Faster Clearance Rate Research Articles

Croconaine-based NIR-II fluorescence imaging-guided tumor photothermal therapy induces long-term antitumor immune memory

Photothermal therapy (PTT) for cancers guided by optical imaging has recently shown great potential for precise diagnosis and efficient therapy. The second near-infrared window (NIR-II, 1000–1700 nm) fluorescence imaging (FLI) is highly desirable owing to its good spatial and temporal resolution, deep tissue penetration, and negligible tissue toxicity. Organic small molecules are attractive as imaging and treatment agents in biomedical research because of their low toxicity, fast clearance rate, diverse structures, ease of modification, and excellent biocompatibility. Various organic small molecules have been investigated for biomedical applications. However, there are few reports on the use of croconaine dyes (CRs), especially NIR-II emission CRs. To our knowledge, there have been no prior reports of NIR-II emissive small organic photothermal agents (SOPTAs) based on CRs. Herein, we report a croconaine dye (CR-TPE-T)-based nanoparticle (CR NP) with absorption and fluorescence emission in the NIR-I and NIR-II windows, respectively. The CR NPs exhibited intense NIR absorption, outstanding photothermal properties, and good biological compatibility. In vivo studies showed that CR NPs not only achieved real-time, noninvasive NIR-II FLI of tumors, but also induced significant tumor ablation with laser irradiation guided by imaging, without apparent side effects, and promoted the formation of antitumor immune memory in a colorectal cancer model. In addition, the CR NPs displayed efficient inhibition of breast tumor growth, improved longevity of mice and triggered efficient systemic immune responses, which further inhibited tumor metastasis to the lungs. Our study demonstrates the great potential of CRs as therapeutic agents in the NIR-II region for cancer diagnosis.

Discovery and Evaluation of Imidazo[2,1-b][1,3,4]thiadiazole Derivatives as New Candidates for α-Synuclein PET Imaging.

α-synuclein (α-syn) pathologies are central to the development of synucleinopathies including Parkinson's disease (PD). Positron emission tomography (PET) imaging of α-syn pathologies is one strategy to facilitate the diagnosis, understanding, and treatment of synucleinopathies, but has been restricted by the lack of specific α-syn PET probes. In this work, we identified 2,6-disubstituted imidazo[2,1-b][1,3,4]thiadiazole (ITA) as a new α-syn-binding scaffold. Through autoradiography studies, we discovered an iodinated lead compound [125I]ITA-3, with moderate binding affinity (IC50 = 55 nM) to α-syn pathologies in human PD brain sections. Modified from [125I]ITA-3, we developed a potential PET tracer, [18F]FITA-2 (radiochemical yield >25%, molar activity >110 GBq/μmol), which demonstrated clear signals in α-syn-rich regions in human PD brain tissues (IC50 = 245 nM), good brain uptake (SUVpeak = 2.80 ± 0.45), and fast clearance rate in rats. Overall, [18F]FITA-2 appears to be a promising candidate for α-syn PET imaging and merits further development.

Management of Moderate to Severe Plaque Psoriasis with Brodalumab in Daily Practice: Real-World Evidence from the LIBERO Study in the Czech Republic.

Psoriasis is a chronic, immune-mediated inflammatory skin disease. Despite the availability of several therapies, many patients affected by this disease remain untreated, do not have adequate response, or suffer from treatment-related toxic effects. It has been shown that the interleukin (IL)-17 pathway plays a key role in the immunopathogenesis of psoriasis. Brodalumab, the first human monoclonal IgG2 antibody that selectively binds to subunitA of the human IL-17 receptor, blocking interactions with a number of cytokines of the IL-17 family, has confirmed fast onset of action, high complete clearance rates, and sustained efficacy. Nevertheless, there is only a limited amount of published real-world evidence (RWE) data. This was an open-label, multicenter, real-world, prospective, non-interventional, non-controlled (single-arm) observational study (LIBERO-CZ) assessing the management of moderate to severe psoriasis with brodalumab in daily practice for up to 52weeks of treatment. Fifty-four patients (70.4% male, mean age 46.9 ± 13.4years, weight 95.6 ± 22.7kg, disease duration 18.6 ± 12.7years) were enrolled and included in the final analysis. Forty-nine of the patients completed the study and five discontinued prematurely; 51.8% of all the enrolled patients were biologic-naïve. At baseline, 28% patients were classified as severe (psoriasis area severity index (PASI) ≥ 20). Overall, the mean PASI decreased by 15.6 from 16.1 (± 5.0) at baseline to 0.5 (± 1.2) at the last visit. The primary endpoint of an absolute PASI ≤ 3 at week12 (as observed analysis) was achieved by 95.9% of patients. The static Physician's Global Assessment (sPGA) success (defined as clear = 0 and almost clear = 1) at week52 was achieved by 92.1% of patients. PASI75, PASI90, and PASI100 were achieved by 98.0%, 87.8%, and 75.5% of patients, respectively, after approximately 52weeks of treatment. The study also recorded very positive results concerning patient-reported outcomes. LIBERO-CZ confirms the fast onset and high clearance rates of brodalumab in real life in both biologic-naïve and biologic-experienced patients.

