Improved pharmacokinetics and enhanced efficacy of the vancomycin derivative FU002 using a liposomal nanocarrier.

Antibiotic resistance still represents a global health concern which diminishes the pool of effective antibiotics. With the vancomycin derivative FU002, we recently reported a highly potent substance active against Gram-positive bacteria with the potential to overcome vancomycin resistance. However, the translation of its excellent antimicrobial activity into clinical efficiency could be hampered by its rapid elimination from the blood stream. To improve its pharmacokinetics, we encapsulated FU002 in PEGylated liposomes. For PEG-liposomal FU002, no relevant cytotoxicity on liver, kidney and red blood cells was observed. Studies in Wistar rats revealed a significantly prolonged blood circulation of the liposomal antibiotic. In microdilution assays it could be demonstrated that encapsulation does not diminish the antimicrobial activity against staphylococci and enterococci. Highlighting its great potency, liposomal FU002 exhibited a superior therapeutic efficacy when compared to the free form in a Galleria mellonella larvae infection model.

Virus-Derived Peptides for Hepatic Enzyme Delivery.

Recently, a lipopeptide derived from the hepatitis B virus (HBV) large surface protein has been developed as an HBV entry inhibitor. This lipopeptide, called MyrcludexB (MyrB), selectively binds to the sodium taurocholate cotransporting polypeptide (NTCP) on the basolateral membrane of hepatocytes. Here, the feasibility of coupling therapeutic enzymes to MyrB was investigated for the development of enzyme delivery strategies. Hepatotropic targeting shall enable enzyme prodrug therapies and detoxification procedures. Here, horseradish peroxidase (HRP) was conjugated to MyrB via maleimide chemistry, and coupling was validated by SDS-PAGE and reversed-phase HPLC. The specificity of the target recognition of HRP-MyrB could be shown in an NTCP-overexpressing liver parenchymal cell line, as demonstrated by competitive inhibition with an excess of free MyrB and displayed a strong linear dependency on the applied HRP-MyrB concentration. In vivo studies in zebrafish embryos revealed a dominating interaction of HRP-MyrB with scavenger endothelial cells vs xenografted NTCP expressing mammalian cells. In mice, radiolabeled 125I-HRP-MyrBy, as well as the non-NTCP targeted control HRP-peptide-construct (125I-HRP-alaMyrBy) demonstrated a strong liver accumulation confirming the nonspecific interaction with scavenger cells. Still, MyrB conjugation to HRP resulted in an increased and NTCP-mediated hepatotropism, as revealed by competitive inhibition. In conclusion, the model enzyme HRP was successfully conjugated to MyrB to achieve NTCP-specific targeting in vitro with the potential for ex vivo diagnostic applications. In vivo, target specificity was reduced by non-NTCP-mediated interactions. Nonetheless, tissue distribution experiments in zebrafish embryos provide mechanistic insight into underlying scavenging processes indicating partial involvement of stabilin receptors.

Reporter cell assay-based functional quantification of TNF-α-antagonists in serum - a proof-of-principle study for adalimumab.

Quantification of therapeutic antibodies is commonly based on physico-chemical assays such as enzyme-linked immunoabsorption assays (ELISA) and lately on mass spectrometry. However, the functional integrity of evaluated immunoglobulins is yet not assessed. Consequently, a commercially available reporter cell line was used to quantify the functional concentration of the anti-tumor necrosis factor alpha (TNF-α) antibody adalimumab present in serum of a healthy beagle dog treated with 3 mg intravenous adalimumab (Humira®). HEK-Blue™-hTLR3 cells express a secreted alkaline phosphatase under the control of a nuclear factor kappa B (NF-κB) response element. Its enzymatic activity can be recorded using colorimetry, which reports activity of extracellular NF-κB stimuli such as TNF-α. Using an adalimumab concentration-response calibration curve, the functional concentration of serum adalimumab was estimated to be 4.9 ± 1.4 µg/ml, which was in excellent agreement with ELISA results (4.8 µg/ml). The obtained data suggest that this simple, easy-to-handle reporter cell assay can be used for the functional quantification of adalimumab present in samples from in vitro or pre-clinical in vivo experiments. Moreover, this assay could be used in vitro to compare the pharmacodynamics of adalimumab biosimilars or different anti-TNF-α compounds, respectively.

