Pharmacodynamic measures within tumors expose differential activity of PD(L)-1 antibody therapeutics.

Macromolecules such as monoclonal antibodies (mAbs) are likely to experience poor tumor penetration because of their large size, and thus low drug exposure of target cells within a tumor could contribute to suboptimal responses. Given the challenge of inadequate quantitative tools to assess mAb activity within tumors, we hypothesized that measurement of accessible target levels in tumors could elucidate the pharmacologic activity of a mAb and could be used to compare the activity of different mAbs. Using positron emission tomography (PET), we measured the pharmacodynamics of immune checkpoint protein programmed-death ligand 1 (PD-L1) to evaluate pharmacologic effects of mAbs targeting PD-L1 and its receptor programmed cell death protein 1 (PD-1). For PD-L1 quantification, we first developed a small peptide-based fluorine-18-labeled PET imaging agent, [18F]DK222, which provided high-contrast images in preclinical models. We then quantified accessible PD-L1 levels in the tumor bed during treatment with anti-PD-1 and anti-PD-L1 mAbs. Applying mixed-effects models to these data, we found subtle differences in the pharmacodynamic effects of two anti-PD-1 mAbs (nivolumab and pembrolizumab). In contrast, we observed starkly divergent target engagement with anti-PD-L1 mAbs (atezolizumab, avelumab, and durvalumab) that were administered at equivalent doses, correlating with differential effects on tumor growth. Thus, we show that measuring PD-L1 pharmacodynamics informs mechanistic understanding of therapeutic mAbs targeting PD-L1 and PD-1. These findings demonstrate the value of quantifying target pharmacodynamics to elucidate the pharmacologic activity of mAbs, independent of mAb biophysical properties and inclusive of all physiological variables, which are highly heterogeneous within and across tumors and patients.

The amyloid-ß1-42-oligomer interacting peptide D-AIP possesses favorable biostability, pharmacokinetics, and brain region distribution.

We have previously developed a unique 8-amino acid Aß42 oligomer-Interacting Peptide (AIP) as a novel anti-amyloid strategy for the treatment of Alzheimer's disease. Our lead candidate has successfully progressed from test tubes (i.e., in vitro characterization of protease-resistant D-AIP) to transgenic flies (i.e., in vivo rescue of human Aß42-mediated toxicity via D-AIP-supplemented food). In the present study, we examined D-AIP in terms of its stability in multiple biological matrices (i.e., ex-vivo mouse plasma, whole blood, and liver S9 fractions) using MALDI mass spectrometry, pharmacokinetics using a rapid and sensitive LC-MS method, and blood brain barrier (BBB) penetrance in WT C57LB/6 mice. D-AIP was found to be relatively stable over 3 h at 37 °C in all matrices tested. Finally, label-free MALDI imaging showed that orally administered D-AIP can readily penetrate the intact BBB in both male and female WT mice. Based upon the favorable stability, pharmacokinetics, and BBB penetration outcomes for orally administered D-AIP in WT mice, we then examined the effect of D-AIP on amyloid "seeding" in vitro (i.e., freshly monomerized versus preaggregated Aß42). Complementary biophysical assays (ThT, TEM, and MALDI-TOF MS) showed that D-AIP can directly interact with synthetic Aß42 aggregates to disrupt primary and/or secondary seeding events. Taken together, the unique mechanistic and desired therapeutic potential of our lead D-AIP candidate warrants further investigation, that is, testing of D-AIP efficacy on the altered amyloid/tau pathology in transgenic mouse models of Alzheimer's disease.

Evaluation of the Pharmacokinetics of the Pancreastatin Inhibitor PSTi8 Peptide in Rats: Integration of In Vitro and In Vivo Findings.

PSTi8 is a pancreastatin inhibitory peptide that is effective in the treatment of diabetic models. This study investigates the pharmacokinetic (PK) properties of PSTi8 in Sprague Dawley rats, for the first time. In vitro and in vivo PK studies were performed to evaluate the solubility, stability in plasma and liver microsomes, plasma protein binding, blood-plasma partitioning, bioavailability, dose proportionality, and gender difference in PK. Samples were analyzed using the validated LC-MS/MS method. The solubility of PSTi8 was found to be 9.30 and 25.75 mg/mL in simulated gastric and intestinal fluids, respectively. The protein binding of PSTi8 was estimated as >69% in rat plasma. PSTi8 showed high stability in rat plasma and liver microsomes and the blood-plasma partitioning was >2. The bioavailability of PSTi8 after intraperitoneal and subcutaneous administration was found to be 95.00 ± 12.15 and 78.47 ± 17.72%, respectively, in rats. PSTi8 showed non-linear PK in dose proportionality studies, and has no gender difference in the PK behavior in rats. The high bioavailability of PSTi8 can be due to high water solubility and plasma protein binding, low clearance and volume of distribution. Our in vitro and in vivo findings support the development of PSTi8 as an antidiabetic agent.

