US 2003O157197A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0157197 A1 OKUNUI et al. (43) Pub. Date: Aug. 21, 2003

(54) PLANTDERVED ANTI-PARASITIC AND (21) Appl. No.: 09/428,203 ANTIFUNGAL COMPOUNDS AND METHODS OF EXTRACTING THE (22) Filed: Oct. 27, 1999 COMPOUNDS Related U.S. Application Data (76) Inventors: CHRISTOPHERO. OKUNJI, SILVER SPRING, MD (US); (60) Provisional application No. 60/105,888, filed on Oct. MAURICE M. IWU, SILVER 27, 1998. SPRING, MD (US); JOAN E. JACKSON, ROCKVILLE, MD (US); JOHN D. TALLY JR., Publication Classification WASHINGTON, DC (US); CYRUS BACCHI, EAST NORTH POINT, NY (51) Int. Cl." ...... A61K 35/78 (US); JOHNSON F. AYAFOR, DSCHANG (CM) (52) U.S. Cl...... 424/725 Correspondence Address: ELIZABETH A, ARWINE USAMRMC (57) ABSTRACT FORT DETRICK BUILDING 521 Provided are biologically active extracts from Aframomum FREDERICK, MD 21701 (US) aulocacarpus, Aframomum danellii, Dracaena arborea, (*) Notice: This is a publication of a continued pros Eupatorium Odoratum, GloSSOcalyx brevipes and Napoleo ecution application (CPA) filed under 37 naea imperialis which are Suitable for use in treating fungal CFR 1.53(d). and protozoa diseases. Patent Application Publication Aug. 21, 2003 Sheet 1 of 21 US 2003/0157197 A1

Fig. 1

ANTIFUNGAL ACTIVITY OF Labda-8 (l7) l2-diene-l5, 16 - dia (AD-1) AGAINST Cladosporium cuculiner inun AT DIFFERENT CONCENTRATIONS

Patent Application Publication Aug. 21, 2003 Sheet 2 of 21 US 2003/0157197 A1

Fig. 2

SCHEME FOR THE ISOLATION OF ANTIFUNGAL CONSTITUENTS OF Afra Inonum danielli i K. Schum (Fam. Zingibe raceae)

Pet ... ether extract of Rhizome (3g) cc "Flash" silica gel 60 l, MeOH in CHCl

l-2 (-) Fr. --3 (620mg) (+) Fr 4-7 (+) Fr. 8-ll ( - ) (2 x 200mg) Lobar Silica gel 60 Pet. ether : EtOAC (6 : l)

Fr. 1.6-l9 (+) Fr. 26-36 (-) (l77mg) Lobar Silica gel Lobar Silica gel Pet. ether EtOAC (6 : ) Pet. ether : EtOAC (6 : . ) Tol. EtOAC (95:5) Tol: EtOAC (95 : 5)

AD-li (93 mg) (+) AD-2 (23 mg) (-)

+ active - inactive Patent Application Publication Aug. 21, 2003 Sheet 3 of 21 US 2003/0157197 A1

undance TIC: AF-2. O --- 796 i.e. 07:

SOOOOOO

l 4. OO 6. O. O. 1800

undance TIC: AF-29 - 8. D ... 4 e -- 07

1.2e -- 07

le + O.

BOOOOOO

6 OOOOOO

4 OOOOOO

2OOOOOO

O itne--> S. OO 0.00 S.O.O. -- 2O. OO Fig 2B.

TIC: AF-29-18.)

5000000

fine --> 00 12.00 l.00 400 15 OO 600 l7.00 18. oo 19 OO 2000 21.00

Figure 2C Patent Application Publication Aug. 21, 2003. Sheet 4 of 21 US 2003/0157197 A1

L.(L) chagasi, MHOM/BR/84/BA-3 MM Medium 96 hrs.A. danielli Rhizome Pet. ether Ext. Fr2(9-18). (50mg/ml) 0.6% DMSO Final Concentration 25000

20000 sK 15000 R s S.s 0000 S S. Patent Application Publication Aug. 21, 2003 Sheet 5 of 21 US 2003/0157197 A1

L. (L.) chagasi, MHOM/BR/84/BA-13 MM Medium

96 hrs. Aframomum danielli Stem Pet, ether Extract. 50 mg/ml), 0.6% DMSO Final Concentration 40000 - TEST CONTROL

30000

S.

20000 & &X s & 8: s 8 &: s : 10000 3.

s

14C-SUBSTRATES p. (LLé-El c Patent Application Publication Aug. 21, 2003 Sheet 6 of 21 US 2003/0157197 A1

Molecular Representation of Labda-8 (17), 2-diene-15, 16- dia (Diterpene Dialdehyde)

space-filling model total electron density Surface

MEP Onto total electron density surface LUMO Surface

isopotential surface at - A - A 1998am. Isopotential surface at - a- a -a -- a a - -5.0 kcal/mol

FIG. a 3 Patent Application Publication Aug. 21, 2003 Sheet 7 of 21 US 2003/0157197 A1

L(L) chagasi, MHOM. IBA-13 MM Medium 'Scià, s. erratt 96 hrs. NI(EtOAc) isola. om Napoleonaea imperialis ten cyni e SatomiSO Fiat Cescentration (50mg/ml) 0.6% DMSO Final Concentration 48086 rest control 30000

i.

20000 0000

0000 0000

fi, 2 A c-suBSAtts 67, 9 B c-SUBSTRATES

L. (L.) chagasi, MHOM/BR84 BA-13 MM Medium L.(L.) chagasi, MHOM/BR184BA-13 MM Medium 96hrs. NI-Glyco Fraction from Napoleonaea, imperialis 96hrs. NI-Glyco Fraction from Napoleonaea. imperialis

(50mg/ml) 0.6% DMSO Final Concentration (50mg/ml) 0.6% DMSO Final Concentration 30000- 500

25000 400

20000 300 S000

200 0000

S000 100

Figure 3 Test results from Napoleonaea imperialis. At 50 g/ml suppression of parasite catabolism of 8 of C-Substrates occurred Patent Application Publication Aug. 21, 2003 Sheet 8 of 21 US 2003/0157197 A1

L. (L.) chagasi, MHOM/BR/84/b., 13 MM Medium 96 hrs. Non-sap isolated from Napoleonaea imperialis L.(L) chagasi, MHOM/BR/84/8A-13 MM Medium (50mg/ml) 0.6% DMSO Final Concentration 96(50ngful) hrs. NI-3 0.6% solated DMSO from Final Napoleonaea Concentration imperials 0000

2SOOO 30000

20000 g O C 15000 so 5 O 3 0000 s -- O000

S000 fy, E. a?...... ZZ; ?y 7 0 F - -WY (N- s *C-SUBSTRATES l4c-SUBSTRATES L. (L.) chagasi, MHOM/BR/84/BA-13 MM Medium L(L.) chagasi, MHOM/BR/84/BA-3MM Medium 96 hrs. N1-4 isolated from Napoleonaea imperialis 96 hrs. NI-5 isolated from Napoleonaea imperialis (50mg/ml) 0.6% DMSO Final Concentration (50mg/ml) 0.6% DMSO Final Concentration 30000-3

l l 20000 3 2 s g10000 2:

O t o e r O e a s : f9.3 G 4C-SUBSTRATES fy,? H CSUBSTRATES

Figure 3 Test results from Napoleonaea imperialis. - At 50 g/ml suppression of parasite catabolism of 8 of C-Substrates occurred Patent Application Publication Aug. 21, 2003 Sheet 9 of 21 US 2003/0157197 A1

Data File C: WHPCHEMV l\DATAWOO50396C. D Sample Name: EOO 60. 69 O.O- 4 O 3 O: 70 atter ACN long C18 column ... On l/rain DAD 254

Acq. Method RDBAYM Seq. Line : - Acq. Operator : dgbg Wial : 100 W Inj : - Sample Name : EOO 60/45 In Volume : 10 Ll Analysis Method : C:\HPCHEMV1\METHODSVIRDBAY.M (modified after loading) OADA, Sig=254,4 Ref=450,80 of OOSQ396C,D A. 8 8

Figure 3. Liquid Chromatographic Separation of Eupatorium odoratum Antileishmania. Fraction EO-O 60-04. Patent Application Publication Aug. 21, 2003 Sheet 10 of 21 US 2003/0157197A1

... . . Until ad Rate Oa t d ------......

2 O C) d 3 VEHICLE CONTROLS (RED & BLUE & LT. GREEN) V) al O O D O 9 CS Car 4 its "1" list-so-scists: griss-to-a. st: -5-3-3, 3-tit, , , 2.5 UG/ML "N" CRUDE PLANT EXTRACT (PURPLE & AQUA)

il- a-...-a dra'a-a-a-heard its dra-al-rihrilrdrdrdr"thrihrheid its “Ax'"my." akra was ifAt f. '- 25 UG/ML"NI" (DARK GREEN)

n-site:-- - - r:-size:------first-startisetsers s pa s: rtist-actitizeta-a-actiti. 9 10:0 2:1) 50.9 4): S: :: :(): Chanbir R 1st Fuld TME (HH: MM: SS) Canbit 8 Chatter C fathbir C s t Canber El st Fluid 50 UG/ML"NI" (YELLOW & PINK- SUPERIMPOSED) is

Figure 4A Patent Application Publication Aug. 21, 2003 Sheet 11 of 21 US 2003/0157197 A1

as e?o cane Eel F. Ga. He LEISHMANIA (LEISHMANIA) CHAGASl ONTR

5 G/MLA"I" (AGUA) G/MLVNI" (PURPLE)

CONTROL (DK. BLUE)

.

