Porcine Intestinal Mucosa Research Articles

Phosphate decorated lipid-based nanocarriers providing a prolonged mucosal residence time

The aim of this study was to develop phosphate decorated lipid-based nanocarriers including self-emulsifying drug delivery systems (SEDDS), solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) to extend their mucosal residence time. All nanocarriers contained tetradecyltrimethylammonium bromide (TTAB) and polyoxyethylene (9) nonylphenol monophosphate ester (PNPP) for surface decoration. Zeta potential, cytotoxicity, charge conversion and phosphate release studies using isolated intestinal alkaline phosphatase (IAP) and Caco-2 cells were performed. Moreover, the residence time of nanocarriers was determined on porcine intestinal mucosa. Results showed a shift from negative to positive zeta potential due to the addition of TTAB and charge conversion back to a negative zeta potential when also PNPP was added. Up to a concentration of 0.3 %, lipid-based nanocarriers were not toxic. Charge conversion studies with IAP revealed the highest zeta potential shift for NLCTTAB-PNPP with almost Δ22 mV. Phosphate release studies using isolated IAP as well as Caco-2 cells showed a fast phosphate release for SEDDSTTAB-PNPP, SLNTTAB-PNPP and NLCTTAB-PNPP. SLN TTAB-PNPP and NLC TTAB-PNPP provided the highest increase in mucosal residence time that was 4-fold more prolonged than that of blank formulations. In conclusion, phosphate modified lipid-based nanocarriers can essentially prolong the intestinal residence time of their payload.

USP Potency Adjusted Bovine Mucosal Heparins are Comparable to Porcine Mucosal Heparin and May be Interchangeable for Anticoagulation

IntroductionMost of the heparins used clinically are derived from porcine intestinal mucosa. In contrast to the porcine mucosal heparin (PMH), the active pharmaceutical ingredient (API) of bovine mucosal heparin (BMH) exhibit a somewhat weaker USP potency as cross‐referenced against PMH. We hypothesized that at equivalent potencies as adjusted by using the USP reference, the BMH may exhibit comparable effects.Materials & MethodsMolecular weight profile and in vitro anticoagulant and antiprotease assays were used to compare the BMH with PMH. USP potency was measured in the amidolytic assays. Potency adjustments in reference to USP standard were made by weight basis. Potency adjusted BMH and PMH were injected at identical dosages (50 U/kg and 100 U/kg) to groups of human primates (n=4) via intravenous route and blood samples were collected for a 6 hour period of time.ResultsBMH exhibited higher molecular weight profiles compared to PMH as determined by size exclusion chromatography {BMH (Mw) 18.6 ± 0.5 kDa and PMH 15.4 ± 0.4 kDa}. BMH showed a potency of 130 U/mg whereas PMH showed a potency of 190 U/mg. When the BMH was compared at a potency adjusted concentration with PMH, it showed identical calibration curves in the aPTT and anti‐protease assays. Mean t1/2 ranged from 54 ± 11 min & to 71 ± 18 min for PMH & BMH respectively (p=>0.05). Mean AUC values based on anti‐Xa or anti‐IIa activities were comparable for both heparins. Mean Vd (~60 ml/kg) and Cl (~0.75 ml/kg/min) were also comparable for both heparins.ConclusionPotency adjusted based dosing results in comparable anticoagulants and pharmacokinetic profiles for BMH and PMH. Therefore, such dosing may provide uniform levels of anticoagulation for the parenteral indications for heparins. These observations suggest that potency equated BMH and PMH produced comparable anticoagulant effects and may be interchangeable.

Synthesis and Characterization of a Cationic Thiomer Based on Ethyl Cellulose for Realization of Mucoadhesive Tablets and Nanoparticles.

