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Vol. 56, Issue 4, 693-704, October 1999
Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium (D.S., L.N., E. De C., J.B.); and Welsh School of Pharmacy, University of Wales, Cardiff, United Kingdom (D.C., A.S., R.P., S.V., C.M.)
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Summary |
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 Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The phosphoramidate triester prodrugs of anti-human HIV 2',3'-dideoxynucleoside analogs (ddN) represent a convenient approach to bypass the first phosphorylation to ddN 5'-monophosphate (ddNMP), resulting in an improved formation of ddN 5'-triphosphate and, hence, higher antiviral efficacy. Although phosphoramidate derivatization markedly increases the anti-HIV activity of 2',3'-didehydro-2',3'-dideoxythymidine (d4T) in both wild-type and thymidine kinase-deficient CEM cells, the concept is far less successful for the 3'-azido-2',3'-dideoxythymidine (AZT) triesters. We now investigated the metabolism of triester prodrugs of d4T and AZT using pure enzymes or different biological media. The efficiency of the first activation step, mediated by carboxylesterases, consists of the formation of the amino acyl ddNMP metabolite. The efficiency of this step was shown to be dependent on the amino acid, alkyl ester, and ddN moiety. Triesters that showed no conversion to the amino acyl ddNMP accumulated as the phenyl-containing intermediate and had poor, if any, anti-HIV activity. In contrast to the relative stability of the triesters in human serum, carboxylesterase-mediated cleavage of the prodrugs was found to be remarkably high in mouse serum. The subsequent conversion of the amino acyl ddNMP metabolite to ddNMP or ddN was highest in rat liver cytosolic enzyme preparations. Although L-alaninyl-d4TMP was efficiently converted to d4TMP, the main metabolite formed from L-alaninyl-AZTMP was the free nucleoside (AZT), thus explaining why d4T prodrugs, but not AZT prodrugs, retain anti-HIV activity in HIV-infected thymidine kinase-deficient cell cultures. The rat liver phosphoramidase responsible for the formation of ddNMP was shown to be distinct from creatine kinase, alkaline phosphatase, and phosphodiesterase.
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Introduction |
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Top
Abstract
 Introduction
 Materials and Methods
Results
Discussion
References
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2',3'-Dideoxynucleoside
analogs (ddN) that are active against HIV [i.e., zidovudine (AZT),
stavudine (d4T), didanosine, zalcitabine, and lamivudine] must
be converted after cell penetration to their corresponding
5'-triphosphate metabolites to act as inhibitors of HIV reverse
transcriptase (Balzarini and De Clercq, 1999
). However, for several
ddNs, the first phosphorylation catalyzed by cellular kinases [i.e.,
thymidine kinase (TK) in the case of d4T and AZT] is the rate-limiting
step that determines the eventual antiviral activity. In vitro studies
on the metabolism of ddN in tumor cell lines or mitogen-stimulated
lymphocytes may not fully reflect the in vivo situation. According to
the findings of Jacobsson et al. (1995), the TK activity in peripheral
blood lymphocytes from HIV-infected persons is about 3-fold lower than that seen in seronegative individuals. In addition, the in vitro and ex
vivo data of Antonelli et al. (1996) strongly suggest that long-term
treatment with ddN may result in a reduction of TK activity and, hence,
reduced phosphorylation efficiency of the lymphocytes. Circumvention of
this initial activation step is possible by the design of
membrane-soluble prodrugs that deliver directly the ddN
5'-monophosphate (ddNMP) into the HIV-infected cells. Among the several
types of nucleotide prodrugs that have already been synthesized, a
series of phosphoramidate triesters have emerged as highly promising
antiviral agents (Farrow et al., 1990; Valette et al., 1996; Winter et
al., 1996; Balzarini et al., 1997; Meier et al., 1997). These triesters
consist of a ddNMP for which the phosphate is linked, on one side, to a
lipophilic (aryl) group and, on the other side, to an amino acid
moiety, via a phosphoramidate (P---N) bond. The
L-alaninyl-d4TMP phosphotriester 2 (Fig. 1) can
be considered the prototype compound of the phosphoramidate prodrug
concept (Balzarini et al., 1996a; McGuigan et al., 1996). Our previous
metabolism studies with radiolabeled 2 in human lymphocyte
CEM cells revealed that this phosphoramidate triester is able to
deliver d4TMP intracellularly (Balzarini et al., 1996a). Consequently,
the independence of this prodrug from cellular TK resulted in a
markedly improved antiviral activity in TK-deficient cells
(CEM/TK
) compared with the parent nucleoside
d4T (Balzarini et al., 1996b; McGuigan et al., 1996).
The phosphoramidate prodrug technology has been used for the synthesis
of a series of closely related amino acyl aryloxyphosphoramidate triester derivatives of d4T, AZT, and lamivudine (Devine et al., 1990;
McGuigan et al., 1993; Valette et al., 1996). The antiviral activity of
these nucleoside prodrugs is determined by the different structural
parts of the molecule (i.e., the nature of nucleoside, the amino acid,
and the alkyl group). However, it is not fully understood how the
antiviral data can be correlated to the intracellular decomposition
pathway followed by the phosphoramidate derivatives.
