Busulfan Administration Research Articles

Biosynthesized poly(3-hydroxybutyrate-co-3-hydroxyvalerate) as biocompatible microcapsules with extended release for busulfan and montelukast

An extended release dosage form based on encapsulating the challenging drug busulfan within microspheres of the biodegradable, biocompatible and biosynthesized poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) polyester was achieved. The used (PHBV) polymer was biosynthesized by the halophilic archaeon Haloferax mediterranei from date waste biomass as feed-stock. PHBV microspheres of 1.2–2.1 μm diameter were successfully fabricated and loaded with busulfan with an encapsulation efficiency of 29.2 ± 0.2%. In addition, PHBV microspheres of 1.5–3.5 μm diameter and loaded with montelukast sodium (MK) drug were also fabricated with an encapsulation efficiency of 16.0 ± 0.4%. The double-emulsion solvent evaporation method was used to fabricate the drug-loaded microspheres. The drug-loaded microspheres have been characterized by XRD, FTIR and SEM, and confirmed to be successfully fabricated. The drugs in vitro release profiles have shown extended release for up to 3 days in case of busulfan and 8 h in case of montelukast sodium. The in vitro release profiles for busulfan and montelukast suggest that these drug-loaded microcapsules can be efficiently used as new dosage forms to solve the current issues of busulfan administration protocols, and to introduce a new dosage form for montelukast with extended release performance.

Fixed-dose administration and pharmacokinetically guided adjustment of busulfan dose for patients undergoing hematopoietic stem cell transplantation: a meta-analysis and cost-effectiveness analysis.

This study aims to evaluate the efficacy, safety, and long-term cost-effectiveness of fixed-dose busulfan (Bu) administration and pharmacokinetically (PK) guided adjustment of Bu dose for patients who underwent hematopoietic stem cell transplantation. The efficacy and safety of both dosing strategies were compared using a systematic review and meta-analysis. A Markov model was used in estimating relevant cost and health outcomes from the perspective of the health system. The primary outcomes of interest were lifetime cost, quality adjusted life-years (QALYs) gained, and incremental cost-effectiveness ratio (ICER) in dollar per QALY gained. Results showed that progression-free survival and overall survival in the PK-guided group were higher than that in the fixed-dose group, and the PK-guided group was associated with low non-relapse mortality and relapse rate. In contrast to safety, the incidence of acute graft-versus-host disease (GVHD) was the same in the two groups (P > 0.05). Cost-effectiveness analysis showed that the QALY of the PK-guided group (12.8135 QALYs and $582,475.07) increased by 2.0609 relative to that in the fixed-dose group (10.7526 QALYs and $562,833.20), and the ICER was $9530.72/QALY. One-way and probability sensitivity analyses confirmed the reliability of the results. In conclusion, the PK-guided approach has higher efficacy and is safer.

Kolaviron, a flavonoid-rich extract ameliorates busulfan-induced chemo-brain and testicular damage in male rats through inhibition of oxidative stress, inflammatory, and apoptotic pathways.

Previous reports revealed that increased oxidative stress with up-regulated inflammatory proteins and apoptotic factors have serious implications in busulfan-induced chemo-brain and testicular damages. Hence, we investigated the potential reversal effects of kolaviron (KV), a neuro-active extract rich in flavonoids with proven anti-oxido-inflammatory and anti-apoptotic properties, on busulfan-induced oxidative damage, inflammatory proteins, and apoptosis in the brains and testes of male rats. In the treatment-regimen, animals in groups 1 and 2 had saline (10ml/kg/p.o./day) and dimethyl sulfoxide (DMSO; 10ml/kg/p.o./day), group 3 received KV extract (200mg/kg/p.o./day), group 4 was given busulfan (50mg/kg/p.o./day) and animals in group 5 were pretreated with busulfan (50mg/kg/p.o./day) successively for 56days in addition to KV extract (200mg/kg/p.o./day) from days 29-56. Non-spatial memory function was valuated with a novel-object recognition memory test. Thereafter, testicular and brain oxidative/antioxidant status, proinflammatory- and apoptotic-related proteins, testicular enzymatic markers were evaluated respectively. Kolaviron extract improved cognitive function by increasing exploration of novel-object of busulfan-treated rats. Kolaviron extract reversed busulfan-mediated increased malondialdehyde, 8-hydroxy-2'-deoxyguanosine, and decreased superoxide dismutase, catalase, glutathione, and glutathione-peroxidase in brains and testes as well as the testicular enzyme markers. Increased brain and testicular weights, and TNF-α, IL-1β, and NF-κβ productions due to busulfan administration were also reduced by the extract. The reduced testicular B-cell lymphoma-2, sperm mitochondrial cytochrome-C, and membrane potential, increased DNA fragmentation, caspases -3 and -9 levels were also profoundly reversed by KV. Additionally, KV extract ameliorated busulfan-induced testicular histopathological changes in rats. Conclusively, KV extract reverses busulfan-induced neuroendopathobiological derangements via oxidative stress inhibition, down-regulation of inflammatory and apoptotic mediators in rats' brains and testes. PRACTICAL APPLICATIONS: Busulfan is an orally effective chemotherapy drug widely used for cancer treatment. It has been reported that chronic usage of busulfan increases the tendency for carcinogenic and teratogenic activities with severe side effects on the functions of the gonads and other body organs such as brain, popularly regarded as chemo-brain. When taken over a prolonged period of time, busulfan causes sterility in animals and destroyed spermatogonial stem cells along with the seminiferous tubules and sperm morphology of animals as well as memory impairments. The findings from the study revealed that KV extract prevent busulfan-induced cognitive and testicular impairments following kolaviron supplementation. Thus, the findings from this scientific investigation suggest that KV extract could improve the quality of reproductive life and cognitive functions of male patients during busulfan chemotherapy, particularly during prolonged therapy.

