APP family member dimeric complexes are formed predominantly in synaptic compartments.

BACKGROUND: The amyloid precursor protein (APP), a key player in Alzheimer's disease (AD), is part of a larger gene family, including the APP like proteins APLP1 and APLP2. They share similar structures, form homo- and heterotypic dimers and exhibit overlapping functions. RESULTS: We investigated complex formation of the APP family members via two inducible dimerization systems, the FKBP-rapamycin based dimerization as well as cysteine induced dimerization, combined with co-immunoprecipitations and Blue Native (BN) gel analyses. Within the APP family, APLP1 shows the highest degree of dimerization and high molecular weight (HMW) complex formation. Interestingly, only about 20% of APP is dimerized in cultured cells whereas up to 50% of APP is dimerized in mouse brains, independent of age and splice forms. Furthermore, we could show that dimerized APP originates mostly from neurons and is enriched in synaptosomes. Finally, BN gel analysis of human cortex samples shows a significant decrease of APP dimers in AD patients compared to controls. CONCLUSIONS: Together, we suggest that loss of full-length APP dimers might correlate with loss of synapses in the process of AD.

Differential effects of familial Alzheimer's disease-causing mutations on amyloid precursor protein (APP) trafficking, proteolytic conversion, and synaptogenic activity.

The amyloid precursor protein (APP) is a key player in Alzheimer`s disease (AD) and the precursor of the Aß peptide, which is generated by consecutive cleavages of ß- and γ-secretases. Familial Alzheimer's disease (FAD) describes a hereditary subgroup of AD that represents a low percentage of AD cases with an early onset of the disease. Different APP FAD mutations are thought to have qualitatively different effects on its proteolytic conversion. However, few studies have explored the pathogenic and putative physiological differences in more detail. Here, we compared different FAD mutations, located at the ß- (Swedish), α- (Flemish, Arctic, Iowa) or γ-secretase (Iberian) cleavage sites. We examined heterologous expression of APP WT and FAD mutants in non-neuronal cells and their impact on presynaptic differentiation in contacting axons of co-cultured neurons. To decipher the underlying molecular mechanism, we tested the subcellular localization, the endocytosis rate and the proteolytic processing in detail by immunoprecipitation-mass spectrometry. Interestingly, we found that only the Iberian mutation showed altered synaptogenic function. Furthermore, the APP Iowa mutant shows significantly decreased α-secretase processing which is in line with our results that APP carrying the Iowa mutation was significantly increased in early endosomes. However, most interestingly, immunoprecipitation-mass spectrometry analysis revealed that the amino acid substitutions of APP FAD mutants have a decisive impact on their processing reflected in altered Aß profiles. Importantly, N-terminally truncated Aß peptides starting at position 5 were detected preferentially for APP Flemish, Arctic, and Iowa mutants containing amino acid substitutions around the α-secretase cleavage site. The strongest change in the ratio of Aß40/Aß42 was observed for the Iberian mutation while APP Swedish showed a substantial increase in Aß1-17 peptides. Together, our data indicate that familial AD mutations located at the α-, ß-, and γ-secretase cleavage sites show considerable differences in the underlying pathogenic mechanisms.

Converging roles of PSENEN/PEN2 and CLN3 in the autophagy-lysosome system.

