Development of a low background liquid scintillation counter for a shallow underground laboratory.

Pacific Northwest National Laboratory has recently opened a shallow underground laboratory intended for measurement of low-concentration levels of radioactive isotopes in samples collected from the environment. The development of a low-background liquid scintillation counter is currently underway to further augment the measurement capabilities within this underground laboratory. Liquid scintillation counting is especially useful for measuring charged particle (e.g., ß and α) emitting isotopes with no (or very weak) gamma-ray yields. The combination of high-efficiency detection of charged particle emission in a liquid scintillation cocktail coupled with the low-background environment of an appropriately designed shield located in a clean underground laboratory provides the opportunity for increased-sensitivity measurements of a range of isotopes. To take advantage of the 35m-water-equivalent overburden of the underground laboratory, a series of simulations have evaluated the scintillation counter's shield design requirements to assess the possible background rate achievable. This report presents the design and background evaluation for a shallow underground, low background liquid scintillation counter design for sample measurements.

Anti-tumour necrosis factor treatment increases circulating T helper type 17 cells similarly in different types of inflammatory arthritis.

We investigated changes in circulating T helper type 17 (Th17) cells following anti-tumour necrosis factor (TNF) in rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA) patients. Peripheral blood mononuclear cells (PBMC) were isolated from 25 RA, 15 AS and eight PsA patients at baseline 4 and 12 weeks after treatment, and Th17 cell frequencies were analysed using interleukin (IL)-17 enzyme-linked immunospot (ELISPOT) and flow cytometry. A significant increase in IL-17-producing cells was observed by ELISPOT in RA and AS patients at 12 weeks. Flow cytometry confirmed significant increases in CD4(+) IL-17(+) cells at 12 weeks in RA and AS and 4 weeks in PsA patients. Anti-TNF treatment increases circulating Th17 cells in three different diseases.

Blockade of tumour necrosis factor-α in experimental autoimmune encephalomyelitis reveals differential effects on the antigen-specific immune response and central nervous system histopathology.

In various autoimmune diseases, anti-tumour necrosis factor (TNF)-α treatment has been shown to reduce both clinical disease severity and T helper type 1 (Th1)1/Th17 responses. In experimental autoimmune encephalomyelitis (EAE), however, the role of TNF-α has remained unclear. Here, C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 and treated with anti-TNF-α, control antibody or vehicle. The clinical disease course, incidence and severity were assessed. On day 20 after immunization the antigen-specific Th1/Th17 response was evaluated by enzyme-linked immunospot (ELISPOT) in spleen and central nervous system (CNS). Also, the extent of spinal cord histopathology was analysed on semi- and ultrathin sections. Our results demonstrate that anti-TNF-α treatment reduced the incidence and delayed the onset of EAE, but had no effect on disease severity once EAE had been established. Whereas anti-TNF-α treatment induced an increase in splenic Th1/Th17 responses, there was no effect on the number of antigen-specific Th1/Th17 cells in the spinal cord. Accordingly, the degree of CNS histopathology was comparable in control and anti-TNF-α-treated mice. In conclusion, while the anti-TNF-α treatment had neither immunosuppressive effects on the Th1/Th17 response in the CNS nor histoprotective properties in EAE, it enhanced the myelin-specific T cell response in the immune periphery.

Amorphous solid dispersions and nano-crystal technologies for poorly water-soluble drug delivery.

Poor water-solubility is a common characteristic of drug candidates in pharmaceutical development pipelines today. Various processes have been developed to increase the solubility, dissolution rate and bioavailability of these active ingredients belonging to BCS II and IV classifications. Over the last decade, nano-crystal delivery forms and amorphous solid dispersions have become well established in commercially available products and industry literature. This article is a comparative analysis of these two methodologies primarily for orally delivered medicaments. The thermodynamic and kinetic theories relative to these technologies are presented along with marketed product evaluations and a survey of commercial relevant scientific literature.

Atmospheric freeze drying for the reduction of powder electrostatics of amorphous, low density, high surface area pharmaceutical powders.