Several data indicate that the first step in the activation pathway
consists of carboxylesterase-mediated hydrolysis of the carboxylic
ester function in the amino acid part (McGuigan et al., 1998; Naesens
et al., 1998). This esterase cleavage is thought to be followed by an
intramolecular nucleophilic attack of the phosphorus by the carboxyl
group with spontaneous elimination of phenol after transient formation
of a five-membered cyclic intermediate (Fig.
1). This is followed by the conversion of
the ddNMP amino acyl metabolite (AAM) to free ddNMP. It has not been clarified whether cleavage of the P---N bond is predominantly catalyzed by one or more less specific phosphatases (that normally use phosphate esters as a substrate) or by a distinct and specific phosphoramidase (Holzer et al., 1962; Holzer et al., 1966; Fernley, 1971; Snyder and
Wilson, 1972; Kelly et al., 1975; Nishino et al., 1994).
Phosphoramidases that catalyze the hydrolysis of phosphoramidate
compounds have been described in mammalian cells and bacteria (Singer
and Fruton, 1957; Stevens-Clark et al., 1968; Parvin and Smith, 1969;
Kuba et al., 1994; Müller, 1995; Abraham et al., 1996) and have
been characterized in more detail by Shabarova and coworkers
(Shabarova, 1970; Ledneva et al., 1967, 1970, 1971; Dudkin et al.,
1971a,b; McIntee et al., 1997).
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We now investigated the activation pathway of a series of phosphoramidate prodrugs of d4TMP and AZTMP in different biological media (i.e., CEM cell extracts, human serum, mouse serum, and rat liver). The purpose of this study was to reveal the influence of the nature of nucleoside, amino acid, and alkyl moiety on the conversion of the triester to ddNMP, with the aim of optimizing the design of new phosphoramidate derivatives.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cells and Viruses.
Wild-type CEM cells (CEM/0) were obtained
from the American Type Culture Collection (Rockville, MD). The
TK-deficient cell line (CEM/TK) was kindly
provided by Prof. Staffan Eriksson (Swedish University of Agricultural
Sciences, Uppsala, Sweden) and Prof. Anna Karlsson (Karolinska
Institute, Stockholm, Sweden). CEM/0 and CEM/TK
cells were grown in 75-cm2 flasks in RPMI 1640 medium (GIBCO, Paisley, Scotland) supplemented with 10% FCS (GIBCO), 2 mM glutamine (GIBCO), and 0.075% sodium bicarbonate (GIBCO). HIV-1
(strain IIIB) was a generous gift from Dr.
R. C. Gallo (at that time at the National Cancer Institute, Bethesda, MD). HIV-2 (strain ROD) was kindly provided by Dr. L. Montagnier (Pasteur Institute, Paris, France).
Enzymes. Pig liver carboxylesterase (E.C. 3.1.1.1), 5'-nucleotidase (E.C. 3.1.3.5, from Crotalus adamanteus), and phosphodiesterase I Type VI (E.C. 3.1.4.1, from C. adamanteus) were purchased from Sigma Chemical Co. (St. Louis, MO). Alkaline phosphatase (E.C. 3.1.3.1, from calf intestine) and creatine phosphokinase (E.C. 2.7.3.2, from rabbit muscle) were obtained from Boehringer Mannheim Gmbh (Mannheim, Germany) and SERVA Feinbiochemica (Heidelberg, Germany), respectively.
Anti-HIV Assays.
CEM/0 or CEM/TK cells
were suspended at 250,000 cells/ml cell culture medium and infected
with approximately 100 CCID50 (1 CCID50 is the 50% cell culture infective dose)
of HIV-1 or HIV-2. Then, 100 µl of the infected cell suspension was
added to the wells of a 96-well microtiter plate containing 100 µl of
an appropriate dilution of the test compounds (250, 100, 20, 4, 0.8, 0.16, 0.032, 0.006, and 0.001 µM).
). The
50% effective concentration (EC50) was
determined as the compound concentration required to inhibit syncytium
formation by 50%. In parallel, the cytopathic activity of the
compounds was determined in mock-infected cells. The 50% cytopathic
concentration (CC50) was defined as the compound
concentration that causes 50% reduction in cell proliferation, as
determined by automated cell counting in a Coulter Counter (Harpenden,
Hertz, UK) on day 4 after the addition of test compound.
Preparation of Crude CEM Cell Extract.
To prepare
concentrated CEM cell extracts, CEM/0 cells were grown in 2-liter
spinner flasks, with the frequent addition of fresh culture medium to
ensure exponential growth. When a total amount of
109 cells was reached (i.e., 5 × 105 cells/ml), the suspension was centrifuged for
10 min (1200 rpm, 4°C), and the supernatant was withdrawn. The cell
pellet was resuspended in PBS and recentrifuged. Then, the cell pellet
was resuspended in a small volume of PBS to obtain
108 cells/ml. After cell lysis by sonication
(3 × 20 s) and centrifugation to remove cells debris
(25,000g, 4°C, 10 min), the supernatant was collected.
When not used immediately, the cell extracts were stored at 
80°C.
Rat Liver Enzyme Preparation.