PSENEN/PEN2 is the smallest subunit of the γ-secretase complex, an intramembrane protease that cleaves proteins within their transmembrane domains. Mutations in components of the γ-secretase underlie familial Alzheimer disease. In addition to its proteolytic activity, supplementary, γ-secretase independent, functions in the macroautophagy/autophagy-lysosome system have been proposed. Here, we screened for PSENEN-interacting proteins and identified CLN3. Mutations in CLN3 are causative for juvenile neuronal ceroid lipofuscinosis, a rare lysosomal storage disorder considered the most common neurodegenerative disease in children. As mutations in the PSENEN and CLN3 genes cause different neurodegenerative diseases, understanding shared cellular functions of both proteins might be pertinent for understanding general cellular mechanisms underlying neurodegeneration. We hypothesized that CLN3 modulates γ-secretase activity and that PSENEN and CLN3 play associated roles in the autophagy-lysosome system. We applied CRISPR gene-editing and obtained independent isogenic HeLa knockout cell lines for PSENEN and CLN3. Following previous studies, we demonstrate that PSENEN is essential for forming a functional γ-secretase complex and is indispensable for γ-secretase activity. In contrast, CLN3 does not modulate γ-secretase activity to a significant degree. We observed in PSENEN- and CLN3-knockout cells corresponding alterations in the autophagy-lysosome system. These include reduced activity of lysosomal enzymes and lysosome number, an increased number of autophagosomes, increased lysosome-autophagosome fusion, and elevated levels of TFEB (transcription factor EB). Our study strongly suggests converging roles of PSENEN and CLN3 in the autophagy-lysosome system in a γ-secretase activity-independent manner, supporting the idea of common cytopathological processes underlying different neurodegenerative diseases.Abbreviations: Aß, amyloid-beta; AD, Alzheimer disease; APP, amyloid precursor protein; ATP5MC, ATP synthase membrane subunit c; DQ-BSA, dye-quenched bovine serum albumin; ER, endoplasmic reticulum; GFP, green fluorescent protein; ICC, immunocytochemistry; ICD, intracellular domain; JNCL, juvenile neuronal ceroid lipofuscinosis; KO, knockout; LC3, microtubule associated protein 1 light chain 3; NCL, neuronal ceroid lipofuscinoses; PSEN, presenilin; PSENEN/PEN2: presenilin enhancer, gamma-secretase subunit; TAP, tandem affinity purification; TEV, tobacco etch virus; TF, transferrin; WB, Western blot; WT, wild type.

Artesunate restores the levels of inhibitory synapse proteins and reduces amyloid-ß and C-terminal fragments (CTFs) of the amyloid precursor protein in an AD-mouse model.

Alzheimer's disease (AD) is the most frequent form of dementia, characterized histopathologically by the formation of amyloid plaques and neurofibrillary tangles in the brain. Amyloid ß-peptide (Aß) is a major component of amyloid plaques and is released together with carboxy-terminal fragments (CTFs) from the amyloid precursor protein (APP) through proteolytic cleavage, thought to contribute to synapse dysfunction and loss along the progression of AD. Artemisinins, primarily antimalarial drugs, reduce neuroinflammation and improve cognitive capabilities in mouse models of AD. Furthermore, artemisinins were demonstrated to target gephyrin, the main scaffold protein of inhibitory synapses and modulate GABAergic neurotransmission in vitro. Previously, we reported a robust decrease of inhibitory synapse proteins in the hippocampus of 12-month-old double transgenic APP-PS1 mice which overexpress in addition to the Swedish mutated form of the human APP a mutated presenilin 1 (PS1) gene and are characterized by a high plaque load at this age. Here, we provide in vivo evidence that treating these mice with artemisinin or its semisynthetic derivative artesunate in two different doses (10 mg/kg and 100 mg/kg), these compounds affect differently inhibitory synapse components, amyloid plaque load and APP-processing. Immunofluorescence microscopy demonstrated the rescue of gephyrin and γ2-GABAA-receptor protein levels in the brain of treated mice with both, artemisinin and artesunate, most efficiently with a low dose of artesunate. Remarkably, artemisinin reduced only in low dose the amyloid plaque load correlating with lower levels of mutated human APP (hAPPswe) whereas artesunate treatment in both doses resulted in significantly lower plaque numbers. Correspondingly, the level of APP-cleavage products, specifically the amount of CTFs in hippocampus homogenates was reduced significantly only by artesunate, in line with the findings in hAPPswe expressing cultured hippocampal neurons evidencing a concentration-dependent inhibition of CTF-release by artesunate already in the nanomolar range. Thus, our data support artemisinins as neuroprotective multi-target drugs, exhibiting a potent anti-amyloidogenic activity and reinforcing key proteins of inhibitory synapses.

Multidisciplinary approach to paediatric aerodigestive disorders: A single-centre longitudinal observational study.