2:23 2:35:88 287 2:18:88 22:39 25:33

Tine (MMSS

Figure 4B Patent Application Publication Aug. 21, 2003 Sheet 12 of 21 US 2003/0157197 A1

(jii is: it at a ii.,

LESHMANIA (LEISHMANIA) CHAGAS

3VEH. CONTROLS

. . " " - .2 et 'i's

12.5 UGIML"NI" PURPLE & AQUA iii- t --4----ti-et--th-E-R-birth E- -- a--- is 4...t- : th 1 ti i te: ' ' ' ti A'. -- ...... is a 16c -is ?ix it is at if... iki -- * : * 'a. it. “ -6. ' ' ' ' ' k. k. 4r 30- 25 UGIML"N." DARKGREEN w

15

-- 50 UGML. "N ------T-6:5): :: :19: 7:2) 5") - YELLOW & PINK-- TIME (HH: MM.SS) -...-- laber E -or-minary won-r--a-- +7 HRS ------

laber ------a-a-a------Chamber ) ------...... i- -- chamber 1. ....I.I.I.I.I.I.I.I. 1 hamberints ri: ... ---...--i: - - -

Figure 4C Patent Application Publication Aug. 21, 2003 Sheet 13 of 21 US 2003/0157197 A1

A

3NWASROW33 NSC

Qin??ag

O

1. Patent Application Publication Aug. 21, 2003 Sheet 14 of 21 US 2003/0157197 A1 - 12.

L.(L.) chagasi, MHOM/BR/84/BA-3 MM Medium

96 hrs, MeOH extract (EOMeOH) of Eodoratum (50mg/ml) 0.6% oMSO Final Concentration 30000 TEST CONTROL

20000

As 10000

14C-SUBSTRATES Patent Application Publication Aug. 21, 2003 Sheet 15 of 21 US 2003/0157197 A1 3, 1.a

L. (L.) chagasi, MHOM/BR/84/BA-13 MM2 Medium 96 hrs.Fraction EOO60-04/BN59168 from Eodoraftint 6% DMSO Final Concentration 15000 TEST E CONTROL

10000

Aa 5000

en 14C-SUBSTRATES Patent Application Publication Aug. 21, 2003 Sheet 16 of 21 US 2003/0157197 A1

s

NWSNOLINISO Patent Application Publication Aug. 21, 2003 Sheet 17 of 21 US 2003/0157197 A1 Fig. 6 ABLE - 1 Nulleric Code abbreviations of 'C-substrates used for drug tests * Numeric Commercial Code 14C-Substratest Source

2 L-Arginine (guanidino-14C) A ---- 3. L-Aspartic Acid (4-14C) L-Asparagine (U-14C) L-Glutamic Acid (U-14C) L-Glutamine (U-14C) Glycine (U-14C) L-Isoleucine (U-14C) O L-Leucine (1-14C) 2 L-Methionine (1-14C) l3 L-Ornithine (1-14C) 5 L-Proline (U-14C) 7 Taurine (U-14C) 18 L-Threonine (U-14C) 2O Tyramine (7-14C) 24 L-Fucose (1-14C) 25 D-Galactose (1-14C) 28 D-Mannose (1-14C) 42 Orotic Acid (carboxyl-14C) Nss

44 Succinic Acid (1, 4-l4C) N

46 Na-n-Butyric Acid (1-14C) 49 D-Glucosamine (1-14c) 52 Na-Glycocholic Acid (1-14C)

53 L-Methionine (methyl-14C) 19 Patent Application Publication Aug. 21, 2003 Sheet 18 of 21 US 2003/0157197 A1

TABLE 2.

In vitro Antimalarial Activity of Isolated Compounds Against Clones of Plasnodium falciparum

ICso (pig/ml)

Corpounds Plant Source W clone D6 clone Labda-8(l7), 12-diene-5, 16-dial (I) Aframomum danielli 96.66 280.18 Sakurenatin (IT) Eupatorium odoratum 164.95 2388 Aulacocarpin (ED Aframomum aulocacarpus 2224. 72 14672

Paufueh Patent Application Publication Aug. 21, 2003 Sheet 19 of 21 US 2003/0157197 A1

Table 3. In vitro Activity of Plant Extract vs Growth of African Trypanosomes.

EATRO 110 KETR 243 KETR 269 KETR 243 AS-03 Picraimanitida pfr2 SU-367 9.2 5. 8.4 8.5 Picralimanitida pfr3 SU-369 1. 6. 8.2 Picralimanitida pfr4 SU-370 64 5 500g/ml-22% 5OOmg/ml-1.3% Aframomum melegueta hex SU-766 102 215 5OOg/m-22% 47 Aframomum autocacarpus SU-787 9.0 8.5 12.6 14.9 Aframomum melegueta MeOHSU-798 8.4 7.2 S 30 Aframomum melegueta aq SU-813 500ug/ml-38% 500 g/ml-14% 5OOug/mi-44% 5OOug/mi-22% Gongronema latifolium CHCl, SU-614 134 74. 79 5 Gongronema latifolium exit SU-105 5OOg/ml-1.6% 500 g/ml-8% SOOughmi-7% S00 g/m3% Grape seed2032 SU 719 1.9 2.0 16 34 Albizia ferruginea hexSU-679 18.0 9.6 28.9 40 SS varia chanae rt DCM SU-799 15 229 4. 7 AMorinda lucida DCM SU-740 33 32.5 30.0 39 O Dracaena mannii pDM-X SU-175 6.5 5.4 6.8 62 Apicralina initida PNP-2 SU-846 S.O. 16.9 8.0 3 S Picralina initida PNP-4 SU-847 13.5 8.3 12.S 2.6 Picralina initida PNP-8 SU-848 14. 6.O 18.0 15 Kigelia africana MeOHSU-769 9 730 74 78 Araliopsis tabouensis MeOH frSU-724 6.4 640 59 Araliopsis labouensis AT6compd SU1459 500 Araliopsis tabouensis.AT7compd SU1458 OO Aframomum autocacarpus (aulacocarpin) AZ2 SU-1460 0.86 Dracaena mannii Mannispirostan ASU14616.4 Napoleonaea imperialis Su-1462 1.75 Mezoneurum benthamianum SU-749 44 19.5 8.5 SU-1750 9 76 37 Eupatorium odoratum L. MeOHSU-1466 50 g/ml compound sakuranetin SU-175 20 20.5 73 Gnetum africanum SU-1752 2O2 190 225 Picralimantida CompoundBN79508 SU-753 Planter velous SU-1756 75 8.5 3.5 Planiex veilous SU-1757 15 3 Fagara lemairei SU-1758 2.2 2 2.05 Fagara emairei SU-1759 20.5 170 130 Erythrina senegalensis SU-1760 7.2 9. 15.5 Erythrina Senegalensis SU-1761 18.9 20 22 Mitracarpus scaber SU-762 98 105 71 Oiar viride SU-763 195 32%@ 500g/ml 235 Chasmanthera dependens. SU-1764 225 225 Glossocalyx brevipes ext SU-1464 0.77 Glossocalyx brevipes Neutra fraction SU-1768 0.78 O.76 O.75 - Cau-lé-É. Patent Application Publication Aug. 21, 2003 Sheet 20 of 21 US 2003/0157197 A1

Dorsternia barteri SU-769 7.5 7.3 S.25 SU-1770 6.5 9.5 16 SU-177 S4 60 SU-1772 SO 47 Garcinia kola Heckel SU. 773 2O 210

Pentamidine 0.00048 0.0036 OOO192 O OO3 Melarsen Oxide 0.00077 O.0025 0.0066 OOO72 (-) not tested SU Submitter number.

4-Gauf- g Catwo Patent Application Publication Aug. 21, 2003 Sheet 21 of 21 US 2003/0157197 A1 Fig. 9 Table 4 Minimum Inhibitory concentration (MIC) of Plant Extracts against Trichomonas vaginalis strain CI-NIH MIC ml CI-NIH CDC-085 KV-1 Lab. No 48 hrs 48 hrs 48 hrs Gongronema latifolium SU-105 2.50 2.50

Dracaena mannii SU-175 250 2.50 2.50

Picralina niiida SU-367 12.50 12.50 0.78

Picralima nitida SU-369 0.62 1.25 25

Picralima nifida SU-370 2.50 250 2.50 Gongronema latifolium CHCl3 SU-614 125 0.62 125 Albizia ferruginea hex SU-679 0.62 0.62 0.62 Grape fruit seed 2032 SU-719 0.31 0.01 0.15 Araliopsis tabouensis MeOH fr SU-724 0.62 0.62 2.50

Morinda lucida DCM SU-740 125 25 1.25 Aframomum melegueta hex SU-766 25 1.25 2.50 Kigelia africana MeOH SU-769 0.3 0.62 0.62 Aframomum melegueta CHCl3 SU-787 0.62 2.50 Aframomum melegueta MeOH SU-798 1.25 0.62 25 Uvaria chamae rt DCM SU-799 0.15 0.3 0.62 Aframomum melegueta aqueousSU-813 2.50 2.50 0.15 Picralina nitida PNP-2 SU-846 2.50 1.25 2.50 Picralina initida PNP-4 SU-847 2.50 2.50 250

