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Despite the ease of production and improved safety profiles of recombinant vaccines, the inherently low immunogenicity of unadjuvanted proteins remains an impediment to their widespread adoption. The covalent tethering of TLR agonists to antigenic proteins offers a unique approach to co-deliver both constituents to the same cell-enhancing vaccine efficacy while minimizing reactogenicity. However, the paucity of simple and effective linker chemistries continues to hamper progress. Here, we present a modular, PEG-based linker system compatible with even extremely lipophilic and challenging TLR7/8 agonists. To advance the field and address previous obstacles, we offer the most straightforward and antigen-preserving linker system to date. These antigen-adjuvant conjugates enhance antigen-specific immune responses in mice, demonstrating the power of our approach within the context of modern vaccinology.

To increase vaccine immunogenicity, modern vaccines incorporate adjuvants, which serve to enhance immune cross-protection, improve humoral and cell-mediated immunity, and promote antigen dose sparing. Pattern recognition receptors (PRRs), including the Toll-like receptor (TLR) family are promising targets for development of agonist formulations for use as vaccine adjuvants. Combinations of co-delivered TLR4 and TLR7/8 ligands have been demonstrated to have synergistic effects on innate and adaptive immune response. Here, we create liposomes that stably co-encapsulate CRX-601, a synthetic TLR4 agonist, and UM-3004, a lipidated TLR7/8 agonist, within the liposomal bilayer in order to achieve co-delivery, allow tunable physical properties, and induce in vitro and in vivo immune synergy. Co-encapsulation demonstrates a synergistic increase in IL-12p70 cytokine output in vitro from treated human peripheral blood mononuclear cells (hPBMCs). Further, co-encapsulated formulations give significant improvement of early IgG2a antibody titers in BALB/c mice following primary vaccination when compared to single agonist or dual agonists delivered in separate liposomes. This work demonstrates that co-encapsulation of TLR4 and lipidated TLR7/8 agonists within the liposomal bilayer leads to innate and adaptive immune synergy which biases a Th1 immune response. Thus, liposomal co-encapsulation may be a useful and flexible tool for vaccine adjuvant formulation containing multiple TLR agonists.

Subunit vaccines require an adjuvant, as protein antigens alone provoke a very poor immune response. Liposomes can be used to deliver an adjuvant, particularly when it is an amphipathic molecule such as a toll-like receptor (TLR) 4 agonist. TLR4 engagement causes the release of cytokines known to skew the immune response towards Th1-mediated cellular immunity, making it attractive for use in viral vaccines. Additionally, liposomes formulated with a TLR4 agonist can be neutral, positive, or negative in charge, further affecting the immune response. We tested the effect of liposome composition and charge on the immune response against the SARS-CoV-2 Spike protein following vaccination with recombinant Spike trimers and liposomes containing a synthetic TLR4 agonist in mice. Anti-Spike antibody titers were modestly boosted by neutral liposomes, but charged liposomes increased it further. Control groups given liposomes without the TLR4 agonist demonstrated that cationic liposomes alone boosted antibody titers, but gave a strikingly different T cell response. These liposomes evoked a strongly Th2-associated immune profile, with high IgG1 levels and T cells that released IL-5 when stimulated ex vivo. In contrast, the TLR4-stimulating cationic liposomes resulted in high IgG2c levels, along with strong production of IFNγ, some IL-17, and no IL-5 by restimulated T cells. Interestingly, despite similar antibody titers, serum from groups given the TLR4-stimulating liposomes more effectively prevented Spike binding to ACE-2 than that of the control groups in pseudoneutralization assays. These data show a beneficial effect of TLR4-stimulating cationic liposomes on the anti-viral cellular immune response in subunit vaccines. Supported by a supplement to NIH Adjuvant Discovery Contract 75N93019C00045

