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Poster Abstracts

 

2020 Poster Abstracts

Does the FDA enforce regulations at virtual and fully integrated pharma and biotech companies the same?


Cassie Alexander University of Georgia BACKGROUND Over the past several decades, virtual companies have become prevalent within the pharmaceutical and biotech industries. Virtual companies utilize a business model where they directly hire employees in supportive roles while outsourcing critical business activities that are highly regulated by the United States Food and Drug Administration (U.S. FDA). Since the corporate structures for virtual and fully integrated companies differ significantly, it is important to understand how virtual companies are regulated by the FDA so management teams can understand how to appropriately staff organizations to mitigate potential compliance risks while maintaining a lean organization. METHODS The study had three areas of focus: public information (websites and databases), articles and personal interviews. For the public information analysis, a randomized sample of 40 companies was created, including virtual and fully integrated pharmaceutical and biotech companies, analytical laboratories and third-party logistics providers. Data was gathered for the 40 companies from websites and databases like Registration and Listing, Inspections, Inspection Citations, Warning Letters and the Orange Book. Data was tabulated to provide a qualitative and quantitative analysis of the trends and frequency of occurrence. For the second area of focus, five online articles/blogs were analyzed to gather common trends in compliance activities relating to virtual companies. Finally, five people with experience with compliance activities were surveyed through a questionnaire to assess personal experiences and perspectives on how the FDA regulates virtual and fully integrated companies. RESULTS The findings from this study demonstrate that the FDA enforces regulations at virtual and fully integrated companies differently. Analysis of data from the companies show that fully integrated pharmaceutical and biotech companies experienced more FDA inspections, inspection citations and escalated regulatory enforcement actions. Analysis of industry the articles pointed to the importance of virtual companies building a strong relationship with contract facilities through robust Quality Agreements and close oversight. Feedback from questionnaires of industry professionals showed a concern that virtual companies have less rigorous quality systems and inadequate oversight of contract facilities, which would lead to FDA enforcement actions. CONCLUSIONS While subject to the same U.S. Regulations, this study demonstrates that virtual companies experience less regulatory oversight and enforcement actions than fully integrated companies. It is likely the contract partners are inspected for compliance and it is incumbent upon the management teams of virtual companies to ensure they have adequate processes and resources in place to appropriately oversee contract partners and mitigate risks. This will reduce the risk of FDA enforcement actions on virtual companies while ensuring safe and effective products are provided to patients.





Does the FDA enforce regulations at virtual and fully integrated pharma and biotech companies the same?


Cassie Alexander University of Georgia BACKGROUND Over the past several decades, virtual companies have become prevalent within the pharmaceutical and biotech industries. Virtual companies utilize a business model where they directly hire employees in supportive roles while outsourcing critical business activities that are highly regulated by the United States Food and Drug Administration (U.S. FDA). Since the corporate structures for virtual and fully integrated companies differ significantly, it is important to understand how virtual companies are regulated by the FDA so management teams can understand how to appropriately staff organizations to mitigate potential compliance risks while maintaining a lean organization. METHODS The study had three areas of focus: public information (websites and databases), articles and personal interviews. For the public information analysis, a randomized sample of 40 companies was created, including virtual and fully integrated pharmaceutical and biotech companies, analytical laboratories and third-party logistics providers. Data was gathered for the 40 companies from websites and databases like Registration and Listing, Inspections, Inspection Citations, Warning Letters and the Orange Book. Data was tabulated to provide a qualitative and quantitative analysis of the trends and frequency of occurrence. For the second area of focus, five online articles/blogs were analyzed to gather common trends in compliance activities relating to virtual companies. Finally, five people with experience with compliance activities were surveyed through a questionnaire to assess personal experiences and perspectives on how the FDA regulates virtual and fully integrated companies. RESULTS The findings from this study demonstrate that the FDA enforces regulations at virtual and fully integrated companies differently. Analysis of data from the companies show that fully integrated pharmaceutical and biotech companies experienced more FDA inspections, inspection citations and escalated regulatory enforcement actions. Analysis of industry the articles pointed to the importance of virtual companies building a strong relationship with contract facilities through robust Quality Agreements and close oversight. Feedback from questionnaires of industry professionals showed a concern that virtual companies have less rigorous quality systems and inadequate oversight of contract facilities, which would lead to FDA enforcement actions. CONCLUSIONS While subject to the same U.S. Regulations, this study demonstrates that virtual companies experience less regulatory oversight and enforcement actions than fully integrated companies. It is likely the contract partners are inspected for compliance and it is incumbent upon the management teams of virtual companies to ensure they have adequate processes and resources in place to appropriately oversee contract partners and mitigate risks. This will reduce the risk of FDA enforcement actions on virtual companies while ensuring safe and effective products are provided to patients.





 

Drug Discovery & Development

Heterologous Prime Boost Influenza Vaccination Provides a Strategy to Induce More Effective Cross Protection Than Repeat Vaccination


Noopur Bhatnagar Institute for Biomedical Sciences, Georgia State University BACKGROUND Recent clinical studies have reported that repeat annual vaccination diminishes vaccine efficacy. Therefore, it is critical to better understand the impact of prior vaccine immunity and to develop an effective vaccination strategy. METHODS In this study, we designed an experimental strategy to determine the impact of heterologous prior vaccine immunity on inducing cross protection in comparison with mimicking homo seasonal repeat vaccination. Groups of mice were intramuscularly primed with a different strain of an inactivated influenza virus and then boosted with homo or heterologous inactivated virus (H1N1, H3N2, H5N1, H7N9, H9N2). RESULTS A wide range of strain specific hemagglutination inhibition (HAI) titers was induced in primed immune sera, depending on the vaccine strain used. When the primed mice were intramuscularly boosted with a homo or heterologous subtype of inactivated virus vaccine, HAI titers increased up to 8 folds. Strain specific HAI titers against prime and boost vaccine strains were induced. Cross-reactive HAI titers against a virus not included in prime and boost as well as enhanced cross protection were induced in certain heterologous prime boost influenza vaccinations. CONCLUSIONS The results in this study suggest that heterologous vaccine-induced prior immunity promotes the induction of homo and cross-reactive HAI titers against prior as well as new hetero virus vaccine strains compared to repeat homologous vaccination. These results support a promising strategy of heterologous prime boost influenza vaccination.




Assessing the role of dopamine in the differential neurotoxicity patterns of stimulants


Neha Chitre Mercer University, College of Pharmacy BACKGROUND Drug-induced Parkinsonism (DIP) is the second most common etiology of Parkinsonism after Parkinson's disease (PD). Stimulants like amphetamine (AMPH) that are known to cause long term dysregulation of the brain dopaminergic system are top contributors to DIP. Methamphetamine, a very commonly abused AMPH is an addictive psychostimulant that has very strong neurotoxic effects on the central nervous system (CNS), and specifically the brain dopaminergic system. 3,4-methylenedioxymethamphetamine (MDMA) recently achieved breakthrough status from the Food and Drug Administration (FDA) for post-traumatic stress disorder (PTSD). However, evidence indicates that exposure to toxic doses of MDMA can lead to long-lasting dysregulation of brain monoaminergic neurotransmitters, primarily from studies conducted in young adult rodents. Additionally, the abuse of synthetic cathinones, which are the β-ketone analogs of AMPH has also been on the rise. Despite their structural similarity to AMPH, not many studies have evaluated their neurotoxic potential and the detrimental effects they could be having on the dopaminergic system. Thus, the aim of the current study is to evaluate the dopaminergic neurotoxicity exhibited by stimulants to study their potential to cause DIP using prototypical AMPH, methcathinones and pyrrolidines METHODS

Physiological effects of stimulants on lethality, body weight and body temperature were assessed using binge-models of dosing in Swiss- Webster mice. Behavioral assays such as Open-Field Testing and stride length evaluation were conducted for motor deficits. Cognitive impairments were assessed by Passive Avoidance testing. Neurochemical analysis of dopamine levels and its major metabolites DOPAC and HVA in the striatum was performed using ultra high performance liquid chromatography (UHPLC-ED).

RESULTS Study results indicate that several key analogs such as methcathinone, 2-fluoromethcathinone, 3-methylmethcathinone, and Alpha-PPP cause behavioral deficits such as decreased locomotor activity and impaired cognitive performance in passive avoidance testing. Additionally, binge-dosing of these stimulants in mice causes significant dopamine depletion in the striatum, which are characteristic hallmarks of Parkinsonism. CONCLUSIONS Taken together, these results suggest that some prototypical AMPH, methcathinones and pyrrolidines may have the potential to cause DIP, warranting further studies and investigation.




Needle-free Measles Immunization using Microparticulate Formulation


Devyani Joshi Mercer University College of Pharmacy BACKGROUND Measles, a highly contagious infectious disease, is a major cause of death among children globally; despite the availability of the vaccine. It is particularly common in developing countries, like parts of Africa and Asia. More than 140,000 people died of measles in 2018. With the current vaccination regimen, two doses of measles vaccine are prescribed: first, before the age of 12 months and a booster dose between 5 to 10 years of age. Since children are the primary targets for the vaccine, we aimed at delivering the vaccine via a needle-free transdermal route. We compared the efficacy of vaccine administered transdermally to that of the traditional subcutaneous administration. We propose a microparticulate formulation of the vaccine. The traditional soluble vaccine is taken up via antigen-presenting cells (APC) and the antigenic epitopes are presented via major histocompatibility complex MHC II. The vaccine thus effectively stimulates the humoral immune response but fails to activate the cellular immune response. The particulate vaccine is taken up via APCs and epitopes are presented via both, MHC I and MHC II complexes; stimulating both, humoral and cellular adaptive immune response. Particulate formulation of vaccine thus increases the immunogenicity of an antigen. METHODS

The measles antigen was incorporated into the biodegradable, crosslinked-albumin matrix and spray dried using Buchi mini spray dryer B-290 to formulate the vaccine loaded microparticles. The microparticles were characterized for size, charge, and polydispersity index (PDI). The surface morphology of microparticles was visualized by Scanning Electron Microscopy. The induction of an immune response by the microparticulate vaccine was confirmed via spectroscopic Griess's assay. The expression of antigen-presenting molecules, MHC I and MHC II, and their co-stimulatory molecules CD80 and CD40 was assessed on the surface of dendritic cells using BD Accuri C6 plus flow cytometer. The equivalent amount of blank microparticles (without antigen and adjuvant) was used as control. The cytotoxicity of microparticles was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. The uptake of microparticles by APCs was studied as a function of time. The in vivo efficacy of the microparticulate vaccine was studied in the mouse model. For the transdermal administration of the microparticulate vaccine, P.L.E.A.S.E. ablative laser was used. This laser creates the micropores of defined size on the surface of the skin, allowing transdermal delivery of vaccine. Seven groups of animals (n=6) were used in the study: naïve group as a control, the second group received measles vaccine solution subcutaneously, the third group received blank microparticles subcutaneously, the fourth group received vaccine microparticles subcutaneously, the fifth group received vaccine and adjuvant (alum and MPL A®) microparticles subcutaneously, the sixth group received vaccine microparticles transdermally via ablative laser and the last group received vaccine and adjuvant (alum and MPL A®) microparticles transdermally via ablative laser. The animals were administered with one prime and one booster dose at weeks 0 and 2. The serum was collected from the animals bi-weekly and IgG and IgM antibody titers were measured using ELISA.

