Journal of Virological Methodr, 30 (1990) 89-9X Elsevier

VIRh4ET 01069

89

Serum levels and catabolism of 3’-azido- 3’-deoxythymidine in vivo measured using a specific radioimmunoassay

Susan Cox”*, Ulla Rud&$, K&oly Nagy”, Jan Albert’.*, Viveca Holmberg4, Erik Sandstr6m4 and Britta Wahren’

‘Department of Virology, National Bacteriological Laboratory, Stockholm, Sweden, 2Department of Virology, Karolinska Institute, Stockholm, Sweden,

31nstitute of isotopes, Hungarian Academy of Sciences, Budapest, Hungary and ‘department of Der~ta~enereaI#~y, Southern Hospital, Stockholm, Sweden

(Accepted 22 June 1990)

Summary

The thymidine analogue 3’-azido-3’-deoxythymidine is an effective inhibitor of HIV replication in vitro and is used in the treatment of acquired immun~eficiency syndrome. We report here upon a rapid sensitive radioimmunoassay for the detec- tion of azidothymidine in serum or plasma. The assay is simple to perform and levels as low as 1 ng azidothymidine/ml can be detected. The assay is specific for azidothymidine and shows almost no cross-reaction with closely related nucleoside analogues or with naturally occurring nucleosides.

Using this radioimmunoassay we were able to measure the azidothymidine levels in the serum of monkeys and acquired immunodeficiency syndrome patients treated with AZT. Individual variation in tbe peak serum level and clearance rate of azidothymidine were seen, which emphasizes the need to tailor the dose to the individual.

Radioimmuuoassay; Azidothymidine; AIDS; Catabolism

_- ~~rres~~~nde~ce to: B. Wahren, ~pa~ment of Virology, National Bacteriological Laboratory, S-105 21 Stockholm, Sweden.

0168-85 10/90/$03.50@ 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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Introduction

The thymidine analogue 3’-azido-3’-deoxythymidine (AZT) is an effective inhibitor of human immunodeficiency virus (HIV) replication in vitro (Mitsuya et al., 1985). AZT is phosphorylated intracellularly and the triphosphate inhibits the viral reverses transcriptase by chain termination, since there is no 3’-hydroxyl group to allow further polymerisation (Furman et al., 1986). AZT is also clinically effective, reducing the mortality rate in HIV-infected AIDS patients who received the drug (Fischl et al., 1987). It is the only compound licensed for the treatment of AIDS to date.

AZT is rapidly absorbed after oral administration and reaches peak serum con- centrations within one hour (Yarchoan et al., 1986). It is rapidly metabolised in human liver to 3’-azido-3’-deo.xy-5’-O-P_D-glucopyranuronisyl-thymidine (Rese- tar and Spector, 1989). AZT and glucuronidated AZT are eliminated from the body by renal excretion (Resetar and Spector, 1989; Good et al., 1986). The plasma clearance of AZT has a half life of approximately one hour (Yarchoan et al., 1986). The rate of breakdown of AZT may vary from patient to patient, especially if there is any impairment of renal function; this may partly explain the different degree of clinical benefit and toxic side effects seen in different patients (Fischl et al., 1987; Yarchoan et al., 1986; Richman et al., 1986). Fur- thermore, the rate of breakdown of AZT may be expected to be influenced by compounds which either stimulate or compete for hepatic glucuronidation. Such compounds might be co-administered with AZT to treat the many secondary in- fections which occur in AIDS patients (Richman et al.., 1987). This will lead to plasma concentrations of AZT either lower or higher* than expected, which may result in reduced antiviral activity or increased toxicity, respectively. Thus, the ability to measure the precise peak plasma concentration .and half life of AZT in individual patients is an important factor in designing an appropriate treatment regime which will provide maximum benefit and minimum toxicity.