The limited efficacy of seasonal influenza vaccines is usually attributed to ongoing variation in the major antigenic targets for protective antibody responses including hemagglutinin (HA) and neuraminidase (NA). Hence, vaccine development has largely focused on broadening antigenic epitopes to generate cross-reactive protection. However, the vaccine adjuvant components which can accelerate, enhance and prolong antigenic immune responses, can also increase the breadth of these responses. We previously demonstrated that the combination of synthetic small-molecule Toll-like receptor 4 (TLR4) and TLR7 ligands is a potent adjuvant for recombinant influenza virus HA, inducing rapid, and sustained antibody responses that are protective against influenza viruses in homologous and heterologous murine challenge models. To further enhance adjuvant efficacy, we performed a structure-activity relationship study for the TLR4 ligand, N-cyclohexyl-2-((5-methyl-4-oxo-3-phenyl-4,5-dihydro-3H-pyrimido[5,4-b]indol-2-yl)thio)acetamide (C25H26N4O2S; 1Z105), and identified the 8-(furan-2-yl) substituted pyrimido[5,4-b]indole analog (C29H28N4O3S; 2B182C) as a derivative with higher potency in activating both human and mouse TLR4-NF-κB reporter cells and primary cells. In a prime-boost immunization model using inactivated influenza A virus [IIAV; A/California/04/2009 (H1N1)pdm09], 2B182C used as adjuvant induced higher serum anti-HA and anti-NA IgG1 levels compared to 1Z105, and also increased the anti-NA IgG2a responses. In combination with a TLR7 ligand, 1V270, 2B182C induced equivalent levels of anti-NA and anti-HA IgG1 to 1V270+1Z105. However, the combination of 1V270+2B182C induced 10-fold higher anti-HA and anti-NA IgG2a levels compared to 1V270+1Z105. A stable liposomal formulation of 1V270+2B182C was developed, which synergistically enhanced anti-HA and anti-NA IgG1 and IgG2a responses without demonstrable reactogenicity after intramuscular injection. Notably, vaccination with IIAV plus the liposomal formulation of 1V270+2B182C protected mice against lethal homologous influenza virus (H1N1)pdm09 challenge and reduced lung viral titers and cytokine levels. The combination adjuvant induced a greater diversity in B cell clonotypes of immunoglobulin heavy chain (IGH) genes in the draining lymph nodes and antibodies against a broad spectrum of HA epitopes encompassing HA head and stalk domains and with cross-reactivity against different subtypes of HA and NA. This novel combination liposomal adjuvant contributes to a more broadly protective vaccine while demonstrating an attractive safety profile.

An effective adjuvant for the induction of humoural and cellular immunity is achieved by a Pickering emulsion formulation that allows pliability and mobility of loaded antigens.

When designing an effective and safe vaccine, the induction of a protective immune response to a pathogen without unacceptable levels of immunopathology is critical. The use of purified or recombinant antigens in combination with adjuvants that elicit a defined immune response is a promising strategy for eliciting a productive immune response while minimizing toxicity. Our group has developed libraries of novel toll-like receptor (TLR)7 and 8 ligands with a range of activity. Previous clinical trials have shown that the high potency of TLR7/8 adjuvants can lead to unacceptable toxicity. We hypothesized that direct conjugation of the adjuvant to the antigen will limit systemic distribution of the adjuvant, while boosting the immune response by ensuring co-delivery of antigen and adjuvant to antigen presenting cells. We demonstrate that covalent conjugation of TLR7/8 agonists to antigens results in greatly improved humoral and cell-mediated responses, without apparent systemic or local inflammation. This likely improves safety by preventing off-target immune responses from the engagement of PAMPs in the absence of appropriate antigens. When mice were immunized with an antigen/adjuvant conjugate we observed a switch towards IgG2a and away from IgG1 antigen-specific antibodies, plus greater Th1 cytokine production after restimulation of immune cells ex vivo. We hypothesize that co-delivery of a TLR7/8 agonist with antigen leads to activation of CD4+ T cells in the presence of Th1-skewing cytokines. The TLR7/8 agonist also appears to improve cross-presentation, inducing CD8+ T cells. We find this approach has great potential to enhance vaccine efficacy, while reducing potential immunopathology by TLR7/8 adjuvants.

Pseudomonas aeruginosa (Pa) is a Gram-negative bacterium which is associated with chronic diabetic wounds as well as potentially deadly lung infections in cystic fibrosis patients. Approximately 51,000 cases of Pa infections occur annually; of these cases, 13% are resistant to antibiotics which results in the deaths of ~400 individuals every year. Diversity between Pa strains presents a challenge in developing a vaccine or therapeutic capable of resolving infections caused by different strains. Pf phage (a filamentous bacteriophage) has been found in Pa from chronic diabetic wound isolates and has been show to increase virulence of Pa. Recent findings by our team indicate that a Pf phage-targeted vaccine or monoclonal antibody provide protection from the establishment of Pa infection in mice. To further improve this innovative new vaccine targeting Pa, adjuvant systems were used to enhance humoral response to the vaccine. A consensus peptide from Pf Phage coat protein was conjugated to the carrier protein CRM197 and combined with novel adjuvants and delivery systems. Selected adjuvants significantly enhanced humoral immunity to the Pf Phage peptide in a dose-dependent manner. Combination adjuvant systems were also used to further enhance antigen-specific immunity to Pf Phage and cell-mediated immunity to the carrier protein. The development of an adjuvanted Pf phage vaccine to protect against Pa infections is a new and innovative strategy with the potential to protect patients at risk for opportunistic bacterial infections.

