This Article Discuss About:
- Etiology of Sleeping sickness
- The Forms And Geographic Prevalence Of Sleeping Sickness
- The Scenario For Treatment Of Sleeping Sickness
- Can Eflornithine Treat Sleeping Sickness or African Trypanosomiasis?
- Physiological Effect Of Eflornithine On The Sleeping Sickness Parasite
- Clinical Experience Of Treating Sleeping Sickness With Eflornithine
- Further Advances In Eflornithine-Based Therapy For Sleeping Sickness
Etiology of Human African Trypanosomiasis (HAT) or Sleeping Sickness
African trypanosomes are unicellular protozoons that belong to the species Trypanosoma brucei. They are hemoflagellate blood parasites of man, animals and the tse-tse fly. The bite of the tse-ste fly transmits the trypanosomes to the mammalian host, where these extracellular parasites propagate by cell division in the blood, lymph nodes, interstitial fluids and the cerebrospinal fluid. In humans the disease is called sleeping sickness. Infection is indicated by classic symptoms of sleeping sickness such as recurrent fever, chills, severe headaches, rigor, muscular pain, edema including swelling of the face, skin lesions, erythematous rashes, lymphadenopathy, encephalitis, and extreme weakness. The disease owes its name to the symptoms late in infection when the parasite crosses the blood-brain barrier and multiplies in the brain, resulting first in lethargy followed by malfunctioning of the brain, stupor, eventually leading to coma and if untreated in time, death. Occassionally the parasite is contracted through blood transfusion , organ transplant, and vertical transmission in- utero from mother to foetus.
A bite from the tse-tse fly releases the trypanosomes into the host bloodstream. While the parasites replicate by cell division in the bloodstream and extracellular fluids, the host launches a defensive immune response to control the infection by producing parasite-specific antibody response. The parasite is covered by a coat of a tightly packed glycoprotein which switches to a different antigenic form on encountering selection pressure in form of neutralizing host antibody in order to escape immune surveillance. Hence it is called the variant surface glycoprotein or VSG. The parasite is completely masked in this VSG coat, and hence there is no access for antibodies to underlying parasite molecules. Switching of the VSG coat to a different antigenic type renders the antibodies irrelevant, while the switched form multiples and launches a new antibody response. This interplay between the host immune system and the parasite's evasion of the response occurs repeatedly. Thus the parasite always remains ahead of the immune response. The parasites continue to expand in the bloodstream, while some escape to extracellular tissue spaces of various organs including the heart, the lymphatics, the cerebrospinal fluid and eventually cross the blood-brain barrier. There are two forms of the infection- the slow or chronic; and the acute or rapid form. In the chronic form the infection lasts over several months, while it lasts a few weeks in the acute form. Infection of the central nervous system causes severe headaches, lassitude, continuous drowsiness, ataxia followed by deep coma. In any case without successful intervention, death is inevitable. The tse-tse fly gets infected upon taking a blood meal from a host, and transmits it to other hosts. As mentioned earlier the host can be human or animal especially bovids both domestic and wild.
The Forms And Geographic Prevalence Of Sleeping Sickness
As mentioned earlier, sleeping sickness can manifest as s a slow or chronic form, or a rapid, fulminant infection. The difference is due to the two subspecies of the Trypanosoma parasite, namely Trypanosoma brucei gambinese and Trypansoma brucei rhodesiense. While both subspecies infect both man and animals, the animals are better able to hold the Typanosoma brucei rhodesiense parasite in check, serving as reservoirs for the organism, while it is lethal for humans. Trypanosoma brucei gambiense infection can progress slowly over years and therefore man too is a reservoir for these parasites. A third subspecies Trypanosoma brucei brucei infects bovids and man, but is effectively controlled by man and wild bovids. In cattle it causes a lethal wasting disease, also called Nagana which has a major economic impact on livestock agriculture. Since this article discusses sleeping sickness and treatments available, Trypanosoma brucei brucei will not be discussed further.
