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Antimicrobial Agents and Chemotherapy, June 2004, p . 2337-2340, Vol . 48, No . 6

Lack of Effect of Recombinant Human Growth Hormone on the In Vitro Activities of Antiretroviral Drugs against Human Immunodeficiency Virus Type 1

Mark A Wainberg,1* Bluma G . Brenner,1 Eric Daar,2 Joseph M . Gertner,3 Clement Olivier,4 and Susan Kenley3

McGill University AIDS Center, Lady Davis Institute, Jewish General Hospital, Montreal, Quebec, Canada,1 Department of Medicine, Cedars-Sinai Medical Center Research Institute, University of California at Los Angeles School of Medicine, Los Angeles, California,2 Serono Inc., Rockland, Maryland,3 Serono International SA, Geneva, Switzerland4

Received 23 July 2003/ Returned for modification 9 October 2003/ Accepted 3 February 2004


   ABSTRACT

 
We studied the effects of recombinant human growth hormone (r-hGH) on human immunodeficiency virus type 1 replication by growing both wild-type and drug-resistant variants of virus in the presence of various concentrations of eight different antiretroviral drugs . r-hGH had no significant effect on either viral replication or the 50% inhibitory concentrations of these compounds .


   TEXT

 
Although highly active antiretroviral therapy (HAART) has led to declines in the rates of mortality and AIDS-related opportunistic infections in the developed world, there remains a need to prevent the loss of lean body tissue (wasting) in patients with human immunodeficiency virus (HIV) infection and AIDS (12, 16) . Human growth hormone (hGH) increases lean body mass and muscle nitrogen retention and thus represents a potential approach to the treatment of AIDS-related wasting (9, 13) . In randomized double-blind placebo-controlled trials performed in the pre-HAART and HAART eras, recombinant hGH (r-hGH) was shown to increase total body weight and lean body mass and to decrease body fat content (9, 15, 17, 20) . These changes are associated with improvements in physical performance and quality of life (15, 17, 20) . Although optimization of nutritional status and exercise may also provide benefits in many cases, treatment with hGH is an excellent option in patients with severe wasting .

However, concern exists that r-hGH might stimulate viral replication or inhibit the activities of antiretroviral drugs (ARVs) (4), since one early study actually found that r-hGH increased the levels of p24 antigen released from peripheral blood mononuclear cells (PBMCs) in tissue culture at concentrations of 50 or 100 ng/ml, but not at concentrations of 1, 5, 10, or 250 ng/ml (7) . Importantly, r-hGH did not affect the antiviral activity of zidovudine in that study . Subsequent experiments (E . Daar, unpublished data) showed that r-hGH did not increase the level of HIV replication in PBMCs at concentrations of up to 250 ng/ml and had no effect on the antiviral activities of a variety of ARVs used in the pre-HAART era (zidovudine, didanosine, zalcitabine, and stavudine) . The present experiments confirm and extend these findings through the use of currently approved ARVs in each of the three major drug classes .

Drugs. r-hGH (Serostim), obtained from Serono Inc . (Rockland, Mass.), was reconstituted in an accompanying vial of sterile water (for injection, USP; provided by the company) and further diluted to final concentrations of 10 and 50 ng/ml in tissue culture medium . Tenofovir was provided by Gilead Sciences (Foster City, Calif.) . Abacavir and amprenavir were provided by GlaxoSmithKline Inc . (Research Triangle Park, N.C.), and delavirdine and nelfinavir were provided by Agouron Inc . (San Diego, Calif.) . Efavirenz, nevirapine, and lopinavir were obtained from Bristol-Myers Squibb Inc . (Wallingford, Conn.), Boehringer-Ingelheim Inc . (Ridgefield, Conn.), and Abbott Laboratories Inc . (North Chicago, Ill.), respectively .

Viral isolates. We used two wild-type (wt) and five drug-resistant clinical isolates of HIV type 1 (HIV-1), the genotypes of which are shown in Table 1 . The wt isolates were from patients who had not received therapy . The concentrations of all approved ARVs that inhibit viral replication by 50% (IC50s) for the isolates were confirmed to be the same as those for other wt isolates, and the phenotypes of the isolates were the same as those of other wt isolates . Genotyping for the detection of mutations associated with drug resistance was performed by sequencing analysis with the Tru-Gene system (Bayer Diagnostics Inc., Toronto, Ontario, Canada) . The resistant isolates were from patients who had been extensively treated with antiviral agents .


TABLE 1 . Effects of r-hGH at 10 or 50 ng/ml on levels of p24 antigen in PBMC culture fluids

 
In vitro studies. IC50s were determined by growing wt or drug-resistant variants of HIV in cultured PBMCs in the presence of various concentrations of the different ARVs, ranging from 0.001 to 10 µM, as described previously (3, 18) . Viral p24 concentrations in culture fluids were measured (Abbott Laboratories Inc., North Chicago, Ill.) to generate IC50s (8) . The effects of r-hGH on viral replication and antiretroviral activity were assessed by adding the hormone to cultures at a concentration of 10 or 50 ng/ml; these represent the midrange and maximum concentrations, respectively, expected in plasma after subcutaneous injection of a standard daily dose of 6 mg of r-hGH . To generate viral stocks, patient PBMCs were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum . Viral replication was enhanced by coculture of HIV-infected PBMCs with phytohemagglutinin-stimulated donor PBMCs in complete medium containing interleukin-2, followed by serial passage with fresh uninfected cells to generate further rounds of replication . Viral production during each passage was monitored by measurement of reverse transcriptase (RT) activity and p24 antigen levels . Amplified viral stocks were assayed for p24 antigen, RT activity, and infectivity . Infectivity was assessed by determining the 50% tissue culture infective dose in PBMCs . Viral stocks were stored at –135°C until they were required .

