Pharmaceutics

This study aimed to fabricate new variations of glycerol-based polyesters by grafting poly(glycerol adipate) (PGA) with hydrophobic bioactive moieties, tocopherol (TOC), and cholesterol (CHO). Their effects on nanoparticle (NP) formation, drug release, and cellular responses in cancer and normal cells were evaluated. CHO and TOC were successfully grafted onto PGA backbones with 30% and 50% mole grafting. Increasing the percentage of mole grafting in both molecules increased the glass transition temperature and water contact angle of the final polymers but decreased the critical micelle concentration of the formulated particles. PGA-TOC NPs reduced the proliferation of MDA-MB-231 cancer cells. However, they enhanced the proliferation of primary dermal fibroblasts within a specific concentration range. PGA-CHO NPs minimally affected the growth of cancer and normal cells. Both types of NPs did not affect apoptosis or the cell cycle of cancer cells. PGA-CHO and PGA-TOC NPs were able to entrap SN-38, a hydrophobic anticancer drug, with a particle size <200 nm. PGA-CHO NPs had a higher drug loading capacity and a greater drug release than PGA-TOC NPs. However, SN-38-loaded PGA-TOC NPs showed higher toxicity than SN-38 and SN-38-loaded PGA-CHO NPs due to the combined effects of antiproliferation and higher cellular uptake. Compared with SN-38, the drug-loaded NPs more profoundly induced sub-G1 in the cell cycle analysis and apoptosis of cancer cells in a similar pattern. Therefore, PGA-CHO and PGA-TOC polymers have potential applications as delivery systems for anticancer drugs. Full article

Outpatient parenteral antimicrobial therapy (OPAT) with continuous infusion pumps is postulated as a very promising solution to treat complicated infections, such as endocarditis or osteomyelitis, that require patients to stay in hospital during extended periods of time, thus reducing their quality of life and increasing the risk of complications. However, stability studies of drugs in elastomeric devices are scarce, which limits their use in OPAT. Therefore, we evaluated the stability of ampicillin in sodium chloride 0.9% at two different concentrations, 50 and 15 mg/mL, in an elastomeric infusion pump when stored in the refrigerator and subsequently in real-life conditions at two different temperatures, 25 and 32 °C, with and without the use of a cooling device. The 15 mg/mL ampicillin is stable for up to 72 h under refrigeration, allowing subsequent dosing at 25 °C for 24 h with and without a cooling device, but at 32 °C its concentration drops below 90% after 8 h. In contrast, 50 mg/mL ampicillin only remains stable for the first 24 h under refrigeration, and subsequent administration at room temperature is not possible, even with the use of a cooling system. Our data support that 15 mg/mL AMP is suitable for use in OPAT if the volume and rate of infusion are tailored to the dosage needs of antimicrobial treatments. Full article

Proteolysis-Targeting Chimeras (PROTACs) are a promising new technology in drug development. They have rapidly evolved in recent years, with several of them in clinical trials. While most of these advances have been associated with monovalent protein degraders, bivalent PROTACs have also entered clinical trials, although progression to market has been limited. One of the reasons is the complex physicochemical properties of the heterobifunctional PROTACs. A promising strategy to improve pharmacokinetics of highly lipophilic compounds, such as PROTACs, is encapsulation in liposome systems. Here we describe liposome systems for intravenous administration to enhance the PK properties of two bivalent PROTAC molecules, by reducing clearance and increasing systemic coverage. We developed and characterized a PROTAC-in-cyclodextrin liposome system where the drug was retained in the liposome core. In PK studies at 1 mg/kg for GNE-01 the PROTAC-in-cyclodextrin liposome, compared to the solution formulation, showed a 80- and a 380-fold enhancement in AUC for mouse and rat studies, respectively. We further investigated the same PROTAC-in-cyclodextrin liposome system with the second PROTAC (GNE-02), where we monitored both lipid and drug concentrations in vivo. Similarly, in a mouse PK study of GEN-02, the PROTAC-in-cyclodextrin liposome system exhibited enhancement in plasma concentration of a 23× increase over the conventional solution formulation. Importantly, the lipid CL correlated with the drug CL. Additionally, we investigated a conventional liposome approach for GNE-02, where the PROTAC resides in the lipid bilayer. Here, a 5× increase in AUC was observed, compared to the conventional solution formulation, and the drug CL was faster than the lipid CL. These results indicate that the different liposome systems can be tailored to translate across multiple PROTAC systems to modulate and improve plasma concentrations. Optimization of the liposomes could further improve tumor concentration and improve the overall therapeutic index (TI). This delivery technology may be well suited to bring novel protein targeted PROTACs into clinics. Full article

