The in Vitro Characterization and Biostability of 99mTc-Dextran and Its Accumulation Within the Inflamed Paws of Adjuvant-Induced Arthritic Rats

The in vitro and in vivo stability in normal and adjuvant-induced arthritic rats of ^99mTc-dextrans (10, 40 and 500 kDa) have been investigated. The circulation half-lives were molecular weight dependent, with 10 and 40 kDa fractions being cleared relatively rapidly due to their ability to cross the glomerular basement membrane. The 500 kDa dextran was eliminated more slowly although 79% had been removed from the circulation 4 h post injection which probably was due to its degradation by dextranases and subsequent glomerular excretion. Dextran accumulation by the RES was found to be similar for all molecular weight preparations with no significant differences found. The sequestration of the dextrans by tissues of the RES (liver, spleen and lung) was independent of clearance rate. No differences were seen between normal and arthritic groups. Accumulation of the polymers by inflamed paws greatly exceeded that of normal paws for the 10 kDa (5-fold) and 500 kDa (6-fold) although no differences were seen with the 40 kDa dextran.     

Function of reticuloendothelial system on ethionine induced liver injury in mice

Phagocytic activity as a function of the reticuloendothelial system (RES) has been studied in ethionine induced liver injury by using the carbon clearance test. Liver damage in male and female mice was induced by DL- and L-ethionine injections (1000 mg/kg/day, i.p.). In both female and male mice, a single dose or three injections of DL- or L-ethionine caused increases in liver/body weight ratio, A/G ratio, GOT and GPT levels, and BSP retention. There was the decrease of the total protein levels in the serum. The degree of liver injury was more severe after three injections of DL- and L-ethionine than after a single injection of them. After a single injection of ethionine, the L-isomer induced a slightly greater response than the racemic mixture, except for BSP retention. On the other hand, phagocytic activities by the carbon clearance test were increased after a single injection or three injections of DL- and L-ethionine. That is, the K value was increased in all ethionine treated mice except for females with three injections of DL-ethionine. The alpha value was increased after three injections in DL- and L-ethionine treated males and DL-ethionine treated females. In addition, the increase in carbon uptake by Kupffer cells can be seen by light microscopy after a single injection or three injections of DL- or L-ethionine. These findings indicate that ethionine injections induce the enhancement of RES phagocytosis, although the biochemical parameters indicating liver injury are changed severely. These results support the data indicating no correlation between the alteration of RES activity and the degree of liver injury.     

Prediction of in vivo potential for metabolic activation of drugs into chemically reactive intermediate: correlation of in vitro and in vivo generation of reactive intermediates and in vitro glutathione conjugate formation in rats and humans

The covalent binding of reactive intermediates to macromolecules might have potential involvement in severe adverse drug reactions. Thus, quantification of reactive metabolites is necessary during the early stage of drug discovery to avoid serious toxicity. In this study, the relationship between covalent binding and glutathione (GSH) conjugate formation in rat and human liver microsomes were investigated using 10 representative radioactive compounds that have been reported as hepatotoxic or having other toxicity derived from their reactive intermediates: acetaminophen, amodiaquine, carbamazepine, clozapine, diclofenac, furosemide, imipramine, indomethacin, isoniazid, and tienilic acid, all at a concentration of 10 microM. The GSH conjugate formation rate correlates well with the covalent binding of radioactivity (both rat and human, r2 = 0.93), which suggests that quantification of the GSH conjugate can be used to estimate covalent binding. To quantify the GSH-conjugation rate with non-radiolabeled compounds in vitro, the validation study for the determination of GSH conjugate formation using 35S-GSH by radio-HPLC was useful to predict metabolic activation. Following oral administration of 20 mg/kg of the radiolabeled compounds to rats, radioactivity that covalently bound to plasma and liver proteins was determined. The in vivo maximum covalent binding level in liver based on the free fraction of plasma area under the concentration curve (AUC) and in vitro covalent binding rate was found to correlate well (r2 = 0.79). Therefore, this model for in vitro covalent binding studies in human and rat and in vivo rat studies might be useful in predicting human metabolic activation of compounds.     

Fasting increases the sensitivity of hepatic harmol glucuronidation to hypoxia.

