Hematopoietic Toxicity Research Articles

Damage and recovery of bone marrow microenvironment after acute Methotrexate chemotherapy

Disruptions to interactions between cells of stromal and haematopoietic lineages caused by chemotherapy treatment alter steadystate proliferation, differentiation and maintenance of stem cell niches, and are associated with skeletal defects known to develop after intensive chemotherapy treatment. The anti-metabolite dihydrofolate reductase inhibitor Methotrexate (MTX) is used commonly to treat Acute Lymphoblastic Leukemia and some solid tumours, known to cause haematopoietic toxicity and bone loss in cancer patients. However, the cellular/molecular mechanisms associated with MTX damage to the bone and bone marrow are yet to be fully elucidated. In this study, short-term MTX chemotherapy (five oncedaily injections at 0.75 mg/kg/day) was conducted in rats to investigate the damage to and subsequent recovery of the bone marrow microenvironment and the potential role of the CXCL12/ CXCR4 axis. Marrow cell density 6, 9 and 10 days after the initial MTX dose was significantly reduced, which was accompanied by an increase in marrow adipocyte number, although both returned to normal by day 14. Consistent with the reduced cellularity, MTX caused a reduction in marrow cell proliferation on days 6 and 9 as measured by BrdU labelling. In addition, a CFU-GM assay of isolated marrow cells was used to estimate contents of committed granulocyte-macrophage progenitor cells, which revealed a reduction in colonies on day 6 but recovery on day 9. Consistent with the increase in adipocyte number, there was an increase in adipocyte formation in an ex vivo assay of isolated bone marrow stromal cells and an increase in adipogenic differentiation regulator PPAR-g expression in bone. To investigate the effects of MTX on marrow stromal progenitor numbers, a CFU-F assay plus Alkaline Phosphatase staining was performed, which revealed a significant decrease in positive colonies on days 6 and 9 returning to near normal by day 14. This study illustrates that acute MTX chemotherapy transiently depletes the bone marrow of its steady-state haematopoietic and stromal progenitors, reducing haematopoietic cellularity and osteogenesis and increasing marrow fat content, which have the capacity to recover by day 14 subsequent to damage.

In Vitro Screening of 50 Highly Prescribed Drugs for Thiol Adduct Formation—Comparison of Potential for Drug-Induced Toxicity and Extent of Adduct Formation

Reactive metabolite formation has been associated with drug-induced liver, skin, and hematopoietic toxicity of many drugs that has resulted in serious clinical toxicity, leading to clinical development failure, black box warnings, or, in some cases, withdrawal from the market. In vitro and in vivo screening for reactive metabolite formation has been proposed and widely adopted in the pharmaceutical industry with the aim of minimizing the property and thus the risk of drug-induced toxicity (DIT). One of the most common screening methods is in vitro thiol trapping of reactive metabolites. Although it is well-documented that many hepatotoxins form thiol adducts, there is no literature describing the adduct formation potential of safer drugs that are widely used. The objective of this study was to quantitatively assess the thiol adduct formation potential of 50 drugs (10 associated with DIT and 40 not associated) and document apparent differences in adduct formation between toxic and safer drugs. Dansyl glutathione was used as a trapping agent to aid the quantitation of adducts following in vitro incubation of drugs with human liver microsomes in the presence and absence of NADPH. Metabolic turnover of these drugs was also monitored by LC/UV. Overall, 15 out of the 50 drugs screened formed detectable levels of thiol adducts. There were general trends toward more positive findings in the DIT group vs the non-DIT group. These trends became more marked when the relative amount of thiol adducts was taken into account and improved further when dose and total daily reactive metabolite burdens were considered. In conclusion, there appears to be a general trend between the extent of thiol adduct formation and the potential for DIT, which would support the preclinical measurement and minimization of the property through screening of thiol adduct formation as part of an overall discovery optimization paradigm.

Normal thiopurine methyltransferase phenotype testing in a Crohn disease patient with azathioprine induced myelosuppression

Severe cytopenias in patients with autoimmune conditions treated with azathioprine are well-recognized. Thiopurine methyltransferase (TPMT) enzymatic activity is subject to individual and ethnic variability. Patients with low TPMT activity (poor metabolizers) are at high risk of developing severe and potentially fatal haematopoietic toxicity. Studies have shown that essentially all TPMT-deficient patients will develop haematopoietic toxicity on administration of conventional thiopurine dosages (6-mercaptopurine, azathioprine). Therefore, screening for TPMT polymorphisms in patients before prescribing thiopurine drugs has been proposed. However, despite normal in vitro enzymatic activity, cytopenia may still occur in vivo. This is the case report of an Asian patient with Crohn disease harbouring a rare TPMT mutation on DNA sequencing, who developed neutropenic sepsis and anaemia after a flare of Crohn disease. The report illustrates the importance of monitoring for cytopenia in the setting of active inflammatory disease despite prior normal phenotyping, the role of predictive pharmacogenetics and the limitations of TPMT phenotype assays that may result in misclassification of at-risk patients.

