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The International Mouse Phenotyping Consortium reports the generation of new mouse mutant strains for over 5,000 genes from targeted embryonic stem cells on the C57BL/6N genetic background. This includes 2,850 null alleles for which no equivalent mutant mouse line exists, 2,987 novel conditional-ready alleles, and 4,433 novel reporter alleles. This nearly triples the number of genes with reporter alleles and almost doubles the number of conditional alleles available to the scientific community. When combined with more than 30 years of community effort, the total mutant allele mouse resource covers more than half of the genome. The extensively validated collection is archived and distributed through public repositories, facilitating availability to the worldwide biomedical research community, and expanding our understanding of gene function and human disease.

The cancer burden is increasing worldwide and is expected tobecome the most important barrier to increasing life expectancy 21stcentury. Low- and middle-income countries are expected to showthe biggest increase in incidence. Cisplatin is a frequently usedchemotherapy, however, challenges like drug resistance and sideeffects have yet to be overcome. A promising solution to thisproblem is the co- administration of cisplatin with other compounds.Many cancer patients in sub-Saharan countries co-administer herbaland prescription drugs. African potato (AP) is a popular choice for itsnegligible side effects and immune-boosting properties. One problemassociated with orally administered drug is poor bioavailability. ThePheroid® delivery system has shown to improve drug bioavailabilityand efficacy. No studies have been done on the interaction between AP and cisplatin, hence, this study aims to formulate a combinationof AP and cisplatin in Pheroids® and evaluate its anticancerproperties. Cytotoxicity of AP and cisplatin will be testedin vitroagainst breast (MCF-7) and prostate (PC-3) cancer cell lines todetermine the combination (CI) and dose reduction index of AP withcisplatin. Pheroids® will be examined for their optimal formulation.Anticancer activity of Pheroid® formulations will be tested throughvarious assays. For the pharmacokinetic study, Sprague- Dawley ratswill be used due to the volume of the blood samples required. Ratswill be administered with cisplatin, unformulated AP-cisplatin orPheroid® formulated AP-cisplatin (N= 10). For the efficacy study,nude mice that will be used as the models have already beenestablished. Animals will be implanted with either PC-3 or MCF-7cells. Once tumor volume has reached≥150 mm3 they will be treatedwith cisplatin, unformulated AP-cisplatin, Pheroid® formulated AP-cisplatin or empty Pheroids® (N= 10). AP and cisplatin have asimilar mode of action, hence synergistic activity whenco-administered is expected. As a result, lower concentrations ofcisplatin are expected to yield potent activity compared to individualadministration, for both Pheroid® formulations and free drugformulations

To evaluate the efficacy of newly synthesized therapies prior to clinical trials, valid and true cancer models should be used [1]. For modelling cancer in vivo, transplantation of rodent (allograft) and human cancer cells (xenograft) in immune-competent and immunedeficient mice, respectively is common [2]. The aim of this study was to develop and characterize breast cancer allograft and human ovarian xenograft cancer rodent models. The allograft model was established by injection of E0771 cells suspended in Matrigel, subcutaneously into the mammary fat pad of female C57BL/6 mice. For xenograft model, human OVCAR-3 cells suspended in Matrigel were injected subcutaneously into the hind flank of athymic nude female mice. Once a tumor was palpable, tumor growth was monitored 2–3 times a week using a digital caliper and the animals were euthanized once tumor volume of ≥300 mm3 was attained. Finally, haematoxylin and eosin staining of the tumors was conducted to confirm malignancy. For the E0771 derived allograft model (Fig. 1), tumors were detectable within a week post inoculation with a tumor take rate of 14/14 (100%). For OVCAR-3 derived xenograft model (Figure1B), tumors were detected approximately 5 weeks post inoculation with tumor take rate of 3/4 (75%). Histological analysis of both models revealed mitotic figures indicating that the tumors were actively proliferating and malignant. The rodent models of breast and ovarian cancer were successfully established and characterized and; can be used to evaluate novel clinical compounds and formulations

Goserelin is a peptide drug that suppresses levels of testosterone and oestradiol [1]. The current formulation is presented in a sustained-release depot with several approved indications such as treatment of endometriosis, prostate cancer and breast cancer. The depots are placed subcutaneously with a large bore needle, hence there is a need for less invasive administration. An oral formulation in a drug delivery system such as Pheroid® would offer ease of administration to the patient, improved bioavailability as well as reduced manufacturing costs [2]. Therefore, the aim of this study was to determine the effect of entrapment of goserelin in Pheroid® on the pharmacokinetics (PK) of goserelin as well as the pharmacodynamic (PD) effects, in mice. Goserelin was entrapped in Pheroid® and characte- rized in terms of size, zeta potential and morphology. Male Balb/c mice, 6–8 weeks old were administered goserelin subcutaneously and goserelin entrapped in Pheroid® orally. At selected time points, mice were euthanised, blood collected and analysed for goserelin and testosterone levels by LCMS/MS to determine the PK and PD effects respectively. The size and zeta potential of Pheroid® remained in the expected ranges suggesting that entrapment of goserelin had no effect on Pheroid® vesicle size and stability. Confocal imaging showed satisfactory quality of the Pheroid® and goserelin did not have any effect on the Pheroid® structure. Entrapment of goserelin in Pheroid led to detectable levels of goserelin in plasma of the mice and a corresponding decrease in testosterone levels. The results suggest that Pheroid® may improve the oral bioavailability of goserelin and could be a potential strategy for an oral formulation which would be less invasive for patients