Effects of single and multiple transplantations of human umbilical cord mesenchymal stem cells on the recovery of ovarian function in the treatment of premature ovarian failure in mice

BackgroundCurrently, there is no effective treatment for premature ovarian failure (POF), and stem cell therapy is considered the most promising treatment. Human umbilical cord blood mesenchymal stem cells (hUC-MSCs) have shown good regenerative ability in various diseases, including POF; however, their underlying mechanism and dosage for POF treatment remain unclear. This study aimed to compare the effect of single and multiple injections of hUC-MSCs on ovarian function repair in chemotherapy-induced POF.MethodsFemale mice were intraperitoneally injected with 30 mg/kg busulfan and 120 mg/kg cyclophosphamide (CTX) to induce POF. In the single hUC-MSC injection group, hUC-MSCs were transplanted into mice D7 after CTX and busulfan administration, while in the multiple injection group, hUC-MSCs were transplanted on D7, D14, and D21 after CTX and busulfan administration. We evaluated the ovarian morphology, fertility, follicle-stimulating hormone and estradiol concentrations, follicle count, POF model, and cell transplantation results. In addition, real-time polymerase chain reaction, immunohistochemistry, and miRNA and mRNA chips were used to evaluate the effect of the cell therapy.ResultsOvary size, number of follicle at all developmental stages, and fertility were significantly reduced in the POF group compared with the control. Under hUC-MSC treatment, the ovarian morphology and follicle count were significantly restored, and fertility was significantly increased. By comparing the single and multiple hUC-MSC injection groups, we found that the anti-Müllerian hormone and Ki-67 levels were significantly increased in the multiple hUC-MSC group on D60 after chemotherapy. The expression of stimulating hormone receptors, inhibin α, and inhibin β was significantly restored, and the therapeutic effect was superior to that of the single hUC-MSC injection group.ConclusionThese results indicate that hUC-MSCs can restore the structure of injured ovarian tissue and its function in chemotherapy-induced POF mice and ameliorate fertility. Multiple hUC-MSC transplantations have a better effect on the recovery of ovarian function than single hUC-MSC transplantation in POF.

Pharmacokinetically guided, once-daily intravenous busulfan in combination with fludarabine for elderly AML/MDS patients as a conditioning regimen for allogeneic stem cell transplantation.

The efficacy of pharmacokinetically (PK) guided, once-daily administration of busulfan (BU) was evaluated in elderly patients with acute myeloid leukemia/myelodysplastic syndrome (AML/MDS). Twenty-one patients (median age 61) received 30mg/m2 fludarabine for 6days and BU for 4days, starting from 3.2mg/m2 and subsequently adjusted to the target area under the curve (AUC) of 6000µmol-min/L. The median AUC of day 1 (AUC1), AUC4, and their average were 4871.3, 6021.0, and 5368.1µmol-min/L, respectively. Veno-occlusive disease/sinusoidal obstructive syndrome (VOD/SOS) occurred in five patients (24%) but all recovered well. Four patients (20%) had non-infectious pulmonary complications (NIPCs). Patients with high AUC1 had frequent gastrointestinal adverse events, but similar incidence of VOD/SOS and NIPCs. Two-year overall survival (OS), non-relapse mortality (NRM), and relapse rates were 44.4%, 28.6%, and 29.1%, respectively. Patients with high AUC1 had significantly high NRM (57.1% vs. 14.3%, P = 0.04) and inferior OS (14.3% vs. 60.1%, P = 0.002), while patients with high AUC4 had a significantly low relapse rate (8.3% vs. 55.6%, P = 0.02). In conclusion, once-daily BU and a PK-guided dose intensification were beneficial for reducing relapse in elderly patients with AML/MDS. However, caution should be exercised as rapid BU dose elevation may contribute to NRM.

Pulmonary hypertension associated with busulfan.

Busulfan is widely used to treat malignant diseases, particularly for therapeutic intensification prior to an autologous stem cell graft. Numerous side effects consecutive to busulfan are described, but few descriptions of pulmonary hypertension exist, while bronchiolitis obliterans remains a rare complication. We report the clinical observations of four patients from the French Pulmonary Hypertension Registry who experienced subacute pulmonary hypertension after receiving busulfan as preparation regimen before an autologous stem cell graft for malignancies (Hodgkin’s disease, Ewing’s sarcoma and primary large B cell lymphoma of the brain). Patients experienced severe pulmonary arterial hypertension 2 to 4.5 months after busulfan administration. Pulmonary hypertension improved after treatment with approved drugs for pulmonary arterial hypertension and/or corticosteroids. During the follow-up period, two patients developed chronic respiratory insufficiency due to interstitial lung disease, leading to double lung transplantation. The pathological assessment of explanted lungs revealed interstitial lung fibrosis with advanced bronchiolar lesions and severe pulmonary vascular damage. Three of the four patients were still alive after 36 to 80 months and the fourth died unexpectedly and suddenly after 5 months. In conclusion, PAH is a rare but severe complication associated with busulfan chemotherapy in adults. Histological examinations provide evidence for diffuse pulmonary vascular damage combined with interstitial lung injury in most cases.