Amorphous itraconazole (ITZ) was prepared by Thin Film Freezing (TFF) utilizing 1,4-dioxane as the solvent with subsequent solvent removal via conventional tray lyophilization (ITZ LYO) or atmospheric freeze drying (ITZ AFD). ITZ AFD was prepared under various drying conditions to assess the influence of drying parameters on powder properties. XRD analysis confirmed all products were amorphous and DSC analysis revealed both drying processes resulted in the formation of the nematic mesophase of ITZ. SEM revealed a larger pore size and agglomerate size with fewer fine particles (i.e. less than 10 microns in diameter) for ITZ AFD compared to ITZ LYO. Residual solvent analysis revealed a primary drying temperature of -10°C resulted in residual solvent levels above the acceptable limits set by the International Conference on Harmonization as a result of microcollapse. Primary drying temperatures of less than -10°C resulted in acceptable residual solvent levels. The extent of microcollapse did not alter the macrostructure of the resulting powder. Powder flowability was determined to be similar for ITZ AFD and ITZ LYO based on Carr's index and the Hausner ratio, as well as by dynamic angle of repose. All powders displayed poor flowability. Chargeability measurements demonstrated a lower charge transfer for ITZ AFD powders compared to ITZ LYO due to a combination of factors including differences in residual solvent level, particle size, pore size, surface area, and fine particles content. The reduction in chargeability as a result of AFD is highly desirable because it allows for improved powder handling and use post-production.

What have we learnt from targeted anti-TNF therapy?

Anti-tumour necrosis factor (anti-TNF) therapy of patients with rheumatoid arthritis dates back to 1992, when the first proof-of-principle trials were performed in London by Maini and Feldmann. Considerable studies of the mechanism of action were performed, and insights into the way in which anti-TNF therapy delivers its benefit were obtained. In this brief review, certain aspects of knowledge acquired and the many gaps will be reviewed. Focus will be on the TNF-dependent cytokine cascade and what it means, and potential new approaches to treatment. Finally, an entertaining challenge: might many or even all unmet clinical needs be dealt with through cytokine analysis?

The anti-allergic drug, N-(3',4'-dimethoxycinnamonyl) anthranilic acid, exhibits potent anti-inflammatory and analgesic properties in arthritis.

OBJECTIVES: The degradation of tryptophan by indoleamine 2,3-dioxygenase yields a number of immunomodulatory metabolites, including 3-hydroxyanthranilic acid, 3-hydroxykynurenic acid and quinolinic acid. N-(3',4'-dimethoxycinnamonyl) anthranilic acid (3,4-DAA) is a synthetic anthranilic acid derivative that has been used therapeutically in Japan for many years as an anti-allergic drug and has recently been shown to be effective in a murine model of multiple sclerosis. METHODS: In the present study, we tested the efficacy of 3,4-DAA in collagen-induced arthritis, a mouse model of rheumatoid arthritis, and analysed its mechanism of action. RESULTS: Administration of 3,4-DAA after arthritis onset reduced clinical and histological severity of arthritis and reduced pain. It completely abrogated thermal and mechanical hyperalgesia. 3,4-DAA also suppressed Th1 cell activity in lymph node cell cultures and raised serum levels of IL-10. In vitro, 3,4-DAA suppressed IFNgamma production and proliferation of both T and B lymphocytes in a manner comparable with the endogenous tryptophan metabolite, 3-hydroxyanthranilic acid, suggesting similar mechanisms of action. CONCLUSION: It is concluded that 3,4-DAA has both anti-inflammatory and analgesic properties, and may therefore be useful in filling an unmet need, in the treatment of rheumatoid and other forms of arthritis, especially in the light of its analgesic properties.

Remission of collagen-induced arthritis is associated with high levels of transforming growth factor-beta expression in the joint.