PurposeEthyl cellulose (EC) based nanoparticles are being extensively studied for their ability to achieve prolonged drug release and improve drug stability. Within this study, the thiolation of unmodified EC using cysteamine as a ligand was carried out to design nanoparticles with mucoadhesive properties and comparatively strong lipophilic properties.MethodsThe thiolation was performed via oxidation and reductive amination, whereas the nanoparticles were generated via the ionic gelation followed by the precipitation method.ResultsThe number of free thiol groups on EC-cysteamine was in the range of 210–261 µmol per gram of polymer. Tablets based on EC-cysteamine demonstrated mucoadhesive properties 16.7-fold improved compared with those comprising unmodified EC. The mean diameter of the particles was in the range of 94–123 nm and the zeta potential was determined to be −7.97 to −14.70 mV. The nanoparticles remained attached to porcine intestinal mucosa for up to 36% after 3 h of incubation. The formation of nanoparticles improved the stability of EC-cysteamine conjugate against cellulase and provided a zero-order release. Moreover, both EC-cysteamine and the nanoparticles did not show any pronounced cytotoxicity.ConclusionAccordingly, EC-cysteamine nanoparticles could be a specific type of nanoparticulate delivery system with mucoadhesive properties. The amount of free thiol groups within EC-cysteamine nanoparticles together with their lipophilic properties could be further modified and modulated for a desired release behavior.

Implications of Heparanase on Heparin Synthesis and Metabolism in Mast Cells.

Heparin is a polysaccharide expressed in animal connective tissue-type mast cells. Owing to the special pentasaccharide sequence, heparin specifically binds to antithrombin (AT) and increases the inhibitory activity of AT towards coagulation enzymes. Heparin isolated from porcine intestinal mucosa has an average molecular weight of 15 kDa, while heparins recovered from rat skin and the peritoneal cavity were 60–100 kDa and can be fragmented by the endo-glucuronidase heparanase in vitro. In this study, we have examined heparin isolated from in vitro matured fetal skin mast cells (FSMC) and peritoneal cavity mast cells (PCMC) collected from wildtype (WT), heparanase knockout (Hpa-KO), and heparanase overexpressing (Hpa-tg) mice. The metabolically 35S-labeled heparin products from the mast cells of WT, Hpa-KO, and Hpa-tg mice were compared and analyzed for molecular size and AT-binding activity. The results show that PCMC produced heparins with a size similar to heparin from porcine intestinal mast cells, whilst FSMC produced much longer chains. As expected, heparanase overexpression resulted in the generation of smaller fragments in both cell types, while heparins recovered from heparanase knockout cells were slightly longer than heparin from WT cells. Unexpectedly, we found that heparanase expression affected the production of total glycosaminoglycans (GAGs) and the proportion between heparin and other GAGs but essentially had no effect on heparin catabolism.

Effect of Sulodexide on Circulating Blood Cells in Patients with Mild COVID-19.

Background. Despite the fact that COVID-19 usually manifests with severe pneumonia, there is a growing body of evidence that life-threatening multiorgan damage is caused by vascular and hemostatic abnormalities. Since there is no established therapy, assessing antithrombotics is indeed important. Sulodexide, a compound derived from porcine intestinal mucosa is a mixture of fast-moving heparin fraction (80%) and dermatan sulfate (20%), is approved in Europe and currently in trials for COVID-19 indication. Methods. This single-center, prospective, observational study included 28 patients with mild COVID-19 hospitalized in the Central Clinical Hospital of the Presidential Administration of the Russian Federation. Patients in the control group (n = 14) were treated using routine therapy according to current guidelines, while patients in the experimental group (n = 14) had the routine treatment supplemented with daily intravenous injections of sulodexide in 600-unit doses. Scanning electron microscopy was utilized to examine the blood specimens derived from the cubital vein at admission and at 10 days after hospitalization, which was approximately the average duration of patients’ treatment in the hospital (11.6 ± 0.4 days). Results. Sulodexide significantly (by 40%) diminished the score of circulating endothelial cells, potentially indicating its antiviral endothelium-protective properties. It also prevented the extra activation of the platelets and the formation of erythrocytic sludges. Among patients in the control group, the share of activated platelets rose from 37 ± 5% to 45 ± 6% (p = 0.04) over the course of the study period, whereas among patients in the experimental group, the share of activated platelets remained practically unchanged (43 ± 6% vs. 38 ± 4%, p = 0.22). The score of erythrocytic sludges in the control group remained practically the same (4.8 ± 1.1 at admission vs. 3.9 ± 0.9 after 10 days, p = 0.67), whereas in the experimental group, it significantly decreased (from 5.7 ± 1.7 to 2.4 ± 0.9, p = 0.03). Conclusions. Sulodexide is able to defend endothelium, normalize blood, and, seemingly, prevent thrombosis. Therefore, it may be considered as a promising and effective agent for the treatment of patients with mild COVID-19. Broader randomized trials are needed to assess whether the observed findings will transform into sustained long-term clinical benefit.