RESULTS The yield of the microparticulate vaccine formulation was 91.37±3.1%. The size of the microparticles was 3.197±0.8361 um, PDI was 0.447 with a charge of -29.4±6.2 mV. The microparticulate vaccine with adjuvants showed significantly higher release of nitric oxide (p<0.05), a hallmark of an innate immune response, compared to the blank microparticles. It resulted in a significantly higher (p<0.05) cell-surface expression of MHC I and MHC II and their co-stimulatory molecules CD80 and CD40 respectively on the surface of dendritic cells. MTT assay showed that the vaccine and adjuvant microparticles were non-cytotoxic compared to dimethyl sulfoxide (DMSO) which is cytotoxic. The uptake of microparticles by APCs increased with time and was maximum at 4 h. The in vivo studies demonstrated elevated humoral immune responses in the mice receiving vaccine and adjuvant microparticles via both, subcutaneous and transdermal routes; as seen with the serum titers of IgG and IgM antibodies (p<0.001). There was no significant difference between the antibody titers in the mice that received the vaccine and adjuvant microparticles subcutaneously and transdermally, indicating that the transdermal route is as efficacious as the subcutaneous route for vaccination. CONCLUSIONS The microparticulate formulation augments the immunogenic properties of the soluble vaccine as seen from better induction of innate immune response. The use of adjuvants potentiates the immune response to the antigen. Higher expression of antigen-presenting molecule, MHC I, and its co-stimulatory molecule CD80 indicates the induction of cytotoxic response by the microparticulate vaccine, required for overcoming acute measles infection. Higher expression of MHC II and its co-stimulatory molecule CD40 indicates the induction of humoral immune response, critical in controlling viral replication and conferring protection. Transdermal administration of the particulate vaccine using a laser epidermal system produced comparable results as that of the subcutaneous administration indicating the potential of the transdermal route for vaccination.




Assessing Adjuvant-effect of Microparticulate DPD and its Analogs


Devyani Joshi Mercer University College of Pharmacy BACKGROUND Autoinducer (AI)-2 is a signaling molecule involved in bacterial quorum sensing (QS): a form of interspecies communication in bacteria. The reduction of QS is a potential therapeutic approach to fight against bacterial diseases. However, targeting of the AI-2 based QS remains challenging because of the rapid interconversion of the AI-2 precursor (S)-DPD (4,5-dihydroxypentane-2,3-dione) to several linear and cyclic forms recognized by different bacteria. (S)-DPD can thus be used as an adjuvant to induce and boost an immunogenic response in humans. Adjuvants are the substances used in combination with antigens to produce a more robust immune response. Since their discovery, the aluminum compounds continue to monopolize the human vaccines as adjuvants. Despite the discovery of more potent adjuvants (lipopolysaccharide etc), they are unsuitable for human use due to their toxicity. Adjuvants are classified as particulate delivery systems and immunostimulatory adjuvants (derived from pathogens), based on their mechanism. We propose to consolidate the advantages of these mechanisms by developing a microparticulate formulation of DPD and its synthetic analogs. METHODS

Various polymers were screened via molecular docking technique for the microparticulate formulation of DPD. Bovine Serum Albumin (BSA) was selected as the polymer of choice based on its docking score. DPD was mixed with the crosslinked-albumin matrix and spray dried using Buchi mini spray dryer B-290 to obtain DPD loaded microparticles. The microparticles were characterized for size, charge, and polydispersity index. The surface morphology was visualized by scanning electron microscopy. The induction of innate immune response, as indicated by the production of nitric oxide by antigen-presenting cells (APCs), was analyzed by Griess's assay. The ability of DPD microparticles to induce cellular and humoral adaptive immune response was evaluated in vitro by measuring the expression of antigen-presenting molecules MHC I and MHC II and their co-stimulatory molecules, CD80 and CD40, on the surface of APCs. The cytotoxicity of DPD microparticles on APCs was assessed by MTT assay. Further evaluation of adjuvant potential of DPD microparticles and comparison of its efficacy as an adjuvant to the traditional FDA approved adjuvants was done by combining DPD microparticles with the particulate vaccines for measles and gonorrhea. Griess's assay was performed to analyze the induction of innate immune response by the particulate vaccines with and without combination with different particulate adjuvants i.e. alum, MF59, MPL A and DPD. Future studies will involve analyzing the expression of antigen-presenting molecules MHC I and MHC II and their co-stimulatory molecules CD80 and CD40 on the surface of APCs on exposure to various particulate vaccines in combination with FDA approved adjuvants and comparing the adjuvant potential of microparticulate DPD to that of the traditional adjuvants. Various synthetic analogs of DPD including ent-DPD, isobutyl-DPD, n-butyl-DPD, and phenyl-DPD will be formulated into the microparticles. The adjuvant potential of these analogs will be compared to that of microparticulate DPD and to other FDA approved adjuvants.

RESULTS BSA was selected as the polymer of choice for the formulation of microparticles based on its BSA was selected as the polymer of choice for the formulation of microparticles based on its docking interactions with DPD. The yield of the microparticulate formulation was 89.52 ± 2.3 %. The particle size was 3.851 ± 0.9769 um with a charge of -31.5 ± 7.4 mV. The Griess's assay showed a significantly higher (p<0.01) release of nitric oxide, a hallmark of the innate immune response, by dendritic cells upon exposure to DPD microparticles alone and in combination with the traditional adjuvants (alum and MF59) as compared to the blank microparticles. The APCs showed significantly higher (p<0.05) expression of antigen-presenting molecules MHC I and MHC II and their co-stimulatory molecules CD80 and CD40 upon exposure to DPD microparticles with and without adjuvant microparticles as compared to the blank microparticles. The DPD microparticles were found to non-cytotoxic to the APCs as compared to dimethyl sulfoxide (DMSO) which is cytotoxic. Microparticulate DPD was found to have an adjuvant effect upon combination with the gonorrhea vaccine as indicated by nitric oxide release by APCs. However, there was no adjuvant effect of DPD observed upon its combination with measles vaccine. These results will further be confirmed by evaluating the expression of MHC I, MHC II, CD40, and CD80 by APCs upon exposure to DPD microparticles in combination with various viral and bacterial vaccines. CONCLUSIONS

Griess's assay and Flow cytometric analysis revealed that the microparticulate DPD is immunogenic and can stimulate innate and adaptive immune response. A combination of DPD with various particulate vaccines reveals that microparticulate DPD shows an adjuvant effect with the bacterial vaccines. These encouraging preliminary results have led us to further test microparticulate DPD and its synthetic analogs to explore their potential as probable vaccine adjuvants.




4-Ethoxybenzoic Acid is an Anti-Pathogenic Anti-Biofilm Compound


Brooke Martin Georgia State University BACKGROUND Microbial biofilms are responsible for a wide variety of medically relevant infections. An issue with treating them is their inherent multidrug resistance, which makes treatment difficult and contributes to drug resistance. Anti-pathogenic drugs control microbial infections by targeting virulence without being biocidal and are less likely to foster drug-resistant strains. We identified several anti-biofilm anti-pathogenic compounds from Rhamnus prinoides (gesho), a plant used in traditional medicine, and a structure-activity analysis identified 4-ethoxybenzoic acid (4EB) as a compound with good activity. METHODS

The colorimetric method was used to determine the abundance of biofilms treated with and without 4EB. Crystal violet staining was used to determine anti-biofilm concentrations. The results from two independent studies were combined and statistically analyzed using ANOVA and t-test.

RESULTS 4EB concentrations from 0.4 to 0.8 mg/mL inhibited biofilm formation of Candida albicans. 4EB concentrations from 0.8 to 7 mg/mL were inhibitory to Streptococcus mutans biofilm formation. Pseudomonas aeruginosa biofilm activity was not impacted by 4EB. CONCLUSIONS

4EB is interesting as an anti-pathogenic anti-biofilm compound and may help prevent biofilm formation on surfaces including medical devices, bandages, and hospital surfaces. It may lead to the identification of additional compounds that have greater potency. Considerable evidence has shown that anti-pathogenic agents that inhibit bacterial virulence are suitable alternatives to antibiotics or help re-sensitizing bacteria to antibiotics.




Evaluation of a novel oxytocin nanoformulation for cocaine-use disorder


Aboagyewaah Oppong-Damoah Mercer University BACKGROUND Cocaine abuse is a significant global public health concern. In 2016 alone, 18 million people reported cocaine use worldwide. Currently, there are no FDA approved pharmacotherapies for treating cocaine dependence. Oxytocin (OT), a highly prosocial neuropeptide has shown recent promise for the treatment of disorders with known social deficits such as autism spectrum disease and cocaine-use disorder (CUD), but concerns remain as to its clinical relevance due to poor blood brain barrier penetration and short half-life. METHODS

In this study, we used the open field locomotor activity assay, a well validated preclinical model for assessing stimulant induced locomotor hyperactivity to investigate the potential of OT and a novel OT nanoformulation for reversing cocaine induced locomotor stimulant effects.

RESULTS OT administered both intranasally and intraperitoneally dose dependently decreased cocaine induced locomotor hyperactivity when given as a 10-minute pretreatment but showed no such effect when administered as 2-hour pretreatment. OT nanoparticles showed sustained effects in reversing cocaine induced locomotor stimulant effects both at 10 minutes and 2-hour pretreatment times. CONCLUSIONS

This study can now be used to support the use of OT nanoformulation and potentially other neuropeptides as promising therapeutic candidates for disorders of the central nervous system such as CUD.




Novel Strategies for Developing Universal Influenza Virus Vaccines


Bo Ryoung Park Institute for Biomedical Sciences, Georgia State University BACKGROUND Current live attenuated and inactivated influenza vaccines based on immunity to the hemagglutinin (HA) hypervariable protein are effective when vaccine strains and circulating viruses are well matched. However, HA-based current vaccines are ineffective in providing cross protection against antigenically distinct drift and new pandemic viruses. Universal influenza virus vaccines based on the highly conserved antigenic targets such as the extracellular domain of M2 (M2e) confer cross protection but are not sufficient to serve as a standalone vaccine due to low efficacy. Neuraminidase is the 2nd major surface protein and suggested to be an additional target for inducing cross protection. Virus-like particles (VLP) are known to be an immunogenic delivery vehicle. METHODS

As a new approach to improve the efficacy of HA-based current vaccines by inducing both HA and cross protective M2e immunity, we generated recombinant influenza virus vaccine platforms, which express chimeric H3 HA-M2e conjugate proteins in the N-terminus (HA-4xM2e). In addition, we developed multimeric VLP vaccine as a single entity containing consensus N1 neuraminidase NA, N2 NA, influenza B virus NA as well as tandem repeat 5xM2e.

RESULTS The live reassortant H3N2 chimeric virus vaccine was highly attenuated in mice, as shown by restricted replication in the upper respiratory but not in the lower respiratory track lung tissues of the mice. Recombinant H3N2 virus containing chimeric H3 HA-4xM2e was effective in inducing cross protective M2e specific IgG antibody responses as well as HA immunity in mice. Mice that intranasally primed with chimeric H3 HA-4xM2e vaccine showed improved cross protection against antigenically different viruses (H1N1, H3N2, H5N1, H7N9, H9N2). As an additional novel approach, multi-NA + 5xM2e VLP vaccination of mice was found to be highly effective in inducing cross protection against influenza A and B viruses. CONCLUSIONS

The results in this study present novel approaches to improve the efficacy of cross protection against seasonal and pandemic viruses by recombinant influenza virus and VLP vaccines inducing immunity to HA, NA, and M2e antigens.