3D printing injectable microbeads using a composite liposomal ink for local treatment of peritoneal diseases

The peritoneal cavity offers an attractive administration route for challenging-to-treat diseases, such as peritoneal carcinomatosis, post-surgical adhesions, and peritoneal fibrosis. Achieving a uniform and prolonged drug distribution throughout the entire peritoneal space, though, is difficult due to high clearance rates, among others. To address such an unmet clinical need, alternative drug delivery approaches providing sustained drug release, reduced clearance rates, and a patient-centric strategy are required. Here, we describe the development of a 3D-printed composite platform for the sustained release of the tyrosine kinase inhibitor gefitinib (GEF), a small molecule drug with therapeutic applications for peritoneal metastasis and post-surgical adhesions. We present a robust method for the production of biodegradable liposome-loaded hydrogel microbeads that can overcome the pharmacokinetic limitations of small molecules with fast clearance rates, a current bottleneck for the intraperitoneal (IP) administration of these therapeutics. By means of an electromagnetic droplet printhead, we 3D printed microbeads employing an alginate-based ink loaded with GEF-containing multilamellar vesicles (MLVs). The sustained release of GEF from microbeads was demonstrated. In vitro studies on an immortalized human hepatic cancer cell line (Huh-7) proved concentration-dependent cell death. These findings demonstrate the potential of 3D-printed alginate microbeads containing liposomes for delivering small drug compounds into the peritoneum, overcoming previous limitations of IP drug delivery.Graphical abstract

Evaluation of (2S,4S)-4-[18F]FEBGln as a Positron Emission Tomography Tracer for Tumor Imaging.

Glutamine metabolism-related tracers have the potential to visualize numerous tumors because glutamine is the second largest source of energy for tumors. (2S,4S)-4-[18F]FEBGln was designed by introducing [18F]fluoroethoxy benzyl on carbon-4 of glutamine. The aim of this study was to investigate the pharmacokinetic properties and tumor positron emission tomography (PET) imaging characteristics of (2S,4S)-4-[18F]FEBGln in detail. The biodistribution results of nude mice bearing MCF-7 tumor showed that (2S,4S)-4-[18F]FEBGln had high initial tumor uptake, and a fast clearance rate, resulting in a high tumor-to-muscle ratio at 30 min postinjection. There was no obvious defluorination in vivo. The micro-PET-CT imaging results of (2S,4S)-4-[18F]FEBGln orthotopic MCF-7 tumor-bearing nude mice were consistent with the biological distribution results. Compared with (2S,4R)-4-[18F]FGln, (2S,4S)-4-[18F]FEBGln showed poor tumor retention, but its clearance in normal tissues was also fast, so it had better PET image contrast than the former. Unlike poor retention in MCF-7-bearing nude mice, (2S,4S)-4-[18F]FEBGln has good retention in NCI-h1975 and 22Rv1 tumor models. Since (2S,4S)-4-[18F]FEBGln has low uptake in normal lungs and high uptake in the bladder, it is expected to be used in the accurate diagnosis of lung cancer but cannot accurately determine prostate cancer. Consistent with the advantages of radiolabeled amino acids in the application of brain tumors, (2S,4S)-4-[18F]FEBGln accurately diagnoses U87MG glioma with higher contrast than [18F]FET and [18F]FDG, and there is a correlation between (2S,4S)-4-[18F]FEBGln uptake and tumor growth cycle. Further kinetic model analysis showed that (2S,4S)-4-[18F]FEBGln was similar to (2S,4R)-4-[18F]FGln, conforming to the one-compartment model and the Logan graphical model, and was expected to assess the size of the glutamine pool of the tumor. Therefore, (2S,4S)-4-[18F]FEBGln is expected to provide a strong imaging basis for the diagnosis, formulation of personalized plans, and efficacy evaluation of glioma, lung cancer, and breast cancer.

Hydroxyethyl-Modified Cycloheptatriene-BODIPY Derivatives as Specific Tau Imaging Probes.

Near-infrared fluorescence (NIRF) imaging as an exquisite sensitive, high spatial-resolution, and real-time tool plays an important role in visualizing pathologies in the brain. In this study, we designed and synthesized a series of NIR probes of hydroxyethyl cycloheptatriene-BODIPY derivatives that have demonstrated strong binding specificity to native neurofibrillary tangles (NFTs) in Alzheimer's disease (AD) brain sections. The improved hydrophilicity of TNIR7-9 and TNIR7-11 resulted in faster clearance rates from healthy brains (4.2 and 10.9, respectively) compared to previously reported compounds. Furthermore, TNIR7-13, which features a fluorinated modification, exhibited a high binding affinity to Tau aggregates (Kd = 11.8 nM) and held promise for future PET studies.