NMR spectroscopy and chemometric models to detect a specific non-porcine ruminant contaminant in pharmaceutical heparin

Heparin has been used successfully as a clinical antithrombotic for almost one century. Its isolation from animal sources (mostly porcine intestinal mucosa) involves multistep purification processes starting from the slaughterhouse (as mucosa) to the pharmaceutical plant (as the API). This complex supply chain increases the risk of contamination and adulteration, mainly with non-porcine ruminant material. The structural similarity of heparins from different origins, the natural variability of the heparin within samples from each source as well as the structural changes induced by manufacturing processes, require increasingly sophisticated methods capable of detecting low levels of contamination. The application of suitable multivariate classification approaches on API 1H NMRspectra serve as rapid and reliable tools for product authentication and the detection of contaminants. Soft Independent Modeling of Class Analogies (SIMCA), Discriminant Analysis (DA), Partial Least Square Discriminant Analysis (PLS-DA) and local classification methods (kNN, BNN and N3) were tested on about one hundred certified heparin samples produced by 14 different manufacturers revealing that Partial Least Squares Discriminant Analysis (PLS-DA) provided the best discrimination of contaminated batches, with a balanced accuracy of 97%.

Efficient Heparin Recovery from Porcine Intestinal Mucosa Using Zeolite Imidazolate Framework-8.

Heparin is one of the most valuable active pharmaceutical ingredients, and it is generally isolated from porcine intestinal mucosa. Traditionally, different types of commercial resins are employed as an adsorbent for heparin uptake; however, using new, less expensive adsorbents has attracted more interest in the past few years to enhance the heparin recovery. Zeolite imidazolate framework-8 (ZIF-8), as a metal–organic framework (MOF) with a high surface area, porosity, and good stability at high temperatures, was selected to examine the heparin recovery. In this research, we demonstrate that ZIF-8 can recover up to ~70% (37 mg g−1) of heparin from porcine intestinal mucosa. A mechanistic study through kinetic and thermodynamic models on the adsorption revealed appropriate surface conditions for the adsorption of heparin molecules. The effect of different variables such as pH and temperature on heparin adsorption was also studied to optimize the recovery. This study is the first to investigate the usage of MOFs for heparin uptake.

The kinetics of heparin adsorption with Dowex 1x1 ion exchange resin

The recovery of heparin from the porcine intestinal mucosa by using the ion exchange resin was studied. The procedure involved the following steps: alkaline hydrolysis, ion exchange resin adsorption, and elution. The activity of heparin was measured by an antifactor Xa assay. The ammonium chloride/ammonium hydroxide was used as a buffer, and the effect of salt concentration on the adsorption efficiency was evaluated. The results showed that the highest heparin yield (86.17%) was achieved at the lowest concentration of ammonium chloride. The pseudo-first-order model was shown as more adequate to describe heparin adsorption onto the resin.

Highly sialylated mucin-type glycopeptide from porcine intestinal mucosa after heparin extraction: O-glycan profiling and immunological activity evaluation.