Higher Order Protein Catenation Leads to an Artificial Antibody with Enhanced Affinity and In Vivo Stability.

The chemical topology is a unique dimension for protein engineering, yet the topological diversity and architectural complexity of proteins remain largely untapped. Herein, we report the biosynthesis of complex topological proteins using a rationally engineered, cross-entwining peptide heterodimer motif derived from p53dim (an entangled homodimeric mutant of the tetramerization domain of the tumor suppressor protein p53). The incorporation of an electrostatic interaction at specific sites converts the p53dim homodimer motif into a pair of heterodimer motifs with high specificity for directing chain entanglement upon folding. Its combination with split-intein-mediated ligation and/or SpyTag/SpyCatcher chemistry facilitates the programmed synthesis of protein heterocatenane or [n]catenanes in cells, leading to a general and modular approach to complex protein catenanes containing various proteins of interest. Concatenation enhances not only the target protein's affinity but also the in vivo stability as shown by its prolonged circulation time in blood. As a proof of concept, artificial antibodies have been developed by embedding a human epidermal growth factor receptor 2-specific affibody onto the [n]catenane scaffolds and shown to exhibit a higher affinity and a better pharmacokinetic profile than the wild-type affibody. These results suggest that topology engineering holds great promise in the development of therapeutic proteins.

Polyethylene Glycol 40-Modified Peptide with High Therapeutic Efficacy in Simian-Human Immunodeficiency Virus-Acutely Infected Rhesus Monkeys.

Anti-human immunodeficiency virus type 1 (anti-HIV-1) fusion peptides have been studied for nearly 2 decades, but few candidates have found useful clinical applications. One factor underlying the failure of such agents to reach the clinic is their poor pharmacokinetic properties, and many efforts have been made to overcome this problem. In this study, we modified C34, a peptide inhibitor of HIV-1 fusion, at its conserved glycosylation site using polyethylene glycols (PEGs) of different molecular weights. PEG40-NC, a conjugate of C34 and branched PEG 40 kDa (PEG40), which has been previously shown to improve the pharmacokinetic profiles of proteins, showed a significantly extended half-life (t1/2; 10.39 h in rats), which compensated for decreased in vitro activity (50% effective concentration [EC50] of 18.51 nM). PEG40-NC also showed a mechanism of action similar to that of C34. PEG40-NC monotherapy in acutely simian-human immunodeficiency virus (SHIV)-infected rhesus monkeys significantly suppressed viral load compared with a control treatment. Efficacy was linked to the extended half-life and lymphatic exposure conferred by attached PEG40. These results highlight the potential of further clinical investigations of PEG40-NC in combination with antiretroviral therapy or other anti-HIV agents.IMPORTANCE Poor pharmacokinetics have severely hindered the clinical use of anti-HIV peptides. Different small molecules, such as lipid, cholesterol, and small PEG, were designed to modify peptides to improve their pharmacokinetics. In this study, we incorporated large branched PEG to anti-HIV peptide and obtained a conjugate with extended half-life and improved in vivo efficacy. The strategy we developed in this study can also be applicable for the development of other peptide candidates.

Delivery of doxorubicin loaded P18 conjugated-poly(2-ethyl-oxazoline)-DOPE nanoliposomes for targeted therapy of breast cancer.