Functional liposome loaded curcumin for the treatment of Streptococcus mutans biofilm.

Introduction: Plaque biofilms, mainly formed by Streptococcus mutans (S. mutans), play an important role in the occurrence and development of dental caries. Antibiotic treatment is the traditional way to control plaque. However, problems such as poor drug penetration and antibiotic resistance have encouraged the search for alternative strategies. In this paper, we hope to avoid antibiotic resistance through the antibacterial effect of curcumin, a natural plant extract with photodynamic effects, on S. mutans. However, the clinical application of curcumin is limited due to its low water solubility, poor stability, high metabolic rate, fast clearance rate, and limited bioavailability. In recent years, liposomes have become a widely used drug carrier due to their numerous advantages, such as high drug loading efficiency, high stability in the biological environment, controlled release, biocompatibility, non-toxic, and biodegradability. So, we constructed a curcumin-loaded liposome (Cur@LP) to avoid the defect of curcumin. Methods: Cur@LP functioned with NHS can adhere to the surface of the S. mutans biofilm by condensation reaction. Liposome (LP) and Cur@LP was characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The cytotoxicity of Cur@LP was evaluated by CCK-8 assay and LDH assay. The adhesion of Cur@LP to S. mutans biofilm was observed by confocal laser scanning microscope (CLSM). The antibiofilm efficiency of Cur@LP were evaluated by crystal violet staining, CLSM, and scanning electron microscope (SEM). Results: The mean diameter of LP and Cur@LP were 206.67 ± 8.38nm and 312 ± 18.78nm respectively. The ζ-potential of LP and Cur@LP were ∼-19.3mV and ∼-20.8mV respectively. The encapsulation efficiency of Cur@LP was (42.61 ± 2.19) %, and curcumin was rapidly released up to ±21% at 2h. Cur@LP has negligible cytotoxicity, and can effectively adhered to the S. mutans biofilm and inhibited its growth. Discussion: Curcumin has been widely studied in many fields such as cancer, which can be attributed to its antioxidant and anti-inflammatory effects. At present, there are few studies on the delivery of curcumin to S. mutans biofilm. In this study, we verified the adhesion and antibiofilm of Cur@LP to S. mutans biofilm. This biofilm removal strategy has the potential to be translated into the clinic.

In Vitro Properties and Pharmacokinetics of Temporarily PEGylated Onc72 Prodrugs.

The favorable properties of antimicrobial peptides (AMPs) to rapidly kill pathogens are often limited by unfavorable pharmacokinetics due to fast degradation and renal clearance rates. Here, a prodrug strategy linking proline-rich AMP Onc72 to polyethylene glycol (PEGs) with average molecular weights of 5 and 20kDa via a peptide linker containing a protease cleavage site is tested for the first time in vivo. Onc72 is released from these 5k- and 20k-prodrugs in mouse serum with half-life times (t1/2 ) of 8 and 14h, respectively. Importantly, PEGylation protects Onc72 from proteolytic degradation providing a prolonged release of Onc72, balancing the degradation of free Onc72, and leading to relatively stable Onc72 concentrations and high antibacterial activities. The prodrugs are not hemolytic on human erythrocytes and show only slight cytotoxic effects on human cell lines indicating promising safety margins. When administered subcutaneously to female CD-1 mice, the prodrugs elimination t1/2 are 66min and ≈5.5h, respectively, compared to 43min of free Onc72. The maximal Onc72 plasma levels are obtained ≈1 and ≈8h postadministration, respectively. In conclusion, the prodrugs provide extended elimination t1/2 and a constant release of Onc72 in mice, potentially limiting adverse effects and increasing efficacy.

A Review of Salinity's Impact on Relevant Aquatic Drug Approval Technical Sections.

Currently, in the USA, there are no aquatic animal drugs approved for use in saltwater-reared finfish. As U.S. aquaculture expands into the marine environment, it is imperative to maximize the current knowledge base to make new approvals for saltwater use as efficient as possible. This article reviews literature relevant to how the saltwater environment may affect the Effectiveness, Target Animal Safety, and Human Food Safety major technical sections of a new animal drug approval. Though research on this subject is limited, there are pharmacokinetic and toxicological studies that have utilized euryhaline species to isolate salinity as the independent variable. I summarize these studies to demonstrate patterns that could facilitate more effective and efficient transfer of data previously used for freshwater drug approvals to be used for potential saltwater approvals. Research suggests that drug residue clearance rates are generally faster or at least similar in salt water if other factors are held constant, suggesting that consistent or shorter withdrawal times may be appropriate for saltwater application. However, faster clearance rates may cause a reduction in efficacy in some cases. The effects of salt water on drug toxicity are much less clear, are possibly highly compound dependent, and deserve further research. This suggests that any future drug approvals for saltwater use should begin by identifying the appropriate effective dose in salt water before addressing the other technical sections.