Three-dimensional-printed customizable drug-loaded implants provide promising opportunities to improve the current therapy options. In this study, we present a modular implant in which shape, dosage, and drug release can be individualized independently of each other to patient characteristics to improve parenteral therapy with triamcinolone acetonide (TA) over three months. This study focused on the examination of release modification via fused deposition modeling and subsequent prediction. The filaments for printing consisted of TA, ethyl cellulose, hypromellose, and triethyl citrate. Two-compartment implants were successfully developed, consisting of a shape-adaptable shell and an embedded drug-loaded network. For the network, different strand widths and pore size combinations were printed and analyzed in long-term dissolution studies to evaluate their impact on the release performance. TA release varied between 8.58 ± 1.38 mg and 21.93 mg ± 1.31 mg over three months depending on the network structure and the resulting specific surface area. Two different approaches were employed to predict the TA release over time. Because of the varying release characteristics, applicability was limited, but successful in several cases. Using a simple Higuchi-based approach, good release predictions could be made for a release time of 90 days from the release data of the initial 15 days (RMSEP ≤ 3.15%), reducing the analytical effort and simplifying quality control. These findings are important to establish customizable implants and to optimize the therapy with TA for specific intra-articular diseases. Full article

Tuberculosis (TB), one of the top ten causes of death globally induced by the infection of Mycobacterium tuberculosis (Mtb), remains a grave public health issue worldwide. With almost one-third of the world’s population getting infected by Mtb, between 5% and 10% of these infected individuals are predicted to develop active TB disease, which would not only result in severe tissue damage and necrosis, but also pose serious threats to human life. However, the exact molecular mechanisms underlying the pathogenesis and immunology of TB remain unclear, which significantly restricts the effective control of TB epidemics. Despite significant advances in current detection technologies and treatments for TB, there are still no appropriate solutions that are suitable for simultaneous, early, rapid, and accurate screening of TB. Various cellular events can perturb the development and progression of TB, which are always associated with several specific molecular signaling events controlled by dysregulated gene expression patterns. Long non-coding RNAs (lncRNAs), a kind of non-coding RNA (ncRNA) with a transcript of more than 200 nucleotides in length in eukaryotic cells, have been found to regulate the expression of protein-coding genes that are involved in some critical signaling events, such as inflammatory, pathological, and immunological responses. Increasing evidence has claimed that lncRNAs might directly influence the susceptibility to TB, as well as the development and progression of TB. Therefore, lncRNAs have been widely expected to serve as promising molecular biomarkers and therapeutic targets for TB. In this review, we summarized the functions of lncRNAs and their regulatory roles in the development and progression of TB. More importantly, we widely discussed the potential of lncRNAs to act as TB biomarkers, which would offer new possibilities in novel diagnostic strategy exploration and benefit the control of the TB epidemic. Full article

Depression is a serious mental disorder and the most prevalent cause of disability and suicide worldwide. Quercetin (QER) demonstrated antidepressant effects in rats exhibiting anxiety and depressive-like behaviors. In an attempt to improve QER’s antidepressant activity, a QER-loaded transferosome (QER-TFS) thermosensitive gel for intranasal administration was formulated and optimized. The therapeutic effectiveness of the optimized formulation was assessed in a depressed rat model by conducting a behavioral analysis. Behavioral study criteria such as immobility, swimming, climbing, sucrose intake, number of crossed lines, rearing, active interaction, and latency to feed were all considerably enhanced by intranasal treatment with the QER-TFS in situ gel in contrast to other formulations. A nasal histopathological study indicated that the QER-TFS thermosensitive gel was safe for the nasal mucosa. An immunohistochemical analysis showed that the animals treated with the QER-TFS thermosensitive gel had the lowest levels of c-fos protein expression, and brain histopathological changes in the depressed rats were alleviated. According to pharmacodynamic, immunohistochemical, and histopathological experiments, the intranasal administration of the QER-TFS thermosensitive gel substantially alleviated depressive symptoms in rats. However, extensive preclinical investigations in higher animal models are needed to anticipate its effectiveness in humans. Full article

