Altered pharmacokinetics of a novel anticancer drug, UCN-01, caused by specific high affinity binding to alpha1-acid glycoprotein in humans

The large species difference in the pharmacokinetics/pharmacodynamics of 7-hydroxystaurosporine (UCN-01) can be partially explained by the high affinity binding of UCN-01 to human alpha1-acid glycoprotein (AGP) (Fuse et al, Cancer Res., 58: 3248-3253, 1998). To confirm whether its binding to human AGP actually changes the in vivo pharmacokinetics, we have studied the alteration in its pharmacokinetics after simultaneous administration of human AGP to rats: (a) the protein binding of UCN-01 was evaluated by chasing its dissociation from proteins using dextran-coated charcoal. The UCN-01 remaining 0.1 h after adding dextran-coated charcoal to human plasma or AGP was approximately 80%, although the values for other specimens, except monkey plasma (approximately 20%), were <1%, indicating that the dissociation from human AGP was specifically slower than from other proteins; and (b) the pharmacokinetics of UCN-01 simultaneously administered with human AGP has been determined. The plasma concentrations after i.v. administration of UCN-O1 with equimolar human AGP were much higher than those after administration of UCN-01 alone. The steady-state distribution volume and the systemic clearance were reduced to about 1/100 and 1/200, respectively. Human AGP thus reduced the distribution and elimination of UCN-01 substantially. On the other hand, dog AGP, which has a low binding affinity for UCN-01, did not change the pharmacokinetics of UCN-01 so much. Furthermore, human AGP markedly reduced the hepatic extraction ratio of UCN-01 from 0.510 to 0.0326. Also, human AGP (10 microM) completely inhibited the initial uptake of UCN-01 (1 microM) into isolated rat hepatocytes, whereas the uptake of UCN-01 was unchanged in the presence of human serum albumin (10 microM). In conclusion, the high degree of binding of UCN-01 to human AGP causes a reduction in the distribution and clearance, resulting in high plasma concentrations in humans.     

In vitro and in vivo properties of novel nucleoside transport inhibitors with improved pharmacological properties that potentiate antifolate activity.

The activity of antimetabolite inhibitors of de novo deoxyribonucleotide biosynthesis can be compromised by the salvage of extracellular preformed nucleosides and nucleobases. Dipyridamole (DP) is a nucleoside transport inhibitor that has been used clinically in an attempt to increase antimetabolite activity; however, DP binds tightly to the serum protein alpha1-acid glycoprotein (AGP) thereby rendering this therapeutic strategy largely ineffective. Four novel DP analogues (NU3076, NU3084, NU3108, and NU3121) have been developed with substitutions at the 2,6- and 4,8-positions of the pyrimidopyrimidine ring. The novel DP analogues inhibit thymidine (dThd) uptake into L1210 cells in vitro (NU3076 IC(50), 0.25 microM; NU3084 IC(50), 0.27 microM; NU3108 IC(50), 0.31 microM; NU3121 IC(50), 0.26 microM; and DP IC(50), 0.37 microM), but, unlike DP, their activity remains largely unaffected in the presence of 5 mg/ml AGP. The four DP analogues inhibit dThd and hypoxanthine rescue from Alimta (multitargeted antifolate)-induced growth inhibition in A549 and COR L23 human lung carcinoma cell lines in the presence of 2.5 mg/ml AGP, whereas the activity of DP is completely abolished. i.p. administration of 10 mg/kg NU3108, NU3121, and DP produced peak plasma concentrations of 4.4, 2.1, and 6.7 microM, respectively, and levels were sustained above 1 microM for approximately 45 min (DP) and 120 min (NU3108 and NU3121). [3H]thymidine incorporation into COR L23 xenografts grown in CD1 nude mice was reduced by 64% (NU3108), 44% (NU3121), and 65% (DP) 2 h after administration of the nucleoside transport inhibitors. In conclusion, two novel DP analogues (NU3108 and NU3121) have been identified that do not bind to AGP and that display superior pharmacokinetic profiles in comparison to DP and inhibit [3H]thymidine incorporation into human tumor xenografts in vivo.     

