Population pharmacokinetics of oral high-dose busulfan in adult patients undergoing hematopoietic stem cell transplantation

Many factors have been reported that contribute to the wide intra- and inter-patient variability of Busulfan [Bu] disposition. The purpose of this study was to develop a population pharmacokinetic model and to determine the covariates affecting the pharmacokinetics [PK] of Bu in Iranian adult patients who received oral high-dose as a conditioning regimen before Hematopoietic Stem Cell Transplantation [HSCT]. A population PK analysis was performed in 30 patients who received an oral Bu and cyclophosphamide regimen before HSCT. Bu was given orally according to the protocol of the institution. In order to prevent seizures caused by Bu, phenytoin was administered orally one hour before each dose of Bu. A total of 180 blood samples were analyzed by HPLC and PK parameters were estimated by the non-linear mixed effect model by MONOLIX 3.1 program. A one-compartment model with an additive error model was used to describe the concentration-time profile of Bu. CL=13.4[1+ [0.141xDisease]], Vd=42.6[1+0.010x [Weight - 63.9]] Patients' disease and weight was found to be the determinant factors for clearance [CL] and the volume of distribution [Vd] according to Monolix analysis. The covariate entered in final model followed by these equations: In this limited study, the age [15-43 years] had no significant effect. For a patient weighting 60 kg, the typical CL and Vd were estimated to be 13.4 l/hr and 42.6 L, respectively. The interindividual variability of CL and Vd were 13.6 and 6.3%, respectively. There was no significant metabolic induction in these four days as is evident by comparing the trough levels of Bu. However it should be mentioned that, one tailed t-test p-values of the days of two and three, two and four and three and four were 0.083, 0.069 and 0.388, respectively. Results of this study showed that the type of disease was a determinant of CL and the weight of patient was a determinant of Vd for Bu population PK parameters. A reliable PK parameters and Css, estimated from only one plasma concentrations [5 hrs after the first dose], were validated. Since these methods require few sampling and are easy to be used, the limited sampling methods might be advantageous in the routine clinical practice

Cell-based regenerative therapy as an alternative to liver transplantation for end-stage liver disease: experience from Iran

Several types of cells including mature hepatocytes, adult liver progenitor cells and human embryonic stem cells, fetal liver progenitor cells, bone marrow derived hematopoietic or mesenchymal stem cells, and um- bilical cord blood cells both in rodents and humans have been reported to be capable of self-replication, giving rise to daughter hepatocytes, both in vivo and in vitro. They have been shown to be able to repopulate liver in both animal models of liver injury and in patients with liver disease and to improve liver function. Human embryonic stem cell therapy seems to be a great promise for the treatment of liver cirrhosis, but there is no human clinical application due to ethical concerns or difficulties in harvesting or safely and efficiently expanding sufficient quantities. In contrast, adult bone marrow-derived hematopoietic or mesen- chymal stem cells, which can be easily and safely harvested, have been used in clinical trials to treat several chronic diseases including chronic liver disease. Cell therapy offers exciting promise for future treatment of cirrhosis and metabolic liver diseases, but significant technical hurdles remain that will only be overcome through years of intensive research. There is also serious concern about the long-term safety of stem cell therapy and the possibility of tumor development. Herein, we present our experience with cell therapy in treatment of chronic liver disease in Iran

[Evaluation of the association of NOD2 gene polymorphisms with the occurrence of GVHD in acute myelogenous leukemia patients]