To date there is no clinically approved adjuvant to drive a protective T-helper cell 17 (Th17) immune response against Mycobacterium tuberculosis (Mtb). Trehalose Dimycolate (TDM) is a glycolipid molecule found in the cell wall of Mtb and similar species. Our team has discovered novel synthetic TDM derivatives that target Mincle receptors and when presented on the surface of amine functionalized silica nanoparticles (A-SNPs) adopt the requisite supramolecular structure for Mincle receptor agonism. Here we describe the preparation and characterization methods for these critical silica nanoparticles (SNPs) co-loaded with Mincle agonists (MAs) and a model antigen. In this work, A-SNPs with a particle diameter of 246 ± 11 nm were prepared and examined for co-adsorption of two synthetic MAs along with ovalbumin (OVA). Due to the insolubility of the studied MAs in aqueous environment, aggregation of the MAs made separation of the adjuvant-loaded A-SNPs from the free-form MAs via centrifugation very challenging. To facilitate separation, we synthesized modified SNPs with comparable amine surface functionalization to the original A-SNPs, but with a superparamagnetic iron oxide core (M-A-SNPs), to allow for magnetic separation. We also substituted Alexa Fluor 488-labeled ovalbumin (AF 488 OVA) for the un-tagged OVA to improve the sensitivity of our quantitation method. A RP-HPLC method was developed to simultaneously determine the amount of adsorption of both the Mincle adjuvant and the model antigen to the A-SNPs. AF488 OVA demonstrated higher than 90% adsorption, with or without the co-adsorption of MAs. Likewise, MAs exhibited higher than 80% adsorption in the presence or absence of antigen. The developed formulations were tested in vitro using murine RAW cells and human peripheral blood mononuclear cells, exhibiting good cytokine induction in both cell lines. Results from these studies indicate that A-SNPs could be used as a customizable presentation platform to co-deliver antigens along with different MAs of varying structural features and biophysical properties.

The mucosa is the primary point of entry for pathogens making it an important vaccination site to produce a protective mucosal immune response. While the sublingual (SL) mucosa presents several barriers to vaccine penetration, its unique anatomy and physiology makes it one of the best options for mucosal vaccination. Efficient and directed delivery of adjuvants and antigens to appropriate immune mediators in the SL tissue will aid in development of effective SL vaccines against infectious diseases. Herein we demonstrate a robust immune response against influenza antigens co-delivered sublingually with engineered liposomes carrying the synthetic Toll-like receptor-4 agonist, CRX-601. Liposome modification with PEG copolymers (Pluronics), phospholipid-PEG conjugates and chitosan were evaluated for their ability to generate an immune response in a SL murine influenza vaccine model. Phospholipid-PEG conjugates were more effective than Pluronic copolymers in generating stable, surface neutral liposomes. SL vaccination with surface modified liposomes carrying CRX-601 adjuvant generated significant improvements in flu-specific responses compared with unmodified liposomes. Furthermore, the coating of modified liposomes with methylglycol chitosan produced the most effective flu-specific immune response. These results demonstrate efficient SL vaccine delivery utilizing a combination of a muco-adhesive and surface neutral liposomes to achieve a robust mucosal and systemic immune response.

When designing an effective and safe vaccine, inducing a protective immune response to a pathogen without also inducing unacceptable levels of immunopathology is a major challenge. One promising strategy is the use of purified or recombinant antigens along with adjuvants that specifically engage pathogen-associated molecular pattern (PAMP) receptors. This targeted approach aims to minimize immune-mediated reactogenicity. Our group has developed libraries of novel toll-like receptor (TLR)7 and/or TLR8 ligands which show a range in receptor activity. Previous studies have shown that the high potency of TLR7/8 adjuvants leads to unacceptable toxicity in human clinical trials. One strategy to overcome this toxicity is direct adjuvant-antigen conjugation, which serves to limit systemic distribution of the adjuvant through the codelivery of the antigen and adjuvant to APCs. We have demonstrated that covalent conjugation of a TLR7/8 agonist to an antigen results in improved humoral and cell-mediated responses, with decreased systemic and local inflammation. This approach allows for the use of a reduced adjuvant dose by preventing the engagement of PAMPs in the absence of antigen. We hypothesize that antigen/adjuvant codelivery boosts the engagement of CD4+ T cells and elicits the necessary cytokines to promote a Th1-related switch to antigen-specific IgG2a antibodies. In addition, engagement of TLR7/8 appears to improve cross-presentation, thereby boosting the CD8+ T cell response, which is desirable for improved immunity to influenza and other viruses. We find this approach has great potential to enhance vaccine efficacy, while reducing the immunopathology often associated with highly active TLR7/8 adjuvants.

