"See text for explanation.
bX = one or more halogens, usually CI.
Figure 7-11. Synthetic evolution of antifungal imidazoles.'
Extensive SAR studies showed that the imidazole ring is crucial for broad-spectrum, high-potency antimycotic activity in vivo. It is ironic that even replacement with benzimi-dazole, the original lead compound, exhibited very low effectivness. Exchanging one nitrogen with carbon (i.e., pyrazoles and piperazines) also resulted in less active, or even inactive, compounds.
Additional imidazoles have since been introduced into clinical use (Fig. 7-10). Butaconazole has been marketed primarily in vulvovaginal anticandidal cream. Tioconazole, where the benzylic phenyl ring has been isosterically exchanged with chlorinated thiophene, has been introduced as a topical product (England) against Candida and dermatophytes; bifonazole has been marketed in Germany for several years against topical dermatophyte infections including tinea versicolor. Its structure is not only simpler than most active imidazoles, but it is also unique as it is devoid of any aryl halogen atoms (Fig. 7-10). Although also a relatively simple-structured imidazole drug, clocanazole nevertheless seems to have the necessary features for treatment of topical human dermatomycoses. Fluconazole has the unique structural features of triazole rings (instead of imidazole) and predictably effectiveness-enhancing fluorine atoms on the only benzene ring in the molecule. It is currently utilized in AIDS patients who have succumbed to life-threatening systemic fungal infections. Finally, oxiconazole has become available in topical dosage forms.
The previously mentioned ergosterol content of fungal cell membranes differentiates it from membranes where cholesterol is the sterol involved in permeability regulation. The imidazoles have been shown to inhibit the biosynthesis of ergosterol in all susceptible organisms including C. albicans. It was established that ketoconazole affected sterol metabolism significantly more in yeast cells than it did in mammalian cells. Furthermore, ergosterol synthesis was 67 times more sensitive to miconazole inhibition than was cholesterol. These differences may well account for the selective toxicity of the imidazoles (and triazoles) observed between fungal and mammalian cells.
Figure 7-12 outlines the terminal steps in the complex sequence of both the biosynthesis of cholesterol and ergosterol. The synthesis, starting with acetate, leads to the common steroid precursor lanosterol.9 Thus, in the case of cholesterol, the scheme involves steps for the reduction of the A24 double bond, and the oxidation and removal of the 14a methyl group and of both methyls at C-4. This is followed by isomerization of the A8 double bond of the resultant zygmosterol to the A5 position, affording desmesterol, and finally cholesterol.
Ergosterol also arises from lanosterol by a one-carbon addition to C-24 followed by a shift of the A24 double bond to the exo position, forming a A24 methylene function. There follows oxidative removal of the three methyls at C-14 and C-4 with concomitant shift of the A24 double bond to A22 position; isomerization of A8 position to A7 and formation of an additional A5 double bond yielding ergosterol.
Ergosterol synthesis inhibition has been found to coincide with an accumulation of 14 a-methylsterols in yeast cells. The corresponding inhibition in rat liver cells could only be achieved at a sixfold increase in ketoconazole concentration. The enzyme initiating the oxidation of the 14a-CH3 function is lanosterol- 14a-methyldemethylase. It has been
9 For review of cholesterol biosynthesis, see Zubay, 1983.
established to be a cytochrome P-450-dependent enzyme, and, furthermore, that miconazole and ketoconazole affect this enzyme in yeast microsomes. The corresponding enzyme obtained from rat liver microsomes was much less sensitive. Of course, this relates well with the selective toxicity observed with these drugs. That lanosterol binds to (i.e., is a substrate for) the microsomal cytochrome P-450 system had already been previously demonstrated for Saccharomyces cerevisiae (a yeast). It remained to be established that increased levels of methylated sterols and decreased availability of ergosterol had the deleterious effects on fungal membranes observed; namely, increased permeability and loss of cell components such as glucose.
It is now apparent that accumulation of methylated steroids, particularly 14a-methyl, accounts for the detrimental alterations in membrane structure and functions induced by the imidazole drugs. The changes in membrane permeability with the resulting leakage of cellular components is the primary cause of cellular death. It has been suggested the axial position of the 14a-methyl group of lanosterol prevents interaction between the flat face of the sterol and the fatty acid chains in the phospholipid membrane.
In addition to inhibition of ergosterol biosynthesis, it is now reasonably well established that the imidazole drugs also have a direct action on fungal membranes. This may in part explain the existence of certain Gm-t- bacteria susceptible to imidazole drugs. Spectrophotometric evidence indicated interactions between imidazoles and mixtures of saturated and unsaturated fatty acyl groups occurring naturally as part of a fungal membrane as phospholipid complement. The imidazole compounds have a duality of antimicrobial action. One involves interference with the iron atom of fungal cytochrome P-450 enzyme. The result is a blockade of oxidative removal of lanosterol's C-14 methyl group (Fig. 7-12). This mechanism is probably responsible for the fungistatic effect. The fungicidal effect observable at higher drug concentrations is likely the result of direct interactions between these compounds and unsaturated fatty acids in the membranes. Ketoconazole does not appear to share this interaction, thereby helping to explain the absence of fungicidal effects of this particular agent.