Drugs may undergo costly preclinical studies but still fail to demonstrate their efficacy in clinical trials, which makes it challenging to discover new drugs. Both in vitro and in vivo models are essential for disease research and therapeutic development. However, these models cannot simulate the physiological and pathological environment in the human body, resulting in limited drug detection and inaccurate disease modelling, failing to provide valid guidance for clinical application. Organs-on-chips (OCs) are devices that serve as a micro-physiological system or a tissue-on-a-chip; they provide accurate insights into certain functions and the pathophysiology of organs to precisely predict the safety and efficiency of drugs in the body. OCs are faster, more economical, and more precise. Thus, they are projected to become a crucial addition to, and a long-term replacement for, traditional preclinical cell cultures, animal studies, and even human clinical trials. This paper first outlines the nature of OCs and their significance, and then details their manufacturing-related materials and methodology. It also discusses applications of OCs in drug screening and disease modelling and treatment, and presents the future perspective of OCs. Full article

Peptides and proteins, two important classes of biomacromolecules, play important roles in the biopharmaceuticals field. As compared with traditional drugs based on small molecules, peptide- and protein-based drugs offer several advantages, although most cannot traverse the cell membrane, a natural barrier that prevents biomacromolecules from directly entering cells. However, drug delivery via cell-penetrating peptides (CPPs) is increasingly replacing traditional approaches that mediate biomacromolecular cellular uptake, due to CPPs’ superior safety and efficiency as drug delivery vehicles. In this review, we describe the discovery of CPPs, recent developments in CPP design, and recent advances in CPP applications for enhanced cellular delivery of peptide- and protein-based drugs. First, we discuss the discovery of natural CPPs in snake, bee, and spider venom. Second, we describe several synthetic types of CPPs, such as cyclic CPPs, glycosylated CPPs, and D-form CPPs. Finally, we summarize and discuss cell membrane permeability characteristics and therapeutic applications of different CPPs when used as vehicles to deliver peptides and proteins to cells, as assessed using various preclinical disease models. Ultimately, this review provides an overview of recent advances in CPP development with relevance to applications related to the therapeutic delivery of biomacromolecular drugs to alleviate diverse diseases. Full article

The neurotensin subtype 1 receptor (NTS₁R) is overexpressed in a number of human tumors, thereby representing a valid target for cancer theranostics with radiolabeled neurotensin (NT) analogs like [^99mTc]Tc-DT1 (DT1, N₄-Gly⁷-NT(8-13)). Thus far, the fast degradation of intravenously injected NT–radioligands by neprilysin (NEP) and angiotensin-converting enzyme (ACE) has compromised their clinical applicability. Aiming at metabolic stability enhancements, we herein introduce (i) DT7 ([DAsn¹⁴]DT1) and (ii) DT8 ([β-Homoleucine¹³]DT1), modified at the C-terminus, along with (iii) DT9 ([(palmitoyl)Lys⁷]DT1), carrying an albumin-binding domain (ABD) at Lys⁷. The biological profiles of the new [^99mTc]Tc–radioligands were compared with [^99mTc]Tc-DT1, using NTS₁R-expressing AsPC-1 cells and mice models without or during NEP/ACE inhibition. The radioligands showed enhanced in vivo stability vs. [^99mTc]Tc-DT1, with [^99mTc]Tc-DT9 displaying full resistance to both peptidases. Furthermore, [^99mTc]Tc-DT9 achieved the highest cell internalization and tumor uptake even without NEP/ACE-inhibition but with unfavorably high background radioactivity levels. Hence, unlike C-terminal modification, the introduction of a pendant ABD group in the linker turned out to be the most promising strategy toward metabolic stability, cell uptake, and tumor accumulation of [^99mTc]Tc-DT1 mimics. To improve the observed suboptimal pharmacokinetics of [^99mTc]Tc-DT9, the replacement of palmitoyl on Lys⁷ by other ABD groups is currently being pursued. Full article