Vaccinology is one of the most important cornerstones in modern medicine, providing better quality of life. The human immune system is composed of innate and adaptive immune processes that interplay when infection occurs. Innate immunity relies on pathogen-associated molecular patterns which are recognized by pathogen recognition receptors localized in antigen presenting cells. After antigen processing and presentation, CD4+ T cell polarization occurs, further leading to B cell and CD8+ activation and humoral and cell-mediated adaptive immune responses. Liposomes are being employed as vaccine technologies and their design is of importance to ensure proper immune responses. Physicochemical parameters like liposome size, charge, lamellarity and bilayer fluidity must be completely understood to ensure optimal vaccine stability and efficacy. Liposomal vaccines can be developed to target specific immune cell types for the induction of certain immune responses. In this review, we will present promising liposomal vaccine approaches for the treatment of important viral, bacterial, fungal and parasitic infections (including tuberculosis, TB). Cationic liposomes are the most studied liposome types due to their enhanced interaction with the negatively charged immune cells. Thus, a special section on the cationic lipid dimethyldioctadecylammonium and TB is also presented.

Doxazolidine (doxaz) is a new anthracycline anticancer agent. While structurally similar to doxorubicin (dox), doxaz acts via a distinct mechanism to selectively enhance anticancer activity over cardiotoxicity, the most significant clinical impediment to successful anthracycline treatment. Here, we describe the synthesis and characterization of a prodrug platform designed for doxaz release mediated by secreted proteolytic activity, a common association with invasiveness and poor prognosis in cancer patients. GaFK-Doxaz is hydrolyzable by the proteases plasmin and cathepsin B, both strongly linked with cancer progression, as well as trypsin. We demonstrate that activation of GaFK-Doxaz releases highly potent doxaz that powerfully inhibits the growth of a wide variety of cancer cells (average IC(50) of 8 nM). GaFK-Doxaz is stable in human plasma and is poorly membrane permeable, thereby limiting activation to locally secreted proteolytic activity and reducing the likelihood of severe side effects.

Infection is the most common cause of mortality early in life, in large measure due to suboptimal early life vaccination strategies as compared to older age groups. New adjuvants are absolutely cardinal to further optimize current immunization approaches. However, only a few classes of adjuvants are presently incorporated in vaccines approved for human use. Recent advances in the discovery and delivery of Toll-like receptor (TLR) adjuvants have provided a new toolbox for vaccinologists. Prominent among these candidate adjuvants are synthetic small molecule TLR7/8 agonists. The development of an effective infant Bordetella pertussis vaccine is now urgent because of the resurgence of pertussis in many countries, contemporaneous and likely related to the switch from whole cell to acellular vaccines. In this context, age-specific adjuvant strategies may be key to enhance early life immunogenicity by creating a vaccine formulation that induces both robust and persistent immunity (i.e., overcome waning immunity) to B. pertussis. In the present study, we firstly optimized the a) formulation delivery system, b) stability, and c) immunologic activity of novel small molecule imidazoquinoline TLR7/8 adjuvants towards human infant leukocytes. Next, in both adult and neonatal mouse models, we demonstrate that this TLR7/8 adjuvant can overcome neonatal hyporesponsiveness to acellular pertussis vaccination by driving Th1 favoring responses to a licensed acellular vaccine (DTaP). This potent immunization strategy may be of fundamental importance in vaccine development and could represent a new paradigm for effective pertussis immunization in early life.

Influenza is a highly communicative viral pathogen that infects 9–35 million individuals and results in 12,000–56,000 deaths in the US annually. For the most commonly used seasonal influenza vaccine, historical efficacy rates range from 10–60% thus improvements to the current vaccine are needed. We identified a novel series of synthetic TLR7/8 ligands that improve influenza vaccine efficacy when used alone or in combination with our synthetic TLR4 ligand. In human PBMCs, TLR7/8 ligands elicited secretion of the inflammatory cytokines TNFα, IL-6, and IL-1β as well as the Th1-polarizing cytokine IL12-p70. These responses can be altered by modification of novel structural features of the TLR7/8 ligands. The addition of a synthetic TLR4 ligand modified the cytokine expression profile, indicating that the structure of a TLR7/8 ligand and/or addition of a TLR4 ligand can drive substantive changes in the innate immune response. To evaluate the adjuventicity of these TLR ligands, Balb/c mice were vaccinated with split-flu vaccine containing TLR7/8 ligands alone or in combination with our TLR4 ligand. TLR7/8 ligands increased influenza-specific total IgG and IgG2a, but not IgG1, over antigen alone. Addition of a TLR4 ligand further increased influenza-specific total IgG and IgG2a. These data demonstrate that these ligands act as adjuvants to promote a Th1 skewed humoral response. The combination of TLR7/8 and TLR4 ligands elicited superior cell mediated immunity compared to either TLR7/8 or TLR4 ligand alone. These data demonstrate that heightened innate and adaptive immune responses can be elicited through a combination adjuvant using novel synthetic TLR7/8 and TLR4 ligands.

