Aminoalkyl Glucosaminide 4-phosphates Research Articles

Development of an ultra-sensitive assay for the determination of an aminoalkyl glucosaminide 4-phosphate, GSK1795091, in plasma to support a first time in human study

A chemical derivatization approach for the determination of an aminoalkyl glucosaminide 4-phosphate, GSK1795091 (or CRX-601).

Characterization of TRIF selectivity in the AGP class of lipid A mimetics: Role of secondary lipid chains

TLR4 agonists that favor TRIF-dependent signaling and the induction of type 1 interferons may have potential as vaccine adjuvants with reduced toxicity. CRX-547 (4), a member of the aminoalkyl glucosaminide 4-phosphate (AGP) class of lipid A mimetics possessing three (R)-3-decanoyloxytetradecanoyl groups and d-relative configuration in the aglycon, selectively reduces MyD88-dependent signaling resulting in TRIF-selective signaling, whereas the corresponding secondary ether lipid 6a containing (R)-3-decyloxytetradecanoyl groups does not. In order to determine which secondary acyl groups are important for the reduction in MyD88-dependent signaling activity of 4, the six possible ester/ether hybrid derivatives of 4 and 6a were synthesized and evaluated for their ability to induce NF-κB in a HEK293 cell reporter assay. An (R)-3-decanoyloxytetradecanoyl group on the 3-position of the d-glucosamine unit was found to be indispensable for maintaining low NF-κB activity irrespective of the substitutions (decyl or decanoyl) on the other two secondary positions. These results suggest that the carbonyl group of the 3-secondary lipid chain may impede homodimerization and/or conformational changes in the TLR4–MD2 complex necessary for MyD88 binding and pro-inflammatory cytokine induction.

Synthetic TLR4 agonist as a potential immunotherapy for melioidosis

Melioidosis, an infection caused by Gram-negative Burkholderia pseudomallei (Bp), has high clinical recurrence and mortality rates associated with pneumonia and sepsis. With the limitations in current therapeutic options and the lack of available human vaccines, development of novel countermeasures against Bp infection is vital. In this study, we evaluated the efficacy of an aminoalkyl glucosaminide 4-phosphate (AGP), a synthetic toll like receptor 4 agonist (CRX-527), in conferring protection against melioidosis in a murine model. Survival data showed 66% of mice treated with AGP prior to lethal intranasal Bp challenge survived and presented no signs of illness over a 3 months period. In contrast, all control mice succumbed to infection within 4 days. Kinetic study on organ bacterial burden demonstrated mice treated with AGP had dramatically reduced bacterial loads in both the lungs and spleens as compared to control mice. Notably, all but one AGP-treated mouse had no Bp growth in the blood as compared to overwhelming bacteraemia found in all control mice. The protective effect of CRX-527 was associated with a transient increase in pulmonary cytokine/ chemokine levels, which boosted the host’s innate immunity. This enabled rapid clearance of the pulmonary and systemic bacterial burden and prevented the development of sepsis. This study demonstrated the potential use of TLR4 agonist as a prophylactic immunotherapy in preventing melioidosis.

A Synthetic Lipid A Mimetic Modulates Human TLR4 Activity

Innate immunity recognition relies on a diverse set of germ line encoded receptors, termed pattern recognition receptors (PRR), which recognize broad classes of molecular structures common to groups of microorganisms. One of the largest and best studied families of PRR are the Toll family of receptors (Toll-like receptors, TLRs) that detect microbial components with high sensitivity and selectivity[1]. Among TLRs, TLR4 selectively responds to bacterial endotoxin (E) (Gram-negative bacterial lipopolysaccharides (LPS) or lipooligosaccharides (LOS)),[2] resulting in the rapid triggering of pro-inflammatory processes necessary for optimal host immune responses to invading Gram-negative bacteria (GNB). TLR4 does not bind directly to endotoxin: LBP,[3] CD14,[4] MD-2[5] are required for efficient extraction and transfer of endotoxin monomers from the GNB outer membrane or aggregates of purified endotoxin to MD-2. The resulting monomeric E·MD-2 complex is the ligand that, depending on the structural properties of E and MD-2, specifies TLR4 activation or antagonism.[6] Although TLR4 plays a key physiologic role in host response to Gram-negative bacterial infection, an excessively potent and/or prolonged TLR4 response can promote life-threatening pathology such as septic shock.[7] TLR4 activation has also been associated with certain autoimmune diseases, non-infectious inflammatory disorders, and neuropathic pain, suggesting a wide range of possible clinical settings for application of TLR4 antagonists.[8] Conversely, agonists of TLR4 can be useful as adjuvants in vaccine development and in cancer immunotherapy [9]. Lipid A[10] (Scheme 1), the hydrophobic part of LPS, is responsible for TLR4-dependent proinflammatory activity.[11] Underacylated lipid A variants, such as tetraacylated lipid IVa[12] and E5564 (Eritoran)[13] are potent LPS antagonists (Scheme 1). The β(1→6) diglucosamine backbone of lipid A can be replaced by an aminoalkyl glucosamine moiety in aminoalkyl glucosaminide 4-phosphates (AGPs)[14] or by other non-carbohydrate structures[15] and the lipid A analogue retains TLR4 agonist or antagonist activity. One or two phosphates are typically present in synthetic lipid A mimics, but these groups could be, in principle, substituted by negatively charged isosteres. A carboxylic acid group replaces the C-1 phosphate in AGP derivatives,[14a] while a sulfate group is present in the monosaccharide lipid A mimic ONO-4007 (Scheme 1) developed by Ono Pharmaceutical Co (Osaka, Japan).[16] This compound showed TLR4 agonist activity inducing TNF-α production in tumour cells, but further clinical development was precluded by the compound's limited water solubility.

