Enzymatic Assay for Measurement of Intracellular DXG Triphosphate Concentrations in Peripheral Blood Mononuclear Cells from Human Immunodeficiency Virus Type 1-Infected Patients

Abstract

DXG {[2R-cis]-2-amino-1,9-dihydro-9-[2-[hydroxymethyl]-1,3-dioxolan-4-yl]-6H-purin-6-one} and its prodrug DAPD ([2R-cis]-4-[2,6-diamino-9H-purin-9-yl]-1,3-dioxolane-2-methanol; amdoxovir) are novel 2′,3′-dideoxynucleosides (ddNs) displaying activity against human immunodeficiency virus type 1 (HIV-1). In this paper, we describe the development of an enzymatic assay for determining the intracellular active metabolite of DXG and DAPD, DXG triphosphate (DXGTP), in peripheral blood mononuclear cells (PBMCs) from HIV-infected patients. The assay involves inhibition of HIV reverse transcriptase (RT), which normally incorporates radiolabeled deoxynucleoside triphosphates (dNTPs) into a synthetic template primer. DXGTP (0.6 pmol) inhibited control product formation with or without a preincubation step. Inhibition was greatest when the template primer was most diluted. DAPDTP inhibited control product formation only at very high levels (50 pmol) and when a preincubation procedure was used. However, reduced template primer stability in assays using preincubation steps, coupled with potential interference by DAPDTP, led to the current assay method for DXGTP being performed without preincubation. Standard DXGTP inhibition curves were constructed. The presence of PBMC extracts or endogenous dGTP did not interfere with the DXGTP assay. Intracellular DXGTP and dGTP concentrations were determined in PBMCs from HIV-infected patients receiving oral DAPD (500 mg b.i.d.). Peak concentrations of DXGTP were obtained 8 h after dosing and were measurable through 48 h postdose. Levels of endogenous dGTP were also determined over 48 h. No direct relationship was observed between concentrations of DXGTP and dGTP. Quantification of DXGTP concentrations in PBMCs from patients receiving a clinically relevant dose of DAPD is possible with this enzymatic assay.

Current therapy for the treatment of human immunodeficiency virus (HIV) infection involves the use of two 2′,3′-dideoxynucleoside (ddN) reverse transcriptase (RT) inhibitors in combination with nonnucleoside RT inhibitors and protease inhibitors. However, continued use of these compounds can lead to the development of resistant virus as well as decreased tolerability. Therefore, the production of novel agents that possess drug-resistant viral activity while incorporating favorable toxicity profiles is justified.

DXG {[2R-cis]-2-amino-1,9-dihydro-9-(2-[hydroxymethyl)-1,3-dioxolan-4-yl]-6H-purin-6-one} and its prodrug, DAPD ([2R-cis]-4-[2,6-diamino-9H-purin-9-yl]-1,3-dioxolane-2-methanol; amdoxovir), are novel ddNs displaying activity against HIV type 1 (HIV-1) (8, 9, 14, 22). The chemical structures of these two compounds are shown in Fig. 1. The use of DAPD (and DXG) in antiretroviral therapy is advantageous, because cross-resistance with other ddNs is minimal. For example, studies have shown that zidovudine (ZDV)-resistant virus remained sensitive to DAPD and DXG, whereas both compounds are only marginally less potent against lamivudine (3TC)-resistant virus (3, 9). Furthermore, DAPD and DXG display synergy with other antiretroviral agents and have favorable safety profiles (8, 9), suggesting that these compounds are good candidates in salvage therapy regimens.

FIG. 1.

Chemical structures of DXG (A) and DAPD (B).

DAPD is deaminated to DXG by the ubiquitous enzyme adenosine deaminase (18), which is then sequentially phosphorylated by intracellular kinases to its active anabolite, DXG-5′-triphosphate (DXGTP). In vitro studies have shown that DXGTP is much more potent than DAPDTP against HIV-1 RT (K_i = 0.019 μM for DXGTP and 250 μM for DAPDTP) (L. H. Wang, J. W. Bigley, M. Brosnan-Cook, N. D. Sista, F. Rousseau, and the DAPD-101 Clinical Trial Group, Abstr. 8th Conf. Retrovir. Opportun. Infect., abstr. P752, 2001). DXGTP competes with its corresponding endogenous substrate, dGTP, for binding to HIV-1 RT. Once incorporated into the nascent DNA strand, the absence of a 3′-hydroxyl group prevents the formation of 3′,5′-phosphodiester linkages, resulting in chain termination (7, 9, 10, 15).

