USOO7566465B2

(12) United States Patent (10) Patent No.: US 7,566.465 B2 Kubata et al. (45) Date of Patent: Jul. 28, 2009

(54) IN THE CLINICAL AND 2004/0067875 A1 4/2004 Lai et al...... 514.6 VETERINARY MANAGEMENT OF 2005/024.0034 A1* 10/2005 Avery et al...... 549,349 KINETOPLASTD INFECTIONS 2007/0032460 A1 2/2007 Stella et al...... 514,154 (75) Inventors: Bruno K. Kubata, Nairobi (KE): FOREIGN PATENT DOCUMENTS Samuel K. Martin, Burtonsville, MD WO WOO3,O95444 11, 2003 (US); Wilbur K. Milhous, Germantown, WO WO 2006/097.472 9, 2006 MD (US) OTHER PUBLICATIONS (73) Assignee: The United States of America as Yang D Metal, Effects of Qinghaosu (artimisinin) and its derivatives represented by the Secretary of the on Experimental cutaneous leishmaniasis, Parasitology, Cambridge Army, Washington, DC (US) Univ. Press, London, GB, vol. 106, No. 1, 1993, p. 7-11. Dardonville C. Recent advances in antitrypanosomal chemotherapy, (*)c Notice:- r Subject to any disclaimer, the term of this Patent literature 2002-2004, Experper UpOpinion on Therapeuticp Patents patent is extended or adjusted under 35 2005, United Kingdom, vol. 15, No. 9, 2005, pp. 1241-1257. Mishina Y V et al: Artemisinins inhibit trypanosoma brucei U.S.C. 154(b) by 0 days. rhodesiense in vitro growth, Antimicrobial Agents and Chemo (21) Appl. No.: 11/644,494 therapy 2007, United States, vol. 51, No. 5, 2007, pp. 1852-1854. y x- - - 9 * cited by examiner (22) Filed: Dec. 21, 2006 (Under 37 CFR 1.47) Primary Examiner Christopher R. Tate Assistant Examiner Deborah A. Davis (65) Prior Publication Data (74) Attorney, Agent, or Firm—Elizabeth Arwine US 2008/O152729 A1 Jun. 26, 2008 (57) ABSTRACT (51) Int. Cl. The invention relates to the treatment of kintoplastid infec A6 IK 36/282 (2006.01) tions by administering a pharmaceutical composition con (52) U.S. Cl...... 424/740 taining an extract from the plant . The inven (58) Field of Classification Search ...... None tion also relates to isolated, semi-synthetic and synthetic See application file for complete search history. artemisinins that show improved efficacy in treating kineto (56) References Cited plastid infections. This invention also relates to a method of treating kintoplastid infections with and arte U.S. PATENT DOCUMENTS misinins and where Artelinic acid is administered orally. 6,461,603 B2 * 10/2002 Bentley et al...... 424/78.19 2003/0171424 A1* 9/2003 Lin et al...... 514,452 6 Claims, 7 Drawing Sheets 13 CH,

C-OH U.S. Patent US 7,566.465 B2 U.S. Patent Jul. 28, 2009 Sheet 2 of 7 US 7,566.465 B2

HOHC

NH2 melarSOprol Fig. 1c

F-So-S-N-N-,F O NH2 difluoromethyl Ornithine / DFMO Fig. 1d U.S. Patent Jul. 28, 2009 Sheet 3 of 7 US 7,566.465 B2

A s - s X w s w o D ea { A. hinhibitionbruce (%) against Plan (123Extraction kg) wiith 80% EtOH of it.) (24hr x1 and 3 hrx2 atroom temperature) ------80% EOH extract (240g)

50 g/mL. 30g/mL.

Partition in EtOAc, n-BuOH, and HO

EtOAcext. n-BuOHext. HO ext. (74 g) (94g) 50gmL OO OO a BO 5gmL > 80 80 K80 1 gmL <80 C8O a80 SiO, gel column chromatography

A B C D E (280mg) (341 mg) (1.72 g) (1.84 g) (690mg) 50 pg/mL 100 100 100 OO 100 10 pg/mL 280 (80 100 100 280 1 g/mL < 80 c80 e 80 a8O C80 ODS column chromatography

C-1 C-2 C-3 C4 C-5 C-6 C-7 (47 mg) (25 mg) (200mg) (629 mg) (16mg) (139 mg) (280mg) 50gm L. 280 >80 CO 100 33.2 100 308 100 40s Sugin C80 <80 OO 100 14.9 OO 272 100 228 0.5g/mL C80 <80 <8O 100 S.S 80 OS 80 14.9 normal phase HPLC

C41 C-42 C-43 C-44 C4S C-46 (1 mg) (0.5 mg) (2.1 mg) (7mg) (3.1 mg) (7.1 mg)

50 pg/mL <80 e8O 80 280 OO 100 Sa 5gm. C80 <80 CBO k80 80 c80 737 13 Fig. 2 U.S. Patent Jul. 28, 2009 Sheet 4 of 7 US 7,566.465 B2

growth inhibition (%) against Plant C-1 (2.3 kg) Extraction with 80% EtOH 1. (24 hrx 1 and 3 hrx 2 at room temperature) ------80%EtOH extract (240 g) 50 g/mL 100 30g/mL 100 10 pg/mL C80 Partition in EtOAc, n-BuOH, and HO