Renaissance of vancomycin: approaches for breaking antibiotic resistance in multidrug-resistant bacteria.

The emergence of multidrug-resistant bacteria demands innovations in the development of new antibiotics. For decades, the glycopeptide antibiotic vancomycin has been considered as the "last resort" treatment of severe infections caused by Gram-positive bacteria. Since the discovery of the first vancomycin-resistant enterococci strains in the late 1980s, the number of resistances has been steadily rising, with often life-threatening consequences. As an alternative to the generation of completely new substances, novel approaches focus on structural modifications of established antibiotics such as vancomycin to overcome these resistances. Here, we provide an overview of several promising modifications of vancomycin to restore its efficacy against vancomycin-resistant enterococci.

An Underestimated Factor: The Extent of Cross-Reactions Modifying APIs in Surface-Modified Liposomal Preparations Caused by Comprised Activated Lipids.

Despite the nowadays available plentitude of strategies to selectively introduce functional surface modification of liposomes, in preclinical research this process is still primarily performed after liposomal preparation utilizing comprised activated phospholipids with functionalized head groups. However, because these activated lipids are present during the liposomal preparation process, they can cross-react with incorporated drugs, especially the particularly often utilized active esters and maleimide groups. Macromolecular drugs, being composed of amino acids, are particularly prone to such cross-reactions due to their often multiple reactive functionalities such as amino and disulfide groups. To demonstrate this impact on the formulation in liposomal surface modification, we assessed the extent of cross-reaction during the liposomal preparation of two activated phospholipids with typically used head group functionalized phospholipids, with the two peptide drugs vancomycin and insulin comprising disulfide and amino functionalities. Both drugs revealed a considerable fraction of covalent modification (estimated 2 to 12%) generated during the liposome preparation process with comprised activated lipids. Modification of the active pharmaceutical ingredients (APIs) was determined by high-resolution mass spectrometric analysis. These findings clearly demonstrate the non-negligibility of potential cross reactions using the post preparation liposomal surface modification strategy in preclinical research.

Vancomycin Resistance Is Overcome by Conjugation of Polycationic Peptides.

Multidrug-resistant bacteria represent one of the biggest challenges facing modern medicine. The increasing prevalence of glycopeptide resistance compromises the efficacy of vancomycin, for a long time considered as the last resort for the treatment of resistant bacteria. To reestablish its activity, polycationic peptides were conjugated to vancomycin. By site-specific conjugation, derivatives that bear the peptide moiety at four different sites of the antibiotic were synthesized. The most potent compounds exhibited an approximately 1000-fold increased antimicrobial activity and were able to overcome the most important types of vancomycin resistance. Additional blocking experiments using d-Ala-d-Ala revealed a mode of action beyond inhibition of cell-wall formation. The antimicrobial potential of the lead candidate FU002 for bacterial infection treatments could be demonstrated in an in vivo study. Molecular imaging and biodistribution studies revealed that conjugation engenders superior pharmacokinetics.

Zebrafish as a predictive screening model to assess macrophage clearance of liposomes in vivo.

Macrophage recognition of nanoparticles is highly influenced by particle size and surface modification. Due to the lack of appropriate in vivo screening models, it is still challenging and time-consuming to characterize and optimize nanomedicines regarding this undesired clearance mechanism. Therefore, we validate zebrafish embryos as an emerging vertebrate screening tool to assess the macrophage sequestration of surface modified particulate formulations with varying particle size under realistic biological conditions. Liposomes with different PEG molecular weights (PEG350-PEG5000) at different PEG densities (3.0-10.0 mol%) and particle sizes between 60 and 120 nm were used as a well-established reference system showing various degrees of macrophage uptake. The results of in vitro experiments, zebrafish embryos, and in vivo rodent biodistribution studies were consistent, highlighting the validity of the newly introduced zebrafish macrophage clearance model. We hereby present a strategy for efficient, systematic and rapid nanomedicine optimization in order to facilitate the preclinical development of nanotherapeutics.

