Nanoparticles for Improved Local Retention after Intra-Articular Injection into the Knee Joint

Purpose

To evaluate using cationic polymeric nanoparticles that interact with hyaluronate to form ionically cross-linked hydrogels to increase the intra-articular retention time of osteoarthritis drugs in the synovial cavity.

Methods

In vitro tests included nanoparticle release from cross-linked hydrogels using syringe and membrane dissolution tests, viscosity measurement of synovial fluid containing hydrogels, and release-rate measurement for a model active conjugated to a cationically substituted dextran using a hydrolyzable ester linkage in a sink dissolution test. Nanoparticle retention after intra-articular injection into rat knees was measured in vivo using fluorescence molecular tomography.

Results

Diffusional and convective transport of cationic nanoparticles from ionically cross-linked hydrogels formed in synovial fluid was slower in vitro than for uncharged nanoparticles. Hydrogels formed after the nanoparticles were mixed with synovial fluid did not appreciably alter the viscosity of the synovial fluid in vitro. In vitro release of a conjugated peptide from the cationic nanoparticles was approximately 20% per week. After intra-articular injection in rat knees, 70% of the nanoparticles were retained in the joint for 1 week.

Conclusions

This study demonstrates the feasibility of using cationic polymeric nanoparticles to increase the retention of therapeutic agents in articular joints for indications such as osteoarthritis.     

Safety and Systemic Exposure of Triamcinolone Acetonide Following Ultrasound-Guided Intra-Articular Injection of Triamcinolone Extended-Release or Standard Triamcinolone Acetonide in Patients with Shoulder Osteoarthritis: An Open-Label,Randomized Study

Background and ObjectivesOsteoarthritis (OA) is a major public health burden. While knee and hip joints are most commonly affected, the glenohumoral (shoulder) joint is also frequently involved. We evaluated the pharmacokinetics and safety/tolerability of triamcinolone acetonide extended-release (TA-ER) and triamcinolone acetonide crystalline suspension (TAcs) in patients with shoulder OA.MethodsIn this phase 2, randomized, open-label, single-dose study ({"type":"clinical-trial","attrs":{"text":"NCT03382262","term_id":"NCT03382262"}}NCT03382262), adults with moderately-to-severely symptomatic shoulder OA for ≥ 6 months randomly received a single ultrasound-guided intra-articular (IA) injection of TA-ER 32 mg or TAcs 40 mg. Safety was evaluated throughout 12 weeks post-injection; blood samples for pharmacokinetic evaluations were collected pre-injection and through Day 85 post-injection.ResultsAmong 25 randomized patients, 12 received TA-ER and 13 received TAcs. Most patients were female (60%), and all had moderate (72%) or severe (28%) shoulder OA. Adverse events (AEs) were reported by four (33%) patients following TA-ER and three (23%) following TAcs injection. No AE was serious or led to study discontinuation. Systemic exposure following TAcs was approximately 1.5-fold higher than that following TA-ER injection (geometric mean [GM] AUC_0–last 873,543 vs 557,602 h × pg/mL). GM C_max was also higher in TAcs- than TA-ER-treated patients (2034 vs 1283 pg/mL). Bioequivalence testing confirmed lower systemic TA exposure following TA-ER than TAcs IA injection.ConclusionThese pharmacokinetic data confirm protracted release of TA from TA-ER following IA injection in patients with shoulder OA. Lower peak and systemic TA exposure following TA-ER suggests TA-ER could potentially confer an improved systemic safety profile over TAcs.Trial Registration Number{"type":"clinical-trial","attrs":{"text":"NCT03382262","term_id":"NCT03382262"}}NCT03382262 (December 22, 2017 retrospectively registered).     

Pharmacokinetic/pharmacodynamic (PK/PD) differentiation of native and PEGylated recombinant human growth hormone (rhGH and PEG-rhGH) in the rat model of osteoarthritis

1. Osteoarthritis (OA) is a degenerative joint disease that has no FDA-approved treatment. The current standard of care does not address the regeneration of the damaged cartilage.