Chemotactic activity for mononuclear phagocytes of culture supernatants from murine and human tumor cells: Evidence for a role in the regulation of the macrophage content of neoplastic tissues

Culture supernatants from 14 mouse solid tumors, 2 mouse lymphomas, 8 human solid tumor lines and 3 human leukemia-lymphomas were examined for chemotactic activity in blind-well chemotaxis chambers using murine peritoneal macrophages and human blood monocytes as indicator cells. Culture supernatants from various murine and human solid tumors had appreciable chemotactic activity for mononuclear phagocytes. Chemotactic activity was found in murine tumors of different histology and transplantation history, including autochthonous mammary carcinomas and chemically-induced sarcomas. Chemotactic activity was not unique to neoplastic cells because is was detected also in supernatants of two preparations of mouse embryo fibroblasts and two human lung embryo fibroblast lines. Production of tumor-derived chemo-attractants did not require serum in the culture medium, was inhibited by the protein synthesis inhibitor Cycloheximide, but was unaffected by the DNA synthesis inhibitor Mitomycin C. Murine supernatants were active on human monocytes and vice-versa. A first preliminary characterization of the chemotactic activity of the human sarcoma line 8387 revealed that it was not dialyzable, that most of the activity was retained at 56deg;C for 30 min, but not at 100deg;C, and that it was susceptible to proteolytic enzymes (trypsin and proteinase K) but unaffected by DNase and RNase. Upon fractionation on a Sephadex G-75 column, the activity eluted in a single, relatively broad peak in the cytocrome C region, corresponding to an apparent molecular weight of about 12,000. In the heterogeneous series of murine solid tumors studied, a significant, though far from absolute, correlation (r=0.71, p=0.013) was found between chemotactic activity in culture supernatants (expressed as area under the chemotaxis titration curve) and the percentage of tumor-associated macrophages. It is suggested that tumor-derived chemotactic factor(s) play a role in the regulation of the macrophage content of neoplastic tissues.     

恶性阻塞性黄疸患者减黄术前后免疫功能变化的研究

检测１８例恶性阻塞性黄疸患者减黄术前１天，术后１、３、７、１４和２１天免疫功能变化另设２０例良性阻塞黄疸患者为对照。结果显示：总补体、补体Ｃ３增高，ｓＩＬ－２Ｒ术后先上升后下降，而后又再次上升，表明减黄术对改善恶性阻塞性黄疸患者免疫功能有效。     

Alpha1 acid glycoprotein binds to imatinib (STI571) and substantially alters its pharmacokinetics in chronic myeloid leukemia patients.

PURPOSE: Imatinib (Glivec) is a potent inhibitor of bcr/abl, an oncogenic fusion protein that causes chronic myelogenous leukemia (CML). alpha1 acid glycoprotein (AGP) binds to imatinib with high affinity and inhibits imatinib activity in vitro and in vivo in an animal model. A pharmacokinetics analysis of imatinib was undertaken in CML patients. EXPERIMENTAL DESIGN: Imatinib plasma concentrations were measured in 19 CML patients treated with imatinib (400 or 600 mg/day). Five patients received a concomitant short-term course of clindamycin (CLI). RESULTS: A positive correlation between AGP and imatinib plasma levels was observed. CLI administration decreased imatinib plasma concentrations, evaluated as area under the curve (AUC) and peak concentrations (C(max)). The effects of a bolus of CLI was studied in three patients on imatinib 23 h after the last imatinib dose. Within 5-10 min in three of three cases, CLI caused a decrease in imatinib plasma concentrations of 2.6-, 2.7-, and 4.7-fold, respectively. In vitro experiments using fresh blasts from CML patients showed that AGP, at concentrations observed in the patients, decreased imatinib intracellular concentrations up to 10 times and blocked imatinib activity. The incubation with CLI restored imatinib intracellular concentrations and biological activity. CONCLUSION: AGP exerts significant effects of the pharmacokinetics, plasma concentrations, and intracellular distribution of imatinib in CML patients; these data indicate that plasma imatinib levels represent unreliable indicators of the cellular concentrations of this molecule.     

alpha1-acid glycoprotein fucosylation as a marker of carcinoma progression and prognosis