Replacement of l-Amino Acids by d-Amino Acids in the Antimicrobial Peptide Ranalexin and Its Consequences for Antimicrobial Activity and Biodistribution.

Infections caused by multidrug-resistant bacteria are a global emerging problem. New antibiotics that rely on innovative modes of action are urgently needed. Ranalexin is a potent antimicrobial peptide (AMP) produced in the skin of the American bullfrog Rana catesbeiana. Despite strong antimicrobial activity against Gram-positive bacteria, ranalexin shows disadvantages such as poor pharmacokinetics. To tackle these problems, a ranalexin derivative consisting exclusively of d-amino acids (named danalexin) was synthesized and compared to the original ranalexin for its antimicrobial potential and its biodistribution properties in a rat model. Danalexin showed improved biodistribution with an extended retention in the organisms of Wistar rats when compared to ranalexin. While ranalexin is rapidly cleared from the body, danalexin is retained primarily in the kidneys. Remarkably, both peptides showed strong antimicrobial activity against Gram-positive bacteria and Gram-negative bacteria of the genus Acinetobacter with minimum inhibitory concentrations (MICs) between 4 and 16 mg/L (1.9-7.6 µM). Moreover, both peptides showed lower antimicrobial activities with MICs ≥32 mg/L (≥15.2 µM) against further Gram-negative bacteria. The preservation of antimicrobial activity proves that the configuration of the amino acids does not affect the anticipated mechanism of action, namely pore formation.

Current status in the therapy of liver diseases.

Hepatic diseases, like viral hepatitis, autoimmune hepatitis, hereditary hemochromatosis, non-alcoholic fatty liver disease (NAFLD) and Wilson's disease, play an important role in the development of liver cirrhosis and, hence, hepatocellular carcinoma. In this review, the current treatment options and the molecular mechanisms of action of the drugs are summarized. Unfortunately, the treatment options for most of these hepatic diseases are limited. Since hepatitis B (HBV) and C (HCV) infections are the most common causes of liver cirrhosis and hepatocellular carcinoma, they are the focus of the development of new drugs. The current treatment of choice for HBV/HCV infection is an interferon-based combination therapy with oral antiviral drugs, like nucleos(t)ide analogues, which is associated with improving the therapeutic success and also preventing the development of resistances. Currently, two new protease inhibitors for HCV treatment are expected (deleobuvir, faldaprevir) and together with the promising drug, daclatasvir (NS5A-inhibitor, currently in clinical trials), adequate therapy is to be expected in due course (circumventing the requirement of interferon with its side-effects), while in contrast, efficient HBV therapeutics are still lacking. In this respect, entry inhibitors, like Myrcludex B, the lead substance of the first entry inhibitor for HBV/HDV (hepatitis D) infection, provide immense potential. The pharmacokinetics and the mechanism of action of Myrcludex B are described in detail.

Forward Light Scattering of the Vitreous Gel After Enzymatic Aging: An In Vitro Model to Study Vitreous Opacification.

Purpose: Symptomatic vitreous opacifications, so-called floaters, are difficult to objectively assess majorly limiting the possibility of in vitro studies. Forward light scattering was found previously to be increased in eyes with symptomatic floaters. Using an objective setup to measure forward light scattering, we studied the effects of enzymatically digesting the components of the vitreous body on straylight to develop an in vitro model of vitreous opacifications. Methods: Fifty-seven porcine vitreous bodies were digested using hyaluronidase, collagenase, trypsin, and bromelain, as well as using a combination of hyaluronidase + collagenase and hyaluronidase + bromelain. A modified C-Quant setup was used to objectively assess forward light scattering. Results: Depletion of hyaluronic acid majorly increased vitreous straylight (mean increase 34.4 deg2/sr; P = 0.01), whereas primarily digesting the vitreous gel with collagenase or trypsin did not significantly affect straylight. When collagenase or bromelain is applied in hyaluronic acid depleted vitreous gels, the increase in forward light scattering is reversed partially. Conclusions: The age-related loss of hyaluronic acid primarily drives the increase in vitreous gel straylight induced by conglomerates of collagen. This process can be reversed partially by digesting collagen. This in vitro model allows the objective quantification and statistical comparison of straylight burden caused by vitreous opacities and, thus, can serve as a first testing ground for pharmacological therapies, as demonstrated with bromelain.