2. Human growth hormone (hGH) is part of the insulin-like growth factor (IGF)-1 axis. There has been preclinical data that suggest its potential regenerative property in the joint.

3. However, unformulated recombinant hGH (rhGH) is short-lived in the joint, and does not provide a desirable pharmacokinetic (PK) profile to support a clinical treatment paradigm.

4. Polyethylene glycol (PEG)ylation is a potential method to extend the half-life of rhGH in the joint.

5. The purpose of this study was to delineate the PK/PD profile of PEG-rhGH in the knee joint in a rat preclinical model of OA.

6. After intra-articular (IA) injection of 100 μg into a rat knee joint that underwent medial meniscectomy, PEG-rhGH exhibits 2-fold longer half-lives in joint than native hGH. However, PEG-rhGH has a much longer systemic exposure. IA injections of PEG-rhGH also resulted in higher levels of IGF-1 in the joint and serum when compared with native rhGH.

7. In order to develop PEG-rhGH as an IA therapeutic treatment for OA, careful dose selection is necessary to avoid systemic effects while retaining its anabolic efficacy in the joint.

     

Exploring the Solid-Form Landscape of Pharmaceutical Hydrates: Transformation Pathways of the Sodium Naproxen Anhydrate-Hydrate System

Purpose

To understand the transformation pathways amongst anhydrate/hydrate solid forms of sodium naproxen and to highlight the importance of a polymorphic dihydrate within this context.

Methods

Multi-temperature dynamic vapour sorption (DVS) analysis combined with variable-humidity X-ray powder diffraction (XRPD) to establish the transformation pathways as a function of temperature and humidity. XRPD and thermogravimetric analysis (TGA) to characterise bulk samples. Monitoring of in-situ dehydration using solid-state ¹³C CP/MAS spectroscopy.

Results

At 25°C, anhydrous sodium naproxen (AH) transforms directly to one dihydrate polymorph (DH-II). At 50°C, AH transforms stepwise to a monohydrate (MH) then to the other dihydrate polymorph (DH-I). DH-II transforms to a tetrahydrate (TH) more readily than DH-I transforms to TH. Both dihydrate polymorphs transform to the same MH.

Conclusions

The properties of the polymorphic dihydrate control the transformation pathways of sodium naproxen.     

Physicochemical Modulation of Skin Barrier by Microwave for Transdermal Drug Delivery

Purpose

To investigate mechanism of microwave enhancing drug permeation transdermally through its action on skin.

Methods

Hydrophilic pectin-sulphanilamide films, with or without oleic acid (OA), were subjected to drug release and skin permeation studies. The skins were untreated or microwave-treated, and characterized by infrared spectroscopy, Raman spectroscopy, thermal, electron microscopy and histology techniques.

Results

Skin treatment by microwave at 2450?MHz for 5?min promoted drug permeation from OA-free film without incurring skin damage. Skin treatment by microwave followed by film loaded with drug and OA resulted in permeation of all drug molecules that were released from film. Microwave exerted spacing of lipid architecture of stratum corneum into structureless domains which was unattainable by OA. It allowed OA to permeate stratum corneum and accumulate in dermis at a greater ease, and synergistically inducing lipid/keratin fluidization at hydrophobic C-H and hydrophilic O-H, N-H, C-O, C=O, C-N regimes of skin, and promoting drug permeation.

Conclusion

The microwave technology is evidently feasible for use in promotion of drug permeation across the skin barrier. It represents a new approach in transdermal drug delivery.     

The impact of treatment with hylan G-F 20 on progression to total hip arthroplasty in patients with symptomatic hip OA: a retrospective study

Abstract

Introduction:

There is scarce data available on intra-articular hyaluronan’s ability to modify the progression of osteoarthritis (OA).     

The effects of the β-lactam antibiotic,ceftriaxone, on forepaw stepping and l-DOPA-induced dyskinesia in a rodent model of Parkinson’s disease

Rationale

Glutamate receptor antagonists can improve the symptoms of Parkinson’s disease (PD) and reduce l-3,4-dihydroxyphenylalanine (l-DOPA)-induced dyskinesia (LID) in both animal models and humans, but usually produce intolerable side effects. Recent evidence suggests that upregulation of the major glutamate transporter, GLT-1, by the β-lactam antibiotic, ceftriaxone, can increase the removal of synaptic glutamate without producing noticeable side effects, and may provide an effective alternative to receptor antagonists for several neurodegenerative diseases.