Bio-Evaluation of 99mTc-Labeled Homodimeric Chalcone Derivative as Amyloid-β-Targeting Probe.

Chalcone derivatives have been successfully utilized for a range of biological applications and can cross the blood–brain barrier easily. β-amyloid-specific bis-chalcone derivative, 6,9-bis(carboxymethyl)-14-(4-[(E)-3-(4-(dimethylamino)phenyl)acryloyl]phenoxy)-3-(2-[(2-(4-[(E)-3-(4-(dimethylamino)phenyl)acryloyl]phenoxy)ethyl)amino]-2-oxoethyl)-11-oxo-3,6,9,12-tetraazatetradecanoic acid, DT(Ch)2, was analyzed using molecular modeling to explain the binding modes of the ligand with amyloid fibril and monomer followed by 99mTc-complexation in 95% yield and 98.7% efficiency. High-binding specificity of the radiocomplex was established following in vitro evaluation against 100-fold excess of DT(Ch)2. 99mTc–DT(Ch)2 exhibited <3% trans-complexation in human serum after 24 h, indicating high stability. A fast clearance rate in pharmacokinetics studies displayed a biphasic pattern with t1/2(F) = 30 min ± 0.09 and t1/2(S) = 4 h 20 min ± 0.06. In vivo single-photon emission computed tomography (SPECT) imaging in rabbits reiterated the pharmacokinetics data with initially high brain uptake followed by rapid washout. Biodistribution studies confirmed the initial brain uptake as 1.16 ± 0.02% ID/g after 2 min and the brain2min/brain30min ratio was 3.74. Radioactivity distribution in the brain was >40% in the cingulate cortex followed by >25% in the hippocampus, a distribution pattern aligned to Alzheimer’s affected brain regions. Radiocomplex also displayed rapid plasma clearance followed by hepatobolic and renal modes of excretion.

An intravitreal implant injection method for sustained drug delivery into mouse eyes

SummaryUsing small molecule drugs to treat eye diseases carries benefits of specificity, scalability, and transportability, but their efficacy is significantly limited by a fast intraocular clearance rate. Ocular drug implants (ODIs) present a compelling means for the slow and sustained release of small molecule drugs inside the eye. However, methods are needed to inject small molecule ODIs into animals with small eyes, such as mice, which are the primary genetic models for most human ocular diseases. Consequently, it has not been possible to fully investigate efficacy and ocular pharmacokinetics of ODIs. Here, we present a robust, cost-effective, and minimally invasive method called "mouse implant intravitreal injection" (MI3) to deliver ODIs into mouse eyes. This method will expand ODI research to cover the breadth of human eye diseases modeled in mice.

Desynchrony and synchronisation underpinning sleep–wake cycles

This paper studies mechanisms of synchronisation and loss of synchrony among the three key oscillatory processes controlling sleep–wake cycles in the human brain: the 24-h circadian oscillator, the homeostatic sleep drive, and the environmental light–dark cycle. Synchronisation of these three rhythms promotes sleep and brain clearance and is critical for human health. Their desynchrony, on the other hand, is associated with impaired performance and disease development, including cancer, cardiovascular disease, and mental disorders. A biophysical model of arousal dynamics simulating sleep–wake cycles and circadian rhythms is used as the study system. It is based on established neurobiological mechanisms controlling sleep–wake transitions and incorporates the three oscillatory processes. Nonlinear dynamics methods and synchronisation theory are used to numerically investigate model dynamics under conditions that are not easily achievable in experiments. The role of homeostatic brain clearance rate in synchronisation is investigated, and selective turning on and off of coupling strengths between the oscillators allows us to determine their role in oscillators’ dynamics. We find that, in the absence of coupling between the circadian and homeostatic oscillators, the default state of the model corresponds to the endogenous homeostatic period that is far from $$\sim $$ 24-h rhythm of the circadian and light–dark cycles. Combined action of light and circadian oscillator on the homeostatic rhythm is required to achieve the typical sleep–wake pattern that is observed in young healthy people. Under 12-/12-h light–dark conditions with light at 80 lux, change of homeostatic clearance rate is found to induce two types of desynchronisation: (i) fast clearance rates $$\tau _H<58.1$$ h desynchronise the homeostatic oscillator from the circadian, while the circadian rhythm remains entrained to the light–dark cycle, and (ii) slow clearance rates $$\tau _H>69$$ h maintain synchronisation between the homeostatic and circadian oscillators, but the period of both is different from that of the light–dark cycle. Between these regimes, all three rhythms are synchronised under the studied conditions. The model predicts that the system is highly sensitive to external inputs to the neuronal populations of the sleep–wake switch, which affect the endogenous period of the homeostatic oscillator and can lead to complete loss of sleep. Model dynamics show that loss of synchronisation, which is traditionally ascribed solely to impairment of the circadian oscillator, can be caused by changes in the homeostatic clearance rate of the brain or external input to the neuronal populations of the sleep–wake switch. Thus, changes in circadian, homeostatic, and external factors (combined or specific) may be responsible for conditions of desynchronisation. This has significant implications for understanding individual variability in sleep–wake patterns and in mechanisms of sleep and circadian disorders, indicating that both the homeostatic and circadian mechanisms can be responsible for the same clinical or behavioural presentation of a disease.