Influenza is a vaccine-preventable contagious respiratory illness caused by influenza (flu) viruses which can lead to hospitalization and sometimes even death. Current flu vaccines delivered intramuscularly (IM) or intradermally (ID) are less effective at eliciting protective mucosal immune responses and vaccines delivered intranasally (IN) possess potential safety concerns. Sublingual (SL) vaccination is a promising alternative route for vaccine delivery which has been indicated as safe and effective at inducing protective immune responses in both systemic and mucosal compartments. We evaluated the efficacy of methylglycol chitosan (MGC) and a synthetic toll-like receptor 4 agonist (CRX-601), alone or in combination, for improving systemic and mucosal immune responses to a monovalent detergent-split flu virus vaccine delivered SL. SL vaccination of mice with split-flu vaccine formulated with either MGC or CRX-601 resulted in specific serum IgG and mucosal IgA titers that were significantly greater than titers from non-adjuvanted vaccination and equivalent to or greater than titers in mice vaccinated IM. Our results demonstrate that SL vaccination utilizing MGC or CRX-601 as adjuvants is a viable alternative route of vaccination for flu which can elicit systemic immune responses equivalent to or greater than IM vaccination with the added benefit of stimulating a robust specific mucosal immune response.

We have reported the synthesis and biological evaluation of a prodrug to a doxorubicin active metabolite. Under physiologic conditions, release of the active metabolite, a conjugate of doxorubicin with formaldehyde, occurs with a half-life of 1 hour. To direct this prodrug to tumor, we designed two conjugates of the prodrug, doxsaliform, with the αvβ3-targeting peptides, CDCRGDCFC (RGD-4C) and cyclic-(N-Me-VRGDf) (Cilengitide). We now report the synthesis of these doxsaliform-peptide conjugates and their evaluation using MDA-MB-435 cancer cells. A hydroxylamine ether tether was used to attach 5″-formyldoxsaliform to RGD-4C in its acyclic form via an oxime functional group. The construct acyclic-RGD-4C-doxsaliform showed good binding affinity for αvβ3 in the vitronection cell adhesion assay (IC50 = 10 nmol/L) and good growth inhibition of MDA-MB-435 breast cancer cells (IC50 = 50 nmol/L). In its bicyclic forms, RGD-4C showed less affinity for αvβ3 and significantly less water solubility. Cyclic-(N-Me-VRGDf) was modified by substitution of d-4-aminophenylalanine for d-phenylalanine to provide a novel attachment point for doxsaliform. The conjugate, cyclic-(N-Me-VRGDf-NH)-doxsaliform, maintained a high affinity for αvβ3 (IC50 = 5 nmol/L) in the vitronectin cell adhesion assay relative to the peptide bearing only the tether (0.5 nmol/L). The IC50 for growth inhibition of MDA-MB-435 cells was 90 nmol/L. Flow cytometry and growth inhibition experiments suggest that the complete drug construct does not penetrate through the plasma membrane, but the active metabolite does on release from the targeting group. These drug conjugates could have significantly reduced side effects and are promising candidates for in vivo evaluation in tumor-bearing mice.

A novel synthesis of the potent glutamate neurotransmitter agonist (RS)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl) propionic acid (ACPA) provides access to numerous analogues as drug candidates for neurological disorders. The one-pot synthesis of an alpha amino phosphonate from aldehyde 4 was successful using ErCl3 as a catalyst. Molecular modeling of the new amino phosphonic acid with the (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl) propionic acid (AMPA) receptor crystal structure suggests this should be an effective receptor binder.