Trypanosoma brucei gambiense occurs in the West and Central regions of the continent of Africa, while Trypanosoma brucei rhodesiense occurs in the Eastern regions of Africa. Both subspecies are found in the country of Uganda. Trypanosoma brucei gambiense is responsible for over 95% of reported cases of sleeping sickness ( Brun, R., Don, R., Jacobs, R.T. et al., 2011. Development of noveldrugs for human African trypanosomiasis. Future Microbiol. 6 (6), 677–691.; Simarro, P.P., Diarra, A., Ruiz Postigo, J.A., et al., 2011. The human African trypanosomiasis control and surveillance programme of the World Health Organization 2000–2009: the way forward. PLoS Negl. Trop. Dis. 5 (2), e1007. ref ). Over 60 million humans are at risk from African trypanosomiasis in sub-Saharan Africa. While the disease has been controlled with comparative success due to the unrelenting efforts of the World Health Organization, 9000 deaths occurred in 2010; with 0.6 million Deaths and disability adjusted life years (DALYs) in that year alone ( Lozano et al., 2012. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet 380 (9859), 2095–2128. ref ). As parasite forms resistant to current treatment drugs continue to arise, there is much concern that the number of deaths and DALYs due to sleeping sickness will soar again ( Vincent, I.M., Creek, D., Watson, D.G., et al., 2010. A molecular mechanism for eﬂornithine resistance in African trypanosomes. PLoS Pathog. 6 (11), e1001204.; ker, N., de Koning, H.P., Maser, P., Horn, D., 2013. Drug resistance in African trypanosomiasis: the melarsoprol and pentamidine story. Trends Parasitol. 29 (3), 110–118. ref ).
The Scenario For Treatment Of Sleeping Sickness
Although attempts have been continuously made to create vaccines, thus far, the antigenic variation of the variant surface glycoprotein coat of the African trypanosome makes vaccination futile. Chemotherapy has been the mainstay of treatment against sleeping sickness over a century and continues to remain so, with many challenges still unresolved. Most of the drugs available have only limited efficacy and are limited to one or the other form of the disease (Trypansoma brucei gambiense or rhodesiense) and/ or to the stage of clinical infection. Further limitations are posed by the scarcity of supplies and logistics to deliver the drugs. However the main and most aggravating limitation is that all the traditional drugs known to work against the sleeping sickness parasites are toxic to man, some far surpassing the benefit of efficacy. Often patients succumb to the toxic profiles of these drugs, rather than the disease per se.
Parenteral administration of Suramin or pentamidine are used to treat early stage infection by both Trypansoma brucei rhodesiense or gambiense ( Lourie, E., Yorke, W., 1937. Studies in chemotherapy. XVI. The trypanocidal action of synthalin. Ann. Trop. Med. Parasitol. 31, 435–445.; Steverding, D., 2010. The development of drugs for treatment of sleeping sickness: a historical review. Parasit. Vectors 3 (1), 15.; Murthy, S., Keystone, J., Kissoon, N., 2013. Infections of the developing world. Crit. Care Clin. 29 (3), 485–507. ref ). Eflornithine and melarsoprol are the drugs used to control late stage infection however parasite resistance has emerged against both melarsoprol and eflornithine( Burri, C., Brun, R., 2003. Eﬂornithine for the treatment of human African trypanosomiasis. Parasitol. Res. 90 (Supp 1), S49–S52.; Friedheim, E.A., 1949. Mel B in the treatment of human trypanosomiasis. Am. J. Trop. Med. Hyg. 29 (2), 173–180.; Pepin, J., Milord, F., Guern, C., Schechter, P.J., 1987. Diﬂuoromethylornithine for arseno-resistant Trypanosoma brucei gambiense sleeping sickness. Lancet 2 (8573), 1431–1433.) (Pepin, J., Milord, F., 1994. The treatment of human African trypanosomiasis. Adv. Parasitol. 33, 1–47.; Burri, C., Keiser, J., 2001. Pharmacokinetic investigations in patients from northern Angola refractory to melarsoprol treatment. Trop. Med. Int. Health 6 (5), 412–420.; Robays, J., Nyamowala, G., Sese, C. et al., 2008. High failure rates of melarsoprol for sleeping sickness, Democratic Republic of Congo. Emerg. Infect. Dis. 14 (6), 966–967.; Barrett, M.P., Vincent, I.M., Burchmore, R.J., et al., 2011. Drug resistance in human African trypanosomiasis. Future Microbiol. 6 (9), 1037–1047. ref ). Although in use for treating sleeping sickness for over eighty years, melarsoprol is an extremely toxic arsenic derivative, and can induce gastrointestinal and renal toxicity, exfoliative dermatitis, cardiac arrest, encephalopathy and death ( Brun, R., Don, R., Jacobs, R.T., et al., 2011. Development of novel drugs for human African trypanosomiasis. Future Microbiol. 