LIPOSOMAL HYBRID SILICON - ORGANIC MEANS OF DELIVERY OF MEDICINAL PREPARATIONS

Nowadays, chemotherapy is an important mean for cancer treatment. Despite many benefits, patients receiving anticancer drugs often suffer unfavorable side effects due to the non-specific toxicity of anticancer drugs. Most anticancer drugs can kill cancer cells with non-selective killing of the normal human cells, which brings great pains to patients. One of the most important tasks facing pharmacology is the creation of such systems that would minimize the harm caused by therapeutic drugs. One way to overcome these problems is to create drug delivery systems. Much attention is attracted the liposomes as delivery systems. They consist of natural components that can minimize toxicity in relation to the human body, so liposomes are interest to study. However, one of the limitations preventing the wide use of liposomes is their insufficient stability under physiological conditions. This property can lead to the fact that the drug will be released from the delivery system until the desired cells or tissues are reached, which means that it damages healthy cells. The problem of stability can be solved by systems called cerasomes. These are nanosized spherical particles consisting of the lipid bilayer as well as liposomes, but their surface is modified by a silicon polymer network. Liposomal nanohybrid cerasomes have been developed based on organoalkoxysilane through a sol-gel reaction in combination with self-assembly process. Among inorganic materials, silicon is an excellent choice to form hydrophilic surface shell due to its high chemical resistance, optical transparency and low physiological toxicity. In addition, cerasomes have better biocompatibility than silicon nanoparticles that have a similar size. They are inert and exhibit less cytotoxicity. Cerasomes are very convenient to store for a certain time due to their physical and chemical properties. Equally important is the fact that cerasomes are capable of encapsulate a wide range of drug molecules. Water-soluble drugs are built into the internal field of the vesicles, and hydrophobic drugs are built into the bilayer lipid membrane. So cerasomes can solve many problems associated with drug molecules like low drug solubility, fast clearance rates, non-specific toxicity, thereby enhancing therapeutic efficiency and reducing side effects. Also, various functional molecules can be included to cerasomes that show thermo-, light-, pH- or multi sensitive properties by chemical conjugation with different molecules in order to modulate the release behavior of the drug. Therefore, cerasomes, serving as delivery carriers, possess great potential for clinical applications due to their unique advantages. This review will summarize the progress of liposomal nanohybrid cerasomes and their applications as drug nanocarriers, transfection of gene materials, systems for visualization and diagnosis of diseases using MRI and PDT. It presents some methods of the synthesis of cerasome-forming lipids to create stable systems of cerasomes. Various approaches of the formation of a siloxane network on their surface are considered. Various variants of modifications of cerasome-forming lipids are presented.

Development of nanostructured lipid carriers containing the antimicrobial ciprofloxacin for topical application

Ciprofloxacin (CIP), a broad spectrum antibacterial agent from the fluoroquinolone family, blocks the activity of the bacterial DNA gyrase. CIP has a limited aqueous solubility and fast clearance rate. Nanostructured lipid carriers (NLC) are drug delivery carriers very appropriate to upload hydrophobic drugs, improving their bioavailability, stability and potency. This work describes the development of NLC containing CIP, their physicochemical characterization and antimicrobial activity, in vitro. NLC particles (215-355 nm) of low size polidispersion (PDI 0.08-0.31) and negative zeta potentials (|20.7-38.7| mV) were prepared; scanning electron microscopy revealed their spherical shapes and regular contour. The particles remained stable during 365 days of storage at 25°C. The encapsulation efficiency of CIP ranged from 72.5 - 95.2%, with a sustained release profile (48 h). Preliminary antimicrobial susceptibility tests revealed a 30% increase in CIP action, when encapsulated in NLC. These proof-of-concept results show the great pharmaceutical and technological potential of the developed nanoformulations. After further tests we believe that NLC-CIP can come to be a novel pharmaceutical form to potentiate the antibiotic effect, reducing its dosage and undesirable side effects.