BACKGROUND: Serum alpha1-acid glycoprotein (AGP), an acute-phase protein secreted by the liver, carries alpha(1,3)-fucosylated structures on its 5 highly branched, N-linked sugar chains. METHODS: Serum AGP levels in patients with various types of malignancies (n=214 patients) were measured using an enzyme-linked immunosorbent assay with anti-AGP antibody. To investigate glycoforms that differed in their degree of branching and extent of fucosylation, serum AGP samples were analyzed by crossed affinoimmunoelectrophoresis (CAIE) with concanavalin A, and Aleuria aurantia lectin (AAL), and anti-AGP antibody. RESULTS: A significant difference (P <0.001) in serum AGP levels was observed in preoperative patients compared with levels in the healthy control group, but the levels in individual patients did not reflect their clinical status. Conversely, it was found not only that the patterns of AGP glycoforms differed widely in the patient group compared with the healthy control group, but they also changed depending on each patient's clinical status. Furthermore, AGP glycoforms seemed to be appropriate markers of disease progression and prognosis according to follow-up studies of 45 patients during prolonged preoperative and postoperative periods. CONCLUSIONS: Patients with advanced malignancies who had AGP glycoforms that contained highly fucosylated triantennary and tetraantennary sugar chains for long periods after surgery were likely to have a poor prognosis. However, patients who had AGP glycoforms without such changes were expected to have a good prognosis.     

Release of a cytotoxic factor by macrophages stimulated with adjuvant-active peptidoglycans

Bacterial peptidoglycans (PG) possess various immunomodulating activities, including the ability to inhibit the growth of some experimental tumors. We report here that PG induce the release by mouse (DBA/2 and C3H/HeJ) peritoneal macrophages of a cytotoxic factor (CF) that is active on L-929 cells and a 3-methylcholanthrene-induced tumor cell line but inactive on normal fibroblasts. Only the adjuvant-active PG (extracted from Bacillus megaterium and Staphylococcus aureus) were good inducers of the CF, whereas the adjuvant inactive PG (extracted from Micrococcus lysodeikticus and Corynebacterium poinsettiae) exhibited only weak activity. The CF was released within 2 hours of contact with PG either in serum-free or serum-containing media. The CF was inhibited neither by serum nor by the protease inhibitors that have been tested. It was stable for 30 minutes at 56 degrees C but inactivated after being heated for 10 minutes at 80 degrees C. After gel filtration, a single peak of activity at 80-90 kilodaltons was found. Chromatofocusing showed that the isoelectric point of the CF was 4.8.     

Suppressive effect of 3-methylcholanthrene on phagocytic activity of mouse peritoneal macrophages for Torulopsis glabrata.

The effect of 3-methylcholanthrene (MCA) on the phagocytic activity of mouse peritoneal macrophages for Torulopsis glabrata was investigated. Macrophages were maintained in glass scintillation vials or on cover slips in Leighton tubes with the use of Hanks' balanced salt solution plus 30% horse serum. Graded amounts of MCA were incorporated into the medium and the macrophages were parasitized with viable cells of T. glabrata. Macrophages from C3H mice, a strain highly susceptible to MCA carcinogenesis, were more prone to the suppressive effect of MCA than were the macrophages from CFW mice, a relatively resistant strain. Significant suppressive effect on phagocytosis of macrophages from C3H mice was observed with 5 micrograms MCA/ml, whereas up to 50 micrograms MCA/ml did not alter the phagocytic activity of CFW macrophages. However, 100 micrograms MCA/ml also suppressed the phagocytosis of CFW macrophages. Suppression in phagocytosis of C3H macrophages was observed after 6 hours' exposure to MCA, whereas a similar effect on CFW macrophages was seen after 12 hours. Treatment with 100 micrograms MCA/ml imparied the fungicidal activity of both C3H and CFW macrophages. These results indicate a correlation between the suppressive effect of MCA on macrophage activity and the strain susceptibility of mice to chemical carcinogenesis.     

Plasma protein profiles and prognosis in gastric cancer.

