Enhancement of sensitivity and quantification quality in the LC-MS/MS measurement of large biomolecules with sum of MRM (SMRM).

Liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) provides a simple and efficient means for the measurement of analytes in biological matrices with high selectivity and specificity. LC-MS/MS plays an important role in the pharmaceutical industry and biomedical research, but it requires analytes to be in an ionized form in order to be detected. This can pose a challenge for large molecules such as proteins and peptides, because they can exist in multiple charged forms, and this will reduce the total analyte signal by distributing it into multiple ion peaks with a different number of charges in a mass spectrum. In conventional LC-MS/MS analysis of such macromolecules, one charged form is selected as the precursor ion which is then fragmented by collision-induced dissociation (CID) in MS/MS to generate product ions, a process referred to as multiple-reaction monitoring (MRM). The MRM method minimizes interference from endogenous molecules within biological matrices that share the same molecular weight of the precursor ion, but at the expense of signal intensity as compared to precursor ion intensity. We describe here an approach to boost detection sensitivity and expand dynamic range in the quantitation of large molecules while maintaining analytical specificity using summation of MRM (SMRM) transitions and LC separation technique. Protein image from PDB-101 (PDB101.rscb.org).

Selecting an anti-malarial clinical candidate from two potent dihydroisoquinolones.

BACKGROUND: The ongoing global malaria eradication campaign requires development of potent, safe, and cost-effective drugs lacking cross-resistance with existing chemotherapies. One critical step in drug development is selecting a suitable clinical candidate from late leads. The process used to select the clinical candidate SJ733 from two potent dihydroisoquinolone (DHIQ) late leads, SJ733 and SJ311, based on their physicochemical, pharmacokinetic (PK), and toxicity profiles is described. METHODS: The compounds were tested to define their physicochemical properties including kinetic and thermodynamic solubility, partition coefficient, permeability, ionization constant, and binding to plasma proteins. Metabolic stability was assessed in both microsomes and hepatocytes derived from mice, rats, dogs, and humans. Cytochrome P450 inhibition was assessed using recombinant human cytochrome enzymes. The pharmacokinetic profiles of single intravenous or oral doses were investigated in mice, rats, and dogs. RESULTS: Although both compounds displayed similar physicochemical properties, SJ733 was more permeable but metabolically less stable than SJ311 in vitro. Single dose PK studies of SJ733 in mice, rats, and dogs demonstrated appreciable oral bioavailability (60-100%), whereas SJ311 had lower oral bioavailability (mice 23%, rats 40%) and higher renal clearance (10-30 fold higher than SJ733 in rats and dogs), suggesting less favorable exposure in humans. SJ311 also displayed a narrower range of dose-proportional exposure, with plasma exposure flattening at doses above 200 mg/kg. CONCLUSION: SJ733 was chosen as the candidate based on a more favorable dose proportionality of exposure and stronger expectation of the ability to justify a strong therapeutic index to regulators.

In Vitro and In Vivo Activity of Amiodarone Against Ebola Virus.

At the onset of the 2013-2016 epidemic of Ebola virus disease (EVD), no vaccine or antiviral medication was approved for treatment. Therefore, considerable efforts were directed towards the concept of drug repurposing or repositioning. Amiodarone, an approved multi-ion channel blocker for the treatment of cardiac arrhythmia, was reported to inhibit filovirus entry in vitro. Compassionate use of amiodarone in EVD patients indicated a possible survival benefit. In support of further clinical testing, we confirmed anti-Ebola virus activity of amiodarone in different cell types. Despite promising in vitro results, amiodarone failed to protect guinea pigs from a lethal dose of Ebola virus.

Efficacy of Tilorone Dihydrochloride against Ebola Virus Infection.

