Biodegradation of inorganic drug delivery systems in subcutaneous conditions.

Despite extensive efforts to develop delivery systems for oral administration, subcutaneous (s.c.) injection remains the most common way to administer peptide drugs. To limit the number of frequent injections, sustained release systems that are easy to produce, suitable for various drugs, safe and biodegradable are urgently needed. Porous silicon (PSi) has been recognized to be one of the most promising materials for s.c. peptide delivery, but its biodegradation in s.c. tissue has not been studied in vivo, despite extensive in vitro research. In the present study, differently modified PSi microparticles were injected s.c. in mice, after which the morphology of the particles was thoroughly studied with transmission electron microscopy, micro-computed tomography and X-ray diffraction. Furthermore, histopathology of the s.c. tissue was analyzed to evaluate biocompatibility. To the best of our knowledge, this is the first systematic study which reveals the degradation behavior of various PSi materials in vivo. The PSi surface chemistry significantly affected the biodegradation rate of the s.c. injected microparticles. The most hydrophobic PSi microparticles with hydrocarbonized surface showed the lowest biodegradation rate while the hydrophilic microparticles, with oxide surface, degraded the fastest. The results from different empirical methods complemented each other to deduce the biodegradation mechanism of the inorganic delivery system, providing useful information for future development of s.c. carriers.

The effect of poly (lactic-co-glycolic) acid composition on the mechanical properties of electrospun fibrous mats.

The aim of this study was to investigate the influence of polymer molecular structure on the electrospinnability and mechanical properties of electrospun fibrous mats (EFMs). Polymers with similar molecular weight but different composition ratios (lactic acid (LA) and glycolic acid (GA)) were dissolved in binary mixtures of N,N-dimethylformamide (DMF) and tetrahydrofuran (THF). The intrinsic viscosity and rheological properties of polymer solutions were investigated prior to electrospinning. The morphology and mechanical properties of the resulting EFMs were characterized by scanning electron microscope (SEM) and dynamic mechanical analysis (DMA). Sufficiently high inter-molecular interactions were found to be a prerequisite to ensure the formation of fibers in the electrospinning process, regardless the polymer composition. The higher the amount of GA in the polymer composition, the more ordered and entangled molecules were formed after electrospinning from the solution in THF-DMF, which resulted in higher Young's modulus and tensile strength of the EFMs. In conclusion, this study shows that the mechanical properties of EFMs, which depend on the polymer molecule-solvent affinity, can be predicted by the inter-molecular interactions in the starting polymer solutions and over the drying process of electrospinning.

Cardiac vagal tone, a non-invasive measure of parasympathetic tone, is a clinically relevant tool in Type 1 diabetes mellitus.

AIMS: To compare a novel index of parasympathetic tone, cardiac vagal tone, with established autonomic variables and to test the hypotheses that (1) cardiac vagal tone would be associated with established time and frequency domain measures of heart rate and (2) cardiac vagal tone would be lower in people with Type 1 diabetes than in a matched healthy cohort and lower still in people with established neuropathy. METHODS: Cardiac vagal tone is a validated cardiometrically derived index of parasympathetic tone. It is measured using a standard three-lead electrocardiogram which connects, via Bluetooth, to a smartphone application. A 5-min resting recording of cardiac vagal tone was undertaken and observational comparisons were made between 42 people with Type 1 diabetes and peripheral neuropathy and 23 without peripheral neuropathy and 65 healthy people. In those with neuropathy, 24-h heart rate variability values were compared with cardiac vagal tone. Correlations between cardiac vagal tone and clinical variables were also made. RESULTS: Cardiac vagal tone was lower in people with established neuropathy and Type 1 diabetes in comparison with healthy participants [median (interquartile range) linear vagal scale 3.4 (1.6-5.5 vs 7.0 (5.5-9.6); P < 0.0001]. Cardiac vagal tone was positively associated with time (r = 0.8, P < 0.0001) and frequency domain markers of heart rate variability (r = 0.75, P < 0.0001), representing established measures of parasympathetic function. Cardiac vagal tone was negatively associated with age (r=-0.32, P = 0.003), disease duration (r=-0.43, P < 0.0001) and cardiovascular risk score (r=-0.32, P = 0.006). CONCLUSIONS: Cardiac vagal tone represents a convenient, clinically relevant method of assessing parasympathetic nervous system tone, potentially facilitating the earlier identification of people with Type 1 diabetes who should undergo formal autonomic function testing.