Graft-versus-host disease [GVHD] is one of life-threatening post-transplantation complications. Several recent studies have described a significant correlation between transplantation outcome and three single nucleotide polymorphisms [SNPs] in the NOD2 gene. This study was conducted to evaluate the association of NOD2 gene polymorphisms with the occurrence of GVHD in acute myelogenous leukemia patients who underwent HSCT from their HLA-matched sibling donors. We examined retrospectively NOD2 genotypes by PCR-SSP both in 124 patients who underwent HSCT and in their donors; then, the association of the genetic polymorphisms on acute and chronic GVHD was evaluated. Median follow up of patients was 40 months [range of 28-77 months]. Statistical analyses were performed using Chi-square test and SPSS software. Mutation incidence were the same between donors and recipients as 12.1%. In three of the patient-donor pairs [2.4%] SNPs occurred in both resulting in an overall frequency of 21.8% in patient-donor pairs. There weren't any significant differences between aGVHD and cGVHD incidence rates when donor/recipient pairs with SNPs were compared with the pairs without SNPs. aGVHD and cGVHD incidence rates in the former pairs were 52% and 56% and in the latter pairs 50.5% and 55%, respectively. No impact of NOD2 SNPs on incidence of acute and chronic GVHD was observed. Further studies are required to ascertain whether the findings of this study can be extended to other disease groups. In addition, further studies are required to identify the relevance of other SNPs

[ effect of arsenic trioxide treatment on mitochondrial apoptotic gene expression in acute promyelocytic leukemia cell line]

Acute promyelocytic leukemia [APL] is one of the most malignant forms of acute leukemia with a fatal course of only weeks which represents 10-15% of AML in adults. Arsenic trioxide as a single agent factor [without chemotherapy] is the treatment of choice for APL patients; it induces cell death through apoptosis but the mechanism by which arsenic targets apoptosis and dramatically affects gene expression remains poorly understood. Since arsenic is used as first line treatment in Iran, it is worth investigating its effect on expression of genes involved in APL. In this descriptive study, to understand the underlying mechanisms of cell death induction by arsenic, we treated NB4 cell line in a dose and time dependent manner. Extracting RNA and synthesis of cDNA, gene expression of apoptotic genes in mitochondrial pathway including caspase3, Mcl-1 and Bcl-2 was analyzed through Real-Time PCR. Our findings showed that As[2]O[3]-induced cell death was paralleled by reduced expression of the antiapoptotic protein Bcl-2 but the expression of Caspase3 and Mcl-1 did not change after arsenic treatment. These results suggest that changes in Bcl-2 gene expression may be one of the mechanisms of action of arsenic in induction of apoptosis, while Caspase3 and Mcl-1 gene expression are not affected by arsenic at the transcriptional level

Results of hematopoietic cell transplantation in pediatric leukemia

Hematopoietic cell transplantation [HCT] is an accepted treatment for acute myeloid leukemia [AML] in first remission, the treatment of choice for chronic myeloid leukemia [CML] and high risk groups of ALL who relapse with conventional chemotherapy. We assessed results of HCT for pediatric leukemia in our center. A total of 92 children, 63 with diagnose of AML, 23 with ALL and 6 with CML received allogeneic transplantation from HLA full matched siblings [57.6%] and autologous transplantation [42.4%]. Source of hematopoietic cells were peripheral blood 83.7%, bone marrow 15.2% and cord blood 1.6%. The median transplanted nucleated cells were 6.4 +/- 4.7 X 10[8] /Kg [body weight of patients] and mononuclear cells were 5.5 +/- 2.9 X 10[8]/Kg. The most common conditioning regimens were cyclophosphamide + busulfan. Prophylaxis regimen for GVHD was cyclosporin +/- methotrexate. GVHD occurred in 50 [54.3%] patients. Eighty five of children had engraftment, 26 [28.6%] relapsed and 57 [62%] are alive. The most common cause of death was relapse [68.6%]. Five years overall survival of patients with AML and ALL were 49% and 44% respectively and disease free survival of them were 52% and 49%. One year overall survival and disease free survival of CML was 57%. Overall survival increased with increasing age of patients at transplantation time [P = 0.06]. Longer survival significantly related to earlier WBC and platelet recovery [P < 0.0001 and P = 0.006 respectively]. Considering acceptable overall and disease free survival of patients after HCT, we concluded that is a good modality in treatment of leukemia of children

CMV detection and monitoring in bone marrow transplant [BMT] recipients by Real-Time PCR [RQ-PCR]

