H. L. PRINGLE, S. G. BRADLEY,* AND L. S. HARRIS
Departments of Microbiology and Pharmacology, Virginia Commonwealth University,
Richmond, Virginia 23298
Received for publication 6 June 1979
Growth of the pathogenic amoeboflagellate Naegleria fowleri is inhibited by A9-tetrahydrocannabinol (A9-THC). A9-THC is amoebostatic at 5 to 50 µg/ml. e-THC prevents enflagellation and encystment, but does not impair amoeboid movement. Calf serum at 10 and 20% (vol/vol) reduces the antiamoeba activity of A9-THC. Only 1-methoxy A8-tetrahydrocannabinol, of 17 cannabinoids tested, failed to inhibit growth of N. fowleri. Antinaeglerial activity was not markedly altered by opening the pyran ring, by converting the cyclohexyl ring to an aromatic ring, or by reversing the hydroxyl and pentyl groups on the benzene ring. A9-THC prevented the cytopathic effect of N. fowleri on African green monkey (Vero) cells and human epithelioma (HEp-2) cells in culture. A9-THC afforded modest protection to mice infected with N. fowleri.
Naegleria fowleri is the etiological agent of primary amoebic meningoencephalitis in hu¬mans (6, 10). To date, amphotericin B is the most effective antinaeglerial drug in vitro, is able to protect mice infected with N. fowleri (9), and appears to alter favorably the outcome of pri¬mary amoebic meningoencephalitis in humans (3). Because of the serious nature of naeglerial infection in humans (7) and the toxicity of am¬photericin B (2, 17), a satisfactory chemothera¬peutic agent for the treatment of primary amoe¬bic meningoencephalitis in humans is yet to be developed.
A9-Tetrahydrocannabinol (A9-THC) has been reported to inhibit the growth of several proto¬zoa, including Dictyostelium discoideum (5) and Tetrahymena pyriformis (14). In addition, A9-THC has been reported to retard the prolifera¬tion of tumor cells in vivo (16) and in vitro (18). Because A9-THC accumulates in the central nervous system (13) and N. fowleri invades the brain (8), the cannabinoids merit examination as potential drugs for the treatment of primary amoebic meningoencephalitis.
MATERIALS AND METHODS
N. fowleri LEE was grown in Nelson medium, con¬sisting of Page saline (0.12 g of NaCl, 0.142 g of Na2HPO4, 0.136 g of KH2PO4, 0.004 g of MgSO4 7H20, and 0.004 g of CaC12 per liter of distilled water) supplemented with 0.1% (wt/vol) Pan Meade liver digest (Harrison and Grosfield, Bronxville, N.Y.), 0.1% (wt/vol) glucose, and 2 or 5% (vol/vol) calf serum (GIBCO, Grand Island, N.Y.). Tissue culture flasks (25-cm2 size; Falcon Plastics, Oxnard, Calif.) contain¬ing 10 ml of medium were inoculated to give 1 x 10' to
5 x 10' amoebae per ml and were incubated for 96 h at 37°C (1).
A9-THC and selected cannabinoids were obtained from the National Institute on Drug Abuse (Rockville, Md.) and dissolved in Emulphor-ethanol (EL-620; GAF Corp., New York, N.Y., and ethanol, 1:1) at a concentration of 20 mg/ml. This preparation was di¬luted in Page saline as appropriate.
To evaluate the effect of A9-THC and related can¬nabinoids on growth, amoebae from rapidly growing cultures were inoculated into growth medium (supple¬mented with 2% calf serum) to give 1 x 10' to 5 x 10' trophozoites per ml. The cannabinoids were added, and the cultures were incubated at 37°C. When indi¬cated, cells were suspended by vigorous agitation or by chilling the culture to 5°C for 10 min; 1-ml samples were removed for counting, and cultures were returned to 37°C. For cell counts, 0.2 ml of the culture was added to 9.8 ml of an electrolyte solution (consisting of NaCl, 0.4% [wt/vol] and Formalin, 0.5% [vol/vol] in distilled water). Cell counts were made using an elec¬tronic cell counter (Coulter Counter model Zs; Coulter Electronics, Inc., Hialeah, Fla.).