Buffer A consisted of 50 mM
Tris · HCl (pH 7.4) supplemented with 0.25 M sucrose and 1 mM EDTA;
buffer B consisted of buffer A with 10 mM 
-mercaptoethanol; buffer C
consisted of buffer B without EDTA; and the corresponding buffers
without sucrose were designated A', B', and C'.
-mercaptoethanol were resuspended in 15 ml of buffer A' and
buffer B', respectively. These preparations, called 3 and
4, were dialysed overnight against buffer A' and buffer B',
respectively. Aliquots were stored at 80°C until further use.
A different cytosolic enzyme preparation 5 was obtained by
the same method as described for preparations 3 and 4, with the exception that buffer C was used during liver homogenization and centrifugation and that the final enzyme extract was
prepared in buffer C'. Finally, preparation 6 was made with
the same procedure as 5, except that the 60-min
centrifugation (105,000g) was omitted. The pellet was
resuspended in buffer C', dialyzed overnight against the same buffer,
and stored in aliquots at 80°C.
Enzymatic Hydrolysis of Phosphoramidate Prodrugs with Pig Liver Carboxylesterase. The 200-µl reaction mixture was made up in 50 mM Tris · HCl buffer (pH 7.4). The final concentration of the prodrug was 200 µM. The reaction was initiated by the addition of the enzyme. For each determination, control samples lacking either enzyme or prodrug were included. For inhibition assays, pig liver carboxylesterase (16 U/ml) was preincubated for 20 min in buffer containing phenylmethylsulfonyl fluoride (PMSF) before addition of the prodrugs. After 2- or 16-h incubation at 37°C, the reaction was stopped by the addition of 300 µl of ice-cold methanol. After 20 min, the precipitate was removed by centrifugation, and the supernatant was subjected to HPLC analysis. All assays were performed in triplicate.
Inhibition of Phosphoramidase Activity by Iodobenzene. Partially purified liver enzyme extract was preincubated with several concentrations of iodobenzene (1, 0.1, 0.01, 0.001, 0.0001, and 0.00001 µM) for 10 min at 37°C. Then, substrate (L-alaninyl-d4TMP) was added to the reaction mixture at 2 mM. After overnight incubation (i.e., ~16 h), the remaining substrate and the reaction products (i.e., d4TMP, d4T) were quantified by HPLC analysis.
Incubation of Phosphoramidate Prodrugs in CEM Cell Extract, Human Serum, and Mouse Serum. Stock solutions of the compounds at a concentration of 50 or 100 mM and prepared in DMSO were diluted in Tris · HCl buffer (50 mM; pH 7.4) to obtain a working stock at 1.4 mM.
The 200-µl incubation mixture contained 160 µl of biological medium, 20 µl of stock solution in Tris · HCl buffer (final prodrug concentration, 200 µM), and 20 µl of MgCl2 plus-mercaptoethanol (both at a final
concentration of 10 mM). For the inhibition studies, the biological
media were preincubated for 30 min at 37°C with 10 mM PMSF before the
addition of prodrug. At the end of the incubation period (0 min, 2 h, or 16 h), the samples were put on ice and deproteinized with
ice-cold methanol (300 µl) for 20 min. Then, samples were centrifuged
for 5 min at 15,000g, and the supernatant was analyzed by
HPLC. When not assayed immediately, the extracts were stored at
20°C. All experiments were performed in duplicate.
Enzymatic Preparation of L-Alaninyl-d4TMP and L-Alaninyl-AZTMP. L-Alaninyl-d4TMP triester 2 and L-alaninyl-AZTMP triester 14 were incubated during 4 days in Tris · HCl buffer (pH 7.4) containing 100 U/ml pig liver carboxylesterase. Fresh enzyme was added daily, and complete conversion to L-alaninyl-ddNMP was verified by HPLC.
Next, 1-ml aliquots were loaded onto C18 Sep-Pak cartridges (Waters Associates, Milford, MA) preactivated with 2 ml of methanol and 4 ml of water. L-Alaninyl-d4TMP and L-alaninyl-AZTMP were adsorbed by the cartridge while most impurities remained in the solvent. After washing with 0.5 ml of distilled water, the L-alaninyl-ddNMP derivatives were eluted with 2 ml of methanol. The eluates were pooled and evaporated to dryness in vacuo, after which the residues were resuspended in water.Chromatographic Conditions. The phosphoramidate prodrugs of d4TMP and AZTMP and their metabolites were quantified by HPLC. A Superspher 100 RP-18 endcapped column (250 × 4 mm, 5 µm; Merck, Darmstadt, Germany) fit into a LiChroCART cartridge (Merck) and protected with a LiChrospher 100 RP-18 guard column (Merck) was used. The chromatographic system consisted of a Waters 600E Pump, a Waters 717 plus Autosampler, and a Waters 996 photodiode array detector and was controlled by Millennium software.
