Biased Agonism at β-Adrenoceptor Subtypes: A Drug Development Perspective.

Based on the conformationally constrained D-Trp-Phe-D-Trp (wFw) core of the prototype inverse agonist [D-Arg(1),D-Phe(5),D-Trp(7,9),Leu(11)]substance P, a series of novel, small, peptide-mimetic agonists for the ghrelin receptor were generated. By using various simple, ring-constrained spacers connecting the D-Trp-Phe-D-Trp motif with the important C-terminal carboxyamide group, 40 nm agonism potency was obtained and also in one case (wFw-Isn-NH(2), where Isn is isonipecotic acid) ~80% efficacy. However, in contrast to all previously reported ghrelin receptor agonists, the piperidine-constrained wFw-Isn-NH(2) was found to be a functionally biased agonist. Thus, wFw-Isn-NH(2) mediated potent and efficacious signaling through the Gα(q) and ERK1/2 signaling pathways, but in contrast to all previous ghrelin receptor agonists it did not signal through the serum response element, conceivably the Gα(12/13) pathway. The recognition pattern of wFw-Isn-NH(2) with the ghrelin receptor also differed significantly from that of all previously characterized unbiased agonists. Most importantly, wFw-Isn-NH(2) was not dependent on GluIII:09 (Glu3.33), which otherwise is an obligatory TM III anchor point residue for ghrelin agonists. Molecular modeling and docking experiments indicated that wFw-Isn-NH(2) binds in the classical agonist binding site between the extracellular segments of TMs III, VI, and VII, interacting closely with the aromatic cluster between TMs VI and VII, but that it does so in an opposite orientation as compared with, for example, the wFw peptide agonists. It is concluded that the novel peptide-mimetic ligand wFw-Isn-NH(2) is a biased ghrelin receptor agonist and that the selective signaling pattern presumably is due to its unique receptor recognition pattern lacking interaction with key residues especially in TM III.

The phenomenon of “biased agonism” presents an attractive avenue for drug development as it allows the separation of therapeutic effects from side effects mediated by the same target. A prototypical G protein-coupled receptor at which biased agonism has been extensively studied is the dopamine D₂ receptor, an important therapeutic target for current treatments of Parkinson’s disease and schizophrenia. There is increasing evidence that biased agonism is important for the antipsychotic efficacy of dopamine D₂ receptor partial agonists, such as aripiprazole and cariprazine. However, a clear relationship between biased agonism at the dopamine D₂ receptor and antipsychotic efficacy remains elusive, not least due to discrepancies in literature describing aripiprazole as ‘biased’ or ‘unbiased’, despite the same signalling endpoints being studied using the same cell background. To clarify such conflicts, and to aid the drug discovery efforts aimed at identifying novel dopamine D₂ receptor biased agonists, the focus of this thesis is to gain greater insight into the mechanisms that mediate biased agonism at the dopamine D₂ receptor. Through the utilization of both mutagenesis-based and structure-activity-based approaches, a secondary binding pocket was identified for being crucial in the affinity, efficacy, and bias of different ligands at the dopamine D₂ receptor. A structure-activity relationship study indicated that both efficacy and biased agonism can be finely tuned by minor structural modifications to the head group, the tail group, and the orientation and length of a spacer region of cariprazine. In particular, it was demonstrated that modifications to the tail region, and thus the interaction with a potential secondary binding site, alter the orientation of the head group within the orthosteric binding site regulating both efficacy and biased agonism. These results were corroborated with a mutagenesis study, in which mutations within a putative secondary binding site significantly impacted the affinity and efficacy of a number of dopamine D₂ receptor agonists. Finally, it was demonstrated that “kinetic context”, as determined by both ligand-binding kinetics and the kinetics intrinsic to different cellular signalling processes, can dramatically impact observations of biased agonism. Such findings illustrate, for the first time, the importance of incorporating kinetic profiling in future studies focussed on biased agonism to allow a more informed selection of preclinical candidates and thus an improved foundation for drug discovery of biased agonists.