The concentration of AZT in plasma has previously been measured by high performance liquid chromatography (Yarchoan et al., 1986). However, the method is rather slow and technically complex; furthermore, the presence of other drugs (especially other thymidine nucleoside analogues) with which the patient may be treated may interfere with the resolution and accurate measurement of AZT. We report here a competitive radioimmunoassay (RIA) for the determination of AZT concentrations in serum or plasma. The assay is rapid, simple, and specific for AZT, and should be useful in the clinical management of AIDS patients. Using this RIA we measured the serum levels and rate of clearance of AZT in monkeys and AIDS patients treated ‘with AZT.

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Materials and Methods

Chemicals

AZT, thymidine and uridine were of the highest purity available and were obtained from Sigma Chemical Co (St. Louis, MO). FLT, AZU, the threo isomer of AZT and AZT-TP were gifts from Prof. Bo &erg, (Medivir, Stockholm, Sweden).

AZT monophosphate was purified by HPLC from lymphocytes incubated with AZT. Briefly, CEM cells (a human CD4 + lymphocyte cell line) were grown in RPM1 medium with antibiotics and 10% fetal calf serum in a humidified atmosphere of 5% CO2 in air at 37’C, in the presence of 10 PM AZT. After 24 h incubation the cells were harvested by centrifugation and the nucleotides extracted by the addition of cold 12% trichloroacetic acid. The acidic extract was neutralised by the amine-freon ion pairing method (Khym, 1975), and ribonucleotides were destroyed using the periodate oxidation method (Garret and Santi, 1979). AZT monophosphate was purified by ion exchange chromatography on a 4.6 x 25 mm partisil 10 SAX column (Whatman, Clifton, NJ) with 15 mM KH2PG4 (adjusted to pH 3.5 with phosphoric acid) at 1.5 ml/min as the eluting solvent. AZT monophosphate was identified by retention time and ratio of absorbance at 280 nm to that at 254 nm.

Serum samples

Serum samples from patients receiving AZT treatment were obtained from the Southern Hospital, Stockholm. Normal human serum was obtained from healthy human volunteers. Monkeys (Macaca fascicularis) treated with subcutaneous doses of AZT or FLT were bled for serum collection. Serum samples were heat treated to inactivate HIV before use in the assay.

A.27 RIA Ki!

The AZT RIA Kit used is commercially available from the Institute of Isotopes, The Hungarian Academy of Sciences, H-1525 Budapest, Hungary, P.O. Box 77. Briefly, AZT was coupled to bovine serum albumin at the N3 position on the base and purified by dialysis and lyophilization. New Zealand white rabbits were immunised with 1 mg AZT-BSA conjugate in Freund’s complete adjuvant for 5 weeks which produced a high titre antiserum. “51-labelled AZT was prepared by coupling AZT to ‘Ylabelled tyrosine at the N3 position on the base and purifying the product by reverse phase HPLC.

Assay of A.27

A volume of 100 ,~l of ‘251-labelled AZT (10 nCi) was mixed with 100 ~1 of either a standard solution of AZT, or a solution of an analogue, or a serum sample

(diluted if necessary) in duplicate. Serum samples taken O-3 h after a dose 2200 mg of AZT were diluted 10 x with normal human serum so that the values fell on the linear portion of the standard curve; other samples were measured undiluted. A volume of 100 //,I of the rabbit anti-AZT antiserum was added, the solution mixed and incubated at room temperature for 2 h. A volume of 1 ml of a cold aqueous 20% solution of polyethylene glycol was then added and the solution mixed. The AZT-antibody precipitate was pelleted by centrifugation at 2000 x I: for 30 min at 0°C and the supematant gently removed by aspiration. The higher the concentration of AZT in the sample, the less ‘2’1-labelled AZT is bound to the antibody and therefore the less radioactivity is found in the pellet. The radioactivity of the pellet was measured in a Packard Cobra 5000 series gamma counter. All assays were performed in duplicate.

