Synergistic cytotoxic effect of azidothymidine and recombinant interferon alpha on normal human bone marrow progenitor cells

Azidothymidine (AZT) and interferon alpha (IFN-alpha) are among the drugs showing strong in vitro activity against the human immunodeficiency virus type-1 (HIV-1). Each drug, however, has significant toxicity against normal marrow progenitor cells that frequently proves dose-limiting in patients. In this study, AZT and recombinant IFN-alpha 2a (rIFN-alpha 2a) were tested as single agents and in combination against normal myeloid (CFU-GM) and erythroid (BFU- E) colony forming cells in a standard methylcellulose culture assay. The data were analyzed using a quantitative computerized analysis based on the median-effect principle and the isobologram equation as described by Chou and Talalay (Adv Enz Regul 22:27, 1984). The ED90 for BFU-E and CFU-GM inhibition was then compared with previously measured in vivo plasma levels of each drug and the ED90 for the anti-HIV-1 effect in vitro. We demonstrate that (a) the drugs are strongly synergistic in inhibiting marrow progenitor cell growth and that this synergism occurs at drug levels that are within the range of measured plasma levels in phase I clinical trials, (b) BFU-E are more sensitive than CFU-GM to the inhibiting effects of AZT, rIFN-alpha 2a or both drugs in combination, (c) the drug concentrations in combination that synergistically inhibit bone marrow progenitors are much higher than those required to inhibit HIV-1 replication in vitro, and (d) the anti- HIV-1 effect for the combination of AZT and rIFN-alpha 2a was clearly superior to the effect of AZT or rIFN-alpha 2a alone as indicated by the combination index and the dose-reduction index. These data suggest that substantially lower doses of AZT and rIFN-alpha than those currently being tested in clinical trials might not only maintain a strong synergistic anti-HIV-1 effect but might also avoid significant hematologic toxicity.

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Synergistic cytotoxic effect of azidothymidine and recombinant interferon alpha on normal human bone marrow progenitor cells

Blood ◽

10.1182/blood.v74.4.1281.1281 ◽

1989 ◽

Vol 74 (4) ◽

pp. 1281-1286 ◽

Cited By ~ 32

Author(s):

E Berman ◽

R Duigou-Osterndorf ◽

SE Krown ◽

MP Fanucchi ◽

J Chou ◽

...

Keyword(s):

Bone Marrow ◽

Clinical Trials ◽

Progenitor Cells ◽

Interferon Alpha ◽

Plasma Levels ◽

Ifn Alpha ◽

Drug Concentrations ◽

Anti Hiv ◽

Hiv 1

Abstract Azidothymidine (AZT) and interferon alpha (IFN-alpha) are among the drugs showing strong in vitro activity against the human immunodeficiency virus type-1 (HIV-1). Each drug, however, has significant toxicity against normal marrow progenitor cells that frequently proves dose-limiting in patients. In this study, AZT and recombinant IFN-alpha 2a (rIFN-alpha 2a) were tested as single agents and in combination against normal myeloid (CFU-GM) and erythroid (BFU- E) colony forming cells in a standard methylcellulose culture assay. The data were analyzed using a quantitative computerized analysis based on the median-effect principle and the isobologram equation as described by Chou and Talalay (Adv Enz Regul 22:27, 1984). The ED90 for BFU-E and CFU-GM inhibition was then compared with previously measured in vivo plasma levels of each drug and the ED90 for the anti-HIV-1 effect in vitro. We demonstrate that (a) the drugs are strongly synergistic in inhibiting marrow progenitor cell growth and that this synergism occurs at drug levels that are within the range of measured plasma levels in phase I clinical trials, (b) BFU-E are more sensitive than CFU-GM to the inhibiting effects of AZT, rIFN-alpha 2a or both drugs in combination, (c) the drug concentrations in combination that synergistically inhibit bone marrow progenitors are much higher than those required to inhibit HIV-1 replication in vitro, and (d) the anti- HIV-1 effect for the combination of AZT and rIFN-alpha 2a was clearly superior to the effect of AZT or rIFN-alpha 2a alone as indicated by the combination index and the dose-reduction index. These data suggest that substantially lower doses of AZT and rIFN-alpha than those currently being tested in clinical trials might not only maintain a strong synergistic anti-HIV-1 effect but might also avoid significant hematologic toxicity.