AIM: Aerodigestive clinics (ADCs) are multidisciplinary programmes for the care of children with complex congenital or acquired conditions affecting breathing, swallowing and growth. Our objective was to describe the demographic, clinical, etiological and investigational profile of children attending the inaugural ADC at a tertiary paediatric centre in Queensland. METHODS: Children referred to the ADC at Queensland Children's Hospital from August 2018 to December 2019 were included. Data on clinical, growth and lung function parameters, bronchoscopy and upper gastrointestinal endoscopy findings, thoracic imaging and comorbidities were retrospectively analysed. RESULTS: Fifty-six children (median (range) age 4 years (3 months-15 years); 18 female) attended the ADC during this 17-month period. Forty-six (82%) children had previous oesophageal atresia with tracheo-oesophageal fistula; 43 of these were type C. Previous isolated oesophageal atresia, congenital diaphragmatic hernia and congenital pulmonary malformation were the underlying disorder in three (5%) children each, with one child having a repaired laryngeal cleft. Vertebral Anal Tracheo Esophageal Renal Limb anomalies (VACTERL)/Vertebral Anal Tracheo Esophageal renal anomalies (VATER) association was seen in 21 (38%) children. Growth was adequate (median weight and body mass index z-score -0.63 and -0.48, respectively). Thirty-four (61%) children reported ongoing wet cough, with 12 (21%) requiring previous hospital admission for lower respiratory tract infection. Fourteen (25%) had bronchiectasis on computed tomography chest and 33 (59%) had clinical tracheomalacia, apparent on bronchoscopic examination in 21 patients. Dysphagia was reported in 15 (27%) children, 11 (20%) were gastrostomy feed-dependent and 5 (9%) had biopsy-proven eosinophilic oesophagitis. CONCLUSION: High proportion of children attending the ADC have ongoing respiratory symptoms resulting in chronic pulmonary suppuration and bronchiectasis. Potential benefits of this model of care need to be studied prospectively to better understand the outcomes.

Respiratory management of infants with chronic neonatal lung disease beyond the NICU: A position statement from the Thoracic Society of Australia and New Zealand.

Chronic neonatal lung disease (CNLD) is defined as continued need for any form of respiratory support (supplemental oxygen and/or assisted ventilation) beyond 36 weeks PMA. Low-flow supplemental oxygen facilitates discharge from hospital of infants with CNLD who are hypoxic in air and is widely used despite lack of evidence on the most appropriate minimum mean target oxygen saturations. Furthermore, there are minimal data to guide the home monitoring, titration or weaning of supplemental oxygen in these infants. The purpose of this position statement is to provide a guide for the respiratory management of infants with CNLD, with special emphasis on role and logistics of supplemental oxygen therapy beyond the NICU stay. Reflecting a variety of clinical practices and infant comorbidities (presence of pulmonary hypertension, retinopathy of prematurity and adequacy of growth), it is recommended that the minimum mean target range for SpO2 during overnight oximetry to be 93-95% with less than 5% of total recording time to be below 90% SpO2 . Safety of short-term disconnection from supplemental oxygen should be assessed before discharge, with majority of infants with CNLD not ready for discharge until supplemental oxygen requirement is ≤0.5 L/min. Sleep-time assessment of oxygenation with continuous overnight oximetry is recommended when weaning supplemental oxygen. Palivizumab is considered safe and effective for the reduction of hospital admissions with RSV infection in this group. This statement would be useful for paediatricians, neonatologists, respiratory and sleep physicians and general practitioners managing children with CNLD.

Is out-patient based treatment of bronchiectasis exacerbations in children comparable to inpatient based treatment?