Moderate Coverage Vaccination Eradicates Oncogenic Human Papillomaviruses if a Gender-Neutral Strategy is Applied

Background: Very high coverage human papillomavirus (HPV) vaccination of girls has the potential to eradicate some HPV types but low coverage vaccination does not. The protection of unvaccinated individuals by herd effect (HE) varies by HPV type and vaccination strategy. By combining community-randomized trial data and mathematical modeling we show that gender-neutral vaccination is superior for achieving eradication of oncogenic HPVs. Methods: We randomized 33 communities into gender-neutral HPV16/18 vaccination (A), HPV16/18 vaccination of girls only (B), and hepatitis B-virus vaccination of boys and girls (C). From 2007-2010 11,622/40,852 resident boys and 20,513/39,420 resident girls born 1992-1995 consented to the study. At follow-up in 2010-14, cervicovaginal samples from vaccinated and unvaccinated girls were typed for HPV6/11/16/18/31/33/35/39/45/51/52/56/58/59/66/68. Trial-based vaccine efficacy (VE) and HE, estimated among non-HPV vaccinated arm A/B girls using arm C controls, were weighted for mean protective effectiveness (PE) estimation, and used for modeling eradication. Findings The HPV18/31/33 HE and PE were significant in younger birth cohorts: four years post vaccination HE and PE were 150% and 40% stronger in the gender-neutral than in the girls-only arm. HPV18/31/33 have moderate to fast clearance rates enabling eradication, and corresponding HEs from vaccinated to unvaccinated arose rapidly. HPV18/31/33 eradication in adolescents/young adults is predicted in 20 years with 75% coverage of gender-neutral vaccination, eradication of HPV16 is predicted within 30 years. Interpretation While low coverage with a girls-only vaccination strategy leaves most females unprotected, HPV-disease eradication of oncogenic HPVs is certain with moderate coverage gender-neutral vaccination strategy. Funding: This ancillary study was partially funded by Academy of Finland, Finnish Cancer Organizations and EU FP7 networks PREHDICT and CoheaHR. GlaxoSmithKline Biologicals SA funded the primary study HPV-040 (NCT00534638, data to be published in a separate manuscript) but was not involved in the conduct of this ancillary study. GlaxoSmithKline Biologicals SA was provided the opportunity to review this manuscript for accuracy of primary HPV-040 data but the authors are solely responsible for final content and interpretation. V.N.P. was supported by a personal grant from the Ministry of Health, Government of Catalonia (PERIS SLT002/16/00496). Declaration of Interest: D.A., J.D., G.G., M.L. and J.P. have received grants from Merck & Co. Inc. or the GSK group of companies through their employers [Family Federation Finland (DA), Karolinska Institute (J.D., M.L.), Universities of Tampere (M.L.) or Helsinki (J.P.) or Imperial College London (G.G.)] for HPV vaccination studies. G.D. is currently a full time employee of Takeda Vaccines, but was working for GSK Biologicals at the time the study was planned and conducted. He holds several patents in the HPV field which have been assigned to the GSK groups of companies and has stock shares in both the GSK groups of companies and Takeda. Ethical Approval: The HPV-040 study (Eudra-CT-2007-001731-55, NCT00534638), and parallel ancillary studies on Chlamydia trachomatis screening (Dnro 111/2009) and HPV screening (ETL-code 13149, NCT02149030) were approved by Finnish ethical committees of the Pirkanmaa and PohjoisPohjanmaa hospital districts in 2007, 2009 and 2013, respectively.

Short-Acting Anti-VWF Aptamer Improves the Recovery and Survival, and Hemostatic Functions of Refrigerated Platelets