ORAL MUCOSITIS – SIGNIFICANT COMPLICATION OF PEDIATRIC PATIENTS UNDERGOING HEMATOPOIETIC STEM CELL TRANSPLANTATION

Introduction. The prognosis of pediatric solid tumors has improved in recent years through the use of multimodal treatment – chemotherapy, local control (surgery and radiotherapy) and hematopoietic stem cell transplantation (HSCT) for high-risk forms. A limiting factor of these transplant procedures is short-term toxicity, oral mucositis (OM) being associated with potential severe evolution. The introduction of tandem stem cell transplant procedures, which increase the intensity of chemotherapy treatment and thus increase the response rate, is associated with increased toxicity. In this study we evaluated the impact of multiple transplant procedures on the severity and evolution of OM. Materials and methods. This retrospective study included 57 pediatric patients diagnosed with solid tumors (neuroblastoma, germ cell tumor, Ewing sarcoma, nephroblastoma) who underwent HSCT in the Pediatric Bone Marrow Transplantation Unit of Fundeni Clinical Institute, between January 2002 and December 2020. Patients were divided into 2 study groups: the first group included patients who had one HSCT procedure (43 patients) and the second group included patients who benefited from multiple HSCT procedures (14 patients). We evaluated the degree and average extent of OM, associated risk factors, duration of analgesic treatment and parenteral nutrition, as well as infectious risk. Conditioning regimens were based on administration of busulfan, thiotepa or ifosfamide. All patients received prophylactic antimicrobial therapy and followed a daily oral care protocol. Results. The 57 patients underwent 73 HSCT procedures for the following diagnoses: 36 neuroblastoma, 6 germinal cell tumor, 12 Ewing sarcoma and 3 Wilms tumor. Patients were diagnosed, staged and treated according to international protocols. The study highlights the influence on the degree of OM: delayed platelet engraftment and increased parenteral nutrition requirements correlated with the number of HSCT procedures per patient. Infectious risk was higher for patients from the second study group. Conclusions. OM treatment remains prevention-centered and identifying effective measures, encouraging modern treatment strategies and developing practical evidence-based guidelines are imperative.

Therapeutic Drug Monitoring of Busulfan in Patients Undergoing Hematopoietic Cell Transplantation: A Pilot Single-Center Study in Taiwan.

Busulfan has been used as a conditioning regimen in allogeneic hematopoietic cell stem transplantation (HSCT). Owing to a large inter-individual variation in pharmacokinetics, therapeutic drug monitoring (TDM)-guided busulfan dosing is necessary to reduce graft failure and relapse rate. As there exists no TDM of busulfan administration for HCT in Taiwan, we conducted a pilot study to assess the TDM-dosing of busulfan in the Taiwanese population; Seven patients with HCT from The Koo Foundation Sun Yat-Sen Cancer Center, Taipei, Taiwan, received conditioning regimens consisting of intravenous busulfan and other chemotherapies. After the initial busulfan dose, blood samples were collected for busulfan TDM at 5 min, 1 h, 2 h, and 3 h. Busulfan was extracted and detected by performing stable-isotope dilution LC–MS/MS. Plasma busulfan concentration was quantified and used for dose adjustment. Potential adverse effects of busulfan, such as mucositis and hepatic veno-occlusive disease (VOD), were also evaluated; The LC–MS/MS method was validated with an analyte recovery of 88–99%, within-run and between-run precision of <15%, and linearity ranging from 10 to 10,000 ng/mL. Using TDM-guided busulfan dosing, dose adjustment was necessary and performed in six out of seven patients (86%) with successful engraftments in all patients (100%). Mild mucositis was observed, and VOD was diagnosed in only one patient; This single-center study in Taiwan demonstrated the importance of busulfan TDM in increasing the success rate of HCT transplantation. It is also necessary to further investigate the optimal busulfan target value in the Taiwanese population in the future.

Use of Busulfan in Conditioning for Allogeneic Hematopoietic Stem Cell Transplantation in Adults: A Survey By the Complications and Quality of Life Working Party of the EBMT

Background : With the introduction of an intravenous formulation, options for the administration of busulfan in conditioning for allogeneic hematopoietic stem cell transplantation have increased. Practical issues in which the policies of transplant centers are likely to vary include the route of administration, number of daily doses, and the use of pharmacokinetic (PK) measurements for dose adjustment. Following the general policy of the European Society for Blood and Marrow Transplantation (EBMT) to aim at standardizing transplantation procedures, the Complications and Quality of Life Working Party has carried out a survey among EBMT centers about their practice in the use of busulfan for conditioning in allogeneic transplantation.Results : All allogeneic transplant centers reporting to the EBMT and treating adult patients were invited to participate. One hundred and nine centers (28 %) sent their report. One hundred and five of these centers used busulfan for conditioning, 4 did not. One hundred and one centers used it in full conventional doses, 92 centers in lower doses for reduced intensity conditioning (RIC). The oral dose in conventional conditioning was 16 mg/kg, the intravenous dose usually 12.8 mg/kg. Six centers determined the dose according to body surface area. The most common indications for conventional dose myeloablative conditioning were AML (99 centers), MDS (87), CML (75), ALL (56), lymphoma (26), thalassemia and other hemoglobinopathies (22), CLL (17), and myeloproliferative disorders (MPD, 6 centers). Lower doses of busulfan (RIC) were most commonly used for MDS (69 centers), AML (66), CML (49), ALL (37), lymphoma (36), CLL (31), and MPD (11 centers). The busulfan dose in RIC conditioning was usually 8 mg/kg in oral administration, in i.v. administration most often (66% of the centers) 6.4 mg/kg. Four centers determined the dose according to body surface area. Eleven centers gave full conventional dose busulfan orally and 94 i.v., while in RIC transplantations the drug was given orally in 10 and i.v. in 77 centers. Overall, 17 of the 105 centers used PK measurements to adjust busulfan doses. Busulfan concentration was measured using liquid chromatography (+ mass spectrometry in 8 centers). With one exception, the parameter used for dose adjustment was AUC. The details of the sampling for PK measurements as well as the method of AUC calculation varied. There was no difference between centers giving oral and those giving i.v. busulfan in the use of PK for dose adjustment, in full dose conditioning 2/11 vs. 14/94 centers, respectively. In RIC transplantations, PK-based dose adjustment was used in 1/10 centers giving oral busulfan and 8/77 using i.v. busulfan. The centers using oral busulfan or those adjusting the doses according to PK did not differ significantly from the rest of the centers in center experience, transplant numbers, JACIE accreditation, or gross national income of their country.Full conventional doses were always given in 4 days. Oral busulfan was given in 4 daily doses with one exception (2 doses). In i.v. administration, the number of daily doses was one in 46 centers, four in 42 centers, and two in 4 centers. In RIC conditioning, busulfan was in most cases given in 2 days, i.v. busulfan usually in one (35 centers) or four (28 centers) daily doses. Twenty-seven centers reported having alternative busulfan administration schedules depending on the disease, 20 centers depending on other components of the conditioning regimen. In myeloablative conditioning, 73 centers adjusted the dose of busulfan in obese patients, whereas 25 centers did not. Thirty-two centers reported defining obesity based on body mass index, whereas 28 centers used actual body weight in relation to ideal body weight. In obese patients, the busulfan dose was determined according to actual body weight (12 centers), ideal body weight (15), AIBW-25 (ideal body weight + 0.25 x (actual body weight - ideal body weight)) (46), AIBW-40 (10), or other (11 centers). In RIC, busulfan doses were adjusted because of obesity in 52 centers but not in 32 centers.Conclusions : There is marked variation between centers in the details of busulfan administration for conditioning in allogeneic transplantation. The clinical impact of this variation remains uncertain. The present results are useful in the production of recommendations toward a more standardized use of busulfan in the conditioning. DisclosuresMohty:Sanofi: Honoraria, Speakers Bureau.