The solvent system consisted of acetonitrile (HPLC grade; Fluka, Buchs, Switzerland) and two buffers: buffer A containing 2.5 mM ammonium dihydrogen phosphate plus 5 mM tetrabutylammonium hydrogen sulfate in water (pH 3.5), and buffer B containing 75 mM ammonium dihydrogen phosphate plus 5 mM tetrabutylammonium hydrogen sulfate in water (pH 3.5). The column was equilibrated with 100% buffer A. The samples were separated at a flow rate of 1 ml/min by a linear gradient from 100% A to 97% B plus 3% acetonitrile (0-30 min) and then to 30% buffer B plus 70% acetonitrile (30-60 min). The last conditions were maintained isocratically (60-70 min), followed by a linear gradient to 100% buffer A (70-75 min), and ended by a reequilibration step (75-90 min). The peaks were identified based on comparison with synthetic standards. The retention times for the phosphoramidate prodrugs of d4TMP and AZTMP were in the range of 56 to 64 min. For compound 2, the retention times were 56, 52, 49, 31, and 29 min, for the prodrug, the intermediate metabolite (IM), the AAM, d4TMP, and d4T, respectively. For compound 14, the retention times for the prodrug, the IM, the AAM, AZTMP, and AZT were 59, 57, 52, 49, and 47 min, respectively. The other prodrugs and their corresponding metabolites had similar elution patterns. In addition, the samples were analyzed on an anion exchange Partisphere SAX column (5 µm, 4.6 × 125 mm; Whatman) to quantify d4TMP and AZTMP and to allow better identification of AAM and IM. The column was equilibrated with 50% buffer A (5 mM ammonium dihydrogen phosphate, pH 5.0) plus 50% water. The samples were separated at a flow rate of 2 ml/min by the following gradient: 50% buffer A and 50% water (0-5 min), linear gradient to 90% buffer A plus 10% buffer B (0.25 M ammonium dihydrogen phosphate, pH 5.0; 5-20 min), then isocratic conditions (20-25 min), followed by a linear gradient to 50% buffer A, plus 50% water (25-45 min), and finally reequilibration during 13 min. Under these conditions, the retention times for L-alaninyl-d4TMP, L-alaninyl-AZTMP, d4TMP, and AZTMP were 21, 20, 15, and 13 min, respectively.| |
Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Anti-HIV Activity in CEM/0 and CEM/TK Cells.
A
selection of phosphoramidate triester derivatives of d4TMP and AZTMP,
carrying different amino acids (Fig. 2),
were evaluated for their antiviral activity against HIV-1 and HIV-2 in
CEM/0 and CEM/TK cells (Table
1). For both the d4TMP and AZTMP
phosphoramidate derivatives, L-alanine was shown to be the
preferred amino acid. Among the d4TMP triesters, the
L-alaninyl derivatives 1, 2, and
3, carrying different ester moieties on the alanine part,
ranked among the most active compounds, with the EC50 value against HIV-1 and HIV-2 in CEM/0 cells
being 0.02 to 0.08 µM. Modification of the amino acid moiety resulted
in partial or virtually complete loss of antiviral activity compared
with the L-alaninyl prodrug derivative. Relatively small
structural changes of the amino acids had a marked effect on the
eventual antiviral activity. For instance, the L-alanine
compound 2 is 40-, >3000-, and 80-fold more active than the
corresponding D-alanine 5, -alanine
12, or glycine 6 prodrugs. As a rule for all of
the d4TMP triesters tested, the anti-HIV activity was not markedly
different in CEM/0 and CEM/TK cells. By
contrast, the anti-HIV activity of the triesters of AZTMP was
significantly lower in CEM/TK than CEM/0 cells,
and the effect of the different amino acids on the eventual antiviral
activity of the AZTMP prodrugs was less pronounced than that observed
for the d4TMP prodrugs. From these data, it can be concluded that the
antiviral activity of the phosphoramidate prodrug derivatives is
strongly dependent on the nature of the nucleoside moiety (d4T or AZT)
and the amino acid substituent.
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Conversion by Esterase, CEM Cell Extract, Human Serum, or Mouse Serum. The metabolism of the phosphoramidate triesters of d4TMP and AZTMP was studied using pig liver carboxylesterase (E.C. 3.1.1.1) and different biological media (i.e., CEM cell extract, human serum, and mouse serum). The results are shown in Table 2.
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-alanine derivative
12, in which case an IM accumulated. Finally, an incomplete
conversion of the triester to AAM was observed with the compounds
containing L-leucine (7),
L-methionine (9), and
methyl-L-glutamic acid (11). For these three
compounds, a mixture was obtained containing intact prodrug, AAM,
and/or IM.
Similar to what was seen with the d4T prodrugs, complete conversion to
AAM was obtained for the AZT derivatives containing L-alanine (14), D-alanine
(16), L-phenylalanine (17), and
glycine (13). The AZT derivatives containing L-leucine (15) and methyl-L-glutamic
acid (18) showed an incomplete conversion to AAM, giving a
mixture of AAM and IM. For these two compounds, formation of AAM was
found to be somewhat less efficient than that for the corresponding d4T compounds.
Next, we determined the carboxylesterase-mediated metabolism of the d4T
and AZT prodrugs in different biological media (i.e., CEM cell extract,
human serum, and mouse serum). As can be seen in Table 2, the relative
conversion patterns in these biological media were fairly comparable to
those observed for pig liver carboxylesterase. In all cases,
8 was found to be fully stable. For 12, no AAM
was formed, due to the stability of the IM. For the other compounds
studied, the conversion to AAM was most pronounced in mouse serum and
least efficient in human serum, whereas an intermediate enzyme activity
was present in CEM cell extract. In all three media, the
L-alanine derivatives of d4TMP and AZTMP (2 and
14, respectively) were among the best converters to their
AAMs. For all d4T prodrugs except for 11 and 12, no accumulation of the IM was seen.
