Versatile Click Linker Enabling Native Peptide Release from Nanocarriers upon Redox Trigger.

Nanocarriers have shown their ability to extend the circulation time of drugs, enhance tumor uptake, and tune drug release. Therapeutic peptides are a class of drug compounds in which nanocarrier-mediated delivery can potentially improve their therapeutic index. To this end, there is an urgent need for orthogonal covalent linker chemistry facilitating the straightforward on-the-resin peptide generation, nanocarrier conjugation, as well as the triggered release of the peptide in its native state. Here, we present a copper-free clickable ring-strained alkyne linker conjugated to the N-terminus of oncolytic peptide LTX-315 via standard solid-phase peptide synthesis (SPPS). The linker contains (1) a recently developed seven-membered ring-strained alkyne, 3,3,6,6-tetramethylthiacycloheptyne sulfoximine (TMTHSI), (2) a disulfide bond, which is sensitive to the reducing cytosolic and tumor environment, and (3) a thiobenzyl carbamate spacer enabling release of the native peptide upon cleavage of the disulfide via 1,6-elimination. We demonstrate convenient "clicking" of the hydrophilic linker-peptide conjugate to preformed pegylated core-cross-linked polymeric micelles (CCPMs) of 50 nm containing azides in the hydrophobic core under aqueous conditions at room temperature resulting in a loading capacity of 8 mass % of peptide to polymer (56% loading efficiency). This entrapment of hydrophilic cargo into/to a cross-linked hydrophobic core is a new and counterintuitive approach for this class of nanocarriers. The release of LTX-315 from the CCPMs was investigated in vitro and rapid release upon exposure to glutathione (within minutes) followed by slower 1,6-elimination (within an hour) resulted in the formation of the native peptide. Finally, cytotoxicity of LTX CCPMs as well as uptake of sulfocyanine 5-loaded CCPMs was investigated by cell culture, demonstrating successful tumor cell killing at concentrations similar to that of the free peptide treatment.

Mechanistic Study on the Degradation of Hydrolysable Core-Crosslinked Polymeric Micelles.

Core-crosslinked polymeric micelles (CCPMs) are an attractive class of nanocarriers for drug delivery. Two crosslinking approaches to form CCPMs exist: either via a low-molecular-weight crosslinking agent to connect homogeneous polymer chains with reactive handles or via cross-reactive handles on polymers to link them to each other (complementary polymers). Previously, CCPMs based on methoxy poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide-lactate] (mPEG-b-PHPMAmLacn) modified with thioesters were crosslinked via native chemical ligation (NCL, a reaction between a cysteine residue and thioester resulting in an amide bond) using a bifunctional cysteine containing crosslinker. These CCPMs are degradable under physiological conditions due to hydrolysis of the ester groups present in the crosslinks. The rapid onset of degradation observed previously, as measured by the light scattering intensity, questions the effectiveness of crosslinking via a bifunctional agent. Particularly due to the possibility of intrachain crosslinks that can occur using such a small crosslinker, we investigated the degradation mechanism of CCPMs generated via both approaches using various analytical techniques. CCPMs based on complementary polymers degraded slower at pH 7.4 and 37 °C than CCPMs with a crosslinker (the half-life of the light scattering intensity was approximately 170 h versus 80 h, respectively). Through comparative analysis of the degradation profiles of the two different CCPMs, we conclude that partially ineffective intrachain crosslinks are likely formed using the small crosslinker, which contributed to more rapid CCPM degradation. Overall, this study shows that the type of crosslinking approach can significantly affect degradation kinetics, and this should be taken into consideration when developing new degradable CCPM platforms.

CINOVA: a phase II study of CPC634 (nanoparticulate docetaxel) in patients with platinum resistant recurrent ovarian cancer.

