INTRODUCTORY
`
`MEDICINAL CHEMISTRY
`
`J. B. TAYLOR, B.Sc., D.I.C., Ph.D.
`Director of Pharmaceutical Research
`Roussel UCLAF
`Paris
`
`and
`
`P. D. KENNEWELL, B.A., M.A., M.Sc., Ph.D.
`Deputy Head of Chemical Research and
`Head of Orientative Research
`Roussel Laboratories Ltd.
`
`Swindon, Wiltshire
`
`
`
`ELLIS HORWOOD LIMITED
`Publishers - Chichester
`
`Halsted Press: a division of
`JOHN WILEY & SONS
`New York - Brisbane -Chichester - Toronto
`
`Mylan Exhibit 1023, Page 1
`
`
`
`
`
`Mylan Exhibit 1023, Page 1
`
`
`
`
`
`
`
`
`
`

`
`First published in 1981 by
`ELLIS HORWOOD LIMITED
`
`Market Cross House, Cooper Street, Chichester, West Sussex, PO19 IEB, England
`
`The publisher’s colophon is reproduced from James Gillz'son’s drawing of the
`ancient Market Cross, Chichester,
`
`Distributors:
`Australia, New Zealand, South-east Asia:
`Jacaranda-Wiley Ltd.,Jacaranda Press,
`JOHN WILEY & SONS INC.,
`
`G.P.O. Box 859, Brisbane, Queensland 40001, Australia
`
`Canada:
`JOHN WILEY & SONS CANADA LIMITED
`
`.
`
`G, \/
`.1"
`M - -‘". y’,/,
`_}
`/ ~/’
`/' L /
`(,3,
`
`/~—~
`
`2
`
`2 /
`
`'1
`
`/ ? E3, /
`
`22 Worcester Road, Rexdale, Ontario, Canada.
`
`Europe, Africa:
`JOHN WILEY & SONS LIMITED
`
`Baffins Lane, Chichester, West Sussex, England.
`
`North and South America and the rest of the world:
`Halsted Press: a division of
`JOHN WILEY & SONS
`
`605 Third Avenue, New York, N.Y. 10016, U.S.A.
`
`© J. B. Taylor, P. D. Kennewell/Ellis Horwood Limited, Publishers 1981
`
`British Library Cataloguing in Publication Data
`Taylor, J. B.
`Introductory medicinal chemistry.
`1. Chemistry, Medical and pharmaceutical
`I. Title
`II. Kennewell, I’.D.
`540’.2461
`RS403
`
`Library of Congress Card No. 81-6703 AACR2
`
`ISBN 0—85312—207—5 (Ellis Horwood Limited, Publishers — Library Edn.)
`ISBN 0-85312-311-X (Ellis Horwood Limited, Publishers ~ Student Edn.)
`ISBN 0-470-27252—X (Halsted Press)
`
`Typeset in Press Roman by Ellis Horwood Limited, Publishers
`Printed in the U.S.A. by The Maple-Vail Book Manufacturing Group, New York
`
`COPYRIGHT NOTICE -
`All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval
`system, or transmitted, in any form or by any means, electronic, mechanical, photocopying,
`recording or otherwise, without the permission of Ellis Horwood Limited, Market Cross
`House, Cooper Street, Chichester, West Sussex, England.
`
`Mylan Exhibit 1023, Page 2
`
`
`
`Mylan Exhibit 1023, Page 2
`
`
`

