Naloxone and Naltrexone as Potential Adjuvants for Vaccination Against Intracellular Pathogens: A Narrative Review

The opioid growth factor (OGF) is an autocrine produced and constitutively expressed peptide that interacts with the OGF receptor (OGFr) and subsequently targets the cyclin dependent kinase inhibitory (CKI) pathways to inhibit proliferation at the G1/S phase of the cell cycle. Naltrexone (NTX) is a nonselective opioid receptor antagonist that blocks this interaction. In vivo administration of a low dosage of NTX (LDN, 0.1 mg/kg) blocks opioid receptors for 4-6 h, resulting in a compensatory upregulation of OGF and OGFr. Following clearance of NTX, the elevated levels of OGF and OGFr interact to decrease cell proliferation. To investigate whether LDN inhibits cancer cell proliferation independent of immune function, a tissue culture model using short term NTX in human ovarian cancer cell lines (SKOV-3, OVCAR-3) was established. Cell replication and cell number were determined over 96 h in cultures treated with a single 6 h application of NTX (10−6 M). In SKOV-3 and OVCAR-3 cultures, cell number was decreased by 21-28% and 27-32%, respectively, between 48 and 96 h; DNA synthesis was inhibited ∼ 33% at 72 h. Peptide specificity studies using a variety of classical μ, δ, or κ opioid receptor agonists confirmed that only OGF inhibited cell number. Antibody neutralization of OGF in cultures revealed that endogenous OGF invoked the inhibitory growth effects of short term NTX. To evaluate which opioid receptor mediated the inhibitory action of short term NTX on growth, cultures were treated with siRNAs against the μ, δ, κ, and OGFr receptors; only OGFr was associated with short term NTX treatment. Immunocytochemical studies and semiquantitative analyses documented an upregulation of OGF and OGFr expression following short term NTX exposure in cancer cells; western blot analysis and receptor binding for OGFr revealed 2-fold increases of receptors 48 h after a brief exposure to NTX. NTX's inhibitory action was not related to apoptosis (TUNEL) or necrosis (trypan blue staining). To investigate whether short term NTX's action on cell proliferation was transduced by the p16 and/or p21 CKI pathways, siRNAs for p16 and p21 were utilized in combination with short term NTX. Silencing of p16 and/or p21 blocked the inhibitory action of short term NTX, indicating a mechanism directed toward the CKI pathways. Independent of immune function, short term opioid receptor antagonism has been demonstrated to inhibit cancer cell proliferation by upregulation of the OGF-OGFr axis. Upregulation of the OGF-OGFr axis by a brief opioid receptor blockade may provide a novel target for biotherapy of cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4153.

We developed a method for applying HIV-1 DNA vaccine topically in mice. Topical application of DNA vaccine to the skin is useful against infections. To find a less expensive and less cumbersome vaccination method, we administered HIV-1 DNA vaccine to the skin of mice after elimination of keratinocytes using a fast-acting adhesive. HIV-1 DNA vaccine induced high levels of both humoral and cell-mediated immune activity against HIV-1 envelope antigen. A high level of HIV-1-specific cytotoxic T lymphocyte response was also observed, and a high level of IFN-gamma and IL-4 production was induced by the improved skin application of DNA vaccine. High levels of both HIV-specific cytotoxic T lymphocyte and delayed type hypersensitivity in topical application were induced by coadministration of the DNA vaccine with IL-12 expression plasmids and granulocyte-macrophage colony-stimulating factor expression plasmids. These immune responses were inhibited by intradermal injection of anti-CD11c or anti-I-A/I-E antibody. Therefore, topical administration of DNA vaccine is an effective route, and may be very useful for the prevention of infectious diseases.

DNA vaccines can induce both humoral and cellular immune responses in animals. However, DNA vaccines suffer from limited vaccine potency due to low immunogenicity. Therefore, different strategies are required for significant improvement of DNA vaccine efficacy such as inclusion of strong adjuvants. The aim of the present study was to investigate the effects of using α-Galactosylceramide (α-GalCer) as an adjuvant to enhance the immune responses induced by a DNA vaccine, encoding influenza A virus matrix protein 2 (M2), against influenza A challenge. BALB/c mice were immunized three times by intramuscular inoculations of DNA vaccine encoding M2 alone or in combination with α-GalCer adjuvant. The adjuvant effect was evaluated by measuring the serum antibody titers, using ELISA, lymphocyte proliferation, using MTT assay as well as Th1 (IFN-γ and IL-12) and Th2 (IL-4) cytokines. The results showed that co-administration of α-GalCer with the vaccine exert protective effects by influencing the magnitude and quality of humoral responses. Adjuvanted DNA-vaccinated mice revealed a higher IgG titer and IgG2a/IgG1 ratio than mice vaccinated with DNA alone. Furthermore, analysis of M2-specific responses revealed that the DNA vaccine triggered predominately IgG1 and IL-4 responses indicating a Th2 bias. The data also showed that α-GalCer is a potent adjuvant for activation of cellular immune responses to DNA vaccine. This was supported by a higher IgG2a/IgG1 ratio, significantly increased IFN-γ and IL-4 production and CD4+ proliferation, compared with mice receiving the DNA vaccine alone, suggesting a mixed Th1/Th2-type cellular immune response with a Th1 bias. The findings of this study indicate that α-GalCer has the potential to be used as a potent adjuvant for a DNA vaccine encoding M2, since it enhances humoral and cellular immune response and improves immune protection against influenza challenge in mice.