Mucins are the major proteins that distributed on the intestinal mucosa layer and protect the intestine from pathogens infection. The composition of intestinal mucin O-glycans can affect the health of the gastrointestinal tract in pigs. Porcine intestinal mucosa is widely used as the main raw material of heparin extraction. The heparin extraction residues rich in mucins were usually wasted. The structure of mucin derived O-glycans in porcine intestinal mucosa are currently unknown. In this study, we isolated the mucins from the heparin extraction residues and profiled the O-glycans. After heparin extraction, mucin was digested with trypsin, and separated by strong anion exchange chromatography. The mucin derived O-glycans were release by alkaline β elimination, and analyzed by ultra high performance liquid chromatography-porous graphitized carbon-Fourier transform mass spectrometry (UPLC-PGC-FTMS/MS). Thirty five kinds of O-glycans were identified, most of which were Core 3-derived glycans. In particular, the O-glycans containing sialic acid Neu5Ac accounted for 71.93% of the total O-glycans, which were different from that of other species, including mouse intestine, fish intestine, and porcine colon. The high content sialylated mucin may explain its effect in biological processes. Furthermore, the immunological activity results indicated that the porcine intestinal mucin could promote phagocytosis and proliferation without any cytotoxic effects, which may aid in the development of immunomodulators.

In Vitro Investigation of Thiolated Chitosan Derivatives as Mucoadhesive Coating Materials for Solid Lipid Nanoparticles.

In the present study, chitosan (CS) was thiolated by introducing l-cysteine via amide bond formation. Free thiol groups were protected with highly reactive 6-mercaptonicotinic acid (6-MNA) and less-reactive l-cysteine, respectively, via thiol/disulfide-exchange reactions. Unmodified CS, l-cysteine-modified thiolated CS (CS-Cys), 6-MNA-S-protected thiolated CS (CS-Cys-MNA), and l-cysteine-S-protected thiolated CS (CS-Cys-Cys) were applied as coating materials to solid lipid nanoparticles (SLN). The strength of mucus interaction followed the rank order plain < CS < CS-Cys-Cys < CS-Cys < CS-Cys-MNA, whereas mucus diffusion followed the rank order CS-Cys < CS-Cys-Cys < CS < CS-Cys-MNA < plain. In accordance with lower reactivity, CS-Cys-Cys-coated SLN were immobilized to a lower extent than CS-Cys-coated SLN, while CS-Cys-MNA-coated SLN dissociated from their coating material resulting in a similar diffusion behavior as plain SLN. Consequently, CS-Cys-Cys-coated SLN and CS-Cys-MNA-coated SLN showed the highest retention on porcine intestinal mucosa by enabling a synergism of efficient mucus diffusion and strong mucoadhesion.

Selective and efficient extraction of heparin by arginine-functionalized flowered mesoporous silica nanoparticles with high capacity

Arginine-functionalized amino flowered mesoporous silica (A-FMS-Arg) nanoparticles are fabricated via the amidation reaction, and taken as efficient sorbent for refined extraction of heparin. Attributed by the multiple H-bond interaction between the sulfate group of heparin and the guanidine group of Arginine, A-FMS-Arg nanoparticles exhibit favorable adsorption selectivity towards heparin. The three-dimensional flowered structure provides A-FMS-Arg with abundant adsorption sites, and the adsorption capacity is high to 173.71 mg/g. 1HNMR assay indicate that A-FMS-Arg nanoparticles can selectively adsorb heparin from the mixture of heparin/chondroitin, and the practicability of A-FMS-Arg is well demonstrated by the efficient extraction of heparin from digested porcine intestinal mucosa.

Protamine Sulfate Neutralization Profile of Various Dosages of Bovine, Ovine and Porcine UFHs and Their Depolymerized Derivatives in Non-Human Primates.

Introduction:Currently used unfractionated heparins (UFHs) and low molecular weight heparins (LMWHs) are derived from porcine intestinal mucosa. However, heparins have also been manufactured from tissues of other mammalian species such as cow (Bovine) and sheep (Ovine). Protamine sulphate (PS) is an effective inhibitor of heparin and is used clinically to neutralize both LMWH and UFH. In this study, we determined the PS neutralization profile of these agents in non-human primate model using anti-Xa and anti-IIa methods.Material and Methods:UFHs obtained from bovine, ovine and porcine mucosal tissues and their respective depolymerized LMWHs were administered at both, gravimetric (0.5 mg/kg) and potency adjusted (100 U/kg) dosages regimen intravenously to individual groups of primates in cross over studies. PS was administered at a fixed dosage and the relative neutralization of these anticoagulants was measured utilizing amidolytic anti-Xa and anti-IIa methods.Results:These studies have demonstrated that, the equi-gravimetric dosages of BMH, PMH and OMH have comparable PS neutralization profiles. At potency adjusted dosages, all UFHs were completely neutralized by PS. Although comparable, the LMWHs were not fully neutralized by PS in both the anti-Xa and anti-IIa assays. PS was more efficient in neutralizing the anti-IIa effects of LMWHs.Conclusion:Heparins of diverse origins showed comparable neutralization profiles by PS in the amidolytic anti-Xa and anti-IIa assays.