Tilorone dihydrochloride (tilorone) is a small-molecule, orally bioavailable drug that is used clinically as an antiviral outside the United States. A machine-learning model trained on anti-Ebola virus (EBOV) screening data previously identified tilorone as a potent in vitro EBOV inhibitor, making it a candidate for the treatment of Ebola virus disease (EVD). In the present study, a series of in vitro ADMET (absorption, distribution, metabolism, excretion, toxicity) assays demonstrated the drug has excellent solubility, high Caco-2 permeability, was not a P-glycoprotein substrate, and had no inhibitory activity against five human CYP450 enzymes (3A4, 2D6, 2C19, 2C9, and 1A2). Tilorone was shown to have 52% human plasma protein binding with excellent plasma stability and a mouse liver microsome half-life of 48 min. Dose range-finding studies in mice demonstrated a maximum tolerated single dose of 100 mg/kg of body weight. A pharmacokinetics study in mice at 2- and 10-mg/kg dose levels showed that the drug is rapidly absorbed, has dose-dependent increases in maximum concentration of unbound drug in plasma and areas under the concentration-time curve, and has a half-life of approximately 18 h in both males and females, although the exposure was ∼2.5-fold higher in male mice. Tilorone doses of 25 and 50 mg/kg proved efficacious in protecting 90% of mice from a lethal challenge with mouse-adapted with once-daily intraperitoneal (i.p.) dosing for 8 days. A subsequent study showed that 30 mg/kg/day of tilorone given i.p. starting 2 or 24 h postchallenge and continuing through day 7 postinfection was fully protective, indicating promising activity for the treatment of EVD.

(+)-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.

Evaluation of Cardiac Toxicity Biomarkers in Rats from Different Laboratories.

There is a great need for improved diagnostic and prognostic accuracy of potential cardiac toxicity in drug development. This study reports the evaluation of several commercially available biomarker kits by 3 institutions (SRI, Eli Lilly, and Pfizer) for the discrimination between myocardial degeneration/necrosis and cardiac hypertrophy as well as the assessment of the interlaboratory and interplatform variation in results. Serum concentrations of natriuretic peptides (N-terminal pro-atrial natriuretic peptide [NT-proANP] and N-terminal pro-brain natriuretic peptide [NT-proBNP]), cardiac and skeletal troponins (cTnI, cTnT, and sTnI), myosin light chain 3 (Myl3), and fatty acid binding protein 3 (FABP3) were assessed in rats treated with minoxidil (MNX) and isoproterenol (ISO). MNX caused increased heart-to-body weight ratios and prominent elevations in NT-proANP and NT-proBNP concentrations detected at 24-hr postdose without elevation in troponins, Myl3, or FABP3 and with no abnormal histopathological findings. ISO caused ventricular leukocyte infiltration, myocyte fibrosis, and necrosis with increased concentrations of the natriuretic peptides, cardiac troponins, and Myl3. These results reinforce the advantages of a multimarker strategy in elucidating the underlying cause of cardiac insult and detecting myocardial tissue damage at 24-hr posttreatment. The interlaboratory and interplatform comparison analyses also showed that the data obtained from different laboratories and platforms are highly correlated and reproducible, making these biomarkers widely applicable in preclinical studies.

Tenofovir disoproxil fumarate: toxicity, toxicokinetics, and toxicogenomics analysis after 13 weeks of oral administration in mice.

Tenofovir disoproxil fumarate (TDF) is a prodrug of tenofovir that exhibits activity against HIV and hepatitis B. The goals of this study were to evaluate the molecular mechanism of TDF-induced toxicity in mice after 13 weeks of daily oral administration (50-1000 mg/kg) by correlating transcriptional changes with plasma drug levels and traditional toxicology end points. Plasma levels and systemic exposure of tenofovir increased less than dose proportionally and were similar on days 1 and 91. No overt toxicity was observed following the completion of TDF administration. The kidneys of TDF-treated mice were histopathologically normal. This result is consistent with the genomic microarray results, which showed no significant differences in kidney transcriptional levels between TDF-treated animals and controls. In liver, after 4 and 13 weeks, cytomegaly was observed in mice treated with 1000 mg/kg of TDF, but mice recovered from this effect following cessation of administration. Analysis of liver transcripts on day 91 reported elevated levels of Cdkn1a in TDF-treated animals compared with controls, which may have contributed to the inhibition of liver cell cycle progression.

Preclinical studies on the pharmacokinetics, safety, and toxicology of oxfendazole: toward first in human studies.