Immunopeptidomics for next-generation bacterial vaccine development.

The poor oral bioavailability of the opioid receptor antagonist naloxone (NA) when compared with naltrexone (NX) may be related to a greater interaction of naloxone with the efflux drug transporter P-glycoprotein (P-gp). We studied the involvement of P-gp in the transepithelial transport of the two opioid receptor antagonists, using a validated human in vitro Caco-2 cell monolayer model. The bidirectional transport of NA and NX (1, 50 and 100 microm) across the monolayers was investigated in the presence and absence of the specific P-gp inhibitor GF120918 (4 microm). NA and NX showed equal transport rates between the apical-to-basolateral (A-B) and the basolateral-to-apical (B-A) directions and neither the influx nor the efflux transport was affected by the P-gp inhibitor (P > 0.05). In conclusion, NA and NX are not P-gp substrates. The differential oral bioavailability of the two opioid antagonists is P-gp independent.

Chemotherapy Enhances CD8+ T Cell-mediated Antitumor Immunity Induced by Vaccination With Vaccinia Virus

Naltrexone (NTX) is a long-acting opiate antagonist. Low-dose naltrexone (LDN) therapy has shown promising results in the treatment of several autoimmune disorders. Our aim was to formulate NTX into a cream for the delivery of LDN and develop an analytical technique for the quantification of NTX and its active metabolite 6-β-naltrexol (NTXol) during transdermal diffusion cell permeation studies. A 1% w/w NTX cream was formulated and drug permeation was examined over 24 h using static Franz diffusion cells mounted with pig skin. A Liquid Chromatography Quadrupole-Time of Flight Mass Spectrometry (LC-MS Q-ToF) method was developed for the detection of NTX and NTXol in the receptor solution, skin membrane and residual cream on the donor chamber after completion of the diffusion studies. The cream formulation exhibited steady state release of NTX over 24 h after an initial lag time of 2.74 h. The bioconversion of NTX to NTXol in the skin membrane was 1.1%. It was concluded that the cream may be an effective formulation for the sustained transdermal delivery of LDN. The novel LC Q-ToF MS method allowed the accurate measurement of NTX and NTXol levels across the diffusion cell assemblies and the quantification of NTX metabolism in the skin.

Opioid Antagonists

Objective To investigate the effects of orexin-A on firing activity of gastric distension-sensitive (GD) neurons in the basomedial amygdala (BMA) and food intake in diet-indaced obese rats. Methods Healthy male Wistar rats were selected, and the diet-induced obesity (DIO) rat model and diet-induced resistant (DR) rat model were established by high-fat diet. The effects of orexin-A and an opioid receptor antagonist naloxone on BMA GD neurons were observed by recording the extracellular potentials of single neurons.The effects of orexin-A and naloxone on the food intake of different rats were observed by using BMA catheterization.The mRNA expression and protein expression of orexin-1 receptor (OX-1R) and μ opioid receptor were detected by real-time PCR and Elisa, respectively. Results After microinjection of orexin-A into the BMA, the firing frequency of GD-sensitive neurons in the normal rats was significantly increased (GD-E: (78.3±6.9)%, GD-I: (55.5±4.7)%, P<0.01), and this effect was completely blocked by OX-1R receptor antagonist SB334867, and naloxone partially blocked the discharge-promoting effect of orexin-A; Compared with the normal rats, the firing frequency of GD-sensitive neurons in the DIO(GD-E: (91.6±7.1)%, GD-I: (67.9±8.1)%) and DR(GD-E: (87.9±6.8)%, GD-I: (69.2±5.8)%)rats was significantly increased after BMA injection of orexin-A (P<0.05). After administration of orexin-A into the BMA, food intake of the normal rats, DIO rats and DR rats ((2.38±0.34) g, (3.75±0.32) g, (4.01±0.38) g, respectively) was significantly increased (P<0.01), and the food intake of DR and DIO rats were significantly higher than that of normal rats (P<0.05). After BMA was injected with naloxone, the food intake of rats was inhibited, and the food intake of the DIO rats was significantly lower than that of the DR rats (P<0.05), food intake of the DR rats was significantly lower than that of the normal rats (P<0.05). The results of real-time PCR showed that the mRNA levels of OX-1R in DIO and DR rats were( 5.85±0.45 )and (6.03±0.42) were higher than that of normal rats, and the difference was significant (P<0.05); and mRNA levels of μ-opioid receptors in DIO and DR rats((4.51±0.42) and (8.31±0.41)times) were higher than those in normal rats (P<0.05). The results of Elisa showed that the protein levels of OX-1R in DIO ((2.98±0.28) ng/μl) and DR rats ((3.05±0.31) ng/μl) were higher than those in normal rats ((1.53±0.31)ng/μl, P<0.05). The content of μ-opioid receptor protein in DR rats ((4.21±0.35)ng/μl) was higher than that of DIO rats ((2.77±0.27) ng/μl), and higher than that of normal rats((1.48±0.32) ng/μl), the difference was significant (P<0.05). Conclusion BMA orexin-A promotes the spontaneous discharge of GD-sensitive neurons and food intake in normal rats, DIO rats and DR rats, μ-opioid receptors may be involved in the regulation of this process. Key words: Orexin-A; Food intake; Gastric distension-sensitive neurons; Diet-induced obesity rat; Amygdala