Livers from fasted (N = 16) and fed (N = 22) rats were perfused with harmol (50 microM) for an initial 30 min with normal oxygen delivery (6-10 mumol/min/g liver), then for 45 min with perfusate equilibrated with O2/N2 mixtures, which reduced hepatic oxygen delivery to 0.9-6 mumol/min/g liver, and finally for a further 30 min period of normal oxygenation. Seventy per cent of the harmol eliminated was accounted for as the glucuronide conjugate and approximately 5% as the sulphate conjugate. During the hypoxia phase with fed preparations, decreasing oxygenation did not reduce harmol clearance or harmol glucuronide formation clearance until oxygen delivery was less than 2.5 mumol/min/g liver, whereas with fasted preparations this hypoxic threshold was much higher (5 mumol/min/g liver). Below the hypoxic threshold, harmol clearance was linearly related to oxygen delivery in both groups. Hepatic tissue concentrations of unchanged harmol at the end of the hypoxia phase were double those after the same period of normal oxygenation, whereas tissue harmol glucuronide concentrations were similar. By establishing a hypoxic threshold for reduced oxygen availability this study shows that harmol glucuronidation is relatively insensitive to hypoxia, but sensitivity increases markedly in fasted animals.     

Cell-polarity dependent effect of chelation on the paracellular permeability of confluent caco-2 cell monolayers

To investigate the effect of extracellular chelation at the apical, basolateral or both sides on the resistance and permeability of epithelial cell layers, we used 15 days cultures of a human intestinal adenocarcinoma cell line (Caco-2) and hydrophilic FITC-labeled dextran model compounds of various molecular weights. Transport of these hydrophilic compounds is restricted to the paracellular pathway in which the tight junctions form a barrier. Tight junctions are dependent on extracellular calcium and magnesium for their integrity and function. Calcium and magnesium chelation with 2.5 mM EDTA at the apical and basolateral side of the monolayer resulted in a drastic drop, up to 80% of the initial value, in trans-epithelial electrical resistance after 60 min. Application at the basolateral side resulted in a drop of 40% in resistance, while application on the apical side almost did not give any effect. The same pattern was also found in transepithelial clearance studies with fluorescein-Na and FITC-labeled dextran model compounds with molecular weights ranging from 4000 to 500 000. After 2.5 mM EDTA treatment on both sides a maximal (1400-fold) enhancement in transport clearance occurred for the dextran molecule with molecular weight 20 000 (Stokes-Einstein molecular radius 30 Å). For basolateral calcium and magnesium chelation similar results were found, however, with lesser maximal effects. For apical application no transport enhancement could be found with 2.5 mM EDTA. These results have shown that transport of hydrophilic compounds through epithelial monolayers is enhanced more effectively by basolateral application of EDTA than by apical application.     

Dextran-methylprednisolone succinate as a prodrug of methylprednisolone: plasma and tissue disposition.

Plasma and tissue disposition of a macromolecular prodrug of methylprednisolone (MP), dextran (70 kDa)-methylprednisolone succinate (DMP), was studied in rats. Single 5-mg/kg doses of DMP or unconjugated MP were administered into the tail veins of different groups of rats (n = 4/group/time point). Blood (cardiac puncture) and tissues (liver, spleen, kidney, heart, lung, thymus, and brain) were collected at various times after DMP (0-96 h) or MP (0-2 h) injections. Concentrations of DMP and MP in samples were analyzed by size-exclusion chromatography (SEC) and reversed-phase high-performance liquid chromatography (HPLC), respectively. Conjugation of MP with 70-kDa dextran resulted in 22-, 300-, and 30-fold decreases in the steady-state volume of distribution, clearance, and terminal plasma rate constant of the steroid, respectively. As for tissue distribution, the conjugate delivered the steroid primarily to the spleen and liver as indicated by 19- and 3-fold increases, respectively, in the tissue/plasma area under the curve (AUC) ratios of the steroid. On the other hand, the tissue/plasma AUC ratios of the prodrug in other organs were negligible. Active MP was released from DMP slowly in the spleen and liver, and AUCs of the regenerated MP in these tissues were 55- and 4.8-fold, respectively, higher than those after the administration of the parent drug. In contrast, no parent drug was detected in the plasma of DMP-injected rats. These results indicate that DMP may be useful for the targeted delivery of MP to the spleen and liver where the active drug is slowly released.     

