Monoclonal Antibodies Follow Distinct Aggregation Pathways During Production-Relevant Acidic Incubation and Neutralization

Purpose

Aggregation aspects of therapeutic monoclonal antibodies (mAbs) are of common concern to the pharmaceutical industry. Low pH treatment is applied during affinity purification and to inactivate endogenous retroviruses, directing interest to the mechanisms of acid-induced antibody aggregation.

Methods

We characterized the oligomerization kinetics at pH 3.3, as well as the reversibility upon neutralization, of three model mAbs with identical variable regions, representative of IgG1, IgG2 and IgG4 respectively. We applied size-exclusion high performance liquid chromatography and orthogonal analytical methods, including small-angle X-ray scattering and dynamic light scattering and supplemented the experimental data with crystal structure-based spatial aggregation propensity (SAP) calculations.

Results

We revealed distinct solution behaviors between the three mAb models: At acidic pH IgG1 retained monomeric, whereas IgG2 and IgG4 exhibited two-phase oligomerization processes. After neutralization, IgG2 oligomers partially reverted to the monomeric state, while on the contrary, IgG4 oligomers tended to aggregate. Subclass-specific aggregation-prone motifs on the Fc fragments were identified, which may lead to two distinct pathways of reversible and irreversible aggregation, respectively.

Conclusions

We conclude that subtle variations in mAb sequence greatly affect responses towards low-pH incubation and subsequent neutralization, and demonstrate how orthogonal biophysical methods distinguish between reversible and irreversible mAb aggregation pathways at early stages of acidic treatment.

     

Probing Conformational Diversity of Fc Domains in Aggregation-Prone Monoclonal Antibodies

Purpose

Fc domains are an integral component of monoclonal antibodies (mAbs) and Fc-based fusion proteins. Engineering mutations in the Fc domain is a common approach to achieve desired effector function and clinical efficacy of therapeutic mAbs. It remains debatable, however, whether molecular engineering either by changing glycosylation patterns or by amino acid mutation in Fc domain could impact the higher order structure of Fc domain potentially leading to increased aggregation propensities in mAbs.

Methods

Here, we use NMR fingerprinting analysis of Fc domains, generated from selected Pfizer mAbs with similar glycosylation patterns, to address this question. Specifically, we use high resolution 2D [¹³C-¹H] NMR spectra of Fc fragments, which fingerprints methyl sidechain bearing residues, to probe the correlation of higher order structure with the storage stability of mAbs. Thermal calorimetric studies were also performed to assess the stability of mAb fragments.

Results

Unlike NMR fingerprinting, thermal melting temperature as obtained from calorimetric studies for the intact mAbs and fragments (Fc and Fab), did not reveal any correlation with the aggregation propensities of mAbs. Despite >97% sequence homology, NMR data suggests that higher order structure of Fc domains could be dynamic and may result in unique conformation(s) in solution.

Conclusion

The overall glycosylation pattern of these mAbs being similar, these conformation(s) could be linked to the inherent plasticity of the Fc domain, and may act as early transients to the overall aggregation of mAbs.

     

Formulation Stabilization and Disaggregation of Bevacizumab,Ranibizumab and Aflibercept in Dilute Solutions

Purpose

Studies were conducted to investigate dilute solutions of the monoclonal antibody (mAb) bevacizumab, mAb fragment ranibizumab and fusion protein aflibercept, develop common procedures for formulation of low concentration mAbs and identify a stabilizing formulation for anti-VEGF mAbs for use in in vitro permeation studies.

Methods

Excipient substitutions were screened. The most stabilizing formulation was chosen. Standard dilutions of bevacizumab, ranibizumab and aflibercept were prepared in PBS, manufacturer’s formulation, and the new formulation. Analysis was by SE-HPLC and ELISA. Stability, disaggregation and pre-exposure tests were studied.