Immunization of genetically susceptible strains of mice with heterologous type II collagen leads to the induction of a self-limiting polyarthritis that begins to subside around 10 days after onset of clinical disease. The aims of this study were to compare pro- and anti-inflammatory cytokine expression in the joints during the course of arthritis in order to identify cytokines involved in spontaneous remission of arthritis. DBA/1 mice were immunized with type II collagen and an immunohistochemical analysis of expression of proinflammatory cytokines [tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-6] and anti-inflammatory cytokines [IL-10, IL-1ra, transforming growth factor (TGF)-beta1, TGF-beta2 and TGF-beta3] in joints was carried out over the course of the disease. Both pro- and anti-inflammatory cytokines were found to be expressed in early arthritis. However, around 10 days after onset of arthritis, the level of expression of proinflammatory cytokines declined while the level of expression of anti-inflammatory cytokines, particularly TGF-beta1 and TGF-beta2, increased. Surprisingly, TNF-alpha continued to be expressed at low levels during the period of disease remission (30 days after onset). Blockade of TNF-alpha during the period of disease remission had no effect on TGF-beta expression. This study confirms that the level of inflammation in arthritis correlates strongly with the balance of pro- and anti-inflammatory cytokine expression in the joints. Of the anti-inflammatory cytokines studied, TGF-beta1 and TGF-beta2 predominate during the time of disease remission, suggesting that these cytokines are involved in regulating disease activity.

Activation of NF-kappaB by the intracellular expression of NF-kappaB-inducing kinase acts as a powerful vaccine adjuvant.

There is a pressing need for adjuvants that will enhance the effectiveness of genetic vaccines. This is particularly important in cancer and infectious disease such as HIV and malaria for which successful vaccines are desperately needed. Here, we describe an approach to enhance immunogenicity that involves the activation of NF-kappaB by the transgenic expression of an intracellular signaling molecule, NF-kappaB-inducing kinase (NIK). In vitro, NIK increases dendritic cell antigen presentation in allogeneic and antigen-specific T cell proliferation assays by potently activating NF-kappaB and consequently up-regulating the expression of cytokines (TNF-alpha, IL-6, IL-12, IL-15, and IL-18), chemokines [IL-8, RANTES (regulated on activation, normal T cell expressed and secreted), macrophage inflammatory protein-1alpha, monocyte chemoattractant protein-1, and monocyte chemoattractant protein-3], MHC antigen-presenting molecules (class I and II), and costimulatory molecules (CD80 and CD86). In vivo, NIK enhances immune responses against a vector-encoded antigen and shifts them toward a T helper 1 immune response with increased IgG2a levels, T cell proliferation, IFN-gamma production, and cytotoxic T lymphocyte responses more potently than complete Freund's adjuvant, a very efficacious T helper 1-inducing adjuvant. These findings define NIK, and possibly other inducers of NF-kappaB activation, as a potent adjuvant strategy that offers great potential for genetic vaccine development.

Pathogenesis and therapy of rheumatoid arthritis.

Rheumatoid arthritis is a chronic disabling disease affecting at least 1% of the population on a worldwide basis. Research aimed at understanding the pathogenesis of this disease led to the identification of TNFalpha as a major pro-inflammatory cytokine expressed in the inflamed joints of patients with rheumatoid arthritis. Subsequently, in vitro studies provided evidence to suggest that TNFalpha played an important role in driving the expression of additional pro-inflammatory cytokines, such as IL-1, GM-CSF, IL-6, and IL-8, in synovial cell cultures. Another important finding that confirmed the pathological significance of TNFalpha was that mice genetically engineered to overexpress TNFalpha spontaneously developed arthritis. Subsequently, the therapeutic effect TNFalpha blockade was tested in animal models prior to clinical trials in human patients, which provided unequivocal verification of the validity of TNFalpha as a therapeutic target. Anti-TNFalpha therapy is now accepted as a fully-validated treatment modality for rheumatoid arthritis.

Inflammation is preceded by tumor necrosis factor-dependent infiltration of mesenchymal cells in experimental arthritis.