Function of reticuloendothelial system on CCl4 induced liver injury in mice

Phagocytic activity as a function of the reticuloendothelial system (RES) has been studied in CCl4-induced liver injury by using the carbon clearance test. Liver damage in mice was induced by administration of 20% CCl4 in olive oil (p.o.). After a single administration of CCl4, significant increases in liver/body weight ratio, serum GOT and GPT levels, alpha, beta and gamma-globulins and BSP retention, and decreases in serum albumin, an activity of the hepaplastintest and the correct phagocytic activity, alpha value, were found. After 15 administrations of CCl4 (3 times a week), mild increases in serum GPT level and BSP retention and decreases in the activity of the hepaplastintest and both phagocytic indices, K and alpha values, were observed. However, zymosan treatment 3 days before sacrifice induced an increase in K value depressed by multiple administrations of CCl4. The depression of carbon uptake by Kupffer cells can be seen by light microscopy after multiple administrations of CCl4 compared with that of saline and olive oil. These findings indicate that the RES phagocytosis is suppressed more strongly in chronic liver injury by 15 CCl4 administrations than in acute injury by a single one, although the biochemical parameters indicating liver injury are shown to have an opposite tendency. A clear correlation between the alteration of RES activity and the degree of liver injury was not noted.     

Soluble dextran complexes of kallikrein. Bradykinin and enzyme inhibitors.

Pig pancreatic kallikrein, the protease inhibitor aprotinin (Trasylol), SQ 21541 (Arg-Pro-Gln-Ile-Pro-Pro, an angiotensin I converting enzyme or kininase II inhibitor), and bradykinin were each coupled covalently to soluble dextran with an average molecular weight of 500,000. Dextran had been activated either with cyanogen bromide (CNBr) or sodium meta-periodate (SMP). Of the reactants, 56 per cent of kallikrein, 35 per cent of aprotinin and 23 per cent of bradykinin had been bound to CNBr-activated dextran, while 38 per cent of SQ 21541 and 45 per cent of bradykinin had been bound to SMP-activated dextran. The activities of the complexes were determined in vitro. Kallikrein CNBr-dextran had 72 per cent of the esterase activity of non-coupled kallikrein. Aprotinin CNBr-dextran had 41 per cent of the kallikrein inhibitory activity of free aprotinin, and SQ 21542 SMP-dextran had 24 per cent of the converting enzyme inhibitory activity of the free peptide in vitro. The relative potencies of bradykinin CNBr-dextran and bradykinin SMP-dextran on the isolated rat uterus were 6 and 29 per cent those of native bradykinin. Their relative immunological potencies, however, were 92 and 80 per cent as determined by radioimmunoassay. Free and bound bradykinin inhibited the hydrolysis of hippuryl-glycylglycine by converting enzyme, but the coupled peptide inhibited less than the free kinin. Bradykinin coupled covalently to dextran was inactivated more slowly by converting enzyme than free bradykinin.     

Biliary excretion of FITC metabolites after administration of FITC labeled asialo orosomucoid as a measure of lysosomal proteolysis

An isolated perfused rat liver system was used to study the hepatic uptake and degradation of asialo orosomucoid (asialo alpha 1-acid glycoprotein). To this aim we coupled the fluorochrome FITC to the asialoglycoprotein. The covalent attachment of FITC to the glycoprotein did not affect its perfusate disappearance. The disappearance rate was characterized by a t1/2 approximately equal to 6.1 min, the clearance being 11.2 ml/min. The internalized ligand was probably extensively degraded in the lysosomes as demonstrated by the appearance of low molecular weight fluorescent compounds in the bile, having a higher fluorescence yield than the native conjugate. Lysosomal degradation of ASOR-FITC was shown to be the rate limiting step in FITC excretion into the bile. Treatment of a perfused liver with varying doses of the protease inhibitor leupeptin did not influence the perfusate disappearance rate of the protein. However, leupeptin inhibited the biliary output of FITC metabolites in a dose dependent fashion, half maximal inhibition occurring at 210 nM (in the perfusion medium), corresponding with a dose of 0.05 mg leupeptin per 10 g liver. It is concluded that the rate of lysosomal degradation of proteins in vivo can be determined by measuring the biliary excretion of fluorescent material originating from fluorescent probes covalently coupled to the particular protein.     