6 (6), 677–691. ref ). 5-10% of patients on melarsoprol suffer drug-induced reactive encephalopathy of which 10-70% die ( Griffin CA, Slavik M, Chien SC, Hermann J, Thompson G, Blanc O, et al. Phase I trial and pharmacokinetic study of intravenous and oral alpha-difluoromethylornithine. Invest New Drugs 1987;5:177-86.; Griffin CA, Slavik M, Chien SC, Hermann J, Thompson G, Blanc O, et al. Phase I trial and pharmacokinetic study of intravenous and or alpha- difluoromethylornithine. Invest New Drugs 1987;5:177-86.; Blum, J., Nkunku, S. and Burri, C. (2001). Clinical description of encephalopathic syndromes and risk factors for their occurrence and outcome during melarsoprol treatment of human African trypanosomiasis. Tropical Medicine and International Health 6, 390 – 400.; Seixas, J. (2004). Investigations on the encephalopathic syndrome during melarsoprol treatment of human African trypanosomiasis. Ph.D. thesis, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa.; Burri, C. (2010). Chemotherapy against human African trypanosomiasis: Is there a road of success? Parasitology 137, 1987 – 1994. ref )
Although eflornithine and melarsoprol are the two drugs available to control trypanosmiasis once the central nervous system is involved due to their ability to traverse blood-brain barrier to enter the CNS, eflornithine is preferred due to two crucial factors- first: slower emergence of drug resistant forms is associated with eflornithine ( Brun, R., Schumacher, R., Schmid, C., et al., 2001. The phenomenon of treatment failures in Human African Trypanosomiasis. Trop. Med. Int. Health 6 (11), 906–914.; Bernhard, S.C., Nerima, B., Maser, P. et al., 2007. Melarsoprol- and pentamidine-resistant Trypanosoma brucei rhodesiense populations and their cross-resistance. Int. J. Parasitol. 37 (13), 1443–1448.; Barrett, M.P., Vincent, I.M., Burchmore, R.J.et al., 2011. Drug resistance in human African trypanosomiasis. Future Microbiol. 6 (9), 1037–1047. ref ).; and second: it is far less toxic than other trypanosome targeted drugs including melarsoprol. The emergence of eflornithine-resistant parasites is largely overcome by the introduction of a combination therapy of eflornithine with the antibiotic nifurtimox. However there is an unfortunate limitation to the best option treatment with eflornithine as it is not successful in control of Trypanosoma brucei rhodesiense ( Iten, M., Mett, H., Evans, A., et al., 1997. Alterations in ornithine decarboxylase characteristics account for tolerance of Trypanosoma brucei rhodesiense to D , L -alpha-diﬂuoromethylornithine. Antimicrob. Agents Chemother. 41 (9), 1922–1925.; Priotto, G., Kasparian, S., Ngouama, D., et al., 2007. Nifurtimox–eﬂornithine combination therapy for second-stage Trypanosoma brucei gambiense sleeping sickness: a randomized clinical trial in Congo. Clin. Infect. Dis. 45 (11), 1435–1442.; Priotto, G., Kasparian, S., Mutombo, W., et al., (2009). Nifurtimox-eﬂornithine combination therapy for second-stage African Trypanosoma brucei gambiense trypanosomiasis: a multicentre, randomised, phase III, non-inferiority trial. Lancet 374, 56 – 64.); Murthy, S., Keystone, J., Kissoon, N., 2013. Infections of the developing world. Crit. Care Clin. 29 (3), 485–507. ref ). Moreover there are certain challenges to be overcome in use of eflornithine such as the high cost of the drug, limited availability due to limited manufacture, , limited access due to logistics of distribution, and difficulty of administration ( Barrett, M.P., Vincent, I.M., Burchmore, R.J.et al., 2011. Drug resistance in human African trypanosomiasis. Future Microbiol. 6 (9), 1037–1047; Simarro, P.P., Franco, J., Diarra, A., et al., 2012. Update on ﬁeld use of the available drugs for the chemotherapy of human African trypanosomiasis. Parasitology 139 (7), 842–846. ref ). Meanwhile, in lieu of eflornithine the use of melarosoprol continues even for Trypanosoma brucei gambiense infections. Eflornithine is a relatively new drug. It has a high success rate for treatment of sleeping sickness due to low toxicity, relatively safe profile, and significant efficacy in elimination of CNS infection, based on a mechanism of action distinct from other trypanosome targeting drugs. Eflornithine is made available in Africa largely due to the efforts of the World Health Organization. As the best available treatment choice and the most recent, development of eflornithine as a drug to treat sleeping sickness is discussed further in detail.
