(+)-SJ733, a clinical candidate for malaria that acts through ATP4 to induce rapid host-mediated clearance of Plasmodium.

Drug discovery for malaria has been transformed in the last 5 years by the discovery of many new lead compounds identified by phenotypic screening. The process of developing these compounds as drug leads and studying the cellular responses they induce is revealing new targets that regulate key processes in the Plasmodium parasites that cause malaria. We disclose herein that the clinical candidate (+)-SJ733 acts upon one of these targets, ATP4. ATP4 is thought to be a cation-transporting ATPase responsible for maintaining low intracellular Na(+) levels in the parasite. Treatment of parasitized erythrocytes with (+)-SJ733 in vitro caused a rapid perturbation of Na(+) homeostasis in the parasite. This perturbation was followed by profound physical changes in the infected cells, including increased membrane rigidity and externalization of phosphatidylserine, consistent with eryptosis (erythrocyte suicide) or senescence. These changes are proposed to underpin the rapid (+)-SJ733-induced clearance of parasites seen in vivo. Plasmodium falciparum ATPase 4 (pfatp4) mutations that confer resistance to (+)-SJ733 carry a high fitness cost. The speed with which (+)-SJ733 kills parasites and the high fitness cost associated with resistance-conferring mutations appear to slow and suppress the selection of highly drug-resistant mutants in vivo. Together, our data suggest that inhibitors of PfATP4 have highly attractive features for fast-acting antimalarials to be used in the global eradication campaign.

Utilizing native fluorescence imaging, modeling and simulation to examine pharmacokinetics and therapeutic regimen of a novel anticancer prodrug.

BACKGROUND: Success of cancer prodrugs relying on a foreign gene requires specific delivery of the gene to the cancer, and improvements such as higher level gene transfer and expression. Attaining these objectives will be facilitated in preclinical studies using our newly discovered CNOB-GDEPT, consisting of the produrg: 6-chloro-9-nitro-5-oxo-5H-benzo-(a)-phenoxazine (CNOB) and its activating enzyme ChrR6, which generates the cytotoxic product 9-amino-6-chloro-5H-benzo[a]phenoxazine-5-one (MCHB). MCHB is fluorescent and can be noninvasively imaged in mice, and here we investigated whether MCHB fluorescence quantitatively reflects its concentration, as this would enhance its reporter value in further development of the CNOB-GDEPT therapeutic regimen. PK parameters were estimated and used to predict more effective CNOB administration schedules. METHODS: CNOB (3.3 mg/kg) was injected iv in mice implanted with humanized ChrR6 (HChrR6)-expressing 4T1 tumors. Fluorescence was imaged in live mice using IVIS Spectrum, and quantified by Living Image 3.2 software. MCHB and CNOB were quantified also by LC/MS/MS analysis. We used non-compartmental model to estimate PK parameters. Phoenix WinNonlin software was used for simulations to predict a more effective CNOB dosage regimen. RESULTS: CNOB administration significantly prolonged mice survival. MCHB fluorescence quantitatively reflected its exposure levels to the tumor and the plasma, as verified by LC/MS/MS analysis at various time points, including at a low concentration of 2 ng/g tumor. The LC/MS/MS data were used to estimate peak plasma concentrations, exposure (AUC0-24), volume of distribution, clearance and half-life in plasma and the tumor. Simulations suggested that the CNOB-GDEPT can be a successful therapy without large increases in the prodrug dosage. CONCLUSION: MCHB fluorescence quantifies this drug, and CNOB can be effective at relatively low doses. MCHB fluorescence characteristics will expedite further development of CNOB-GDEPT by, for example, facilitating specific gene delivery to the tumor, its prolonged expression, as well as other attributes necessary for successful gene-delivered enzyme prodrug therapy.

Biodistribution of the multidentate hydroxypyridinonate ligand [(14) C]-3,4,3-LI(1,2-HOPO), a potent actinide decorporation agent.