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2'-Deoxycytidine protects normal human bone marrow progenitor cells in vitro against the cytotoxicity of 3'-azido-3'-deoxythymidine with preservation of antiretroviral activity

Blood ◽

10.1182/blood.v74.6.1923.1923 ◽

1989 ◽

Vol 74 (6) ◽

pp. 1923-1928 ◽

Cited By ~ 12

Author(s):

K Bhalla ◽

M Birkhofer ◽

GR Li ◽

S Grant ◽

W MacLaughlin ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

High Dose ◽

Normal Bone Marrow ◽

Normal Bone ◽

Bone Marrow Progenitor Cells ◽

Bone Marrow Progenitor ◽

High Concentrations ◽

Anti Hiv

Abstract Bone marrow cytotoxicity of 3′-azido-3′-deoxythymidine (AZT), an anti- human immunodeficiency virus (anti-HIV) drug, has been attributed to deoxyribonucleotide pool perturbations that might result in impaired DNA synthesis in normal bone marrow elements. We examined, in vitro, the effect of high, but clinically achievable and nontoxic, concentrations of 2′-deoxycytidine (dCyd) (greater than or equal to 100 mumol/L) on high-dose AZT mediated growth inhibition and intracellular biochemical perturbations in normal bone marrow progenitor cells. Colony formation by bone marrow progenitor cells in semisolid medium was significantly protected by dCyd against the inhibitory effects of co-administered, high concentrations of AZT (10 mumol/L). Also, dCyd significantly corrected AZT mediated depletion of intracellular thymidine triphosphate (dTTP) and dCyd triphosphate (dCTP) levels in normal bone marrow mononuclear cells (BMMC). Moreover, dCyd reduced the intracellular accumulation of AZT triphosphate (AZT-TP) and its DNA incorporation in BMMC. In contrast, co-administration of dCyd (100 mumol/L to 1 mmol/L) did not reverse AZT (10 mumol/L) mediated suppression of HIV infectivity in HUT-102 cells in culture, although a partial reduction in intracellular AZT-TP pools and its DNA incorporation as well as a correction of AZT mediated depletion of dTTP and dCTP pools was observed in these cells. These studies suggest that dCyd at high concentrations might ameliorate the bone marrow cytotoxicity of high-dose AZT without impairing its anti-HIV effect.

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2'-Deoxycytidine protects normal human bone marrow progenitor cells in vitro against the cytotoxicity of 3'-azido-3'-deoxythymidine with preservation of antiretroviral activity

Blood ◽

10.1182/blood.v74.6.1923.bloodjournal7461923 ◽

1989 ◽

Vol 74 (6) ◽

pp. 1923-1928

Author(s):

K Bhalla ◽

M Birkhofer ◽

GR Li ◽

S Grant ◽

W MacLaughlin ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

High Dose ◽

Normal Bone Marrow ◽

Normal Bone ◽

Bone Marrow Progenitor Cells ◽

Bone Marrow Progenitor ◽

High Concentrations ◽

Anti Hiv

Bone marrow cytotoxicity of 3′-azido-3′-deoxythymidine (AZT), an anti- human immunodeficiency virus (anti-HIV) drug, has been attributed to deoxyribonucleotide pool perturbations that might result in impaired DNA synthesis in normal bone marrow elements. We examined, in vitro, the effect of high, but clinically achievable and nontoxic, concentrations of 2′-deoxycytidine (dCyd) (greater than or equal to 100 mumol/L) on high-dose AZT mediated growth inhibition and intracellular biochemical perturbations in normal bone marrow progenitor cells. Colony formation by bone marrow progenitor cells in semisolid medium was significantly protected by dCyd against the inhibitory effects of co-administered, high concentrations of AZT (10 mumol/L). Also, dCyd significantly corrected AZT mediated depletion of intracellular thymidine triphosphate (dTTP) and dCyd triphosphate (dCTP) levels in normal bone marrow mononuclear cells (BMMC). Moreover, dCyd reduced the intracellular accumulation of AZT triphosphate (AZT-TP) and its DNA incorporation in BMMC. In contrast, co-administration of dCyd (100 mumol/L to 1 mmol/L) did not reverse AZT (10 mumol/L) mediated suppression of HIV infectivity in HUT-102 cells in culture, although a partial reduction in intracellular AZT-TP pools and its DNA incorporation as well as a correction of AZT mediated depletion of dTTP and dCTP pools was observed in these cells. These studies suggest that dCyd at high concentrations might ameliorate the bone marrow cytotoxicity of high-dose AZT without impairing its anti-HIV effect.

