Preparation of Internalizing RGD-Modified Recombinant Methioninase Exosome Active Targeting Vector and Antitumor Effect Evaluation.

BACKGROUND/AIMS: Targeted drug delivery vehicles with low immunogenicity and toxicity are needed for cancer therapy. Here, we prepare an active targeting drug carrier of low immunogenicity and toxicity for targeted therapy. METHODS: Immature dendritic cells (imDCs) from BALB/c mice were used as donor cells of exosomes (Exos) that were transfected with the plasmids expressing fusion proteins of a tumor-targeting peptide known as internalizing RGD (iRGD) to construct a type of tumor-targeting iRGD-Exos and observe the interaction between these iRGD-Exos. Also, recombinant methioninase (rMETase) was loaded into the iRGD-Exos by electroporation to construct iRGD-Exos-rMETase and to assess the tumor-targeting function of the iRGD-Exos-rMETase. Finally, 30 BALB/c were randomly divided into five groups (n = 6), to observe tumor growth in vivo. RESULTS: The iRGD-Exos-rMETase was 99.58 nm in diameter and presented a unique "goblet" structure under transmission electron microscopy (TEM), with the encapsulation efficiency (EE) of 19.05%. iRGD-Exos-rMETase group has the strongest tumor suppressive effect. Compared to the iRGD-Exos-rMETase group, rMETase group and the blank-Exos-rMETase group were less effective, while the PBS group and the iRGD-Exos group showed no inhibitory effect on tumor growth. After treatment, the iRGD-Exos-rMETase group had gastric tumors significantly smaller and lighter than the other groups (P < 0.05). CONCLUSION: The iRGD-Exos-rMETase is an effective antitumor therapy that delivers rMETase to tumor tissue using the iRGD-Exos. With its favorable inhibitory effect and tumor-targeting function, the iRGD-Exos-rMETase shows excellent potential value and exciting prospects in clinical applications.

Diagnostic pitfalls of drug-induced immune hemolytic anemia.

Immune hemolytic anemia (IHA) is a rare complication of drug administration. However, its true incidence remains obscure, as there are a number of factors that may lead to misdiagnosis. The clinical and serologic pictures are variable, and there is a great deal of unawareness that certain drugs can cause IHA. Furthermore, serologic results can be easily misinterpreted, resulting in a wrong diagnosis.

Effects of 5-fluorouracil chemotherapy on fatigue: role of MCP-1.

Chemotherapy has been known to cause severe side effects, including fatigue. While the mechanisms for chemotherapy induced fatigue (CIF) are likely to be multi-factorial in origin, it is thought that inflammation and anemia may play a role. The purpose of this study was to examine the effect of chemotherapy on fatigue in mice, and further, to begin to determine if inflammation and anemia may contribute to this response. For experiment 1, C57BL/6 mice were assigned to: vehicle (PBS), low (20 mg/kg), medium (40 mg/kg), or high (60 mg/kg) doses of 5-fluorouracil (5-FU). Voluntary physical activity (PA) was measured throughout the treatment period (day 1-5) as well as during the recovery period (day 6-14). In experiment 2, we examined the effects of 5-FU (60 mg/kg) on the inflammatory mediator MCP-1 and on markers of anemia (RBC, Hct and Hb). Finally, using MCP-1(-/-) mice we examined the role of MCP-1 on CIF (experiment 3). 5-FU reduced voluntary PA in a dose response manner (p<0.05). Plasma MCP-1 was increased following 5-FU treatment on both days 5 (p=0.10) and 14 (p<0.05). In addition, RBCs, Hct and Hb were reduced with 5-FU on days 5 and 14 (p<0.05). Both C57BL/6 and MCP-1(-/-) mice saw similar decrements in PA through the duration of the treatment period (days 1-5), however the MCP-1(-/-) mice recovered much earlier than wildtype mice. This study provides evidence of the dose response effect of a standard chemotherapy agent on fatigue and demonstrates a potential role of MCP-1 and presumably inflammation, and anemia.

Fatal immune haemolysis due to antibodies to individual metabolites of 5-fluorouracil.

