Seren B Christensen

Halki Diabetes Remedy

How I Healed my Diabetes

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CONTENTS

21.1 Introduction 341

21.2 Infections Caused by Helminthic Parasites 342

21.2.1 Schistosomiasis 342

21.2.2 Filariasis 342

21.3 Infections Caused by Protozoan Parasites Other Than Plasmodium 343

21.3.1 Trypanosomiasis 343

21.3.2 Leishmaniasis 343

21.4 Malaria 345

21.5 Drugs against Malaria 346

21.5.1 Drugs Targeting Hemozoin Formation 346

21.5.1.1 4-Aminoquinolines 347

21.5.2 Drugs Targeting a Ca2+ Pump of Plasmodium Parasites 349

21.5.3 Drugs Targeting Deoxyxylulosephosphate-Reductoisomerase 351

21.5.4 Drugs Targeting Mitochondrial Functions 351

21.5.4.1 Naphtoquinones 352

21.5.5 Drugs with Nonestablished Targets 353

21.5.5.1 8-Aminoquinolines 353

21.5.5.2 4-Quinolinemethanols 353

21.5.5.3 Phenanthrenemethanols 355

21.5.6 Drugs Targeting Folate Synthesis 355

21.6 Resistance 357

21.6.1 Chloroquine Resistance 357

21.6.2 4-Quinolinemethanol Resistance 357

21.6.3 Antifolate Resistance 357

21.7 Concluding Remarks 357

Further Readings 358

21.1 INTRODUCTION

Infections including parasitic diseases account for approximately one-third of the worldwide disease burden but only 5% of the disease burden in high-income countries. Pharmaceutical companies assume that only a marginal profit can be made from drugs against diseases in low-income countries and consequently less than 2% of new chemical entities marketed in the last 30 years have been anti-infective drugs. The majority of the few marketed anti-infective agents were antiretroviral drugs, the development of which benefited from a serious political commitment from high-income countries for finding drugs against acquired immune deficiency syndrome (AIDS).

The limited interest for development of drugs against infectious diseases like malaria, African trypanosomiasis, Chagas disease, schistosomiasis, leishmaniasis, and tuberculosis has led to the use of the term neglected diseases, even though infectious diseases worldwide are responsible for a heavier burden than cardiovascular or central nervous system (CNS) diseases.

Medicine for Malaria Venture is a nonprofit organization founded in 1999, which has set up a public private partnership (PPP) in order to develop drugs according to the highest international standards against diseases of the developing countries. The organization includes academic institutions like Yale University, the University of Oxford, the University of California, San Francisco, private companies like F. Hoffmann-La Roche, Novartis Pharma, Korea Shin Poong Pharm, Holleykin Pharmaceutical Company (China), and international organizations like the World Health Organization (WHO).

In the past, the major inspiration for development of drugs against infectious diseases has come from natural products (see Chapter 6).

21.2 INFECTIONS CAUSED BY HELMINTHIC PARASITES

A parasite is an organism that lives in or on and takes its nourishment from another organism. A parasite cannot live independently. A helminth is a multicellular parasitic worm. In general, a helminth is visible to the naked eye in its adult stages. Parasites might have more hosts. The smaller host is generally called the vector. Many neglected diseases are caused by parasites. In contrast to bacteria, which are prokaryotes, parasites are eukaryotes.

21.2.1 Schistosomiasis

Schistosomiasis (bilharziasis) is caused by infection with flatworms belonging to the genus Schistosomas, S. mansoni is found in South America and Africa, S. haematopium found throughout Africa, in particular in Egypt, and S. japonicum is confined to the Far East. Approximately 200 million people in more than 70 developing countries suffer from the diseases, 20 million suffer severe consequences, such as colonic polyposis with bloody diarrhoea (S. mansoni), splenome-gami, and portal haematemesis with vomiting of blood (S. japonicum and S. mansoni), cystitis, and ureteritis, which might lead to bladder cancer (S. haematopium), and CNS lesions. The diseases are estimated to cause 280,000 deaths each year. Three safe, effective drugs, praziquantel, oxam-niquine, and metrifonate, are now available for schistosomiasis and are included in the WHO model list of essential drugs. WHO has defined essential drugs as "those drugs that satisfy the health care needs of the majority of the population; they should therefore be available at all times in adequate amounts and in appropriate dosage forms, at a price the community can afford."

21.2.2 Filariasis

Filariasis is caused by parasites belonging to the order Filaroidea. Wuchereria bancrofti and Brugia malayi both cause lymphatic filariasis (elephantiasis), Loa loa causes fugitive swelling, in particular, around the eyes, and Onchocerca volvulus causes onchocerciasis (river blindness). All the diseases are caused by helminthic worms transmitted through bites of insects belonging to the order Diptera. The symptoms pertaining to the diseases are caused by the presence of parasites restricting the flow of lymph fluid. Approximately, 120 million people are infected with the parasites and 40 millions are severely disabled. Onchocerciasis is estimated to have infected 17.7 millions people, of which 500,000 have visual impairment and 270,000 are blinded. The disease is limited to the vicinity of rivers where the vector, blackflies of the genus Similium, is endemic. Unfortunately the burden of the disease often forces the population to leave these areas uninhabited. The infection can be treated with ivermectin (21.1) (Figure 21.1). Ivermectin, a dihydro derivative of avermectin B1a, acts by opening invertebrate specific glutamate-gated chloride ion channels in the nerve end and muscles

H3C HO

L-Oleandrose

21.2

FIGURE 21.1 Ivermectin (21.1) is a mixture containing at least 80% of the analog in which R = C2H5, and not more than 20% of the analog in which R = CH3. Configuration of melarsopol (21.2).

of the parasites. This leads to death of microfilariae, the first larval stage. The drug does not cause immediate death of the adult parasite but reduces the worm's life span.

21.3 INFECTIONS CAUSED BY PROTOZOAN PARASITES OTHER THAN PLASMODIUM

Protozoan parasites are single-celled organisms, which have an animal-like nutrition (they cannot perform photosynthesis). The life cycle of protozoan parasites involves two hosts; the smaller of which typically is named the vector. Important genera are Plasmodium, Trypanosoma, and Leishmania.