EtOAc ext, n-BuOH ext, HO ext. (72 g) (74 g) (94g) 50 pg/mL. OO 100 <8O 5 g/mL 280 80 a80 C80 €80 (8O SiO gel column chromatography

C D E (280mg) (341 mg) (1.72 g) (1.84 g) (690 mg) 50 pg/mL OO OO 100 1CO 100 10 g/mL 280 80 OO OO >80 lug/n < 80 <80 280 C8O s80 ODS column chromatography

C-1 C-2 C-3 C-4 C-5 C-6 C-7 (4.7 mg) (25 mg) (200mg) (629 mg) (16mg) (139mg) (280mg) 50 g/mL (280 > 80 OO 100 332 OO 30.8 100 405 100 5ugmL C80 C80 100 100 14.9 100 272 100 22.8 e 80 0.5g/mL C80 C8O C80 OO SS 80 10.S. e80 14.9 (80 normal phase HPLC

C-S C-S2 C-53 C-SA C-SS (0.9 mg) (0.7mg) (1.1 mg) (1.1 mg) (0.8mg) 50 g/mL, 10 pg/mL.

Fig. 5 U.S. Patent Jul. 28, 2009 Sheet 5 Of 7 US 7,566.465 B2

U.S. Patent Jul. 28, 2009 Sheet 6 of 7 US 7,566.465 B2

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U.S. Patent Jul. 28, 2009 Sheet 7 Of 7 US 7,566.465 B2