Self-immolative nitrogen mustards prodrugs cleavable by carboxypeptidase G2 (CPG2) showing large cytotoxicity differentials in GDEPT

Nineteen novel potential self-immolative prodrugs and their corresponding drugs have been synthesized for gene-directed enzyme prodrug therapy (GDEPT) with carboxypeptidase G2 (CPG2) as the activating enzyme. The compounds are derived from o- and p-amino and p-methylamino aniline nitrogen mustards. Their aqueous stability, kinetics of drug release by CPG2, and cytotoxicity in the colon carcinoma cell line WiDr, expressing either surface-tethered CPG2 (stCPG2(Q)3) or control beta-galactosidase, are assessed. The effect of various structural features on stability, kinetics of activation, and biological activity is discussed. The p-methylamino prodrugs are the most stable compounds from this series, with the largest cytotoxicity differentials between CPG2-expressing and nonexpressing cells. The most potent compounds in all series are prodrugs of bis-iodo nitrogen mustards. 4-[N-[4'-Bis(2' '-iodoethyl)aminophenyl]-N'-methylcarbamoyloxymethyl]phenylcarbamoyl-l-glutamic acid, compound 39b, is 124-fold more cytotoxic to WiDr cells expressing CPG2 than to cells expressing beta-galactosidase. An additional six compounds show better cytotoxicity differential than the published N-[4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl]-l-glutamic acid (CMDA) prodrug.     

Biodistribution and metabolism of immunostimulatory oligodeoxynucleotide CPG 7909 in mouse and rat tissues following subcutaneous administration

To evaluate pharmacokinetics (PK) and biodistribution, CPG 7909, a 24-mer immunostimulatory fully phosphorothioated oligodeoxynucleotide (PS-ODN), was administered by subcutaneous injection at 2, 5 and 12.5mg/kg to mice and at 9mg/kg to rats. Parent compound and metabolites were isolated from plasma and tissues and quantified by capillary gel electrophoresis with UV detection (CGE-UV) and molecular masses were determined by matrix-assisted-laser-desorption-ionization time of flight detection (MALDI-TOF). An established method for PS-ODN isolation from plasma and tissue was modified to prevent oxidation of the phosphorothioate bonds during the extraction process, significantly increasing sensitivity in the subsequent MALDI-TOF analysis. Concentrations of CPG 7909 and metabolites were highest at the injection site (>600mg/kg at 4h). Maximal concentrations in local (draining) lymph nodes (LLN), kidney and liver were 10-15% of that at the injection site. The highest total amount of PS-ODN (percentage of administered dose) was found in the liver (32% at 4h), followed closely by the injection site (23% at 4h). Only very low levels of CPG 7909 and metabolites were found in plasma and only during the first hours. Metabolites identified by MALDI-TOF were similar for both species and all analyzed tissues, although the relative amounts of the different metabolites varied with tissue and over time. Degradation of CPG 7909 in vivo occurred predominantly by 3'exonucleases with additional cleavage by endonucleases.     

Body distribution profile of polysaccharides after intravenous administration

Abstract

The body distribution of pullulan and dextran, which are nonionic polysaccharides, was studied. After intravenous injection of ¹²⁵I-labeled polysaccharides with different molecular weights, the half-life period in the blood circulation and the body distribution were pharmacokinetically investigated and compared with those of polyethylene glycol (PEG) and polyvinyl alcohol (PVA). Polysaccharides of higher molecular weights circulated in the bloodstream for a longer period than those of lower molecular weights. The half-life period of pullulan ranged from 15 to 90 min, changing remark-ably around molecular weight 30,000, and was shorter than that of dextran. Body distribution studies demonstrated that pullulan tended to accumulate in the liver to a greater extent than dextran, whereas PEG and PVA were hardly disposed there. This preferable accumulation of polysaccharides in the liver was observed over the whole molecular weight range studied. Eighty percent of pullulan with large molecular weights was localized in the liver without any accumulation in other organs, suggesting an inherent affinity of pullulan for the liver. In addition, the excretion clearance of pullulan and dextran decreased with an increase in molecular weight, although the clearance of pullulan was smaller than that of dextran over the high-molecularweight range. It is likely that the remarkable affinity of pullulan for liver causes a short half-life period in the circulation compared with that of dextran. These findings indicate that pullulan is promising as a polymeric carrier for drugs targeted to the liver.     

