ISCOMs and related colloidal particles prepared by the lipid film hydration method for antigen delivery

Purposes: The aims of this thesis were to physicochemically investigate the formation of ISCOM matrices and other types of colloidal particles formed in aqueous dispersion as a function of different mass ratios of Quil A, cholesterol and phospholipid (phosphatidylcholine (PC) or phosphatidylethanolamine (PE)) prepared by the hydration method and to investigate the delivery of subunit vaccines to antigen presenting cells using these colloids. Methods: The hydration method, recently developed for the preparation of ISCOMs or ISCOM matrices, was used to produce these colloids and other related structures. Factors such as effects of buffer salts, equilibration time and type of phospholipid on the formation of ISCOM matrices and other colloidal particles prepared were investigated. The standard dialysis method for the preparation of ISCOMs was also used for comparison to prepare various colloidal particles. Colloidal particles were characterized by negative staining transmission electron microscopy (TEM). Polarized light microscopy (PLM) was used to identify samples containing cholesterol crystals. Incorporation of a model antigen (modified ovalbumin) into various colloidal particles was investigated by fluorescence spectroscopy following analytical sucrose density gradient ultracentrifugation. Physical properties of solid Quil A-cholesterol-phospholipid formulations (as powder mixtures or compressed to pellets) with or without model antigen were characterized by X-ray powder diffractometry (XRPD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), and PLM. Release of model antigen from compressed pellets was investigated in vitro. Flow cytometric analysis (FACS) was used to investigate the in vitro delivery of antigen incorporated into various colloidal structures to murine bone-marrow derived dendritic cells (BMDC). Results: Pseudo-ternary diagrams revealed that depending on the mass ratio of Quil A, cholesterol and phospholipid, various colloidal particles including ISCOM matrices, liposomes, lipidic/layered structures, ring-like micelles, and worm-like micelles could be identified in the different regions of the diagrams. In the presence of these predominant colloids, helices and lamellae (hexagonal pattern of ring-like micelles) structures were also formed as minor structures. Buffer salts and equilibration time were important factors for the formation of ISCOM matrices and liposomes. The type of phospholipid affected the morphology of ISCOM matrices and lamellae. ISCOM matrices were predominantly found near the phospholipid apex of the pseudo-ternary diagram following sample preparation by the hydration method. On the other hand, samples prepared by the dialysis method produced ISCOM matrices that were predominantly found near the Quil A apex of the pseudo-ternary diagram. No ISCOM matrices could be formed in any binary mixtures prepared by the hydration method in contrast to the dialysis method. Worm-like micelles could only be formed if samples were prepared by the hydration method. An incorporation study demonstrated that the various colloidal particles formed as a result of hydrating phospholipid/cholesterol lipid films with different amounts of Quil A are capable of incorporating antigen, provided it is amphipathic. Freeze-dried lipid powder mixtures were found to contain a lower degree of crystalline cholesterol compared to physically mixed powders. Consequently, physically mixed powders (with or without model antigen) and pellets prepared from the same powders did not spontaneously form ISCOM matrices and related colloidal structures upon hydration as expected from the pseudo-ternary diagram. Release of antigen incorporated into ISCOM particles was relatively slower from the pellets made using freeze-dried powders in contrast to pellets prepared from the physically mixed powders. Using ISCOMs, liposomes and ring-like micelles, it was demonstrated that the model antigen incorporated into these particles could be delivered to dendritic cells leading to activation and proliferation of transgenic T cells. Conclusions: Depending on the mass ratio of Quil A, cholesterol and phospholipid, ISCOM matrices and other types of colloidal structures such as liposomes, lipidic/layered structures, ring-like micelles, lamellae (hexagonal array of ring-like micelles) and wormlike micelles prepared by the hydration method could be identified in the different regions of pseudo-ternary diagram. All the colloids containing Quil A were capable of incorporating an antigen, provided it is amphipathic. Delivery of antigen to DC and immunestimulatory effects of various colloidal particles could be demonstrated

Perceptions of Board Members on the Presence of Pharmacists as Strategic Leaders of Manufacturing Pharmaceutical Companies Operating in South Africa: A Qualitative Study

