From: PLOS Neglected Tropical DiseaseJanuary 2014 Presented by Pavitra Charoensrisakkul and Peeraya Permkarnjaroen 3 rd year medical cadet Phramongkutklao college of medicine

Fatal Disease M people/year Sodium stibogluconate + Paromomycin = First-line 2

AmBisome® Easy managementSafest ↓Renal toxicity High efficacy in India & Kenya Background 3

Study design Objectives Determine the minimum efficacious and safe single dose of AmBisome® Evaluate the efficacy and safety of a single dose compared to the multiple dose regimen 4

Multi-centre, open-label, non-inferiority, Randomized controlled trial Study design 5

Figure 1. Patient flow chart. Consort Patient flow chart: AmBisome® multi dose vs. single dose, ITT Intention to treat, PP per protocol, LTFU Lost to Follow-up. Inclusion criteria age > 4 years HIV-negative non-severe VL Inclusion criteria age > 4 years HIV-negative non-severe VL 6

Figure 1. Patient flow chart. Consort Patient flow chart: AmBisome® multi dose vs. single dose, ITT Intention to treat, PP per protocol, LTFU Lost to Follow-up. Exclusion criteria Severe VL Anti-leishmanial within six months Chronic disease Abnormal serum creatinine Liver function tests > 3 times the normal range Platelet < 40,000/mm3 Alcohol abuse Pregnancy or lactation Concomitant acute drug usage for malaria and bacterial infection Pneumonia within last 7 days Known hypersensitivity to AmBisome® or Amphotericin B Exclusion criteria Severe VL Anti-leishmanial within six months Chronic disease Abnormal serum creatinine Liver function tests > 3 times the normal range Platelet < 40,000/mm3 Alcohol abuse Pregnancy or lactation Concomitant acute drug usage for malaria and bacterial infection Pneumonia within last 7 days Known hypersensitivity to AmBisome® or Amphotericin B 7

Figure 1. Patient flow chart. Consort Patient flow chart: AmBisome® multi dose vs. single dose, ITT Intention to treat, PP per protocol, LTFU Lost to Follow-up. 1 st Multiple Doses Single Dose 7.5 mg/kg 8

Figure 1. Patient flow chart. Consort Patient flow chart: AmBisome® multi dose vs. single dose, ITT Intention to treat, PP per protocol, LTFU Lost to Follow-up. 2 nd Multiple Doses Single Dose 10mg/kg 9

Figure 1. Patient flow chart. Consort Patient flow chart: AmBisome® multi dose vs. single dose, ITT Intention to treat, PP per protocol, LTFU Lost to Follow-up. 2 interim analysis: Early detection of inefficacious single doses Worsening clinical conditions 1 st Multiple Doses Single Dose 7.5 mg/kg 2 nd Multiple Doses Single Dose 10mg/kg 10

= Single Dose 10.0 mg/kg (2 nd interim) = Multiple Doses 3.0 mg/kg (Both interims) Parasite clearance was measured by quantitative reverse transcriptase (qRT) PCR targeting Leishmania 18S ribosomal RNA Method Primary (definitive / final cure) efficacy endpoint Secondary (initial cure) efficacy endpoint SD 10 mg/kg SD 7.5 mg/kg MD 3.0 mg/kg = Single Dose 7.5 mg/kg (1 st interim) 11

Figure 1. Patient flow chart. Consort Patient flow chart: AmBisome® multi dose vs. single dose, ITT Intention to treat, PP per protocol, LTFU Lost to Follow-up. Multiple Doses n = 63 Single Dose 7.5 mg/kg n = 21 Single Dose 10 mg/kg n = 40 12

Statistical Analysis If the single dose arm showed significantly poorer efficacy (P<0.05) The single dose was increased Intention-to-Treat (ITT) Population Difference between the parasite clearance rate 13