CD14‐independent responses induced by a synthetic lipid A mimetic

CRX-527 belongs to a new family of synthetic lipid A mimetics, the aminoalkyl glucosaminide 4-phosphates, which are considered as potential vaccine adjuvants or stand-alone immunotherapeutics to harness innate immune defenses. Since natural lipid A from bacterial LPS depends on membrane-bound (mCD14) or soluble CD14 for its TLR4 ligand activity, we investigated the involvement of both forms of CD14 in the responses elicited by CRX-527. First, we found that CRX-527 induces NF-kappaB and interferon regulatory factor-3 (IRF-3) activation in human embryonic kidney cells transfected with TLR4 and MD-2 genes alone, whereas the responses to LPS require either co-transfection of the gene encoding mCD14 or addition of soluble CD14. We then observed that monocyte-derived DC, which are devoid of mCD14 respond to CRX-527 but not to LPS in serum-free medium. Furthermore, we found that, in contrast to LPS, CRX-527 induces the production of cytokines in whole blood of a patient with paroxysmal nocturnal hemoglobinuria, a disease in which mCD14-dependent responses are defective. Finally, we demonstrated that splenocytes from CD14-deficient mice produce cytokines in response to CRX-527 but not to LPS. We conclude that the lipid A mimetic CRX-527 does not require the CD14 co-receptor to elicit TLR4-mediated responses.

Lipid A mimetics are potent adjuvants for an intranasal pneumonic plague vaccine

An effective intranasal (i.n.) vaccine against pneumonic plague was developed. The formulation employed two synthetic lipid A mimetics as adjuvant combined with Yersinia pestis-derived V- and F1-protective antigens. The two nontoxic lipid A mimetics, classed as amino-alkyl glucosaminide 4-phosphates (AGPs) are potent ligands for the Toll-like receptor (TLR) 4. Using a murine (BALB/c) pneumonic plague model, we showed a single i.n. application of the vaccine provided 63% protection within 21 days against a Y. pestis CO92 100 LD 50 challenge. Protection reached 100% by 150 days. Using a homologous i.n. 1°/2° dose regimen, with the boost administered at varying times, 63% protection was achieved within 7 days and 100% protection was achieved by 21 days after the first immunization. Little or no protection was observed in animals that received antigens alone, and no protection was observed when the vaccine was administered to BALB/c TLR4 mutant mice. Vaccine-induced serum IgG titers to F1 and V-antigen were reflected in high titers for IgG1 and IgG2a, the latter reflecting a bias for a cell-mediated (T H1) immune response. This intranasal vaccine showed 90% protection in Sprague–Dawley rats challenged with 1000 LD 50. We conclude that lipid A mimetics are highly effective adjuvants for an i.n. plague vaccine.

Induction of innate immunity by lipid A mimetics increases survival from pneumonic plague

This study analysed the effect of priming the innate immune system using synthetic lipid A mimetics in a Yersinia pestis murine pulmonary infection model. Two aminoalkyl glucosaminide 4-phosphate (AGP) Toll-like receptor 4 (TLR4) ligands, delivered intranasally, extended time to death or protected against a lethal Y. pestis CO92 challenge. The level of protection was dependent upon the challenge dose of Y. pestis and the timing of AGP therapy. Protection correlated with cytokine induction and a decreased bacterial burden in lung tissue. AGP protection was TLR4-dependent and was not evidenced in transgenic TLR4-deficient mice. AGP therapy augmented with subtherapeutic doses of gentamicin produced dramatically enhanced survival. Combined, these results indicated that AGPs may be useful in protection of immunologically naive individuals against plague and potentially other infectious agents, and that AGP therapy may be used synergistically with other therapies.