`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`CHAPTER 3
`
`The Pharmacokinetic Phase
`
`
`
`The passage of a drug from its point of entry into the body until it reaches its
`site of action is covered by the pharmacokinetic phase. During this journey the
`drug will encounter many tissues which need to be entered and numerous
`membranes which need to be traversed. It is thus necessary to consider the
`structures of these membranes which are ubiquitous in tissues and, further,
`the structure of the fundamental building block of the body, the cell. From the
`structural features of the cell, and its means of assimilating materials, can be
`derived the physico-chemical properties of a drug desirable for passage through
`membranes. This knowledge can then be used to attempt to quantitatively relate
`the biological activity of a series of molecules to their chemical structures.
`Such relationships, especially their predictive uses, have occupied the dreams and
`ambitions of all medicinal chemists who desire to change the reliance of their art
`on serendipity to that of a logical science.
`
`3.1 THE CELL
`
`The cell is the fundamental building block of all independently viable forms of
`life. The cell may be described as being a unit of biological activity contained
`within a semi-permeable membrane and capable of self-reproduction in a medium
`free from other living systems. Thus the lowest forms of life on this earth consist
`of simple cells, which are capable of performing all essential functions, for
`example,
`ingestion of food, excretion of waste materials and reproduction,
`necessary to maintain their viability. Whilst numerous unicellular organisms
`thrive, such systems are very much at the mercy of the external environment,
`particularly its pH, temperature and ionic strength, over which they can have
`little control.
`
`Grouping together individual cells to produce a multi-cellular organism has
`a number of advantages. It is no longer necessary for all cells to be able to
`perform all the essential functions for life and some can specialise to the mutual
`advantage of the whole organism. The internal environment surrounding the
`
`Mylan Exhibit 1023, Page 3
`
`
`
`Mylan Exhibit 1023, Page 3
`
`

`
`48
`
`The Pharmacokinetic Phase
`
`[Ch. 3
`
`constituent cells can be more easily controlled and maintained separately from
`the external environment. A major disadvantage of co-operative units is that
`damage in a section of the organism can affect the whole organism, but despite
`this, multi-cellular organisms have reached some very sophisticated levels.
`An idea of the complexity achieved can be envisaged in the human body
`which contains an estimated 10” different types of cell ranging in size from
`that of nerve cells which can be 0.5-lm in length to the red blood cell which
`has a diameter of 7pm.
`It is perhaps useful at this stage, especially for organic chemists accustomed
`to thinking in molecular terms, to give an approximate idea of the scales involved
`(Fig. 3.1). A simple amino-acid with a molecular weight of approximately 200
`has a radius of 0.5 nm, whilst a small macromolecule, for example, egg albumin,
`
`1 metre height of 3-year old human child
`
`diameter of ostrich egg (single cell)
`
`10
`
`10
`
`diameter of human ovum
`
`diameter of human red blood cell
`
`(micron)l0_6 diameter of Staphylococcus albus (a bacterium)
`
`_7
`
`-8
`
`10
`
`(nanometer)l0—
`
`9
`
`length of tobacco mosaic virus (a virus)
`
`diameter of haemoglobin molecule (a protein)
`
`(X,angStr0m)10-10
`
`size of glycine (an amino acid)
`length of a C-C bond
`
`Fig. 3.1 — Scale of sizes in the biological world.
`
`has. a radius of 4nm. A large macromolecule, the haemoglobin molecule, has a
`radius of 12mm, whilst
`the smallest unicellular bacteria are some ten times
`larger than this. As seen in the table, some viruses are even smaller than this but
`as viruses can only reproduce inside a host cell, they are differentiated from cells.
`Using one of the smallest bacteria, Dialester pneumosintes, which has the
`dimensions 0.5 X 0.5 X l.0um, it is possible to calculate from its material
`composition and its size that it contains about 800 different proteins, having an
`
`Mylan Exhibit 1023, Page 4
`
`Mylan Exhibit 1023, Page 4
`
`
`