Results

When Avastin, Lucentis and Eylea are diluted in PBS or manufacturer’s formulation, there is a 40–50% loss of monomer concentration and drug activity. A formulation containing 0.3% NaCl, 7.5% trehalose, 10 mM arginine and 0.04% Tween 80 at a pH of 6.78 stabilized the mAbs and minimized the drug loss. The formulation also disaggregates mAb aggregation while preserving the activity. Degassing the formulation increases recovery.

Conclusions

We developed a novel formulation that significantly stabilizes mAbs under unfavorable conditions such as low concentration or body temperature. The formulation allows for tissue permeation experimentation. The formulation also exhibits a disaggregating effect on mAbs, which can be applied to the manufacture/packaging of mAbs and bioassay reagents.

     

High Throughput Differential Scanning Fluorimetry (DSF) Formulation Screening with Complementary Dyes to Assess Protein Unfolding and Aggregation in Presence of Surfactants

Purpose

The purpose was to evaluate DSF for high throughput screening of protein thermal stability (unfolding/ aggregation) across a wide range of formulations. Particular focus was exploring PROTEOSTAT® – a commercially available fluorescent rotor dye – for detection of aggregation in surfactant containing formulations. Commonly used hydrophobic dyes (e.g. SYPRO? Orange) interact with surfactants, complicating DSF measurements.

Methods

CRM197 formulations were prepared and analyzed in standard 96-well plate rT-PCR system, using SYPRO? Orange and PROTEOSTAT® dyes. Orthogonal techniques (DLS and IPF) are employed to confirm unfolding/aggregation in selected formulations. Selected formulations are subjected to non-thermal stresses (stirring and shaking) in plate based format to characterize aggregation with PROTEOSTAT®.

Results

Agreement is observed between SYPRO? Orange (unfolding) and PROTEOSTAT® (aggregation) DSF melt temperatures across wide range of non-surfactant formulations. PROTEOSTAT® can clearly detect temperature induced aggregation in low concentration (0.2 mg/mL) CRM197 formulations containing surfactant. PROTEOSTAT® can be used to explore aggregation due to non-thermal stresses in plate based format amenable to high throughput screening.

Conclusions

DSF measurements with complementary extrinsic dyes (PROTEOSTAT®, SYPRO? Orange) are suitable for high throughput screening of antigen thermal stability, across a wide range of relevant formulation conditions – including surfactants –with standard, plate based rT-PCR instrumentation.

     

Comprehensive Characterization of Relationship Between Higher-Order Structure and FcRn Binding Affinity of Stress-Exposed Monoclonal Antibodies

Purpose

In biopharmaceutical development, information regarding higher-order structure (HOS) is important to verify quality and characterize protein derivatives. In this study, we aimed to characterize the association between HOS and pharmacokinetic property of a stress-exposed monoclonal antibody (mAb).

Methods

Purity, primary structure, thermal stability, and HOS were evaluated for mAbs exposed to heat, photo-irradiation, and chemical oxidation. To investigate conformation of stress-exposed mAbs, hydrogen/deuterium exchange coupled with mass spectrometry (HDX–MS) was utilized.

Results

No distinct difference in secondary or tertiary structure between stress-exposed and non-stressed samples was found by conventional spectroscopic techniques. In binding activity with the neonatal Fc receptor (FcRn), however, a marked decline was observed for force-oxidized mAb and a slight decline was observed for heat- and photodegraded mAbs. Using differential scanning calorimetry, a change in thermal stability was observed in the C_H2 domain for all the stress-exposed samples. Using HDX–MS analyses, individual regions with altered conformation could be identified for heat-degraded and force-oxidized samples.

Conclusions

These findings indicate that comprehensive study is important for detecting conformational changes and helpful for predicting biophysical property, and that the evaluation of HOS using several analytical techniques is indispensable for confirming biopharmaceutical quality.

     

Challenges in Determining Intrinsic Viscosity Under Low Ionic Strength Solution Conditions

Purpose

To determine the intrinsic viscosity of several monoclonal antibodies (mAbs) under varying pH and ionic strength solution conditions.