BACKGROUND AND OBJECTIVE: Children with bronchiectasis have recurrent exacerbations and may require hospitalization. "Hospital in the home (HITH)" is used as an alternative to hospitalization for children with cystic fibrosis (CF) but to date, there is no published data on children without CF. We describe our experience of HITH (intravenous [IV] antibiotics and at least once-daily physiotherapy-treated airway clearance therapy) in a cohort of children with bronchiectasis, comparing outcomes between hospital and HITH-based pathways. METHODS: Medical records were retrospectively reviewed in children with bronchiectasis who were hospitalized in our center from July 2016 to July 2018. We compared treatment duration, symptom resolution, adverse events, oral antibiotic prescription on discharge and "time-to-next hospitalization" between children managed with the two treatment pathways. RESULTS: Exacerbations in 63 children (median age = 6 years [range: 1-17]; females = 33, indigenous = 8) with bronchiectasis treated with IV antibiotic therapy were analyzed (HITH n = 45, 71.5%). Duration of treatment and symptom resolution was similar between groups (hospital: median = 14 days [interquartile range {IQR}: 14-14] and 12/18 [66.6%], respectively vs HITH: 14 [14-15.5] and 31/45 [69%]; P = .53 and .85, respectively). There was no significant difference in adverse events (16.6% vs 9%), prescription of oral antibiotics on discharge (44% vs 24%), or "time-to-next hospitalization" (median 42 [IQR: 24-100] vs 67 [IQR: 32-95] weeks) between hospital and HITH groups, respectively. CONCLUSIONS: In children with bronchiectasis treated for a severe exacerbation, receiving treatment in the home setting with HITH does not compromise short-term clinical outcomes compared to hospital only treatment. Prospective studies are required to provide more robust evidence in this under-researched area.

LRP1 Modulates APP Intraneuronal Transport and Processing in Its Monomeric and Dimeric State.

The low-density lipoprotein receptor-related protein 1, LRP1, interacts with APP and affects its processing. This is assumed to be mostly caused by the impact of LRP1 on APP endocytosis. More recently, also an interaction of APP and LRP1 early in the secretory pathway was reported whereat retention of LRP1 in the ER leads to decreased APP cell surface levels and in turn, to reduced Aß secretion. Here, we extended the biochemical and immunocytochemical analyses by showing via live cell imaging analyses in primary neurons that LRP1 and APP are transported only partly in common (one third) but to a higher degree in distinct fast axonal transport vesicles. Interestingly, co-expression of LRP1 and APP caused a change of APP transport velocities, indicating that LRP1 recruits APP to a specific type of fast axonal transport vesicles. In contrast lowered levels of LRP1 facilitated APP transport. We further show that monomeric and dimeric APP exhibit similar transport characteristics and that both are affected by LRP1 in a similar way, by slowing down APP anterograde transport and increasing its endocytosis rate. In line with this, a knockout of LRP1 in CHO cells and in primary neurons caused an increase of monomeric and dimeric APP surface localization and in turn accelerated shedding by meprin ß and ADAM10. Notably, a choroid plexus specific LRP1 knockout caused a much higher secretion of sAPP dimers into the cerebrospinal fluid compared to sAPP monomers. Together, our data show that LRP1 functions as a sorting receptor for APP, regulating its cell surface localization and thereby its processing by ADAM10 and meprin ß, with the latter exhibiting a preference for APP in its dimeric state.

APLP1 Is a Synaptic Cell Adhesion Molecule, Supporting Maintenance of Dendritic Spines and Basal Synaptic Transmission.

The amyloid precursor protein (APP), a key player in Alzheimer's disease, belongs to the family of synaptic adhesion molecules (SAMs) due to its impact on synapse formation and synaptic plasticity. These functions are mediated by both the secreted APP ectodomain that acts as a neurotrophic factor and full-length APP forming trans-cellular dimers. Two homologs of APP exist in mammals: the APP like proteins APLP1 and APLP2, exhibiting functions that partly overlap with those of APP. Here we tested whether APLP1 and APLP2 also show features of SAMs. We found that all three family members were upregulated during postnatal development coinciding with synaptogenesis. We observed presynaptic and postsynaptic localization of all APP family members and could show that heterologous expression of APLP1 or APLP2 in non-neuronal cells induces presynaptic differentiation in contacting axons of cocultured neurons, similar to APP and other SAMs. Moreover, APP/APLPs all bind to synaptic-signaling molecules, such as MINT/X11. Furthermore, we report that aged APLP1 knock-out mice show impaired basal transmission and a reduced mEPSC frequency, likely resulting from reduced spine density. This demonstrates an essential nonredundant function of APLP1 at the synapse. Compared to APP, APLP1 exhibits increased trans-cellular binding and elevated cell-surface levels due to reduced endocytosis. In conclusion, our results establish that APLPs show typical features of SAMs and indicate that increased surface expression, as observed for APLP1, is essential for proper synapse formation in vitro and synapse maintenance in vivoSIGNIFICANCE STATEMENT According to the amyloid-cascade hypothesis, Alzheimer's disease is caused by the accumulation of Aß peptides derived from sequential cleavage of the amyloid precursor protein (APP) by ß-site APP cleaving enzyme 1 (BACE1) and γ-secretase. Here we show that all mammalian APP family members (APP, APLP1, and APLP2) exhibit synaptogenic activity, involving trans-synaptic dimerization, similar to other synaptic cell adhesion molecules, such as Neuroligin/Neurexin. Importantly, our study revealed that the loss of APLP1, which is one of the major substrates of BACE1, causes reduced spine density in aged mice. Because some therapeutic interventions target APP processing (e.g., BACE inhibitors), those strategies may alter APP/APLP physiological function. This should be taken into account for the development of pharmaceutical treatments of Alzheimer's disease.

