Objectives: Neurokinin-1 receptor (NK1R) antagonists interfere with binding of neuropeptide substance P to NK1R and exhibit novel anti-HIV-1 activities. Since NK1R antagonists effectively penetrate the blood–brain barrier to reduce the inflammatory response within the brain, we wished to evaluate their potential as anti-HIV-1 candidates for targeting HIV-1 infections of the central nervous system.
Design: A series of small molecule agents were evaluated for anti-NK1R and anti-HIV-1 activity using peripheral blood mononuclear cells (PBMCs). The most promising of these, aprepitant (Emend, Merck and Co. Inc.), was investigated for potential synergies with other antiretroviral drugs.
Methods: Anti-NK1R activity was tested by measuring intracellular calcium increase triggered by substance P. Anti-HIV-1 activity was evaluated by measuring p24 antigen in culture supernatants of PBMC following exposure to HIV. The concentration of drug which produced 50% reduction in intracellular calcium levels or viral production in 7-day PBMC cultures was determined. The combined effect of aprepitant with each of the major classes of anti-HIV-1 drugs was evaluated in synergy studies.
Results: Aprepitant had the highest anti-HIV-1 activity of the NK1R antagonists examined and was equally active against all major HIV-1 subtypes. Aprepitant acted synergistically with protease inhibitors (ritonavir and saquinavir), but not with nucleoside reverse transcriptase, non-nucleoside reverse transcriptase, or viral entry inhibitors.
Conclusion: The ability of aprepitant to penetrate the blood–brain barrier, its safety record as an FDA-approved drug for reducing nausea and vomiting in chemotherapy, and synergistic activity with other anti-HIV-1 drugs make it a promising candidate for treatment of HIV infection.
aSeraCare Life Sciences Inc., Gaithersburg, Maryland, USA
bDivision of Allergy and Immunology, The Children's Hospital of Philadelphia Research Institute, USA
cDivision of Infectious Diseases, University of Pennsylvania School of Medicine, USA
dDepartment of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
Received 18 December, 2009
Revised 27 August, 2010
Accepted 8 September, 2010
Correspondence to Mark Manak, PhD, SeraCare Life Sciences, 217 Perry Parkway, Gaithersburg, MD 20877, USA. Tel: +1 240 306 4109; fax: +1 240 306 4209; e-mail: firstname.lastname@example.org
The neuropeptide substance P modulates the interaction between the immune and nervous systems and is a major mediator of neurogenic inflammation [1,2]. The biologic responses to substance P are mediated by neurokinin-1 receptor (NK1R), a G-protein-coupled receptor. Drugs which interfere with binding of substance P to NK1R reduce the inflammatory response . Substance P enhances inflammatory cytokine (TNF-α, IL-1 and IL-6) production by immune cells such as macrophages through activation of NF-κB . Aprepitant (Emend, Merck & Co., Inc.), an NK1R antagonist, is approved by the FDA for the prevention of both acute and delayed chemotherapy-induced nausea and vomiting . The substance P–NK1R interaction is also important in viral infections, including HIV-1 infection of human immune cells. Infection of cells by HIV-1 requires the use of the chemokine receptors CCR5, CXCR4, or both (R5X4) . The nonpeptide substance P antagonist CP-96,345 [7,8] and aprepitant [3,9,10], down-regulate CCR5, the principal co-receptor for HIV-1 entry into macrophages, and inhibit HIV-1 infection of macrophages in vitro [8,9]. The addition of substance P to macrophages ex vivo leads to enhanced HIV-1 expression in macrophages isolated from blood of healthy individuals [8,9,11–13] and activates HIV-1 replication in latently infected immune cells . In nearly all individuals, the CCR5-tropic variants are responsible for transmission and establishment of infection, whereas the emergence of R5X4-tropic, and ultimately the shift to predominantly CXCR4-tropic variants, is frequently associated with disease progression and accelerated pathogenesis [6,15]. NK1R antagonists inhibit substance P-induced T-lymphocyte activation , and infection by HIV-1 .