OBJECTIVE: Recurrent platinum-resistant ovarian cancer has a poor prognosis with limited therapeutic options. Sub-therapeutic intra-tumoral drug concentrations may add to therapy resistance. CPC634 (docetaxel entrapped in CriPec nanoparticles) was designed to enhance tumor accumulation of drug with localized drug release at the target site to increase therapeutic efficacy. This study investigated the therapeutic effect of CPC634 in patients with platinum-resistant ovarian cancer. METHODS: According to a Simon 2-stage design trial, the first stage included 13 patients, and 12 patients were enrolled in the second stage. Eligible patients had measurable disease and had progressed ≤6 months after the last platinum-based therapy. Platinum-refractory disease was excluded. In stage 1, the number of previous treatment lines was unlimited; in the second stage, a maximum of two prior lines altogether were allowed. The primary endpoint was the objective response rate by Response Evaluation Criteria in Solid Tumor (RECIST) V1.1. Secondary endpoints included safety, progression-free survival at 6 months, cancer antigen 125 (CA125) response, and disease control rate. RESULTS: The patients' median age was 66 years (range 22-77) and most were International Federation of Gynecology and Obstetrics (FIGO) stage III (56%). The median number of previous treatment lines was 3 (range 3-5) in stage I and 2 (range 1-4) in stage II of the study. None of the patients had an objective response, one patient had a CA125 response (5%), and seven patients had stable disease at first evaluation (35%). Median progression-free survival was 1.4 months in stage 1 and 3.0 months in stage 2. Adverse events (all grades) were mainly gastrointestinal in 24 patients (96%), fatigue in 11 (44%), dyspnea in 10 (40%), and infections in 10 (40%) of patients. Grade 3 or higher adverse events occurred in 14 patients (36%), including gastrointestinal in 4 (16%), anemia in 3 (12%), and febrile neutropenia, fatigue, chronic kidney disease, dehydration, and hypertension each in 1 (4%) patient. The trial was stopped prematurely due to futility. CONCLUSIONS: Treatment with CPC634 was feasible, but without apparent clinical activity in patients with recurrent platinum-resistant ovarian cancer. Side effects were mainly gastrointestinal in 24 (96%) patients, including nausea, vomiting, and decreased appetite, fatigue, anemia, and dyspnea.

A New Class of Tunable Acid-Sensitive Linkers for Native Drug Release Based on the Trityl Protecting Group.

Core-cross-linked polymeric micelles (CCPMs) are a promising nanoparticle platform due to favorable properties such as their long circulation and tumor disposition exploiting the enhanced permeability and retention (EPR) effect. Sustained release of covalently linked drugs from the hydrophobic core of the CCPM can be achieved by a biodegradable linker that connects the drug and the core. This study investigates the suitability of trityl-based linkers for the design of acid-triggered native active pharmaceutical ingredient (API) release from CCPMs. Trityl linker derivatives with different substituent patterns were synthesized and conjugated to model API compounds such as DMXAA-amine, doxorubicin, and gemcitabine, and their release kinetics were studied. Hereafter, API release from CCPMs based on mPEG-b-pHPMAmLac block copolymers was investigated. Variation of the trityl substitution pattern showed tunability of the API release rate from the trityl-based linker with t1/2 varying from <1.0 to 5.0 h at pH 5.0 and t1/2 from 6.5 to >24 h at pH 7.4, all at 37 °C. A clear difference in release kinetics was found between gemcitabine and doxorubicin, with gemcitabine showing no detectable release for 72 h at pH 5.0 and doxorubicin showing a t1/2 of less than 1 h. Based on these findings, we show that the reaction mechanism of trityl deprotection plays an important role in the API release kinetics. The first step in this mechanism, which is protonation of the trityl-bound amine, is pKa-dependent, which explains the difference in release rate. In conclusion, acid-sensitive and tunable trityl linkers are highly promising for the design of linker-API conjugates and for their use in CCPMs.

Specific N-terminal attachment of TMTHSI linkers to native peptides and proteins for strain-promoted azide alkyne cycloaddition.

The site specific attachment of the reactive TMTHSI-click handle to the N-terminus of peptides and proteins is described. The resulting molecular constructs can be used in strain-promoted azide alkyne cycloaddition (SPAAC) for reaction with azide containing proteins e.g., antibodies, peptides, nanoparticles, fluorescent dyes, chelators for radioactive isotopes and SPR-chips etc.

Exploring the Chemical Properties and Medicinal Applications of Tetramethylthiocycloheptyne Sulfoximine Used in Strain-Promoted Azide-Alkyne Cycloaddition Reactions.

