Accelerated Blood Clearance of PEGylated PLGA Nanoparticles Following Repeated Injections: Effects of Polymer Dose, PEG Coating, and Encapsulated Anticancer Drug

Purpose

To investigate accelerated blood clearance (ABC) induction upon repeated injections of PLGA-PEG nanoparticles as a commonly used polymeric drug carrier.

Methods

Etoposide-loaded PLGA-PEG NPs were developed and administered as the test dose to rats pre-injected with various NP treatments at certain time intervals. Pharmacokinetic parameters of etoposide and production of anti-PEG IgM antibody were evaluated.

Results

A notable ABC effect was induced by a wide range of polymer doses (0.1 to 20 mg) of empty NPs, accompanied by IgM secretion. However, a further increase in polymer dose resulted not only in the abrogation of the observed ABC induction but also in distinctly a higher value for AUC of the NPs relative to the control. The data from the PEG-negative group verified the fundamental role of PEG for ABC induction. The first injection of etoposide-containing PEGylated nanoparticles (a cell cycle phase-specific drug) produced a strong ABC phenomenon. Three sequential administrations of etoposide-loaded NPs abolished ABC, although a high level of IgM was still detected, which suggests saturation with insignificant poisoning of immune cells.

Conclusion

The presented results demonstrate the importance of clinical evaluations for PLGA-PEG nanocarriers that consider the administration schedule in multiple drug delivery, particularly in cancer chemotherapy.     

The Co-Delivery of Oxaliplatin Abrogates the Immunogenic Response to PEGylated siRNA-Lipoplex

Purpose

In vivo application of siRNA/PEGylated cationic liposome complex (lipoplex) is impeded by two main obstacles: cytokine responses and anti-PEG IgM responses to PEGylated siRNA-lipoplex. Here, we investigated whether co-administration of oxaliplatin (l-OHP) abrogates the cytokine release and anti-PEG IgM production by PEGylated siRNA-lipoplex.

Methods

Free l-OHP was administered either simultaneously or 30 min prior to PEGylated siRNA-lipoplex administration, and cytokine response and anti-PEG IgM production were evaluated. In addition, the effect of the liposomal encapsulation of l-OHP on the immunogenic response of PEGylated siRNA-lipoplex was investigated.

Results

Simultaneous co-administration of free l-OHP with PEGylated siRNA-lipoplex caused a significant reduction in anti-PEG IgM production, along with an increase in the cytokine response. Free l-OHP injected prior to the lipoplex injection, however, successfully reduced cytokine release and anti-PEG IgM response. Platination of siRNA by simultaneously administered free l-OHP might facilitate the dissociation of double-stranded siRNA to single-stranded siRNA, resulting in the inducement of a potent immuno-stimulation of siRNA via endosomal toll-like receptors (TLRs). On the other hand, encapsulation of l-OHP into the siRNA-lipoplex resulted in a reduction of both anti-PEG IgM production and cytokine responses.

Conclusions

Our results suggest that, besides the expected therapeutic efficacy of co-administration, encapsulation of l-OHP into the PEGylated siRNA-lipoplex has great potential for minimizing the immunostimulation of PEGylated siRNA-lipoplex, resulting in a safe, applicable, and compliant treatment regimen for sequential clinical administration.     

Time Interval of Two Injections and First-Dose Dependent of Accelerated Blood Clearance Phenomenon Induced by PEGylated Liposomal Gambogenic Acid: The Contribution of PEG-Specific IgM