Setting the stage Drug research relies on classical scienti!c methods and statistical inferences across the spectrum of preclinical discovery to clinical practice: formulate a hypothesis, test the hypothesis experimentally, analyze the data, and make informed decisions to accept, reject, or re!ne the hypothesis. Drug discovery and development is iterative, collaborative, and multidisciplinary, culminating in pivotal clinical trials that provide evidence of ecacy and safety to support market access. Through the mid-1990s, this process worked remarkably well in some disease areas with the rapid identi!cation of validated drug targets, such as with antiretroviral drugs for treating HIV and statins for managing high cholesterol in cardiovascular diseases, leading to the launch of many new medicines that reduced morbidity and mortality and increased life expectancy. But in other diseases, such as cancer, progress has been more modest. Over the past 15 years, this success story has been threatened by expiring patents, poorly understood disease mechanisms, a high rate of attrition, and burgeoning drug development costs. As a result, the pace of new medicines coming out of the pharmaceutical industry pipelines has slowed considerably. Translational bioinformatics (TBI) has recently emerged as an important technology to address these challenges. At the risk of being oversold, TBI is expected to help bridge the gap between pathogenic pathways and disease phenotypes and guide molecular measurements that can improve target identi!cation, drug selection, and clinical trial design. #e quotation by Einstein above emphasizes the importance of “thinking about thinking” and the need to better understand human cognition. In the context of scienti!c thinking, cognition is the thought process that describes how data acquired from drug research are transformed into information and stored as knowledge for future decision making. In today’s world of rapid technological and computational advances, it is easy to get lost in the ubiquitous and dicult problem of cognitive overload and workload timelines. TBI is a novel approach to solving these problems and is designed to avoid getting “lost in the data.” It can help decision makers answer important questions by integrating pertinent information beyond that which could be achieved by human memory, intuition, and pattern thinking alone. Today, pharmaceutical companies, academia, and others involved in drug discovery and development have access to a far more sophisticated understanding of disease pathways and biological networks to decode complex clinical disease phenotypes. #is has changed the way drug research is conducted. It also has led to recent breakthroughs in targeted therapies for cancer such as vemurafenib, a B-Raf enzyme inhibitor for the treatment of late-stage melanoma, and crizotinib, an anaplastic lymphoma kinase inhibitor for treating some types of non–small cell lung carcinomas. Federal agencies have become part of the solution to problems in drug discovery and development. #e US Food and Drug Administration (FDA) had taken notice of the trend toward lower productivity when it launched the Critical Path Initiative in 2004, which was intended to frame a national strategy for driving innovation in scienti!c tools and processes that would foster a turnaround in the success of drug development. More recently, the FDA published a strategic plan for advancing regulatory science and emphasized the importance of TBI in several priority areas and implementation strategies.1 #e Obama administration started a government drug development center, called the National Center for Advancing Translational Sciences, to partner with pharmaceutical companies and other organizations to apply scienti!c advances and develop new tools for drug research. Both initiatives acknowledge the rapid growth of biomedical knowledge and the urgent need for new predictive bioinformatics tools in drug discovery, development, and postmarketing clinical practice. This article provides a macroscopic view of TBI and a perspective on the future of knowledge development in the drug discovery, development, regulatory, and clinical practice continuum.

A major portion of both the time and cost of new drug and biological product development is the series of human clinical studies required to determine the safety and effectiveness of the drug before it can be marketed. This paper analyses data from 68 new drug and biological product development projects sponsored by US-based biotechnology companies over a period of 19 years, from 1979 to 1998. The type of collaboration in place for each project and the size of the sponsoring company were assessed for potential effects on the projects' clinical development times. While there was no statistical difference among the type of collaboration, the data shows evidence of a trend toward shorter clinical development times for drug development projects without a collaborative partner. Findings also indicated a significantly shorter clinical development time for projects sponsored by medium size versus small or large biotechnology companies. The results offer insight into the process of new drug development, along with pertinent information regarding resources that may help lead to success in the effort to reduce time involved in human clinical studies.