The recently developed compound, tetramethylthiocycloheptyne sulfoximine (TMTHSI), has shown to be a promising strained alkyne for strain-promoted azide-alkyne cycloaddition (SPAAC), metal-free click chemistry. This research explores the properties of TMTHSI-based compounds via three aspects: (1) large-scale production, (2) unique stability in acidic conditions and its subsequent use in peptide synthesis, and (3) the functionalization of antibodies. Here, it is shown that (1) scale-up is achieved on a scale of up to 100 g. (2) TMTHSI is remarkably stable against TFA allowing for the site-specific functionalization of peptides on resin. Finally, (3) the functionalization of an antibody with a model payload is very efficient, with antibody conjugation demonstrating more beneficial features such as a high yield and limited hydrophobicity as compared to other alkyne reagent conjugates. These results illustrate the high potential of TMTHSI for diverse bioconjugation applications, with production already being GMP-compatible and a highly efficient conversion resulting in attractive costs of goods.

Profiling target engagement and cellular uptake of cRGD-decorated clinical-stage core-crosslinked polymeric micelles.

Polymeric micelles are increasingly explored for tumor-targeted drug delivery. CriPec® technology enables the generation of core-crosslinked polymeric micelles (CCPMs) based on thermosensitive (mPEG-b-pHPMAmLacn) block copolymers, with high drug loading capacity, tailorable size, and controlled drug release kinetics. In this study, we decorated clinical-stage CCPM with the αvß3 integrin-targeted cyclic arginine-glycine-aspartic acid (cRGD) peptide, which is one of the most well-known active targeting ligands evaluated preclinically and clinically. Using a panel of cell lines with different expression levels of the αvß3 integrin receptor and exploring both static and dynamic incubation conditions, we studied the benefit of decorating CCPM with different densities of cRGD. We show that incubation time and temperature, as well as the expression levels of αvß3 integrin by target cells, positively influence cRGD-CCPM uptake, as demonstated by immunofluorescence staining and fluorescence microscopy. We demonstrate that even very low decoration densities (i.e., 1 mol % cRGD) result in increased engagement and uptake by target cells as compared to peptide-free control CCPM, and that high cRGD decoration densities do not result in a proportional increase in internalization. In this context, it should be kept in mind that a more extensive presence of targeting ligands on the surface of nanomedicines may affect their pharmacokinetic and biodistribution profile. Thus, we suggest a relatively low cRGD decoration density as most suitable for in vivo application.

Glucocorticoid-loaded core-cross-linked polymeric micelles with tailorable release kinetics for targeted therapy of rheumatoid arthritis.

Polymerizable and hydrolytically cleavable dexamethasone (DEX, red dot in picture) derivatives were covalently entrapped in core-cross-linked polymeric micelles that were prepared from a thermosensitive block copolymer (yellow and gray building block). By varying the oxidation degree of the thioether in the drug linker, the release rate of DEX could be controlled. The DEX-loaded micelles were used for efficient treatment of inflammatory arthritis in two animal models.

PET-CT Imaging of Polymeric Nanoparticle Tumor Accumulation in Patients.

Several FDA/EMA-approved nanomedicines have demonstrated improved pharmacokinetics and toxicity profiles compared to their conventional chemotherapeutic counterparts. The next step to increase therapeutic efficacy depends on tumor accumulation, which can be highly heterogeneous. A clinical tool for patient stratification is urgently awaited. Therefore, a docetaxel-entrapping polymeric nanoparticle (89 Zr-CPC634) is radiolabeled, and positron emission tomography/computed tomography (PET/CT) imaging is performed in seven patients with solid tumors with two different doses of CPC634: an on-treatment (containing 60 mg m-2 docetaxel) and a diagnostic (1-2 mg docetaxel) dose (NCT03712423). Pharmacokinetic half-life for 89 Zr-CPC634 is mean 97.0 ± 14.4 h on-treatment, and 62.4 ± 12.9 h for the diagnostic dose (p = 0.003). At these doses accumulation is observed in 46% and 41% of tumor lesions with a median accumulation in positive lesions 96 h post-injection of 4.94 and 4.45%IA kg-1 (p = 0.91), respectively. In conclusion, PET/CT imaging with a diagnostic dose of 89 Zr-CPC634 accurately reflects on-treatment tumor accumulation and thus opens the possibility for patient stratification in cancer nanomedicine with polymeric nanoparticles.

Design, development and clinical translation of CriPec®-based core-crosslinked polymeric micelles.