21.3.1 Trypanosomiasis

Two major tropical diseases, American trypanosomiasis (Chagas disease) and African trypanosomiasis (sleeping sickness) are caused by T. cruzi and subspecies of T. brucei, respectively. African trypanosomiasis is spread with tsetse flies (Glossina species). If untreated, the disease may be lethal since the parasites enter the CNS causing coma (explaining the name sleeping disease) and death. Approximately 48,000 persons are estimated to die from the disease each year. The only drug available for treatment of the disease in a late stage is the arsenical drug melarsoprol (21.2) (Figure 21.1), developed more than 50 years ago. It is known to cause a range of side effects including convulsions, fever, loss of consciousness, rashes, nausea, and vomiting. It is fatal in a significant fraction of cases. Early stages of the disease are treated with pentamidin and suramin.

21.3.2 Leishmaniasis

Leishmaniasis is caused by parasites of the genus Leishmania. The diseases vary from simple self-healing skin ulcers (cutaneous leishmaniasis), severe disfiguring of nose, throat, and mouth cavities (mucocutaneous leishmaniasis) to life-threatening infections (visceral leishmaniasis). Visceral leishmaniais can be fatal if untreated. Approximately 12 million humans are infected with leishmaniasis and it is estimated that 59,000 die each year. The parasites nourish in the macrophages. The life cycle is illustrated in Figure 21.2.

Until recently no orally active drug was known for leishmaniasis but the treatment was based on amphotericin B (21.3) (Figure 21.3), pentamidine or antimony containing drugs like sodium stibogluconate and meglumine antimonate. Liposomal formulations of amphotericin B have increased the efficiency of the drug. Application of a drug in vesicles as liposomes will target the drug

Human

FIGURE 21.2 Life cycle of leishmania parasites: by taking a blood meal sandflies, belonging to the genus Phlebotomus, introduce promastigotes into the blood. The promastigotes are phagocytized by macrophages and converted into amastigotes in the blood cells. The amastigotes multiply in the macrophages. A sandfly feeding on the infected person will ingest parasites and thereby conclude the cycle.

Human

FIGURE 21.2 Life cycle of leishmania parasites: by taking a blood meal sandflies, belonging to the genus Phlebotomus, introduce promastigotes into the blood. The promastigotes are phagocytized by macrophages and converted into amastigotes in the blood cells. The amastigotes multiply in the macrophages. A sandfly feeding on the infected person will ingest parasites and thereby conclude the cycle.

Amphotericin Cancer Cell Membrane

FIGURE 21.3 Mechanism of action of amphotericin B (21.3). The polyene region interacts with the double bonds of ergosterol, which is found in the cell membrane of parasites. Mammalian cells contain cholesterol, in which the presence of only one double bond causes the formation of a weaker complex with amphotericin B. The orientation of the amphotericin B-ergosterol complexes creates an ion channel, through which an unregulated flux of small inorganic ion passes. Inability to control the concentration of inorganic ions eventually kills a cell.

FIGURE 21.3 Mechanism of action of amphotericin B (21.3). The polyene region interacts with the double bonds of ergosterol, which is found in the cell membrane of parasites. Mammalian cells contain cholesterol, in which the presence of only one double bond causes the formation of a weaker complex with amphotericin B. The orientation of the amphotericin B-ergosterol complexes creates an ion channel, through which an unregulated flux of small inorganic ion passes. Inability to control the concentration of inorganic ions eventually kills a cell.

21.4

FIGURE 21.4 Configuration of miltefosin (21.4).

against cells performing phagocytosis such as the macrophages. Since the macrophages host the parasites some selectivity in activity is obtained. The main mechanism of action of amphotericin B is based on the amphiphilic nature of the molecule consisting of a lipophilic heptaene region and a hydrophilic polyol region. The polyene region complexes with steroids in the parasite's membrane. The hydrophilic polyol region form an ion channel permeable to small ions (Figure 21.3). Some selectivity is obtained because the drug has higher affinity for the double bonds of ergosterol dominating in the cell membrane of the parasites than for cholesterol in the membrane of mammalian cell.

Serendipitously it was discovered that the cancer drug miltefosine (21.4) (Figure 21.4) is an orally active drug against visceral leishmaniasis. Growth inhibition of leishmania parasites induced by miltefosine is correlated with a change in the phosphatidylcholine to the phosphatidylethanolamine ratio in the parasite's membrane. The selectivity might reside on different ways of formation of phosphatidylcholine in vertebrates and in leishmania parasites.

21.4 MALARIA

Malaria is a leading cause of morbidity and mortality in the tropical world; some 300-500 million of the world population are infected with malaria parasites, presenting 120 million clinical cases each year. It is estimated that between 1.5 and 2.7 million persons die from malaria each year and that 1 million of those are African children younger than 5 years. Among the more than 100 species of Plasmodium parasites, only four can infect humans: P. falciparum (causing malignant tertian malaria), P. malariae (quartan malaria), P. ovale (ovale tertian malaria), and P. vivax (benign tertian malaria). P. falciparum is responsible for the majority of deaths.

The life cycle of the malaria parasite encompassing several stages is depicted in Figure 21.5. A bite from an infected female mosquito belonging to the genus Anopheles introduces malarial parasites in the sporozoite stage into human with the salvia, which contains agents that prevent clotting of the blood. The sporozoites grow and multiply in the liver for about 5-15 days depending on the species. During this period, the patient has no symptoms. After having multiplied in the liver, the parasites enter the bloodstream as merozoites and invade the red blood cells (the erythrocytes). In the erythrocytes, the parasites proliferate and emerge as merozoites in a synchronous manner in about 48 h (tertian malaria) or 72 h (quartan malaria). This results in the clinical symptoms of the disease, namely, chills with rising temperatures, followed by fever and intense sweating. In addition, there might be severe headache, fatigue, dizziness, nausea, lack of appetite, and vomiting. Since the sporozoites catabolize the hemoglobin of the erythrocytes, a heavy infection will also induce anemia. After eruption, some merozoites reinvade erythrocytes and complete a new erythrocytic cycle. In the erythrocytes, some parasites change into game-tocytes. After entering a mosquito stomach, blood meal gametocytes undergo another cycle in the mosquitoes. Merozoites will be digested in the stomach. The falciparum parasites cause the erythrocytes to adhere to the walls of capillary vessels resulting in reduced blood flow to organs. Reduced blood flow to brain contributes to cerebral malaria, which can be fatal. Because of the symptoms, some persons chronically infected with malaria, such as, the majority of Africans perform poorly. Studies suggest that national income in some African countries was suppressed by much as 18% because of malaria.

