Solvent-Assisted Hot Melt Extrusion of a Thermally Labile, High Melting Point Compound.

Molecular dispersions are a highly effective method of increasing bioavailability for a poorly soluble active pharmaceutical ingredient (API) and can be prepared on a large scale by hot melt extrusion (HME). Processing thermally labile active pharmaceutical ingredients (APIs) via HME is generally more difficult, with operating temperatures limited to below that of the API melting point. API melting is considered essential to facilitate the formation of a fully homogeneous amorphous system. Processing below the melting point renders the system much more susceptible to residual crystalline content; hence, HME is not suitable for APIs which degrade upon melting. In the following work, meloxicam (MEL) was used as a model API, possessing properties of high melting temperature and thermal lability. In this proof of concept work, a modified HME method, termed solvent-assisted HME, was used to overcome this issue and prepare an amorphous solid dispersion using HME, wherein a solvent was incorporated in the formulation blend during extrusion and removed post-processing. Formulations containing 10%wt meloxicam (MEL) and 90%wt polyvinylpyrrolidone vinyl acetate (PVPVA) copolymer were extruded using a twin-screw extruder at temperatures below the melting point of MEL. Dimethylformamide (DMF) solvent was added directly into the extruder barrel through a liquid addition port, resulting in extrudate products having a higher conversion of API to the amorphous form. The incorporation of solvent allowed a significant reduction in processing temperatures due to its increased mobility, while also driving the conversion of the API to its amorphous form. The solvent was successfully reduced through a secondary drying step using a vacuum oven. This advancement has demonstrated the potential for thermally labile APIs to be processed via HME expanding the applications of this technology.

Optimizing Small, Low-Risk, Unruptured Intracranial Aneurysm Treatment Using Game Theory.

The incidental diagnosis of unruptured intracranial aneurysms has been increasing in the past several decades. A significant proportion represent small, low-risk, unruptured intracranial aneurysms for which there is equipoise on whether to offer treatment or conservative management. Given this uncertainty, patients may not always be comfortable with their physicians' recommendations. Herein, we use game theory to study the interactions between physicians and patients to determine how conflict and cooperation affect the management of small, low-risk, unruptured intracranial aneurysms. We constructed a game theory model of the interaction between physicians and patients with respect to decision-making for a small, low-risk, unruptured intracranial aneurysm in an asymptomatic patient when there is perceived equipoise between whether to treat or manage conservatively. Assuming that both the physician and patient are rational and eliciting individual patient preferences is not practical, the physician should play the game based on an ex ante probability of meeting a patient with a certain type of preference. This recommendation means that the expectations of the physician regarding the patient's preferences should guide the decision to offer treatment or conservative management as a first option for a small, asymptomatic, low-risk, unruptured intracranial aneurysm for which there is clinical equipoise.

Rescan Time Delays in Ischemic Stroke Imaging: A Retrospective Observation and Analysis of Causes and Clinical Impact.

BACKGROUND AND PURPOSE: Delays to reperfusion negatively impact outcomes of patients with ischemic stroke, yet current guidelines recommend selective sequential imaging for thrombectomy candidates. We aimed to quantify and analyze time delays associated with rescanning in sequential acute stroke imaging. MATERIALS AND METHODS: This was a retrospective cohort study of consecutive patients with acute ischemic stroke who underwent imaging for treatment decision-making from January 1, 2017, to June 30, 2020. Rescan time delay was defined as ≥10-minute difference between initial NCCT and CTA ± CTP. Mean rescan time delays in comprehensive and primary stroke centers were compared. Bivariate and multivariable regression analyses assessed clinical and imaging factors associated with rescanning time delays and early outcomes. RESULTS: A total of 588 patients with acute ischemic were included in statistical analyses. Rescanning occurred in 27.9% (164/588 patients), with a mean time delay of 53.7 (SD, 43.4) minutes. For patients presenting at primary compared with comprehensive stroke centers, rescan time delays were more common (59.6% versus 11.8%, P < .001), with longer delays (65.4 [SD, 45.4] minutes versus 23.6 [SD, 14.0] minutes, P < .001). Independent predictors of rescan time delays included primary stroke center presentation, intravenous thrombolysis administration, black race, admission NIHSS ≥10, baseline independent ambulation, and onset-to-comprehensive stroke center arrival in ≥6 hours. Protocols for early simultaneous comprehensive CT (NCCT + CTA + CTP) were associated with lower odds of time delays (OR = 0.34; 95% CI, 0.21-0.55). Rescanning was associated with lower odds of home discharge (OR = 0.53; 95% CI, 0.30-0.95). CONCLUSIONS: A sequential approach to CT-based imaging may be significantly associated with prolonged acute stroke evaluations. Adoption of early simultaneous comprehensive CT could minimize treatment delays and improve outcomes.