In previous studies, aprepitant inhibited HIV-1 infection of monocyte-derived macrophages [8,9] and inhibited infection of azidothymidine (AZT)-resistant virus strains. Aprepitant treatment also led to decreased CCR5 expression on monocyte-derived macrophages . In other studies, aprepitant acted synergistically with other antiviral agents, and significantly enhanced the anti-HIV-1 activity of the antiretrovirals zidovudine, efavirenz, indinavir and enfuvirtide in macrophages [10,11]. Whereas either aprepitant or enfuvirtide treatment alone inhibited HIV-1 BaL infection of macrophages, the cells treated with both aprepitant and enfuvirtide had the lowest levels of HIV-1 reverse transcriptase among the cell cultures tested and at these concentrations exhibited no cytotoxic effects on macrophages. The results of these in-vitro studies suggest that aprepitant can act synergistically with other antiviral drugs to give significantly greater inhibition of HIV-1 infection than either drug alone [9,10]. In this study, we tested additional substance P antagonists for potential anti-HIV-1 activity in vitro, and examined the inhibition of infection by CCR5 vs. CXCR4-tropic HIV-1 isolates representing a cross-section of major world-wide subtypes. Synergies with additional anti-HIV-1 drugs were further evaluated and strong synergies were found with protease inhibitors.
Materials and methods
Inhibition of HIV-1 infectivity of peripheral blood mononuclear cells (PBMCs) in vitro was measured according to published methods . Briefly, 96-well plates containing 200 000 PBMCs per well and decreasing concentrations of drug were prepared. The PBMCs used in these studies were obtained from uninfected healthy blood donors. Fresh blood samples were separated by Ficoll-Hypaque centrifugation, and PBMCs were frozen in liquid nitrogen in media containing dimethyl sulfoxide (DMSO). Cells were thawed, washed and stimulated for 3 days with phytohemagglutinin (PHA). After a 1-h preincubation in the presence or absence of varying concentration of drug, cells were infected with 200 TCID50 of HIV-1 BaL (0.8 ng) or NL4–3 (3.5 ng) for a multiplicity of infection (m.o.i.) of 0.001. Infected cells were incubated for 7 days at 37oC in the same concentrations of anti-HIV-1 drug. Viral replication in PBMC from three different healthy donors was measured by quantifying p24 levels of culture supernatant following 7 days incubation at 37oC (Zeptometrix p24 ELISA). Serial dilutions of supernatant were tested in triplicate to establish a tissue culture infectious dose 50% (TCID50). The inhibitory concentration of drug which resulted in 50% reduction of virus (IC50) was calculated as the ratio of p24 production in test sample divided by the p24 production in the positive control. The cytotoxic concentration which resulted in 50% cell death in the absence of virus infection (CC50) was determined from the optical density (OD) readings of the MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay.
Synergy studies were performed in 96-well plates containing 2 × 105 PBMCs per well and infected with BaL virus at m.o.i. = 0.01. Serial dilutions of aprepitant and HIV-1 antiviral drugs were added to the wells at 0, 0.1X, 0.5X, and 2X the TCID50 for each drug. The plates were incubated for 5 days at 37oC and virus infection was determined by a tetrazolium assay in which 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) is metabolically reduced in viable cells to a water-soluble formazan product, phenazine methosulfate (PMS) .
A series of NK1R antagonists, including aprepitant (Merck & Co, Inc., Whitehouse Station, New Jersey, USA), L-733,060 (Sigma–Aldrich, St. Louis, Missouri, USA), RP-67,580 (Tocris Bioscience, Ellisville, Missouri, USA), L-759,274 (Merck & Co, Inc., Whitehouse Station, New Jersey, USA), CP-96,345 (Pfizer, Inc, New York, New York, USA), and CP-99,994 (Pfizer, Inc, New York, New York, USA) were tested for potential in-vitro antiviral activity against CCR5-tropic (HIV-1 BaL) or CXCR4-tropic (HIV-1 NL4-3) viral isolates.
The inhibitory activity of aprepitant was also tested against a panel of primary HIV-1 isolates (SeraCare Life Sciences, Inc.) grown in PBMC. These included a broad range of HIV-1 subtypes, representing both T-cell-tropic (CXCR4) and macrophage-tropic (CCR5) primary isolates. Synergistic effects of combinations of aprepitant with representatives of each of the major classes of approved anti-HIV-1 drugs were evaluated for inhibition of HIV-1 BaL in PBMC.