OBJECTIVE: To determine the involvement of mesenchymal progenitor cells in the induction of collagen-induced arthritis (CIA). METHODS: DBA/1 mice were immunized with type II collagen in adjuvant or adjuvant alone, and the presence of mesenchymal cells in the joints of prearthritic mice was studied by immunohistochemistry. RESULTS: An analysis of the joints on day 10 postimmunization (at least 10 days before the onset of arthritis) revealed synovial hyperplasia without leukocytic infiltration. Large, round cells expressing bone morphogenetic protein receptors (BMPRs), which serve as markers for primitive mesenchymal cells, were present in increased numbers in the bone marrow adjacent to the joint, in the synovium itself, and within enlarged bone canals that connect the bone marrow to the synovium. Similar changes were observed in mice given adjuvant without collagen. Adjuvant-induced infiltration of BMPR(+) cells and enlargement of bone canals were abrogated by anti-tumor necrosis factor (anti-TNF) treatment and were absent in TNFR p55/p75(-/-) mice. Increased numbers of bone marrow cells and enlarged bone canals were observed in nonimmunized TNF transgenic mice (which spontaneously develop arthritis). CONCLUSION: These findings suggest that in CIA, there is an antigen-independent (innate) prearthritic phase that prepares the joint for the subsequent immune-mediated arthritis. The induction phase involves marrow-derived mesenchymal cells and requires the presence of TNF.

Immunotherapy for rheumatoid arthritis.

Recently published studies confirm that the long-term use of biological agents targeting TNF-alpha in therapy for rheumatoid arthritis (RA) gives rise to sustained improvement in symptoms and signs of disease, and in the quality of life. Furthermore, it has emerged that anti-TNF therapy protects joints from structural damage, which unexpectedly is also observed in the patient population showing no apparent benefit in control of signs and symptoms. Therapeutic benefit is observed in established disease that is unresponsive to conventional DMARDS and in early DMARDS-naïve RA patients. Thus, for patients with RA, anti-TNF therapies set a new standard for symptom control and joint protection.

Investigation of some commercially available spacer devices for the delivery of glucocorticoid steroids from a pMDI.

Five commercially available spacers were investigated to determine their influence on the percentage of drug retained in the spacer device, percentage fine particle fraction (FPF), percentage deposited in the induction port, mass median aerodynamic diameter (MMAD), and geometric standard deviation (GSD). Betamethasone valerate (BMV) and triamcinolone acetonide (TAA) were used as model drugs in the pressurized metered dose inhaler (pMDI) formulations containing the propellant HFA 134a. The BMV was dissolved in an ethanol/HFA 134a system, and the TAA was suspended in HFA 134a using ethanol as a dispersing agent. The metering chamber volume of the valve was either 50 microl or 150 microl. The spacer devices investigated included the ACE, Aerochamber, Azmacort, Easivent, and Ellipse spacers. Each spacer device was attached to an Andersen Cascade Impactor powered by a vacuum pump. Cascade impaction data were used to derive the percentage drug deposited in the induction port, MMAD, GSD, and FPF. The BMV particles emitted from the spacers were finer than the TAA particles because the dissolved drug precipitated as the cosolvent evaporated. The TAA particles had significantly larger MMADs because many undissolved drug particles were contained within each droplet following actuation. After evaporation of the liquid continuous phase, the suspended drug aggregated to form larger agglomerates than those particles precipitated from the BMV pMDI solution droplets. The addition of a spacer device lowered the MMAD to less than 4.7 microm for particles from both the BMV pMDI solution and the TAA pMDI suspension. The addition of a spacer device also lowered the percentage drug deposited in the induction port. The FPF was significantly increased when a spacer device was used. The MMAD significantly decreased when a spacer device was added for the two model drugs when using the 150-microl metering valves, but the difference was not statistically significant when the 50-microl valves were used (P < .05). The GSD was not influenced by the use of a spacer device. The use of a spacer device will enhance pMDI therapy by reducing the amount of drug deposited in the oropharyngeal region, which will lead to fewer instances of local and systemic side effects. In addition, the spacer devices investigated will allow a higher dose of drug to reach the deep lung, which may permit the use of lower dosage regimens with increased therapeutic efficacy.

Chronic relapsing homologous collagen-induced arthritis in DBA/1 mice as a model for testing disease-modifying and remission-inducing therapies.