The Special Programme for Research and Training in Tropical Diseases (TDR) was established in 1975 by the United Nations’ Development Programme, the World Bank and the World Health Organization (WHO), at a time when only minimal scientific worldwide effort was dedicated to research into tropical diseases (Morel, 2000). TDR was therefore created with a core mission of fighting these diseases and has two specific goals: first, it seeks to identify and develop new tools and methods to control tropical diseases; and second, it seeks to develop research capacities in developing countries so that their investigators are able to establish their own research activities and contribute to the control of diseases that affect their countries. Because of its mission, TDR has always been involved in product research and development (RD Trouiller et al ., 2001). Of 1,450 new chemicals introduced to the global market, only 13 were specifically for treating neglected infectious diseases, and many of these substances came out of R&D for other disease indications, such as veterinary medicine and cancer. Through its work with industry, TDR was crucially involved in the development of about half of these new drugs. In this article, I highlight how TDR has worked effectively with industry over the past 27 years, identify several key achievements and indicate how future activities might develop in this area. > Of 1,450 new chemical entities introduced to the global market, only 13 were specifically for treating neglected infectious diseases TDR consists of four main functional areas: …

Product R&D for neglected diseases: Twenty-seven years of WHO/TDR experience with public-private partnerships

Trypanosoma brucei is the parasite that causes African sleeping sickness in humans and the related disease nagana in cattle. The development of drugs is hampered by the many similarities between this parasite and the cells of its host. Until now advanced drug-design strategies have focussed on the differences between the three-dimensional structure of trypanosome and human enzymes (Verlinde & Hol, 1994). We propose that the selectivity of a drug can be further enhanced by choosing a target enzyme with a high flux control coefficient in the parasite and a low flux control coefficient in the host cells. Trypanosome glycolysis is a very suitable model system for this approach. In the mammalian bloodstream T. brucei depends completely on glycolysis (Michels, 1988). In many of its host cells glycolysis is also essential. The organization of this pathway and the regulation of its enzymes differ greatly between the two organisms (Michels, 1988). Most conspicuously, in trypanosomes part of glycolysis takes place in specialized organelles (glycosomes), whereas in the host the corresponding pathway is localized in the cytosol. In view of this and other differ-ences there is a fair chance that the distributrion of the control of the glycolytic flux over the glycolytic enzymes will also be different.

Food Effect on Oral Bioavailability: Old and New Questions.

Strategies to Modulate Immune Responses: A New Frontier for Gene Therapy

During the last decade, despite increased investment in drug research and development related activity, stagnation in new drug discovery has been documented. Despite a 70% increase in investment in research and development-related activities, a 40% fall in launch of new chemical entities was seen during 1994–2004. A steep rise in the attrition rate of drug development has complicated the matter. Rising cost and increased attrition rates proved major barriers to investment in higher risk drugs or in therapies for uncommon diseases or diseases that predominantly afflict the poor. This prompted Food and Drug Administration (FDA) to highlight this problem in a 2004 white paper classified as “Critical Path Initiative” (CPI) and to initiate steps to target stagnation and rise in attrition rates. Many new drug development projects have started worldwide taking cue from CPI; adopting microdosing, adaptive designs and taking advantage of newly developed biomarkers under the CPI. This review discusses the various strategies adopted under CPI to decrease attrition rate and stagnation of new drug development, and the challenges and controversies associated with CPI.