Nanomedicines are used to improve the efficacy and safety of pharmacotherapeutic interventions. Unraveling the biological behavior of nanomedicines, including their biodistribution and target site accumulation, is essential to establish design criteria that contribute to superior performance. CriPec® technology is based on amphiphilic methoxy-poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide lactate] (mPEG-b-pHPMAmLacn) block copolymers, which are designed to upon self-assembly covalently entrap active pharmaceutical ingredients (API) in core-crosslinked polymeric micelles (CCPM). Key features of CCPM are a prolonged circulation time, high concentrations at pathological sites, and low levels of accumulation in the majority of healthy tissues. Proprietary hydrolysable linkers allow for tunable and sustained release of entrapped API, including hydrophobic and hydrophilic small molecules, as well as peptides and oligonucleotides. Preclinical imaging experiments provided valuable information on their tumor and tissue accumulation and distribution, as well as on uptake by cancer, healthy and immune cells. The frontrunner formulation CPC634, which refers to 65 nm-sized CCPM entrapping the chemotherapeutic drug docetaxel, showed excellent pharmacokinetic properties, safety, tumor accumulation and antitumor efficacy in multiple animal models. In the clinic, CPC634 also demonstrated favorable pharmacokinetics, good tolerability, signs of efficacy, and enhanced localization in tumor tissue as compared to conventional docetaxel. PET imaging of radiolabeled CPC634 showed quantifiable accumulation in âˆ¼50 % of tumors and metastases in advanced-stage cancer patients, and demonstrated potential for use in a theranostic setting even when applied at a companion diagnostic dose. Altogether, the preclinical and clinical results obtained to date demonstrate that mPEG-b-pHPMAmLacn CCPM based on CriPec® technology are a potent, tunable, broadly applicable and well-tolerable platform for targeted drug delivery and improved anticancer therapy.

Acoustic Cluster Therapy (ACT®) enhances accumulation of polymeric micelles in the murine brain.

The restrictive nature of the blood-brain barrier (BBB) prevents efficient treatment of many brain diseases. Focused ultrasound in combination with microbubbles has shown to safely and transiently increase BBB permeability. Here, the potential of Acoustic Cluster Therapy (ACT®), a microbubble platform specifically engineered for theranostic purposes, to increase the permeability of the BBB and improve accumulation of IRDye® 800CW-PEG and core-crosslinked polymeric micelles (CCPM) in the murine brain, was studied. Contrast enhanced magnetic resonance imaging (MRI) showed increased BBB permeability in all animals after ACT®. Near infrared fluorescence (NIRF) images of excised brains 1 h post ACT® revealed an increased accumulation of the IRDye® 800CW-PEG (5.2-fold) and CCPM (3.7-fold) in ACT®-treated brains compared to control brains, which was retained up to 24 h post ACT®. Confocal laser scanning microscopy (CLSM) showed improved extravasation and penetration of CCPM into the brain parenchyma after ACT®. Histological examination of brain sections showed no treatment related tissue damage. This study demonstrated that ACT® increases the permeability of the BBB and enhances accumulation of macromolecules and clinically relevant nanoparticles to the brain, taking a principal step in enabling improved treatment of various brain diseases.

Lyophilization stabilizes clinical-stage core-crosslinked polymeric micelles to overcome cold chain supply challenges.

BACKGROUND: CriPec technology enables the generation of drug-entrapped biodegradable core-crosslinked polymeric micelles (CCPM) with high drug loading capacity, tailorable size, and drug release kinetics. Docetaxel (DTX)-entrapped CCPM, also referred to as CPC634, have demonstrated favorable pharmacokinetics, tolerability, and enhanced tumor uptake in patients. Clinical efficacy evaluation is ongoing. CPC634 is currently stored (shelf life > 5 years) and shipped as a frozen aqueous dispersion at temperatures below -60°C, in order to prevent premature release of DTX and hydrolysis of the core-crosslinks. Consequently, like other aqueous nanomedicine formulations, CPC634 relies on cold chain supply, which is unfavorable for commercialization. Lyophilization can help to bypass this issue. METHODS AND RESULTS: Freeze-drying methodology for CCPM was developed by employing CPC634 as a model formulation, and sucrose and trehalose as cryoprotectants. We studied the residual moisture content and reconstitution behavior of the CPC634 freeze-dried cake, as well as the size, polydispersity index, morphology, drug retention, and release kinetics of reconstituted CPC634. Subsequently, the freeze-drying methodology was validated in an industrial setting, yielding a CPC634 freeze-dried cake with a moisture content of less than 0.1 wt%. It was found that trehalose-cryoprotected CPC634 could be rapidly reconstituted in less than 5 min at room temperature. Critical quality attributes such as size, morphology, drug retention, and release kinetics of trehalose-cryoprotected freeze-dried CPC634 upon reconstitution were identical to those of non-freeze-dried CPC634. CONCLUSION: Our findings provide proof-of-concept for the lyophilization of drug-containing CCPM and our methodology is readily translatable to large-scale manufacturing for future commercialization.

