The enhancement of interstitial transport of a doxorubicin-lactosaminated albumin conjugate by imatinib: In rat hepatocellular carcinoma it is not preferentially higher than that in liver and bone marrow

The finding that imatinib enhances the drug transport from bloodstream to neoplastic cells suggested a possible role of this drug as an adjuvant to the chemotherapeutics given in the treatment of solid malignancies. The present experiments aimed to verify whether imatinib can selectively increase the penetration of a doxorubicin-lactosaminated human albumin conjugate (L-HSA-DOXO) in chemically induced rat hepatocellular carcinomas (HCCs). We observed that imatinib increased the uptake of L-HSA-DOXO by HCCs but at the same time caused a similar enhanced penetration of the conjugate in liver and bone marrow. To our knowledge, this is the first demonstration that the enhancing effect of imatinib on interstitial drug transport is not restricted to the tumors, but can be also displayed in normal tissues. This observation casts some doubts about the possibility that the value of anticancer agents with toxic side effects on liver and bone marrow can be improved by imatinib.     

Doxorubicin coupled to lactosaminated human albumin: a hepatocellular carcinoma targeted drug

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide. There is a substantial need for new chemotherapeutic drugs effective against this tumor. Doxorubicin (DOXO), used for chemoembolization of HCCs, is poorly efficacious when administered systemically at conventional doses; dose escalation is hindered by unacceptable toxicity. Here, we review preclinical experiments showing that the efficacy of DOXO against HCCs and its safety increased following conjugation to lactosaminated human albumin (L-HSA). L-HSA-DOXO was initially prepared to improve the anticancer activity of the drug on well-differentiated HCCs, which actively internalize L-HSA by means of the asialoglycoprotein receptor. Unexpectedly, it was found that the conjugate enhanced DOXO concentrations in all forms of HCCs, independently of their differentiation grade.     

A novel method for coupling doxorubicin to lactosaminated human albumin by an acid sensitive hydrazone bond: synthesis, characterization and preliminary biological properties of the conjugate.

The expression of the asialoglycoprotein receptor on the cells of the large majority of the well differentiated hepatocellular carcinomas can be exploited to improve the chemotherapy of these tumours by coupling anticancer agents to macromolecules taken up by the receptor. In line with this approach, in previous experiments we coupled doxorubicin (DOXO) to lactosaminated human albumin (L-HSA) using the (6-maleimidocaproyl)hydrazone derivative of the drug as an acid sensitive linker. Encouraging results were obtained in laboratory animals using L-HSA-DOXO. This conjugate, however, has the disadvantage of a difficult synthesis, which requires protein thiolation with iminothiolane and can hinder its preparation on a large scale. Here we describe a very simple method of coupling. The HS-groups required for the reaction with the maleimide moiety of DOXO-EMCH are made available in L-HSA by a cleavage of the protein disulphides achieved with tris(2-carboxyethyl) phosphine (TCEP). Contrary to thiolic reducing agents, the use of TCEP eliminates the need of an inert atmosphere and allows a one-step coupling reaction, without purification of the reduced protein before the addition of DOXO-EMCH. As the previous L-HSA-DOXO conjugate, the new conjugate accomplishes a very efficient liver targeting of the drug. This novel method of synthesis should facilitate the preparation of L-HSA-DOXO in the amounts required for clinical studies.     

Doxorubicin coupled to lactosaminated albumin: effect of heterogeneity in drug load on conjugate disposition and hepatocellular carcinoma uptake in rats.