US 7,566.465 B2 1. 2 ARTEMISINNS IN THE CLINICAL AND genus Trypanosoma, are incriminated as the causative agents VETERINARY MANAGEMENT OF 1-71. Trypanosoma bruceii rhodesiense and Trypanosoma KNETOPLASTD INFECTIONS bruceii gambiense provoke human African trypanosomiasis ("sleeping sickness') while Trypanosoma Congolense, Try GOVERNMENT INTEREST 5 panosoma simiae, Trypanosoma vivax and Trypanosoma bruceii bruceii cause animal African trypanosomiasis (“na The invention described herein may be manufactured, used gana) http://www.vet.uga.edu/vpp/gray book/FAD/ and licensed by or for the U.S. Government. AAT.htm. In the particular case of human African trypanosomiasis, BACKGROUND OF THE INVENTION 10 the trypanosomes multiply in the blood and lymph glands of the infected persons, therefore, defining the first-stage of 1. Field of the Invention sleeping sickness 5,13. The symptoms in this early stage are This invention relates to methods of treating and managing characterized by bouts of fever, headaches, skin itching, pain kinetoplastid infections by administering artemisimins and in the joints, gradual loss of weight, nausea and Vomiting artelinic acid. 15 6.9. Later, in the second stage, the trypanosomes cross the 2. Brief Description of Related Art blood-brain barrier and invade the central nervous system to Trypanosomiasis is a re-emerging 1-3 tropical infectious cause sleeping sickness. Sleeping sickness is characterized disease that poses a real challenge to public health counter by neurological disorders such as mental confusion, sensory measures. According to the World Health Organization disturbances and poor muscular coordination, and reversal of (WHO)4, about 36 sub-Saharan countries in West, Central, the circadian sleep/wake cycle (insomnia in the night, drowsi and East Africa and some 22 Latin countries in Central and ness in the daytime) 5.9.13, 14 hence, the nickname “sleep South America delimit its geographic prevalence Zone, thus, ing sickness'. In the absence of effective treatment, sleeping leading to the establishment of two distinct manifestations of sickness invariably leads to death 3.6. the disease: African trypanosomiasis and American trypano Control measures for African trypanosomiasis are directed Somiasis. 25 either against the transmission vector through eradication of Trypanosoma species pathogenic to human beings and the tsetse fly, or against the causative pathogen, trypano domestic animals in Africa, cause one of the world's most Somes. Although vector control strategies had been effective neglected tropical infections—African trypanosomiasis 3. in the past, they have been virtually abandoned because of Nearly eliminated in the 1960s, African trypanosomiasis has their harmful effects on the environment 7. Treatment of been making an alarming comeback due to civil wars, popu 30 infected persons with the few available synthetic trypanocidal lation displacements, and the collapse of public health sys agents have shown significant drawbacks 15-17 related to tems mainly due to political instability (www.accessmed high cost, host toxicity, limited oral , and a msforg/documents/SSfactsheet.pdf) 1. Human African requirement for hospitalization during the entire course of trypanosomiasis (HAT) threatens 60 million 1.3-6 men, treatment. The emergence of drug resistance has also limited women, and children among, principally, the rural popula 35 the choice and effectiveness of affordable agents in clinical tions, but actually even the citadin populations, in countries of use. Moreover, the type of treatment depends on the stage of high endemicity Such as Angola, Southern Sudan, the Demo the disease: hemolymphatic (first stage) or cerebral (second cratic Republic of Congo, and northern Uganda. The inci stage). Effectiveness in the second stage relies on the ability dence rate in HAT is estimated between 300,000-500,000 of the drug to cross the blood-brain barrier and reach concen cases annually 3-6 and only 3 to 4 million people at risk are 40 trations high enough to kill the infective trypanosome. The under regular medical surveillance (http://www.who.int/inf four trypanocidal drugs, shown below 5.6, 13 that have been fs/en/fact259.htlm). In animal African trypanosomiasis clinically used up-to-date against sleeping sickness are, in (AAT), the infection threatens about 50 million head of cattle chronological order—: Suramin (developed in 1921 against with an estimation of 3 million deaths per year in livestock T. b. rhodesiense in the first-stage infection), Pentamidine (http://www.fao.org/ag/againfo/programmes/en/paat/dis 45 (discovered in 1941 against first-stage T. b. gambiense infec ease.htlm). tion), Melarsoprol (developed in 1949 against both human American trypanosomiasis ("Chagas disease') occurs infective Trypanosoma Subspecies in cerebral infection), and mainly in countries Such as Brazil, Chile, Mexico, Uruguay, Difluoromethyl ornithine (developed in 1981 as an alternative Paraguay, Bolivia, and Argentina 10, 11. Over 13 million to Melarsoprol treatment failure in cerebral sleeping sick persons in the Southern American region are at risk of infec 50 ness). FIGS. 1a and b show the known trypanocidal drugs for tion and the annual incidence rates of the disease reaches early stage infection which are Suramin and pentamidine, 200,000 cases in 15 endemic countries 10. The bloodstream respectively. Suramin has no oral bioavailability and causes protozoan Trypanosoma Cruzi (10-12 is the etiologic agent and kidney disease. Pentamidine has no oral bio of Chagas disease. availability, is an immunosuppressive and causes bleeding. Host-to-host transmission is mediated by blood-sucking 55 FIGS. 1c and 1d show the known trypanocidal drugs for triatomine bugs such as Triatoma infestans (11. Moreover, cerebral infection which are melarsoprol and difluoromethyl blood transfusion and congenital transmission have been ornithine (DFMO), respectively. Melarsoprol has no oral bio encountered, particularly, in humans 10. These pathogens availability and causes tumors in the brain, blood in the urine are all cyclically transmitted to mammalian hosts through the and stomach pain. DFMO has no oral bioavailability and has bite of haematophagus tsetse flies (Glossina morsitans, Glos 60 a high Susceptibility to resistance. sina palpalis) 9 Serving as vectors of the disease. In the absence of prospective vaccine candidates for the The consistent decimation of human populations and cattle disease, the limitations and drawbacks of these drugs empha by African trypanosomiasis has reached dramatic proportions size the crucial need to develop new safe, effective and afford and represents a social and economical obstacle for develop able drugs (trypanocides) against all forms of human and ment 6.7. In AAT. breeding animal losses are estimated to 65 Veterinary trypanosomiasis. cost African farmers US$4.5 billion per year 8). Blood The Chinese plant Artemesia annua has been used to treat stream flagellated protozoan, members of the taxonomic for centuries. Research in the past three decades have US 7,566.465 B2 3 4 uncovered derivatives like artemisinin, dihy FIG. 1d is the chemical structure for difluoromethyl orni droartemisinin, , that have played a thine (DFMO: critical role in the management of infectios caused by the FIG. 2 is a Flow diagram showing the bioassay-guided multi-drug resistant malaria parasite, Plasmodium falci isolation of active constituents of plant C-1 leading to C-46; parum. Artemisinin has also been reported to be selectively FIG. 3 is a flow diagram showing the bioassay-guided active against cancer cells in vitro 39. Utzinger et al. have isolation of active constituents of plant C-1 leading to C-53; demonstrated the use of artemisinin derivatives against tropi FIG. 4a is the chemical structure of artemisinin C-46; cal parasite species Schistosoma, responsible for Schistoso FIG. 4b is the chemical structure of Artelinic acid; miasis 40. The effect of artemisinin and its derivatives on FIG. 5a is a photograph showing untreated Leishmania Leishmania major, another tropical parasite provoking leish 10 major amastigotes; maniasis has also been reported 41. However, the wide FIG. 5b is a photograph showing the effect of 1 g/ml spread use of this class of semi-synthetic artemisinin deriva MeOH extract of Artemisia annua on Leishmania major tives have been limited by the high cost of production, low amastigotes: bioavailability and long treatment regimens. FIG. 5c is a photograph showing the effect of 10 g/ml African, Asian and Amerindian Societies have a rich tradi 15 MeOH extract of Artemisia annua on Leishmania major tion 17 in the use of plants for medical care. However, only amastigotes: few reports exist on the phytochemical treatment of sleeping FIG. 5d is a photograph showing the effect of 100 g/ml sickness 17 and other kinetoplastid infections. Artemisinins MeOH extract of Artemisia annua on Leishmania major have never before been used to treat human and veterinary amastigotes: trypanosomiasis. Until now, no experimental study has been FIG. 6 is a photograph showing the foot of an untreated carried out to establish the effectiveness of artemisinin or its mouse; and derivatives on any Trypanosoma species. FIG. 7 is a photograph showing amouse foot with the effect The inventors are the first report on the trypanocidal of 100 ug/ml Me?oH extract treatment for 14 days. potency of artemisinin and by extension, artemisinin-derived compounds, including artelinic acid. 25 DETAILED DESCRIPTION Therefore, an object of the invention is to prepare a phar maceutical composition containing artemisinin lead com The present invention relates to treatment ofkinetoplasmid pounds from natural resources for the treatment of trypano infections such as Leishmaniasis