Results

AZT standard curve

Fig. I shows a standard curve of radioactivity bound to the pellet when different concentrations of AZT are added to the incubation mix. The response is linear between 2 to 256 ng AZT/ml (7.5 to 960 nM). The presence of human serum or cell culture medium does not affect the linearity of the response but does raise slightly the amount of radioactivity bound to the pellet.

“.

; 1'0 100 1000

AZT concentration (ng/ml)

Fig. I. Standard curve of cpm bound vs AZT concentration. RIA was performed as described in Materials and Methods. The mean of duplicate experiments is shown. The standard deviation was always <IO%. AZT ‘samples were tested in aqueous solution (0). normal human serum (0) or in cell culture medium

(0).

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F

FLT

0

N3 AZU

0

HO

uridlne

N3

AZT

CH3

HO

HNA C”3

I II

00 N’

HO

thymidine

Fig. 2. Structures of the analogues tested in the RIA.

Cross-reactivity with related nucleoside analogues

Several nucleoside analogues with similar structures to AZT were tested to determine whether the antiserum displayed any cross-reactivity. Fig. 2 shows the structures of these analogues. Fig. 3 shows the radioactivity bound to the pellet in the presence of various concentrations of AZT or other nucleoside analogue. It can be seen that very few of the analogues can successfully compete with the ‘2’I-labelled AZT for binding to the antiserum. Only the threo isomer of AZT, and AZU, at the highest concentration tested (256 &ml), show any degree of cross reactivity. They bind to the antiserum with an efficiency of 27 and 32%, respectively, compared to AZT at the same concentration. Neither thymidine nor

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p 1925

P E 140 8

* 0 0,75

,”

concentration (nglml)

Fig. 3. Cross-reactivity of anti-AZT antiserum. RIA was performed as in Materials and Methods with AZT or analogues dissolved in cell culture medium. Cl: AZT, 0: FLT, 0: thymidine; A: uridine;

A: AZT, n : AZU. The mean of duplicate experiments is shown.

uridine show any cross-reaction with the anti-AZT antiserum.

Cross-reactivity with AZT nucleotides

AZT monophosphate (AZT-MP) and AZT triphosphate (AZT-TP) were tested to determine the specificity of the antiserum towards the 5’C of AZT. The results are shown in Table 1. Neither the monophosphate (up to 200 nM) nor the triphosphate (up to 200 nM) showed any reaction with the antiserum.

Serum levels of AZT in monkeys

Six cynomolgus monkeys were treated daily with subcutaneous AZT, PLT or a 1O:l mixture of both. Table 2 shows the serum levels of AZT at day 8 after

TABLE 1 Reactivity of anti-AZT antiserum to the monophosphate and triphosphate of AZT; mean of duplicate experiments is shown; standard deviation was <lo%

Nucleoside Concentration analogue WV

cpm bound %AZT reactivity

none 15872 0 AZT 5602 100 AZT-MP 16626 0 AZT-TP 16445 0

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TABLE 2 Serum AZT levels in cynomolgus monkeys after subcutaneous injection of a single dose of AZT or FLT; mean of two separate determinations. is shown

Treatment Serum-AZT (t&ml)

Untreated 0 AZT 15 mgPtg 1600 AZT 2.5 m&g monkey A 260

monkey B 210 FLT15mgIkg !Y FLT 0.25 mg/kg 0 Combination-AZT 1.25 mgkg 103

plus FLT 0.125mgkg

“A small degree of cross-reactivity with FLT was seen in this test where the dose of FLT was very high.

initiation of treatment. AZT was clearly measurable, while FLT showed little cross-reaction.

The serum levels of AZT measured using the RIA at various times after an oral dose of AZT in two patients who were undergoing AZT therapy are shown in Fig. 4. Peak serum concentrations were reached within 30 min of administration of AZT, but then declined quickly. After 50 min there seemed to be a slower decline of AZT levels. The peak levels were comparable for both patients despite the different doses given. AZT was also easily measured in the urine, but no kinetic study was made.