The pharmacokinetics and biodistribution of the (14) C-labeled actinide decorporation agent 3,4,3-LI(1,2-HOPO) were investigated in young adult Swiss Webster mice and Sprague Dawley rats, after intravenous, intraperitoneal, and oral dose administration. In all routes investigated, the radiolabeled compound was rapidly distributed to various tissues and organs of the body. In mice, the 24 h fecal elimination profiles suggested that the biliary route is the predominant route of elimination. In contrast, lower fecal excretion levels were observed in rats. Tissue uptake and retention of the compound did not differ significantly between sexes although some differences were observed in the excretion patterns over time. The male mice eliminated a greater percentage of (14) C through the renal pathway than the female mice after receiving an intravenous or intraperitoneal dose, while the opposite trend was seen in rats that received an intravenous dose. Metabolite profiling performed on selected rat samples demonstrated that a putative major metabolite of [(14) C]-3,4,3-LI(1,2-HOPO) is formed, accounting for approximately 10% of an administered oral dose. Finally, to improve its oral bioavailability, 3,4,3-LI(1,2-HOPO) was coformulated with a proprietary permeability enhancer, leading to a notable increase in oral bioavailability of the compound.

Elucidation of the time course of adenosine deaminase APOBEC3G and viral infectivity factor vif in HIV-2(287) -infected infant macaques.

BACKGROUND: Although the interactions of cellular cytidine deaminase A3G and viral infection factor (vif) of human immunodeficiency virus (HIV) were reported, regulation of A3G after in vivo HIV infection and disease progression is not known. METHODS: Time courses of plasma virus, CD4(+) T lymphocyte Macaca levels, and concentrations of A3G and vif transcripts were determined in infant macaques infected with HIV-2(287) . These in vivo results were compared with those collected in vitro in HIV-2-infected T cells. RESULTS: Human immunodeficiency virus-infected macaques exhibited plasma viremia (≥10(8) copies/ml) followed by a precipitous CD4(+) T-cell (from 40-70 to ≤5%) decline. An initial increase in A3G transcripts coincides with early increases in virus and vif RNA. As virus load continues to increase, A3G RNA decreases but recovers at a later phase as virus level stabilizes. Pearson correlation analysis revealed strong interactions of A3G-CD4, vif-CD4, and A3G-vif. CONCLUSIONS: There is a time-dependent A3G and vif RNA interaction throughout the course of HIV infection.

First in Human Evaluation and Dosimetry Calculations for Peptide 124I-p5+14-a Novel Radiotracer for the Detection of Systemic Amyloidosis Using PET/CT Imaging.

PURPOSE: Accurate diagnosis of amyloidosis remains a significant clinical challenge and unmet need for patients. The amyloid-reactive peptide p5+14 radiolabeled with iodine-124 has been developed for the detection of amyloid by PET/CT imaging. In a first-in-human evaluation, the dosimetry and tissue distribution of 124I-p5+14 peptide in patients with systemic amyloidosis. Herein, we report the dosimetry and dynamic distribution in the first three enrolled patients with light chain-associated (AL) amyloidosis. PROCEDURES: The radiotracer was assessed in a single-site, open-label phase 1 study (NCT03678259). The first three patients received a single intravenous infusion of 124I-p5+14 peptide (≤37 MBq). Serial PET/CT imaging was performed during the 48 h post-infusion. Dosimetry was determined as a primary endpoint for each patient and gender-averaged mean values were calculated. Pharmacokinetic parameters were estimated from whole blood radioactivity measurements and organ-based time activity data. Lastly, the biodistribution of radiotracer in major organs was assessed visually and compared to clinically appreciated organ involvement. RESULTS: Infusion of the 124I-p5+14 was well tolerated with rapid uptake in the heart, kidneys, liver, spleen, pancreas, and lung. The gender-averaged whole-body effective radiation dose was estimated to be 0.23 (± 0.02) mSv/MBq with elimination of the radioactivity via renal and gastrointestinal routes. The whole blood elimination t1/2 of 21.9 ± 7.6 h. Organ-based activity concentration measurements indicated that AUClast tissue:blood ratios generally correlated with the anticipated presence of amyloid. Peptide uptake was observed in 4/5 clinically suspected organs, as noted in the medical record, as well as six anatomic sites generally associated with amyloidosis in this population. CONCLUSION: Peptide 124I-p5+14 rapidly distributes to anatomic sites consistent with the presence of amyloid in patients with systemic AL. The dosimetry estimates established in this cohort are acceptable for whole-body PET/CT imaging. Pharmacokinetic parameters are heterogeneous and consistent with uptake of the tracer in an amyloid compartment. PET/CT imaging of 124I-p5+14 may facilitate non-invasive detection of amyloid in multiple organ systems.

