The Urine Metabolome of R6/2 and zQ175DN Huntington's Disease Mouse Models.

Huntington's disease (HD) is caused by the expansion of a polyglutamine (polyQ)-encoding tract in exon 1 of the huntingtin gene to greater than 35 CAG repeats. It typically has a disease course lasting 15-20 years, and there are currently no disease-modifying therapies available. Thus, there is a need for faithful mouse models of HD to use in preclinical studies of disease mechanisms, target validation, and therapeutic compound testing. A large variety of mouse models of HD were generated, none of which fully recapitulate human disease, complicating the selection of appropriate models for preclinical studies. Here, we present the urinary liquid chromatography-high-resolution mass spectrometry analysis employed to identify metabolic alterations in transgenic R6/2 and zQ175DN knock-in mice. In R6/2 mice, the perturbation of the corticosterone metabolism and the accumulation of pyrraline, indicative of the development of insulin resistance and the impairment of pheromone excretion, were observed. Differently from R6/2, zQ175DN mice showed the accumulation of oxidative stress metabolites. Both genotypes showed alterations in the tryptophan metabolism. This approach aims to improve our understanding of the molecular mechanisms involved in HD neuropathology, facilitating the selection of appropriate mouse models for preclinical studies. It also aims to identify potential biomarkers specific to HD.

Fatty acid acylated peptide therapeutics: discovery of omega-n oxidation of the lipid chain as a novel metabolic pathway in preclinical species.

We recently described C18 fatty acid acylated peptides as a new class of potent long-lasting single-chain RXFP1 agonists that displayed relaxin-like activities in vivo. Early pharmacokinetics and toxicological studies of these stearic acid acylated peptides revealed a relevant oxidative metabolism occurring in dog and minipig, and also seen at a lower extent in monkey and rat. Mass spectrometry combined to NMR spectroscopy studies revealed that the oxidation occurred, unexpectedly, on the stearic acid chain at ω-1, ω-2 and ω-3 positions. Structure-metabolism relationship studies on acylated analogues with different fatty acids lengths (C15-C20) showed that the extent of oxidation was higher with longer chains. The oxidized metabolites could be generated in vitro using liver microsomes and engineered bacterial CYPs. These systems were correlating poorly with in vivo metabolism observed across species; however, the results suggest that this biotransformation pathway might be catalyzed by some unknown CYP enzymes.

Statin-based endothelial prophylaxis and outcome after allogeneic stem cell transplantation.

BACKGROUND: Allogeneic haematopoietic stem cell transplantation (alloSCT) often remains the only curative therapy for hematologic malignancies. Although the management of transplant-associated adverse events considerably improved over the last decades, nonrelapse mortality (NRM) remains a challenge, and endothelial dysfunction was identified as a major contributor to NRM. METHODS: Statin-based endothelial prophylaxis (SEP) has been implemented in the standard of care in our transplant centre to reduce NRM caused by endothelial injury. Here, we retrospectively analysed the impact of SEP on clinical outcome in a cohort of 347 alloSCT patients. RESULTS: SEP (n = 209) was associated with significantly reduced NRM (hazard ratio 0.61, 95% CI 0.38-0.96) and better overall survival (OS) after acute graft-versus-host disease (HR 0.59, 95% CI 0.37-0.93). Subgroup analyses showed that the NRM benefit was mainly found in patients with an intermediate endothelial activation and stress index (EASIX), while relapse risk was not affected. On day 100 post-alloSCT, patients receiving SEP had significantly higher levels of the rate-limiting enzyme of tryptophan metabolism, indoleamine 2,3-dioxygenase (IDO), higher kynurenine to tryptophan ratios as a proxy of IDO activity and tended to have lower levels of the endothelial injury marker ST2 (p = .055). No significant differences in interferon-gamma or IL18 levels were observed. These biomarker signatures suggest that the beneficial effects of SEP might be mediated by both endothelial protection and immunomodulation. CONCLUSIONS: Together, these data suggest that SEP improves NRM and OS post-alloSCT in particular in patients with intermediate endothelial risk and provide first mechanistic clues about its potential mode of action.

Design and Evaluation of [18F]CHDI-650 as a Positron Emission Tomography Ligand to Image Mutant Huntingtin Aggregates.