`
`Sec. 3.1]
`
`The Cell
`
`49
`
`average molecular weight of 40,000. Thus, ignoring the presence of any non-
`enzymatic proteins,
`this would mean that
`the cell
`is capable of performing
`some 800 enzymatic reactions. It seems that a basic minimum of reactions
`necessary to maintain a functioning living cell
`is between 500 and 1000. The
`lower limit to cell size is therefore set by the physical requirement of the volume
`needed to contain sufficient enzymes to catalyse these reactions.
`At the other end of the scale the upper limit to cell size is set by other
`considerations. A bird or reptile egg just after being laid is essentially a very
`large single cell. This is because the egg has to contain all the food necessary to
`maintain the developing embryo, but once the embryo starts to develop the cell
`is no longer unicellular. Within the body the upper size limit is controlled by the
`fact that, somehow, by processes not yet understood, the nucleus of the cell
`controls the whole activity of the cell. When the cell becomes too big this process
`becomes very inefficient. Cells exist because they exchange nutrients and waste
`materials with their environment through their membrane. As a cell gets bigger
`the ratio of surface area to volume falls rapidly and it becomes more difficult for
`the cell to obtain its nutrient supplies. In some cells this difficulty is overcome
`by the cell surface becoming deeply invaginated, essentially to increase the area,
`and in others by the cells becoming very long and thin.
`Thus the size of the cell needs to be sufficient to contain all the elements
`
`required to maintain viability, but not so large that its functioning becomes
`inefficient. The balance between these two considerations results in most cells
`
`having sizes in the range 1-20pm.
`It was stated earlier that an advantage for multi-cellular organisms is that
`certain cells can become specialised. A result of this specialisation is that the
`overall shapes and sizes of the cells can vary and this is certainly the case in the
`human body (Fig. 3.2). The cells of the gut, kidney and liver all have deeply
`invaginated surfaces, called microvilli, which give the cells enormous surface
`areas. One of the major functions of these cells is to absorb materials from their
`surrounding media, notably blood or intracellular fluids, and a large surface area
`will greatly facilitate this process. Muscle cells, on the other hand, spend their
`lives expanding and contracting and are, therefore, long, relatively thin, and
`packed with fibrils which govern their change in length. Nerve cells control the
`activity of the body in the transmission of electric messages. Thus, they consist
`of a central body with extremely long arms known as axons, which are covered
`with a myelin sheath to insulate them, rather as the rubber covering insulates
`electrical cables. The myelin sheath is designed to be highly impenetrable to
`ions and other materials but to facilitate rapid electrical conduction. The arteries
`and veins of the body are made up of tough smooth muscle cells which facilitate
`the blood circulation, but the walls of the capillaries are only a single cell thick
`to allow ready transfer of gases, nutrients and drugs between the blood and the
`body tissues. Thus the shape and sizes of the millions of cells in the body vary
`enormously depending on the roles they have to play in the functioning of the
`complete system.
`
`Mylan Exhibit 1023, Page 5
`
`
`
`Mylan Exhibit 1023, Page 5
`
`
`

`
`50
`
`The Pharmacokinetic Phase
`
`[Ch- 3
`
`Axon
`
`sheath@ "J-r
`
`Myelin
`
`é3
`
`Cilia
`
`3
`
` Mitochondrion
`
`Nucleus
`
`
`
`Fig. 3.2 — Sketches of representative body cells (not drawn to scale)! (a) Sensory
`nerve cell showing the very long axon; (b) skeletal muscle cell; (c) visceral muscle
`cell; (d) crlrated columnar cells from the nasal respiratory epithelium.
`
`3.1.1 Cell Structure
`
`Since the cell is the basic building block for all forms of life, however disparate
`they may be, it is perhaps not surprising that the key elements of cellular struc-
`ture are also common to all cells. Cells may differ in size and shape, but all have
`in common many of the elements described below.
`
`A generalised picture of the cell is shown in Fig. 3.3 a familiar picture
`which may perhaps give the impression that the cell is a bag of elements in a
`watery liquid contained in another skin-like bag. In fact, a more accurate analogy
`would be that of a honeycomb with a much more structured system, as will
`become clear as the component elements of the cell are discussed. Plate 3 p. 165
`shows an electron micrograph of a representative cell.
`
`3.1.1.1 The Cell Membrane
`
`Since the functioning cell results from a complex interplay between all its con-
`stituent parts it would be invidious, and indeed meaningless, to single out one
`part as being any more important
`than any other. Nevertheless, the cellular
`
`Mylan Exhibit 1023, Page 6
`
`Mylan Exhibit 1023, Page 6
`
`
`