Can Eflornithine Treat Sleeping Sickness or African Trypanosomiasis? Pioneering Studies Of Eflornithine And Its Discovery As A Treatment For Sleeping Sickness
Alpha-Difluoromethylornithine (DFMO) or Eﬂornithine (RMI 71,782), is a compound that irreversibly inhibits polyamine biosynthesis by specific inhibition of a the enzyme ornithine decarboxylase which catalyzes the first step in synthesis of polyamines ( Science. 1980 Oct 17;210(4467):332-4. Polyamine metabolism: a potential therapeutic target in trypanosomes. Bacchi CJ, Nathan HC, Hutner SH, McCann PP, Sjoerdsma A. ref ). Eflornithine was created in the 1970s by the Merrell International Research Center, Strasbourg, France. First identiﬁed for its ability to inhibit the replication of cancer cells, ( Casero Jr., R.A., Woster, P.M., 2009. Recent advances in the development of polyamine analogues as antitumor agents. J. Med. Chem. 52 (15), 4551–4573. ref ) the efficacy of eflornithine as an anticancer agent was tested in clinical trials. But the drug was abandoned due to the adverse effect profile observed ( Abeloff, M.D., Rosen, S.T., Luk, G.D., et al., 1986. Phase II trials of alpha-diﬂuoromethylornithine, an inhibitor of polyamine synthesis, in advanced small cell lung cancer and colon cancer. Cancer Treat. Rep. 70 (7), 843–845. ref ). Eflornithine remains a potential prophylactic anticancer agent against skin cancer, along with the effect of facial hair loss ( Inhibition of the Development of Metastatic Squamous Cell Carcinoma in Protein Kinase C Transgenic Mice by -Difluoromethylornithine Accompanied by Marked Hair Follicle Degeneration and Hair Loss. Deric L. Wheeler, Kristin J. Ness, Terry D. Oberley, and Ajit K. Verma, CANCER RESEARCH 63, 3037–3042, June 15, 2003 ref ). Eﬂornithine was found to be active against P. carinii infection, and has since been used for treating AIDS patients ( Paulson, Y.J., Gilman, T.M., Heseltine, P.N., et al., 1992. Eﬂornithine treatment of refractory Pneumocystis carinii pneumonia in patients with acquired immunodeﬁciency syndrome. Chest 101 (1), 67–74. ref ). In 1989 it was found that in sheeps, intravenously administered eflornithine prevents wool growth by inhibiting polyamine synthesis in hair follicle cells ( Reis, P.J and Hynd, P.I., The influence of -difluoromethylornithine on the activity of wool follicles. AJAS 1989 Vol. 2 (No. 3) 204-205 ref ), and was later found to inhibit human hair growth by the same mechanism. In 2001 FDA granted approval to Bristol-Myers Squibb Company and the Gillette Company to market eflornithine hydrochloride as a prescription drug for hirsutism, under the label Vaniqa. Vaniqa is a topical cream containing eflornithine for removing facial hair in women ( Hickman, J.G., Huber, F., Palmisano, M., 2001. Human dermal safety studies with eﬂornithine HCl 13.9% cream (Vaniqa), a novel treatment for excessive facial hair. Curr. Med. Res. Opin. 16 (4), 235–244.; Shapiro, J., Lui, H., 2001. Vaniqa–eﬂornithine 13.9% cream. Skin Therapy Lett. 6 (7), 1–3, 5. ref ). Clinical trials have shown Vaniqa to be highly effective against hirsutism, with results noticeable after 8 weeks post-treatment; and clinical benefit after twelve months of treatment in 81% patients. On an aside, here is an entertaining quote: "A six-page supplement to the January issue of Cosmopolitan begins with pictures of three gorgeous women with hairless lips and the words: ''If the mustache that prevents you from getting close is yours (not his), it may be time for a beauty about-face. Millions of women like yourself battle unwanted facial hair.'' ( http://www.nytimes.com/2001/02/09/world/cosmetic-saves-a-cure-for-sleeping-sickness.html. ref )
Polyamines are necessary to cell functions such as cell division and cell differentiation, especially spermine and spermidine are needed for synthesis of both nucleic acid and protein that occur at a high rate in actively growing and/or differentiating cells such as cancer cells, hair follicle cells and trypanosomes ( Pegg AE, McCann PP. Am J Physiol. 1982 Nov;243(5):C212-21. Polyamine metabolism and function. ref ). Blocking polyamine synthesis therefore retards the growth of rapidly dividing or differentiating cells. Putrescine is the product of the first step in polyamine synthesis resulting from the decarboxylation of ornithine by ornithine decarboxylase. Putrescine is then converted to spermidine by the transfer of an aminopropyl group derived from S-adenosyl methionine to putrescine by the enzyme spermidine synthase, and further to spermine upon addition of another aminopropyl group to spermidine by spermine synthase. As already mentioned the very first synthesis enzyme ornithine decarboxylase is irreversibly inactivated by its substrate analog eflornithine.