Methods

An online viscosity detector attached to HPLC (Viscotek®) was used to determine the intrinsic viscosity of mAbs. The Ross and Minton equation was used for viscosity prediction at high protein concentrations. Bulk viscosity was determined by a Cambridge viscometer.

Results

At 15 mM ionic strength, intrinsic viscosity of the mAbs determined by the single-point approach varied from 5.6 to 6.4 mL/g with changes in pH. High ionic strength did not significantly alter intrinsic viscosity, while a significant increase (up to 24.0 mL/g) was observed near zero mM. No difference in bulk viscosity of mAb3 was observed around pH 6 as a function of ionic strength. Data analysis revealed that near zero mM ionic strength limitations of the single-point technique result in erroneously high intrinsic viscosity.

Conclusions

Intrinsic viscosity is a valuable tool that can be used to model baseline viscosity at higher protein concentrations. However, it is not predictive of solution non-ideality at higher protein concentrations. Furthermore, breakdown of numerous assumptions limits the applicability of experimental techniques near zero mM ionic strength conditions. For molecules and conditions studied, the single-point approach produced reliable intrinsic viscosity results at 15 mM. However, this approach must be used with caution near zero mM ionic strength. Data analysis can be used to reveal whether determined intrinsic viscosity is reliable or erroneously high.

     

The Use of Surfactants to Solubilise a Glucagon Analogue

Purpose

The peptide hormone glucagon, used to treat hypoglycaemic incidents, is prone to aggregation. Generating alternatives with better stability is of pharmaceutical interest in the treatment of diabetes. Here we investigate the impact of six different surfactants on the solubility and stability of ZP-GA-1, a stable version of glucagon.

Methods

We use chemical surfactants (sodium dodecyl sulphate, dodecyl maltoside and polysorbate 20) and the biosurfactants rhamnolipid, sophorolipid and surfactin. We investigate their interaction with ZP-GA-1 by pyrene fluorescence, circular dichroism and isothermal titration calorimetry.

Results

All six surfactants induce α-helical structure in ZP-GA-1, SDS having the biggest impact and polysorbate 20 the smallest. SDS keeps ZP-GA-1 solubilised over >48 days as opposed to 29 days in DDM, 3 days in polysorbate 20 and 0 days in buffer. Similarly, much less SDS than DDM, polysorbate 20 or biosurfactant is needed to redissolve aggregated ZP-GA-1. ITC confirms this trend, with SDS exhibiting very strong, and polysorbate 20 very weak interactions.

Conclusion

Simple surfactant structures promote stronger peptide interactions. ITC shows promise as a general strategy to predict surfactants’ solubilising powers. Stronger enthalpic interactions improved the absolute solubility of ZP-GA-1 and their strength correlated to the absolute solubility of the peptides though not to the kinetics of precipitation.

     

Experimental Model System to Study pH Shift-Induced Aggregation of Monoclonal Antibodies Under Controlled Conditions

Purpose

To present a convenient screening method for evaluating additive effects on the renaturation of an acid-exposed monoclonal antibody (mAb).

Methods

The assay involves brief incubation of a mAb at acidic pH and subsequent neutralization in the absence or presence of additive to induce mainly aggregation. An increase in absorbance depicted aggregation. The recorded aggregation data traces were fitted with a nucleation-autocatalytic growth model for the extraction of kinetic parameters.

Results

All kinetic data traces were fitted successfully with the selected model and the adjusted R square values were greater than 0.99. Trehalose had strongly stabilizing, proline mildly stabilizing and trimethylamine oxide had destabilizing effects on both the nucleation and growth phase of the reaction. Histidine was strongly stabilizing but was limited by its poor solubility.

Conclusion

The results demonstrate the suitability of the experimental mAb aggregation system and the nucleation-autocatalytic growth fit in the screening and quantification of additive effects on the renaturation of an acid-exposed mAb respectively. This will aid the investigation and derivation of quantitative structure-activity relationships of additive effects on mAb solubility.