Therapeutic interventions are being developed for Huntington's disease (HD), a hallmark of which is mutant huntingtin protein (mHTT) aggregates. Following the advancement to human testing of two [11C]-PET ligands for aggregated mHTT, attributes for further optimization were identified. We replaced the pyridazinone ring of CHDI-180 with a pyrimidine ring and minimized off-target binding using brain homogenate derived from Alzheimer's disease patients. The major in vivo metabolic pathway via aldehyde oxidase was blocked with a 2-methyl group on the pyrimidine ring. A strategically placed ring-nitrogen on the benzoxazole core ensured high free fraction in the brain without introducing efflux. Replacing a methoxy pendant with a fluoro-ethoxy group and introducing deuterium atoms suppressed oxidative defluorination and accumulation of [18F]-signal in bones. The resulting PET ligand, CHDI-650, shows a rapid brain uptake and washout profile in non-human primates and is now being advanced to human testing.

Subcutaneous catabolism of peptide therapeutics: bioanalytical approaches and ADME considerations.

Many peptide drugs such as insulin and glucagon-like peptide (GLP-1) analogues are successfully administered subcutaneously (SC). Following SC injection, peptides may undergo catabolism in the SC compartment before entering systemic circulation, which could compromise their bioavailability and in turn affect their efficacy.This review will discuss how both technology and strategy have evolved over the past years to further elucidate peptide SC catabolism.Modern bioanalytical technologies (particularly liquid chromatography-high-resolution mass spectrometry) and bioinformatics platforms for data mining has prompted the development of in silico, in vitro and in vivo tools for characterising peptide SC catabolism to rapidly address proteolytic liabilities and, ultimately, guide the design of peptides with improved SC bioavailability.More predictive models able to recapitulate the interplay between SC catabolism and other factors driving SC absorption are highly desirable to improve in vitro/in vivo correlations.We envision the routine incorporation of in vitro and in vivo SC catabolism studies in ADME screening funnels to develop more effective peptide drugs for SC delivery.

Oligomerization, albumin binding and catabolism of therapeutic peptides in the subcutaneous compartment: An investigation on lipidated GLP-1 analogs.

Lipidation, a common strategy to improve half-life of therapeutic peptides, affects their tendency to oligomerize, their interaction with plasmatic proteins, and their catabolism. In this work, we have leveraged the use of NMR and SPR spectroscopy to elucidate oligomerization propensity and albumin interaction of different analogs of the two marketed lipidated GLP-1 agonists liraglutide and semaglutide. As most lipidated therapeutic peptides are administered by subcutaneous injection, we have also assessed in vitro their catabolism in the SC tissue using the LC-HRMS-based SCiMetPep assay. We observed that oligomerization had a shielding effect against catabolism. At the same time, binding to albumin may provide only limited protection from proteolysis due to the higher unbound peptide fraction present in the subcutaneous compartment with respect to the plasma. Finally, identification of catabolites in rat plasma after SC dosing of semaglutide showed a good correlation with the in vitro data, with Tyr19-Leu20 being the major cleavage site. Early characterization of the complex interplay between oligomerization, albumin binding, and catabolism at the injection site is essential for the synthesis of lipidated peptides with good pharmacokinetic profiles.

Kynurenine pathway activation and deviation to anthranilic and kynurenic acid in fibrosing chronic graft-versus-host disease.

Fibrosing chronic graft-versus-host disease (cGVHD) is a debilitating complication of allogeneic stem cell transplantation (alloSCT). A driver of fibrosis is the kynurenine (Kyn) pathway, and Kyn metabolism patterns and cytokines may influence cGVHD severity and manifestation (fibrosing versus gastrointestinal [GI] cGVHD). Using a liquid chromatography-tandem mass spectrometry approach on sera obtained from 425 patients with allografts, we identified high CXCL9, high indoleamine-2,3-dioxygenase (IDO) activity, and an activated Kyn pathway as common characteristics in all cGVHD subtypes. Specific Kyn metabolism patterns could be identified for non-severe cGVHD, severe GI cGVHD, and fibrosing cGVHD, respectively. Specifically, fibrosing cGVHD was associated with a distinct pathway shift toward anthranilic and kynurenic acid, correlating with reduced activity of the vitamin-B2-dependent kynurenine monooxygenase, low vitamin B6, and increased interleukin-18. The Kyn metabolite signature is a candidate biomarker for severe fibrosing cGVHD and provides a rationale for translational trials on prophylactic vitamin B2/B6 supplementation for cGVHD prevention.

[11C]CHDI-626, a PET Tracer Candidate for Imaging Mutant Huntingtin Aggregates with Reduced Binding to AD Pathological Proteins.

