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\begin{document}

\title{Methaemoglobinaemia and the radical curative efficacy of
8-aminoquinoline antimalarials}



\author[1]{Nicholas White}%
\author[2]{James Watson}%
\author[3]{Kevin Baird}%
\affil[1]{Faculty of Tropical Medicine, Mahidol Iniversity}%
\affil[2]{Mahidol University Faculty of Tropical Medicine}%
\affil[3]{Eijkman Institute for Molecular Biology}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Methaemoglobin results from the oxidation of ferrous to ferric iron in
the centre of the haem moeity of haemoglobin. The production of
dose-dependent methaemoglobinaemia by 8-aminoquinoline antimalarial
drugs appears to be associated with, but is not directly linked to
therapeutic efficacy against latent vivax and ovale malarias. Iatrogenic
methaemoglobinaemia may be a useful pharmacodynamic measure in
8-aminoquinoline drug and dose optimization.%
\end{abstract}%



\sloppy


\textbf{Methaemoglobinaemia and the radical curative efficacy of
8-aminoquinoline antimalarials}

Nicholas J White\textsuperscript{1,2}, James A
Watson\textsuperscript{1,2}, J Kevin Baird\textsuperscript{2,3}

\begin{enumerate}
\tightlist
\item
  Mahidol Oxford Research Unit, Faculty of Tropical Medicine, Mahidol
  University, Bangkok, Thailand .
\item
  Centre for Tropical Medicine and Global Health, Nuffield Department of
  Medicine, University of Oxford, Oxford, United Kingdom.
\item
  Eijkman-Oxford Clinical Research Unit, Eijkman Institute for Molecular
  Biology, Jakarta, Indonesia
\end{enumerate}

* WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

The 8-aminoquinoline antimalarials are the only drugs which can prevent
relapses of \emph{P. vivax} and \emph{P. ovale} malaria (radical cure)
but no pharmacodynamic correlates of this activity have been identified.
The 8-aminoquinolines cause dose dependent methaemoglobinaemia, and they
cause haemolysis in patients with G6PD deficiency.

* WHAT THIS STUDY ADDS

Review of data accrued over the past 70 years indicates that
methaemoglobinaemia is correlated with radical curative activity.
Methaemoglobinaemia could be used to optimize 8-aminoquinoline
antimalarial dose regimens.

\textbf{Abstract}

Methaemoglobin results from the oxidation of ferrous to ferric iron in
the centre of the haem moeity of haemoglobin. The production of
dose-dependent methaemoglobinaemia by 8-aminoquinoline antimalarial
drugs appears to be associated with, but is not directly linked to
therapeutic efficacy against latent vivax and ovale malarias. Iatrogenic
methaemoglobinaemia may be a useful pharmacodynamic measure in
8-aminoquinoline drug and dose optimization.



\textbf{Introduction}

Hepatic latency in vivax and ovale malarias requires treatment with
8-aminoquinoline drugs in order to prevent recurrent attacks called
relapses (a therapy called radical cure). The first 8-aminoquinoline
(variously named plasmoquine, plasmochin, or pamaquine) was discovered
nearly one hundred years ago. Within four years of the reported
discovery and introduction, Sinton and colleagues working in Northern
India discovered that the combination of quinine and plasmoquine was
effective in preventing late recurrences (presumed relapses)
of\emph{Plasmodium vivax} malaria (1,2). This efficacy against late
attacks of vivax malaria was very slow to be accepted by international
authorities (notably the malaria commission of the League of Nations)
and malaria experts. Plasmoquine was not well tolerated at the doses
required for radical cure-- abdominal pain and vomiting were dose
limiting, and ``cyanosis'' was noted with higher doses (3-5). It was
also observed that about 10\% of patients of African or Asian origin
developed significant haemolytic anaemia (5). The cyanosis following
pamaquine therapy was caused by methaemoglobinaemia, and the sporadic
severe haemolytic anaemia was later identified as resulting from the
oxidant drug susceptibility of erythrocytes with reduced
glucose-6-phosphate dehydrogenase (G6PD) activity. During the Second
World War recurrent vivax malaria in the Indo-Burman and Pacific
theatres of war was a major threat to soldiers on both sides. An
intensive research effort, based in the United States, set out to
discover more effective and better tolerated 8-aminoquinolines.
Pharmacometric studies in volunteers infected with \emph{P.
vivax}(notably the frequent relapse Chesson strain originating in New
Guinea) and in rhesus monkeys infected with \emph{P. cynomolgi}
continued after the end of the Second World War, and ultimately led to
the replacement of pamaquine by primaquine in 1951 during the Korean war
(6). These large and detailed pioneering volunteer studies recorded a
wealth of valuable information.

