Eflornithine derivatives for enhanced oral
bioavailability in the treatment of human
African trypanosomiasis
TT Cloetea, JC Breytenbacha, DD N’Daa, M Ashtonb and C Johanssonb
Pharmaceutical Chemistry, School of Pharmacy, North-West University,
Potchefstroom 2520, South Africa
b
Unit for Pharmacokinetics and Drug Metabolism, Department of Pharmacology,
Institute of Neuroscience and Physiology, Sahlgrenska Academy at
University of Gothenburg, Gothenburg, Sweden.
a
Theunis Cloete
Theunis Cloete was the winner of the 2009 Young Scientist Award, which is sponsored by Adcock Ingram
Introduction
Human African trypanosomiasis (HAT), or sleeping sickness, is
caused by the parasite protozoa Trypanosoma brucei gambiense and T.b. rhodesiense and is transmitted to humans by
the tsetse fly (Glossina) which can only be found in sub-Sahara
Africa between latitudes 14°N and 29°S (WHO,1998). At the
start of Angola’s independence in 1974 only three cases of
HAT were detected amongst 471 588 people screened for the
disease. In 1998, twenty four years later, 6 610 cases were
detected out of only 154 700 people screened (Stanghellini et
al., 2001). The gradual breakdown of countermeasures, war
and civil instability have caused a re-emergence of sleeping
sickness with an estimated 30 000 new cases per year (Delespaux et al., 2007). In the first or haematolymphatic stage
of the disease the parasite is restricted to the bloodstream
and the extracellular tissue; if this stage is left untreated it will
progress to the second or meningoencephalitic stage where the
parasite penetrates the central nervous system. The first stage
is treated with either suramin or pentamidine and has a high
success rate with only minor side effects (Bouteille et al., 2003).
Treatment for the second stage consists of melarsoprol which is
highly toxic (Fairlamb, 2003). Eflornithine which has fewer side
effects is an alternative, but is too hydrophilic for oral administration and has to be given intravenously each day for 14 days
making this drug too expensive and difficult to administer under
African healthcare conditions. Oral eflornithine would greatly
decrease the cost of administering the drug and would make the
drug available to more people (Chappuis et al., 2005).
Aim
The aim of this study was to synthesise a series of eflornithine
derivatives and to evaluate their oral bioavailability with the ultimate goal to obtain a more affordable alternative to melarsoprol.
Methodology
Synthesis
All the compounds were synthesised in three steps or less by
α- and/or δ-amidation and/or esterification processes. Ethyl esters of eflornithine were synthesized using two
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methods. The first method adopted was reported by Perrin and
co-workers (2007), and consists of activating the carboxylic
acid of eflornithine with thionyl chloride as depicted in Scheme
1 (Perrin et al., 2007). This leads in situ to the formation of an
acyl chloride, which is then coupled to ethanol. The second
method was literature reported (March, 1992), and consists of
reacting the sodium carboxylate of an amide derivative of eflornithine with a primary bromide via the SN2 reaction mechanism
at room temperature in DMF as depicted in Scheme 3.
δ-amides of eflornithine were synthesised according to
the general method depicted in Scheme 2. The δ-amine of
eflornithine was deprotected by dissolution in aqueous NaOH
(5M, pH 11) and allowed to react with the acyl chloride portion
wise (one portion a day), first in an ice bath and then at room
temperature over 5 days. During the synthesis hydrochloric
acid was formed as a by product with the potential to induce
acid hydrolysis of the formed amide bond if not neutralized. For
this reason a base was added to trap the formed acid. Thus,
the pH of the medium was kept above 11 during the course of
the synthesis by addition of aliquots aqueous NaOH.
A sodium carboxylate reacts via the SN2 mechanism with
a primary bromide at room temperature to give carboxylic
esters in high yields. This approach was used to esterify
the δ-amides of eflornithine as depicted in Scheme 3, and
outlined as follows. To a solution of a δ-amide eflornithine
derivative dissolved in 40 ml of anhydrous DMF, and stirring
at room temperature, was added ethylbromide. The stirring
was continued for 4 days.