Life-saving medicines are produced by manufacturing pharmaceutical companies (MPCs) with operations worldwide. The COVID-19 pandemic resulted in unequal access to vaccines, which led to advocacy around health rights and MPCs needing to prioritise saving lives over profits. This study aimed to determine the perceptions of board members of the largest listed MPCs in South Africa regarding the presence of pharmacists in the strategic leadership of MPCs, as custodians of medicines. A snowball sampling method was used to identify board members of the listed MPCs on the Johannesburg Stock Exchange (JSE). The board members were approached and requested to participate in their capacity. Data were collected through semi-structured interviews. Transcription, coding and narrative thematic analysis was applied under five (5) themes with emerging themes identified. A theoretical framework was developed to describe pharmacists at the strategic leadership of an MPC. The data collected were from five (5) respondents (80% male and 20% female), with a mean age of 57 years (SD±=2,24), from medical, business and pharmacy professional backgrounds. The respondents indicated a limited presence of pharmacists in the strategic leadership of MPCs, especially the larger ones. The reasons for the limited presence included the narrow diversity of corporate and leadership skills necessary to lead at a strategic level by pharmacists. Some of the barriers to entry into strategic leadership by pharmacists included the lack of recognition of pharmacy specialisations by the regulator of the pharmacy profession, a mismatch of skills of a graduate and what the industry needs and inadequate governance of the pharmacy profession in South Africa. The respondents agreed that having a pharmacist with ambition, business skills, and experience would benefit the MPC and users of products manufactured. The role and need for pharmacists in the strategic leadership of MPCs was established. It is clear, however, that while pharmacists have value in the leadership of MPCs, their entry should be supported by diversifying their skills in business, leadership and corporate management to extend their value beyond the technical level

Strategies towards Empowering Nurses on the Rational Use of Antiretrovirals in Children Initiated and Managed on Therapy in Rural Primary Healthcare Clinics of South Africa

HIV has affected the health and welfare of children and undermined the success of child survival in some countries. The introduction of antiretroviral therapy (ART) in managing HIV is one great public health success story. ART has commanded increased survival for people living with HIV (PLHIV). Barriers to achieving ART outcomes in children have been simplifying the prescribing process for non-paediatricians, such as medical doctors and nurses familiar with prescribing ART for adults but involved in treating children, particularly at the primary health care level. And the lack of appropriate antiretroviral formulations for children. The calculation of individualised doses for liquid oral ARVs for children at each clinic visit is considered complicated and time-consuming. ART failure among children seems to be an under-recognised issue, and adherence to treatment guidelines is reported to be a challenge among nurses caring for children and PLWHIV. Rational medicine use is essential to ensure the success of pharmacologic interventions. The attainment of ART goals depends on the effective use ARVs as recommended in guidelines. It is pivotal that nurses be empowered with strategies aimed at promoting the rational use of antiretrovirals

Straw blood cell count, growth, inhibition and comparison to apoptotic bodies

<p>Abstract</p> <p>Background</p> <p>Mammalian cells transform into individual tubular straw cells naturally in tissues and in response to desiccation related stress <it>in vitro</it>. The transformation event is characterized by a dramatic cellular deformation process which includes: condensation of certain cellular materials into a much smaller tubular structure, synthesis of a tubular wall and growth of filamentous extensions. This study continues the characterization of straw cells in blood, as well as the mechanisms of tubular transformation in response to stress; with specific emphasis placed on investigating whether tubular transformation shares the same signaling pathway as apoptosis.</p> <p>Results</p> <p>There are approximately 100 billion, unconventional, tubular straw cells in human blood at any given time. The straw blood cell count (SBC) is 45 million/ml, which accounts for 6.9% of the bloods dry weight. Straw cells originating from the lungs, liver and lymphocytes have varying nodules, hairiness and dimensions. Lipid profiling reveals severe disruption of the plasma membrane in CACO cells during transformation. The growth rates for the elongation of filaments and enlargement of rabbit straw cells is 0.6~1.1 (μm/hr) and 3.8 (μm³/hr), respectively. Studies using apoptosis inhibitors and a tubular transformation inhibitor in CACO2 cells and in mice suggested apoptosis produced apoptotic bodies are mediated differently than tubular transformation produced straw cells. A single dose of 0.01 mg/kg/day of p38 MAPK inhibitor in wild type mice results in a 30% reduction in the SBC. In 9 domestic animals SBC appears to correlate inversely with an animal's average lifespan (R²= 0.7).</p> <p>Conclusion</p> <p>Straw cells are observed residing in the mammalian blood with large quantities. Production of SBC appears to be constant for a given animal and may involve a stress-inducible protein kinase (P38 MAPK). Tubular transformation is a programmed cell survival process that diverges from apoptosis. SBCs may be an important indicator of intrinsic aging-related stress.</p