Cytomegalovirus [CMV] is an important pathogen in patients undergoing bone marrow transplantation. The prevalence of CMV varies from 30-100% in different countries as shown by seroepidemiological studies. Only 20-25% of patients develop CMV disease. Because of the similarity between CMV and GVHD and the different therapies required, detection of viral load will be effective in patients' survival. 51 recipients of BMT were monitored for 100 days post-BMT during which the samples were collected weekly. The Real-Time PCR was developed for quantitation of CMV viral DNA, using TaqMan tecnnology. For generation of standard curve, UL83 gene from CMV was cloned into a plasmid using a T/A cloning procedure. RQ-PCR assay was preformed in parallel with pp65 antigenemia assay on 415 samples. The results obtained by both techniques were significantly correlated [p < 0.01]. We could detect 13x10[1] -15x10[7] [CMV DNA copies/2x10[5] cells] by RQ-PCR method. About 76% of patients developed at least one episode of CMV reactivation. First positive result of RQ-PCR appeared 13 days earlier than of pp65 antigenemia. After preemptive therapy, 16 days were required to achieve negative result by RQ-PCR. Both assays were highly correlated; however, RQ-PCR was more sensitive than the antigenemia assay. After preemptive therapy, negative results of RQ-PCR were the best indicator to determine the endpoint of treatment and its success

[Cross-resistance to anticancer drugs in acute myeloblastic leukemia Pgp expressing K562 cell line]

Drug resistance remains one of the most important clinical obstacles in the treatment of some cancers. This drug resistance referred to as Multidrug Resistance [MDR] induces cross-resistance to many chemotherapy agents such as anthracyclines, vinca alkaloides, epipodophyllotoxins and Taxol. MDR is most likely due to the reduction of drug accumulation with an energy-dependent drug efflux pump. This drug pump is a 170 kDa transmembrane glycoprotein [Pgp]. We developed a resistance subline of K562 by stepwise increase in concentration of Doxorubicin, and Pgp expression was verified by flowcytometry and RT-PCR methods. Cross resistance of the resistant cell line to Etoposide, Vincristine and Taxol was analyzed by MTT assay. IC[50] [the level of drug concentration inhibiting 50% of cell growth] of Doxorubicin, Etoposide and Taxol of parental K562 came out to be 100ng/ml and it was 50 ng/ml for vincristine. IC[50] levels of these drugs on resistant K562 were 500, 500, 450 and 450ng/ml. These drugs also displayed 5-, 5-, 4.5-, and 9- fold resistance respectively. According to the results, expression of Pgp confers MDR phenotype to the K562 cell line and makes it resistant to most of anticancer drugs including anthracyclines, vinca alkaloides, epipodophyllotoxins and taxans. This MDR phenotype is a major obstacle of cancer treatment and in recent years investigators are trying to reverse it by gene therapy

[Assessment of Wilms tumor gene [WT1] in acute myeloid leukemia patients as a new marker to detect minimal residual disease [MRD]]

WT1 gene encodes a transcription factor that is involved in differentiation and proliferation of hematopoietic precursor cells as well as some other tissues like kidney, ovary, heart, etc. Quantitative assessment of WT1 gene expression is proposed as a useful marker in MRD detection and leukemia management. To assess the relevance of this gene, we analysed peripheral blood mononuclear cells of 62 AML patients [new cases] for the expression level of WT1 mRNA using Real-time quantitative RT-PCR. We followed the analysis up to 3 years, depending on patient availability. This study as a fundamental and applicable one was done cross-sectionnaly. Samples were obtained randomly from the AML patients referred to the BMT center, and selection was based on the diagnostic criteria defined by clinical wards. WT1 expression in MNCs of patients was compared with 24 healthy individuals [K562 cells considered to express WT1 gene equivalent to 10[6]]. Samples for diagnosis showed significantly high levels of WT1 expression [>80%]. After chemotherapy, its expression decreased [diminished about 1-2 log within induction therapy and around 3-4 log after consolidation therapy]. There was a noticeable correlation between the relative expression levels of WT1 and prediction of relapse [lower than gray zone versus higher than]. Patients whose WT1 expression levels remained lower than the gray zone benefit from a better compete remission. On the contrary, 1-6 months prior to overt clinical relapse in 6 patients, their WT1 expression raised to levels upper than gray zone. This study revealed that WT1 is a useful marker for detecting minimal residual disease, assessing chemotherapy effects, and predicting relapse in AML patients