Caspase Mediated Enhanced Apoptotic Action of Cyclophosphamide- and Resveratrol-Treated MCF-7 Cells

Cyclophosphamide (CPA) is a widely used chemotherapeutic drug for neoplasias. It is a DNA and protein alkylating agent having a broad spectrum of activity against a variety of neoplasms including breast cancer. The therapeutic effectiveness of CPA is limited by the high-dose hematopoietic, renal, and cardiac toxicity that accompanies the systemic distribution of liver-derived activated drug metabolites. The present study examines the potential of combining resveratrol (RES) with CPA and aims to increase the understanding of the mechanism of cell killing. Interestingly, we found that RES significantly enhances the caspase-mediated cytotoxic activity of CPA on MCF-7 cells in vitro. RES at 50 μM decreases the IC50 value of CPA from 10 to 5 mM. FACS data reveals CPA or RES alone mediated G0/G1 and S phase arrest, while the combination of these drugs released both the arrests and results in an increase in the sub G0/G1 peak. Additional analyses indicated the significant up-regulation (P = 0.001) of tumor suppressor p53 and p53-regulated pro-apoptotic Bax and Fas in MCF-7 cells following CPA treatment in combination with RES, which may contribute to the enhancement of the antitumor effect of CPA. Furthermore, downregulation of anti-apoptotic Bcl-2 (P = 0.001) was observed in MCF-7 cells treated with CPA with or without RES when compared to untreated MCF-7. These results suggest the possibility of a new combination chemotherapeutic regimen leading to improvements in the treatment of breast cancer.

Hedgehog Signaling Is Dispensable for Adult Murine Hematopoiesis and Leukemogenesis.

Hedgehog (Hh) pathway proteins are a highly conserved family of intracellular signaling molecules that are critical for the development of multiple organs and tissues, and play a role in cell fate determination of self-renewing tissues in the adult. Mutations that impair Hh signaling have been associated with developmental abnormalities, and recent studies indicate that Hh plays an important role in hemangioblast formation and in adult hematopoiesis, as well as in the differentiation and proliferation of hematopoietic stem cells (HSC) and progenitor cells. We used a genetic and pharmacologic approach to define the role of the Hh pathway in adult hematopoiesis and leukemogenesis. We report the unexpected finding that loss of Hh signaling through conditional deletion of Smoothened (Smo) in the adult hematopoietic compartment has no effect on adult hematopoiesis, including peripheral blood count, number or cell cycle status of stem and progenitor cells, hematopoietic colony forming potential, long-term repopulating activity in competitive repopulation assays, or stress-response to serial 5-fluorouracil treatment. In support of these observations based on genetic inactivation of the pathway, we observed that pharmacologic inhibition of Hh signaling with a potent and highly selective small molecule antagonist of Smo has no apparent effect on hematopoiesis in the mouse in vivo. In addition, we observed that Hh signaling is not required for the development of MLL-AF9 mediated leukemia. Taken together, these data indicate that Hh signaling is dispensable for normal hematopoietic development and leukemogenesis, and that pharmacologic inhibition of Hh signaling, as a therapeutic strategy in treatment of solid tumors with constitutive Hh pathway activation is not likely to be associated with unmanageable hematopoietic toxicity.

SHP-2 Knockdown Results in Profound Inhibition of Human CD34+ Hematopoietic Stem/Progenitor Cell Survival, Proliferation and Differentiation in Response to Growth Factor Stimulation