Docetaxel Skin Exposure and Micronucleation Contributes to Skin Toxicity Caused by CPC634.

Docetaxel entrapped nanoparticle CPC634 is associated with dose-related skin toxicity that resembles conventional docetaxel (Cd)-related skin toxicity. This study compared the cutaneous pharmacokinetics and pharmacodynamics of docetaxel and CPC634. In this randomised cross-over study, patients with solid tumours received one cycle of CPC634 and Cd (both at 75 mg/m2). Skin biopsies were taken at baseline and at day 8 of both cycles. Released and total docetaxel (released docetaxel plus entrapped docetaxel) concentrations and histopathological changes in the skin biopsies were evaluated. Twenty patients underwent paired skin biopsies for pharmacokinetic analysis and 10 patients had biopsies available for histopathological assessment. The total skin docetaxel concentration was 369% (95%CI: 229% to 569%, p < 0.001) higher after CPC634 administration compared to Cd while the released docetaxel concentrations were not statistically different (95%CI: -9% to 63%, p = 0.169). The CPC634 released docetaxel concentration in the skin was positively correlated with plasma concentrations (Pearson's correlation 0.48, p = 0.03). Histopathological examination revealed increased apoptosis, mitotic cells with nuclear atypia, and micronucleation with an enhanced Ki-67 index for both compounds. In conclusion, both CPC634 and Cd treatment result in docetaxel exposure in the skin causing cutaneous anti-mitotic effects such as micronucleation, which could induce an inflammatory reaction leading to skin toxicity.

TMTHSI, a superior 7-membered ring alkyne containing reagent for strain-promoted azide-alkyne cycloaddition reactions.

We describe the development of TMTH-SulfoxImine (TMTHSI) as a superior click reagent. This reagent combines a great reactivity, with small size and low hydrophobicity and compares outstandingly with existing click reagents. TMTHSI can be conveniently functionalized with a variety of linkers allowing attachment of a diversity of small molecules and (peptide, nucleic acid) biologics.

A phase I dose-escalation and pharmacokinetic study of a micellar nanoparticle with entrapped docetaxel (CPC634) in patients with advanced solid tumours.

BACKGROUND: CPC634 is docetaxel entrapped in core-cross linked polymeric micelles. In preclinical studies, CPC634 demonstrated enhanced pharmacokinetics and improved therapeutic index. This phase I dose escalation study is the first-in-human study with CPC634. METHODS: adult patients with advanced solid tumours received CPC634 intravenously either 3-weekly (Q3W) (part 1, dose range 15-100 mg/m2), 2-weekly (Q2W) (part 2, 45 mg/m2) or Q3W with dexamethasone premedication (part 3, 60 mg/m2). RESULTS: thirty-three patients were enrolled. Skin toxicity was dose limiting (DLT) at ≥60 mg/m2 in part 1 and at 45 mg/m2 in part 2 and was the most common CPC634 related grade ≥ 3 adverse event (24%). With dexamethasone premedication no DLTs were observed at 60 mg/m2 Q3W. CPC634 exhibited a dose-proportional pharmacokinetic profile. At 60 mg/m2, the plasma area under the curve was 4067.5 ± 2974.0 ng/h/mL and the peak plasma level 217.3 ± 91.9 ng/mL with a half-life of 39.7 ± 9.4 h for released docetaxel. CONCLUSION: CPC634 could be administered safely upon pretreatment with dexamethasone. Cumulative skin toxicity was the main DLT. The recommended phase 2 dose was determined at 60 mg/m2 Q3W with dexamethasone premedication.

Optical imaging of the whole-body to cellular biodistribution of clinical-stage PEG-b-pHPMA-based core-crosslinked polymeric micelles.