Goserelin is a type of hormone therapy used in addition to standard adjuvant therapy. Currently, goserelin is only available as a slow releasing subcutaneous implant but this is an invasive and generally not well- tolerated method of administration. By using a drug delivery system (DDS), peptides can be protected from the harsh environment of the gastrointestinal (GI) tract. Pheroid® technology is one such example of a drug delivery system. This delivery system is a patented, colloidal drug delivery system and is capable of targeting specific areas for treatment, transportation of genetic material to the cellular nucleus and the decrease of drug resistance, all this while improving the delivery of dynamic complexes [1,2]. The prospect of changing to a less invasive administration method will yield a great advantage. Goserelin was investigated in female Balb/c mice. Pheroid® technology was used as a drug delivery system (DDS) to compare the bioavailability of goserelin subcutaneous depot against a peroral formulation with goserelin pro-Pheroid® formulation. The mice were divided into three groups; group one received goserelin subcutaneously, group 2 received an oral dose of 4 mg/kg pro-Pheroid®-goserelin and group 3 being the control group which received only pro- Pheroid®. Vaginal cytology was evaluated to track the disruption of the oestrus cycle Vaginal cytology results indicated a disruption in the oestrus cycle in the subcutaneous groups and the pro- Pheroid®-goserelin oral group. No disruption in the cycle was observed for the pro-Pheroid® oral group. Entrapment in Pheroid® resulted in oral bioavailability of goserelin as indicated by the related effect on the oestrus cycle. No disruption of the cycle was observed in the pro-pheroid® oral group, thus, goserelin was successfully absorbed in the blood after the peroral administration with pro-Pheroid. In conclusion, Pheroid® technology seemed to protect goserelin from the harsh environment of the gastrointestinal tract and could thus be a helpful DDS

Tafenoquine (TQ), a new synthetic analog of primaquine, has relatively poor bioavailability and associated toxicity in glucose-6-phosphate dehydrogenase (G6PD)-deficient individuals. A microemulsion formulation of TQ (MTQ) with sizes

MSc (Pharmaceutical Science), North-West University, Potchefstroom Campus The therapeutic amphiphilic peptide, goserelin, being investigated in these studies, is a type of hormone therapy generally used in addition to standard adjuvant therapy. Goserelin serves as treatment for numerous hormone-dependent disorders, for instance benign conditions (e.g. uterine fibroids, menorrhagia, endometriosis etc.) or malignant tumours (breast, ovarian, endometrial and prostate carcinoma). Goserelin is a synthetic analogue of the naturally occurring gonadotropin-releasing hormone (GnRH), which is also commonly known as a GnRH agonist. Goserelin acts directly on the hypothalamic-pituitary axis, consequently leading to receptor down-regulation, which in turn leads to the inhibition of the secretion of the pituitary gonadotropins. This causes a decrease of endogenous testosterone in males and oestrogen in females. Through this, a hypogonadal status is achieved. Goserelin in combination with menotropins or recombinant follicle-stimulating hormone (FSH) is commonly used for women undergoing in vitro fertilisation (IVF), as it aids in inducing folliculogenesis for controlled ovarian hyperstimulation. Currently, goserelin is administered as a slow releasing subcutaneous depot (slow releases goserelin over a period of 28 days); this is typically a more invasive administration regimen and commonly not usually tolerated well by patients, thus changing the administration route from subcutaneous to oral may yield great advantages. Goserelin, being a peptide, will face several challenges after oral administration, such as the acidic and enzymatic degradation in the gastrointestinal tract (GI) tract; however, these can be overcome by different strategies, such as formulation and chemical technologies. Using a particulate drug delivery system (DDS) to protect peptide drugs is an example of a formulation technology. Pheroid® is a novel colloidal DDS, with the capability of protecting a therapeutic drug and consequently increasing the bioavailability. In this study, goserelin was used in combination with Pheroid® technology to determine the pharmacokinetics and pharmacodynamics of goserelin after administration of an oral dose of a novel pro-Pheroid®-goserelin formulation, in comparison to the existing goserelin subcutaneous implant (Zoladex®) in male and female BALB/c mice. The physicochemical characteristics, i.e. particle size, colloidal stability and morphology, of this novel Pheroid® formulation of goserelin were also investigated. The effect of entrapment of goserelin in Pheroid® on its gastrointestinal stability was determined by comparing the stability of goserelin and pro-Pheroid®-goserelin in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). The characteristics of Pheroid® revealed no change after the addition of goserelin to pro-Pheroid®. The stability study was non-conclusive due to loss of sample during centrifugation of the SIF and SGF of the pro-Pheroid®-goserelin samples. An optimised and revised stability study will be conducted in the near future. The male BALB/c mice were divided into three groups: group 1 received the peptide subcutaneously, group 2 received pro-Pheroid®-goserelin as an oral dose of 2 mg/kg and group 3 received pro-Pheroid®-goserelin as an oral dose of 4 mg/kg. Blood samples were collected at different time points and the plasma was analysed for testosterone and goserelin concentrations using LC-MS/MS. From the goserelin concentration-time profile, the observed peak plasma concentration (Cmax) and time to maximum concentration (Tmax) were reported and statistically compared. An expected significantly higher Cmax and Tmax were observed in the subcutaneous group. The 2 mg/kg pro-Pheroid® goserelin resulted in a significantly higher Cmax compared to the 4 mg/kg dose. An initial (0 - 30 minutes) increase in the testosterone concentration for each group was observed, thus adhering to the flare-up effect. Both the 2 mg/kg and 4 mg/kg pro-Pheroid® groups proved to have an effect on the testosterone concentration. The 4 mg/kg pro-Pheroid®-goserelin was chosen for further investigation in female BALB/c mice. Hence, it can be concluded that goserelin did succeed in reaching the blood and consequently caused a decrease in the testosterone concentration after the oral administration with pro-Pheroid®. The female BALB/c mice were also divided into three groups; group 1 received goserelin subcutaneously, group 2 received 4 mg/kg pro-Pheroid®-goserelin through oral gavage and group 3, the negative control group, received only pro-Pheroid®. Evaluation for the daily vaginal smears was done by vaginal cytology for 21 days, to track the disruption or lack thereof of the 4-stage oetrous cycle. The vaginal cytology revealed disruption in the oestrous cycle in groups 1 and 2. Thus it can be concluded that the oral administration of goserelin in pro-Pheroid resulted in sufficient uptake to cause the hormonal effects. The pro-Pheroid®-goserelin formulation in both the male and female studies resulted in bioavailable goserelin as indicated by the related pharmacodynamic (PD) results. In summary, therefore, Pheroid® technology proved to protect goserelin from the degradation in the GI tract and could thus be a helpful DDS for oral delivery of therapeutic drugs. Masters

