Chemical Composition, but Not Specific Surface Area, Affects Calcium Retention of Nanostructured Calcium Compounds in Growing Rats.

Background: Low dietary calcium intake and bioavailability may adversely affect bone health. Reducing the size of calcium compounds increases their specific surface area (SSA, expressed as m2/g) and may increase calcium dissolution and bioavailability.Objective: We investigated the influence of SSA and chemical composition on the bioavailability of calcium and compared in vitro calcium dissolution with in vivo absorption.Methods: Calcium dissolution was measured in 0.1 M phosphoric acid, whereas color and pH changes of foods were assessed as indicators for potential sensory performance. Calcium absorption, retention, and fractional retention were measured over a 5-d balance study in growing Sprague-Dawley male rats after 21 d of feeding. Femoral and vertebral bone mineral density (BMD) and extensive tissue histology were assessed at study end. The influence of SSA on calcium bioavailability was assessed by comparing the groups fed pure calcium carbonate (CaCO3) with increasing SSAs of 3, 36, and 64 m2/g (CaCO3_3, CaCO3_36 and CaCO3_64), whereas chemical composition was assessed by comparing the smallest CaCO3_64, a 50:50 wt:wt percent solution mixture of CaCO3 and hydroxyapatite_94, and pure hydroxyapatite_100.Results: In vivo, fractional calcium retention from hydroxyapatite_100 (mean ± SEM: 54.86% ± 0.95%/5 d) was significantly greater than from CaCO3_64 (49.66% ± 1.15%/5 d) (P = 0.044). Increasing SSA of the pure CaCO3 did not significantly improve calcium retention. Across all 5 groups, there were no significant differences in BMD or tissue calcification by histology. In vitro calcium dissolution did not correlate with SSA or calcium absorption. In selected food matrixes, hydroxyapatite_100 caused less color change and/or smaller pH increase than did the other calcium compounds.Conclusions: Our findings suggest that chemical composition rather than SSA is a predictor of nanostructured calcium bioavailability and that in vitro dissolution of nanostructured calcium does not predict in vivo absorption. Although its phosphorus content may limit use in some populations, nanostructured hydroxyapatite may be a promising calcium compound for food fortification.

Comparing Antibody Responses in Chickens Against Plasmodium falciparum Lactate Dehydrogenase and Glyceraldehyde-3-phosphate Dehydrogenase with Freund's and Pheroid® Adjuvants.

Pheroid® technology was assessed as an alternative to Freund's adjuvant to raise antibodies in experimental animals. Chickens were immunized with two recombinantly expressed Plasmodium falciparum proteins, lactate dehydrogenase (PfLDH) and glyceraldehyde-3-phosphate dehydrogenase (PfGAPDH), alone or in combination with Freund's adjuvant or Pheroid®. Chicken egg yolk antibodies (IgY) were isolated and compared for specificity, sensitivity and yield. Freund's adjuvant and Pheroid® stimulated prolonged antibody responses in chickens against both antigens. Affinity purified antibodies had specificity for the recombinant and the native proteins on Western blots. Antibodies generated in the presence of Freund's adjuvant had high sensitivity for both antigens. Pheroid® generated antibodies that detected the lowest concentration of recombinant PfLDH. Freund's adjuvant and Pheroid® both improved chicken IgY yields, with Pheroid® showing a 2-fold increase relative to controls. Pheroid® was well-tolerated in chickens and has potential for development as a safe adjuvant for testing alternative stimulatory factors to improve adjuvant formulations.

Assessment of the induction of dormant ring stages in Plasmodium falciparum parasites by artemisone and artemisone entrapped in Pheroid vesicles in vitro.

The in vitro antimalarial activities of artemisone and artemisone entrapped in Pheroid vesicles were compared, as was their ability to induce dormancy in Plasmodium falciparum. There was no increase in the activity of artemisone entrapped in Pheroid vesicles against multidrug-resistant P. falciparum lines. Artemisone induced the formation of dormant ring stages similar to dihydroartemisinin. Thus, the Pheroid delivery system neither improved the activity of artemisone nor prevented the induction of dormant rings.

Trends in malaria admissions at the Mbakong Health Centre of the North West Region of Cameroon: a retrospective study.