In 1979, researchers added eflornithine and radioactive [3H] ornithine to in vitro cultures of Trypanosoma brucei brucei and measured radioactive putrescine. The generation of puterscine diminished significantly indicating inhibition of this step ( Bacchi CJ, Nathan HC, Hutner SH, McCann PP, Sjoerdsma A. Science. 1980 Oct 17;210(4467):332-4. Polyamine metabolism: a potential therapeutic target in trypanosomes. ref ). Trypanosoma brucei brucei causes a virulent infection in mice, which was controlled, and the mice recovered when when treated with eflornithine orally or via intubation. Typanosoma brucei brucei is very closely related to Trypansooma brucei gambiense and Trypanosoma brucei rhodesiense. It is known that under selection pressure the three forms can interconvert, indicating that the intrinsic genome is the same, but modulated to differential expression based on the type of selection pressure.
It is interesting to note that the same researchers screened and found many ornithine and puterscine analogs that inhibit ornithine decarbxylase of trypanosoma brucie brucei , both in trypanosome cultures in vitro, and in mouse infections in vivo, but found only one analog of ornithine more potent in controlling infection than eflornithine: alpha-monofluoromethyldehydroornithine methyl ester( Biochem Pharmacol. 1985 May 15;34(10):1773-7. Catalytic irreversible inhibition of Trypanosoma brucei brucei ornithine decarboxylase by substrate and product analogs and their effects on murine trypanosomiasis. Bitonti AJ, Bacchi CJ, McCann PP, Sjoerdsma A. ref ). However, there is no further development reported on this more potent molecule for its potential use in treating trypanosomiasis in the experimental setting (in animal models), or in man. Further studies are warranted.
The Physiological Effect Of Eflornithine On The Sleeping Sickness Parasite
The effect of eflornithine on parasites in a rat model of trypanosomiasis was studied by comparing parasites harvested from infected, treated animals with infected but untreated controls ( Mol Biochem Parasitol. 1983 Mar;7(3):209-25. In vivo effects of alpha-DL-difluoromethylornithine on the metabolism and morphology of Trypanosoma brucei brucei. Bacchi CJ, Garofalo J, Mockenhaupt D, McCann PP, Diekema KA, Pegg AE, Nathan HC, Mullaney EA, Chunosoff L, Sjoerdsma A, Hutner SH. ref ). Rats infected with Trypanosoma brucei brucei for 60 hrs were treated with eflornithine for 12 hr or 36 hr, then killed to match the infection period of untreated control animals that died at 72-80 hr post-infection. Rats were injected with radioactive ornithine in order to trace polyamine synthesis. At the designated time points, rats were sacrificed, and their blood drawn. Parasites were isolated from the blood and studied for their intracellular polyamine content, and for their cell morphology. As expected, parasites obtained from rats treated with eflornithine had very low content of [3H] spermidine and putrescine at 99% reduction compared to parasites from untreated mice, at 12hr treatment. Parasites from rats treated for 36hr with eflornithine, also had greatly reduced spermidine and putrescine content. They could not synthesize [3H] putrescine from [3H]ornithine, but could produce spermidine if supplied with [3H] putrescine and methionine, indicating that eflornithine specifically inhibited putrescine production.
These parasites accumulated up to four-fold excess polyamines from the extracellular [3H] polyamine pool as a compensatory mechanism, as compared to parasites from rats which were not treated with eflornithine. The decarboxylated form of S-adenosylmethionine accumulated in thousand-fold excess of untreated cells, with decline in S-adenosylmethionine decarboxylase activity. The decarboxylated form of S-adenosylmethionine supplies the aminopropyl group to putrescine and spermidine, with their enzymatic conversion to spermidine and spermine.
The 36 hr treated parasites were unable to synthesize DNA and RNA with upto 100% inhibition, while protein synthesis increased four-fold. Congruent with this observation, the treated parasites occurred as the short, stumpy forms which are the non-replicating form in the parasite life cycle, rather than the actively dividing long, slender forms, which were obtained from untreated rats. This indicates that eflornithine inhibited cell division of the parasite. Trypanosomes contain two organelles that harbor DNA, the nucleus, and an additional body called the kinetoplast. The short, stumpy parasites contained two or more nuclei and kinetoplasts, indicating that cytokinesis was inhibited by action of eflornithine. The authors concluded that eflornithine blocked nucleic acid synthesis, inhibited cytokinesis, and induced conversion from dividing to non-dividing forms.