     

First Infusion Reactions are Mediated by FcγRIIIb and Neutrophils

Purpose

Administration of therapeutic monoclonal antibodies (mAbs) is frequently accompanied by severe first infusion reactions (FIR). The mechanism driving FIR is still unclear. This study aimed to investigate the cellular and molecular mechanisms causing FIR in humanized mouse models and their potential for evaluating FIR risk in patients.

Methods

Mice humanized for Fc gamma receptors (FcγRs) were generated by recombination-mediated genomic replacement. Body temperature, cytokine release and reactive oxygen species (ROS) were measured to assess FIR to mAbs.

Results

Infusion of human mAb specific for mouse transferrin receptor (HamTfR) into FcγR-humanized mice, produced marked transient hypothermia accompanied by an increase in inflammatory cytokines KC and MIP-2, and ROS. FIR were dependent on administration route and Fc-triggered effector functions mediated by neutrophils. Human neutrophils also induced FIR in wild type mice infused with HamTfR. Specific knock-in mice demonstrated that human FcγRIIIb on neutrophils was both necessary and sufficient to cause FIR. FcγRIIIb-mediated FIR was abolished by depleting neutrophils or blocking FcγRIIIb with CD11b antibodies.

Conclusions

Human FcγRIIIb and neutrophils are primarily responsible for triggering FIR. Clinical strategies to prevent FIR in patients should focus on this pathway and may include transient depletion of neutrophils or blocking FcγRIIIb with specific mAbs.

     

Effects of Histidine and Sucrose on the Biophysical Properties of a Monoclonal Antibody

Purpose

Histidine is a commonly used buffer in formulation of monoclonal antibodies (mAb), often with excipients like sucrose. The objective of this study was to examine the effects of both histidine and sucrose on the biophysical characteristics of a mAb.

Methods

The hydrodynamic radius of the mAb was determined by dynamic light scattering and confirmed by size exclusion chromatography. Data were also obtained for the osmotic virial coefficients (from osmotic pressure measurements), the solution viscosity, and the mAb thermal stability (using differential scanning calorimetry) at selected conditions.

Results

There were no significant changes in mAb conformation / stability as determined by DSC. The hydrodynamic radius initially increased with increasing histidine concentration, going through a maximum at a histidine concentration of about 20 mM. The addition of sucrose increased the mAb hydrodynamic radius at all histidine concentrations by about 0.5 nm. The observed effects of histidine and sucrose on the hydrodynamic radius were also reflected in changes in the osmotic pressure and solution viscosity.

Conclusions

These results provide important insights into the effects of both histidine and sucrose on the behavior of concentrated mAb solutions, including the potential impact on ultrafiltration / diafiltration processes.

     

Scanning Electron Microscope Observations of Powder Sticking on Punches during a Limited Number (<Emphasis Type="BoldItalic">N</Emphasis> &lt; 5) of Compactions of Acetylsalicylic Acid

Purpose

To obtain quantitative information and mechanistic insight into the problem of sticking of acetylsalicylic acid tablets on a metallic punch.

Methods

Low voltage scanning electron microscopy was used to observe punch area coverage and morphology of adhered powder on a flat punch used for a limited number of compactions.

Results

Material accumulation in terms of area coverage of the punch per compaction cycle was determined at two pressures over five compactions. The distribution of the adhered material on the punch was non-uniform with more material left on the center of the punch. The sizes of the adhered particles range from 1 to 100 μm, with 50% of the punch surface coverage from particles of an equivalent diameter > 30 μm. Three types of adhered particles were identified after the first compaction: (a) fragments of initial particles with very high aspect ratio, (b) nearly equiaxed fragments with multiple cracks, (c) heavily deformed islands of low profile. Some preliminary ideas that explain these observations are presented and discussed.

Conclusions

The ability of SEM to provide quantitative information on sticking from few compactions presents an interesting possibility for a material sparing technique that provides insight on the propensity of sticking.