OBJECTIVE: To test whether the chronic relapsing arthritis induced by immunizing DBA/1 mice with homologous type II collagen is a valuable model for testing disease-modifying antiarthritic drugs. METHODS: Six-week-old male DBA/1 mice were immunized with murine type II collagen in Freund's complete adjuvant, resulting in a chronic relapsing polyarthritis in >80% of the mice 4 weeks after immunization. At the onset of clinical arthritis, mice were treated for 4 weeks with different treatments, including anti-tumor necrosis factor (anti-TNF) and antiinterleukin-12 (anti-IL-12) antibodies, salbutamol, or indomethacin. Alternatively, treatment was administered as a pulse at the beginning of clinical arthritis. Pulse treatments tested included anti-CD3 in combination with anti-TNF, anti-TNF alone, and anti-CD4, either alone or in combination with anti-TNF. After 4 weeks of arthritis, mice were killed and hind paws were assessed histologically for joint damage. RESULTS: Anti-TNF and salbutamol both suppressed clinical arthritis more effectively than indomethacin and, moreover, protected the joints from damage, whereas indomethacin did not. Anti-IL-12 treatment initiated after the onset of clinical symptoms accelerated disease. Pulse therapy with anti-CD3 plus anti-TNF was found to induce remission, clinically as well as histologically, whereas a pulse with either anti-CD4, anti-TNF, or the combination of anti-CD4 plus anti-TNF was less effective. CONCLUSION: Chronic relapsing homologous collagen-induced arthritis is a valuable model for identifying remission-inducing antiarthritic drugs and has predictive value with respect to their joint-protective potency.

Increased production of intracellular interleukin-1 receptor antagonist type I in the synovium of mice with collagen-induced arthritis: a possible role in the resolution of arthritis.

OBJECTIVE: To examine the patterns of production of interleukin-1 receptor antagonist (IL-1Ra) isoforms and of IL-1beta during arthritis in vivo. METHODS: Arthritis was induced in DBA/1 mice by immunization with type II collagen, and the production of IL-1Ra isoforms was examined in whole joints and in dissected synovial tissues by reverse transcription-polymerase chain reaction (RT-PCR), RNase protection assay, Western blotting, immunostaining, and in situ hybridization. Production of IL-1beta also was examined using similar approaches. RESULTS: Production of IL-1Ra increased in the joints during collagen-induced arthritis (CIA). By RT-PCR, secreted IL-1Ra messenger RNA (mRNA) was detected in normal joints, whereas intracellular IL-1Ra type I (icIL-1Ra1) mRNA was only produced in inflamed joints. Western blot studies showed that icIL-1Ra1 protein levels increased in the joints during the course of CIA and that icIL-1Ra3 protein was also present in low amounts. RNase protection assays showed that the IL-1beta:IL-1Ra mRNA ratio was increased in inflamed joints through day 14 of arthritis, whereas a reverse pattern was present at later time points (from day 20 to day 60). Consistent with this finding, immunohistochemistry and in situ hybridization studies confirmed that icIL-1Ra1 was only present in inflamed joints. The histologic evaluation of CIA during the course of the disease indicated a resolution of acute inflammation, since icIL-1Ra1 production increased and the ratio of IL-1beta to total IL-1Ra decreased. CONCLUSION: Production of IL-1Ra isoforms, particularly icIL-1Ra1, is stimulated in inflamed joints during CIA in mice. The combination of decreased production of IL-1beta and elevated levels of icIL-1Ra1 during the course of CIA was associated with a reduction in inflammatory activity. These results suggest that icIL-1Ra1 may play a role in the resolution of murine CIA.

Influence of water on the solubility of two steroid drugs in hydrofluoroalkane (HFA) propellants.

