Handbook of experimental pharmacology最新文献

The molecular basis of chloride transport varies all along the nephron depending on the tubular segments especially in the apical entry of the cell. The major chloride exit pathway during reabsorption is provided by two kidney-specific ClC chloride channels ClC-Ka and ClC-Kb (encoded by CLCNKA and CLCNKB gene, respectively) corresponding to rodent ClC-K1 and ClC-K2 (encoded by Clcnk1 and Clcnk2). These channels function as dimers and their trafficking to the plasma membrane requires the ancillary protein Barttin (encoded by BSND gene). Genetic inactivating variants of the aforementioned genes lead to renal salt-losing nephropathies with or without deafness highlighting the crucial role of ClC-Ka, ClC-Kb, and Barttin in the renal and inner ear chloride handling. The purpose of this chapter is to summarize the latest knowledge on renal chloride structure peculiarity and to provide some insight on the functional expression on the segments of the nephrons and on the related pathological effects.

High molecular weight actives and cell-based therapy have the potential to revolutionize the prophylaxis and therapy of severe diseases. Yet, the size and nature of the agents - proteins, nucleic acids, cells - challenge drug delivery and thus formulation development. Moreover, off-target effects may result in severe adverse drug reactions. This makes delivery and targeting an essential component of high-end drug development. Loading to nanoparticles facilitates delivery and enables targeted mRNA vaccines and tumor therapeutics. Stem cell therapy opens up a new horizon in diabetes type 1 among other domains which may enhance the quality of life and life expectancy. Cell encapsulation protects transplants against the recipient's immune system, may ensure long-term efficacy, avoid severe adverse reactions, and simplify the management of rare and fatal diseases.The knowledge gained so far encourages to widen the spectrum of potential indications. Co-development of the active agent and the vehicle has the potential to accelerate drug research. One recommended starting point is the use of computational approaches. Transferability of preclinical data to humans will benefit from performing studies first on validated human 3D disease models reflecting the target tissue, followed by studies on validated animal models. This makes approaching a new level in drug development a multidisciplinary but ultimately worthwhile and attainable challenge. Intense monitoring of the patients after drug approval and periodic reporting to physicians and scientists remain essential for the safe use of drugs especially in rare diseases and pave future research.

Considering nucleic acids as the language of life and the genome as the instruction manual of cells, their targeted modulation promises great opportunities in treating and healing diseases. In addition to viral gene transfer, the overwhelming power of non-viral mRNA-based vaccines is driving the development of novel gene transporters. Thereby, various nucleic acids such as DNA (pDNA) or RNA (mRNA, siRNA, miRNA, gRNA, or ASOs) need to be delivered, requiring a transporter due to their high molar mass and negative charge in contrast to classical agents. This chapter presents the specific biological hurdles for using nucleic acids and shows how new materials can overcome these.

Adrenoceptors importantly contribute to the physiological regulation of lower urinary tract (LUT) function and have become a target of several clinically successful treatments for major LUT diseases. In the bladder dome, β-adrenoceptor subtypes are found in multiple cell types and mediate relaxation of detrusor smooth muscle, perhaps partly indirectly by acting on afferent nerves and cells of the mucosa. β₃-adrenoceptor agonists such as mirabegron and vibegron are used to treat overactive bladder syndrome. In the bladder trigone and urethra, α₁-adrenoceptors cause contraction and thereby physiologically contribute to bladder outlet resistance. α₁-adrenoceptors in the prostate also cause contraction and pathophysiologically elevate bladder outlet resistance leading to voiding dysfunction in benign prostatic hyperplasia. α₁-adrenoceptor antagonist such as tamsulosin is widely used as a first-line option to treat LUT symptoms in men, but it remains unclear to which extent and how smooth muscle relaxation contributes to symptom relief.

The sympathetic nervous system plays an important role in the regulation of endocrine pancreatic function, most importantly insulin release. Among the nine adrenoceptor (AR) subtypes, the α_2A-AR appears to be the subtype most abundantly expressed in the human pancreas. While α₂- and β-AR have opposing effects, the net response to sympathetic stimulation is inhibition of insulin secretion mediated by α₂-AR located in the plasma membrane of pancreatic β cells. This inhibition may be present physiologically as evidenced by increased insulin secretion in healthy and diabetic humans and animals in response to α₂-AR antagonists, a finding that was confirmed in all studies. Based on such data and on an association of an α_2A-AR polymorphism, that increases receptor expression levels, with an elevated risk for diabetes, increased α_2A-AR signaling in the pancreatic β cells has been proposed as a risk factor for the development of type 2 diabetes. Thus, the α_2A-AR was proposed as a drug target for the treatment of some forms of type 2 diabetes. Drug research and development programs leveraging this mechanism have reached the clinical stage, but none have resulted in an approved medicine due to a limited efficacy. While β-AR agonists can increase circulating insulin levels in vivo, it remains controversial whether this includes a direct effect on β cells or occurs secondary to general metabolic effects. Therefore, the regulation of endocrine pancreatic function is physiologically interesting but may be of limited therapeutic relevance.