A 2-week study in rats identified target organs of oxfendazole toxicity to be bone marrow, epididymis, liver, spleen, testis, and thymus. Female rats had greater oxfendazole exposure and exhibited toxicities at lower doses than did males. Decreased white blood cell levels, a class effect of benzimidazole anthelmintics, returned to normal during the recovery period. The no observed adverse effect level was determined to be >5 but <25 mg/kg/d and the maximum tolerated dose 100 mg/kg/d. The highest dose, 200 mg/kg/d, resulted in significant toxicity and mortality, leading to euthanization of the main study animals in this group after 7 days. Oxfendazole did not exhibit genetic toxicology signals in standard Ames bacterial, mouse lymphoma, or rat micronucleus assays nor did it provoke safety concerns when evaluated for behavioral effects in rats or cardiovascular safety effects in dogs. These results support the transition of oxfendazole to First in Human safety studies preliminary to its evaluation in human helminth diseases.

Quantitative proteomics for cardiac biomarker discovery using isoproterenol-treated nonhuman primates.

To identify new cardiac biomarkers, a quantitative proteomic analysis has been performed on serum and heart tissue proteins from three species of nonhuman primates following isoproterenol (ISO) treatment. Three serum proteins--serum amyloid A (SAA), α-1-acid glycoprotein (A1AG), and apolipoprotein A-1 (Apo A1)--were consistently identified as changed and remained altered 72 h post dose in all three species post ISO treatment, indicating the potential of including these proteins in preclinical or clinical evaluation of drug-induced cardiac injury. Furthermore, proteomic analysis of heart tissue proteins following ISO treatment demonstrated detrimental effects on calcium signaling and energy generation in cardiac myocytes. It is worth noting that cardiac troponins were not identified in serum but were identified as altered in heart tissue lysate along with other cardiac-specific proteins. This strategy for cardiac biomarker discovery by proteomic screening of heart tissue proteins, followed by verification in serum samples using immunoassays or targeted mass spectrometry, could be applied in future biomarker studies.

Evaluation of Antitrypanosomal Dihydroquinolines for Hepatotoxicity, Mutagenicity, and Methemoglobin Formation In Vitro.

N1-Benzylated dihydroquinolin-6-ols and their corresponding esters display exceptional activity against African trypanosomes in vitro, and administration of members of this class of compounds to trypanosome-infected mice results in cures in a first-stage African trypanosomiasis model. Since a quinone imine intermediate has been implicated in the antiparasitic mechanism of action of these compounds, evaluation of the hepatotoxic, mutagenic, and methemoglobin-promoting effects of these agents was performed. 1-Benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-ol hydrochloride and 1-benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-yl acetate showed outstanding in vitro selectivity for Trypanosoma brucei compared to the HepG2, Hep3B, Huh7, and PLC5 hepatocyte cell lines. 1-Benzyl-1,2-dihydro-2,2,4-trimethylquinolin-6-ol hydrochloride and 1-(2-methoxybenzyl)-1,2-dihydro-2,2,4-trimethylquinolin-6-yl acetate were not mutagenic when screened in the Ames assay, with or without metabolic activation. The latter 2 compounds promoted time- and dose-dependent formation of methemoglobin when incubated in whole human blood, but such levels were below those typically required to produce symptoms of methemoglobinemia in humans. Although compounds capable of quinone imine formation require careful evaluation, these in vitro studies indicate that antitrypanosomal dihydroquinolines merit further study as drug candidates against the neglected tropical disease human African trypanosomiasis.

Serum biomarkers reveal long-term cardiac injury in isoproterenol-treated African green monkeys.