A Novel Diagnostic Method for Detection and Quantitation of Paralytic Shellfish Poisoning in Humans


Mani Deepika Vakkalanka Mercer University BACKGROUND Paralytic shellfish toxins(PSTs) are potent neurotoxins which bind to the voltage gated sodium channels and prevent the conduction of action potentials leading to respiratory paralysis and death. Carbamate toxins are the most potent PSTs and include gonyautoxins(GTXs) 1,2,3,4. Testing these toxins in human biological matrices is the best approach to accurately detect, quantify them and identify the exposures. Here, we describe a solid phase extraction method for extracting GTXs from human plasma using HILIC HPLC-MS/MS analysis for quantitation. The developed method was further optimized and validated so that it can be applied to clinical specimens. METHODS

GTXs were extracted from human plasma using strong cationic exchange cartridges. Sample pH was adjusted with 10mM acetate buffer. Cartridges were conditioned with methanol and acetate buffer and toxins were eluted with 5% ammonium hydroxide in methanol. Experiments were conducted using pooled plasma spiked with a series of concentrations of gonyautoxins. Calibrants were prepared at the following concentration ranges: GTX1 8.13-517.66 ng/mL, GTX2 6.98-473.25 ng/mL, GTX3 2.96-200.68 ng/mL and GTX4 2.56-162.91 ng/mL. Low- and High-quality controls (QCs) were prepared at a concentration of 44 and 124 ng/mL for GTX1 , 40 and 113 ng/mL for GTX2, 17 and 71 ng/mL for GTX3, 14 and 39 ng/mL for GTX4. Eight plasma specimens were collected and spiked with toxin at a concentration of 173.85ng/ml. Calibrants, quality controls and specimen plasmas were then extracted as described above, dried down and reconstituted. Extracted samples were injected onto HPLC-MS/MS for analysis.

RESULTS The developed method was validated according to the FDA guidance for bioanalytical method validation. The calibration curves were linear with the coefficients of determination value (R-squared value) greater than 0.99 for all toxins. The method showed good percent accuracies for all the toxins: GTX1 95-104%, GTX2 92-114%, GTX3 92-117%, GTX4 92-107%. Precision or percent CV ranged from 3.5 to 10.9% for GTX1, 3.03 to 11.25% for GTX2, 3.01 to 12.72 for GTX3 and 2.08 to 10.49 for GTX4. Percent accuracies for quality controls ranged from 90.3-97.7%(Low QC) and 96.7-105.1%(High QC). Recovery of GTXs from specimen plasmas were as follows: GTX1-110.55%, GTX2-103.72%, GTX3-3.27%, GTX4-99.7%. Limits of detection were calculated (by Taylor method) to be 7.08, 7.59, 2.1, 1.98 ng/mL for GTXs 1,2,3 and 4 respectively. Limits of quantitation were the lowest calibrators for each analyte. Selectivity of the method was characterized by analyzing blank samples and there were no peaks seen at the analyte retention times. CONCLUSIONS

We have successfully developed a solid phase extraction method for extracting GTXs 1,2,3,4 from human plasma. This method has all the validation parameters within the limits specified by FDA. This is a fast and simple diagnostic method which is easily accessible to common clinical laboratories and can be applied to clinical specimens.




Development and Evaluation of a Gel-based System for Transdermal Delivery of Heparin via Laser-Generated Micropores


Deepal Vora Mercer University BACKGROUND Heparin is an anticoagulant, commonly administered through intravenous route to treat venous thromboembolism, a condition where inflammation and clotting are observed inside a vein below skin surface. Our study investigates the feasibility of transdermal delivery of heparin, a hydrophilic macromolecule, through laser-microporated skin as an alternative to intravenous administration. METHODS

Microporation was performed using P.L.E.A.S.E.® (Precise Laser Epidermal System; Pantec BioSolutions AG, Liechtenstein) technology and characterized by staining, histology, and confocal microscopy. A poloxamer gel (20% w/v; Kolliphor P407 from BASF) was prepared and loaded with 0.3% w/v heparin. Heparin-loaded gel was then characterized for rheological properties. In vitro permeation on Franz diffusion cells was performed to determine the rate and extent of delivery into and across laser-treated (fluence of 22.7 J/cm2) porcine ear skin. Delivery from poloxamer gel was compared to a 0.3% w/v solution of heparin. Additionally, passive diffusion across untreated porcine ear skin was evaluated. The receptor compartment contained 5 mL of 10 mM phosphate buffered saline (1X PBS; pH 7.4). Samples were collected from the receptor at predetermined time points over 24 h and analyzed by Chromagenix® Coatest Heparin. Skin was extracted using 1X PBS to determine the amount delivered into skin.

RESULTS Characterizations of laser-created micropores by staining, histology and confocal microscopy confirmed the formation of microchannels in the skin. The transition property of poloxamer from solution at room temperature to gel at skin temperature (32°C) was utilized in this study. Amount of heparin delivered for laser treated skin (26.07 ± 1.82 µg/sq.cm) was significantly higher as compared to passive permeation (0 µg/sq.cm). A lower but more sustained delivery was observed for heparin loaded gel (11.28 ± 5.32 µg/sq.cm) as compared to heparin solution (26.07 ± 1.82µg/sq.cm) in the laser-treated group. CONCLUSIONS

A sustained delivery of heparin into and across the skin was demonstrated with gel-based formulation, indicating the feasibility of transdermal delivery of macromolecules using laser treatment.




A First-in-Class Skp1-FBP Inhibitor for the Treatment of Bone Metastatic Castration-Resistant Prostate Cancer


Daqing Wu Clark Atlanta University BACKGROUND Bone metastasis directly contributes to the morbidity and mortality of prostate cancer, but current treatment modalities only temporarily control disease progression. Clearly, it is an urgent and unmet medical need to identify new therapeutic targets and develop novel targeting strategies against this lethal disease. Recent studies have demonstrated a major role of S phase kinase-associated protein 1 (Skp1)-Cullin1-F-Box protein (FBP) (SCF) ubiquitin ligase complexes in human cancer progression. Skp1 stabilizes FBPs (such as Skp2) and enhances their substrate identification, thereby is pivotal in the formation and function of SCF complexes. Compared with FBP-targeted strategies, blocking the interaction between Skp1 and FBPs may provide a general and effective approach to inhibit multiple SCF oncogenic signals and maximize therapeutic efficacy against tumors with highly heterogeneous backgrounds. Therefore, Skp1-FBP interaction represents a promising therapeutic target in metastatic castration-resistant prostate cancer (mCRPC). However, no synthetic Skp1 inhibitor has been developed yet. METHODS

Supported by a Small Business Technology Transfer (STTR) grant from the National Cancer Institute (NCI), we developed GH501, a first-in-class synthetic inhibitor of Skp1-FBP protein-protein interaction. We evaluated the in vitro anticancer activity of GH501 in collaboration with the NCI Developmental Therapeutics program. We investigated the mechanism of action of GH501 in mCRPC cells using molecular and cellular approaches. We evaluated the in vivo toxicity, pharmacokinetic and efficacy against mCRPC in clinically relevant models.

RESULTS GH501 effectively disrupts the protein-protein interaction between Skp1 and FBPs, thereby affecting multiple prominent drivers of mCRPC progression, including Skp2, p21, p27, beta-catenin, E2F1, EZH2, c-Myc, cyclin D1, RUNX2 and survivin. GH501 exhibits potent cytotoxicity against mCRPC cells and a broad spectrum of human cancer cell lines at nanomolar concentrations. Animal studies demonstrated that GH501 is well tolerated in mice and has favorable physicochemical properties as a lead compound. Uniquely, GH501 is preferably distributed to and deposited in bone tissues, making it an ideal and attractive drug candidate for treating bone cancers. Significantly, in vivo efficacy studies demonstrated that GH501 effectively suppresses the skeletal and subcutaneous growth of mCRPC in cell- and patient-derived xenograft models. CONCLUSIONS

Our preclinical studies support the promise of GH501 as a lead compound against bone metastatic prostate cancer. We envision that GH501 is a novel targeted therapy to improve the survival and quality of life of patients with bone metastasis.




Microporation Assisted Topical Delivery of Kojic Acid for the Treatment of Hyperpigmentation


Amruta Dandekar

Center for Drug Delivery Research, Department of Pharmaceutical Sciences, College of Pharmacy, Mercer University

BACKGROUND Hyperpigmentation is a common skin disorder prevalent worldwide. It is caused due to excessive melanin production regulated by the enzyme tyrosinase. Kojic acid is an anti-tyrosinase agent used for the treatment of hyperpigmentation. Due to its hydrophilic nature (logP=-0.5) topical delivery of this molecule is a challenge. Our study demonstrates the use of microporation to enhance the delivery of kojic acid via skin. METHODS

In vitro drug permeation studies were performed on dermatomed porcine ear skin using vertical Franz diffusion cells. Microporation of skin using dissolving maltose microneedles (500 µm; 81 needles) and Dr. PenTM Ultima A6 (5mm needle-length; 13,000 insertions per minute; 5s pre-treatment) was compared to passive diffusion (1% w/v solution). Skin resistance was measured using an arbitrary waveform generator (Agilent 33220A, 20 MHz Function), a 34410A 6 ½ digital multimeter (Agilent Technologies, Santa Clara, CA, USA) and silver/silver chloride electrodes to ensure skin integrity. Samples were collected at predetermined time points from the receptor and amount of drug delivered into skin after 8h and 24h was analysed. Samples were quantified using validated RP-HPLC method and statistical difference was concluded using one way ANOVA with Tukey's post-HOC analysis (p< 0.05).

RESULTS Successful microporation of skin was observed, concluded by reduced skin electrical resistance with both maltose microneedles (74.39±9.21%) as well as Dr. PenTM Ultima A6 (64.86±22.55%). Pre-treatment with maltose microneedles resulted in significantly higher delivery across skin into the receptor compartment (88.22±23.06µg/sq.cm) with a reduced lag time (0.08±0.02h) in comparison to passive diffusion (5.99±1.26µg/sq.cm; 2.11 ± 0.07h) within 4 hours. A reduction in lag time with Dr. PenTM Ultima A6 (0.11±0.03h) was also observed with significantly higher delivery across skin after 8 hours (77.32±5.94 µg/sq.cm). Microporation of skin did not result in significantly higher delivery into skin after 8 hours as compared to passive diffusion. (2.76±0.34% with maltose microneedle; 1.71±0.29% with Dr. PenTM Ultima A6 pre-treatment; 2.25±0.71% with passive diffusion). There was no significant difference observed in skin delivery of kojic acid at the end of 8 hours (35.84±4.44µg/sq.cm) as compared to 24 hours (42.70±9.38µg/sq.cm) in the maltose microneedle pre-treatment group. However, significantly higher amount of kojic acid was delivered into skin at the end of 24 hours (51.85±5.22µg/sq.cm) as compared to 8 hours (24.53±7.125µg/sq.cm) in the case of passive diffusion. CONCLUSIONS

Delivery of kojic acid across skin was enhanced after microporation of skin using dissolving maltose microneedle and Dr. PenTM Ultima A6. Also, lag time for delivery into skin was reduced following pre-treatment with microneedles.





 

Food and Nutrition

Does the FDA enforce regulations at virtual and fully integrated pharma and biotech companies the same?