Membrane-active peptides (MAPs) possess unique properties that make them valuable tools for studying membrane structure and function and promising candidates for therapeutic applications. This review paper provides an overview of the fundamental aspects of MAPs, focusing on their membrane interaction mechanisms and potential applications. MAPs exhibit various structural features, including amphipathic structures and specific amino acid residues, enabling selective interaction with multiple membranes. Their mechanisms of action involve disrupting lipid bilayers through different pathways, depending on peptide properties and membrane composition. The therapeutic potential of MAPs is significant. They have demonstrated antimicrobial activity against bacteria and fungi, making them promising alternatives to conventional antibiotics. MAPs can selectively target cancer cells and induce apoptosis, opening new avenues in cancer therapeutics. Additionally, MAPs serve as drug delivery vectors, facilitating the transport of therapeutic cargoes across cell membranes. They represent a fascinating class of biomolecules with significant potential in basic research and clinical applications. Understanding their mechanisms of action and designing peptides with enhanced selectivity and efficacy will further expand their utility in diverse fields. Exploring MAPs holds promise for developing novel therapeutic strategies against infections, cancer, and drug delivery challenges. Full article

Lung cancer remains one of the major human malignancies affecting both men and women worldwide, with non-small cell lung cancer (NSCLC) being the most prevalent type. Multiple mechanisms have been identified that favor tumor growth as well as impede the efficacy of therapeutic regimens in lung cancer patients. Among tumor suppressor genes that play critical roles in regulating cancer growth, the phosphatase and tensin homolog (PTEN) constitutes one of the important family members implicated in controlling various functional activities of tumor cells, including cell proliferation, apoptosis, angiogenesis, and metastasis. Notably, clinical studies have also documented that lung tumors having an impaired, mutated, or loss of PTEN are associated with low survival or high tumor recurrence rates. To that end, PTEN has been explored as a promising target for anti-cancer agents. Importantly, the ability of PTEN to crosstalk with several signaling pathways provides new approaches to devise effective treatment options for lung cancer treatment. The current review highlights the significance of PTEN and its implications in therapeutic approaches against NSCLC. Full article

Pain is an unpleasant sensory and emotional experience that affects every aspect of a patient’s life and which may be treated through different pharmacological and non-pharmacological approaches. Analgesics are the drugs most commonly used to treat pain, and in specific situations, the use of opioids may be considered with caution. These drugs, in fact, do not always induce optimal analgesia in patients, and several problems are associated with their use. The purpose of this narrative review is to describe the pharmacological approaches currently used for the management of chronic pain. We review several aspects, from the pain-scale-based methods currently available to assess the type and intensity of pain, to the most frequently administered drugs (non-narcotic analgesics and narcotic analgesics), whose pharmacological characteristics are briefly reported. Overall, we attempt to provide an overview of different pharmacological treatments while also illustrating the relevant guidelines and indications. We then report the strategies that may be used to reduce problems related to opioid use. Specifically, we focus our attention on therapeutic drug monitoring (TDM), a tool that could help clinicians select the most suitable drug and dose to be used for each patient. The actual potential of using TDM to optimize and personalize opioid-based pain treatments is finally discussed based on recent scientific reports. Full article