Measurement of plasma HIV RNA levels and determination of CD4 cell number represent the current standards for monitoring the effectiveness of antiretroviral drug regimens. More recently, resistance testing and the measurement of plasma drug concentrations have been shown to be useful tools to further assess effectiveness when used in conjunction with the standard procedures. However, for ddNs that require intracellular activation to the TP anabolite, quantification of the intracellular dideoxynucleoside triphosphate (ddNTP) levels rather than the plasma ddN concentrations better correlates with the efficacy of these compounds. Furthermore, knowledge of intracellular ddNTP pharmacokinetics can aid in determination of dosing regimens for the ddN antiviral drugs.

There are a number of techniques available for quantifying intracellular ddNTP levels in peripheral blood mononuclear cells (PBMCs) from HIV-1-infected patients. These include a combined high-pressure liquid chromatography-radioimmunoassay (HPLC/RIA) procedure (11, 17, 19, 23) and a liquid chromatography tandem mass spectrometry (LC/MS/MS) method (16, 20; F. Becher, R. Landman, A. Canestri, S. Mboup, C. Ndeye Toure Kane, F. Liegeois, M. Vray, C. Dalban, M. H. Prevot, G. Leleu, and H. Benech, Abstr. 9th Conf. Retrovir. Opportun. Infect., abstr. P452-w, 2002). Our approach has been to determine the concentrations of the active ddNTP by using an enzymatic assay. This method involves the competitive inhibition of HIV-1 RT that normally incorporates radiolabeled dNTP into a specific synthetic template primer by the ddNTP of interest. By using known amounts of the ddNTP standards as a competitive inhibitor, a standard inhibition curve can be derived. Concentrations of ddNTP present in cell extracts are then quantified from the standard curve.

We have previously developed enzymatic assays for the determination of intracellular ddNTP concentrations of two ddNs, 3TC and abacavir (ABC) in PBMCs (13). These assays have shown to be an inexpensive alternative for the determination of intracellular ddNTPs, when compared to other methods, such as LC/MS/MS. The relative advantages and disadvantages of the enzymatic assays in comparison with these other types of procedures have been discussed previously (13). Here, we describe the development of an enzymatic assay to quantify DXGTP levels in PBMCs from HIV-1-infected patients receiving oral DAPD.

MATERIALS AND METHODS

Chemicals.

[5′-³H]dGTP (specific activity, 4.0 Ci mmol⁻¹) was purchased from Moravek Biochemicals, Inc., Brea, Calif. HIV RT was obtained from Amersham Pharmacia Biotech U.K., Ltd., Buckinghamshire, United Kingdom (manufactured by the Research Foundation for Microbial Disease of Osaka University, Osaka, Japan). Poly(rC) · p(dG)_12-18 template primer was obtained from Amersham Pharmacia Biotech, Inc., Piscataway, N.J. DXGTP and DAPDTP were provided by Triangle Pharmaceuticals, Inc., Durham, N.C. DE81 filter papers (25-mm DEAE paper) were acquired from Whatman, U.K. Liquid scintillation fluid (Ultima Gold) was obtained from Packard BioScience B.V., Groningen, The Netherlands. Lymphoprep was procured from Nycomed Pharma AS, Oslo, Norway. All other chemicals were purchased from Sigma Chemical Company, Ltd., U.K.

Preparation of PBMC extracts from healthy volunteers and HIV-infected patients.

For preparation of PBMC extracts from healthy volunteers, fresh heparinized venous blood (≥20 ml) was collected, and PBMCs were isolated by the method of density cushion centrifugation with lymphoprep resolving medium (12). PBMCs were then washed in phosphate-buffered saline and an aliquot (10 μl) was counted with a hemocytometer. Cells were centrifuged (2,772 × g, 4 min, 4°C), and the resulting cell pellet was extracted overnight in 60% methanol (2 ml) at 4°C. Following extraction, the cell suspensions were centrifuged (2,772 × g, 4 min, 4°C), and the methanolic supernatant fractions were evaporated to dryness.

The residue of the methanolic extracts was then reconstituted in perchloric acid (0.4 N, 200 μl, 4°C) and extracted on ice for 30 min. Samples were centrifuged (2,772 × g, 4 min, 4°C), and the acid supernatant fractions (200 μl) were transferred to fresh 1.5-ml microcentrifuge tubes.

