Zuzana Hassan

The effect of busulphan on the pharmacokinetics of cyclophosphamide and its 4-hydroxy metabolite: time interval influence on therapeutic efficacy and therapy-related toxicity

Busulphan and cyclophosphamide (Bu/CP) are widely used in preparative regimens for bone marrow transplantation. Many studies have shown a wide variation in busulphan pharmacokinetics. Moreover, higher rates of liver toxicity were reported in Bu/CP protocols than in a total body irradiation (TBI)-containing regimen. In the present paper we investigated the effect of the time interval between the last dose of busulphan and the first dose of cyclophosphamide on the pharmacokinetics of CP and its cytotoxic metabolite 4-hydroperoxycyclophosphamide (4-OHCP). Thirty-six patients undergoing bone marrow transplantation (BMT) were included in the study. We also investigated the occurrence of veno-occlusive disease, mucositis and graft-versus-host disease. Ten patients conditioned with CP followed by TBI served as a control group (TBI). Twenty-six patients were conditioned with Bu/CP. The patients received Bu (1 mg/kg × 4 for 4 days), followed by CP (60 mg/kg for 2 days) administered as a 1-h infusion. Patients received their CP therapy either 7–15 h (group A, n = 12) or 24–50 h (group B, n = 14) after the last dose of Bu. None of the patients were given phenytoin or any other drug known to enhance CP metabolism. The administration of CP less than 24 h after the last dose of Bu resulted in: (1) a significantly (P = 0.003) lower clearance for cyclophosphamide was observed in group A (0.036 l/h/kg) compared to 0.055 and 0.055 l/h/kg, in the B and TBI groups, respectively; (2) significantly (P = 0.002) longer elimination half-life in group A (10.93 h) than in groups B and TBI (6.87 and 7.52 h, respectively); (3) significantly (P < 0.001) lower exposure to the cytotoxic metabolite (4-ohcp), expressed as the ratio auc4-OHCP/AUCCP, in group A (0.0053) than that obtained in group B (0.013) and group TBI (0.012); (4) the patients in group A had a significantly (P < 0.05) higher incidence of vod (seven of 12) than the other groups, b and tbi (2/14 and 1/10, respectively); and (5) mucositis was significantly higher in group a patients (8/12), being seen in only one patient in group b and none in the tbi group. the present study has shown that the interval between busulphan and cyclophosphamide administration can negatively affect the pharmacokinetics of cyclophosphamide and its cytotoxic metabolite. we conclude that the timing of cp administration must be considered in order to improve drug efficacy and reduce conditioning-related toxicity. Bone Marrow Transplantation (2000) 25, 915–924.

Improved Survival after Allogeneic Hematopoietic Stem Cell Transplantation in Recent Years. A Single-Center Study

We analyzed the outcome of allogeneic hematopoietic stem cell transplantation (HSCT) over the past 2 decades. Between 1992 and 2009, 953 patients were treated with HSCT, mainly for a hematologic malignancy. They were divided according to 4 different time periods of treatment: 1992 to 1995, 1996 to 2000, 2001 to 2005, and 2006 to 2009. Over the years, many factors have changed considerably regarding patient age, diagnosis, disease stage, type of donor, stem cell source, genomic HLA typing, cell dose, type of conditioning, treatment of infections, use of granulocyte-colony stimulating factor (G-CSF), use of mesenchymal stem cells, use of cytotoxic T cells, and home care. When we compared the last period (2006-2009) with earlier periods, we found slower neutrophil engraftment, a higher incidence of acute graft-versus-host disease (aGVHD) of grades II-IV, and less chronic GVHD (cGHVD). The incidence of relapse was unchanged over the 4 periods (22%-25%). Overall survival (OS) and transplant-related mortality (TRM) improved significantly in the more recent periods, with the best results during the last period (2006-2009) and a 100-day TRM of 5.5%. This improvement was also apparent in a multivariate analysis. When correcting for differences between the 4 groups, the hazard ratio for mortality in the last period was 0.59 (95% confidence interval [CI]: 0.44-0.79; P < .001) and for TRM it was 0.63 (CI: 0.43-0.92; P = .02). This study shows that the combined efforts to improve outcome after HSCT have been very effective. Even though we now treat older patients with more advanced disease and use more alternative HLA nonidentical donors, OS and TRM have improved. The problem of relapse still has to be remedied. Thus, several different developments together have resulted in significantly lower TRM and improved survival after HSCT over the last few years.

Population pharmacokinetic analysis resulting in a tool for dose individualization of busulphan in bone marrow transplantation recipients.