Oral Delivery of the Vancomycin Derivative FU002 by a Surface-Modified Liposomal Nanocarrier.

Oral delivery of peptide therapeutics faces multiple challenges due to their instability in the gastrointestinal tract and low permeation capability. In this study, the aim is to develop a liposomal nanocarrier formulation to enable the oral delivery of the vancomycin-peptide derivative FU002. FU002 is a promising, resistance-breaking, antibiotic which exhibits poor oral bioavailability, limiting its potential therapeutic use. To increase its oral bioavailability, FU002 is incorporated into tetraether lipid-stabilized liposomes modified with cyclic cell-penetrating peptides on the liposomal surface. This liposomal formulation shows strong binding to Caco-2 cells without exerting cytotoxic effects in vitro. Pharmacokinetics studies in vivo in rats reveal increased oral bioavailability of liposomal FU002 when compared to the free drug. In vitro and in vivo antimicrobial activity of FU002 are preserved in the liposomal formulation. As a highlight, oral administration of liposomal FU002 results in significant therapeutic efficacy in a murine systemic infection model. Thus, the presented nanotechnological approach provides a promising strategy for enabling oral delivery of this highly active vancomycin derivative.

Viscoelastic, Optical, and Surgical Properties of Vitreous Body Replacement Hydrogels After Aging Compared to Porcine Vitreous Bodies And Silicone Oils.

Purpose: First- (monomers), second- (pre-gelated), and third- (in situ gelating after injection) generation hydrogels were previously introduced to replace the vitreous body after vitrectomy surgery. In this study, we evaluated the surgical, optical, and viscoelastic properties of vitreous body replacement hydrogels before and after an accelerated aging protocol previously applied to intraocular implants. Methods: Measurements of injection force, removal speed using a clinically established vitrectomy setup, as well as evaluation of forward light scattering and viscoelastic properties before and after an accelerated aging protocol were conducted. Results were compared to porcine and human vitreous bodies, as well as currently clinically applied lighter- and heavier-than-water silicone oils. Results: Removal speed of all tested hydrogels is substantially lower than the removal speed of porcine vitreous body (0.2 g/min vs. 2.7 g/min for the best performing hydrogel and porcine vitreous body, respectively). Forward light scattering in second-generation vitreous body replacement hydrogels was higher after the aging process than the straylight of the average 70-year-old vitreous body (9.4 vs. 5.5 deg2/sr, respectively). The viscoelastic properties of all hydrogels did not change in a clinically meaningful manner; however, trends toward greater stiffness and greater elasticity after aging were apparent. Conclusions: This study demonstrates surgical weaknesses of the hydrogels that need to be addressed before clinical use, especially low removal speed. Pre-linked hydrogels (second-generation) showed inferior performance regarding surgical properties compared to in situ gelating hydrogels (third-generation). Translational Relevance: This study highlights possible pitfalls regarding surgical and optical properties when applying vitreous replacement hydrogels clinically.

New approaches to explanting high-viscosity silicone oil in retinal surgery-polyimide cannulas vs extraction sleeves vs a luer-trocar.