Breast cancer, a heterogeneous disease, has the highest incidence rate and is a major cause of death in females worldwide. Drug delivery by using nanotechnology has shown great promise for improving cancer treatment. Nanoliposomes are known to have enhanced accumulation ability in tumors due to prolonged systemic circulation. Peptide 18 (P18), a tumor homing peptide targeting keratin-1 (KRT-1), was previously shown to have high binding affinity towards breast cancer cells. In this study, we investigate the ability of P18 conjugated PEtOx-DOPE nanoliposomes (P18-PEtOx-DOPE) for the targeted delivery of doxorubicin to AU565 breast cancer model. Toxicology studies of PEtOx-DOPE nanoliposomes performed on normal breast epithelial cells (MCF10A), showed minimal toxicity. Doxorubicin delivery by P18-PEtOx-DOPE to AU565 cells induces cytotoxicity in a dose and time dependent manner causing mitotic arrest in G2/M phase at 24 h. Anti-cancer activity of P18-PEtOx-DOPE-DOX nanoliposomes on AU565 cells was detected by Annexin V/PI apoptosis assay. In terms of in vivo antitumor efficacy, P18-PEtOx-DOPE-DOX nanoliposomes administration to AU565 CD-1 nu/nu mice model showed significant decrease in tumor volume suggesting that DOX delivered by these nanoliposomes elicited a strong antitumor response comparable to the free delivery of doxorubicin. Overall, our results offered preclinical proof for the use of P18-PEtOx-DOPE-DOX nanoliposomes in KRT-1+ breast cancer therapy.

Improved pharmacokinetics and bone tissue accumulation of Angiotensin-(1-7) peptide through bisphosphonate conjugation.

The renin-angiotensin system (RAS) has a central role in renal and cardiovascular homeostasis. Angiotensin-(1-7) (Ang1-7), one of the RAS active peptides, exerts beneficial effects through different mechanisms. These biological actions suggest that Ang1-7 is an effective therapeutic agent for treating various diseases associated with activated RAS. However, its short half-life and poor pharmacokinetics restrict its therapeutic utility. Our laboratory has successfully synthesized and characterized an Ang1-7 conjugate (Ang Conj.) with a prolonged half-life and improved pharmacokinetics profile. The Ang Conj. has been prepared by PEGylation of Ang1-7 and conjugation with a bisphosphonate using solid-phase peptide synthesis and characterized by HPLC and mass spectrometer. The compound's stability has been tested in different storage conditions. The bone binding capacity was evaluated using a hydroxyapatite assay. Pharmacokinetic and tissue distribution studies were performed using iodinated peptides in rats. Ang Conj. was synthesized with > 90% purity. Bone mineral affinity testing showed Ang Conj. exhibited significantly higher bone mineral affinity than Ang1-7. The Ang Conj. remained stable for more than a month using all tested storage conditions. The Ang Conj. demonstrated higher affinity to bone, a longer half-life, and better bioavailability when compared with the native peptide. These results support that conjugation of Ang1-7 with bisphosphonate enables it to utilize bone as a reservoir for the sustained delivery of Ang1-7 to maintain therapeutic plasma levels. High chemical stability and about five to tenfold prolongation of Ang Conj. plasma half-life after administrations into rats proves the effectiveness of our approach.

Assembling BH3-mimic peptide into a nanocluster to target intracellular Bcl2 towards the apoptosis induction of cancer cell.

Bcl-2, an anti-apoptotic protein, is always overexpressed in tumor cells to suppress the pro-apoptotic function of Bax, thereby prolonging the life of the tumor. However, BH3 proteins could directly activate Bax via antagonizing Bcl-2 to induce apoptosis in response to the stimulation. Thus, mimicking BH3 proteins with a peptide is a potential strategy for anti-cancer therapy. Unfortunately, clinical translation of BH3-mimic peptide is hindered by its inefficacious cellular internalization and proteolysis resistance. Herein, we translated a BH3-mimic peptide into a peptide-auric spheroidal nanocluster (BH3-AuNp), in which polymeric BH3-Auric precursors [Au1+-S-BH3]narein situself-assembled on the surface of gold nanoparticles by a one-pot synthesis. Expectedly, this strategy could improve the anti-proteolytic ability and cytomembrane penetrability of the BH3 peptide. As a result, BH3-AuNp successfully induced the apoptosis of two cancer cell lines by an order of magnitude compared to BH3. This therapeutic and feasible peptide nano-engineering strategy will help peptides overcome the pharmaceutical obstacles, awaken its biological functions, and possibly revive the research about peptide-derived nanomedicine.

Novel brain-targeted nanomicelles for anti-glioma therapy mediated by the ApoE-enriched protein corona in vivo.