The assessment of cardiac toxicity is a major challenge in both drug development and clinical trials, and numerous marketed pharmaceuticals have been removed from the market due to unpredicted cardiac effects. Serum troponins are widely used indicators of cardiac injury; however, they are short-lived and have not been validated in preclinical animal models. In this study, we have used filter-aided sample preparation (FASP) and tandem mass tag (TMT) labeling to investigate serum protein alterations in isoproterenol-treated African green monkeys. Our results showed that the combination of FASP and TMT labeling provided highly reproducible and efficient sample preparation, which enables us to identify and quantify serum proteins with high confidence. We focused on the proteins that exhibit long-term alteration upon isoproterenol injection and discovered nine proteins exhibiting significant changes at 48 and 72 h postdosing. We further chose three proteins, serum amyloid A (SAA), frutose biphosphate aldolase A (FBAA), and fetuin A, for validation using enzyme-linked immunosorbent assay (ELISA). The serum concentration of SAA showed a ∼ 50 fold increase, while concentration of FBAA and fetuin A exhibited a significant decrease accompanying isoproterenol-induced cardiotoxicity. This work provides valuable insights for multimarker evaluation of long-term cardiac injury.

Evaluation of arylimidamides DB1955 and DB1960 as candidates against visceral leishmaniasis and Chagas' disease: in vivo efficacy, acute toxicity, pharmacokinetics, and toxicology studies.

Arylimidamides (AIAs) have shown outstanding in vitro potency against intracellular kinetoplastid parasites, and the AIA 2,5-bis[2-(2-propoxy)-4-(2-pyridylimino)aminophenyl]furan dihydrochloride (DB766) displayed good in vivo efficacy in rodent models of visceral leishmaniasis (VL) and Chagas' disease. In an attempt to further increase the solubility and in vivo antikinetoplastid potential of DB766, the mesylate salt of this compound and that of the closely related AIA 2,5-bis[2-(2-cyclopentyloxy)-4-(2-pyridylimino)aminophenyl]furan hydrochloride (DB1852) were prepared. These two mesylate salts, designated DB1960 and DB1955, respectively, exhibited dose-dependent activity in the murine model of VL, with DB1960 inhibiting liver parasitemia by 51% at an oral dose of 100 mg/kg/day × 5 and DB1955 reducing liver parasitemia by 57% when given by the same dosing regimen. In a murine Trypanosoma cruzi infection model, DB1960 decreased the peak parasitemia levels that occurred at 8 days postinfection by 46% when given orally at 100 mg/kg/day × 5, while DB1955 had no effect on peak parasitemia levels when administered by the same dosing regimen. Distribution studies revealed that these compounds accumulated to micromolar levels in the liver, spleen, and kidneys but to a lesser extent in the heart, brain, and plasma. A 5-day repeat-dose toxicology study with DB1960 and DB1955 was also conducted with female BALB/c mice, with the compounds administered orally at 100, 200, and 500 mg/kg/day. In the high-dose groups, DB1960 caused changes in serum chemistry, with statistically significant increases in serum blood urea nitrogen, lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase levels, and a 21% decrease in body weight was observed in this group. These changes were consistent with microscopic findings in the livers and kidneys of the treated animals. The incidences of observed clinical signs (hunched posture, tachypnea, tremors, and ruffled fur) were more frequent in DB1960-treated groups than in those treated with DB1955. However, histopathological examination of tissue samples indicated that both compounds had adverse effects at all dose levels.

Metabolism, pharmacokinetics, tissue distribution, and stability studies of the prodrug analog of an anti-hepatitis B virus dinucleoside phosphorothioate.

The alkoxycarbonyloxy dinucleotide prodrug R(p), S(p)-2 is an orally bioavailable anti-hepatitis B virus agent. The compound is efficiently metabolized to the active dinucleoside phosphorothioate R(p), S(p)-1 by human liver microsomes and S9 fraction without cytochrome P450-mediated oxidation or conjugation. The conversion of R(p), S(p)-2 to R(p), S(p)-1 appears to be mediated by liver esterases, occurs in a stereospecific manner, and is consistent with our earlier reported studies of serum-mediated hydrolytic conversion of R(p), S(p)-2 to R(p), S(p)-1. However, further metabolism of R(p), S(p)-1 does not occur. The presence of a minor metabolite, the desulfurized product 10 was noted. The prodrug R(p), S(p)-2 was quite stable in simulated gastric fluid, whereas the active R(p), S(p)-1 had a half-life of <15 min. In simulated intestinal fluid, the prodrug 2 was fully converted to 1 in approximately 3 h, whereas 1 remained stable. To ascertain the tissue distribution of the prodrug 2 in rats, the synthesis of (35)S-labeled R(p), S(p)-2 was undertaken. Tissue distribution studies of orally and intravenously administered radiolabeled [(35)S]2 demonstrated that the radioactivity concentrates in the liver, with the highest liver/plasma ratio in the intravenous group at 1 h being 3.89 (females) and in the oral group at 1 h being 2.86 (males). The preferential distribution of the dinucleotide 1 and its prodrug 2 into liver may be attributed to the presence of nucleoside phosphorothioate backbone because phosphorothioate oligonucleotides also reveal a similar tissue distribution profile upon intravenous administration.