The expanded polyglutamine-containing mutant huntingtin (mHTT) protein is implicated in neuronal degeneration of medium spiny neurons in Huntington's disease (HD) for which multiple therapeutic approaches are currently being evaluated to eliminate or reduce mHTT. Development of effective and orthogonal biomarkers will ensure accurate assessment of the safety and efficacy of pharmacologic interventions. We have identified and optimized a class of ligands that bind to oligomerized/aggregated mHTT, which is a hallmark in the HD postmortem brain. These ligands are potentially useful imaging biomarkers for HD therapeutic development in both preclinical and clinical settings. We describe here the optimization of the benzo[4,5]imidazo[1,2-a]pyrimidine series that show selective binding to mHTT aggregates over Aß- and/or tau-aggregates associated with Alzheimer's disease pathology. Compound [11C]-2 was selected as a clinical candidate based on its high free fraction in the brain, specific binding in the HD mouse model, and rapid brain uptake/washout in nonhuman primate positron emission tomography imaging studies.

Personalized Metabolic Profile by Synergic Use of NMR and HRMS.

A new strategy that takes advantage of the synergism between NMR and UHPLC-HRMS yields accurate concentrations of a high number of compounds in biofluids to delineate a personalized metabolic profile (SYNHMET). Metabolite identification and quantification by this method result in a higher accuracy compared to the use of the two techniques separately, even in urine, one of the most challenging biofluids to characterize due to its complexity and variability. We quantified a total of 165 metabolites in the urine of healthy subjects, patients with chronic cystitis, and patients with bladder cancer, with a minimum number of missing values. This result was achieved without the use of analytical standards and calibration curves. A patient's personalized profile can be mapped out from the final dataset's concentrations by comparing them with known normal ranges. This detailed picture has potential applications in clinical practice to monitor a patient's health status and disease progression.

Turnover rate of coenzyme A in mouse brain and liver.

Coenzyme A (CoA) is a fundamental cofactor involved in a number of important biochemical reactions in the cell. Altered CoA metabolism results in severe conditions such as pantothenate kinase-associated neurodegeneration (PKAN) in which a reduction of the activity of pantothenate kinase isoform 2 (PANK2) present in CoA biosynthesis in the brain consequently lowers the level of CoA in this organ. In order to develop a new drug aimed at restoring the sufficient amount of CoA in the brain of PKAN patients, we looked at its turnover. We report here the results of two experiments that enabled us to measure the half-life of pantothenic acid, free CoA (CoASH) and acetylCoA in the brains and livers of male and female C57BL/6N mice, and total CoA in the brains of male mice. We administered (intrastriatally or orally) a single dose of a [13C3-15N-18O]-labelled coenzyme A precursor (fosmetpantotenate or [13C3-15N]-pantothenic acid) to the mice and measured, by liquid chromatography-mass spectrometry, unlabelled- and labelled-coenzyme A species appearance and disappearance over time. We found that the turnover of all metabolites was faster in the liver than in the brain in both genders with no evident gender difference observed. In the oral study, the CoASH half-life was: 69 ± 5 h (male) and 82 ± 6 h (female) in the liver; 136 ± 14 h (male) and 144 ± 12 h (female) in the brain. AcetylCoA half-life was 74 ± 9 h (male) and 71 ± 7 h (female) in the liver; 117 ± 13 h (male) and 158 ± 23 (female) in the brain. These results were in accordance with the corresponding values obtained after intrastriatal infusion of labelled-fosmetpantotenate (CoASH 124 ± 13 h, acetylCoA 117 ± 11 and total CoA 144 ± 17 in male brain).

Identification of Potent and Long-Acting Single-Chain Peptide Mimetics of Human Relaxin-2 for Cardiovascular Diseases.

The insulin-like peptide human relaxin-2 was identified as a hormone that, among other biological functions, mediates the hemodynamic changes occurring during pregnancy. Recombinant relaxin-2 (serelaxin) has shown beneficial effects in acute heart failure, but its full therapeutic potential has been hampered by its short half-life and the need for intravenous administration limiting its use to intensive care units. In this study, we report the development of long-acting potent single-chain relaxin peptide mimetics. Modifications in the B-chain of relaxin, such as the introduction of specific mutations and the trimming of the sequence to an optimal size, resulted in potent, structurally simplified peptide agonists of the relaxin receptor Relaxin Family Peptide Receptor 1 (RXFP1) (e.g., 54). Introduction of suitable spacers and fatty acids led to the identification of single-chain lipidated peptide agonists of RXFP1, with sub-nanomolar activity, high subcutaneous bioavailability, extended half-lives, and in vivo efficacy (e.g., 64).