BACKGROUND: The interactions between nanoparticles (NPs) and plasma proteins form a protein corona around NPs after entering the biological environment, which provides new biological properties to NPs and mediates their interactions with cells and biological barriers. Given the inevitable interactions, we regard nanoparticle‒protein interactions as a tool for designing protein corona-mediated drug delivery systems. Herein, we demonstrate the successful application of protein corona-mediated brain-targeted nanomicelles in the treatment of glioma, loading them with paclitaxel (PTX), and decorating them with amyloid ß-protein (Aß)-CN peptide (PTX/Aß-CN-PMs). Aß-CN peptide, like the Aß1-42 peptide, specifically binds to the lipid-binding domain of apolipoprotein E (ApoE) in vivo to form the ApoE-enriched protein corona surrounding Aß-CN-PMs (ApoE/PTX/Aß-CN-PMs). The receptor-binding domain of the ApoE then combines with low-density lipoprotein receptor (LDLr) and LDLr-related protein 1 receptor (LRP1r) expressed in the blood-brain barrier and glioma, effectively mediating brain-targeted delivery. METHODS: PTX/Aß-CN-PMs were prepared using a film hydration method with sonication, which was simple and feasible. The specific formation of the ApoE-enriched protein corona around nanoparticles was characterized by Western blotting analysis and LC-MS/MS. The in vitro physicochemical properties and in vivo anti-glioma effects of PTX/Aß-CN-PMs were also well studied. RESULTS: The average size and zeta potential of PTX/Aß-CN-PMs and ApoE/PTX/Aß-CN-PMs were 103.1 nm, 172.3 nm, 7.23 mV, and 0.715 mV, respectively. PTX was efficiently loaded into PTX/Aß-CN-PMs, and the PTX release from rhApoE/PTX/Aß-CN-PMs exhibited a sustained-release pattern in vitro. The formation of the ApoE-enriched protein corona significantly improved the cellular uptake of Aß-CN-PMs on C6 cells and human umbilical vein endothelial cells (HUVECs) and enhanced permeability to the blood-brain tumor barrier in vitro. Meanwhile, PTX/Aß-CN-PMs with ApoE-enriched protein corona had a greater ability to inhibit cell proliferation and induce cell apoptosis than taxol. Importantly, PTX/Aß-CN-PMs exhibited better anti-glioma effects and tissue distribution profile with rapid accumulation in glioma tissues in vivo and prolonged median survival of glioma-bearing mice compared to those associated with PMs without the ApoE protein corona. CONCLUSIONS: The designed PTX/Aß-CN-PMs exhibited significantly enhanced anti-glioma efficacy. Importantly, this study provided a strategy for the rational design of a protein corona-based brain-targeted drug delivery system. More crucially, we utilized the unfavorable side of the protein corona and converted it into an advantage to achieve brain-targeted drug delivery.

Impact of CytoSorb on kinetics of vancomycin and bivalirudin in critically ill patients.

CytoSorb is a promising tool to treat severe inflammatory status with multiple mechanisms in the acute care setting. Its effect on drugs is, however, poorly documented in vivo, although removal of small molecules might translate into decreased blood levels of life-saving medications. The aim of this study was to assess the impact of CytoSorb on vancomycin and bivalirudin clearance in a large population of critically ill patients. We performed a single-center analysis of CytoSorb treatments performed between January 2018 and March 2019 in critically ill patients admitted to our intensive care unit. A total of 109 CytoSorb treatments were performed in 89 patients. A decrease in lactate dehydrogenase (P = .007), troponin T (P = .022), and creatine phosphokinase (P = .013) was reported during treatment. Vancomycin dose required significant adjustments during treatment (P < .001), but no significant change was necessary after the first 3 days. Similarly, the requirements of bivalirudin significantly changed over days (P < .001), but no dose adjustment was needed after the first 3 days of treatment. No differences in terms of vancomycin and bivalirudin dose need was observed between patients on extracorporeal membrane oxygenation and those who were not (P = .6 and P = .6, respectively), between patients with and without continuous veno-venous hemofiltration (P = .9 and P = .9, respectively), and between CytoSorb responders or not (P = .4 and P = .7, respectively). CytoSorb is effective in mitigating the systemic inflammatory response and safe with respect to vancomycin and bivalirudin administration. These preliminary data further support the use of CytoSorb as adjunct therapy in critically ill patients.

Thrombin rapidly digests adrenomedullin: Synthesis of adrenomedullin analogs resistant to thrombin.

Human adrenomedullin (AM) functions as a circulating hormone and as a local paracrine mediator with multiple biological activities. We investigated the metabolism of AM by examining its fragmentation in human serum. Adrenomedullin was rapidly cleaved in human serum, but was relatively stable in plasma. We showed that AM was rapidly digested by thrombin in serum, with AM(13-44) as the main product. On the basis of these data, we prepared AM analogs in which Arg-44 was replaced by Ala, Lys, and D-Arg, respectively. These analogs were resistant to thrombin and showed comparable biological activity to native AM. Furthermore, the bioavailabilities of these peptides were improved after subcutaneous administration in rats. These AM analogs may be promising drug candidates for clinical applications.