Background:Refrigerated platelets are rapidly cleared upon transfusion. The mechanism of this fast clearance is not fully understood. It was recently reported that refrigeration-induced binding of von Willebrand factor (VWF) to platelets contributed to the rapid clearance of platelet after transfusion. Adding a peptide that blocks the binding of VWF to platelet GPIbα during refrigeration can improve the post-transfusion recovery and survival of refrigerated murine platelets in vivo. However, since the interaction of VWF with GPIbα is required for primary hemostasis, adding a long-acting inhibitory peptide to stored platelets may impede the hemostatic function of these platelets after transfusion.DNA aptamer ARC1779 binds VWF and inhibits its binding to GPIbα. It has previously been developed as an anti-thrombotic agent. Its half-life is relatively fast, at a reported 2 hours in human.Objective:We aimed to test whether ARC1779 can improve the post-transfusion recovery and survival of refrigerated platelets, and, due to its fast clearance rate, without significantly impeding the hemostatic functions of these platelets in vivo.Methods:Human or murine platelets were stored at 4°C with or without ARC1779 for 48 hours. VWF binding was measured by flow cytometry. After refrigeration, human platelets were retro-orbitally transfused into NOD-SCID mice, while murine platelets into wild-type mice. Post-transfusion, periodic blood draw was performed, and the percentage of infused platelets in the total platelet population was measured by flow cytometry to calculate the recovery and survival over time. To monitor the hemostatic function, refrigerated murine platelets were transfused into transgenic IL4R-IbαTg mice, and a tail bleeding time assay was performed at four hours after transfusion. IL4R-IbαTg platelets lack the extracellular domain of GPIbα and cannot bind VWF and other GPIbα ligands. As a result, hemostasis in IL4R-IbαTg mice is severely impaired.Results and conclusion:Treatment of ARC1779 during 48-hour refrigeration efficiently inhibited VWF binding to platelets. ARC1779-treated refrigerated murine platelets exhibit increased post-transfusion recovery and survival rates compared to untreated refrigerated ones. (Recovery of ARC1779-treated platelets: 76.7±5.5%; Untreated: 63.7±0.8%, N=6, p<0.01. Half-life time of ARC1779-treated platelets: 31.4±2.36 hr; Untreated: 28.1±0.86 hr, N=6, p<0.05). A similar increase was also observed for refrigerated human platelets (Recovery: 49.4±4.4% vs. 36.8±2.1%, N=6, p<0.01; Half-life time: 9.2±1.5 hr vs. 8.7±0.9 hr, N=6, n.s.). Additionally, the tail bleeding time in ARC1779-treated group was significantly shorter than no ARC1779-treated group (160±65 vs. 373±96 seconds, N=7, p<0.01). For comparison, un-transfused IL4R-IbαTg mice have a tail bleeding time longer than 1200 s, and those transfused with freshly prepared platelets 155±80 s. Overall, these results demonstrate that inhibiting the GPIbα-VWF interaction by ARC1779 during cold storage of platelets improves platelet post-transfusion recovery. The treatment also helps to preserve the hemostatic function of refrigerated platelets. These results suggest that an inhibitor of the GPIbα-VWF interaction but with a limited half-life may be a potential therapeutic option to enable or improve the refrigeration of platelets for transfusion treatment. DisclosuresLi:Neoletix: Consultancy, Equity Ownership.

NLS-Cholic Acid Conjugation to IL-5Rα-Specific Antibody Improves Cellular Accumulation and In Vivo Tumor-Targeting Properties in a Bladder Cancer Model.

Receptor-mediated internalization followed by trafficking and degradation of antibody-conjugates (ACs) via the endosomal-lysosomal pathway is the major mechanism for delivering molecular payloads inside target tumor cells. Although a mainstay for delivering payloads with clinically approved ACs in cancer treatment and imaging, tumor cells are often able to decrease intracellular payload concentrations and thereby reduce the effectiveness of the desired application. Thus, increasing payload intracellular accumulation has become a focus of attention for designing next-generation ACs. We developed a composite compound (ChAcNLS) that enables ACs to escape endosome entrapment and route to the nucleus resulting in the increased intracellular accumulation as an interleukin-5 receptor α-subunit (IL-5Rα)-targeted agent for muscle invasive bladder cancer (MIBC). We constructed 64Cu-A14-ChAcNLS, 64Cu-A14-NLS, and 64Cu-A14 and evaluated their performance by employing mechanistic studies for endosome escape coupled to nuclear routing and determining whether this delivery system results in improved 64Cu cellular accumulation. ACs consisting of ∼20 ChAcNLS or NLS moieties per 64Cu-A14 were prepared in good yield, high monomer content, and maintaining high affinity for IL-5Rα. Confocal microscopy analysis demonstrated ChAcNLS mediated efficient endosome escape and nuclear localization. 64Cu-A14-ChAcNLS increased 64Cu cellular accumulation in HT-1376 and HT-B9 cells relative to 64Cu-A14 and 64Cu-A14-NLS. In addition, we tested 64Cu-A14-ChAcNLS in vivo to evaluate its tissue distribution properties and, ultimately, tumor uptake and targeting. A model of human IL-5Rα MIBC was developed by implanting NOD/SCID mice with subcutaneous HT-1376 or HT-B9MIBC tumors, which grow containing high and low IL-5Rα-positive tumor cell densities, respectively. ACs were intravenously injected, and daily blood sampling, biodistribution at 48 and 96 h, and positron emission tomography (PET) at 24 and 48 h were performed. Region of interest (ROI) analysis was also performed on reconstructed PET images. Pharmacokinetic analysis and biodistribution studies showed that 64Cu-A14-ChAcNLS had faster clearance rates from the blood and healthy organs relative to 64Cu-A14. However, 64Cu-A14-ChAcNLS maintained comparable tumor accumulation relative to 64Cu-A14. This resulted in 64Cu-A14-ChAcNLS having superior tumor/normal tissue ratios at both 48 and 96 h biodistribution time points. Visualization of AC distribution by PET and ROI analysis confirmed that 64Cu-A14-ChAcNLS had improved targeting of MIBC tumor relative to 64Cu-A14. In addition, 64Cu-A14 modified with only NLS had poor tumor targeting. This was a result of poor tumor uptake due to extremely rapid clearance. Thus, the overall findings in this model of human IL-5Rα-positive MIBC describe an endosome escape-nuclear localization cholic-acid-linked peptide that substantially enhances AC cellular accumulation and tumor targeting.