MSc (Pharmaceutical Sciences), North-West University, Potchefstroom Campus Background: Diabetes mellitus is a chronic disease that causes high blood sugar. The cost and side effects of modern antidiabetic drugs have become a major burden to patients suffering from diabetes, especially those from developing countries. As a result, medicinal plants are considered a favourable alternative for antidiabetic treatment in developing countries. In South Africa there is anecdotal evidence that the 1:1 combination of Catharanthus roseus (C. roseus) and Portulacaria afra (P. afra) are being used to treat symptoms of diabetes mellitus. There are multiple scientific reports on the in vitro and in vivo antidiabetic potential of C. roseus, whilst there is insufficient data on the antidiabetic effects of P. afra and its combination with C. roseus. Aim: This present study investigated the antidiabetic potential of the aqueous leaf extract of Catharanthus roseus and Portulacaria afra, independently and in combination (1:1), in both in vitro and in vivo models. Methods: In the in vitro study, the safety of the plants was determined by measuring the cytotoxicity against HepG2 cell line. In addition, the antidiabetic potentials of the plant extracts were evaluated by measuring the enzyme activity (α-amylase, α-glucosidase and hexokinase) — common markers of diabetes therapy —in the HepG2 cells. An in vivo study was conducted using a chemically induced diabetic rat model. Diabetes was induced in male Sprague Dawley rats (50) by a single intravenous injection of streptozotocin at 55 mg/kg body weight. Animals varying between seven to nine weeks of age were randomly assigned to six groups; non-diabetic (n = 10), diabetic non-treated (n = 8), metformin (500 mg/kg body weight, n = 8), C. roseus (CR, 100 mg/kg body weight, n = 8), P. afra (PA, 100 mg/kg body weight, n = 8) and 1:1 combination of C. roseus and P. afra (CR:PA, 100 mg/kg body weight, n = 8). The rats were treated by receiving an oral administration of the aqueous leaf extracts of CR, PA and CR:PA for 28 consecutive days. Results: The 1:1 extract combination of C. roseus and P. afra, with a IC50 value of 4.10 μg/mL (i.e. NR assay), had the highest cytotoxicity on the HepG2 cell line, with statistical significance (p < 0.05), compared to P. afra (IC50 = 27.93 μg/mL). Extract of C. roseus (IC50 = 6.02 μg/mL i.e. NR assay) was also cytotoxic towards liver cells, whilst P. afra had the least cytotoxic effect on the liver cells (IC50 = 27.93 μg/mL i.e. NR assay). Further cytotoxic investigation (i.e. MTT assay) showed that the interference of copper compounds with MTT formazan, rendered the MTT assay to be less suitable for cell viability measurements when compared to the neutral red assay. The results of the in vitro assays showed that the plant extracts had no effects on the α-glucosidase activity in HepG2 cells. However, the inhibition activity of P. afra and CR:PA extracts on α-amylase and the activation of the liver hexokinase enzyme by the plant extracts, suggested potential antidiabetic effects that are commonly involved in the reduction of blood glucose levels. The in vivo study demonstrated that daily oral gavage with C. roseus effectively lowered plasma glucose in diabetic rats. In addition, administration of C. roseus showed improved reversal effects in the relative weights of the heart, liver and kidneys, whilst also maintaining the body weights in diabetic rats. In contrast, oral treatment with P. afra resulted in elevated plasma glucose levels and caused a supplemental increase in the relative liver and kidney weights. The treatment with P. afra to diabetic rats resulted in a significant decline in body weights (p < 0.05). The administration of CR:PA to diabetic rats exhibited similar results in the liver and kidneys, thus displaying an antagonistic effect between C. roseus and P. afra. While C. roseus lowered the blood glucose levels in diabetic rats, P. afra raised it. Further investigation showed that the relative weights of the pancreas were unaffected for all treatment groups, except for CR:PA-treated rats, which displayed a significant decrease in comparison to non-diabetic rats (p < 0.05). The reason for this incident is still not well understood and would require further evaluation through organ histology. Understanding the major physiological function of these two plant extracts would be crucial for future studies. Conclusion: The present in vitro data displayed potential antidiabetic effects of aqueous leaf extracts of P. afra and its 1:1 combination with C. roseus, whilst this was not the case for the in vivo study. The leaf extract of P. afra caused hyperglycaemic effects in diabetic rats, meaning that the alleged antidiabetic effects of P. afra, as presented by the inhibition activity of α-amylase and activation of hexokinase (as seen by the in vitro data), had no lowering effects on the blood glucose levels in animal models. The in vivo data demonstrated that the combination of CR:PA displayed antagonistic effects in glycaemic control, as an effect that was produced by the contrasting actions of C. roseus and P. afra. The current study evidently revealed the shortcomings associated with in vitro studies, one of which was the misleading antidiabetic results that was presented by the in vitro data. Our study has conclusively shown that the efficacy of drugs is better evaluated when using complex in vivo models such as animal. However, one of the disadvantages of STZ is that it specifically targets β-cells in the pancreas. Therefore, the STZ model could contain potential limitations to the proposed testing of the antidiabetic effects of the plants. In addition, one would need to perform a carbohydrate-loading test to see whether the increased postprandial blood glucose levels are decreased by the plant extracts. In conclusion, P. afra and its 1:1 combination with C. roseus (CR:PA) leaf extracts have shown no antidiabetic effects in Sprague Dawley rats, whereas leaf extracts of C. roseus did show antidiabetic activities. Masters

