WO2000078268A2

WO2000078268A2 - Potentiation of chloroquine

Info

Publication number: WO2000078268A2
Application number: PCT/SD2000/000004
Authority: WO
Inventors: Elnour Abdel Magid Osman
Original assignee: Elnour Abdel Magid Osman
Priority date: 1999-06-20
Filing date: 2000-06-01
Publication date: 2000-12-28
Also published as: AU4961600A

Abstract

Administration of high doses of the vitamin B members; nicotinamide, thiamine, riboflavine, pyridoxine, pantothenic acid and B12 combined with chloroquine warranted a 100 % sustainable cure in human volunteers infected by malaria.

Description

POTENTIATION OF CHLOROQUINE

INTRODUCTION

The role of vitamin B complex in malaria appears to be

aggravative in general. The deliberate elimination of certain vitamins

from the host results in marked diminution of the multiplication of

malaria parasites in the blood stream, and if these substances are injected

into the depleted animals, the parasitaemia will immediately rise. This

applies particularly to biotin, thiamine, and riboflavine¹. It was

repeatedly demonstrated that the withdrawl of coenzyme A from the host

will abolish parasitaemia . In chicks, R Loyhurae infection was

aggravated by nicotinic acid deficiency whereas this did not affect the

symptoms in infected ducks³.

Such approaches led to the administration of some vitamin B

antagonists in malaria treatment and prophylaxis e.g. d-pantoyltaurine

(pantothenic acid antagonist) , and pyrimethamine (folic acid

antagonist)⁵.

Resistance to chloroquine has become a grave problem.

Chloroquine resistance has been associated with a decrease in drug-

concentrating capacity of red cells infected with malaria parasites⁶, and

changes in high-affinity binding sites for the drug⁷. A hypothesis

describing the mode of membrane permeability alteration , which involves several members of the vitamin B group, was tested, at the

macro level, in this context.

Experimental

Thirty two volunteers from both sexes (age group 20-45 years)

with positive malaria blood film were devided into two equal groups.

The control group (16 patients) recieved only chloroquine whereas the

treated group recieved, in addition to chloroquine, the vitamin B complex

supplement. Blood films for malaria were performed on the second day

of the end of treatment.

Chloroquine:

Chloroquine tablets were obtained from the local market

(Amipharma Co., Sudan). 3 grams of chloroquine were administered to

both group as per clinically practiced.

Vitamin B Complex Supplement

The required high doses of thiamine, riboflavine, pantothenic

acid, nicotinamide, pyridoxine and vitamin B_!2 were formulated from

pharmaceutically available vitamin B preperations (Dumex Ltd,

Danemark). Results and Discussions

Table 1: Effect of vitamin B supplementation on response of

malaria patients to chloroquine.

Descriptive statistics of table (1) indicate a 100% cure of malaria

infection as a result of vitamin B supplementation of chloroquine.

Chloroquine resistance was demonstrated in many areas of Sudan since

1978. It has been shown to vary from one year to the other. The

percentage of resistance (1986-1995) fluctuated between 5-46%,

respectively in Gedarif hospital⁹. However, our results indicate more than

60%) irresponsiveness to chloroquine in Khartoum area. Moreover, the

patients who recieved the vitamin B supplement did not relapse or

contract a new infection up to one month after the end of the treatment.

Malaria Pathogenesis

A malaria toxin, associated with the tissue schizogony, was

identified as lecithinase¹⁰. In vitro, lecithinase, deoxycholate and organic solvents release serum albumin from the liver microsomal fraction,

causing it to appear in the supernatant fluid". Such displacement of

serum albumin from microsomes might be the cruiacial event in malaria

pathogenesis in vivo. It might trigger a cascade of metabolic events thus

establishing the infectious process. A prominant feature of such events is

increased membrane permeability and the consequent invasion of

parasites. Bovine serum albumin lowered the plasmodial penetration and

• 1 ------ infectivity in avian erythocytes in vitro . The molecular basis of the

effect of serum albumin on membrane permeability is discussed in

another report⁸.

Lecithinase releases the unsaturated fatty acids from position 2 of

phospholipid molecules yielding lysophospholipids, the powerful

• 1 "X haemolytic agents able to dissolve cell membranes . This is probably the

mode by which lecithinase displaces serum albumin from microsomes.

Each membrane has its own characteristic spectrum of phospholipid

molecules; the fatty acid residues of these phospholipids have

characteristic degree of unsaturation¹ . There is great specifity in the

composition of phospholipids which is relevant to the properties of

membranes (permeability, stability, etc.). It was surmised that any

alteration of the lipid component of membranes might secondarily affect

the activity of enzymes residing in those membranes . Lecithinase might, therefore, activate membraneous enzymes such as Pyridine

Nucleotides Phosphorylsis Enzymes (PNPE)¹⁶. Infection increases

17 consumption of coenzymes I and II .

