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Trioaolanes: A new generation of compounds with wide ranging activities

Davy K. Koech, James A Haman, Mawuli W. Kofi Tsekpo
and Stephen D. Harman

Kenya Medical Research Institute, Mbagathi Road P.0. Box 51840,
Nairobi, Kenya, 517 S Beach Road, Hobe Sound , Florida 33455, USA.

To whom correspondence should be addressed

SUMMARY

Direct biological activity of a 1,2,4-trioxolane derivative was assessed in vitro using bacteria and fungi causing common infections. The product was found to be uniformly active against all organisms tested. In addition, it is active against certain tumor cells and protozoa and is also an immunomodulator. These observations are discussed in the light of the product's potential use in the clinical management of conditions in which its use is indicated.

TRIOXOLANE

A 1,2,4-trioxolane (KE-091/ATX), an oxygen heterocycle was produced through a patented procedure. The product has a molecular weight of 148. It is soluble in propylene glycol and in polyethylene glycol (PEG 600) up to 40 percent. It is also soluble in other oils. Although we have synthesized several trioxolane derivatives including diperoxide compounds, we report hem the wide ranging activities of KE- 091/ATX whose properties we have studied in greater detail.

KE-091/ATX was synthesized at the facilities of the Kenya Medical Research Institute (KEMRI) and its purity and stability were assessed using chromatographic techniques. It is a colorless liquid and the neat product is viscous with a specific gravity of 1.51. It is tolerated in mice and intraperitoneal administration gave a maximum tolerable dose at 600 mg/kg.

To our knowledge, there is no published report on the experimental or clinical use of this class of compounds, except those disclosed in patent claims, especially these of our own. Accordingly, so far as can be determined, none of the medical uses of such compounds described in prior art have ever made use of substantial quantities of trioxolane or diperoxide compounds. Moreover, none of the prior art compounds
appears to have ever been commercialized for medical applications. Presumably, this lack of commercialization is due to poor yield, impurities, unacceptable side-effects,toxicity, difficulties in storage, or minimal effectiveness. Many of these compounds normally decompose in storage. All the trioxolanes we have synthesized (including KE-091/ATX) have not been previously described. The method we have used leads to substantial amounts of KE09 1/ATX of assured purity, quality and stability.

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IN VITRO ACTIVITY

Direct activity of KE-091/ATX was assessed in vitro using cells, protozoa, bacteria and fungi. The product was dissolved in propylene glycol to give 250 mg/ml as a working a concentration and further diluted in the culture in which the particular organism was grown. Solvent alone and where possible, standard drugs and reference stains were used as controls.

At dilutions of less than 1:2x10 , a myeloma cell line (x63 balb/c line) was killed within 48 hours and human spermatozoa were killed within 1 minute.

Direct biological activity of KE-091/ATX was tested on microorganisms which cause common infections in the community; and the activity was assessed on the basis of minimum inhibition concentration (MIC) as well as minimum bacterial concentration (MBC) after the standard overnight incubation period in culture. The results are summarized in Table 1. From the Table, it is evident that, standard microorganisms that have been identified as resistant to conventional antimicrobials have uniform sensitivity to KE-091/ATX in the same manner as are sensitive organisms. Those isolates known to be resistant to antibiotics are Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coil (ATCC 25922). The antibiotics used in the screening were penicillin, ampicillin, tetracycline, erythromycin, streptomycin, kanamycin, gentamycin, chloramphenicol and sulpharmetrol.

The solvent or any stabilate, did not inhibit the in vitro growth of bacteria, fungi and protozoa used in the studies.

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These observations indicate that KE-091/ATX is more active than conventional drugs against commonly encountered microorganisms (in vitro) such as those causing common infections as diarrhea (Salmonella spp, Shigella spp, enteropathogenic/enterotoxigenic Escherichia coli), urinary tract infections (Neisseria gonorrhoea, Candida spp), respiratory tract infections (Klebsiella spp, Staphylococcus spp, Phialophora spp, Penicillium spp) and protozoal infections (Leishmania spp). These microorganisms were uniformly sensitive to KE-091/ATX and the activity of the product on prior administration of the KE-091/ATX.

After the pentobarbitone sleeping time experiment, the mice were kept under observation for 12 weeks to see if any toxic effects could be detected. Pentobarbitone was used at 40mg/kg of KE-091/ATX at 500mg/kg in a set of 38 mice as the test group and another set of 32 mice as the control group. The test group slept more than the controls by 9.05 minutes (with a range of 0.06 to 18.16 minutes) with a p value of <0.052 which is barely significant. On the other hand, using pentobarbitone at 80 mg/kg in another set of 24 test mice and maintaining the concentration of KE-091/ATX at 500 mg/kg, the controls slept more than the test group by an average of 33.33 minutes (33.33 ±6.89 SE, with a range of 18.79 to 47.87 minutes) with a p value of <0.001 which is highly significant.