Coupling to lactosaminated human albumin (L-HSA) makes doxorubicin (DOXO) an effective drug against chemically induced rat hepatocellular carcinomas (HCCs). In the conjugate there is a large heterogeneity in the number of DOXO molecules bound to one L-HSA molecule. After lyophilization, the molecules with the higher DOXO load form large complexes (C-DOXOL), whereas those with low drug load (C-DOXOS) have the size of the carrier L-HSA. In the present experiments, we demonstrated that in C-DOXOL the molecules are not linked by covalent bonds, but are strongly aggregated probably because of mutual drug-drug interaction between the DOXO residues. In healthy rats and in animals with HCCs which received the same dose (1 microg/g) of DOXO injected in C-DOXOL or in C-DOXOS forms, penetration of the drug in tumors and in tissues was more rapid after administration of the former complex. Three hours after injection of both conjugate forms the intracellular release of DOXO from the carrier was completed. The AUCs from 0.5 to 4h of the levels of the released DOXO in HCCs, surrounding liver and bone marrow of animals injected with C-DOXOL were similar to those calculated in animals given C-DOXOS. This suggests that after administration of the dose of DOXO used in the present experiments the conjugate molecules with lower or higher drug load can exert comparable pharmacological and toxic effects.     

Zidovudine plus sulfamethoxazole-trimethoprim adversely affects B lymphocyte maturation in bone marrow of normal mice

Sulfamethoxazole-trimethoprim and zidovudine (AZT), drugs used often in combination in patients infected with HIV, were investigated for their effects on B cell development in a mouse model. BALB/c mice were randomized to receive oral doses of AZT, sulfamethoxazole-trimethoprim, or the combination via oral gavage for up to 28 days. Immune cell populations in the spleen, lung, and peripheral blood were examined, and toxicity to B lineage subtypes in the bone marrow was investigated by phenotypic analysis via flow cytometry. Pre-pro-B, pro-B, early pre-B, and late pre-B cells were assayed for apoptosis and analyzed for cell cycle profile. Total as well as B cell splenic and bone marrow cellularities were significantly decreased by using the drugs concomitantly, while B cell populations in the lungs and percentage in the peripheral blood were not affected. Combination therapy caused significant increases in apoptosis in B cells and granulocytes in the bone marrow, with the late pre-B cell population being the most depleted. The proliferative expansion and differentiation of early pre-B cells (B220+/CD43+/BP-1+/HSA+) to the late pre-B cell (B220+/CD43-/IgM-) stage was blocked, with early pre-B cells accumulating in the proliferative phases of the cell cycle. This apoptosis increase is likely due to elevated blood sulfamethoxazole concentrations that were observed in mice also receiving AZT. Concurrent sub-chronic administration of AZT and sulfamethoxazole-trimethoprim adversely affected B lymphocyte development in mouse bone marrow.     

Conjugate (MTC-220) of muramyl dipeptide analogue and paclitaxel prevents both tumor growth and metastasis in mice

1 (MTC-220), a conjugate of paclitaxel and a muramyl dipeptide analogue, has been synthesized as a novel agent of dual antitumor growth and metastasis activities. In vitro and in vivo tests show that 1 retains its ability to inhibit tumor growth. It is superior to paclitaxel in its ability to prevent tumor metastasis in Lewis lung carcinoma and 4T1-tumor-bearing mice. The present studies indicate that 1 suppresses myeloid derived suppressor cell accumulation in the spleen and bone marrow of tumor-bearing mice and also represses inflammatory cytokines in tumor tissue. These results demonstrated that 1 could be a potential therapeutic and preventive agent for cancer growth and metastasis.     

Low doses of Bisphenol A have pro-inflammatory and pro-oxidant effects,stimulate lipid peroxidation and increase the cardiotoxicity of Doxorubicin in cardiomyoblasts

Endocrine disrupters are strictly associated to cancer and several cardiovascular risk factors. Bisphenol A (BPA) is an endocrine disrupter commonly used in the manufacturing of plastics based on polycarbonate, polyvinyl chloride and resins. Our study aims to investigate whether BPA may cause pro-oxidative and pro-inflammatory effects on cardiomyoblasts, thus exacerbating the Doxorubicin (DOXO)-induced cardiotoxicity phenomena. We tested the metabolic effects of BPA at low doses analyzing its affections on the intracellular calcium uptake, oxidative stress, lipid peroxidation and production of nitric oxide and interleukins. Co-incubation of BPA and DOXO significantly reduced the cardiomyoblast viability, compared to only DOXO exposure cells. The mechanisms underlying these effects are based on the stimulation of the intracellular calcium accumulation and lipid peroxidation. Notably, BPA increase the production of pro-inflammatory interleukins involved in cardiovascular diseases as well as in DOXO-Induced cardiotoxicity phenomena. This study provides a rationale for translational studies in the field of cardio-oncology.     