COVID-19 Severity and Stroke: Correlation of Imaging and Laboratory Markers.

BACKGROUND AND PURPOSE: Coronavirus disease 2019 (COVID-19) appears to be an independent risk factor for stroke. We hypothesize that patients who develop stroke while hospitalized for severe COVID-19 will have higher inflammatory markers and distinct stroke imaging patterns compared with patients positive for COVID-19 with out-of-hospital stroke onset and milder or no COVID-19 symptoms. MATERIALS AND METHODS: This is a retrospective case series of patients positive for COVID-19 on polymerase chain reaction testing with imaging-confirmed stroke treated within a large health care network in New York City and Long Island between March 14 and April 26, 2020. Clinical and laboratory data collected retrospectively included complete blood counts and creatinine, alanine aminotransferase, lactate dehydrogenase, C-reactive protein, ferritin, and D-dimer levels. All CT and MR imaging studies were independently reviewed by 2 neuroradiologists who recorded stroke subtype and patterns of infarction and intracranial hemorrhage. RESULTS: Compared with patients with COVID-19 with outside-of-hospital stroke onset and milder or no COVID-19 symptoms (n = 45, 52.3%), patients with stroke already hospitalized for severe COVID-19 (n = 41, 47.7%) had significantly more frequent infarctions (95.1% versus 73.3%, P = .006), with multivascular distributions (56.4% versus 33.3%, P = .022) and associated hemorrhage (31.7% versus 4.4%, P = .001). Patients with stroke admitted with more severe COVID-19 had significantly higher C-reactive protein and ferritin levels, elevated D-dimer levels, and more frequent lymphopenia and renal and hepatic injury (all, P < .003). CONCLUSIONS: Patients with stroke hospitalized with severe COVID-19 are characterized by higher inflammatory, coagulopathy, and tissue-damage biomarkers, supporting proposed pathogenic mechanisms of hyperinflammation activating a prothrombotic state. Cautious balancing of thrombosis and the risk of hemorrhagic transformation is warranted when considering anticoagulation.

The public health impact of avian influenza viruses.

Influenza viruses with novel hemagglutinin and 1 or more accompanying genes derived from avian influenza viruses sporadically emerge in humans and have the potential to result in a pandemic if the virus causes disease and spreads efficiently in a population that lacks immunity to the novel hemagglutinin. Since 1997, multiple avian influenza virus subtypes have been transmitted directly from domestic poultry to humans and have caused a spectrum of human disease, from asymptomatic to severe and fatal. To assess the pandemic risk that avian influenza viruses pose, we have used multiple strategies to better understand the capacity of avian viruses to infect, cause disease, and transmit among mammals, including humans. Seroepidemiologic studies that evaluate the frequency and risk of human infection with avian influenza viruses in populations with exposure to domestic or wild birds can provide a better understanding of the pandemic potential of avian influenza subtypes. Investigations conducted in Hong Kong following the first H5N1 outbreak in humans in 1997 determined that exposure to poultry in live bird markets was a key risk factor for human disease. Among poultry workers, butchering and exposure to sick poultry were risk factors for antibody to H5 virus, which provided evidence for infection. A second risk assessment tool, the ferret, can be used to evaluate the level of virulence and potential for host-to-host transmission of avian influenza viruses in this naturally susceptible host. Avian viruses isolated from humans exhibit a level of virulence and transmissibility in ferrets that generally reflects that seen in humans. The ferret model thus provides a means to monitor emerging avian influenza viruses for pandemic risk, as well as to evaluate laboratory-generated reassortants and mutants to better understand the molecular basis of influenza virus transmissibility. Taken together, such studies provide valuable information with which we can assess the public health risk of avian influenza viruses.