Very Short and Stable Lactoferricin-Derived Antimicrobial Peptides: Design Principles and Potential Uses.

The alarming rate at which micro-organisms are developing resistance to conventional antibiotics represents one of the global challenges of our time. There is currently ample space in the antibacterial drug pipeline, and scientists are trying to find innovative and novel strategies to target the microbial enemies. Nature has remained a source of inspiration for most of the antibiotics developed and used, and the immune molecules produced by the innate defense systems, as a first line of defense, have been heralded as the next source of antibiotics. Most living organisms produce an arsenal of antimicrobial peptides (AMPs) to rapidly fend off intruding pathogens, and several different attempts have been made to transform this versatile group of compounds into the next generation of antibiotics. However, faced with the many hurdles of using peptides as drugs, the success of these defense molecules as therapeutics remains to be realized. AMPs derived from the proteolytic degradation of the innate defense protein lactoferrin have been shown to display several favorable antimicrobial properties. In an attempt to investigate the biological and pharmacological properties of these much shorter AMPs, the sequence dependence was investigated, and it was shown, through a series of truncation experiments, that these AMPs in fact can be prepared as tripeptides, with improved antimicrobial activity, via the incorporation of unnatural hydrophobic residues and terminal cappings. In this Account, we describe how this class of promising cationic tripeptides has been developed to specifically address the main challenges limiting the general use of AMPs. This has been made possible through the identification of the antibacterial pharmacophore and via the incorporation of a range of unnatural hydrophobic and cationic amino acids. Incorporation of these residues at selected positions has allowed us to extensively establish how these compounds interact with the major proteolytic enzymes trypsin and chymotrypsin and also the two major drug-binding plasma proteins serum albumin and α-1 glycoprotein. Several of the challenges associated with using AMPs relate to their size, susceptibility to rapid proteolytic degradation, and poor oral bioavailability. Our studies have addressed these issues in detail, and the results have allowed us to effectively design and prepare active and metabolically stable AMPs that have been evaluated in a range of functional settings. The optimized short AMPs display inhibitory activities against a plethora of micro-organisms at low micromolar concentrations, and they have been shown to target resistant strains of both bacteria and fungi alike with a very rapid mode of action. Our Account further describes how these compounds behave in in vivo experiments and highlights both the challenges and possibilities of the intriguing compounds. In several areas, they have been shown to exhibit comparable or superior activity to established antibacterial, antifungal, and antifouling commercial products. This illustrates their ability to effectively target and eradicate various microbes in a variety of settings ranging from the ocean to the clinic.

Human Amyloid-ß40 Kinetics after Intravenous and Intracerebroventricular Injections and Calcitriol Treatment in Rats In Vivo.

Amyloid-ß peptides of 40 and 42 amino acid lengths, which are synthesized in neurons and degraded in the brain and liver, have the potential to aggregate and form neuritic plaques in Alzheimer disease. The kinetics of human amyloid-ß (hAß) 40 were examined in the rat pursuant to intravenous and intracerebroventricular administration after pretreatment with calcitriol, the active vitamin D receptor ligand (6.4 nmol·kg-1 in 0.3 ml corn oil every other day for four intraperitoneal doses) to induce P-glycoprotein (P-gp) and enhance hAß40 brain efflux. The interference of hAß40 by media matrix that suppressed absorbance readings in the ELISA assay was circumvented with use of different calibration curves prepared in Standard Dilution Buffer, undiluted, 10-10,000 or 5-fold diluted plasma, or artificial cerebrospinal fluid. Simultaneous fitting of hAß40 plasma and cerebrospinal fluid (CSF) data after intravenous and intracerebroventricular administration were described by catenary-mammillary models comprising of a central and two peripheral compartments, the brain, and one to four CSF compartments. The model with only one CSF compartment (model I) best fitted the intravenous data that showed a faster plasma decay t1/2 and slower equilibration between plasma and brain/CSF. Calcitriol induction increased the brain efflux rate constant, k41 (1.8-fold), at the blood-brain barrier when compared with the control group, as confirmed by the 2-fold (P < 0.05) increase in brain P-gp relative protein expression. SIGNIFICANCE STATEMENT: An accurate description of the kinetic behavior of human amyloid-ß (hAß) 40 is needed in defining the toxic peptide as a biomarker of Alzheimer disease. Modeling of hAß40 data after intravenous and intracerebroventricular administration to the rat revealed an initially faster plasma half-life that reflected faster peripheral distribution but slower equilibration between plasma and brain/cerebrospinal fluid even with calcitriol pretreatment that increased P-glycoprotein protein expression and enhanced efflux clearance from brain.