The flow properties and presence of crystals in drug-polymer mixtures: Rheological investigation combined with light microscopy.

The presence of solid matter in polymer melts affects the rheological properties of a drug-polymer mixture, and thus the processability of these mixtures in melt-based processes. The particle morphological changes related to dissolution and crystal growth in the mixtures of paracetamol and ibuprofen with polyethylene oxide and methacrylate copolymer (Eudragit® E PO) were observed by polarized microscopy simultaneously while measuring their rheological properties within temperature ranges relevant for melt processes, such as hot melt extrusion and fused deposition modeling 3D printing. The dissolution of solid crystalline matter into the molten polymer and its effects on the rheological parameters showed that the plasticization effect of the drug was highly dependent on the temperature range, and at a temperature high enough, plasticization induced by the small-molecule drugs could enhance the flowability even at very high drug loads. Therefore, even supersaturated mixtures can be plasticized efficiently, enabling their melt processing, such as hot melt extrusion or 3D printing. The combination of rheometry and polarized light microscopy proved to be very useful for studying the link between morphological changes in the drug-polymer and the flow behavior of the drug-polymer mixtures at different temperature ranges and deformation modes.

Visualization and prediction of porosity in roller compacted ribbons with near-infrared chemical imaging (NIR-CI).

The porosity of roller compacted ribbon is recognized as an important critical quality attribute which has a huge impact on the final product quality. The purpose of this study was to investigate the use of near-infrared chemical imaging (NIR-CI) for porosity estimation of ribbons produced at different roll pressures. Two off-line methods were utilized as reference methods. The relatively fast method (oil absorption) was comparable with the more time-consuming mercury intrusion method (R(2)=0.98). Therefore, the oil method was selected as the reference off line method. It was confirmed by both reference methods that ribbons compressed at a higher pressure resulted in a lower mean porosity. Using NIR-CI in combination with multivariate data analysis it was possible to visualize and predict the porosity distribution of the ribbons. This approach is considered important for process monitoring and control of continuously operating roller compaction line.

Dissolution testing of amorphous solid dispersions.

The main purpose of this study was to investigate the effect of different polymers, with varying physicochemical properties and molecular weight on the stability and dissolution of co-milled amorphous solid dispersions (ASDs) of piroxicam (PRX). The stability of amorphous PRX (aPRX) in ASDs was significantly improved by the polymers. In-line Raman spectroscopy revealed that solvent mediated solid state changes occurred in biorelevant medium, however differences between ASDs were found. Thus, the dissolution behavior of ASDs of PRX and the respective polymer during conventional large volume (900ml) and a commercial small volume (20ml) dissolution testing was evaluated. The results of these studies indicated that the molecular weight of the polymer (PVP90 vs PVP25) is influencing the solubility of PRX from ASD. Interestingly the effect of molecular weight of the polymer was different than reported previously in the literature for the similar ASDs prepared by spray drying. Furthermore, the dose related bioavailability was determined by investigating the experimental saturation concentrations for different doses. These studies confirmed the findings of the dissolution studies. The differences are presumably caused by the formation of physically different diffusion layers around the ASD particles.

Investigations on the effect of different cooling rates on the stability of amorphous indomethacin.