`
`Sec. 3.1]
`
`The Cell
`
`51
`
`
`
`Secretory granule
`
`Golgi complex
`Dense body
`
`endoplasmic
`reticulum
`
`Cell membrane
`
`Lipid droplet
`
`Nuclear membrane
`
`Nuclear pore
`
`Fig. 3.3 — Diagram of a cell as it would appear in a thin section viewed in the
`electron microscope. This is in fact a secretory cell, but the major components are
`common to all cells. From W. M. Copenhaver, R. P. Bunge and M. B. Bunge in
`Bailey 3' Textbook of Histology, 16th Ed., (1971), p. 15, Williams and Wilkins Co.
`Ltd., Baltimore; reproduced with permission of the copyright owner.
`
`membrane is a fascinating, perplexing and vitally significant part of the cell, of
`particular importance to the medicinal chemist. The cellular membrane com-
`prises three, possibly interconnected, elements namely the plasma membrane,
`the endoplasmic reticulum and the nuclear membrane. Since most studies have
`been performed on the plasma membrane it is this membrane which will be
`discussed in more detail. All substances, including drugs, entering the cell must
`pass through this membrane and all drugs must pass through or interact with
`many such membranes during their course of action, accounting for its particular
`importance.
`The plasma membrane is an exquisite arrangement of proteins, lipids and
`carbohydrates which not only surrounds and delineates the cell, but also carries
`certain recognition sites which interact with hormones and neurotransmitters.
`These, after binding to the cell, induce fundamental changes in its behaviour.
`Whilst another role for the membrane is to prevent entry of possibly harmful
`materials into the cell,
`it does have mechanisms by which water, nutrients,
`especially sugars, and ions, particularly sodium, potassium and chloride, are
`able to penetrate it rapidly. It is not a rigid structure and severe damage will
`
`Mylan Exhibit 1023, Page 7
`
`Mylan Exhibit 1023, Page 7
`
`
`

`
`52
`
`The Pharmacokinetic Phase
`
`[Ch. 3
`
`allow the cell contents to leak out thus leading to death of the cell. It is thought
`that this may well be the mode of action of a number of toxins.
`Attempts to explain how the plasma membrane performs these and other
`functions have been the focus of extensive research in the last half-century.
`This work is still progressing and involves studies on the outer fringes of both
`practical and theoretical techniques. Thus although much has been discovered,
`or hypothesised, by no means have all the mysteries been unravelled. Much of the
`work has been performed on the membrane of the red blood cell, the erythro-
`cyte, because the erythrocyte, as well as being the smallest cell in the human
`body, does not usually have a nucleus. By placing the erythrocytes in a medium
`of low ionic strength, osmosis results in water entering the cell, and its distension
`to such an extent that all the intra-cellular material, the cytoplasm, eventually
`escapes. All that is then left is the membrane itself, the so-called erythrocyte
`‘ghost’. Studies on such ‘ghosts’ and other membrane preparations have esta-
`blished that the membrane is comprised of three essential elements: lipids,proteins,
`and carbohydrates. Whilst the ratio of these three elements varies within the
`different membranes and tissues of the cell, in general lipid molecules are the
`most abundant. However, the greater molecular mass of the proteins means that
`the relative mass proportions are roughly the same. Carbohydrate is the least
`abundant element and is usually associated with the plasma, rather than the
`internal membranes.
`
`Lipids
`
`Two main classes of lipids are found in membranes, the neutral cholesterol (33)
`and the ionic phospholipids (Fig. 3.4), all having in common the property of
`being amphipathic. This means that within the individual lipid molecules one
`section of the molecule is water soluble, hydrophilic, whilst the rest is insoluble
`in water and more soluble in organic solvents, or hydrophobic. Thus in choles-
`terol, a neutral non-hormonal steroid, the hydroxyl group is hydrophilic and the
`large carbon skeleton hydrophobic. In the ionic phospholipids, phosphatidyl-
`ethanolamine (34), phosphatidylcholine (35), sphingomyelin (36) and phos-
`phatidylserine (37), the glyceryl phosphate part of the molecule is hydrophilic,
`the fatty acid chains hydrophobic. If a solution of a fatty acid, or a phospholipid
`is spread on the surface of a bath of water, then the ionic, hydrophilic, end
`dissolves in the water whilst the long aliphatic chains wave around in the air.
`When forced totally into an aqueous environment, such lipids form spherical
`vesicles with the ionic portions of the molecule on the outside shielding the
`aliphatic chains from the water. Such relatively simple observations and concepts
`have played a considerable role in hypotheses about the architecture of the
`membrane, as will be shown below.
`
`Proteins
`
`The number of different lipids utilised in membrane formulation appears to be
`
`Mylan Exhibit 1023, Page 8
`
`Mylan Exhibit 1023, Page 8
`
`
`