Evaluating interest in an influenza A(H5N1) vaccine among laboratory workers who work with highly-pathogenic avian influenza viruses in the United States.

BACKGROUND: Highly pathogenic avian influenza A (HPAI) viruses found in poultry and wild birds occasionally infect humans and can cause serious disease. In 2014, the Advisory Committee on Immunization Practices (ACIP) reviewed data from one licensed ASO3-adjuvanted influenza A(H5N1) vaccine for consideration of use during inter-pandemic periods among persons with occupational exposure. To guide vaccine policy decisions, we conducted a survey of laboratory workers to assess demand for HPAI vaccination. METHODS: We designed an anonymous web survey (EpiInfo 7.0) to collect information on demographics, type of work and time spent with HPAI viruses, and interest in HPAI vaccination. Eligible participants were identified from 42 entities registered with United States Department of Agriculture's Agricultural Select Agent program in 2016 and emailed electronic surveys. Personnel with Biosafety Level 3 enhanced (BSL-3E) laboratory access were surveyed. Descriptive analysis was performed. RESULTS: Overall, 131 responses were received from 33 principal investigators, 26 research scientists, 24 technicians, 15 postdoctoral fellows, 6 students, and 27 others. The estimated response rate was 15% among the laboratory personnel of responding principal investigators. One hundred respondents reported working in a BSL-3E area where HPAI experiments occurred with a mean time of 5.1-11.7 h per week. Overall, 49% were interested in receiving an A(H5N1) vaccine. By role, interest was highest among students (80%) and among those who spent >50% of their time in a BSL-3E area (64%). Most (61%) of those who said they might be or were not interested in vaccine believed it would not provide additional protection to current safety practices. CONCLUSIONS: Half of responding laboratory workers was interested in receiving an influenza A(H5N1) vaccine. HPAI vaccination of laboratory workers at risk of occupational exposure could be used along with existing safety practices to protect this population.

Influenza vaccines generated by reverse genetics.

Influenza viruses cause annual epidemics and occasional pandemics of acute respiratory disease. Vaccination is the primary means to prevent and control the disease. However, influenza viruses undergo continual antigenic variation, which requires the annual reformulation of trivalent influenza vaccines, making influenza unique among pathogens for which vaccines have been developed. The segmented nature of the influenza virus genome allows for the traditional reassortment between two viruses in a coinfected cell. This technique has long been used to generate strains for the preparation of either inactivated or live attenuated influenza vaccines. Recent advancements in reverse genetics techniques now make it possible to generate influenza viruses entirely from cloned plasmid DNA by cotransfection of appropriate cells with 8 or 12 plasmids encoding the influenza virion sense RNA and/or mRNA. Once regulatory issues have been addressed, this technology will enable the routine and rapid generation of strains for either inactivated or live attenuated influenza vaccine. In addition, the technology offers the potential for new vaccine strategies based on the generation of genetically engineered donors attenuated through directed mutation of one or more internal genes. Reverse genetics techniques are also proving to be important for the development of pandemic influenza vaccines, because the technology provides a means to modify genes to remove virulence determinants found in highly pathogenic avian strains. The future of influenza prevention and control lies in the application of this powerful technology for the generation of safe and more effective influenza vaccines.

