MAJOR ARTICLE Short-Course Paromomycin Treatment of Visceral Leishmaniasis in India: 14-Day vs 21-Day Treatment Shyam Sundar, Neha Agrawal, Rakesh Arora, Dipti Agarwal, Madhukar Rai, and Jaya Chakravarty Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India Background. Treatment of visceral leishmaniasis (VL) is far from satisfactory. There is an urgent need for a therapy that is efficacious, safe, affordable, and of short duration. Methods. A randomized open-label study was conducted to assess the efficacy and safety of 2 regimens of paromomycin administered intramuscularly. Group A received 11 mg/kg/day for 14 days ( n p 217 ) and group B received 11 mg/kg/day for 21 days ( n p 112) for the treatment of VL in India. Results. Mild grade injection site pain was the most common adverse event. There was no nephrotoxicity, but 4 patients in group A had to discontinue treatment because of grade 3 elevation of hepatic enzymes. Initial cure was observed in 91.2% and 96.4% of patients in group A and group B, respectively. Definitive cure at 6 months of follow up was seen in 82% of patients in group A and 92% of patients in group B by intention-to-treat analysis and in 84.3% of patients in group A and 92.8% of patients in group B by per protocol analysis. Conclusions. Although the cure rate in the group of patients who received the 14-day regimen was not optimal, the results with respect to initial cure were encouraging. Further studies that combine a short course of paromomycin with treatment with another antileishmanial agent are warranted. (ClinicalTrials.gov identifier: NCT00629031). Treatment of visceral leishmaniasis (VL; also known as kala-azar) continues to be unsatisfactory in most regions of the developing world in which the disease is endemic. In India, the problem has become acute, because pentavalent antimony compounds, which have been standard of care for VL for 16 decades, have become ineffective. In recent years, the approval of miltefosine and paromomycin for the treatment of VL in India has provided therapeutic alternatives [1, 2]. Paromomycin, although affordable, requires intramuscular injections for 3 weeks. Miltefosine, the only oral agent, is expensive, teratogenic, associated with frequent gastrointestinal adverse events, and needs to be given for 4 weeks. Moreover, because of its long half life, there is apprehension that drug resistance may develop early. Although Gilead, the manufacturer of liposomal amphotericin B, has offered a preferential 10% Received 22 February 2009; accepted 5 May 2009; electronically published 7 August 2009. Reprints or correspondence: Dr. Shyam Sundar, Dept. of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221 005, India (drshyamsundar@hotmail.com). Clinical Infectious Diseases 2009; 49:914 8 2009 by the Infectious Diseases Society of America. All rights reserved. 1058-4838/2009/4906-0013$15.00 DOI: 10.1086/605438 of its price for public sector, the drug remains out of reach for those seeking treatment in the private sector. Thus, treatment of VL has to be given for 3 4 weeks, and compliance with any therapy is known to decrease with longer duration of treatment, increasing the probability of the emergence of drug-resistant parasites. In addition, a regimen with shorter duration would cost less, be easier to administer, and be likely to have increased compliance. A phase 3 study showed that paromomycin at a dosage of 11 mg/kg (15 mg/kg as the sulphate) delivered intramuscularly for 21 days was noninferior to amphotericin B, which is the current standard treatment for VL [2]. It also had a reasonable safety profile. Not only is it the cheapest antileishmanial agent, it can be administered easily in the existing infrastructure of the health care delivery system in India. Paromomycin has been used for 17 days in combination with sodium stibogluconate [3]. Therefore, we decided to generate efficacy data for a shorter-duration, 14-day treatment with paromomycin. This study was conducted to evaluate whether paromomycin administered at a dosage of 11 mg/kg/day for 14 days has efficacy and safety similar to that of paromomycin administered at a dosage of 11 mg/kg/ 914 CID 2009:49 (15 September) Sundar et al