Similar Survival but Increased Toxicity with a Sequential Versus Concurrent FluBu4 Regimen

Combined in vitro treatment with fludarabine and busulfan has been shown to lead to synergistic cytotoxicity. Maximal synergy is achieved when fludarabine is administered immediately prior to busulfan. However, fludarabine and myeloablative busulfan (FluBu4) has been administered in both a concurrent and a sequential, non-overlapping fashion. Because timed sequential regimens are becoming increasingly popular, we sought to compare survival and relapse outcomes as well as toxicity between patients receiving allogeneic stem cell transplant (SCT) with a concurrent 5-day versus a sequential 9-day FluBu4 regimen.We analyzed 102 consecutive patients who received FluBu4 prior to allogeneic SCT between 2003 and 2016. Thirty-seven (36%) patients received the 5-day regimen, which consisted of fludarabine 40 mg/m2 and intravenous busulfan (target AUC 4800 mmol-min per dose) administered daily from Day -5 to Day -2. In 65 (64%) patients, the 9-day regimen was administered, utilizing equivalent doses of fludarabine on day -9 to day -6 and busulfan on day -5 to day -2. All patients received graft-versus-host disease (GVHD) prophylaxis with tacrolimus and methotrexate. Patients undergoing a matched unrelated donor transplant received anti-thymocyte globulin.Baseline characteristics including gender, race, median time to transplant, median lines of prior treatment, disease risk index, hematopoietic cell transplantation-comorbidity index (HCT-CI), donor type, stem cell source, and mean CD34 count were similar between the two groups. Patients in the 9-day group were younger compared to the 5-day group (median age 45 versus 51 years, p=0.003). The majority of patients in both groups were diagnosed with acute myeloid leukemia, myelodysplastic syndrome, or acute lymphoblastic leukemia.The median time to neutrophil and platelet engraftment was not different between the two groups. More patients receiving the 9-day regimen experienced Grade 4 mucositis (53 versus 6 patients, p<0.0001). Thus, 23 (35.4%) patients in the 9-day group versus 5 (13.5%) patients in the 5-day group required a day +11 methotrexate dose reduction or omission (p=0.02). This resulted in a higher cumulative incidence of both Grade 2-4 (27% versus 38%, p=0.01) and Grade 3-4 (11% versus 34%, p=0.006) acute GVHD (aGVHD) in the 9-day group compared to the 5-day group. In a multivariate analysis, receiving the 9-day regimen predicted for a greater risk of grade 3-4 aGVHD (p=0.025). Additonally, extensive chronic GVHD was found to be higher in the 9-day FluBu4 cohort (19% vs. 40% p=0.047).Median one-year GVHD-free, relapse-free survival (GRFS) was significantly worse for patients receiving a 9-day FluBu4 regimen (68.4 versus 144 days, p=0.032). In a multivariate analysis adjusted for HCT-CI, a trend towards significance was observed with the 5-day regimen predicting for longer GRFS compared to the 9-day regimen (p=0.07). Notably, we observed no differences in overall survival, progression-free survival, or relapse rates between the 5- and 9-day regimen.This is the first study to compare outcomes between a concurrent 5-day and sequential 9-day FluBu4 regimen. We demonstrate similar survival and relapse rates, suggesting that concurrent administration of fludarabine and busulfan may not be necessary for synergistic cytotoxicity. At the same time, we observed increased early toxicity, less MTX administration, and higher aGVHD rates in patients receiving sequential 9-day FluBu4. Contrary to what has been observed with timed sequential regimens, our data suggests that a prolonged administration schedule of conditioning chemotherapy leads to greater toxicity resulting in higher GVHD rates. DisclosuresPatel:Celgene: Consultancy, Honoraria.

Safety and Efficacy of Once-Daily Intravenous Busulfan in Allogeneic Transplantation: A Matched-Pair Analysis