Cassie Alexander University of Georgia BACKGROUND Over the past several decades, virtual companies have become prevalent within the pharmaceutical and biotech industries. Virtual companies utilize a business model where they directly hire employees in supportive roles while outsourcing critical business activities that are highly regulated by the United States Food and Drug Administration (U.S. FDA). Since the corporate structures for virtual and fully integrated companies differ significantly, it is important to understand how virtual companies are regulated by the FDA so management teams can understand how to appropriately staff organizations to mitigate potential compliance risks while maintaining a lean organization. METHODS The study had three areas of focus: public information (websites and databases), articles and personal interviews. For the public information analysis, a randomized sample of 40 companies was created, including virtual and fully integrated pharmaceutical and biotech companies, analytical laboratories and third-party logistics providers. Data was gathered for the 40 companies from websites and databases like Registration and Listing, Inspections, Inspection Citations, Warning Letters and the Orange Book. Data was tabulated to provide a qualitative and quantitative analysis of the trends and frequency of occurrence. For the second area of focus, five online articles/blogs were analyzed to gather common trends in compliance activities relating to virtual companies. Finally, five people with experience with compliance activities were surveyed through a questionnaire to assess personal experiences and perspectives on how the FDA regulates virtual and fully integrated companies. RESULTS The findings from this study demonstrate that the FDA enforces regulations at virtual and fully integrated companies differently. Analysis of data from the companies show that fully integrated pharmaceutical and biotech companies experienced more FDA inspections, inspection citations and escalated regulatory enforcement actions. Analysis of industry the articles pointed to the importance of virtual companies building a strong relationship with contract facilities through robust Quality Agreements and close oversight. Feedback from questionnaires of industry professionals showed a concern that virtual companies have less rigorous quality systems and inadequate oversight of contract facilities, which would lead to FDA enforcement actions. CONCLUSIONS While subject to the same U.S. Regulations, this study demonstrates that virtual companies experience less regulatory oversight and enforcement actions than fully integrated companies. It is likely the contract partners are inspected for compliance and it is incumbent upon the management teams of virtual companies to ensure they have adequate processes and resources in place to appropriately oversee contract partners and mitigate risks. This will reduce the risk of FDA enforcement actions on virtual companies while ensuring safe and effective products are provided to patients.





 

Digital Health

Does the FDA enforce regulations at virtual and fully integrated pharma and biotech companies the same?


Cassie Alexander University of Georgia BACKGROUND Over the past several decades, virtual companies have become prevalent within the pharmaceutical and biotech industries. Virtual companies utilize a business model where they directly hire employees in supportive roles while outsourcing critical business activities that are highly regulated by the United States Food and Drug Administration (U.S. FDA). Since the corporate structures for virtual and fully integrated companies differ significantly, it is important to understand how virtual companies are regulated by the FDA so management teams can understand how to appropriately staff organizations to mitigate potential compliance risks while maintaining a lean organization. METHODS The study had three areas of focus: public information (websites and databases), articles and personal interviews. For the public information analysis, a randomized sample of 40 companies was created, including virtual and fully integrated pharmaceutical and biotech companies, analytical laboratories and third-party logistics providers. Data was gathered for the 40 companies from websites and databases like Registration and Listing, Inspections, Inspection Citations, Warning Letters and the Orange Book. Data was tabulated to provide a qualitative and quantitative analysis of the trends and frequency of occurrence. For the second area of focus, five online articles/blogs were analyzed to gather common trends in compliance activities relating to virtual companies. Finally, five people with experience with compliance activities were surveyed through a questionnaire to assess personal experiences and perspectives on how the FDA regulates virtual and fully integrated companies. RESULTS The findings from this study demonstrate that the FDA enforces regulations at virtual and fully integrated companies differently. Analysis of data from the companies show that fully integrated pharmaceutical and biotech companies experienced more FDA inspections, inspection citations and escalated regulatory enforcement actions. Analysis of industry the articles pointed to the importance of virtual companies building a strong relationship with contract facilities through robust Quality Agreements and close oversight. Feedback from questionnaires of industry professionals showed a concern that virtual companies have less rigorous quality systems and inadequate oversight of contract facilities, which would lead to FDA enforcement actions. CONCLUSIONS While subject to the same U.S. Regulations, this study demonstrates that virtual companies experience less regulatory oversight and enforcement actions than fully integrated companies. It is likely the contract partners are inspected for compliance and it is incumbent upon the management teams of virtual companies to ensure they have adequate processes and resources in place to appropriately oversee contract partners and mitigate risks. This will reduce the risk of FDA enforcement actions on virtual companies while ensuring safe and effective products are provided to patients.





 

Medical Technology and Devices

Additive Manufacturing to Create Antimicrobial Nitric Oxide Releasing Surfaces


Manjyot Kaur Chug University of Georgia BACKGROUND Each year 99,000 deaths are reported due to hospital-acquired infections. A great extent of nosocomial infections in the patients' source from medical devices that feature a suitable surface to promote biofilm formation. One approach to addressing these infections is to create bioactive devices with nitric oxide-releasing (NO) properties. Nitric oxide has been extensively utilized for treating medical device infections due to its inherent antimicrobial and anti-inflammatory properties. Polymers releasing NO at endogenous rates (ca. 0.5-4x10-10 mol cm-2 min-1) possess similar antimicrobial properties. To overcome the design of geometrically complex devices, additive manufacturing technology was utilized to print custom designed films (porous and solid) using polycarbonate-based silicone elastomer (ChronoSil). The films were impregnated with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) using a solvent-swelling process to generate antibacterial interface. Based up on the modulation of structure via distinct porosities, the NO-releasing films were studied for real-time NO release, SNAP leaching and impregnation, and antibacterial properties, that are crucial to enable clinical translation. METHODS

Polycarbonate-based silicone elastomer (PCU-Sil) films were fabricated with different porosities (60% porous, 40% porous, solid, and capped) using 3D printer. Films were soaked in SNAP (100 mg/mL) dissolved in 40% methyl ethyl ketone and 60% methanol for 2 h and dried under ambient conditions for 24 h. The NO-releasing PCU-Sil were incubated in PBS-EDTA at 37°C to determine the real-time NO release under physiological conditions using Nitric Oxide Analyzer 280i (Frederick, CO). The SNAP impregnation and leaching studies for the 3D printed films were conducted using UV-Vis spectroscopy. SEM and EDS mapping were used to examine the morphology and elemental composition of the films before and after SNAP impregnation. Finally, NO releasing films were assessed for antibacterial ability using 24 h bacterial adhesion against Staphylococcus aureus strain of bacteria frequently found in medical device related infections.

RESULTS The UV-vis studies demonstrated highest amount of SNAP in porous samples (60% and 40%) followed by solid and capped. All the samples exhibited higher levels of NO release in the initial days. While the porous samples demonstrated continued NO release at relatively higher levels up to 14 days, solid and capped samples had diminished flux levels after day 5. NO releasing films exhibited > 99% suppression of S. aureus adhesion on the film surface (p < 0.05) after 24 h. CONCLUSIONS The SNAP impregnated 3D-printed PCU-Sil films provide > 7 d of NO release at physiologically relevant levels. This study highlights the importance of additive manufacturing in biotherapeutics and paves a new path for developing patient specific antimicrobial medical device with customized NO-releasing capability. The closely regulated personalized bioactive polymers can help overcome the design of geometrically complex devices, the potential toxicity to the patient from the biofilm colonization, and emergence of antimicrobial resistance.




Characterization of the Combined Therapy of a Nitric Oxide (NO) Donor Molecule and Cerium Oxide Nanoparticles for Antimicrobial Applications


Lori Estes

School of Chemical, Materials, and Biomedical Engineering, College of Engineering, University of Georgia

BACKGROUND Broad-spectrum antimicrobials are needed towards mitigating the issues of antibiotic-resistant infections. It is highly important to formulate new antimicrobials by combining agents with different mechanistic and broader microbial targets. A combined antimicrobial solution could be a highly critical step towards developing the strategy to prevent polymicrobial infection. Herein, we have investigated the interaction and antimicrobial potential of a solution that contains cerium oxide nanoparticles (CNP) and a nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine (SNAP). It is hypothesized that these two agents induce synergistic effects and would provide broad antimicrobial effects since CNP is known to be an effective antifungal agent while NO released by SNAP is known to be a potent bactericidal agent. METHODS

Different concentrations of SNAP and CNP were combined in a solution and tested for colloidal stability, NO release, mammalian cell cytotoxicity, and antimicrobial efficacy against Staphylococcus aureus, Escherichia coli, and Candida albicans, accounting for Gram-positive, Gram-negative, and fungi, respectively.

RESULTS

The combination of SNAP and CNP led to decreased colloidal stability but increased biocompatibility compared to CNP and SNAP alone, respectively. The scavenging capabilities of CNP allowed for a prolonged NO release from the combination solution. SNAP and CNP combined in equimolar solution of 3 mM were found to be highly effective for all microbes tested compared to higher amounts of the treatments required individually.

CONCLUSIONS These results hold a promising outlook toward the development of broad-spectrum antimicrobial coatings and films with the potential to prevent polymicrobial infections and further enhance biomedical device usage and applications.




A Recipe for Academic Labs to Produce SARS-CoV-2 RT-qPCR Test Kits


Sara Fakhretaha-Aval

Georgia Institute of Technology

BACKGROUND The global COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 30 million people, claimed many lives, and substantially disrupted activities in the public and private sectors. Widespread testing for the presence of the novel coronavirus SARS-CoV-2 in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise and/or instrumentation necessary to detect the virus by quantitative reverse transcription polymerase chain reaction (RT-qPCR), the most robust, sensitive, and specific assay currently available. METHODS

Here we describe our in-house Master Mix and RT-qPCR assay for detection of SARS-CoV-2. We discuss preparation of singleplex and multiplex primers and probes with CDC sequences that can be used with commercial enzyme Master Mixes. In addition, we present the production of reverse transcriptase (RT), Taq DNA polymerase, and ribonuclease inhibitor (RI) proteins, and the formulation of a working 1-step enzyme Georgia Tech Master Mix (GT-Master Mix) for use with our primers and probes.

RESULTS

We formulated a functional SARSCoV-2 assay that compares favorably to commercially available RT-qPCR kits. Our assay comprises a Master Mix as well as primers and probes identical to validated CDC sequences. GT-Master Mix is composed of affinity-purified GT-rRI, GT-His-Taq, and GT-MMLV reverse transcriptase, a compatible buffer containing cationic cofactors, plus BSA and trehalose for stability and long-term storage. In GT- Master Mix, the efficiencies of our GT primer and probe sequences met (multiplex) or closely approached (singleplex) our efficiency target of 90- 110% with high linearity (r 2 >0.990), indicating minimal primer dimers or non-specific amplification. The efficiency and linearity of our multiplex kit over a five-log concentration range is competitive with other kits that have received EUA for SARS-CoV-2 testing. In sum, our GT RT-qPCR assay, composed of proteins and enzymes produced in house, with either singleplex or multiplexed primers and probes kit exhibits a high level of qPCR efficiency and storage stability.

CONCLUSIONS The goal of our project was to create contingency SARSCoV-2 diagnostic test components in the face of supply line insecurity. We translated published information about RT-qPCR and sophisticated commercial kits into a series of fundamental protocols, executable with consumables and equipment routinely used in academic biochemistry laboratories. Our published blueprint should be readily reproducible by research teams at other academic institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource limited settings. In the long term, our protocols should be adaptable to the detection of other novel or seasonal infectious viral agents.