day for 21 days (ClinicalTrials.gov identifier: NCT00629031). METHODS Study design. This was a randomized, open label study intended to assess the efficacy and safety of 2 regimens of paromomycin administered intramuscularly. Group A received 11 mg/kg/day for 14 days and group B received 11 mg/kg/day for 21 days for the treatment of VL in India. Study site. The study was conducted at the Muzaffarpur, India, field site of Kala-azar Medical Research Center. The Ethics Committee of Kala-azar Medical Research Center approved the protocol. Written informed consent was obtained from all of the patients. In subjects!18 years of age, a parent or legal guardian s consent was taken. The study was conducted in accordance with the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use Guidelines for Good Clinical Practice. Inclusion criteria. Patients of either sex who were 5 55 years of age with signs and symptoms of VL (ie, fever, weight loss, and splenomegaly) who had demonstrable parasites in splenic smears were included in this study. Patients with relapsed VL or who experienced treatment failure while receiving a regimen that did not contain paromomycin were also included. Exclusion criteria. Women with child-bearing potential were excluded from the study if they were breast-feeding, were pregnant, or were not using any contraception. Exclusion criterion included a granulocyte count of!1000 cells/mm 3, a hemoglobin level of!4 g/dl, a platelet count!40,000 platelets/ mm 3, liver function test results 13 times of upper limit of normal, prothrombin time 5 sec more than that for control subjects, serum creatinine level above the upper limit of normal, abnormal audiometric, and/or vestibular function, serological test results positive for HIV, and concomitant severe medical conditions. Patients who were allergic to aminoglycosides, those treated with a parenteral aminoglycoside within 28 days prior to randomization or with paromomycin at any time and those who had taken VL treatment within the past 14 days were excluded from the study. Baseline evaluation. A baseline screening for pregnancy, malaria, and HIV infection was performed. A baseline complete blood count, a biochemical profile including a liver function test and determination of serum creatinine and electrolyte levels, a blood urea nitrogen test, a blood sugar test, and urinalysis were performed. Parasitological diagnosis was made by demonstration of leishmania amastigotes in splenic aspirate smears. Drug. Paromomycin sulfate was supplied as 2-mL ampoules (375 mg/ml of base), manufactured by Gland Pharma (Hyderabad, India). The drug was donated by Institute of One World Health; however, the company had no role in the design, the data analysis, or the decision to publish the results. Treatment protocol. Each subject received injections of paromomycin sulfate solution as a single intramuscular injection into the gluteus muscle (alternating buttock cheeks) daily until completion of treatment, treatment discontinuation, or the subject s withdrawal from the study. The dosing regimen was 11 mg/kg/day for 14 days in group A and 11 mg/kg/day for 21 days in group B. Rescue medication. In cases of premature withdrawal as a result of adverse events, treatment failure, or relapse, patients were given 15 infusions of amphotericin B deoxycholate at a Table 1. Baseline and Post-Treatment Cinical and Laboratory Parameters in a Study of Short-Course Paromomycin Treatment of Visceral Leishmaniasis in India Variable Normal range Group A Group B Day 0 Day 14 Day 0 Day 21 Age, years 22.04 14.64 18.15 12.34 Age 5 14 years, % of patients 43.7 54.4 Weight, kg 32.44 14.4 33.1 14.33 30.25 12.63 30.3 12.59 Palpable spleen size, cm 4.39 3.38 2.1 2.35 a 4.07 2.8 1.68 1.53 a Karnofsky score (0 100) 70 0 86.3 6.8 a 70 0 83.39 8.96 a Hemoglobin level, g/dl Men, 13.0 16.5; women, 11.5 13.5 8.1 2.05 9.44 1.79 a 8.18 1.89 10.83 10.62 b White blood cell count, 10 3 cell/mm 3 3.5 10 3.3 1.6 5.6 2.1 a 3.3 1.5 7.3 2.7 a Platelet count, 10 3 platelets/mm 3 100 400 115.2 56.3 208.4 78.6 a 123.2 55.3 258.6 88.5 a Creatinine level, mg/dl Men, 0.6 1.1; 0.69 0.21 0.66 0.19 a 0.63 0.17 0.61 0.16 b women, 0.5 0.9 Serum aspartate aminotransferase level, U/L 7 35 48.74 21.83 51.46 25.07 48.87 21.75 48.63 20.95 Serum alanine aminotransferase, U/L 9 45 31.64 25.64 44.38 29.66 b 31.75 20.23 41.16 19.78 a NOTE. Data are mean value standard deviation, unless otherwise indicated. Group A received 14 days of treatment. Group B received 21 days of treatment. a P.001. b P p.01. Treatment of VL with Paromomycin CID 2009:49 (15 September) 915