The capability of Q9-THC to prevent the cytopathic effects of N. fowleri on African green monkey (Vero) cells and human epithelioma (HEp-2) cells in culture was assessed. The two established cell cultures were grown in Eagle basal medium with Earle balanced salt solution, supplemented with 10% fetal calf serum (Flow Laboratories, Rockville, Md.) as described pre-viously (4, 15). Antibiotics were not added. N. fowleri at a final population of 10' to 105 amoebae per ml was added to Vero or HEp-2 cultures with confluent cell monolayers. Diluent of A9-THC was added simulta¬neously when indicated, and the cell cultures were incubated at 37°C for 48 h. Cytopathogenicity was scored visually with the aid of a compound light mi¬croscope.
To evaluate the effect of Q9-THC on the suscepti-bility of mice to naeglerial infection, BALB/c male mice (50) were inoculated intranasally with 1 50% lethal dose of rapidly growing Naegleria. One group served as the infected but untreated control; the other group was given 50 mg of Q9-THC per kg intraperito¬neally on 4 consecutive days. The mice were observed daily for 3 weeks.
The growth of Naegleria fowleri was re¬tarded by 5 and 10 µg of t 9-THC per ml and was markedly inhibited by 20 µg of Q9-THC per ml (Fig. 1). Emulphor-ethanol at the concentra¬tion needed to deliver 20 µg of A9-THC per ml had little effect on the growth of N. fowleri. In contrast, 2 mg of A9-THC per ml failed to inhibit the growth of Mycobacterium marinum 437 or Saccharomyces cerevisiae ATCC 9763, nor did 400 µg/ml (maximum concentration tested) in¬hibit Cryptococcus laurentii 9-389. Q9-THC was amoebostatic at 20 ftg/ml, but amoebicidal at greater concentrations (50 to 100 µg/ml). After prolonged incubation in medium containing 20 µg of Q9-THC per ml the amoebae began to proliferate, but with a significantly extended generation time. However, this population of amoebae possessed the same susceptibility to the drug as those that had not been exposed to the drug. Attempts to select for variants of N. fowleri resistant to Q9-THC have not been suc¬cessful.
Most N. fowleri amoebae exposed to 20 µg of Q9-THC per ml became rounded and detached from the surface. The viable spherical cells, whether attached or free, did not differentiate into flagellates or form cysts. Those cells that remained attached to the surface displayed es¬sentially normal amoeboid mobility.
Calf serum added to give 1% final concentra¬tion failed to support growth of N. fowleri (data not presented). Serum concentrations greater than 10% inhibited growth. The inhibitory effect of elevated concentrations of calf serum could be reduced by dialyzing the serum. Q9-THC at concentrations of 10 and 20 µg/ml inhibited growth of N. fowleri in medium containing 5% serum but not in medium containing 10% serum or 20% dialyzed serum (Table 1). Cat+ (3.6 x 10-5 to 3.6 x 10-3 M) and Fe" (10-6 to 10-' M) had little effect on the growth of N. fowleri and did not alter the susceptibility of N. fowleri to A9-THC.
A number of cannabinoids inhibited the growth of N. fowleri. Of 17 cannabinoids tested, only 1-methoxy A8-tetrahydrocannabinol failed to inhibit the proliferation of N. fowleri at 10 to 20 µg/ml (Table 2). The remainder of the cannabinoids may be grouped as those more active than Q9-THC and those with the same activity as Q9-THC. Two cannabinoids some-what more active than Q9-THC were 11-hydroxy A8-tetrahydrocannabinol and 9-nor-9-hydroxy-cannabinol. Both of these molecules differ from the parent Q9-THC at two sites in ring III (Fig. 2). The inactive 1-methoxy Q8-THC differs from the active Q8-THC at only one site in ring I. Cannabinoids with an aromatic ring III retained antinaeglerial activity. Either a methyl group or a hydroxyl group could occupy position 11 of ring III. The antinaeglerial activity of the can¬nabinoids was not markedly altered by opening of ring II or by the removal of the methyl groups from ring II. The antinaeglerial activity was not markedly altered when the hydroxyl group and pentyl chain of ring I were reversed. However, if the hydroxyl group was substituted with a meth¬oxy group, the antinaeglerial activity was lost. N. fowleri was cytopathic for Vero cells and HEp-2 cells in culture. At 105 amoebae per ml, the cultured mammalian cells were destroyed within 24 h; with 10' amoebae per ml, 48 h was required. Growth of N. fowleri was very limited in the mammalian cell cultures and in the cell culture medium. The amoebae displayed normal morphology and were actively mobile in mixed culture with the mammalian cells. A9-THC at 20 to 50 sg/ml protected Vero and HEp-2 cells from the cytopathic action of N. fowleri (Fig. 3). Above 50 pg/ml, A9-THC was cytotoxic for the mammalian cell monolayer.