Site-specific Substitutions Eliminate Aggregation Properties of Hemopressin.

Hemopressin is a naturally occurring and therapeutically relevant peptide with applications in hypertension, pain, addiction, and obesity. We had previously demonstrated that hemopressin converts into amyloid-like fibrils under aqueous conditions. However, the amino acid residues that modulate the aggregation propensity of hemopressin were not identified. In this study, we designed and synthesized 25 different analogs of hemopressin and analyzed their aggregation properties using the principle of dynamic light scattering. As a result, we were able to identify four conservative changes in the peptide sequence (Val(2) →DVal(2), Asn(3) →Gln(3) Leu(7) →Npg(7) and C-OH→C-NH2) that minimize aggregation propensity of hemopressin. The results indicate that hemopressin aggregation is cooperative in nature and involves contribution from multiple amino acids within the peptide chain. The analogs and the corresponding aggregation propensity data reported in this study would be useful for researchers investigating therapeutic properties of hemopressin, which have been hampered due to the tendency of hemopressin to aggregate in aqueous solutions.

Evaluation of Ebola Virus Inhibitors for Drug Repurposing.

A systematic screen of FDA-approved drugs was performed to identify compounds with in vitro antiviral activities against Ebola virus (EBOV). Compounds active (>50% viral inhibition and <30% cellular toxicity) at a single concentration were tested in dose-response assays to quantitate the antiviral activities in replication and viral entry assays as well as cytotoxicity in the Vero cell line used to conduct these assays. On the basis of the approved human dosing, toxicity/tolerability, and pharmacokinetic data, seven of these in vitro hits from different pharmacological classes (chloroquine (CQ), amiodarone, prochlorperazine, benztropine, azithromycin, chlortetracycline, and clomiphene) were evaluated for their in vivo efficacy at a single dose and were administered via either intraperitoneal (ip) or oral route. Initially, azithromycin (100 mg/kg, twice daily, ip), CQ (90 mg/kg, twice daily, ip), and amiodarone (60 mg/kg, twice daily, ip) demonstrated significant increases in survival in the mouse model. After repeat evaluation, only CQ was found to reproducibly give significant efficacy in the mouse model with this dosing regimen. Azithromycin and CQ were also tested in a guinea pig model of EBOV infection over a range of doses, but none of the doses increased survival, and drug-related toxicity was observed at lower doses than in the mouse. These results show the benefits and specific challenges associated with drug repurposing and highlight the need for careful evaluation of approved drugs as rapidly deployable countermeasures against future pandemics.

ß-Carotene Biosynthesis in Probiotic Bacteria.

Susceptibility to deadly diarrheal diseases is partly due to widespread pediatric vitamin A deficiency. To increase vitamin A coverage in malnourished children, we propose to engineer a probiotic bacterium that will produce ß-carotene in the intestine, which will be metabolized to vitamin A. Such a therapy has the potential to broadly stimulate mucosal immunity and simultaneously reduce the incidence and duration of diarrheal disease. To that end, a ß-carotene-producing variant of the probiotic Escherichia coli strain Nissle 1917 (EcN-BETA) was generated. Notably, the strain produces ß-carotene under anaerobic conditions, reflective of the gut environment. EcN-BETA also retains ß-carotene production capability after lyophilization, suggesting that it may be amenable to dry formulation. Moreover, EcN-BETA activates murine dendritic cells in vitro, suggesting that the presence of ß-carotene may not diminish the immunostimulatory capacity of EcN. Finally, we present a framework through which further improvements may enable approaches such as the one described in this report to yield innovative life-saving therapies for the developing world.