General Considerations for Diversifying Heparin Drug Products by Improving the Current Heparin Manufacturing Process and Reintroducing Bovine Sourced Heparin to the US Market.

Heparin is one of the most widely used drugs in the world. It has been described as a lifesaving drug due to its roles in treating many serious diseases and illnesses including kidney dialysis, surgery, cardiac-invasive, heart attack, cardiac arrhythmia, acute coronary syndrome, pulmonary embolism, stroke, deep vein thrombosis, blood clot prevention, and many other related uses. Heparin drug products currently approved in the United States are obtained from porcine intestinal mucosa sourced from pigs, the majority of which is imported from China. However, due to the heparin contamination crisis (2008) and potential shortage and to safeguard the quality of current and future heparin supply chains including raw material, Food and Drug administration (FDA) posted a notification on its website titled “FDA Encourages Reintroduction of Bovine-Sourced Heparin”. This perspective is intended to address the history of regulatory and scientific background of heparin drug products obtained from bovine and porcine sources and general recommendations for improving the quality of current heparin manufacturing process including Critical Quality Attributes (CQA), control management, process control, related tests, limits, etc. Additionally, a general plan with systematic steps is proposed for diversifying heparin supply chains by reintroduction of bovine sourced heparin to the US market.

Enhancement of the gut-retention time of resveratrol using waxy maize starch nanocrystal-stabilized and chitosan-coated Pickering emulsions

To enhance its gut health-promoting activity and bioaccessibility, resveratrol was loaded in emulsions stabilized by octenyl succinic anhydride-modified waxy maize starch nanocrystals and then coated with chitosan. Resveratrol loaded in > 0.1% chitosan-coated emulsions adhered more robustly onto porcine intestinal mucosa than did resveratrol powder (23-fold) and chitosan-uncoated emulsions (7-fold). Unlike chitosan-uncoated emulsions, chitosan-coated emulsions were stable during gastric digestion and slowly hydrolyzed in a small intestinal environment. After in vitro digestion, > 90% of the soluble fractions of resveratrol loaded in chitosan-coated and chitosan-uncoated emulsions were 14-fold greater than that of resveratrol powder. The findings suggest that the chitosan-coating can enhance both the gut-retention time of resveratrol and its bioavailability, resulting in prolonged bioactivity of non-oxidized resveratrol. Moreover, the colloidal stability of resveratrol-loaded and chitosan-coated emulsions was maintained for 2 months (pH 4; 25 °C). This study provides useful information to guide development of an effective oral delivery system for resveratrol.

Sulodexide in the Treatment of Chronic Venous Insufficiency: Results of the All-Russian Multicenter ACVEDUCT Program

Sulodexide was a safe and effective medication in the treatment of a cohort of patients with lower extremity chronic venous insufficiency (CVI). This multicenter prospective observational Russian study enrolled 2263 patients with various stages of CVI, mainly classes C3 and C4, who were routinely prescribed sulodexide. Symptom improvement was seen in 56.4%, and 42.8% reported a decreased number of symptoms, mainly after about 2 weeks of treatment and in those aged 30 to 40 years. Sulodexide, used in many countries outside the United States, is a mixture of glycosaminoglycans extracted from porcine intestinal mucosa including low-molecular-weight heparin and dermatan sulfate. It is a lipid-lowering, antithrombotic, and anti-inflammatory agent, decreasing viscosity. It has been used for the management of venous disease and venous ulcerations and for the prevention of recurrent thromboembolism. There are reports of efficacy in advanced stages of CVI, but not routine use. This study overestimates the effectiveness of this drug, adding the results and stating that 99.2% (56.4% plus 42.8%) of patients had improvements. High-quality data are needed.