Imaging Mutant Huntingtin Aggregates: Development of a Potential PET Ligand.

Mutant huntingtin (mHTT) protein carrying the elongated N-terminal polyglutamine (polyQ) tract misfolds and forms protein aggregates characteristic of Huntington's disease (HD) pathology. A high-affinity ligand specific for mHTT aggregates could serve as a positron emission tomography (PET) imaging biomarker for HD therapeutic development and disease progression. To identify such compounds with binding affinity for polyQ aggregates, we embarked on systematic structural activity studies; lead optimization of aggregate-binding affinity, unbound fractions in brain, permeability, and low efflux culminated in the discovery of compound 1, which exhibited target engagement in autoradiography (ARG) studies in brain slices from HD mouse models and postmortem human HD samples. PET imaging studies with 11C-labeled 1 in both HD mice and WT nonhuman primates (NHPs) demonstrated that the right-hand-side labeled ligand [11C]-1R (CHDI-180R) is a suitable PET tracer for imaging of mHTT aggregates. [11C]-1R is now being advanced to human trials as a first-in-class HD PET radiotracer.

Comparison of different protein precipitation and solid-phase extraction protocols for the study of the catabolism of peptide drugs by LC-HRMS.

LC-HRMS-based identification of the products of peptide catabolism is the key to drive the design of more stable compounds. Because the catabolite of a given peptide can be very different from the parent compound and from other catabolites in terms of physicochemical properties, it can be challenging to develop an analytical method that allows recovery and detection of the parent and all parent-related catabolites. The aim of this study was to investigate how the recovery and the matrix effect of peptidic drugs and their catabolites are affected by different protein precipitation (PP) and solid-phase extraction (SPE) protocols. To this purpose, four model peptides representative of different classes (somatostatin, GLP-2, human insulin and liraglutide) were digested with trypsin and chymotrypsin to simulate proteolytic catabolism. The resulting mixtures of the parent peptides and their proteolytic products covering a wide range of relative hydrophobicity (HR ) and isoelectric points (pI) were spiked in human plasma and underwent different PP and SPE protocols. Recovery and matrix effect were measured for each peptide and its catabolites. PP with three volumes of ACN or EtOH yielded the highest overall recoveries (more than 50% for the four parent peptides and all their catabolites) among all the tested PP and SPE protocols. Mixed-mode anion exchange (MAX) was the only SPE sorbent among the five tested that allowed to extract all the peptides with recoveries more than 20%. Matrix effect was generally lower with SPE. Overall, it was observed that peptides with either high hydrophilicity (e.g., somatostatin catabolites) or hydrophobicity (GLP-2 and lipidated liraglutide catabolites) had a much narrower choice of PP solvent or SPE protocol. Simulation of catabolism using recombinant enzymes together with in silico calculation of the HR and the pI of potential proteolysis products is recommended to select the optimal extraction conditions for the study of peptide catabolism.

Application of an in Vitro Blood-Brain Barrier Model in the Selection of Experimental Drug Candidates for the Treatment of Huntington's Disease.

Huntington's disease (HD) is a neurodegenerative disease caused by polyglutamine expansion in the huntingtin protein. For drug candidates targeting HD, the ability to cross the blood-brain barrier (BBB) and reach the site of action in the central nervous system (CNS) is crucial for achieving pharmacological activity. To assess the permeability of selected compounds across the BBB, we utilized a two-dimensional model composed of primary porcine brain endothelial cells and rat astrocytes. Our objective was to use this in vitro model to rank and prioritize compounds for in vivo pharmacokinetic and brain penetration studies. The model was first characterized using a set of validation markers chosen based on their functional importance at the BBB. It was shown to fulfill the major BBB characteristics, including functional tight junctions, high transendothelial electrical resistance, expression, and activity of influx and efflux transporters. The in vitro permeability of 54 structurally diverse known compounds was determined and shown to have a good correlation with the in situ brain perfusion data in rodents. We used this model to investigate the BBB permeability of a series of new HD compounds from different chemical classes, and we found a good correlation with in vivo brain permeation, demonstrating the usefulness of the in vitro model for optimizing CNS drug properties and for guiding the selection of lead compounds in a drug discovery setting.

A liquid chromatography high-resolution mass spectrometry in vitro assay to assess metabolism at the injection site of subcutaneously administered therapeutic peptides.