To this day, opioids represent the most effective class of drugs for the treatment of severe pain. On a molecular level, all opioids in use today are agonists at the μ-opioid receptor (μ receptor). The μ receptor is a class A G protein-coupled receptor (GPCR). GPCRs are among the biological structures most frequently targeted by pharmaceuticals. They are membrane bound receptors, which confer their signals into the cell primarily by activating a variety of GTPases called G proteins. In the course of the signaling process, the μ receptor will be phosphorylated by GRKs, increasing its affinity for another entity of signaling proteins called β-arrestins (β-arrs). The binding of a β-arr to the activated μ receptor will end the G protein signal and cause the receptor to be internalized into the cell. Past research showed that the μ receptor’s G protein signal puts into effect the desired pain relieving properties of opioid drugs, whereas β-arr recruitment is more often linked to adverse effects like obstipation, tolerance, and respiratory depression. Recent work in academic and industrial research picked up on these findings and looked into the possibility of enhancing G protein signaling while suppressing β-arr recruitment. The conceptual groundwork of such approaches is the phenomenon of biased agonism. It appreciates the fact that different ligands can change the relative contribution of any given pathway to the overall downstream signaling, thus enabling not only receptor-specific but even pathway-specific signaling. This work examined the ability of a variety of common opioid drugs to specifically activate the different signaling pathways and quantify it by means of resonance energy transfer and protein complementation experiments in living cells. Phosphorylation of the activated receptor is a central step in the canonical GPCR signaling process. Therefore, in a second step, expression levels of the phosphorylating GRKs were enhanced in search for possible effects on receptor signaling and ligand bias. In short, detailed pharmacological profiles of 17 opioid ligands were recorded. Comparison with known clinical properties of the compounds showed robust correlation of G protein activation efficacy and analgesic potency. Ligand bias (i.e. significant preference of any path- way over another by a given agonist) was found for a number of opioids in native HEK293 cells overexpressing μ receptor and β-arrs. Furthermore, overexpression of GRK2 was shown to fundamentally change β-arr pharmacodynamics of nearly all opioids. As a consequence, any ligand bias as detected earlier was abolished with GRK2 overexpression, with the exception of buprenorhin. In summary, the following key findings stand out: (1) Common opioid drugs exert biased agonism at the μ receptor to a small extent. (2) Ligand bias is influenced by expression levels of GRK2, which may vary between individuals, target tissues or even over time. (3) One of the opioids, buprenorhin, did not change its signaling properties with the overexpression of GRK2. This might serve as a starting point for the development of new opioids which could lack the ability of β-arr recruitment altogether and thus might help reduce adverse side effects in the treatment of severe pain.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted in response to nutrient ingestion following it’s binding to the GLP-1 receptor (GLP-1R). Long-acting GLP-1R peptidomimetics have become an important class of therapeutics for treating type 2 diabetes (T2D) because of their gluco-regulatory actions. However, they have been associated with adverse side-effects. Pharmaceutical companies are actively pursuing development of small molecule ligands as alternatives, but with little success to date. GLP-1R ligands can stabilize distinct subsets of receptor conformations that can “traffic†stimulus to diverse functional outputs with varying prominence, a concept referred to as biased agonism. Allosteric modulators can also alter the signalling profiles of orthosteric ligands in a ligand-dependent manner, termed probe-dependence. While GLP-1R biased agonism and allosteric probe dependence are established, to date most studies are from recombinant systems overexpressing the GLP-1R. This thesis utilizes a reductionist (cAMP accumulation, glucose-stimulated insulin secretion (GSIS), ERK1/2 phosphorylation, proliferation and apoptosis) and non-reductionist approach (transcriptomics) to understand GLP-1R biased agonism and allosteric modulation in natively expressing cells that display glucose dependence, addressing some of the issues of translation from recombinant to more physiologically relevant systems mimicking β-cell physiology. Extensive and systemic analysis performed using GLP-1R peptides in INS-1 832/3 insulinoma cells, revealed that cAMP accumulation, proliferation and anti-apoptosis were glucose-independent, whereas, insulin secretion, [Ca2+]i mobilization and aspects of the ERK1/2 phosphorylation kinetics were glucose-dependent. Furthermore, attenuation of glucose-mediated activation of ERK1/2 at 5 min by GLP-1R peptides was a novel, unexpected observation. Assessment of ligand signalling profiles revealed that biased agonism occurred with distinct ligands; however the bias profile was different in two physiologically relevant glucose concentrations. One significant observation was a large degree of bias at the therapeutically relevant endpoints of proliferation and apoptosis, where for equivalent amounts of cAMP generated, GLP-1 was more efficacious compared to exendin-4 and oxyntomodulin. For small molecule ligands there was bias relative to GLP-1 between the amount of cAMP production and insulin secretion that was not observed with peptide ligands. In addition, both BETP and Compound 2 allosterically altered the signalling profiles of peptide ligands, in a peptide-dependent manner that differed depending on the glucose concentration assessed, observations that have clinical relevance for the development of allosteric drugs. In the final chapter, analysis of transcriptomics data identified a number of genes associated with functions of cell to cell signalling, signal transduction, proliferation, cell death and survival, along with number genes for GPCRs and GPCR ligands that were up or down-regulated following GLP-1R activation. In addition, a number of genes associated with T2D, but not previously with GLP-1R signalling also emerged. Exendin-4 and oxyntomodulin displayed bias relative to GLP-1 at the level of gene transcription with a number of differentially regulated genes identified. Thus, this thesis expands the existing knowledge around GLP-1R pharmacology and signalling. This will facilitate ligand profiling and better understanding of biased agonism in natively expressing systems that may be useful for future therapeutic development.