For each concentration of virus, ARV, and r-hGH, inhibitory activity against HIV was calculated as the amount p24 obtained with an antiretroviral concentration of x/amount of p24 obtained with an antiretroviral concentration of 0 . The logarithm of (1 – activity)/activity was regressed on the logarithm of the concentration of the antiretroviral agent by least-squares linear regression to predict the IC50 of each antiretroviral agent (1) . IC50s obtained in the presence and the absence of r-hGH were compared by two-way analysis of variance .

Effects of r-hGH on cell replication in vitro. Preliminary experiments first confirmed that r-hGH had no significant effect on cell replication kinetics . After 7 days, the mean number of cells in cultures originally seeded at 2 x 106 cells/ml was 7.4 x 106/ml in the absence of r-hGH, whereas the mean numbers of cells were 8.0 x 106 and 8.5 x 106/ml in the presence of r-hGH at 10 and 50 ng/ml, respectively .

Effects of r-hGH on HIV-1 replication and antiretroviral activity in vitro. p24 antigen concentrations were measured after infection of PBMCs over 7 days with the seven HIV isolates used in these studies in the presence or absence of r-hGH . The results in Table 1 show that r-hGH had no significant effect on the replication of any of the HIV variants studied . Moreover, the presence of r-hGH at either 10 or 50 ng/ml had no significant effect on the IC50s of any of the eight ARVs tested for the two wt isolates (Fig . 1) . Figure 2 shows a similar lack of an effect of r-hGH when the five drug-resistant variants of HIV-1 were studied .


FIG . 1 . IC50s of eight ARVs for two wt clinical HIV-1 isolates (a and b) in the presence and absence of r-hGH (logarithmic scale) . Each experiment was performed by using three replicate samples and was repeated on at least two different occasions . In no instance were significant differences in IC50s observed between studies performed in the presence or absence of r-hGH . Abbreviations and symbols: ABC, abacavir; TDF, tenofovir; EFV, efavirenz; DLV, delavirdine; NVP, nevirapine; NFV, nelfinavir; APV, amprenavir; LPV, lopinavir; solid bars, control; open bars r-hGH at 10 ng/ml; shaded bars, r-hGH at 20 ng/ml.

 

FIG . 2 . IC50s of eight ARVs for five drug-resistant clinical of HIV-1 isolates (a to e) in the presence and the absence of r-hGH (logarithmic scale) . The results presented in panels a to e correspond to those for isolates 3 to 7, respectively, in Table 1 . Each experiment was performed with three replicate samples and was repeated on at least two different occasions . In no instance were significant differences in IC50s observed between studies performed in the presence or absence of r-hGH . Abbreviations and symbols: ABC, abacavir; TDF, tenofovir; EFV, efavirenz; DLV, delavirdine; NVP, nevirapine; NFV, nelfinavir; APV, amprenavir; LPV, lopinavir; solid bars, control; open bars r-hGH at 10 ng/ml; shaded bars, r-hGH at 20 ng/ml.

 
Clinical studies in both the pre-HAART era (15, 17, 19, 20) and the HAART era (11) have shown that r-hGH can be effective and well tolerated as therapy for AIDS-related wasting . hGH has anabolic effects, which result in an increase in lean body mass (6, 10, 14) . In contrast to other anabolic agents, hGH also has anticatabolic actions mediated via the direct effects on cells, the release of insulin-like growth factor type 1, and the protein-sparing effects of lipid oxidation (2, 5, 10, 19, 21) . Given the clinical benefits of r-hGH in patients with AIDS-related wasting, our findings that r-hGH has no effect on viral replication or on the antiretroviral activity of currently available ARVs are reassuring . The use of two wt and five drug-resistant HIV-1 strains contributes to the validity and clinical relevance of the study, as these strains reflect the heterogeneity of viral isolates found in patients with HIV-1 infection today .

Our data are also consistent with other in vitro studies (E . Daar, unpublished data), in which r-hGH had no effect on p24 release in a variety of cell types, including PBMCs that had been acutely infected with primary patient HIV isolates and chronically infected T-cell lines . Furthermore, r-hGH at concentrations as high as 250 ng/ml had no effect on the antiviral activities of commonly used agents .

These findings are also consistent with those of in vivo studies in the pre-HAART era, in which no increase in the plasma viral RNA load or the plasma infectious viral load was noted in patients receiving r-hGH (E . Daar, unpublished) . In addition, a double-blind placebo-controlled clinical trial with 770 patients with AIDS-associated wasting, of whom more than 500 received r-hGH for 12 weeks while continuing with HAART, showed no changes in viral loads after 12 weeks compared with the baseline values (E . Daar, unpublished) . In addition, a recent limited study showed that r-hGH could promote the replication of naïve CD4 cells in HIV-1-infected patients while not affecting the plasma viral load (11) .

Hence, the present results and previous in vivo findings support the clinical use of r-hGH to treat HIV-associated wasting .

 


   ACKNOWLEDGMENTS

 
Research in the laboratories of the McGill AIDS Centre is supported by grants from the Canadian Institutes of Health Research and by a generous donation from Aldo and Diane Bensadoun . We are also grateful to Serono Inc . for a grant in support of this work .


   FOOTNOTES

 
* Corresponding author . Mailing address: McGill AIDS Center, Jewish General Hospital, Lady Davis Institute, 3755 Cote St . Catherine Rd., Montreal, Quebec H3T 1E2, Canada . Phone: (514) 340-8260 . Fax: (514) 340-7502 . E-mail: mark.wainberg{at}mcgill.ca .


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