Nick Oberlies, one of my graduate students, observed that at the Purdue Cell Culture Laboratory the parental nonresistant wild-type (MCF-7/wt) human mammary adenocarcinoma cells and multidrug-resistant (MDR) MCF-7/Adr cells exposed to bullatacin (6) yielded the same sort of result, wherein 6 inhibited the MDR cells at lower doses than were required to inhibit the wild-type cells. After completing cell refeeding experiments, it was concluded that 6 is cytotoxic to MCF-7/Adr cells but is cytotoxic to the MCF-7/wt (wild-type) cells.98 Thus, such MDR cells are more susceptible to ATP depletion than their parental cells; this is another important biochemical difference between parenteral cancer cells and their MDR descendants and should be exploitable by the chemotherapeutic use of the acetogenins to prevent, and even combat, multiple drug resistance.

With most other anticancer agents, a higher dose is required to inhibit resistant cells than normal (wild-type) cells. It is known that MDR cells have a P-170-kDa glycoprotein (P-gp),⁹⁹ which forms a channel or pore in the plasma membrane and pumps out the intracellular xenobiotics. This mechanism is very efficient at keeping the resistant cells functioning and explains why chemotherapy fails as the tumor becomes populated with MDR cells. The P-gp has two ATP-binding sites, and, to provide energy to drive the pump, ATP is cleaved through its ATPase action. Being ATP-dependent, the P-gp causes the MDR cells to be more susceptible to compounds that inhibit ATP production. Hence, when the acetogenins, as potent inhibitors of complex I and NADH oxidase, decrease intracellular levels of ATP, they, therefore, decrease the effectiveness of the P-gp efflux pump, and they should even synergize with other chemotherapeutic agents. Oberlies et al.¹⁰⁰ evaluated 14 acetogenins (seven adjacent bis-THF, two nonadjacent bis-THF, and five mono-THF ring compounds) against the MCF-7/Adr cell line to establish their SARs. All compounds were tested with adriamycin, vincristine, and vinblastine as standard chemotherapeutic agents. Of the 14 acetogenins, 13 were generally more potent than all three of the standard drugs. Bullatacin (6) was 258 times more cytotoxic against the MCF-7/Adr cell line than adriamycin. Acetogenins with the stereochemistry threo-trans-threo-trans-erythro from C-15 to C-24 were the most potent among those having adjacent bis-THF rings. A bullatacin index permitted comparisons of activities, and gigant-tetrocin A (11), a mono-THF compound, was the most potent, being about twice as potent as bullatacin (6).

A part of our research effort has been directed toward the development of the annonaceous acetogenins as new, environmentally friendly, organic, pesticides.17,71 Reasoning that pesticide resistance in insects may, as with drug resistance in cancer, also involve ATP-driven efflux mechanisms, we demonstrated that the acetogenins are equipotent or even more potent against insecticide-resistant versus insecticide-susceptible German cockroaches.101 The speed of kill values (LT50) for six acetogenins and five standard synthetic pesticides were determined against second and fifth instar stages of the insecticide-resistant and insecticide-susceptible roaches. The bis-THF acetogenins showed the highest potency among the three structural classes of acetogenins. Parviflorin (12), a C35 acetogenin of the asimicin subtype and originally found in Asimina parviflora (Michx.) Dunal,102 was the most potent, followed closely by asimicin (1). Both compounds showed more potent activities than the five synthetic pesticides, with the exception of cypermethrin, against both stages of both strains. Chlorpyrifos showed the highest resistance ratio at 8.0. Annomontacin (13), a mono-THF acetogenin, showed particular effectiveness against the resistant strain, acting 5 times faster than against the nonresistant strain. Low resistance ratios for the acetogenins and for hydramethylnon (an inhibitor of ETS complex III rather than complex I) strongly suggest that the inhibition of ATP production, indeed, thwarts insect, as well as tumor cell, resistance.

Two review papers summarize our work with the acetogenins in attempting to thwart ATP-dependent resistance with anticancer

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agents as well as with pesticides.103,104 Since these reviews were written, Fu et al.105 confirmed our observations using bullatacin (6) and resistant KB cells, and Raynaud et al.106 confirmed our observations using squamocin and resistant MCF-7 cells. Fu et al.105 also found that 6 induced an increase of intracellular adriamycin in the treated MCF-7/Adr cells. This group worked also with an acetogenin named 89-2 (which seems to be a 4-deoxy-28,29-erythro-dihydroxyasimicin); compound 89-2 increased, by 4.3-fold, the concentration of a fluorescent xenobiotic (Fura-2) in KBv200 (MDR) cells but not in parental (wild-type) KB cells. Such results again suggest that the acetogenins block the P-gp efflux pump and imply that coadministration of acetogenins, with other anticancer agents, should help to avoid and even circumvent MDR. Thus, the acetogenins are promising in the treatment of both nonresistant and MDR types of tumors.

In further experiments, Fu et al.107 demonstrated that compound 89-2 exhibited similar but potent cytotoxicities against the KBv200 (MDR) and parental (drug sensitive) KB cell lines. They next studied xenografts of the resistant and parental KB cell lines in nude mice. As predicted, the KBv200 (MDR) xenografts were refractory to vincristine, while vincristine successfully inhibited the parental KB xenografts. Compound 89-2, when administered as a treatment of 900 μg/kg every two days for six days, caused significant inhibition, 52.3% and 56.5%, respectively, of both types of xenografts with no significant weight losses or deaths in the treated mice. Thus, the acetogenins have been demonstrated to be effective in vivo against MDR tumors.

Using a modified guinea pig maximization test, a paw paw extract was found to be only a weak skin sensitizer and asimicin (1) was found to be only a weak skin irritant; neither produced the vesication or ulceration typical of urushiol (poison ivy) components.[108] In our extensive work with the Annonaceae over the past 30 years, our researchers have never experienced any form of dermatitis during plant collection, drying, milling, extraction, or isolation of the acetogenins. One researcher rubbed one eye after his finger came in contact with a concentrated solution of mixed acetogenins, and he experienced severe eye irritation and the loss of the outer layer of cells from the cornea but with complete recovery. The Paw Paw Lice Remover Shampoo (described below), which contains 0.5% of standardized paw paw extract, passed the Draize test for eye irritation.[109]

Ames test results for mutagenicity were obtained at Sitek Research Laboratories (Rockville, MD) using a paw paw extract. The tests were negative in nine out of 10 determinations and only slightly positive (2.5% above background reversions) on one histidine mutant of Salmonella typhimurium after enzyme activation of the extract.[110] These results have been confirmed using the purified acetogenins squamocin and annonacin (3); neither acetogenin was mutagenic in three different strains of S. typhimurium, although both were toxic to the bacteria in the absence of a metabolic activation system.[70]

In feeding experiments,71 mice tolerated paw paw extract mixed in their diet at 1% (a no choice diet); the mice ate this for 4 days without lethal effects. However, at 5% and above in their diets, they succumbed after 3 days, showing lethargy but with their internal organs appearing normal. At Asta Laboratories (Germany), bullatacin (6) was emetic, after injection at 185 μg/kg, to pigs; this was our first proof that the acetogenins are the