The acid extracts were neutralized by the addition of freshly prepared 0.5 N trioctylamine (27%) in 1′,1′,2′-trichlorotrifluoroethane (73%) (200 μl) to each tube. After rotary mixing (15 min), the phases were separated by centrifugation (6,000 × g, 10 s). An aliquot (150 to 170 μl) of the upper aqueous phase (of the resulting three-phase system) was carefully aspirated into 1.5-ml microcentrifuge tubes. The resulting extracts were then checked to ensure successful neutralization (pH 7.0) with pH paper and were stored (−20°C) until determination of intracellular levels.

For analysis of PBMC extracts from HIV-infected patients, methanolic extracts (60% methanol; 1 ml) were sent from Triangle Pharmaceuticals, Inc., to the University of Liverpool laboratory. These extracts were centrifuged, dried down, re-extracted in perchloric acid, and stored as described above.

DXGTP enzymatic assay development: comparison of inhibition of HIV RT by DXGTP with and without preincubation.

For the single-incubation procedure, control product formation (i.e., incorporation of [³H]dGTP into the template primer in the absence of inhibitor) was determined by using a reaction mixture containing [³H]dGTP (15.625 pmol); 5 μl of template primer [poly(rC) · p(dG)_12-18 at 2.5 U ml⁻¹] at dilutions of 1:5, 1:10, 1:20, or 1:40; 30 mM KCl; 0.1% (wt/vol) Triton X-100; 0.025% (wt/vol) bovine serum albumin (BSA); 0.5 mM EDTA; 1 mM dithiothreitol (DTT); 6 mM MgCl₂; 50 mM Tris-HCl (pH 8.0); and 0.4 U of HIV RT. The total mixture volume was 50 μl. To assess inhibition of the assay by DXGTP, the same protocol was followed, but with the addition of 0.6 pmol of DXGTP to the reaction mixtures.

The reaction was initiated by the addition of the enzyme and continued for 30 min at 37°C. Duplicate aliquots (20 μl) from each reaction mixture were spotted onto DE81 paper circles, presoaked with cold 5% (wt/vol) trichloroacetic acid-1% (wt/vol) sodium pyrophosphate solution. The paper circles bind oligonucleotides. After drying, the paper circles were washed three times for 5 min each with cold 5% trichloroacetic acid-1% sodium pyrophosphate solution (2 × 5 ml, 2 × 4 ml) to remove unincorporated nucleotides and then rinsed once with 95% ethanol (3 ml). The paper circles were dried and counted in Ultima Gold scintillant (4 ml) to assess the amount of product formed. Each experiment was performed in duplicate on three separate occasions.

For the preincubation procedure, the same reaction mixtures were prepared, but no [³H]dGTP was added. The total mixture volume was 45 μl. The reaction was initiated by the addition of the enzyme, for 30 min at 37°C. Following this period, [³H]dGTP (15.625 pmol) was then added to each reaction mixture, and the reaction was reinitiated for 30 min at 37°C. After the second incubation period, the protocol was the same as that described for the single incubation. Each experiment was performed in duplicate on three separate occasions.

Comparison of inhibition of HIV RT by DAPDTP with and without preincubation.

For the single-incubation procedure, control product formation (i.e. incorporation of [³H]dGTP into the template primer in the absence of inhibitor) was determined with a reaction mixture the same as that described for the DXGTP assay. To assess inhibition of the assay by DAPDTP, the same protocol was followed, but with the addition of 50 pmol of DAPDTP to the reaction mixtures.

For the preincubation procedure, the same reaction mixtures were prepared, but no [³H]dGTP was added. The reaction was initiated by the addition of the enzyme, and following this period, [³H]dGTP (15.625 pmol) was added and the reaction was reinitiated. Spotting, washing, and counting were performed as described for the DXGTP assay. These incubation experiments were also performed in duplicate on three separate occasions.

Optimizing the standard DXGTP inhibition curve: effect of DAPDTP.

DXGTP inhibition was initially assessed (with a template primer dilution of 1:10) with 0, 0.1, 0.2, 0.4, 0.6, and 1.0 pmol of DXGTP by a single-incubation procedure, and with 0, 0.05, 0.1, 0.2, 0.4, and 0.6 pmol of DXGTP by a preincubation procedure. The experiments described above were repeated in parallel, but in the presence of DAPDTP at 1.0 and 0.6 pmol for the single-incubation and preincubation procedures, respectively. The single-incubation procedure was then adopted for all future studies, with standard inhibition curves comprising 0, 0.02, 0.05, 0.1, 0.2, 0.4, and 0.6 pmol of DXGTP and with a template primer dilution of 1:10 only.

Effect of PBMC extracts on the standard DXGTP inhibition curve.