A9-THC afforded some protection in mice to naeglerial infection (Fig. 4). The protection af¬forded by A9-THC involved both extension of life and 25% fewer deaths. Similar results were obtained with mice infected with N. fowleri in¬travenously.
A9-THC inhibits growth of several protozoa (5, 14) and mammalian cells (12, 18), but does not inhibit growth of selected yeast or bacteria. A9-THC prevents enflagellation and encystment by N. fowleri, but not amoeboid mobility, indi¬cating that some energy generating and transfer systems are functional, but that macromolecular synthesis is inhibited directly or indirectly. A9-THC binds to proteins, and the antinaeglerial activity of the O9-THC-protein complex is re-duced. Both serum concentration and inoculum size markedly influenced the degree of inhibition observed with lower (5 to 20 µg/ml) concentra¬tions of A9-THC. N. fowleri will not grow in standard mammalian cell culture media. Several mammalian cell culture media can support nae¬gleria) growth when diluted fivefold or more. The inhibition of growth with higher concentra¬tions (20%, vol/vol) of undialyzed serum appears to be due to the toxicity of inorganic salts; how-ever, calcium salts did not markedly affect growth of naegleria or the action of A9-THC on naegleria. The reported capacity of calcium to alter the effects of A9-THC on continuous cell lines (12) does not seem to be a general effect.
The cannabinoid structure can be substan-tially altered without loss of antinaeglerial activ-ity. Methoxylation of the hydroxyl group of the phenolic ring drastically reduced its activity. The essential role of the phenolic group might indicate that an interaction between cannabi-noids and iron-containing molecules is involved in its mechanism of action, but added inorganic iron salts had no effect on the inhibitory activity of A9-THC. Cannabinoids have a wide variety of biological effects, including psychotomimetic, immunosuppressant, antitumor, and antimicro-bial activities (14, 18). These diverse activities are not absolutely linked, because 1-methoxy A8-THC is an effective immunosuppressant agent, but has little antinaeglerial and little cen-tral nervous system activities. Abnormal can-nabinol has little central nervous system and immunosuppressant activity, but has antinae-glerial activity. These varied activities, however, do not necessarily mean that the molecular mechanisms of action of various cannabinoids are different, but that they may differ in solu-bility, distribution, permeation, availability, and metabolism. Clearly the action of cannabinoids is not limited to highly specialized nerve cells; thus the mechanism of action of cannabinoids may be best pursued in a microbial system.
A9-THC prevented the cytopathic effect of N. fowleri on Vero or HEp-2 cells in culture. The cytopathic effect on the monolayer is presum-ably the result of a cytotoxic phospholipolytic factor and actual phagocytosis (11). i 9-THC was not amoebicidal under our experimental condi¬tions, so the basis of the protection afforded by A9-THC is not known. O9-THC may impair the synthesis or release of a cytopathic phospholi¬pase.
A9-THC provided only modest protection for mice infected intranasally or intravenously with N. fowleri. A9-THC is not the most active can-nabinoid in vitro, so a more effective response in vivo may be achieved with other cannabinoids.
This investigation was supported, in part, by Public Health Service grants AI-00382 from the National Institute of Allergy and Infectious Diseases and DA-00490 from the National Institute on Drug Abuse.