Eflornithine is trypanostatic rather than trypanocidal. It controls only Trypanosoma brucei gambiense but not Trypanosoma brucei rhodesiense as the target enzyme ornithine decarboxylase turnover is much higher in the latter parasite, therefore the enzyme is not suppressed by eflornithine ( Burri, C., Brun, R., 2003. Eﬂornithine for the treatment of human African trypanosomiasis. Parasitol. Res. 90 (Supp 1), S49–S52. ref ), S49–S52.)
The Clinical Experience Of Treating Sleeping Sickness With Eflornithine
In 1990 the U.S. FDA granted Sanofi Aventis approval for Ornidyl - which is an intravenous formulation of eflornithine- as a prescription drug for second line treatment of sleeping sickness when the infection reaches the central nervous system ( Burri, C., Brun, R., 2003. Eﬂornithine for the treatment of human African trypanosomiasis. Parasitol. Res. 90 (Supp 1), S49–S52.; Kennedy, P.G., 2008. The continuing problem of human African trypanosomiasis (sleeping sickness). Ann. Neurol. 64 (2), 116–126. ref ). This is a significant event in treatment of this lethal disease as eflornithine is the only new drug approved in ~60 years ( Burri, C., Brun, R., 2003. Eﬂornithine for the treatment of human African trypanosomiasis. Parasitol. Res. 90 (Supp 1), S49–S52. ref ). The manufacture of eflornithine as an intravenous form is challenging and hence the drug is more expensive than Vaniqa, the cream based formulation for treating hirsutism. The bulk drug is highly corrosive to the manufacturing equipment, and is made by a modification of the process used to make Vaniqa
( http://www.nytimes.com/2001/02/09/world/cosmetic-saves-a-cure-for-sleeping-sickness.html. ref ). Since 2001 eflornithine was widely distributed in Africa in endemic zones due to collaborative efforts of the World Health Organization, and Sanofi ( Kennedy, P.G., 2008. The continuing problem of human African trypanosomiasis (sleeping sickness). Ann. Neurol. 64 (2), 116–126. ref ) It was observed in Kiri, southern Sudan, that the relative risk of death of patients was significantly lower with eflornithine (n = 251, year 2003), adjusted relative risk of death was 0.2, as compared to melarsoprol-treated patients (n=708, year 2001 and 2002). The number of adverse events of cutaneous and neurological nature were much lower for eflornithine than melarosprol indicating eflornithine is far safer with much less morbidity than melarsoprol ( Chappuis F, Udayraj N, Stietenroth K, Meussen A, Bovier PA. Eflornithine is safer than melarsoprol for the treatment of second-stage Trypanosoma brucei gambiense human African trypanosomiasis. Clin Infect Dis 2005;41:748-51. ref ). Reversible adverse reactions are generally associated with eflornithine treatment such as seizures (7%), gastrointestinal symptoms like nausea, vomiting and
diarrhea (10%-39%), myelosuppression resulting in anemia, leucopenia and thrombocytopenia (25-50%), impaired hearing (5% in cancer patients) and alopecia (5-10%) ( Burri, C., Brun, R., 2003. Eﬂornithine for the treatment of human African trypanosomiasis. Parasitol. Res. 90 (Supp 1), S49–S52. ref ). The drug is terratogenic in lab animals such as rodents and rabbits.
