Carbon Monoxide: Promises and Challenges in Its Pharmaceutical Development

Binghe Wang and Leo E. Otterbein

Carbon monoxide (CO), one of the smallest organic natural molecules, is widely known for its toxicity. Formation of CO via incomplete combustion is a major contributing factor to accidental or intentional CO poisoning, leading to severe health consequences or death. In addition, CO is a by-product of tobacco smoking, and has been associated with some of the harmful effects of smoking. However, less known and probably far more important is the recognition of the essential physiological roles of CO as a signaling molecule in mammals. Against over more than a century of negative connotation, the last few decades have proven that CO possesses a multitude of physiological roles and therapeutic functions, including regulation of the immune response, cellular proliferation, and control of cell survival. This concept is supported by the discovery that CO is produced by all cells and more so under conditions of stress. This book comprehensively summarizes key aspects of CO’s endogenous roles, therapeutic functions, and challenges that we face in its development as a therapeutic agent. We hope this preface will provide a thread for reading this book and a bird’s-eye view of the landscape for understanding this field, and more importantly lay out the challenges ahead in understanding the detailed mechanisms of action of CO and in its development as a therapeutic agent. We have divided the book into four sections that provide a framework for the reader to follow the evolution of CO from an accepted poison to a bioactive molecule that may offer enormous clinical benefits.

Section I begins with “foundational” knowledge of the CO field, including a general background and known physiological mechanisms. Endogenous production is a prerequisite for a molecule to be an endogenous signaling molecule and CO is no exception. Therefore, the book commences with a detailed discussion of CO’s endogenous production in all cells, the enzymes involved, and the detailed chemistry of the major pathway leading to endogenous CO production during the degradation of heme by the heme oxygenases (Chapter 1), a principal player in how CO is generated by all cells. Other products from heme degradation are also discussed so as to contextualize CO’s functions (Chapter 10) as an endogenous signaling molecule. Section I also includes a comprehensive examination of the molecular targets of CO (Chapter 2), providing the molecular basis for later discussion on its physiological and therapeutic functions. The physiological, therapeutic, and/or toxicological functions of any molecule are only meaningful in the context of concentrations. Therefore, the examination of the pharmacokinetic and pharmacodynamic characteristics of CO is discussed (Chapter 3). This section also includes information on CO’s role in energy metabolism (Chapter 4), regulation of the circadian clock (Chapter 5), and mitochondrial function (Chapter 6) because these three aspects impact a large number of the reported CO effects. One very important aspect of CO is its ability to signal and its relationship with other gaseous signaling molecules: nitric oxide (Chapter 8) and hydrogen sulfide (Chapter 9). Along a similar line, CO’s functions overlap with that of oxygen in very intriguing ways, which go beyond simply competing for binding with hemoproteins (Chapter 7). Therefore, there are three chapters in this section devoted to the interplay among these four gas molecules.

Section II focuses on the development of various delivery forms of CO, which are critical to studying CO’s mechanism(s) of actions, validating its pharmacological functions, and developing CO-based therapeutics. All such delivery forms focus on going beyond inhalational delivery of CO. Chapter 11 discusses noncarrier formulations, including CO in solution, CO donors encapsulated in various types of materials, and extracorporeal delivery of CO. Chapter 12 focuses on examples of metal-immobilized carbonyls, which are also commonly referred to as CO-releasing molecules (CORMs). For a period of three decades, there was a very high level of activity in this area. Chapter 12 is only able to describe a few select examples of immobilized carbonyls as CO donors. In recent years, there has been a rapid increase in the level of interest in developing metal-free CO donors for reasons of diversity and for avoiding metal-related issues. Chapter 13 discusses metal-free CO donors that rely on photolysis for CO release. A major new direction in the field of CO donors over the past 5 years has been the development of organic CO prodrugs with tunable release rates, triggered release, and the ability to deliver multiple payloads in a single prodrug. Chapter 14 comprehensively examines this area, including discussions of the unique chemistry employed and pharmacological validation studies. This chapter also has a very important section on the proper use of controls in studying CO donors, including both organic prodrugs and metal-based CORMs. Though using proper controls is normally considered routine practice in scientific research, the unique challenges of CO-independent effects observed with some metal-based CORMs indeed elevate this issue to a prominent position. In the field of CO delivery, there is always the question of whether there is the need for targeted CO delivery. Chapter 15 summarizes recent developments in this area. In terms of CO delivery, there is one area that may offer very unique opportunities for efficacy studies and toxicity assessment. Fluorinated general anesthetics such as sevoflurane, desflurane, and isoflurane are known to decompose under basic conditions, leading to the production of CO. Such basic conditions are needed in a ventilator to remove carbon dioxide. Therefore, there is the issue of anesthesia-related CO exposure, which is the focus of Chapter 16. Chapter 17 explores various aspects of natural product-based CO production, including the mechanism(s) of the chemical reactions involved and its potential implications in terms of nutritional and/or therapeutic values. These discussions also bring in a sense of effective concentrations needed for observed activities whenever possible. The last point is very important because discussions of pharmacologic activities outside of the context related to concentration and potency have very little meaning.