[Background] Intravenous administration of busulfan (ivBU) has been widely used in conditioning regimens for allogeneic hematopoietic stem cell transplantation (HSCT), especially in patients with myeloid malignancies. IvBU was originally administered 4-times-daily (ivBU4) at 0.8 mg/kg over 2 hours according to the established way of administration in oral busulfan, but ivBU4 needs frequent preparations and a midnight administration. A once-daily infusion of ivBU (ivBU1) at 3.2 mg/kg over 3 hours seemed to be safe and convenient by resolving these problems, but clinical data of direct comparison of ivBU1 and ivBU4 has been scarce. We demonstrated that ivBU1 was safely performed based on a pharmacokinetic profile in a small cohort of Japanese patients (Int J Hematol. 2015;101:497-504), and therefore we planned a large matched-pair analysis using the nation-wide registry data to demonstrate the safety and efficacy of ivBU1 in Japanese patients.[Methods] Patients aged more than 15 years with myeloid malignancies who underwent HSCT using ivBU1 in conditioning regimen were selected in our institutions. Control patients of ivBU4 matched for age, sex, performance status, disease risk, conditioning regimen, and donor type were selected from the Transplant Registry Unified Management Program (TRUMP), which is the registry data of the Japanese Society for Hematopoietic Cell Transplantation (JSHCT), using optimal matching algorithms in a 1:3 ratio. Patients who had ivBU for 2 days or 4 days were selected separately, and were combined when clinical outcomes were analyzed. Primary outcome measure was an overall survival after HSCT. This study was approved by the data management committee of TRUMP and the Institutional Review Board of Saitama Medical Center, Jichi Medical University.[Results] A total of 91 patients who received ivBU1 for 2 days (n = 18) or 4 days (n = 73) after 2010 in our institutions were matched with 273 control patients who received ivBU4 between 2007 and 2013 (Table 1). In patients who received ivBU for 2 days of both groups, ivBU was mainly combined with fludarabine (Flu). On the other hand, in patients who received ivBU for 4 days of both groups, half of them received ivBU combined with cyclophosphamide (CY), and the remaining patients did combined with Flu. Overall survival (1y OS: 56.8 % vs 57.1 %, P= 0.94, Figure 1), disease-free survival (1y DFS: 51.6 % vs 50.8 %, P= 0.73), relapse rate (1y RR: 28.5 % vs 26.2 %, P= 0.94), non-relapse mortality (1y NRM: 19.9 % vs 23.0 %, P= 0.71), and the incidence of graft-versus-host disease (grade 2-4 acute GVHD: 26.6 % vs 36.8 %, P= 0.07, chronic GVHD: 38.6 % vs 32.5 %, P= 0.46) were not significantly different between the ivBU1 and ivBU4 groups. In patients who received ivBU1, neutrophil recovery was slower (median days: 22 vs 17, P < 0.001), and incidence of veno-occlusive disease (VOD) was lower (2.6% vs 17.4%, P= 0.04) than in those who received ivBU4. The former might have resulted from the less frequent usage of granulocyte-colony stimulating factor (25.3 % vs 84.2 %, P < 0.01), and the latter from the different criteria used for the diagnosis of VOD (Seattle criteria vs Baltimore criteria). [Conclusion] IvBU1 can be safely administered in Japanese patients. Clinical outcomes were similar between ivBU1 and ivBU4 groups. [Display omitted] DisclosuresKako:Otsuka Pharmaceutical Co., Ltd.: Honoraria. Hidaka:Chugai: Research Funding; Eisai: Honoraria. Suzuki:Mochida Pharmaceutical: Honoraria; Novartis: Honoraria; Gilead Sciences: Consultancy; MundiPharma: Consultancy; Meiji Seika Pharma: Honoraria; Shionogi: Honoraria; Takeda Pharmaceuticals: Honoraria; Kyowa-Hakko Kirin: Honoraria; Ohtsuka: Honoraria; Chugai Pharmaceutical: Honoraria; Sawai Pharmaceutical: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Sumitomo Dainippon Pharma: Honoraria; MSD: Honoraria; Jazz Pharmaceuticals: Consultancy. Atsuta:Otsuka Pharmaceutical Co., Ltd.: Honoraria.

Amniotic fluid-derived exosomes improved spermatogenesis in a rat model of azoospermia

AimsThis study aimed to explore the therapeutic effects of amniotic fluid-derived extracellular vesicles including exosomes (AF-Exos) on the recovery of sperm production capacity in a rat model of azoospermia. Main methodsThe non-obstructive azoospermia (NOA) was induced in rats using intratesticular administration of Busulfan. Azoospermia was confirmed by testis histology. AF-Exos samples containing 10 or 40 μg exosomal proteins were injected into testicular tissue of NOA rats. After two months, the recovery of spermatogenesis was monitored via histopathological staining, spermiogram, and hormonal analysis. Immunohistochemistry staining for OCT-3/4 was used to identify of spermatogonial progenitors. The expression of DAZL and VASA, was also measured. Key findingsAF-Exos exhibited sphere-shaped morphology with the mean diameter and zeta potential of 50 ± 7.521 nm and −7.16 mV. Immunoblots revealed that isolated nanoparticles were CD63, CD9, and CD81 positive. Histopathological evaluation revealed that spermatogenesis was improved significantly in NOA rats after AF-Exos injection. Data showed that the sperm parameters and spermatogenesis index were significantly improved after AF-Exos injection compared to azoospermic groups. OCT-3/4+ cells were increased in NOA rats after AF-Exos injection, showing the restoration of spermatogenesis. In the present study, both doses of exosome (10 and 40 μg) restored the testicular function of NOA rats. DAZL and VASA were increased significantly in animals who received 40 μg exosomal protein compared to azoospermic rats. Except in a high dose of AF-Exos (40 μg) for Testosterone and FSH, no statistically significant differences were found regarding hormones post-exosome injection. SignificanceOur study demonstrated that AF-Exos regenerated spermatogenesis and improved sperm quality in NOA rats.

Human umbilical cord perivascular cells prevent chemotherapeutic drug-induced male infertility in a mouse model

To study whether intratesticular (IT) administration of 2 sources of human umbilical cord perivascular cells (HUCPVC), rich and potent sources of mesenchymal stromal cells (MSC), before chemotherapy can prevent infertility in a mouse model. Two control groups of CD1 male mice without busulfan (BUS) administration (untreated and IT media injection groups) were included. Experimental groups included IT administration of media, first trimester (FTM) HUCPVCs or term HUCPVCs (n = 5 each) injected 3 days before BUS treatment (20 mg/kg). All groups were included in a mating time course study over 6 months. Preclinical study in a fertility center research laboratory. Not applicable. IT delivery of FTM or term HUCPVC before BUS treatment. Pregnancies, litter sizes, and gross morphology of offspring were monitored. Caudal epididymal sperm concentration, motility, and progressive motility were assessed by computer-assisted sperm analysis. Spermatogenesis was also assessed histologically in testicular tissue sections. FTM and term HUCPVC displayed an MSC-associated immunophenotype and expressed transcripts encoding paracrine factors known to regulate the testicular cell niche. IT administration of FTM and term HUCPVC before chemotherapy promoted the recovery of spermatogenesis and fertility compared with BUS-treated animals that received a media injection. Although the total number of pups sired over 6 months by males treated with FTM or term HUCPVC was reduced compared with untreated or media-injected controls, litter size and sperm parameters in fertile animals did not differ between control and cell-treated groups. HUCPVC represent a promising source of MSC-based therapy to prevent gonadotoxic chemotherapeutic drug-induced infertility.