SHP-2 (ptpn11), a Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase, is expressed at high levels in hematopoietic cells and regulates downstream signaling from growth factor (GF) receptors. SHP-2 has been shown to play an important role in murine hematopoiesis. Moreover, several SHP-2 activating mutations have been identified in myeloid malignancies and there is interest in the development of SHP-2 inhibitors for cancer treatment. On the other hand previous report suggested that SHP-2 inhibition was associated with enhanced GF responsiveness in human hematopoietic cell lines. However the role of SHP-2 signaling in normal human hematopoietic stem and progenitor cell growth has not been studied. Here we investigated the function of SHP-2 in normal human hematopoiesis by inhibiting SHP-2 expression in cord blood (CB) CD34+ cells with stable SHP-2 shRNA expression. We transduced CB CD34+ cells with lentivirus vectors coexpressing SHP-2 specific shRNAs (Si-1 or Si-2) or a control shRNA (Ctrl) and RFP and selected RFP expressing CD34+ cells by flow cytometry sorting. We observed >80% inhibition of SHP-2 expression by Western blotting in Si-1 or Si-2 shRNA transduced cells compared with Ctrl shRNA transduced cells. We observed that culture with increasing concentrations of GF was associated with markedly reduced GF-induced stimulation of proliferation of SHP-2-knockdown CD34+ cells compared to controls. In addition we observed significantly increased apoptosis of SHP-2-knockdown CD34+ cells cultured under low and high GF conditions compared to controls, but little increase in apoptosis in GF-deprived cells, indicating markedly reduced response of SHP-2-knockdown cells to GF-mediated promotion of cell survival. SHP-2-knockdown CD34+ cells also demonstrated significantly reduced expansion in cell numbers following culture in high GF conditions compared with controls (115.3, 25.5 and 10.4 fold expansion for Ctrl, Si-1 and Si-2 at day 7). Analysis of the nature of cells generated in GF culture showed significantly reduced generation of both myeloid (CD33+, CD11b+ and CD14+) and erythroid cells from SHP-2-knockdown CD34+ cells compared with controls, with relatively greater inhibition of myeloid compared with erythroid differentiation. On the other hand CD34+ cell numbers were retained at levels similar to controls after culture. We also observed significantly reduced cell expansion and differentiation and higher apoptotic rates of SHP-2-knockdown cells cultured under either myeloid promoting (IL-3+SCF+G-CSF+GM-CSF) or erythroid promoting (SCF+EPO) GF conditions. SHP-2-knockdown cells demonstrated reduced activation of MAPK and STAT5 but not Akt on Western blotting that was associated with reduced MCL-1 expression, consistent with their reduced GF mediated proliferation and survival. Expression of the transcription factors SCL1, GATA-1, NF-E2 and FOG-1 was increased in SHP-2 knockdown CD34+ cells compared to controls, consistent with the relatively higher retention of CD34+ and erythroid cells compared with myeloid cells after culture. In conclusion, we show that SHP-2 knockdown in human CD34+ cells results in markedly decreased responsiveness to GF stimulation with significantly increased apoptosis, markedly diminished proliferation and reduced generation of differentiated cells during GF culture. A relative retention of the CD34+ cell population was seen despite increased apoptosis, which may be the result of reduced cell turnover and altered transcription factor expression in SHP-2-knockdown cells, and is in contrast to reduced stem cell self-renewal observed following SHP-2 knockdown in murine models. These results indicate a critical role for SHP-2 in GF mediated signaling responses in human hematopoietic stem/progenitor cells. These studies also caution that therapeutic SHP-2 inhibition could be associated with significant hematopoietic toxicity.

Efficient Gene Marking with a Clinical Gammaretrovirus Vector Expressing MGMTP140K in Baboons and Macaques

Using gene therapy to protect hematopoietic stem cells (HSC) from alkylating agents used in the treatment of malignant disease is an attractive strategy to alleviate prolonged neutropenia and thrombocytopenia that is dose-limiting. Both adult and pediatric patients with glioblastomas urgently need improved therapeutic strategies since even with aggressive treatment the median survival after diagnosis is approximately 12 months. Chemotherapy with the nitrosourea BCNU, the methylating agents procarbazine or temozolomide, and other alkylating agents is effective and can prolong survival but the therapeutic benefit is attenuated due to hematopoietic toxicity which limits dose-escalation of these drugs. We have previously demonstrated in the dog and non-human primate model efficient gene marking, in vivo selection and chemoprotection from temozolomide and BCNU following transplantation with MGMTP140K gene-modified cells. Thus, we wanted to explore the efficacy of autologous transplantation in macaques and baboons using cells gene-modified with an MLV-based gammaretrovirus vector developed for a pending clinical trial. This retroviral vector contains a myeloproliferative sarcoma virus LTR, negative control region deleted, dl587rev primer binding site (MND) vector backbone, which expresses MGMTP140K from the 5′ LTR promoter, and is pseudotyped with the gibbon ape leukemia virus (GALV) envelope produced from Phoenix-GALV packaging cells (PhGALV-MND.GRS.P140K c38). Following transduction the gene marking in pre-infusion colony forming units (CFUs) was 74.3% and 69.8% in the macaque and baboon respectively as determined by CFU-PCR. Intracellular MGMT-staining of cultured baboon cells 4 (76.4%) and 11 (89.9%) days after transduction confirmed high gene transfer levels. Both animals recovered neutrophil and platelet levels within expected time frames relative to historical controls and the average provirus copy number determined by real-time PCR approximately one month after transplantation was 0.14 and 0.72 in the macaque and baboon respectively. Retrovirus integration site analysis in the macaque 90 days after transplantation, and before chemotherapy, confirmed polyclonal hematopoietic reconstitution. There was no indication of progression to a pre-leukemic state. The macaque has been treated twice with O6-benzylguanine (O6BG) and BCNU and the gene marking has stably increased approximately 2.5-fold. Aside from transient elevated liver enzymes following O6BG/BCNU treatment no additional extra-hematopoietic toxicity has been observed. In summary, we have been able to achieve efficient polyclonal gene marking with MGMTP140K gene-modified cells using a vector designed for clinical application in both macaques and baboons and have preliminary evidence of in vivo selection in the macaque. We believe that these large animal studies closely reflect a clinical setting and will help to further improve clinical HSC gene therapy.