Subcutaneous (SC) injection is the most common administration route for peptide therapeutics. Catabolism at the injection site can be a specific and major degradation pathway for many SC administered peptides. In some cases, it can significantly affect pharmacokinetics, particularly bioavailability, and have detrimental effects on the efficacy of the drug. This work describes a liquid chromatography-high resolution mass spectrometry based in vitro assay to assess peptide metabolism in the SC tissue (SCiMetPep assay). The SCiMetPep assay was developed using human, Sprague-Dawley rat and Göttingen minipig SC tissue homogenate supernatant, and allows for both determination of degradation rate (half-life) of the parent peptide and identification of metabolites generated from enzymatic proteolysis. The assay was developed and validated using known peptides including human insulin and four GLP-1 analogues (lixisenatide, exenatide, liraglutide and semaglutide). Different experimental parameters were evaluated in order to optimize the homogenization process of the SC tissue and the peptide incubation conditions. In vitro metabolism of these peptides was in good agreement with in vivo data reported in the literature. Finally, when SCiMetPep assay was applied on a series of structurally related peptides, a fairly good correlation was found between SC metabolic stability and bioavailability, suggesting that catabolism at the injection site can have a major role in the absorption, distribution, metabolism, and excretion (ADME) of peptide therapeutics. The SCiMetPep showed the ability to identify analogs with improved SC metabolic stability and hence higher bioavailability. The assay can be used in the early phases of drug discovery to identify peptide metabolic soft spots at the injection site and guide the peptide drug discovery process.

Determination of acetyl coenzyme A in human whole blood by ultra-performance liquid chromatography-mass spectrometry.

Acetyl coenzyme A is involved in several key metabolic pathways. Its concentration can vary considerably in response to physiological or pathological conditions making it a potentially valuable biomarker. However, little information about the measurement and concentration of acetyl CoA in human whole blood is found in the literature. The aim of this study was the development of an accurate method for the determination of acetyl CoA in human whole blood by LC-MS/MS. The method, involving extraction from whole blood by a rapid protein precipitation procedure was thoroughly validated: limit of quantitation was 3.91 ng mL-1. Accuracy and precision were calculated at five concentrations and were within ±15%. The average endogenous level of acetyl CoA in human whole blood was determined in 17 healthy individuals to be 220.9 ng mL-1 (ranging from 124.0 to 308.0 ng mL-1). This represents, to our knowledge, the first report of acetyl CoA levels in human whole blood, and the first practical and reliable method for its determination.

An efficient liquid chromatography-high resolution mass spectrometry approach for the optimization of the metabolic stability of therapeutic peptides.

In drug discovery, there is increasing interest in peptides as therapeutic agents due to several appealing characteristics that are typical of this class of compounds, including high target affinity, excellent selectivity, and low toxicity. However, peptides usually present also some challenging ADME (absorption, distribution, metabolism, and excretion) issues such as limited metabolic stability, poor oral bioavailability, and short half-lives. In this context, early preclinical in vitro studies such as plasma metabolic stability assays are crucial to improve developability of a peptidic drug. In order to speed up the optimization of peptide metabolic stability, a strategy was developed for the integrated semi-quantitative determination of metabolic stability of peptides and qualitative identification/structural elucidation of their metabolites in preclinical plasma metabolic stability studies using liquid chromatography-high-resolution Orbitrap™ mass spectrometry (LC-HRMS). Sample preparation was based on protein precipitation: experimental conditions were optimized after evaluating and comparing different organic solvents in order to obtain an adequate extraction of the parent peptides and their metabolites and to minimize matrix effect. Peptides and their metabolites were analyzed by reverse-phase liquid chromatography: a template gradient (total run time, 6 min) was created to allow retention and good peak shape for peptides of different polarity and isoelectric points. Three LC columns were selected to be systematically evaluated for each series of peptides. Targeted and untargeted HRMS data were simultaneously acquired in positive full scan + data-dependent MS/MS acquisition mode, and then processed to calculate plasma half-life and to identify the major cleavage sites, this latter by using the software Biopharma Finder™. Finally, as an example of the application of this workflow, a study that shows the plasma stability improvement of a series of antimicrobial peptides is described. This approach was developed for the evaluation of in vitro plasma metabolic stability studies of peptides, but it could also be applied to other in vitro metabolic stability models (e.g., whole blood, hepatocytes). Graphical Abstract Left: trend plot for omiganan and major metabolites. Right: stability plot for five antimicrobial peptidesafter incubation with mouse plasma.