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Lack of evidence for infection of or effect on growth of hematopoietic progenitor cells after in vivo or in vitro exposure to human immunodeficiency virus

Blood ◽

10.1182/blood.v76.12.2476.bloodjournal76122476 ◽

1990 ◽

Vol 76 (12) ◽

pp. 2476-2482 ◽

Cited By ~ 2

Author(s):

JM Molina ◽

DT Scadden ◽

M Sakaguchi ◽

B Fuller ◽

A Woon ◽

...

Keyword(s):

Bone Marrow ◽

Human Immunodeficiency Virus ◽

Progenitor Cells ◽

Bone Marrow Cells ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Marrow Cells

The pathogenesis of the hematologic abnormalities commonly observed in patients with acquired immunodeficiency syndrome (AIDS) is incompletely understood. We report here that in vitro growth of myeloid (CFU-GM) and erythroid (BFU-E) progenitor cells from six patients with AIDS was not significantly different from that of normal human immunodeficiency virus (HIV) seronegative donors: 25.3 +/- 5 CFU-GM per 5 x 10(4) low density marrow cells and 33.5 +/- 5 BFU-E were observed in AIDS patients versus 32.7 +/- 5 CFU-GM and 42.1 +/- 5 BFU-E in controls. Furthermore, no HIV-DNA in individual colonies (CFU-GM and BFU-E) could be detected using the polymerase chain reaction (PCR) technique, although HIV-1 DNA was detected in peripheral blood mononuclear cells from the same patients. Similarly, normal bone marrow cells exposed in vitro to different isolates of HIV or recombinant purified HIV-1 envelope glycoprotein (gp) 120 did not exhibit any difference in growth of CFU-GM or BFU-E as compared with mock exposed bone marrow cells. HIV- 1 DNA could not be detected by the PCR technique in individual colonies derived from HIV exposed marrow. This study suggests that committed myeloid and erythroid progenitors from AIDS patients are responsive to hematopoietic growth factors in vitro and do not appear to contain HIV- 1 DNA. Also, HIV or its envelope gp did not alter the growth of hematopoietic progenitor cells in vitro. No evidence of HIV infection of progenitor cells could be demonstrated. Impaired hematopoiesis in patients with AIDS may not be related to direct effects of HIV on committed progenitor cells.

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Interferon Alpha Subtype-Specific Suppression of HIV-1 InfectionIn Vivo

Journal of Virology ◽

10.1128/jvi.00451-16 ◽

2016 ◽

Vol 90 (13) ◽

pp. 6001-6013 ◽

Cited By ~ 61

Author(s):

Kerry J. Lavender ◽

Kathrin Gibbert ◽

Karin E. Peterson ◽

Erik Van Dis ◽

Sandra Francois ◽

...

Keyword(s):