Interestingly, a few AZTMP prodrugs showed partial accumulation of the
IM (i.e., 15 and 17) that was not observed for
their corresponding d4TMP derivatives. This is presumably due to a
higher chemical stability of the IM.
Finally, when the pig liver carboxylesterase or mouse serum was
preincubated during 30 min in the presence of the serine protease inhibitor PMSF (final concentration, 10 mM), which is known to be also
an inhibitor of carboxylesterase (Shao and Mitra, 1994), followed by
the addition of prototype compounds of the AZTMP prodrug or d4TMP
prodrug and overnight incubation, the formation of their AAM was
inhibited by more than 90%.
As shown in Table 2, the stability of the L-alanine
derivatives of d4T on incubation in CEM cell extracts and in human
serum was also found to depend on the alkyl moiety, with the conversion rate to AAM in human serum being 80, 62, and 23% for the benzyl 1, methyl 2, and ethyl 3 derivatives,
respectively. We therefore extended the stability studies in human
serum to a large series of L-alaninyl-d4TMP prodrug
derivatives, with variations in the alkyl moiety (Fig.
3). To better discriminate between the compounds, the incubation was performed during 3 h (instead of overnight as in the previous studies). The two most striking extremes in stability were the compounds containing phenyl (least stable) and
tert-butyl (most stable). Compared with the prototype
compound 2, which showed an intermediate stability in human
serum, the ethyl derivative was more stable, whereas the benzyl
compound was less stable. These data were confirmed in studies with pig liver carboxylesterase: the percentages of prodrug left after 10 min
incubation were 1.5, 31, 66, 92, and 98% for the derivatives containing phenyl, benzyl, methyl, ethyl, and tert-butyl,
respectively.
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Optimization of Rat Liver Enzyme Preparation. Incubation of the prodrugs in CEM cell extract, human serum, or mouse serum resulted in only trace levels of ddNMP (d4TMP or AZTMP) or free ddN (d4T or AZT). In addition, no ddN or ddNMP was detected after incubation with pig liver carboxylesterase. These data suggest that a distinct enzyme is involved in the cleavage of the phosphoramidate linkage of the AAM ddNMP to ddNMP.
To obtain a partially purified enzyme preparation that is able to convert the AAM to ddNMP (or ddN), we used rat liver as the enzyme source because it is known that liver is rich with hydrolytic enzymes, including amidases (Ledneva et al., 1967, 1970; Shabarova, 1970). Our
procedure was based on the method of Khandwala and Smith (1967). The
enzyme was partially purified by ammonium sulfate precipitation (the
fraction between 32 and 42% was used). The purification was about
50-fold. The enzymatic activity was determined by monitoring the
formation of d4TMP from L-alaninyl-d4TMP. The
L-alaninyl-d4TMP source was obtained from high amounts of
2, exposed to pig liver carboxylesterase. After an
extraction and cleaning step with C18 cartridge columns, we obtained an
AAM yield of 95% and a compound purity of 98%, as assessed by HPLC.
Six different procedures for partial purification of the rat liver
phosphoramidase were performed (see Materials and Methods). The phosphoramidase activity in these enzyme fractions was determined from the percentage of L-alaninyl-d4TMP converted
to d4TMP plus d4T. The values for the different enzyme preparations
were normalized for an equal amount of total protein. Preparations
1 and 2, corresponding to the microsomal fraction
of the liver cells, showed a weak phosphoramidase activity (1.6 and
2.4% conversion, respectively). In contrast, a much higher
phosphoramidase activity (6.8% conversion to ddNMP plus ddN) was
present in preparation 3, obtained through ammonium sulfate
precipitation of the cytosolic fraction. This enzymatic activity could
be further increased to 23.1% by the addition of 10 mM
-mercaptoethanol during preparation (4), whereas EDTA had
no influence (preparation 5; 25% conversion). However, the
highest phosphoramidase activity was recovered from rat liver by
omitting the centrifugation step at 105,000g, thus
considerably shortening the preparation time. This preparation
6, with an enzyme activity of 80%, was stored in aliquots
at 80°C and was routinely used in the incubation studies with the
phosphoramidate derivatives.
Metabolism of AAM in a Rat Liver Enzyme Preparation. Table 3 shows the metabolism of the triester prodrugs of d4T or AZT after overnight incubation in the rat liver enzyme preparation 6. The conversion of the prodrugs to d4TMP was most pronounced for the derivatives containing L-alanine, followed (in decreasing order) by methyl-L-aspartic acid, glycine, and D-alanine.
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Conversion of AAMs to d4TMP or AZTMP.
Pure
L-alaninyl-d4TMP and L-alaninyl-AZTMP (prepared
from 2 and 14 by carboxylesterase-mediated
hydrolysis) were incubated overnight in the rat liver enzyme
preparation. The marked differences in their metabolism are clearly
depicted in Fig. 4. After 2-h incubation,
20% of L-alaninyl-d4TMP was converted, with the main
metabolite being d4TMP, whereas the d4T formation was negligible at
this time point. After 16 h, only 20% of
L-alaninyl-d4TMP was left, 59% was present as d4TMP, and
21% was present as d4T.