In malaria, a direct correlation between the number of parasites

and liver damage on one hand, and elevated levels of alkaline

phosphatase, a member of (PNPE), was reported¹⁸. Alkaline phosphatase

cleaves NADP, an important coenzyme in fatty acid synthesis. It also

hydrolyse glycerophosphate¹ , a key intermediate in phospholipid

synthesis. Alkaline phosphatase might, therefore, increase membrane

permeability and pave the way for entry of parasites. However, with the

view of the central role of the liver in phospholipid synthesis , such

incidence might cause export of defective phospholipids to other tissues

and consequently increased membranes permeability.

The consumption of coenzymes I and II in infection precipitate

9 i deficiency of nicotinic acid . It should be emphasized that such a

deficiency and the following ones might occur at the level of the

physiologic form of the vitamins. Nicotinic acid deficiency, reduces

riboflavine stores; lack of riboflavine then reduces biosynthesis of tissue

folate²². Riboflavin deficiency, in turn, can lead to pantothenate

deficiency²³. Riboflavine is a cofactor in the synthesis of pyridoxal

phosphate²⁴. Nutritional factors might provoke similar vitamin B coenzymes deficiencies. Inadequate dietary protein intake may also lead ^ to a deficiency of vitamins such as thiamine , poor utilization of

riboflavine²⁶ and poor retention of riboflavine in the liver²⁷. The

previously mentioned interrelations might come to action leading to loss

of the physiologic forms of other vitamin B coenzymes. As the most

important aspect in the regulation of albumin production is nitrogen

98 • intake it is conceivable how chronic malnutrition is associated with a

low level of albumin synthesis and this disorder is certainly complicated

9Q by infection . In rodent malaria, during the normal course of infection,

there is a constant decrease in albumin by 33%>³⁰. It is probable that the

inactivation of vitamin B coenzymes, as suggested, might be responsible,

at least in part, for the decreased albumin levels. Pantothenic acid

deficiency caused a marked decrease of serum albumin in rats .

19

Riboflavine deficiency in baboons and absence of pyridoxine in high-

fat diets also lowered serum albumin levels.

The metabolic requirements of the tissue stages of the

Haemospordia in the vertebrate must be closely linked with type of host

cell, the parenchyma cell of the liver is rich in metabloites e.g. vitamin

B₁₂, and actively engaged in bile secretion . It is probable that during

tissue schizogony, liver vitamin Bι₂ is exhaused and/or bile secretion is

altered. Altered bile secretion might elevate deoxycholate levels by the action of intestinal flora. Deoxycholic acid was observed to induce

resistance to the antimicrobial agent furazolidone in vitro²⁵. Pyridoxine

^"deficiency might also contribute to diminished level of Bι₂.

Malabsorption of orally administered vitamin B₁₂ to rats might occur in

pyridoxine deficiency . In vitamin Bι₂ deficiency in the rats there is a

decrease in serum albumin and an increase of beta globulins³⁷. A typical

decreased serum albumin and elevated beta globulins ' are reported

in malaria. Increases in some globulins and decreases in albumin,

increase sedimentation rate of red cells⁴⁰. The ESR is constantly and

significantly elevated in malaria⁴¹. However, in vitamin Bι₂ deficiency;

the incorporation of nicotinamide into rat liver DPN was reduced⁴² and

low values of tissue phospholipids were reported⁴³. Administration of

vitamin Bι₂ to rats increased haemoglobin and serum albumin⁴⁴.

P. Berghei was reported to require large quantities of nicotinic

acid for its metabolism and infected erythrocytes were found to contain

much more of this substance than uninfected ones; the vitamin is

extracted from organs such as the liver and the depletion of nicotinic acid

from their cells constitutes a severe "biochemical lesion⁴⁵". In fact, the

probable Ca⁺²-induced inhibition of liver glutamine synthetase by the

parasite, as described in the hypothesis⁸, might hinder the amination of

dietary nicotinic acid in the process of pyridine nucleotides synthesis ⁶. Nicotinic acid might accumulate as a result of the poor excretion due to

inavailability of the physiologic forms of B coenzymes as described later.