Use of 'dilute-and-shoot' liquid chromatography-high resolution mass spectrometry in preclinical research: application to a DMPK study of perhexiline in mouse plasma.

This work describes a simple, sensitive and rapid liquid chromatography-high resolution mass spectrometry method for the quantitation of perhexiline and the simultaneous detection of perhexiline metabolites in C57bl/6 mice plasma. Only 5 µL of plasma was used for analysis. Pretreatment was limited to a 100-fold dilution ('dilute-and-shoot'). The analyte was detected by high resolution mass spectrometry (Orbitrap™ technology). Three scan events were performed over the entire chromatogram. Targeted single ion monitoring with data dependent acquisition was employed for perhexiline quantitation and confirmation, while full scan was used to perform untargeted detection of perhexiline phase I and phase II circulating metabolites. The calibration curve was linear (r(2)=0.990) ranging from 0.305 ng/mL (LLOQ) to 10000 ng/mL. Matrix effect was limited to 6.1%. The method was applied to a pharmacokinetic study of perhexiline in mouse plasma and the results obtained were compared to a standard sample preparation method based on protein precipitation and liquid chromatography-tandem mass spectrometry (MRM mode) detection. The new approach provided comparable results in terms of pharmacokinetics parameters estimate with a high sensitivity, additional information on perhexiline circulating metabolites and a low consumption of biological sample. The combination of the 'dilute-and-shoot' approach together with HRMS targeted and untargeted detection represents a suitable alternative to classic bioanalytical approaches in preclinical research.

A single-run liquid chromatography mass spectrometry method to quantify neuroactive kynurenine pathway metabolites in rat plasma.

Neuroactive metabolites in the kynurenine pathway of tryptophan catabolism are associated with neurodegenerative disorders. Tryptophan is transported across the blood-brain barrier and converted via the kynurenine pathway to N-formyl-L-kynurenine, which is further degraded to L-kynurenine. This metabolite can then generate a group of metabolites called kynurenines, most of which have neuroactive properties. The association of tryptophan catabolic pathway alterations with various central nervous system (CNS) pathologies has raised interest in analytical methods to accurately quantify kynurenines in body fluids. We here describe a rapid and sensitive reverse-phase HPLC-MS/MS method to quantify L-kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxy-L-kynurenine (3HK) and anthranilic acid (AA) in rat plasma. Our goal was to quantify these metabolites in a single run; given their different physico-chemical properties, major efforts were devoted to develop a chromatography suitable for all metabolites that involves plasma protein precipitation with acetonitrile followed by chromatographic separation by C18 RP chromatography, detected by electrospray mass spectrometry. Quantitation range was 0.098-100 ng/ml for 3HK, 9.8-20,000 ng/ml for KYN, 0.49-1000 ng/ml for KYNA and AA. The method was linear (r>0.9963) and validation parameters were within acceptance range (calibration standards and QC accuracy within ±30%).

Generation and selection of novel fully human monoclonal antibodies that neutralize Dickkopf-1 (DKK1) inhibitory function in vitro and increase bone mass in vivo.

Wnt/LRP5 signaling is a central regulatory component of bone formative and resorptive activities, and the pathway inhibitor DKK1 is a suppressor of bone formation and bone mass accrual in mice. In addition, augmented DKK1 levels are associated with high bone turnover in diverse low bone mass states in rodent models and disease etiologies in human. However, examination of the precise role of DKK1 in the normal skeleton and in higher species requires the development of refined DKK1-specific pharmacological tools. Here, we report the strategy resulting in isolation of a panel of fully human anti-DKK1 antibodies applicable to studies interrogating the roles of mouse, rhesus, and human DKK1. Selected anti-DKK1 antibodies bind primate and human DKK-1 with picomolar affinities yet do not appreciably bind to DKK2 or DKK4. Epitopes mapped within the DKK1 C-terminal domain necessary for interaction with LRP5/6 and consequently effectively neutralized DKK1 function in vitro. When introduced into naïve normal growing female mice, IgGs significantly improved trabecular bone volume and structure and increased both trabecular and cortical bone mineral densities in a dose-related fashion. Furthermore, fully human DKK1-IgG displayed favorable pharmacokinetic parameters in non-human primates. In summary, we demonstrate here a rate-limiting function of physiologic DKK1 levels in the regulation of bone mass in intact female mice, amendable to specific pharmacologic neutralization by newly identified DKK1-IgGs. Importantly the fully human IgGs display a profile of attributes that recommends their testing in higher species and their use in evaluating DKK1 function in relevant disease models.