Mosquito Gametocyte -► Sporozoite

Bite \

Bite

/y^ ~\GametocyteS\

/ Hepatic schizont \

Erythrocytic <—\ Merozoite V-1 I schizonts 1

Erythrocyte

\ Liver /

Blood

Human

FIGURE 21.5 A mosquito belonging to the genus Anopheles pumps salvia into dermis of humans through the proboscis during feeding. If the mosquito is infected with malarial parasites, the salvia also will contain sporozoites, which will infect liver cells. In the liver, the parasites will develop into merozoites, which will be released into the blood by rupture of the liver cells. In the red blood cells, the merozoites will proliferate. At certain intervals, the red blood cells will rupture to release merozoites and male and female gametocytes. If a mosquito takes a blood meal on an infected human, the intraerythrocytic schizonts will be digested but the extraerythrocytic gametocytes will undergo a sexual proliferation in the mosquito enabling the mosquito to infect a new human.

FIGURE 21.5 A mosquito belonging to the genus Anopheles pumps salvia into dermis of humans through the proboscis during feeding. If the mosquito is infected with malarial parasites, the salvia also will contain sporozoites, which will infect liver cells. In the liver, the parasites will develop into merozoites, which will be released into the blood by rupture of the liver cells. In the red blood cells, the merozoites will proliferate. At certain intervals, the red blood cells will rupture to release merozoites and male and female gametocytes. If a mosquito takes a blood meal on an infected human, the intraerythrocytic schizonts will be digested but the extraerythrocytic gametocytes will undergo a sexual proliferation in the mosquito enabling the mosquito to infect a new human.

Today the major burden of malaria is restricted to the tropical world: India, South East Asia, SubSaharan Africa, and Central and South America, but the endemic area of malaria besides the tropics also encompasses the subtropics and the major part of the temperate zones. During the 1950s and 1960s, a combined use of dichlorodiphenyltrichloroethane (DDT) for control of the vector mosquitoes and chloroquine (21.7) (refer to Figure 21.8) for the control of the parasites almost eradicated malaria from the Indian subcontinent. Resistance of the parasites toward chloroquine and of the mosquitoes toward DDT and the environmental consequences of extended use of DDT led to discontinuation of the project and return of the malaria burden.

21.5 DRUGS AGAINST MALARIA

In the absence of vaccines, malaria therapy relies on small molecule drugs. A number of antibiotics are used successfully either individually or more common in combination with other drugs. It may be surprising that antibiotics display considerable activity against the eukaryotic malarial parasite. This contradiction can be explained by the presence of two essential organelles in the parasites, namely, the mitochondria and the apicoplasts (Figure 21.6).

The apicoplast, probably, is a remnant of endosymbiotic cyanobacteria, which in plants have developed into the photosynthetic chloroplasts. Even though the apicoplasts do not perform photosynthesis, their metabolic pathways are still essential for the parasites. Both organelles have their own machinery for replication. Most antibiotics used in malaria therapy affect the apicoplasts.

21.5.1 Drugs Targeting Hemozoin Formation

The ultimate diagnosis of malaria is microscopic observation of parasites in the erythrocytes of a thick blood film. The presence of the malaria pigment, hemozoin in the erythrocytes, unequivocally reveals the presence of parasites. Hemozoin is formed from the heme (ferroprotoporphyrin IX, 21.5) remaining after digestion of the peptide part of hemoglobin (Figure 21.7). The digestion proceeds in

PfMDRI

Pf CRT

Pf CRT

Apicoplast <p

/Terpenoids

Apicoplast <p

/Terpenoids

FIGURE 21.6 Plasmodium parasite: The Ca2+-ATPase (P/ATP6) pumps Ca2+ from the cytosol into the endoplasmic reticulum (ER) maintaining a low cytosolic Ca2+ concentration and a high concentration inside the ER. The single circle outside the ER represents the low cytosolic Ca2+ concentration in contrast to the several order of magnitudes higher ER concentration (many circles). Artemisinin and its analogs block this pump. Blockage of the pump leads to a prolonged high cytosolic Ca2+ concentration, which is lethal for the cell. Hemozoin is accumulated inside the food vacuole (FV). Blockage of hemozoin formation by, e.g., chloroquine is lethal to the cell. Terpenoids are formed in the apicoplasts by the nonmevalonate pathway. Blockage of this pathway by, e.g., fosmidomycin is lethal to the cell. De novo pyrimidine synthesis includes reduction of dihy-roorotate into orotate, a reaction that is dependent on the electron flow in the mitochondria. Atovaquone blocks the electron flow. By removing chloroquine from the FV, the pump PfCRT makes the parasite resistant toward chloroquine. The pump PfM DR1 removes a number of drugs from the cytosol and induces resistance.

the food vacuole of the parasite. The food vacuole is characterized by a pH between 5.0 and 5.5. The parasites use at least three types of proteases for the catabolism of hemoglobin. Since nude heme is toxic to all kind of cells the parasites have to detoxify it by converting it into an insoluble complex.

The heme detoxification process is concluded by precipitation of microcrystalline hemozoin (21.6) (Figure 21.7), which by precipitation loses the effect on the biological system. The mechanism of action behind a series of antimalarial drugs consists in blocking the formation of hemozoin by association with hematin. Two criteria have to be fulfilled for drugs that act by preventing detoxification of heme: (1) the drug must accumulate in the food vacuole of the parasite, and (2) the drug must bind to hemozoin. For a drug to be active in the human patient, several other factors including absorption, metabolism, and distribution need to be taken into account. Thus association with hematin is a necessary, but not sufficient requirement, for an antimalarial drug targeting the hemazoin formation. Criterion 1 might be fulfilled by introduction of a basic aliphatic amine into the molecule, thus taking advantage of the low pH of the food vacuole.

