Engineering synergistically active and bioavailable cost-effective medicines for neglected tropical diseases; the role of excipients

Leishmaniasis is a neglected tropical disease responsible for the ninth largest disease burden in the world threatening 350 million people mostly in developing countries. The lack of efficacy, severe adverse effects, long duration, high cost and parenteral administration of the current therapies result in poor patient compliance and emergence of resistance. Leishmaniasis' unmet need for safer, affordable and more effective treatments is only partly addressed by today's global health product pipeline that focuses on products amenable to rapid clinical development, mainly by reformulating or repurposing existing drugs for new uses. Excipients are necessary for ensuring the stability and bioavailability of currently available antileishmaniasis drugs which in their majority are poorly soluble or have severe side-effects. Thus, selection of excipients that can ensure bioavailability and safety as well as elicit a synergistic effect against the Leishmania parasites without compromising safety will result in a more efficacious, safe and fast to market medicine. We have evaluated the in vitro activity of 30 commercially available generally regarded as safe (GRAS) excipients against different Leishmania spp., their cytotoxicity and potential use for inclusion in novel formulations. Amongst the tested excipients, the compounds with higher selectivity index were Eudragit E100 (cationic triblock copolymer of dimethylaminoethyl methacrylate, butyl methacrylate, and methyl methacrylate), CTAB (cetyltrimethylammonium bromide, cationic), lauric acid, Labrasol (non-ionic, caprylocaproyl polyoxyl-8 glycerides) and sodium deoxycholate. An ideal excipient needs to possess amphiphilic nature with ionic/polar groups and possess a short or medium fatty acid chain such as lauric (C12), capric (C10) or caprylicacid (C8). Inclusion of these excipients and identification of the optimal combination of drug and excipients would lead to a more effective and safer antileishmanial therapies

Strategies to deliver peptide drugs to the brain

Neurological diseases such as neurodegeneration, pain, psychiatric disorders, stroke, and brain cancers would greatly benefit from the use of highly potent and specific peptide pharmaceuticals. Peptides are especially desirable because of their low inherent toxicity. The presence of the blood brain barrier (BBB), their short duration of action, and their need for parenteral administration limits their clinical use. However, over the past decade there have been significant advances in delivering peptides to the central nervous system. Angiopep peptides developed by Angiochem (Montreal, Canada), transferrin antibodies developed by ArmaGen (Santa Monica, USA), and cell penetrating peptides have all shown promise in delivering therapeutic peptides across the BBB after intravenous administration. Noninvasive methods of delivering peptides to the brain include the use of chitosan amphiphile nanoparticles for oral delivery and nose to brain strategies. The uptake of the chitosan amphiphile nanoparticles by the gastrointestinal epithelium is important for oral peptide delivery. Finally protecting peptides from plasma degradation is integral to the success of most of these peptide delivery strategies

Preformulation Studies of a Stable PTEN-PDZ Lipopeptide Able to Cross an In Vitro Blood-Brain-Barrier Model as a Potential Therapy for Alzheimer's Disease

PurposeAmyloid beta (A beta) drives the accumulation of excess Phosphatase and Tensin Homolog Deleted on Chromosome 10 (PTEN) at synapses, inducing synaptic depression and perturbing memory. This recruitment of PTEN to synapses in response to A beta drives its interaction with PSD95/Disc large/Zonula occludens-1 (PDZ) proteins and, indeed, we previously showed that an oligo lipopeptide (PTEN-PDZ) capable of blocking such PTEN:PDZ interactions rescues the synaptic and cognitive deficits in a mouse model of Alzheimer's disease. Hence, the PTEN:PDZ interaction appears to be crucial for A beta -induced synaptic and cognitive impairment. Here we have evaluated the feasibility of using PTEN-PDZ lipopeptides based on the human/mouse PTEN C-terminal sequence, testing their stability in biological fluids, their cytotoxicity, their ability to self-assemble and their in vitro blood-brain barrier (BBB) permeability. Myristoyl or Lauryl tails were added to the peptides to enhance their cell permeability.MethodsLipopeptides self assembly was assessed using electron microscopy and the thioflavin T assay. Stability studies in mouse plasma (50%), intestinal washing, brain and liver homogenates as well as permeability studies across an all human 2D blood-brain barrier model prepared with human cerebral endothelial cells (hCMEC/D3) and human astrocytes (SC-1800) were undertaken.ResultsThe mouse lauryl peptide displayed enhanced overall stability in plasma, ensuring a longer half-life in circulation that meant there were larger amounts available for transport across the BBB (Papp(0-4h): 6.281.85x10(-6) cm s(-1)).ConclusionThis increased availability, coupled to adequate BBB permeability, makes this peptide a good candidate for therapeutic parenteral (intravenous, intramuscular) administration and nose-to-brain delivery.The authors declare that this study received funding from MemoryPlus Ltd. The funder was not involved in the study design, collection, analysis, and interpretation of data. One of the authors of this article (SK) owns the controlling interest in MemoryPlus Ltd., and has filed an application for a U.S. Patent with respect to the compounds, compositions and methods mentioned in this article. SK was involved in the writing of this article and in the decision to submit it for publication