Scaleable manufacture of HIV-1 entry inhibitor griffithsin and validation of its safety and efficacy as a topical microbicide component.

To prevent sexually transmitted HIV, the most desirable active ingredients of microbicides are antiretrovirals (ARVs) that directly target viral entry and avert infection at mucosal surfaces. However, most promising ARV entry inhibitors are biologicals, which are costly to manufacture and deliver to resource-poor areas where effective microbicides are urgently needed. Here, we report a manufacturing breakthrough for griffithsin (GRFT), one of the most potent HIV entry inhibitors. This red algal protein was produced in multigram quantities after extraction from Nicotiana benthamiana plants transduced with a tobacco mosaic virus vector expressing GRFT. Plant-produced GRFT (GRFT-P) was shown as active against HIV at picomolar concentrations, directly virucidal via binding to HIV envelope glycoproteins, and capable of blocking cell-to-cell HIV transmission. GRFT-P has broad-spectrum activity against HIV clades A, B, and C, with utility as a microbicide component for HIV prevention in established epidemics in sub-Saharan Africa, South Asia, China, and the industrialized West. Cognizant of the imperative that microbicides not induce epithelial damage or inflammatory responses, we also show that GRFT-P is nonirritating and noninflammatory in human cervical explants and in vivo in the rabbit vaginal irritation model. Moreover, GRFT-P is potently active in preventing infection of cervical explants by HIV-1 and has no mitogenic activity on cultured human lymphocytes.

3D Spheroids of Human Primary Urine-Derived Stem Cells in the Assessment of Drug-Induced Mitochondrial Toxicity.

Mitochondrial toxicity (Mito-Tox) risk has increased due to the administration of several classes of drugs, particularly some life-long antiretroviral drugs for HIV+ individuals. However, no suitable in vitro assays are available to test long-term Mito-Tox (≥4 weeks). The goal of this study is to develop a 3D spheroid system of human primary urine-derived stem cells (USC) for the prediction of drug-induced delayed Mito-Tox. The cytotoxicity and Mito-Tox were assessed in 3D USC spheroids 4 weeks after treatment with antiretroviral drugs: zalcitabine (ddC; 0.1, 1 and 10 µM), tenofovir (TFV; 3, 30 and 300 µM) or Raltegravir (RAL; 2, 20 and 200 µM). Rotenone (RTNN, 10 µM) and 0.1% DMSO served as positive and negative controls. Despite only mild cytotoxicity, ddC significantly inhibited the expression of oxidative phosphorylation enzyme Complexes I, III, and IV; and RAL transiently reduced the level of Complex IV. A significant increase in caspase 3 and ROS/RNS level but a decrease in total ATP were observed in USC treated with ddC, TFV, RAL, and RTNN. Levels of mtDNA content and mitochondrial mass were decreased in ddC but minimally or not in TFV- and RAL-treated spheroids. Thus, 3D USC spheroid using antiretroviral drugs as a model offers an alternative platform to assess drug-induced late Mito-Tox.

Biodistribution, toxicology, and radiation dosimetry of 5-HT1A-receptor agonist positron emission tomography ligand [11C]CUMI-101.