`
`Sec. 3.1]
`
`The Cell
`
`53
`
`H0
`
`(33) CHOLESTEROL
`
`+
`
`Me3N
`
`0
`O=|l"'0—
`ll
`o
`9 H
`CH2(‘3CH2OCC15H31
`9
`0=‘|3
`C15H31
`
`+
`
`Me3N
`
`8
`o:g_ _
`6
`éH2E‘NHC0R
`¢HOH
`cH=cHc,3H2,
`
`(35) PHOSPHATIDYLCHOLINE
`
`(35) smueomvsun
`
`_
`
`_
`9
`9
`+ ‘F02
`IH3NCHCH20—l: -ocH2gHocc,5H3,
`0
`cH2ogc,5H31
`0
`
`(37)
`
`PHOSPHATIDYLSERINE
`
`+
`""3
`
`hydmphmc
`
`9
`O: - —
`3°
`0
`u
`I
`H
`CH2-('2-CH20(l:C15H3‘
`o
`04";
`C15H31
`
`hydrophobic
`
`(34)
`
`PHOSPHATIDYLETHANOLAMINE
`
`Fig. 3.4 — Typical membrane lipids.
`
`Mylan Exhibit 1023, Page 9
`
`Mylan Exhibit 1023, Page 9
`
`
`

`
`54
`
`The Pharmacokinetic Phase
`
`[Ch- 3
`
`restricted to around six,
`the structures of which, at least in the case of the
`phospholipids, are very similar. The proteins, however, are much less homo-
`geneous both in structure and in occurrence. The largest proteins appear to have
`molecular weights of around 300,000 and some of them appear to be present
`only in very small numbers indeed. The problems of structure determination of
`large molecular weight proteins, especially those which occur in very small
`quantities mean that these structures are not nearly so well defined as those of
`the lipids. However, it is known that many proteins are amphipathic and have
`regions which are hydrophilic and others which are hydrophobic. Thus as was
`the case with the lipids, these will also possess regions which are happier in water
`than in organic media and vice versa.
`
`Carbohydrates
`
`Carbohydrates, the minor constituents of the membrane structure, do not appear
`to be present as free structures but combined with either lipids, glycolipids,
`or proteins, as glycoproteins. Further, their glycolated structures seem to be
`confined to the outer surface of plasma membranes where they are conjectured
`to play a role in cell recognition and antibody-antigen interactions.
`Membrane Structure
`
`It is one problem to isolate and identify the individual elements which together
`make up the membrane but
`it
`is quite another to determine how these are
`actually fitted together in the living, intact, three-dimensional system. Many
`models of membrane structure have been proposed, only to be subsequently
`modified in the light of further experimental and theoretical advances, but it is
`fair to say that even today the picture is not totally clear.
`The first clues to their probable organisation came from the results described
`above of spreading fatty acid films on water. Clearly the amphipathic nature of
`lipids requires that the fatty acid part of their structure be maintained in isolation
`from the aqueous medium. The obvious way in which this could be achieved was
`as a bilayer structure (Fig. 3.5) in which the ionic heads of the molecules interact
`
`lipid "tails"
`
`Ionic "heads"
`Fig. 3.5 — Lipid bilayer.
`
`Mylan Exhibit 1023, Page 10
`
`Mylan Exhibit 1023, Page 10
`
`
`