Applications of 3D printing in cancer

The tumour heterogeneity and interindividual variability is a major problem when treating cancer as every patient responds in a different way to the current drug therapies. 3D printing is a tool that can hamper the issues faced in cancer patients allowing for individualisation of treatment by the production of in vitro models with micro-environments mimicking more closely real cancer conditions facilitating complex therapies. Further improvements are required, for example the development of biocompatible bioinks or need for vascularisation. The journey from bench to bedside is challenging from the regulatory point of view where the establishment of manufacturing guidelines, quality systems and safety of use and administration of personalised medicines remains unclear. This review will provide an insight in the major applications of 3D printing in cancer both in the development of in vitro cancer models as well as personalised medicines for cancer patients focused on hydrogels and therapeutic implants

Preformulation studies of a stable PTEN-PDZ lipopeptide able to cross an in vitro blood-brain-barrier model as a potential therapy for Alzheimer's disease

Purpose: Amyloid β (Aβ) drives the accumulation of excess Phosphatase and Tensin Homolog Deleted on Chromosome 10 (PTEN) at synapses, inducing synaptic depression and perturbing memory. This recruitment of PTEN to synapses in response to Aβ drives its interaction with PSD95/Disc large/Zonula occludens-1 (PDZ) proteins and, indeed, we previously showed that an oligo lipopeptide (PTEN-PDZ) capable of blocking such PTEN:PDZ interactions rescues the synaptic and cognitive deficits in a mouse model of Alzheimer’s disease. Hence, the PTEN:PDZ interaction appears to be crucial for Aβ-induced synaptic and cognitive impairment. Here we have evaluated the feasibility of using PTEN-PDZ lipopeptides based on the human/mouse PTEN C-terminal sequence, testing their stability in biological fluids, their cytotoxicity, their ability to self-assemble and their in vitro blood-brain barrier (BBB) permeability. Myristoyl or Lauryl tails were added to the peptides to enhance their cell permeability. Methods: Lipopeptides self assembly was assessed using electron microscopy and the thioflavin T assay. Stability studies in mouse plasma (50%), intestinal washing, brain and liver homogenates as well as permeability studies across an all human 2D blood-brain barrier model prepared with human cerebral endothelial cells (hCMEC/D3) and human astrocytes (SC-1800) were undertaken. Results: The mouse lauryl peptide displayed enhanced overall stability in plasma, ensuring a longer half-life in circulation that meant there were larger amounts available for transport across the BBB (Papp0-4h: 6.28 ± 1.85 × 10−6 cm s−1). Conclusion: This increased availability, coupled to adequate BBB permeability, makes this peptide a good candidate for therapeutic parenteral (intravenous, intramuscular) administration and nose-to-brain delivery