Pretargeted Immuno-PET Based on Bioorthogonal Chemistry for Imaging EGFR Positive Colorectal Cancer.

Pretargeted immuno-PET imaging based on the bioorthogonal chemistry between 18F-labeled Reppe anhydride derivatives and tetrazine conjugates of the EGFR-specific monoclonal antibodies cetuximab and panitumumab was performed. This pretargeting approach yielded high target-to-nontarget ratios. Furthermore, due to the fast clearance rate of the PET probe, the overall radiation burden to nontarget tissues was also substantially decreased.

Self-assembly of Filamentous Cell Penetrating Peptides for Gene Delivery.

Cell penetrating peptides (CPPs) have been proven to be an effective vector to deliver a variety of membrane-impermeable macromolecules, such as DNAs, siRNAs, and proteins. Conventional single-chain CPPs typically suffer from severe protease degradation and fast clearance rate for in vivo therapeutic delivery application. In this chapter, we show that supramolecular assembly of de novo designed cationic multidomain peptides (MDPs) leads to nanostructured filaments with increased proteolytic stability and potent membrane activity necessary for improved transfection efficiency.

Escalating Single Doses of PF-05230907 (Recombinant Factor Xa Variant FXaI16L) Are Safe and Demonstrate Hemostatic Pharmacology in Healthy Volunteers

PF-05230907 (FXaI16L) is a zymogen-like variant of recombinant activated coagulation factor X (FXa), which is more resistant to inactivation by plasma protease inhibitors (compared to endogenous FXa). Previous studies showed that its cofactor, Factor Va (FVa), facilitates its transition to an active conformation, promoting hemostatic activity. The properties of PF-05230907 are consistent with its use as a short acting hemostatic agent to control bleeding and it currently is in clinical development for treatment of individuals with intracerebral hemorrhage (ICH).We conducted a Phase 1 First-in-Human study to determine if single escalating doses of PF-05230907 were safe and tolerable and to characterize pharmacokinetics (PK) and pharmacodynamics (PD). The study was conducted in healthy adult volunteers aged 18-35 years with no risk factors for thrombosis and was approved by the local ethics committee. Subjects were assigned to one of six ascending single dose cohorts, each with 2 placebo treated subjects and 6 subjects treated with PF-05230907 (0.1, 0.3, 1, 2, 3, or 5 µg/kg). Assessments included safety monitoring, PD, PK, and immunogenicity testing. All placebo treated subjects were to be included in the safety and PD analyses. All PF-05230907 treated subjects were to be included in the safety, PK and PD analyses.Forty-nine male subjects were enrolled after informed consent. One subject from the 2 µg/kg cohort received less than 80% of the planned PF-05230907 dose and was replaced. This subject was included in the safety analysis, but excluded in PK and PD analysis. PF-05230907 exhibited a fast clearance rate with a terminalhalf-life of approximately 4 minutes. Exposure of PF-05230907 appeared to increase proportionally with dose. Treatment-related PD changes were observed for activated partial thromboplastin time (aPTT), thrombin-anti-thrombin III complexes, prothrombin fragment 1+2, D-dimer and thrombin generation assay (lag time and peak thrombin). PF-05230907 was safe and well tolerated at all dose levels tested with no dose limiting toxicity. There were no serious adverse events. One subject treated with PF-05230907 had a weak and transient non-neutralizing anti-drug antibody response, which did not cross react with native FX or native FXa.PF-05230907 was safe and tolerated and exhibited pharmacologic effect in healthy adults when administered at doses up to 5 µg/kg. The safety profile, PK, and PD observed in this study support further development of PF-05230907 for hemostatic treatment in individuals with acute hemorrhagic conditions. DisclosuresArkin:Pfizer Inc: Employment. Hua:Pfizer Inc: Employment. Kantaridis:Pfizer Inc: Employment. Li:Pfizer Inc: Employment. Parsons-Rich:Pfizer Inc: Employment. Pittman:Pfizer: Employment.