Intracellular calcium measurements
U373MG astrocytoma cells were used to assess the potency of NK1R antagonists. Intracellular calcium recordings were performed as previously described . Briefly, U373MG cells were cultured in 96-well plates and incubated with Fura-2 AM (Molecular Probes, Eugene, Oregon, USA) for 45 min at room temperature. Cells were washed with Hank's Balanced Salt Solution containing calcium chloride (1 mmol/l) and intracellular calcium measurements were performed using Tsien's ratiometric method [16,20]. Peak intracellular calcium increases elicited by 100 nmol/l substance P were measured in the presence of different concentrations of antagonists (as indicated in each figure). The concentrations of antagonists were increased on a semi-logarithmic scale until the intracellular calcium response induced by 100 nmol/l substance P was completely blocked. Concentration–response curves were constructed and curves were fitted to data using the equation:
where E is the measured effect (the increase in intracellular calcium concentration), max is the intracellular calcium increase measured in the absence of antagonist (the maximal response), min is baseline intracellular calcium level before addition of substance P, C is the concentration of antagonist, and p is the slope of the concentration–response curve. Logistic curves were fitted to data and used to calculate the IC50 values for each antagonist.
Initial studies were performed in order to determine the effect of aprepitant on the infection of PBMC cultures with well characterized laboratory strains of HIV-1 with defined coreceptor tropism. For these studies, the viral isolate HIV-1 BaL was used as the reference CCR5-tropic virus, and the HIV-1 NL4-3 isolate as the CXCR4-tropic virus. The mean anti-HIV-1 activity (IC50) of AZT on PBMCs obtained from three different healthy adult donors was 0.008 +/− 0.002 μmol/l for HIV-1 BaL isolates and 0.005 +/− 0.004 μmol/l for HIV-1 NL4-3 virus isolates. Similar studies with 2′-3′-dideoxycytidine (ddC) showed IC50 values of 0.011 +/− 0.004 μmol/l for HIV-1 BaL, and 0.012 +/− 0.003 μmol/l for HIV-1 NL4-3 virus. These results demonstrate that antiviral activities can be measured with reasonable precision, and that the nucleoside RT inhibitors AZT and ddC inhibit HIV-1 infection by both CCR5 and CXCR4-tropic viruses in the PBMC system. The assays have excellent reproducibility between runs and operators. We selected AZT as a control for subsequent studies because of its consistency of results, wide availability and extensive characterization in the literature [21,22].
The inhibitory effect of aprepitant on HIV-1 infection was initially tested on human PBMC from three different donor cells infected ex vivo with HIV-1 BaL and HIV-1 NL4-3 isolates. The average antiretroviral activity for aprepitant was IC50 5.4 μmol/l against the BaL isolate, and 5.7 μmol/l against the NL4-3 isolate. All three donors gave very similar results with both viruses (Fig. 1). The average concentration that resulted in 50% of virus inhibition (IC50) was 6.4 μmol/l for BaL and 8.0 μmol/l for NL4-3. The cellular cytotoxicity in both cases was very low, with a CC50 above 50 μmol/l. Thus, aprepitant inhibits viral replication of both CCR5 and CXCR4-tropic viruses in PHA-stimulated PBMC, and has very limited cytotoxicity.
The effect of NK1R antagonists on substance P-induced intracellular calcium increase in U373MG cells
In order to assess the potency of the NK1R antagonists selected for this study, we measured their ability to inhibit the intracellular calcium increase induced by substance P in U373MG astrocytoma cells which endogenously express NK1R receptors . Concentration–response curves (Fig. 2) were obtained by incubating different concentration of antagonist with U373MG cells prior to addition of substance P. Among the tested compounds, aprepitant, L-733,060, CP-96,345, and CP-99,994 were most potent (Table 1), with IC50 values below 100 nmol/l (0.1 μmol/l), whereas RP-67,580 and L-759,274 were less potent with IC50 values above 200 nmol/l (0.2 μmol/l).