Repeated injection of PEGylated liposomes can cause the disappearance of long circulating property because of the induction of anti-PEG IgM antibody referred to as “accelerated blood clearance (ABC) phenomenon.” Although ABC phenomenon typically occurs when entrapped drugs are chemotherapeutic agent with low cytotoxic, there is little evidence of accelerated blood clearance of PEGylated herbal-derived compound on repeated injection. Herein, we investigated the blood concentration of PEGylated liposomal gambogenic acid (PEG-GEA-L), a model PEGylated liposomal herbal extract, on its repeated injection to rats. We found time interval between injections had considerable impact on the magnitude of ABC phenomenon induced by PEG-GEA-L. When time interval was prolonged from 3 days to 7 days, ABC phenomenon could be attenuated. Furthermore, its magnitude was enhanced accompanied by a marked rise in the accumulation of PEG-GEA-L in the liver and spleen in a first-dose–dependent manner. Consistently, the level of anti-PEG IgM significantly increased with the first dose of PEG-GEA-L and decreased with the extended time interval between injections, which implies anti-PEG IgM is a major contributor to the ABC phenomenon. Notably, the increased expression of liver anti-PEG IgM was accompanied by an increased expression of efflux transporters in the induction process of the ABC phenomenon.     

A Cell Assay for Detecting Anti-PEG Immune Response against PEG-Modified Therapeutics

Purpose

Immunogenicity of PEGylated proteins and nanomedicines represents a potential impediment against their development and use in clinical settings. The purpose of this study is to develop a method for detecting anti-PEG immunity of PEGylated proteins and/or nanomedicines using flow cytometry.

Methods

The binding of fluorescence-labeled mPEG-modified liposomes to HIK-G11 cells, PEG-specific hybridoma cells, or spleen cells was evaluated by flow cytometry for detecting immunogenicity of PEGylated therapeutics.

Results

The fluorescence-labeled methoxy PEG (mPEG)-modified liposomes were efficiently bound to HIK-G11 cells. Such staining with fluorescence-labeled mPEG-modified liposomes was significantly inhibited in the presence of either non-labeled mPEG-modified liposomes or mPEG-modified ovalbumin (OVA) but not polyglycerol-modified liposomes. In addition, we found that mPEG-modified liposomes, highly immunogenic, caused proliferation of PEG-specific cells, while hydroxyl PEG-modified liposomes, less immunogenic, scarcely caused. Furthermore, after intravenous injection of mPEG-modified liposomes, the percentage of PEG-specific cells in the splenocytes, as determined by flow cytometry, corresponded well with the production level of anti-PEG antibodies, as determined by ELISA.

Conclusions

PEG-specific B cell assay we introduced may become a useful method to detect an anti-PEG immune response against PEGylated therapeutics and clarify the mechanism for anti-PEG immune responses.

     

The Mystery of Antibodies Against Polyethylene Glycol (PEG) - What do we Know?

Purpose

Recent findings demonstrated anti-PEG antibody formation in some healthy individuals and patients who have not received PEGylated biotherapeutics. Some of these findings evoked criticism because of shortcomings in the antibody assays used. To better understand this topic, we established robust antibody analytics and screened two cohorts of healthy individuals and one cohort of hemophilia patients for the expression of anti-PEG antibodies.

Methods

A flow cytometry approach and a fully validated ELISA platform were established to detect specific anti-PEG antibodies. Immunohistochemistry was used to test for potential binding of anti-PEG antibodies to human tissues.

Results

IgM and/or IgG anti-PEG antibodies are expressed by some healthy individuals and by some patients with hemophilia who have not received PEGylated biotherapeutics. These antibodies can be either transient or persistent and recognize PEGs of different sizes with or without terminal methoxy groups. Age and location of healthy individuals influence the prevalence of IgG but not of IgM antibodies. Anti-PEG antibodies do not cross-react with human tissues supporting the safety of the antibodies.

Conclusion

We confirm that some healthy individuals and some patients with hemophilia express specific antibodies against PEG which are not associated with any pathology and do not bind to human tissues.

     

Influence of cocaine administration patterns on dopamine receptor regulation

Rationale

Chronic exposure to drugs of abuse induces important modifications on neuronal systems. Increasing evidence shows that the consequences to chronic cocaine exposure can be different depending on the administration pattern.