Table 2. Response to Treatment and the Final Outcome at 180 Days for Patients Participating in a Study of Short-Course Paromomycin Treatment of Visceral Leishmaniasis in India Variable Group A (n p 217) Group B (n p 112) Failure at the end of treatment 12 3 Lost to follow up a 3 1 Treatment discontinued because of high SGOT:SGPT 4 0 Initial cure 198 (91.2) 108 (96.4) Death 0 0 Relapse 21 5 Definitive cure Per protocol analysis b, % of patients (95% CI) 84.3 (79 89) 92.8 (88 98) Intention-to-treat analysis c No. of patients 177 103 Percentage of patients (95% CI) 81.6 (76 88) 91.96 (84 99.9) NOTE. Data are no. or no. (%) of patients, unless otherwise indicated. CI, confidence interval; SGOT, serum glutamic oxaloacetic transaminase; SGPT, serum glutamic pyruvic transaminase. a Three patients in group A and 1 patient in group B had a splenic score of 1+ at the end of treatment but were lost to follow up and did not return for the repeat splenic aspiration scheduled after 28 days. b P p.03. c P p.007. dosage of 1 mg/kg diluted in 5% dextrose solution on alternate days. Patients were assessed daily for adverse events. A complete blood count, a biochemical profile including a liver function test and determination of serum creatinine and electrolyte levels, a blood urea nitrogen test, a blood sugar test, and urinalysis were repeated on day 7 and day 14 or 21 of treatment. Pain at the injection site was assessed 30 min after each injection. The Common Terminology Criteria for Adverse Events, version 3.0, was used to assess all adverse events. Treatment with the study drug was discontinued in the event of a serious adverse event attributable to the study drug, an increase in serum creatinine level 2.5 mg/dl, or a doubling of the baseline serum creatinine level to 2.0 mg/dl, increase in alanine transaminase and/or aspartate aminotransferase to grade 3 and above, treatment with a medication that is not allowed by protocol, serious or repeated noncompliance of the subject with protocol specifications, and pregnancy. Efficacy. Post-treatment parasitic evaluation was performed on day 15 for group A and day 22 for group B by Splenic aspirate smear examination. Initial cure was defined as parasite-negative splenic aspirate findings at the end of treatment (day 14 for group A and day 21 for group B) with improvement in clinical signs and symptoms. If the aspirate score was equal to 1 at the end of treatment, patients were reassessed for parasitological cure by examination of an additional splenic aspirate obtained 28 days later. If the aspirate score was 2, then the subject was considered to have experienced treatment failure, and rescue medication (amphotericin B deoxycholate administered at a dosage of 1 mg/kg for 15 infusions) was given. Final cure was defined as the absence of clinical signs and symptoms of VL at the follow-up visit on day 180 without clinical relapse before this time (with the presence of VL confirmed by splenic aspirate findings positive for parasites). Statistical analysis. Assuming a 95% cure rate after 21 days of treatment with paromomycin, 327 patients were needed in a 2:1 ratio to support a 1-sided noninferiority analysis without stratification and with 80% power to detect a type I error rate of 5%. Primary efficacy was calculated as the proportion of patients who achieved a final cure. An exact confidence interval for that proportion was computed; the exact, 1-sided, upper bound of the 97.5% confidence interval for the difference in success probabilities was compared with the use of d p 0.07 (the chosen margin for noninferiority). Patients were allocated to a randomization arm from a random table generated for this purpose. Statistical significance of the baseline parameters between the groups was calculated by independent Student s t test for quantitative variables and x 2 test for qualitative variables. Paired Student s t test was used to compare the pretreatment and posttreatment effects in case of quantitative variables. RESULTS Baseline characteristics were similar in both groups with the exception of serum creatinine level, which was higher in group A than in group B (table 1). Efficacy. There was improvement in most of the clinical and hematological parameters at the end of treatment (table 916 CID 2009:49 (15 September) Sundar et al