Microtubule Active Agents: Beyond the Taxane Frontier

Microtubules are essential to cell transport, signaling, and mitosis. An increasing range of anticancer drugs interferes with the normal formation and function of microtubules. Vinca alkaloids act as microtubule destabilizers and the taxanes act as microtubule stabilizers. Taxanes are widely used cytotoxic agents that are active in a range of solid tumor malignancies and are routinely used in a variety of settings. Significant limitations with the taxanes exist, including acquired and intrinsic tumor resistance through the expression of multidrug resistance proteins such as P-glycoprotein, risk of hypersensitivity reactions, dose-limiting hematopoietic toxicity, and cumulative neurotoxicity. Hence, there is a need to develop novel agents that act on the microtubules. Epothilones are macrolide antibiotics that bind near the taxane-binding site on microtubules and have been extensively studied in recent and ongoing clinical trials. A variety of other agents that act on the microtubules at different sites with a variety of structures are at varying stages of development.

Transporter-Mediated Protection against Thiopurine-Induced Hematopoietic Toxicity

Thiopurines are effective immunosuppressants and anticancer agents, but intracellular accumulation of their active metabolites (6-thioguanine nucleotides, 6-TGN) causes dose-limiting hematopoietic toxicity. Thiopurine S-methyltransferase deficiency is known to exacerbate thiopurine toxicity. However, many patients are highly sensitive to thiopurines for unknown reasons. We show that multidrug-resistance protein 4 (Mrp4) is abundant in myeloid progenitors and tested the role of the Mrp4, an ATP transporter of monophosphorylated nucleosides, in this unexplained thiopurine sensitivity. Mrp4-deficient mice experienced Mrp4 gene dosage-dependent toxicity caused by accumulation of 6-TGNs in their myelopoietic cells. Therefore, Mrp4 protects against thiopurine-induced hematopoietic toxicity by actively exporting thiopurine nucleotides. We then identified a single-nucleotide polymorphism (SNP) in human MRP4 (rs3765534) that dramatically reduces MRP4 function by impairing its cell membrane localization. This SNP is common (>18%) in the Japanese population and indicates that the increased sensitivity of some Japanese patients to thiopurines may reflect the greater frequency of this MRP4 SNP.

Benzene-induced hematopoietic toxicity transmitted by AhR in wild-type mouse and nullified by repopulation with AhR-deficient bone marrow cells: Time after benzene treatment and recovery

Previously, we found an aryl hydrocarbon receptor (AhR)-transmitted benzene-induced hematotoxicity; that is, AhR-knockout (KO) mice did not show any hematotoxicity after benzene exposure [Yoon, B.I., Hirabayashi, Y., Kawasaki, Y., Kodama, Y., Kaneko, T., Kanno, J., Kim, D.Y., Fujii-Kuriyama, Y., Inoue, T., 2002. Aryl hydrocarbon receptor mediates benzene-induced hematotoxicity. Toxicol. Sci. 70, 150–156]. Furthermore, our preliminary study showed a significant attenuation of benzene-induced hematopoietic toxicity by AhR expression, when the bone marrow (BM) of mice was repopulated with AhR-KO BM cells [Hirabayashi, Y., Yoon, B.I., Li, G., Fujii-Kuriyama, Y., Kaneko, T., Kanno, J., Inoue, T., 2005a. Benzene-induced hematopoietic toxicity transmitted by AhR in the wild-type mouse was negated by repopulation of AhR deficient bone marrow cells. Organohalogen Comp. 67, 2280–2283]. In this study, benzene-induced hematotoxicity and its nullification by AhR-KO BM cells were further precisely reevaluated including the duration of the effect after benzene treatment and recovery after the cessation of exposure. Exposure routes, namely, intraperitoneal ( i.p.) injection used in our previous study and intragastric ( i.g.) administration used in this study, were also compared in terms of their toxicologic outcomes. From the results of this study, mice that had been lethally irradiated and repopulated with BM cells from AhR-KO mice essentially did not show any benzene-induced hematotoxicity. The AhR-KO BM cells nullified benzene-induced toxicities in notably different hematopoietic endpoints between the i.p. treatment and the i.g. treatment; however, the number of granulo-macrophage colony-forming unit in vitro (CFU-GM) was a common target parameter, the benzene-induced toxicity of which was nullified by the AhR-KO BM cells.

Intensive anti-inflammatory therapy with dexamethasone in patients with non-small cell lung cancer: effect on chemotherapy toxicity and efficacy

Our preclinical and clinical data suggest that pretreatment with dexamethasone 4 days prior to chemotherapy increased the efficacy and decreased the toxicity of carboplatin and gemcitabine. To translate these findings to patients, we have undertaken a Phase 1/2 clinical trial. Thirty patients with advanced non-small cell lung cancer (NSCLC) received gemcitabine, 1,000 mg/m(2) on days 1 and 8, and carboplatin, AUC 5.5 on day 1. Patients were randomized (1:2:2) to receive, no dexamethasone (cohort 1), or oral dexamethasone at 8 mg (cohort 2) or 16 mg (cohort 3) twice per day, 4 days before and of the day of chemotherapy. Dexamethasone was administered to patients in cohorts 2 and 3 during courses 2-4. In cohorts 1, 2, and 3, patients completing four planned courses of therapy were: 1/6, 6/12, 9/12. Partial responses (RECIST) were: 2/6, 6/12, and 7/12. Overall, dexamethasone significantly improved AGC and platelet nadirs and recovery times. There were no significant differences in non-hematologic toxicities between cohorts and no significant differences in pharmacokinetic parameters between course 1 and 2 in any cohort. These data support our previous preclinical and clinical observations that dexamethasone pre-treatment decreases hematopoietic toxicity and improves efficacy of this chemotherapeutic regimen in patients with metastatic non-small cell lung cancer and suggests that further randomized trials should be undertaken.