Increasing the immunogenicity of a trivalent influenza virus vaccine with adjuvant-active nonionic block copolymers for potential use in the elderly.

High molecular weight nonionic block copolymers consisting of a large hydrophobic core made from repeat oxypropylene units and smaller hydrophilic blocks of oxyethylene repeat units were evaluated as adjuvants in experimental influenza virus vaccine formulations. The goal was to identify a copolymer that would increase the immunogenicity of the commercial Fluogen trivalent influenza virus vaccine. Vaccine experiments done using BALB/c mice provided data that allowed us to identify a copolymer that increased both antibody titers specific for total virus proteins as well as antibodies with hemagglutination inhibition (HAI) activity. This copolymer, termed CRL1005, increased the production of IgG1, IgG2a and IgG2b which suggested it increased the activity of both Type-1 and Type-2 T-helper lymphocytes. The CRL1005 copolymer was tested further in rhesus monkeys with similar results. Levels of antibodies specific for total virus protein preparations were increased as were HAI antibody titers following vaccination with the copolymer-supplemented Fluogen vaccine. Thus, the CRL1005 copolymer adjuvant appears to be compatible for use with the current generation of inactivated viron-based influenza vaccines and useful for increasing the immunogenicity. A more potent influenza virus vaccine could well be more efficacious in the aged segment of our population than current vaccines.

Effects of ovarian steroid hormones on esterases in the rat endometrium.

A technique in which uterine luminal epithelium is separated from the remainder of the endometrium by rapidly vibrating everted uterine cornua in a 3-5 mM solution of EDTA has been developed. Examination of the hormonal sensitivities and physiological roles of the tissue components of the endometrium is thus facilitated. Biochemical and histochemical studies of epithelial, stromal and endometrial esterases have shown that the rate of hydrolysis of alpha-naphthyl acetate is significantly higher in epithelial and endometrial tissue extracts during pro-oestrus than at any other stage of the oestrous cycle. In ovariectomized animals, oestradiol-17 beta caused a 60% increase in the rate of esterase activity over that of control animals, whereas medroxyprogesterone acetate had no effect. These findings suggest that the variations in the levels of neutral lipids in the uterine luminal epithelium of non-pregnant mature female rats result from the periodic stimulation of the epithelial esterases by the cyclically increased levels of plasma oestrogens.

Educational impact of faculty review on radiology residents' radiographic interpretations.

The objective of this study was to analyze the improvements in film reading performance made by radiology residents during their first six months of training. Five first-year residents and eight radiologic technology students each interpreted two of three matched sets of 39 films under two conditions. One set's readings were reviewed by a staff radiologist, while the other's were unreviewed. Six months later, each observer read all three sets. After the first six months of training, residents improved their reporting of findings. There was less improvement in technologists' readings. Review and instruction by staff, both in the laboratory and daily work settings, appeared to contribute to improved performance. Accuracy of residents' final diagnosis did not improve significantly. We conclude that a training system in which residents' film interpretations are reviewed by staff can lead to improved resident performance. When studied in a longitudinal fashion, these improvements are detectable within six months. This teaching system is used in many radiology departments.

Pigmented basal cell cancer masquerading as superficial spreading malignant melanoma.

A 49-year-old woman of Spanish descent had a pigmented lesion on the thigh present for many years, which enlarged recently. The lesion showed notching and black, brown, and blue colors that led to the initial diagnosis of superficial spreading malignant melanoma. Results of skin biopsy disclosed the lesion was a basal cell epithelioma with large amounts of melanin in the dermis. This report illustrates the importance of considering pigmented basal cell epithelioma in the differential diagnosis of superficial spreading malignant melanoma.

Occurrence of bullous pemphigoid after furosemide therapy.