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), was determined. A complete
inhibition of L-alaninyl-d4TMP conversion to d4TMP in the
rat liver preparation was achieved with iodobenzene at a concentration
of 1 µM (Fig. 5). At lower
concentrations, iodobenzene inhibited the phosphoramidase activity in a
concentration-dependent manner, with a 50% inhibitory concentration of
10 nM (defined as the concentration at which the formation of d4TMP
plus d4T was inhibited by 50%). It should be mentioned that
iodobenzene was inactive as an inhibitor of phosphodiesterase and
carboxylesterase at 1 mM (data not shown).
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Physicochemical Properties of Rat Liver Phosphoramidase. The partially purified rat liver enzyme was found to display a markedly enhanced activity on the addition of MgCl2 (10 mM), and this cofactor was routinely used in phosphoramidase assays.
The rat liver phosphoramidase showed an optimal enzymatic activity at pH 7.4 that was 8-fold reduced at pH 5.4 and 9.4. Metal-chelating agents such as EDTA had no effect on the phosphoramidase activity of the rat liver enzyme preparation. By using ultrafiltration membranes with different molecular mass cut-off values (from 10 to 100 kDa), we determined that the highest phosphoramidase activity was present in the rat liver enzyme fraction with a molecular mass ranging from 50 to 100 kDa.| |
Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The phosphoramidate derivatives of ddN were designed to act as
membrane-soluble nucleotide prodrugs that enable intracellular delivery
of the ddNMP, thus bypassing the first activation step by cellular
kinases (TK in the case of d4T and AZT; McGuigan et al., 1996). The
ddNMP is then further phosphorylated to ddN 5'-triphosphate, the active
metabolite that inhibits HIV reverse transcriptase (Balzarini et al.,
1998; Balzarini and De Clercq, 1999). In this study, we focused on a
series of phosphoramidate triesters of d4TMP and AZTMP, with variations
in the amino acid moiety and the attached alkyl group. The antiviral
activity in HIV-infected CEM cells was found to be determined by three
structural parameters: the nature of the nucleoside (d4T or AZT), the
amino acid moiety, and the carboxyl ester group. Most importantly, the
phosphoramidate derivatives of d4TMP were found to be equally active in
wild-type and TK-deficient CEM/TK cells, thus
proving that the TK bypass concept is fully successful with these d4T
prodrugs. This is in sharp contrast to the failure of the AZTMP
triesters to afford pronounced antiviral activity in
CEM/TK cells. In addition, depending on the
amino acid moiety, large differences were seen in the antivirally
effective EC50 values of the d4TMP triesters,
with L-alanine being the preferred amino acid.
To better understand the structure-activity relationship of the
phosphoramidate derivatives of d4TMP and AZTMP, we performed a detailed
study on their metabolism, using purified enzymes as well as crude and
partially purified enzyme preparations. Several groups have suggested
that the activation is initiated by the carboxylesterase-mediated
hydrolysis of the carboxyl ester in the amino acid part (Fig. 1;
Valette et al., 1996; Winter et al., 1996; McGuigan et al., 1998). This
esterase cleavage is followed by a nucleophilic attack of the
phosphorus by the carboxyl group, with elimination of phenol after the
formation of a five-membered cyclic intermediate. The resulting AAM is
then converted to ddNMP, which is either further phosphorylated to
ddNTP by kinases or cleaved to free ddN by phosphatases and/or nucleotidases.
We first concentrated on the carboxylesterase-dependent formation of
the AAM. Our previous studies on the metabolism of radiolabeled 2 in intact cells have pointed to the intermediary formation of a key metabolite (AAM), which markedly accumulates intracellularly (Balzarini et al., 1996a). We now performed direct incubations of
several phosphoramidate derivatives of d4TMP and AZTMP with high
amounts of pig liver carboxylesterase. AAM was very efficiently formed
from the L-alanine-containing triesters of d4TMP or AZTMP. The L-valine- and -alanine-containing d4TMP derivatives
did not convert to AAM. This is consistent with the low or marginal
antiviral activity of these two compounds. Qualitatively, a similar
pattern for AAM formation was observed when the triesters were
incubated in biological media (i.e., human serum, CEM cell extract, or
mouse serum). Overall, the conversion to AAM proved to be highest in mouse serum, lowest in human serum, and intermediate in CEM cell extract. These data are in agreement with the ubiquitous presence of
carboxylesterases in mammalian tissues, albeit at enzyme levels that
are highly dependent on tissue type and species (Robbi and Beaufay,
1983; Hosokawa et al., 1990).
Some triesters showed a significant conversion to a metabolite, of
which the retention time on HPLC was between those of the triester and
the AAM. We hypothesize that this is the IM that is formed after
hydrolysis of the carboxyl ester in the amino acid moiety and that may
be assumed to have a high chemical instability (Fig. 1). The nature of
the side chain of the amino acid and the nature of the sugar moiety, in
particular the azido group at the 3'-position in the case of AZT, may
play an essential role in the formation of the AAM through the
hypothetical cyclic intermediate. The only exception seen here was the
-alanine triester of d4TMP, of which the IM proved fully stable. The
most logical explanation is that due to the extra carbon in the
-alanine chain, this IM is unable to form a six-membered cyclic
intermediate to allow further conversion to AAM.