Well-characterized and scalable downstream processes for the purification of biologics are extremely demanding for delivering quality therapeutics to patients at a reasonable price. Erythropoietin (EPO) is a blockbuster biologic with diverse clinical applications, but its application is limited to financially well-off societies due to its high price. The high price of EPO is associated with the technical difficulties related to the purification challenge to obtain qualified products with a cost-effective defined process. Though there are reports for the purification of EPO there is no report of a well-characterized downstream process with critical process parameters (CPPs) that can deliver EPO consistently satisfying the quality target product profile (QTPP), which is a critical regulatory requirement. To advance the field, we applied the quality by design (QbD) principle and design of experiment (DoE) protocol to establish an effective process, which is scalable up to 100× batch size satisfying QTPP. We have successfully transformed the process from static mode to dynamic mode and validated it. Insignificant variation (p > 0.05) within and between 1×, 10×, and 100× batches showed that the process is reproducible and seamlessly scalable. The biochemical analysis along with the biofunctionality data ensures that the products from different scale batches were indifferent and comparable to a reference product. Our study thereby established a robust and scalable downstream process of EPO biosimilar satisfying QTPP. The technological scheme presented here can speed up the production of not only EPO but also many other life-saving biologics and make them available to the mass population at a reduced cost. Full article

Blood-brain barrier (BBB) dysfunction is prevalent in Alzheimer’s disease and other neurological disorders. Restoring normal BBB function through RNA therapy is a potential avenue for addressing cerebrovascular changes in these disorders that may lead to cognitive decline. Although lipid nanoparticles have been traditionally used as drug carriers for RNA, bicelles have been emerging as a better alternative because of their higher cellular uptake and superior transfection capabilities. Cationic bicelles composed of DPPC/DC₇PC/DOTAP at molar ratios of 63.8/25.0/11.2 were evaluated for the delivery of RNA in polarized hCMEC/D3 monolayers, a widely used BBB cell culture model. RNA-bicelle complexes were formed at five N/P ratios (1:1 to 5:1) by a thin-film hydration method. The RNA-bicelle complexes at N/P ratios of 3:1 and 4:1 exhibited optimal particle characteristics for cellular delivery. The cellular uptake of cationic bicelles laced with 1 mol% DiI-C18 was confirmed by flow cytometry and confocal microscopy. The ability of cationic bicelles (N/P ratio 4:1) to transfect polarized hCMEC/D3 with FITC-labeled control siRNA was tested vis-a-vis commercially available Lipofectamine RNAiMAX. These studies demonstrated the higher transfection efficiency and greater potential of cationic bicelles for RNA delivery to the BBB endothelium. Full article

The pharmacokinetic variability of nifedipine widely observed in the clinic cannot be fully explained by pharmacogenomics. As a new factor affecting drug metabolism, how the gut microbiota affects the pharmacokinetics of nifedipine needs to be explored. Spontaneously hypertensive rats (SHRs) have been commonly used in hypertension-related research and served as the experimental groups; Wistar rats were used as control groups. In this study, the bioavailability of nifedipine decreased by 18.62% (p < 0.05) in the SHRs compared with the Wistar rats. Changes in microbiota were associated with the difference in pharmacokinetics. The relative abundance of Bacteroides dorei was negatively correlated with AUC_0–t (r = −0.881, p = 0.004) and C_max (r = −0.714, p = 0.047). Analysis of serum bile acid (BA) profiles indicated that glycoursodeoxycholic acid (GUDCA) and glycochenodeoxycholic acid (GCDCA) were significantly increased in the SHRs. Compared with the Wistar rats, the expressions of CYP3A1 and PXR were upregulated and the enzyme activity of CYP3A1 increased in the SHRs. Spearman’s rank correlation revealed that Bacteroides stercoris was negatively correlated with GUDCA (r = −0.7126, p = 0.0264) and GCDCA (r = −0.6878, p = 0.0339). Moreover, GUDCA was negatively correlated with C_max (r = −0.556, p = 0.025). In primary rat hepatocytes, GUDCA could induce the expressions of PXR target genes CYP3A1 and Mdr1a. Furthermore, antibiotic treatments in SHRs verified the impact of microbiota on the pharmacokinetics of nifedipine. Generally, gut microbiota affects the pharmacokinetics of nifedipine through microbial biotransformation or by regulating the enzyme activity of CYP3A1. Full article