Standard DXGTP inhibition curves were determined (0, 0.1, and 0.6 pmol only) in the absence and presence of PBMC extracts from four healthy volunteers (no DXGTP present). The extracts contained 10 and 40 × 10⁶ cells for each standard curve sample. Extracts (5 μl from a total volume of 200 μl) were added to the reaction mixtures.

Effect of dGTP on the standard DXGTP inhibition curve.

Standard DXGTP inhibition curves were repeated (0, 0.1, and 0.6 pmol only) in the absence and presence of dGTP (0.1 and 0.5 pmol).

Determination of variability and recovery for the DXGTP assay.

Interassay variability of the DXGTP assay was determined by quantifying DXGTP concentrations from the same PBMC extracts on five separate occasions. PBMCs were isolated from blood taken from HIV-infected patients receiving DAPD (500 mg b.i.d.), and extracted in methanol and perchloric acid. Interassay variability of the DXGTP assay was also determined by quantifying DXGTP quality control (QC) standards (0.05, 0.075, 0.12, 0.3, and 0.6 pmol) on four separate occasions.

Intra-assay variability of the DXGTP assay was determined by quantifying DXGTP concentrations from the same PBMC extracts five times with the same assay. PBMCs were isolated from blood taken from HIV-infected patients receiving DAPD (500 mg b.i.d.) and extracted in methanol and perchloric acid.

Recovery of the assay was determined by quantifying DXGTP from previously prepared PBMC extracts alone and in the presence of approximately 0.2, 0.3, or 0.5 pmol of DXGTP QC standards. Only 5 μl of extract was added to the reaction mixture from the total 200 μl. Recovery of the QC standards was calculated by subtracting the amount of DXGTP present in the extract alone from the amount of DXGTP obtained in the extract plus QC standard. All QC standards were prepared independently of the standard curves. Assay recovery was also investigated by spiking PBMCs from healthy volunteers with DXGTP (5.0 pmol) and then extracting the samples as previously described. DXGTP levels were then determined in these extracts in the usual manner and compared to a DXGTP standard of the same amount present in the extract (0.125 pmol of DXGTP, because only 5 μl of extract was added to the reaction mixture from the 200 μl in total).

Quantification of DXGTP and dGTP concentrations in PBMC extracts from HIV-infected patients.

DXGTP concentrations in PBMC extracts from eight HIV-infected patients were quantified by using the enzymatic assay described above. All patients received oral DAPD (500 mg b.i.d.) for 14 days, with blood samples (2 × 8 ml CPT tubes) collected over 48 h after the last dose (0, 1, 3, 6, 8, 12, 24, and 48 h). PBMCs were isolated and extracted in methanol and perchloric acid as described earlier.

Concentrations of dGTP were also quantified in these samples with a specific primer extension assay as previously published by Sherman and Fyfe (21) and routinely used in our laboratory (13). This assay is similar to the type described above, except elongation, not inhibition, is determined. The basic premise is to use a DNA template primer where elongation can only occur through the incorporation of two dNTPs. The dNTP to be determined (in this case dGTP) will be the limiting factor in the reaction, while the other dNTP not only will be present in excess, but also will be radiolabeled. Therefore, the amount of radioactivity incorporated into the template primer will be proportional to the amount of limiting dNTP. By using known amounts of the limiting dNTP standard, a standard curve can be derived. Concentrations of dNTP present in PBMC extracts are then quantified from the standard curve.

RESULTS

DXGTP assay development.

As the concentration of template primer was diluted, the amount of control product formation decreased when the single-incubation procedure was used. For example, at a 1:20 template primer dilution, control product formation was reduced to approximately 40% of the 1:5 template primer dilution levels (Table 1). However, at each template primer dilution, the addition of 0.6 pmol of DXGTP inhibited control product formation (greatest at a 1:40 template primer dilution [Table 1]).

TABLE 1.

Effect of altering the concentration of template primer on control product formation and inhibition by DXGTP and DAPDTP, with and without preincubation