     

Modulating Sticking Propensity of Pharmaceuticals Through Excipient Selection in a Direct Compression Tablet Formulation

Purpose

To investigate how excipient matrix affects punch sticking propensity of active pharmaceutical ingredients (API), with the focus on the effect of bonding interactions between API-API (F2) and API-excipient (F3).

Method

Sticking kinetics of direct compression formulations, consisting of 20% of celecoxib (CEL) or ibuprofen (IBN) in different excipient matrices, i.e., microcrystalline cellulose (Avicel PH102 and Avicel PH105 dry coated with nano-sized silica (PH105(n)), hypromellose (K15 M), and a 3:1 mixture between starch and Avicel PH102 (S3P1), was assessed using a removable punch tip on a compaction simulator. The amount of material transferred to punch was determined gravimetrically every 10 compressions up to 50 compactions.

Results

CEL exhibited higher F2 than IBN. CEL also exhibited more sticking under otherwise identical compaction conditions in the same excipient matrix. Among different excipient matrices, sticking propensity of both APIs followed the ascending order: PH105(n)?<?PH102?<?K15 M?<?S3P1. This order was exactly opposite to the order of F3, confirming that greater bonding strength of the formulation favors lower sticking propensity of a given API.

Conclusion

For an API prone to punch sticking, judicious use of excipients to render higher tablet mechanical strength can mitigate severity of punch sticking.

     

Bilateral Effects of Excipients on Protein Stability: Preferential Interaction Type of Excipient and Surface Aromatic Hydrophobicity of Protein

Purpose

Understanding the mechanism of protein-excipient interaction and illuminating the influencing factors on protein stability are key steps in the rational design of protein formulations. The objective of this study was to assess effects of preferential interaction type of excipient and surface aromatic hydrophobicity of protein on protein solution stability.

Methods

The preferential interaction between excipient and aromatic hydrophobic area of protein was investigated by solubility and fluorescence studies of amino acid derivatives in excipient solutions. We examined conformational, colloidal and mechanical stabilities of model proteins with different surface aromatic hydrophobicities, including bovine serum albumin (BSA) and ovalbumin (OVA), and then stability data were visualized by three-index empirical phase diagram.

Results

The result showed that preferentially excluded excipients (trehalose, sucrose and sorbitol) protected protein conformation against damage, but they could accelerate mechanical stress-induced aggregation. Preferentially bound excipients (propanediol and arginine) suppressed BSA aggregation, but arginine failed to inhibit OVA aggregation, which might be attributed to the disparate conformational perturbing effects of arginine on aromatic hydrophobic regions of BSA and OVA.

Conclusions

These findings provided strong evidence that excipient possessed bilateral effects, and its application should be determined on different preferential interaction behaviors of excipients with protein, especially with the aromatic hydrophobic region.

     

Drug Interaction Between Clopidogrel and Ranitidine or Omeprazole in Stable Coronary Artery Disease: A Double-Blind,Double Dummy,Randomized Study

Background

Proton-pump inhibitors (PPIs) are often prescribed to patients receiving dual antiplatelet therapy (DAPT). However, this class of medication, especially omeprazole, has been associated with a reduction in clopidogrel efficacy, leading many clinicians to substitute omeprazole with ranitidine.

Objectives

Our objective was to compare the antiplatelet effect of clopidogrel before and after the addition of omeprazole or ranitidine.

Methods

We measured platelet aggregability at baseline and after 1 week of clopidogrel 75 mg daily. Subjects were then randomized in a double-blinded, double-dummy fashion to omeprazole 20 mg twice daily (bid) or ranitidine 150 mg bid. We repeated aggregability tests after 1 additional week, using VerifyNow P2Y12? (Accumetrics; San Diego, CA, USA), depicting aggregability as percent inhibition of platelet aggregation (IPA).