Less Reactive Thiol Ligands: Key towards Highly Mucoadhesive Drug Delivery Systems.

As less reactive s-protected thiomers can likely interpenetrate the mucus gel layer to a higher extent before getting immobilized via disulfide bond formation with mucins, it was the aim of this study to develop a novel type of s-protected thiomer based on the less reactive substructure cysteine-N-acetyl cysteine (Cys-NAC) in order to obtain improved mucoadhesive properties. For this purpose, two types of s-protected thiomers, polyacrylic acid-cysteine-mercaptonicotinic acid (PAA-Cys-MNA) and polyacrylic acid-cysteine-N-acetyl cysteine (PAA-Cys-NAC), were synthesized and characterized by Fourier-transform infrared spectroscopy (FT-IR) and the quantification of attached disulfide ligands. The viscosity of both products was measured in the presence of NAC and mucus. Both thiomers were also evaluated regarding swelling behavior, tensile studies and retention time on the porcine intestinal mucosa. The FT-IR spectra confirmed the successful attachment of Cys-MNA and Cys-NAC ligands to PAA. The number of attached sulfhydryl groups was in the range of 660–683 µmol/g. The viscosity of both s-protected thiomers increased due to the addition of increasing amounts of NAC. The viscosity of the mucus increased in the presence of 1% PAA-Cys-MNA and PAA-Cys-NAC 5.6- and 10.9-fold, respectively, in comparison to only 1% PAA. Both s-protected thiomers showed higher water uptake than unmodified PAA. The maximum detachment force (MDF) and the total work of adhesion (TWA) increased in the case of PAA-Cys-MNA up to 1.4- and 1.6-fold and up to 2.4- and 2.8-fold in the case of PAA-Cys-NAC. The retention of PAA, PAA-Cys-MNA, and PAA-Cys-NAC on porcine intestinal mucosa was 25%, 49%, and 76% within 3 h, respectively. The results of this study provide evidence that less reactive s-protected thiomers exhibit higher mucoadhesive properties than highly reactive s-protected thiomers.

Thiolated chitosans: Are Cys-Cys ligands key to the next generation?

The potential of Cys-Cys ligands for the development of a novel type of S-protected thiomers was evaluated. S-protected thiomers chitosan-N-acetylcysteine-mercaptonicotinamide (CS-NAC-MNA) and chitosan-N-acetylcysteine-N-acetylcysteine (CS-NAC-NAC) were synthesized and characterized. Viscosity of polymers in presence of various concentrations of S-amino acids was monitored. Mucoadhesive properties were evaluated. FT-IR characterization confirmed the covalent attachment of NAC-MNA and NAC-NAC. Attached sulfhydryl groups were found in the range of 550 μmol/g. In the presence of amino acids bearing a free thiol group viscosity of both polymers increased. This increase in viscosity depended on the amount of added free thiols. Maximum force required to detach CS-NAC-MNA and CS-NAC-NAC from porcine intestinal mucosa was 1.4- and 2.7-fold higher than that required for chitosan, respectively. CS-NAC-MNA adhered up to 3 h, whereas CS-NAC-NAC adhered even for 8 h on this mucosa. Accordingly, the Cys-Cys substructure could be identified as highly potent ligand for the design of mucoadhesive polymers.

Synthesis and in vitro characterization of a preactivated thiolated acrylic acid/acrylamide-methylpropane sulfonic acid copolymer as a mucoadhesive sprayable polymer.