and Trypanosomaisis with Somiasis. Medicinal plants such as Artemesia annua have artemisinins such as artelinic acid and/or trioxolane com secondary metabolites of diverse molecular structures, 30 pounds. physico-chemical properties, and pharmacological activities The antimalarial activity of the artemisinins is believed to and offer an invaluable reservoir for new remedies. reside in the generation of toxic free oxygen radicals subse Another object of the invention is to provide a cost effective quent to the interaction with released from the metabo treatment for kinetoplastid infections. lism of hemoglobin. The inventors have found that artemisi Another object of the present invention is to provide a 35 nins and synthetic artemisinins, in particular, artelinic acid method of treating humans and other mammals with kineto and trioxolane compounds, are effective in a method of treat plastid infections with artemisimin compounds such as arte ment of kinetoplasmid infections, particularly against Leish misinin and artelinic acid. mania and Trypanosoma. Other objects, features and advantages of the present The structural identity, and biological activity of com invention will become apparent from the following detailed 40 pounds extracted from the medicinal plant Artemesia annua description. It should be understood, however, that the “Plant C-1 was established. For these experiments, chro detailed description and specific examples, while indicating matographic techniques were used for total purification of the preferred embodiments of the invention, are given by way of active principles. Various spectroscopic methods and conven illustration only, since various changes and modifications tional database analysis were also utilized for chemical struc within the spirit and scope of the invention will become 45 ture elucidation. The identified candidates were subjected to apparent to those skilled in the art from this detailed descrip standard bioassay protocols for in vitro characterization of tion. their trypanocidal activity and also selective toxicity against trypanosomes Versus mammalian host cells. SUMMARY OF THE INVENTION The extract of the plant Artemesia annua was shown to kill 50 Leishmania parasited cultivated within mouse macrophages The invention relates to the treatment of kintoplastid infec in vitro in a dose-dependant manner without significant tox tions by administering a pharmaceutical composition con icity to the mouse macrophages. The results were duplictated taining an extract from the plant Artemisia annua. The inven using commercially sourced artemisinin. The active com tion also relates to synthetic artemisinins that show improved pound in the compound was isolated and purified and chemi efficacy in treating kinetoplastid infections. This invention 55 cally identified as artemisinin. also relates to a method of treating kintoplastid infections Materials and Methods with artelinic acid and artemisinins. The accompanying drawings show illustrative embodi a. Plant Material ments of the invention from which these and other of the Artemesia annua can be obtained in countries where it is objectives, novel features and advantages will be readily 60 cultivated such as its native China and Kenya. Freshly cut apparent. leaves of Artemesia annua were collected in Kenya, Sun-dried and then ground into powder that can be stored in a refrigera BRIEF DESCRIPTION OF THE FIGURES tor prior to extraction. In the case of artemisinin, the compound was extracted FIG. 1a is the chemical structure for Suramin; 65 from Artemisia annua with 70% aqueous EtOH at room tem FIG. 1b is the chemical structure for pentamidine: perature for 48 h (first extraction) and then 24 h each for the FIG. 1c is the chemical structure for melarsoprol; second and third extraction. The extracts were combined and US 7,566.465 B2 5 6 concentrated under reduced pressure to obtain the EtOH (DMSO, Wako-for biochemical assay) then diluted in the extract. The EtOH extracts were purified by organic solvent culture medium so that DMSO content decreased to 10% in fractionation and a combination of chromatographic proce the medium. Trypanosomes were harvested at late exponen dures such as ion exchange on a DIAION HP-20 column and tial growth level, counted with a hemocytometer (Erma hydrophobic interaction on an ODS column and gel filtration 5 Tokyo 7059), and resuspended by appropriate dilution in on a Sephadex column. The active fractions were then iden culture medium for achieving a final density of 1x10" cells/ tified. The following experiment confirms that the com mL. Aliquots of 90 uL of T. b. brucei suspension (1x10' pounds that were extracted are efficacious against kinetoplas cells/mL) were transferred in wells of a Becton Dickinson tid infection and are artemisinins. 96-well microculture plate. Then, 10 uL of each sample b. General Experimental Procedures: (Plant C-1) 10 preparation containing 10% DMSO were added to each 'H-NMR (500 MHz) spectra were measured on JEOL inoculum well, achieving a final concentration of 1% DMSO. Lambda 500 spectrometer in CHCl-d as well as MeOH-d The microculture plate was incubated in a humidified, 5% with TMS as an internal standard. 'C-NMR (600 MHz) and CO atmosphere incubator at 37° C. After successively 24, 2D-NMR (600MHz) data were recorded on Varian Inova 600 48, and 72 hr-incubations, parasites viability was determined in CHCl-d. Fast-atom bombardment (FAB) and high-reso 15 by observing directly inside the wells of the microculture lution fast-atom bombardment (HR-FAB) mass spectra were plate with an optical microscope (Injectoscope model IMT recorded in positive ion mode JMS SX-102 spectrometer. For YF/Olympus). The in vitro trypanocidal potency of each column chromatography, silica gel (Fuji Sylisia BW-200, sample was then evaluated and translated into mathematic Merck), ODS (Cosmosil 75Cs OPN, Nacalai) were used. symbols (-): <80% growth inhibition level, (+): 280% Thin-layer chromatography (TLC) analyses were performed growth inhibition level, (+): 100% growth inhibition level. over normal phase pre-coated plates (Kiesel gel 60Fs, Control wells with the commercial drug pentamidine (posi Merck) and reversed-phase high-performance thin-layer tive control) and the solvent DMSO only (negative control) chromatography (HPTLC) plates (RP-18 WFss, Merck). were also achieved in the same conditions as the test samples. Spots on chromatograms were detected under UV light (254 A blank (parasites only in culture medium) was also included and 365 nm) and by spraying with phosphomolybdic acid (5 25 for reference. g, EtOH 100 mL), vaniline/HSO4 (vaniline 5 g, conc. HSO f. Culture Medium for HeLa S3 Cells 95 mL), and p-anisaldehyde/HSO (AcOH 5 mL, conc. An axenic culture of human carcinoma HeLa S3 cell line HSO 25 mL, EtOH 425 mL, p-anisaldehyde 25 mL) fol was established in Dulbecco's Modified Eagle's Medium lowed by heating. For high-performance liquid chromatogra (D-MEM, Sigma) supplemented with 4500 mg glucose/L, phy (HPLC), detection of analytes was carried out with a 30 L-glutamine, NaHCO, and pyridoxine HCl, to which 10% refractive index detector (Shodex RI-71). For in vitro try heat-inactivated (56°C., 40 min) fetal bovine serum (FBS, panocidal assay, Stock solutions of samples were prepared in MultiSer) was added. DMSO (Wako, for biochemical assay) which concentration g. Sample Preparation for Cytotoxic Evaluation. In Vitro in culture medium never exceeded 1%. Pentamidine (Sigma) Plant chromatographic fractions, which have shown potent was used as a positive control. For cytotoxic evaluation in 35 trypanocidal activity in vitro, were selected for cytotoxicity vitro, mitomycin C was used as the positive control. assay. Previously dissolved in neat DMSO (Wako-for bio c. Trypanosome Stocks chemical assay), an aliquot of 10 u, of each test sample was diluted in 490 uL of the cell culture mediumwith the intent of Culture Suspension of Trypanosoma bruceii bruceii (T. b. decreasing the DMSO concentration to 2% in the medium. brucei) was obtained from Research Institute for Microbial 40 Diseases (Osaka University, Japan). Trypanosomes were Sub h. Cytotoxicity Assay In Vitro cultured in appropriate medium (see 5.1.2) as frequently as HeLa S3 cells maintained in culture dish (Sumilon) were needed to avoid overgrowth, and maintained in culture flasks washed with Dulbecco's phosphate saline buffer (-) DPBS at 37°C. in a humidified, 5% CO atmosphere incubator. In (-), Nissui Pharmaceutical Co., Ltd after syphonating the addition, parasite culture stabilates were prepared by Sus 45 culture medium. Adherent cells were released from their dish pending trypanosomes centrifugation (1500 rpm, room tem bottom Substrate by adding trypsin (Nacalai) and gently tap perature, 3 min) pellet in 1 mL of a mixture of 77% (v/v) ping with hand on the round lateral side of the dish. Thus, trypanosome dilution buffer 56 and 33% (v/v) glycerol. The trypsinized cells were harvested, Suspended in culture whole volume was transferred into cryotubes and stored at medium then centrifuged at 800-1000 rpm for 3 min at room -80° C. until further needed. 