Sprague Dawley rats (10/sex/group) were given a single intravenous (iv) dose of CUMI-101 to determine acute toxicity of CUMI-101 and radiation dosimetry estimations were conducted in baboons with [(11)C]CUMI-101. Intravenous administration of CUMI-101 did not produce overt biologically or toxicologically significant adverse effects except transient hypoactivity immediately after dose in the mid- and high-dose groups, which is not considered to be a dose-limiting toxic effect. No adverse effects were observed in the low-dose group. The no observed adverse effect level (NOAEL) is considered to be 44.05 µg/kg for a single iv dose administration in rats. The maximum tolerated dose (MTD) was estimated to be 881 µg/kg for a single iv dose administration. The Medical Internal Radiation Dose (MIRDOSE) estimates indicate the maximum permissible single-study dosage of [(11)C]CUMI-101 in humans is 52 mCi with testes and urinary bladder as the critical organ for males and females, respectively.

Formulation, Stability, Pharmacokinetic, and Modeling Studies for Tests of Synergistic Combinations of Orally Available Approved Drugs against Ebola Virus In Vivo.

Outbreaks of Ebola ebolavirus (EBOV) have been associated with high morbidity and mortality. Milestones have been reached recently in the management of EBOV disease (EVD) with licensure of an EBOV vaccine and two monoclonal antibody therapies. However, neither vaccines nor therapies are available for other disease-causing filoviruses. In preparation for such outbreaks, and for more facile and cost-effective management of EVD, we seek a cocktail containing orally available and room temperature stable drugs with strong activity against multiple filoviruses. We previously showed that (bepridil + sertraline) and (sertraline + toremifene) synergistically suppress EBOV in cell cultures. Here, we describe steps towards testing these combinations in a mouse model of EVD. We identified a vehicle suitable for oral delivery of the component drugs and determined that, thus formulated the drugs are equally active against EBOV as preparations in DMSO, and they maintain activity upon storage in solution for up to seven days. Pharmacokinetic (PK) studies indicated that the drugs in the oral delivery vehicle are well tolerated in mice at the highest doses tested. Collectively the data support advancement of these combinations to tests for synergy in a mouse model of EVD. Moreover, mathematical modeling based on human oral PK projects that the combinations would be more active in humans than their component single drugs.

Biodistribution, toxicity and radiation dosimetry studies of the serotonin transporter radioligand 4-[18F]-ADAM in rats and monkeys.

PURPOSE: 4-[(18)F]-ADAM is a potent serotonin transport imaging agent. We studied its toxicity in rats and radiation dosimetry in monkeys before human studies are undertaken. METHODS: Single and multiple-dosage toxicity studies were conducted in Sprague-Dawley rats. Male and female rats were injected intravenously with 4-F-ADAM as a single dose of 1,023.7 microg/kg (1,000 times the human dose) or as five consecutive daily doses of 102.37 microg/kg (100 times the human dose). PET/CT scans were performed in seven Formosa Rock monkeys (four males and three females) using a Siemens Biograph scanner. After injection of 4-[(18)F]-ADAM (182+/-8 MBq), a low dose CT scan and a series of eight whole-body PET scans were performed. Whole-body images were acquired in 3-D mode. Time-activity data of source organs were used to calculate the residence times and estimate the absorbed radiation dose using OLINDA/EXM software. RESULTS: In the rats neither the single dose nor the five daily doses of 4-F-ADAM produced overt adverse effects clinically. In the monkeys the radiation doses received by most organs ranged between 7.1 and 35.7 microGy/MBq, and the urinary bladder was considered to be the critical organ. The effective doses extrapolated to male and female adult humans were 17.4 and 21.8 microSv/MBq, respectively. CONCLUSION: Toxicity studies in Sprague-Dawley rats and radiation dosimetry studies in Formosa Rock monkeys suggested that 4-[(18)F]-ADAM is safe for use in human PET imaging studies.

Orally bioavailable anti-HBV dinucleotide acyloxyalkyl prodrugs.

The acyloxyalkyl derivatives of a model anti-HBV dinucleotide were synthesized and evaluated as orally bioavailable prodrugs. Our studies have led to the identification of the first orally bioavailable dinucleotide prodrugs for further therapeutic development against the hepatitis B virus (HBV).