Flucytosine (5-Fluorocytosine, Ancobon, Fig. 7-10) was first synthesized as a potential antimetabolite for use in leukemia treatment, but it exhibited no anticancer effects. It was found to have in vivo antifungal activity, however. In vitro activity includes Cryptococcus neoformans, many isolates of C. albicans, and other Candida species and some isolates of Aspergillus spp. Fungicidal concentrations range from 0.2 to 12.5 |Xg/mL. The activity of flucytosine against filamentous fungi is only fungistatic. The drug is not toxic in experimental animals and is readily absorbed. It can therefore be orally administered. Clinical use of flucytosine is limited to susceptible systemic infections and cryptococcal meningitis.
Resistance emerging during therapy can be a major problem, so that even though the drug is less toxic than amphotericin B, the two drugs should be prudently used together in serious mycoses to minimize the likelihood of resistance emergence and to take advantage of the additive, or even synergistic, effects.
The reason for the relative safety of flucytosine, and the lack of carcinolytic effect in humans, is that the enzyme cytosine deaminase does not appear to be present. Conversion to 5-FU occurs to an insignificant degree of about 4% (Eq. 7.10). However, following per-mease-catalyzed uptake by the fungal cell, the deaminase present there effectively converts the drug to the cytotoxic 5-FU through several steps to 5-fluorouridine triphosphate (see Fig. 4-18). This results in its incorporation into the fungal cell's RNA, leading to the synthesis of miscoded proteins. This faulty RNA (containing 5-FU) ultimately accounts for a major portion of the cell's nucleic acid. In addition, 5-fluorouridine monophosphate can also be reduced to the corresponding 5-fluorodeoxyuridilic acid inhibiting the cell's thymidylate synthetase, thus affecting DNA synthesis as well (see Fig. 4-19).
nh2 oh cytosine
5-fluorocytosine 5-fluorouracil (7 1Q)
Parasitic helminth (worm) infections are widespread and may be the most common disease in the world. Estimates by the WHO and others are that one type of schistosomiasis, the blood flukes, has 200 million people infected. Ascaris, a large roundworm, accounts for at least 650 million, hookworm 450 million, and the several filarial worm species add another 250 million victims to the total. The situation has improved little in the past decade.
Table 7-9 is a condensed classification and nomenclature of the important parasitic helminths that affect humans. The worms that infect humans are the flatworms and the roundworms. Helminths are the only parasites discussed here that are not microscopic. Quite the opposite, their dimensions are of the order of millimeters and, in the case of certain tapeworms, reach lengths of meters.
There are two types of flatworms; the flukes, whose appearance can best be described as lanceolate or leaflike, and the tapeworms, which are ribbonlike. The latter consist of repeating egg-containing segments, or proglottids (numbering up to several thousand) linked in a chain called a strobilia and attached to a "head" or scolex. They have no digestive system. Their nutrients are absorbed directly from the host's intestines; they are true parasites.
The second helminth type are the roundworms. Their shape is cylindrical. They vary in proportions, size, and structure.
There is no totally broad-spectrum antihelminthic drug. There are groups of drugs that are useful against blood flukes; others against liver flukes or lung-infesting flukes. There are some agents well suited to the treatment of tapeworms. Another category is useful against intestinal roundworms and still another against roundworms infesting tissues or blood. There may be considerable overlaps of activities.
Parasitic helminths infect by transmission of eggs or larvae. Once developed into adult worms, they reproduce by releasing their own eggs or larvae, which then leave the host, where they develop "on the outside," and/or intermediate hosts (e.g., snails). When the larvae become infective and again enter the human host, the life cycle is complete (Fig. 7-13).
Pathology in the host can be produced by the adult worms, the larvae, or the eggs, and the intensity is usually directly related to the worm load. Unlike protozoal disease, clinical symptoms with worm infections are not well defined. Diagnosis usually depends on recovery and identification of the parasite or its eggs. Immunoserological tests are sometimes available.