The treatment regimen for eflornithine was labor-intensive, consisting of 56 intravenous infusions of strength 100 mg/kg body weight (an infusion every 6 h for 14 days) in adults. The strength was higher for children at 150mg/kg body weight. The treatment drug was modified to suppress resistance to eflornithine in the parasite by combining eflornithine with the antibiotic nifurtimox. Nifurtimox is a nitrofuran that kills Trypanosoma cruzi which causes Chagas disease ( Simarro, P.P., Diarra, A., Ruiz Postigo, J.A., et al., 2011. The human African trypanosomiasis control and surveillance programme of the World Health Organization 2000–2009: the way forward. PLoS Negl. Trop. Dis. 5 (2), e1007.; Simarro, P.P., Franco, J., Diarra, A., et al., 2012. Update on ﬁeld use of the available drugs for the chemotherapy of human African trypanosomiasis. Parasitology 139 (7), 842–846 ref ). Besides fewer relapses due to less frequency of drug resistance in parasite, the combination therapy is well tolerated, and is better for both healthcare givers and patients, as the infusions are decreased to 1 to 2 per day ( Priotto, G., Kasparian, S., Mutombo, W, et al.; 2009. Nifurtimox–eﬂornithine combination therapy for second-stage African Trypanosoma brucei gambiense trypanosomiasis: a multicentre, randomised, phase III, non-inferiority trial. Lancet 374 (9683), 56–64.; Lutje, V., Seixas, J., Kennedy, A., 2013. Chemotherapy for second-stage human African trypanosomiasis. Cochrane Database Syst. Rev. 6, CD006201.Lutje et al., 2013 ref )
Eflornithine has a bioavailability of 54% upon oral administration. The drug half-life is 1.5 to 5 hr on intravenous injection, and is manily (> 80%) cleared by the kidney at 2ml/min/kg. Eflornithine volume of distribution is 0.35 litres/kilogram. The critical drug concentration for effectiveness in treatment of sleeping sickness is 50 micro mol/litre of cerebrospinal fluid ( Burri, C., Brun, R., 2003. Eﬂornithine for the treatment of human African trypanosomiasis. Parasitol. Res. 90 (Supp 1), S49–S52.; Haegele KD, Alken RG, Grove J, Schechter PJ, Koch-Weser J. Kinetics of alpha-difluoromethylornithine: an irreversible inhibitor of ornithine decarboxylase. Clin Pharmacol Ther 1981; 30:210-7.; Abeloff MD, Slavik M, Luk GD, Griffin CA, Hermann J, Blanc O, et al. Phase I trial and pharmacokinetic studies of alpha-difluoromethylornithine — an inhibitor of polyamine biosynthesis. J Clin Oncol 1984;2:124-30.; Griffin CA, Slavik M, Chien SC, Hermann J, Thompson G, Blanc O, et al. PhaseI trial and pharmacokinetic study of intravenous and oral alpha-ifluoromethylornithine. Invest New Drugs 1987;5:177-86. ref ).
A major trial of eflornithine was conducted in disease endemic southern Sudan to assess its safety and efficacy in treatment of sleeping sickness ( Priotto, G., Pinoges, L., Badi Fursa, I., et al., (2008). Safety and eﬀectiveness of ﬁrst line eﬂornithine for Trypanosoma brucei gambiense sleeping sickness in Sudan. British Medical Journal 336, 705 – 708. ref ). The main inclusion criterion was newly diagnosed second stage infection, screened over a 16 month period. Second stage infection was determined by trypanosome- positive cerebrospinal fluid; or positive blood and lymph with 5×10 9 leucocytes/l of cerebrospinal fluid; or positive by agglutination test (titre 1:4 or greater) with 20×10 9 leucocytes /l of cerebrospinal fluid. Eflornithine was administered by intravenous infusions lasting 2 hours, at a dose of 100 mg/kg every six hours (400 mg/kg/day) for 14 days for adults. Children were dosed at 150 mg/kg every six hours (600 mg/kg/day) for 14 days. The higher dose in children was well-tolerated but did not show greater efficacy.
Primary endpoints were deaths, serious drug-related adverse events, and cure, measured at 24 months. 1055 adults and children were included in the trial. Serious adverse events were low. Serious adverse
events were seen in 138 (13.1%) patients- convulsions, fever, diarrhoea, and soft tissue bacterial infections, of which 15 deaths occurred. Prevention or early monitoring, detection and prompt treatment of soft tissue infections enhanced safety. Cerebrospinal fluid leucocyte counts ≥ 100 × 10 9 /l (adults: odds ratio 2.6, 95% confidence interval 1.5 to 4.6), seizures (adults: 5.9, 2.0 to 13.3), and stupor (children: 9.3, 2.5 to 34.2) were beacons for oncoming serious adverse events.
Clinical evaluation and parasitology assessments were done 6, 12 and 24 months post-treatment follow-up (n=924 (87.6%)). Of these, 15 (1.6%) succumbed to disease, 16 (1.7%) died during treatment, 823 recovered of which, 403 (43.6%) were confirmed cured, and 420 (45.5%) were probably cured. 70 (7.6%) relapsed. Relapse was defined by trypanosome-positive lymph, blood or cerebrospinal fluid; or high leucocyte count in the cerebrospinal fluid. The most number of relapses and deaths combined occurred at 12 months follow-up (65.8%, 52/79). Since late relapses and deaths occurred, the authors stress that long term follow-up periods should be practiced once eflornithine treatment is used. They also considered that eflornithine should replace melarsoprol as first line treatment in sleeping sickness.