Confusion still exists in the diagnosis of drug-induced immune haemolysis (DIH). The aim of this study was to demonstrate antibodies specific to 5-fluorouracil (5-FU) in a patient with fatal immune haemolysis (IH). The case of a patient who died due to protracted IH is described. A 57-year-old female underwent treatment with oxaliplatin, 5-FU and folinic acid due to cholangiocarcinoma. Following drug administration, she was transfused because of a mild non-haemolytic anaemia and died following haemolysis. Serological testing including antibody screening, direct antiglobulin test and detection of drug-dependent antibodies was performed using standard techniques. The patient's serum was observed to be red in colour due to the presence of free haemoglobin prior to and following blood transfusion, and contained antibodies reactive with RBCs only in the presence of urine from several patients treated with 5-FU (ex vivo antigens). Drug-induced immune haemolysis (DIH) and metabolite-dependent antibodies should always be taken into consideration when a patient being administered any type of drug develops haemolysis.

Mercaptopurine-induced fever: hypersensitivity reaction in a patient with acute lymphoblastic leukemia.

The antimetabolite mercaptopurine is commonly used as part of treatment regimens for acute lymphoblastic leukemia and as treatment for inflammatory bowel diseases. Adverse effects associated with mercaptopurine include myelosuppression, hepatotoxicity, and hyperpigmentation. We describe a 36-year-old man with Philadelphia chromosome-negative pre-B-cell acute lymphoblastic leukemia who experienced a serious mercaptopurine-induced hypersensitivity reaction requiring prolonged hospitalization and extensive laboratory testing and imaging. He was treated with a multiagent chemotherapy regimen. Mercaptopurine 60 mg/m(2)/day orally was started as part of his third course of chemotherapy. On day 9 of mercaptopurine therapy, the patient developed persistent fevers, skaking chills, and rigors that persisted in the absence of documented infection, consistent with drug fever. All symptoms and signs resolved after discontinuation of mercaptopurine. Use of the Naranjo adverse drug reaction probability scale indicated a probable relationship between the patient's development of fever and mercaptopurine therapy. Mercaptopurine-induced fever has been reported in patients with inflammatory bowel diseases, but this case report is the first, to our knowledge, in a patient with acute lymphoblastic leukemia. Health care professionals should be aware of the possible development of fever as a hypersensitivity reaction in patients with acute lymphoblastic leukemia treated with mercaptopurine.

Pharmacogenomic studies of the anticancer and immunosuppressive thiopurines mercaptopurine and azathioprine.

AIMS: To examine the allelic variation of three enzymes involved in 6-mercaptopurine/azathioprine (6-MP/AZA) metabolism and evaluate the influence of these polymorphisms on toxicity, haematological parameters and metabolite levels in patients with acute lymphoblastic leukaemia (ALL) or inflammatory bowel disease (IBD). METHODS: Clinical data and blood samples were collected from 19 ALL paediatric patients and 35 IBD patients who were receiving 6-MP/AZA therapy. All patients were screened for seven genetic polymorphisms in three enzymes involved in mercaptopurine metabolism [xanthine oxidase, inosine triphosphatase (C94-->A and IVS2+21A-->C) and thiopurine methyltransferase]. Erythrocyte and plasma metabolite concentrations were also determined. The associations between the various genotypes and myelotoxicity, haematological parameters and metabolite concentrations were determined. RESULTS: Thiopurine methyltransferase variant alleles were associated with a preferential metabolism away from 6-methylmercaptopurine nucleotides (P = 0.008 in ALL patients, P = 0.038 in IBD patients) favouring 6-thioguanine nucleotides (6-TGNs) (P = 0.021 in ALL patients). Interestingly, carriers of inosine triphosphatase IVS2+21A-->C variants among ALL and IBD patients had significantly higher concentrations of the active cytotoxic metabolites, 6-TGNs (P = 0.008 in ALL patients, P = 0.047 in IBD patients). The study confirmed the association of thiopurine methyltransferase heterozygosity with leucopenia and neutropenia in ALL patients and reported a significant association between inosine triphosphatase IVS2+21A-->C variants with thrombocytopenia (P = 0.012). CONCLUSIONS; Pharmacogenetic polymorphisms in the 6-MP pathway may help identify patients at risk for associated toxicities and may serve as a guide for dose individualization.

The DNA demethylating agent 5-aza-2'-deoxycytidine activates NY-ESO-1 antigenicity in orthotopic human glioma.