Ethyl 4-acetyl-5-methyl-3-isoxazolyl carboxylate was smoothly lithiated at the 5-methyl position, when the 4-acetyl group was protected with a 5,5-dimethyl-1,3-dioxanyl group. The lithio anion was quenched with a variety of electrophiles such as alkyl halides, aldehydes, TMSCl, and Me3SnCl in good to excellent yields. The lithiation of the unprotected compound and the 4-acetyl group protected as 1,3-dioxolanyl both failed. The effects of different bases have been investigated and the addition of LiCl significantly increased yields. Based on variable temperature NMR studies the 5,5-dimethyl-1,3-dioxanyl group appears to occupy a single chair conformation which may facilitate lateral metalation. This represents a facile entry into 5-functionalized 3-isoxazolyl carboxylic acid derivatives as prodrugs for the AMPA glutamate neurotransmitters of the central nervous system.

The reaction of 4-acetyl-5-methyl-3-isoxazolyl carboxylate with a variety of hydrazines and semicarbazides yielded molecules which are viable antagonist candidates for the AMPA receptor. Molecular modeling studies used in conjunction with the x-ray crystal structures of these derivatives show a close correlation between the hydrogen bonding characteristics of AMPA with that of the hydrazone and semicarbazone isoxazole derivatives. Uncyclized hydrazones and semicarbazones (1–5) were formed by using corresponding hydrazines and semicarbazides containing strong electron withdrawing groups to prevent cyclization with the ethyl ester. The crystals of 1 are orthorhombic with a = 14.2997(3), b = 15.4112(4),c = 16.0153(4) A, Z = 8, and space group Pbca; 2 monoclinic with a = 19.738(2), b = 10.4155(7), c = 15.583(1) A, β = 92.348(2)°, Z = 8, and space group C2/c; 3 triclinic with a = 8.3365(5), b = 8.4930(5), c = 12.2379(7) A, α = 92.568(2), β = 102.229(2), γ = 104.449(1)°, Z = 2, and space group P $$\bar 1$$ .

A method is described for the analysis of the rodenticide zinc phosphide (Zn3P2, CAS Registry No. 1314-84-7) in various agricultural food commodities as well as for certain animal stomach contents. The method involves reacting the sample with aqueous H2SO4 and toluene in a volatile organic analysis vial. The H2SO4 hydrolyzes Zn3P2 to phosphine gas (PH3), which is trapped in toluene. The sample vials are centrifuged to break the emulsion formed by mixing, and the toluene layer is then analyzed by gas chromatography. PH3 is detected with a thermionic nitrogen-phosphorus detector or a flame photometric detector. This method is capable of detecting quantities as low as 5n g of Zn3P2/g of sample. The method has been validated for blueberry, cantaloupe, cucumber, hay grass, potato, raspberry, snap bean, soybean, spinach, and squash.

The sequence of research leading to a proposal for anthracycline cross-linking of DNA is presented.The clinical anthracycline antitumor drugs are anthraquinones, and as such are redox active. Their redoxchemistry leads to induction of oxidative stress and drug metabolites. An intermediate in reductive glycosidiccleavage is a quinone methide, once proposed as an alkylating agent of DNA. Subsequent research nowimplicates formaldehyde as a mediator of anthracycline-DNA cross-linking. The cross-link at 5'-GC-3'sites consists of a covalent linkage from the amino group of the anthracycline to the 2-amino groupof the G-base through a methylene from formaldehyde, hydrogen bonding from the 9-OH to the G-base onthe opposing strand, and hydrophobic interactions through intercalation of the anthraquinone. The combinationof these interactions has been described as a virtual cross-linkof DNA. The origin of the formaldehyde in vivo remains a mystery. In vitro, doxorubicin reacts withformaldehyde to give firstly a monomeric oxazolidine, doxazolidine, and secondly a dimeric oxazolidine,doxoform. Doxorubicin reacts with formaldehyde in the presence of salicylamide to give the N-Mannich baseconjugate, doxsaliform. Doxsaliform is several fold more active in tumor cell growth inhibition than doxorubicin,but doxazolidine and doxoform are orders of magnitude more active than doxorubicin. Exploratory researchon the potential for doxsaliform and doxazolidine as targeted cytotoxins is presented. A promisinglead design is pentyl PABC-Doxaz, targeted to a carboxylesterase enzyme overexpressed in liver cancercells and/or colon cancer cells.

The Dess-Martin periodinane (DMP) oxidation of alcohols, proceeds in good to excellent yield in the presence of the isoxazole ring, and other nitrogen-containing functional groups. The DMP route allows for the preparationof highly branched polyketide synthetic equivalents.

A novel synthesis of the potent glutamate neurotransmitter agonist (RS)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl) propionic acid (ACPA) provides access to numerous analogues as drug candidates for neurological disorders. The one-pot synthesis of an alpha amino phosphonate from aldehyde 4 was successful using ErCl3 as a catalyst. Molecular modeling of the new amino phosphonic acid with the (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl) propionic acid (AMPA) receptor crystal structure suggests this should be an effective receptor binder.