\emph{Relapse in experimental and naturally acquired vivax malaria}

In the experimental malaria studies, non-immune volunteers were infected
either by blood taken from a malaria patient or by infected mosquito
bites. The volunteers were then observed daily, usually for at least one
year (many were prison volunteers). Building on the extensive
experiences of malariatherapy of neurosyphilis during the early to
mid-20\textsuperscript{th} century, which had refined and standardized
the methods of infection and clinical management, at least five and
usually ten infected (i.e. sporozoite positive) laboratory reared
anopheline mosquitoes were used to initiate the infection. This ensured
a substantial sporozoite inoculum was delivered to the recipient. With
the Chesson strain of \emph{P. vivax} the consequent early relapse rate
was 100\% (7).

This high relapse rate in experimental malaria should be contrasted with
natural infections in which a single mosquito, with an infection of
variable age (and thus transmission potential), infects an individual
who may already have significant natural immunity dampening illness (8).
This distinction is important because it has a substantial bearing on
the binary outcome therapeutic response to 8-aminoquinolines i.e.
relapse or no relapse. Very high relapse rates usually result from
multiple inoculations (e.g. in soldiers fighting in the Pacific theatre
of World War 2) (9). Overall, the relapse prevention (i.e. radical
curative) efficacy of 8-aminoquinolines was substantially better in
clinical practice compared with the stern test applied in the volunteer
infections. For example, the combination of quinine and plasmoquine at
doses \emph{\textgreater{}} 30mg base/day was considered highly
effective in preventing relapse in clinical studies in endemic areas,
but doses below 90mg base/day were relatively ineffective in the
experimental challenge model (2, 3, 10, 11). Thus, to compare radical
curative efficacies it is necessary to consider both drug dosage, and
thus exposure, and also the likely hypnozoite burden (12).

\emph{Predictors of relapse}

Several different factors determine whether vivax or ovale malarias will
relapse. The hypnozoite burden, geographic origin (i.e. ``strain''), and
degree of immunity are all important determinants. Liver stage immunity
may affect hepatic schizont development and blood stage immunity
suppresses the relapse parasitaemia to an extent that it is
asymptomatic, subpatent, or both (13, 14). Although there is no evidence
of acquired 8-aminoquinoline resistance in hypnozoites, parasites do
differ in intrinsic susceptibility. For example, the long
latency\emph{P. vivax} ``strains'' prevalent in temperate regions are
more susceptible to 8-aminoquinolines than tropical frequent relapse
strains, and \emph{P. vivax} parasites in SE Asia and Oceania appear to
be less susceptible (i.e. require a larger primaquine dosage) than other
tropical ``strains'' (15).

From a therapeutic perspective exposure to the biologically active
metabolites of the 8-aminoquinoline is the critical factor determining
relapse prevention (radical cure) efficacy (16, 17). This exposure
results from the total dose absorbed, and the degree of
biotransformation. Patients with cytochrome P450 2D6 polymorphisms
conferring reduced function have reduced production of primaquine's
bioactive metabolite(s) and correspondingly reduced radical curative
efficacy (18,19). The oxidative activity of the 8-aminoquinoline
treatment is also reflected in the intraerythrocytic concentrations of
methaemoglobin. The association of methaemoglobinaemia with radical
curative efficacy of 8-aminoquinolines is discussed.