`
`Sec. 3.1]
`
`The Cell
`
`55
`
`with water and the hydrophobic tails interact with each other. In one of the
`more celebrated of membrane models, Danielli and Davson suggested the
`triple layer structure shown in Fig. 3.6 where the inner lipid bilayer is sandwiched
`
`lipid bilayer
`
`Fig. 3.6 —- Schematic representation of the Davson—Daniel1i model of the membrane
`structure.
`
`by two layers of proteins whose function was to provide the hydrophilic inter-
`action. This model survived for many years and even seemed to be verified by
`the early electron micrographs which appeared to show just such a sandwich
`structure, although it does possess three glaring faults. The protein layer is
`implied to be entirely hydrophilic when it is clear that many membrane proteins
`are amphiphatic and possess lipophilic regions which could not favour this distri-
`bution. Secondly, many of the proteins are known to be globular and if they
`were attached to the lipid bilayer in this conformation then the whole sandwich
`would be too thick to account for the electron micrographs. Finally, the l1ydro-
`philic ends of the lipid chains are insulated from the aqueous medium in which
`they would be most stable.
`These, and other considerations, led to S. J. Singer suggesting the replace-
`ment of the sandwich model by the fluid mosaic model (Fig. 3.7).
`This envisages that the main feature of the membrane is the lipid bilayer in
`which float the globular proteins. Some of these proteins are integral and pass
`completely through the membrane, others are peripheral and are only associated
`with one membrane surface. The difference from the previous model is at once
`obvious and is quite profound. No longer are the proteins evenly and symmetri-
`cally spread over the surface, now they are concentrated and asymmetrically
`distributed. The integral proteins are envisaged to be globular proteins in which
`two hydrophilic regions interact with the aqueous media on either side of the
`membrane whilst
`the hydrophobic backbones interact with the lipid bilayer.
`Peripheral proteins were originally proposed to be rather like icebergs with
`hydrophobic regions floating in the lipid, but subsequent research suggests that
`they are restricted to the inner cytoplasmic membrane and are always found in
`
`Mylan Exhibit 1023, Page 11
`
`Mylan Exhibit 1023, Page 11
`
`
`

`
`56
`
`The Pharmacokinetic Phase
`
`[Ch~ 3
`
`association with the integral proteins. Such proteins can be readily detached
`from both the proteins and the lipids of the membranes whilst the integral
`proteins are difficult
`to isolate entirely free of lipid. On the other hand, no
`proteins seem to be entirely buried in the membrane.
`
`
`
`Fig. 3.7 — The Fluid Mosaic model of the membrane. Adapted from S. J. Singer
`and G. L. Nicholson, Science, 175, 720, (1972); Copyright 1972 by the American
`Association for the Advancement of Science.
`
`This simple model thus allows both major elements to exist in their preferred
`conformations. However, unique though it is, it does have its profound com-
`plexities. A major difference from the earlier model which had a plane of
`symmetry down the plane between the lipid tails is that the fluid mosaic model
`is asymmetric. Thus the integral proteins have different hydrophilic chain
`lengths sticking out of both sides of the membrane. Also, and it was some time
`before this was realised,
`the lipids are also asymmetric. In fact the choline-
`derived phospholipids appear to comprise the outer layer and the amino phos-
`pholipids the inner one with the cholesterol relatively concentrated in the
`outer layer. This asymmetry is very significant since it imposes uncertainties
`about the mode of formation of the membrane and the mechanism of main-
`tenance of the asymmetry.
`If a membrane is broken down into its constituent parts by means of a
`detergent and then allowed to reform, it is found that in the reformed membrane
`the proteins are arranged purely randomly. Thus the asymmetry is deliberately
`engineered into the membrane during its synthesis and then prevented from
`subsequent rearrangement.
`Possibly the simplest question to answer concerns the maintenance of the
`lipid asymmetry, in that it simply requires too much energy to re-orientate this
`arrangement. It has been established that whilst the lipid molecules can readily
`
`Mylan Exhibit 1023, Page 12
`
`Mylan Exhibit 1023, Page 12
`
`
`

`
`Sec. 3.1]
`
`The Cell
`
`57
`
`exchange position within their mono-layer by a process called lateral diffusion,
`only rarely does exchange take place between the layers via ‘flip—flop’ motion
`(Fig. 3.8).
`
`F|ip—f|Op(difficult)
`
`
`
`W l
`
`stlplfitlil
`
`Lateral diffusion (easy)
`
`Fig. 3.8 -— Motion available in a lipid bilayer.
`
`On reflection this is only to be expected, since lateral diffusion merely
`requires the lipid molecules to shuffle around with the major hydrophobic
`interactions of the fatty acid tails being largely unaffected. Flip—flop motion, on
`the other hand, will be much more difficult since it requires the hydrophilic
`phosphate head of the lipid to traverse entirely the lipid central core of the
`membrane. Similarly, an integral protein, once established in the membrane, can
`only reverse its position by passing its hydrophilic ends through the lipid layers.
`As stated above, simple mixing of proteins and lipids gives a random orien-
`tation. Thus the very process of cell membrane synthesis must produce the final
`orientation. Protein must be inserted into a section oflipid bilayer in its correct
`orientation before this is inserted into the membrane. The carbohydrate frag-
`ments must also be attached before insertion takes place. A possible mechanism
`of how this marvellously complicated process could occur is shown in Fig. 3.9.
`Essentially a mini-cell, a lipid vesicle, can be synthesized within the cell with
`the exterior fragments of the integral proteins held in the centre of the vesicle.
`The vesicle then travels through the cytoplasm to the inner wall of the membrane.
`Here the vesicle opens, rather like a flower, at the same time as it inserts into the
`membrane so that the inner wall of the vesicle now becomes the outer wall of
`
`the same time, of course, the outer portion of the integral
`the membrane. At
`membrane is placed in the outer wall of the membrane. This is a very simple,
`neat model which does provide a method by which the membrane structure
`could be established.
`
`The peripheral proteins of course pose nothing like the same problem since
`they can be synthesised within the cell and transfer through the cytoplasm to
`the inner wall of the membrane.
`
`Mylan Exhibit 1023, Page 13
`
`Mylan Exhibit 1023, Page 13
`
`
`