Objectives

The aim of the present study was to evaluate the consequences of two cocaine administration patterns on dopaminergic receptor regulation.

Methods

Male Sprague–Dawley rats were injected with cocaine (20 mg/kg, i.p.) for 14 days according to an intermittent (one daily injection) or a binge (three daily injections) pattern. By autoradiography, we compared the modifications of dopamine D₁ and D₂ receptor densities in the dopaminergic systems (mesocorticolimbic and nigrostriatal) 1 (WD1) and 14 (WD14) days after the last cocaine injection.

Results

On WD1, we observed modifications of D₁ receptors after the binge cocaine treatment pattern while no modification was observed after the intermittent pattern, suggesting that multiple daily injections are needed to induce early D₁ receptor modifications. On the contrary, densities of the D₂ receptors were modified by both cocaine administration patterns, and interestingly, they were opposite depending on the administration pattern. On WD14, we observed different modifications of D₁ and D₂ receptors depending on the administration pattern, suggesting that the cocaine administration pattern promoted long-term regulations of the dopaminergic system.

Conclusion

Two cocaine administration patterns induce different modifications of the dopaminergic receptor densities.     

Milnacipran enhances the control of impulsive action by activating D1-like receptors in the infralimbic cortex

Rationale

Elevated impulsivity is often observed in patients with depression. We recently found that milnacipran, an antidepressant and a serotonin/noradrenaline reuptake inhibitor, could enhance impulse control in rats. However, the neural mechanisms underlying the effects of milnacipran on impulsive action remain unclear. Milnacipran increases not only extracellular serotonin and noradrenaline but also dopamine specifically in the medial prefrontal cortex, which is one of the brain regions responsible for impulsive action.

Objectives

Our goal was to identify whether D₁- and/or D₂-like receptors in the infralimbic cortex (IL), the ventral portion of the medial prefrontal cortex, mediates the milnacipran-enhanced impulse control in a three-choice serial reaction time task.

Methods

The rats were bilaterally injected with SCH23390, a selective D₁-like receptor antagonist (0.3 or 3 ng/side) or eticlopride, a selective D₂-like receptor antagonist (0.3 or 1 μg/side) into the IL after acute intraperitoneal administration of milnacipran (10 mg/kg).

Results

Intra-IL SCH23390 injections reversed the milnacipran-enhanced impulse control, whereas injections of eticlopride into the IL failed to block the effects of milnacipran on impulsive action.

Conclusions

This is the first report that demonstrates a critical role for D₁-like receptors of the IL in milnacipran-enhanced control of impulsive action.     

Pharmacology of intra-articular triamcinolone

Introduction

The inflammatory joint diseases of juvenile inflammatory arthritis (JIA), rheumatoid arthritis (RA) and osteoarthritis (OA): and also mild to moderate joint injury, all require a multidisciplinary approach to management. Intra-articular injections of corticosteroids have been shown to be a very beneficial adjunctive treatment in the management of the above disorders. It is, therefore, important that clinicians have a good understanding of the clinical actions of intra-articular injections.

Objective

This article explores the pharmacokinetics, pharmacodynamics, and clinical pharmacology of triamcinolone acetonide (TA) and triamcinolone hexacetonide (TH) in JIA, RA, and OA.

Methods

Literature search of TA and TH articles was conducted using key word searches in the PubMed and Google Scholar databases and through references within found articles.

Results

TA and TH intra-articular injections have been shown to provide good clinical benefit for up to 6 months and even longer. TH has been shown to decrease in the expression of citrullinated proteins, the monoclonal antibody F95, and peptidylarginine deiminase 4 in RA synovium. TA and TH intra-articular injections have a low side effect profile which is similar to other corticosteroid. They have minimal to no mineralocorticoid adverse effects and facial flushing 2–3 days post injections is the most common side effect recorded, and in almost all cases is no worse than nuisance.

Conclusion

TA and TH are useful adjunct therapies in the management of JIA, RA, OA, and mild to moderate joint injury.     