Purpurinimide carbohydrate conjugates: effect of the position of the carbohydrate moiety in photosensitizing efficacy

A lactose moiety was regioselectively introduced at various positions of N-hexyl-mesopurpurinimide (a class of chlorin containing a fused six-membered imide ring system, lambda(max): 700 nm) to investigate the effect of its presence and position on photosensitizing efficacy. The resulting novel structures produced a significant difference in in vitro and in vivo efficacy. Among the positional isomers in which the lactose moiety was introduced at positions 3, 8, and 12, the 3-lactose purpurin-18-N-hexylimide produced the best efficacy. Compared to these analogues, the lactose moiety joined with an amide bond at position 17(2), and with an N-benzyl group bearing a -C[triple bond]C- linkage at position 13(2) showed reduced in vitro/in vivo photosensitivity. A noticeable difference between lactose conjugates in cell uptake (RIF tumor cells) was observed at 3 and 24 h postincubation. Replacing the lactose (Galbeta1 --> 4Glc) with beta-galactose and glucose moieties at position 3 of purpurinimide produced an increase in both cell uptake and in in vitro efficacy, but with reduced in vivo efficacy. Sites of intracellular localization differed among photosensitizers with and without carbohydrate moieties. Molecular modeling shows favorable interactions of 3- and 12-lactose-purpurinimide analogues with both galectin-1 and galectin-3, but clear contributions were not found for the conjugate containing lactose moiety at position 8. In a comparative ELISA study of the lactose conjugates with free lactose, all carbohydrate-purpurinimides showed binding to both galectins with a significant variation between the batches of galectins.     

Biotin/Folate‐decorated Human Serum Albumin Nanoparticles of Docetaxel: Comparison of Chemically Conjugated Nanostructures and Physically Loaded Nanoparticles for Targeting of Breast Cancer

Docetaxel (DTX) is a widely used chemotherapeutic agent with very low water solubility. Conjugation of DTX to human serum albumin (HSA) is an effective way to increase its water solubility. Attachment of folic acid (FA) or biotin as targeting moieties to DTX‐HSA conjugates may lead to active targeting and specific uptake by cancer cells with overexpressed FA or biotin receptors. In this study, FA or biotin molecules were attached to DTX‐HSA conjugates by two different methods. In one method, FA or biotin molecules were attached to remaining NH₂ residues of HSA in DTX‐HSA conjugate by covalent bonds. In the second method, HSA‐FA or HSA‐biotin conjugates were synthesized separately and then combined by DTX‐HSA conjugate in proper ratio to prepare nanoparticles containing DTX‐HSA plus HSA‐FA or HSA‐biotin. Cell viability of different nanoparticle was evaluated on MDA‐MB‐231 (folate receptor positive), A549 (folate receptor negative), and 4T1 (biotin receptor positive) and showed superior cytotoxicity compared with free docetaxel (Taxotere^®). In vivo studies of DTX‐HSA‐FA and DTX‐HSA‐biotin conjugates in BULB/c mice, tumorized by 4T1 cell line, showed the conjugates prepared in this study were more powerful in the reduction in tumor size and increasing the survival rate when compared to free docetaxel.     

Interaction of folate-conjugated human serum albumin (HSA) nanoparticles with tumour cells

Folic acid has been previously demonstrated to mediate intracellular nanoparticle uptake. Here, we investigated cellular uptake of folic acid-conjugated human serum albumin nanoparticles (HSA NPs). HSA NPs were prepared by desolvation and stabilised by chemical cross-linking with glutaraldehyde. Folic acid was covalently coupled to amino groups on the surface of HSA NPs by carbodiimide reaction. Preparation resulted in spherical HSA NPs with diameters of 239 ± 26 nm. As shown by size exclusion chromatography, 7.40 ± 0.90 μg folate was bound per mg HSA NPs. Cellular NP binding and uptake were studied in primary normal human foreskin fibroblasts (HFFs), the human neuroblastoma cell line UKF-NB-3, and the rat glioblastoma cell line 101/8 by fluorescence spectrophotometry, flow cytometry, and confocal laser scanning microscopy. Covalent conjugation of folic acid to HSA NPs increased NP uptake into cancer cells but not into HFFs. Free folic acid interfered with cancer cell uptake of folic acid-conjugated HSA NPs but not with uptake of folic acid-conjugated HSA NPs into HFFs. These data suggest that covalent linkage of folic acid can specifically increase cancer cell HSA NP uptake.     