ISCOMs and related colloidal particles prepared by the lipid film hydration method for antigen delivery

Purposes: The aims of this thesis were to physicochemically investigate the formation of ISCOM matrices and other types of colloidal particles formed in aqueous dispersion as a function of different mass ratios of Quil A, cholesterol and phospholipid (phosphatidylcholine (PC) or phosphatidylethanolamine (PE)) prepared by the hydration method and to investigate the delivery of subunit vaccines to antigen presenting cells using these colloids. Methods: The hydration method, recently developed for the preparation of ISCOMs or ISCOM matrices, was used to produce these colloids and other related structures. Factors such as effects of buffer salts, equilibration time and type of phospholipid on the formation of ISCOM matrices and other colloidal particles prepared were investigated. The standard dialysis method for the preparation of ISCOMs was also used for comparison to prepare various colloidal particles. Colloidal particles were characterized by negative staining transmission electron microscopy (TEM). Polarized light microscopy (PLM) was used to identify samples containing cholesterol crystals. Incorporation of a model antigen (modified ovalbumin) into various colloidal particles was investigated by fluorescence spectroscopy following analytical sucrose density gradient ultracentrifugation. Physical properties of solid Quil A-cholesterol-phospholipid formulations (as powder mixtures or compressed to pellets) with or without model antigen were characterized by X-ray powder diffractometry (XRPD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), and PLM. Release of model antigen from compressed pellets was investigated in vitro. Flow cytometric analysis (FACS) was used to investigate the in vitro delivery of antigen incorporated into various colloidal structures to murine bone-marrow derived dendritic cells (BMDC). Results: Pseudo-ternary diagrams revealed that depending on the mass ratio of Quil A, cholesterol and phospholipid, various colloidal particles including ISCOM matrices, liposomes, lipidic/layered structures, ring-like micelles, and worm-like micelles could be identified in the different regions of the diagrams. In the presence of these predominant colloids, helices and lamellae (hexagonal pattern of ring-like micelles) structures were also formed as minor structures. Buffer salts and equilibration time were important factors for the formation of ISCOM matrices and liposomes. The type of phospholipid affected the morphology of ISCOM matrices and lamellae. ISCOM matrices were predominantly found near the phospholipid apex of the pseudo-ternary diagram following sample preparation by the hydration method. On the other hand, samples prepared by the dialysis method produced ISCOM matrices that were predominantly found near the Quil A apex of the pseudo-ternary diagram. No ISCOM matrices could be formed in any binary mixtures prepared by the hydration method in contrast to the dialysis method. Worm-like micelles could only be formed if samples were prepared by the hydration method. An incorporation study demonstrated that the various colloidal particles formed as a result of hydrating phospholipid/cholesterol lipid films with different amounts of Quil A are capable of incorporating antigen, provided it is amphipathic. Freeze-dried lipid powder mixtures were found to contain a lower degree of crystalline cholesterol compared to physically mixed powders. Consequently, physically mixed powders (with or without model antigen) and pellets prepared from the same powders did not spontaneously form ISCOM matrices and related colloidal structures upon hydration as expected from the pseudo-ternary diagram. Release of antigen incorporated into ISCOM particles was relatively slower from the pellets made using freeze-dried powders in contrast to pellets prepared from the physically mixed powders. Using ISCOMs, liposomes and ring-like micelles, it was demonstrated that the model antigen incorporated into these particles could be delivered to dendritic cells leading to activation and proliferation of transgenic T cells. Conclusions: Depending on the mass ratio of Quil A, cholesterol and phospholipid, ISCOM matrices and other types of colloidal structures such as liposomes, lipidic/layered structures, ring-like micelles, lamellae (hexagonal array of ring-like micelles) and wormlike micelles prepared by the hydration method could be identified in the different regions of pseudo-ternary diagram. All the colloids containing Quil A were capable of incorporating an antigen, provided it is amphipathic. Delivery of antigen to DC and immunestimulatory effects of various colloidal particles could be demonstrated