The objective of this research work was to investigate the influence of water level, temperature, and propellant composition on the solubility of two hydrophobic steroid drugs, triamcinolone acetonide (TAA) and beclomethasone diapropionate (BDP). pMDIs containing TAA or BDP, spiked water, and propellant blend with different ratios of HFA 134a and HFA 227 were prepared. The contents of the prepared pMDIs were filtered through a 0.22 mm Acrodisc, syringe filter into a receiving canister after the pMDIs were equilibrated at 15 degrees C, 25 degrees C, 30 degrees C, and 40 degrees C. The drug concentration in the receiving canisters was determined by HPLC and the drug solubility in the propellant blend was calculated. Also, the drug crystal collected on the filter from the donor pMDIs were characterized by x-ray diffraction. The solubility of TAA and BDP varied with propellant composition at all experimental temperatures investigated. The solubility of TAA and BDP increased as the temperature was increased at all propellant compositions and water levels studied, but decreased as the water level in the propellant system was increased at all compositions and temperatures. The x-ray diffraction results indicated that the water in the propellant system had no significant influence on the crystal characteristics of TAA in HFA propellant system, but had a significant impact on the crystal characteristics of BDP was higher than TAA at all propellant compositions, experimental temperatures and water levels investigated. The solubility of TAA and BDP was not only influenced by propellant composition and storage temperature, but also depended on the water level in the propellant system. As a consequence, the crystallinity of the drugs formulated in HFA propellant was influenced by the temperature, propellant composition and the water level in the propellant system. The impact of these factors on the crystallinity of formulated drugs.

Method to recover a lipophilic drug from hydroxypropyl methylcellulose matrix tablets.

A reverse-phase high-performance liquid chromatographic (HPLC) method for recovery of the lipophilic drug, alprazolam, from matrix tablets containing the hydrophilic polymer hydroxypropyl methylcellulose (HPMC) was developed. Lipophilic drugs, such as alprazolam, are difficult to completely extract and quantitate from tablets containing HPMC polymer. The percentage of recoveries of alprazolam from placebo powder spiked with alprazolam stock solution and from placebo powder mixed with alprazolam powder were about 100% and 85% to 95%, respectively. The validated method using water to completely dissolve HPMC before the addition of a strong solvent to dissolve and extract the drug from the HPMC solution was shown to be the most reproducible method. Different molecular weight distributions of the HPMC polymer, such as HPMC-K4M and HPMC-K100LV, did not influence the dissolution results of alprazolam using this validated method. Similarly, the excipients composing the matrix tablet formulations, such as dicalcium phosphate dihydrate, dicalcium phosphate anhydrous, calcium sulfate dihydrate, sucrose, dextrose, and lactose monohydrate, did not influence the percent recovery of alprazolam. The recovery method reported herein was shown to be the most efficient to achieve complete recovery of alprazolam from powder blends and tablets containing a variety of excipients and different grades of HPMC.

The influence of plasticizer on heat-humidity curing of cellulose acetate phthalate coated beads.

The objectives of the present study are to investigate the effect of plasticizer type and level on the curing of cellulose acetate phthalate (CAP) coated beads with and without the presence of humidity. Theophylline beads were coated in a fluidized-bed with CAP dispersion (Aquacoat CPD) plasticized by a water-insoluble plasticizer, diethyl phthalate (DEP), or a water-soluble plasticizer, triethyl citrate (TEC), at various levels. The coated heads were cured at a heat-only condition (50 degrees C for 24 hr) and a heat-humidity condition (50 degrees C/75% RH for 24 hr). Rapid drug release in the acidic media was found for both heat-only and heat-humidity cured beads when plasticizer was not used in the coating dispersion, indicating that the heat-humidity curing is ineffective without the presence of plasticizers. When plasticizer was incorporated in the coating formulations, heat-humidity curing effectively improved the acid resistance of the coated films at all plasticizer levels investigated. The minimum plasticizer level required to obtain enteric release profiles for heat-humidity cured beads coated at an outlet coating temperature of 46 degrees C was 15%. This limit was further decreased when the beads were coated at a lower temperature due to a less plasticizer loss at the lower coating temperature. Between the two plasticizers, less TEC was lost during the coating process, and TEC was more effective compared to DEP with regards to heat-humidity curing at the 10% plasticizer level. The enteric release profiles were reproducible following a 7-day drying period at 40 degrees C for all heat-humidity cured beads that had initially passed the enteric release dissolution test. The rapid leaching of TEC and DEP into the.