Dermal Microdialysis Technique to Evaluate the Trafficking of Surface-Modified Lipid Nanoparticles upon Topical Application

Purpose

To evaluate the skin pharmacokinetics and tissue distribution of cell penetrating peptides (CPP) modified nano-structured lipid carrier (NLC) using an in vivo dermal microdialysis (MD) technique.

Methods

Celecoxib (Cxb) encapsulated NLCs (CXBN), CPP modified CXBN (CXBN-CPP) and Cxb-Solution (CXBS) formulations were prepared and tested for in vitro skin distribution. MD was used to assess pharmacokinetic parameters of Cxb after topical application of Cxb formulations. The effect of pre-treatment with Cxb formulations was evaluated for expression of prostaglandin-E2 (PGE₂) and Interleukin-6 (IL-6) after exposure of xylene using MD. Allergic contact dermatitis (ACD) model was used to confirm in vivo therapeutic response of Cxb formulations.

Results

The cumulative permeation of Cxb in MD dialysate after 24 h for CXBN-CPP was significantly higher (p?2 and IL-6 expression compared to CXBS and CXBN (p?p?Conclusions Surface modification of NLC with CPPs can enhance the skin permeation of Cxb and MD can be used to investigate pharmacokinetics of Cxb nanoparticles in the skin.     

Effects of chronic clozapine administration on markers of arachidonic acid cascade and synaptic integrity in rat brain

Background

The mode of action of clozapine, an atypical antipsychotic approved for treating schizophrenia (SZ) and used for bipolar disorder (BD) mania, remains unclear. We tested for overlap with the actions of the mood stabilizers, lithium, carbamazepine and valproate, which downregulate arachidonic acid (AA) cascade markers in rat brain and upregulate BDNF. AA cascade markers are upregulated in BD and SZ postmortem BD brain in association with neuroinflammation and synaptic loss, while BDNF is decreased.

Methods

Rats were injected intraperitoneally with a therapeutically relevant dose of clozapine (10?mg/kg/day) or with saline for 30?days, and AA cascade and synaptic markers and BDNF were measured in the brain.

Results

Compared with saline-injected rats, chronic clozapine increased brain activity, mRNA and protein levels of docosahexaenoic acid (DHA)-selective calcium-independent phospholipase A₂ type VIA (iPLA₂), mRNA and protein levels of BDNF and of the postsynaptic marker, drebrin, while decreasing cyclooxygenase (COX) activity and concentration of prostaglandin E₂ (PGE₂), a proinflammatory AA metabolite. Activity and expression of AA-selective calcium-dependent cytosolic cPLA₂ type IVA and of secretory sPLA₂ Type II were unchanged.

Conclusions

These results show overlap with effects of mood stabilizers with regard to downregulation of COX activity and PGE₂ and to increased BDNF and suggest a common action against the reported neuropathology of BD and SZ. The increased iPLA₂ expression following clozapine suggests increased production of anti-inflammatory DHA metabolites, and, with increased BDNF and drebrin, clear neuroprotective action.     

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.     

Chronic cannabidiol treatment improves social and object recognition in double transgenic APPswe/PS1∆E9 mice

Rationale

Patients suffering from Alzheimer’s disease (AD) exhibit a decline in cognitive abilities including an inability to recognise familiar faces. Hallmark pathological changes in AD include the aggregation of amyloid-β (Aβ), tau protein hyperphosphorylation as well as pronounced neurodegeneration, neuroinflammation, neurotoxicity and oxidative damage.

Objectives

The non-psychoactive phytocannabinoid cannabidiol (CBD) exerts neuroprotective, anti-oxidant and anti-inflammatory effects and promotes neurogenesis. CBD also reverses Aβ-induced spatial memory deficits in rodents.

