivered into the solid tumors was 2- to 3-fold of that without nitroglycerin [68]. 

Figure 1. Accumulation of Evans blue-albumin complex in the tumor tissue. Tumor S-I80 was injected into mice skin. A to D provide a macroscopic picture of the tumor in the skin taken at 0,6, 24 and 72h, respectively, alto- i.v. injection of Evans blue (lOmg/kg).

Figure 2. Clearance of Evans blue-albumin complex from blood plasma and its accumulation in tumor tissue and normal skin in tumor-bearing mice. Quantification of concentration of die Evans blue-albumin complex at different times for plasma ( ), normal skin ( fl ), and tumor ( ).

Figure 20.1 Distribution volume of Evans blue-labeled albumin in a rat fibrosarcoma as a function of (A) perfusion pressure or (B) perfusion rate. The ratio of distribution volume (Fd)/infused volume ( ] ) was quantified at the infusion pressures of 36, 50, 94, and 163 in cmH20, respectively. Symbols represent data from individual experiments N= 2 for pressure of 36 cmH20 and N=5 for other pressures. The line in (B) was obtained through linear curve-fitting of the data. Reproduced with permission (McGuire and Yuan, 2001).

Distribution volume of Evans blue-labeled albumin in a rat fibrosarcoma as a function of 400... 

Figure 8.6 shows a diagram of a general model of blood-tissue solute transport, used to analyze data on the transport of labeled solutes introduced in the blood or perfusate flow supplied to individual organs. The development and analysis of models of this sort to analyze solute transport in physiological systems is a field pioneered by Sangren and Sheppard [178], Renkin [172], and Crone [40], Optically detectable probes (such as Evans Blue dye bound to albumin) can be used in conjunction with model analysis to probe the intravascular transport of... 

Another method of Pgp regulation has been demonstrated by adrenomedullin (AM) (89). It is produced by endothelial cells in the brain and acts as a vasodilator in the cerebral circulation. It was shown that AM antisense decreased the transendo-thelial electrical resistance across endothelial monolayers. Treatment of these cells with AM activated Pgp, suggesting that AM acts as an autocrine mediator in the regulation of the properties of BBB endothelial cells. In addition, AM incubation decreased BBB permeability for sodium fluorescein (376 Da) but not for Evan s blue albumin (67 kDa). An interesting observation was that it also attenuated fluid-phase endocytosis. 

Figure 5 Diagram of staining of the bronchial circulation in the cat. The bronchial and pulmonary circulations of the cat were perfused separately with aerated physiological salt solution containing bovine serum albumin (4% wt/vol) maintained at 37°C. Perfusates from the bronchial and pulmonary circulations were collected from cannulae positioned in the right and left ventricles, respectively. Infusion of Evans blue dye (30mg/Kg) into the systemic circulation resulted in deep blue staining of the central airways (black) with no staining of the parenchymal tissues (dotted). Further, 75-80% of the dye was collected from the cannula from the right heart.

The closely related dyes Evans blue (T 1824), trypan blue (T 1936), and Congo red show bathychromic shifts in their spectra in the presence of albumin (B26). Of the three dyes, only the former has no affinity for globulins at neutral pH. 

The possibility of measuring plasma volume using BSP has been described earlier (see Section 5.1). More commonly, I-albumin (B44, C16, 12, Z6) or the dyes Evans blue (A7, B44, G22, SIO) or vital red (G22, R2, S44) have been employed, but rose bengal (B4, S30), and indocyanine green (C16) have also been used. 

Figure 3. Effect of atropine in blocking muUiple-dose, DSCG-induced tachyphylaxis in rats. Sensitized animals were given atropine 25 mg/kg ip 20 min before jo (DSCG). After 1.0 hr the animals were given another iv dose of DSCG cotaaining 2 mg egg albumin and 5 mg Evans blue. Eight controls of nondrug-predosed animals were also used to calculate inhibition of the PC A assay after 30-min development of the skin sites. Six animals were used for each variable. The variability of repeated assays of DSCG in these animals is approximately 8%.

Figure 4. Effect of muscarinic blockers atropine and QNB on multiple-dose lodoxamide tachyphylaxis. Sensitized rats were given atropine 25mglkg ip or QNB 0.01 mg/kg ip 20 min before the first iv dose of lodoxamide. One hr Utter the animals were given another iv dose of lodoxamide (0.2 mg/kg containing 2 mg egg albumin and S mg Evans blue), and 30 min later the skin sites of sensitization were scored and compared to nonpredosed rats as well as control rats. Numbers in parenthesis refer to number of animals per variable.

The relative proportion of extra- and intracellular fluid has been determined by the dilution technique. Chemicals of known distribution in the body fluids are administered, and the volume of a given fluid compartment can be measured by determining the concentration of these chemicals. For example, Evans blue, bromosulftalein, iodinated albumin, and iodine 131 are used to determine plasma volume. Urea, thiourea, deuterium, and tritiated water are useful for determining the total body fluids. Thiocyanate, iodide, sulfate, mannitol, sucrose, raffmose, chloride 36, chloride 38, and bromide 32 are used to determine extracellular space. 

The characteristics of vascular pathophysiology Usted in Table 1 contribute to the selective, enhanced accumulation and prolonged retention of macro-molecular drugs or lipid particles in tumor tissue (EPR effect). Evans blue dye, which forms complexes with albimiin (67 kDa) or lipiodol (a lipid contrast agent), clearly revealed the presence of the EPR effect in rodent tumors after intravenous injection of Evans blue bound albumin, or after arterial... 

Serum albumin, mouse Evans blue dye - 2 hr 30 hr Mouse... 

Figures 1 and 2 explain some examples of macroscopic derangement of solid tumor, which are now generally accepted as an effect named EPR (Enhanced Permeability and Retention) effecr . In the experiment shown in Figure 1, a blue dye, Evans blue was injected into the tail vein of tumorbearing mice at a dosage of lOmg/kg. At this dose level there was no free dye in the plasma it was mostly bound to albumin, as confirmed by molecular sieve chromatography. These four pictures illustrate tumor tissue...

Z6. Zipf, R. E., Webber, J. M., and Grove, G. R., A comparison of routine plasma volume method using radioidinated human serum albumin and Evan s blue (T-1824) J. Lab. Clin. Med. 46, 800-805 (1955). 

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