Objectives

We examined whether repeated i.p. injections of ceftriaxone would, like glutamate antagonists, reduce the deficits in contralateral forepaw stepping produced by unilateral injections of 6-OHDA into the medial forebrain bundle of rats and reduce LID (as measured by abnormal involuntary movements).

Methods and results

In Experiment 1, daily injections of 100 mg/kg ceftriaxone improved contralateral forepaw stepping by 44 %, and these therapeutic effects were still apparent 29 days following the cessation of treatment. In Experiment 2, daily injections of 50 mg/kg ceftriaxone were as effective as daily injections of 10 mg/kg?l-DOPA in increasing contralateral forepaw stepping by 40 %. These therapeutic effects of ceftriaxone were decreased by an injection of 10 mg/kg of the selective GLT-1 antagonist, dihydrokainate (DHK), and were still evident 69 days after the cessation of ceftriaxone injections. Furthermore, ceftriaxone did not produce dyskinesia by itself and reduced the development, but not the expression, of LID.

Conclusions

These data suggest that ceftriaxone, by producing a long-term increase in GLT-1 function and increasing the removal of synaptic glutamate, may offer several advantages over l-DOPA as therapy for PD.     

Optimization of Stability, Encapsulation, Release, and Cross-Priming of Tumor Antigen-Containing PLGA Nanoparticles

Purpose

In order to investigate Poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NP) as potential vehicles for efficient tumor antigen (TA) delivery to dendritic cells (DC), this study aimed to optimize encapsulation/release kinetics before determining immunogenicity of antigen-containing NP.

Methods

Various techniques were used to liberate TA from cell lines. Single (gp100) and multiple (B16-tumor lysate containing gp100) antigens were encapsulated within differing molecular weight PLGA co-polymers. Differences in morphology, encapsulation/release and biologic potency were studied. Findings were adopted to encapsulate fresh tumor lysate from patients with advanced tumors and compare stimulation of tumor infiltrating lymphocytes (TIL) against that achieved by soluble lysate.

Results

Four cycles of freeze-thaw + 15 s sonication resulted in antigen-rich lysates without the need for toxic detergents or protease inhibitors. The 80KDa polymer resulted in maximal release of payload and favorable production of immunostimulatory IL-2 and IFN-γ. NP-mediated antigen delivery led to increased IFN-γ and decreased immunoinhibitory IL-10 synthesis when compared to soluble lysate.

Conclusions

Four cycles of freeze-thaw followed by 15 s sonication is the ideal technique to obtain complex TA for encapsulation. The 80KDa polymer has the most promising combination of release kinetics and biologic potency. Encapsulated antigens are immunogenic and evoke favorable TIL-mediated anti-tumor responses.     

Neural substrates of acupuncture in the modulation of cravings induced by smoking-related visual cues: an FMRI study

Rationale

Cue reactivity is a key factor in modulating motivational and goal-directed behaviors associated with compulsive drug intake and relapse. Smoking-associated cues produce smoking urges and cravings and are accompanied by the activation of brain regions involved in attention, motivation, and reward.

Objectives

We investigated whether acupuncture ameliorates cravings induced by smoking-related visual cues, and we explored the neural mechanisms underlying the effects of acupuncture on modulating smoking urges.

Methods

After 36 h of smoking abstinence, 25 right-handed male smokers underwent fMRI, during which smoking-related and neutral visual cues were presented. Twelve subjects were treated with real acupuncture (RA) at HT7 and 13 subjects received sham acupuncture (SA). During the scanning sessions, craving scores to smoking-related visual cues were assessed before and after RA or SA treatment. The differences in brain responses to smoking vs. neutral cues after treatment between the RA and SA groups were detected using three-way ANOVAs (Cue × Session × Group).