BACKGROUND: Malaria is the leading cause of death worldwide. It is urgent to assess the impact of interventions and scaled-up control efforts. Despite reported reduction in malaria prevalence in Africa, the trends in Cameroon are not yet fully understood. The aim of this study was to investigate the trends in malaria admissions among febrile patients seeking treatment over a seven-year period (2006-2012) in an endemic area in Cameroon, hypothesizing a declining trend. This period followed changes in malaria treatment policy. The objectives were to identify possible trends in malaria admissions and to evaluate the impact of changes to treatment guidelines on the prevalence. METHODS: Data was collected through consultation and perusal of laboratory and prescription registers of the Mbakong Health Centre. Data analysis was conducted using SPSS and SAS Statistics. RESULTS: Analysis revealed that 4,230 febrile patients were received from 2006-2012. Of these febrile cases, 29.30% were confirmed positive. Between 2006 and 2012 confirmed malaria positive cases of those tested fluctuated, dropping from 53.21% in 2006 to 17.20% in 2008; then rising to 35.00% in 2011 and, finally, dropping to 18.2% of those tested in 2012. The prevalence in females and males across all age groups were similar: a slightly higher risk of males to have malaria (OR = 1.08, 95% CI 0.94-1.25) were not practically significant. Of those tested, the 5 to < 15 years and the 1 to < 5 years age groups were the hardest hit by malaria in the area. A practically visible and significant association was observed between the age and gender with regards to the number of malaria positive results (Pearson ×2 = 153.675, p < 0.00001, Cramer's V = 0.352). Malaria prevalence exhibited a fluctuating yet declining trend, as observed over the 28 quarters between January, 2006 and December, 2012. CONCLUSIONS: The changes to the treatment guidelines appear to result in a declining trend as was observed between 2006 and 2008. However, malaria admissions fluctuated between 2008 and 2012. There is, therefore, a need to step up control efforts of especially the vulnerable groups, such as the very young.

Elucidating the effect of specific surface area on the gastrointestinal absorption of nanostructured calcium through Calcium-45 in vivo radiotracing.

Low dietary calcium intake and absorption may increase the risk of hypocalcaemia disease states. Reducing the particle size of calcium-containing powders and increasing the specific surface area (SSA), may have high oral calcium bioavailability. The absorption of a single dose of different sized calcium carbonate nanoparticles was traced in Sprague-Dawley rats with radioactive calcium-45 (half-life = 162.6 days, ß- endpoint = 258 keV; 100%). Four calcium carbonate formulations (calcium-45) were administered to Sprague-Dawley rodents (6 per treatment; n = 24). The groups were [45Ca]CaCO3 SSA 3 m2/g, [45Ca]CaCO3 36 m2/g, [45Ca]CaCO3 64 m2/g and a separate [45Ca]CaCO3 36 m2/g formulation produced by flame assisted pyrolysis. Blood and urine were sampled periodically, and organs collected and analysed after euthanasia. No changes in SSA or crystallinity were observed when powders before or after irradiation were compared. The [45Ca]CaCO3 64 m2/g formulation presented with higher levels in blood 2 h after administration and a higher liver and femur concentration. These findings suggest [45Ca]CaCO3 64 m2/g could lead to increased oral bioavailability.

Twenty years of safety pharmacology model validation and the wider implications of this to drug discovery.

This editorial summarizes the content of the current themed issue of J Pharm Tox Methods derived from the 2019 Annual Safety Pharmacology Society (SPS) meeting held in Barcelona, Spain, and reflects on 20 years of innovation in the elaboration of methods for evaluating adversity, particularly during the nonclinical research phase. Given the success of safety pharmacology (SP) in the last 20 years, we propose that the rubric for SP method invention and validation be examined in more detail to explore whether it may have wider relevance to the drug discovery process. Articles arising from the Barcelona meeting are summarized here. They reflect current areas of controversy and innovation in SP. Not for the first time in recent years, the suitability of the No Observable Adverse Effect Level (NOAEL) as a variable in SP was considered in an article derived from a survey of SPS members. It was found from the survey and concluded from the analysis that the NOAEL is not necessary for assessing the safety of a New Chemical Entity (NCE). The meeting included scientific content from more than 190 abstracts (reproduced in the current volume of J Pharm Tox Methods). The impact of the INSPIRE program on the educational endeavor of SP, cardiovascular SP with regard to hERG and advances in CiPA and stem cells assays, the use of the echocardiogram in SP, the applicability of deep learning methods in SP and toxicology studies, the role of biomarkers in renal SP studies, and advances in CNS SP are highlighted in this issue of the Journal. This continued innovation reflects a rubric in SP that identifies problems, seeks solutions and, importantly, validates the solutions. If there is a lesson to be learned from the 20 years of annual SP methods themed issues it is that drug discovery efforts may benefit from a more rigorous validation process for discovery methods, using positive and negative controls for validation, as is done in SP method validation.

Drug safety Africa: An overview of safety pharmacology & toxicology in South Africa.