It might induce many metabolic events favourable for the parasite and

adverse to the host. More information on altered nicotinic acid

metabolism in diseases in discussed in the hypothesis⁸. However, malaria

parasites were observed to mediate the transport mechanisms controlling

the influx of L-glutamine across the host cell membrane in erythrocytes

infected with human malaria . Such descsribed diminshed synthesis of

pyridine nucleotides might consequently impair the synthesis of fatty

acids, phospholipids, and vitamin B coenzymes. Nicotinic acid, when

injected I.V., induces a striking rise in indirect serum bilirubin in normal

and those with hepatic diseases . Increased urinary urobilin was also

noted⁴⁹.Typically, in malaria, the content of direct bilirubin in the blood

serum elevates⁵⁰, and increased urinary excretion of urobilin occurs⁵¹. As

serum albumin binds strongly to bilirubin prior to its conjugation,

nicotinic acid might, therefore, depress the levels of free serum albumin

necessary for other biological functions. It is claimed that nicotinic acid

possesses blood coagulation properties possibly through its haemolytic

action⁵². Haemolytic anaemia⁵ and parasitogenic thrombi⁵⁴ in malaria

might, therefore, be attributed to nicotinic acid. Consequently,

haemoglobin becomes available for the parasite. However, the excretion of nicotinic acid and its metabolites is affected in certain diseases and is

greatly reduced in most conditions⁵⁵.

Another metabolic event which contributes to increased

membrane permeability is the observed hypocholesterolaemia which

associates malaria infection ' . The common role of cholesterol in

membranes is to reduce the permeability of the lipid barrier of

membranes, and erythrocytes depleted 30-40%> of their membrane

cholesterol become more permeable and also less stable⁵⁸. The possible

involvement of displaced and decreased serum albumin in

hypocholesterolaemia is discussed in the hypothesis .

However, the infectous parasite by deactivating coenzymes I and

II of the host cell, as described, might induce inavailability of the

physiologic forms of the other vitamin B cofactors. The host cell is,

therefore, rendered unable to utilize the nutrients whereas the parasite is

capable of that. Such arguements might probably materialize the concept

of parasitism. They also explain why the addition of several of the B

vitamins aggravates parasitaemia as they appear to be inconvertable to

the physiologic form by the host and readily available for the parasite,

unless cleavage of coenzymes I and II is arrested. However, the

symptoms of the inavailability of the physiologic forms of vitamin B

coenzymes might resemble that of dietary deficiency. The involvement of organs and systems in malaria might reveal a

more clear picture of a multiple vitamin B complex deficiency. Similarity

of the clinical picture of algid malaria to that seen in acute adrenal

insufficiency was reported . Adrenals from pantothenic acid-deficient

rats were functionally insufficient⁶⁰. A typical pantothenic acid

deficiency symptom is the adrenal necrosis and haemorrhage⁶¹; which

was observed in malaria

.

The malaria associated leucopenia⁶³ might be attributed to

riboflavine deficiency⁶⁴. Riboflavine deficiency⁶⁵ might also explain the

eye lesions of malaria⁶⁶.

Malaria has an immunosuppressive action . Severe impairment

of antibody response was observed in pyridoxine deficiency⁶⁸. The same

deficiency impaired erythropoiesis⁶⁹. A temporary depression of

7fϊ erythropoiesis was reported in malaria . A similar mode of parasitism

might probably be valid at the level of the invertiberate host as supported

by the identification of xanthurenic acid as the putative inducer of

malaria development in the mosquito . Pyridoxine deficiency increased

719 1 xanthurenic acid exretion in rat and man .

Psychic manifestations⁷ , nausea and diarrhoea⁷⁵ in malaria might

reflect nicotinic acid deficiency as its administration controls these

symptoms⁷⁶. Malaria patients display bradycardia or tachycarida⁷⁷. The heart

78 rate of vitamin Bi deficient rats was lower than normal animals , while

7Q tachycardia is relieved by thiamine . However, owing to the complex

nature of vitamin B interrelations, the symptoms of such a multiple

deficiency in different tissues might be atypical to the symptoms of the

difficiency of a single vitamin.

Chloroquine is preferentially accumulated within parasitized

erythrocytes to the extent of at least two orders of magnitude greater than

• SO the concentration occurring outside . The mechanism of this

accumulation is unclear, the hypothesis envision the importance of ionic

or potential gradient , or the participation of high affinity receptor

R9 sites . Chloroquine resistance has been associated with a decrease in

drug contentrating capacity of red cells infected with malaria parasites

and changes in high affinity binding sites of the drug⁸⁴. Drug resistant

parasites lose the capacity to bind chloroquine to a great degree: thus, in

the red blood cells invaded by them the chloroquine concentration is six

times lower as compared to the erythrocytes infected with sensitive

cells⁸⁵. It is most likely that chloroquine resistance might be regarded as

type I resistance (exclusion from the site). The physical basis for type I

resistance is suggested by the observation that the cell membrane can

adjust its net charge by varying the proportion of phosphatidyl-glycerol (anionic) to lysophosphatidyl-glycerol (cationic) . Anionic or cationic

drug can be excluded by the operation of Coulomb's law.