Tuberculosis (TB) is a major cause of childhood death. Despite the startling statistics, it is neglected globally as evidenced by treatment and clinical care schemes, mostly extrapolated from studies in adults. The objective of this study was to formulate and evaluate a reconstitutable dry suspension (RDS) containing isoniazid, a first-line anti-tubercular agent used in the treatment and prevention of TB infection in both children and adults. The RDS formulation was prepared by direct dispersion emulsification of an aqueous-lipid particulate interphase coupled with lyophilization and dry milling. The RDS appeared as a cream-white free-flowing powder with a semi-crystalline and microparticulate nature. Isoniazid release was characterized with an initial burst up to 5 minutes followed by a cumulative release of 67.88% &plusmn, 1.88% (pH 1.2), 60.18% &plusmn, 3.33% (pH 6.8), and 49.36% &plusmn, 2.83% (pH 7.4) over 2 hours. An extended release at pH 7.4 and 100% drug liberation was achieved within 300 minutes. The generated release profile best fitted the zero order kinetics (R2 = 0.976). RDS was re-dispersible and remained stable in the dried and reconstituted states over 4 months and 11 days, respectively, under common storage conditions.

MSc (Pharmaceutical), North-West University, Potchefstroom Campus Introduction: Herpes simplex virus type 2 (HSV-2) has been a global epidemic for many years. This disease is prevalent in Europe, North America, and Africa. HSV-2 is one of the most common sexually transmitted infections (STIs) in low-income countries with high incident rates of HIV/AIDS. With more than 20 million cases reported annually, it has become an issue of great concern. As an incurable STI, preventative alternatives are preferred over the development of treatments. There is a need to develop preventative strategies to effectively reduce HSV infection. Animal models are the preferred biological systems for the testing of safety and efficacy of the newly developed products, including vaccines and novel microbicides. In our study, we developed and characterised a murine model which can be applied in the evaluation of Bi-SP, a novel bismuth (III) complex which has previously been shown to display anti-microbial activity in vitro against HSV-2 and other STI causing agents. Methods: For model establishment, six to eight-week old fasted male (n = 8) and medroxyprogesterone pretreated female (n = 8) BALB/c mice were administered 10 μl of sterile phosphate buffered saline (PBS) rectally and vaginally respectively. Ten minutes after PBS administration, the mice were infected with 16.6 μl of HSV-2 strain G at a viral titer of 1 x 106 plaque forming units. The mice were monitored for infection twice a day for the duration of the study. Vaginal and rectal swab samples were collected from all animals 2 days post infection, immediately before euthanasia and after euthanasia. The swab samples were assayed for viable virus by virus plaque assay and CD4+ and CD8+ analysis using flow cytometry. Rectal and vaginal tissue samples from male and female mice respectively were collected from euthanised animals for histology. The tissue samples were fixed in formalin and were processed through a series of dehydration and elucidation steps and embedded in histoplast wax. The tissues were transversely sectioned with a microtome at 5 – 7 μm sections. Sectioned bands were mounted on glass microscopic slides and were stained with hematoxylin & eosin. The stained tissues were evaluated for epithelial cell disruption and evidence of inflammation under a compound microscope. Safety assessment of Bi-SP was conducted in male (n = 5) and female BALB/c mice (n = 5) at 5 μg/animal. Administration of the novel microbicide was done rectally and vaginally using a 2 – 20 μl pipette. Rectal and vaginal tissue samples were collected 2 hours after Bi-SP administration for histopathology. In determining the efficacy of the novel microbicide, female mice were administered Bi-SP at 5 μg/animal in PBS (n = 10), 5 μg/animal in a hypo-osmotic 2.7% HEC gel (n = 10) and 2.7% HEC gel only as control (n = 10), vaginally. The male mice were administered Bi-SP at 5 μg/animal (n = 20) and 10 μg/animal (n = 10) in PBS, rectally. Ten minutes after microbicide administration, the mice were infected with HSV-2 strain G at a titer of 1 x 106 PFU. The male (n = 1) and female (n = 1) control mice were administered with sterile PBS. The experiments were conducted in a biosafety level (BSL)-3 laboratory and the mice were monitored for infection twice a day over a period of 19 days. During the course of the study, the animals were monitored and clinical scores were assigned. Once the animals reached the humane endpoint clinical score, they were euthanised. The rectal and vaginal swab samples were collected 2 days post-infection and immediately before euthanasia. For virus plaque assay, only swabs collected 2 days post infection were used while CD4+ and CD8+ analysis was done using swabs from 2 days post infection and immediately before euthanasia. Vaginal and rectal tissues were excised from the female and male mice respectively, after euthanasia. The tissues were sliced open longitudinally and stored in formalin for histological analysis. Results: The survival rate of male and female