Picralina rifida PNP-8 SU-848 250 2.50 2.50

Metronidazole 0.003 0.40 0.004 US 2003/O157197 A1 Aug. 21, 2003

PLANT-DERVED ANTI-PARASITIC AND Pentostam(E) (sodium antimony gluconate) can be adminis ANTIFUNGAL COMPOUNDS AND METHODS OF tered only via “investigational protocol” in the USA. There EXTRACTING THE COMPOUNDS is no chemoprophylaxis for any leishmanial disease. An aminoquinoline, WR6026, having showed initial promising 0001. This application claims priority to U.S. provisional results in animal models for Visceral leishmaniasis, has not patent application serial No. 60/105,888, filed on Oct. 27, proven effective in human clinical testing either in Africa or 1998, the complete disclosure of which is incorporated South America (Division of Experimental Therapeutics, herein by reference. Walter Reed Army Institute of Research, unpublished obser 0002 Part of this work was completed while holding a Vations). Liposomal Amphotericin B has not proven effica National Research Council Associateship at the Walter Reed cious against Leishmania Species known to cause mucocu Army Institute of Research. taneous disease (Wortmann, et al., 1997). Topical treatment for leishmanial disease is not effective even for cutaneous FIELD OF THE INVENTION disease forms because leishmaniasis is a Systemic disease (Neva, et al., 1997). There is no general vaccine for leish 0003. The present invention relates to biologically active maniases, although a live vaccine is used in the Middle East compounds prepared from Aframomum au locacarpus, Afra for certain Leishmania (Leishmania) tropica/Leishmania momum danellii, Dracaena arborea, Dracaena mannii, (Leishmania) major to prevent facial Scarring. Eupatorium Odoratum, GloSSOcalyx brevipes, Napoleonaea imperialis, for use in the treatment of fungal and protozoa 0007 Drug resistance is so severe in certain endemic diseases. The invention also relates to methods of extracting regions that thousands are dying in India of untreatable, the biologically active compounds from these plants. multi-drug resistant Visceral leishmaniasis, and in Northern Africa as a result of malnutrition exacerbated disease (Anonymous, 1993; Cerf, et al., 1987; de Beer, et al., 1991; BACKGROUND OF INVENTION Sundar, 1997). 0004 Available drugs for the treatment of diseases due to 0008 Immunodeficiency, either as the result of leishma various protozoal infections are inadequate due to increasing nial tubercular- or HIV co-infections, poses Serious thera parasite resistance and Serious toxicity associated with Some peutic difficulties as leishmanial coinfection is reported to of them. There is therefore a need for new and effective potentiate the pathology of both these bacterial and viral therapeutic agents. In general, antiprotozoals are not given infections (Alvar, et al., 1997; Bernier R, et al., 1995; high priority for commercial development because the per Bryceson, 1987; Faraut-Gamarelli, et. al., 1997). Terra and capita health expenditure in many tropical countries is leSS coworkers (1996) proposed to include visceral leishmaniasis than the cost of one course of drug therapy. Thus, many as an opportunistic infection in the IVC-2 group of clinical "modern' antiparasitic drugs were initially marketed more classification of HIV infection; and Montalban, et al., than 40 years ago. (1990b), in the CDC group IVC-1 as indicative of AIDS 0005 Clinical intervention in the treatment of limited of (also, Federico, et al., 1989; Montalban, et al., 1990). leishmaniasis, for example is limited to the use of pentava 0009 Global travel and commerce result in patients hav lent antimonials (SbV), sodium stilbogluconate and N-me ing complex disease exposure history, and transportation of thylglucamine antimonate, and Secondarily, amphotericin or leishmanial parasites far from their anticipated endemic pentamidine. Croft, S. L., 1988, Recent developments in the regions making both diagnosis and patient management chemotherapy of leishmaniasis, Trends Pharmacol. Sci. 9. difficult (Albrecht, et al., 1996). Leishmaniases have an 376., Bryceson, A., 1987, Therapy in man. In The Leishma annual incidence of 2-3 million new cases per year with 12 niases in Biology and Medicine, Vol. 2, Clinical Aspects and million infected and 350 million at risk in 88 countries Control, W. Peters, and R. Killick-Kendrick, eds., Academic worldwide (Anonymous, 1990; Croft, 1988; World Report Press, New York, 847. Treatment with these agents is not on Tropical Diseases, 1990). However, these figures do not consistently effective particularly for the most virulent leish take into consideration that leishmanial disease transmission manial disease forms (Jha, T. K., 1983, Evaluation of may result in asymptomatic infections which when the diamidine compound (pentamidine isethionate) in the treat immunologic cell mediated response is depressed due for ment of resistant cases of kala-azar occurring in North Bihar, example to co-infection (HIV), natural ageing, pregnancy, India, Trans. Roy. Soc. Trop. Med. Hyg. 77:167., Rocha, R. immunodepressant drugs, malnutrition, etc. The disease A. A., Sampaio, R. N., Guerra, M., Magalhaes, A., Cuba, C. Symptoms can recur, Sometimes more than 30 years post C., Barreto, A. C., and Marsden, P. D., 1980, Apparent infection. Glucantime failure in five patients with mucocutaneous 0010) Two major groups of diseases caused by flagellate leishmaniasis, J. Trop. Med. Hyg. 83:131-139. Mebrahtu, Y. protozoa are African sleeping sickness (Trypanosoma brucei B., Lawyer, P., Githure, J., Were, J. B., Muigai, R., Hen spp.) and trichomoniasis (Trichomonas/Tritrichomonas). dricks, L., Leeuwenburg, J., Koech, D., Roberts, C., 1989, African trypanoSomiasis affects both domestic and wild Visceral leishmaniasis unresponsive to pentostam caused by animals as well as humans in mainly rural Settings (Kuzoe, Leishmania tropica in Kenya, Am. J. Trop. Med. Hyg. 1993; WHO, 1995) while trichomoniasis is a cosmopolitan 41:289. disease in men as well as women, and a threat to cattle 0006 The four drugs most frequently used to treat leish breeding in most agricultural areas of the world (Hammill, maniasis all require parenteral administration, use dates 1989; Levine, 1985). Treatment of the organisms causing back to 40 to more than 50 years, and all have such severe these diseaseS presents problems, in part, due to lack of new, Side-effects that treatment only in a hospital Setting is inexpensive agents, the toxicity of existing agents, and the recommended (Bryceson, 1968, 1987). No antileishmanial development of resistance to existing drugs (Kuzoe, 1993; is Food and Drug Administration (FDA) approved, and Lossick, 1989). US 2003/O157197 A1 Aug. 21, 2003