50 temperature. After removing the Supernatant, the remaining d. Culture Medium for Trypanosoma bruceii brucei pellet was resuspended in the culture medium. Cells were The axenic cultivation of the bloodstream forms of T. b. counted with a hemocytometer (Erma Tokyo 7059), and then brucei was performed in Iscove’s Modified Dulbecco's diluted appropriately to achieve a final density of 1x10 cells/ Medium (Gibco) supplemented with L-glutamine and 25 mM mL in the culture medium. Later on, cells were seeded in a HEPES buffer. Additionally, 15%heat-inactivated (56°C., 40 55 96-well microtiter plate (Becton Dickinson), each well con min) fetal bovine serum (FBS, MultiSer) and also 0.05 mM taining 100 uL (1x10 cells/mL) of cell suspension. Then, bathocuproine disulfonic acid disodium salt (Dojin), 1.5 mM 100 uL of test samples preparation (see 5.2.2) were added in L-cystein (Nacalai), 1.0 mM hypoxanthine (Nacalai), 0.16 triplicate for each concentration into respective wells, thus mM thymidine (TCI), 0.2 mM 2-mercaptoethanol (Sigma), achieving a final concentration of 1% DMSO. Respective 60 triplicate wells for the positive control (mitomycin C) and the and 1.0 mM pyruvate (Nacalai) were aseptically negative control (1% DMSO only in culture medium) were incorporated in the culture medium. also included. After 72 hr-incubation at 37° C. in a humidi e. Trypanocidal Assay fied, 5% CO, atmosphere incubator (Sanyo), 25uL of MTT All fractions obtained stepwise in the course of the sepa 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bro ration process of the plant crude extracts were assessed for 65 mide 57-59 reagent were added to each well including their in vitro activity against T. b. brucei. Various concentra controls. The microtiter plate was returned in the same incu tions oftest samples were prepared in neat dimethylsulfoxide bator for an additional 3 hr-exposure at 37° C. to allow the US 7,566.465 B2 7 8 formazan 57-59 crystals to accumulate. Then, supernatants vitro against T. b. bruceii after 24-hr incubation at 1 ug/mL, were syphonated from the wells and 200 uL of DMSO were and only 3.1% inhibition ratio against HeLa cells in vitro at 5 added to solubilize the formazan crystals. After shaking (MS1 Lig/mL. Minishaker IKA) for 3-5 min, the absorbance in each well 5 j. Chemical Structure of C-46 was measured at 540 nm in a microtiter plate reader (Immu Comparison of H- and 'C-NMR, H-H COSY, HMBC, noMini NJ-2300). The percentage of cytotoxicity (growth FAB-MS, and specific rotation data in our hands with those inhibition level) was calculated as (A-B)/Ax100, where A is reported in the literature 34.35 led to conclusion that C-46 the mean optical density of negative control wells and B is the was (+)-artemisinin (FIG. 4). mean optical density of test sample wells. H-NMR spectrum analysis for structure elucidation of 10 C-46 indicated the presence of doublet peaks, respectively, at i. Bioassay-guided Isolation of Active Constituents of Arte 81.19 (3H, d, J–74 Hz) and 80.98 (3H, d, J=6.0 Hz), and a misinin singlet peak at 61.43 (3H, s). This set of peaks was indicative Dried and pulverized powder from “plant C-1” (2.3 kg) of the three methyl groups at C-13, C-14, and C-15, respec were extracted three times with 80% ethanol after 24-hr mac tively. The signal observed at 65.84 was assignable to H-5, eration (first time) and 3-hr maceration (second and third 15 which corresponding carbon signal was displayed at 693.9 on time) at room temperature. The liquid extracts were com the 'C-NMR spectrum. The signal that appeared at 8.172.2 bined, filtered, and concentrated under reduced pressure with was assignable to the carbonyl moiety of the lactone group at a rotary evaporator below 30°C. The ethanolic residue (240 C-12. The signal that appeared at 8105.6 was due to the g) obtained exhibited a complete inhibition of trypanosomes quartenary carbon C-4 activated by two geminal oxygen growth at 30 ug/mL after 24-hr exposure and 100% growth atoms. The peak observed at 879.7 was due to the quartenary inhibition in vitro against T. b. bruceiiat 100 ug/mL after 24-hr carbon C-6 connected to an oxygen atom. In addition, the exposure. This residue was later on dissolved in double positive ion mode FAB-MS displayed M--Li at m/z 289 distilled water, then subjected to solvent-solvent partition in (65%), M+Na" at m/z 305 (100%), and M+H" at m/z 283 EtOAc and n-BuOH to yield, after solvent evaporation, 72 g 25 (12%). Additionally, the optical rotation parameter revealed of EtOAc extract, 74 g of n-BuOH extract, and 94 g of H2O Ol' +41.5 (c=0.2, CHCl). Comparison between spectral extract. The strongest trypanocidal activity in vitro was data in our hands with those in the literature database led to observed in the EtOAc extract exhibiting 100% and 280% conclusion that C-46 corresponded to (+)-artemisinin growth inhibition against T. b. brucei, respectively, at the (CHOs, Mw-282). concentrations of 50 ug/mL and 5 ug/mL after 24 hr-expo 30 k. Chemical Structure of C-53 sure. This most potent EtOAc residue was applied to SiO gel Relevant H and 'C-NMR, H-H COSY, HMOC, column and eluted successively with binary solvent mixtures HMBC, NOESY, FAB-MS, and HRFAB-MS data were of hexane: EtOAc=4:1-> 1:1-> 1:1.5-s1:2->100% MeOH. recorded; the specific rotation value was calculated. The eluted fractions were monitored by normal phase TLC C-53 has trypanocidal potency in vitro but is slightly and similar fractions were pooled into five main fractions 35 weaker than that of (+)-artemisinin. Nevertheless, from its named A (280 mg), B (341 mg), C (1.72 g), D (1.84g), and E chemical characterization, 1 D- and 2D-NMR, FAB- and (690 mg). Among them, fraction C was the only one that HRFAB-MS, and specific rotation data that have been col showed trypanocidal activity until the low concentration lected, spectral data analysis reveals that C-53 might be poly value of 1 g/mL, thus featuring 280% growth inhibition alicyclic; containing three characteristic methyl Substituents after 24-hr incubation. Further separation of fraction C car 40 and an unsaturated lactone ring; displaying a molecular ried out by reversed-phase ODS column chromatography and weight of 234 associated to the chemical formula C5H2O. eluting gradually with 60% MeOH->80% MeOH->90% Accordingly, C-53 is presumed to be a sesquiterpene lactone MeOH->100% MeOH yielded seven fractions named from congenere of artemisinin, but obviously lacking the endoper C-1 to C-7. Fractions C-4 (629 mg), C-5 (16 mg), and C-6 oxide bridge. This activity is of more general interest because (139 mg) showed the highest biological potency and an 45 the of artemisinins, as an anti-plasmo encouraging selectivity toward HeLa cell in vitro at 0.5 dial or anti-tumoricidal agent, is believed by some to be ug/mL with, respectively, 100% trypanocidal level and 5.5% related to the presence of the endoperoxide bridge. cytotoxic level for C-4, 280% trypanocidal level and 10.5% The information obtained by careful examination of C-53 cytotoxic level for C-5, and 280% trypanocidal level and and C46 as well as other eluted compounds in the above 14.9% cytotoxic level for C-6. 50 experiment verify that artemisinins are active ingredients in Firstly, C-4 was subjected to final purification with HPLC artemeseia annua that hinder the growth of kintoplastid infec (column: 5 SL-II type Waters, 10x250 mm i.d.; flow rate: 3.0 tions. mL/min: detection: refractive index detector at range 512; Both compounds Artemisinin and Artelinic acid belong to eluent: hexane/EtOAC=8/1) leading to isolation of C-46 (7.1 the same class of compounds and both have been found by the mg) at at of 21.27 min (FIG. 2), whose trypanocidal activity 55 inventors in testing to be effective against leishmaniasis and in vitro was 280% growth inhibition after 24-hr incubation other kinteplastid infections. and the cytotoxic level in vitro was 17.3% at 1 lug/mL. Sec ondly, C-5 was purified by normal phase HPLC (column: 5 EXAMPLE 1. SL-II type Waters, 10x250 mm i.d.; flow rate: 3.0 mL/min: detection: refractive index detector at range 512; eluent: hex 60 ane/EtOAC=7/1) affording C-53 (1.1 mg) at to 12.99 min Preliminary drug screening (in vitro amastigote/macroph (FIG. 3). Because of the small amount yielded by C-53, age culture) of artemisinin and artelinic acid compounds. extensive spectral data measurement was inconvenient. Only Four compounds were obtained. 'H-NMR data could be obtained. Thus, purification was BN: 97.471 artelinic acid repeated Starting off with a larger quantity of EtOAc extract. 65 ZW: 60909 Fortunately, 11.0 mg of C-53 were obtained. According to the BN: BL 48816 WRil 255131 (Beta-Arteether) bioassay outcome, C-53 showed 280% growth inhibition in BN: BL 50129 WRit: 249309 (Artemisinin QHS) US 7,566.465 B2 10 The four compounds were dissolved in the respective sol had no compound added. The effectiveness of the WRAIR vents as indicated in the table below (Table 1) and were tested compounds tested in the screen was judged to be: against Leishmania major ATCC 50122 amastigotes in vitro in a mouse macrophage system. A positive (Pentostam) and Artelinic acidds-Dihydroartemisinin>Beta negative (culture medium) controls were set for each plate. 5 Arteethers Artemisinin. Compounds were tested at 2.0 uM in a total volume of 2-0 Because of the high potency of Artelinic acid relative to the mls. Drug was added to well every 4 hoursx1, 2 and 3, and other compounds, the experiments were repeated with differ results shown in (Table 2) below. ent doses of Artelinic acid alone. Additionally, the addition of drug to culture wells q4h did not seem to affect the results for TABLE 1. '' this compound (Table 2). Hence, Artelinic acid was added Compound. Solvent. Conc. Used. Volume. once in the dose response experiments (Table 3). 1 Artelinic Methanol. 2.0 M 5.6 l/2 ml. The initial observation implicating Artemisinins was acid obtained with crude extracts from the plant (Artemesia 3 Dihydro DMSO. 2.0 M 1.1 ul/2 ml 15 annua. On the strength of these results Artemisinin-derived arteniSinn compounds that had been synthesized previously as potential SB-Arteether DMSO 2.0 M 1.18 ul/2 ml 6 Artemisinin Methanol. 2.0 M 1.2 l/2 ml anti-malarials were secured from the WRAIR inventory and tested (See U.S. Pat. No. 4,791,135, incorporated in its entirety by reference for the preparation of synthetic artemisi Results nins, dihydroartemisinins, and artesunic acid.). The active