Inhibitor	Template primer dilution	Control product formation (dpm)a	Control product formation with inhibitor (dpm)a	% Inhibition
0.6 pmol of DXGTP
No preincubation	1:5	42,150 ± 4,126	20,696 ± 2,897	50.9
	1:10	36,002 ± 4,068	16,633 ± 2,611	53.8
	1:20	17,989 ± 1,883	7,789 ± 1,106	56.7
	1:40	10,056 ± 1,002	4,263 ± 571	57.6
Preincubation	1:5	21,908 ± 2,056	6,529 ± 764	70.2
	1:10	10,143 ± 984	1,876 ± 285	81.5
	1:20	5,126 ± 554	218 ± 63	89.9
	1:40	2,358 ± 289	184 ± 25	92.2
50 pmol of DAPDTP
No preincubation	1:5	41,158 ± 4,028	41,323 ± 3,967	−0.4
	1:10	33,682 ± 2,560	34,625 ± 4,813	−2.8
	1:20	16,989 ± 1,598	16,021 ± 2,675	5.7
	1:40	9,410 ± 999	9,598 ± 931	−2.0
Preincubation	1:5	22,690 ± 2,051	15,928 ± 2,246	29.8
	1:10	10,002 ± 1,176	8,182 ± 1,628	18.2
	1:20	4,942 ± 554	3,286 ± 575	33.5
	1:40	2,294 ± 389	1,457 ± 220	36.5

^aData are expressed as the mean ± standard deviation for three separate experiments.

A preincubation procedure was adopted to assess whether the degree of inhibition by DXGTP observed was different to that found with the single-incubation procedure. The results are also shown in Table 1. As observed with the single-incubation procedure, there was a steady decline in control product formation as the template primer was diluted. There was a concurrent increase in the degree of inhibition of control product formation by 0.6 pmol of DXGTP as the template primer was diluted (e.g., 0.6 pmol of DXGTP inhibited control product formation by 70.2 and 92.2% at 1:5 and 1:40 template primer dilutions, respectively). Overall, the degree of inhibition was greater in incubations with a preincubation procedure than when in the single-incubation procedure. However, it should be noted that the amount of control product formation was decreased (approximately by half) when comparing the preincubation procedure with a single-incubation procedure.

Similar results were observed in studies with DAPDTP as the inhibitor, with a decrease in control product formation noted as the template primer was diluted. A reduction in control product formation was also observed when a preincubation procedure was adopted, in comparison to the single-incubation procedure (Table 1). However, DAPDTP (50 pmol) did not inhibit control product formation at any template primer dilution, with the single-incubation procedure. In contrast, inhibition by 50 pmol of DAPDTP was observed at all template primer dilutions when a preincubation procedure was adopted. For example, 50 pmol of DAPDTP inhibited control product formation by 33.5%, at a 1:20 template primer dilution (Table 1). Additional studies with DAPDTP at 0.6-pmol amounts gave rise to no discernible effects on control product formation, with or without the use of a preincubation procedure.

Inhibition of control product formation by DXGTP was unaffected by the presence in the incubation of DAPDTP, at amounts of 1.0 and 0.6 pmol for single-incubation and preincubation procedures, respectively (Table 2). It was decided to proceed with the single-incubation procedure for future development of the assay.

TABLE 2.

Effect of DAPDTP on the DXGTP inhibition curve^a

Incubation conditions	Amt of DXGTP (pmol)	Result with (105 dpm)b:
		DXGTP alone	+ DAPDTP
No preincubation	0	3.10	3.11
	0.1	3.55 (12.8)	3.60 (13.6)
	0.2	4.61 (32.8)	4.48 (30.6)
	0.4	6.57 (52.8)	6.59 (52.8)
	0.6	8.53 (63.7)	9.03 (65.5)
	1.0	13.65 (77.3)	13.87 (77.6)
Preincubation	0	5.96	5.92
	0.05	7.07 (15.7)	6.30 (6.0)
	0.1	7.72 (22.8)	7.27 (18.6)
	0.2	8.72 (31.7)	8.73 (32.2)
	0.4	18.13 (67.1)	17.77 (66.7)
	0.6	32.03 (81.4)	35.25 (83.2)

^aExperiments were performed with and without preincubation in the presence and absence of 1.0 and 0.6 pmol of DAPDTP, respectively.

^bResults are expressed as 1/dpm (10⁵) with the corresponding inhibition shown in parentheses. Data are expressed as mean values from two separate experiments.

Figure 2A and B show typical standard DXGTP inhibition curves (0, 0.02, 0.05, 0.1, 0.2, 0.4, and 0.6 pmol) in terms of product formed in 1/dpm and percent inhibition, respectively. The coefficients of regression (r) values were usually greater than 0.99. The standard curves displayed low interassay variability over time (<15%), indicating the stability of the DXGTP standards. In addition, the stability of the stock DXGTP solution was routinely checked by HPLC with UV detection (at a wavelength of 254 nm) and found to be stable for at least 12 months when stored at −20°C.

FIG. 2.

Typical standard inhibition curves for the determination of DXGTP. Data from each point are expressed as the mean ± standard deviation for 15 separate curves performed over a period of several months. Panel A shows the variation in product formed as 1/dpm, while panel B shows the variation in percent inhibition. Assay conditions utilized a single-incubation procedure of 30 min at 37°C. The amount of [³H]dGTP was 15.625 pmol, with the template primer at a 1:10 dilution.

