Background. Acute cutaneous graft-versus-host disease (acGVHD) following haematopoietic stem cell transplant (HSCT) is common but difficult to distinguish from other causes of rash. Plasma elafin has been proposed as a diagnostic and prognostic biomarker of skin GVHD. Aim. To evaluate the role of plasma elafin as a biomarker in acGVHD in an Indian population. Methods. Plasma elafin was evaluated in a prospective study of HSCT recipients, conducted over 2 years, taking measurements at baseline and at onset of skin rash after HSCT. Patients were categorized into those with GVHD rash, those with non-GVHD rash and those with no rash and the three groups were compared. Results. Two hundred and sixty-one patients with a median age of 16 years (range 1-61 years) and a male predominance (175 : 86 M/F) underwent HSCT during the study period: 56 patients in the GVHD group, 49 in the non-GVHD group and 156 in the no-rash group. The median baseline elafin was similar in all three groups. At the onset of rash, median elafin level was similar between GVHD and non-GVHD rash (34 549 vs. 32 077 pg/mL; P = 0.58) and between GVHD and no rash (34 549 vs. 26 197 pg/mL; P = 0.08). A rise in elafin from baseline was significantly different between GVHD and no rash (P < 0.001) but not between GVHD and non-GVHD rash (P = 0.44). Conclusion. The utility of plasma elafin as a biomarker of skin GVHD is very limited. Plasma elafin, although elevated in cutaneous GVHD, is not helpful in distinguishing between GVHD rash and other causes of rash following HSCT.
A toxicity reduced conditioning regimen containing Treosulfan (Treo), fludarabine (Flu), thiotepa for high risk Thal Major (TM) has been used since 2009 at our centre that has significantly improved transplant outcomes of these patients compared to the historical cohort of patients receiving busulfan/ cyclophosphamide based myeloablative regimen (Mathews et al, 2013). Limited knowledge is available on the pharmacokinetics (PK), pharmacogenetics (PG) and pharmacodynamics of fludarabine and treosulfan, especially in non-malignant hematological disorders like TM. We describe here the PK of Flu and Treo in patients with TM undergoing HSCT, the factors influencing the inter-individual variability in PK and the role of these factors on HSCT outcome. Seventy one patients diagnosed with TM undergoing HSCT with Flu/Treo based conditioning regimen between January 2012 and January 2015 were included (Table: Patient demographics). Selected functional polymorphisms in the NT5E, DCK, hENT1 and GST genes that are involved in fludarabine or treosulfan metabolism were screened. All patients received Flu 40mg/m2/day x 4 days as an 1hr infusion on days 1 and 4 and Treo as 14g/m2/day x 3 days at the rate 5g/hr. Plasma was separated from the peripheral blood collected at predetermined time points after the infusion of Flu and Treo PK analysis. Plasma Flu was analyzed using a LC-MS/MS method and the concentration was expressed as mMole/ml while Treo was analyzed using a HPLC-RI method and concentration was expressed as mg/L. Flu and Treo PK was estimated using nonlinear mixed effects modeling via Monolix 4.3.3. The covariates tested for both PK were: age, sex, body weight, BSA, ferritin, and polymorphisms in NT5E, hENT1, dCK and GST genes. The PK parameters AUC, CL, V and k were estimated on day 1 for Treo and on day 1 and day 4 for Flu (Table). The influence of Flu and Treo PK and PG on graft rejection, early transplant related mortality (TRM) & chimerism status was estimated using logistic regression analysis. Wide inter-individual variation in Flu and Treo PK was noted (7 and 9 fold Vs 5 and 8 fold respectively for Day 1 & 4 Flu AUC & Cl; 33 & 31 fold variation in Treo AUC and Cl) (Table). Flu CL was significantly higher on day 4 compared to day1 (Figure A). The variation in Flu PK was explained by genetic variants in NT5E and dCK. Patients having variant genotype for the SNPs in NT5E (rs2295890) and dCK (rs11544786) showed significantly lower plasma Flu clearance compared to those with wild type genotype (p=0.006 & p=0.05 respectively) (Figure B). This is consistent with our previous report in patients with aplastic anemia undergoing HSCT (Mohanan et al. 2014; Blood: 124 (21)). None of the genetic variants in the GST genes explained the variation in Treo PK. Day21 mortality was seen in 6/71 patients (8.5%) and graft rejection in 3/66 evaluable patients (4.5%). Analysis of the influence of PK and PG variables on transplant outcome showed significantly high first dose Flu AUC to be associated with D21 mortality upon Univariate analysis (median 42.5, range 32.1-63.7 compared to 31.8, range 15.2-111 mMole*h/mL, in those with and without TRM respectively; p=0.043); none of these parameters were significantly associated with graft rejection or mixed chimerism. There was no association between Treo PK parameters and graft rejection or TRM. The influence of Flu and Treo PK on regimen related toxicity is yet to be evaluated. The lack of the influence of PK on transplant outcome could be due to lower incidence of rejection and TRM in this cohort. Further analysis in a larger cohort of patients will be done once we enroll more patients for PK analysis. Our results demonstrate that Flu PK is influenced by genetic variants in NT5E and dCK, the enzymes involved in Flu biotransformation. The relationship between high-plasma Flu exposure and TRM and given the fact that multiple factors influence TRM, we can extrapolate that the plasma Flu AUC may be a surrogate marker of overall preparative regimen intensity as reported previously (Long-Boyle et al, Bone Marrow Transplant, 2011). The lack of association of genetic variants in GST genes in explaining the inter-patient variability in treosulfan exposure suggests the involvement of other drug metabolizing genes on treosulfan PK. We are currently evaluating the role of genetic variants in a large panel of drug metabolizing genes on explaining this inter-individual variability in Treo PK. Disclosures No relevant conflicts of interest to declare.