Evaluation of the best condition for ex vivo expansion of hematopoietic stem cells for the propose of cord blood transplantation

Umbilical cord blood [CB] has been identified as a rich source for hematopoietic stem cells [HSCs], and has provided an alternative to bone marrow transplantation. The use of ex vivo expanded cells has been suggested as a possible means to accelerate the speed of engraftment in cord blood [CB] transplantation. The main aim of our study is to find the best culture media and condition to increase number of CD34+/CD38- hematopoietic stem cells in cord blood for transplantation. Mononuclear cells [MNCs] were seperated from cord blood and cultured in RPMI1640 with 10% fetal calf serum [FCS] or 10% cord blood plasma [CBP] or serum free media [SF]. Culture media contained 50ng/ml of Interlukin 6 [IL6], IL3, Thrombopoietin [TPO] Stem cell factor [SCF] and flt3-ligand. Cells were cultured for two weeks and number of CD34+/CD38- cells and total MNCs measured at days 0, 7 and 14. At 14 days culture mean fold of expansion of CD34+ and CD34+/CD38- cells was 20.4 and 57.4 for FCS, 5.6 and 10.3 for SF and 10.8 and 4.7 for CBP culture media. Due to efficacy and predictability of SF media for cell expansion and because of its better safety for allergic reactions and microbial contamination [in comparison to animal products containing media] and enough expansion for clinical applications, we suggest that SF media is better than CBP or SF from clinical points of view

Frequency of BCR-ABL fusion transcript in Iranian patients with chronic myeloid leukemia

Reverse transcriptase-polymerase chain reaction [RT-PCR] assay is a useful tool for the detection of fusion transcript resulting from specific chromosomal translocation of the leukemia cells. A specific chromosomal abnormality, the Philadelphia chromosome [Ph], is present in 90% to 95% of CML patients. The aberration results from a reciprocal translocation between chromosome 9 and 22, creating a BCR-ABL fusion gene. There are two major forms of the BCR/ABL fusion gene, involving ABL exon 2, but including different exons of BCR gene. The transcripts b2a2 or b3a2 code for a p210 protein. Another fusion gene leads to the expression of an e1a2 transcript, which codes for a p190 pro-tein. Another, less common fusion genes are b3a3 or b2a3 [p203] and e19a2 [p230]. The incidence of one or other rearrangement in chronic myeloid leukemia [CML] patients varies in different reported series. In general, fusion transcripts are determined individually, a process which is labor intensive in or-der to detect all major fusion transcripts. This study was designed to determine the frequency of different fusion genes in 75 iranian patients with CML. peripheral blood samples were analyzed by multiplex reverse transcriptase poly-merase chain reaction [RT-PCR] from adult patients to detect all types of BCR-ABL transcripts of the t [9:22] and found that all cases were positive for some type of BCR/ABL rearrangement. Most of our patients showed b3a2 fusion gene [62%], while the remaining showed one of the transcripts of b2a2, b3a3, b2a3, e1a2 or coexpression of b3a2 and b2a2. The rate of coexpression of the b3a2 and b2a2 was 5%. In contrast to the other reports, we did not see any coexpression of p210/p190. This may reflect either the sensitivity of the detection techniques used or the possibility of genetic differences be-tween the populations studied. Coexpression may be due to alternative splicing or to phenotypic varia-tion, with clinical course different from classical CML

Quantitative analysis of Epstein - Barr virus DNA load in bone marrow transplant recipients by using real-time PCR

