Preface

We are grateful for the opportunity to compile and edit this book, composed of two volumes, on materials and methods for conducting experiments to study the many aspects of the disease, ocular hypertension (OHT) and glaucoma, a constellation of potentially blinding diseases that afflict millions of patients worldwide. Additionally, it was important to guide readers about the drug / device discovery process and the basic aspects of drug development to treat the afore-mentioned eye diseases. Thus, salient features of Investigational New Drug (IND)-enabling studies for new compounds and devices are presented to underscore the ultimate purpose of the ocular research that is conducted in academia and industry, namely, to unearth mechanisms of ocular hypertensive disease and then find treatment options to mitigate and retard the disease process(es).

We have been conducting and supervising basic and applied ocular research for advancing knowledge about the pathogenesis of eye diseases, and drug discovery and development for more than three decades. Working in the pharmaceutical and academic arenas, respectively, and frequently guiding and mentoring junior staff, we realized there was a deficiency of a single source of key methods utilized for performing ocular pharmacology, disease mechanisms and therapeutics discovery research. To capture existing procedures and rapidly emerging techniques and technologies, we decided to develop this book. This project would not have been possible without the kind and generous help and support of our family members, Bentham Science Publishers and our many colleagues who have contributed to this endeavor. We have deliberately focused on OHT, which develops from elevated intraocular pressure (IOP) in the anterior chamber of the eye (ANCe) as a case study, since lowering and controlling IOP by pharmaceuticals, aqueous humor drainage devices and ocular surgery are the only currently approved medical treatments for OHT and for many forms of glaucoma. The topic of glaucomatous optic neuropathy (GON), which has several risk factors of which OHT is one, and that encompasses the many factors and events occurring at the optic nerve head, retina, optic nerve and the brain leading to potential blindness, will be covered in a follow-up book.

Protocols described in the various sections and chapters in the current two volumes cover a wide range of techniques that are currently being used in studying the anatomy, morphology, pathology, biochemistry, physiology, pharmacology, and molecular biology of the cells/tissues within the anterior chamber of the eye (ANCe). In Volume 1, Section 1, beginning with a brief review of the basic anatomy and physiology of the eye (Chapter 1), a treatise on research and development of drugs, their sources and screening paradigms in vitro and in vivo are described in Chapters 2 and 3. Since the majority of drugs used to treat elevated IOP engage with membranous and /or cytoplasmic receptors and/or enzymes, it is important to determine whether the tissues/cells contain the requisite drug targets. This is accomplished using specific protocols and a range of techniques (reverse transcriptase polymerase chain reaction [RT-PCR]; receptor binding; autoradiography and immunohistochemistry) that are described in Section 2 (Chapters 4-7). Once cellular binding site for target receptor/enzyme verification has been accomplished, it is necessary to demonstrate the functional activity associated with the target protein through drug-receptor engagement using well characterized reference compounds in suitable cell-based assays. Additional relevant cell types are also needed to potentially define off-target effects of test compounds and/or to find ocular cells that may contain the drug receptor/enzyme/transporter protein(s). This necessitates isolation, propagation and utility of cells from appropriate ANCe uveal tissues (e.g. cells from the ciliary muscle, ciliary processes, trabecular meshwork, Schlemm’s canal) and other cells derived from the sclera, corneal and conjunctival tissues. Suitable protocols needed for such endeavors are described in Section 3 (Chapters 8-13).

Cell- and tissue-based assays to permit interrogation of receptor/enzyme activity for compounds of interest are crucial in the quest to find new drugs for treating elevated IOP, and these are described in Section 4 (Chapters 14-24). Drug discovery screening, whether conducted manually or using automated high-through-screening platforms, requires the use of a broad range of biochemical and pharmacological techniques and technologies. Agonist or antagonist activity of reference and test compounds can be determined in isolated cells or tissues using a variety of functional parameters such as quantifying second messengers, cell impedance, cell volume changes, tissue contraction/relaxation, release of transmitters, cellular ionic changes, etc. The concentration-response data, including receptor/enzyme affinity, compound potency and intrinsic activity, can then help rank order compounds and find suitable hits for further optimization by the medicinal chemists. Once all the in vitro studies have been completed and suitable compounds selected to progress for testing in normal animals for ocular safety and tolerability, and then for efficacy, the more labor-intensive and expensive portion of drug/device discovery and development begins.

Although the in vitro pharmacological characterization of drug targets and test compounds is important, ultimately appropriate response(s) in animal models of disease dictates the final selection of a suitable lead compound, and back-ups, that can be further characterized via additional IND-enabling studies. These aspects are the subject matter covered in Volume 2 of this book. In the case of glaucoma/OHT, compounds should be safe (not causing eye irritation or other systemic/ central nervous system adverse effects) and be able to significantly lower and control IOP. Thus, Volume 2, Section 5 (Chapters 25-36) provides numerous protocols for such in vivo studies in various mammalian species (rodent, rabbit, dog, monkey) using induced methods to raise IOP in various animals. Since the aim of the IOP-reducing drugs for treating OHT/glaucoma is to promote AQH outflow from the ANCe and/or to reduce AQH production, a specific protocol dealing with AQH drainage devices (Chapter 36) is also provided. In the modern age the use of genetic medicine has come to fruition. Along the way, the discovery of naturally occurring mouse models of glaucoma and development of new animal models of OHT using genetic engineering has also been accomplished, and these are described in Section 6 (Chapters 37-39).

Once compound ocular safety and tolerability has been established, and some relative efficacy in a few models of OHT demonstrated, the lead compound and any back-up compounds can now be progressed to a stage where an ultimate formulation is selected for all remaining studies. Typically, a safe and comfortable drug formulation suitable for topical ocular dosing is developed and appropriate pharmacokinetic, pharmacodynamic and toxicological studies performed on the lead compound. Such investigations can be performed using protocols described in Section 7 (Chapters 40-42). The results from the in vitro and in vivo studies performed for the IND-ready compound(s) or device(s) using validated protocols and standard operating procedures (SOPs) using Good Laboratory Practices (GLP) and/or Good Manufacturing Practices (GMP) can then be assembled into dossiers for submission to the regulatory authority under an IND application. These and other key regulatory considerations need to be addressed through appropriate communications and interactions with the health agency and are out of scope for the current book.

Finally, we surmised that in this technologically advanced age we should provide the modern researchers in ocular physiology and pharmacology new methods to help advance basic and applied eye research and thus help discover new drugs and treatments for OHT/glaucoma. To that end, Section 8 (Chapters 43-48) provides some new information on modern techniques and technologies addressing some novel avenues of eye research delving into gene and cell therapies and studying cell-to-cell communication via nanotubes and study of metabolomics of cells in the ANCe.

In conclusion, both clinical and basic science ocular researchers, graduate students, postdoctoral fellows, optometrists, and ophthalmologists should find this book to be a comprehensive and one-stop-resource for methodologies needed to study ocular structure and function of cells and tissues of the ANCe. The preservation of our most precious sense, eyesight, and its potential restoration in patients afflicted with blinding eye diseases such as OHT/glaucoma is a goal worthy of pursuit. We hope that this collection of protocols helps eye scientists achieve such audacious goals. We extend our thanks and gratitude to all the authors and the publisher for making this book a reality.