Cancer/testis antigens (CTAs) are considered to be suitable targets for the immunotherapy of human malignancies. It has been demonstrated that in a variety of tumors, the expression of certain CTAs is activated via the demethylation of their promoter CpG islands. In our study, we have shown that while the composite expression of 13 CTAs in 30 human glioma specimens and newly established cell lines from the Japanese population was nearly imperceptible, the DNA-demethylating agent 5-aza-2'-deoxycytidine (5-aza-CdR) markedly reactivated CTA expression in glioma cells but not in normal human cells. We quantified the diminished methylation status of NY-ESO-1-one of the most immunogenic CTAs-following 5-aza-CdR treatment by using a novel Pyrosequencing technology and methylation-specific PCR. Microarray analysis revealed that 5-aza-CdR is capable of signaling the immune system, particularly, human leukocyte antigen (HLA) class I upregulation. (51)Cr-release cytotoxicity assays and cold target inhibition assays using NY-ESO-1-specific cytotoxic T lymphocyte (CTL) lines demonstrated the presentation of de novo NY-ESO-1 antigenic peptides on the cell surfaces. In an orthotopic xenograft model, the systemic administration of 5-aza-CdR resulted in a significant volume reduction of the transplanted tumors and prolonged the survival of the animals after the adoptive transfer of NY-ESO-1-specific CTLs. These results suggested that 5-aza-CdR induces the expression of epigenetically silenced CTAs in poorly immunogenic gliomas and thereby presents a new strategy for tumor immunotherapy targeting 5-aza-CdR-induced CTAs.

Activity and toxicity of 2-CDA in Langerhans cell histiocytosis: a single institutional experience.

BACKGROUND: Langerhans cell histiocytosis (LCH) is a rare disorder characterized by clonal proliferation of immature and abnormal bone marrow derived langerhans cells. Treatment is usually multimodal. Potent anti-monocyte as well as immunomodulatory activity of 2-CDA and its proven efficacy in many lymphoproliferative disorders has made 2-CDA a rational choice in treatment of LCH. AIM: To evaluate the efficacy and toxicity profile of 2-CDA in children with relapsed or refractory LCH. SETTING AND DESIGN: This is a pilot study and we present the initial data of the first seven patients treated at our institution. MATERIALS AND METHODS: Seven patients of relapsed and refractory LCH were enrolled from July 2000 to June 2004. The cohort of seven patients included six males and one female with a median age at initiation of cladribine was 2.25 years (range, 1.67 to 7.0 years). Three patients had received one prior chemotherapy regimen while the rest were heavily pretreated. Cladribine was administered over two hours IV daily for five days and repeated every four weeks. RESULTS: After a median of six courses of cladribine (range, 2 to 9), two (33%) patients achieved PR and two (33%) patients have SD on imaging but are clinically better. None experienced grade 3 or 4 hematologic toxicity. At a median follow-up of 19 months (range, 8 to 52 months), five patients remain alive and one patient has died. CONCLUSION: Our study shows that single agent 2-CDA is active and well-tolerated in children with relapsed or refractory LCH.

Application of anti-methotrexate Fab fragments for the optimization of intraperitoneal methotrexate therapy in a murine model of peritoneal cancer.

Anti-drug antibodies may be used to impart regio-specific alterations in drug disposition, potentially enhancing the therapeutic selectivity of intracavitary chemotherapy. In the present study, we tested the hypotheses that systemic therapy with anti-methotrexate antibodies would allow increases in the maximum tolerated dose of intraperitoneal methotrexate (MTX) and allow increases in the therapeutic efficacy of intraperitoneal MTX in a murine model of peritoneal cancer. Monoclonal anti-MTX Fab antibody fragments (AMF) were produced, purified, and characterized. AMF pharmacokinetics were determined following i.v. bolus injection (0.4 g/kg) and s.c. bolus injection (0.4, 0.8, 2.2 g/kg). MTX efficacy was investigated in mice bearing peritoneal sarcoma 180 tumors, following administration of MTX via 72 h i.p. infusion at 1.9, 2.8, 3.8 mg/kg, and following combination therapy of 7.5 or 10 mg/kg i.p. MTX (72 h infusion) and 4.2 g/kg s.c. AMF. The mean terminal half-life of AMF was found to be 10.9 +/- 3.3 h and was not dose-dependent, and s.c. bioavailability was 28% +/- 7% at 2.2 g/kg. In mice bearing peritoneal tumors, the maximally tolerated dose of i.p. MTX increased from 1.9 mg/kg (following i.p. MTX alone) to 10 mg/kg (with co-administration of s.c. AMF). Median survival times for saline-treated control animals and animals receiving i.p. MTX (1.9, 2.8, 3.8 mg/kg) were 9, 12, 10, and 7 days, respectively. However, for animals receiving combination therapy with i.p. MTX 7.5 or 10 mg/kg and 4.2 g/kg s.c. AMF, median survival time increased to 17 and 14 days, respectively. As such, the present data suggest that systemic administration of AMF may allow increases in the maximally tolerated dose of i.p. MTX, and allow increases in the therapeutic efficacy of i.p. MTX chemotherapy of peritoneal tumors.