BALB/c mice infected with HSV-2 was reduced to 0%, 9 days post infection. All the animals reached severe clinical symptoms between days seven and nine post infection. The symptoms included severe inflammation perianally and perivaginally in male and female mice respectively. Severe swelling of the bladder and constipation in the digestive tract were observed. Virus plaque assays confirmed the presence of viable virus in the collected swab samples. Histological evaluation revealed elevated levels of inflammatory infiltrate in the submucosa and severe disruption on the mucosa, more specifically the glands and the glandular crypts in rectal tissues collected from male mice. A thinned squamous epithelium and an infiltration of inflammatory cells was observed in vaginal tissue collected from female mice, which was indicative of tissue damage. The presence of the virus from swab samples and the effect HSV-2 had on the vaginal and rectal mucosa is confirmation of the susceptibility of BALB/c mice to HSV-2 strain G. Bi-SP at 5 μg/animal did not cause tissue damage. The microbicide demonstrated significant activity (p

MSc (Pharmaceutical Sciences), North-West University, Potchefstroom Campus Cancer is a global health burden; of which lung cancer is the most frequently diagnosed type and has the highest mortality. The survival profile of lung cancer patients at all stages is dismal despite the availability of various treatment options. This underscores the dire need for alternative treatment options that have better treatment modalities. In this regard, phytochemicals have been used as an emerging treatment strategy to combat all cancer types. Curcumin and ginger extract (GE) phenolics — particularly [6]-shogaol (6SG) — have been shown to have promising chemopreventive activity, the latter being the most potent one. A combination of phytochemicals often produces a more profound anticancer activity than single agent treatment. However, the clinical utility of phytochemicals is restricted owing to their poor physicochemical properties, which can be enhanced by using drug delivery systems such as the Pheroid® technology. In this study, the therapeutic activity of combined Meriva®; a curcumin phytosome, and GE Pheroid® formulations were investigated against the human lung cancer adenocarcinoma A549 cell line both in vitro and in vivo. The contents of curcumin in Meriva® and 6SG in GE were quantified. In the in vitro study, cellular uptake, cell viability, apoptosis, oxidative stress markers and mitochondrial health were assessed. Furthermore, the formulation administered to the animals was characterised in terms of particle size and distribution, zeta potential and compatibility assays. An in vivo study was conducted using an established athymic nude mice xenograft model. Accordingly, male and female athymic nude mice were inoculated with viable A549 cancer cells. Once the tumour volume reached a palpable size, mice were allocated into four groups and received a daily oral gavage of saline, Pheroid® only and phytochemical combination in Pheroid® for 14 days. Cisplatin was injected intraperitonially once a week. The amounts of principal actives — curcumin and 6SG — in Meriva® and GE were found to be 400 mg/g and 11 mg/g, respectively. Zeta potential and compatibility studies indicated that the phytochemicals were stable in Pheroid® and that no drug-excipient interactions were observed. Confocal microscopy revealed co-localisation of phytochemicals within the Pheroid® vesicles. In vitro results indicated that Pheroid® significantly enhanced cellular uptake, anti-proliferative and apoptotic effects of phytochemical combination compared to individual actives and the free active DMSO formulations. From the mitochondrial health assessment, it was noted that Meriva® but not GE was responsible for the mitochondrial dysfunction, and the effect was more pronounced in the Pheroid® formulation than in the free forms. In addition, the anticancer activity observed with combined phytochemicals in Pheroid® was without induction of oxidative stress, indicating the potential safe use of the formulation. The in vivo study demonstrated that daily oral gavage with the Pheroid® formulated phytochemical combination non-significantly reduced the tumour growth and burden in mice compared to the Pheroid® only treatment. However, cisplatin significantly reduced tumour growth compared to the saline negative control. The sub-therapeutic effect observed with the phytochemical combination treatment can be attributed to the suboptimal dose of curcumin and 6SG principal actives administered in the formulation. In addition, treatment with cisplatin was accompanied with a reduction in body mass. Research has indicated that the side effects of chemotherapeutic drugs such as cisplatin can be overcome by co-administration of phytochemicals. The present in vitro and in vivo study conclusively show the potential anticancer activity of combined Meriva® and ginger extract phenolic compounds. Pheroid® significantly improved the biological activity of the phytochemicals. In addition, the study opens an opportunity to further investigate the anticancer activity of cisplatin co-administered with therapeutic doses of curcumin and 6SG phytochemicals against lung cancer. Masters