21.5.1.1 4-Aminoquinolines

The 4-aminoquinolines are characterized by the presence of an amino group in the 4-position of the quinoline nucleus.

It is claimed that the drug, which with no comparison has saved most human lives, is chloroquine (21.7) (Figure 21.8). For more than 40 years, chloroquine was the first-line therapeutic and prophylactic agent for malaria. In the last three decades, however, chloroquine resistant P. falciparum and P. vivax strains have developed, but the drug is still believed to be efficient toward infections by P. ovale and P. malariae. The resonance interaction between the electron pair of the exocyclic amino group and the quinoline nitrogen atom (Figure 21.8) will give the protonated 4- and 2-aminoquinolines higher pKa values (8.1 and 10.2) than other aminoquinolines.

Hematin

FIGURE 21.7 Digestion of hemoglobin, which is a tetramer consisting of four protein strings and four heme (21.5) molecules, leads to liberation of the heme molecules. Oxidation of iron(II) to iron(III) converts heme into hematin. Ionic interactions between the propanoate side chain and the iron(III) yield the poorly soluble b-hematin = hemozoin (21.6), which precipitates (shown as a dimer, but in the cell hemozoin will precipitate as a polymer).

Hematin

FIGURE 21.7 Digestion of hemoglobin, which is a tetramer consisting of four protein strings and four heme (21.5) molecules, leads to liberation of the heme molecules. Oxidation of iron(II) to iron(III) converts heme into hematin. Ionic interactions between the propanoate side chain and the iron(III) yield the poorly soluble b-hematin = hemozoin (21.6), which precipitates (shown as a dimer, but in the cell hemozoin will precipitate as a polymer).

81 The quinoline nucleus

.ch3

FIGURE 21.8 The quinoline nucleus and configurations of chloroquine (21.7) and hydroxychloroquine and resonance structures revealing the delocalization of the electrons in the electron-rich pyridine ring. Protonation of the two basic amino groups ensures accumulation in the food vacuole.

Chloroquine 21.7

FIGURE 21.8 The quinoline nucleus and configurations of chloroquine (21.7) and hydroxychloroquine and resonance structures revealing the delocalization of the electrons in the electron-rich pyridine ring. Protonation of the two basic amino groups ensures accumulation in the food vacuole.

The lipophilicity of the neutral molecule enables it to cross the membranes of the erythrocytes and the parasites. Having entered the food vacuole with a pH ~ 5.2, the two amino groups will become protonated preventing chloroquine from leaving the vacuole by passive penetration. Furthermore, the protonation of quinoline nitrogen enables a cation-p interaction between the quinoline and the hemozoin (Figure 21.9).

FIGURE 21.9 Two orthogonal views of a suggested binding mode of chloroquine (green) to hemozoin (yellow). Hetero-atoms are colored red, blue, and orange for oxygen, nitrogen, and iron, respectively. (Figure prepared by Dr. F. S. J0rgensen, University of Copenhagen, Denmark.)

The complexation between chloroquine and hemozoin further contributes to the concentration of choroquine in the food vacuole. Actually, the concentration of chloroquine, in sensitive parasites, is four orders of magnitude higher inside the vacuole than outside. The presented model also confirms the finding that variations in the side chain, only to a minor extent, influence the strength of the association. Notice the absence of stereocenters in the porphyrin nucleus. The achirality of this target explains that the two enantiomers of chloroquine have the same binding affinity. In agreement with the target being heme, chloroquine affects the erythrocytic stages of the parasite only, the only stages in which hemozoin is formed.

21.5.2 Drugs Targeting a Ca2+ Pump of Plasmodium Parasites

Whereas European physicians did not get access to an efficient antimalarial drug until the sixteenth century, Chinese authors described the effect of qing hao and cao hao against intermittent fever two thousand years ago. Since the Chinese way of describing symptoms of diseases is very different from the Western terminology, intermittent fever cannot in a simple way be translated into a well-known term. However, intermittent fever could include the fever caused by malaria. Quing hao has later been identified as Artemisia apiacea (Asteraceae) and cao hao as A. annua (sweet wormwood). In the 1970s, Chinese scientists isolated artemisinin (21.8) (Figure 21.10) from both these species and showed that the compound was a potent antimalarial agent. Artemisinin is an irregular sesquit-erpene lactone containing an endoperoxide bridge. Synthetic analogs, in which the peroxide bridge has been removed, show no activity toward Plasmodium parasites. The amount of artemisinin that may be extracted from the wormwood varies between 0.01% and 0.8% of dry weight, a serious limitation for the commercialization of the drug.

Originally, it was suggested that artemisinin by reaction with the iron (II) ion of heme generates a radical, which reacted with the heme skeleton and thereby prevented this from precipitation (Scheme 21.1). Although this idea still is favored in some laboratories, later studies indicate that artemisinin inhibits a plasmodial intracellular calcium pump (PfATP6). All cells maintain a low cytosolic Ca2+ concentration by removing Ca2+ from the cytosol with a pump sitting in the membrane of the endoplasmic reticulum. Blocking this pump yields a high cytosolic Ca2+ concentration, which eventually leads to cell death (Figure 21.6). A crucial difference between the mammalian pump, the sarco-/endoplasmic calcium ATPase (SERCA), and the PfATP6 pump is the presence of Glu255 in SERCA, whereas a Leu is located in the equivalent position 263 in PfATP6.

hCH3

o 21.12

h ch h ch3

21.10 r=ch3

21.11 r=ch2ch3

21.10 r=ch3

21.11 r=ch2ch3

FIGURE 21.10 Treatment of artemisinin (21.8) with sodium borohydride selectively reduces the ester to the semiacetal dihydroartemisinin (21.9). The semiacetal can be converted into the lipid-soluble artemether (21.10) or arteether (21.11). Monoesterification with dicarboxylic acids results in the formation of water-soluble esters of the hemiacetal, e.g., artesunate (21.12).

FIGURE 21.10 Treatment of artemisinin (21.8) with sodium borohydride selectively reduces the ester to the semiacetal dihydroartemisinin (21.9). The semiacetal can be converted into the lipid-soluble artemether (21.10) or arteether (21.11). Monoesterification with dicarboxylic acids results in the formation of water-soluble esters of the hemiacetal, e.g., artesunate (21.12).