Pharmacokinetic effects of 4C9, an anti-FcRn antibody, in rats: implications for the use of FcRn inhibitors for the treatment of humoral autoimmune and alloimmune conditions.

FcRn protects immune gamma globulin (IgG) from intracellular catabolism, and thereby contributes to the long plasma half-life associated with this class of antibody. The present study tested the hypothesis that 4C9, an anti-FcRn antibody, would increase the in vivo systemic clearance of a model antibody, anti-methotrexate IgG (AMI), in rats. Hybridomas secreting 4C9 and AMI were grown in serum free medium, and monoclonal 4C9 and AMI were purified via protein-G chromatography. Rats were instrumented with jugular vein cannulas 2-3 days prior to investigation, and 4C9 was administered intravenously at doses of 3, 15, and 60 mg/kg. AMI was then administered 4, 24, and 48 h after administration of 4C9. Blood samples were collected and assayed to determine AMI concentrations. The anti-FcRn antibody, 4C9, increased AMI systemic clearance in a dose-dependent manner (from 0.99+/-0.14 mg/h/kg in control animals to 1.27+/-0.05, 1.73+/-0.50, and 1.97+/-0.49 mL/h/kg in animals treated with 3, 15, and 60 mg/kg 4C9; p<0.05). These data were well-captured with an indirect-effect pharmacokinetic-pharmacodynamic model. The effect of 4C9 was found to be transient; no significant effects on AMI systemic clearance were observed when pre-treatment time was increased to 24 or 48 h. As such, the data demonstrate that 4C9, a monoclonal anti-FcRn antibody, induces a transient, dose-dependent increase in the elimination of IgG. The results suggest that FcRn inhibitors may have utility in the treatment of antibody-mediated autoimmune and alloimmune conditions.

Proinflammatory cytokines mediate the systemic inflammatory response associated with high-dose cytarabine treatment in children.

BACKGROUND: Treatment with high-dose cytarabine (1-beta-D-arabinofuranosylcytosine) is often associated with an acute febrile reaction sometimes including abdominal pain, myalgia, and rash. The similarity of these symptoms to those caused by hypersecretion of cytokines in the systemic inflammatory response syndrome (SIRS) prompted us to investigate the plasma levels of proinflammatory cytokines during treatment of children with high-dose cytarabine. PROCEDURE: Sixteen children treated for hematological malignancies and in clinical remission were studied during treatment with six infusions of cytarabine given every 12 hr at a dose of 2 g/m(2). Blood samples for analysis of tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-1gamma (IL-1gamma), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10) and interleukin-1 receptor antagonist (IL-1ra) were obtained prior to treatment and subsequently at 12, 36 and 60 hr. Additional samples were collected as soon as fever occurred. RESULTS: Thirteen of 16 patients developed fever at a median time of 30 hr following start of treatment. At 12 hr levels of TNF-alpha were elevated followed by a rise in IL-6, IFN-alpha, and IL-1ra, peaking at the onset of fever. Thereafter these levels slowly declined whereas low IL-10 levels became detectable. CONCLUSIONS: We conclude that high-dose cytarabine treatment often induces release of TNF-alpha followed by the sequential release of other proinflammatory cytokines. Most likely these cytokines mediate the development of symptoms comprising the cytarabine syndrome.

[Immunological markers of disease activity in the course of 2-CDA treatment of multiple sclerosis]. / Immunologiczne markery aktywnosci choroby w przebiegu leczenia kladrybina stwardnienia rozsianego.

52 clinically definite multiple sclerosis (MS) patients were treated with subcutaneous injection of 5 mg 2-CDA in 5 consecutive days. The injection courses were repeated 6 times in one month interval. The MRI pattern and immunological markers were studied in serum and CSF before and after 6 months of treatment. The obtained results suggest that treatment with 2-CDA has not any significant effect on humoral immunological events in multiple sclerosis, what is in contrast to some normalization of cellular immunopathological processes.

[Antigenicity tests of a new antineoplastic agent S-1].