Clinical Trials ◽

Interferon Alpha ◽

T Cell Activation ◽

Cell Activation ◽

Antiretroviral Drugs ◽

Human Interferon ◽

Human Immune System ◽

Antiviral Protein ◽

Hiv 1

ABSTRACTAlthough all 12 subtypes of human interferon alpha (IFN-α) bind the same receptor, recent results have demonstrated that they elicit unique host responses and display distinct efficacies in the control of different viral infections. The IFN-α2 subtype is currently in HIV-1 clinical trials, but it has not consistently reduced viral loads in HIV-1 patients and is not the most effective subtype against HIV-1in vitro. We now demonstrate in humanized mice that, when delivered at the same high clinical dose, the human IFN-α14 subtype has very potent anti-HIV-1 activity whereas IFN-α2 does not. In both postexposure prophylaxis and treatment of acute infections, IFN-α14, but not IFN-α2, significantly suppressed HIV-1 replication and proviral loads. Furthermore, HIV-1-induced immune hyperactivation, which is a prognosticator of disease progression, was reduced by IFN-α14 but not IFN-α2. Whereas ineffective IFN-α2 therapy was associated with CD8+T cell activation, successful IFN-α14 therapy was associated with increased intrinsic and innate immunity, including significantly higher induction of tetherin and MX2, increased APOBEC3G signature mutations in HIV-1 proviral DNA, and higher frequencies of TRAIL+NK cells. These results identify IFN-α14 as a potent new therapeutic that operates via mechanisms distinct from those of antiretroviral drugs. The ability of IFN-α14 to reduce both viremia and proviral loadsin vivosuggests that it has strong potential as a component of a cure strategy for HIV-1 infections. The broad implication of these results is that the antiviral efficacy of each individual IFN-α subtype should be evaluated against the specific virus being treated.IMPORTANCEThe naturally occurring antiviral protein IFN-α2 is used to treat hepatitis viruses but has proven rather ineffective against HIV in comparison to triple therapy with the antiretroviral (ARV) drugs. Although ARVs suppress the replication of HIV, they fail to completely clear infections. Since IFN-α acts by different mechanism than ARVs and has been shown to reduce HIV proviral loads, clinical trials are under way to test whether IFN-α2 combined with ARVs might eradicate HIV-1 infections. IFN-α is actually a family of 12 distinct proteins, and each IFN-α subtype has different efficacies toward different viruses. Here, we use mice that contain a human immune system, so they can be infected with HIV. With this model, we demonstrate that while IFN-α2 is only weakly effective against HIV, IFN-α14 is extremely potent. This discovery identifies IFN-α14 as a more powerful IFN-α subtype for use in combination therapy trials aimed toward an HIV cure.

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Lack of evidence for infection of or effect on growth of hematopoietic progenitor cells after in vivo or in vitro exposure to human immunodeficiency virus

Blood ◽

10.1182/blood.v76.12.2476.2476 ◽

1990 ◽

Vol 76 (12) ◽

pp. 2476-2482 ◽

Cited By ~ 73

Author(s):

JM Molina ◽

DT Scadden ◽

M Sakaguchi ◽

B Fuller ◽

A Woon ◽

...

Keyword(s):

Bone Marrow ◽

Human Immunodeficiency Virus ◽

Progenitor Cells ◽

Bone Marrow Cells ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Immunodeficiency Virus ◽

Hiv 1 ◽

Marrow Cells

Abstract The pathogenesis of the hematologic abnormalities commonly observed in patients with acquired immunodeficiency syndrome (AIDS) is incompletely understood. We report here that in vitro growth of myeloid (CFU-GM) and erythroid (BFU-E) progenitor cells from six patients with AIDS was not significantly different from that of normal human immunodeficiency virus (HIV) seronegative donors: 25.3 +/- 5 CFU-GM per 5 x 10(4) low density marrow cells and 33.5 +/- 5 BFU-E were observed in AIDS patients versus 32.7 +/- 5 CFU-GM and 42.1 +/- 5 BFU-E in controls. Furthermore, no HIV-DNA in individual colonies (CFU-GM and BFU-E) could be detected using the polymerase chain reaction (PCR) technique, although HIV-1 DNA was detected in peripheral blood mononuclear cells from the same patients. Similarly, normal bone marrow cells exposed in vitro to different isolates of HIV or recombinant purified HIV-1 envelope glycoprotein (gp) 120 did not exhibit any difference in growth of CFU-GM or BFU-E as compared with mock exposed bone marrow cells. HIV- 1 DNA could not be detected by the PCR technique in individual colonies derived from HIV exposed marrow. This study suggests that committed myeloid and erythroid progenitors from AIDS patients are responsive to hematopoietic growth factors in vitro and do not appear to contain HIV- 1 DNA. Also, HIV or its envelope gp did not alter the growth of hematopoietic progenitor cells in vitro. No evidence of HIV infection of progenitor cells could be demonstrated. Impaired hematopoiesis in patients with AIDS may not be related to direct effects of HIV on committed progenitor cells.