Phase I trial of yttrium 90 ibritumomab tiuxetan (90Y-RIT) with autologous stem cell transplantation (ASCT) in patients with relapsed or refractory B-cell non-Hodgkin’s lymphoma (NHL)

7535 Background: 90YIT is an effective agent for patients with lymphoma. Unfortunately, dose escalation is limited by hematopoetic toxicity. In order to determine the non-hematopoetic maximum tolerated dose, we are conducting a dose finding study of 90YITwith ASCT. Methods: Eligible patients had relapsed or refractory NHL, rituximab sensitive disease, age ≥18 years, platelet count ≥75,000/mm3, WBC >3,000/mm3, bone marrow involvement ≤35%, and acceptable renal and hepatic function. Patients receive weekly rituximab (375 mg/m2) for 4 consecutive weeks followed by a single dose of cyclophosphamide (2.5 g/m2). Filgrastim 10 mcg/kg is administered post cyclophosphamide until stem cell collection. Following successful collection of at least 2 X 106 CD34/kg, patients receive a dosimetric dose of indium 111 (111In) ibritumomab tiuxetan (5 mCi) on day 1, quantitative planar and SPECT/CT scans followed by cohort specific dose of 90Y IT on day 15. Stem cell infusion occurs when estimated marrow dose rate is < 1 cGy/...

Characterisation of novel defective thiopurine S-methyltransferase allelic variants

Human thiopurine S-methyltransferase (TPMT, EC 2.1.1.67) is a key enzyme in the detoxification of thiopurine drugs widely used in the treatment of various diseases, such as inflammatory bowel diseases, acute lymphoblastic leukaemia and rheumatic diseases. The TPMT gene is genetically polymorphic and the inverse relationship between TPMT activity and the risk of developing severe hematopoietic toxicity is well known. In this study, the entire coding sequence of TPMT, together with its 5′-flanking promoter region, was analysed in patients with an intermediate phenotype for thiopurine drug methylation. Four polymorphisms were identified, two previously described, c.356A>C (p.Lys 119Thr, TPMT*9) and c.205C>G (p.Leu 69Val, TPMT*21), and two novel missense mutations, c.537G>T (p.Gln 179His, TPMT*24) and c.634T>C (p.Cys 212Arg, TPMT*25). Structural investigations, using molecular modeling, were undertaken in an attempt to explain the potential impact of the amino acid substitutions on the structure and activity of the variant proteins. Additionally, in order to determine kinetic parameters ( K m and V max) of 6-thioguanine (6-TG) methylation, the four variants were expressed in a recombinant yeast expression system. Assays were performed by HPLC and the results were compared with those of wild-type TPMT. The p.Leu 69Val and the p.Cys 212Arg substitutions encode recombinant enzymes with a significantly decreased intrinsic clearance compared to that of the wild-type protein, and, consequently, characterise non-functional alleles of TPMT. The p.Lys 119Thr and the p.Gln 179His substitutions do not affect significantly the catalytic activity of the corresponding variant proteins, which prevents to unambiguously describe these latter alleles as defective TPMT variants.

Is inhalation exposure to formaldehyde a biologically plausible cause of lymphohematopoietic malignancies?

The United States Environmental Protection Agency (EPA) recently proposed a hypothetical mode of action (MOA) to explain how inhaled formaldehyde (FA) might induce leukemia, lymphoma and a variety of other lymphohematopoietic (LHP) malignancies in occupationally exposed workers. The central hypothesis requires that B lymphocytes or hematopoietic progenitor cells (HPC) present at the “portal of entry (POE)” undergo sustained mutagenic change as a result of direct FA exposure. These modified cells would then migrate back to the bone marrow or primary lymphatic tissue and subsequently develop into specific LHP disease states. Chemical interaction at the POE is an absolute requirement for the hypothesized MOA as there is no convincing evidence that inhaled FA causes distant site (e.g., bone marrow) toxicity. The purpose of this review is to critically evaluate this proposed MOA within the context of the existing data concerning the toxicokinetic and biological properties of FA, the current understanding of the induction of chemically-induced leukemias and lymphomas, as well as within EPA’s specific guidelines for evaluating the MOA of chemically-induced cancers. Specifically, we examine the scientific support for the hypothesis that FA exposure may induce carcinogenic transformation of localized lymphocytes or peripheral hematopoietic progenitor cells (HPC) in the absence of discernable systemic hematopoietic toxicity (i.e., peripheral transformation). While little or no empirical evidence exists upon which to fully evaluate the proposed hypothesis, available data does not support the proposed concept of “peripheral transformation” at the chemical entry site. Numerous animal bioassays evaluating chronic inhalation of FA clearly do not support this hypothesis since no properly conducted study as ever shown an increase in any LHP malignancy. Moreover, the notion that FA can cause any LHP malignancy is not supported with either epidemiologic data or current understanding of differing etiologies and risk factors for the various hematopoietic and lymphoproliferative malignancies. It is therefore concluded that existing science does not support the proposed MOA as a logical explanation for proposing that FA is a realistic etiological factor for any LHP malignancy.