Table 1. Baseline data on patient demographics, clinical characteristics and laboratory values. Age in years Mean(SD) 16(9.0)21(9.2) 14(7.4) Sex Female, n(%) 16(9.0)21(9.2) 14(7.4) Male, n(%) 53(84)19(91) 30(75) Spleen Size Mean(SD) 9.5(5.3)12.0(6.0) 9.1(5.3) Nutritional Status Weight(kg), Mean (SD) 36(14.5)44(12.2) 33(14.4) Multiple Doses n = 63 Single Dose 7.5 mg/kg n = 21 Single Dose 10 mg/kg n = 40 Baseline characteristics 14

Table 1. Baseline data on patient demographics, clinical characteristics and laboratory values. Hemoglobin (g/dl) Mean(SD) 7.9(1.7)7.7(1.6) 7.7(1.4) AST Mean(SD) 52(27.1)48(28.5) 54(29.0) ALT Mean(SD) 32(20.7)35(21.3) 33(20.0) Multiple Doses n = 63 Single Dose 7.5 mg/kg n = 21 Single Dose 10 mg/kg n = 40 15

Table 2. Interim analyses and non-comparative efficacy analysis for primary (day 210) and secondary (day 30) end points. Overall 62 53(85) (50) (73) Overall 54 46(85) (40) (58) Cure at Day 30 Cure at Day 210 (6 months follow-up) Multiple Doses n = 63 Single Dose 7.5 mg/kg n = 21 Single Dose 10 mg/kg n = 40 16

95%CI 73-93% 95%CI 27-73% 95%CI 19-64% 95%CI 56-85% 95%CI 46-83% ≈ India & Kenya 17

Table 3. Infusion-related and drug-related treatment emergent AEs. Pyrexia 17(27) 0(0) 14(35) Multiple Doses n = 63 Single Dose 7.5 mg/kg n = 21 Single Dose 10 mg/kg n = 40 Safety 18

Table 3. Infusion-related and drug-related treatment emergent AEs. Alanine aminotransferase increased 17(27) 13(62) 11(28) Aspartate aminotransferase increased 22(35) 14(67) 13(33) Hypokalaemia 30(48) 4(19) 16(40) Multiple Doses n = 63 Single Dose 7.5 mg/kg n = 21 Single Dose 10 mg/kg n = 40 19

Discussion Prematurely terminated because low efficacy in both arm Patient characteristics leading to variation in treatment response  Geographical variation  Severely underweight  Hemoglobin  Immunity Results were not conclusive On day 3  peripheral parasite clearance were significantly decreased 20

Figure 3. Parasite clearance from peripheral blood. Comparison of parasite clearance rates from peripheral blood in single (10 mg/kg) and multiple dose (763 mg/kg) regimens of AmBisome®. Data are from 5 consenting patients in each of the 10 mg/kg single-dose and multiple-dose arms. doi: /journal.pntd g003 21

Conclusion Optimal single dose  Not identified Efficacy of multiple dose > single dose AmBisome® regimens not be suitable for Eastern Africa Efficacy of AmBisome® in Eastern Africa < India 22

Clearly focused question Clinical Appraisal 23

Randomized assignment of subjects  Computer-generated randomization list Clinical Appraisal 24

Adequate concealment method  Seal and opaque envelopes of treatment allocations  Opened after patient entered the trial Clinical Appraisal 25

Blinding  Not possible because nature of intervention Clinical Appraisal 26

Similar treatment and control groups  Randomization  No differences between groups Clinical Appraisal 27

Outcomes  Absence of parasite in tissue aspirates Clinical Appraisal 28

Percentage of dropped out individuals before the study completed Clinical Appraisal Multiple Doses n = 63 Single Dose 7.5 mg/kg n = 21 Single Dose 10 mg/kg n = %4.8% 0% 29

Intention to treat analysis Clinical Appraisal 30

Not comparable results for all sites (Multi-centre) Clinical Appraisal 31

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