Immunosuppressive effects of methylglyoxal-bis(guanylhydrazone) on mouse bone marrow and spleen cells and their antagonism by spermidine

Changes in immunological function and in the activity of S-adenosylmethionine decarboxylase were studied in bone marrow and spleen cells from mice immunized with sheep red blood cells and given multiple doses of methylglyoxal-bis(guanylhydrazone) (CH₃-G) or CH₃-G in combination with spermidine. Inhibition by CH₃-G was found in the complement-dependent and complement-independent immune responses of cells from both tissue sources; however, this inhibition was greater and more persistent in bone marrow than in spleen cells. Co-administration of spermidine with CH₃-G completely prevented this inhibition in the spleen at all the time points studied, whereas in the bone marrow prevention appeared later and was lower in magnitude. The increase in the activity of S-adenosylmethione decarboxylase, a key enzyme in spermidine biosynthesis, was greater in bone marrow than in spleen after the administration of CH₃-G. Co-administration of spermidine with CH₃-G consistently reduced this increase in enzyme activity in spleen but not in bone marrow. In addition to prevention, appropriate scheduling of spermidine subsequent to CH₃-G resulted in reversal of both the immunosuppression and the increase in S-adenosylmethionine decarbooxylase activity produced by CH₃-G in spleen cells.     

Effects of neopterin on the hematopoietic microenvironment of senescence-accelerated mice (SAM)

The pteridine neopterin (NP) is produced by monocytes and is known to be a useful marker of immunological activation, although, it remains elusive whether neopterin itself exhibits biological functions. Recently, we found that NP stimulates hematopoietic cell proliferation and differentiation by activating bone marrow stromal cell function. In order to elucidate the biological effect of NP on stromal cells, its effects on hematopoiesis was determined in the mouse model of age-related stromal impairment, senescence-accelerated mice (SAMs). An intraperitoneal administration of NP increased the number of peripheral leukocytes and CFU-GM in the bone marrow and spleen of young SAMs, however, no increase of CFU-GM in old SAMs (stromal impairment) was observed when compared with young SAMs. NP also increased the CFU-GM colony formation of bone marrow and spleen cells from young SAMs in a soft agar culture system, but it did not enhance CFU-GM colony formation of cells from old SAMs cultured in this system. Treatment with NP induced the production of hematopoietic stimulating factors, including IL-6 and GM-CSF, by bone marrow stromal cells from young SAMs but stromal cells from old SAMs did not respond to NP stimulation. Further studies will be required to clarify the mechanism by which NP stimulates the production of hematopoietic growth factors from stromal cells, the results of this study indicate that NP is a potent hematopoietic regulatory factor by activating stromal cell function(s).     

Conjugates of daidzein-alliinase as a targeted pro-drug enzyme system against ovarian carcinoma

Human ovarian cancer cells specifically bind the isoflavone daidzein. A chemical conjugate between daidzein and the garlic enzyme alliinase was prepared. The conjugate specifically bound to ovarian cancer cells and upon addition of the prodrug alliin, it effectively produced cytotoxic allicin molecules which killed the cancer cells. In vivo targeting and antitumor effect was confirmed by NIR and bioluminescence imaging using daidzein-alliinase-CyTE-777 conjugates and luciferase-expressing ovarian cancer cells. Co-localization of the fluorescent conjugate with bioluminescence was observed for intraperitoneal tumors while nonconjugated alliinase did not accumulate. Biodistribution studies with Europium-labeled conjugate revealed a five fold higher uptake in tumors as compared to other tissues. Treatment of tumor bearing mice with daidzein-alliinase and alliin effectively attenuated tumor progression during the first 12 days while a 5-fold increase in bioluminescence was detected in placebo-treated animals. Autopsy revealed only small individual foci of luminescence at the site of tumor cells inoculation. Histological examination of organs and tissues did not reveal any additional foci of carcinoma or signs of toxicity. These results suggest that the targeted alliinase conjugates in the presence of alliin, generated therapeutically effective levels of allicin which were capable of suppressing tumor progression of intraperitoneal ovarian cancer in an animal model.     