Calibrating the In Vitro-In Vivo Correlation for OATP-Mediated Drug-Drug Interactions with Rosuvastatin Using Static and PBPK Models.

Organic anion-transporting polypeptide (OATP) 1B1/3-mediated drug-drug interaction (DDI) potential is evaluated in vivo with rosuvastatin (RST) as a probe substrate in clinical studies. We calibrated our assay with RST and estradiol 17-ß-D-glucuronide (E217ßG)/cholecystokinin-8 (CCK8) as in vitro probes for qualitative and quantitative prediction of OATP1B-mediated DDI potential for RST. In vitro OATP1B1/1B3 inhibition using E217ßG and CCK8 yielded higher area under the curve (AUC) ratio (AUCR) values numerically with the static model, but all probes performed similarly from a qualitative cutoff-based prediction, as described in regulatory guidances. However, the magnitudes of DDI were not captured satisfactorily. Considering that clearance of RST is also mediated by gut breast cancer resistance protein (BCRP), inhibition of BCRP was also incorporated in the DDI prediction if the gut inhibitor concentrations were 10 × IC50 for BCRP inhibition. This combined static model closely predicted the magnitude of RST DDI with root-mean-square error values of 0.767-0.812 and 1.24-1.31 with and without BCRP inhibition, respectively, for in vitro-in vivo correlation of DDI. Physiologically based pharmacokinetic (PBPK) modeling was also used to simulate DDI between RST and rifampicin, asunaprevir, and velpatasvir. Predicted AUCR for rifampicin and asunaprevir was within 1.5-fold of that observed, whereas that for velpatasvir showed a 2-fold underprediction. Overall, the combined static model incorporating both OATP1B and BCRP inhibition provides a quick and simple mathematical approach to quantitatively predict the magnitude of transporter-mediated DDI for RST for routine application. PBPK complements the static model and provides a framework for studying molecules when a dynamic model is needed. SIGNIFICANCE STATEMENT: Using 22 drugs, we show that a static model for organic anion-transporting polypeptide (OATP) 1B1/1B3 inhibition can qualitatively predict potential for drug-drug interaction (DDI) using a cutoff-based approach, as in regulatory guidances. However, consideration of both OATP1B1/3 and gut breast cancer resistance protein inhibition provided a better prediction of the magnitude of the transporter-mediated DDI of these inhibitors with rosuvastatin. Based on these results, we have proposed an empirical mechanistic-static approach for a more reliable prediction of transporter-mediated DDI liability with rosuvastatin that drug development teams can leverage.

Development of Radiogallium-Labeled Peptides for Platelet-Derived Growth Factor Receptor ß (PDGFRß) Imaging: Influence of Different Linkers.

The purpose of this study is to develop peptide-based platelet-derived growth factor receptor ß (PDGFRß) imaging probes and examine the effects of several linkers, namely un-natural amino acids (D-alanine and ß-alanine) and ethylene-glycol (EG), on the properties of Ga-DOTA-(linker)-IPLPPPRRPFFK peptides. Seven radiotracers, 67Ga-DOTA-(linker)-IPLPPPRRPFFK peptides, were designed, synthesized, and evaluated. The stability and cell uptake in PDGFRß positive peptide cells were evaluated in vitro. The biodistribution of [67Ga]Ga-DOTA-EG2-IPLPPPRRPFFK ([67Ga]27) and [67Ga]Ga-DOTA-EG4-IPLPPPRRPFFK ([67Ga]28), which were selected based on in vitro stability in murine plasma and cell uptake rates, were determined in BxPC3-luc-bearing nu/nu mice. Seven 67Ga-labeled peptides were successfully synthesized with high radiochemical yields (>85%) and purities (>99%). All evaluated radiotracers were stable in PBS (pH 7.4) at 37 °C. However, only [67Ga]27 and [67Ga]28 remained more than 75% after incubation in murine plasma at 37 °C for 1 h. [67Ga]27 exhibited the highest BxPC3-luc cell uptake among the prepared radiolabeled peptides. As regards the results of the biodistribution experiments, the tumor-to-blood ratios of [67Ga]27 and [67Ga]28 at 1 h post-injection were 2.61 ± 0.75 and 2.05 ± 0.77, respectively. Co-injection of [67Ga]27 and an excess amount of IPLPPPRRPFFK peptide as a blocking agent can significantly decrease this ratio. However, tumor accumulation was not considered sufficient. Therefore, further probe modification is required to assess tumor accumulation for in vivo imaging.