Amorphous forms of indomethacin have previously been prepared using various preparation techniques and it could be demonstrated that the way the material was prepared influenced the physicochemical properties of the amorphous form of the drug. The aim of this study was to use one preparation technique (transformation via the melt) to prepare amorphous indomethacin and to investigate the influence of the cooling rate (as a processing parameter) on the physical stability of the resulting amorphous form. The amorphous materials obtained were analysed for their structural characteristics using Raman spectroscopy in combination with multivariate data analysis. The onset of crystallisation was determined as an indicator of the physical stability of the materials using differential scanning calorimetry (DSC) and polarising light microscopy. The Johnson-Mehl-Avrami (JMA) model and Sestak-Berggren (SB) model were used in this study to describe the non-isothermal crystallisation behaviour. All differently cooled samples were completely X-ray amorphous. Principal component analysis of the Raman spectra of the various amorphous forms revealed that the samples clustered in the scores plot according to the cooling rate, suggesting structural differences between the differently cooled samples. The minimum cooling rate required to obtain amorphous indomethacin was 1.2 K min(-1), as assessed from the time-temperature-transformation (TTT) diagram. The physical stability of the samples was found to increase as a function of cooling rate in the order of 30 K min(-1) > 20 K min(-1) > 10 K min(-1) > 5 K min(-1) > 3 K min(-1) ≈ 1.2 K min(-1) and was in agreement with calculated descriptors for the glass forming ability (GFA), including the reduced glass transition temperature (T(rg)) and the reduced temperature (T(red)). The JMA model could not be applied to describe the crystallisation process for the differently cooled melts of indomethacin in this study. The kinetic exponent M from the autocatalytic SB model however, showed a positive correlation with glass stability.

Crystallization of a polymorphic hydrate system.

Nitrofurantoin can form two monohydrates, which have the same chemical composition and molar ratio of water, but differ in the crystal arrangements. The two monohydrates (hydrates I and II) could be produced independently via evaporative crystallization, where supersaturation and solvent composition were both found to have an effect. Hydrate I showed much slower crystallization than hydrate II. During cooling crystallization, the nucleation and growth of hydrate II was again dominant, consuming all supersaturation and leading to no hydrate I formation. Seeding of hydrate I during cooling crystallization was also applied, but the hydrate I seeds were not able to initiate its nucleation rather than dissolving into crystallizing solution. Although solubility tests revealed that hydrate II is more stable than hydrate I due to its lower solubility (110 +/- 4 and 131 +/- 12 microg/mL for hydrates II and I, respectively), this difference is rather small. Therefore, the small free energy difference between the two hydrates, together with the slow crystallization of hydrate I, both lead to a hindrance of hydrate I formation. Furthermore, the crystal structure of hydrate II demonstrated a higher H-bonding extent than hydrate I, suggesting its more favorable crystallization. This is in good agreement with experimental results.

Implementation of a process analytical technology system in a freeze-drying process using Raman spectroscopy for in-line process monitoring.

The aim of the present study was to propose a strategy for the implementation of a Process Analytical Technology system in freeze-drying processes. Mannitol solutions, some of them supplied with NaCl, were used as models to freeze-dry. Noninvasive and in-line Raman measurements were continuously performed during lyophilization of the solutions to monitor real time the mannitol solid state, the end points of the different process steps (freezing, primary drying, secondary drying), and physical phenomena occurring during the process. At-line near-infrared (NIR) and X-ray powder diffractometry (XRPD) measurements were done to confirm the Raman conclusions and to find out additional information. The collected spectra during the processes were analyzed using principal component analysis and multivariate curve resolution. A two-level full factorial design was used to study the significant influence of process (freezing rate) and formulation variables (concentration of mannitol, concentration of NaCl, volume of freeze-dried sample) upon freeze-drying. Raman spectroscopy was able to monitor (i) the mannitol solid state (amorphous, alpha, beta, delta, and hemihydrate), (ii) several process step end points (end of mannitol crystallization during freezing, primary drying), and (iii) physical phenomena occurring during freeze-drying (onset of ice nucleation, onset of mannitol crystallization during the freezing step, onset of ice sublimation). NIR proved to be a more sensitive tool to monitor sublimation than Raman spectroscopy, while XRPD helped to unravel the mannitol hemihydrate in the samples. The experimental design results showed that several process and formulation variables significantly influence different aspects of lyophilization and that both are interrelated. Raman spectroscopy (in-line) and NIR spectroscopy and XRPD (at-line) not only allowed the real-time monitoring of mannitol freeze-drying processes but also helped (in combination with experimental design) us to understand the process.