This meeting report is based on presentations given at the first Drug Safety Africa Meeting in Potchefstroom, South Africa from November 20-22, 2018 at the North-West University campus. There were 134 attendees (including 26 speakers and 34 students) from the pharmaceutical industry, academia, regulatory agencies as well as 6 exhibitors. These meeting proceedings are designed to inform the content that was presented in terms of Safety Pharmacology (SP) and Toxicology methods and models that are used by the pharmaceutical industry to characterize the safety profile of novel small chemical or biological molecules. The first part of this report includes an overview of the core battery studies defined by cardiovascular, central nervous system (CNS) and respiratory studies. Approaches to evaluating drug effects on the renal and gastrointestinal systems and murine phenotyping were also discussed. Subsequently, toxicological approaches were presented including standard strategies and options for early identification and characterization of risks associated with a novel therapeutic, the types of toxicology studies conducted and relevance to risk assessment supporting first-in-human (FIH) clinical trials and target organ toxicity. Biopharmaceutical development and principles of immunotoxicology were discussed as well as emerging technologies. An additional poster session was held that included 18 posters on advanced studies and topics by South African researchers, postgraduate students and postdoctoral fellows.

A molecular analysis of the GBA gene in Caucasian South Africans with Parkinson's disease.

BACKGROUND: The molecular basis of Parkinson's disease in South African population groups remains elusive. To date, substitutions in the GBA gene are the most common large-effect genetic risk factor for Parkinson's disease. The primary objective of this study was to determine the prevalence of GBA substitutions in South Africans with idiopathic Parkinson's disease. METHODS: Participants were recruited from tertiary hospitals in the Gauteng Province in South Africa. All participants were screened for substitutions in GBA exon 8-11 and the full coding region was analysed in 20 participants. Peripheral ß-glucocerebrosidase enzymatic activity of GBA-carriers was measured in mixed leukocytes. RESULTS: Of 105 Caucasian Parkinson's disease participants (82.7% Afrikaner) with an average age of disease onset of 61.9 ± 12.2 years and 40 controls (age 73.4 ± 12.4 years) were included. Heterozygous GBA substitutions were identified in 12.38% of affected participants (p.G35A, p.E326K, p.I368T, p.T369M, p.N370S, p.P387L and p.K441N) and 5.00% of controls (p.E326K and p.T369M). The substitutions ranged from predicted benign to moderately damaging; with p.E326K and p.T369M most prevalent, followed by the Afrikaner Gaucher disease substitution p.P387L. Severe Gaucher disease mutations, like p.L444P, were absent in this cohort. Enzyme activity analysis revealed a nonsignificant reduction in the GBA-Parkinson's disease individuals (14.49 ± 2.30 nmol/h/mg protein vs. 15.98 ± 3.06 nmol/h/mg in control samples). GBA substitutions occur in both young-onset and late-onset Parkinson's cases in the cohort. CONCLUSION: Mild GBA substitutions that may not cause Gaucher disease were a common risk factor for Parkinson's disease in the participant group.

Advances in GBA-associated Parkinson's disease--Pathology, presentation and therapies.

GBA mutations are to date the most common genetic risk factor for Parkinson's disease. The GBA gene encodes the lysomal hydrolase glucocerebrosidase. Whilst bi-allelic GBA mutations cause Gaucher disease, both mono- and bi-allelic mutations confer risk for Parkinson's disease. Clinically, Parkinson's disease patients with GBA mutations resemble idiopathic Parkinson's disease patients. However, these patients have a modest reduction in age-of-onset of disease and a greater incidence of cognitive decline. In some cases, GBA mutations are also responsible for familial Parkinson's disease. The accumulation of α-synuclein into Lewy bodies is the central neuropathological hallmark of Parkinson's disease. Pathologic GBA mutations reduce enzymatic function. A reduction in glucocerebrosidase function increases α-synuclein levels and propagation, which in turn inhibits glucocerebrosidase in a feed-forward cascade. This cascade is central to the neuropathology of GBA-associated Parkinson's disease. The lysosomal integral membrane protein type-2 is necessary for normal glucocerebrosidase function. Glucocerebrosidase dysfunction also increases in the accumulation of ß-amyloid and amyloid-precursor protein, oxidative stress, neuronal susceptibility to metal ions, microglial and immune activation. These factors contribute to neuronal death. The Mendelian Parkinson's disease genes, Parkin and ATP13A2, intersect with glucocerebrosidase. These factors sketch a complex circuit of GBA-associated neuropathology. To clinically interfere with this circuit, central glucocerebrosidase function must be improved. Strategies based on reducing breakdown of mutant glucocerebrosidase and increasing the fraction that reaches the lysosome has shown promise. Breakdown can be reduced by interfering with the ability of heat-shock proteins to recognize mutant glucocerebrosidase. This underlies the therapeutic efficacy of certain pharmacological chaperones and histone deacetylase inhibitors. These therapies are promising for Parkinson's disease, regardless of mutation status. Recently, there has been a boom in studies investigating the role of glucocerebrosidase in the pathology of Parkinson's disease. This merits a comprehensive review of the current cell biological processes and pathological pictures involving Parkinson's disease associated with GBA mutations.