New drug development can be made by providing products of higher "selectivity for the drug" for medical treatment. There are two ways for the approach to get higher "selectivity of drug": 1) discovery of new compounds with high selectivity of drug; 2) innovation of new drug administration, that is new formulation and/or method with high selectivity of drug by integration and harmonization of various hard/soft technologies. An extensive increase of biological information and advancement of surrounding science and technology may modify the situation as the latter overcomes the former in the 21 century. As the science and technology in the 21 century is said to be formed on "3H", that is, 1. hybrid; 2. hi-quality; 3. husbandry, the new drug development by innovative drug administration is exactly based on the science and technology of 3H. Its characteristic points are interdisciplinary/interfusion, international, of philosophy/ethics, and systems of hard/hard/heart. From these points of view, not only the advance of unit technology but also a revolution in thinking way should be "must" subjects. To organize this type of research well, a total research activity such as ROR (research on research) might take an important and efficient role. Here the key words are the "Optimization technology" and "Change in Pharmaceutical Fields." As some examples of new drug innovation, our trials on several topical mucosal adhesive dosage forms and parenteral administration of peptide drugs such as insulin and erythropoietin will be described.

Thymidine Kinase (TK) plays a basic role in DNA synthesis and cell proliferation. The enzyme is highly cell cycle dependent and correlates with cell growth state. Numerous studies have consistently shown elevated TK activity (TKa) in the blood of patients with solid tumors and active disease. DiviTum® TKa, a clinically and analytically validated FDA cleared assay, measures and quantifies TKa in serum or plasma in humans and animals. TKa as a biomarker offers valuable information at all stages of drug development, from drug efficacy and dose response studies in cell culture and xenograft models, to patient selection and treatment monitoring in clinical trials. The launch of FDA Project Optimus highlights the need for new biomarkers to assist in drug development to identify optimal dose. Using TKa to measure the impact on cell proliferation as a drug evaluation endpoint can confirm mechanism of action efficacy and a minimally effective dose of a new compound rather than a maximally tolerated dose. TKa can be used as a pharmacodynamic tool to improve study endpoints. TKa data in cultured cells and mouse xenografts demonstrates that a complete shutdown of tumor cell proliferation can be observed well before and at much lower drug-doses than when cell death or toxicity is used as the dose determination endpoint. Increased dosing of CDK4/6 inhibitors demonstrates immediate impact on TKa levels in cell lines and provides signals of drug effect at doses where cell viability is unaffected. After initiation of samuraciclib treatment, a CDK7 inhibitor, a significant reduction in TKa levels in blood from metastatic breast cancer (MBC) patients was observed, indicating inhibition of cell cycle progression. For patients on samuraciclib therapy and with stable disease before progression, TKa levels were significantly lower than pre-treatment (p=0.0038). Post-progression TKa levels increased to significantly higher levels than during therapy including the last sample measured before progression (p=0.014). Baseline blood TKa levels are predictive for progression free survival (PFS) allowing for the early stratification of patients with long vs short PFS. In patients with MBC, TKa levels during the first treatment cycle are strongly predictive for PFS. Suppressed TKa levels after 2 and 4 weeks of CDK4/6 inhibitor therapy predict longer PFS than high TKa levels at the same time points (HR 5.65; p< 0.0001). MBC patients monitored >3 months and showing low TKa levels have very low likelihood of progression within 30- to 60 days, <2% and <6%, respectively. TKa is a translational liquid biomarker that bridges results between preclinical and clinical studies, providing fundamental information for drug development decision making. Citation Format: Mattias Bergqvist, Hanna Ritzén, Amy Williams. The liquid biomarker thymidine kinase activity reflecting proliferation rate can provide guidance in drug development and address Project Optimus [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7625.

The impossible task of developing a new treatment for heart failure