Rapid Susceptibility Testing from Positive Blood Cultures


Alexandra Filbrun

Georgia Institute of Technology

BACKGROUND In large part due to the rapid development of antimicrobial resistance, bacterial bloodstream infections (BSIs) remain a major cause of mortality and morbidity despite the wide availability of antibiotics. With exceedingly low bacterial burdens of 1-100 colony-forming-units per mL blood, conventional diagnosis relies on lengthy blood culturing and purification steps prior to already slow identification and antibiotic susceptibility testing (AST). This >60-hour time-to-result imposed on actionable treatment determinations negatively impacts patient outcomes and increases proliferation of antimicrobial resistance through the misuse and overuse of broad-spectrum antibiotics. Consequently, the development of novel technologies capable of rapidly recovering bacteria originally present in high blood backgrounds is increasingly important. METHODS

To address this need, we developed a novel bacterial separation technology from positive blood cultures that couples selective lysis with direct, centrifugation-based bacterial recovery.

RESULTS

Evaluated against the most common BSI-causing bacteria pathogens: Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, near-pure bacteria can be recovered in <15 minutes with minimal sample handling. ASTs are readily performed with excellent correspondence to much slower ASTs performed with VITEK-2 Instrumentation.

CONCLUSIONS Overall, this direct from positive blood culture AST enables susceptibility determinations to be assessed in 3-6 hours from blood culture positivity, with minimal sample handling and processing.




Functional Outcome and Self-reported Outcome Comparison Between Osseointegrated and Socket Prosthesis for Lower Extremity Amputees: A Meta Analysis


Jacob Lonowski

Georgia State University Department of Physical Therapy

BACKGROUND

Osseointegrated (OI) prostheses are becoming a more attractive option for patients with an amputation, especially for those who have had chronic skin issues and recurrent infections with socket prosthetic use. Despite the mounting number of publications which support OI prostheses, a meta-analysis of related outcomes has not yet been conducted. Thus, this study's aim is to provide a meta-analysis of self-reported and functional outcome comparisons between OI prostheses and traditional socket prostheses in lower extremity (LE) amputees. The selected functional outcomes for comparison, the 6 Minute Walk Test (6MWT) and Timed Up and Go (TUG), are staples for assessing the functionality of an individual and are valid and reliable measurements. The self-reported outcomes, the Questionnaire for Persons with a Transfemoral Amputation (QTFA) and the Short Form 36 (SF-36), are both valid and reliable measures for the assessment of this population. Both the QTFA and the SF-36 are questionnaires which assess the status of an individual across a spectrum of physical and mental qualities.

METHODS

Six databases were searched including Cochrane, Pubmed, Medline, EMBASE, CINAHL, and Web of Science. Literature search was independently conducted by two reviewers. 1434 studies met the search criteria, 277 studies screened after the removal of duplicates, 23 full text articles were reviewed, and 15 studies were eligible for final inclusion. Two reviewers assessed study quality and extracted data independently. Analysis of the extracted data was performed using 'Comprehensive Meta-Analysis.'

RESULTS

The 15 included studies compared outcomes of 6MWT, TUG, QTFA, and SF-36 between OI prostheses and socket prostheses. The analysis favored OI prostheses with large effect sizes of statistical significance for all noted outcomes; 6MWT (ES=2.576, P<0.001), TUG (ES=2.156, P<0.001), QTFA (ES=2.981, P<0.001), and SF-36 (ES=2.068, P<0.001) in LE amputees.

CONCLUSIONS OI prostheses were found to produce superior functional outcomes in both 6MWT and TUG as well as superior self-reported metrics in both QTFA and SF-36 scores in LE amputees.




A Review of Fall Detection and Fall Prediction Systems


Lorna Migiro

Kennesaw State University

BACKGROUND

Falls threaten seniors' safety, their independence and generate enormous economic and personal costs. Methodologies for anticipating falls are essential as the benefit of prevention far outweigh those of rehabilitation. There is a current surge of smart sensors and Internet of Things being incorporated in critical fall detection and prediction safety applications. However, in recent years, there is a lack of survey reviewing and discussing on these novel sensors, technologies and algorithms introduced and employed as well as the emerging challenges and new opportunities. This paper is a survey of the state of the art of technology-based fall detection and prediction systems, which are identified from a systematic review of studies presented in contemporary research literature. It aims to serve as a point of reference for future research on the mentioned systems

METHODS

The review of the current literature was performed to explore sources of literature from ACM, IEEE digital library, PubMed, web of science and science direct. We have performed searches using the keyword "fall detection", "fall prevention", "smart fall detection", "smart fall prevention". We limit the articles between year 2010 and 2020. We gave priority to papers whose intention was broad characterization of the tech - based fall detection and prediction systems

RESULTS

This review identified 41 projects that used wearable systems and 23 projects using non-wearable systems. Context aware and vision-based systems were found to be less popular with the elderly as they lack privacy. 90 % of approaches reviewed leverage on assimilating the smartphone sensor data in generating fall prediction machine learning algorithms. Multi sensor approaches that combine acceleration magnitude, sensor velocity, and body posture give the highest fall sensitivity and the lowest false positive rate

CONCLUSIONS Fall detection and prediction systems whether physiology or kinematics both rely on sensors and fall factors. With smart sensors and Internet of Things (IoT) developing rapidly, this field has made great progress. Whereas fall prediction systems are typically developed to estimate real-time or future fall risk, real time emulations among the elderly is difficult to obtain and all studies reviewed use young adults whose representation still remains controversial, thus poses challenge on how to put this studies into practice




A Portable Optoelectronic Device for Lung Disease Diagnosis Through an Exhaled Breath


Sanjay Sarma Oruganti Venkata

University of Georgia

BACKGROUND

Diagnosis of lung diseases in both adults and children begins with the physical observation of the patient's respiratory efforts and comparing them to the respiratory rate thresholds set forth by the WHO. Beyond breath rate identification, other diagnostic measures, including auscultation by stethoscope, cultures, blood serum examination, complete blood cell count, and chest radiography (x-rays). Regardless of the identification through these methods, they lack the capability of quickly and accurately identifying the infection-causing pathogen-a critical aspect to consider when deciding treatment course as there are a wide variety of treatments available.

METHODS

Considering the shortcomings in the current diagnostic procedures, we developed a highly portable online device for detecting the particle densities and pathogen composition in a breath. Our invention under progress is a highly portable optoelectronic device that detects a breath's optical scatter characteristics passed/ blown through it. To assimilate the breathing in humans, we conducted preliminary experiments by blowing aerosol particles into the device for detecting the particle density and flow characteristics.

CONCLUSIONS We further plan to conduct experiments with various bacteria types suspended in the aerosols similar to an exhaled breath. In the current poster, we introduce the device and the results obtained from our preliminary experiments.




Characterization of E-Cigarette Aerosol Deposition in the Human Lung Airways With a Comparison to Conventional Tobacco Cigarettes


Sara Spalding

School of Engineering, Mercer University

BACKGROUND

In recent years, electronic cigarettes (e-cigarettes) have been under scrutiny due to their adverse health effects, although they had been advertised as a healthier option for current smokers. The effects of e-cigarette flavors and various flow rates on aerosol particle concentrations were evaluated. Analysis of inhaled aerosol particles in human airways using analytical lung morphometry software was performed. Additionally, a comparison of aerosol characteristics of the e-cigarette and the conventional tobacco cigarette was performed.

METHODS

Aerosol characteristics from e-cigarettes and tobacco cigarettes were measured using a Wide Range Particle Spectrometer (WPS, 1 LPM), which can measure the concentration of aerosols in the range of 10 nm to 10 microns. The Multiple-Path Particle Dosimetry Model (MPPD) was used to calculate the amount of aerosol deposited in the lung airways.

RESULTS

The maximum particle number concentrations in ambient air, filtered air, and e-cigarette aerosols during the experiment were 57, 7, and 113,428 number of particles/cc, respectively. The influence of ambient and filtered air is negligible for particle concentration measurements. It was found that there is no statistical difference in the particle concentrations of different flavors and flow rates. The experimental data used in the MPPD analysis was that of the 5% nicotine, 1.7 LPM, mint e-cigarette. Within the MPPD analysis, we compared the Stochastic Model, Stochastic 25 bpm (lung disease), Stochastic 6 bpm (breathing impairment), Yeh Schum 5 Lobe, Age-Specific 14 years old, and Age-Specific 21 years old. In the pulmonary region Age 21 and Stochastic 6 bpm had higher particle deposition, Stochastic 25 bpm had lower particle deposition. In the whole lung, Age 21 and Stochastic 6 bpm models had higher particle deposition, Age 14 had lower particle deposition. It was found that 13.04% of the cigarette smoke was deposited in the lungs, of that amount 62.89% deposited in the pulmonary region. In comparison, 6.97% of E-cigarette smoke deposited in the lungs, of that amount 44.28% deposited in the pulmonary region.

CONCLUSIONS When comparing the different MPPD models, the amount of particles deposited by the e-cigarette aerosol is dependent on the age and breathing ability of the user. Young adults had higher deposition in the whole lung and the pulmonary region. In reference to the e-cigarette aerosol, the highest amount of particles were deposited in the pulmonary airways of the lungs where O2-CO2 exchange occurs. This potentially causes inflammation of the pulmonary airways, which would hinder breathing. When comparing conventional cigarette smoke to e-cigarette aerosol, the conventional cigarette smoke deposits more particles in the lungs than e-cigarettes.




Assessing Improvements in the Quality of Life of Parents of Children with Down Syndrome After Using MapHabit's Visual Mapping Software


Kaylin White

MapHabit Inc.

BACKGROUND

There is a growing body of evidence that assistive digital technology may improve the Quality of Life (QoL) of individuals with intellectual disabilities. The MapHabit System (MHS) is an example of such a technology for it provides visual-mapping, step-by-step cues containing photos and audio, to assist users in completing tasks. The MHS has previously been used amongst memory-impaired individuals and was shown to facilitate the accomplishment of their activities of daily living (ADLs) more independently and improve the overall quality of life. Given the MHS promotes independence, we decided to explore the potential benefits of the MHS for both individuals with developmental disability and their caregivers.

METHODS

To address this possibility, a focus group of 6 parents was provided the MHS's visual-mapping software to implement into the daily routines of their children with Down syndrome for a 4-week period. The children of the parents within this focus group were between the ages of 5 and 17 years old. Throughout the trial period, participants were interviewed on a weekly basis to assess progress with the MapHabit app and to assist with map building and customization. At the end of the 4-week period, the parents completed an 18 item QoL assessment and an assessment of user satisfaction.

RESULTS

Each of the parents included in the trial reported some positive change or much positive change in one or more of the in the following areas: Completed activities of daily living more quickly, increased independence, increased social interaction, decreased frustration, increased social engagement, and/or overall improved QoL. The net promotor scores, indicative of participant satisfaction, obtained showed that parents found using visual maps with their children was satisfying and that they would recommend the MHS to a friend or colleague.

CONCLUSIONS The positive findings reported in this proof-of-concept study is the first instance of the MHS being utilized by individuals with developmental disability. These initial findings demonstrate that the MHS addresses the need for a personalized low-risk, low-cost, readily available, and portable assistive technology tool that assists with independence amongst families with children with Down syndrome. Furthermore, the MHS may be applicable to a broader range of neurodiverse individuals across the lifespan.