The results revealed that the effect of KE-091/ATX on pentobarbitone sleeping time in mice appeared to be biphasic: at lower doses of pentobarbitone (40 mg/kg), KE- 091/ATX appears to potentiate the depressant effect of the barbiturate. The test group slept more than the controls. However, at higher doses of the hypnotic (80 mg/kg), the same dose of KE-091/ ATX appears to act in the reverse. The controls slept more than the test group. These results therefore show that there is a definite biphasic effect, and further experimental work is being done to prove this conclusively.

The central nervous system effects of KE-09l/ATX, as shown in the present study, support the neuroimmunomodulatory properties of this compound which as been demonstrated. Further experimental work, including in vitro animal tissue studies, are planned in order to throw more light on the mechanisms of action of this interesting compound.

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CLINICAL SIGNIFICANCE

On the basis of its tolerability in mice, the amount required to clear common pathogens in vitro and on the basis of observations made, KE-091/ATX is considered of high clinical potential and significance. Using appropriately formulated forms, KE- 091/ATX has great potential in the clinical treatment and management of infections caused by the microorganisms tested in vitro. In addition, its high in vitro activity on
spermatozoa makes it a good candidate as a contraceptive as well as for preventing

TREATMENT OF MICE INJECTED WITH LEISHMANIA MAJOR

Experimental cutaneous leishmaniasis was achieved when 1x10 promastigotes of Leishmania major strain NLB 144 were inoculated sub-cutaneously in the nasal dorsum of inbred balb/c mice weighing approximately 20g each. The mice were fed with water and food ad libitum. The infection was allowed to develop until visible lesions of a least 2mm diameter are noticeable. Some mice developed large lesions earlier than others but treatment was started when all lesions were of approximately the same size.

Efficacy of KE-091/ATX in the treatment of cutaneous leishmaniasis was then assessed in these balb/c mice experimentally infected with Leishmania major. The compound was administered intraperitoneally (IP) and topically and further compared with the standard anti-leishmanial regimen (Pentostam, an antimony-based formulation) and controls.

The topical preparation was formulated as an ointment containing 4mg/g of compound and using 100µg per lesion per mouse, whereas the IP preparation contained 0.4mg in 0.5ml of compound per mouse. There were a total of 15 mice per treatment group.

The summary of observations is given in Table 2. It is clear that mice treated with the topical preparation responded better than those in any of the other treatment groups, and that KE-091/ATX was superior to Pentostam in treating Leishmania major. infection in mice.

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IMMUNOMODULATORY ACTIVITY

A group of 15 balb/c mice which had been used as negative controls (with KE- 091/ATX but without infection) in the previous experiments on Leishmania infection were studied further to assess the effect of KE-091/ATX on the immunological status of these mice.

Bone marrow examination revealed that there was an increase in new clones of lymphocytes in mice put on KE-091/ ATX as compared to mice without any drug or those with Pentostam alone. This observation strongly suggests that KE-091/ATX is an immunomodulator, a property which is of critical consideration in the treatment of diseases in an immunocompromised host and of autoimmune disorders. Studies in
this area are still continuing.

PENTOBARBITONE SLEEPING TIME IN MICE

The pentobarbitone sleeping time experiment is usually an indicator of whether a drug directly or indirectly produces effects on the CNS. A depressant effect of the agent would synergize that of pentobarbitone, whereas an excitant effect of the agent would reduce the depressant effect of the barbiturate.

Balb/c mice aged 8 to 10 weeks old were used in these experiments. Two dose regimens of pentobarbitone were used: 40 mg/kg and 80 mg/kg. The dose of KE- 09l/ATX was 500 mg/kg which is just below the maximum tolerable dose determined in the previous toxicity experiments. In the experiments, the drug KE- 091/ATX was administered IP first, followed 30 minutes later by the dose of pentobarbitone, also IP. The 30 minute interval appeared to be the most practical interval. An interval of 5hrs was tried but it gave no satisfactory result. Sets of mice
were used for the test drug at 40 mg/kg and at 80 mg/kg and another set were used as controls. The control group was given the same dose of pentobarbitone but without being antimicrobial, antiprotozoal, immunomodulator, spermicidal and tumoricidal. This could be true as with other trioxolanes. The product is stable under specified conditions and can be administered in various formulations. It is likely to be acting through different pathways. However. since its possible sites, modes and mechanisms of action are. currently being studied, it would be
premature to speculate over them.

ACKNOWLEDGEMENTS: We thank James Muthotho, Alex Wamachi and Jim Kagai of the Kenya Medical Research Institute for their technical assistance as well as Jean Herman for her support and encouragement. This study was supported in part by The Davy Koech Foundation and published with the permissions of the Director, Kenya Medical Research Institute.

(This paper was received on 8 July, 1994 and accepted for publication on 13 October, 1994)