1. Adams, A., D. T. John, and S. G. Bradley. 1976. Modification of resistance of mice to Naegleria fowleri infections. Infect. Immun. 13:1387-1391.
2. Andriole, V. T., and H. M. Kravetz. 1962. The use of amphotericin B in man. J. Am. Med. Assoc. 180:269-272.
3. Apley, J., S. K. R. Clarke, A. P. C. H. Roome, S. A. Sandry, G. Sygi, B. Silk, and D. C. Warhurst. 1970. Primary amoebic meningoencephalitis in Britian. Br. M. J. 1:596-599.
4. Bradley, S. G., and D. B. Howe. 1976. Perturbation by bacterial lipopolysaccharide of the metabolic processes of human cells in continuous culture. J. Reticuloendo¬thel. Soc. 20:135-145.
5. BraS., and P. Brachet. 1976. Inhibition of prolifera¬tion and differentiation of Dictyostelium discoideum by tetrahydrocannabinol and cannabinol, p. 207-211. In G. G. Nahas (ed.), Marihuana: chemistry, biochemistry and cellular effects. Springer-Verlag, New York.
6. Butt, C. G., C. Baro, and R. W. Knorr. 1968. Naegleria (sp.) identified in amebic encephalitis. Am. J. Clin. Pathol. 50:574.
7. Callicott, J. H., Jr. 1968. Amebic meningoencephalitis due to free-living amebae of the Hartmannella (Acan¬thamoeba)-Naegleria group. Am. J. Clin. Pathol. 49: 84-91.
8. Carter, R. F. 1968. Primary amoebic meningoencephali¬tis: clinical, pathological and epidemiological features of six fatal cases. J. Pathol. Bacteriol. 96:1-25.
9. Carter, R. F. 1969. Sensitivity to amphotericin B of a Naegleria (sp.) isolated from a case of primary amoebic meningoencephalitis. J. Clin. Pathol. 22:470-474.
10. Carter, R. F. 1970. Description of a Naegleria (sp.) isolated from two cases of primary amoebic meMngoen¬cephalitis and of the experimental pathological changes induced by it. J. Pathol. 100:217-244.
11. Cursons, R. T. M., and T. J. Brown. 1978. Use of cell cultures as an indicator of pathogenicity of free-living amoebae. J. Clin. Pathol. 31:1-11.
12. Huot, J. 1976. Cellular and biochemical alterations in-duced in vitro by A9-tetrahydrocannabinol: effects on cell proliferation, nucleic acid, plasma cell membrane, ATPase and adenylate cyclase, p. 313-327. In G. G. Nahas (ed.), Marihuana: chemistry, biochemistry and cellular effects. Springer-Verlag, New York.
13. Just, W. W., G. Erdman, G. Werner, M. Wiechmann, and S. Mel. 1976. Forensic metabolic and radiographic studies of A" and A' tetrahydrocannabinol, p. 123-138. In G. G. Nahas (ed.), Marihuana: chemistry, biochem¬istry and cellular effects. Springer-Verlag, New York.
14. McClean, D. K., and A. M. Zimmerman. 1976. Action of A9-tetrahydrocannabinol on cell division and macro-molecular synthesis in division-synchronized protozoa. Pharmacology 14:307-321.
McGivney, A., and S. G. Bradley. 1979. Effects of bacterial endotoxin on lysosomal and mitochondria) enzyme activities of established cell cultures. J. Retic-uloendothel. Soc. 26:307-316.
Munson, A. E., L S. Harris, M. A. Friedman, W. L Dewey, and R. A. Carchman. 1975. Antineoplastic activity of cannahinoids. J. Natl. Cancer Inst. 55:597-602.
Sternberg, T. H., E. T. Wright, and M. Oura. 1956. A new antifungal antibiotic, amphotericin B. Antibiot. Annu. 1955-56:566-573.
White, A. C., J. A. Munson, A. E. Munson, and R. A. Carchman. 1976. Effects of A9-tetrahydrocannabinol in Lewis lung adenocarcinoma cells in tissue culture. J. Natl. Cancer Inst. 56:655-668.
Source: Susceptibility of Naegleria fowleri to delta 9-tetrahydrocannabinol