Both pumps are inhibited by the sesquiterpene lactone thapsigargin, but P/ATP6 only by artemisinin and analogs. A mutation of Leu263 into Glu in P/ATP6 preserves thapsigargin but abolish artemisinin sensitivity. Very recent results, however, question the effect of artemisinin on P/ATP6.

The poor solubility of artemisin allows only oral or rectal administration of the drug. Since oral administration is impossible for severely sick patients, lipid-soluble derivatives (artemether [21.10], and arteether [21.11]) and water-soluble derivatives (e.g., artesunate [21.12]) have been developed by selective reduction of the ester carbonyl group followed by ether or ester formation (refer to Figure 21.10).

To avoid recrudescense (reappearance of a disease after it has been quiescent), artemisinin and artemisinin derivatives are preferentially given in combination therapy. Some examples of combination therapies are artesunate in combination with chlorproguanil and dapsone (21.34) (refer to Figure 21.18), artesunate in combination with mefloquine (21.27) (refer to Figure 21.15), and artesunate in combination with sulphadoxine (21.33) (refer to Figure 21.18) and pyrimethamine (21.31) (refer to Figure 21.16).

21.5.3 Drugs Targeting Deoxyxylulosephosphate-Reductoisomerase

In plants, the biosynthesis of isopentenyl diphosphate (21.16), the precursor of all terpenoids, follows two independent pathways: (1) the mevalonate pathway and (2) the 1-deoxy-D-xylulose-5-phosphate pathway (the nonmevalonate—or the Rohmer pathway). In plants, the mevalonate pathway occurs in the cytosolic compartment, but the deoxyxylulose pathway takes place in the plastids, which are analogous to apicoplasts. A search in the genome of Plasmodium parasites revealed the presence of genes encoding the enzymes of the deoxyxylulose pathway including the genes encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DOXP-reductoisomerase). This enzyme catalyses a crucial step in the deoxyxylulose pathway (Scheme 21.2): the reductive rearrangement of 1-deoxy-D-xylulose 5-phosphate (21.13) into 2-C-methyl-D-erythritol 4-phosphate (21.15).

Fosmidomycin (21.17) (Scheme 21.2), an antibiotic and herbicidal agent isolated from cultures of Streptomyces lavendulae, efficiently inhibits the plants' carotenoid and phytol synthesis. Fosmidomycin is a structural analog to 2-methylerythrose 4-phosphate (21.14), a never isolated compound but likely to be an intermediate in the conversion of (21.13) into (21.15). Combination therapy using fosmidomycin and clindamycin has revealed high antimalarial activity and only mild gastrointestinal side effects. The drug, however, is still in development.

OH 21.14

DOXP-reductoisomerase

21.16

OH OH 21.15

21.17

SCHEME 21.2 Reductive rearrangement of 1-deoxy-D-xylulose 5-phosphate (21.13) to 2-C-methyl-D-erythritol 4-phosphate (21.15) in the deoxyxylulose pathway via the never isolated 2-methylerythrose 4-phosphate (21.14). Isopentenyl diphoshate (21.16) is the precursor for terpenoids. Configuration of the DOXP-reductoisomerase inhibitor, fosmidomycin (21.17).

21.5.4 Drugs Targeting Mitochondrial Functions

In contrast to the hosts, the mitochondrial electron transport of Plasmodium parasites is not coupled to the synthesis of ATP. An important function of the mitochondria is to maintain an electron transport needed for nucleotide synthesis. Parasites are dependent on de novo synthesis of the nucleotides. The mitochondrial cytochrome bcj complex is a part of the electron transport. The enzyme consists of a cytochrome and a Rieske protein bound to an iron sulfur subunit. In the ubiquinol binding pocket, two electrons are transferred from ubiquinol via the subunit to cytochrome c heme iron (Scheme 21.3).

21.5.4.1 Naphtoquinones

The antimalarial naphtoquinones are developed from naturally occurring naphtoquinones such as, lapachol (21.18) (Figure 21.11). The problem of fast metabolism, however, prevented the clinical use. Among the several hundreds of napthoquinones synthesized and tested atovaquone (21.18) was finally selected for use. Atovaquone is assumed to bind to the ubiquinol oxidation pocket of the parasite and thereby prevent the electron transfer. Model studies performed on the yeast bc} complex suggest that a hydrogen bond between the hydroxyl group of atovaquone and nitrogen of His181 of yeast Rieske-protein and a hydrogen bond between Glu272 of bc} complex via a water molecule and one of the carbonyls of atovaquone stabilize the complex and thereby prevent transfer of the electrons to the iron-sulfur complex. Replacement of Leu275 with the more bulky Phe275 as found in bovine bc} prevents the binding of atovaquone in the pocket (Figure 21.12). Similar atovaquone only possesses a poor affinity for human cytochrome bc}.

Rapid development of resistance and a high rate of recrudescence necessitated the use of combination therapy. Proguanil (21.29) (refer to Figure 21.16)-atovaquone combination (Malarone®) is at the present an effective therapy for multidrug resistant falciparum malaria. Unfortunately, the high costs of this treatment limit its use.

FIGURE 21.11 Configurations of lapachol (21.18) and atovaquone (21.19).

21.19

21.19

FIGURE 21.11 Configurations of lapachol (21.18) and atovaquone (21.19).

His181

Rieske protein

His181

Rieske protein

Glu272

FIGURE 21.12 Suggested binding of atovaquone to the ubiquinol binding site.

Cytochrome i

Leu275

Glu272

FIGURE 21.12 Suggested binding of atovaquone to the ubiquinol binding site.

hn nh2

21.20

21.21

21.22

21.20

21.21

21.22

FIGURE 21.13 Configurations of methylene blue (21.20), primaquine (21.21), and tafenoquine (21.22).

21.5.5 Drugs with Nonestablished Targets

21.5.5.1 8-Aminoquinolines

Approximately 100 years ago, Paul Ehrlich (1854-1915) noticed a selective uptake and staining of tissues with dyes such as methylene blue (21.20) (Figure 21.13). Based on the pioneering idea, at that time, that this selective staining was caused by selective receptors for the dyes, he discovered that methylene blue had antimalarial activity. Elaborating of this idea led to the development of the 8-aminoquinolines, among which primaquine (21.21) is the more important.