Results

After treatment, the craving scores were significantly decreased in the RA group, as compared to the SA group. When we explored the neural substrates of acupuncture on the modulation of cravings induced by smoking cues, significant differences were found in the medial prefrontal cortex, the premotor cortex, the amygdala, the hippocampus, and the thalamus.

Conclusions

These findings suggest that acupuncture alleviates cue-induced cravings through the regulation of activity in brain regions involved in attention, motivation, and reward relative to craving scores in the initial abstinence phase.     

Repeated cocaine administration increases nitric oxide efflux in the rat dorsal striatum

Rationale

Repeated injections of cocaine alter extracellular nitric oxide (NO) efflux via interactions between dopamine and glutamate receptor-coupled signaling cascades.

Objectives

Putative cellular mechanisms underlying changes in NO efflux following repeated cocaine administration were investigated.

Methods

Real-time detection of NO efflux using a NO biosensor was mainly performed in the rat dorsal striatum in vivo.

Results

Repeated exposure to cocaine (20 mg/kg), once a day for seven consecutive days, increased NO levels. Repeated injections of cocaine also increased the phosphorylation of neuronal nitric oxide synthase (nNOS), and inhibition of nNOS decreased the repeated cocaine-evoked increases in NO levels. Inhibition of protein kinase A, but not protein phosphatases, synergistically increased NO levels elevated by repeated cocaine injections. Blockade of dopamine D1 (D1) receptors or stimulation of dopamine D2 (D2) receptors decreased the repeated cocaine-evoked increases in NO levels. Similarly, blockade of N-methyl-d-aspartate (NMDA) receptors and group I metabotropic glutamate receptors (mGluRs) or stimulation of group III mGluRs also decreased the repeated cocaine-evoked increases in NO levels.

Conclusion

Stimulation of D1 receptors or group I mGluRs following repeated cocaine administration upregulates NO efflux via an NMDA receptor-evoked Ca²⁺ influx, while stimulation of D2 receptors or group III mGluRs downregulates NO efflux. Dephosphorylation of phosphorylated nNOS by protein phosphatases is necessary for upregulating NO efflux in the dorsal striatum after repeated cocaine administration.     

Mifepristone treatment affects the response to repeated amphetamine injections, but does not attenuate the expression of sensitization

Rationale

Glucocorticoid hormones facilitate sensitization to repeated administration of psychostimulants, an effect that is mediated by glucocorticoid receptors (GRs). It is still unclear, however, at which stage of psychomotor sensitization are stress and GR-mediated effects involved.

Objectives

In the present study, we have tested the hypothesis that GR-mediated effects during the phase of repeated amphetamine injections play a crucial role in the long-term expression of sensitization. For this purpose, we used DBA/2 mice, an inbred strain commonly used for the study of stress effects on psychostimulant sensitization.

Methods

Animals were treated with the GR antagonist mifepristone (200 mg/kg) at 2.5 h before each daily injection of amphetamine (2.5 mg/kg) or saline in a 5-day protocol. The amphetamine or saline injections were given in the home or a novel context. This was followed by a 2.5-week withdrawal period, without any drug delivery. Following the withdrawal period, two low-dose amphetamine challenges (1.25 mg/kg) were given subsequently, without additional mifepristone.

Results

The animals receiving amphetamine in the novel context showed a higher expression of sensitization at challenge as compared to those in the home condition. Mifepristone treatment influenced locomotor response to repeated amphetamine injections, but this effect during the initial phase did not affect the expression of sensitization after a withdrawal period.

Conclusion

Our results indicate that GR-related processes during the initial phase of sensitization are involved in, but not crucial for, the development of long-term sensitization.     

Inhibition of CaV3.2 T-type calcium channels in peripheral sensory neurons contributes to analgesic properties of epipregnanolone

Rationale

T-type calcium channels (T-channels) play an important role in controlling excitability of nociceptors. We have previously shown that a synthetic series of 5β-reduced steroids induce a voltage-dependent blockade of T-currents in rat dorsal root ganglia (DRG) cells in vitro and induce potent analgesia to thermal stimuli in rats in vivo (Mol Pharmacol 66:1223–1235, 2004).