Core-crosslinked polymeric micelles (CCPM) based on PEG-b-pHPMA-lactate are clinically evaluated for the treatment of cancer. We macroscopically and microscopically investigated the biodistribution and target site accumulation of CCPM. To this end, fluorophore-labeled CCPM were intravenously injected in mice bearing 4T1 triple-negative breast cancer (TNBC) tumors, and their localization at the whole-body, tissue and cellular level was analyzed using multimodal and multiscale optical imaging. At the organism level, we performed non-invasive 3D micro-computed tomography-fluorescence tomography (µCT-FLT) and 2D fluorescence reflectance imaging (FRI). At the tissue and cellular level, we performed extensive immunohistochemistry, focusing primarily on cancer, endothelial and phagocytic immune cells. The CCPM achieved highly efficient tumor targeting in the 4T1 TNBC mouse model (18.6 %ID/g), with values twice as high as those in liver and spleen (9.1 and 8.9 %ID/g, respectively). Microscopic analysis of tissue slices revealed that at 48 h post injection, 67% of intratumoral CCPM were localized extracellularly. Phenotypic analyses on the remaining 33% of intracellularly accumulated CCPM showed that predominantly F4/80+ phagocytes had taken up the nanocarrier formulation. Similar uptake patterns were observed for liver and spleen. The propensity of CCPM to primarily accumulate in the extracellular space in tumors suggests that the anticancer efficacy of the formulation mainly results from sustained release of the chemotherapeutic payload in the tumor microenvironment. In addition, their high uptake by phagocytic immune cells encourages potential use for immunomodulatory anticancer therapy. Altogether, the beneficial biodistribution, efficient tumor targeting and prominent engagement of PEG-b-pHPMA-lactate-based CCPM with key cell populations underline the clinical versatility of this clinical-stage nanocarrier formulation.

Core-shell structure of degradable, thermosensitive polymeric micelles studied by small-angle neutron scattering.

The structure of assemblies of block copolymers composed of thermosensitive, biodegradable poly(N-(2-hydroxypropyl) methacrylamide-dilactate) and poly(ethylene glycol) (pHPMAmDL-b-PEG) has been studied by small-angle neutron scattering (SANS). Three amphiphilic copolymers with a fixed PEG of 5 kDa and a partially deuterated pHPMAmDL(d) block of 6700, 10400, or 21200 Da were used to form micelles in aqueous media by heating the polymeric solution from below to above the cloud point temperature (around 10 degrees C) of the thermosensitive block. Simultaneous and quantitative analysis of the scattering cross sections obtained at three different solvent contrasts is expedited using core-shell model, which assumed a homogeneous core of uniform scattering length density. The mean core radius increased from 13 to 18.5 nm with the molecular weight of the pHPMAmDL(d) block, while the thickness of the stabilizing PEG layer was around 8 nm for the three investigated assemblies. In addition, the volume fraction values of the stabilizing PEG chains in the shell are low and decreased from 31% to 14% with increasing the size of pHPMAmDL(d) block which shows that the shell layer of the assemblies is highly hydrated. The corresponding PEG chain grafting densities decreased from 0.22 to 0.11 nm-2 and the distance between PEG chains on the nanoparticles surface increased from 2.4 to 3.4 nm. The pHPMAmDL-b-PEG micelles showed a controlled instability due to hydrolysis of the lactic acid side groups in the thermosensitive block; that is, an increase of the degradation time leads to an increase of the size of the core which becomes less hydrophobic and consequently more hydrated. Neutron experiments supplied accurate information on how the size of the core and the micelle's aggregation number changed with the incubation time. This feature and the initially small size and dense structure in aqueous solution make the polymeric micelles suitable as carriers for hydrophobic drugs.

High systemic availability of core-crosslinked polymeric micelles after subcutaneous administration.