PBMC extracts from blood samples obtained from four healthy volunteers were investigated for the potential of cell extract to alter the standard DXGTP inhibition curve. Extracts containing 10 × 10⁶ and 40 × 10⁶ cells were studied. There was no evidence of any interference on either control product formation itself or on the degree of inhibition by DXGTP at either cell number investigated.

Additional studies investigated the standard DXGTP inhibition curve in the absence and presence of dGTP (0.1 and 0.5 pmol). The three-point standard DXGTP inhibition curve was unaffected by dGTP.

Determination of variability and recovery of the DXGTP assay.

The limit of quantification of the DXGTP assay is dependent on the cell number from which the extract is obtained. More than 10 × 10⁶ cells are optimal for the assay to detect DXGTP levels. However, from the standard curve itself, the limit of quantification was set at 0.02 pmol of DXGTP. Therefore, for an extract containing 10 × 10⁶ cells, the limit of quantification is 0.08 pmol/10⁶ cells.

Interassay variability of the DXGTP assay determined from the same PBMC extract on five separate occasions was 13% (range, 7 to 23%). Interassay variability was also determined by repeat analysis of various DXGTP QC standards (0.05, 0.075, 0.12, 0.3, and 0.6 pmol; n = 4) and found to be <10%. Quantification of the QC standards was within 10% of the theoretical values (accuracy). Intra-assay variability determined from analyzing PBMC extracts five times in the same assay was 12% (range, 8 to 21%).

Variability in the recovery of three independently prepared QC standards (approximately 0.2, 0.3, and 0.5 pmol) in the presence of PBMC extracts is shown in Table 3, Quantification of these QC standards alone was again within 10% of the theoretical values (e.g., 0.470 pmol compared to 0.5 pmol). Recovery of each of the QC standards was ≥95%. For example, variability of recovery of the 0.470 pmol of QC standard was 6% (0.486 ± 0.030; n = 7), with a mean recovery of 103% when compared to the actual value.

TABLE 3.

Recovery of DXGTP (in QC samples) when spiked into PBMC extracts containing DXGTP^a

Theoretical amt in spiked QC (pmol)	(n)	Actual amt in spiked QC (pmol)	Amt recovered (pmol)	% of actual recovered
0.2	(5)	0.220	0.208 ± 0.031 (15)	95 ± 14
0.3	(6)	0.276	0.265 ± 0.033 (12)	96 ± 11
0.5	(7)	0.470	0.486 ± 0.030 (6)	103 ± 7

^aExtrapolated values are means ± standard deviation with values in parentheses representing the percent variability in the recovery of these QC standards.

Recovery of the 0.125 pmol of DXGTP standard in a PBMC sample from a healthy volunteer when 5 pmol of DXGTP was added to the sample initially (because only 5 μl was added to the reaction mixtures from a total of 200 μl), was determined to be 91% (0.114 ± 0.012 pmol; n = 4), when compared to standard containing 0.125 pmol of DXGTP alone. Furthermore, repeated thawing and analysis of a DXGTP-spiked PBMC extract over a period of 6 months gave rise to only 6% variability (0.126 ± 0.007 pmol).

Quantification of DXGTP and dGTP concentrations in PBMC extracts from HIV-infected patients.

Figure 3 shows a 48-h pharmacokinetic profile of intracellular DXGTP in PBMC extracts from eight HIV-infected patients receiving oral DAPD (500 mg b.i.d.) for 14 days. DXGTP concentrations were measurable throughout 48 h following the last dose of DAPD, ranging from 0.012 to 1.838 pmol/10⁶ cells. There was considerable interpatient variability. Peak concentrations were observed at the 8-h time point. The intracellular levels of endogenous dGTP in these samples were also measurable throughout 48 h following the last dose of DAPD, ranging from 0.013 to 0.424 pmol/10⁶ cells (Fig. 4). Considerable interpatient variability was also observed with these levels.

FIG. 3.

Intracellular pharmacokinetic profile of DXGTP in PBMC extracts from blood samples obtained from HIV-infected patients, over 48 h after administration of the last dose following 14 days of the 500-mg b.i.d. DAPD oral regimen. The results shown are the mean ± standard error for eight patients.

FIG. 4.

Intracellular pharmacokinetic profile of dGTP in PBMC extracts from blood samples obtained from HIV-infected patients over 48 h after administration of the last dose following 14 days of a 500-mg b.i.d. DAPD oral regimen. The results shown are the mean ± standard error for eight patients.