PSII-36 The application of busulfan to inhibit the spermatogenesis in quail testis

Abstract The use of testicular stem cells (spermatogonia) is of most interest for obtaining individuals with predetermined traits and genome genetic modification and for conservation of poultry gene pool. A significant population of mature donor germ cells (sperm) is formed upon successful spermatogonia cells transplantation into the testes of male recipients. Obtained sperm can be used to produce offspring with the desired traits. A key step in this technology is the removal of own spermatogenic cells (inhibition of spermatogenesis) in male recipients. The aim of research was to develop and optimize methodological approaches to inhibit the spermatogenesis in quail using busulfan. This drug was injected directly into the testes parenchyma of mature males by multiple injection at the concentration from 20 to 100 mg per 1kg of body weight (n = 25). Histological preparations of testes from the experimental quails were obtained to study composition of spermatogenic cells in the seminiferous tubules after busulfan administration. The male peers who were not injected with busulfan were used as a control. Experimental quails showed a decrease in the number of spermatogenic cells in the seminiferous tubules 32, 75, 111, 119 and 118 times compared with the control when using busulfan in concentrations 20, 40, 60, 80 and 100 mg/kg of weight, respectively (P &amp;lt; 0.001). The cells composition in the seminiferous tubules from experimental quails was represented mainly by Sertoli cells and spermatogonia. After busulfan introduction at the concentrations 20, 40, 60, 80 and 100 mg/kg, the percentage of spermatogonia was 55±5 %, 24±4 %, 6±2 %, 5±2 % and 4±1 %, respectively. The use of busulfan at the concentration of 80–100 mg/kg led to high mortality of quails. Thus, it was found that the optimal busulfan concentration for elimination of quail spermatogenic cells was 60 mg/kg. Supported by RFBR within Project №18-29-07079.

Prediction of Acute Graft versus Host Disease and Relapse by Endogenous Metabolomic Compounds in Patients Receiving Personalized Busulfan-Based Conditioning

Busulfan-based conditioning is the most commonly used high-dose conditioning regimen for allogeneic hematopoietic cell transplant (HCT). The alkylating agent busulfan has a narrow therapeutic index, with busulfan doses personalized to a target plasma exposure (targeted busulfan). Using a global pharmacometabonomics approach, we sought to identify novel biomarkers of relapse or acute graft versus host disease (GVHD) in a cohort of 84 patients receiving targeted busulfan before allogeneic HCT. A total of 763 endogenous metabolomic compounds (EMCs) were quantitated in 230 longitudinal blood samples before, during, and shortly after intravenous busulfan administration. We performed both univariate linear regression and pathway enrichment analyses using global testing. The cysteine/methionine pathway and the glycine, serine, and threonine metabolism pathway were most associated with relapse. The latter be explained by the fact that glutathione S-transferases conjugate both busulfan and glutathione, which contains glycine as a component. The d-arginine and d-ornithine metabolism pathway and arginine and proline metabolism pathway were most associated with acute GVHD. None of these associations were significant after correcting for false discovery rate (FDR) with a strict cutoff of FDR-adjusted p < 0.1. Although larger studies are needed to substantiate these findings, the results show that EMCs may be used as predictive biomarkers in HCT patients.

137 Recovery of quail spermatogenesis by donor spermatogonia transplantation

Spermatogonia, the stem cell precursors of male germ cells, are used as convenient biological material for the preservation of genetic resources (cryobanks) and the introduction of recombinant DNA (transgenesis). Donor spermatogonia subsequently differentiate into mature germ cells (spermatozoa), which are used to produce offspring. Our laboratory is investigating methods to improve the efficiency of spermatogonial germ cell transplantation in quail. The objectives of this study were to (1) determine the optimal age for spermatogonia isolation from the testes of donor male quail and (2) identify the most appropriate concentration of busulfan for treatment of recipient quail testes. Statistical analysis was performed using SPSS ver. 15.0 (IBM Corp.; analysis of variance test). In order to determine the optimal age for spermatogonia isolation, testes from male quail at 1-4 weeks of age were isolated and histological studies were performed on a population of spermatogenic cells in the seminiferous tubules. Histological studies of quail testes isolated at different ages showed that the optimal age for obtaining a culture of spermatogonia is a period from 1-2 weeks of age. During this period, spermatogenic cells were represented mainly by spermatogonia (P&amp;lt;0.01). Therefore, testes of 1-week-old quails were used to obtain a culture of spermatogonia. The resulting cell culture consisted mainly of spermatogonia (85%) with a small number of Sertoli cells. Next, a series of experiments introducing busulfan into quail testes was carried out using concentrations from 10-150mg kg−1 of liveweight. Experiments showed that an effective dose to remove the recipient male's own spermatogenic cells was a concentration of 100mg kg−1 of liveweight (P&amp;lt;0.05). Finally, using the optimal parameters described above, spermatogonia cultures were obtained and introduced into the testes of quail recipients (n=6), following administration of busulfan for 2-3 weeks before donor spermatogonia were introduced. The effectiveness of spermatogenesis recovery was assessed based on the analysis of sperm from quail recipients at 3 months after the injection of donor cells. The presence of donor germ cells in the testes of quail recipient drakes was confirmed by microsatellite analysis of DNA isolated from the blood and sperm of recipients as well as the donor cells (spermatogonia). The microsatellite profiles of the blood and sperm DNA in quail recipient males were different, which confirms the restoration of spermatogenesis in the studied recipients due to the development of donor germ cells. The reported study was funded by RFBR, project number 18-29-07079.

