Table 3.

Rabbit and horse ATG pharmacokinetic/pharmacodynamic studies in alloHCT

Study	Patient & alloHCT characteristics	ATG Dosing, PK Sampling & Analytes	Pharmacokinetic and -dynamic results
Waller et al., 2003131	N=28 Age 21–56 yr Regimens MA: N=28, varied Donors Related: N=28, all partially mismatched Graft sources PBSC: N=28 Diagnoses Malignant: N=28 Additional IS None	Rabbit or horse ATG Both rabbit and horse ATG dosing Horse: 60 mg/kg total dose horse ATG, given as 20 mg/kg/day over last 3 days of conditioning regimen Rabbit: 6–10 mg/kg total dose rabbit ATG, given as 1.5 mg/kg/day or 2.5 mg/kg/day over last 4 days of conditioning PK sampling Days +1,+ 2, +3, +4, +7, +14, +28, +45, +60, +75, +100 Total or active ATG Both total and active	Data analysis Pharmacokinetic samples collected in a subset of 21 patients, evaluable in 19 patients Pharmacokinetic samples collected in recipients of rabbit ATG only Pharmacokinetics Biphasic elimination for both total and active <1 μg/mL of active rabbit ATG is subtherapeutic After administration of 6 mg/kg rabbit ATG, total Cmax was 64±20 μg/mL and active Cmax was 9.2±5.8 μg/mL Clearance of active rabbit ATG (t1/2=6 days) to sub-therapeutic concentrations (<1 μg/mL) by a median (range) of 15 days (8–38) after transplantation Engraftment Not evaluated Rejection Not evaluated Chimerism Not evaluated Acute GVHD All 3 patients who received horse ATG had acute GVHD One of 25 patients who received rabbit ATG had acute GVHD Chronic GVHD Not evaluated NRM Not evaluated Relapse Not evaluated OS Not evaluated Other Not evaluated concentration
Eiermann et al., 1999140	N=12 Age 21–55 yr Regimens MA: N=12, CY/TBI or BU/CY/etoposide Donors Related: N=6 URD: N=6 Graft sources Marrow: N=12 Diagnoses Malignant: N=12 Additional IS CSA/MTX, CSA TCI not specified	Rabbit or horse ATG Rabbit only (Fresenius) ATG dosing 30mg/kg total dose, given from day −3 to day −1 PK sampling Various time points between days −1 and +22 Total or active ATG Total	Data analysis No PK modeling Pharmacokinetics At 10 mg/mL ATG-F, 3H-labeled thymidine incorporation was effectively blocked. This effect was lost at an ATG-F concentration of 10 ng/mL Pharmacodynamics Not evaluated
Kakhniashvili et al., 2005141	N=30 Age 18–66 yr Regimens MA: N=30, FLU/cytarabine/melphalan Donors Related: N=21, some partially mismatched URD: N=9, some partially mismatched Graft sources PBSC: N=30 Diagnoses Malignant: N=30 Additional IS Not specified	Rabbit or horse ATG Rabbit only ATG dosing 20 mg/kg total, given as 4 mg/kg/day on days −3, −2, +2, +4, +6 for first 14 patients 16 mg/kg total, given as 4 mg/kg/day on days −3, −2, +2, +4 for remaining 16 patients PK sampling Days −3, 0, +7, and approximately weekly thereafter Total or active ATG Active only	Data analysis PK sampling evaluable in 24 patients Pharmacokinetics All values mean ± standard deviation ATG clearance was 3.41 ± 1.72 L/day Overall ATG half-life was 6.8 ± 2.9 days ATG Volume of distribution was 47.2 ± 24.9 L Fourteen patients’ data best described by biexponential model Ten patients with sparser data best described by monoexponential, one-compartmental model Pharmacodynamics Not evaluated
Seidel et al., 2005138	N=32 Age 0.34 –18 yr Regimens MA: N=18, varied NMA: N=14, varied Donors Related: N=5, some partially mismatched URD: N=27, some partially mismatched Graft sources Marrow: N=11 PBSC: N=21 Diagnoses Malignant: N=18 Non-malignant: N=14 Additional IS None in patients with leukemia who received TCD grafts; all others received CSA/MTX, CSA TCI not specified	Rabbit or horse ATG Rabbit only ATG dosing 7.5 to 40 mg/kg total, given as 2.5–10mg/kg/day from day −4 or day −3 to day −1 PK sampling Days −4 to +30, initially daily and later every other day or three times a week At least 20 samples collected in total Total or active ATG Both total and active	Data analysis For pharmacodynamic studies, patients divided into low-dose group (cumulative dose of 7.5–10mg/kg) and high-dose