In 104 patients with gastric cancer the serum proteins carcinoembryonic antigen (CEA), C-reactive protein (CRP), alpha 1-acid glycoprotein (AGP) (orosomucoid) and alpha 1-antichymotrypsin (ACT) were measured pre-operatively. The estimated median survival of patients with both raised CEA and ACT was only 5 weeks in contrast to 64 weeks for those with both proteins normal. An intermediary group with one of these proteins raised and the other normal had an estimated median survival of 15 weeks. Similar results were obtained by considering a combination of CEA with either AGP or CRP. For these data the results were not explicable in terms of associations between survival time and patient''s age, stage, operative procedure, histological classification or site of primary tumour.     

Pseudouridine determination in blood serum as tumor marker

A new method to measure pseudouridine (psi rd) in blood serum has been used to study patients with cancer of breast, lung, colon, and ovaries, and patients with hepatomas, non-Hodgkin lymphomas, and acute lymphoblastic leukemias, at different stages. The results indicate: (1) a significant (p less than 0.01) difference between the psi rd serum level of the cancer patient group and the normal control group; (2) that the increase of the level of serum psi rd is correlated both to the stage of the disease and to the spreading of the tumor tissue; (3) a correlation between the response to therapy and the behaviour of psi rd serum levels.     

Immune complexes, serum proteins, cell-mediated immunity, and immune regulation in patients with squamous cell carcinoma of the head and neck

A collaborative study of the humoral and cellular immune status of patients with carcinoma of the Head and Neck (H&N) was conducted at the West Virginia University (WVU) hospital. In addition, blind-coded serum panels were supplied on H&N cancer patients being treated at the National Cancer Institute (NCI). Serum protein analysis of the WVU study groups revealed that at the pretreatment sampling, the alpha-1 acid glycoprotein (AGP), total complement, and IgA levels were significantly elevated. The AGP levels and total complement levels declined to normal levels in the post-treatment period, whereas the IgA levels remained elevated throughout the entire observation period. Levels of serum immune complexes (SIC) were measured in both the WVU and NCI H&N cancer populations using the polyethylene glycol (PEG) precipitation method. In both survey populations all cancer groups had significantly elevated levels of SIC when compared to any of the control populations. The SIC levels never returned to comparative normal values even in cases after successful treatment. A subpopulation of the WVU-H&N cancer study group underwent a short course of intravenous hyperalimentation prior to their treatment regimen. These patients demonstrated a transient decrease in their SIC levels as well as a concomitant increase in their in vitro cell-mediated immune (CMI) correlates. The analysis of in vitro CMI correlates of the WVU study group using both polyclonal mitogens and specific antigens demonstrated a significant depression in these parameters pretreatment and post-treatment. In addition, it was observed that the time course for elevation of selected serum proteins (i.e., IgA and SIC) correlated with concomitant drops in CMI activity. Investigations were also conducted into the effects of immune complex-rich serum fractions upon selected in vitro CMI correlates. Significant blockage of a normal donor leukocyte migration-inhibition assay was demonstrated. Also, a similar inhibition of the ability of normal human lymphocytes to form high affinity rosettes was accomplished with serum from H&N cancer patients.     

Induction of prostaglandin production by hyperthermia in murine peritoneal exudate macrophages

In response to inflammatory stimuli, macrophages synthesize and secrete prostaglandins (PGE) along with other potent inflammatory mediators. We have studied the effect of hyperthermia on the production of PGE by murine mononuclear phagocytes. Exposure to high temperature induced PGE production by cultured C3H/HeJ exudate macrophages in a time- and temperature-dependent manner. Increase in PGE production was detected when macrophages were treated at 41 degrees C and above for 1 h with a much greater increase at 42 and 43 degrees C. The secretion of PGE into culture supernatants by heat-treated macrophages reached a maximum approximately 24 h after heat treatment. The production of PGE by macrophages after hyperthermia was inhibited either by the addition of 5 X 10(-7) M indomethacin or by the subsequent incubation at 4 degrees C, suggesting that the elevated PGE production by macrophages is mediated through the activation of cyclooxygenase. Heat treatment under the same conditions failed to stimulate the production of PGE by either a human monocyte-like tumor cell line (U-937) or a mouse fibroblast cell line (L-929).     

Radiation leukemia virus (RadLV)-induced leukemogenesis is associated with an increased number and activity of thymic macrophages.