Objectives

Here, we investigated the effects of the endogenous 5β-reduced neuroactive steroid molecule, epipregnanolone [(3β,5β)-3-hydroxypregnan-20-one], on peripheral nociception.

Methods

We used acutely dissociated DRG cells in vitro from adult rats as well as in vivo pain studies in mice and rats to investigate the effects of epipregnanolone on DRG T-channels.

Results

We found that epipregnanolone reversibly blocked DRG T-currents with a half-maximal inhibitory concentration (IC₅₀) of 2 μM and stabilized the channel in the inactive state. However, sodium, potassium, and gamma-aminobutyric acid (GABA)-gated ionic currents were not sensitive to the blocking effects of epipregnanolone even at 10 μM. In ensuing in vivo studies, we found that intraplantar (i.pl.) injections of epipregnanolone directly into peripheral receptive fields reduced responses to nociceptive heat stimuli in rats in a dose-dependent fashion. Furthermore, i.pl. epipregnanolone injections effectively reduced responses to peripheral nociceptive thermal and mechanical stimuli in wild-type mice but had no effect on the responses of Ca_V3.2 knockout mice.

Conclusions

We conclude that the inhibition of peripheral Ca_V3.2 T-channels contributes to the potent analgesic effect of the endogenous steroid epipregnanolone.     

The GABAB receptor agonist baclofen administered into the median and dorsal raphe nuclei is rewarding as shown by intracranial self-administration and conditioned place preference in rats

Rationale

The midbrain raphe regions have long been implicated in affective processes and disorders. There is increasing evidence to suggest that the median (MR) and dorsal raphe nuclei (DR) tonically inhibit reward-related processes.

Objectives

Stimulation of GABA_B receptors in the midbrain raphe nuclei is known to inhibit local neurons, especially serotonergic neurons. We sought to determine if injections of the GABA_B receptor agonist baclofen into the MR or DR are rewarding, using intracranial self-administration and conditioned place preference.

Results

Rats quickly learned to lever press for infusions of baclofen (0.1–2.5 mM) into the MR, but not the ventral tegmental area or central linear nucleus. Rats increased lever pressing associated with intra-DR baclofen infusions, but not readily. Baclofen self-administration into the MR or DR was attenuated by coadministration of the GABA_B receptor antagonist SCH 50911 (1 mM) or systemic pretreatment with the dopamine receptor antagonist SCH 23390 (0.025 mg/kg, i.p.). In addition, intra-DR and intra-MR injections of baclofen induced conditioned place preference; injection into DR was more effective.

Conclusions

Baclofen injections into the midbrain raphe nuclei are rewarding. Baclofen was more readily self-administered into the MR than into the DR, while baclofen injections into the DR more readily induced conditioned place preference than those into the MR. These sites may be differentially involved in aspects of reward. These findings suggest that MR or DR neurons containing GABA_B receptors are involved in tonic inhibitory control over reward processes.     

An investigation of flumazenil to antagonize gamma-hydroxybutyrate intoxication in a murine model

Objective

Prior animal studies have suggested that flumazenil may blunt GHB’s sedative-hypnotic affects. We hypothesized that flumazenil would decrease the affects of GHB in a murine model of intoxication.

Methods

We performed a controlled, pilot experiment using 32 mice divided into 3 groups. All mice received intraperitoneal injections of GHB (1.5 g/kg). Group I received sham injections at time 0, and then GHB at 5 minutes. Group II received flumazenil (0.3 mg/kg) at time 0, and then GHB at 5 minutes. Group III received sham injection at time 0, then GHB at 5 minutes, and then 4 escalating flumazenil doses administered at 3-minute intervals (0.003 to 1 mg/kg). We measured certain functions: time to loss/recovery of righting reflexes (RR), time to sprawl/recovery of sprawl (postural tone [PT]), and death.

Results

There were statistically significant delays in the loss of PT and shortened recovery time to RR in pre-treated mice (group II) versus controls (group I). There were no differences in group III versus group I for any outcome parameters.