Next, Priotto et al. assessed the efficacy and safety of NECT as compared to eflornithine monotherapy for treatment of second stage sleeping sickness in a multicentre, randomised, phase III, non-inferiority trial ( Priotto, G., Kasparian, S., Mutombo, W., et al., (2009). Nifurtimox-eﬂornithine combination therapy for second-stage African Trypanosoma brucei gambiense trypanosomiasis: a multicentre, randomised, phase III, non-inferiority trial. Lancet 374, 56 – 64. ref ) The patients on NECT received infusions every 12 h for 7 days, while those on eflornithine monotherapy received the standardized treatment (described earlier). In the follow-up period according to intent-to-treat analysis, 131/143 i.e. 91.6% patients on eflornithine and 138/143 i.e. 96.5% on NECT were cured of the disease at 18 months, with a difference of -4.9%, (one-sided 95% CI -0.3; p<0.0001) to the advantage of NECT over monotherapy. The per-protocol analysis favored NECT even more: 122/133 i.e. 91.7% patients on eflornithine and 129 /132 i.e. 97.7% on NECT were cured at 18 months follow-up , with a difference of -6.0% (one-sided 95% CI -1.5; p<0.0001) in favor of NECT. Thus both ITT and PP analysis support greater efficacy of NECT. While major adverse events were similar in both groups, the significant advantage of NECT over monotherapy was the lack of infections. The major adverse events shared by eflornithine and NECT groups were fever (n=18) and seizures (n=6) in patients on eflornithine , and fever (n=7) and seizures (n=6) in patients on NECT. In addition eflornithine patients suffered from infections (n=5). The common drug-related serious adverse events were double in the eflornithine monotherapy group compared to NECT at 41 (28.7%) patients while and 20 (14.0%) in the NECT group. Drug-related deaths were higher in monotherapy (n=3) than NECT (n=1). Thus NECT has a significantly safer profile than monotherapy. The authors concluded that NECT can be used safely for not only second-line, but first-line therapy in sleeping sickness.
Since 2010, World Health Organization has replaced the eflornithine monotherapy (and melarsoprol therapy) with the nifurtimox and eflornithine combination therapy or NECT ( Simarro, P.P., Franco, J., Diarra, A., et al., 2012. Update on ﬁeld use of the available drugs for the chemotherapy of human African trypanosomiasis. Parasitology 139 (7), 842–846 ref ) as first line treatment for sleeping sickness. As discussed earlier, the combination drug treatment decreases the emergence of resistant mutant parasites significantly. The combination therapy has additional advantages. First, much more facile treatment administration and regimen resulting in more widespread use than monotherapy, thereby replacing melarsoprol and preventing toxicity to the patient. Second, cheaper than monotherapy at half the cost. Third, the regimen requires shorter hospitalization, further reducing cost to patient. Note that NECT has its limitations, since it cannot treat infection with Trypanosoma brucei rhodesiense.
Further Advances In Eflornithine-Based Therapy For Sleeping Sickness: Monitoring For Resistant Parasites
In spite of NECT, resistant parasites do emerge, confounding therapy of sleeping sickness, although the rates are lower than eflornithine monotherapy. Researchers isolated resistant parasites by in vitro selection in the lab, and confirmed the phenotype in vivo ( Vincent, I. M., Creek, D., Watson, D. G., Kamleh, M. A., Woods, D. J., Pui EeWong, P. E., Burchmore, R. J. S. and Barrett, M. P. (2010). A Molecular Mechanism for E ﬂ ornithine Resistance in African Trypanosomes. PLoS Pathogens 6, e1001204. doi: 10.1371/journal.ppat.1001204. ref ). The researchers found that ornithine decarboxylase levels were comparable to non-resistant parasites, and so were metabolite levels of all compounds involved in polyamine synthesis. The resistant parasite differed from the eflornithine-sensitive ones by the absence of an amino acid transporter gene, TbAAT6 (Tb927.8.5450). Ablation of the gene from sensitive parasites conferred resistance on them, while ecptopic expression of the gene restored eflornithine-sensitivity. In drug-resistant parasites, uptake of eflornithine does not occur, indicating that the product of the gene TbAAT6 functions as the carrier for uptake of eflornithine into the parasite cell. A PCR test designed to identify deletion of this gene in parasites recovered from the patient is recommended for identifying prior to, and monitoring emergence of resistance during eflornithine-based therapy in the field setting. Implementing such a test would allow physicians to take quick measures on optimization of therapy to overcome the resistant parasites. Most important, such a practice would help to curb resurgence of resistant parasites.