Shedding of APP limits its synaptogenic activity and cell adhesion properties.

The amyloid precursor protein (APP) plays a central role in Alzheimer's disease (AD) and has essential synapse promoting functions. Synaptogenic activity as well as cell adhesion properties of APP presumably depend on trans-cellular dimerization via its extracellular domain. Since neuronal APP is extensively processed by secretases, it raises the question if APP shedding affects its cell adhesion and synaptogenic properties. We show that inhibition of APP shedding using cleavage deficient forms of APP or a dominant negative α-secretase strongly enhanced its cell adhesion and synaptogenic activity suggesting that synapse promoting function of APP is tightly regulated by α-secretase mediated processing, similar to other trans-cellular synaptic adhesion molecules.

Amyloid precursor protein dimerization and synaptogenic function depend on copper binding to the growth factor-like domain.

Accumulating evidence suggests that the copper-binding amyloid precursor protein (APP) has an essential synaptic function. APP synaptogenic function depends on trans-directed dimerization of the extracellular E1 domain encompassing a growth factor-like domain (GFLD) and a copper-binding domain (CuBD). Here we report the 1.75 Å crystal structure of the GFLD in complex with a copper ion bound with high affinity to an extended hairpin loop at the dimerization interface. In coimmunoprecipitation assays copper binding promotes APP interaction, whereas mutations in the copper-binding sites of either the GFLD or CuBD result in a drastic reduction in APP cis-orientated dimerization. We show that copper is essential and sufficient to induce trans-directed dimerization of purified APP. Furthermore, a mixed culture assay of primary neurons with HEK293 cells expressing different APP mutants revealed that APP potently promotes synaptogenesis depending on copper binding to the GFLD. Together, these findings demonstrate that copper binding to the GFLD of APP is required for APP cis-/trans-directed dimerization and APP synaptogenic function. Thus, neuronal activity or disease-associated changes in copper homeostasis likely go along with altered APP synaptic function.

Use of highly compressible Ceolus™ microcrystalline cellulose for improved dosage form properties containing a hydrophilic solid dispersion.

The development of amorphous solid dispersions containing poorly soluble drug substances has been well-documented; however, little attention has been given to the development of the finished dosage form. The objective of this study was to investigate the use of Ceolus(™) microcrystalline cellulose, a highly compressible excipient, for the production of rapidly disintegrating tablets containing a hydrophilic solid dispersion of a poorly soluble drug, indomethacin. Solid dispersions of indomethacin and Kollidon(®) VA64 were prepared by hot melt extrusion and characterized for amorphous nature. Milled dispersion particles at 500 mg/g drug loading were shown to be amorphous by differential scanning calorimetry and provided rapid dissolution in sink conditions. Physical characterization of the milled extrudate showed that the particle size of the intermediate was comparable with Ceolus(™) PH-102 and larger than the high compressibility grades of microcrystalline cellulose selected for the trial (Ceolus(™) KG-802, Ceolus(™) UF-711). Preliminary tableting trials showed that dissolution performance was significantly reduced for formulations at dispersion loadings in excess of 50%. Using a mixture design of experiments (DOE), the levels of PH-102, KG-802, UF-711, and PH-301 were optimized. Trials revealed a synergistic relationship between conventional grades (PH-102 and PH-301) and highly compressible grades (KG-802 and UF-711) leading to improved compression characteristics and more rapid dissolution rates. The formulation and resulting compressibility were also shown to have an impact on in vitro supersaturation indicating tablet formulation could impact oral bioavailability. Through the use of highly compressible microcrystalline cellulose grades such as Ceolus(™) KG-802 and UF-711, it may be possible to maximize the bioavailability benefit of amorphous solid dispersions administered as tablet dosage forms.