Crocin Reduces Oxidative Stress Produced by Busulfan Administration on Fertility of Male Rats

Crocin Reduces Oxidative Stress Produced by Busulfan Administration on Fertility of Male Rats

Preclinical Evaluation of ALS20, a New and Improved Lentiviral Vector for Beta-Globinopathies

The ongoing clinical trials, based on the use of lenti-globin vectors for beta-globinopathies, indicate that current vectors require high number of integrations (~3-4 copies per genome) in a pancellular fashion to make a patient transfusion independent, regardless of the genotype. This increased VCN requirement could increase the risk of genome toxicity, limiting the application of these vectors and preventing their use in a reduced myeloablative regimen. To overcome this limitation we designed new vectors, using modification or inclusion of a variety of regulatory genomic elements aiming to increase expression of the beta-globin gene. We performed in vitro and in vivo studies to compare the ability of these constructs to express the therapeutic gene with a low number of integrations and reduced chimerism. We screened new constructs using a CRISPR-Cas9 modified clonal cell line, HUDEPM#13, which derives from the previously described erythroid HUDEP-2 cell line (Kurita, 2013). Upon differentiation, HUDEPM#13 cells produce a hemoglobin variant (HbMut) that can be discriminated by liquid chromatography from the adult hemoglobin (HbA) produced by the transgene in the lentiviruses. Among our candidates we identified a construct, indicated as ALS20, that synthesizes HbA at high level at a single integration (VCN). Moreover, ALS20 produced significantly more HbA per copy than constructs currently utilized in clinical trials, which were reproduced based on the literature (Negre, 2015; Miccio, 2008; and Boulad, 2014) and designated CV-1, CV-2, and CV-3, respectively. In erythroblasts differentiated in vitro from patients with SCD ALS20 produces, on average, 21% HbA at VCN=1(P&lt;0.001). CV1, which proved to be the most powerful among the clinical vectors tested in HUDEP#M13, produces, on average, 14.9% HbA at VCN=1 (P&lt;0.001). Overall, ALS20 expresses 40% more HbA per VCN in patients' cells, confirming results observed in the mutant HUDEP cell line. In a parallel potency assay, we tested ALS20 using beta0/beta0 thalassemic specimens, which represent the most severe phenotype to correct, due to complete absence of HbA production. Statistical analyses indicate that ALS20 produces, on average, 32.5% HbA at VCN=1 (P&lt;0.001). All results were obtained targeting a range between 0 and 3 integrations, using dilutions of the viral product. Our ongoing bone marrow transplantation studies, using a semi-myeloablative conditioning based on busulfan administration on the beta thalassemia Hbbth3/+mouse model, indicate that ALS20 is curative at VCN lower than 1. In fact, mice with an average of 0.8 copies per genome and 65% transgenic chimerism, present Hb levels of 13.6g/dL, reiterating the potential of this new lentivirus in vivo. In immunocompromised NSG mice, injection of human CD34+cells transduced with ALS20 did not trigger tumor formation or meaningful pathological changes. The safety of our vector has been further assessed using an in vitro immortalization assay on primary mouse BM cells, in which no clonal survival has been observed. Finally, genome integration analyses on human CD34+cells infected with ALS20 showed that none of the examined samples had clones that exceeded the 20% abundance threshold, in accordance with the expected level of safety for gene therapy-based approaches. In summary, we have identified a powerful new lentiviral vector with an enhanced ability to synthesize hemoglobin with a low number of integrations. ALS20's performance has been demonstrated in specimens from patients with hemoglobinopathies as well as in mice affected by thalassemia. Treatment of thalassemia and SCD with ALS20 could reduce the risk of cytotoxic events due to high levels of integration, and also lessen the intensity of the myeloablative regimen to correct anemia in patients. This, in addition with its confirmed safety features, makes this construct an outstanding candidate for clinical trial. Disclosures Kwiatkowski: Imara: Consultancy; Apopharma: Research Funding; Terumo: Research Funding; Novartis: Research Funding; bluebird bio, Inc.: Consultancy, Research Funding; Agios: Consultancy; Celgene: Consultancy. Rivella:Meira GTx, Ionis Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Disc medicine, Protagonist, LIPC, Meira GTx: Consultancy.

Pharmacokinetic-Guided Busulfan Based Myeloablative Versus Fixed Dose Reduced Intensity Conditioning Regimen in Patients Older Than 55 Years Undergoing Allogeneic Stem Cell Transplantation for High Risk Hematological Malignancies