Development and characterization of aqueous amylose-rich maize starch dispersion for film formation.

Development and characterization of amylose-rich starch dispersion for film forming was performed. The influence of dispersion preparation temperature on amylose-rich maize starch (Hylon VII) film formation, and the physical properties of the films were investigated. The film-forming ability of the dispersions was evaluated with free films plasticized with glycerol and sorbitol, and the films were prepared at an elevated temperature (70 degrees C) by a casting technique. The solid-state and particle properties of dispersions were investigated by means of X-ray diffraction (XRD), Fourier transform near infrared (FT-NIR) spectroscopy and laser diffraction particle size analysis. Free films were characterized with respect to their appearance, by FT-NIR spectroscopy, and by XRD. Mechanical stress-strain properties were also studied. Increasing the temperature of dispersion preparation results in higher crystallinity, thus affecting the film forming ability. Mechanically strong and elastic films can be formed from amylose-rich starch dispersion formed at 40 degrees C. The more crystalline precipitate complex (obtained at 80 degrees C) and the entirely amorphous gel (obtained at 10 degrees C) formed non-continuous and cloudy films. The better film-forming properties of the dispersion formed at 40 degrees C are probably due to the highly amorphous structure and smaller particle size. The study shows the possibility of using ambient tempered amylose-starch dispersion for film forming.

Acute traumatic extension deficit of the knee.

The purpose of this study was to evaluate the events leading to acute traumatic extension deficit of the knee and the arthroscopic findings in these patients. A total of 78 consecutive patients treated in the Turku university hospital during the years 1994-1996 were included. The mean annual incidence of acute traumatic extension deficits of the knee in our study was 1.1 per 10 000 inhabitants. The single most common (33%) event causing the extension deficit was non-sports related twisting of the knee. Various sports related activities accounted for 42% of the extension deficits, and soccer was the most common sport in this group. In conclusion, acute traumatic extension deficit of the knee is usually a sign of serious intra-articular damage, and the most likely finding (in 82% of the patients in our study) is either a meniscal rupture, an anterior cruciate ligament (ACL) rupture, a patellar dislocation, or a combination of these. The lesions in these knees require prompt evaluation by an orthopaedic surgeon mainly because of the high number of bucket-handle and menisco-capsular insertion ruptures of the menisci, which are possibly suitable for repair.

Acute traumatic hemarthrosis of the knee. Is routine arthroscopic examination necessary? A study of 320 consecutive patients.

BACKGROUND AND AIMS: Distortions and contusions of the knee are common. Fairly often they will result in hemarthrosis. The purpose of this study was to evaluate the findings in patients with acute traumatic hemarthrosis of the knee and based on these findings estimate the necessity of acute arthroscopic examination. MATERIAL AND METHODS: A total of 320 patients were examined arthroscopically between the years 1994 to 96 in the Turku University Hospital. Patients with other than chondral or osteochondral fractures were excluded. RESULTS: The most common arthroscopic findings were rupture of the ACL (45%), dislocation of the patella (23%) and meniscal tear (21%). In only 113 (35%) cases an immediate therapeutic procedure was performed in addition to the arthroscopic examination. CONCLUSIONS: Based on our findings we believe that routine arthroscopic examination is not necessary in patients with acute traumatic hemarthrosis of the knee. In our opinion the patient should be examined and followed by an orthopedic surgeon and if a lesion requiring operative treatment is diagnosed or suspected, an arthroscopic examination should be scheduled. The timing of the procedure should be such that all possible lesions encountered can be treated in the same operation.

The long-term results of Lubinus interplanta hemiarthroplasty in 228 acute femoral neck fractures. A retrospective six-year follow-up.