Figure 1. Disposition of patients in a study of short-course paromomycin treatment of visceral leishmaniasis in India. ALT, alanine aminotransferase level; AST; aspirtate transaminase level. 1). All patients were afebrile, with improvement in performance status and statistically significant decreases in the size of the spleen at the end of treatment. There was improvement in each of the biochemical parameters except serum alanine transaminase level, which became elevated in several patients. Initial cure was observed in 91.2% and 96.4% of patients. Definitive cure at the end of 6 months was observed in 82% and 92% of patients in group A and group B, respectively, using intentionto-treat analysis and in 84.3% and 92.8% of patients in group A and group B, respectively, using per protocol analysis (table 2 and figure 1). Safety. Injection site pain was the most common adverse event, with 44.4% patients reporting pain of mild grade. No patient developed nephrotoxicity in either group. Elevation of hepatic transaminase levels was seen in both groups. A grade 2 or greater elevation in alanine aminotrasferase levels occurred in 6.0% and 6.3% patients of group A and group B, respectively. Similarly, a grade 2 or greater elevation in aspartate aminotransferase was seen in 14.7% and 16.9% of patients in group A and group B, respectively. However, 20.7% and 19.6% of patients in group A and group B, respectively, had grade 1 elevations of alanine aminotransferase levels at baseline. Therapy was discontinued for 4 patients in group A because of increases in hepatic enzyme levels to grade 3; these patients were given rescue medication. Each of these patients had grade 1 elevation of liver enzyme levels at baseline. DISCUSSION Paromomycin administered at a dosage of 11 mg/kg for 21 days demonstrated safety and efficacy similar to that reported in our previous study [2]. The safety profile was also excellent; none of the patients developed nephrotoxicity. A small proportion of patients developed elevated hepatic transaminases levels, although in most cases, these elevations were mild and did not necessitate a discontinuation of the study drug. Four patients (1.2%) had significant elevations in hepatic enzyme levels that lead to a discontinuation of the study drug, but it is pertinent to note that these patients had elevated hepatic enzyme levels at baseline. However, 20.7% and 19.6% of patients in group A Treatment of VL with Paromomycin CID 2009:49 (15 September) 917
and group B, respectively, had grade 1 elevation of alanine aminotransferase levels at baseline, suggesting that this elevation of transaminase levels may not be related to the study drug. Elevation of hepatic enzyme levels was also observed in few patients in the phase III trial, as well [2]. Although this adverse event may or may not be related to paromomycin, monitoring of hepatic enzyme levels should nevertheless be done in patients who receive paromomycin therapy. Treatment with 14 days of therapy resulted in a statistically significantly lower cure rate. However, if the rate of initial parasitic clearance is considered, the difference between 14-day and 21-day regimens was not statistically significant, and at the end of 14 days of treatment, 91% of patients had spleen smears with undetectable parasite loads. There was also significant improvement in clinical parameters (eg, fever, spleen size, and karnofsky score) and biochemical parameters (eg, hemoglobin levels, white blood cell counts, and platelet counts) at the end of therapy, even with only 2 weeks of treatment. It is obvious that, with such low cure rates, this short regimen cannot be used as monotherapy. Similar low cure rates have been observed with other antileishmanial drugs when lower total doses have been used [4, 5]. The high rate of initial parasitic clearance and improvement in clinical and biochemical parameters can be used and should be tested in combination with other drugs. As is the case with the treatment of other diseases, such as tuberculosis and leprosy, combining multiple drugs offers several advantages, such as reduced drug pressure and a shorter duration of treatment, with better compliance, reduced toxicity, reduced cost, and shorter