Materials and methods

Thus we determined the therapeutic-like effects of chronic CBD treatment (20 mg/kg, daily intraperitoneal injections for 3 weeks) on the APP_swe/PS1?E9 (APPxPS1) transgenic mouse model for AD in a number of cognitive tests, including the social preference test, the novel object recognition task and the fear conditioning paradigm. We also analysed the impact of CBD on anxiety behaviours in the elevated plus maze.

Results

Vehicle-treated APPxPS1 mice demonstrated impairments in social recognition and novel object recognition compared to wild type-like mice. Chronic CBD treatment reversed these cognitive deficits in APPxPS1 mice without affecting anxiety-related behaviours.

Conclusions

This is the first study to investigate the effect of chronic CBD treatment on cognition in an AD transgenic mouse model. Our findings suggest that CBD may have therapeutic potential for specific cognitive impairments associated with AD.     

NIR-/pH-Responsive Drug Delivery of Functionalized Single-Walled Carbon Nanotubes for Potential Application in Cancer Chemo-Photothermal Therapy

Purpose

To establish a NIR (near infrared)-/pH-responsive and sustained-release tumor-targeting drug delivery system (SWNT-PEI/DOX/NGR).

Methods

Functionalized SWNTs with polymerised polymeric poly(ethylene imine) was linked NGR (Asn-Gly-Arg) tumor-targeting peptide by DSPE-PEG2000-Maleimide via the maleimide group and sulfhydryl group of cysteine, in the end, doxorubicin (DOX) was attached to SWNT-PEI to obtain a SWNT-PEI/DOX/NGR delivery system.

Results

The SWNT-PEI/DOX/NGR delivery system has significantly sustained-release effect and the slow release of DOX in normal tissues contribute to reduced systemic toxicity, while under 808 nm NIR laser irradiation or under lower pH environment the release of DOX can be accelerated.

Conclusions

Due to hyperthermia sensitizer effect of DOX, chemo-photothermal exemplified by SWNT-PEI/DOX/NGR tumor-targeting delivery system is a promising approach to anticancer therapy in vivo or in vitro.     

Factors Influencing in Vivo Disposition of Polymeric Micelles on Multiple Administrations

l-lactic acid)₃₀ (AB-type), which accumulates in solid tumors through the enhanced permeability and retention (EPR) effect. However, lactosome on multiple administrations changed its pharmacokinetics from accumulation in tumors to liver due to the production of antilactosome IgM, which was triggered by the first administration. This phenomenon is called the accelerated blood clearance (ABC). In order to reduce the production of antilactosome IgM, a novel nanoparticle composed of (poly(sarcosine)₂₃)₃-block-poly(l-lactic acid)₃₀ (A₃B-type) was prepared. The A₃B-type lactosome at the second administration showed an in vivo disposition similar to that at the first administration due to suppression of antibody production. This study involving the AB- and A₃B-type lactosomes, with variation of conditions, revealed that the high local density of poly(sarcosine) chains of the A₃B-type lactosome should relate to the prevention of a polymeric micelle from interacting B-cell receptors.     

Effects of Ionic Strength and Sugars on the Aggregation Propensity of Monoclonal Antibodies: Influence of Colloidal and Conformational Stabilities

Purpose

To develop a general strategy for optimizing monoclonal antibody (MAb) formulations.

Methods

Colloidal stabilities of four representative MAbs solutions were assessed based on the second virial coefficient (B ₂) at 20°C and 40°C, and net charges at different NaCl concentrations, and/or in the presence of sugars. Conformational stabilities were evaluated from the unfolding temperatures. The aggregation propensities were determined at 40°C and after freeze–thawing. The electrostatic potential of antibody surfaces was simulated for the development of rational formulations.

Results

Similar B ₂ values were obtained at 20°C and 40°C, implying little dependence on temperature. B ₂ correlated quantitatively with aggregation propensities at 40°C. The net charge partly correlated with colloidal stability. Salts stabilized or destabilized MAbs, depending on repulsive or attractive interactions. Sugars improved the aggregation propensity under freeze–thaw stress through improved conformational stability. Uneven and even distributions of potential surfaces were attributed to attractive and strong repulsive electrostatic interactions.