Pharmacokinetic (PK)-targeted dose adjustment of oral Bu has been shown to minimize the toxicity and treatment related mortality following hematopoietic stem cell transplantation (HSCT). Intravenous busulfan (IV Bu) based myeloablative conditioning regimen has significantly reduced the inter-patient variability, ease of administration and reduced toxicities compared to oral Bu. However due to challenges in availability and cost of original formulation (Busulfex, Otsuka Pharmaceuticals) has resulted in the increased use of generic versions of the drug worldwide. Also, in many parts of the world, regulatory approval for generic drugs is obtained only with bioequivalence studies without a clinical trial or PK data. From June 2010 to December 2014, our center has used generic IV busulfan Bucelon™ (Celon Labs, Hyderabad, India) with routine therapeutic drug monitoring and dose adjustment. Since January 2015, Buslera™ (Biem Pharmaceuticals, Ankara, Turkey), a newer generic IV busulfan formulation, is being used in patients undergoing HSCT in our centre due to non-availability of Bucelon™. We prospectively analyzed the PK of IV Buslera™ and compared the systemic exposure and targeted dose adjustment pattern with our experience with Bucelon™. Eighteen patients underwent HSCT with IV Buslera™ based conditioning regimen between January and July 2015. Twelve patients received once daily Bu dose (Q24H; 40mg/m2/day x 4 days) and 6 received every 6 hour dosing (Q6H; 0.8mg/kg/dose x 4 days). In the historical cohort, 135 patients (30 Q6H and 105 Q24H) received Bucelon™ (Mohanan et al, Blood 2013; 122:3280). Demographics of the patients in both groups are depicted in the Table. Blood samples were collected at predetermined time points on day 1 and 3 & busulfan plasma concentrations analyzed using previously published method (Desire et al, 2013). Further doses of busulfan were adjusted to achieve target busulfan AUC (4500-5500 mmoles*min for Q24H and 900-1350 mmoles*min for Q6H). In patients receiving Q24H IV Buslera, the median busulfan AUC on day 1 was 6516 mMoles*min (3908-14064 mMoles*min). The Buslera dose was reduced in 8 patients (median dose reduction 13%; range: 7-18%), increased in one (9%) and 3 patients did not require any dose adjustment. In patients receiving Q6H IV Bu, the AUC on day 1 was 784 mMoles*min (446-960 mMoles*min). Five out of 6 patients required a moderate increase in dose (median dose increase 5%; range: 5-8%) and one did not require any dose adjustment. This data is strikingly different when compared to the historical cohort of patients receiving IV Bucelon™ where 60% patients receiving Q24h dose required dose increase, while with Buslera™ only 9% of patients needed dose increase (p<0.001) to achieve target AUC of busulfan. In general, we noticed that the median clearance of Buslera™ was significantly lower compared to Bucelon™ (Figure) Our study strongly suggests the need for routine therapeutic drug monitoring and targeted dose adjustment of busulfan, especially when different generic busulfan formulations are used. TDM with generic busulfan is even more relevant in markets where approval of generics is done only based on bioequivalence studies with PK data in an appropriate clinical setting and without a clinical trial. Disclosures No relevant conflicts of interest to declare.