To obtain optimal delivery of the ddNMP inside the target cells (i.e., the HIV-infected lymphocytes), an efficient conversion rate by carboxylesterase may be considered as favorable. However, in the in vivo situation, the prodrugs can reach the target cells only if they are resistant to hydrolysis by extracellular carboxylesterases (such as in serum). If not, partial conversion of the prodrugs to AAM would result in a lower cell penetration and, hence, reduced antiviral response. Thus, a compromise must be reached between the extracellular stability of the prodrugs and their conversion to the AAM once they have been taken up intracellularly.
Our studies have revealed that the carboxylester group linked to the amino acid moiety has pronounced influence on the pharmacokinetics of the triester and its associated stability. Indeed, the stability in human serum of L-alaninyl-containing phosphoramidates of d4TMP proved to be highly dependent on the nature of the alkyl ester group, with, for instance, an ethyl providing higher stability than the methyl group. Because an additional concern may be the safety of the alcohol that is released by carboxylesterase, the ethyl derivative may seem preferential over the methyl derivative. Moreover, the more stable ethyl ester derivative showed a slight advantage in antiviral activity.
Next, we investigated the second part of the activation pathway, consisting of the cleavage of AAM to ddNMP or free ddN. Although incubation of the triesters in CEM cell extract, human serum, or mouse serum resulted in limited formation of ddNMP and ddN, this conversion was considerably higher when a rat liver enzyme preparation was used. The metabolism of AAM to ddNMP and ddN was found markedly depending on the amino acid moiety, with L-alanine being the preferred amino acid, thus fully agreeing with the superior antiviral activity of the L-alaninyl-containing phosphoramidate triesters.
Moreover, the d4TMP triesters were found to be superior to the
corresponding AZTMP triesters in two aspects: a higher total amount of
ddNMP plus ddN release and a markedly higher ratio of ddNMP to ddN
(Fig. 6). These results were further confirmed in incubation studies
with purified AAM compounds. After overnight incubation, the percentage
of AAM left was 20 and 84% for L-alaninyl-d4TMP and
L-alaninyl-AZTMP, and the ratio of ddNMP to ddN was 2.4 and 0.6, respectively. The latter result can be explained by the higher sensitivity of AZTMP than d4TMP to nonspecific phosphatases
and/or 5'-nucleotidases in this preparation. Similar observations were obtained for the prodrug derivatives of d4TMP (3 and
4) and AZTMP (13 and 16). These data
are fully consistent with the observation that the d4TMP triesters, but
not the AZTMP triesters, keep their anti-HIV activity in TK-deficient
CEM cells. Clearly, the nature of the nucleoside in the prodrug
determines the degree at which the kinase-bypass concept is successful.
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The last part of our study was focused on the partial purification and
characterization of the enzyme that hydrolyzes the phosphoramidate
(P---N) linkage in the AAM. The original definition of phosphoramidase
(E.C. 3.9.1.1) as given by Dixon and Webb (1979) refers to an enzyme
that is acting on a phosphorus-nitrogen (P---N) bond. However,
different enzymes with phosphoramidase activity have been isolated from
various sources (i.e., rat liver, spleen, or kidney), and the enzyme
has been associated with both microsomal and cytosolic fractions
(Holzer et al., 1966; Snyder and Wilson, 1972; Kuba et al., 1994;
Nishino et al., 1994). In these studies, the phosphoramidase activity
was determined based on release of free phosphate from the P---N
substrate. In our studies, we describe an enzyme activity that
hydrolyzes a P---N bond with the release of a substituted phosphate
(i.e., a phosphate attached to a nucleoside moiety). After
fractionation of a rat liver homogenate by centrifugal separation, the
different subcellular fractions (mitochondrial, microsomal, and
cytosolic) were evaluated for phosphoramidase activity by incubation
with L-alaninyl-d4TMP and measurement of the d4TMP
formation. The highest enzymatic activity was found in the cytosolic
fraction. Reduction in the preparation time and the addition of
-mercaptoethanol in the isolation buffer and magnesium chloride in
the incubation mixture resulted in a significantly higher enzyme yield
and activity. Such an enzyme activity has been described by Shabarova
and coworkers (Ledneva et al., 1967, 1970; Shabarova, 1970, 1971;
Dudkin et al., 1971a,b) and recently by McIntee et al. (1997).
Shabarova (1970) reported on the discovery of a nucleoside
5'-phosphoramidase in some animal tissues (i.e., rabbit liver). This
enzyme hydrolyses the phosphoramide bond to form the nucleotide and the
amino acid. Nucleotide 5'-amidates were the most readily hydrolyzed
compounds, and the enzyme preparation proved capable of hydrolyzing
both L- and D-amino acid derivatives of
nucleotides (Shabarova, 1970). McIntee et al. (1997) recently found
that the 3-indolyl aminoacyl phosphoramidate prodrugs of AZT and
3'-fluoro-2',3'-dideoxythymidine were also substrates for
phosphoramidase activity in peripheral blood mononuclear cell extracts.