0.011 African trypanosomiasis is endemic in over 10 0014 Chemotherapy for human trichomoniasis relies on million Square kilometers of Sub-Saharan Africa, affecting a group of 5-nitroimidazoles, with metronidazole (Flagyle) humans and all domesticated livestock (WHO, 1995). There being the most utilized. In the United States, metronidazole are an estimated 25,000 new cases of human disease yearly is the only available agent, although other derivatives are and an animal incidence of 250-300,000 cases but these used in Europe and other areas. Since metronidazole has estimates are low, based on recent civil unrest and lapses in been in continuous use Since 1955, there has been increasing local tsetse fly control and medical surveillance (WHO, reports of metronidazole-resistant vaginitis (Meingassner & 1995; F. Kuzoe, pers. commun.). The primary drugs for Thurner, 1979; Wong et al., 1990; Voolman & Boreham, human and Veterinary trypanoSomiasis have been in use for 1993). Because of its potential to produce free radicals upon >50 years. Resistance is spreading, especially to the dia reduction, it is potentially mutagenic and not given to midines, pentamidine and Berenile, and melarSoprol (ArSo pregnant women (LOSsick, 1989). At present, there is no bal(R), the only available agent for late stage (CNS) human alternative to the 5'-nitroimidazoles for therapy of metron disease (van Nieuwenhove, 1992; Kuzoe, 1993). Melarso idazole-refractory disease, nor for treatment of pregnant prol is also toxic, with a 3-5% incidence of cerebral episodes WOC. reported (Pepin, & Milord 1994; Wery, 1994). A recent 0015 Trichomonas foetus is the agent of bovine tri FDA-approved drug, difluoromethylornithine (DFMO. chomoniasis, causing reproductive failure. Parasites are Eflornithine(R), is effective but expensive for use in eco Spread by infected bulls, multiply in the vagina and invade nomically deprived areas (S450/patient: WHO, 1995). Vet the cervix and uterus. One to 16 weeks after breeding, erinary trypanocides include diminaZene (Berenile) and abortion of the fetus occurs (Levine, 1985). If the placenta isometamidium (Samorine) which are used prophylacti and fetal membranes are eliminated following abortion, the cally for control of disease in cattle herds (WHO, 1995; cow may spontaneously recover. If Some of these tissues Kaminsky et al., 1993). Resistance to both agents has been remain inside the animals, permanent Sterility may result. documented in field studies (Kuzoe, 1993; Schoenfeld et al., There is no Satisfactory treatment for treatment of diseased 1987; Williamson, 1970). For these reasons, there is an cows, while treatment of bulls is tedious and expensive. urgent need to develop new trypanocides. Aminoquinuride (Surfen(R) or acriflavine (Trypaflavine(R) 0012. In Africa, traditional medicine with herbal treat may be used topically, with dimetridazole injected into the ment has a long history and is used routinely in medical care urethra. Unless the bull is valuable, it is usually destroyed (Harley, 1941; Feierman, 1981; Assi & Guinko, 1991), (Levine, 1985). however only a few reports document activity of plant 0016. The disease is common in open range breeding extracts against African trypanosomes. One study demon ranches and may reach epidemic levels. In Australia, strated activity of extracts of Khaya spp seeds (West African 40-65% of cattle were reported to be infected, while the hardwoods) in vitro vs. T. b. bruceii (Owolabi et al., 1990). prevalence in California was reported to be 14% (Yule et al., The active agents were a group of furanodlimonoids related 1989). The economic losses due to bovine trichomoniasis to quassinods, plant products found to block protein Syn have been estimated to be S665 per infected dairy cow, while thesis (Kirby et al., 1989). Another report indicated that the widespread prevalence of the disease would account for gossypol, a quassinoid, blocked respiration and destroyed T. tens of millions of dollars annually (Yule et al., 1989).The b. bruceii blood forms in vitro at micromolar concentrations overall Situation for chemotherapy of trichomoniasis there (Eid et al. 1988). Igweh and Onabanjo (1989) cured mice fore, is the reliance on a single drug as drug class for infected with T. b. bruceii using aqueous root extracts of chemotherapy of human disease, and no effective control Annona Senegalensis. Recently, a Series of Studies by measures for bovine trichomoniasis. Freiburghaus et al. (1996a, 1996b, 1997) evaluated extracts of traditional medicinal plants from Uganda, Tanzania and 0017 Another protozoan disease, malaria, remains the the Ivory Coast, against T. b. rhodesiense blood forms in greatest human killer among parasitic infections, despite the vitro. Of those tested, 42 of 310 extracts (13.5%) were found world-wide effort to combat the disease and attempts at the to have significant growth inhibitory activity at 10 mg/ml or eradication of the causative organisms. The emergence of leSS. The active agents generally had modest Selectivity multi-drug resistant Strains of Plasmodium falciparum, the indicies, as compared to commercially available agents, but, most lethal of the malaria parasites, poses a Serious health overall, these studies confirmed the potential for ethnobo care problem, not only in the malaria-endemic countries but tanically Selected plants as potential Sources of agents also among international travellers. against Sleeping sickness (Freiburghaus et al., 1996a, 1996b, 0018 Similarly, fungal and yeast infections are becoming 1997). increasingly resistant to modern drugs. In immunologically 0013 Trichomonas vaginalis is a sexually transmitted compromised individuals, for example, complications aris pathogen of the human urogenital tract. It infects the vaginal ing from uncontrollable fungal infections are among the epithelium, causing Severe irritation and the development of leading cause of death. There is, therefore, a need for new a discharge. Trichomoniasis is one of the most prevalent and effective alternative treatment. STD’s in the Western world, accounting for a large number 0019 Protozoan infections are also a major cause of of Visits to private gynecologists and public clinics (Ham mortality and morbidity in immunosuppressed patients, as in mill, 1989). In addition to social distress caused by the acquired immunodeficiency syndrome (AIDS). A single disease, recent evidence Suggests a high incidence rate therapeutic agent active against different types of protozoa between cervical cancer and trichomoniasis (Gram et al., would be a major innovation in the treatment of these 1992). The disease is widespread, with about 3 million cases diseases. It would therefore be useful to develop more in women annually in the United States alone (Hammill, effective, leSS toxic and orally active antiprotozoal agents. 1989). There are reports on the potential of plants as Sources of new US 2003/O157197 A1 Aug. 21, 2003 antiprotozal agents. A Series of investigations on the anti vehicle-control-treated parasite respiration is represented by protozoal activity of plant from West and central Africa had the light grey vertical bars; the Labda-8(17),12-diene-15, be conducted. For example the antileishmanial and antimi 16-dial (50 ug ml for 96 h) treated parasites, by the solid crobial activities of Nigerian medicinal plants, have been black bars. The 'C-substrate numeric codes (X-axis) are evaluated by Iwu (1992), Okunji, et al. (1990, 1991, 1996, provided. pg. 9(3)). For example, this method relies on drug inhibition 0029 FIGS. 5A-5H illustrate radiorespirometric (RAM) of parasite production of "CO from a battery of "C- data showing markedly reduced respiration of Leishmania Substrates to detect drug-mediated parasite damage at low (Leishmania) chagasi, a visceral disease parasite after Eupa drug concentration within a short time (Jackson et al., 1989, torium Odorantum treatment in vitro. The vehicle-control 1990). treated parasite respiration is represented by the light grey 0020 Thus, there is an urgent need for new biologically vertical bars; the, Labda-8(17), 1 2-diene-15, 16-dial (50 ug active compounds for use in treating protozoa diseases ml for 96 h) treated parasites, by the solid black bars. The which avoid the harmful side-effects of conventional phar "C-Substrate numeric codes (X-axis) are provided. maceuticals. (All documents cited herein Supra and infra are hereby incorporated in their entirety by reference thereto.) 0030 FIGS. 6-9 illustrate Tables 1-4. SUMMARY OF THE INVENTION DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 0021. An objective is to provide new biologically active compounds for use in treating fungal and protozoa diseases 0031. The therapeutic potential of medicinal plants based which avoid the harmful side-effects of conventional phar on a battery of biologic test Systems, including radiorespiro maceuticals. metric, Cytosensor(R), bioautography, agar dilution methods, 0022. The above objective and other objectives are and MALARIA test method & Trypanosomails test method, obtained by novel biologically active extract from at least etc., are described. Bioassay-guided chromatographic frac one plant Selected from the group consisting of Aframomum tionation of the active crude extracts led to the isolation, aulocacarpus, Aframomum danellii, Dracaena arborea, identification and structure elucidation of bioactive com Eupatorium Odoratum, GloSSOcalyx brevipes and Napoleo pounds. Exemplary Sources of active ingredients include labdane-dial from Aframomum danellii, aframodial from naea imperialis. Aframomum au locacarpus, Mannispirostan A from Dra 0023 The present invention also provides methods of caena mannii, and Sakuranetin from Eupatorium Odoratum. obtaining the novel biologically active extracts. Other sources include GloSSOcalyx brevipes and Napoleo naea imperialis. BRIEF DESCRIPTION OF THE DRAWINGS 0032 Compounds having antiprotozoal properties are 0024 FIG. 1 illustrates a thin layer chomatography identified and described using bioassay-directed fraction (TLC)-bioassay on a silica gel plate, showing inhibition of ation. Previously, we reported the Evaluation of Plant the fungus, Cladosporium cucumerinum, by, Labda-8017), Extracts for Antileishmanial Activity. Using a Mechanism 12-diene-15, 16-dial at four concentration levels. Based Radiorespirometric Microtechnique (RAM). Iwu, M. 0025 FIGS. 2, 2A-2F illustrate radiorespirometric M., Jackson, J. E., Tally, J. D., and Klayman, D. L., (RAM) data showing markedly reduced respiration of Leish Evaluation of Plant Extracts for Antileishmanial Activity mania (Leishmania) chagasi, a Visceral disease parasite Using a Mechanism-Based Radiorespirometric Microtech after, Labda-8(17),11-diene-15, 16-dial treatment in vitro. nique (RAM Planta Medica, (1992) 58: 436-441., Recently The vehicle-control-treated parasite respiration is repre the biological activity of Saponins from two Dracaena Spe sented by the light grey vertical bars; the Labda-8(17), 12 cies,was reported by our group Okunji C. O., Iwu M. M., diene-15,16-dial (50 ug/ml for 96 h) treated parasites, by the Jackson J. E. and Tally J. D., Biological Activity of Saponins solid black bars. The "C-substrate numeric codes (X-axis) from Two Dracaena Species, Advances in Experimental are provided. Medicine & Biology, 404:415-28, 1996, antifungal and sev eral molluscicidal constituents of D. mannii (Okunji et al., 0.026 FIGS. 3A-3I illustrate cytosensor microphysiom 1990; 1991). eter (CMS) antileishmanial promastigote results after 17.5h Napoleonaea imperialis treatment. 0033) Materials and Methods 0027. The duplicate control parasite (i.e. parasites treated 0034 Plant Materials with drug solvent, 0.6% DMSO) tests, represented as upper 0035 All plant materials utilized were collected near the most lines, “G” and “H”, have a consistently higher meta Nsukka campus of the University of Nigeria with the bolic rate during the 11 h of observation. ParasiteS preincu exception of Aframomum au locacarpus which was collected bated in parallel with controls for 17.5 h with 6.3- (lines “A” at Bamenda, Cameroon. The collection in Nigeria was and “B”), 12.5- (lines “C” and “D”), and 50 lug ml chosen primarily from plants listed in an ethnomedicinal Napoleonaea imperialis(lines “E” and “F”), manifest lower Survey carried out among the Igbo people (Iwu, 1981/82, metabolic rates, with the two highest drug concentrations 1993). The plants were taxonomically identified by Mr. A. resulting in metabolic rates very close to Zero. Ozioko of the Department of Botany, University of Nigeria, 0028 FIGS. 4A-4C illustrate radiorespirometric (RAM) Nsukka and the identities confirmed by Dr. J. C. Okafor of data showing markedly reduced respiration of Leishmania the Forestry Herbarium, Enugu. Voucher Specimens have (Leishmania) chagasi, a visceral disease parasite after, been deposited at the Bioresources Development and Con Labda-8(17),12-diene-1 5, 16-dial treatment in vitro. The servation programme Herbarium at Nsukka, Nigeria. Prior US 2003/O157197 A1 Aug. 21, 2003 to extraction, the plant materials were oven dried at 40 C. Spore Suspension of Cladosporium cucumerinum, and Sub and the dried vegetable drugs were ground to coarse powder. Sequent plates with Spore Suspensions of Cladosporium carrionii, Cladosporium cladosporioides, Cladosporium 0036 For column chromatography (CC), Flash CC silica tennuisimum (ATCC 623337) and Fonsecaea pedrosoi gel 60 size 0.063-0.200 mm (70-230 mesh ASTM, EM (ATCC 52593), in some cases to determine the spectrum of Science, was used, and SephadeX LH-20, Sigma, for gel activity. The plates were then incubated in sealed humid filtration. Lichro-prep silica gel 60 (40-60 um, Merck), chambers at room temperature for four days in the dark. Low-pressure liquid chromatography (Lobar) was done Antifungal activity was manifested by the appearance of a using a LichroPrep RP-8 column (40-63 mm 2.5x25 Merck) white Spot, corresponding to the position of the active equipped with an FMI pump. DCCC equipment consisted of compound, Surrounded by a grey-black fungal growth all (Tokyo Rikakikai Nishikawa Bldg Toyama-Cho Kanda over the plates To illustrate the bioassay-directed fraction Chiyoda, Tokyo, type 300 glass tubes (length 400 mm, I.D. ation protocol, an example was drawn from the fractionation 2 mm), solvent system: CHCl:MeOH:HO (7:13:8). of rhizome extract of Aframomum danellii which showed 0037. Thin layer chromatographic (TLC) was performed Strong antifungal activity. The powdered rhizome of A. on Uniplate HPTLC-HLF normal phase silica gel (150 danellii (200 g)was continously extracted with petroleum microns) Analtech while preparative thin-layer chromatog ether (bp 40-60° C.) to exhaustion in as Soxhlet extractor. raphy was carried out on pre-coated thin-layer chromato The petroleum ether extracts when combined and concen graphic plates silica gel GF (2000 microns). The plates were trated to dryneSS in vacuo gave a reddish brown residue visualized with UV-lamp (Chromato-Vue Model CC-20) (11.41 g). The air dried marc from the above extract was and Sprayed with appropriate Spray reagents to detect the again extracted with ethanol in a SOXhlet eXtractor, extracts spots. were concentrated to dryneSS under reduce pressure using the rotary evaporator at 40 C. Both extracts were tested for 0038 GC-MS A Hewlett Packard HP 5890 CC was antifungal activity using bioautography (TLC-bioassay) as interfaced with a quadrupole mass spectrometer (MS) described above. Screening for antifungal activity showed (HP5970). OV351 and BP1 fused silica columns (25x0.20 that the petroleum ether extract of A. danellii were fungi mm i.d.) were used with helium as carrier gas (0.5 ml/min). The temperature was programmed from 70° C. to 200 C. cidal against Cladosporium cucumerinum on a TLC bioas (50° C./min.). MS operating parameter were: ionization Say voltage -700V, scan rate -1100 amu?sec, electron multiplier 0044) The active petroleum spirit extract (3 g) was sub energy -1600V, and ion source temperature 250 C. On-line jected to bioassay-directed fractionation by flash chroma acquisition and computation of mass spectra data were tography with a gradient elution of 1% methanol in chlo performed using Hewlett Packard HP 9827 computer roform. Fractions (20 ml) were collected and pooled on the equipped with a disc memory (HP9145). The constituents basis of their TLC profiles. Low pressure liquid chromatog were identified by comparison of their mass spectral data raphy (Lobar) separation of the most antifungal fraction with the computer library (400 mg) was performed in two Successive separations on Lichro-prep silica gel 60 (40-60 um, Merck) eluted first with 0039) Preparation of Test Material petroleum spirit/ethylacetate (6:1) and then with toluenel 0040 Extracts of each plant parts, including roots, stem ethylacetate (95:5). This yielded major two UV active bark, leaves, fruits, or Seeds, was prepared by either extract compounds of which only one inhibited the growth of ing 200 g of the dried coarsely powdered plant using SOXhlet Cladosporium cucumerinum spores at a minimum concen extractors or macerating at room temperature overnight with tration of 0.5 lug. The flow chart of this procedure is shown Shaking. The bulk extracts from each plant was then filtered in FIG. 2. and evaporated to dryneSS in vacuousing the rotary evapo 0045. In Vitro Antileishmanial Activity rator. The crude extracts were Screened for biological activi 0046) An in vitro radiorespirometric microtest (RAM) ties using a battery of test Systems to detect antifungal, technique was used to evaluate the extracts/fractions or pure antimalarial, antileishmanial and antitrypanoSmal activities compounds for possible antileishmanial activity. This etc. Each plant extract was intially Submitted for a particular method, as already noted, relies on drug inhibition of test based on their ethnomedical usage. Bioassay-directed parasite production of ''CO from a battery of 'C-Sub fractionation of the active extracts/fractions using a combi Strates by promastigotes to detect drug-mediated parasite nation of chromatographic techniques, gel filtration by damage at low drug concentration within a short time. The Sephadex, droplet countercurrent chromatography (DCCC), test is quantitative, rapid, consistent, and is conducted in and low-pressure liquid chromatography (Lobar), open col Serum-free medium in which prior adaptation is not neces umn chromatography or preparative thin layer chromatog Sary to cultivate the So-called “difficult to grow species. raphy(PTLC) led to the isolation and characterization of 0047 Leishmania Species/Strains: biologically active compounds. 0048. A clinical isolate of visceral Leishmania (Leishma 0041 Bioassay-Directed Fractionation Protocol nia) chagasi, MHOM/BR/84/BA-13, was used for this study. This isolate was selected because sensitivity to SbV 0.042 Antifungal Tests-Bioautographic was previously determined using RAM. MHOM/BRI84/ 0.043 A method similar to that of Homans and Fuchs BA-13 is sensitive to Pentostam(R), sodium antimony glu (1970) was employed. This technique involves direct spray conate, at 6 ug ml. Sb (20 ug ml drug); and to Glucan ing of thin layer chromatograms with conidial Suspensions time(B, N-methylglucamine antimonate, at 80 ug ml. Sb of a test organism. About 100 lug of extract was spotted on (286 ug ml drug). silica gel TLC plates and developed with suitable solvent 0049. The 'C-labeled substrates as shown in Table 1 of System. Dried plates were separately Sprayed with either a FIG. 6, are (numerical codes given in the x-axis of FIGS. US 2003/O157197 A1 Aug. 21, 2003