Novel application of hot-melt extrusion for the preparation of monolithic matrices containing enteric-coated particles.

The objective was to investigate a novel application of hot-melt extrusion for the preparation of multiparticulate matrices comprising delayed-release particles. Multiparticulates of different mechanical strengths (theophylline granules, wet-mass extruded/spheronized pellets and drug-layered microcrystalline cellulose spheres) were coated with Eudragit(®) L30D-55 and characterized regarding potency, moisture content, dissolution properties and tensile strength. The coated particles were incorporated into a water-soluble matrix using hot-melt extrusion. Six hydrophilic polymers including polyethylene glycols, poloxamers and polyethylene oxides were studied as the carrier material for the extrusion. Dissolution testing showed that the maintenance of the delayed-release properties of the incorporated particles was independent of the particle tensile strength, but influenced by the nature of the carrier polymer. High miscibility between the carrier and the coating polymer correlated with increased film permeability and higher drug release in acidic media. Of the materials tested, poloxamer 407 exhibited lower miscibility with the Eudragit(®) L polymer and matrices containing up to 40% enteric pellets were compliant with the USP dissolution requirements for delayed-release dosage forms. The potential advantages of hot-melt extrusion over direct compression for the processing of soft drug granules coated with Eudragit(®) L polymer were demonstrated.

Influence of plasticizer type and level on the properties of Eudragit S100 matrix pellets prepared by hot-melt extrusion.

Matrix-type pellets with controlled-release properties may be prepared by hot-melt extrusion applying a single-step, continuous process. However, the manufacture of gastric-resistant pellets is challenging due to the high glass transition temperature of most enteric polymers and an unacceptably high, diffusion-controlled drug release from the matrix during the acidic phase. The objective was to investigate the influence of three plasticizers (triethyl citrate, methylparaben and polyethylene glycol 8000) at two levels (10% or 20%) on the properties of hot-melt extruded Eudragit S100 matrix pellets. Extrusion experiments showed that all plasticizers produced similar reductions in polymer melt viscosity. Differential scanning calorimetry and powder X-ray diffraction demonstrated that the solid state plasticizers were present in the amorphous state. The drug release in acidic medium was influenced by the aqueous solubility of the plasticizer. Less than 10% drug was released after 2 h at pH 1.2 when triethyl citrate or methylparaben was used, independent of the plasticizer level. Drug release at pH 7.4 resulted from polymer dissolution and was not influenced by low levels of plasticizer, but increased significantly at the 20% level. Mechanical testing by diametral compression demonstrated the high tensile strength of the hot-melt extruded pellets that decreased when plasticizers were present.

Properties of melt extruded enteric matrix pellets.

The objective of this study was to investigate the properties of enteric matrix pellets that were prepared by hot-melt extrusion in a one-step, continuous process. Five polymers (Eudragit) L100-55, L100 and S100, Aqoat grades LF and HF) were investigated as possible matrix formers, and pellets prepared with Eudragit S100 demonstrated superior gastric protection and acceptable processibility. Extruded pellets containing Eudragit S100 and up to 40% theophylline released less than 10% drug over 2h in acid, however, the processibility and yields were compromised by the high amounts of the non-melting drug material in the formulation. Efficient plasticization of Eudragit S100 was necessary to reduce the polymer's glass transition temperature and melt viscosity. Five compounds including triethyl citrate, methylparaben, polyethylene glycol 8000, citric acid monohydrate and acetyltributyl citrate were investigated in terms of plasticization efficiency and preservation of the delayed drug release properties. The aqueous solubility of the plasticizer and its plasticization efficiency impacted the drug release rate from the matrix pellets. The use of water-soluble plasticizers resulted in a loss of gastric protection, whereas low drug release rates in acid were found for pellets containing insoluble plasticizers or no plasticizer, independent of the extent of Eudragit S100 plasticization. The release rate of theophylline in buffer pH 7.4 was faster for pellets that were prepared with efficient plasticizers. The microstructure and solid-state properties of plasticized pellets were further investigated by scanning electron microscopy and powder X-ray diffraction. Pellets prepared with efficient plasticizers (TEC, methylparaben, PEG 8000) exhibited matrices of low porosity, and the drug was homogeneously dispersed in its original polymorphic form. Pellets containing ATBC or citric acid monohydrate had to be extruded at elevated temperature and showed physical instabilities in the form of recrystallization at room temperature. Enteric matrix pellets with a diameter below 1mm and containing 30% theophylline could be successfully prepared by hot-melt extrusion when Eudragit S100 plasticized with either TEC or methylparaben was employed as the matrix material.