The radiation leukemia virus (RadLV) is a chronic leukemia retrovirus that induces thymic lymphomas in C57BL/6 mice after a latency of 3 to 6 months. During the pre-leukemic (PL) period, the number of thymic macrophages gradually increased up to 100 fold. Of the cells in a RadLV-induced lymphoma, 0.3% were large macrophages packed with infected lymphoma cells. These thymic lymphoma macrophages (TLM) also ingested RadLV-induced lymphoma cells in vitro. Cultured RadLV-induced lymphoma lines could activate and fix C3 fragments through the alternative complement pathway (ACP). C3-bound lymphoma cells elicited an oxidative burst (OB) response in TLM but not in bone-marrow macrophages (BMM). However, IL4 treatment of BMM rendered them capable of responding with an OB following triggering by C3-opsonized cells. Thymic macrophages (TM) responded moderately with OB to C3-opsonized cells and this response was elevated if the TMs were treated by rIL4. The OB reaction of the TLMs could be partially inhibited by anti-LFA-I or anti-MALA-2 antibodies, and was completely inhibited by anti-CR3 antibodies. These results suggest that IL4 can prime macrophages for triggering an OB reaction and that the interaction between C3-opsonized cells and IL4-primed macrophages is mediated primarily through CR3.     

Differential induction of suppressor macrophages by cloned Lewis lung carcinoma variants in mice

The splenic T-lymphocyte blastogenic and natural killer cell (NK) activities of C57BL/6 mice bearing cloned metastatic C3 or nonmetastatic C8 variants of the Lewis lung carcinoma were suppressed. The suppression was greater in mice bearing the metastatic C3 tumors than the nonmetastatic C8 tumors, although primary tumor sizes were similar. Macrophages were shown to cause this differential in immune responsiveness, while depletion of splenic macrophages by adherence restored the T-cell and NK responses. Also, splenic macrophages from C3 tumor bearers were more suppressive to normal spleen cell activities than were splenic macrophages from C8 tumor bearers. The suppression by the macrophages of C3 bearers was indomethacin sensitive and was associated with an increased secretion of prostaglandin E2 (PGE2). Normal macrophages incubated with C3 culture supernatants were more suppressive to NK and T-cell activities and secreted more PGE2 than did macrophages incubated with the C8 supernatants or with medium. This finding suggested that the immune suppression in mice bearing C3 tumors was initiated by a soluble tumor factor(s) that stimulated the development of prostaglandin-dependent suppressor macrophages. An in vivo study examined if treating C3-bearing mice with indomethacin to prevent the prostaglandin-dependent macrophage suppressor activity would influence host survival. The survival time of C3-bearing mice treated with indomethacin was prolonged. These results suggest that the macrophage-mediated immune suppression induced by tumor cells may facilitate tumor growth and metastasis.     

Quantitative viremia test for early prediction of a biochemical response in patients with chronic hepatitis C treated with interferon

To evaluate the importance of the changes in viremia as an early predictor of the outcome of interferon (IFN) therapy, we assayed the levels of hepatitis C virus (HCV)-RNA in stored serum samples obtained from 34 patients with chronic hepatitis C who showed different biochemical responses to therapy. Serum samples obtained before the start of therapy and after 1 and 3 months were used, and viremia levels were determined by "branched DNA (bDNA)" technique. Viremia levels at 1 month of therapy were lower than pre-therapy levels in all 19 patients who had shown a persistent normalization of ALT during therapy (responder patients). The bDNA test was negative, i.e. the levels of viremia were below the sensitivity threshold of the method, in 12 (63.1%) patients at 1 month and in 13 (68.4%) at the 3rd month of therapy, whereas the bDNA test was negative in none of the 15 non-responder patients at the 1st month and in only 2 (13.3%) of them at the 3rd month of therapy. The bDNA test was superior to the ALT test both in predicting the non-response and the biochemical response to IFN after 1 month of therapy. The bDNA test results, instead, were not predictive of the duration of the response to IFN, either at the 1st or 3rd months of therapy. These results seem to indicate the usefulness of measuring the HCV-RNA levels at the beginning and after 1 month of IFN therapy in order to envisage or exclude a possible biochemical response early on in treatment.     