BACKGROUND: The removal of high-viscosity silicone oil in retinal surgery is time-intensive. In this laboratory and porcine eye study, we evaluated the efficiency of novel extraction techniques, namely shortened polyimide-cannulas, an extraction sleeve and an experimental luer-trocar system, for commercially available silicone oils. METHODS: The volume of silicone oil after a 5-min removal time was quantified. The feasibility of all studied techniques was additionally tested in vitrectomized porcine eyes. Siluron 1000, 2000, 5000 and Xtra, as well as Densiron 68 and Densiron Xtra were examined. RESULTS: Shortening the 23G-polyimide-cannula from 6 mm up to 1.5 mm led to a significantly higher mean aspirated volume (e.g., 1 ml/min to 1.7 ml/min for Siluron Xtra). The extraction sleeve and luer-trocar increased the flow threefold compared to the shortest polyimide cannulas at 23G (5.2 ml/min and 5.2 ml/min vs 1.7 ml/min for Siluron Xtra, respectively). The extraction sleeve and luer-trocar system reached a similar flow at 27G as the best performing 23G-cannula. Angulation and movement of the eye was possible with the 2 mm-cannula, no angulation was possible when using the extraction sleeve. Stability and control were not impaired with the experimental luer-trocar setup. CONCLUSION: Shorter polyimide-cannulas and extraction sleeves increase the extraction flow but may decrease stability during surgery. The luer-trocar system facilitates rapid removal while offering great stability and control. Small 27G silicone oil removal is possible and time efficient.

ApoE-functionalization of nanoparticles for targeted brain delivery-a feasible method for polyplexes?

The blood-brain barrier (BBB) poses a major obstacle in the treatment of all types of central nervous system (CNS) diseases. Small interfering RNA (siRNA) offers in principle a promising therapeutic approach by downregulating disease-related genes via RNA interference. However, the BBB is a formidable barrier for macromolecules such as nucleic acids. In an effort to develop a brain-targeted strategy for siRNA delivery systems formed by electrostatic interactions with cationic polymers (polyplexes (PXs)), we investigated the suitability of the well-known surfactant-based approach for Apolipoprotein E (ApoE)-functionalization of nanoparticles (NPs). The aim of this present work was to investigate if ApoE coating of siRNA PXs formed with cationic branched 25-kDa poly(ethyleneimine) (b-PEI) and nylon-3 polymers without or after precoating with polysorbate 80 (PS 80) would promote successful delivery across the BBB. We utilized highly hydrophobic NM0.2/CP0.8 nylon-3 polymers to evaluate the effects of hydrophobic cyclopentyl (CP) subunits on ApoE binding efficacy and observed successful ApoE binding with and without PS 80 precoating to the nylon-3 but not the PEI polyplexes. Accordingly, ApoE-coated nylon-3 polyplexes showed significantly increased uptake and gene silencing in U87 glioma cells but no benefit in vivo. In conclusion, further optimization of ApoE-functionalized polyplexes and more sophisticated in vitro models are required to achieve more successful in vitro-in vivo translation in future approaches.

Forward Light Scattering of First to Third Generation Vitreous Body Replacement Hydrogels after Surgical Application Compared to Conventional Silicone Oils and Vitreous Body.

To treat certain vitreoretinal diseases, the vitreous body, a hydrogel composed of mostly collagen and hyaluronic acid, must be removed. After vitrectomy surgery, the vitreous cavity is filled with an endotamponade. Previously, pre-clinical hydrogel-based vitreous body substitutes either made from uncrosslinked monomers (1st generation), preformed crosslinked polymers (2nd generation), or in situ gelating polymers (3rd generation) have been developed. Forward light scattering is a measure of Stray light induced by optical media, when increased, causing visual disturbance and glare. During pinhole surgery, the hydrogels are injected into the vitreous cavity through a small 23G-cannula. The aim of this study was to assess if and to what extent forward light scattering is induced by vitreous body replacement hydrogels and if Stray light differs between different generations of vitreous body hydrogel replacements due to the different gelation mechanisms and fragmentation during injection. A modified C-Quant setup was used to objectively determine forward light scattering. In this study, we found that the 1st and 3rd generation vitreous body replacements show very low stray light levels even after injection (2.8 +/- 0.4 deg2/sr and 0.2 +/- 0.2 deg2/sr, respectively) as gel fragmentation and generation of interfaces is circumvented. The 2nd generation preformed hydrogels showed a permanent increase in stray light after injection that will most likely lead to symptoms such as glare when used in patients (11.9 +/- 0.9 deg2/sr). Stray light of the 2nd generation hydrogels was 3- and 2-fold increased compared to juvenile and aged vitreous bodies, respectively. In conclusion, this significant downside in the forward light scattering of the 2nd generation hydrogels should be kept in mind when developing vitreous body replacement strategies, as any source of stray light should be minimized in patients with retinal comorbidities.