The nine G protein-coupled adrenoceptor subtypes are where the endogenous catecholamines adrenaline and noradrenaline interact with cells. Since they are important therapeutic targets, over a century of effort has been put into developing drugs that modify their activity. This chapter provides an outline of how we have arrived at current knowledge of the receptors, their physiological roles and the methods used to develop ligands. Initial studies in vivo and in vitro with isolated organs and tissues progressed to cell-based techniques and the use of cloned adrenoceptor subtypes together with high-throughput assays that allow close examination of receptors and their signalling pathways. The crystal structures of many of the adrenoceptor subtypes have now been determined opening up new possibilities for drug development.

People with cystic fibrosis (CF) suffer from a multi-organ disorder caused by loss-of-function variants in the gene encoding the epithelial anion channel cystic fibrosis transmembrane conductance regulator (CFTR). Tremendous progress has been made in both basic and clinical sciences over the past three decades since the identification of the CFTR gene. Over 90% of people with CF now have access to therapies targeting dysfunctional CFTR. This success was made possible by numerous studies in the field that incrementally paved the way for the development of small molecules known as CFTR modulators. The advent of CFTR modulators transformed this life-threatening illness into a treatable disease by directly binding to the CFTR protein and correcting defects induced by pathogenic variants. In this chapter, we trace the trajectory of structural and functional studies that brought CF therapies from bench to bedside, with an emphasis on mechanistic understanding of CFTR modulators.

Various human tissues express the calcium-activated chloride channel Anoctamin 1 (ANO1), also known as TMEM16A. ANO1 allows the passive chloride flux that controls different physiological functions ranging from muscle contraction, fluid and hormone secretion, gastrointestinal motility, and electrical excitability. Overexpression of ANO1 is associated with pathological conditions such as hypertension and cancer. The molecular cloning of ANO1 has led to a surge in structural, functional, and physiological studies of the channel in several tissues. ANO1 is a homodimer channel harboring two pores - one in each monomer - that work independently. Each pore is activated by voltage-dependent binding of two intracellular calcium ions to a high-affinity-binding site. In addition, the binding of phosphatidylinositol 4,5-bisphosphate to sites scattered throughout the cytosolic side of the protein aids the calcium activation process. Furthermore, many pharmacological studies have established ANO1 as a target of promising compounds that could treat several illnesses. This chapter describes our current understanding of the physiological roles of ANO1 and its regulation under physiological conditions as well as new pharmacological compounds with potential therapeutic applications.

The transmembrane protein 16 (TMEM16) family consists of Ca²⁺-activated chloride channels and phospholipid scramblases. Ten mammalian TMEM16 proteins, TMEM16A-K (with no TMEM16I), and several non-mammalian TMEM16 proteins, such as afTMEM16 and nhTMEM16, have been discovered. All known TMEM16 proteins are homodimeric proteins containing two subunits. Each subunit consists of ten transmembrane helices with Ca²⁺-binding sites and a single ion-permeation/phospholipid transport pathway. The ion-permeation pathway and the phospholipid transport pathway of TMEM16 proteins have a wide intracellular vestibule, a narrow neck, and a smaller extracellular vestibule. Interestingly, the lining wall of the ion-permeation/phospholipid transport pathway may be formed, at least partially, by membrane phospholipids, though the degree of pore-wall forming by phospholipids likely varies among TMEM16 proteins. Thus, the biophysical properties and activation mechanisms of TMEM16 proteins could differ from each other accordingly. Here we review the current understanding of the structure and function of TMEM16 molecules.

The contribution of electron microscopy, and here, in particular transmission electron microscopy (TEM), to the formulation and understanding of the biological action of drug delivery systems has led to a better insight into the design principles of drug delivery systems. TEM can be applied for particle characterization, for the visualization of the uptake and intracellular pathways of drug vehicles in cells and tissues and more recently can be also applied for the high-resolution investigation of drug-receptor interactions with near-atomic resolution. This chapter introduces basic techniques to optimize imaging quality of soft matter samples, highlights possibilities to study certain aspects of drug delivery applications, and finally provides a short introduction to high-resolution characterization possibilities which recently emerged.