The purpose of this reported study was to develop and validate an LC-MS/MS method for the quantification of goserelin in a Pheroid (R) formulation simulated intestinal fluid. Biopharmaceuticals are formulated in drug delivery systems to improve their gastrointestinal stability. Goserelin, a peptide drug was formulated in Pheroid (R) delivery system and its gastrointestinal stability assessed using simulated intestinal fluid, which required an assay to determine the varying amounts of goserelin remaining after a specific time. Several extraction methods and solvents investigated to extract goserelin from complex matrix led to either poor recovery, peak shape or high background interference. A rapid gradient reversed-phase method coupled to tandem mass spectrometry detection was optimized for the separation and quantification of the extracted peptide. A simple, reproducible and good recovery extraction procedure for goserelin quantification was achieved through simultaneous acetonitrile protein precipitation and water-saturated n-butanol liquid-liquid extraction with water dilution. The method was found to be rapid, specific, precise and accurate, and successfully applied to determine goserelin remaining content in a simulated intestinal fluid, with potential use in other lipid-based formulation evaluated in simulated intestinal fluids. (C) 2019 Elsevier B.V. All rights reserved

Microemulsions (MEs) are reported to improve the efficacy of a drug, minimise side effects and toxicity of the encapsulated compound. MEs are hypothesized to aid in making the encapsulated compound, which has been incorporated within the ME, safe in vivo due to reduced side effects and toxicity to the kidneys and other non-target organs. This study aimed to contribute to knowledge on the toxicity of the ME delivery system and ME containing [68Ga]Ga-PSMA-11 with specific reference to intravenous tail vein administration. ME and [68Ga]Ga-PSMA-11-ME were synthesized by homogenisation under high temperature and a dose of 150 μl per mouse was administered intravenously in 10 healthy male BALB/c mice. The mice were monitored and evaluated for 14 days for clinical signs, mortality, body weights, and gross necropsy findings. The mice were euthanized after the respective 14-day period followed by isolation of respective organs and blood samples. Blood test results included assessment of alanine transaminase, aspartate transaminase, total protein, urea, creatinine and serum lipid levels. There were no abnormal changes observed in the body weight, coat condition, respiration, mobility and behaviour of any of the mice during the study. The survival rate was 10/10 mice (100%) after 14 days. None of the treatment groups showed any treatment related toxicity to either ME or [68Ga]Ga-PSMA-11-ME. Blood testing showed that liver and kidney function was in the normal range in both mice groups. This treatment regimen of the administered ME and [68Ga]Ga-PSMA-11-ME concentration caused no acute toxicity to small rodents but further investigations are required in larger animal models to justify the safety of [68Ga]Ga-PSMA-11-ME injections into humans

Herpes simplex virus −2 (HSV-2) is a serious global epidemic with an estimated 400 million people infected and about 20 million cases reported annually. The prevalence in South Africa is at 31% in females aged 15–29 years old. Microbicides are self-administered topical agents which provide women-controlled protection against sexually transmitted infections (STIs). There is an urgent need for microbicides which not only inhibit HSV-2 and other STIs but also reduce the risk of HIV contraction. There is a lack of in vivo models to evaluate the safety and efficacy of novel microbicides, hence this study aims to provide an HSV-2 challenge model which can be used to evaluate the safety and efficacy of novel microbicides. Safety assessment of the microbicide was done at 500 μg/mL in male and female Balb/c mice (n = 8) and were inoculated intrarectally and intravaginally respectively. Rectal and vaginal tissue samples were collected 2 h post-inoculation for histology. For model establishment, 1×06 PFU of the HSV-2 strain G virus was used. In the efficacy of the microbicide, male and female mice (n = 10) were inoculated with 10 μl microbicide 10 min before being inoculated with the virus and the control group were inoculated with sterile phosphate buffered saline. The experiments were conducted in a BSL3 lab. The mice were monitored for infection twice a day over 10 days. Histological analysis showed that the microbicide was safe to use as there were no signs of tissue damage. The amounts of virus used was sufficient to induce a definite infection within 8 days. Application of the novel microbicide delayed mortality rate in the challenge group up to day 19 in both male and female groups compared to day 8 in the control group. Balb/c mice are highly susceptible to the HSV-2 virus and are an appropriate model for microbicide studies