Results

We enrolled 41 patients in the omeprazole group and 44 in the ranitidine group. IPA was significantly decreased after the addition of omeprazole to clopidogrel (from 26.3 ± 32.9 to 17.4 ± 33.1 %; p = 0.025), with no statistical significant changes observed in the ranitidine group (from 32.6 ± 28.9 to 30.1 ± 31.3 %; p = 0.310). The comparison of IPA in both groups at the end of the follow-up showed a trend toward significance (p = 0.07, 95 % confidence interval [CI] ?1.19 to 26.59); after excluding homozygous patients for 2C19*2 genotype, the comparison of IPA between the groups reached statistical significance (32.7 ± 30.8 vs. 17.7 ± 33.4 %, respectively, for ranitidine and omeprazole groups; p = 0.04).

Conclusions

Unlike omeprazole, ranitidine did not influence platelet aggregability response to clopidogrel.

Clinical Trial Registration

NCT01896557.

     

Identification of D-Amino Acids in Light Exposed mAb Formulations

Purpose

We previously demonstrated that D-amino acids can form as a result of photo-irradiation of a monoclonal antibody (mAb) at both λ?=?254 nm and λ?>?295 nm (λ_max?=?305 nm), likely via reversible hydrogen transfer reactions of intermediary thiyl radicals. Here, we investigate the role of various excipients (sucrose, glucose, L-Arg, L-Met and L-Leu) on D-amino acid formation, and specifically the distribution of D-amino acids in mAb monomers and aggregates present after light exposure.

Methods

The mAb-containing formulations were photo-irradiated at λ?=?254 nm and λ_max?=?305 nm, followed by fractionation of aggregate and monomer fractions using size exclusion chromatography. These aggregate and monomer fractions were subjected to hydrolysis and subsequent amino acid analysis.

Results

Both aggregate and monomer fractions collected from all formulations showed the formation of D-Glu and D-Val, whereas the formation of D-Ala was limited to the aggregate fraction collected from an L-Arg-containing formulation. Interestingly, quantitative analysis revealed higher yields of D-amino acids in the L-Arg-containing formulation.

Conclusions

Generally, D-amino acids accumulated to similar extents in monomers and aggregates.

     

Photokinetic Drug Delivery: Near infrared (NIR) Induced Permeation Enhancement of Bevacizumab,Ranibizumab and Aflibercept through Human Sclera

Purpose

Permeation studies, with near infrared (NIR) light and anti-aggregation antibody formulation, were used to investigate the in vitro permeation of bevacizumab, ranibizumab and aflibercept through human sclera.

Methods

A vertical, spherical Franz cell diffusion apparatus was used for this scleral tissue permeation model. A photokinetic ocular drug delivery (PODD) testing device accommodated the placement of NIR LEDs above the donor chambers. An adjustable LED driver/square wave generator provided electrical energy with a variable pulse rate and pulse width modulation (duty cycle).

Results

Exposure to non-thermal NIR light had no effect on mAbs with regard to monomer concentration or antibody binding potential, as determined by SE-HPLC and ELISA. The optimal LED wavelength was found to be 950 nm. Duty cycle power of 5% vs 20% showed no difference in permeation. When compared to controls, the combination of non-aggregating antibody formulation and NIR illumination provided an average transscleral drug flux enhancement factor of 3X.

Conclusion

Narrow wavelength incoherent (non-laser) light from an NIR LED source is not harmful to mAbs and can be used to enhance drug permeation through scleral tissue. The topical formulation, combined with pulsed NIR light irradiation, significantly improved scleral permeation of three anti-VEGF antibody drugs.

     

A Multiparticulate Delivery System for Potential Colonic Targeting Using Bovine Serum Albumin as a Model Protein

Purpose

There are many important diseases whose treatment could be improved by delivering a therapeutic protein to the colon, for example, Clostridium difficile infection, ulcerative colitis and Crohn’s Disease. The goal of this project was to investigate the feasibility of colonic delivery of proteins using multiparticulate beads.