Quantification of Straylight Induced by Silicone Oil Adherent to Intraocular Lenses of Different Materials.

PURPOSE: A complication of using silicone oil as an intraocular endotamponade is its adhesion to intraocular lenses (IOLs). Forward light scattering is a measure to quantify the optical disturbance caused by adherent oil droplets. We tested the straylight caused by silicone oil adhesion to different IOLs and examined whether an approved cleaning solution, F4H5, reverses the induced straylight. DESIGN: An experimental study. METHODS: Two hydrophobic acrylic IOL models and 1 hydrophilic model with a hydrophobic surface (n = 8 per model: 24 lenses) had straylight measured before contact with silicone oils, providing a baseline for subsequent testing: 12 lenses with lighter-than-water silicone oil (Siluron 2000) and 12 with heavier-than-water oil (Densiron 68). The final measurement was performed after cleansing with F4H5 when we used scanning electron and light microscopy to detect surface changes. RESULTS: Straylight was majorly increased in IOLs with adherent silicone oil (baseline vs adherent oil median 3.1 [2.1, 3.9] and 39.7 [22.7, 87.8] deg2/sr, respectively; P < .001). No difference was seen between heavier- and lighter-than-water silicone oils. Between IOL types, induced straylight varied significantly, with 1 hydrophobic model reaching the highest average straylight. F4H5 significantly reduced straylight values in all IOL types (median 9.4 [5.4, 13.8] deg2/sr). The microscopy revealed surface changes on the IOLs even after cleaning. CONCLUSIONS: Silicone oil adhesion to IOLs can induce amounts of straylight known to cause severe optical disturbance. F4H5 cleansing solution reversed straylight values to only slightly increased values. We found no difference in straylight formation between the lighter- and heavier-than-water silicone oils.

Overcoming the blood-brain barrier? - prediction of blood-brain permeability of hydrophobically modified polyethylenimine polyplexes for siRNA delivery into the brain with in vitro and in vivo models.

The blood-brain barrier (BBB) is a highly selective biological barrier that represents a major bottleneck in the treatment of all types of central nervous system (CNS) disorders. Small interfering RNA (siRNA) offers in principle a promising therapeutic approach, e.g., for brain tumors, by downregulating brain tumor-related genes and inhibiting tumor growth via RNA interference. In an effort to develop efficient siRNA nanocarriers for crossing the BBB, we utilized polyethyleneimine (PEI) polymers hydrophobically modified with either stearic-acid (SA) or dodecylacrylamide (DAA) subunits and evaluated their suitability for delivering siRNA across the BBB in in vitro and in vivo BBB models depending on their structure. Physicochemical characteristics of siRNA-polymer complexes (polyplexes (PXs)), e.g., particle size and surface charge, were measured by dynamic light scattering and laser Doppler anemometry, whereas siRNA condensation ability of polymers and polyplex stability was evaluated by spectrophotometric methods. The composition of the biomolecule corona that absorbs on polyplexes upon encountering physiological fluids was investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and by a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method. Cellular internalization abilities of PXs into brain endothelial cells (hCMEC/D3) was confirmed, and a BBB permeation assay using a human induced pluripotent stem cell (hiPSC)-derived BBB model revealed similar abilities to cross the BBB for all formulations under physiological conditions. However, biodistribution studies of radiolabeled PXs in mice were inconsistent with in vitro results as the detected amount of radiolabeled siRNA in the brain delivered with PEI PXs was higher compared to PEI-SA PXs. Taken together, PEI PXs were shown to be a suitable nanocarrier to deliver small amounts of siRNA across the BBB into the brain but more sophisticated human BBB models that better represent physiological conditions and biodistribution are required to provide highly predictive in vitro data for human CNS drug development in the future.

Antibody-drug conjugates: What drives their progress?