2-5) "C-substrates: (3) L-aspartic acid (4-"C); (7) glycine 0055 Bioassay-Directed Fractionation of Napoleonaea (U-'C); (10) L-leucine (1-'C); (13) L-omithine (1-'C); imperialis P. Beauv. (Fam Lecythida Caec) Seed Extract. (25) D-galactose (1-'C); (28) D-mannose (1-'C); (44) succinic acid (1,4-'C); (46) Na-n-butyric acid (1-'C). All 0056. The effect of Napoleonaea imperialis seed extract "C-substrates were selected with specific activities as close and isolated pure compounds on promatigotes were assessed to 40 mCi mM per carbon atom as obtainable from by in vitro Radiorespirometric Microtest (RAM) technique commercial Sources. The quantitative promastigote growth as described above. inhibition assay was used as a guide to identify isolates 0057 The powdered seed of Napoleonaea imperialis was exhibiting antileishmanial activity. SOXhlet extracted with Solvents of increasing order of polar ity in three batches, starting with hexane (48 h), chloroform 0050 RAM Drug Test Procedure: (48 h), ethyl acetate (48 h) and methanol (48 h). Each extract 0051. The procedure was conducted as previously was concentrated to dryneSS in vacuo using the rotary described (Jackson et al., 1989, 1990). Promastigotes were evaporator at 40 C. The promastigote growth assay was maintained in log phase growth for 3 Successive transfers used to identify fractions exhibiting antileishmanial activity. (48-72 h apart) prior to radiorespirometric (RAM) testing. The methanol eXtract was the most active fraction. A portion Test samples (or PBSS, 0.1 M phosphate-buffered balanced of the methanol extract (20g) was first partitioned between salt solution, plus drug solvent, DMSO, for parallel control chloroform-methanol-water mixture (2:2:1, 1000 ml) to cultures) was added 24 h after the third promastigote transfer yield a Saponin-enriched lower organic layer which was to fresh growth medium. Incubation in the presence of plant concentrated to dryneSS in vacuo and lyophilized. Both samples was continued for 96 additional hours while the factions showed antileishmanial activity were Subjected to parasites remained in mid-log phase growth. The test Sample fractionation by column chromatography on Silica gel with was tested at 50 g ml. Drug sensitivity or resistance was a gradient elution of chloroform methanol. Preparative low based on 'C-Substrate(s) (listed in Table 1) for which 'CO preSSure Liquid chromatography of the antileishmanial frac release was decreased for drug-treated parasites compared to tion was performed on Silica gel with CM (1:), yielding an parallel tests of phosphate-buffered balanced Salt Solution antileishmanial Substance, consisting of a mixture of three and vehicle (PBSS+DMSO) controls. Each experiment con Saponins. Sisted of parallel: (a) duplicate tests of drug-treated para 0058 Screening for antileishmanial activity showed that Sites; plus (b) duplicate tests of drug vehicle control-treated the methanol extract of N imperialis showed inhibitory parasites; plus (c) one “nonbiological” Sterility control. The effects against the growth of Leishmania promatigotes. A nonbiological control consisted of each "C-Substrate (one Second method of generating active components from N substrate per microtiter tray well), and PBSS (the same imperialis was carried as follows. A portion of the methanol PBSS batch used to wash, to suspend the parasites, and to extract (11.35g) was dissolved in minimum amount of make drug Solution). Since there were no parasites in the methanol and Subsequently precipitated with diethyl ether, nonbiological control, any "CO detected was attributed to yield a 59% of saponin-rich portion(6.73 g) and 4.54 g% either to biologic contamination (or, less likely, chemical of non-Saponin portions. Both fractions were Submitted for contamination) of the "C-substrates resulting in breakdown antileishmanial Screening. The Saponin portion showed of the 'C-substrates. If radioactivity above background (10 remarkably more antileishmanial activity than the non dpm) was detected in the nonbiological control, the Suspect Saponin portion. The active Saponin-rich fraction (6.03 g) Solution(s) was replaced and the experiment was repeated. was Subjected to fractionation by column chromatography 0.052 Promastigote Viability was Assessed by Morpho on Silica gel with a gradient elution of chloroform methanol. logical Criteria Such as Flagellar Mobility and Parasite Fractions (20 ml) were collected and pooled on the basis of Morphology. their TLC profiles. The resulting fractions (1-7) were then screened for antileishmanial activity. Fraction 13-15 (1.48 g) 0053. The powdered rhizome (208g) of Aframomum eluted with chloroform:methanol (5:2) gave the most active daniellii was soxhlet extracted with petroleum ether (b.p fraction and Subsequent re-chromatographed on Silica gel 40-60 C.) and ethanol. The petroleum ether extract of A. column eluted with chloroform:methanol (3:2) to yield danielli which showed pronounced antileishmanial activity Several Sub-fractions. The residues of the fractions possess was further investigated to isolate the active constituents ing the major activity were re-chromatographed over Silica The active petroleum ether extract (3 g) was Subjected to gel by using eluents comprising chloroform:methanol to bioassay-directed fractionation by flash chromatography which increasing amounts of methanol were added. After with a gradient of 1% methanol in chloroform as described Screening these fractions for antileishmanial activity, Sub earlier. Fractions (20 ml) were collected and pooled on the fraction36-51 (352.6 g), was found to be the most active and basis of their TLC profiles. was Subsequently purified by low pressure preparative liquid 0.054 The resulting four fractions were then screened for chromatography using on Rp-8 lobar column eluted with antileishmanial activity and the most active fraction (Fr.3) methanol: water(7:3).This yielded three active fraction; was analyzed by GC-MS to access its purity and identifi imperialiside (A-C) (NI-3 (152 mg), NI4 (30 mg) & NI-5 cation as shown in FIGS. 2A-C. Low pressure liquid chro (102 mg)). matography (Lobar) separation of the antileishmanial frac 0059 Bioassay-Directed Fractionation of Eupatorium tion was performed as described above in two Successive Separations using Lichro-prep Silica gel 60 (40-60 um, Odoratum P. Beav. (Fam. Lecythidacaea) Leaf Extract. Merck) This yielded the same Labda8(17),12-diene-15,16 0060. By the same process used, the powdered seeds of dial identified above as the antileishmanial compound. The Eupatorium Odoratum was Successively extracted with identity was established based on Spectra analysis and petroleum ether bp (40-60° C.) (48 h) and methanol (48 h) comparison with the literature data. using SOXhlet extractor. Each extract was concentrated to US 2003/O157197 A1 Aug. 21, 2003 dryness in vacuousing the rotary evaporator at 40 C. Both visceral leishmaniasis, and Leishmania (Viannia) panamen extract showed antileishmanial activity with the methanol sis, MHOM/PA/83/WR539, an etiological agent of simple extract showing greater inhibition of the growth of promas cutaneous leishmaniasis. Imperiside A was tested in each in tigotes. A portion of the methanol extract (20.0 g) was first Vivo leishmanial model by the oral, intramuscular, and partitioned between chloroform-methanol-water mixture Subcutaneous routes of administration. (2:2:1) to yield a lower organic layer and a more polar aqueous layer. Both fractions were concentrated to dryneSS 0066 Cytosensor Microphysiometer System in vacuo and lyophilized. The more active organic fraction 0067. The rate at which cell excrete acids into their (8.10 g) was Subjected to further bioassay-directed fraction environment is closely linked to the rate which they convert ation by column chromatography on Silica gel. The column food to energy, i.e. metabolic rate. The Cytosensor Micro was eluted with chloroform:methanol (19:1, 1:3, ethyl physiometer System (CMS) measures the rate at which cells acetate, ethylacetate:MeOH 17:1). Fractions (20 ml) were acidify their immediate environment. The CMS monitors collected and pooled on the basis of their TLC profiles. The these metabolic changes as changes in the rate of cellular monitoring of the fractions was carried out with TLC acidification. In this way, the System provides a real-time, aluminum sheet silica gel 60-F254 in solvent system I and noninvasive means of measuring cellular responses to a II. After Screening these fractions for antileishmanial activ wide variety of agents (McConnell et al., 1992). ity, fraction 60-104 was found to be the most active and was 0068 Extracts of Napoleonaea imperialis seeds were Subsequently purified by combination of gel filtration on tested for antileishmanial activity in vitro using CMS. Pro Sephadex LH-20 column (2.0x50 cm) LC-No. 5, FIG. 3. mastigote leishmanial forms were exposed to N. imperialis and preparative thin layer chromatography, leading to the in the chemically defined, Serum-free medium (Jackson et identification of Sakarentin as the antileishmanial com a/., 1989) for 17.5 h during logarithmic growth phase. To pound. The structure of sakurenatin (II) was established by prepare cells for CMS, the non-adherent cell protocol was GC-MS and Nuclear magnetic resonance spectroScopes and utilized. Briefly, the cells were centrifugally concentrated, by comparison with published data, as shown in the follow counted by hemacytometer, and re-Suspended in 0.2% low ing formula: temperature agarose in balanced Salt Solution. Leishmanial promastigotes, a 10 ul Suspension containing 1-2x10 cells (II) in agarose, were placed in each of 8 Cytosensor flow OH chambers and the low-buffer formulation of RPMI medium (pH 7.4, Molecular Devices Corporation) was pumped over the cells. The repetitive pump cycle time was 2.0 min (88 sec OHC O of medium flow followed by 32 sec of pump off). During the 32 Sec the peristaltic pump was not operating, the rate of leishmanial acidification of RPMI medium in each of 8 Separate cell chambers was measured. Acidification rates OH O during the two-min cycle resulted in less than 0.1 pH unit change and were not detrimental to the leishmanial cells. C17H13O4 286.32 The CMS leishmanial acidification rates (representative data 286.12O509 given in FIG. 4) were relatively constant for each drug C 71.3% H 6.3% O 22.4% treatment concentration (6.3, 12.5, 50 ug ml) and vehicle control (0.6% DMSO) duplicate pair, tested in parallel 0061 Antimalarial Bioassay Simultaneously, over the 11 h observation period. 0062) The in vitro antimalarial assays were performed by 0069 Drug Development for Parasitic Protozoa using a modification of the Semi-automated microdilution 0070 Haskins Laboratories has focused on the nutrition technique described earlier (Desjardins et al., 1979, Milhous and biochemistry of protozoa and the development of novel et al., 1985). Two Plasmodium falciparum malaria parasite leads to chemotherapy of parasitic protozoa for >40 years. clones, designated Indochina (W-2) and Sierra Leone (D-6), Research initially stressed nutritional and Vitamin-require were utilized in Susceptibility testing. The W-2 clone is ment Studies leading to development of chemically defined resistant to chloroquine, pyrimethamine, Sulfadoxine, and media for many free-living and parasitic protozoa. Recent quinine, and the D-6 clone is resistant to mefloquine. Studies have emphasized African trypanoSomes, tri chomonads, and the opportunistic pathogens Cryptospo 0.063. The test extracts/compound, was dissolved in ridium and Microsporidia. DMSO and serially diluted using malarial growth medium. Drug-induced reduction in uptake of titriated hypoxanthine 0071. The biochemical studies surrounding drug devel was used as an index of inhibition of parasite growth. opment have centered on the Synthesis and metabolism of polyamines in these organisms, with biochemical peculiari 0064. In Vivo Antileishmanial Activity ties in the protozoa Serving as focal points for targeting antiparasitic agents. 0065. The in vivo antileishmanial activity was deter mined by administering various doses of the Napoleonaea 0072) Our study of polyamine synthesis in African try imperialis extracted to golden hamsters and determining the panosomes led to the development of DFMO, an enzyme effect on laboratory-induced Visceral and cutaneous leish activated inhibitor of ornithine decarboxylase, for clinical manial of the animals. For this assay, the compounds were use in human trypanosomiasis (Bacchi et al., 1980; Bacchi tested against Leishmania (Leishmania) donovani, MHOM/ & McCann, 1987; Sjoerdsma & Schechter, 1989). This SD/43/Khartoum, a causative organism of kala azar or agent was approved by the U.S. Food and Drug Adminis US 2003/O157197 A1 Aug. 21, 2003 tration in 1990 and has been used clinically in greater than Zoan-caused disease ug ml was used as reference standard 2,000 cases of West African disease with a 95% cure rate and 0.1 ml DMSO as a control. The plates were incubated (Kuzoe, 1993; pers. commun., WHO, 1995; van Nieuwen at 37 C. and the diameter of Zones of inhibition was hove, 1992). The mechanism of DFMO action in part has measured across each well after 24 h. The MIC for bacteria been investigated extensively; our Studies indicate it acts in was determined in trypticase Soy broth to which were added trypanoSomes by causing overproduction of S-adenosylme serial 2-fold concentrations (0.025-200 ug ml) of Man thionine (AdoMet), a precursor of spermidine (Yarlett & nispirostan A. The tubes were inoculated in triplicate with Bacchi, 1988a). Recentwork indicates that the over synthe 0.01 ml quantities of 6th broth cultures of the test isolates. sized AdoMet is rapidly used in the methylation of proteins The tubes were incubated at 37 C. for 24 h and examined and lipids (Bacchi et al., 1995; Goldberg et al., 1997), aiding Spectrophotometrically at 530 nm. The lowest drug concen in the blockage of cell division. tration that showed no turbidity was taken as the MIC. 0.073 Polyamine synthesis in T vaginalis was found to Streptomycin was used as the Standard reference drug. differ from mammalian cells in that AdoMet decarboxylase 0.077 Results and Discussion was lacking and that Spermidine was obtained from conver Sion of exogenous Spermine, taken up through an amine 0078 Rapid methods for screening, isolation, and char transport System, and then converted through a polyamine acterization of antiprotozoal compounds are described. oxidase (Yarlett & Bacchi, 1994). Although putrescine was These methods utilize relatively Small quantities of plant Synthesized from arginine through the arginine dihydrolase Sample or compounds. By these methods, the major com pathway, this process also functions in part to generate ATP pounds identified from the antileishmanial Study include and to generate putrescine which is exchanged for Spermine labdane-dial from Aframomum danielli, Mannispirostan A through an antiporter (Yarlett & Bacchi, 1988b; Yariett & from Dracaena mannii and D. arborea and Sakuranetin Bacchi, 1994; Yarlett et al., 1994, 1996). These findings from Eupatorium Odorantum and Imperialisides from Napo indicate that T vaginalis (and T. foetus) obtain polyamines leOnaea imperialis;-antimalarial and antitrichomoniasis by processes differing from mammalian cells (Yarleft et al., and antitrypanoSomiasis 1992,1993; Bacchi & Yarlett, 1995). We are examining the 0079. In an activity-directed investigation of the extracts effects of amine analogs for ability to enter through the of leaves, Stem and rhizome of Aframomum daniellii, the polyamine transport system (Yarlett et al., 1992; Woster et petroleum ether extract of the rhizome of A. daniellii exhib al., 1993), and for amine oxidase inhibitors to reduce ited Strong antifungal and antileishmanial activities, as polyamine interconversion. shown in FIG. 1. Bioassay directed fractionation of this 0.074. Our most recent work has also focused on two active fraction led to the isolation of a labda-8(17),12-diene opportunistic pathogens frequently associated with chronic 15, 16-dial (i), as shown in the following formula: intractable diarrhea in AIDS, Cryptospoidium and Micro Spoidia. The diseases caused by these agents are presently (I) incurable. Biochemical Studies on the apicomplexan, CHO Cryptosporidium parvum, have indicated that polyamine production is initiated by conversion of arginine to agmatine via arginine decarboxylase (ADC), an enzyme normally CHO asSociated with plants. This enzyme has been partly purified and characterized from C. parvum (Keithly et al., 1997), and the efficacy of polyamine analogs and ADC inhibitors are now being assessed in C. parvum growth. 0075 Microspondia spp. are a group of parasites encom passing at leastfive genera. These organisms are thought to 0080 Characterization of labda-8(17),12-diene-15,16 be protozoans on the basis of Structural features of the dial Zoospore. This group of organisms has been implicated in numerous pathological conditions including kerato conjunc 0081) TLC (SiO, Tol/EtoAC 6: 1) Rf=0.39. m.p. 90-92oC tivitis, brain lesions and intense diarrhea in immunoSup 0082) UV ). CHCl 240 nmi; IRV, (KBr) 2940, pressed patients, especially those with AIDS (Wittner et al., 1730, 1993). We have begun studies with Enterocytozoon cuniculi, examining polyamine metabolism and the ability of 0083) 1680 and 1640 cm-1; DCI-MS m/z (relint.) polyamine analogs to inhibit growth of this intracellular 302(21), intestinal parasite. Our critical observations indicate that two 0084) 287(11) 273(9), 258(18), 241(4), 231(4), 190(7), polyamine analogs 1,11-diethylnorspermine and 1,19-bis 177(10), 161(8), 147913), 137(100), 123(54),109(29), (ethylamino)5,10,15-triazanonadecane at 100 mM sterilize 95(45).91(31), 83(66), 69(52), 55(30). the culture of this parasite, are without toxicity to the feeder 0085 Labda-8(17),12-diene-15, 16-dial showed strong layer RK13 cells (Coyle et al., 1996). absorption at 1680 cm which is assigned as an 8-unsatur 0.076 These studies plus the collaboration of the Haskins ated carbonyl group. The UV absorption maximum at 240 Laboratories group with >10 Synthetic chemists at eight nm supported this assignment. DCI-MS showed a molecular institutions (Auburn Univ., Cornell Univ., Johns Hopkins ion peak of m/z. 302 and base peak of 137, Suggesting a Univ., Roswell Park Cancer Inst., Tennessee State Univ., molecular formula of CHO. There is good agreement Univ. of Michigan, Univ. of Wisconsin, Wayne State Univ.) between the above spectroscopic data and those reported for indicates the focus of the group on chemotherapy of proto labdane dialdehyde ( Kimbu et al., 1979 and Hideji et al US 2003/O157197 A1 Aug. 21, 2003

