Int. J. lmmunopharmac., Vol. 12, No. 7, pp. 737-741, 1990. Printed in Great Britain.

0192-0561/90 $3.00 + .00 Pergamon Press plc.

© 1990 International Society for lmmunopharmacology.

EFFECT OF AZIDOTHYMIDINE (AZT) ON HIV P24 ANTIGEN, BETA2-MICROGLOBULIN, NEOPTERIN, SOLUBLE CD8,

SOLUBLE INTERLEUKIN-2 RECEPTOR AND TUMOR NECROSIS FACTOR ALPHA LEVELS IN PATIENTS WITH

AIDS-RELATED COMPLEX OR AIDS

MOHAN M. REDDY,* GEORGE McKINLEY, ARTHUR ENGLARD and MICHAEL H. GRIECO

R. A. Cooke Institute of Allergy, St Luke's - Roosevelt Hospital Center New York, New York, U.S.A.

(Received 22 December 1989 and in final form 13 March 1990)

A b s t r a c t - - Circulating HIV P24 antigen, beta2-microglobulin, neopterin, soluble CD8, soluble interleukin- 2 receptor and TNF alpha levels were measured in 20 patients (9 with ARC and 11 with AIDS) treated with azidothymidine (AZT) and in 12 patients (3 with ARC and 9 with AIDS) who were in a placebo group. Mean levels of HIV P24 antigen, beta2-microglobulin, neopterin and SCD8 decreased significantly (P<0.05) after 12 to 16 weeks of AZT administration. SIL-2R and TNF alpha serum levels did not appear to change in association with AZT therapy. No changes were observed in the placebo group except that TNF alpha levels appeared to increase after 12 to 16 weeks. These results suggest that AZT administration may have led to reduced HIV P24 antigen, beta2-microglobulin, neopterin and SCD8 mean levels in these patients.

Acquired immunodeficiency syndrome (AIDS) is caused by the human immunodeficiency virus (HIV). It infects primarily the cells of the immune system and leads to a progressive decline of CD4 cells (Fauci, 1988). In addition, HIV P24 antigen, beta2- microglobulin (beta2-M), neopterin, soluble CD8 (SCD8), soluble interleukin-2 receptor (SIL-2R) and tumor necrosis factor alpha (TNF alpha) levels have been reported to be elevated in patients with AIDS- related complex (ARC) and AIDS (Reddy, Lange & Grieco, 1989; Kloster, John, Miller, Rubin, Nelson, Blair & Tomar, 1987; Reddy & Grieco, 1988; Reddy, Sorrell, Lange & Grieco, 1988; Fuchs, Hausen, Reibnegger, Werner, Dierich & Wachter, 1988; Moss, 1988). Azidothymidine (AZT, 3 '-azido-2' , 3'-dideoxythymidine) was first synthesized in 1964 as a potential antineoplastic agent by Horwitz and his associates. Mitsuya, Weinhold, Furman, St Clair, Lehrman, Gallo, Bolognesi, Barry & Broder (1985) observed antiviral activity against HIV by AZT in vitro. In 1986, 12 Medical Centers began a Phase II evaluation of AZT. St. Luke 's-Roosevel t Hospital Center was a participant in this multicenter trial of AZT (Fischl, Richmann, Grieco, Gottlieb, Volberding, Laskin, Leedom, Groopman, Mildvan, Schooley, Jackson, Durack, King & the AZT

Collaborative Working Group, 1987). Significant decreases in mortality and the frequency of opportunistic infections were observed in patients with ARC or AIDS treated with AZT (Fischl et al., 1987; Yarchoan, Mitsuya & Broder, 1989). We previously reported that the HIV P24 levels in patients with ARC or AIDS in the multicenter trial decreased significantly following AZT treatment (Reddy, McKinley, Englard & Grieco, 1989). This report summarizes data concerning circulating HIV P24 antigen, beta2-microglobulin, neopterin, SCD8, SIL-2R and TNF alpha levels at baseline and following AZT therapy in 20 patients enrolled in the AZT multicenter trial as well as in the subsequent AZT treatment IND and 12 patients who were in the original multicentral trial placebo group.