Antibody-drug conjugates (ADCs) are on the brink of widespread use for the targeted treatment of cancer. ADCs manage the toxicity of drugs with unacceptable narrow therapeutic windows by guiding highly toxic compounds to the target cells, thereby sparing healthy cells. In this review, we describe approved ADCs and discuss their modes of action, together with medicinal chemical aspects, to evaluate the potential for improvement and to combat tumor-acquired resistance. A recent research focus has centered on the stimulation of immune responses to induce immunogenic cell death and the influence on the tumor microenvironment to enhance bystander effects.

Intravitreal Application: Physicochemical Properties of Drugs Dissolved in Silicone Oils of Different Density in Comparison to the Porcine Vitreous Body.

Silicone oil endotamponades provide a reservoir for drugs in the eye. Following vitrectomy surgery to treat retinal detachments, extensive diabetic retinopathy or endophthalmitis, they can be used as long-term lipophilic depots. This study aimed to investigate the physicochemical properties of intravitreally applied drugs of different lipophilicity, namely vancomycin, ceftazidime and voriconazole. For this purpose, an in vitro model of the silicone-oil-filled eye compared to porcine vitreous bodies (PVBs) was used. In a glass container, either light or heavy silicone oil or PVB was set into equilibrium with an aqueous fluid. Vancomycin, voriconazole and ceftazidime were added in concentrations commonly applied in clinical practice. The time course of the concentration of the drugs was determined in the hydrophilic phase for up to 24 h. With silicone oil present, the concentrations of vancomycin, voriconazole and ceftazidime were elevated in the aqueous humor when compared to the vitreous body (p < 0.001 for all drugs). With increasing lipophilicity, higher concentrations of the drug dissolved in silicone oil after 24 h (52.7%, 49.1% and 34.3% for vancomycin, ceftazidime and voriconazole, respectively). While no difference between lighter- and heavier-than-water silicone oil was apparent for vancomycin and ceftazidime (p = 0.17 and p = 0.72), voriconazole dissolved significantly better in the heavier-than-water silicone oil (p = 0.002). A higher-than-expected percentage of the glycopeptide vancomycin dissolved in the porcine vitreous body, possibly due to protein binding. In conclusion, silicone oils influence the drug concentration and distribution of intravitreally applied drugs depending on their lipophilicity. The addition of F6H8 used to create heavy silicone oils attenuates these effects for lipophilic drugs. Knowledge of the distribution of these intravitreally applied drugs is crucial to ensure the desired anti-infectious effect.

Impact of Linker Modification and PEGylation of Vancomycin Conjugates on Structure-Activity Relationships and Pharmacokinetics.

As multidrug-resistant bacteria represent a concerning burden, experts insist on the need for a dramatic rethinking on antibiotic use and development in order to avoid a post-antibiotic era. New and rapidly developable strategies for antimicrobial substances, in particular substances highly potent against multidrug-resistant bacteria, are urgently required. Some of the treatment options currently available for multidrug-resistant bacteria are considerably limited by side effects and unfavorable pharmacokinetics. The glycopeptide vancomycin is considered an antibiotic of last resort. Its use is challenged by bacterial strains exhibiting various types of resistance. Therefore, in this study, highly active polycationic peptide-vancomycin conjugates with varying linker characteristics or the addition of PEG moieties were synthesized to optimize pharmacokinetics while retaining or even increasing antimicrobial activity in comparison to vancomycin. The antimicrobial activity of the novel conjugates was determined by microdilution assays on susceptible and vancomycin-resistant bacterial strains. VAN1 and VAN2, the most promising linker-modified derivatives, were further characterized in vivo with molecular imaging and biodistribution studies in rodents, showing that the linker moiety influences both antimicrobial activity and pharmacokinetics. Encouragingly, VAN2 was able to undercut the resistance breakpoint in microdilution assays on vanB and vanC vancomycin-resistant enterococci. Out of all PEGylated derivatives, VAN:PEG1 and VAN:PEG3 were able to overcome vanC resistance. Biodistribution studies of the novel derivatives revealed significant changes in pharmacokinetics when compared with vancomycin. In conclusion, linker modification of vancomycin-polycationic peptide conjugates represents a promising strategy for the modulation of pharmacokinetic behavior while providing potent antimicrobial activity.