`
`58
`
`The Pharmacokinetic Phase
`
`[Ch. 3
`
`eExterior Surface
`el‘'
`“'u“§Plasma Membrane
`
`Cytoplasm
`
`
`
`en
`Protein/
`Lumen
`
`Vesicle
`Membrane
`
`Fig. 3.9 —- The passage of essential proteins synthesised inside the cell through the
`external membrane, from H. F. Lodish and J. E. Rothman,Scz'. American, (January
`1979), 240, 56; reproduced with the permission of the copyright owner.
`
`Mylan Exhibit 1023, Page 14
`
`
`
`Mylan Exhibit 1023, Page 14
`
`
`

`
`Sec. 3.1]
`
`The Cell
`
`59
`
`Overall, the picture is one of a largely lipid membrane through which the
`proteins are inserted in a manner which is almost certainly not random. It is
`entirely possible that individual and groups of proteins come together to produce
`recognition sites for hormones, neurotransmitters, etc. The essential feature,
`however,
`is that the lipid component provides the structural basis of the mem-
`brane whilst the less numerous protein molecules form the active elements.
`Finally, the caution stated at the beginning of this section should be reiterated.
`Much of the work described here has been conducted, of necessity, on a limited
`number of membranes, particularly that of the erythrocyte, and it is not intended
`to suggest
`that all membranes have the same structure. Further,
`the picture
`presented is an overall one and it is entirely possible that regions of the membrane,
`especially those close to the proteins, may have different structures due to strong
`protein-lipid interactions.
`In summary, the main functions of cell membranes are to limit the cell and
`protect
`it
`from its external environment
`(whilst permitting the passage of
`essential materials) and to catalyse digestive and energy-producing reactions
`using enzymes present within its structure. Of great importance to the medicinal
`chemist
`is the presence in the cell membranes of receptor sites not only for
`many hormones and neurotransmitters but the sites of action of many drugs.
`It is for this reason that the membrane has been dealt with in some detail.
`
`3.1.1.2 Protoplasm — Nucleoplasm — Cytoplasm
`The whole material contained within the plasma membrane is known as the
`protoplasm; which can be further sub-divided into the material contained within
`the nucleus, namely the nucleoplasm, and its exterior, the cytoplasm.
`
`3.1.1.3 The Endoplasmic Reticulum (Fig. 3.10)
`This is a complex system of fluid-filled tubes with a membrane very similar in
`
`Tubular
`
`endoplasmic
`reticulum
`
`“"
`
`‘
`
`' '
`
`"'3"
`
`- -
`
`Ribosome
`
`
`
`Cisternae of
`
`
`endoplasmic reticulum
`
`(9 9
`Vesicular
`
`
`
`endoplasmic reticulum
`Fig. 3.10 — Schematic drawing of the interconnected cisternae and tubules of
`granular endoplasmic reticulum. From W. M. Copenhaver, R. P. Bunge and M. B.
`Bunge in Bailey's Textbook of Histology, 16th Ed., (1971), p. 23, Williams and
`Wilkins Co. Ltd., Baltimore; reproduced with permission of the copyright owner.
`
`Mylan Exhibit 1023, Page 15
`
`Mylan Exhibit 1023, Page 15
`
`
`