E X P E R I M E N T A L P R O C E D U R E S

Subjects

The study population consisted of 9 patients with ARC and 11 AIDS patients with Pneumocystis carinii pneumonia. A capsule consisting of 250 mg of AZT was administered orally every 4 h through- out the 24-h day, while 2 patients in the treatment

*Author to whom correspondence should be addressed at: R. A. Cooke Institute of Allergy, St. Luke's-Roosevelt Hospital Center, 428 West 59th Street, New York, New York 10019, U.S.A.

737

738 M.M. REDDY et al.

H I V P 2 4 A N T I G E N

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F i g . 1. Bar graph showing the effect of AZT treatment on mean values at The following numbers of patients were studied: 20, 12, 18, 14 and 13 at 0,

obtained by dependent Student's t-test. NS

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different intervals in the AZT treatment group. 4, 8, 12 and 16 weeks respectively. P values were = not significant.

IND received only 1200mg/day with capsules containing 100 mg of A Z T per capsule for 16 weeks. Subsequently the dose was reduced to 600 m g / d a y in some patients. In addition, 3 patients with A R C and 9 AIDS patients with P. carini i pneumonia in the placebo group were also studied.

M e t h o d s

The sera obtained f rom these patients at different intervals were stored at - 7 0 ° C until analyzed. All the serum samples f rom each patient were tested at the same time in each assay. HIV P24 antigen levels were measured using a sandwich solid phase enzyme immunoassay (Abbott Laboratories, Nor th Chicago, IL) and expressed as pg /ml (Reddy et al. , 1989). Beta2-microglobulin was quanti tated by utilizing the Phadebas Beta2 Microtest radioimmunoassay (Pharmacia Diagnostics, Piscataway, N J) and expressed as ug/1 (Reddy et al. , 1988). Neopterin

concentrations were analyzed using a radioimmuno- assay [DRG International (Hennig, Berlin), Mountainside, NJ] and expressed as ng /ml . SCD8 levels in sera were quantitated using a Cell Free Test Enzyme Immunoassay (T Cell Sciences, Inc., Cambridge, MA), as previously described and expressed as uni ts /ml (Reddy et al. , 1989). SIL-2R levels were determined by using a Cell Free IL-2R ELISA test assay (T Cell Sciences) and expressed as uni ts /ml (Reddy et al. , 1988). TNF alpha levels were quantitated by using a sandwich enzyme immuno- assay using the Biokine TNF Test Kit (T Cell Sciences) and expressed as pg /ml (Reddy et al. , 1988). The following within run coefficients of variat ion were obtained: HIV P24 Ag - - 3.3%; beta2-microglobulin - - 7%; neopterin - - 5%; SCD8 - - 6.8%; SIL-2R - - 10.8%; and TNF alpha - - 13%. The following normal ranges were obtained: HIV P24 Ag - - 0 pg /ml ; beta2-microglobulin - -

AZT and Laboratory Predictors

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Fig. 2. Bar graph showing the mean values at different intervals in the placebo group. The following numbers of patients were studied: 12, 11, 11, 10 and 9 at 0, 4, 8, 12 and 16 weeks respectively. P values were obtained by dependent Student's

t-test. NS = not significant.

1302-2520 ug/1; neopterin - - 0 . 2 - 2 . 2 ng /ml ; SCD8 - - 85 -795 units/ml; SIL-2R - - 71 -348 units/ml and T N F - - 0 - 2 5 pg /ml .

Statistical analysis was performed by dependent two-tailed Student 's t-test of change f rom baseline.

RESULTS

The mean values and standard errors o f means for HIV P24 antigen, beta2-microglobulin, neopterin, SCD8, SIL-2R and TNF alpha for patients treated with A Z T are depicted in Fig. 1, and those for patients in the placebo group are shown in Fig. 2. The mean levels in the placebo group for HIV P24 antigen, beta2-microglobulin, neopterin, SCD8 and SIL-2R did not differ significantly by paired Student 's t-test when baseline values were compared

with results at different t ime intervals. However , TNF mean levels did appear to have increased significantly at the 12 (P<0.10) to 16-week (P<0.05) intervals compared to the baseline value (Fig. 2).