group (cumulative dose of 15mg/kg or more) Pharmacokinetics PK of active rabbit ATG in children was similar to that of adults when total dose < 20mg/kg; non-linear PK when total dose > 20 mg/kg Average half-life ranged from 2.04 – 3.5 h with total dose of 7.5 – 20 mg/kg (N=27) and from 6.32 – 7.92 h with total dose of 30 – 40 mg/kg (N=5) Engraftment Not evaluated Rejection Not evaluated Chimerism Not evaluated Acute GVHD Acute GVHD <10% in both low-dose and high-dose groups Chronic GVHD Not evaluated NRM Not evaluated Relapse Not evaluated OS Not evaluated Other Higher one-year cumulative incidence of EBV in high-dose group than in low-dose group (0.56±0.12 in high-dose group, 0.19±0.10 in low-dose group, p=0.018)
Remberger et al., 2005134	N=61 Ages 1–61 yr Regimens MA: N=52, BU/CY or CY/TBI RIC: N=9, FLU/TBI Donors URD: N=61 Graft sources Marrow: N=28 PBSC: N=33 Diagnoses Malignant: N=53 Non-malignant: N=8 Additional IS CSA/MTX for 53 patients, CSA/prednisolone for 3 patients, CSA/MMF for 3 patients; all CSA with TCI to C0 of 200–300ng/mL Tacrolimus/sirolimus for 2 patients, tacrolimus or sirolimus TCI not specified	Rabbit or horse ATG Rabbit only ATG dosing 4–10mg/kg total dose, given at 2mg/kg/day over 2–5 days (last dose always on day −1) PK sampling Weekly through week 5 Total or active Total only in serum	Data analysis Pharmacodynamic analysis only Pharmacokinetics No pharmacokinetic modeling Engraftment No correlation between ATG concentrations and engraftment Rejection Not evaluated Chimerism Not evaluated Acute GVHD Incidence of acute GVHD was inversely correlated with dose (lower dose, more acute GVHD) Patients with concentrations >70 μg/mL on day 0 had lower risk of developing moderate to severe acute GVHD than those with concentrations <70 μg/mL (p=0.006) Patients with week 1 concentrations <45 μg/mL had more grades II–IV acute GVHD (p=0.01) Chronic GVHD Not evaluated NRM Not evaluated Relapse Not evaluated OS Not evaluated Other Rate of CMV infection higher in patients with day 0 rabbit ATG level >150 μg/mL (70% vs 52%, p=ns)
Remberger et al., 2009144	N=76 Ages 1.5–67 yr Regimen MA: N=37, BU/CY or CY/TBI RIC: N=29, varied Donors URD: N=76 Graft sources Marrow: N=16 PBSC: N=60 Diagnoses Malignant: N=64 Non-malignant: N=12 Additional IS CSA/MTX for 60 patients, CSA TCI to C0 of 200–300 ng/mL Tacrolimus/sirolimus in 16 patients, tacrolimus or sirolimus TCI not specified	Rabbit or horse ATG Rabbit only ATG dosing 4–8mg/kg total dose, given as 2mg/kg/day over 2–4 doses with last dose on day −1 PK sampling Days 0, +1, and +7 Total or active Total only	Data analysis Pharmacodynamic studies only No pharmacokinetic modeling Pharmacokinetics Estimated half-life of 9 days Engraftment Not evaluated Rejection No correlation between total concentrations and rejection Chimerism Not evaluated Acute GVHD Total dose was inversely correlated with grades II–IV acute GVHD Patients with concentrations ≤70 μg/mL on day 0 had significantly higher cumulative incidence of acute GVHD compared to patients with concentrations >70 μg/mL (33% vs 6%, p=0.023) Chronic GVHD Not evaluated NRM Not evaluated Relapse No correlation between total concentrations and relapse OS Not evaluated Other Not evaluated
Call et al., 2009142	N=13 Ages 2–16 yr Regimens MA: N=13, TBI/thiotepa/CY Donors URD: N=13 with ≥ 7 of 8 allele match Graft sources Marrow: N=13, non-TCD Diagnoses Malignant: N=13 Additional IS CSA/MTX, CSA TCI to C0 of 170–250 ng/mL by EMIT	Rabbit or horse ATG Rabbit only ATG dosing 10 mg/kg total, given as1mg/kg on dayminus;4, then 3mg/kg/day on days −3 to −1 PK sampling Days−4, −3, −2, −1, 0, +1, +3, +5, +7; weeks 1, 2, 4, 8, 16, and 24 Total or active Both total and active	Data analysis Samples analyzed by FACS for both total (LOQ 3.9 μg/mL) and active plasma ATG (LOQ 0.2 μg/mL) Pharmacokinetic data modeled with NONMEM No pharmacodynamic analysis Pharmacokinetics Biphasic elimination and large interpatient variability Mean CL for total rabbit