Covalent linkage of carboxypeptidase G2 to soluble dextrans--I. Properties of conjugates and effects on plasma persistence in mice

The covalent attachment of the therapeutic enzyme carboxypeptidase G2 to soluble dextrans of varying molecular weight resulted in a 5-15-fold increase in plasma persistence in normal and tumour-bearing mice. The molecular weight of the dextran used markedly affected the number of dextran molecules present in the conjugate, resulting in a molecular weight distribution between 6 and 12 X 10(5) daltons. The isoelectric point of the conjugates varied between 4.1 and 4.8 compared to native enzyme 7.8. Conjugates were resistant to proteolysis by trypsin and chymotrypsin, but showed little difference in their affinity for substrate.     

Significant differences in biological parameters between prodrugs cleavable by carboxypeptidase G2 that generate 3,5-difluoro-phenol and -aniline nitrogen mustards in gene-directed enzyme prodrug therapy systems

Nine new nitrogen mustard compounds derived from 2,6-difluoro-4-hydroxy- (3a-e) and 2,6-difluoro-4-amino- (4a-d) aniline were synthesized as potential prodrugs. They were designed to be activated to their corresponding 3,5-difluorophenol and -aniline (4)-nitrogen mustards by the enzyme carboxypeptidase G2 (CPG2) in gene-directed enzyme prodrug therapy (GDEPT) models. The compounds were tested for cytotoxicity in the MDA MB-361 breast adenocarcinoma. The cell line was engineered to express stably either CPG2 tethered to the cell surface stCPG2-(Q)3 or beta-galactosidase (beta-Gal) as control. The cytotoxicity differentials were calculated between CPG 2-expressing and -nonexpressing cells and yielded different results for the two series of prodrugs despite their structural similarities. While the phenol compounds are ineffective as prodrugs, their aniline counterparts exhibit outstanding activity in the tumor cell lines expressing CPG2. [3,5-Difluoro-4-[bis(2-chloroethyl)amino]phenyl]carbamoyl-l-glutamic acid gave a differential of >227 in MDA MB361 cells as compared with 19 exhibited by 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-l-glutamic acid, 1a, which has been in clinical trials.     

Studies on biliary excretion mechanisms of drugs—IV: Inhibitory studies on sulfobromophthalein and glucuronides in the rat

After the administration of 50μmoles sulfobromophthalein (BSP) or BSP glutathione conjugate (BSP-GSH) to rats, the maximal dye excretion was approximately 50 per cent higher for the latter as compared to the former. BSP or BSP-GSH and three glucuronides, thiamphenicol glucuronide (TPG). chloramphenicol glucuronide (CPG) and phenolphthalein glucuronide (PPG), depressed each other's bilary excretion. Competition between BSP or BSP-GSH and these glucuronides was not observed in the first phase of disappearance from blood. Transport of these drugs from blood to bile occurs rapidly. From these results, it can be inferred that in the transport of these drugs from blood to bile at least one common step is involved, but that this step is not the uptake from blood to liver. The binding affinity of these organic anions to hepatic cytoplasmic proteins was examined by Sephadex G-75 gel filtration and by ultraliltration. In the former experiment. BSP showed an apparent binding affinity to the cytosol fraction containing ligandin and Z protein. BSP-GSH showed a lower affinity than that of BSP. On the other hand, three glucuronides were not eluted with the fraction. In the latter experiment, 41 per cent of BSP was bound to the eytosol fraction, whereas only 10 per cent of BSP-GSH and PPG and none of CPG and TPG were bound. These results also suggest that the competition in biliary excretion observed between BSP or BSP-GSH and the glucuronides does not occur at the binding sites of ligandin or Z protein, and that binding to these proteins may not be required for the overall transport of these glucuronides from blood to bile.     

Development of age-dependent resistance to sindbis virus encephalitis: Correlation with inactivation of virus within the blood stream

The clearance of Sindbis virus from the blood stream and its localization in the reticuloendothelial system (RES) was studied in mice susceptible (2 weeks old) and resistant (6 weeks old) to fatal Sindbis virus encephalitis. No significant differences in the relative capacity of 2-week-old and 6-week-old mice to remove ¹²⁵I-labeled virus from the blood stream and to localize virus within the liver were observed. However, the decline of infectious virus was more rapid in the blood of adult mice. These studies suggest that, in addition to physical removal of virus by the RES, inactivation of virus in the blood stream plays an important role in limiting viremia during infections with Sindbis virus.     