\emph{Methaemoglobinaemia}

Methaemoglobin results when the haem iron in haemoglobin is converted
from the ferrous state (Fe\textsuperscript{++}), the reduced form, to
the (Fe\textsuperscript{+++}) ferric ion, the oxidized form (20). This
process is coupled to redox cycles in the red cell. In the main cycle,
driven by the NAD-cytochrome b5 reductase (the main methaemoglobin
reductase), haemoglobin and methaemoglobin are cycled (Figure 1). In the
second, a cell redox cycle system is driven by the oxidation of
haemoglobin, with methaemoglobin as the product. Although the main
enzyme responsible for intraerythrocytic methaemoglobin reduction is the
NADH-cytochrome b5-reductase, there are alternative pathways. These
include an NADPH-dependent methaemoglobin reductase (which has
substantially reduced activity in G6PD deficiency), and direct reduction
by intracellular ascorbate and glutathione (Figure 1). Methaemoglobin
reduction is a first order process. Under normal steady state conditions
approximately 3\% of the body's haemoglobin is oxidized each day to
methaemoglobin but, because of back-conversion, the average proportion
of methaemoglobin is less than 2\% of the corresponding haemoglobin
concentration. Numerous factors influence this balance (including foods,
drugs, exercise, smoking, hypoxia).

Methaemoglobin is a dark blue-brown colour compared with the bright red
of oxygenated haemoglobin, so the skin colouration in
methaemoglobinaemia resembles that in cyanosis caused by increased
concentrations of deoxygenated haemoglobin. Methaemoglobin has a
distinct spectrum from haemoglobin. Methaemoglobinaemia can be measured
by spectrophotometry of fresh blood samples in the laboratory or,
utilizing the same principle, by continuous transcutaneous oximetry
devices. During exposure to the oxidizing agent methaemoglobinaemia
increases, although there is substantial interindividual variation in
iatrogenic methaemoglobinaemia with a skew distribution of steady state
values. Interquartile ranges typically extend from approximately 60 to
140\% of the median values and, for a given drug exposure, individual
values can range tenfold in large series. With daily primaquine dosing
methaemoglobinaemia has an estimated elimination half-life of
approximately 1.5 days (Figure 2) i.e. 90\% of the new steady state is
reached in approximately 6 days.

Several different oxidant chemicals and drugs can cause
methaemoglobinaemia. Although methaemoglobin production (i.e. haem
oxidation) by 8-aminoquinolines is generally considered not to lie in
the causal pathway to haemolysis or antimalarial activity (26), it does
provide an approximate correlate of these activities within the
8-aminoquinoline class. Under the rigorous test of the experimental
challenge studies conducted in the USA in the late 1940s and early 1950s
drugs, or drug concentrations, which produced less than 6\% steady state
methaemoglobinaemia were associated with sub-optimal radical cure rates
(23-25) (Figure 3). As for iatrogenic haemolysis, there is a slight
delay before intraerythrocytic methaemoglobin levels begin to rise
following 8-aminoquinoline administration. This presumably reflects
depletion of the oxidant defences (notably reduced glutathione) (27). In
a large recent study of vivax malaria conducted on the Thailand-Myanmar
border the relationships between primaquine and carboxyprimaquine plasma
concentrations, relapses, CYP2D6 polymorphisms and methaemoglobinaemia
were investigated. After adjusting for age and partner drug, the day 7
concentrations of primaquine and carboxyprimaquine were not associated
with the risk of recurrence, but a 1\% absolute increase in day 7
methemoglobin was associated with a hazard ratio for recurrence of 0.9
(95\% CI: 0.85-0.99, p=0.02) (28).