A 78-year-old woman with Parkinson disease developed tense bullous lesions on the chest, arms, and in the groin that were diagnosed as bullous pemphigoid. Histologic examination, as well as immunofluorescence tests, confirmed this diagnosis. The possibility of a drug-induced disease was considered because she was taking seven different medications. Furosemide (Lasix) was suspected primarily. Complete clearing occurred with prednisone therapy, but readministration of furosemide resulted in bulla formation.

Successful use of cyclophosphamide and prednisone for initial treatment of pemphigus vulgaris.

Previous reports showed that cyclophosphamide used late in the treatment of patients with pemphigus vulgaris reduced the complications due to steroids. In this study, cyclophosphamide was used in the initial treatment of five patients with pemphigus vulgaris in combination with prednisone to avoid side-effects of prednisone. Two patients had no prior treatment with steroids for pemphigus. Results indicate that initial combined program may be a highly effective treatment method for pemphigus vulgaris. Followup from one to four years after treatment shows all five patients in remission; four require no medication.

The in vitro effect of gold complexes on bone resorption.

Mouse calvaria were maintained in organ culture without serum additives. The effects of three gold complexes--aurothioglucose, aurothiomalate, and auranofin--on active bone resorption (45Ca release) and hydroxyproline synthesis were determined. The influence of these compounds on DNA and protein synthesis and lysosomal enzyme release from calvaria was also assessed. All gold complexes reduced bone resorption to some extent, with auranofin being the most potent within a narrow concentration range (10(-6) M). This concentration of auranofin also significantly inhibited collagen synthesis, although DNA and protein synthesis were unaffected. None of the compounds tested appeared to mediate their action via significant inhibition of lysosomal enzyme release.

Pathogenesis of and immunity to avian influenza A H5 viruses.

In 1997 in Hong Kong, 18 human cases of respiratory illness were caused by an avian influenza A H5N1 virus. Although avian influenza viruses had not previously been known to cause respiratory illness in humans, the H5N1 viruses caused severe illness and death, primarily in individuals aged > 12 years. The introduction of H5N1 viruses into humans raised concerns about the potential of these viruses to cause a pandemic. We have used the BALB/c mouse to better understand the pathogenesis of and immunity to the H5N1 viruses in a mammalian model. Previously, we demonstrated that H5N1 viruses isolated from humans replicated efficiently in the lungs of mice without prior adaptation to this host. Two general phenotypes of pathogenicity of H5N1 viruses, based on high and low lethality for mice, were observed. We now demonstrate that in addition to a lethal outcome, H5N1 viruses with a high pathogenicity phenotype exhibit additional features that include rapid and uncontrolled replication in the lungs of infected mice, dissemination and replication of the virus in other organs, and depletion of peripheral blood leukocytes. The BALB/c mouse model was also used to better understand the parameters of protective immunity to the H5N1 viruses. Prior infection with H5N1 viruses of low pathogenicity or an antigenically related non-pathogenic H5N3 virus protected mice from death by infection with a highly pathogenic HK/483 virus. Serum hemagglutination-inhibition antibody titers of 40 or greater were associated with protection of mice from death. Immunization of mice with baculovirus-expressed recombinant H5 hemagglutinin protein or a previously defined HS-specific synthetic peptide induced MHC class II restricted CTL activity. Mice that had CTL activity but no serum hemagglutination-inhibition antibody were not protected from a lethal challenge with H5N1 virus. These results suggest that antibody is required for protection of mice against lethal challenge with H5N1 viruses of the high pathogenicity phenotype.

A histochemical study of muscle in club foot.

A histochemical analysis was made of 103 muscle biopsies taken from 62 patients with idiopathic club feet. Any reduction in the diameter of the muscle fibres associated with wasting of the calf muscle was recorded. Histochemical abnormalities existing in these biopsies were revealed by comparison with normal biopsies obtained from the normal legs of 13 children with unilateral deformities. No significant difference was found between the diameter of the muscle fibres taken from normal and affected legs aged under six months. This indicates that wasting of the calf muscle is due to a reduction in the number of fibres rather than their size. The muscle structure was normal excluding denervation and reinnervation. The soleus muscle in patients aged under six months contained 61 per cent Type 1 fibres in the affected legs, compared to 44.3 per cent in normal legs. Similar values were found in the normal and abnormal tibialis posterior muscles, long flexors of the toe and peroneal muscles. The change in composition of the soleus muscle and the reduction in the number of fibres may be caused by a defective neural influence on the development of the limb in club foot.