Methods

In this work, bovine serum albumin (BSA) was adopted as a model protein. BSA was spray layered onto beads, followed by coating of an enteric polymer EUDRAGIT® FS 30 D to develop a colonic delivery system. The secondary and tertiary structure change and aggregation of BSA during spray layering process was examined. The BSA layered beads were then challenged in an accelerated stability study using International Council for Harmonization (ICH) conditions. The in vitro release of BSA from enteric coated beads was examined using United States Pharmacopeia (USP) dissolution apparatus 1.

Results

No significant changes in the secondary and tertiary structure or aggregation profile of BSA were observed after the spray layering process. Degradation of BSA to different extents was detected after storing at 25°C and 40°C for 38 days. Enteric coated BSA beads were intact in acidic media while released BSA in pH 7.4 phosphate buffer.

Conclusion

We showed the feasibility of delivering proteins to colon in vitro using multiparticulate system.

     

Humid Heat Autoclaving of Hybrid Nanoparticles Achieved by Decreased Nanoparticle Concentration and Improved Nanoparticle Stability Using Medium Chain Triglycerides as a Modifier

Purpose

Humid heat autoclaving is a facile technique widely used in the sterilization of injections, but the high temperature employed would destroy nanoparticles composed of biodegradable polymers. The aim of this study was to investigate whether incorporation of medium chain triglycerides (MCT) could stabilize nanoparticles composed of poly (ethylene glycol)-b-polycaprolactone (PEG-b-PCL) during autoclaving (121°C, 10 min).

Methods

Polymeric nanoparticles with different MCT contents were prepared by dialysis. Block copolymer degradation was studied by GPC. The critical aggregation concentrations of nanoparticles at different temperatures were determined using pyrene fluorescence. The size, morphology and weight averaged molecular weight of pristine/autoclaved nanoparticles were studied using DLS, TEM and SLS, respectively. Drug loading content and release profile were determined using RP-HPLC.

Results

The protecting effect of MCT on nanoparticles was dependent on the amount of MCT incorporated. Nanoparticles with high MCT contents, which assumed an emulsion-like morphology, showed reduced block copolymer degradation and particle disassociation after incubation at 100°C for 24 h. Nanoparticles with high MCT content showed the lowest critical aggregation concentration (CAC) under either room temperature or 60°C and the lowest particle concentration among all samples. And the particle size, drug loading content, physical stability and release profile of nanoparticles with high MCT contents remained nearly unchanged after autoclaving.

Conclusion

Incorporation of high amount of MCT changed the morphology of PEG-b-PCL based nanoparticles to an emulsion-like structure and the nanoparticles prepared could withstand autoclaving due to improved particle stability and decreased particle concentration caused by MCT incorporation.

     

Reevaluating the nicotine delivery kinetics hypothesis

Rationale

The view of smoking as an addiction to nicotine implies that nicotine is an addictive drug and a primary reinforcer. However, nicotine other than in tobacco does not appear to be very rewarding for smokers. This potential anomaly to the nicotine addiction thesis is resolved by the proposition that the reward associated with smoking depends on “high-nicotine boli.” According to the nicotine delivery kinetics hypothesis, smoked nicotine reaches the brain in 5–10 s in high concentrations, which provide reinforcing “hits” of nicotine to the brain.

Objectives

Because of its essential role in the nicotine addiction thesis, this review set out to examine the current empirical basis of the nicotine delivery kinetics hypothesis.

Materials and methods

We reviewed studies that bear on two questions: First, does nicotine from cigarettes reach the brain significantly faster than from other nicotine delivery devices? Second, is there a relationship between delivery kinetics and any rewarding effects of nicotine?

Results

There is little empirical support for the nicotine delivery kinetics hypothesis. Several studies found that arterial nicotine levels associated with smoking are much lower than predicted by the nicotine delivery kinetics thesis and not higher than with other nicotine delivery devices. More importantly, comparisons of nicotine delivery devices with varying speeds of delivery do not suggest any correlation between nicotine delivery profile and subjective reward.

Conclusions

This review indicates that the wide endorsement of the nicotine delivery kinetics hypothesis is unjustified. Critical research is required to resolve the anomalies within the nicotine addiction theory of smoking.