1980) The presence of this compound is being reported for 0092) Molecular Model the first time in the genus Aframomum. However, Labda 0.093 Molecular representation of the Labda-8(17), 12 8(17),12-diene-15, 16-dial was first reported in Alpinia spe diene-15,16-dial showing ball and Stick model, Space-filling ciosa (Itokawa et al., 1980) and related diterpene (E)-8b,17 model, total electron density Surface including Lumo and epoxy-12-ene-15,16-dial from A. daniellii. The bioassay Homo Surfaces as well as isopotential Surfacaes at both -10 directed chromatographic separation of Labda-8(17), 12 kcal/mol and -5.0 kcal/mol are shown in FIG. 2F. The diene-15,16-dial from the petroleum ether extract monitored figure revealed Several interesting electronic Surface of the by gas chromatographic analysis are shown in FIGS. 2A-C. molecule Such as total electron density, location of the most 0.086 The antifungal activity of extracts of this plants nucleophilic and electrophilic Sites, Sites for nucleophilic was originally detected by direct Spraying of TLC plates and electrophilic attacks and with a Spore Suspension of the test fungus Cladosporium cucumerinum. A clearly visible inhibition Zone, even at the 0094) molecular recognition patterns for receptor bind lowest concentration of 0.5 lug was observed after using ing. labdane-dial, as shown in FIG. 1. The most striking result 0.095 Napoleonaea imperialis (Lecythidaceae) was obtained with Labda-8(17), 12-diene-15, 16-dial which inhibited the Leishmania promastigotes at a concentration of 0096. The ethylacetate and alcoholic extracts of Napo 50 ug or leSS. leOna imperialis Seeds were effective in vitro at concentra tion of 50 tug/ml or less using Visceral Leishmania isolates as 0087. The result of the RAM test showed that the lab shown in FIG. 3. Bioassay-directed chromatographic frac dane-dial completely inhibited the growth of the leishmania tionation of the active extracts led to the isolation of three Strain at a dose of 50 tug/ml. The test compounds also promising antileishmanial compounds tentatively identified inhibited the catabolism of various Substrates. The results of as imperialisides(A-C). The extracts and pure compounds the RAM test for leishmanial parasites are shown in FIGS. showed significant activity in Vivo (Suppression of lesion 2D-E. After a 96 hr incubation with labdane-dial, no live Size & L. donovani units) on hamster challenged with parasites were observed in culture and RAM respiratory cutaneous or Visceral Leishmania isolates as shown in Table rates for all "C-substrates reflect this lack of parasite 2. There was no apparent toxicities during the experiment. viability. The metabolic rate for every 'C-substrate by the The results of the activity of the Napoleonaea imperialis labdane-dial-treated parasites is near Zero (Solid pink bars). administered through the intramuscular route to hamsters The drug-treated results are in Sharp contrast to the vehicle infected with cutaneous L. panamensis represent an example control (0.6% DMSO) treated promastigote "C-substrate of dose-dependent in Vivo activity of the compound. At a catabolism, which show high respiratory rates during the 30 dose of 104 mg kg total dose (equivalent to 26 mg kg per min test period (Solid green bars). day) of the Napoleonaea imperialis, administered by intra 0088. Most of our preliminary work utilized radiorespiro muscular route twice a day for 4 days, the test Substance metric in vitro methodology (Jackson, et al., 1989; Jackson produced a 73% inhibition of lesion caused by L. panamen et al., 1990). Radiorespirometry is based on drug inhibition sis in hamsters. A dose of 52 mg kg (13 mg kg per day) of parasite catabolism of a variety of diverse, simple 'C- by the Same regimen gave a 51% reduction of the lesion Substrates (simple Sugars, amino acids, amines, fatty acid area, and at a dose of 13 mg kg (3.25 mg kg per day) 7% precursors, nucleic acid precursors, etc.) to 'CO2. We have reduction of the lesion area was observed. data on >55 different "C-substrates. Thus, radiorespirom etry offers the advantage of checking Specific drug inhibition 0097. The results of antileishmanial activity shown above of a variety of physiologic pathways Simultaneously, (RAMs and in vivo method) corroborated the results from depending on the type and number of "C-substrates utilized cytosensor. FIGS. 4A-4C illustrate Cytosensor Microphysi for each drug test. This compound being a lipophilic diter ometer of promatigote incubated in the presence of 50 mg of pene dialdehyde would be expected to associate Strongly N. imperialis per ml or medium alone. Confirmed antileish manial activity was observed using cytosensor, which is a with membrane lipids. new nonradioisotopic microchip-based method. 0089 Radiorespirometry is a very sensitive method, effective in detection of drug activities to <1 mg/ml using 0098. Furthermore the results using the Cytosensor as cultured promastigotes. Controls are two-fold, one non shown in FIG. 4 agree well with visual observation of the biologic control in which parasites are omitted from the test parasites by light microscopy and the growth inhibition to verify sterility of the "C-substrates; and parallel culti curve. The vehicle control parasites, manifest the typical Vated promastigotes treated only with the Solvent Solution Spindle-shaped monoflagellate form of leishmanial promas (containing no drug) as a drug "vehicle' control. However, tigotes. Cell density of the control parasites in culture was use of radioactive material has Some very pronounced 5x10 ml. At 50 ug ml drug, no intact parasites are disadvantages, biohazard and cost. Visible, only hollow parasite membranes, with no cytoplasm. 0090 Labda-8(17),12-diene-15, 16-dial also inhibited the Likewise, an ICso of approximately 10 ugml was observed in vitro growth of both a chloroquine-susceptible (D6) and for the growth inhibition data, Maximum achievable serum a chloroquine-resistant (D2) strain of Plasmodium falci level for SbV drugs, current “drugs-of-choice” for antile parum in a Hhyproxanthine uptake assay, as shown in ishmanial therapy, has been determined to be 20 ugml. 1-2 Table 2. h post-administration (references reviewed in Jackson, et al., 0091. In this assay, the Labda-8(17),12-diene-15, 16-dial 1989, 1990). treatment resulted in an ICso value of 280.18 ng/ml for the 0099 Comparative analyses of the polar extracts from W-2 clone, and 96.66 ng/ml for the D-6 Plasmodium falci Napoleonaea imperialis demonstrated that the imperaliside parum clone. This compound has been known to exhibit analogues are the major biologically active components with Strong antifungal activity. imperialiside A & B being the most active compounds. US 2003/O157197 A1 Aug. 21, 2003