Addition of cyclic angiotensin-(1-7) to angiotensin-converting enzyme inhibitor therapy has a positive add-on effect in experimental diabetic nephropathy.

The Renin-Angiotensin System (RAS) possesses a counter-regulatory axis composed of angiotensin converting enzyme (ACE)2, angiotensin-(1-7) [Ang-(1-7)] and the Mas receptor, which opposes many AT1-receptor-mediated effects of ligand angiotensin II. Ang-(1-7), as a ligand of the Mas receptor, has inhibitory effects on renal inflammation and fibrosis in experimental diabetes. However, Ang-(1-7) has a short half-life in plasma, which may render it unsuitable for use in clinics. Here, we investigated the effects of the lanthionine-stabilized Ang-(1-7), cyclic (c)Ang-(1-7), a lanthipeptide that is more peptidase-resistant than the linear peptide, in BTBR ob/ob mice with type 2 diabetic nephropathy. BTBR ob/ob mice received vehicle, cAng-(1-7), or the ACE inhibitor lisinopril. The treatment started at ten weeks of age, when the animals had already developed albuminuria, and ended at 19-20 weeks of age. cAng-(1-7) limited albuminuria progression, and limited podocyte dysfunction similarly to lisinopril. cAng-(1-7), unlike lisinopril, reduced glomerular fibrosis and inflammation, and counteracted glomerular capillary rarefaction. Furthermore, when cAng-(1-7) was combined with lisinopril, a superior antiproteinuric effect than with lisinopril alone was found, in association with better preservation of podocyte proteins and amelioration of capillary density. Thus, adding cAng-(1-7) to ACE-inhibitor therapy could benefit those diabetic patients who do not respond completely to ACE-inhibitor therapy.

A Novel Angiotensin-(1-7) Glycosylated Mas Receptor Agonist for Treating Vascular Cognitive Impairment and Inflammation-Related Memory Dysfunction.

Increasing evidence indicates that decreased brain blood flow, increased reactive oxygen species (ROS) production, and proinflammatory mechanisms accelerate neurodegenerative disease progression such as that seen in vascular contributions to cognitive impairment and dementia (VCID) and Alzheimer's disease and related dementias. There is a critical clinical need for safe and effective therapies for the treatment and prevention of cognitive impairment known to occur in patients with VCID and chronic inflammatory diseases such as heart failure (HF), hypertension, and diabetes. This study used our mouse model of VCID/HF to test our novel glycosylated angiotensin-(1-7) peptide Ang-1-6-O-Ser-Glc-NH2 (PNA5) as a therapy to treat VCID and to investigate circulating inflammatory biomarkers that may be involved. We demonstrate that PNA5 has greater brain penetration compared with the native angiotensin-(1-7) peptide. Moreover, after treatment with 1.0/mg/kg, s.c., for 21 days, PNA5 exhibits up to 10 days of sustained cognitive protective effects in our VCID/HF mice that last beyond the peptide half-life. PNA5 reversed object recognition impairment in VCID/HF mice and rescued spatial memory impairment. PNA5 activation of the Mas receptor results in a dose-dependent inhibition of ROS in human endothelial cells. Last, PNA5 treatment decreased VCID/HF-induced activation of brain microglia/macrophages and inhibited circulating tumor necrosis factor α, interleukin (IL)-7, and granulocyte cell-stimulating factor serum levels while increasing that of the anti-inflammatory cytokine IL-10. These results suggest that PNA5 is an excellent candidate and "first-in-class" therapy for treating VCID and other inflammation-related brain diseases.

Evaluation of a Novel Pb-203-Labeled Lactam-Cyclized Alpha-Melanocyte-Stimulating Hormone Peptide for Melanoma Targeting.