DISCUSSION

DAPD and DXG belong to a novel class of ddNs that display different resistance profiles to other commonly known ddNs, such as ZDV and 3TC. DXGTP is the active antiviral form of both DAPD and DXG in vivo (18). However, due to the poor solubility and oral bioavailability of DXG (4, 5), the prodrug DAPD was developed to improve these characteristics (14). DAPD is converted to DXG, which is then sequentially phosphorylated to DXGTP. Studies have shown that DAPD phosphates have not been detected in vitro (8), but it is not known whether DAPDTP can be formed from DAPD in vivo. In vitro studies have shown that DXGTP is much more potent than DAPDTP in inhibiting HIV-1 RT, and that the anti-HIV activity of DAPD in cell culture decreases by a factor of 50 in the presence of an adenosine deaminase inhibitor (L. H. Wang, J. W. Bigley, M. Brosnan-Cook, N. D. Sista, F. Rousseau, and the DAPD-101 Clinical Trial Group, Abstr. 8th Conf. Retrovir. Opportun. Infect., abstr. P752, 2001). These findings support that DXG(TP) not DAPD(TP) is responsible for the antiviral activity of DAPD.

Due to the importance of the formation of intracellular DXGTP from either DAPD or DXG, measurement of concentrations in PBMCs from HIV-infected patients will better delineate the pharmacokinetics of the ddNs and help optimize dosing regimens. Therefore, we have developed an enzymatic assay for the quantification of intracellular DXGTP concentrations that may be used as a tool to investigate intracellular pharmacokinetics. We have previously developed enzymatic assays for the determination of intracellular 3TCTP and carbovir TP (CBVTP, the active anabolite of ABC) (13). Therefore, an enzymatic assay for the quantification of DXGTP has been developed here, by modifying the existing assay procedure for CBVTP. The enzymatic assays have been shown to provide viable alternatives to other procedures, such as HPLC/RIA and LC/MS/MS, for the quantification of ddNTP concentrations.

Preliminary studies centered on the effects of template primer concentration on control product formation. By using a single-incubation procedure, when the amount of template primer was diluted, there was a concurrent decline in control product formation. These results show that the amount of template primer is very important to the amount of product that can be formed, because the number of binding sites present will restrict the incorporation of [³H]dGTP. The addition of 0.6 pmol of DXGTP resulted in inhibition of this control product formation, with the greatest inhibition observed when the template primer was most diluted. Similarly to the CBVTP assay, a 1:10 template primer dilution was chosen for the ongoing studies, because product formation was sufficiently high while still allowing for a good degree of inhibition by DXGTP.

Although incorporation of [³H]dGTP was inhibited by DXGTP by the single-incubation procedure, the study was repeated to include a preincubation step in order to assess whether sensitivity could be improved further. Diluting the template primer again led to a fall in the amount of control product formation, while the degree of inhibition was greater with the preincubation procedure than with the single-incubation procedure. These results are as expected, because there is no initial competition between DXGTP and [³H]dGTP when the preincubation procedure is used, and DXGTP is allowed to become incorporated into the template primer before [³H]dGTP.

The observed decrease in control product formation when the preincubation procedure is used may involve a reduction in template primer stability as the length of the incubation is increased from 30 min to 1 h (overall).

The above studies were repeated, but with DAPDTP as the inhibitor (50 pmol). The ability of DAPDTP to inhibit control product formation must be investigated, because if DAPDTP does inhibit control product formation, the assay will not be able to distinguish between DAPDTP and DXGTP already present in PBMC extracts. Reassuringly, when a single-incubation procedure was used, there was no evidence of inhibition by DAPDTP at 50 pmol. However, with a preincubation procedure, there was inhibition observed by DAPDTP. This finding confirms that DAPDTP is a much weaker substrate than DXGTP for HIV-1 RT.

The ability of DAPDTP to inhibit control product formation was further investigated at smaller amounts (0.6 and 1.0 pmol with and without using a preincubation procedure, respectively). DAPDTP had no effect on the DXGTP inhibition curves at these amounts, suggesting that by both procedures, the assay is specific for measuring DXGTP. Although the use of the preincubation procedure led to greater inhibition by DXGTP, due to the reduction in template primer stability and potential interference by DAPDTP at larger amounts, the single-incubation procedure was adopted for routine use. Repeated analysis of the standard DXGTP inhibition curve over time showed the assay to be highly reproducible in terms of product formed and inhibition observed.