Analogues of amino methylisoxazole propionic acid (AMPA), were prepared from a common intermediate 12, including lipophilic analogues using lateral metalation and electrophilic quenching, and were evaluated at System x c - . Both the 5-naphthylethyl-(16) and 5-naphthylmethoxymethyl-(17) analogues adopt an E-conformation in the solid state, yet while the former has robust binding at System x c - , the latter is virtually devoid of activity. The most potent analogues were amino acid naphthyl-ACPA 7g, and hydrazone carboxylic acid, 11e Y = Y′ = 3,5-(CF3)2, which both inhibited glutamate uptake by the System x c - transporter with comparable potency to the endogenous substrate cystine, whereas in contrast the closed isoxazolo[3,4-d] pyridazinones 13 have significantly lower activity. A preliminary pharmacophore model has been constructed to provide insight into the analogue structure–activity relationships.

The sequence of research leading to a proposal for anthracycline cross-linking of DNA is presented.The clinical anthracycline antitumor drugs are anthraquinones, and as such are redox active. Their redoxchemistry leads to induction of oxidative stress and drug metabolites. An intermediate in reductive glycosidiccleavage is a quinone methide, once proposed as an alkylating agent of DNA. Subsequent research nowimplicates formaldehyde as a mediator of anthracycline-DNA cross-linking. The cross-link at 5′-GC-3′sites consists of a covalent linkage from the amino group of the anthracycline to the 2-amino groupof the G-base through a methylene from formaldehyde, hydrogen bonding from the 9-OH to the G-base onthe opposing strand, and hydrophobic interactions through intercalation of the anthraquinone. The combinationof these interactions has been described as a virtual cross-linkof DNA. The origin of the formaldehyde in vivo remains a mystery. In vitro, doxorubicin reacts withformaldehyde to give firstly a monomeric oxazolidine, doxazolidine, and secondly a dimeric oxazolidine,doxoform. Doxorubicin reacts with formaldehyde in the presence of salicylamide to give the N-Mannich baseconjugate, doxsaliform. Doxsaliform is several fold more active in tumor cell growth inhibition than doxorubicin,but doxazolidine and doxoform are orders of magnitude more active than doxorubicin. Exploratory researchon the potential for doxsaliform and doxazolidine as targeted cytotoxins is presented. A promisinglead design is pentyl PABC-Doxaz, targeted to a carboxylesterase enzyme overexpressed in liver cancercells and/or colon cancer cells.

The synthesis and tumor cell growth inhibition by doxazolidine carbamate prodrugs are reported. The carbamates were designed for selective hydrolysis by one or more human carboxylesterases to release doxazolidine (Doxaz), the formaldehyde-oxazolidine of doxorubicin that cross-links DNA to trigger cell death. Simple butyl and pentyl, but not ethyl, carbamate prodrugs inhibited the growth of cancer cells that overexpress carboxylesterase CES1 (hCE1) and CES2 (hiCE). Relative CES1 and CES2 expression levels were determined by reverse transcription of the respective mRNAs, followed by polymerase chain reaction amplification. More complex structures with a p-aminobenzyl alcohol (PABA) self-eliminating spacer showed better growth inhibition (IC50=50 nM for Hep G2 liver cancer cells) while exhibiting reduced toxicity toward rat cardiomyocytes, relative to the parent drug doxorubicin. Pentyl 4-(N-doxazolidinylcarbonyloxymethyl)phenylcarbamate, the lead compound for further investigation, appears to be activated in Hep G2 cells that express both CES1 and CES2.

A crystal structure establishes doxoform as a dimeric formaldehyde conjugate of the oxazolidine of doxorubicin. Doxoform is a prodrug of doxazolidine, a monomeric doxorubicin formaldehyde-oxazolidine. Both doxoform and doxazolidine inhibit the growth of cancer cells at 1-4 orders of magnitude lower concentration than doxorubicin. They also inhibit the growth of cancer cells better than doxsaliform, a prodrug for an acyclic doxorubicin-formaldehyde conjugate. Doxoform rapidly hydrolyzes to doxazolidine, which then hydrolyzes to doxorubicin with a half-life of 3 min in human serum at 37 degrees C. Both doxoform and doxazolidine are taken up by multidrug-resistant MCF-7/Adr cells 3- to 4-fold better than doxorubicin. A molecular model suggests that doxazolidine can cross-link DNA by direct reaction with a G-base in a tautomeric form with synchronous ring opening of the oxazolidine. These results point to doxoform being a prodrug for doxazolidine that is the reactive species that directly cross-links DNA.