Tafenoquine is a slowly eliminated 8-aminoquinoline which has been
introduced recently. It is notable that the currently recommended dose
of tafenoquine (adult dose 300mg), which provides radical cure rates
which are inferior to the lower dose of primaquine (total dose 3.5mg/kg)
in South-East Asia (29), is associated with methaemoglobin
concentrations which are less than half those associated with the lower
dose primaquine regimen (30,31) (Figure 4).

\emph{Drug interactions}

The 8-aminoquinolines have important pharmacokinetic and pharmacodynamic
interactions with other structurally related antimalarial drugs.
Coadministration of mepacrine (atebrin, quinacrine) substantially
increases plasma concentrations of pamaquine and also reduces
tolerability. It substantially increases methaemoglobinaemia associated
with pamaquine (33). Chloroquine, piperaquine and pyronaridine all
increase plasma concentrations of primaquine by approximately 20\%, but
they do not affect tolerability (34). Pamaquine and primaquine radical
curative activity is increased by concomitant (but not sequential)
administration with quinine (35, 36). Chloroquine has also been shown to
potentiate the radical curative efficacy of primaquine. During the
clinical development of primaquine it was observed that coadministration
of primaquine with quinine or chloroquine attenuated the consequent
methaemoglobinaemia. This was not observed in comparable studies with
pamaquine (plasmoquine) (Figure 5).

However larger, more recent, studies have not confirmed this early
finding (Figure 6). They do not suggest any substantial differences
between methaemoglobin levels with or without concomitant medicines
(quinine, chloroquine, artesunate, artesunate-pyronaridine,
dihydroartemisinin-piperaquine have all been evaluated), nor do they
suggest differences between methemoglobin levels in the treatment of
malaria (in which steady state methaemoglobin levels are reached well
after resolution of symptoms), or in healthy subjects. Thus the impact
of blood schizontocides on 8-aminoquinoline induced methaemoglobineamia
is specific to the drugs and is not a general class effect.

\emph{Methaemoglobinaemia and G6PD deficiency}

The discrepancy between 8-aminoquinoline haemolytic toxicity and
methaemoglobinemia has been noted widely (26). In the research which led
to registration of primaquine, Edgcomb et al (24) noted no correlation
between haemolysis and methaemoglobinaemia following pamaquine or
primaquine. The erythrocytes in G6PD deficiency have a reduced ability
to reduce methaemoglobin in the presence of ``electron-donors'', such as
the bioactive metabolites of primaquine or methylene blue (this is the
basis of the methylene blue methaemoglobin reduction test developed to
diagnose G6PD deficiency). This is because of the reduced
intraerythrocytic activity of the NADPH dependent methemoglobin
reductase. Brewer et al showed that whereas G6PD deficient individuals
had \emph{increased} levels of methaemoglobinaemia following oral sodium
nitrite (an oxidizing agent which does not cause haemolysis), they
had\emph{decreased} levels of methaemoglobinemia following primaquine
(26) (Figure 7). This apparent paradox was explained by the iatrogenic
haemolysis of the older erythrocytes which contained the highest
concentrations of methaemoglobin. Other mechanisms are also possible,
such as the sequestration of the primaquine oxidant metabolites in the
oxidized haemoglobin Heinz bodies. The mechanism of 8-aminoquinoline
haemolytic toxicity has not been explained satisfactorily but clearly
involves more than broad oxidant stress.

\emph{Conclusions}

Methaemoglobinaemia is associated with exposure to therapeutic doses of
8-aminoquinoline drugs. The proportion of methaemoglobin to haemoglobin
correlates with the efficacy of anti-relapse therapy, but methaemoglobin
appears to be uninvolved directly either in that activity or in
haemolytic toxicity in G6PD-deficient patients. This suggests that the
oxidative processes which result in methaemoglobinaemia are necessary,
but they are not sufficient, for the radical curative activity of the
8-aminoquinoline antimalarial drugs. Although there is substantial
inter-individual variation in iatrogenic methaemoglobinaemia, the
overall clinical data suggest that methaemoglobinaemia is a
pharmacodynamic correlate of radical curative activity. Measurement of
methaemoglobinaemia might be useful in drug screening and dose
evaluation.

