Robustness of a continuous direct compression line against disturbances in feeding.

The aim of the current study was to characterize the robustness of an integrated continuous direct compression (CDC) line against disturbances from feeding, i.e. impulses of API and short step disturbances. These disturbances mimicked typical variations that can be encountered during long-term manufacture. The study included a primary formulation, with API of standard particle size, which was manufactured at 5 and 10 kg/h production rates, and a modified formulation, with API of large particle size, which was manufactured at 5 kg/h production rate. Overall, the CDC line smoothened all the disturbances, fulfilling the USP uniformity of dosage units (UDU) limit for single tablets. However, runs with the modified formulation failed the pharmacopoeia UDU requirements for the entire run due to high variation between tablets. The primary formulation passed the requirements in all cases. The residence time distribution (RTD) results indicated that the primary formulation allowed better smoothening ability, and an increase in production rate led to poorer smoothening due to shorter RTD. The RTDs revealed that a substantial part of back-mixing took place after the blender. Thus, the tablet press has an important role in smoothening disturbances longer than the mean residence time of the blender, which was very short.

Assessing tissue damage in multiple sclerosis: a biomarker approach.

OBJECTIVES: Magnetic resonance imaging (MRI) of the brain and spinal cord is the gold standard for assessing disease activity in multiple sclerosis (MS). MRI is an excellent instrument for determination of accumulated damage to the brain and spinal cord, but tells us little about ongoing tissue damage. In this study, biomarkers of oligodendrocyte, axonal and astrocyte injury were related to MRI and clinical findings and used to assess tissue damage in MS. MATERIALS AND METHODS: Cerebrospinal fluid from 44 patients with relapsing-remitting MS, 20 with secondary progressive MS and 15 controls were investigated with ELISA to determine levels of myelin basic protein (MBP), neurofilament light (NFL) and glial fibrillary acidic protein (GFAp). Patients underwent MRI of the brain and spinal cord, and gadolinium enhancing lesions, T1 lesions and T2 lesions were counted. RESULTS: Patients in clinical relapse and patients with nonsymptomatic gadolinium enhancing lesions had high levels of MBP and NFL, indicating ongoing damage to oligodendrocytes and axons. The level of MBP dropped quickly within a week from the onset of a relapse, whereas NFL remained elevated for several weeks and GFAp slowly rose during the course of a relapse. Relapsing-remitting MS patients without gadolinium enhancing lesions had values of MBP, NFL and GFAp similar to controls, while patients with secondary progressive disease had moderately increased values of all biomarkers. CONCLUSIONS: Analysis of MBP, NFL and GFAp provides direct means to measure tissue damage and is a useful addition to our methods for evaluation of MS.

Combining CAR T cells and the Bcl-2 family apoptosis inhibitor ABT-737 for treating B-cell malignancy.

B-cell malignancies upregulate the B-cell lymphoma 2 (Bcl-2) family inhibitors of the intrinsic apoptosis pathway, making them therapy resistant. However, small-molecule inhibitors of Bcl-2 family members such as ABT-737 restore a functional apoptosis pathway in cancer cells, and its oral analog ABT-263 (Navitoclax) has entered clinical trials. Gene engineered chimeric antigen receptor (CAR) T cells also show promise in B-cell malignancy, and as they induce apoptosis via the extrinsic pathway, we hypothesized that small-molecule inhibitors of the Bcl-2 family may potentiate the efficacy of CAR T cells by engaging both apoptosis pathways. CAR T cells targeting CD19 were generated from healthy donors as well as from pre-B-ALL (precursor-B acute lymphoblastic leukemia) patients and tested together with ABT-737 to evaluate apoptosis induction in five B-cell tumor cell lines. The CAR T cells were effective even if the cell lines exhibited different apoptosis resistance profiles, as shown by analyzing the expression of apoptosis inhibitors by PCR and western blot. When combining T-cell and ABT-737 therapy simultaneously, or with ABT-737 as a presensitizer, tumor cell apoptosis was significantly increased. In conclusion, the apoptosis inducer ABT-737 enhanced the efficacy of CAR T cells and could be an interesting drug candidate to potentiate T-cell therapy.

Systemic up-regulation of TLR4 causes lipopolysaccharide-induced augmentation of nasal cytokine release in allergic rhinitis.

BACKGROUND: Allergic rhinitis is a systemic disorder, and it is clinically well recognized that it can be aggravated by infection. Activation of the innate immune system constitutes a critical element in the process. Toll-like receptors (TLRs) comprise a part of the innate immune system, and lipopolysaccharide (LPS)-induced activation of TLR4 represents bacterial-induced interactions in various model systems. The present study examines how TLR2 and TLR4 expression is affected by symptomatic allergic rhinitis, and if LPS added upon allergen affects nasal cytokine release. METHODS: In patients with pollen-induced allergic rhinitis and healthy non-allergic volunteers, nasal lavage (NAL), peripheral blood and bone marrow were sampled before and during the pollen season. TLR2 and TLR4 expression was determined flow cytometrically. Changes in the TLR receptor expression pattern were evaluated by a nasal challenge with allergen followed by LPS, or vice versa. Symptoms along with cells and cytokines in NAL were analyzed. RESULTS: TLR4 expression increased in leukocytes in NAL, peripheral blood and bone marrow during symptomatic allergic rhinitis. A similar increase was seen for TLR2 in neutrophils in blood. Nasal challenge with allergen followed by LPS augmented the release of IL-4, IL-5, IL-10, IL-13, IFN-γ and TNF-α. CONCLUSION: A systemic up-regulation of TLR4 in symptomatic allergic rhinitis may explain why LPS preceded by allergen increases nasal cytokine release.