In this respect, the 3'-fluoro-2',3'-dideoxythymidine-MP derivative was
a better substrate than the AZTMP derivative. The d4TMP derivative was
not included in this study.
The inhibitory effect of iodobenzene on the phosphoramidase activity is
similar to that previously reported for the closely related compound
iodosobenzene (Singer and Fruton, 1957). At high concentrations, the
naturally occurring phosphoramidate compound phosphocreatine was shown
to be able to partially inhibit the phosphoramidase-mediated hydrolysis
of L-alaninyl-d4TMP. However, L-alaninyl-d4TMP
proved not to be a substrate for creatine phosphokinase, the enzyme
that catalyzes the phosphorylation of creatine. In addition, we
incubated the AAM compounds with phosphodiesterase, alkaline
phosphatase, and 5'-nucleotidase. Phosphodiesterase I was able to
hydrolyze L-alaninyl-d4TMP and
L-alaninyl-AZTMP, yet this reaction was not inhibited by
iodobenzene. Taken together, these results suggest that the
phosphoramidase enzyme in the rat liver fraction that recognizes the
phosphoramidate ddNMP prodrugs is distinct from known esterases. We
also found that 10 µM benzylalcohol is able to completely block the
conversion of L-alaninyl-d4TMP to d4TMP. However, when
benzylalcohol is released in the intact cells on conversion of the
L-alaninyl-d4TMP prodrug to L-alaninyl-d4TMP, it will immediately be spread over the whole cell content, and it is
even expected to diffuse out of the cells to the extracellular medium.
Therefore, it is reasonable to assume that the benzylalcohol released
from the phosphoramidate prodrug has no chance to efficiently inhibit
the phosphoramidase-catalyzed intracellular release of d4TMP. The
potent antiviral activity of the benzyl prodrug ester derivative is in
agreement with this hypothesis. We are currently planning the isolation
of the phosphoramidase enzyme by ion-exchange or affinity
chromatography to identify its physicochemical properties, substrate
specificity, and physiological role. These insights should help to
design new phosphoramidate prodrugs with improved biochemical and
therapeutic properties.
In Fig. 1, we proposed as the main metabolic pathway of the prodrugs
the release of the alkyl (methyl) ester group by carboxylesterases before the release of the aryl part of the molecule. Indeed, we recently revealed that an 
amino acid is necessary for biological activity and consistently results in the formation of the amino acyl
diester (McGuigan et al., 1998a). In contrast, (and longer) amino
acids also show efficient ester cleavage but are biologically inert and
show no phenyl loss. This strongly implies that 1) the amino acyl
liberation is necessary for biological action, 2) an amino acid is
necessary for the phenyl cleavage (by intramolecular catalysis), and 3)
phenyl loss proceeds after ester cleavage. Similarly, in a recent
report (McGuigan et al., 1998b), we noted that replacement of methyl by
t-butyl as the carboxylate ester lead to a significant
reduction in antiviral potency. This directly correlated with the high
stability of the t-butyl ester to any esterase-mediated
degradation. Because the stability of the phenyl phosphate group per se
should be unaffected by such a modification at the carboxyl terminus,
the "apparent" stabilization of the phenyl phosphate toward
cleavage (and resulting reduction in antiviral potency) can only arise
from the carboxylate ester stabilization. Again, these data strongly
support the suggestion that carboxyl ester cleavage is a necessary
prerequisite for phenyl loss and for eventual antiviral activity.
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Acknowledgments |
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We thank Ann Absilis, Lizette van Berckelaer, and Ria Van Berwaer for excellent technical assistance.
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Footnotes |
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Received April 14, 1999; Accepted July 14, 1999
This work was supported by Biomedical Research Programme of the European Commission, Belgian Geconcerteerde Onderzoeksacties (Project 95/5), and Fondation Singer-Polignac, Paris, France.
Send reprint requests to: Dr. Lieve Naesens, Rega Institute for Medical Research, Minderbroedersstraat 10, B-3000 Leuven, Belgium. E-mail: lieve.naesens{at}rega.kuleuven.ac.be
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Abbreviations |
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ddN, 2',3'-dideoxynucleoside analog; ddNMP, 2',3'-dideoxynucleoside 5'-monophosphate; ddNTP, 2',3'-dideoxynucleoside 5'-triphosphate; d4T, 2',3'-didehydro-2',3'-dideoxythymidine; PMSF, phenylmethylsulfonyl fluoride; AZT, 3'-azido-2',3'-dideoxythymidine; CC50, 50% cytotoxic concentration; HIV, human immunodeficiency virus; IM, intermediate metabolite; AAM, amino acyl metabolite.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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-arabinofuranosylcytosine: Evidence of phosphoramidase activity.
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4569-4575[Medline].
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Arch Biochem Biophys
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101-106[Medline].This article has been cited by other articles:
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) and the metabolites ddNMP (
) and free
ddN (
).
) and of formation of the metabolites (d4TMP plus
d4T; 
). The dashed line represents the IC50 value,
defined as the iodobenzene concentration at which the conversion
of L-alaninyl-d4TMP to d4TMP plus d4T is inhibited by
50%.