Effect of NK1R antagonists on inhibition of HIV-1 infection in PBMC
A series of substance P antagonists, including aprepitant, L733060, RP-67,580, CP-96345, and CP-99,994, were tested for potential in-vitro antiviral activity against CCR5-tropic (BaL) or CXCR4-tropic (NL4-3) viral isolates. The ability of the NK1R antagonists to block HIV-1 BaL infection of PBMC was measured and the IC50 concentrations determined as described above. The cytotoxicity was also measured using the MTS assay and expressed as CC50. The results of these studies are shown in Table 1, which shows antiviral activity and toxicity of these drugs compared to that of AZT and ddC. The NK1R antagonists showed varying degrees of anti-HIV-1 activity, and did not have cytotoxic effect at concentrations as high as 25 μmol/l. Aprepitant showed the highest antiretroviral activity (IC50 5.4 μmol/l) of the NK1R antagonists tested, followed by L-733,060. The other NK1R antagonists had lower IC50 values, or failed to show significant antiviral activity. The control drugs AZT and ddC performed as expected.
Efficacy against primary HIV-1 isolates in PBMC
Our results with NK1R antagonists confirm our previously published findings that aprepitant inhibits HIV-1 viral replication (8, 9, 10). In contrast to the results of previous studies in monocyte-derived macrophages, when aprepitant showed a strong inhibition of CCR5 virus infectivity, but lower inhibition by CXCR4 isolate , in PBMC, both the CCR5 (BaL) and CxCR4 (NL4-3) isolates were inhibited to approximately the same extent. We further extended these studies to determine whether other HIV-1 isolates of varying tropism would be differentially inhibited by aprepitant. The inhibitory activity of aprepitant was tested against a panel of primary HIV-1 isolates cultivated in PBMC, which included representatives of a broad range of subtypes, representing both T lymphotropic (CXCR4) and monocytotropic (CCR5) primary isolates (Table 2).
The results of duplicate measurements are shown in Table 2 and indicate that aprepitant showed similar efficacy against each of the primary HIV-1 subtypes tested, including subtypes A, B, C, D, F, G, H, CRF01AE, CRF02AG, and group O isolates when propagated in human PBMC. Furthermore, no significant differences were observed against T-lymphotropic (CXCR4) and M-tropic (CCR5) primary isolates. The IC50 ranged between 3.86 and 19.86 μmol/l in the various isolates, which is within a half log of the mean for all viruses and not significant. As shown in Table 2, the mean IC50 values showed very similar results for virus categories with usage of CCR5, CXCR4 or R5/X4 coreceptors.
Synergy of aprepitant with antiretrovirals
An important consideration in studying any antiviral is the possible emergence of drug resistant strains and the requirement to switch therapies to alternate drugs. Current highly active antiretroviral therapies (HAARTs) often utilize combinations of drugs, and certain combinations are much more effective in reducing viral load than either drug alone. Combinations of drugs also greatly extend the time at which drug resistance is likely to emerge. Since the IC50 dose of aprepitant is much higher than for many of the currently approved anti-HIV drugs, it was of particular interest to see whether aprepitant may act synergistically with other drugs, allowing better inhibition than by either drug alone.
Drug synergies studies were conducted in the PBMC system in assays employing aprepitant in combination with representatives of each of the major approved classes of HIV inhibitors. For these studies, we used the HIV-1 BaL strain propagated in PBMC at different concentrations of aprepitant with currently approved antiretroviral drugs. The drugs evaluated in these studies represent a variety of different mechanisms of action, and are shown in Fig. 3 along with their IC50s as determined in triplicate studies using our HIV-PBMC infection system.
Each drug was incubated alone or in combination with aprepitant at varying concentrations from 0 to 2.0× its previously determined IC50. The results were scored as % inhibition of HIV-1 infectivity as shown in Fig. 3. Each chart in the figure shows the inhibition by the drug alone, or in combination with aprepitant. The first two panels show aprepitant alone for comparison. Synergistic effects were those in which the combination of drug showed a greater inhibition than simple additive effect. We observed significant synergistic effects for aprepitant with the protease inhibitors ritonavir (RTV) and saquinavir (SQV), with a more than two-fold greater inhibition when both drugs are used together at 0.5× and 2× TCID50 than observed for either drug alone. A lesser degree of enhancement was also observed with nucleoside reverse transcriptase inhibitors AZT and 3TC. No significant synergies were observed with the nucleoside reverse transcriptase inhibitor (NRTI), non-nucleoside reverse transcriptase inhibitor (NNRTI) and entry inhibitor drugs, and combinations of these drugs with aprepitant showed parallel levels of viral inhibition to that of the index drug alone. These in-vitro PBMC results indicate that aprepitant is most effective in reducing HIV-1 infectivity if coupled with a protease inhibitor.