Nod1, Nod2 and Nalp3 receptors, new potential targets in treatment of allergic rhinitis?

BACKGROUND: Recently, a new set of pattern-recognition receptors, the nucleotide-binding oligomerization domain (Nod)-like receptors (NLRs), have emerged. Their activation, either by allergens or microbes, triggers an inflammatory response. The knowledge about NLRs in human airways is limited. AIM OF THE STUDY: To investigate presence of NLRs in the human nose of healthy individuals and patients with intermittent allergic rhinitis outside and during pollen season. METHODS: The expression of Nod1, Nod2, and Nalp3 in nasal biopsies was determined with real-time RT-PCR and immunohistochemistry. Cultured primary human nasal epithelial cells (HNECs) were analyzed using real-time RT-PCR and flow cytometry to further verify the presence of NLRs in the epithelium. RESULTS: Immunohistochemical analysis revealed presence of Nod1, Nod2, and Nalp3 in the nasal epithelium. This was corroborated in cultured HNECs. Patients suffering from symptomatic allergic rhinitis exhibited lower Nod1 and Nalp3 mRNA levels than both controls and patients during pollen season. Nod2 expression was found in all specimens tested, but no differences were seen between the three groups. CONCLUSION: Nod1, Nod2, and Nalp3 receptors were found to be present in the human nose. The expression of Nod1 and Nalp3 were down-regulated during pollen season among patients with allergic rhinitis. This opens up for new insights and novel therapeutic strategies in inflammatory airway disease.

Inverse relation between nasal fluid Clara Cell Protein 16 levels and symptoms and signs of rhinitis in allergen-challenged patients with intermittent allergic rhinitis.

BACKGROUND: Decreased levels of the anti-inflammatory Clara Cell Protein 16 (CC16) are found in intermittent allergic rhinitis (IAR) and asthma. In asthma this decrease has been associated with hyperreactivity and the A38G single nucleotide polymorphism (SNP). The aim of this study was to examine if IAR is associated with signs and symptoms of rhinitis and the A38G SNP. METHODS: Nasal fluid CC16 was analyzed in 20 patients with IAR before allergen challenge and 1 and 6 h after challenge, and from 28 healthy controls. The A38G SNP was analyzed in 80 patients with IAR and 106 controls. Nasal biopsies were obtained from three subjects in each group for immunohistochemical analysis of CC16. RESULTS: In the allergen-challenged patients symptoms and rhinoscopic signs of rhinitis increased after 1 h and normalized after 6 h. In contrast, nasal fluid CC16 decreased 1 h after allergen challenge and returned to baseline after 6 h. Nasal fluid CC16 levels did not differ from controls before and 6 h after challenge. Immunohistochemical investigation showed intense CC16 staining in the nasal epithelium of both patients before season and healthy controls, but weak staining in symptomatic patients during season. No significant association between the A38G SNP and IAR was found. CONCLUSION: There was an inverse relation between nasal fluid CC16 levels and symptoms and signs of rhinitis in allergen-challenged patients with IAR. However, there was no association between IAR and the A38G SNP.

Laboratory evaluation of a new evidential breath-alcohol analyser designed for mobile testing--the Evidenzer.

The Evidenzer is a new kind of forensic breath-alcohol analyser, designed for use both at a police station (stationary) and also in a police vehicle (mobile) at the roadside. In this paper we report the accuracy and precision of the Evidenzer, determined under controlled laboratory conditions. The results were compared with a well-established breath-alcohol instrument (Intoxilyzer 5000S) and also with the concentration of alcohol in venous blood. Twenty healthy volunteers (10 men and 10 women) consumed ethanol (0.4 g/kg) in 15 minutes starting two to three hours after their last meal. Venous blood and breath were obtained for determination of ethanol at 15-30 minute intervals for up to four hours post-dosing. There was a good overall agreement between the two breath-alcohol instruments and the mean bias was only 0.003 mg/L (95% limits of agreement of -0.016 to 0.023 mg/L). The standard deviation (SD) of measuring ethanol in breath was about the same for both instruments, being 0.006 mg/L, and this corresponds to a relative precision or coefficient of variation (CV) of 4.7%. When the Evidenzer was used to analyse ethanol vapour (0.50 mg/L) generated from a wet-bath simulator, i.e. in-vitro conditions, the coefficient of variation was 0.7% indicating high analytical precision. The concentration of ethanol in venous blood and breath were highly correlated (r = 0.95) although systematic differences existed depending on time after drinking when comparisons were made. Both breath-alcohol instruments gave results higher than venous blood alcohol in tests made at 15 minutes after the end of drinking whereas at all later times the venous blood-alcohol concentration was higher

Blood analysis by headspace gas chromatography: does a deficient sample volume distort ethanol concentration?