Antiradiation vaccine : immunoprophylaxis of acute radiation syndromes - radioprotective efficiency.

Current medical management of Acute Radiation Syndromes does not include immune prophylaxis based on Antiradiation Vaccine. Existing principles for the treatment of acute radiation syndromes are based on the replacement therapy and supportive therapy. A large amount of antigens isolated from bacterias (flagellin and derivates), plants, different types of venoms (honeybees, scorpions, snakes) can produce a nonspecific stimulation of immune system of mammals and protect against of irradiation. But only radiation toxins stimulate a specific antigenic stimulation of antibody synthesis. Active immunization by non-toxic doses of radiation toxins includes a complex of radiation toxins that we call the Specific Radiation Determinant (SRD). Immunization must be provided not less than 30 days before irradiation and it is effective up to three years and more. Active immunization by radiation toxins significantly diminishes mortality rate (100%) and improves survival rate up to 60% compare with 0% survival rate among irradiated animals in control groups. The SRD molecules were isolated from Lymphatic Systems of animals that were irradiated with high doses of irradiation and had clinical and laboratory picture of Cerebral Acute Radiation Syndrome, Cardiovascular Acute Radiation Syndrome, Gastrointestinal Acute Radiation Syndrome and Hematological Acute Radiation Syndrome. Our classification of radiation toxins include 4 major groups: 1.SRD-1, neurotoxic radiation toxins; 2.SRD-2, neurovascular radiation toxins; 3.SRD-3, non-Bacterial Gastrointestinal Radiation Toxins; 4.SRD-4, Hematopietic Radiation Toxins. Radiation toxins possess both toxic and immunological properties. But mechanisms of immune-toxicity by which radiation toxins stimulate development of ARS are poorly understood. We have compared lethal toxicity of radiation toxins and a potential for neutralization of their toxic activity by specific antibodies to radiation toxins. Blocking antiradiation antibodies induce an immunologically specific effect and possess inhibiting effects to radiation induced neuro-toxicity, vascular-toxicity, gastrointestinal toxcity, hematopoietic toxicity and radiation induced cytolysis of selected sensitive to radiation groups of cells. Blocking Antiradiation Antibodies are immunologically specific and can be produced by immunization with different radiation toxins isolated from irradiated mammals. We propose that Specific Antiradiation Antibodies targeted to radiation induced Toxins. Specific Antiradiation Antibodies neutralize toxic properties of radiation toxins. Antiradiation Antibodies in different phases of Acute Radiation Syndromes can compete with cytotoxic lymphocytes and prevent cytolysis mediated by cytotoxic lymphocytes .

Response to Oral Forodesine in Refractory Cutaneous T-Cell Lymphoma: Interim Results of a Phase I/II Study.

Background: Forodesine is a rationally designed, potent inhibitor of purine nucleoside phosphorylase (PNP) that leads to intracellular accumulation of dGTP and then apoptosis. Intravenous forodesine has demonstrated activity in treatment of cutaneous T-cell lymphoma (CTCL) and served as the basis for the design of an oral forodesine Phase I/II trialMethods: An open label dose escalation study of oral forodesine (40 mg/m2 to 320 mg/m2 QD) for 4 weeks was performed to determine the maximum tolerated dose (MTD) and/or the optimal biologic dose (OBD) based on PK, and PNP inhibition as evidenced by elevation of plasma deoxyguanosine (dGuo) levels. Additional subjects were accrued at the optimal dose (80 mg/m2) to further assess clinical safety and efficacy. Patients with previously treated, refractory CTCL with stage IB disease or higher were eligible. The primary efficacy endpoint (objective response rate [ORR]) for this analysis was defined as at least a 50% decrease in modified severity-weighted assessment tool (mSWAT) from baseline maintained for at least 28 days. Only subjects who had at least 6 months follow-up as of March 1, 2007 were included in this analysis.Results: Although an MTD was not reached, based on plateau of the AUC versus dose plot at and above 80 mg/m2, and the same observation for plasma dGuo versus dose, 80 mg/m2 was judged as an OBD. The 36 subjects treated at 80 mg/m2 are the main subject of this report. Median age was 61.6 years (range 28.4–81.1) and 67% were males and were exposed to a median of 3 prior systemic therapies (range 0, 8). The ORR using mSWAT was 39% (14/36) with a median duration of response of 127 days (25%–75%, 71 - NA). Response by stage was: IB 3/9, IIA 1/1, IIB 3/5, III 4/12, IVA 2/5, IVB 1/4. Median time to response was 42 days (25%–75%, 29–58). The median time on treatment was 131 days (range 1, 479) with 6 subjects remaining on treatment. For subjects with Sezary Syndrome (n=20, defined by ISCL B2 classification), the ORR by mSWAT was 40%, and 65% by erythroderma score. More than a 50% reduction in Sezary cells (detected by flow cytometry) was observed in 9/20 (45%) subjects with SS. For all 56 forodesine-treated subjects, the only grade 3 or higher non-laboratory adverse events (without regard to attribution and observed in at least 2 subjects) were diarrhea, acute renal failure (not related), cellulitis, and rash (2 subjects each). The only grade 3 or higher related non-laboratory AEs were vertigo, diarrhea, generalized edema, and pneumonia (1 each). For laboratory events, a single grade 3 elevation for each of the following liver-related parameters was noted: AST, ALT, bilirubin, and alkaline phosphatase. There were no grade 3 or higher elevations of creatinine. Grade 3 or higher lymphopenia and low CD4 counts were observed in 71% and 31% of subjects and these rates were similar across dose groups. Median baseline, nadir, and last visit lymphocytes counts (1000/mm3) were 0.8 (95%CI: 0.0, 6.0), 0.2 (95% CI: 0.0, 0.8), and 0.6 (95% CI: 0.0, 2.9) respectively. Hematopoietic toxicity was limited to 1 episode of grade 3 neutropenia, and 1 episode of grade 3 anemia.Conclusion: Oral forodesine demonstrates clinical activity in subjects with refractory CTCL, including those with SS, with minimal toxicity to date.