Primaquine remains the only drug approved for the cure of vivax malaria. The 8-aminoquino-lines possess activity toward all stages of the parasite, including the hypnozoites in the liver and the gametocytes in the blood. Killing of hypnozoites prevents relapse, which is caused by the activation of hypnozoites resting in the liver. Relapse is pronounced for vivax malaria. Killing of gameto-cytes prevents transmission. The ability to affect all stages reveals that the mechanism of action of the 8-aminoquinolines must differ from that of the 4-aminoquinolines, which only affect parasites digesting hemoglobin (Section 21.5.1.1). Drawbacks of primaquine include a narrow therapeutic window, a short half-life (4-6 h), which requires repeated administration for 14 days to achieve a cure, and hemolysis and methemoglobin formation. The latter side effect is particularly pronounced in patients with an inborn deficiency of glucose-6-phosphate dehydrogenase, a genetic abnormality common in areas where malaria is endemic. Structure-activity relationships (SARs) have revealed that an appropriate substitution in the 2-position improved efficacy and decreased general systemic toxicity, a methyl group in the 4-position improved not only the therapeutic activity but also toxicity, and that a phenoxy group in the 5-position decreased toxicity and maintained activity. The studies led to synthesis of tafenoquine (21.22). The substituent in the 5-position provides tafenoquine with a half-life of 2-3 weeks. A long half-life is essential for the development of a single-dose oral cure for malaria.

The mechanism of action of the 8-aimonquinolines has not yet been established. It is suggested that the compounds might affect the calcium homeostasis, affect the mitochondria by causing oxi-dative stress, or act by a combination of these effects.

21.5.5.2 4-Quinolinemethanols

The quinolinemethanols might be considered as methanol substituted with 4-quinoline and an aliphatic substituent (Figure 21.14).

21.5.5.2.1 The Cinchona Alkaloids

The oldest representatives for the quinolinemethanols are (-)-quinine (21.23), (-)-cinchonidine (21.24), (+)-quinidine (21.25), and (+)-cinchonine (21.26). All four alkaloids are isolated from the bark of trees belonging to the genus Cinchona (Rubiaceae), often referred to as fever trees. Until isolation of quinine in large scale in 1820, the crude bark was the only efficient drug in Europe for

4-Quinolinemethanol

4-Quinolinemethanol

21.23 R=OCH3

21.23 R=OCH3

21.25 R=OCH3

21.25 R=OCH3

FIGURE 21.14 4-Quinoline methanols. Absolute configurations of (-)-quinine (21.23), (-)-cinchonidine (21.24), (+)-quinidine (21.25), and (+)-cinchonine (21.26).

N CF3 CF3

h3c h3c

f3c cl

21.27 21.28

FIGURE 21.15 Absolute configuration of (+)-mefloquine (21.27) and configuration of halofantrine (21.28).

treatment of malaria. The narrow therapeutic window of the cinchona alkaloids in combination with different concentrations of the alkaloids in the bark depending on species and time of harvesting made access to the homogenous compounds as a major therapeutic improvement. In spite of the several severe side effects including cardiac arrhythmia, insulin release causing hypoglycemia, and peripheral vasodilatation the cinchona alkaloids are still used for treatment of multiresistant malaria, most frequently in combination with tetracyclines. The treatment, however, requires careful supervision.

The mechanism of action of the cinchona alkaloids is unknown, but the target might be situated in the cytosol of the parasite. Clinically quinidine and cichonine (both 8R,9S) are two- to threefold more active than quinine and cinchonidine (8S,9R). Quinidine and cinchonine, however, should be used only in the case of shortage of quinine because of a narrow therapeutic window. The importance of the stereochemistry at C8 and C9 is illustrated by the lack of activity of 9-epiquinine (8S,9S).

21.5.5.2.2 Mefloquine

Mefloquine (21.27) (Figure 21.15) was selected among 300 quinolinemethanols prepared in order to develop agents that are effective against chloroquine resistant agents. Mefloquine is marketed as a 1:1 mixture of the two diastereomeric racemic pairs. The four stereoisomers show similar ability to inhibit hemazoin formation in vitro. The pharmacokinetic, however, is different for the two since the half-life of the (-)-isomer (enantiomer of 21.27) is significantly longer than that of the (+)-isomer of 21.27.

The 2,8-bis-trifluoromethyl arrangement proved to be the most active of the series. Compounds that possessed 2-aryl groups were found to have augmented antimalarial activity, but at the same time unacceptable phytotoxic side effects. Mefloquine has been marketed as Lariam, a drug that has serious hallucinogenic side effects in some patients. Resistance against mefloquine has led to the use of combination therapy using mefloquine and arteminisin derivatives.

21.5.5.3 Phenanthrenemethanols

The dibutylaminopropyl groups of halofantrine (21.28) (Figure 21.15) were found to give optimal antimalarial effect in the 9-phenanthrene system. Even though the evidence for the mechanism of action for this compound is less convincing than that for chloroquine the findings that mefloquine only affects erythrocytic stages of the parasite and that some studies show association with hema-zoin support the suggestion that halofantrine acts by preventing detoxification of hemazoin. Like quinine halofantrine can induce cardiac arrhythmias.

21.5.6 Drugs Targeting Folate Synthesis

Tetrahydrofolic acid is an important coenzyme in parasites as well as their hosts. The coenzyme is involved in the biosynthesis of thymine, pyrine nucleotide, and several amino acid syntheses. Malaria parasites are dependent on de novo folate synthesis (Scheme 21.4) whereas mammalian cells take up fully formed folic acid as vitamin B9. Consequently, dihydropteroate synthase is absent in humans. In the mammalian as well as in parasitic cells, the precursors [folate or dihydrofolate (21.32), respectively] have to be reduced to the enzymatically active tetrahydrofolate, a reaction that is catalyzed by dihydrofolate reductase (DHFR). DHFR and thymidylate synthase are separate enzymes in mammalians, whereas they are covalently linked to one bifunctional enzyme (DHFR-TS) in protozoan parasites. The binding site of dihydrofolate in DHFR-TS is sufficiently different from the binding site in the human DHFR to allow selectivity. The binding site of DHFR-TS inhibitors like cycloguanil (21.30) (Figure 21.16) and pyrimethamine (21.31) and the enzyme is illustrated in Figure 21.17 using pyrimethamine as an example. Proguanil (21.29) will metaboli-cally be converted into cycloguanil in the liver. The negatively charged carboxylate of Asp54 of the enzyme binds to the positively charged amino group of pyrimethamine. The 4-amino group forms hydrogen bonds with the backbone carbonyl groups of Ile14 and Ile164. The coenzyme of DHFR, NADPH, is oriented through a hydrogen bond to Ser108.