Design and characterization of novel peptide-coated lipid nanoparticles for targeting anti-HIV drug to CD4 expressing cells.

Human immunodeficiency virus (HIV) persists in lymph nodes and lymphoid tissues even during aggressive drug treatment, likely due to insufficient drug concentrations at this site. Therefore, to eliminate this residual virus, methods that enhance lymph node drug concentrations are currently being evaluated. Although enhanced drug concentrations in tissue have been achieved with drug-associated lipid nanoparticles, targeting these particles to CD4(+) cells may provide specific delivery of drug to HIV target cells and further enhance drug efficacy. We have evaluated four candidate peptides with reported binding specificity to CD4 for anchoring on lipid nanoparticle preparations previously shown to localize in lymph nodes. Terminal cysteine containing candidate peptides were conjugated to lipid nanoparticles through maleimide-linked phospholipids for targeting to CD4 cells. Using fluorescently labeled lipid nanoparticle binding to cells with varying degree of CD4 expression (CEMx174, Molt-4, Jurkat, and Ramos), we indentified two peptide sequences that provided CD4 selectivity to nanoparticles. These two peptide candidates on lipid nanoparticles bound to cells corresponding to the degree of CD4 expression and in a peptide dose dependent manner. Further, binding of these targeted lipid nanoparticles was CD4 specific, as pre-exposure of CD4(+) cells to anti-CD4 antibodies or free peptides inhibited the binding interactions. These results indicate targeting of lipid nanoparticles for specific binding to CD4 can be accomplished by tagging CD4 binding peptides with peptides, and these results provide a basis for further evaluation of this targeted delivery system to enhance antiviral drug delivery to CD4(+) HIV host cells, particularly those in lymph nodes and lymphoid tissues.

Enhanced anti-HIV efficacy of indinavir after inclusion in CD4-targeted lipid nanoparticles.

BACKGROUND: Combination drug therapy has reduced plasma HIV to undetectable levels; however, drug-sensitive virus persists in patients' lymphoid tissue. We have reported significant lymphoid tissue drug localization with indinavir-associated lipid nanoparticles (LNPs). Our current objective is to evaluate whether additional enhancement is achievable by targeting these particles to CD4-HIV host cells. METHODS: We characterized 2 peptide-coated (CD4-BP2 and CD4-BP4) drug-associated LNPs and demonstrated CD4-cell specificity. Drug-associated LNPs expressing polyethyleneglycol were exposed on HIV-2-infected cells under dynamic conditions that emulated lymph node physiology for 15, 30, and 60 minutes at concentrations from 0 to 25 µM and evaluated for antiviral activity and cell-associated drug concentrations. The specificity of CD4-mediated enhancement of indinavir LNPs antiviral activity was evaluated by blocking with anti-CD4 antibody. RESULTS: Inclusion of CD4-binding peptides on LNPs enhanced antiviral activity for all incubation conditions, compared with control particles or soluble drug (eg, 60 minutes exposure, EC50 = 0.12-0.13 vs. 0.46 µM for targeted nanoparticles vs. soluble drug). The CD4-BP4 peptide exhibited higher efficiency in eliciting antiviral activity than CD4-BP2-coated particles (EC50 = 7.5 µM vs. >25 µM at 15 minutes drug exposure). This enhancement seems to be driven by CD4 availability and cell-associated indinavir concentrations, as blocking of CD4 significantly ablated indinavir efficacy in targeted particles and indinavir concentrations reflected the observed anti-HIV activity. CONCLUSIONS: We constructed CD4-targeted LNPs that provide selective binding and efficient delivery of indinavir to CD4-HIV host cells. Inclusion of polyethyleneglycol in LNPs would minimize immune recognition of peptides. The enhancement of anti-HIV effects is effective even under limited time exposure.

Design considerations for liposomal vaccines: influence of formulation parameters on antibody and cell-mediated immune responses to liposome associated antigens.