\textbf{Acknowledgements:} We are very grateful to our many colleagues
who have contributed extensively to the clinical studies.

\textbf{Declaration of Interests.} We have no conflicts of interest.

\textbf{Ethics approval and consent to participate.} Not relevant.

\textbf{Availability of data and material} : All data used are available
from the original publications.

\textbf{Funding} : Wellcome Trust



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\end{enumerate}

Figure 2: The figures show increasing levels of methaemoglobinaemia (as
a proportion of the corresponding haemoglobin concentration) during
radical curative treatment of vivax malaria with primaquine. On the left
(Figure 2A) primaquine 30mg base/day was given to adults for 14 days
either concurrently with quinine, or 28 days after a treatment dose of
dihydroartemisinin-piperaquine (from Sutanto et al (21)). On the right
(Figure 2B) primaquine was given to adults and children either at a dose
of 0.5mg base/kg /day for 14 days or 1mg/kg/day for 7 days and patients
were randomized to receive concurrent chloroquine or
dihydroartemisinin-piperaquine (from Chu et al (22))

Figure 3: During the clinical investigations, conducted over 70 years
ago in the USA, to develop new 8-aminoquinolines 18 different compounds
were evaluated in addition to pamaquine, and later primaquine (23-25).
Each was administered for 14 days concurrently with quinine. The
volunteers were infected by multiple bites of \emph{A.
quadrimaculatus}infected with the Chesson strain of \emph{P. vivax} .
The figure shows the relationship between the radical cure proportion
and the average blood methaemoglobin concentration (\%) measured in the
last 4 days of treatment. The size of the circles is proportional to the
number recruited as shown in the inset box.

Figure 4: Dose-response relationships for primaquine and tafenoquine in
generating methaemoglobinaemia (21,22, 24-26, 28-32). The dose shown is
the daily adult dose of primaquine, whereas for tafenoquine the dose
shown is the total dose administered. The size of the circles is
proportional to the number of patients or volunteers recruited as shown
in the inset box.

Figure 5: Studies conducted during the development of primaquine which
assessed the average methaemoglobin concentration (\%) as a proportion
of the haemoglobin concentration in the last 4 days of treatment in
relation to the dosing of plasmoquine (pamaquine) (left) and primaquine
(right) (11,33,37). These initial observations suggested that
methaemoglobinaemia following primaquine, but not plasmoquine, was
attenuated by concomitant administration of chloroquine or quinine.
However, later studies (summarized in Figure 6) did not confirm this.
The size of the circles is proportional to the number of patients or
volunteers recruited as shown in the inset box.

Figure 6: More recent studies in which methaemoglobinaemia has been
assessed after different dose regimens of primaquine whether given alone
(26,39-41), or in combination with antimalarial drugs (18, 34, 37, 38),
either to healthy volunteers, in antimalarial prophylaxis, or in the
treatment of vivax malaria (15,19,21,22,24-26,28-32,37,42,43). These do
not suggest attenuation of primaquine induced methaemoglobinaemia by
concomitant antimalarial drugs (as reported by Clayman et al (37)), nor
are there differences between healthy subjects and patients with
malaria. The size of the circles is proportional to the number of
patients or volunteers recruited as shown in the inset box.

Figure 7: Mean values of haemoglobin (g/dL) and methaemoglobin (\%) in
12 normal and 12 African A- G6PD deficient volunteers given primaquine
(from Brewer et al (26)).

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\verb`Fig 7.pptx`  available at \url{https://authorea.com/users/312614/articles/549411-methaemoglobinaemia-and-the-radical-curative-efficacy-of-8-aminoquinoline-antimalarials}

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