 

Molecular and Biological Research

Heterologous Prime Boost Influenza Vaccination Provides a Strategy to Induce More Effective Cross Protection Than Repeat Vaccination


Noopur Bhatnagar Institute for Biomedical Sciences, Georgia State University BACKGROUND Recent clinical studies have reported that repeat annual vaccination diminishes vaccine efficacy. Therefore, it is critical to better understand the impact of prior vaccine immunity and to develop an effective vaccination strategy. METHODS In this study, we designed an experimental strategy to determine the impact of heterologous prior vaccine immunity on inducing cross protection in comparison with mimicking homo seasonal repeat vaccination. Groups of mice were intramuscularly primed with a different strain of an inactivated influenza virus and then boosted with homo or heterologous inactivated virus (H1N1, H3N2, H5N1, H7N9, H9N2). RESULTS A wide range of strain specific hemagglutination inhibition (HAI) titers was induced in primed immune sera, depending on the vaccine strain used. When the primed mice were intramuscularly boosted with a homo or heterologous subtype of inactivated virus vaccine, HAI titers increased up to 8 folds. Strain specific HAI titers against prime and boost vaccine strains were induced. Cross-reactive HAI titers against a virus not included in prime and boost as well as enhanced cross protection were induced in certain heterologous prime boost influenza vaccinations. CONCLUSIONS The results in this study suggest that heterologous vaccine-induced prior immunity promotes the induction of homo and cross-reactive HAI titers against prior as well as new hetero virus vaccine strains compared to repeat homologous vaccination. These results support a promising strategy of heterologous prime boost influenza vaccination.




Assessing the role of dopamine in the differential neurotoxicity patterns of stimulants


Neha Chitre Mercer University, College of Pharmacy BACKGROUND Drug-induced Parkinsonism (DIP) is the second most common etiology of Parkinsonism after Parkinson's disease (PD). Stimulants like amphetamine (AMPH) that are known to cause long term dysregulation of the brain dopaminergic system are top contributors to DIP. Methamphetamine, a very commonly abused AMPH is an addictive psychostimulant that has very strong neurotoxic effects on the central nervous system (CNS), and specifically the brain dopaminergic system. 3,4-methylenedioxymethamphetamine (MDMA) recently achieved breakthrough status from the Food and Drug Administration (FDA) for post-traumatic stress disorder (PTSD). However, evidence indicates that exposure to toxic doses of MDMA can lead to long-lasting dysregulation of brain monoaminergic neurotransmitters, primarily from studies conducted in young adult rodents. Additionally, the abuse of synthetic cathinones, which are the β-ketone analogs of AMPH has also been on the rise. Despite their structural similarity to AMPH, not many studies have evaluated their neurotoxic potential and the detrimental effects they could be having on the dopaminergic system. Thus, the aim of the current study is to evaluate the dopaminergic neurotoxicity exhibited by stimulants to study their potential to cause DIP using prototypical AMPH, methcathinones and pyrrolidines METHODS

Physiological effects of stimulants on lethality, body weight and body temperature were assessed using binge-models of dosing in Swiss- Webster mice. Behavioral assays such as Open-Field Testing and stride length evaluation were conducted for motor deficits. Cognitive impairments were assessed by Passive Avoidance testing. Neurochemical analysis of dopamine levels and its major metabolites DOPAC and HVA in the striatum was performed using ultra high performance liquid chromatography (UHPLC-ED).

RESULTS Study results indicate that several key analogs such as methcathinone, 2-fluoromethcathinone, 3-methylmethcathinone, and Alpha-PPP cause behavioral deficits such as decreased locomotor activity and impaired cognitive performance in passive avoidance testing. Additionally, binge-dosing of these stimulants in mice causes significant dopamine depletion in the striatum, which are characteristic hallmarks of Parkinsonism. CONCLUSIONS Taken together, these results suggest that some prototypical AMPH, methcathinones and pyrrolidines may have the potential to cause DIP, warranting further studies and investigation.




Needle-free Measles Immunization using Microparticulate Formulation


Devyani Joshi Mercer University College of Pharmacy BACKGROUND Measles, a highly contagious infectious disease, is a major cause of death among children globally; despite the availability of the vaccine. It is particularly common in developing countries, like parts of Africa and Asia. More than 140,000 people died of measles in 2018. With the current vaccination regimen, two doses of measles vaccine are prescribed: first, before the age of 12 months and a booster dose between 5 to 10 years of age. Since children are the primary targets for the vaccine, we aimed at delivering the vaccine via a needle-free transdermal route. We compared the efficacy of vaccine administered transdermally to that of the traditional subcutaneous administration. We propose a microparticulate formulation of the vaccine. The traditional soluble vaccine is taken up via antigen-presenting cells (APC) and the antigenic epitopes are presented via major histocompatibility complex MHC II. The vaccine thus effectively stimulates the humoral immune response but fails to activate the cellular immune response. The particulate vaccine is taken up via APCs and epitopes are presented via both, MHC I and MHC II complexes; stimulating both, humoral and cellular adaptive immune response. Particulate formulation of vaccine thus increases the immunogenicity of an antigen. METHODS

The measles antigen was incorporated into the biodegradable, crosslinked-albumin matrix and spray dried using Buchi mini spray dryer B-290 to formulate the vaccine loaded microparticles. The microparticles were characterized for size, charge, and polydispersity index (PDI). The surface morphology of microparticles was visualized by Scanning Electron Microscopy. The induction of an immune response by the microparticulate vaccine was confirmed via spectroscopic Griess's assay. The expression of antigen-presenting molecules, MHC I and MHC II, and their co-stimulatory molecules CD80 and CD40 was assessed on the surface of dendritic cells using BD Accuri C6 plus flow cytometer. The equivalent amount of blank microparticles (without antigen and adjuvant) was used as control. The cytotoxicity of microparticles was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. The uptake of microparticles by APCs was studied as a function of time. The in vivo efficacy of the microparticulate vaccine was studied in the mouse model. For the transdermal administration of the microparticulate vaccine, P.L.E.A.S.E. ablative laser was used. This laser creates the micropores of defined size on the surface of the skin, allowing transdermal delivery of vaccine. Seven groups of animals (n=6) were used in the study: naïve group as a control, the second group received measles vaccine solution subcutaneously, the third group received blank microparticles subcutaneously, the fourth group received vaccine microparticles subcutaneously, the fifth group received vaccine and adjuvant (alum and MPL A®) microparticles subcutaneously, the sixth group received vaccine microparticles transdermally via ablative laser and the last group received vaccine and adjuvant (alum and MPL A®) microparticles transdermally via ablative laser. The animals were administered with one prime and one booster dose at weeks 0 and 2. The serum was collected from the animals bi-weekly and IgG and IgM antibody titers were measured using ELISA.

RESULTS The yield of the microparticulate vaccine formulation was 91.37±3.1%. The size of the microparticles was 3.197±0.8361 um, PDI was 0.447 with a charge of -29.4±6.2 mV. The microparticulate vaccine with adjuvants showed significantly higher release of nitric oxide (p<0.05), a hallmark of an innate immune response, compared to the blank microparticles. It resulted in a significantly higher (p<0.05) cell-surface expression of MHC I and MHC II and their co-stimulatory molecules CD80 and CD40 respectively on the surface of dendritic cells. MTT assay showed that the vaccine and adjuvant microparticles were non-cytotoxic compared to dimethyl sulfoxide (DMSO) which is cytotoxic. The uptake of microparticles by APCs increased with time and was maximum at 4 h. The in vivo studies demonstrated elevated humoral immune responses in the mice receiving vaccine and adjuvant microparticles via both, subcutaneous and transdermal routes; as seen with the serum titers of IgG and IgM antibodies (p<0.001). There was no significant difference between the antibody titers in the mice that received the vaccine and adjuvant microparticles subcutaneously and transdermally, indicating that the transdermal route is as efficacious as the subcutaneous route for vaccination. CONCLUSIONS The microparticulate formulation augments the immunogenic properties of the soluble vaccine as seen from better induction of innate immune response. The use of adjuvants potentiates the immune response to the antigen. Higher expression of antigen-presenting molecule, MHC I, and its co-stimulatory molecule CD80 indicates the induction of cytotoxic response by the microparticulate vaccine, required for overcoming acute measles infection. Higher expression of MHC II and its co-stimulatory molecule CD40 indicates the induction of humoral immune response, critical in controlling viral replication and conferring protection. Transdermal administration of the particulate vaccine using a laser epidermal system produced comparable results as that of the subcutaneous administration indicating the potential of the transdermal route for vaccination.




Assessing Adjuvant-effect of Microparticulate DPD and its Analogs


Devyani Joshi Mercer University College of Pharmacy BACKGROUND Autoinducer (AI)-2 is a signaling molecule involved in bacterial quorum sensing (QS): a form of interspecies communication in bacteria. The reduction of QS is a potential therapeutic approach to fight against bacterial diseases. However, targeting of the AI-2 based QS remains challenging because of the rapid interconversion of the AI-2 precursor (S)-DPD (4,5-dihydroxypentane-2,3-dione) to several linear and cyclic forms recognized by different bacteria. (S)-DPD can thus be used as an adjuvant to induce and boost an immunogenic response in humans. Adjuvants are the substances used in combination with antigens to produce a more robust immune response. Since their discovery, the aluminum compounds continue to monopolize the human vaccines as adjuvants. Despite the discovery of more potent adjuvants (lipopolysaccharide etc), they are unsuitable for human use due to their toxicity. Adjuvants are classified as particulate delivery systems and immunostimulatory adjuvants (derived from pathogens), based on their mechanism. We propose to consolidate the advantages of these mechanisms by developing a microparticulate formulation of DPD and its synthetic analogs. METHODS

Various polymers were screened via molecular docking technique for the microparticulate formulation of DPD. Bovine Serum Albumin (BSA) was selected as the polymer of choice based on its docking score. DPD was mixed with the crosslinked-albumin matrix and spray dried using Buchi mini spray dryer B-290 to obtain DPD loaded microparticles. The microparticles were characterized for size, charge, and polydispersity index. The surface morphology was visualized by scanning electron microscopy. The induction of innate immune response, as indicated by the production of nitric oxide by antigen-presenting cells (APCs), was analyzed by Griess's assay. The ability of DPD microparticles to induce cellular and humoral adaptive immune response was evaluated in vitro by measuring the expression of antigen-presenting molecules MHC I and MHC II and their co-stimulatory molecules, CD80 and CD40, on the surface of APCs. The cytotoxicity of DPD microparticles on APCs was assessed by MTT assay. Further evaluation of adjuvant potential of DPD microparticles and comparison of its efficacy as an adjuvant to the traditional FDA approved adjuvants was done by combining DPD microparticles with the particulate vaccines for measles and gonorrhea. Griess's assay was performed to analyze the induction of innate immune response by the particulate vaccines with and without combination with different particulate adjuvants i.e. alum, MF59, MPL A and DPD. Future studies will involve analyzing the expression of antigen-presenting molecules MHC I and MHC II and their co-stimulatory molecules CD80 and CD40 on the surface of APCs on exposure to various particulate vaccines in combination with FDA approved adjuvants and comparing the adjuvant potential of microparticulate DPD to that of the traditional adjuvants. Various synthetic analogs of DPD including ent-DPD, isobutyl-DPD, n-butyl-DPD, and phenyl-DPD will be formulated into the microparticles. The adjuvant potential of these analogs will be compared to that of microparticulate DPD and to other FDA approved adjuvants.