Analogs of the excitatory neurotransmitter glutamate are potential medicinal agents for a wide variety of neurological disorders. The isoxazole glutamate derivatives represent an important class of compounds because of their receptor specificity and binding affinity. Since the discovery of (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl) propionic acid (AMPA) in 1980, numerous analogs built around the isoxazole scaffold have shown remarkable selectivity for specific ionotropic glutamate receptors, but strong side effects in human clinical trials have shown the need for improvement. Trends revealed by structure activity relationship and crystallographic studies indicate the role of stereochemistry may be important in uncovering the prerequisite selectivity, which would give rise to effective therapeutics for neurological dysfunction of the glutamate receptor.

We have reported the synthesis and biological evaluation of a prodrug to a doxorubicin active metabolite. Under physiologic conditions, release of the active metabolite, a conjugate of doxorubicin with formaldehyde, occurs with a half-life of 1 hour. To direct this prodrug to tumor, we designed two conjugates of the prodrug, doxsaliform, with the alphavbeta3-targeting peptides, CDCRGDCFC (RGD-4C) and cyclic-(N-Me-VRGDf) (Cilengitide). We now report the synthesis of these doxsaliform-peptide conjugates and their evaluation using MDA-MB-435 cancer cells. A hydroxylamine ether tether was used to attach 5''-formyldoxsaliform to RGD-4C in its acyclic form via an oxime functional group. The construct acyclic-RGD-4C-doxsaliform showed good binding affinity for alphavbeta3 in the vitronection cell adhesion assay (IC50 = 10 nmol/L) and good growth inhibition of MDA-MB-435 breast cancer cells (IC50 = 50 nmol/L). In its bicyclic forms, RGD-4C showed less affinity for alphavbeta3 and significantly less water solubility. Cyclic-(N-Me-VRGDf) was modified by substitution of D-4-aminophenylalanine for D-phenylalanine to provide a novel attachment point for doxsaliform. The conjugate, cyclic-(N-Me-VRGDf-NH)-doxsaliform, maintained a high affinity for alphavbeta3 (IC50 = 5 nmol/L) in the vitronectin cell adhesion assay relative to the peptide bearing only the tether (0.5 nmol/L). The IC50 for growth inhibition of MDA-MB-435 cells was 90 nmol/L. Flow cytometry and growth inhibition experiments suggest that the complete drug construct does not penetrate through the plasma membrane, but the active metabolite does on release from the targeting group. These drug conjugates could have significantly reduced side effects and are promising candidates for in vivo evaluation in tumor-bearing mice.

Utilizing our lateral metalation coupled with Jacobsen's catalytic asymmetric amino nitrile synthesis, we have demonstrated the ability to synthesize isoxazole-containing amino acid glutamate analogues in high yield and high enantiomeric excesses. Chiral centers alpha or beta at the C-5 position do not detract from diastereoselectivity of the Jacobsen-Strecker reaction. [reaction: see text]

Antibody-mediated capture of amyloid-beta (Aβ) in peripheral blood was identified as an attractive strategy to eliminate cerebral toxic amyloid in Alzheimer's disease (AD) patients and murine models. Alternatively, defective capacity of peripheral monocytes to engulf Aβ was reported in individuals with AD. In this report, we developed different approaches to investigate cellular uptake and phagocytosis of Aβ, and to examine how two immunological devices – an immunostimulatory Adjuvant System and different amyloid specific antibodies – may affect these biological events. Between one and thirteen months of age, APPswe X PS1.M146V (TASTPM) AD model mice had decreasing concentrations of Aβ in their plasma. In contrast, the proportion of blood monocytes containing Aβ tended to increase with age. Importantly, the TLR-agonist containing Adjuvant System AS01B primed monocytes to promote de novo Aβ uptake capacity, particularly in the presence of anti-Aβ antibodies. Biochemical experiments demonstrated that cells achieved Aβ uptake and internalization followed by Aβ degradation via mechanisms that required effective actin polymerization and proteolytic enzymes such as insulin-degrading enzyme. We further demonstrated that both Aβ-specific monoclonal antibodies and plasma from Aβ-immunized mice enhanced the phagocytosis of 1μm Aβ-coated particles. Together, our data highlight a new biomarker testing to follow amyloid clearance within the blood and a mechanism of Aβ uptake by peripheral monocytes in the context of active or passive immunization, and emphasize on novel approaches to investigate this phenomenon.