Development of a preactivated thiomer as sprayable excipient for mucoadhesive formulations. CG4500 (acrylic acid/acrylamide-methyl propane sulfonic acid copolymer) was thiolated by conjugation with L-cysteine and preactivated by further modification with 2-mercaptonicotinic acid (MNA) in a two-step synthesis and characterized regarding degree of modification and cytotoxicity on Caco-2 cells. The mucoadhesive properties of this novel thiomer were evaluated via rheological synergism, tensile and mucosal residence time studies. Furthermore, the sprayability of the thiomer was evaluated. The newly synthesized derivatives CG4500-SH and CG4500-S-S-MNA showed mean coupling rates of 651µmol thiol groups and 264µmol MNA per gram polymer, respectively. Even for the unmodified polymer a rheological synergism was observed with isolated porcine intestinal mucus, which was 2.81-fold higher in case of the preactivated thiomer. Mucoadhesion studies on freshly excised porcine intestinal mucosa confirmed these results via a 2.43-fold higher total work of adhesion and a 2.31-fold higher mucosal residence time of the preactivated thiomer. In sprayability tests it was shown that solutions of the preactivated thiomer could be sprayed in concentrations up to 12% (m/V). The novel polymer CG4500-S-S-MNA is a promising sprayable excipient for mucoadhesive formulations.

Phenytoin-loaded lipid-core nanocapsules improve the technological properties and in vivo performance of fluidised bed granules.

Lipid-core nanocapsules (LNCs) were recently reported by our group as a suitable binder system to produce fluidised bed granules. However, there is still a lack of knowledge about the influence of using these nanocarriers loaded with a drug on the properties of the granules and their in vivo performance. Therefore, this study was designed to produce innovative fluidised bed granules containing phenytoin-loaded LNCs (LNCPHT) as a strategy to evaluate the influence of the presence of the drug-loaded nanocarriers on their in vitro and in vivo properties. Granules were produced using a mixture of maltodextrin and phenytoin (1:0.004 w/w) as substrate. They were prepared by fluid bed granulation using water or LNCPHT as the liquid binder, affording good yields (73-82%) of granules with low moisture content (<5%). Granules prepared with LNCPHT had larger mean size (122μm) compared to maltodextrin primary particles (50μm) due to the formation of solid bridges. Moreover, the use of LNCPHT as the liquid binder improved their powder flow properties. The nanocarriers were recovered after aqueous dispersion (3.00mg.mL-1 of PHT) with a redispersibility close to 90%. After reconstitution in water, granules containing LNCPHT showed an improved dissolution behaviour compared to those prepared without them. In addition, they showed a higher mucoadhesive effect due to a combined effect of the LNCPHT and maltodextrin in the interactions with porcine intestinal mucosa. Regarding the in vivo studies, granules containing the combination of non-encapsulated PHT and PHT-loaded lipid-core nanocapsules increased the latency to seizures compared to placebo granules, showing effective anticonvulsant effect in mice. In conclusion, the use of drug-loaded nanocapsules as binder is an encouraging approach to produce fluidised bed mucoadhesive granules with improved technological properties and in vivo performance.

Quantitative Analysis of Impurities in Unfractionated Heparin of Bovine Origin.

The USP heparin sodium monograph lists impurities with specifications developed for porcine derived products. Most of these impurities are of biological origin and are present in porcine intestinal mucosa, the tissue source used in the production of porcine heparin. One of the specified impurities, oversulfated chondroitin sulfate (OSCS), has been introduced in the monograph to detect intended adulteration of heparin products with this impurity. The evaluation of bovine intestinal heparin as an alternative source of pharmaceutical heparin included an evaluation of bovine heparin with the current USP heparin sodium monograph methods. This evaluation included a comparison of impurity quantities observed in multiple bovine intestinal heparin samples against the specifications found in the USP heparin sodium monograph. The impurities investigated in this study were protein, galactosamine, nucleotidic impurities, and OSCS. Bovine intestinal heparin met the requirements in the tests for protein, galactosamine, and nucleotidic impurities. A potential issue was observed with the strong anion exchange high performance liquid chromatography (SAX-HPLC) used to analyze for the presence of OSCS. While the OSCS was well-resolved from the bovine heparin peak, the resolution of dermatan sulfate from heparin did not consistently meet system suitability requirements in the current USP Heparin sodium monograph. The overall levels of impurities observed in bovine intestinal mucosal heparin were comparable to those observed in porcine intestinal mucosal heparin. Bovine intestinal mucosal heparin can be produced with acceptable impurity levels that align with these important quality attributes found in porcine heparin.