Sulfadoxine (21.33) (Figure 21.18) and dapsone (21.34) act as antimetabolites of para-aminobenzoic acid (21.35) (Scheme 21.4), which is a building block in the dihydrofolate synthesis. An antimetabolite is an agent, which prevents the incorporation of a structural related endogenic metabolite.

Melarsopol Charged

Tetrahydrofolic acid

Dihydrofolic acid (21.32)

SCHEME 21.4 Simplified folate pathway.

Tetrahydrofolic acid

Dihydrofolic acid (21.32)

SCHEME 21.4 Simplified folate pathway.

ch3 21.29

Liver

h2n^x

21.30

21.31

FIGURE 21.16 Metabolic conversion of proguanil (21.29) into cycloguanil (21.30) and configurations of pyrimethamine (21.31).

Asp54

FIGURE 21.17 Binding of pyrimethamine (21.31, Figure 21.16) to the dihydrofolate binding site of dihy-drofolate reductase (DHFR). The binding is stabilized through hydrogen bondings between the carboxylate of Asp54 and the positively charged NH group of the 2-amino group and the nitrogen of the pyrimidine ring, of hydrogen bonds between 4-amino group and the backbone carbonyl groups of Ile14 and Ile164. In addition, a charge-transfer interaction between the chlorophenyl residue and the dihydropyridine ring of the NADPH and finally a hydrogen bond between the Ser108 and one of the hydrogen acceptors at the NADPH molecule stabilize the complex. (R = the remaining part of the NADPH molecule.)

H ILe164 H

FIGURE 21.17 Binding of pyrimethamine (21.31, Figure 21.16) to the dihydrofolate binding site of dihy-drofolate reductase (DHFR). The binding is stabilized through hydrogen bondings between the carboxylate of Asp54 and the positively charged NH group of the 2-amino group and the nitrogen of the pyrimidine ring, of hydrogen bonds between 4-amino group and the backbone carbonyl groups of Ile14 and Ile164. In addition, a charge-transfer interaction between the chlorophenyl residue and the dihydropyridine ring of the NADPH and finally a hydrogen bond between the Ser108 and one of the hydrogen acceptors at the NADPH molecule stabilize the complex. (R = the remaining part of the NADPH molecule.)

21.33

OCH, h2n

OCH,

FIGURE 21.18 Configurations of sulfadoxine (21.33) and dapsone (21.34). Compare these structures with para-aminobenzoic acid (21.35 Scheme 21.4).

21.6 RESISTANCE

Treatment of malaria frequently fails because of the development of resistance. In fact, regular mutations of the parasites force continued development of new drugs. Resistance develops in different ways e.g. mutations cause changes in the target proteins preventing interaction with the drug, or a transport system develops, which decreases the concentration of the drug at the target site.

21.6.1 Chloroquine Resistance

Chloroquine resistance has been correlated to a mutation in a wild-type food vacuolar membrane protein termed P. falciparum chloroquine resistance transporter (PfCRT). A mutation replacing Lys76 with Thr enables the protein, in an energy-dependent manner, to transport chloroquine out of the food vacuole thus, decreasing the chloroquine concentration to below the pharmacologically active concentration. Other 4-aminoquinolines might also be substrates for the transporter explaining cross resistance. Chloroquine analogs with a modified side chain are developed in order to make analogs, which are not substrates for the transporter.

A number of other hypotheses for resistance including an increased value of the pH in the food vacuole or prevention of association between chloroquine and heme cannot be excluded.

21.6.2 4-Quinolinemethanol Resistance

The membrane transport P-glycoprotein pump, PfMDR1, which is an analog of the mammalian ABC multidrug-transporter, has a central role in the resistance development of P. falciparum parasites. An increased number of PfMDR1 transporters facilitate removal of the drug from the putative target in the cytosol.

21.6.3 Antifolate Resistance

Resistance toward antifolate drugs is caused by mutations that alter the active site resulting in different binding affinities for different drugs. The resistance conferring mutations occur in a stepwise sequential fashion with a higher level of resistance occurring in the presence of multiple mutations. The decreased affinity for the drug often is followed with a decreased activity for the natural substrate, suggesting the parasites containing mutated forms of the enzyme might be selected against in the absence of drug. Mutation of Ser108 into Asn causes steric interaction between the Asn side chain and the chlorophenyl group of pyrimethamine and thereby reduces the affinity of pyrimethamine to PfDHFR (Figure 21.17). This mutation, however, only causes a moderate loss of susceptibility to cycloguanil (21.30), in which the side chain is shorter. Additional replacement of Asn51 into Ile results in a higher pyrimethamine resistance but only a moderate effect of cyclogua-nil. On the contrary, replacement of Ser108 into Thr coupled with Ala16 into Val confers resistance to cycloguanil but only modest loss of susceptibility to pyrimethamine.

21.7 CONCLUDING REMARKS

A series of examples of drugs targeting biological systems present only in parasites has been given. In principle, addressing targets not present in the host but essential for the survival of the parasite should give a therapy without side effects. Unfortunately, very few drugs are truly selective. Quinine, as an example, has targets in the parasite cytosol, but does also cause a number of effects in the patient such as insulin release, inducing severe hypoglycemia, and cardiac arrhythmia. Another serious problem in the treatment of parasitic diseases is the development of resistance, which is addressed by giving a combination of drugs. Artemisin and derivatives of artemisinin are given in combination with, e.g., proguanil. A further advantage of combination therapy is prevention of recrudescence, which in particular is a problem after treatment with artemisinin.