TABLE 2 Compound added Compound added Compound added Compound q4h x1 q4h x 2 q4h x 3 Artelinic Acid 1 No intracellular No amastigote was S8ile amastigote was seen in the seen in the macrophages. ctoplasm of the Apart from staining macrophages. pinkish, the cells Cytoplasm stained cytoplasm looked pinkish instead of mushy and seem to purplish which is desentegrate. an indication of cell cytotoxicity. Dihydro About 50% of About 30% of About 5-7% of artemisinin 3 macrophages had macrophages had macrophages had intracellular intracellular intracellular ieishmania ieishmania ieishmania amastigotes. Cells amastigotes. Cells amastigotes. About morphology was morphology was 30% of macrophages O8. normal. had intracellular ieishmania amastigotes BArteether About 90% of About 90% of About 50% of macrophages had macrophages had macrophages had ieishmania ieishmania ieishmania amastigotes similar amastigotes similar to amastigotes similar to to the Negative the Negative control the Negative control control Cytoplasm Cytoplasm stained Cytoplasm stained stained poorly poorly (pinkish). poorly (pinkish) with (pinkish). mild cytotoxicity. 6 Artemisinin No difference between control and the macrophages Subjected to the test compound. About 80% of the cell had between 4-7 parasites per macrophage. Cell morphology was normal.