RESULTS BSA was selected as the polymer of choice for the formulation of microparticles based on its BSA was selected as the polymer of choice for the formulation of microparticles based on its docking interactions with DPD. The yield of the microparticulate formulation was 89.52 ± 2.3 %. The particle size was 3.851 ± 0.9769 um with a charge of -31.5 ± 7.4 mV. The Griess's assay showed a significantly higher (p<0.01) release of nitric oxide, a hallmark of the innate immune response, by dendritic cells upon exposure to DPD microparticles alone and in combination with the traditional adjuvants (alum and MF59) as compared to the blank microparticles. The APCs showed significantly higher (p<0.05) expression of antigen-presenting molecules MHC I and MHC II and their co-stimulatory molecules CD80 and CD40 upon exposure to DPD microparticles with and without adjuvant microparticles as compared to the blank microparticles. The DPD microparticles were found to non-cytotoxic to the APCs as compared to dimethyl sulfoxide (DMSO) which is cytotoxic. Microparticulate DPD was found to have an adjuvant effect upon combination with the gonorrhea vaccine as indicated by nitric oxide release by APCs. However, there was no adjuvant effect of DPD observed upon its combination with measles vaccine. These results will further be confirmed by evaluating the expression of MHC I, MHC II, CD40, and CD80 by APCs upon exposure to DPD microparticles in combination with various viral and bacterial vaccines. CONCLUSIONS

Griess's assay and Flow cytometric analysis revealed that the microparticulate DPD is immunogenic and can stimulate innate and adaptive immune response. A combination of DPD with various particulate vaccines reveals that microparticulate DPD shows an adjuvant effect with the bacterial vaccines. These encouraging preliminary results have led us to further test microparticulate DPD and its synthetic analogs to explore their potential as probable vaccine adjuvants.




4-Ethoxybenzoic Acid is an Anti-Pathogenic Anti-Biofilm Compound


Brooke Martin Georgia State University BACKGROUND Microbial biofilms are responsible for a wide variety of medically relevant infections. An issue with treating them is their inherent multidrug resistance, which makes treatment difficult and contributes to drug resistance. Anti-pathogenic drugs control microbial infections by targeting virulence without being biocidal and are less likely to foster drug-resistant strains. We identified several anti-biofilm anti-pathogenic compounds from Rhamnus prinoides (gesho), a plant used in traditional medicine, and a structure-activity analysis identified 4-ethoxybenzoic acid (4EB) as a compound with good activity. METHODS

The colorimetric method was used to determine the abundance of biofilms treated with and without 4EB. Crystal violet staining was used to determine anti-biofilm concentrations. The results from two independent studies were combined and statistically analyzed using ANOVA and t-test.

RESULTS 4EB concentrations from 0.4 to 0.8 mg/mL inhibited biofilm formation of Candida albicans. 4EB concentrations from 0.8 to 7 mg/mL were inhibitory to Streptococcus mutans biofilm formation. Pseudomonas aeruginosa biofilm activity was not impacted by 4EB. CONCLUSIONS

4EB is interesting as an anti-pathogenic anti-biofilm compound and may help prevent biofilm formation on surfaces including medical devices, bandages, and hospital surfaces. It may lead to the identification of additional compounds that have greater potency. Considerable evidence has shown that anti-pathogenic agents that inhibit bacterial virulence are suitable alternatives to antibiotics or help re-sensitizing bacteria to antibiotics.




Evaluation of a novel oxytocin nanoformulation for cocaine-use disorder


Aboagyewaah Oppong-Damoah Mercer University BACKGROUND Cocaine abuse is a significant global public health concern. In 2016 alone, 18 million people reported cocaine use worldwide. Currently, there are no FDA approved pharmacotherapies for treating cocaine dependence. Oxytocin (OT), a highly prosocial neuropeptide has shown recent promise for the treatment of disorders with known social deficits such as autism spectrum disease and cocaine-use disorder (CUD), but concerns remain as to its clinical relevance due to poor blood brain barrier penetration and short half-life. METHODS

In this study, we used the open field locomotor activity assay, a well validated preclinical model for assessing stimulant induced locomotor hyperactivity to investigate the potential of OT and a novel OT nanoformulation for reversing cocaine induced locomotor stimulant effects.

RESULTS OT administered both intranasally and intraperitoneally dose dependently decreased cocaine induced locomotor hyperactivity when given as a 10-minute pretreatment but showed no such effect when administered as 2-hour pretreatment. OT nanoparticles showed sustained effects in reversing cocaine induced locomotor stimulant effects both at 10 minutes and 2-hour pretreatment times. CONCLUSIONS

This study can now be used to support the use of OT nanoformulation and potentially other neuropeptides as promising therapeutic candidates for disorders of the central nervous system such as CUD.




Novel Strategies for Developing Universal Influenza Virus Vaccines


Bo Ryoung Park Institute for Biomedical Sciences, Georgia State University BACKGROUND Current live attenuated and inactivated influenza vaccines based on immunity to the hemagglutinin (HA) hypervariable protein are effective when vaccine strains and circulating viruses are well matched. However, HA-based current vaccines are ineffective in providing cross protection against antigenically distinct drift and new pandemic viruses. Universal influenza virus vaccines based on the highly conserved antigenic targets such as the extracellular domain of M2 (M2e) confer cross protection but are not sufficient to serve as a standalone vaccine due to low efficacy. Neuraminidase is the 2nd major surface protein and suggested to be an additional target for inducing cross protection. Virus-like particles (VLP) are known to be an immunogenic delivery vehicle. METHODS

As a new approach to improve the efficacy of HA-based current vaccines by inducing both HA and cross protective M2e immunity, we generated recombinant influenza virus vaccine platforms, which express chimeric H3 HA-M2e conjugate proteins in the N-terminus (HA-4xM2e). In addition, we developed multimeric VLP vaccine as a single entity containing consensus N1 neuraminidase NA, N2 NA, influenza B virus NA as well as tandem repeat 5xM2e.

RESULTS The live reassortant H3N2 chimeric virus vaccine was highly attenuated in mice, as shown by restricted replication in the upper respiratory but not in the lower respiratory track lung tissues of the mice. Recombinant H3N2 virus containing chimeric H3 HA-4xM2e was effective in inducing cross protective M2e specific IgG antibody responses as well as HA immunity in mice. Mice that intranasally primed with chimeric H3 HA-4xM2e vaccine showed improved cross protection against antigenically different viruses (H1N1, H3N2, H5N1, H7N9, H9N2). As an additional novel approach, multi-NA + 5xM2e VLP vaccination of mice was found to be highly effective in inducing cross protection against influenza A and B viruses. CONCLUSIONS

The results in this study present novel approaches to improve the efficacy of cross protection against seasonal and pandemic viruses by recombinant influenza virus and VLP vaccines inducing immunity to HA, NA, and M2e antigens.




A Novel Diagnostic Method for Detection and Quantitation of Paralytic Shellfish Poisoning in Humans


Mani Deepika Vakkalanka Mercer University BACKGROUND Paralytic shellfish toxins(PSTs) are potent neurotoxins which bind to the voltage gated sodium channels and prevent the conduction of action potentials leading to respiratory paralysis and death. Carbamate toxins are the most potent PSTs and include gonyautoxins(GTXs) 1,2,3,4. Testing these toxins in human biological matrices is the best approach to accurately detect, quantify them and identify the exposures. Here, we describe a solid phase extraction method for extracting GTXs from human plasma using HILIC HPLC-MS/MS analysis for quantitation. The developed method was further optimized and validated so that it can be applied to clinical specimens. METHODS

GTXs were extracted from human plasma using strong cationic exchange cartridges. Sample pH was adjusted with 10mM acetate buffer. Cartridges were conditioned with methanol and acetate buffer and toxins were eluted with 5% ammonium hydroxide in methanol. Experiments were conducted using pooled plasma spiked with a series of concentrations of gonyautoxins. Calibrants were prepared at the following concentration ranges: GTX1 8.13-517.66 ng/mL, GTX2 6.98-473.25 ng/mL, GTX3 2.96-200.68 ng/mL and GTX4 2.56-162.91 ng/mL. Low- and High-quality controls (QCs) were prepared at a concentration of 44 and 124 ng/mL for GTX1 , 40 and 113 ng/mL for GTX2, 17 and 71 ng/mL for GTX3, 14 and 39 ng/mL for GTX4. Eight plasma specimens were collected and spiked with toxin at a concentration of 173.85ng/ml. Calibrants, quality controls and specimen plasmas were then extracted as described above, dried down and reconstituted. Extracted samples were injected onto HPLC-MS/MS for analysis.

RESULTS The developed method was validated according to the FDA guidance for bioanalytical method validation. The calibration curves were linear with the coefficients of determination value (R-squared value) greater than 0.99 for all toxins. The method showed good percent accuracies for all the toxins: GTX1 95-104%, GTX2 92-114%, GTX3 92-117%, GTX4 92-107%. Precision or percent CV ranged from 3.5 to 10.9% for GTX1, 3.03 to 11.25% for GTX2, 3.01 to 12.72 for GTX3 and 2.08 to 10.49 for GTX4. Percent accuracies for quality controls ranged from 90.3-97.7%(Low QC) and 96.7-105.1%(High QC). Recovery of GTXs from specimen plasmas were as follows: GTX1-110.55%, GTX2-103.72%, GTX3-3.27%, GTX4-99.7%. Limits of detection were calculated (by Taylor method) to be 7.08, 7.59, 2.1, 1.98 ng/mL for GTXs 1,2,3 and 4 respectively. Limits of quantitation were the lowest calibrators for each analyte. Selectivity of the method was characterized by analyzing blank samples and there were no peaks seen at the analyte retention times. CONCLUSIONS

We have successfully developed a solid phase extraction method for extracting GTXs 1,2,3,4 from human plasma. This method has all the validation parameters within the limits specified by FDA. This is a fast and simple diagnostic method which is easily accessible to common clinical laboratories and can be applied to clinical specimens.




Development and Evaluation of a Gel-based System for Transdermal Delivery of Heparin via Laser-Generated Micropores


Deepal Vora Mercer University BACKGROUND Heparin is an anticoagulant, commonly administered through intravenous route to treat venous thromboembolism, a condition where inflammation and clotting are observed inside a vein below skin surface. Our study investigates the feasibility of transdermal delivery of heparin, a hydrophilic macromolecule, through laser-microporated skin as an alternative to intravenous administration. METHODS

Microporation was performed using P.L.E.A.S.E.® (Precise Laser Epidermal System; Pantec BioSolutions AG, Liechtenstein) technology and characterized by staining, histology, and confocal microscopy. A poloxamer gel (20% w/v; Kolliphor P407 from BASF) was prepared and loaded with 0.3% w/v heparin. Heparin-loaded gel was then characterized for rheological properties. In vitro permeation on Franz diffusion cells was performed to determine the rate and extent of delivery into and across laser-treated (fluence of 22.7 J/cm2) porcine ear skin. Delivery from poloxamer gel was compared to a 0.3% w/v solution of heparin. Additionally, passive diffusion across untreated porcine ear skin was evaluated. The receptor compartment contained 5 mL of 10 mM phosphate buffered saline (1X PBS; pH 7.4). Samples were collected from the receptor at predetermined time points over 24 h and analyzed by Chromagenix® Coatest Heparin. Skin was extracted using 1X PBS to determine the amount delivered into skin.