The purpose of this study is to examine the melanocortin-1 receptor (MC1R) targeting and specificity of 203Pb-DOTA-GGNle-CycMSHhex in melanoma cells and tumors to facilitate its potential therapeutic application when labeled with 212Pb. The MC1R-specific targeting and imaging properties of 203Pb-DOTA-GGNle-CycMSHhex were determined on B16/F1 and B16/F10 murine melanoma cells and in B16/F1 flank melanoma-, B16/F10 flank melanoma-, and B16/F10 pulmonary metastatic melanoma-bearing C57 mice. 203Pb-DOTA-GGNle-CycMSHhex displayed MC1R-specific binding on B16/F1 and B16/F10 melanoma cells and tumors. B16/F1 flank melanoma, B16/F10 flank melanoma, and B16/F10 pulmonary metastatic melanoma lesions could be clearly imaged by single photon emission computed tomography (SPECT) using 203Pb-DOTA-GGNle-CycMSHhex as an imaging probe. The favorable melanoma targeting and imaging properties highlighted the potential of 203Pb-DOTA-GGNle-CycMSHhex as a MC1R-targeting melanoma imaging probe and warranted the evaluation of 212Pb-DOTA-GGNle-CycMSHhex for melanoma therapy in future studies.

Pharmacokinetic and Pharmacodynamic Modeling and Simulation Analysis of CTB-001, a Recently Developed Generic of Bivalirudin.

PURPOSE: CTB-001, a recently developed generic version of bivalirudin, an FDA-approved anticoagulant used for prophylaxis and treatment of cardiovascular diseases, has shown good efficacy and safety in clinical trials. We characterized the pharmacokinetics (PK) and pharmacodynamics (PD) of CTB-001 by modeling and simulation analysis. METHODS: PK/PD data were collected from a randomized, double-blind, placebo-controlled, single-dose, dose-escalation phase 1 study conducted in 24 healthy Korean male subjects. PK/PD analysis was conducted sequentially by nonlinear mixed-effects modeling implemented in NONMEM®. Monte-Carlo simulations were conducted for PK, activated partial thromboplastin time (aPTT), prothrombin time (PT), and thrombin time (TT). RESULTS: The CTB-101 PK was best described by a three-compartment linear model with a saturable binding peripheral compartment. All PD endpoints showed dose-response relationship, and their changes over time paralleled those of CTB-101 concentrations. A simple maximum effect model best described the aPTT, PT in INR, PT in seconds, and TT, whereas an inhibitory simple maximum effect model best described PT in percentages. The maximum duration of effect of CTB-001 on aPTT prolongation was 52.1 s. CONCLUSIONS: The modeling and simulation analysis well-characterized the PK and PD of CTB-001 in healthy Koreans, which will be valuable for identifying optimal dosing regimens of CBT-001.

Comparison of 99mTc-UBI 29-41, 99mTc-ciprofloxacin, 99mTc-ciprofloxacin dithiocarbamate and 111In-biotin for targeting experimental Staphylococcus aureus and Escherichia coli foreign-body infections: an ex-vivo study.

BACKGROUND: Diagnosis of implant-associated infection is challenging. Several radiopharmaceuticals have been described but direct comparisons are limited. Here we compared in vitro and in an animal model 99mTc-UBI, 99mTc-ciprofloxacin, 99mTcN-CiproCS2 and 111In-DTPA-biotin for targeting E. coli (ATCC 25922) and S. aureus (ATCC 43335). METHODS: Stability controls were performed with the labelled radiopharmaceuticals during 6 hours in saline and serum. The in vitro binding to viable or killed bacteria was evaluated at 37 °C and 4 °C. For in vivo studies, Teflon cages were subcutaneously implanted in mice, followed by percutaneous infection. Biodistribution of i.v. injected radiolabelled radiopharmaceuticals were evaluated during 24 h in cages and dissected tissues. RESULTS: Labelling efficiency of all radiopharmaceuticals ranged between 94% and 98%, with high stability both in saline and in human serum. In vitro binding assays displayed a rapid but poor bacterial binding for all tested agents. Similar binding kinetic occurred also with heat-killed and ethanol-killed bacteria. In the tissue cage model, infection was detected at different time points: 99mTc-UBI and 99mTcN-CiproCS2 showed higher infected cage/sterile cage ratio at 24 hours for both E. coli and S. aureus; 99mTc-Ciprofloxacin at 24 hours for both E. coli and at 4 hours for S. aureus; 111In-DTPA-biotin accumulates faster in both E. coli and S. aureus infected cages. CONCLUSIONS: 99mTc-UBI, 99mTcN-CiproCS2 showed poor in vitro binding but good in vivo binding to E. coli only. 111In-DTPA-biotin showed poor in vitro binding but good in vivo binding to S. aureus and poor to E. coli. 99mTc-Ciprofloxacin showed poor in vitro binding but good in vivo binding to all tested bacteria. The mechanism of accumulation in infected sites remains to be elucidated.