Conclusions

Assessment of colloidal stability at the lowest ionic strength is particularly effective for the development of formulations. If necessary, salts are added to enhance the colloidal stability. Sugars further improved aggregation propensities by enhancing conformational stability. These behaviors are rationally predictable according to the surface potentials of MAbs.     

Prefrontal stimulation of GABAA receptors counteracts the corticolimbic hyperactivity produced by NMDA antagonists in the prefrontal cortex of the rat

Rationale

The hypofunction of NMDA receptors in the prefrontal cortex (PFC) has been suggested to produce corticolimbic hyperactivity through the reduction of cortical GABA transmission.

Objectives

The present study investigates the effects of injections of the NMDA antagonist 3-[(R)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP) into the PFC on (1) the release of dopamine and/or acetylcholine in the amygdala and hippocampus, (2) the levels of corticosterone in the hippocampus and (3) spontaneous motor activity. Also, the stimulation of GABA_A receptors, by prefrontal injections of muscimol, on the effects produced by NMDA antagonists on these same neurochemical, hormonal and behavioural parameters was evaluated.

Methods

Male Wistar rats were implanted with guide cannulae to perform bilateral microinjections into the PFC and microdialysis experiments in the amygdala and/or ventral hippocampus, simultaneously. Spontaneous motor activity was monitored in the open field.

Results

Injections of CPP (1???g/0.5???l) into the PFC increased dialysate concentrations of dopamine and acetylcholine in the amygdala, acetylcholine and free corticosterone in the hippocampus and also motor activity. Simultaneous injections of muscimol (0.5???g/0.5???l) into the PFC counteracted the increases of dopamine and acetylcholine in the amygdala and hippocampus and also significantly reduced the peak increase of corticosterone in the hippocampus. Injections of muscimol (0.05 and 0.5???g/0.5???l) reduced the increases of motor activity produced by prefrontal NMDA antagonists.

Conclusions

These results suggest that the hypofunction of NMDA receptors in the PFC produces corticolimbic hyperactivity through the activation of prefrontal efferent projections to subcortical/limbic areas.     

The Effect of Metal-Chelating Polymers (MCPs) for 111In Complexed via the Streptavidin-Biotin System to Trastuzumab Fab Fragments on Tumor and Normal Tissue Distribution in Mice

Purpose

To study the effects of backbone composition and charge of biotin-functionalized metal-chelating polymers (Bi-MCPs) for ¹¹¹In complexed to streptavidin (SAv)-trastuzumab Fab fragments on tumor and normal tissue localization.

Methods

Bi-MCPs were synthesized with a polyacrylamide [Bi-PAm(DTPA)₄₀], polyaspartamide [Bi-PAsp(DTPA)₃₃] or polyglutamide [Bi-PGlu(DTPA)₂₈] backbone and harboured diethylenetriaminepentaacetic acid (DTPA) chelators for ¹¹¹In. Bi-PAm(DTPA)₄₀ had a net negative charge; Bi-PAsp(DTPA)₃₃ and Bi-PGlu(DTPA)₂₈ were zwitterionic with a net neutral charge. Binding to HER2+ SKOV-3 human ovarian carcinoma cells was determined. Tissue uptake was studied in Balb/c mice by MicroSPECT/CT imaging and biodistribution studies. Tumor and normal tissue uptake of ¹¹¹In-labeled Bi-PAsp(DTPA)₃₃ or Bi-PGlu(DTPA)₂₈ complexed to SAv-Fab was evaluated 48?h post-injection in athymic mice with subcutaneous SKOV-3 xenografts.