FVIII Tolerance Following Nonmyeloablative Transplantation of Hematopoietic Stem Cells Engineered To Encode a High-Expression Factor VIII Transgene in Hemophilia A Mice.

Clinical gene therapy trials for hemophilia A have failed due to insufficient target cell transduction or expression of factor VIII (fVIII). Animal studies have documented the extreme difficulty of expressing therapeutic levels of human fVIII from recombinant viral vectors without eliciting an anti-fVIII humoral response. We previously demonstrated curative fVIII activity levels in hemophilia A mice following a nonmyeloablative, genetically-modified bone marrow transplantation (Ide et al., Blood 2007). In these studies recipient hemophilia A mice received 3×105 bone marrow-derived sca-1+ cells transduced with a retrovirus encoding B-domain-deleted high-expression porcine fVIII (BDDpfVIII) after pre-transplantation conditioning with reduced-intensity TBI or busulfan followed by minimal immunosuppression with either costimulation blockade or antithymocyte serum (ATS). Using our high-expression fVIII transgene and busulfan or 3Gy TBI coupled with ATS resulted in sustained fVIII activity of >100% normal human levels. The main hematopoietic toxicity observed was transient T-cell suppression. High-level fVIII activity (>2 units/ml) was achieved without induction of anti-BDDpfVIII humoral immune responses. Furthermore because the development of inhibitory antibodies directed against fVIII remains a significant clinical complication associated with the treatment of hemophilia A, we tested the effectiveness of genetically-modified cells encoding BDDpfVIII in the setting of pre-existing fVIII immunity. Transplantation of genetically-modified HSCs into 5.5 Gy TBI/ATS or busulfan/ATS-conditioned hemophilia A mice that had significant anti-human fVIII inhibitory titers induced sustained, high-level fVIII activity. Following transplantation and stable engraftment, both naïve and pre-immunized mice were challenged with six weekly injections of 10 units human fVIII, an immunization regimen that results in antibody formation in 100% of naïve hemophilia A mice. All mice sustained their pre-challenge BDDpfVIII activity levels, and anti-fVIII antibodies were not detected. Surface markers of T-cell activation were the same between naïve hemophilia A mice and transplanted mice, for both naïve and preimmunized animals. In a mixed lymphocyte reaction, T-cells were found to be equally capable of reacting to allogenic antigens (i.e. irradiated Balb/C splenocytes) when isolated from mice with stable fVIII expression compared to nontransduced controls, demonstrating that the lack of anti-fVIII inhibitors is not due to a lack of immunocompetent T-cells. Further studies are underway assessing T-cell nonresponsiveness to BDDpfVIII in these transplanted mice. By demonstrating that transplanted mice are tolerant to BDDpfVIII, even in the context of pre-existing inhibitory antibodies and nonmyeloablative conditioning, this data further supports our previous studies showing that retroviral gene transfer using high-expression porcine fVIII elements is a compelling treatment for hemophilia A.

Phase I trial of escalating doses of total marrow irradiation (TMI) with helical tomotherapy and peripheral blood progenitor cell rescue (PBPC) following high-dose melphalan and PBPC as part of tandem high-dose therapy (THDT) for patients with multiple myeloma (MM)