P-glycoprotein (P-gp), expressed at the blood-brain barrier (BBB), is critical in preventing brain access to substrate drugs and effluxing amyloid beta (Aβ), a contributor to Alzheimer’s disease (AD). Strategies to regulate P-gp expression therefore may impact central nervous system (CNS) drug delivery and brain Aβ levels. As we have demonstrated that the copper complex copper diacetyl bis(4-methyl-3-thiosemicarbazone) (Cu(ATSM)) increases P-gp expression and function in human brain endothelial cells, the present study assessed the impact of Cu(ATSM) on expression and function of P-gp in mouse brain endothelial cells (mBECs) and capillaries in vivo, as well as in peripheral organs. Isolated mBECs treated with Cu(ATSM) (100 nM for 24 h) exhibited a 1.6-fold increase in P-gp expression and a 20% reduction in accumulation of the P-gp substrate rhodamine 123. Oral administration of Cu(ATSM) (30 mg/kg/day) for 28 days led to a 1.5 & 1.3-fold increase in brain microvascular and hepatic expression of P-gp, respectively, and a 20% reduction in BBB transport of [3H]-digoxin. A metallomic analysis showed a 3.5 and 19.9-fold increase in Cu levels in brain microvessels and livers of Cu(ATSM)-treated mice. Our findings demonstrate that Cu(ATSM) increases P-gp expression and function at the BBB in vivo, with implications for CNS drug delivery and clearance of Aβ in AD. Full article

Acne is a chronic inflammatory skin disease that affects the quality of life of patients. Several treatments exist for acne, but their effectiveness tends to decrease over time due to increasing resistance to treatment and associated side effects. To circumvent these issues, a new approach has emerged that involves combating the pathogen Cutibacterium acnes while maintaining the homeostasis of the skin microbiome. Recently, it was shown that the use of a G2 lysine dendrigraft (G2 dendrimer) could specifically decrease the C. acnes phylotype (IAI) involved in acne, compared to non-acne-causing C. acnes (phylotype II) bacteria. In the present study, we demonstrate that the efficacy of this technology is related to its 3D structure, which, in contrast to the linear form, significantly decreases the inflammation factor (IL-8) linked to acne. In addition, our in-vitro data confirm the specific activity of the G2 dendrimer: after treatment of bacterial cultures and biofilms, the G2 dendrimer affected neither non-acneic C. acnes nor commensal bacteria of the skin (Staphylococcus epidermidis, S. hominis, and Corynebacterium minutissimum). In parallel, comparative in-vitro and in-vivo studies with traditional over-the-counter molecules showed G2’s effects on the survival of commensal bacteria and the reduction of acne outbreaks. Finally, metagenomic analysis of the cutaneous microbiota of volunteers who applied a finished cosmetic product containing the G2 dendrimer confirmed the ability of G2 to rebalance cutaneous acne microbiota dysbiosis while maintaining commensal bacteria. These results confirm the value of using this G2 dendrimer to gently prevent the appearance of acne vulgaris while respecting the cutaneous microbiota. Full article

Lipid nano-systems were prepared and characterized in a series of well-established in vitro tests that could assess their interactions with the hCMEC/D3 and SH-SY5Y cell lines as a model for the blood–brain barrier and neuronal function, accordingly. The prepared formulations of nanoliposomes and nanostructured lipid carriers were characterized by z-average diameters of ~120 nm and ~105 nm, respectively, following a unimodal particle size distribution (PDI < 0.3) and negative Z-potential (−24.30 mV to −31.20 mV). Stability studies implied that the nano-systems were stable in a physiologically relevant medium as well as human plasma, except nanoliposomes containing poloxamer on their surface, where there was an increase in particle size of ~26%. The presence of stealth polymer tends to decrease the amount of adsorbed proteins onto a particle’s surface, according to protein adsorption studies. Both formulations of nanoliposomes were characterized by a low cytotoxicity, while their cell viability was reduced when incubated with the highest concentration (100 μg/mL) of nanostructured lipid formulations, which could have been associated with the consumption of cellular energy, thus resulting in a reduction in metabolic active cells. The uptake of all the nano-systems in the hCMEC/D3 and SH-SY5Y cell lines was successful, most likely following ATP-dependent internalization, as well as transport via passive diffusion. Full article