BACKGROUND Reduced intensity conditioning (RIC) combining fludarabine, busulfan and ATG is commonly used in patients with advanced age and/or comorbid conditions, thus deemed unfit for standard myeloablative conditioning (MAC) regimens (i.e. BuCy or CyTBI12). Although non-relapse mortality (NRM) is dramatically reduced with RIC, relapse remains a major concern. In younger patients, myeloablative doses of busulfan in combination with fludarabine (i.e. myeloablative reduced toxicity conditioning [MA-RTC] regimen) produce similar antitumor effect compared with standard MAC while keeping low NRM. The feasibility of such an approach for older patients is not known. We hypothesized that a pharmacokinetics (PK) guided strategy could improve the safety of busulfan administration, allowing the use of myeloablative doses in older patients. STUDY DESIGN AND METHODS We compared the outcome of patients older than 55 years with hematological malignancies included in a prospective monocentric single arm trial (BxPK: NCT02483325) evaluating the feasibility of PK-guided busulfan doses in myeloablative conditioning regimen (BxPK group: fludarabine 150 mg/m² + IV busulfan targeted AUC 5300 x 4 days + ATG 5 mg/kg) before matched sibling (MSD) or unrelated (URD) donor AlloSCT, with a retrospectively enrolled control cohort of patients with same inclusion criteria, treated during the same period but receiving fixed busulfan dose RIC regimen including fludarabine (150 mg/m²), IV busulfan (260 mg/m²) and ATG (5 mg/kg) [Bx2 group]. The primary objective was 2-year progression-free survival (PFS). Hazard ratio of BxPK vs. Bx2 were adjusted by age, disease risk index (DRI), donor type and hematopoietic cell transplantation comorbidity index (HCT-CI). RESULTS We analyzed 83 consecutive patients (BxPK: n=27; Bx2: n=56) without significant difference in baseline characteristics between both conditioning groups. In the BxPK and Bx2 groups: median age was 65 (range: 57-70) and 63 (range: 55-70) years, respectively (p = 0.552); 10/10 URDs were used for 15 (56%) and 37 (66%) patients, respectively (p = 0.493); HCT-CI was ≥ 3 in 18 (67%) and 28 (50%) patients , respectively (p = 0.232); and DRI was high in 5 (19%) and 5 (9%) patients, respectively (p = 0.369). Based on PK data from day-6 busulfan administration, 22 (81%) patients had dose adjustment (increased and decreased dose in 12 and 10 patients, respectively) in the BxPK group. Median busulfan AUC on day-2 (post adjustment) was 5287 µmol.min (range: 3326-7556). Four patients had AUC &gt; 6000 µmol.min on day-2. Grade 3-4 mucositis (BxPK vs Bx2: 52% vs. 11%, p &lt; 0.001) and nausea (BxPK vs Bx2: 11% vs 0%, p = 0.050) were more frequently observed in the BxPk group. No difference was observed between BxPK and Bx2 patients in grade 3-4 liver (BxPK vs Bx2: 15% vs 11%, p = 0.810), renal (BxPK vs Bx2: 0% vs 1.8%, p = 1) and gastrointestinal tract (BxPK vs Bx2: 11% vs 0%, p = 0.450) toxicities. There was a trend to higher cumulative incidence of grades 2-4 acute graft-versus-host disease (GVHD) at 100 days in the BxPK group (BxPK vs Bx2: 41% vs 29%, p = 0.058) while no difference was observed in 2-year chronic GVHD (BxPK vs Bx2: 41% vs. 36%, p = 0.547). Those trends were confirmed by multivariate analyses (acute GVHD: HR 2.22, 95Cl [1.10-4.48], p = 0.026; chronic GVHD: HR 1.30, 95Cl [0.60-2.80], p=0.502) With a median follow up of 33 months (range: 9-60), 2-year NRM was 19% and 18% (p=0.960) in the BxPK and Bx2 group, respectively. Multivariate analysis confirmed the absence of significant increase in the risk of NRM in the BxPK group (HR 1.11, 95CI [0.36-3.45], p = 0.859). The 2-year cumulative incidence of relapse was 19% and 31% (p=0.269) in the BxPK and Bx2 group, respectively (HR 0.55, 95IC [0.20-1.56], p = 0.264). No significant difference in 2-year PFS (BxPK vs Bx2: 62% vs. 51%, p = 0.391) and OS (BxPK vs Bx2: 70% vs. 61%, p = 0.667) was observed. CONCLUSION In patients older than 55 years who are usually candidates for RIC regimen, the use of PK-guided busulfan myeloablative dose allows delivering higher antitumor intensity without increasing NRM. Beyond the feasibility, we observed low incidence of relapse (19%) and promising OS (70%) suggesting that conditioning intensification may lead to long term disease control. These trends need to be confirmed in a larger comparative study for validating the optimal conditioning intensity in older and/or unfit patients. Disclosures Stoppa: takeda: Other: travel fees; celgene: Other: travel fees, lecture fees. Vey:Novartis: Consultancy, Honoraria; Janssen: Honoraria. Chabannon:Novartis: Other: speaker's fees; Celgene: Other: speaker's fees; Sanofi SA: Other: research support, speaker's fees, hospitalities; Gilead: Other: speaker's fees, hospitalities; EBMT: Other: Working Party Chair, Board member; Fresenius Kabi: Other: research support; Miltenyi Biotech: Other: research support; Terumo BCT: Other: speaker's fees. Blaise:Pierre Fabre medicaments: Honoraria; Sanofi: Honoraria; Molmed: Consultancy, Honoraria; Jazz Pharmaceuticals: Honoraria.

Maternal exposure to busulfan reduces the cell number in the somatosensory cortex associated with delayed somatic and reflex maturation in neonatal rats

Busulfan is a bifunctional alkylating agent used for myeloablative conditioning and in the treatment of chronic myeloid leukemia due to its ability to cause DNA damage. However, in rodent experiments, busulfan presented a potential teratogenic and cytotoxic effect. Studies have evaluated the effects of busulfan on fetuses after administration in pregnancy or directly on pups during the lactation period. There are no studies on the effects of busulfan administration during pregnancy on offspring development after birth. We investigated the effects of busulfan on somatic and reflex development and encephalic morphology in young rats after exposure in pregnancy. The pregnant rats were exposed to busulfan (10 mg/kg, intraperitoneal) during the early developmental stage (days 12–14 of the gestational period). After birth, we evaluated the somatic growth, maturation of physical features and reflex-ontogeny during the lactation period. We also assessed the effects of busulfan on encephalic weight and cortical morphometry at 28 days of postnatal life. As a result, busulfan-induced pathological changes included: microcephaly, evaluated by the reduction of cranial axes, delay in reflex maturation and physical features, as well as a decrease in the morphometric parameters of somatosensory and motor cortex. Thus, these results suggest that the administration of a DNA alkylating agent, such as busulfan, during the gestational period can cause damage to the central nervous system in the pups throughout their postnatal development.