Quantification of Epstein - Barr virus [EBV] in peripheral blood mononuclear cells [PBMNC] of allogenic bone marrow transplant [BMT] recipients is important because EBV-associated posttransplant Lymphoproliferative disease [PTLD] can occur after transplantation due to immunosup-pression therapy. To this end we chose Real-Time PCR using TaqMan probe. For the standard curve, we cloned BALF5 gene of EBV into a plasmid vector. After purification of the EBV-clone and calculation of plasmid copy number, the standard curve was constructed by using serial dilution of the plasmid clone. We were able to detect from 2 to 107 copies per 2x105 PBMNC with wide linear range. The mean EBV DNA copy number was 103.7 copies per 2x105 PBMNC. In this study, No patient of 35 BMT recipients [275 PBMNC samples] developed PTLD during five months follow up post transplant. EBV copy numbers in 22 samples [3 patients] out of 35 BMT recipients were higher than cut off value with symptoms like fever and pulomonary noddes [9%]. The virus load in one patient in the last sample obtained was 72400 copies. We detected low levels of EBV DNA in 20 BMT patients [57/1%]. Real-Time PCR is useful to measure virus load in PBMNC. Detection of EBV in PBMNC samples may be valuable predictive marker to prognosis PTLD. Further studies need to determine ac-curate viral cut off value for treatment patients at risk for PTLD

Effects of chimerism on graft versus host disease recurrence and survival after HLA identical marrow transplantation in Iran

The co-existence of recipient's hamatopoietic systems after allogeneic marrow transplantation is called mixed chimerism. Chimerism analysis provides a national method of different conditioning regimens, graft-versus-host disease [GVHD], prophylactic regimens, and cellular therapy to promote engraftment. The association of mixed chimerism with acute graft-versus-host disease [GVHD], disease recurrence, survival, and relapse free survival was investigated in 91 patients 12 and 79 of whom underwent either bone or peripheral blood HLA-identical marrow transplantation respectively. Chimerism was assessed using multiplex amplification of shorty tandem repeats [STR-PCR].cases included thalassemics [19 subjects], AML [29], ALL [20], CMT [18] and others [5].Median age was 21 [age range of 3-50]. There were 38 females [41.8%] and 53 males [58.2%]. Conditioning was busulfan plus cyclophosphamide in 34 patients, busulfan plus fludarabin in 51 patients and busulfan plus fludarabin plus anti-thymocyte globulin in 6 patients. Median of follow up was 13 months. Data was analyzed using SPSS statistical software. On day 30 after transplantation, mixed chimerism [MC] was observed in 15 patients [16.5%], complete donor chimerism [CC] in 72 patients [79%], and no chimerism in 4 patients. The incidence of acute GVHD was significantly lower in mixed chimeras that in complete chimeras [p=0.01] but there was no significant difference in acute GVHD grade [I, II vs. III, IV] between two groups. The incidence of relapse and overall survival were 17.6% and 88.9% respectively showing no significant difference between MC and CC. Relapse free survival was 80.2% and significantly different between two groups. Despite some previous reports, we found no significant difference in survival and relapse rate between MC and CC. Relapse free survival was 80.2% and not significantly different between tw ogroup

[Enrichment of intact peripheral blood dendritic cells by immunomagnetic depletion]

Introduction: Enrichment of intact peripheral blood dendritic cells [DCs] has been done using rosette and immunomagnetic depletion techniques

Material and Methods: Peripheral blood mononuclear cells were separated using ficoll density gradient centrifugation and T lymphocytes were depleted through reaction with AET- treated sheep red blood cells. The remaining cells were depleted from lineage marker positive cells using monoclonal antibodies against CD3, CD11b, CD14, CD16, CD19, CD56 and anti- mouse IgG magnetic beads. Flowcytometric analysis was used to determine the purity of the obtained DCs through HLA-DR expression and lack of lineage markers

Results: The obtained results showed that a significant population of isolated cells [58.5% +/- 4.5, n=5] fail to react with anti-lineage marker monoclonal antibodies but express the HLA-DR antigen

Conclusion Immunomagnetic depletion is a new method for DCs separation that unlike classic methods, can isolate DCs in intact form. According to our information, this is the first report of DCs separation in Iran and purity of the obtained DCs is comparable with similar studies in other countries. Significant enrichment of intact DCs could facilitate the study of DCs natural functions in different fields such as autoimmunity, cancers and infectious diseases