In this chapter, no attempt has been made to include all of the drugs or putative targets.

FURTHER READINGS

Azzouz, S., Maache, M., Garcia, R. G., and Osuma, A. 2005. Leishmanicidal activity of edelfosine, miltefosine and ilmofosine. Pharmacology & Toxicology 96: 60-65.

Cook, G. C. and Zumla, A. I. (eds.). 2003. Manson's Tropical Diseases, 21st edn. London: Elsevier Science.

Croft, S. L., Barrett, M. P., and Urbina, J. A. 2005. Chemotherapy of trypanosomiasis and leishmaniasis. Trends in Parasitology 21: 508-512.

Egan, T. J. 2004. Haemozoin formation as a target for the rational design of new antimalarials. Drug Design Reviews—Online 1: 93-110.

Rosenthal, P. J. (ed.). 2001. Antimalarial Chemotherapy. Mechanism of Action, Resistance, and New Directions in Drug Discovery. Totowa, NJ: Humana Press Inc.

Schlitzer, M. 2007. Malaria chemotherapeutics. ChemMedChem 2: 944-986.

Willcox, M., Bodeker, G. and Rasoanaivo, P. (ed.). 2004. Traditional Medicinal Plants and Malaria. Boca Raton, FL: CRC Press.

Immunomodulating Agents

Ulla G. Sidelmann CONTENTS

22.1 Introduction 359

22.2 Brief Introduction to the Immune System 360

22.2.1 Cells of the Immune System 360

22.2.1.1 B Cells 361

22.2.1.2 T Cells 361

22.2.2 Antibodies 363

22.2.3 Natural Killer Cells 363

22.3 Diseases of the Immune System 364

22.3.1 Autoimmune Diseases 364

22.3.2 Inflammation 365

22.3.3 Cancer Immunology 365

22.4 Immunosuppressive Agents 366

22.5 Immunomodulating Biologies 368

22.5.1 Recombinant Protein and Engineered Proteins 368

22.5.2 Monoclonal Antibodies 369

22.5.3 Cytokines 369

22.5.3.1 Interferons 369

22.5.3.2 Tumor Necrosis Factor 370

22.5.3.3 Interleukins 370

22.5.4 B Cell Depletion 372

22.5.5 Targeting T Cells 373

22.6 Prospects of Immune Modulating Agents 373

Further Readings 373

22.1 INTRODUCTION

The purpose of the immune system is to combat infectious diseases caused by bacteria and viruses; however, evidence suggests that the immune system plays a central role in protecting the body against cancer and in combating cancer that has already developed. When the immune system is weakened, it can be more easily overwhelmed by cancerous cells. Cancer may occur when the immune system can no longer defend against invading tumor cells. There is evidence in many cancer patients that rebuilding the immune system slows down the growth and spread of tumors.

The immune system can also lead to pathological consequences for the individual. The first example is the normal attack of a healthy immune response on a transplant leading to transplant rejection. The second example is when tolerance to self break downs and self tissues are attacked by the immune system leading to autoimmune disease. The third example is when the immune system responds dramatically to otherwise harmless antigens leading to allergy or hypersensitivity.

Immunomodulating agents can be separated into agents that suppress or block the immune system when it is reacting against self antigens, hyperreacting or in relation to transplantation (autoimmune

diseases, allergy/asthma, inflammation, and transplantation), agents that stimulate or activates the immune system to work harder or smarter (virus infections and cancer), and finally the removal of unwanted cellular subtypes of the immune system by depletion via specific surface antigens (autoimmune disease and cancer).

This chapter will give an overview of the key cellular and molecular drivers of the immune system. The following molecular approaches to treat diseases will then be covered: general immune suppression, depletion of B cells, modulation of T cells, and finally cytokines. Focus will be on autoimmune diseases, inflammation, and cancer.

22.2 BRIEF INTRODUCTION TO THE IMMUNE SYSTEM

The immune system has evolved to fight antigens invading the human body. Antigens are nonself entities, e.g., parts of bacteria, virus, parasites, foreign materials such as splinters and host generated threats such as cancer. An immune response can be divided into two phases: (1) the recognition of the antigen and (2) the elimination of the antigen.

There are two types of immune responses, namely, an innate response and an adaptive response both capable of distinguishing between self and nonself antigens but with different mechanism and specificity. The innate and the adaptive immune responses are interdependent and elements of one response enhance the other. The innate response is nonspecific and is designed to recognize molecular patterns common to a wide variety of pathogens. On second encounter of a pathogen, the response is exactly the same as the first. The response involves physical, chemical, and molecular barriers that distinguish and exclude the antigens. The adaptive immune response involves highly specific recognition and effector actions involving a variety of cells in the body. Unique antigens from the foreign entity will be specifically recognized by receptors expressed on B and T cells and an effector action against the entity is initiated. On second attack by the same entity, a faster and much stronger response called a memory adaptive response is induced, which will prevent disease from occurring the second time.

22.2.1 Cells of the Immune System

The cells mediating the immune response are the leukocytes or white blood cells. The innate immune response is constituted by (1) granulocytes, that by phagocytosis, can engulf and destroy pathogens; (2) monocytes that can differentiate into macrophages; and (3) dendritic cells (DCs) that are also capable of phagocytosis and secretion of cytokines and growth factors that activate T cells in the adaptive immune system. Macrophages and DCs can present antigens to the T and B cells and fall into the category of antigen presenting cells (APCs).

B cells and T cells are lymphocytes, and are mediators of the adaptive immune response. Although mature lymphocytes look alike, they are diverse in their functions. B cells are produced and mature in the bone marrow, whereas the precursors of T cells leave the bone marrow and mature in the thymus (which accounts for their designation). The specificity of binding is defined by their respective receptors for antigens, the B cell receptor (BCR) and the T cell receptor (TCR), respectively.

Both BCRs and TCRs are integral membrane proteins, present in many identical copies, and exposed at the cell surface. They are present before the cell encounters an antigen and characterized by a unique binding site. A portion of the antigen called an epitope binds to the binding site, through noncovalent interactions. Successful binding of the antigen receptor to the epitope (accompanied by additional signals) will result in stimulation of the cell to enter the cell cycle. Repeated mitosis leads to the development of a clone of cells be

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