Tropical corticosteroid bioequivalence testing comparison of chromameter and visual data

The major criticism of the human skin blanching assay is the subjective nature of grading the response. Recently the American FDA released a Guidance document for topical corticosteroid bioequi valence testing. The guidelines require the use of a chromameter as a reliable method to estimate skin blanching. The purpose of this study was to evaluate the recommendations of this document for appropriateness by comparing visual and chromameter data. The visually-assessed blanching assay methodology routinely practised in our laboratories was modified to comply with the specifications of the Guidance. The preliminary trial indicated that the training period that is required for a novice to be classified as an experienced observer is not a major problem. The major trend that emerged from the pilot study was that visual assessment was better than the chromameter. Longer dose durations were found to be more discriminatory than shorter durations. The visual data were best described by the sigmoid Emax model and the chromameter data were best described by the simple Emax model. The pivotal results indicated that the D2/Dj criterion to determine sample size of "acceptable blanchers" produced only few subjects suggesting that the validity of this criterion requires extensive investigations. The estimates of the Locke's confidence interval method were simiJar to those for the general simple formula. However, due to undefined parameters of the Locke's method in the Guidance, the validity of the Locke's method requires evaluation. The chromameter b-scale parameter was the least sensitive in estimating skin blanching whereas the a- and L-scale parameters produced similar results. Poor correlation between visual and chromameter was noted indicating that the visual method is still the best method

Tropical corticosteroid bioequivalence testing comparison of chromameter and visual data

The major criticism of the human skin blanching assay is the subjective nature of grading the response. Recently the American FDA released a Guidance document for topical corticosteroid bioequi valence testing. The guidelines require the use of a chromameter as a reliable method to estimate skin blanching. The purpose of this study was to evaluate the recommendations of this document for appropriateness by comparing visual and chromameter data. The visually-assessed blanching assay methodology routinely practised in our laboratories was modified to comply with the specifications of the Guidance. The preliminary trial indicated that the training period that is required for a novice to be classified as an experienced observer is not a major problem. The major trend that emerged from the pilot study was that visual assessment was better than the chromameter. Longer dose durations were found to be more discriminatory than shorter durations. The visual data were best described by the sigmoid Emax model and the chromameter data were best described by the simple Emax model. The pivotal results indicated that the D2/Dj criterion to determine sample size of "acceptable blanchers" produced only few subjects suggesting that the validity of this criterion requires extensive investigations. The estimates of the Locke's confidence interval method were simiJar to those for the general simple formula. However, due to undefined parameters of the Locke's method in the Guidance, the validity of the Locke's method requires evaluation. The chromameter b-scale parameter was the least sensitive in estimating skin blanching whereas the a- and L-scale parameters produced similar results. Poor correlation between visual and chromameter was noted indicating that the visual method is still the best method

An assessment of positions, perceptions and valued leadership competencies of pharmacists that work in manufacturing pharmaceutical companies (MPCs) in South Africa