基于液质联用的人血清代谢组学样品前处理方法研究

目的 建立一种稳定、可靠且适合大样本分析的血清代谢组学前处理方法.方法 采用超高效液相色谱-四级杆飞行时间串联质谱(ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry,UPLC-QTOF-MS)...     

Prognosis and lung-cancer - the contribution of plasma-proteins

In this prospective study we aimed at assessing the potential prognostic significance of the serum protein content. We studied 388 consecutive patients, newly diagnosed for lung cancer during the last 6 years, by measuring the serum concentration of immunoglobulins (IgG, IgA, IgM), transferrin (T), haptoglobin (HP), alpha-1-acid glycoprotein (AGP), alpha-1-antitrypsin (AT) and alpha-2-macroglobulin (MG). In all patients, the values of other 9 clinical variables (age, sex, weight loss, ECOG performance status, stage- of disease, TNM factors and histology) were also available. Univariate analysis showed that patients with transferrin above 218 mg/dl had a median survival of 9 months, as compared to the 7 months for the remaining subjects (p<0.01). Patients with AT above 281 mg/dl had a significantly shorter survival than patients with low AT (11 months vs 6 months, p<0.01). High AP and AGP were also significantly associated with a poor prognosis (p<0.01 and 0.05 respectively). A multivariate analysis of survival (the Cox's proportional hazards regression analysis), selected, in decreasing order of significance, the following variables: (i) stage of disease; (ii) ECOG performance status; (iii) sex; (iv) alpha-1-acid glycoprotein; (v) weight loss; (vi) histology. We conclude that serum proteins (particularly AGP, T and AT) do have some prognostic significance in lung cancer.     

Apoptosis induced by ultraviolet B irradiation of leukemia cells and macrophages

In this study, we examined the effects of ultraviolet-B (UV-B) irradiation on a variety of cultured cells including human (HL-60, U937) and mouse (MI, WEHI3B/D) leukemia cells, and mouse macrophages (P388-D1, J744). UV-B irradiation for 15 min variably induced apoptosis in all of the cell lines examined, except for J744. Apoptosis was apparently prevented by the treatment of a panel of anti-oxygen reagents. WEHI3B/D, M1 and HL-60 induced cell differentiation showed delayed induction of apoptosis. Protein kinase C and bcl-2 protein expression did not change during the process of apoptosis. These results indicate that (1) UV-B irradiation induces apoptosis of leukemia cells and macrophages via direct and/or indirect DNA damage, and (2) cell differentiation results in less frequent apoptosis.     

Augmented uptake of neuraminidase-treated sheep red blood cells: participation of opsonic factors.

Neuraminidase from Vibrio cholerae (VCN) was used to treat sheep red blood cells (SRBC) which were then incubated in vitro with murine peritoneal macrophages. The uptake of VCN-treated SRBC by macrophages was greater than the uptake of SRBC not treated with VCN. SRBC opsonized with normal mouse serum (NMS) were taken up to a greater extent than untreated SRBC. SRBC treated with VCN and opsonized with NMS were phagocytosed to a greater extent than untreated SRBC, VCN-treated SRBC, or opsonized SRBC. Evidence demonstrated that factors in serum from normal C3H/HeJ mice augmented the uptake of VCN-treated SRBC in greater amounts than of normal SRBC. These findings were discussed in relation to the increased immunogenicity of neuraminidase-treated cells.     

Macrophage-induced cytotoxicity and anti-metastatic activity of a 43-kDa human urinary protein against the lewis tumor

Resident, inflammatory or bone-marrow macrophages from C57BI/6 mice incubated in vitro with a pure human urinary protein (HGP.43) decreased the growth rate of Lewis tumor cells (3LL). This inhibition of 3LL growth was the result of a cytotoxic activity of these macrophages which was independent of oxygen metabolites and nitrous oxide. Murine monoclonal antibodies (MAbs) against HGP.43 inhibited macrophage-mediated cytotoxicity. This cytotoxic activity was not due to the release of cytotoxic factors in the culture supernatant, showing that a contact between macrophages and tumor cells was required to express cytotoxicity. The presence of HGP.43 was absolutely necessary during the incubation of macrophages with target cells. In vivo, in HGP.43-treated mice, the growth of the primary tumor was not delayed but the size and number of lung metastases were significantly reduced 21 days after tumor inoculation.