RESULTS Characterizations of laser-created micropores by staining, histology and confocal microscopy confirmed the formation of microchannels in the skin. The transition property of poloxamer from solution at room temperature to gel at skin temperature (32°C) was utilized in this study. Amount of heparin delivered for laser treated skin (26.07 ± 1.82 µg/sq.cm) was significantly higher as compared to passive permeation (0 µg/sq.cm). A lower but more sustained delivery was observed for heparin loaded gel (11.28 ± 5.32 µg/sq.cm) as compared to heparin solution (26.07 ± 1.82µg/sq.cm) in the laser-treated group. CONCLUSIONS

A sustained delivery of heparin into and across the skin was demonstrated with gel-based formulation, indicating the feasibility of transdermal delivery of macromolecules using laser treatment.




A First-in-Class Skp1-FBP Inhibitor for the Treatment of Bone Metastatic Castration-Resistant Prostate Cancer


Daqing Wu Clark Atlanta University BACKGROUND Bone metastasis directly contributes to the morbidity and mortality of prostate cancer, but current treatment modalities only temporarily control disease progression. Clearly, it is an urgent and unmet medical need to identify new therapeutic targets and develop novel targeting strategies against this lethal disease. Recent studies have demonstrated a major role of S phase kinase-associated protein 1 (Skp1)-Cullin1-F-Box protein (FBP) (SCF) ubiquitin ligase complexes in human cancer progression. Skp1 stabilizes FBPs (such as Skp2) and enhances their substrate identification, thereby is pivotal in the formation and function of SCF complexes. Compared with FBP-targeted strategies, blocking the interaction between Skp1 and FBPs may provide a general and effective approach to inhibit multiple SCF oncogenic signals and maximize therapeutic efficacy against tumors with highly heterogeneous backgrounds. Therefore, Skp1-FBP interaction represents a promising therapeutic target in metastatic castration-resistant prostate cancer (mCRPC). However, no synthetic Skp1 inhibitor has been developed yet. METHODS

Supported by a Small Business Technology Transfer (STTR) grant from the National Cancer Institute (NCI), we developed GH501, a first-in-class synthetic inhibitor of Skp1-FBP protein-protein interaction. We evaluated the in vitro anticancer activity of GH501 in collaboration with the NCI Developmental Therapeutics program. We investigated the mechanism of action of GH501 in mCRPC cells using molecular and cellular approaches. We evaluated the in vivo toxicity, pharmacokinetic and efficacy against mCRPC in clinically relevant models.

RESULTS GH501 effectively disrupts the protein-protein interaction between Skp1 and FBPs, thereby affecting multiple prominent drivers of mCRPC progression, including Skp2, p21, p27, beta-catenin, E2F1, EZH2, c-Myc, cyclin D1, RUNX2 and survivin. GH501 exhibits potent cytotoxicity against mCRPC cells and a broad spectrum of human cancer cell lines at nanomolar concentrations. Animal studies demonstrated that GH501 is well tolerated in mice and has favorable physicochemical properties as a lead compound. Uniquely, GH501 is preferably distributed to and deposited in bone tissues, making it an ideal and attractive drug candidate for treating bone cancers. Significantly, in vivo efficacy studies demonstrated that GH501 effectively suppresses the skeletal and subcutaneous growth of mCRPC in cell- and patient-derived xenograft models. CONCLUSIONS

Our preclinical studies support the promise of GH501 as a lead compound against bone metastatic prostate cancer. We envision that GH501 is a novel targeted therapy to improve the survival and quality of life of patients with bone metastasis.




Microporation Assisted Topical Delivery of Kojic Acid for the Treatment of Hyperpigmentation


Amruta Dandekar

Center for Drug Delivery Research, Department of Pharmaceutical Sciences, College of Pharmacy, Mercer University

BACKGROUND Hyperpigmentation is a common skin disorder prevalent worldwide. It is caused due to excessive melanin production regulated by the enzyme tyrosinase. Kojic acid is an anti-tyrosinase agent used for the treatment of hyperpigmentation. Due to its hydrophilic nature (logP=-0.5) topical delivery of this molecule is a challenge. Our study demonstrates the use of microporation to enhance the delivery of kojic acid via skin. METHODS

In vitro drug permeation studies were performed on dermatomed porcine ear skin using vertical Franz diffusion cells. Microporation of skin using dissolving maltose microneedles (500 µm; 81 needles) and Dr. PenTM Ultima A6 (5mm needle-length; 13,000 insertions per minute; 5s pre-treatment) was compared to passive diffusion (1% w/v solution). Skin resistance was measured using an arbitrary waveform generator (Agilent 33220A, 20 MHz Function), a 34410A 6 ½ digital multimeter (Agilent Technologies, Santa Clara, CA, USA) and silver/silver chloride electrodes to ensure skin integrity. Samples were collected at predetermined time points from the receptor and amount of drug delivered into skin after 8h and 24h was analysed. Samples were quantified using validated RP-HPLC method and statistical difference was concluded using one way ANOVA with Tukey's post-HOC analysis (p< 0.05).

RESULTS Successful microporation of skin was observed, concluded by reduced skin electrical resistance with both maltose microneedles (74.39±9.21%) as well as Dr. PenTM Ultima A6 (64.86±22.55%). Pre-treatment with maltose microneedles resulted in significantly higher delivery across skin into the receptor compartment (88.22±23.06µg/sq.cm) with a reduced lag time (0.08±0.02h) in comparison to passive diffusion (5.99±1.26µg/sq.cm; 2.11 ± 0.07h) within 4 hours. A reduction in lag time with Dr. PenTM Ultima A6 (0.11±0.03h) was also observed with significantly higher delivery across skin after 8 hours (77.32±5.94 µg/sq.cm). Microporation of skin did not result in significantly higher delivery into skin after 8 hours as compared to passive diffusion. (2.76±0.34% with maltose microneedle; 1.71±0.29% with Dr. PenTM Ultima A6 pre-treatment; 2.25±0.71% with passive diffusion). There was no significant difference observed in skin delivery of kojic acid at the end of 8 hours (35.84±4.44µg/sq.cm) as compared to 24 hours (42.70±9.38µg/sq.cm) in the maltose microneedle pre-treatment group. However, significantly higher amount of kojic acid was delivered into skin at the end of 24 hours (51.85±5.22µg/sq.cm) as compared to 8 hours (24.53±7.125µg/sq.cm) in the case of passive diffusion. CONCLUSIONS

Delivery of kojic acid across skin was enhanced after microporation of skin using dissolving maltose microneedle and Dr. PenTM Ultima A6. Also, lag time for delivery into skin was reduced following pre-treatment with microneedles.





 

Nanotechnology

Does the FDA enforce regulations at virtual and fully integrated pharma and biotech companies the same?


Cassie Alexander University of Georgia BACKGROUND Over the past several decades, virtual companies have become prevalent within the pharmaceutical and biotech industries. Virtual companies utilize a business model where they directly hire employees in supportive roles while outsourcing critical business activities that are highly regulated by the United States Food and Drug Administration (U.S. FDA). Since the corporate structures for virtual and fully integrated companies differ significantly, it is important to understand how virtual companies are regulated by the FDA so management teams can understand how to appropriately staff organizations to mitigate potential compliance risks while maintaining a lean organization. METHODS The study had three areas of focus: public information (websites and databases), articles and personal interviews. For the public information analysis, a randomized sample of 40 companies was created, including virtual and fully integrated pharmaceutical and biotech companies, analytical laboratories and third-party logistics providers. Data was gathered for the 40 companies from websites and databases like Registration and Listing, Inspections, Inspection Citations, Warning Letters and the Orange Book. Data was tabulated to provide a qualitative and quantitative analysis of the trends and frequency of occurrence. For the second area of focus, five online articles/blogs were analyzed to gather common trends in compliance activities relating to virtual companies. Finally, five people with experience with compliance activities were surveyed through a questionnaire to assess personal experiences and perspectives on how the FDA regulates virtual and fully integrated companies. RESULTS The findings from this study demonstrate that the FDA enforces regulations at virtual and fully integrated companies differently. Analysis of data from the companies show that fully integrated pharmaceutical and biotech companies experienced more FDA inspections, inspection citations and escalated regulatory enforcement actions. Analysis of industry the articles pointed to the importance of virtual companies building a strong relationship with contract facilities through robust Quality Agreements and close oversight. Feedback from questionnaires of industry professionals showed a concern that virtual companies have less rigorous quality systems and inadequate oversight of contract facilities, which would lead to FDA enforcement actions. CONCLUSIONS While subject to the same U.S. Regulations, this study demonstrates that virtual companies experience less regulatory oversight and enforcement actions than fully integrated companies. It is likely the contract partners are inspected for compliance and it is incumbent upon the management teams of virtual companies to ensure they have adequate processes and resources in place to appropriately oversee contract partners and mitigate risks. This will reduce the risk of FDA enforcement actions on virtual companies while ensuring safe and effective products are provided to patients.





 

Regulatory Affairs

Does the FDA enforce regulations at virtual and fully integrated pharma and biotech companies the same?


Cassie Alexander University of Georgia BACKGROUND Over the past several decades, virtual companies have become prevalent within the pharmaceutical and biotech industries. Virtual companies utilize a business model where they directly hire employees in supportive roles while outsourcing critical business activities that are highly regulated by the United States Food and Drug Administration (U.S. FDA). Since the corporate structures for virtual and fully integrated companies differ significantly, it is important to understand how virtual companies are regulated by the FDA so management teams can understand how to appropriately staff organizations to mitigate potential compliance risks while maintaining a lean organization. METHODS The study had three areas of focus: public information (websites and databases), articles and personal interviews. For the public information analysis, a randomized sample of 40 companies was created, including virtual and fully integrated pharmaceutical and biotech companies, analytical laboratories and third-party logistics providers. Data was gathered for the 40 companies from websites and databases like Registration and Listing, Inspections, Inspection Citations, Warning Letters and the Orange Book. Data was tabulated to provide a qualitative and quantitative analysis of the trends and frequency of occurrence. For the second area of focus, five online articles/blogs were analyzed to gather common trends in compliance activities relating to virtual companies. Finally, five people with experience with compliance activities were surveyed through a questionnaire to assess personal experiences and perspectives on how the FDA regulates virtual and fully integrated companies. RESULTS The findings from this study demonstrate that the FDA enforces regulations at virtual and fully integrated companies differently. Analysis of data from the companies show that fully integrated pharmaceutical and biotech companies experienced more FDA inspections, inspection citations and escalated regulatory enforcement actions. Analysis of industry the articles pointed to the importance of virtual companies building a strong relationship with contract facilities through robust Quality Agreements and close oversight. Feedback from questionnaires of industry professionals showed a concern that virtual companies have less rigorous quality systems and inadequate oversight of contract facilities, which would lead to FDA enforcement actions. CONCLUSIONS While subject to the same U.S. Regulations, this study demonstrates that virtual companies experience less regulatory oversight and enforcement actions than fully integrated companies. It is likely the contract partners are inspected for compliance and it is incumbent upon the management teams of virtual companies to ensure they have adequate processes and resources in place to appropriately oversee contract partners and mitigate risks. This will reduce the risk of FDA enforcement actions on virtual companies while ensuring safe and effective products are provided to patients.