The aims of the present study were (1) to investigate and quantify the pharmacokinetics, including inter-occasion variability and covariate relationships, of busulphan in BMT patients and (2) to develop a user-friendly initial dosing and therapeutic drug monitoring (TDM) strategy for the treatment of those patients with busulphan. The pharmacokinetics of busulphan was studied in 64 adults and 12 children who received busulphan (1 mg/kg) four times daily for 4 days. A one-compartment model with first order absorption and a lag time was sufficient in describing the concentration-time profile. Oral clearance (CL/F) was found to be correlated to weight (+1.2%/kg), ALT (−13%/μcat/l) and concomitant phenytoin treatment (+21%). CL/F and the volume of distribution (V/F) were estimated to 9.23 l/h and 39.3 l, respectively, in a typical individual. Inter-occasion variability (9.4%) in CL/F was estimated to be less than inter-individual variability (28%), a prerequisite for the value of TDM. Bayesian CL/F estimates based on three samples were in good accordance with those based on all samples. The final population model was implemented into the program Excel. The resulting flexible and easy to use dosing program might be used for both initial and, requiring only three plasma samples, maintenance dose individualization of busulphan therapy. Bone Marrow Transplantation (2001) 28, 657–664.

Simultaneous Determination of Busulphan in Plasma Samples by Liquid Chromatography‐electrospray Ionization Mass Spectrometry Utilizing Microextraction in Packed Syringe (MEPS) as On‐line Sample Preparation Method

Abstract Busulphan is an alkylating agent used in high doses as preparative regimen before stem cell transplantation (SCT). Busulphan has a narrow therapeutic window and under‐ or overdosing may have a fatal outcome for the patient. Therapeutic drug monitoring followed by dose adjustment is currently used to adjust the exposure to busulphan. This is an important issue to optimise and individualise high dose therapy with busulphan. However, this approach is limited to centres with experienced personal measuring busulphan plasma concentrations. An automated and easy‐to‐handle method for measurement of busulphan plasma concentrations may facilitate and wide‐spread drug monitoring approach and thus improve the outcome of the patients undergoing SCT. Microextraction in packed syringe (MEPS) in combination with liquid chromatography and electrospray ionization mass spectrometric detection (LC‐MS/LC‐MS‐MS) to quantify busulphan in human plasma samples without derivatization was developed. MEPS is a new miniaturised, solid‐phase extraction technique that can be connected on‐line to GC or LC without any modifications. In MEPS approximately 1 mg of the solid packing material is inserted into a syringe (100–250 µL) as a plug. The validation of the method showed that the selectivity, accuracy and precision for the method were satisfactory. This is well in line with the international criteria for the study validation. The present method has shortened extraction time considerably and the method is fully automated, which benefits therapeutic drug monitoring of busulphan in SCT.

The effect of modulation of glutathione cellular content on busulphan-induced cytotoxicity on hematopoietic cells in vitro and in vivo.

Busulphan is used in conditioning regimens prior to SCT. A relationship between exposure to busulphan, expressed as an area under the plasma concentration time curve (AUC), and effect and/or adverse effects, such as veno-occlusive disease (VOD), was reported. Exhaustion of glutathione (GSH) contributes to VOD and modulation of intracellular levels of GSH influences bulsulphan-induced toxicity in hepatocytes. Thus, increase of GSH might serve as prophylaxis against VOD. However, it should not interfere with the myeloablative effects of busulphan. We investigated the relationship between exposure to busulphan, and its in vitro toxicity to CD34+ hematopoietic progenitors from volunteers using clonogenic assays. Busulphan inhibited colony formation by CD34+ cells in an AUC-dependent manner. Myeloid progenitors were more sensitive than erythroid progenitors, expressed as 100% inhibition of colony formation (68.7 ± 7.5 μg.h/ml and 140.3 ± 35.7, respectively). The observed exposure corresponds to the total AUC obtained in patients treated with busulphan (1 mg/kg/day) for 4 days. Secondly, we studied the effect of modulation of GSH cellular levels on busulphan-induced toxicity in vitro in CD34+ cells from volunteers, and in vivo in bone marrow cells from Balb/c mice. The intracellular concentration of GSH was increased or decreased by treatment with N-acetylcysteine or buthionine sulfoximine, respectively. Neither in vitro nor in vivo treatment with GSH modulators affected the hematological toxicity of busulphan. Thus, N-acetylcysteine would not interfere with the myeloablative effect of busulphan and therefore it is a potential candidate for VOD prophylaxis during busulphan-based conditioning regimens.

GVHD after chemotherapy conditioning in allogeneic transplanted mice

GVHD is a major complication in allogeneic SCT. Available GVHD models are mainly based on radiotherapy-conditioning and/or immune deficient mice. GVHD models based on chemotherapy-based regimens remain poorly studied, despite 50% of all transplantations being chemotherapy based. Our aim was to develop a GVHD model using chemotherapy as conditioning. Female BALB/c (H-2Kd) were conditioned with BU–CY and transplanted with 2 × 107 BM and 3 × 107 spleen cells from either C57BL/6 (H-2 Kb) mice (allogeneic setting) or from male BALB/c to serve as a control group for regimen-related toxicity and engraftment. GVHD manifestations and histopathological changes were evaluated. Chimerism and donor T cells presence in skin, intestine and liver were studied using FACS-, FISH analysis and immunohistochemistry. Allogeneic transplanted mice developed lethal GVHD starting from day+7 with both histological and clinical signs. Donor T cells accumulated in recipient skin and intestine with GVHD progression. BM-failure, apoptosis and T-lymphocyte infiltration into target organs were significantly higher in allogeneic when compared with the syngeneic group. No toxicity or GVHD signs were observed in the syngeneic setting. We report a mouse model of GVHD using BU–CY conditioning that represents the most common myeloablative-conditioning regimen in clinical SCT. This model can be utilized to study the role of conditioning on mechanisms underlying GVHD.