On the other hand, HIV P24 antigen mean levels were significantly decreased by 8 weeks of treatment with A Z T and remained low after 16 weeks (Fig. 1). Similarly mean levels of beta2-microglobulin, neopterin, and SCD8 decreased after 12 to 16 weeks of A Z T administration. A Z T treatment had no apparent effect on SIL-2R and T N F alpha mean levels.

DISCUSSION

Azidothymidine, also known as zidovudine or Retrovir (Burroughs Wellcome, Research Triangle Park, NC) is the first drug to be licensed for

740

treatment of patients with ARC and AIDS (Yarchoan et al. , 1989; Hirsch, 1988; Bartlett, 1988). AZT therapy has been associated with increased survival in ARC and AIDS (Fischl et al. , 1987; Creagh-Kirk, Doi, Andrews, Nusinoff-Lehrman Tilson, Hoth & Barry, 1988) and has been shown to be associated with decrease in HIV P24 antigen levels in serum of patients with initially elevated levels (Reddy et al. , 1988; Jackson, Paul, Falk, Rubenis, Despotes, Mack, Knigge & Emeson, 1988).

Recently several laboratory parameters (CD4 lymphocytes, HIV P24 antigen, beta2-microglobulin and neopterin levels) have been suggested as potentially useful in predicting progression to AIDS after HIV infection (Moss, 1988). In addition, SCD8, SIL-2R and TNF in serum have been noted to be elevated in some patients with ARC and AIDS (Reddy et al. , 1989; Kloster et al. , 1987; Reddy & Grieco, 1988; Reddy et al . , 1988). Conversely these markers which might predict progression to AIDS could possibly be used to monitor the effectiveness of treatment. HIV P24 antigen, beta2-microglobu- lin, neopterin and SCD8 levels decreased in the AZT treatment group whereas in the placebo group the levels remained about the same as at baseline. Although HIV P24 antigen, beta2-microglobulin, neopterin and SCD8 levels increase in circulation after HIV infection and in development of AIDS, AZT appears to reverse these processes, effectively reducing these values of predictors in a manner similar to its effect which results in a decrease in mortality and the frequency of opportunistic infections (Fischl et al. , 1987; Yarchoan et al. , 1989). Baseline HIV P24 antigen was higher in the AZT group and was still higher after 16 weeks of treatment compared to the placebo group.

M. M. REDDY et al.

Jacobson, Abrams, Volberding, Bacchetti, Wilber, Chaisson, Crowe, Howard & Moss (1989) reported that serum beta2-microglobulin levels decreased in patients with AIDS or ARC treated with AZT, and they thought beta2-microglobulin to be a better marker than P24 antigen for early intervention trials involving antiretroviral drugs.

In the placebo group, the TNF alpha mean levels appeared to increase. TNF alpha changes may be significant only in the placebo group because of the very low baseline level. Lower TNF alpha levels at the beginning could result when the molecule had been destroyed during storage. On the other hand, AZT may have prevented increase in the mean level in those patients treated with the drug. Recently TNF alpha has been demonstrated to induce the expres- sion of HIV in a chronically infected T-cell clone through induction of nuclear factor binding to the NF-kB sites in the long terminal repeat (Folks, Clouse, Justement, Rabson, Duh, Kehrl & Fauci, 1989; Duh, Maury, Folks, Fauci & Rabson, 1989).

CONCLUSIONS

Our results suggest that: (1) AZT treatment decreases HIV P24 antigen, beta2-microglobulin, neopterin and SCD8 mean values after 12 to 16 weeks of treatment; and (2) AZT administration had no detectable effect on SIL-2R and TNF alpha levels.

Acknowledgements - - We thank Jim O'Connor, RN, for his help and Mary Reilingh, Gary Jelich and Mark Sydlo for their excellent technical assistance.

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