Novel anti-HIV-1 activity produced by conjugating unsulfated dextran with polyL-lysine

A conjugate of polyL-lysine (PLL) with unsulfated dextran produced by reductive amination was found to have remarkable anti-HIV-1 activity against both the macrophage-tropic R5 virus Ba-L and T-cell line tropic X4 virus IIIB strains, although neither PLL nor dextran has such activity. The conjugate is a pseudoproteoglycan (pseudoPG) that simulates the structure of a proteoglycan. Conjugation with dextran was found to produce an antiviral effect in three kinds of assay systems including a human CD4(+) T-cell line, and the pseudoPG synthesized using 10 kDa PLL and 10 kDa dextran showed EC(50) 4-40 times lower than that of sulfated dextran or heparin against Ba-L and EC(50) equal to that against IIIB, indicating that PLL-dextran (PLL-Dex) was more effective against R5 virus than sulfated polysaccharides. PLL-Dex significantly suppressed a clinically isolated R5 virus from primary peripheral blood mononuclear cells. PLL-Dex interacted with the virus during adsorption to the cell and also decreased virus entry into the cell, suggesting PLL-Dex has multiple preventive mechanisms against HIV-1.     

Apoptosis of lymphocytes in allograft in a rat liver transplantation model induced by resveratrol.

AIM: To study the effect of resveratrol (RES) on the apoptosis of lymphocytes in allograft in a rat liver transplantation model. METHODS: Male Sprague-Dawley (SD) rats were selected as donors and male Wistar rats as recipients for a rejection model. The recipients were divided into four groups after orthotopic liver transplantation (OLTx). In the RES A, B, and C groups, RES was given intraperitoneally once a day (25, 50, and 100 mgkg(-1), respectively) after OLTx, whereas in the control group vehicle buffer was given intraperitoneally once a day. The survival period, lymphocytes apoptosis, expressions of Bcl-2/Bax proteins in lymphocytes, and histopathological findings were then compared among these groups. RESULTS: The mean survival period after OLTx in RES C group was significantly longer than that in control group (P < 0.05). On the seventh post-transplant day, the apoptosis index (AI) of lymphocytes in portal area and the positive rate of Bax protein in lymphocytes in RES C group were significantly increased in comparison with those in control group (both P < 0.05), whereas there is no obvious difference in the expression of Bcl-2 protein in lymphocytes between the control group and various drug groups (all P < 0.05), and a histological examination revealed apparent difference in the severity of rejection between the RES C group and control group (P < 0.05). CONCLUSION: RES has an immunosuppressive effect on lymphocytes under allograft rejection in rat. Inducing apoptosis of lymphocytes and upregulating the ratio of Bax/bcl-2 proteins in lymphocytes in allograft liver may be part of the mechanisms.     

Immunoconjugates of Soybean Bowman-Birk Protease Inhibitor as Targeted Antitumor Polymeric Agents

To enhance the antitumor potential of soybean Bowman-Birk inhibitor (BBI), the conjugate of BBI with an antibody via a macromolecular carrier was prepared. Clinical dextran (D) was used as a biocompatible biodegradable carrier for co-immobilization of BBI and antibody. A model immunoglobulin isolated from sheep serum (slgG), raised against human IgM was utilized to develop the procedure of immunoconjugate synthesis. The molar ratio of the ingredients in the conjugate was the following BBI:D:sIgG=9:1:1. Comparison of the dose response curves for the native sIgG and the BBI-D-sIgG conjugate indicated that sIgG completely retained its specific activity (>90%) after modification with dextran. The determination of the K_i, values for chymotrypsin interaction with the native BBI and the BBI-D-sIgG conjugate indicated high anti-chymotrypsin activity. In the next step, the monoclonal antibody (ICO 25 MAb) against the mucin-like human epithelial membrane antigen was used for conjugation as it is the most universal vector for targeting different agents to human tumors of epithelial origin. The influence of conjugation on the specificity of the Mab reaction with its antigen was studied. The conjugated MAb reacted with tumor cells of different epithelial genesis (breast, lung, gastric, ovarian and uterus tumors), but did not react with tumor cells of non-epithelial origin. It was shown that BBI-D-ICO 25 MAb conjugate has almost the same immunohistochemical activity as non-conjugated MAb. These results demonstrated the feasibility of exploiting the activities of covalently bound BBI and ICO 25 MAb for anticarcinogenic agent targeting.