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Interferon-alpha overrides the deficient adhesion of chronic myeloid leukemia primitive progenitor cells to bone marrow stromal cells

Blood ◽

10.1182/blood.v78.2.499.499 ◽

1991 ◽

Vol 78 (2) ◽

pp. 499-505 ◽

Cited By ~ 77

Author(s):

C Dowding ◽

AP Guo ◽

J Osterholz ◽

M Siczkowski ◽

J Goldman ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Progenitor Cells ◽

Interferon Alpha ◽

Stromal Cells ◽

Myeloid Leukemia ◽

Neuraminidase Treatment ◽

Recombinant Interferon ◽

Ifn Alpha

Abstract Primitive blast colony-forming cells (BI-CFC) from chronic myeloid leukemia (CML) patients are defective in their attachment to bone marrow-derived stromal cells compared with normal BI-CFC. We investigated the effect of recombinant interferon-alpha 2a (IFN-alpha) on this interaction between hematopoietic progenitor cells and bone marrow-derived stromal cells by culturing normal stromal cells with IFN- alpha (50 to 5,000 U/mL). At 50 U/mL we found that: (1) the capacity of stromal cells to bind two types of CML primitive progenitor cells (BI- CFC and long-term culture-initiating cells) was increased; and (2) the amount of sulfated glycosaminoglycans (GAGs) in the stromal layer was increased. However, sulfated GAGs were not directly involved in binding CML BI-CFC, unlike binding by normal BI-CFC, which is sulfated GAG- dependent. Neuraminidase-treated control stromal cells bound an increased number of CML BI-CFC, reproducing the effect of IFN-alpha, whereas the binding to IFN-alpha-treated stromal cells was unaffected by neuraminidase treatment. Thus, the enhanced attachment by primitive CML progenitor cells to INF-alpha-treated stromal cells might be due to changes in the neuraminic acid composition in the stromal cell layer. Our in vitro evidence may provide insights into the mechanism of action of IFN-alpha in vivo. Prolonged administration may alter the marrow microenvironment in some patients such that it can restrain the aberrant proliferation of Philadelphia chromosome (Ph)-positive stem cells while permitting Ph-negative stem cells to function normally.

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Interferon-alpha overrides the deficient adhesion of chronic myeloid leukemia primitive progenitor cells to bone marrow stromal cells

Blood ◽

10.1182/blood.v78.2.499.bloodjournal782499 ◽

1991 ◽

Vol 78 (2) ◽

pp. 499-505

Author(s):

C Dowding ◽

AP Guo ◽

J Osterholz ◽

M Siczkowski ◽

J Goldman ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Progenitor Cells ◽

Interferon Alpha ◽

Stromal Cells ◽

Myeloid Leukemia ◽

Neuraminidase Treatment ◽

Recombinant Interferon ◽

Ifn Alpha

Primitive blast colony-forming cells (BI-CFC) from chronic myeloid leukemia (CML) patients are defective in their attachment to bone marrow-derived stromal cells compared with normal BI-CFC. We investigated the effect of recombinant interferon-alpha 2a (IFN-alpha) on this interaction between hematopoietic progenitor cells and bone marrow-derived stromal cells by culturing normal stromal cells with IFN- alpha (50 to 5,000 U/mL). At 50 U/mL we found that: (1) the capacity of stromal cells to bind two types of CML primitive progenitor cells (BI- CFC and long-term culture-initiating cells) was increased; and (2) the amount of sulfated glycosaminoglycans (GAGs) in the stromal layer was increased. However, sulfated GAGs were not directly involved in binding CML BI-CFC, unlike binding by normal BI-CFC, which is sulfated GAG- dependent. Neuraminidase-treated control stromal cells bound an increased number of CML BI-CFC, reproducing the effect of IFN-alpha, whereas the binding to IFN-alpha-treated stromal cells was unaffected by neuraminidase treatment. Thus, the enhanced attachment by primitive CML progenitor cells to INF-alpha-treated stromal cells might be due to changes in the neuraminic acid composition in the stromal cell layer. Our in vitro evidence may provide insights into the mechanism of action of IFN-alpha in vivo. Prolonged administration may alter the marrow microenvironment in some patients such that it can restrain the aberrant proliferation of Philadelphia chromosome (Ph)-positive stem cells while permitting Ph-negative stem cells to function normally.

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