Liposomes (phospholipid bilayer vesicles) are versatile and robust delivery systems for induction of antibody and T lymphocyte responses to associated subunit antigens. In the last 15 years, liposome vaccine technology has matured and now several vaccines containing liposome-based adjuvants have been approved for human use or have reached late stages of clinical evaluation. Given the intensifying interest in liposome-based vaccines, it is important to understand precisely how liposomes interact with the immune system and stimulate immunity. It has become clear that the physicochemical properties of liposomal vaccines - method of antigen attachment, lipid composition, bilayer fluidity, particle charge, and other properties - exert dramatic effects on the resulting immune response. Here, we present a comprehensive review of the physicochemical properties of liposomal vaccines and how they influence immune responses. A discussion of novel and emerging immunomodulators that are suitable for inclusion in liposomal vaccines is also presented. Through a comprehensive analysis of the body of liposomal vaccine literature, we enumerate a series of principles that can guide the rational design of liposomal vaccines to elicit immune responses of a desired magnitude and quality. We also identify major unanswered questions in the field, pointing the direction for future study.

Comparative analysis of the substrate preferences of two post-proline cleaving endopeptidases, prolyl oligopeptidase and fibroblast activation protein α.

Post-proline cleaving peptidases are promising therapeutic targets for neurodegenerative diseases, psychiatric conditions, metabolic disorders, and many cancers. Prolyl oligopeptidase (POP; E.C. 3.4.21.26) and fibroblast activation protein α (FAP; E.C. 3.4.24.B28) are two post-proline cleaving endopeptidases with very similar substrate specificities. Both enzymes are implicated in numerous human diseases, but their study is impeded by the lack of specific substrate probes. We interrogated a combinatorial library of proteolytic substrates and identified novel and selective substrates of POP and FAP. These new sequences will be useful as probes for fundamental biochemical study, scaffolds for inhibitor design, and triggers for controlled drug delivery.

Combining drug and immune therapy: a potential solution to drug resistance and challenges of HIV vaccines?

According to the World Health Organization's 2007 estimates, close to 33 million people worldwide are living with HIV/AIDS. Over the past two decades, significant progress has been made in understanding HIV pathogenesis and disease progression, which has allowed the identification of a multitude of drug and vaccine targets. Although currently available drug therapies have greatly increased the time from HIV infection to development of AIDS, drug resistance is an inevitable consequence that limits the duration of successful treatment. Consequently, a preventative vaccine remains the top priority; however, no vaccine trial performed to date has shown efficacy in human trials. Therefore, we must use all of our current resources in new creative therapies and strive to develop new methods to reduce persistent viral levels until an effective preventative vaccine is developed. One possible strategy is to use therapeutic vaccination or immune modulators to augment the immune response while antiretroviral chemotherapy limits viral replication. This combination approach is being utilized with success in the treatment of Hepatitis B infections and several trials have been completed and others are ongoing to determine the potential of combination immunological and chemical therapies for HIV infection. We will review the progress to date of anti-HIV drugs, preventative vaccines, and therapeutic vaccines and discuss the future strategies of combination drug and vaccine therapeutic strategies in the fight against HIV.

Recent developments in drug targets and delivery of anti-HIV drugs.

With almost 40 million people infected with human immunodeficiency virus (HIV), it is one of the most devastating diseases with no cure in sight. Over the past two decades, significant progress has been made to identify and validate drug targets for HIV. However, most of the 20 FDA approved drugs are targeted to two enzymes; reverse transcriptase and protease. Other drug targets derived from HIV and host factors are being validated, and novel compounds are being developed to overcome drug resistance. Recent data indicate that low and residual virus found in tissues of the lymphoid and central nervous system (CNS) is likely due to insufficient drug levels. Thus, improvement in the delivery of anti-HIV drugs to these tissues with limited drug penetration or accumulation, is equally important to maximally suppress viral replication. Novel lipid associated drugs (i.e. indinavir) targeted to the lymphoid tissue have been shown to overcome limited drug exposure in the lymph nodes and to further reduce residual virus in tissue. This review discusses viral and cellular targets that could interrupt viral replication, as well as novel and proven strategies to enhance the delivery of anti-HIV drugs to the lymphoid, CNS, and cells where low viral replication and limited drug levels exist.