Conclusions

In this model, pre-dosing flumazenil prior to GHB administration delayed clinical intoxication.     

Quinaldic acid in synovial fluid of patients with rheumatoid arthritis and osteoarthritis and its effect on synoviocytes in vitro

Background

Previously, we have demonstrated that kynurenic acid (KYNA), an endogenous metabolite of tryptophan formed along kynurenine pathway, is present in synovial fluid of rheumatoid arthritis (RA) and osteoarthritis (OA) patients. In this study, the goal was to investigate the presence of quinaldic acid (QUDA), a putative metabolite of KYNA, in synovial fluid of RA and OA patients.

In Vitro Efficacy of Polysaccharide-Based Nanoparticles Containing Disease-Modifying Antirheumatic Drugs

Purpose

To evaluate the therapeutic efficacy of dexamethasone (DM) and methotrexate (MTX) entrapped within polysialic acid (PSA)-trimethyl chitosan (TMC) nanoparticles using an in vitro model of rheumatoid arthritis (RA).

Methods

The loading capacity of the PSA-TMC nanoparticles was determined. An RA in vitro model was developed by stimulating a synovial cell line with a proinflammatory mediator. Multiplex immunoassay was used to determine changes in the secretion of interleukin-6 (IL-6), interleukin-8 (IL-8), and granulocyte-macrophage colony-stimulating factor (GM-CSF) by the in vitro model following administration of the DM- and MTX-loaded nanoparticles.

Results

The loading capacity of the PSA-TMC nanoparticles was approximately 0.1 mg of drug/mg of nanoparticle. When applied to our in vitro model of RA, there were no significant differences in the concentrations of IL-6 and IL-8 when comparing the free drugs and drug-loaded nanoparticles, administered at concentration of 0.1 mg/ml and 1.0 mg/ml, respectively.

Conclusions

The present study verified that MTX and DM are able to retain bioactivity when loaded into PSA-TMC nanoparticles. Although in vitro efficacy was not increased, the in vivo efficacy will likely be enhanced by the site-specific targeting conferred by nanoparticle entrapment.     

Acquisition of responding with a remifentanil-associated conditioned reinforcer in the rat

Rationale

Drug-associated environmental stimuli may serve as conditioned reinforcers to enhance drug self-administration behaviors in humans and laboratory animals. However, it can be difficult to distinguish experimentally the conditioned reinforcing effects of a stimulus from other behavioral processes that can change rates of responding.

Objectives

To characterize the conditioned reinforcing effects of a stimulus paired with the μ-opioid agonist, remifentanil, using a new-response acquisition procedure in the rat.

Methods

First, in Pavlovian conditioning (PAV) sessions, rats received response-independent IV injections of remifentanil and presentations of a light-noise compound stimulus. In paired PAV groups, injections and stimulus presentations always co-occurred. In random PAV control groups, injections and stimulus presentations occurred with no consistent relationship. Second, in instrumental acquisition (ACQ) sessions, all animals could respond in an active nose-poke that produced the stimulus alone or in an inactive nose-poke that had no scheduled consequences.

Results

During ACQ, rats made significantly more active nose-pokes than inactive nose-pokes after paired PAV, but not after random PAV. Between groups, rats also made more active nose-pokes after paired PAV than after random PAV. After paired PAV, increased active responding was obtained under different schedules of reinforcement, persisted across multiple ACQ sessions, and depended on the number of PAV sessions conducted.

Conclusions

The remifentanil-paired stimulus served as a conditioned reinforcer for nose-poking: responding depended on both the contingency between the stimulus and remifentanil and the contingency between the nose-poke and the stimulus. Generally, new-response acquisition procedures may provide valid, flexible models for studying opioid-based conditioned reinforcement.     

Effects of acute and sub-chronic nicotine on impulsive choice in rats in a probabilistic delay-discounting task

Rationale

Cigarette smokers typically display impulsivity by preferring immediate rewards over larger, delayed rewards at shorter delays than do non-smokers. Suggesting causality, nicotine injections in rats increase the choice for an immediate reward over a larger, delayed reward.