ATG was 198 mL/day (CV of 47%) and for active rabbit ATG was 4530mL/day (CV of 61%) Median (range) beta half-life for total ATG was 27.3 days (25.7–30.4) and for active ATG was 12.5 days (5.8–22.4) Mean (range) actual Cmax for total ATG was 57.7 μg/mL (23.7–80.7) and for active ATG was 4.0 μg/mL (1.6–8.0) Day 0 mean (range) total ATG was 53 μg/mL (23.7–80.7) and active ATG was 4.0 μg/mL (1.6–8.0) Day +28 mean (range) total ATG was 15.9 μg/mL (5.1–20.6) and active ATG was 1.13 μg/mL (<0.2–4.64) Engraftment Not evaluated Rejection Not evaluated Chimerism Not evaluated Acute GVHD No occurrences of grades III-IV acute GVHD despite the majority of patients having day 0 concentrations <70 μg/mL Chronic GHVD Not evaluated NRM Not evaluated Relapse Not evaluated OS Not evaluated Other Not evaluated
Podgorny et al., 2010145	N=153 Ages 19–66 yr Regimens MA: N=153, FLU/BU or FLU/BU/TBI Donors Mismatched: N=26, related or unrelated not specified Related: N=76 URD: N=51 Graft sources Marrow: N=10 PBSC: N=143 Diagnoses Malignant: N=147 Non-malignant: N=3 Unknown: N=3 Additional IS CSA/MTX, CSA TCI not specified	Rabbit or horse ATG Rabbit only ATG dosing 4.5 mg/kg total, given as 0.5mg/kg on day −2, then 2mg/kg/day on days −1 and 0 PK sampling Days +7 and +28 Total or active Active only	Data analysis Pharmacodynamic studies only No pharmacokinetic modeling Pharmacokinetics Large interpatient variability in drug concentrations Median (range) day +7 ATG concentrations were 1.109 mg/L (undetectable to 4.401 mg/L) Median (range) day +28 concentrations were 0.053 mg/L (undetectable to 0.733 mg/L) Engraftment Not evaluated Rejection Not evaluated Chimerism Not evaluated Acute GVHD At low doses ATG has anti-GVHD effects ATG concentrations >1.45 mg/L on day +7 were associated with a 0.35-fold risk of developing grades II–IV acute GHVD (p=0.030) ATG concentrations >0.029 mg/L on day +28 were associated with 0.52-fold risk of developing grades II–IV acute GVHD (p=0.035) Chronic GVHD Not evaluated NRM Not evaluated Relapse Observed no effect of ATG concentrations on relapse OS Not evaluated Other Patients who developed PTLD had higher ATG concentrations than those who did not; association observed with both day +7 concentrations (p=0.039) and day +28 concentrations (p=0.014) Observed no effect of ATG concentrations on non-PTLD infections
Remberger et al., 2012139	N=43 Ages 0.4–65 yr Regimens MA: N=27, BU/CY or CY/TBI RIC: N=16, varied Donors URD: N=43 Graft sources UCB: N=43 Diagnoses Malignant: N=27 Non-malignant: N=16 Additional IS CSA/prednisolone for 38 patients, CSA TCI to C0 of 200–300 ng/mL Other for 5 patients, regimen or TCI not specified	Rabbit or horse ATG Rabbit only ATG dosing 6 or 8mg/kg total dose, given as 2mg/kg/day for 3–4 days, last dose on day −1 PK sampling Days 0, +11, +25 Total or active Total only	Data analysis Pharmacodynamic studies only Reported outcomes in patients with low ATG concentrations (≤ 40 μg/mL) to those with high ATG concentrations (> 40 μg/mL) on day +11 Stated that similar but weaker correlations were found between day +25 concentrations and outcomes, but data not shown Pharmacokinetics No pharmacokinetic modeling Engraftment No relationship between ATG concentrations and platelet or neutrophil engraftment Rejection No relationship between ATG concentrations and rejection Chimerism Not evaluated Acute GHVD Patients with low ATG concentrations had higher incidence of grades III–IV acute GVHD than those with high ATG concentrations (32% vs 0%, p<0.01) Chronic GVHD Similar incidence in those with low and high ATG concentrations (p=ns) NRM Patients with low concentrations had higher than those with high concentration (69% vs 7%, p=0.005) Relapse Patients with low concentration had lower relapse than those with high concentration (17% vs 82%, p<0.01) OS Similar incidence in those with low and high concentrations Other No relationship between ATG concentration and EBV lymphoma or relapse-free survival