Citric acid monohydrate as a release-modifying agent in melt extruded matrix tablets.

Incomplete drug release and particle size-dependent dissolution performance can compromise the quality of controlled release matrix systems. The objective of the current study was to investigate the ability of citric acid monohydrate (CA MH) to enhance the release of diltiazem hydrochloride from melt extruded Eudragit RS PO tablets and to eliminate drug particle size effects. Preformulation studies demonstrated the thermal stability of all components, drug insolubility in the polymer but miscibility with the CA MH. Tablets with either constant polymer levels or constant drug-to-polymer ratios and containing different drug particle size fractions and increasing amounts of CA MH were manufactured by melt extrusion and characterized by dissolution testing, powder X-ray diffraction and scanning electron microscopy. The addition of CA MH to the formulation promoted the thermal processibility and matrix integrity by plasticization of the polymer. The drug release from systems with constant drug-to-polymer ratio was significantly increased when CA MH was added as a result of enhanced pore formation. Particle size effects were eliminated when large amounts of CA MH were used due to the loss of drug crystallinity. Matrix tablets with CA MH furthermore showed a faster and more complete drug release compared to systems with drug only or alternative pore formers (sucrose, NaCl, or PEG 3350). The enhanced drug release was attributed to the amorphous character of the soluble components, improved drug dispersion in the plasticized polymer along with increased polymer permeability. In summary, CA MH promoted the miscibility between the drug and Eudragit RS PO during hot-melt extrusion, resulting in the extrusion of an amorphous system with improved dissolution characteristics.

Citric acid as a solid-state plasticizer for Eudragit RS PO.

The use of solid-state plasticizers for the hot-melt extrusion of pharmaceutical dosage forms has been shown to be beneficial compared with liquid plasticizers. The purpose of this study was to determine the suitability of citric acid (CA) as a solid plasticizer for the preparation of Eudragit RS PO extended-release matrix systems by a melt extrusion technique. The influence of increasing levels of CA monohydrate (CA MH) or anhydrous CA in the powder blend on the extrusion process parameters (screw speed and motor load) was determined as a function of temperature. The solubility of CA MH in extruded tablets was studied by means of modulated differential scanning calorimetry (MDSC) and powder X-ray diffraction (PXRD). Films were cast from organic solutions to demonstrate the plasticizing effect of CA MH as a change in physico-mechanical properties (tensile strength, elastic modulus and elongation). The CA release from extruded tablets was studied over 12 h. The monohydrate form was found to distinctly facilitate the extrusion of Eudragit RS PO, whereas the addition of anhydrous CA to the polymer powder was less effective. This divergent behaviour in plasticization of Eudragit RS PO was attributed to the higher solubility of the monohydrate in the acrylic polymer. The plasticizing effect of the CA MH reached a plateau at 25% during hot-melt extrusion, which coincided with the solubility limit of the organic acid in the polymer as shown by MDSC and PXRD results. The CA MH increased the flexibility of Eudragit RS PO films, as demonstrated by a decrease in tensile strength and elastic modulus and an increase in elongation as a function of CA MH concentration. The dissolution of CA from the matrix tablets followed an extended-release profile, with CA MH exhibiting a faster dissolution rate than the anhydrous form. In conclusion, CA MH was found to be an effective plasticizer for Eudragit RS PO that facilitates the production of controlled-release matrix systems by hot-melt extrusion.