Section III has only one chapter, but it represents a very important aspect of the CO field. One unique challenge in studying a gaseous signaling molecule is difficulty in detection and concentration determination. Chapter 18 describes in detail available fluorescent probes for CO detection, including intracellular detection. The chapter is meticulously written with information on detection limits and signal to noise ratios for various probes. This chapter also includes discussions of reported “CO probes” that are only able to detect certain metal-based CORMs because of metal-mediated reactions, but not CO itself.

Section IV examines the pharmacologic effects and mechanistic understandings of CO in various cell culture and animal models, including organ transplantation (Chapter 19), lung injury (Chapter 20), brain injury (Chapter 21), liver injury (Chapter 22), cancer (Chapter 23), diabetes (Chapter 24), kidney injury (Chapter 25), platelet function (Chapter 26), gastrointestinal protection (Chapter 27), sickle cell disease (Chapter 28), and pain management (Chapter 29). The title for each chapter is sufficiently self-explanatory; collectively, these chapters show the breadth of clinical applications of CO. The last chapter (Chapter 30) summarizes all human clinical trials reported so far.

With its vast therapeutic potential, major challenges remain in understanding CO’s molecular mechanism(s) of action and in its pharmaceutical development. Here, we would like to highlight some of these challenges to aid future studies. First, studying the dose–response relationship of a gaseous molecule (CO) is much more challenging than that of a traditional small molecule. The volatility of CO means that the concentration of delivery may or may not be directly related to the effective concentration of CO under a given set of conditions. Second, the mode in which it is administered, the route of delivery, the resulting tissue distribution, and the elimination are standard development matters, but fortunately CO is not metabolized to an appreciable degree, making standard ADME (absorption, distribution, metabolism, and excretion) studies unique. This delivery issue becomes more complex, however, when administering CO in a form other than a gas or a saturated liquid. For instance, the release kinetics of a CO donor or prodrug is known to affect the CO concentration and duration profiles, even in simple buffer solution. In animals, the effects may appear as the same as that observed with CO gas, but with added complexity, including the need to deconvolute the effects of CO from that of the CO donor molecule or its metabolic by-product. There have been a number of reports in recent years that attributed some of the widely reported pharmacological effects of certain CORMs to CO-independent effects. The absorption and tissue distribution may differ depending on physiology or pathophysiology, e.g., lung disease where inhaled CO diffusion will be different or liver disease where metabolism of CO donor molecules and therefore the release of CO may be altered. Other considerations include the fact that some metal-based CORMs have a wide range of chemical reactivities driven by the carrier molecule. The third challenge is the unique difficulty in studying the pharmacokinetic properties of CO. Current studies use carboxyhemoglobin (COHb) as a surrogate indicator of CO concentration. This may not be sufficient. The free concentration of CO in the blood is a very important factor to consider. There is a widely held perception that free CO concentration is always low. However, this is not correct. Though Chapter 3 addresses some of these issues, more work is needed to understand what it means to use COHb as an indicator of CO concentrations. A fourth challenge is the availability of a large number of hemoproteins in a cell, which are all potential targets for CO. How to deconvolute the effects of engaging such a large number of targets that are in constant flux is an important question, but has hardly been examined. Moreover, there are likely differences in hemoprotein distributions across different species. A fifth challenge is the issue of allometric scaling. CO’s efficacy has been widely validated in various pharmacological models in different animal species such as mice and rats and large animals including pigs and dogs. However, the large number of clinical trials has yet to demonstrate efficacy in humans in a convincing fashion. In one kidney transplant study, the results seem to be very positive (Chapter 25); however, the trials were terminated prematurely without conclusive demonstration of efficacy in a statistically significant manner. The need to understand allometric scaling is a critical first step in translating success in animal models to humans.

We hope that this book will allow the readers to see the vast potential of CO and stimulate much needed research to assess its therapeutic potential. Collectively, we would like to thank all the authors for their contributions as experts in their respective fields related to CO as well as their diligence and patience in working with us. We would also like to express our sincere appreciation of Ms Andrea Mahone in the office of B. Wang for her assistance in coordinating all aspects of this book project as well as the Wiley team for their support.

May 2021