Chawla et al., 2014126	N=180 Ages 18–66 yr Regimens MA: N=180, varied Donors Related: N=67, all HLA-matched Other: N=113, at least 8 of 10 alleles matched Graft sources PBSC: N=180, non-TCD Diagnoses Malignant: N=180 Additional IS CSA/MTX, CSA TCI to C0 of 200–400 μg/L	Rabbit or horse ATG Rabbit only ATG dosing 4.5 mg/kg total, given as 0.5mg/kg on day −2, 2mg/kg/day on days −1 and 0 PK sampling Day 0, +7, +28 Total or active Total only	Data analysis Study originally enrolled N=185, but patients who did not engraft, relapsed, or died before day +30 were excluded from subsequent analysis Measured serum by using flow cytometry-based assay of Kakhniashvili141 Pharmacokinetics Median (range) clearance of 0.023 L/kg/day (0.005–0.26) Median (range) half-life of 5 days (2.2–16.2) Engraftment Not evaluated Rejection Not evaluated Chimerism Not evaluated Acute GVHD ATG concentrations on day 0 were not associated with acute GVHD High ATG concentrations on days +7 and +28 were associated with a lower likelihood of acute GVHD Chronic GVHD Patients with an ATG level >8.12 mg/L on day 0 were less likely to have chronic GVHD than patients with an ATG level <8.12mg/L (p<0.001) Patients with an ATG level >1.26 mg/L on day +7 were less likely to have chronic GVHD than patients with an ATG level <1.26 mg/L (p<0.001) Patients with an ATG level >0.14 mg/L on day +28 were less likely to have chronic GVHD than patients with an ATG level <0.14 mg/L (p=0.008) NRM NRM not associated with ATG concentrations Relapse Relapse not associated with ATG concentrations OS Not evaluated Other Low day +28 ATG concentration were associated with CMV reactivation in univariate analysis but not in multivariate analysis Day 0 ATG concentration were not associated with PTLD High ATG concentrations on days +7 and +28 were associated with PTLD
Hannon et al., 2015143	N=25 Ages 45–71 yr Regimens NMA: N=25, TLI/ATG Donors Related: N=14 URD: N=11 Graft sources PBSC: N=25 Diagnoses Malignant: N=25 Additional IS Tacrolimus/MMF; tacrolimus TCI to C0 of 15–20 ng/mL through day +28 and 10–15 ng/mL after day +28	Rabbit or horse ATG Rabbit only ATG dosing 7.5 mg/kg total, given as 1.5 mg/kg/day on days −11 through −7 PK sampling Days −7, −4, 0, +3, +7, +10, +14, +17, +20 Total or active Active only	Data analysis Pharmacokinetic data available from 15 of the 25 patients Pharmacokinetics Median functional ATG concentration were 4.0 mg/L (range, 3.2–5.6) on day 0, 2.2 mg/L (range, 0.6–3.9) on day +3, and 0.95 mg/L (range, 0.34–1.49) on day +10 Engraftment Not evaluated Rejection Not evaluated Chimerism Not evaluated Acute GVHD Not evaluated Chronic GVHD Authors note that day 0 concentration were below 8.12 mg/L, the threshold of ATG concentration associated with a lower incidence of chronic GVHD as reported by Chawla et al. NRM Not evaluated Relapse Not evaluated OS Not evaluated Other Not evaluated
Yamane et al., 2011149 reported day 0 total and unbound ATG concentrations were not correlated in two adults

Abbreviations: alloHCT: allogeneic hematopoietic cell transplantation; ATG: antithymocyte globulins; ATG-F: Fresenius ATG: BU: busulfan; C₀: trough concentration; CL: clearance; C_max: maximum plasma concentration; CMV: cytomegalovirus; CSA: cyclosporine; CV: coefficient of variation (expressed as percentage, calculated as mean/standard deviation *100); CY: cyclophosphamide; EBV: Epstein-Barr virus; EMIT: enzyme multiplied immunoassay technique; FACS: fluorescein-activated cell sorting; GVHD: graft-versus-host disease; HLA: human leukocyte antigen; IS: immunosuppression; LOQ: limit of quantitation; MA: myeloablative; MTX: methotrexate; NMA: nonmyeloablative; NRM: non-relapse mortality; PBSC: peripheral blood stem cells; PD: pharmacodynamics; PK: pharmacokinetics; PTLD: post-transplant lymphoproliferative disorder; RIC: reduced-intensity conditioning; TBI: total body irradiation; TCD: T-cell depletion; TCI: target concentration intervention; TLI: total lymphoid irradiation; URD: unrelated donor