The effects of varying oxygen tensions upon bone resorption in vitro.

Calvaria from six-day-old infant mice were grown on a grid culture in a chemically defined medium under varying oxygen tensions. Quantitative isotope studies demonstrated a linear association between bone resorption and oxygen tension in the physiological range. This result was supported by histological, histochemical and vital staining experiments. The clinical finding of osteoporosis in areas of hyperaemia could therefore be attributed to a rise in oxygen tension causing increased bone resorption.

Relationship between human immunodeficiency virus-1 RNA identification in placenta and perinatal transmission.

OBJECTIVES: The objectives of this study were to identify human immunodeficiency virus (HIV)-associated nucleic acids in placenta with 35S RNA in situ hybridization and to correlate the presence of virus with perinatal HIV transmission. STUDY DESIGN: Blood from nine mother-infant pairs was collected for CD4+ lymphocyte count and p24 antigen analysis. Placental specimens were obtained for in situ hybridization, viral culture, and hematoxylin-eosin histologic analysis. Neonatal infection was diagnosed by p24 antigen analysis, polymerase chain reaction, and viral culture. Pediatric follow-up was obtained by personal communication and review of chart records. RESULTS: Three of 10 placentas from nine HIV-positive pregnant women (one set of twins) were found to contain evidence of HIV infection by RNA in situ hybridization. Maternal age, gravidity, parity, previous number of terminations, duration HIV-seropositive, maternal CD4+ at delivery, and neonatal cord CD4+ count were similar in those whose placentas contained virus versus those in whom virus was not identified. The incidence of histologic changes was similar in the HIV-positive and negative placentas from seropositive subjects and was similar to the incidence of histologic changes in placentas from known seronegative subjects. Placental culture failed to demonstrate the presence of virus in vitro. Of the three placentas that had positive in situ hybridization, two infants became HIV-positive. CONCLUSIONS: 35S RNA in situ hybridization identified the presence of nucleic acids in syncytiotrophoblasts from formalin-fixed, paraffin-embedded placentas. This technique is a useful tool to screen placentas at birth for HIV-RNA to judge the likelihood of perinatal HIV transmission.

The impact of avian influenza viruses on public health.

In the late 1990s, H5N1 and H9N2 avian influenza viruses caused respiratory infections in humans in Hong Kong. Exposure to domestic poultry in live-bird markets was significantly associated with human H5N1 disease. Seroepidemiologic studies conducted among contacts of H5N1-infected persons determined that human-to-human transmission of the avian H5N1 viruses occurred but was rare. The relatively high rates of H5 and H9 antibody seroprevalence among Hong Kong poultry workers in 1997 highlight the potential for avian viruses to transmit to humans, particularly those with occupational exposure. Such transmission increases the likelihood of reassortment between a currently circulating human virus and an avian virus and thus the creation of a strain with pandemic potential.

Mechanisms of pathogenicity of influenza A (H5N1) viruses in mice.

Avian-like H5N1 influenza viruses isolated from humans in 1997 were shown to have two distinct pathogenic phenotypes in BALB/c mice, after intranasal inoculation and without prior adaptation to this host. To further understand the mechanisms of H5N1 pathogenicity, we investigated the consequences of the mute of viral inoculation on morbidity and mortality, viral replication in pulmonary and systemic organs, and lymphocyte depletion. This study demonstrates the importance of extrapulmonary spread and replication, particularly in the brain, for the lethality of H5N1 viruses.