hospital stay. There is a growing interest in using multidrug therapy for the treatment of visceral leishmaniasis. Paromomycin was one of the first drugs to be used in combination with pentavalent antimony compounds in the 1990s in Kenya, India, and Sudan [3, 6, 7]. These combinations not only reduced the duration of treatment but also enhanced the overall efficacy. The combination of pentavalent antimony compounds with paromomycin is currently in use in Sudan [8]. Recently, a short-course sequential regimen with singledose liposomal amphotericin B followed by 7 14 days of treatment with miltefosine showed excellent results in the Indian subcontinent [9]. Another multicenter, multidrug study that includes paromomycin has been initiated in India; the results of our study could help in designing and interpreting the results of this and other future studies involving short-course paromomycin [10]. The drawback of any combination with paromomycin will be the need for intramuscular injections and monitoring of liver enzyme levels; these drawbacks seem to be inevitable, because miltefosine also causes elevation of hepatic enzyme levels and is teratogenic. The advantages associated with the use of paromomycin are low cost, easy availability, and indigenous production. In conclusion, there is an urgent need to rethink the strategy for the VL elimination program [11] and to shift from monotherapy to a multidurg therapy that is effective, cheap, and feasible in the existing infrastructure. Acknowledgment We thank the Institute of One World Health for donating paromomycin. Financial support. Sitaram Memorial Trust. Potential conflicts of interest. S.S. has received support from the Institute of One World Health for presenting scientific data at international meetings and for conducting clinical trials of paromomycin and has also received support for clinical trials and travel funds to attend scientific meetings from Zentaris, Paladin Labs, and NeXstar/Gilead Sciences. All other authors: no conflicts. References 1. Sundar S, Jha TK, Thakur CP, et al. Oral miltefosine for Indian visceral leishmaniasis. N Engl J Med 2002; 347:1739 46. 2. Sundar S, Jha TK, Thakur CP, Sinha PK, Bhattacharya SK. Injectable paromomycin for visceral leishmaniasis in India. N Engl J Med 2007; 356:2571 81. 3. Seaman J, Pryce D, Sondorp HE, Moody A, Bryceson AD, Davidson RN. Epidemic visceral leishmaniasis in Sudan: a randomized trial of aminosidine plus sodium stibogluconate versus sodium stibogluconate alone. J Infect Dis 1993; 168:715 20. 4. Sundar S, Jha TK, Thakur CP, Mishra M, Singh VR, Buffels R. Lowdose liposomal amphotericin B in refractory Indian visceral leishmaniasis: a multicenter study. Am J Trop Med Hyg 2002; 66:143 6. 5. Sundar S, Makharia A, More DK, et al. Short-course of oral miltefosine for treatment of visceral leishmaniasis. Clin Infect Dis 2000; 31:1110 3. 6. Chunge CN, Owate J, Pamba HO, Donno L. Treatment of visceral leishmaniasis in Kenya by aminosidine alone or combined with sodium stibogluconate. Trans R Soc Trop Med Hyg 1990; 84:221 5. 7. Thakur CP, Olliaro P, Gothoskar S, et al. Treatment of visceral leishmaniasis (kala-azar) with aminosidine (p paromomycin)-antimonial combinations, a pilot study in Bihar, India. Trans R Soc Trop Med Hyg 1992; 86:615 6. 8. Melaku Y, Collin SM, Keus K, Gatluak F, Ritmeijer K, Davidson RN. Treatment of kala-azar in southern Sudan using a 17-day regimen of sodium stibogluconate combined with paromomycin: a retrospective comparison with 30-day sodium stibogluconate monotherapy. Am J Trop Med Hyg 2007; 77:89 94. 9. Sundar S, Rai M, Chakravarty J, et al. New treatment approach in Indian visceral leishmaniasis: single-dose liposomal amphotericin B followed by short-course oral miltefosine. Clin Infect Dis 2008;47: 1000 6. 10. ClinicalTrials.gov National Institutes of Health. Clinical trials registry. Available at: http://www.clinicaltrials.gov/ct2/search. Accessed 12 January 2009. 11. World Health Organization on behalf of the Special Programme for Research and Training in Tropical Diseases. Research to support the elimination of visceral leishmaniasis. TDR Business Line 10. Available at: http://www.who.int/tdr/svc/publications/about-tdr/annual -reports/visceral-leishmaniasis-elimination-2008. Accessed 12 January 2009. 918 CID 2009:49 (15 September) Sundar et al