Selective uptake of a toxic lipophilic anthracycline derivative by the low-density lipoprotein receptor pathway in cultured fibroblasts

N-(N-Retinoyl)-L-leucyldoxorubicin 14-linoleate (r11-DOX), a new lipophilic derivative of doxorubicin, was synthesized and incorporated into low-density lipoprotein (LDL). The drug-LDL complex contained 100-200 drug molecules/LDL particle. When cultured normal human fibroblasts were incubated with 125I-LDL-incorporated drug, there was a perfect correlation between the cellular uptake plus degradation of 125I-LDL and the cellular drug accumulation. The presence of excess native LDL inhibited the cellular uptake and degradation of 125I-LDL and the drug accumulation to the same extent. In contrast, methylated LDL, which does not bind to the LDL receptor, did not alter the cellular uptake and degradation of 125I-LDL nor did it alter the drug accumulation. When LDL receptor negative fibroblasts from a patient with the homozygous form of familial hypercholesterolemia were incubated with the drug-125I-LDL complex, cellular drug accumulation was very low. The drug-LDL complex inhibited the growth of cultured normal human fibroblasts. The drug incorporated into methylated LDL was much less toxic. These findings suggest that r11-DOX incorporated into LDL is delivered to cells selectively by the LDL receptor pathway. This might be of value in the treatment of leukemia, since we have previously found that leukemic cells exhibit higher LDL receptor activity than white blood cells and bone marrow cells from healthy subjects.     

Plasma cell leukemia accompanied by polyclonal gammopathy and congenital tumor in the atrioventricular node

A 32-year-old man with plasma cell leukemia and congenital tumor of the atrioventricular node is described. Although M protein could not be detected by immunoelectrophoresis, the examination of bone marrow atypical plasma cells with immunofluorescent and immunoperoxidase showed that most of these plasma cells produced IgG or IgA. Also some plasma cells possessed concomitantly both IgG and IgA. An electrocardiogram revealed complete atrioventricular block. The patient died of aspergillosis and bleeding in the lung. Postmortem examination revealed the infiltration of atypical plasma cells in the bone marrow, spleen, liver and lungs, and infiltration of mesothelial tumor cells in the atrioventricular node.     

Selectivity in the effects of indomethacin on BCG-activated suppressor cell populations

Intravenous inoculation of BCG into C57 B1/6 mice activated natural suppressor cells in the bone marrow and induced suppressor cells in the spleen. The suppressor activity of these cell populations was determined by co-cultivating them with normal lymphocytes being immunized against allogeneic P815 cells in vitro. Six million cells from the spleen or bone marrow of BCG treated mice inhibited by more than 50% the alloimmunization of twenty million normal syngeneic lymphocytes. The suppressor cells were found in the nylon wool adherent population in spleen and in both the nylon wool adherent and non-adherent populations in bone marrow. Indomethacin, at a concentration of 10(-6)M, completely blocked the suppression generated by unfractionated spleen or adherent spleen from BCG treated mice. However, 10(-6)M and higher concentrations of indomethacin only partially blocked the suppression generated by unfractionated marrow or adherent marrow from BCG treated mice. The suppression generated by non-adherent marrow from BCG treated mice was completely insensitive to indomethacin.     

芪术口服液对环磷酰胺所致小鼠骨髓造血功能损伤的保护作用

目的:观察芪术口服液对环磷酰胺所致小鼠骨髓造血功能损伤的保护作用.方法:对小鼠腹腔注射100 mg/kg环磷酰胺后,观察芪术口服液对小鼠外周血象、骨髓有核细胞和胸腺、脾脏指数的影响.结果:芪术口服液能升高受损小鼠白细胞、红细胞、血小板、血红蛋白和骨髓有核细胞数量,并能使小鼠胸腺、脾脏指数升高.结论:芪术口服液对环磷酰胺引起的小鼠骨髓造血功能损伤有一定的保护作用.     