Pharmacists are custodians of medicines and play a significant role in the healthcare system as the profession responsible for managing medicines. A self-administered online questionnaire was distributed to pharmacists who are members of the South African Association for Pharmacists in Industry (SAAPI). A majority (66%) of responses received was from females, 87.9% identified experience as the attribute necessary for pharmacists to enter positions of strategic leadership as opposed to availability of opportunities (69.7%) and having an additional business-related qualification (60.6%). Pharmacists that work in the manufacturing sector of the industry were mostly working at technical levels. They were in positions such as responsible pharmacist (RP) (24,1%, n=66), regulatory affairs (18,5%, n=66), quality assurance (16,7%, n=66) production (11,1%, n=66) pharmacists. The pharmacists felt that experience was the key attribute required for entry into strategic leadership positions of manufacturing pharmaceutical companies (MPCs), the attribute for possessing business-related qualifications followed this. The need for a quota system that makes provision for a pharmacist to be present in certain strategic leadership positions of an MPC, was supported by respondents (58%, n=66), 91% of the respondents deemed it necessary for all MPCs to have a pharmacist in the strategic leadership of MPCs. The presence of pharmacists in the strategic leadership of MPCs was limited. The pharmacists who participated in the study felt that their presence could add value as strategic leaders of MPCs

Pharmacists as Strategic Leaders of Manufacturing Pharmaceutical Companies with Operations in South Africa

The manufacturing sector of the pharmaceutical industry has faced criticism for disparities in access to pharmaceuticals, especially within the context of past incidents and the COVID-19 pandemic. Balancing profitability with the public responsibility to produce affordable, safe and effective medicines is challenging. The World Health Organisation (WHO) recognises the significant role pharmacists play in discovering, manufacturing and dispensing medicines. Pharmacists are responsible for safeguarding pharmaceuticals at all levels of care and where medicines are used. The research aimed to assess the involvement of pharmacists in the strategic leadership of Multinational Pharmaceutical Companies (MPCs) operating in South Africa. The study assessed the presence of pharmacists, recognised as custodians of medicines, in the strategic leadership of pharmaceutical companies operating in South Africa but headquartered globally. A desktop review was done to assess the company profiles, including revenue, size, number of employees and professional backgrounds of the persons in strategic leadership, including board and executive levels. The pharmaceutical companies were headquartered in eleven countries across Asia (3), Africa (1), North America (1), and Europe (6). On average, these companies operated in 86.6 countries (SD ±46.2). The strategic leadership roles within MPCs were comprised of individuals with backgrounds in commerce, sciences, and engineering. Predominantly, professionals with backgrounds in commerce held significant representation in both board membership and executive leadership within these companies. Notably, only 3.2% (33 out of 1023) of leaders possessed a pharmacy qualification, with a mere 27% (9 out of 33) being female. This was the least represented professional background among the strategic leaders, and the likelihood was affected by gender. The pharmacists more likely to hold strategic positions were predominantly male, had additional qualifications, and were situated in specific countries like India and South Africa. Pharmaceutical companies are essential in producing medicines to address global healthcare needs, functioning as healthcare service providers. Strategic leaders in these companies guide the manufacturing sites’ strategic goals of the companies. The study’s outcomes revealed a restricted presence of pharmacists in leadership roles despite their typical responsibility for manufacturing sites. These pharmacists were often found to have limited authority and were excluded from pivotal decision-making processes, resulting in significant implications for patient welfare

Current Advances in Specialised Niosomal Drug Delivery: Manufacture, Characterization and Drug Delivery Applications

Development of nanomaterials for drug delivery has received considerable attention due to their potential for achieving on-target delivery to the diseased area while the surrounding healthy tissue is spared. Safe and efficiently delivered payloads have always been a challenge in pharmaceutics. Niosomes are self-assembled vesicular nanocarriers formed by hydration of a non-ionic surfactant, cholesterol or other molecules that combine to form a versatile drug delivery system with a variety of applications ranging from topical delivery to targeted delivery. Niosomes have advantages similar to those of liposomes with regards to their ability to incorporate both hydrophilic and hydrophobic payloads. Moreover, niosomes have simple manufacturing methods, low production cost and exhibit extended stability, consequently overcoming the major drawbacks associated with liposomes. This review provides a comprehensive summary of niosomal research to date, including the types of niosomes and critical material attributes (CMA) and critical process parameters (CPP) of niosomes and their effects on the critical quality attributes (CQA) of the technology. Furthermore, physical characterisation techniques of niosomes are provided. The review then highlights recent applications of specialised niosomes in drug delivery. Finally, limitations and prospects for this technology are discussed