The results of the initial screen showed that Artelinic acid principle in the plant extract was prepared into pharmaceuti at 2 uM cleared all of the amastigates in macrophages. cal compositions Suitable for treating kinetoplastid infec Observer also noted pinkish staining of the cytoplasm of the is tions. macrophages that was interpreted as cell toxicity. However, Pharmaceutical compositions containing the active ingre similar changes were also noted in the negative control that dients from the plant artenesia annua, including artemisinins US 7,566.465 B2 11 12 and artelinic acid can be prepared using any known pharma selected and treated daily with test article for one week. The ceutical carrier Suitable for i.p. injection including but not first doses were administered on 18 May. Lesions were mea limited to saline. Carriers for oral administration can be cap Sured with calipers prior to dosing and 9 days after com Sules or pills made by any known and accepted pharmaceu mencement of treatment (27 May) and the difference in lesion tical composition used for carrying active ingredients to the size calculated. The thickness of the uninfected footpad was digestive tract. Acceptable doses for oral administration are also measured. Only Artelinic acid and the crude methanol 4-8 ul/kg for ip or oral administration. Concentrations for in extract of the plant Artemesia annua, that had shown marked vitro testing are 0.01 to 2.0 uM potency in the mouse macrophagefamastigote in vitro Screen Effects of Different Concentrations of Artelinic Acid (Dose were tested in vivo. Testarticles were administered orally and Response) on Leishmania Amastigotes. intra-peritoneally (ip).