These studies were designed to further characterize the in-vitro anti-HIV-1 activity of aprepitant in a cultured PBMC system. Of several NK1R antagonists tested, all had low cytotoxicity (CC50 >25 μmol/l). Aprepitant exhibited the highest antiretroviral activity (IC50 5.4 μmol/l). Aprepitant had reproducible antiviral effect on a broad range of HIV-1 isolates in PBMC culture with notable efficacy (IC50 7.95 μmol/l +/− 3.88) against primary infection by each major HIV-1 subtype (A, CRF02-AG; B, C, D, CRF02-AE; F, G, H and O), including CXCR4 and CCR5-tropic primary isolates propagated in PBMC, with low cytotoxicity above 50 μmol/l. Isolates which primarily utilize CXCR4 and CCR5 or both coreceptors were inhibited, with no apparent differences based on receptor usage. These results in PBMC are in contrast with those for monocyte-derived macrophages, in which a strong preference on inhibition by CCR5-mediated infection was seen . These studies suggest that inhibition of virus infectivity by aprepitant may function by more than one mechanism, depending on target cell. Strong synergistic effects were observed for aprepitant with the protease inhibitors RTV and SQV, but no significant synergies were observed with AZT, ddC, ddI, 3TC, NPV and T-20. This study extends our observations on the antiviral activities of the neurokinin-1 antagonist aprepitant. Previous studies demonstrated that aprepitant, as well as CP96,345, inhibit HIV-1 infection of monocyte-derived macrophages of HIV-1 R5-isolates (subtype A, F, and H), but showed a lower degree of inhibition for infection with the X4-tropic isolates (B and D subtype) . Those studies showed that aprepitant treatment also inhibited HIV-1 infection of GAGO or JRFL HIV-1 strains in 7-day cultured microglia, whereas in the macrophage system, aprepitant enhanced the antiviral activity of T20, enfuvirtide, the HIV-fusion inhibitor. Thus although aprepitant may act by modulating CCR5 expression, the current studies show that additional antiviral effects not involving CCR5 may also operate in the PBMC system.
We observed that the concentrations of NK1R antagonists required to induce inhibition of HIV-1 infection were higher than the corresponding concentrations required for inhibiting substance P-induced intracellular calcium increase. It is known that the full length NK1R intracellular signaling mechanisms involve Gq activation and intracellular calcium increase ). This observation suggests that more complex mechanisms of virus inhibition than the simple blockade of the full length NK1R are involved. These mechanisms may be related to the modulation of expression of the HIV-1 coreceptors on the target cells  and they may require the engagement of the truncated NK1R. This hypothesis is supported by the observation that the affinity of substance P for the truncated receptor is about 10-fold lower than the affinity for the full-length receptor ). Furthermore, interactions of the studied antagonists with molecular targets other than the NK1R cannot be excluded based on our observations, and they might contribute to the antiviral effect.
The present findings extend earlier observations and now demonstrate that aprepitant, L-733,060, RP-67,580, L-759,274, and CP-96,345 have low-potency antiviral effect against the HIV-1 BaL strain. Furthermore, we have shown that aprepitant is active against a broad range of HIV-1 isolates in PBMC cultures and we have observed that there is synergy between aprepitant and the protease inhibitors in the PBMC virus culture system. The fact that this drug is already used clinically to control nausea in chemotherapy and has demonstrable anti-HIV-1 activity which is synergistic with other antiretrovirals, make it an excellent candidate as a potential anti-HIV-1 therapeutic agent for control of neurologic infections. The average peak levels of aprepitant (Cmax) after a single oral dose of 240 mg can reach values as high as 3.8 μg/ml , which is equivalent to 7 μmol/l. This concentration is above the EC50 value determined in our study. Furthermore, it is reasonable to assume that higher doses of aprepitant will yield even higher plasma concentrations, which are expected to have measurable anti-HIV effect in vivo.