BACKGROUND AND AIMS: We retrospectively studied survival of patients treated with Lubinus interplanta (IP) semiendoprosthesis in acute hip fracture. MATERIALS AND METHODS: The long-term results of Lubinus interplanta (IP) hemiarthroplasty in 228 acute medial fractures of the femoral neck in 222 patients were reviewed. RESULTS AND CONCLUSIONS: The mean age of the patients was 80.3 years. The mean duration of post-operative follow-up was 6.3 years (range 5-8 years). There were 12 reoperations (5.3%), of which 8 were revisions (3.5%). Dislocation rate was 3.5%. Survival was 93% at three years and 88% at six years (Kaplan-Meier-survival analysis). CONCLUSIONS: These results surpass those previously published after Thompson and Moore hemiarthroplasties. The Lubinus prosthesis has a greater CCD (caput,collum, diaphyse) angle and a longer stem compared to Thompson and Moore implants. The need for resection of calcar cortex is also limited. These biomechanical facts may explain the good long-term results of Lubinus hemiarthroplasty.

Near infrared reflectance spectroscopy for the fast identification of PVC-based films.

Near infrared (NIR) reflectance spectroscopy was used to develop a non-destructive and rapid qualitative method for the analysis of plastic films used by the pharmaceutical industry for blistering. Three types of films were investigated: 250 microm PVC [poly(vinyl chloride)] films, 250 microm PVC films coated with 40 g m(-2) of PVDC [poly(vinylidene dichloride)] and 250 microm PVC films coated with 5 g m(-2) of TE (Thermoelast) and 90 g m(-2) of PVDC. Three analyses were carried out using different pre-treatment options and a PLS (partial least squares) algorithm. Each analysis was aimed at identifying one type of film and rejecting all types of false sample (different thickness, colour or layer). True and false samples from four plastics manufacturers were included in the calibration sets in order to obtain robust methods that were suitable regardless of the supplier. Specificity was demonstrated by testing validation sets against the methods. The tests showed 0% of type I (false negative identification) and 1% of type II errors (false positive identification) for the PVC method, 13 and 3%, respectively, for the PVC-PVDC method and no error for the PVC-TE-PVDC method. Type II errors, mostly due to the slight sensitivity of the methods to film thickness, are easily corrected by simple thickness measurements. This study demonstrates that NIR spectroscopy is an excellent tool for the identification of PVC-based films. The three methods can be used by the pharmaceutical industry or plastics manufacturers for the quality control of films used in blister packaging.

Phosphorylation of serine 230 promotes inducible transcriptional activity of heat shock factor 1.

Heat shock factor 1 (HSF1) is a serine-rich constitutively phosphorylated mediator of the stress response. Upon stress, HSF1 forms DNA-binding trimers, relocalizes to nuclear granules, undergoes inducible phosphorylation and acquires the properties of a transactivator. HSF1 is phosphorylated on multiple sites, but the sites and their function have remained an enigma. Here, we have analyzed sites of endogenous phosphorylation on human HSF1 and developed a phosphopeptide antibody to identify Ser230 as a novel in vivo phosphorylation site. Ser230 is located in the regulatory domain of HSF1, and promotes the magnitude of the inducible transcriptional activity. Ser230 lies within a consensus site for calcium/calmodulin-dependent protein kinase II (CaMKII), and CaMKII overexpression enhances both the level of in vivo Ser230 phosphorylation and transactivation of HSF1. The importance of Ser230 was further established by the S230A HSF1 mutant showing markedly reduced activity relative to wild-type HSF1 when expressed in hsf1(-/-) cells. Our study provides the first evidence that phosphorylation is essential for the transcriptional activity of HSF1, and hence for induction of the heat shock response.

The expression of intermediate filament protein nestin as related to vimentin and desmin in regenerating skeletal muscle.