The antigenicity was tested of a new antineoplastic agent S-1 (a combination of tegafur (FT), CDHP and potassium oxonate (Oxo)) in mice and guinea pigs. 1. Male BALB/c or C3H/He mice were sensitized with S-1, CDHP, Oxo, and conjugates of CDHP (or Oxo) and human serum albumin (HSA). S-1 was administered by oral gavage, and the other compounds were administered intraperitoneally with adjuvant (alum). In the heterologous passive cutaneous anaphylaxis (PCA) test, using Sprague-Dawley rats as recipients, no IgE antibodies against S-1, CDHP, or Oxo were detected to any serum obtained from the sensitized mice, and no eliciting antigenicities were seen for CDHP or Oxo. 2. Male Hartley guinea pigs were sensitized with S-1, CDHP, Oxo, and conjugates of CDHP (or Oxo) and HSA. S-1 was administered by oral gavage, and the other compounds were administered subcutaneously with Freund's complete adjuvant (FCA). The homologous PCA test, active systemic anaphylaxis test, and passive hemagglutination test showed no production of antibodies against S-1, CDHP, or Oxo in any sensitized guinea pig, and no eliciting antigenicities for CDHP or Oxo. 3. Female Hartley guinea pigs were sensitized with S-1 subcutaneously with FCA. The active cutaneous anaphylaxis test revealed that S-1 did not induce cell-mediated delayed type hypersensitivity. 4. These results indicated that S-1, Oxo, and CDHP were not antigenic in mice and guinea pigs.

Inverse targeting of peritoneal tumors: selective alteration of the disposition of methotrexate through the use of anti-methotrexate antibodies and antibody fragments.

We have hypothesized that antidrug antibodies (ADAb) may be employed to impart site-specific alterations in the disposition of drug molecules, potentially allowing for targeted drug therapy. We are specifically interested in minimizing systemic exposure to free drug and systemic toxicities resultant from regional chemotherapy through the intravenous administration of ADAb. In this report, we present the production and purification of anti-methotrexate Fab fragments, and we present investigations of the effects of anti-methotrexate Fab and anti-methotrexate immunoglobulin G on the disposition of methotrexate in the rat. Pharmacokinetic studies revealed that intravenous anti-methotrexate immunoglobulin G (anti-MTX IgG) and anti-methotrexate Fab (anti-MTX Fab) administration produced dramatic alterations in the plasma pharmacokinetics of methotrexate (MTX), following intraperitoneal MTX administration (area under the total MTX concentration vs time curve for anti-MTX IgG relative to control, 420 +/- 90 (p < 0.05); for anti-MTX Fab relative to control, 46 +/- 6.1 (p < 0.05); area under the free MTX concentration vs time curve for anti-MTX IgG relative to control, 0.64 +/- 0.16; for anti-MTX Fab relative to control, 0.45 +/- 0.20 (p < 0.05)). Additional studies conducted in anesthetized rats revealed no significant alterations in the area under the total peritoneal MTX concentration vs time curves, free MTX peritoneal concentration vs time curves, or peritoneal exit rate of MTX in anti-MTX Fab treated animals relative to controls. Therefore, our pharmacokinetic studies demonstrate that ADAb may produce site-specific alterations in drug pharmacokinetics, potentially enhancing the site specificity of drug distribution and drug action following regional chemotherapy.

Antibodies to 4-hydroxyandrostenedione--a new anti-breast cancer agent suitable for use in a radioimmunoassay.

A specific antiserum suitable for radioimmunoassay (RIA) of 4-hydroxy-4-androstene-3,17 dione (4-OHA) has been developed. Sheep antiserum was raised by injecting two different conjugates prepared by coupling 4-OHA to ovalbumin. Antisera obtained from a sheep immunised with 4-hydroxy-testosterone-17-hemisuccinate ovalbumin conjugate were of higher titre and more specific than antisera obtained from sheep immunised with 4-hydroxyandrostenedione-7 alpha-carboxyethylthioether. The antiserum bound 50% of 20 picograms of [6,7-3H]-4-OHA at an initial dilution of 1:270. The most relevant steroids, androstenedione (AD) and testosterone (T) were tested and showed cross reactivity of 2.7% and 5.1% respectively. The lower limit of detection was 4.5 pg/tube. Antisera from this animal will prove useful as the basis of a sensitive and specific RIA for clinical pharmacokinetic studies of 4-OHA.