Tlmo (mm)

Fig. 4. Serum levels of AZT in two AIDS patients at various times after a single oral dose of 200 mg (patient A) or 300 mg (patient B). Tbe mean of two separate determinations is shown. O: patient

A; 0: patient B.

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Discussion

The AZT RIA was shown to be a simple, fast and specific method of measuring the AZT content of serum, plasma or urine. The assay relies upon competition between AZT in the sample and “‘1-1abelled AZT for binding to anti-AZT anti- serum. The amount of radioactivity bound to the pelleted antibody-AZT complex decreased linearly with increasing sample AZT concentration. The presence of human serum or cell culture medium increased slightly the radioactivity in the pellet, presumably owing to nonspecific binding of ‘251-labelled AZT. An AZT standard curve was therefore always performed under the same conditions as the samples to be tested - that is, in the presence of serum or medium as appro- priate. The standard curve was reproducible with the standard deviation always <lo% and usually 4%. The specificity of the AZT antiserum was determined by testing for crossreactivity with a number of structurally related analogues. Only AZU and the threo isomer of AZT reacted at the top concentrations tested to approximately 30% of the AZT reactivity. The complete lack of reactivity of FLT, uridine and thymidine indicates the importance of the azido group in recog- nition by the antiserum. The poor reactivity of AZU shows that the C5 methyl group is also important for recognition. The poor reactivity of the threo isomer of AZT is to be expected since we have previously shown that this compound has a quite different conformation to AZT (Bazin et al., 1989). The 5’OH is obviously also involved in the interaction since neither AZT-MP nor AZT-TP react with the antiserum.

Having determined the reproducibility and specificity of the method we then measured the AZT concentration in the serum of monkeys who were treated with AZT in a controlled fashion by injection. The results showed that AZT could be clearly and easily measured, even when used in combination with the related analogue FLT.

We also studied the peak concentration and clearance rate of AZT in the serum of two AIDS patients who were undergoing AZT therapy. Several factors might affect the serum AZT level in vivo, including individual variations in uptake, metabolism and compliance to the dose prescribed. In these patients high levels of AZT were rapidly achieved. After 30 min the peak serum concentration had been reached. The concentration of AZT then fell rapidly followed by a slower decline. The clearance rate of AZT differed to some extent between the two patients in this second slower phase. These results emphasize the need to tailor the treatment regime to the individual patient. The use of heat treatment of the sera as an inactivating step prior to the RIA did not affect the result.

AZT has also been measured in serum using enzyme linked immunosorbent assay (ELISA; Tadepalli and Quinn, 1990), time resolved fluoroimmunoassay (Tadepalli and Quinn, 1990) and fluorescence polarization (Granich et al., 1989). Although the latter two are as sensitive and reproducible as the RIA they are more complex to perform. The ELISA is 25fold less sensitive than these assays and the RIA. RIA has previously been shown to be a useful technique for the measurement of AZT (Tadepalli and Quinn, 1990). The specificity of RIA

91

towards closely related analogues of AZT has not previously been described. In conclusion, RIA represents a useful technique for the determination of AZT

levels in serum. It is fast and simple to perform and specific for AZT. This specificity is a distinct advantage with the growing trend toward combination chemotherapy where there is a need for accurate measurement of AZT levels in serum in the presence of other drugs which might be co-administered. It will also be useful in studies of clinical resistance to AZT, in deciding whether there is a changed metabolism of the compound. The variation in AZT metabolism we observed between individuals emphasizes the need to monitor and adjust the treatment regime as appropriate for each individual patient.

Acknowledgements

We thank Ewa Ljungdal-Stlhle for excellent technical assistance, Staffan Eriksson and Bjiim Jacobson for helpful discussions and Tibor Leidecher for the development of the AZT RIA kit.

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