Hydrophobic ion pair for the oral delivery of leucine-enkephalin

1.Purpose The global therapeutic peptides market was valued at US$26.98 billion in 2019 and is projected to reach US$51.24 billion by 2027, growing at a compound annual growth rate (CAGR) of 8.7% from 2020 to 2027 (Research 2021). The peptide market is bound to grow by the increase in metabolic diseases andcancers but their translation remains challenging due to pre-systemic degradation, short plasma half-lives, and poor permeability across physiological barriers. Ion-pairing has been proposed as a method for the oraldelivery of peptides (Griesser, Hetenyi et al. 2017) as it enables increase stability to gastrointestinal enzymedegradation and enhanced permeability across physiological barriers. In this study, we explore the formationof an ion-pair of leucine-enkephalin (LENK), an endogenous opioid pentapeptide with applications as an oraltherapeutic for the treatment of pain (Lalatsa, Lee et al. 2012), Chron’s disease, and other gastrointestinalinflammatory conditions, as a strategy to enhance its oral bioavailability (Owczarek, Cibor et al. 2011). 2.Method Standard orthogenic solid phase peptide synthesis was utilized to synthesize LENK (0.5 mmole scale) (Lalatsa, Lee et al. 2012) and the peptide was obtained in high yield ( >85%) and highpurity ( >95%) as determined by HPLC and LC-MS. LENK (2.73 mmole, 1.52 mg/mL, 3mL) andsodium docusate (3mL) in deionized water (pH: 2.9) were mixed at different molar ratio (1:1,1:3, 1:5 respectively) to understand optimal ratio for pair formation and vortexed over 1 minute prior centrifugation or ultracentrifugation for 90 minutes at 4oC at 40,000 rpm (Hitachi Ultracentrifuge CP1000 NX). The supernatant was separated and the pellet was frozen withliquid nitrogen and lyophilized for 24 hours (Teslar, -50oC, 0.2mbar pressure). The LENK content was characterized by a previously validated HPLC method (Lalatsa, Lee et al. 2012). Intestinal fluid (50mM phosphate buffer, pH 6.6) was prepared from excised mouse intestine(C57BL/6, 8 weeks old, male) as previously described and characterized for protein contentusing the Bradford assay and diluted (1mg mL) (Lalatsa, Lee et al. 2012). Stability studies (37oC, 50 rpm) were undertaken and the remaining LENK was analyzed using HPLC after dilution in ice-cold acetonitrile (1:1) and centrifugation. Permeability studies across a Caco-2 cell monolayer were undertaken for LENK, the ion-paired LENK, and antipyrine with FITC dextran (3-5 kDa) as an internal control as previously described (Hubatsch, Ragnarsson et al. 2007). Caco-2 seed density was 10,000 cells/cm in this experiment. 3.Results The 1:1 and the 1:5 ratios resulted in low ion-pairing yields of 37% and 19%. The optimal ratio for pairing was identified to be 1:3 which resulted in an ion-pairing yield of 56% and this ratio was further tested in stability and permeability studies. Intestinal wash was selected as it more closely describes in vivo data scenarios (McConnell, Basit et al. 2008). LENK degraded rapidly in the intestinal wash (< 60 minutes) while the ion pair showed a 3.5-fold increase in half-life and showed levels that were significantly different after the first initial 10 minutes (Student t-test, p< 0.05) (Figure 1). Permeability across the Caco-2 cells indicate a trend for higher uptake for ion-pair LENK, but due to low TEER values obtained in our experiments due to low cell seed density (Figure 2). 4.Conclusion Ion-paired LENK has shown a significant enhancement in oral gastrointestinal stability and further studies are underway to assess its oral bioavailability across Caco-2 monolayers. Combining ion-pair technology with solid state or additively manufactured formulations can enable the production of an oral LENK formulation for the treatment of pain and inflammatory diseases such as Chron’s disease

Engineering butylglyceryl-modified polysaccharides towards nanomedicines for brain drug delivery

Colloidal systems prepared from carbohydrates are subject of intense research due to their potential to enhance drug permeability through biological membranes, however their characteristics and performance are never compared directly. Here we report the results of a comparative investigation of a series of butylglyceryl-modified polysaccharides (chitosan, guar gum, and pullulan) that were formulated into nanoparticles and loaded with a range of model actives (Doxorubicin, Rhodamine B, Angiotensin II). Butylglyceryl-modified guar gum and corresponding pullulan nanocarriers were more stable at physiological pH compared to those obtained from modified chitosan, and studies of the in-vitro interactions with mouse brain endothelial cells (bEnd3) indicated an increased biological membrane permeability and lack of toxicity at application-relevant concentrations. No significant haemolytic effect was observed, and confocal microscopy and flow cytometry studies confirmed the efficient cellular uptake and cytoplasmic localisation of NPs. Most promising characteristics for brain drug delivery applications were demonstrated by butylglyceryl pullulan nanocarriers

Harnessing the antibacterial properties of fluoridated chitosan polymers against oral biofilms

Dental caries are a worldwide endemic chronic disease affecting people of all ages. Due to the limitations of daily used oral hygiene products, there is an unmet need for new, effective, safe, and economic oral products. We have recently demonstrated that N-(2(2,6-diaminohexanamide)-chitosan (CS3H Lys) has enhanced antibacterial properties against Streptococcus mutans, the main cariogenic bacterium, and here we investigated the effect of fluoridation of this polymer (CS3H Lys F) on its antibacterial properties and the ability to protect teeth from acid demineralization. We further formulated this polymer into mouthwash preparations and studied their cytocompatibility and physicochemical stability over 6 months. CS3H Lys F was 1.6-fold more effective than the highest tested oral NaF dose in preventing acid demineralization. CS3H Lys F has a 3-to 5-fold lower minimum inhibitory concentration value against S. mutants than the values reported for chitosan polymers and showed negligible cell toxicity. The mouthwashes were stable at both 25 and 40◦C. Further work is under way towards other CS3H Lys F oral hygiene products such as a toothpaste