`
`60
`
`The Pharmacokinetic Phase
`
`[Ch. 3
`
`constitution to the plasma membrane which permeates all parts of the cytoplasm.
`It is the endoplasmic reticulum which justifies the earlier description of the cell
`as a honeycomb rather than as a bag. A function of the endoplasmic reticulum is
`to carry substances through the cytoplasm within the tubes (which are filled
`with fluid derived from the extra-cellular environment); in some cases it is con-
`nected directly to the external environment via pores in the plasma membranes
`and this provides a route for substances to pass deep into the cell.
`Two varieties of endoplasmic reticulum have been identified, a rough and a
`smooth variety. The roughness, which can be clearly seen in electron micro-
`graphs, is caused by the presence of ribosomal particles which are missing in the
`smooth forms. These ribosomes are the principal sites of protein synthesis from
`amino acids in the cell. Smooth endoplasmic reticulum is believed to be the
`site of the synthesis of lipids, steroids and carbohydrates. The membranes of the
`endoplasmic reticulum penetrating, so completely as they do, the cytoplasm
`of the cell form a truly vast surface area on which are absorbed many of the
`enzymes involved in the synthesis of necessary cell materials. The absorption of
`enzymes on this surface, especially consecutive ones held in close proximity,
`helps to make the enzymatic processes so efficient.
`
`3.1.1.4 The Golgi Apparatus (Fig. 3.11)
`The Golgi apparatus is a specialised portion of the endoplasmic reticulum found
`near to the nucleus and comprises four or more layers of thin vesicles surrounded
`by a smooth membrane. The principle function of the Golgi apparatus appears
`to be in the preparation of materials prior to secretion from the cell. Proteins
`synthesised by the ribosomes on the endoplasmic reticulum and which have to
`be secreted from the cell, pass through the vesicles of the reticulum to the Golgi
`apparatus. There they are surrounded by a membrane to prevent dissipation as
`they pass through the cytoplasm and on to eventual secretion. Glycoproteins,
`glycolipids and protein polysacharides are also believed to be synthesised by the
`Golgi apparatus and these can also be packaged and secreted.
`Since the Golgi apparatus is so intimately involved in secretory processes,
`the extent of its development is dependent on the function of the cell. Thus it is
`
`C)___,_tj;Secretory granules
`
` Agranular membranes
`Q
`(fixes;°°o
`0 0 I0 0
`
`o
`
`\
`
`Vesicles
`
`Fig. 3.11 ~ Schematic representation of the Golgi Apparatus.
`
`Mylan Exhibit 1023, Page 16
`
`Mylan Exhibit 1023, Page 16
`
`
`

`
`Sec. 3.1]
`
`The Cell
`
`61
`
`well developed in secretory cells, notably in the mucosal glands which secrete
`digestive juices into the gastro-intestinal tract, and poorly developed in muscle
`cells.
`
`3.1.1.5 Lysosomes
`
`These are sacs of hydrolytic enzymes bound by a membrane whose function is
`to digest large molecules ingested by the cell. In a very real sense the lysosomes
`are the seeds of the cells’ own destruction since their enzymes, if released into
`the cytoplasm, are perfectly capable of digesting the cell itself. Indeed, a number
`of venoms including African Rhingals cobra venom destroy cells by disrupting
`the lysosomal membrane thus releasing the enzymes.
`
`3.1.1.6 Mitochondria
`
`Mitochondria are rod-like objects of 0.5-1 pm in diameter and vary in length
`up to 400 pm. They have a smooth outer membrane and an inner membrane
`much folded into projections known as cristae (Fig. 3.12). In the vast surfaces
`of the inner membrane thus produced are found enzymes whose function it is
`to metabolise the cell nutrients to produce adenosine triphosphate (ATP), the
`main energy source of the cell. The mitochondrion is, therefore, the energy
`source of the cell without which it would soon cease to function.
`
`Outer
`
` membrane
`
`Fig. 3.12 — Schematic view of a mitochondrion showing proposed inner structure.
`
`3.1.1.7 Centrioles
`
`Close to the nucleus of all animal cells in a clear non—granular form of pr0t0-
`
`plasm called th