Such modifications of chloroquine high affinity receptors might

be induced under our hypothetical conditions, by diminshsed fatty acid

synthesis, elongation and desaturation owing to the inactivation of the

codehydrogenases. Lecithinase might also induce such modifications.

Modified phospholipid synthesis might prevail. It was observed that

Pneumococci which had become resistant to mepacrine or acriflavine

readily lost dehydrogenase activity on dilution or warming and that the

activity was restored on addition of riboflavine; susceptible cells did not

exhibit this behavior. Riboflavine content of both types of cells were

H7 approximately the same . Such a finding is suggestive to the link

between resistance, drug binding sites and electron transfer chain. It also

points out to the inavailability of the physiologic form of riboflavine and

not the vitamin precursor of the coenzyme. It might be concluded that the

drug binding sites might be modified in the infected tissue and the

resistant strains.

However, chloroquine increased serum albumin levels in

rehumatoid arthrititis which is characterized by low serum albumin

levels⁸⁹. Similarly, chloroquine might hinder the invasion of erythrocytes

by malaria parasites as bovine serum albumin lowered plasmodial penetration and infectivity in avian erythrocytes in vitro⁹⁰. A new mode

of chloroquine antimalarial activity, via elevation of serum albumin

levels, is suggested.

The mode of chloroquine potentiation by the administered

vitamin B members include the previously discussed deficiency disorders

in addition to other specific reactions. Nicotinamide is capable of

inhibiting DPNase which cleaves the pyridine nucleotides at the

nicotinamide riboside linkage⁹¹. The enzyme is localized in the

membranous structures, being present in tissues in microsomes and in

> 99 erythrocytes in the stroma . Nicotinamide is also capable of inhibiting

Q9 nicotinamide riboside phosphorylase which is present in mammalian

livers and the soluble cytoplasm of human erythrocytes⁹⁴. Such dual

inhibition of the two members of PNPEs might arrest inactivation of

coenzymes I and II and the consequent inavailability of B coenzymes.

Nicotinamide might be utilized for the synthesis of pyridine nucleotides

thus improving fatty acid and membrane phospholipids synthesis.

Nicotinamide was shown to decrease the permeability of human

erythrocytes to glucose⁹⁵. Glucose is essential for malaria parasites in

vitro⁹⁶ and extra glucose tends to enhance parasitaemia in vitro .

Nicotinamide has the ability to increase gastric secretion, including free and total acid . It might, therefore, depress intestinal synthesis of

deoxycholic acid⁹⁹.

Pyridoxine, riboflavine, thiamine¹⁰⁰; pantothenic acid¹⁰¹ and

109 • nicotinamide were administered to accelerate disposal of nicotinic acid

thus depriving the parasite of this essential nutrient. However, it is

probable that the suppressive effect of milk diet on parasitaemia¹⁰³, might

be related to its content of the B vitamins¹⁰⁴. It might also be related to

the trytophan present in lactalbumin which appears readily available for

conversion into niacin . Moreover, tryptophan promotes serum albumin

synthesis¹⁰⁶. With growth of malaria parasites inhibited by milk diet,

changes in albumin is less marked

. The loss of protection against

malaria with age in off-springs of immune mice, might be explained by

I DS the fall of serum albumin levels with age . The decreases in serum

albumin caused by organic solvents¹⁰⁹ and different types of irradiation

¹¹⁰ might incriminate the environmental crisis in the resistance

phenomenon. However, the previous discussionson serum albumin are

oversimplified and might be tricky. The molecular mechanisms described

in the hypothesis are certainly more complex. Funds and molecular

techniques are needed to expand the project. Acknowledgement

This research was completely financed and mostly carried out by

the integrated clinic of the International Faith Research Centre in

Khartoum.

Conclusions

- Malaria pathogenesis is related, in no small way,, to the malaria toxin

lecithinase which hydrolyse membrane phospholipid and dislocate

serum albumin.

- Lecithinase induces inavailability of the physiologic forms of vitamin

B cofactors mainly by increasing membrane permeability.

- Loss of B coenzymes might lead to many metabolic disorders among

which altered membrane phospholipid synthesis. Chloroquine high

affinity binding sites are thus affected and resistance occurs.

- Chloroquine resistance, might therefore, be induced by persistant low

parasitaemia, altered bile acid metabolism, environmental and

nutritional factors.

- Pathogenesis and treatment of infectous diseases might follow a similar

line of reasoning. The concept might also be valid in other non-

infectous diseases. The whole issue might have been unintegerated.

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Claims

C A I M S

Formulation and Commercial Production of the new vitamin recipe alone or combined with chloroquine.