Development of a CTL vaccine for Her-2/neu using peptide-microspheres and adjuvants

With the ultimate goal of developing a therapeutic cancer vaccine, we encapsulated the Her-2/neu peptide p369–377 in poly(lactide-co-glycolide) microspheres. This formulation was found to effectively elicit CD8+ cytotoxic T cell (CTL) responses in an HLA-A*0201 transgenic mouse model. In contrast, immunization with either peptide alone or peptide formulated in incomplete Freund's adjuvant (IFA) failed to elicit such CTL responses. Responses induced by the peptide-microsphere formulation were found to peak at approximately 6 weeks post-immunization, and were enhanced by delivering increased doses of peptide and with repeated administrations over time. Co-administration of the peptide-microspheres with adjuvants, including granulocyte–macrophage colony stimulating factor, MPL ® adjuvant and select synthetic Toll-Like Receptor 4 ligands, the aminoalkyl glucosaminide-4 phosphates, significantly augmented CTL responses. These studies provide important guidance for the design of human clinical trials of microsphere vaccines in terms of optimal peptide-microsphere formulation, vaccination regimen, vaccine dose, and adjuvant selection.

Immunostimulatory activity of aminoalkyl glucosaminide 4-phosphates (AGPs): induction of protective innate immune responses by RC-524 and RC-529

Earlier we showed that the structural requirements for adjuvanticity among the aminoalkyl glucosaminide 4-phosphate (AGP) class of synthetic immunostimulants may be less strict than those for other endotoxic activities, including the induction of nitric oxide synthase in murine macrophages and cytokine production in human whole blood. The known role of nitric oxide and pro-inflammatory cytokines in the activation of host defenses against infection prompted us to examine the ability of certain AGPs to enhance non-specific resistance in mice to Listeria monocytogenes and influenza infections as well as to stimulate the production of pro-inflammatory cytokines in mouse splenocytes, human PBMCs, and human U937 histiocytic lymphoma cells. Intranasal administration of RC-524 or RC-529 to mice 2 days prior to a lethal influenza challenge provided significant protection in each case. Similarly, the intravenous administration of these AGPs induced resistance to L. monocytogenes infection as measured by survival or reduction of bacteria in the spleen. Activation of the innate immune response by AGPs appears to involve activation of Toll-like receptor 4 (TLR4) because RC-524 failed to elicit a protective effect in C3H/HeJ mice which have a defect in TLR4 signaling or induce significant cytokine levels in C3H/HeJ splenocytes. Both AGPs also stimulated pro-inflammatory cytokine release in human cell cultures in a dose-dependent manner.

Immunostimulatory activity of aminoalkyl glucosaminide 4-phosphates (AGPs): induction of protective innate immune responses by RC-524 and RC-529.

Earlier we showed that the structural requirements for adjuvanticity among the aminoalkyl glucosaminide 4-phosphate (AGP) class of synthetic immunostimulants may be less strict than those for other endotoxic activities, including the induction of nitric oxide synthase in murine macrophages and cytokine production in human whole blood. The known role of nitric oxide and pro-inflammatory cytokines in the activation of host defenses against infection prompted us to examine the ability of certain AGPs to enhance non-specific resistance in mice to Listeria monocytogenes and influenza infections as well as to stimulate the production of pro-inflammatory cytokines in mouse splenocytes, human PBMCs, and human U937 histiocytic lymphoma cells. Intranasal administration of RC-524 or RC-529 to mice 2 days prior to a lethal influenza challenge provided significant protection in each case. Similarly, the intravenous administration of these AGPs induced resistance to L. monocytogenes infection as measured by survival or reduction of bacteria in the spleen. Activation of the innate immune response by AGPs appears to involve activation of Toll-like receptor 4 (TLR4) because RC-524 failed to elicit a protective effect in C3H/HeJ mice which have a defect in TLR4 signaling or induce significant cytokine levels in C3H/HeJ splenocytes. Both AGPs also stimulated pro-inflammatory cytokine release in human cell cultures in a dose-dependent manner.

Synthesis and biological evaluation of a new class of vaccine adjuvants: aminoalkyl glucosaminide 4-phosphates (AGPs)

A novel series of acylated ω-aminoalkyl 2-amino-2-deoxy-4-phosphono-β-D-glucopyranosides (aminoalkyl glucosaminide 4-phosphates) was synthesized and screened for immunostimulant activity. Several of these compounds enhance the production of tetanus toxoid-specific antibodies in mice and augment vaccine-induced cytotoxic T cells against EG.7-ova target cells.