Risk factors for acute graft-versus-host disease grades II–IV after reduced intensity conditioning allogeneic stem cell transplantation with unrelated donors—a single centre study

We analysed factors associated with moderate to severe acute GVHD in 111 patients treated with fludarabin-based reduced intensity conditioning (RIC) and allogeneic haematopoietic stem cell transplantation (HSCT). Most patients had a haematological malignancy. Donors were 97 HLA-A, -B and -DRβ1 identical unrelated and 14 HLA-A, -B or -DRβ1 allele mismatched unrelated donors. In the univariate analysis, we found ten factors associated with acute GVHD. These were diagnosis (P=0.06), GVHD prophylaxis with combinations other than CsA+MTX (P=0.006), graft nucleated (P<0.001) and CD34 (P<0.001) cell-dose, bidirectional ABO mismatch (P=0.001), conditioning (P=0.002), hospital vs home-care (P=0.06), ATG dose (P<0.001), donor herpes virus serology (P=0.07) and an immunized female donor to male recipient (P=0.05). In the multivariate analysis, three factors remained significant: a high CD34 cell dose (P<0.001), low dose (4 mg/kg) ATG (P<0.001), and an immunized female donor to male recipient (P<0.01). Patients receiving a CD34 cell dose ⩾17.0 × 106 per kg had a higher incidence of GVHD, 53.7%, compared to 22.3% in patients receiving a lower dose (P=0.002). In patients without any of these risk factors (n=70), the incidence of acute GVHD was 14.1%, while it was 38.0 and 85.0% in patients with one (n=29) or two (n=10) risk factors (P<0.001). We concluded that risk factors for acute GVHD using RIC are similar as using myeloablative conditioning.

A prospective randomized study using N-acetyl-L-cysteine for early liver toxicity after allogeneic hematopoietic stem cell transplantation

Allogeneic hematopoietic stem cell transplantation (ASCT) and its conditioning with chemoradiotherapy often results in liver toxicity, the most severe form being veno-occlusive liver disease (VOD). N-acetyl-L-cysteine (NAC), an antioxidant glutathione precursor, may provide protection from liver toxicity. Patients with elevated bilirubin (>26 mmol/l) and/or elevated (ALT) (>1.4 μkat/l) and/or aspartate aminotransferase (AST) (>1.4 μkat/l) levels were randomized to treatment with NAC or no treatment. Among 522 transplanted patients, 160 were included in the trial. NAC was given, 100 mg/kg per day, as a 6-h i.v. infusion until normalization of bilirubin, ALT and AST values. Maximum bilirubin level was the same in patients randomized to NAC (n=72) or controls (n=88). Increase and recovery of ALT and AST were the same in patients randomized to NAC or controls. There were two patients in the NAC group who developed VOD, as compared to three of the controls. To conclude, NAC does not improve liver toxicity after ASCT.

A phase II trial of liposomal busulphan as an intravenous myeloablative agent prior to stem cell transplantation: 500 mg/m 2 as a optimal total dose for conditioning

We conducted a phase I/II trial, to evaluate the efficacy and safety of an intravenous liposomal formulation of busulphan (LBu) as a myeloablative agent for stem cell transplantation (SCT). The liposomal busulphan was administered as a 3 h infusion twice daily over 4 consecutive days. Six adults received 1.6–2 mg/kg/dose and 18 children received 1.8–3 mg/kg/dose. Pharmacokinetic parameters were studied after the first and the last dose of busulphan. No significant difference in clearance, AUC, elimination half-lives or distribution volume between the first and the last dose was found in either groups. A significantly (P < 0.005) higher clearance was observed in children after the first and the last dose (3.61 and 3.79 ml/min/kg, respectively) compared to adults (2.40 and 2.33 ml/min/kg, respectively). The elimination half-lives after the first and the last dose were significantly (P < 0.005) shorter in children (2.59 and 2.72 h, respectively) compared to adults (3.35 and 3.61 h, respectively). Clearance correlated significantly with age. However, no significant correlation with age was observed when clearance was adjusted to the body surface area. Two cases of VOD following a total dose of 24 mg/kg were observed. Six patients experienced mucositis. No other organ toxicity was observed. We conclude that intravenous liposomal busulphan pharmacokinetics is age dependent. A dosage schedule based on body surface area should be used especially in young children to reduce the age-dependent difference in kinetics. An intravenous liposomal dose of busulphan of 500 mg/m2 is suggested to reach a similar systemic exposure and myeloablative effect in both children and adults. Moreover, the novel liposomal form of busulphan showed a favorable toxicity profile and seems safe as a part of the high-dose therapy prior to SCT.

Management of refractory hemorrhagic cystitis following hematopoietic stem cell transplantation in children

Hassan Z. Management of refractory hemorrhagic cystitis following hematopoietic stem cell transplantation in children.
Pediatr Transplantation 2011: 15: 348–361.