Objectives

To examine the generality of this latter effect, the present study employed a delay-discounting task to determine if acute and sub-chronic nicotine will also increase impulsive choice when subjective reward value is manipulated by changes in the probability, rather than magnitude, of reward.

Materials and methods

Rats were presented with two levers, one of which delivered an immediate water reward on half of the trials, while the other lever delivered the same reward on every trial, but only after one of five increasing delays.

Results

Acute injections of 1.2 mg/kg, but not 0.8 mg/kg, of nicotine increased the preference for the immediate (but less certain) reward lever at intermediate delays. Moreover, twice-daily injections of 0.8 mg/kg of nicotine for 6 days progressively increased the preference for the immediate reward. Latency to make the first response on each trial was not affected by nicotine.

Conclusions

The similar increases in impulsive choice produced by both acute and sub-chronic nicotine in delay-discounting paradigms whether subjective reward value is manipulated by changes in reward magnitude or probability suggests that nicotine may be increasing what is common to these paradigms, namely delay discounting. Whatever the mechanism, these data indicate that both acute and sub-chronic nicotine may help develop and maintain an addiction by increasing impulsivity.     

Accelerated Blood Clearance Phenomenon Upon Repeated Injection of PEG-modified PLA-nanoparticles

Purpose

We recently developed prostaglandin E₁ (PGE₁)-encapsulated nanoparticles, prepared with a poly(lactide) homopolymer (PLA, Mw?=?17,500) and monomethoxy poly(ethyleneglycol)-PLA block copolymer (PEG-PLA) (NP-L20). In this study, we tested whether the accelerated blood clearance (ABC) phenomenon is observed with NP-L20 and other PEG-modified PLA-nanoparticles in rats.

Methods

The plasma levels of PGE₁ and anti-PEG IgM antibody were determined by EIA and ELISA, respectively.

Results

Second injections of NP-L20 were cleared much more rapidly from the circulation than first injections, showing that the ABC phenomenon was induced. This ABC phenomenon, and the accompanying induction of anti-PEG IgM antibody production, was optimal at a time interval of 7 days between the first and second injections. Compared to NP-L20, NP-L33s that were prepared with PLA (Mw?=?28,100) and have a smaller particle size induced production of anti-PEG IgM antibody to a lesser extent. NP-L20 but not NP-L33s gave rise to the ABC phenomenon with a time interval of 14 days. NP-L33s showed a better sustained-release profile of PGE₁ than NP-L20.

Conclusions

This study revealed that the ABC phenomenon is induced by PEG-modified PLA-nanoparticles. We consider that NP-L33s may be useful clinically for the sustained-release and targeted delivery of PGE₁.     

Breaking the Aggregation of the Monoclonal Antibody Bevacizumab (Avastin®) by Dexamethasone Phosphate: Insights from Molecular Modelling and Asymmetrical Flow Field-Flow Fractionation

Purpose

To investigate the mechanism behind the aggregation breaking properties of dexamethasone phosphate and related corticosteroids on the IgG1 antibody bevacizumab (Avastin®).

Methods

An in silico 3D dimer model is developed to identify the bevacizumab-bevacizumab interface, and different corticosteroids are docked onto the model to distinguish preferred binding sites. In silico predictions are validated by in vitro stability studies, where the antibody is stressed in presence or absence of each corticosteroid and formed aggregates are quantified by asymmetrical flow field-flow fractionation.

Results

The dimer model features one close crystal contact area: Lys445 on the Fc region interacts with one Fab arm of the second bevacizumab. Docking reveals an interaction between the phosphate group of dexamethasone phosphate and Lys445, while the rest of the molecule is hindering dimer formation. Predictions are confirmed in vitro, demonstrating that dexamethasone phosphate and betamethasone phosphate partly prevent antibody aggregation, whereas triamcinolone acetonide phosphate does not.

Conclusions

Results suggest that bevacizumab monomers follow a specific mechanism to form dimers in which a protein-protein interaction hotspot can be distinguished. The dimer formation can be hindered by corticosteroids in a specific way. This approach allows a simple way to stabilize IgG1 antibodies.