The endogenous opioid system has been implicated in mediating the reinforcing effects of ethanol (EtOH). Naltrexone (NTX), an opioid antagonist with concentration-dependent selectivity for the mu receptor, naltrindole (NTI), a selective delta receptor antagonist, and U50,488H, a selective kappa receptor agonist were examined in both alcohol-preferring (P) and nonselected (Long Evans (LE)) rats to determine whether they differentially affected the seeking and consumption of EtOH and sucrose. Using the sipper-tube model, rats reinforced with either 2% sucrose or 10% EtOH were injected with vehicle and either NTI (2.5, 5.0, or 10.0 mg/kg), U50 (2.5, 5.0, or 10.0 mg/kg), low-dose NTX (0.1, 0.3, or 1.0 mg/kg), or high-dose NTX (1.0, 3.0, or 10.0 mg/kg). Subsequent intakes (consummatory) or lever responses (seeking) were assessed. Overall, NTI, U50, and NTX attenuated intake and responding for sucrose and EtOH, with EtOH-reinforced P rats being the most sensitive to the effects of NTI on intake and seeking. U50 treatment decreased intake and seeking in both P and LE rats but did not selectively reduce EtOH intake or seeking in either line. P rats were more sensitive than LE rats to lower doses of NTX, and these doses more selectively attenuated responding for EtOH than sucrose. Higher doses of NTX suppressed intake and responding across both lines and reinforcers. These results suggest that drugs selective for the opioid receptors may be good pharmacotherapeutic targets, particularly in those with an underlying genetic predisposition for greater EtOH preference/intake.

Effects of opioid antagonists and their quaternary derivatives on locomotor activity and fixed ratio responding for brain self-stimulation in rats

The opioid antagonists, naloxone (NOX) and naltrexone (NTX), were found to produce dose-dependent increases in fetal mean arterial pressure over a dose range of 5-80 mg/kg. There was a concomitant decrease in fetal heart rate up to 40 mg/kg. Above this dose, NOX and NTX caused an increase in heart rate as well as blood pressure. NTX produced similar effects in maternal ewes, although at lower doses (mg/kg) than those needed for fetal lambs. There were no age-related differences in antagonist effects in two fetal age groups studied (100-116 and 124-144 days of gestation). The partial antagonist, levallorphan (LVL), produced effects which were qualitatively similar to those produced by NOX and NTX in doses up to 20 mg/kg. These effects were not stereospecific, as the enantiomer of LVL, dextrallorphan, produced similar effects at equal doses. Pretreatment with the alpha 1-adrenoreceptor antagonist, prazosin, abolished the opioid antagonist effects on fetal blood pressure. We postulate that high doses of opioid antagonists activate sympathetic systems to increase fetal blood pressure through mechanisms which do not involve interactions with mu, delta or kappa opioid receptors.

Metronomic Chemotherapy Enhances Antitumor Effects of Cancer Vaccine by Depleting Regulatory T Lymphocytes and Inhibiting Tumor Angiogenesis

Treatment of psoriasis vulgaris using low-dose naltrexone

Soluble multi-trimeric TNF superfamily ligand adjuvants enhance immune responses to a HIV-1 Gag DNA vaccine