Before a determination of intracellular DXGTP in PBMC extracts from HIV-infected patients could be performed, it was essential that any potential interference with the assays by cell extracts be identified and then minimized. Previous studies have shown that the standard CBVTP inhibition curve was not affected in the presence of healthy volunteer PBMC extracts. In contrast, there was observed interference with the standard 3TCTP inhibition curve by the presence of PBMC extracts (13). This is probably caused by certain active enzymes present in these samples. As with the CBVTP assay, the presence of PBMC extracts had no effects on the standard DXGTP inhibition curves or product formation. Furthermore, the addition of dGTP, even at levels that are not observed in PBMC extracts, had no effect on the standard DXGTP inhibition curve. This apparent lack of interference observed suggests that DXGTP levels in PBMC extracts from HIV-infected patients can be directly quantified without the need for any correction to the standard inhibition curve.

The intracellular pharmacokinetic profile of DXGTP in PBMC extracts from eight HIV-infected patients was determined. There was considerable variation in DXGTP concentrations between patients, consistent with data from other ddNs used to treat HIV infection (1, 11, 13, 16, 17, 19, 20, 23). Concentrations of DXGTP were measurable throughout 48 h following the last 500-mg dose, being highest at the 8-h time point (t_1/2 = 27.3 h). More rigorous pharmacokinetic analysis will be performed with a larger cohort of patients.

Endogenous dGTP concentrations were variable between patients and displayed no obvious pattern over time, suggesting that there is no direct relationship between intracellular DXGTP and dGTP concentrations. Quantification of the respective dNTPs is important as the efficacy of the ddNs depends not only on the concentrations of active ddNTP formed, but also on that of the competing endogenous dNTP.

Previous studies with ZDV have shown that there is a poor relationship between levels of the parent compound in plasma and their efficacy and toxicity (1, 2, 6). Furthermore, levels of ZDV phosphates (including ZDVTP) were not appreciably changed by an increase in drug dosage (2). Similar results were observed with 3TC (17). This is probably due to a saturation of the intracellular kinases involved in the phosphorylation (and thus activation) of these drugs at higher doses. The effect of the dosage of guanosine ddNs (such as DAPD, DXG, and ABC) on their activation and efficacy has yet to be fully elucidated. However, previous findings have shown that CBVTP levels were lower in patients receiving a dose of 300 mg of ABC (13), when compared to patients receiving a 600-mg dose (M. Harris, S. Jutha, D. J. Back, S. Kewn, R. Marina, and J. S. G. Montaner, Abstr. 8th Conf. Retrovir. Opportun. Infect., abstr. P746, 2001). These observations suggest that the pathways for activation of these compounds may not be so readily saturated. Therefore, antiviral effect may be altered by a change in dosage. This demonstrates the importance of quantifying intracellular ddNTP levels in terms of the correlation between pharmacokinetics and efficacy.

The quantification of intracellular DXGTP levels in PBMCs from HIV-infected patients is possible by using the enzymatic procedure described above and should have clinical utility. The assay requires a relatively low number of cells to determine DXGTP (≥10 × 10⁶ cells) and is reproducible, displaying acceptable inter- and intra-assay variabilities. One drawback to the enzymatic approach is that specific individual assays are required to determine each ddNTP. In contrast, LC/MS/MS procedures can determine more than one ddNTP in the same assay (16, 20; F. Becher, R. Landman, A. Canestri, S. Mboup, C. Ndeye Toure Kane, F. Liegeois, M. Vray, C. Dalban, M. H. Prevot, G. Leleu, and H. Benech, Abstr. 9th Conf. Retrovir. Opportun. Infect., abstr. P452-w, 2002). Furthermore, ddNTPs from the same class (e.g., the guanosine analogues DXGTP and CBVTP) cannot be present in the same extract, because they will both compete for incorporation into the template primer. However, ddNs from the same class are rarely if ever coadministered. Interference resulting from other classes of ddNTP is unlikely due to the unique template primers used for each assay, which are specific for one class of ddNTP. Although ddNTPs are described as inhibitors of RT, they do not inhibit the function of the enzyme itself, only the elongation of the DNA chain by acting as chain terminators. Therefore, the assays can determine the concentration of a specific ddNTP in extracts containing multiple classes of ddNTP, without any interference. Additionally, other inhibitors of RT (e.g., nevirapine, a nonnucleoside RT inhibitor) do not interfere with the assay unless present at extremely high concentrations (data not shown).

In conclusion, quantification of DXGTP in PBMCs from HIV-infected patients receiving DAPD has been demonstrated by and achieved with an enzymatic assay.