Furthermore, administration of aprepitant with a CYP3A4 inhibitor such as ritonavir, is expected to result in increased plasma levels of the drug due to inhibition of the induction phenomenon that has been reported with extended aprepitant administration [27,28]. A phase 1B clinical trial evaluating the pharmacokinetic profile and safety of aprepitant in HIV-infected individuals is underway.
The work was supported by NIH Grant #PO1 MH-076388, entitled ‘Neurokinin-1R (SP Receptor) Antagonists for HIV-1 Therapy’ (Core B, Mark Manak), Steven D. Douglas, Principal Investigator. CP-96.345 and CP-99,994 were obtained from Pfizer, Inc.; Aprepitant, and L-759,274 was obtained from Merck & Co, Inc. We thank Dr Richard Hargreaves from Merck Research Laboratories, West Point, Pennsylvania, USA, for his review of the manuscript and insightful comments.
Author contributions: M.M.M., Principal Investigator, headed up the study, designed experiments and lead author of the paper. D.A.M. assisted M.M.M. in planning infectivity and synergy experiments. L.T.N. executed all virus infectivity experiments, assisted in experimental design and particularly details of infection. J.M. carried out the NK-1R activity studies. P.T. coordinated clinical studies of aprepitant and provided information on clinical utility of the drug and provided guidance on in-vitro studies. F.T. assisted S.D.D. in coordination of overall aprepitant program providing all drugs used in study and performing NK-1R activity studies. S.D.D. is the Lead Investigator of aprepitant study for clinical evaluation and coordination of various aspects of investigation.
1. McGillis JP, Organist ML, Payan DG. Substance P and immunoregulation. Fed Proc 1987; 46:196–199.
2. Tuluc F, Lai JP, Kilpatrick LE, Evans DL, Douglas SD. Neurokinin 1 receptor isoforms and the control of innate immunity. Trends Immunol 2009; 30:271–276.
3. Kennedy PG, Rodgers J, Jennings FW, Murray M, Leeman SE, Burke JM. A substance P antagonist, RP-67,580, ameliorates a mouse meningoencephalitic response to Trypanosoma brucei brucei
. Proc Natl Acad Sci U S A 1997; 94:4167–4170.
4. Lai JP, Lai S, Tuluc F, Tansky MF, Kilpatrick LE, Leeman SE, Douglas SD. Differences in the length of the carboxyl terminus mediate functional properties of neurokinin-1 receptor. Proc Natl Acad Sci U S A 2008; 105:12605–12610.
5. Patel L, Lindley C. Aprepitant: a novel NK1-receptor antagonist. Expert Opin Pharmacother 2003; 4:2279–2296.
6. Bjorndal A, Deng H, Jansson M, Fiore JR, Colognesi C, Karlsson A, et al
. Coreceptor usage of primary human immunodeficiency virus type 1 isolates varies according to biological phenotype. J Virol 1997; 71:7478–7487.
7. Lieb K, Fiebich BL, Berger M, Bauer J, Schulze-Osthoff K. The neuropeptide substance P activates transcription factor NF-kappa B and kappa B-dependent gene expression in human astrocytoma cells. J Immunol 1997; 159:4952–4958.
8. Lai JP, Ho WZ, Zhan GX, Yi Y, Collman RG, Douglas SD. Substance P antagonist (CP-96,345) inhibits HIV-1 replication in human mononuclear phagocytes. Proc Natl Acad Sci U S A 2001; 98:3970–3975.
9. Wang X, Douglas SD, Lai JP, Tuluc F, Tebas P, Ho WZ. Neurokinin-1 receptor antagonist (aprepitant) inhibits drug-resistant HIV-1 infection of macrophages in vitro. J Neuroimmune Pharmacol 2007; 2:42–48.
10. Wang X, Douglas SD, Song L, Wang YJ, Ho WZ. Neurokinin-1 receptor antagonist (aprepitant) suppresses HIV-1 infection of microglia/macrophages. J Neuroimmune Pharmacol 2008; 3:257–264.
11. Ho WZ, Kaufman D, Uvaydova M, Douglas SD. Substance P augments interleukin-10 and tumor necrosis factor-alpha release by human cord blood monocytes and macrophages. J Neuroimmunol 1996; 71:73–80.