Drugs. Several formerly popular remedies, while they may be effective in some case, have been abandoned as obsolete due to unacceptable toxicity. The natural lactone san-
Table 7-9. Classification and Nomenclature of Helminths8
Ascaris lumbricoides Ancylostoma duodenale Enterobius vermicularis
(also called Oxyuris vermicularis) Necator americanus Strongyloides stercoral is Trichinella spiralis Trichuris trichiura Loa loa
Onchocerca volvulus Wuchereria species
Fasciola hepatica Fasciolopsis buski Heterophyes heterophyes Metagonimus yokogawai Cloriorchis sinensis flukes Paragonimum species Schistosoma haematobium Schistosoma japonicum Schistosoma mansoni roundworm hookworm pinworm or seatworm hookworm threadworm trichina (pork roundworm)
liver fluke intestinal flukes, giant intestinal flukes, small
Chinese liver lung flukes blood flukes
Diphyllobothrium latum Hymenolepsis nana Taenia saginata Taenia soilum fish tapeworm dwarf tapeworm beef tapeworm pork tapeworm
" Selected for the purpose of this discussion. Families listed alphabetically. b When sexually differentiated the female is invariably longer than the male. c Commonly encountered in Africa, Asia, and Mediterranean Europe, but not in U.S.
tonin is one example. Other popular vermifuges were the harsh oleoresin from Aspidium (male fern) and chenopodium (American wormseed oil), which contained an organic peroxide that exploded on heating. In spite of some new developments, many anthelmintics still in use are anything but "modern" drugs.
Whether particular drugs are well tolerated or not, anthelmintic therapy frequently requires adjunctive therapy. Thus allergic reactions to tissue infestation by parasites may require antihistamines, or even steroids. Iron-deficiency anemia (due to blood losses) sometimes has to be counteracted. Gastric effects have to be treated traditionally, as do any secondary bacterial infections. With some agents cathartics may be indicated to expel dead or incapacitated worms.
Certain drugs, although safe and effective, are not available in the United States. They can be obtained by physicians for their patients from the Parasitic Disease Drug Service, Center for Disease Control (CDC), U.S. Public Health Service (USPHS), Atlanta, Georgia.
Anthelmintics as a group share few common features of chemical similarity or mechanism of action. Table 7-10 lists many of the available drugs and the disease or organism against which they have been found useful. (Structures given elsewhere are not repeated.)
Schistosomiasis (Bilharziasis). The schistosome species listed in Table 7-9 parasitize humans widely in Africa, South America, the Far East, and the Middle East. S. mansoni is the most widespread species, S. haematobium resides mostly in Africa, and S.japonicum is found primarily in the Far East. The latter is the most difficult to treat. The intermediate host, the snail, is not prevalent in urban areas of the U.S. mainland; the disease is not endemic here. However, S. mansoni, for example, is seen in New York City because of its prevalence in the Caribbean Islands.
Historically, the first treatments were antimonial compounds. The medicinal properties of antimony have been known (but not understood) for thousands of years. Paracelsus prescribed it in the sixteenth century. Antimony potassium tartrate was probably the first specific antischistosomal treatment. Organic antimony compounds are effective. However, the need for lengthy treatment and high toxicity makes their obsolescence a desirable goal, if safer and more effective agents can be developed. Coadministration of mercapto compounds such as penicillamine (dimethylcysteine) (Chapter 5) can reduce toxicity. The chelate with tartar emetic is apparently better tolerated. At the molecular level antimony compounds produce an accumulation of glucose-6-phosphate and fructose-6-phosphate while the level of fructose- 1,6-diphosphate decreases in the parasites. This chemical alteration therefore implicates the enzyme phosphofructokinase (PFC) as the target being inhibited. Since schistosomes (and possibly other antimony-sensitive parasites) appear to have their major energy source—glycolysis—inhibited, the parasiticidal activity of these drugs becomes explicable.
The basis of the selective toxicity depends on the enzyme's source. The schistosomal enzyme has been shown to be 80 times more responsive to antimonial inhibition than is PFC from a mammalian source. It is also of interest that the schistosomal enzyme is activated by -SH compounds such as cysteine, penicillamine, glutathione, and even 2-mercap-toethane, and that the inhibitory effect on the enzyme is not lessened by these compounds. This would rule out the likelihood of irreversible inhibition of enzyme SH groups as the underlying mechanism of drug action. Nevertheless, by taking advantage of the attenuated toxicity via its complexation with three molecules like dimercaptosuccinate [H02CCH(SH)CH(SH)C02H], drugs like stibocaptate (Table 7-10) remain as useful, though secondary, antischistosomal drugs.
Niridazole, a nitrothiazole, is another toxic drug that was selected from a drug development program. The drug has some effect on all three species; however, it is primarily useful clinically against S. hematobium, especially in children. The antiparasitic effect is directly on the reproductive system: disruption of and abnormal egg productions and
Table 7-10. Anthelmintic Drugs
Antimony potassium Fig. 7-9
tartrate (tartar emetic)
Chinese liver fluke (Clonorchis sinensis)
Diethylcarbamazine'' citrate (Hetrazan)
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