Targeted intravenous (IV) Busulfan (TBu) in combination with cyclophosphamide (Cy) is widely used as a preparative regimen in children with thalassemia major undergoing matched allogeneic haematopoietic stem cell transplantation (HSCT) to reduce toxicity. At our centre, we have been using TBu/Cy regimen since 2011 for very young children (Pesaro class I/II risk status) and with matched related donors. We have compared the outcome of HSCT in this group of patients with a retrospective cohort (from 1995-2009) of age and risk status matched patients receiving fixed dose oral Bu without PK monitoring. All patients with beta thalassaemia major undergoing HSCT with matched related donor between January, 2011 - May, 2018, receiving IV Bu (0.8mg/kg Q6H days x 4 days) / Cy (50mg/kg x 4 days) with anti-thymocyte globulin based conditioning regimen were included in this study. Busulfan levels were monitored after the 1st dose of busulfan and further doses adjusted to achieve a target range of 900-1300um*min. The Bu plasma levels achieved on day 1 and on day 3 were compared with HSCT outcome endpoints including chimerism status on D28, overall and event-free survival (OS, EFS), and graft rejection. There were 52 children, median age of 3 years (range:1-6); 44 class I/II and 8 class III low risk. Different proprietary Bu products were used: 26 received Bucelon™ (Celon Labs, Hyderabad, India), 23 patients received Buslera™ (Biem Pharma, Ankara, Turkey), and 3 received Bufatas™ (Intas Pharma, Ahmedabad, India). The 3rd dose of Bu was increased in 35, decreased in 2 and unchanged in 15 patients. The median 1st dose Bu AUC was 625 um*min (range: 115- 2466) while the 9th dose AUC was 1105 um*min (range: 543-2656). Target Bu AUC was achieved in 40 patients (77%) as assessed by Bu AUC on Day 3 while the AUC in the remaining 12 patients (23%) was lower than 900 um*min (range: 543-872). Twenty-three of the 50 evaluable patients showed mixed chimerism (MC) or rejection on day 28 (46%; MC level 1 - 14; MC level 2 - 3; MC level 3 - 2 and >90 recipient chimerism - 4, as per our previous data- Fouzia et al, BMT, 2017). 11/23 (47%) had graft rejection; The OS and EFS were 96% and 79% respectively. Two patients died - 1 class III, of diffuse alveolar hemorrhage/ idiopathic pulmonary syndrome and the other of complications related to primary graft failure. Correlation of PK with all demographic variables by univariate analysis did not reveal any significant associations. However, while 11 out of 39 patients (28%) with 9th dose Bu AUC in the lower three quartiles rejected the graft (Q1: <913 (543-906)-3rej; Q2: 936-1100 -2rej; Q3: 1110-1271- 6rej), none of the 13 patients (0%) in the highest quartile (>1292 (1299-2656) um*min) rejected the graft (p=0.034). While none of the 13 patients in Q4 died, 7 had hepatotoxicity (grade 2 and above) and mucositis (grade 2 and above). The incidence of hepatotoxicity in the first three quartiles were as follows: Q1: 11 had grade 2-3 hepatotoxicity and 2 had grade 2-3 mucositis; in Q2: 5 had grade 2-3 hepatotoxicity while 7 had grade 2-3 mucositis; Q3: 3 had grade 4 hepatotoxicity while 4 had grade 2-3 mucositis (p=ns with respect to toxicity in Q4 vs. the other 3 quartiles). There was no significant association between the busulfan formulation used and the incidence of graft rejection. We then compared the HSCT outcome parameters in these patients with age and Pesaro class matched retrospective cohort of thalassemia patients (n=79) who had received a similar conditioning regimen but with oral Bu without PK guided dose adjustment. There was a significantly better OS (Fig A) in the TBu cohort compared to the oral Bu (p=0.03) but this did not translate to better EFS (Fig B) due to increased incidence of graft rejection (Figure C). In conclusion, our data suggests that targeting higher Bu AUCs within the therapeutic window could reduce the risk of graft rejection and improve OS without increasing toxicity. Strategies for rapid dose adjustments after the first dose PK are needed to better achieve these target values to reduce rejections and improve outcome. Figure. Figure. Disclosures No relevant conflicts of interest to declare.
Vitiligo is a chronic inflammatory autoimmune disease resulting in skin depigmentation and a high quality of life burden. Vitiligo pathogenesis and progression is driven, in part, by increased IFN-gamma activation and the subsequent downstream signaling through the Janus Kinases (JAK). Ruxolitinib cream (Rux Cream) is a potent JAK inhibitor designed for topical administration. Treatment with Rux Cream was associated with significant skin repigmentation in vitiligo patients (NCT03099304). This study investigated the effects of topical treatment with Rux Cream on inflammatory mediator expression in circulation. Sera from 130 subjects (n¼23 Vehicle, n¼26 0.15% QD, n¼27 0.5% QD, n¼24 1.5% QD, n¼30 1.5% BID) with baseline and week 24 samples were analyzed for broad proteomic changes. Paired t-tests established significant changes within treatment groups at a cutoff of p<0.05. Baseline biomarkers and facial Vitiligo Area Scoring Index (FVASI) were assessed for significance using Spearman's correlation. Expression of 1104 proteins was evaluated for each subject. From baseline to week 24; 204 proteins were significantly (p<0.05) modulated in the 1.5% BID, 162 proteins in 1.5% QD, 71 in 0.5% QD, and 29 proteins in 0.15% QD compared with 56 proteins in the vehicle cohort. Chemokine C-X-C Motif Chemokine Ligand 10 (CXCL10) was significantly down-regulated in both 1.5% QD and BID and plays a central role in pathogenesis by recruiting T cells to the site of inflammation to target melanocytes. Other proteins modulated in 1.5% QD and 1.5% BID included Chemokine (C-C Motif) Ligand 18 (CCL18), Matrix Metalloprotease 12 (MMP12), and CD27. Interleukin 20 receptor subunit alpha (IL-20RA) and Paraoxonase 2 (PON2) were associated with FVASI at baseline. Overall, topical treatment with rux cream, and the associated skin improvement, corresponded with dose dependent modulation of circulating inflammatory mediators.
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