12. Douglas SD, Cnaan A, Lynch KG, Benton T, Zhao H, Gettes DR, Evans DL. Elevated substance P levels in HIV-infected women in comparison to HIV-negative women. AIDS Res Hum Retroviruses 2008; 24:375–378.
13. Douglas SD, Ho WZ, Gettes DR, Cnaan A, Zhao H, Leserman J, et al
. Elevated substance P levels in HIV-infected men. AIDS 2001; 15:2043–2045.
14. Li Y, Douglas SD, Song L, Sun S, Ho WZ. Substance P enhances HIV-1 replication in latently infected human immune cells. J Neuroimmunol 2001; 121:67–75.
15. Stalmeijer EH, Van Rij RP, Boeser-Nunnink B, Visser JA, Naarding MA, Schols D, Schuitemaker H. In vivo evolution of X4 human immunodeficiency virus type 1 variants in the natural course of infection coincides with decreasing sensitivity to CXCR4 antagonists. J Virol 2004; 78:2722–2728.
16. Santoni G, Perfumi MC, Spreghini E, Romagnoli S, Piccoli M. Neurokinin type-1 receptor antagonist inhibits enhancement of T cell functions by substance P in normal and neuromanipulated capsaicin-treated rats. J Neuroimmunol 1999; 93:15–25.
17. Castro BA, Weiss CD, Wiviott LD, Levy JA. Optimal conditions for recovery of the human immunodeficiency virus from peripheral blood mononuclear cells. J Clin Microbiol 1988; 26:2371–2376.
18. Scudiero DA, Shoemaker RH, Paull KD, Monks A, Tierney S, Nofziger TH, et al
. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res 1988; 48:4827–4833.
19. Chernova I, Lai JP, Li H, Schwartz L, Tuluc F, Korchak HM, et al
. Substance P (SP) enhances CCL5-induced chemotaxis and intracellular signaling in human monocytes, which express the truncated neurokinin-1 receptor (NK1R). J Leukoc Biol 2009; 85:154–164.
20. Tsien RY. New tetracarboxylate chelators for fluorescence measurement and photochemical manipulation of cytosolic free calcium concentrations. Soc Gen Physiol Ser 1986; 40:327–345.
21. Yarchoan R, Klecker RW, Weinhold KJ, Markham PD, Lyerly HK, Durack DT, et al
. Administration of 3′-azido-3′-deoxythymidine, an inhibitor of HTLV-III/LAV replication, to patients with AIDS or AIDS-related complex. Lancet 1986; 1:575–580.
22. Broder S, Fauci AS. Progress in drug therapies for HIV infection. Public Health Rep 1988; 103:224–229.
23. Palma C, Nardelli F, Manzini S, Maggi CA. Substance P activates responses correlated with tumour growth in human glioma cell lines bearing tachykinin NK1 receptors. Br J Cancer 1999; 79:236–243.
24. Regoli D, Boudon A, Fauchere JL. Receptors and antagonists for substance P and related peptides. Pharmacol Rev 1994; 46:551–599.
25. Fong TM, Yu H, Huang RR, Strader CD. The extracellular domain of the neurokinin-1 receptor is required for high-affinity binding of peptides. Biochemistry 1992; 31:11806–11811.
26. Bergman AJ, Marbury T, Fosbinder T, Swan S, Hickey L, Bradstreet TE, et al
. Effect of impaired renal function and haemodialysis on the pharmacokinetics of aprepitant. Clin Pharmacokinet 2005; 44:637–647.
27. Sanchez RI, Wang RW, Newton DJ, Bakhtiar R, Lu P, Chiu SH, et al
. Cytochrome P450 3A4 is the major enzyme involved in the metabolism of the substance P receptor antagonist aprepitant. Drug Metab Dispos 2004; 32:1287–1292.
28. Eagling VA, Back DJ, Barry MG. Differential inhibition of cytochrome P450 isoforms by the protease inhibitors, ritonavir, saquinavir and indinavir. Br J Clin Pharmacol 1997; 44:190–194.
Keywords:© 2010 Lippincott Williams & Wilkins, Inc.
antiretroviral drugs; aprepitant; human immunodeficiency virus type 1; neurokinin-1 receptor antagonists; synergy