Intermediate filament (IF) proteins show specific spatial and temporal expression during development of skeletal muscle. Nestin, the least known muscle IF, has an important role in neuronal regeneration. Therefore, we analyzed the expression pattern of nestin as related to that of vimentin and desmin during skeletal muscle regeneration. Nestin and vimentin appear at 6 h post-injury in myoblasts, with maximum expression around day 3-5 post-injury. Thereafter, vimentin expression ceases completely, whereas that of nestin is downregulated to remain only in the sarcoplasm next to neuromuscular and myotendinous junctions. Desmin appears at 6-12 h post-injury and becomes the predominant IF in myofibers simultaneously with the appearance of cross-striations. The expression pattern and colocalization of nestin and vimentin, known to form heteropolymers, suggests that they are essential during the early dynamic phase of the myofiber regeneration when migration, fusion, and structural modeling of myogenic cells occurs, whereas desmin is responsible for keeping myofibrils in register in mature myofibers. In conclusion, the expression of nestin is dynamically orchestrated with that of vimentin and desmin during skeletal muscle regeneration and recapitulates that seen during myogenesis, i.e. these IFs have key functional roles in the construction and restoration of skeletal myofibers.

Characterization of wet massing behavior of silicified microcrystalline cellulose and alpha-lactose monohydrate using near-infrared spectroscopy.

The purpose of this study was to investigate the energetic state of water in silicified microcrystalline cellulose (SMCC) and alpha-lactose monohydrate wet masses using near-infrared (NIR) spectroscopy. The applicability of NIR spectroscopy to studying pharmaceutical wet masses at a wide moisture range was evaluated in comparison with mixer torque rheometry (MTR). With increasing moisture content changes in the physical properties of the samples resulted in an apparent increase in log (1/R) throughout the whole spectrum. The upward displacement of baseline and the relative height of water bands were greatest with materials that had a poor liquid-retention capacity. In the case of SMCC and 1:1 mixture of SMCC and alpha-lactose monohydrate, the height of the baseline-corrected water bands increased linearly at low moisture contents, thereafter achieving a plateau stage. According to the MTR results, the plateau stage of the band heights indicated a capillary state of liquid saturation. The second derivative spectrum was capable of distinguishing monohydrate, absorbed, and adsorbed water, which overlapped in the absorbance spectrum. When water was absorbed to the internal structure of the material (SMCC), the water bands were first seen at higher wavelengths, then followed by a shift to lower wavelengths. When water was only adsorbed onto the surface of the particles (glass ballotini), the water bands were seen directly in the region of bulk water.

Visualization of fluid-bed granulation with self-organizing maps.

The degree of the instrumentation of pharmaceutical unit operations has increased. This instrumentation provides information of the state of the process and can be used for both process control and research. However, on-line process data is usually multidimensional, and is difficult to study with traditional trends and scatter plots. The Self-Organizing Map (SOM) is a recognized tool for dimension reduction and process state monitoring. The basics of the SOM and the application to on-line data collected from a fluid-bed granulation process are presented. As a batch process, granulation traversed through a number of process states, which was visualized with SOM as a two-dimensional map. In addition, it is demonstrated how the differences between granulation batches can be studied. The results suggest that SOM together with new in-line process analytical solutions support the in-process control of the pharmaceutical unit operations. Further, a novel research tool for understanding the phenomena during processing is achieved.

Novel identification of pseudopolymorphic changes of theophylline during wet granulation using near infrared spectroscopy.

The purpose of this study was to demonstrate the efficiency of near infrared (NIR) spectroscopy in studying the pseudopolymorphic changes and the state of water during the wet granulation process. Anhydrous theophylline was granulated in a planetary mixer using water as granulation liquid. NIR spectra and differential scanning calorimetric (DSC) and wide-angle X-ray scattering (WAXS) patterns of theophylline granules, anhydrous theophylline, and theophylline monohydrate were measured. At a low level of granulation liquid (0.3 mol of water per mole of anhydrous theophylline), water absorption maxima in the NIR region occurred first at around 1475 and 1970 nm. These absorption maxima were identical to those of theophylline monohydrate. At higher levels of granulation liquid (1.3-2.7 mol of water per mole of anhydrous theophylline), the increasing absorption maxima occurred at 1410 and 1905 nm due to OH vibrations of free water molecules. X-ray diffraction patterns confirmed the transformation of anhydrous theophylline to theophylline monohydrate during wet granulation. NIR spectroscopy was able to detect different states of water molecules during the wet granulation process faster and in a more flexible manner than conventional methods.