MSc (Pharmaceutical Science), North-West University, Potchefstroom Campus Introduction: To improve early detection of breast and ovarian cancer, characterised animal models of cancer are required for screening novel tumour-specific imaging radiopharmaceuticals. The purpose of this study was to establish and characterise allograft and xenograft tumour mouse models of breast and ovarian malignancies, respectively, for evaluation of 64Cu-GluCAB, a novel imaging radiopharmaceutical intended to target tumours through their high expression of glucose receptor (GLUT-1) and their increased vascularisation. Methods: The breast tumour allograft model was established by subcutaneous inoculation of E0771 cells suspended in Matrigel into the mammary fat pad of female C57BL/6 mice. The ovarian tumour xenograft model was established by subcutaneous inoculation of OVCAR-3 cells, with and without Matrigel, above the proximal tibia of the female athymic nude (nu/nu) mice. Tumour growth was monitored using a digital calliper and the tumours were excised after reaching the end-point tumour volume (≥300 mm3) to determine the tumour growth rate and confirm malignancy using haematoxylin and eosin (H &E) staining. To illustrate the application of the tumour models established, the E0771 derived allograft model was used for investigation of the ex vivo biodistribution and in vivo imaging of 64Cu-GluCAB. The mice were administered intravenously with 64Cu-GluCAB precursor (without albumin) and images acquired at 1, 2, 6 and 24 hours using microPET/CT. After 24 hours, blood, tumours and several organs and tissues were collected to determine the compound biodistribution using a gamma counter. Flow cytometry and immunofluorescence staining were conducted in order to evaluate the expression of the GLUT-1 receptor in E0771 cells and E0771 derived tumours. Results: Palpable tumours were detected within one-week post inoculation for the E0771 derived allograft model, with a tumour take rate of 100% (26/26) and average tumour growth rate of 0.03 g/day based on the final ex vivo tumour weight. For the OVCAR-3 derived xenograft model, tumours were palpable within approximately one month and two months with and without Matrigel, respectively, however, the tumour growth rate (based on the final ex vivo tumour weight) with or without Matrigel was statistically insignificant (p>0.05). Histological analysis revealed that the tumours of both models were malignant and actively proliferating. The biodistribution profile of 64Cu-GluCAB illustrated high accumulation of radioactivity in the plasma (4.07 ± 0.21%ID/g), confirming that 64Cu-GluCAB precursor (without albumin) bound to albumin in vivo thereby increasing the biological half-life of the compound. In correlation with the microPET/CT images, high uptake was observed in the liver (3.63 ± 0.80 %ID/g) and large intestine (2.82 ± 1.29 %ID/g), suggesting hepatobiliary excretion of the compound. In contrast, uptake of 64Cu-GluCAB by tumours (0.95 ± 0.30 %ID/g) and other organs was minimal. Moreover, the tumours could not be visualised using microPET/CT. Evaluation of GLUT-1 receptor expression in E0771 cells and E0771 derived tumour, yielded inconclusive results. Conclusion: The tumour-bearing mouse models of breast and ovarian cancers were successfully developed and characterised. Although the expression of the GLUT-1 receptor could not be confirmed, the biodistribution profile of 64Cu-GluCAB indicated a minimal amount of uptake by the tumour. The low radioactivity signal could however not be used for localisation and visualisation of the tumour by microPET/CT. Masters

PhD (Pharmaceutics), North-West University, Potchefstroom Campus It has been reported that microemulsion (ME) delivery systems provide an opportunity to enhance the bioavailability and efficacy of a therapeutic agent whilst minimising side effects. The prostate-specific membrane antigen (PSMA) targeting agents PSMA-11 and PSMA-617, which accumulate in prostate tumours, allows for [68Ga]Ga3+-radiolabelling and PET imaging of PSMA-expression in vivo. Radiolabelled [68Ga]Ga-PSMA-617 can be encapsulated in a ME delivery system which is hypothesized to enhance its pharmacokinetic properties. This study investigated the synthesis of [68Ga]Ga-PSMA-617 and [68Ga]Ga-PSMA-617 contained within a ME, the toxicity profile, and microPET/CT imaging and biodistribution in PC3 tumour xenograft male BALB/c mice. [68Ga]Ga-PSMA-617 was synthesized in a combined solid phase and solution chemistry strategy. The formulation of [68Ga]Ga-PSMA-617 into a ME was then evaluated for in vitro and in vivo toxicity and biodistribution. The cytotoxicity of [68Ga]Ga-PSMA-617-ME was tested in HEK293 and PC3 cells. [68Ga]Ga-PSMA-617-ME indicated negligible cellular toxicity at different concentrations. HEK293 cells showed a statistically higher tolerance towards the [68Ga]Ga-PSMA-617-ME compared to PC3 cells. [68Ga]Ga-PSMA-617 and [68Ga]Ga-PSMA-617-ME was administered intravenously in BALB/c mice with or without PC3-tumours followed by microPET/CT imaging and ex vivo biodistribution determination. The ex vivo biodistribution in PC3-tumour bearing BALB/c mice showed the highest amounts of [68Ga]Ga-PSMA-617 radioactivity accumulation in the kidneys and the lowest uptake was seen in the brain. Both the [68Ga]Ga-PSMA-617 and [68Ga]Ga-PSMA-617-ME followed an expected clearance profile for small-sized polar radiopharmaceuticals, with predominant renal clearance. The ME did alter the biodistribution pattern of [68Ga]Ga-PSMA-617 but maintained distribution to the kidneys, albeit at statistically significant higher levels. Similarly, encapsulation in the ME may have resulted in delayed uptake into tumours as can be seen from the higher blood pool values. The incorporation of [68Ga]Ga-PSMA-617 into ME was successfully demonstrated and resulted in a stable non-toxic formulation as evaluated by in vitro and in vivo means. Both the [68Ga]Ga-PSMA-617 and [68Ga]Ga-PSMA-617-ME showed enterohepatic metabolism of [68Ga]Ga-PSMA-617. Doctoral