Very small superparamagnetic iron oxide nanoparticles: Long-term fate and metabolic processing in atherosclerotic mice

We investigated the biotransformation of very small superparamagnetic iron oxide nanoparticles (VSOP) in atherosclerotic LDLR^?/? mice. Transmission electron microscopy revealed an uptake of VSOP not only by macrophages but also by endothelial cells in liver, spleen, and atherosclerotic lesions and their accumulation in the lysosomal compartment. Using magnetic particle spectroscopy (MPS), we show that the majority of VSOP's superparamagnetic iron was degraded within 28?days. MPS spectrum shape indicated changes in the magnetic properties of VSOP during the biodegradation process. Experiments with primary murine bone marrow derived macrophages, primary murine liver sinusoidal endothelial cells, and primary human aortic endothelial cells demonstrated that loading with VSOP induced a differential response of cellular iron homeostasis mechanisms with increased levels of ferritin and iron transport proteins in macrophages and increased levels of ferritin in endothelial cells.     

Differential surface expression of CD18 and CD44 by neutrophils in bone marrow and spleen contributed to the neutrophilia in thalidomide-treated female B6C3F1 mice

Previously, we have reported that thalidomide (Thd) can enhance neutrophil function in female B6C3F1 mice. The present study was intended to evaluate the mechanisms underlying the enhanced neutrophil responses following Thd treatment intraperitoneally (100 mg/kg) for 14 or 28 days. Treatment with Thd increased the numbers of neutrophils in the spleen, peripheral blood, bone marrow, peritoneal cavity and lungs of female B6C3F1 mice when compared to the vehicle control mice. Thd treatment for 14 days increased the percentage and the number of neutrophils in the spleen in the first 8 h (peaking at 2 h) after the last Thd treatment, and it returned to the baseline after 24 h. However, Thd treatment for 28 days increased the percentage and number of neutrophils in the spleen even at the 24-h time point after the last Thd treatment. These neutrophils were demonstrated to be functional by the myeloperoxidase activity assay. Further studies have ruled out the possibility of an increased bone marrow granulopoiesis following Thd treatment. Flow cytometric analysis of the surface expression of adhesion molecules suggested that Thd treatment for either 14 or 28 days decreased the surface expression of either CD18 or CD44 by bone marrow neutrophils. On the other hand, the surface expression of both CD18 and CD44 by splenic neutrophils was increased following Thd treatment for 28 days but not for 14 days. No effect was produced for other cell surface molecules such as CD62L and CD11a. It was possible that decreased surface expressions of CD18 and CD44 facilitated neutrophils' release from the bone marrow; increased surface expressions of CD44 and CD18 by splenic neutrophils after 28 days of Thd treatment increased their ability to remain in the periphery. Taken together, Thd treatment increased neutrophils in female B6C3F1 mice, at least partially, through differentially modulating the surface expression of CD18 and CD44 by the neutrophils in the bone marrow and spleen.     

Effect of sinomenine on antibody responses in mice

Sinomenine, an epimorphinan alkaloid, was tested for the immunosuppressive effect in mice. This compound produced a decrease of plaque-forming cells (PFC) to a T cell-dependent antigen, sheep red blood cells, in vivo. The depression of the PFC response induced with sinomenine was dose and time dependent. On the other hand, it failed to suppress the PFC response to a T cell-independent antigen, lipopolysaccharide. The immunosuppressive dose of sinomenine did not alter the cellularity of spleen, thymus, bone marrow and peripheral blood leucocytes, the DNA synthesis activity of bone marrow cells nor the proliferative responses of spleen cells induced by T cell and B cell mitogens in unprimed mice. These data suggest a selective effect of sinomenine on lymphoid cells. This compound has a potential for use in studies of immuno-deficiencies or clarifying some aspect of immunity.