The trisubstituted derivatives were obtained by acylation
on the α-amine of the preceding disubstituted compounds.
The acylation occurred in an acetonitrile medium between the
disubstituted eflornithine and the acyl chloride. Triethylamine
(TEA), an aprotic base was used to trap the acid during the reaction. The reaction is depicted in Scheme 4, and described as
follows: The disubstituted eflornithine was dissolved in 20 ml of
acetonitrile together with the base triethylamine to trap any acid
that will form. The acyl chloride was then added in two portions
over a period of 2 days (a portion was added each day).
The structures of all compounds were confirmed by
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nuclear magnetic resonance spectroscopy (NMR) and mass
spectrometry (MS).
In vivo biological study
The in vivo biological study was done on male Sprague-Dawley
rats. The animals were anesthetized by inhalation of isoflurane
(2.9 - 3.7%) in air. A catheter was inserted into the left jugular
vein and tunnelled subcutaneously to emerge at the back of the
neck. A total of 20 rats were administered the derivatives orally by
gavage at an equimolar dose to eflornithine of 100 mg/kg of body
weight. Blood samples were drawn from the jugular vein catheter
and flushed with heparinized saline solution (20 IU/ml) after each
sampling occasion. The HPLC method developed to analyse the
samples detects the amount of eflornithine in each sample. It is
thus important to note that this study tests each derivative for the
capability to act as a prodrug and cannot determine the amount of
compound in the blood (Jansson et al, 2008).
Results
The chromatogram of eflornithine given per se is shown in Fig
1.1. The first peak, with a retention time of 11.87 minutes, represents the L-isomer of eflornithine and the second peak, with
a retention time of 13.49 minutes, the D-isomer.
On the chromatograms of compounds (2) and (3) certain
anomalies occurred. It appears that both compounds are hydrolysed producing the parent drug eflornithine. This initial results
were however not constantly reproducible. Another anomaly was
that only one sample on each chromatogram gave this massive
increase in abundance of eflornithine, with only a small increase
witnessed in other samples. Normally when observing metabolites the increase in abundance can be seen in more than one
sample. A logical explanation for this would be that no hydrolysis took place, and that the observed increase of eflornithine
was a carry over effect on the analysis. This implies that both
the ester and amide bonds proved to be more stable then
previously thought. Chollet and co-workers also believed that
their compounds’ low activity against trypanosomes were the
Scheme 1: Esterification of eflornithine using thionyl chloride
Scheme 2: Coupling of an acyl chloride to the δ-amine of eflornithine
Scheme 3: Esterification of δ-amides of eflornithine using ethylbromide
Scheme 4: Synthesis of trisubstituted derivatives of eflornithine
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Fig 1.1: Chromatogram of eflornithine concentrations in the blood after oral administration
result of the ester not being cleaved. They ascribed this to the
steric bulk of the α-amine next to the carboxylic acid group that
protected the ester bond from esterases preventing the bond
form being cleaved (Chollet et al., 2009).
Unfortunately solubility problems were experienced with compounds (4)-(10). These compounds were much more lipophilic
than compounds (2) and (3) and had to be dissolved in different
solvents. Because this was an in vivo study done on rats we were
limited to certain non toxic solvents and could only administer
a small volume of solution to each rat. The solubility of these
compounds proved to be too great a problem and could not be
administered without causing unnecessary harm to the animals.
Conclusion
Both compounds (2) and (3), after further inspection of the results, showed no increase in the concentration of eflornithine in
the blood. It is now clear that new compounds synthesised from
eflornithine are proving to be much more stable then previously
thought. Future research on the synthesis of new prodrugs of
eflornithine should thus be done bearing this in mind.r
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