Purpose Mycobacterium tuberculosis which causes tuberculosis, is primarily resident within macrophages. 1,3-β-glucan has been proposed as a ligand to target drug loaded nanoparticles (NPs) to macrophages. In this study we characterized the intracellular pharmacokinetics of the anti-tubercular drug rifampicin delivered by 1,3-β-glucan functionalized PLGA NPs (Glu-PLGA). We hypothesized that Glu-PLGA NPs would be taken up at a faster rate than PLGA NPs, and consequently deliver higher amounts of rifampicin into the macrophages. Methods Carbodiimide chemistry was employed to conjugate 1,3-β-glucan and rhodamine to PLGA. Rifampicin loaded PLGA and Glu-PLGA NPs as well as rhodamine functionalized PLGA and Glu-PLGA NPs were synthesized using an emulsion solvent evaporation technique. Intracellular pharmacokinetics of rifampicin and NPs were evaluated in THP-1 derived macrophages. A pharmacokinetic model was developed to describe uptake, and modelling was performed using ADAPT 5 software. Results The NPs increased the rate of uptake of rifampicin by a factor of 17 and 62 in case of PLGA and Glu-PLGA, respectively. Expulsion of NPs from the macrophages was also observed, which was 3 fold greater for Glu-PLGA NPs than for PLGA NPs. However, the ratio of uptake to expulsion was similar for both NPs. After 24 h, the amount of rifampicin delivered by the PLGA and Glu-PLGA NPs was similar. The NPs resulted in at least a 10-fold increase in the uptake of rifampicin. Conclusions Functionalization of PLGA NPs with 1,3-β-glucan resulted in faster uptake of rifampicin into macrophages. These NPs may be useful to achieve rapid intracellular eradication of Mycobacterium tuberculosis

The combination of polymeric nanoparticles (NPs) as a core and lipid vesicles as a shell has emerged to be a robust and promising drug delivery strategy. This study explores the development of a novel combined delivery system where poly d,l, lactic-co-glycolic acid (PLGA) NPs are entrapped within Pheroid® drug delivery system. The solid NPs were combined with the Pheroid® vesicles using two different methods: pre-mix and post-mix. The surface properties of the PLGA NPs were altered through the inclusion (pos-NPs) and exclusion (neg-NPs) of chitosan (CT) and polyethylene glycol (PEG), to evaluate their interaction with the Pheroid® Vesicles. The average particle size of the novel NP–Pheroid® combined system ranged from approximately 1990–2450 nm while the zeta potential (ZP) ranged from −18 to −30 mV, measured using dynamic light scattering (DLS) and electrophoretic velocity techniques, respectively. The NP/Pheroid® mixing ratio experiment indicated that a maximum of 2.5% (w/v) NPs can be optimally added to the Pheroid® vesicles without compromising the structure and the stability of the NP–Pheroid® combined system. Visual analysis of this system was done through transmission electron microscopy (TEM), cryogenic (cryo) TEM and confocal laser scanning microscopy (CLSM) techniques to obtain adequate information of this novel combined drug delivery system which includes the localization of the PLGA NPs with the Pheroid ® vesicles.

Colloidal suspensions of 14 nm gold nanoparticles (AuNPs) were repeatedly administered intravenously at three dose levels (0.9, 9 and 90 µg) to male Sprague Dawley rats weekly for 7 weeks, followed by a 14-day washout period. After sacrificing, the amount of gold was quantified in the liver, lungs, spleen, skeleton and carcass using neutron activation analysis (NAA). During the study, pre- and post (24 h) administration blood samples were collected from both the test and control groups, the latter which received an equal injection volume of normal saline. General health indicators were monitored together with markers of kidney and liver damage for acute and subchronic toxicity assessment. Histopathological assessments were done on the heart, kidneys, liver, lungs and spleen to assess any morphological changes as a result of the exposure to AuNPs. The mass measurements of all the groups showed a steady increase with no signs of overt toxicity. The liver had the highest amount of gold (µg) per gram of tissue after 56 days followed by the spleen, lungs, skeleton and carcass. Markers of kidney and liver damage showed similar trends between the pre and post samples within each group and across groups. The histopathological examination also showed no hepatotoxicity and nephrotoxicity. There was accumulation of Au in tissues after repeated dosing, albeit with no observable overt toxicity, kidney or liver damage.