Trends in malaria case management following changes in the treatment policy to artemisinin combination therapy at the Mbakong Health Centre, Cameroon 2006-2012: a retrospective study.

National malaria treatment policies are devised to guide health professionals and to facilitate diagnosis and case management. Following the recommendations of the WHO, Cameroon changed its malaria treatment policy from monotherapy to artemisinin-based combination therapy (ACT) as the first-line treatment for uncomplicated malaria. We report an investigation into trends of case management following this change in policy. Data was collected retrospectively, through consultation and perusal of laboratory and prescription registers of the Mbakong Health Centre. Analysis of data was done using SPSS and SAS Statistics. Data presented herein demonstrate that from 2006 to 2012, a total of 2484 (58.7%) of the total prescriptions included an anti-malarial, 1989 (47.0%) included an antibiotic and 1935(45.7%) included an antipyretic. The anti-malarials prescribed were Anti-malaria combination therapy (ACT) - 1216 (47.6%), quinine 1044 (40.8%) or SP 296 (11.6%). Of the 1216 patients prescribed an ACT, 441(36.3%) had a positive malaria parasite confirmation, 746 (61.3%) were negative for plasmodium. Overall, 29 patients (2.4%) were treated either with an ACT without any test performed. Quinine intake was recorded in 566 (54.2%) patients positive for plasmodium. ACT prescription increased from 23% in 2007 to between 44 and 45% in 2008-2009. During this period there was a corresponding drop in the prescription of quinine from 38% in 2007 to 13% in 2009 (r=-0.43, p>0.05). Sulphadoxine-Pyrimethamine (SP) was restrictively prescribed to women of childbearing age (97.0%) after 2008. Antibiotics prescription dropped from 53.7% to 39.3% from 2010 to 2012. The odds of being prescribed an antibiotic was significantly higher in patients with a malaria negative result compared to malaria positive patients (OR=6.12, CI 4.74-7.91, p<0.00001). Overall, there is an over treatment of malaria, thus departing from the WHO guidelines of appropriate treatment. Although there is an overall increase in the prescription of ACT, less prescription of quinine and a noticeable restrain from prescription of SP to febrile cases, the old practice was still rampant. There is need for healthcare workers to adhere to guidelines in order to enhance the rational use of drugs to achieve appropriate treatment of uncomplicated malaria according to WHO guidelines.

Influence of variation in eumelanin content on absorbance spectra of liquid skin-like phantoms.

The attenuation behavior of two different types of skin-like phantoms representing the range of Fitzpatrick skin Types I-VI was investigated and compared with real human skin. Intralipid (IL) and Pheroid(™) artificial lipid membrane vesicles, respectively, were added to synthetic eumelanin concentrations ranging from 0.0044 to 0.13mgmL(-1) to produce skin-like phantoms. Spectrophotometric absorbance and transmittance measurements were performed. Results indicated some of the nonmonotonic trends observed in real human skin, albeit shifted more toward the visible wavelength range. There exists, however, an underlying difference in interaction between the melanin and the Pheroid(™) and IL skin-like phantoms.

Absorption of the novel artemisinin derivatives artemisone and artemiside: potential application of Pheroid™ technology.

Artemisinins have low aqueous solubility that results in poor and erratic absorption upon oral administration. The poor solubility and erratic absorption usually translate to low bioavailability. Artemisinin-based monotherapy and combination therapies are essential for the management and treatment of uncomplicated as well as cerebral malaria. Artemisone and artemiside are novel artemisinin derivatives that have very good antimalarial activities. Pheroid™ technology is a patented drug delivery system which has the ability to entrap, transport and deliver pharmacologically active compounds. Pharmacokinetic models were constructed for artemisone and artemiside in Pheroid™ vesicle formulations. The compounds were administered at a dose of 50.0mg/kg bodyweight to C57 BL/6 mice via an oral gavage tube and blood samples were collected by means of tail-bleeding. Drug concentrations in the samples were determined using an LC/MS/MS method. There was 4.57 times more artemisone in the blood when the drug was entrapped in Pheroid™ vesicles in comparison to the drug only formulation (p < 0.0001). The absorption of artemiside was not dramatically enhanced by the Pheroid™ delivery system.