Covalent entrapment of drug molecules within core-crosslinked polymeric micelles (CCPM) represents an attractive approach to improve their therapeutic index. As an alternative to the most commonly employed intravenous (i.v.) route, subcutaneous (s.c.) administration offers the possibility of self-administration and thereby may reduce healthcare costs. The aim of this work was to assess the pharmacokinetic profile and systemic availability of drug-containing CCPM following s.c. injection. We here derivatised dexamethasone (DMS) with three different linkers, which enabled covalent attachment of this drug to the core of CCPM. The obtained DMS-containing CCPM exhibited varying drug release kinetics in vitro. Remarkably, a single dose of DMS-containing CCPM resulted in high systemic availability of about 30% following s.c. injection into the flank of healthy mice, as evidenced by an AUC between 26-37% relative to the AUC attained following i.v. injection. Although different linkers resulted in moderate variations in pharmacokinetic parameters, the overall pharmacokinetic profiles of these i.v. or s.c. administered nanomedicines were not substantially different. Next to DMS, we covalently attached paclitaxel (PTX) to the core of CCPM. Similarly, a single s.c. dose of PTX-containing CCPM resulted in high systemic availability of about 40% compared to i.v. injection and PTX (entrapped plus released) was detected in the blood for at least 3days. Importantly, the systemic availability of s.c. administered drug-containing CCPM is substantially higher than that of other nanoformulations as reported in the literature (e.g. 3% in rodents). These results demonstrate that s.c. administration is a promising route to attain high systemic availability of CCPM, enabling a potentially more patient-friendly and cost-effective treatment approach than the i.v. route.

Tailoring the physicochemical properties of core-crosslinked polymeric micelles for pharmaceutical applications.

To optimally exploit the potential of (tumor-) targeted nanomedicines, platform technologies are needed in which physicochemical and pharmaceutical properties can be tailored according to specific medical needs and applications. We here systematically customized the properties of core-crosslinked polymeric micelles (CCPM). The micelles were based on mPEG-b-pHPMAmLacn (i.e. methoxy poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl) methacrylamide-lactate]), similar to the block copolymer composition employed in CriPec® docetaxel, which is currently in phase I clinical trials. The CCPM platform was tailored with regard to size (30 to 100nm), nanocarrier degradation (1month to 1year) and drug release kinetics (10 to 90% in 1week). This was achieved by modulating the molecular weight of the block copolymer, the type and density of the crosslinking agent, and the hydrolytic sensitivity of the drug linkage, respectively. The high flexibility of CCPM facilitates the development of nanomedicinal products for specific therapeutic applications.

Thermosensitive and biodegradable polymeric micelles for paclitaxel delivery.

The preparation, release and in vitro cytotoxicity of a novel polymeric micellar formulation of paclitaxel (PTX) were investigated. The micelles consisted of an AB block copolymer of poly(N-(2-hydroxypropyl) methacrylamide lactate) and poly(ethylene glycol) (pHPMAmDL-b-PEG). Taking advantage of the thermosensitivity of pHPMAmDL-b-PEG, the loading was done by simply mixing of a small volume of a concentrated PTX solution in ethanol and an aqueous polymer solution and subsequent heating of the resulting solution above the critical micelle temperature of the polymer. PTX could be almost quantitatively loaded in the micelles up to 2 mg/mL. By dynamic light scattering and cryo-transmission electron microscopy, it was shown that PTX-loaded micelles have a mean size around 60 nm with narrow size distribution. At pH 8.8 and 37 degrees C, PTX-loaded micelles destabilized within 10 h due to the hydrolysis of the lactic acid side group of the pHPMAmDL. Because the hydrolysis of the lactic acid side groups is first order in hydroxyl ion concentration, the micelles were stable for about 200 h at physiological conditions. The presence of serum proteins did not have an adverse effect on the stability of the micelles during at least 15 h. Interestingly, the dissolution kinetics of pHPMAmDL-b-PEG micelles was retarded by incorporation of PTX, indicating a strong interaction between PTX and the pHPMAmDL block. The PTX-loaded micelles showed a release of the incorporated 70% of PTX during 20 h at 37 degrees C and at pH 7.4. PTX-loaded pHPMAmDL-b-PEG micelles showed comparable in vitro cytotoxicity against B16F10 cells compared to the Taxol standard formulation containing Cremophor EL, while pHPMAmDL-b-PEG micelles without PTX were far less toxic than the Cremophor EL vehicle. Confocal laser-scanning microscopy (CLSM) and fluorescence activated cell sorting (FACS) analysis of fluorescently labelled micelles showed that pHPMAmDL-b-PEG micelles were internalized by the B16F10 cells. The present results suggest that pHPMAmDL-b-PEG block copolymer micelles are a promising delivery system for the parenteral administration of PTX.