TABLE 3

Concentration. Observation Results. 2.0 M All the leishmania amastigotes were cleared but the cell showed some marked pink staining. 1.0 M All the amastigotes were cleared from the macrophages and SOE vacuoles could be seen in the macrophage cytoplasm. Mild cell pink staining was noticed. 0.5 M Cleared all the parasites in the cytoplasm. Some cells showed pinkish staining. 0.25 M Cleared all the Leishmania amastigotes and no noticeable pink staining of cells. The cell morphology was normal as compared to the control cells. 0.1 M There was reduction of intracellular Leishmania amastigotes in the macrophages but unable to clear them completely. No pinkish stain of cells was noticed. 0.05 M No effect of the drug was seen on either the Leishmania parasites or the macrophages. Control. 90% of the macrophages were infected with between 5-7 parasites per cell.

As shown in the results above (Table 3), killing of intrac ellular amastigotes was noted at concentrations as low as 0.1 uM of Artelinic acid and clearance was complete at doses as low as 0.25uM of compound. 35

EXAMPLE 2 40 In Vivo Testing in BALB/c Mice

Because of the marked potency of Artelinic acid in the clearance of intracellular Leishmania amastigotes, an in vivo 45 screen was undertaken using BALB/C mice. One foot pad was infected with infective Leishmania promastigotes while the other foot served as control. After the infected foot pads had developed lesions, mice with similar size lesions were

TABLE 4 Footpad Thickness Lesion Lesion (mm) Size Size Diff Uninfected (mm) (mm) Lesion size Compound Dose Admin Route Foot Pre-Rx Post-Rx (mm)* Artelinic 4 ul/Kg Oral 1.5 2.8 2.0 -0.8 acid Oral 1.5 2.7 2.0 -O.7 Oral 1.5 2.8 Ulcerated IP 1.5 2.6 2.1 -0.5 IP 1.5 2.5 2.2 -0.3 IP 1.6 2.6 2.0 -0.6 Plant extract 8 ul/Kg Oral 1.2 2.9 2.0 -0.9 Oral 1.3 2.8 2.0 -0.8 IP 1.4 2.7 2.2 -0.5 US 7,566.465 B2 13 14

TABLE 4-continued Footpad Thickness Lesion Lesion (mm) Size Size Diff Uninfected (mm) (mm) Lesion size Compound Dose Admin Route Foot Pre-Rx Post-Rx (mm)* IP 1.5 2.8 1.8 -1.0 IP 1.5 2.6 2.7 +0.1 Pentostam 20 mg/Kg Oral 1.4 2.8 2.6 -0.2 Oral 1.5 2.7 2.9 -0.2 Oral 1.5 2.7 2.9 +0.2 IP 1.6 2.8 2.3 -0.5 IP 1.5 2.8 3.0 +0.2 IP 1.6 2.6 2.7 +0.1 Control NA 1.4 2.5 2.8 +0.3 *(positive value indicates worsening and a negative value healing of the lesion with time)

Results (Table 4) show that Artelinic acid and plant extract 11. Panzera, F., et al. Genomic changes of Chagas disease of Artemesia annua, containing artemisinins, either given vector, South America. Emerging Infectious Diseases, orally orip to BALB/c mice for one week led to a decrease in 2004, 10(3): 438-446. the size of Leishmania major lesions induced in the footpad of 12. El-Sayed, N. M.; et al. The genome sequence of Trypa the animals. Both Artelinic acid and plant extract were much nosoma Cruzi, etiologic agent of Chagas disease. Science, more effective than Pentostam, the positive control, at the 2005, 309: 409-410. doses and route of administration tested. 25 13. Kennedy, P. G. E. Human African trypanosomiasis of the The in vivo experiments, are remarkable in that Artelinic CNS: current issues and challenges. The Journal of Clini acid and the active ingredient in the plant extract, artemisi cal Investigation, 2004, 113: 496-504. nins, show potency against Leishmania when administered 14. Legros, D. Treatment of human African trypanosomiasis orally. Moreover the in vitro experiments demonstrate that present situation and needs for research and development. these compounds do not require to be metabolized into an 30 The Lancet Infectious diseases, 2002, 2: 437-440. active moiety by the for them to show potency against 15. Freiburghaus, F.; Steck, A.; Pfander, H.; Brun, R. Bioas intracellular Leishmania amastigotes. say-guided isolation of a diastereoisomer of kolavenol The invention has been described herein with reference to from Entada abyssinica active on Trypanosoma brucei certain preferred embodiments. However, as obvious varia rhodesiense. 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