Results

SAv-Fab complexed to MCPs bound specifically to SKOV-3 cells; but specific binding was decreased 2-fold. Liver uptake was 5?C13 fold higher for Bi-PAm(DTPA)₄₀ than Bi-PAsp(DTPA)₃₃ and Bi-PGlu(DTPA)₂₈ but was reduced by decreasing negative charges by saturation with indium. ¹¹¹In-Bi-PAsp(DTPA)₃₃ complexed to SAv-Fab accumulated in SKOV-3 tumors; low tumor uptake was found for ¹¹¹In-Bi-PGlu(DTPA)₂₈-SAv-Fab.

Conclusions

Zwitterionic MCPs composed of polyaspartamide with a net neutral charge are most desirable for constructing radioimmunoconjugates.     

An MCASE approach to the search of a cure for Parkinson's Disease.

Background

Prostaglandin E₂ (PGE₂) has multiple physiologic roles mediated by G protein coupled receptors designated E-prostanoid, or "EP" receptors. Evidence supports an important role for the EP₂ receptor in regulating fertility, vascular tone and renal function.

Results

The full-length rabbit EP₂ receptor cDNA was cloned. The encoded polypeptide contains 361 amino acid residues with seven hydrophobic domains. COS-1 cells expressing the cloned rabbit EP₂ exhibited specific [³H]PGE₂ binding with a K_d of 19.1± 1.7 nM. [³H]PGE₂ was displaced by unlabeled ligands in the following order: PGE₂>>PGD₂=PGF_2α=iloprost. Binding of [³H]PGE₂ was also displaced by EP receptor subtype selective agonists with a rank order of affinity consistent with the EP2 receptor (butaprost>AH13205>misoprostol>sulprostone). Butaprost free acid produced a concentration-dependent increase in cAMP accumulation in rabbit EP₂ transfected COS-1 cells with a half-maximal effective concentration of 480 nM. RNase protection assay revealed high expression in the ileum, spleen, and liver with lower expression in the kidney, lung, heart, uterus, adrenal gland and skeletal muscle. In situ hybridization localized EP₂ mRNA to the uterine endometrium, but showed no distinct localization in the kidney. EP2 mRNA expression along the nephron was determined by RT-PCR and its expression was present in glomeruli, MCD, tDL and CCD. In cultured cells EP2 receptor was not detected in collecting ducts but was detected in renal interstitial cells and vascular smooth muscle cells. EP2 mRNA was also detected in arteries, veins, and preglomerular vessels of the kidney.

Conclusion

EP2 expression pattern is consistent with the known functional roles for cAMP coupled PGE₂ effects in reproductive and vascular tissues and renal interstitial cells. It remains uncertain whether it is also expressed in renal tubules.     

Liquid Formulations for Stabilizing IgMs During Physical Stress and Long-Term Storage

Purpose

To develop a liquid formulation for IgMs to survive physical stress and storage.

Methods

Stabilizing formulations for 8 monoclonal immunoglobulin (IgMs) were found using differential scanning calorimetry (DSC). In these formulations, the IgMs were subjected to stress and storage and analyzed by size exclusion chromatography and fluorescence activated cell sorting. Structure was analyzed using small-angle X-ray scattering (SAXS).

Results

The highest conformational stability was found near the isoelectric point and further enhanced by addition of sorbitol, sucrose and glycine. For 2 IgMs, the pH optimum for conformational and storage stability did not correspond. Lowering the pH led to the desired storage stability. Optimized formulations prevented aggregation and fragmentation from shear stress, freeze-thaw cycles, accelerated storage and real time storage at 4°C and ?20°C for 12 months. Optimized formulations also preserved immunoreactivity for 12 months. SAXS indicated that IgM in stabilizing conditions was closer to the structural IgM model (2RCJ) and less susceptible for aggregation.

Conclusions

A long-term stabilizing formulation for 8 IgMs was found comprising 20% sorbitol and 1 M glycine at pH 5.0–5.5 which may have broad utility for other IgMs. Formulation development using DSC and accelerated storage was evaluated in this study and may be used for other proteins.