This study was prompted by a recent judgment in the Royal Courts of Justice (Gregory v. Director of Public Prosecutions, 2002) in a case of driving a motor vehicle after consuming too much alcohol (Road Traffic Act 1988). An expert witness for the defence alleged that a deficient volume of blood in the tube sent for analysis meant an excess amount of sodium fluoride (NaF) preservative, which would increase the concentration of ethanol, determined by headspace gas chromatography (HS-GC), owing to a salting-out effect. The prosecution did not produce expert evidence to rebut this argument and the drunk driving suspect was acquitted. A small volume of blood and excess sodium fluoride might have increased the concentration of ethanol in the air-space in the tube sent for analysis but this does not mean that the result of the HS-GC analysis would be higher. This follows because prior to analysis an aliquot of blood is removed and diluted (approximately 10 times) with n-propanol as the internal standard. The dilution lowers the concentration of NaF in the blood and for quantitative analysis the ratio of the ethanol to n-propanol response is measured. The use of a ratio also helps to compensate for any salting-out effect of ethanol. Our experiments showed that a deficient volume of blood and excess NaF actually lowered the concentration of ethanol by 2-3% compared with heparinised blood. Seemingly, n-propanol (n-PrOH) a 3-carbon straight chain alcohol is salted out slightly more effectively than the 2-carbon ethanol (EtOH) causing a lower peak area ratio (EtOH/n-PrOH) and a lower apparent concentration of ethanol. In a separate study, we showed that the concentration of ethanol was lowered even more when a 4-carbon alcohol (t-butanol) was used as the internal standard.

On-line process control of liquid chromatography.

Many analytical methods are based on liquid chromatography and typically the only measure of system stability is standards, injected repeatedly throughout the sequence. In this paper, a novel approach is presented, where the analytical run is treated as a process with the chromatographic data as the product. It is postulated that enhanced quality of the data can be obtained through monitoring the process, i.e., the chromatographic system, during the sequence. For this purpose, a liquid chromatography process control (LCPC) system has been developed. Here, several parameters, e.g., the pressure at the column and the injection valve, are monitored. Chemometrics is used for interpreting the data and producing multivariate statistical process control (MSPC) charts. The chromatographic run is divided into two parts: the dynamic injection phase and the static elution phase. Two principal component analysis (PCA) models, one for each phase, are continuously created and upgraded as the data are collected. The results of the PCA are shown in the MSPC charts, and when an error detection limit is exceeded, the analyst is promptly notified. LCPC, a continuous system suitability test, provides better control of the analysis, allowing a reduction in the number of standards and replicates. Furthermore, troubleshooting is facilitated.

Vibrational spectrometry for the assessment of active substance in metoprolol tablets: a comparison between transmission and diffuse reflectance near-infrared spectrometry.

Near-infrared spectrometry (NIR) was used to quantify metroprolol succinate in controlled release tablets. Metoprolol tablets were made according to an experimental design using different strengths around a central strength of 47.5 mg per tablet. A comparison was made between NIR in the diffuse reflectance mode and the transmission mode. This showed that, although a narrower wavelength range was available in the transmission mode, predictions were much better for models based on transmission spectra than for models based on diffuse reflectance spectra. The main reason for this is that in the reflectance mode NIR spectrometry is very sensitive to the inhomogeneity of the material, while in the transmission mode this problem is less severe. This is due to the larger volume of the material scanned in the transmission mode compared to that in diffuse reflectance. Spectra were taken before and after the tablets were stored under humid conditions. This allowed the final calibration models to be made more robust towards variations in the amount of water in the tablet. Different batches of metoprolol pellets and microcrystalline cellulose were used during the production of the tablets. this resulted in models that were more robust towards possible batch-to-batch differences in the main constituents.

Effects of piracetam on regional cerebral blood flow and mental functions in patients with organic dementia.

The effects of piracetam (Nootropil, UCB6215) on mental functions and on regional cerebral blood flow (rCBF) were investigated in eight patients in the presenile age who displayed symptoms of moderate dementia. The double-blind crossover design included nine measurement occasions, each involving rCBF measurement by the 133-Xe inhalation method, ratings of symptoms of dementia, personality changes, and side effects, and a psychometric investigation. Three investigations were included in each of three treatment periods. The first investigation in a period was made without medication. Then either placebo or piracetam 4.8 g/day or 9.6 g/day was given during four weeks with measurements after 2 weekks and 4 weeks. There were intervals of 4 weeks without medication between the treatment periods. Piracetam had no significant effect on either mental functions or rCBF.