These biological effects can perhaps explain the traditional resistant (W-2) strain of Plasmodium falciparum in a 3H use of the these plant species in treating different skin hypoxanthine uptake assay, as shown in Table 2, with ICso diseases. values of 169.95 ng ml for the W-2 clone, and 123.88 ng 0100 Sakuranetin from Eupatorium Odorantum ml for the D-6. These results demonstrate that Sakuranetin exhibits potent antimalarial activity and might be developed 0101 The methanol extracts of E. Odoratum exhibited very Strong antileishmanial activity. Bioassay guided frac into a new antimalarial drug. tionation of this extract yielded a mixture of yellowish 0107. In Africa, traditional medicine with herbal treat compounds. The activity was concentrated in the flavonoid ment has a long history and is used routinely in medical care fractions which yielded 4 flavanoids. The strongest antile (Harley, 1941; Feierman, 1981; Assi & Guinko, 1991), ishmanial activity were observed with Sakurenatin (II), as however only a few reports document activity of plant shown in FIG. 5. extracts against African trypanoSomes. One Study demon 0102 Available literature indicates that no previous anti strated activity of extracts of Khaya spp seeds (West African leishmanial Study has been carried out on E. Odoratum and hardwoods) in vitro vs. Tb. bruceii (Owolabi et al., 1990). there is Scanty reports on the chemical constituents of the The active agents were a group of furanodlimonoids related local variety of this plant which appears a new comer in the to quassinods, plant products found to block protein Syn West African vegetation. At a concentration of 50 lug/ml, thesis (Kirby et al., 1989). Another report indicated that total inhibition of promastigote growth occurred as shown in gossypol, a quassinoid, blocked respiration and destroyed T. FIGS. 5A and 5B. About 100% inhibition of promatigote b. bruceii blood forms in vitro at micromolar concentrations growth was observed with both petroleum ether and metha nol eXtracts. Partitioning the methanol eXtract into organic (Eid et al. 1988). Igweh and Onabanjo (1989) cured mice and aqueous fractions, the organic fraction showed greater infected with T. b. bruceii using aqueous root extracts of inhibitory activity than the aqueous fraction as shown as Annona Senegalensis. Recently, a Series of Studies by FIGS. 5C and D. The methanol extract displayed maximum Freiburghaus et al. (1996a, 1996b, 1997) evaluated extracts inhibitory activity in the Radiorespirometric microtest when of traditional medicinal plants from Uganda, Tanzania and compared with the petroleum ether and water eXtracts. At this the Ivory Coast, against T. b. rhodesiense blood forms in concentration, about 95% of the promastigotes exhibited vitro. Of those tested, 42 of 310 extracts (13.5%) were found abnormal round morphology. Further bioassay-guided chro to have significant growth inhibitory activity at 10 mg/ml or matographic fractionation of the organic fraction using leSS. The active agents generally had modest Selectivity column chromatography on silica gel eluted with CM(19:1; indicies, as compared to commercially available agents, but, 9:1), EtOAc, EtOAC:MeOH(17:1) yeilded five sub-fraction overall, these studies confirmed the potential for ethnobo with varying activity, as shown in FIGS. 5G and 5H. tanically Selected plants as potential Sources of agents Sub-fraction eluted with EtOAC, as shown in FIG. 5E, against Sleeping sickness (Freiburghaus et al., 1996a, 1996b, showed the greatest inhibitory activity. The most active 1997). antileishmanial constituents of E. Odoratum was isolated from these two fractions. The pure compound was identified 0108. The in vitro activity of 39 plant extracts was tested as Sakurentin by Spectra analysis as well as Co-TLC with against four Strains of animal or human-pathogenic African authentic Samples. trypanoSomes, and three Strains of mammalian-pathogenic 0103) In recent years many effort have rationalize the Trichomonas spp. The trypanoSomes Studied were Trypano bioactivity and the importance of flavonoids (Middleton and Soma bruceii brucei Lab 110 EATRO, which is pathogenic to Kandaswami 1994). One of such activity is the antileish cattle and other livestock, and Several Strains of Trypano manial activity exhibited by Sakurenatin isolated from E. SOma bruceii rhodesiense, a parasite of humans, domestic Odoratm. Very few flavonoids have been reported to process and wild animals. Strains of T. b. rhodesiense included drug antileishmanial activity. resistant clinical isolates KETRI 243 and 269 and KETRI 0104 Sakuranetin was isolated from the most active 243 AS-10-3, a highly melarsen- and diamidine-resistant fraction while Lupeol, a-amyrin, betuletol, 3,5,7,3'-tetra-O- clone of KETRI 243. The 39 extracts were tested in an in methyl quercetagetin, quercetin and two flavonoid glyco Vitro Screen using a Semi-defined medium for growth of Sides based on Sakuranetin and isoSakuranetin moieties were bloodstream trypomastigotes at 37° C. (Hirumi & Hirumi, isolated from inactive extract/fractions. Sakuranetin being a 1989) to determine ICso values (Bacchi et al., 1996). Using flavonoid belongs to a class of natural products generally a cutoff of 100 tug/ml, 28 of the 39 extracts consistently gave known to be non-toxic. These findings Suggest that ICs values in the active range, as shown in Table 3 of FIG. Sakuranetin in concentration that are nontoxic to the host cell 7. Of these, 10 had IC.svalues at or below 10 ug/ml and were may exhibit a Strong antileishmanial activity in Vivo and that considered Sufficiently active to warrant testing of more appropriate Substituted flavonone having basic Sakuranetin purified extracts. Of the four Secondary extracts Supplied, skeleton might provide new class of antileishmanial drugs. one, aulacocarpin (III) (SU1460), derived from the primary extract SU787 of A. aulocacarpus featured a 10-15 fold 0105 Because antiprotozoal and antifungal activities are increase in activity. SU787 had ICso values of 8.5-14.9 frequently associated with the same or chemically similar mg/ml, while the value for SU1460 was 0.86 mg/ml. Eight compounds, we considered it probable that natural products additional primary extracts were also tested in the trypano that show remarkable antifungal activity Such as, imperiali some screen (Table 3). Of these, SU1462from Napoleonaea Side, labdane-dial and Sakuranetin would have antiprotozoal imperialis and SU1464 from GloSSOcalyx brevipes were activity. highly active (ICso-1 mg/ml) and warrant further study. 0106 Sakuranetin also inhibited the in vitro growth of Aulocacarpin (III) is an extract from Aframonum au locac both a chloroquine-Susceptible (D-6) and a chloroquine arpus and has the following formula: US 2003/O157197 A1 Aug. 21, 2003 10