8121 Background: Attempts to combine total body irradiation (TBI) with high-dose melphalan (Mel) resulted in substantial organ toxicities and precluded optimal dosing of Mel. Helical tomotherapy may allow delivery of total marrow irradiation (TMI), while avoiding collateral toxicities. Methods: We designed a phase I/II study for patients (pts) with responding or stable stage I-III MM. Following PBPC mobilization, pts receive THDT first with Mel 200 mg/m2 and PBPC, and, ≥ 6 weeks later, escalating doses of TMI (starting dose: 200 cGy daily × 5 [1,000 cGy], up to 200 cGy twice daily × 5 days [2,000 cGy]) and PBPC. Maintenance consists of dexamethasone 40 mg/day × 4 days every 28 days and thalidomide 50–200 mg/day. Results: The median number of prior chemotherapy regimens is 2 (1- 4). The median duration from diagnosis to the HDT is 8 mos (4–13). Median age of pts is 53 (35–66). Sixteen pts with stages II (6) and III (10) MM have received Mel; 15 of 16 pts (8F/8M) have received treatment at dose levels 1–5 of TMI (1,000 cGy through 1,800 cGy); 1 pt is about to start TMI at 1,800 cGy. The median time between the first and second THCT cycles is 74 days (47–125). Hematopoietic toxicities were independent of TMI dose levels: granulocyte recovery to &gt;1,000/microliter following Mel required 12 days (11–38) versus a median of 10 days after TMI (range; 9–12). Platelet (excluding 6 pts not needing plt transfusion) independence was seen by day 10 (8–13) versus 8 (6–11) following TMI. In the first 15 pts the estimated median radiation dose to normal organs was 15–60% of the targeted bone marrow dose. Reversible grade 3 non-hematologic toxicities by TMI dose levels included fatigue and febrile neutropenia (FN) (level 1: 1 pt each), FN (level 2: 1 pt); none (level 3); fatigue (level 4: 2 pts); anorexia and stomatitis (level 5: 1pt; anorexia: 1 pt). The median follow-up is 8 mos (3–21); 1 pt progressed at 8 mos. Final data on toxicities including patients already in screening for treatment at TMI 2,000 cGy, tolerability of maintenance, and response rate will be presented. Conclusion: TMI is feasible and could potentially be useful as part of THDT for patients with MM. No significant financial relationships to disclose.

The influence of active hexose correlated compound (AHCC) on cisplatin-evoked chemotherapeutic and side effects in tumor-bearing mice

Cisplatin ( cis-diaminedichloroplatinum (II) or CDDP) (a widely used platinum-containing anticancer drug) is nephrotoxic and has a low percentage of tolerance in patients during chemotherapy. The active hexose correlated compound (AHCC) is an extract of Basidiomycotina marketed as a supplement for cancer patients due to its nutrients and fibre content and its ability to strengthen and optimize the capacity of the immune system. The possibility that AHCC could reduce the side effects of cisplatin was assessed in the tumor-bearing BALB/cA mice on the basis of the ability to ameliorate the cisplatin-induced body weight loss, anorexia, nephrotoxicity and hematopoietic toxicity. Although cisplatin (8 mg/kg body weight) reduced the size and weight of the solid tumors, supplementation with AHCC significantly enhanced cisplatin-induced antitumor effect in both the size ( p < 0.05) and weight ( p < 0.05). Food intake in the cisplatin-treated mice were decreased following commencement of treatment and this remained low compared with the cisplatin-untreated group (control) throughout the experiment period. Supplementation with AHCC increased the food intake in the cisplatin-treated mice. The blood urea nitrogen and serum creatinine concentrations, and the ratio of blood urea nitrogen to serum creatinine were significantly increased in the cisplatin alone treated group compared to the control group. Their increased levels were mitigated by supplementation with AHCC (100 mg/kg body weight) in the cisplatin-treated group. AHCC was also able to modulate the suppression of bone marrow due to cisplatin and the improvement was statistically significant. The histopathological examination of the kidney revealed the presence of cisplatin-induced damage and this was modulated by AHCC treatment. The potential for AHCC to ameliorate the cisplatin-evoked toxicity as well as the chemotherapeutic effect could have beneficial economic implications for patients undergoing chemotherapy with cisplatin.

TPMT genotype and its clinical implication in renal transplant recipients with azathioprine treatment

Thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs. Patients with intermediate or deficient TPMT activity are at risk of toxicity after receiving standard doses of these drugs. This study determined the frequencies of TPMT alleles (TPMT*2, *3A, *3B and *3C) and explored the association between TPMT genetic polymorphism and the development of adverse drug reactions in Chinese renal transplant patients receiving azathioprine (AZA). TPMT genotypes were determined using polymerase chain reaction-based assays in 122 renal transplant patients and 210 healthy subjects. Biochemical and clinical data were retrospectively evaluated after renal transplantation. Of 122 patients, eight (allele frequency 3.28%) were heterozygous for TPMT*3C and no TPMT*2, *3A or *3B or homozygous TPMT*3C subjects were identified. The pattern and frequency of the main mutant TPMT alleles were similar in patients and healthy subjects. Four of five patients (80%) with haematopoietic toxicity were heterozygotes. TPMT heterozygosity was associated with significant reductions in haematological indices and a significant decrease in cyclosporine plasma concentrations in the first year after renal transplantation. No association between TPMT genotype and renal rejection was identified. Our results, together with those of others pointing in the same direction, suggest that genotyping the major TPMT variant alleles may be a valuable tool in preventing AZA toxicity and optimization of immunosuppressive therapy.