Species, Aframomum melegulata, Showed moderate activity against TrypanoSoma brucei in Vitro ICso 9.0 mg/ml. How

(III) ever, a third plant Species, Aframomum aulocacarpus, con tains aframodial which showed activity within the highly active drug range, ICso 0.86 mg/ml, a 10-11-fold increase in activity Aframodial has since been shown to be a nontoxic broad spectrum antifungal agent (2)Morita, H and Itokawa H., Planta medica, 54, 117 (1988)The structural modifica tions in active antiparasitic with these botanical Species changes are in progreSS. Numerous similarities in leishma nial and trypanoSomal lipid uptake and metabolism may explain common natural product drug Susceptibility. Proto Zoan diseases, including leishmaniases, offer little commer cial drug development incentive, thus, the readily available, 0109 The trichomonad screen consisted of two human cheaper, oral and leSS toxic traditional medicined frequently pathogenic Trichomonas vaginalis Strains and a livestock prevail in developing countries. parasite Trittichomonas foetus. The T vaginalis isolates 0113 AS can be seen from the test results, compounds include a metronidazole sensitive isolate (C1-NIH: ATCC I-III Surprisingly exhibit potent activity against Leishmania 30001) and a strain highly resistant to metronidazole (CDC (Leishmania) chagasi the cause of cutaneous leishmaniasis 085: ATCC 50143). The screening procedure used is that of as well as exhibiting antimalarial and . This finding Strongly Meingassner et al. (1978) and determines the minimal Supports the tremendous potential that exists in the explo inhibitory concentration (MIC) in mg/ml needed to com ration traditional remedies for lead compounds in the devel pletely inhibit growth. Table 4 of FIG. 9 details data from opment of new anti-protozoal drugs. the initial group of 19 primary extracts. Of these, Seven had MIC values of 1 mg/ml for all three isolates and were 0114 While the claimed invention has been described in considered of interest for further study. Some fractions from detail and with reference to specific embodiments thereof, it the above active extracts were tested along with new will be apparent to one of ordinary skill in the art that various extracts. The results show that the most active extract in this changes and modifications can be made to the claimed group was GloSSOcalyx brevipes which had an MIC value of invention without departing from the Spirit and Scope 0.0125 mg/ml for each isolate and was the most potent of the thereof. primary extracts tested thus far. 0110. The active plant extracts in each screen are listed in Tables 3 and 4. The results are reported on the basis of MIC 1. A biologically active extract from at least one plant levels (<1 mg/ml) for trichomonad Screens and ICso values Selected from the group consisting of Aframomum aulocac (

13. A method of preparing a biologically active extract 26. Method according to claim 23, comprising injecting from at least one plant Selected from the group consisting of an intravenous composition containing Said biologically Aframomum aulocacarpus, Aframomum danelli, Dracaena active extract. arborea, Eupatorium Odoratum, GloSSOcalyx brevipes and Napoleonaea imperialis, the method comprising: 27. A compound comprising: pulverizing at least a part of Said plant; (I) Selecting a Solvent which dissolves or Solubilizes a CHO desired biologically active compound from Said plant, combining Said Solvent and Said pulverized plant to CHO extract Said desired biologically active compound; and removing Said Solvent from Said extract of Said desired biologically active compound. 14. A method according to claim 13, wherein Said plant 28. A compound comprising: comprises Aframomum au locacarpus. 15. A method according to claim 13, wherein Said plant comprises Aframomum danellii. (II) 16. A method according to claim 13, wherein Said plant OH comprises Dracaena arborea. 17. A method according to claim 13, wherein Said plant comprises Eupatorium Odoratum. OHC O 18. A method according to claim 13, wherein Said plant comprises GloSSOcalyx brevipes. 19. A method according to claim 13, wherein said plant comprises Napoleonaea imperialis. OH O 20. A topical composition composition comprising a biologically active extract from at least one plant Selected from the group consisting of Aframomum au locacarpus, 286.120509 Aframomum danellii, Dracaena arborea, Eupatorium Odo C 71.3% H 6.3% O 22.4% ratum, GloSSOcalyx brevipes and Napoleonaea imperialis in a topical carrier. 21. An oral composition comprising a biologically active C17H18O extract from at least one plant Selected from the group consisting of Aframomum au locacarpus, Aframomum 286.32 danellii, Dracaena arborea, Eupatorium Odoratum, GloSSO 286.120509 calyx brevipes and Napoleonaea imperialis in an oral carrier. 22. An intravenous composition comprising a biologically C 71.3% H 6.3% O 22.4% active extract from at least one plant Selected from the group 29. A compound comprising: consisting of Aframomum au locacarpus, Aframomum danellii, Dracaena arborea, Eupatorium Odoratum, GloSSO calyx brevipes and Napoleonaea imperialis in an intrave (III) nous carrier. 23. A method of treating a fungal or protozoal disease in a mammal comprising applying a biologically active extract from at least one plant Selected from the group consisting of Aframomum aulocacarpus, Aframomum danelli, Dracaena arborea, Eupatorium Odoratum, GloSSOcalyx brevipes and Napoleonaea imperialis. 24. Method according to claim 23, comprising applying a topical composition containing Said biologically active eXtract. 25. Method according to claim 23, comprising ingesting an oral composition containing Said biologically active eXtract.