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cuaprer 6 a a | | neo da Biotechnology and Drug Discovery E JOHN M, BEALE, JR BIOTECHNOLOGY: AN OVERVIEW Developments in biotechnology in recem times have been quite dramatic. The years between 1999 and 2001 witnessed a tremendous increase in the number of biotechnology-re- lated pharmaceutical products in development, and a number of important new drugs progressed through trials and into the clinic. À good reflection of the impact of biotechnology 15 the GenBank datubuse. GenBank is an electronic reposi- tory of gene sequence information, specifically the nucleo- tide sequences of complementary DNA (CDNA), represent- ing the messenger RNA (mRNA), and genomic clones that have been isolated and sequenced by scientists world- wide," ? The growth of the GenBank database has been rapid, and it has been increasing steadily since about 1992. Figures 6-1 and 6-2 graphically depict these growth rates In October 2002, the Pharmaceutical Research and Manu facturers Association (PhRRMA) reported that 371 biotech- nology-derived medicines were in testing at various stages and that nearly 200 diseases are being targeted by research conducted by 144 companies and the National Cancer Insti- tute. Of these—all of which are in human trials or awaiting Food and Drug Administration (FDA) approval-—=178 are new drugs for cancer, 47 are new drugs for infectious dis- cases, 26 are new drugs for autoimmune diseases, 22 are new drugs for neurological disorders, and 21 are new drugs for human immunodeficiency virus (HIV) and acquired im- munodeficiency syndrome (AIDS) and related conditions* PhRMA also reported 194 new medicines targeted for pedi- atric use.* Approved drugs derived from biotechnology also treat or help prevent myocardial infarction, stroke, multiple selerosis, leukemia, hepatitis, rhewmatord arthritis, breast cancer, diabetes, congestive heart failure, lymphoma, renal cancer, cystic fibrosis, and other discases. The number of approvals of biotechnology drugs per year has been increas- ing steadily. These data are shown in Figure 6-3, The Human Genome Project, an international effort to ob- tain complete genetic maps, including nucleotide sequences, of each of the 24 human chromosomes, has spawned much new knowledge and technology. K 15 awesome to consider that in the mere 30 years since 1972, the science has reached the stage of atempting genetic cures for some diseases, such as evstic fibrosis and immune deficiency disorders BIOTECHNOLOGY AND PHARMACEUTICAL CARE As it alfects medicine and pharmaceutical care, biotechnol- ogy has forever altered the drug discovery process and the 160 thinking about patient care, Extensive screening programs once drove drug discovery on natural or synthetic com- pounds. Now, the recombinant DNA (DNA j-driven drug discovery process is beginning to yield new avenues for lhe preparation of some old drugs. For example, insulin, once prepared by isolaúon from pancreatic tissue of bovine or porcine species, can now be prepared in a pure form identical with human insulin. Likewise, human growth hormone, once isolated from the pituitary glands of the deceased, can now be prepared in pure form, Recombinant systems such as these provide high-yielding, reproducible batches of the drug and uniform dosing for patients LITERATURE OF BIOTECHNOLOGY Many good literature sources on biotechnology exist for the pharmacist and medicinal chemist. These cover topics such as management issues in biotechnology.** implementation of instruction on biotechnology in education, "2 costs of bivtechnology drugs implementation in a practice set- ting, 2º regulatory issues,“ product evaluation and for- mulation,2-** patient compliance“ and finding informa ton, Additionally, there are a number of general text review urticles,*!"** and a general resource refer ence catalogue."” Any good biochemistry textbook is also a useful resource BIOTECHNOLOGY AND NEW DRUG DEVELOPMENT The tools of biotechnology are also being brought to bear in the search for new biological targets for presently avail- able drugs as well as for the discovery of new biological molecules with therapeutic utility, Molecular cloming of novel receptors can provide access to tremendous tools for the testing of drugs (e.g., the adrenergic receptors), while cloning of à novel growth factor might potentially provide u new therapeutic agent. Biotechnology is also being used to screen compounds for biological activity, By using cloned and expressed genes, it is possible to generate receptor pro- teins to facilitate high-throughput screening of drugs in vitro or in cell culture systems rather than in animals or tissues Biotechnology is being investigated in completely novel ap- proaches to the battle against human disease, including the use of antisense oligonucleotides and gene replacement ther- apies for the treatment of discases such as cystic fibrosis Chapter 6 = Biotechnology and Drug Discovery 161 6-1 = Yeary growth of Gen- km base pars use of monocional antibodies for the treatment of nology encompasses many subdisciplines includ- nomios, proteomics, gene therapy, made-to-order mol- +, computer-assisted drug design, and pharmacogeno- goal of biotechnology in the early 21st century is the “one drug fis all” paradigm for pharma- drugs that are elaborated by biotechnological methods eins and, hence, require special handling, There are sie requirements of pharmaceutical care for the phar- working with biotechnologically derived products:** An understanding of how the handling and stability of bioplhar- maceuticals differs from other drugs that pharmacists dispense Knowledge of preparation of the product for patient use, in elúding reconstitution or compounding 1 required Paticnt education om the discuse, benefits of lhe preseribed bropharmaceutical, potential side effects or drug interacions to be aware of, and the techniques of self-administration Patient counseling on reimbursement issues involving an ex- pensive product Monitoring of the patrent for compliance The pharmacist must maintain an adequate knowledge of agents produced through the methods of biotechnology and re h=2 = Yearly growth of Gen- terms ol gene sequences Chapter 6 = Biviechnology and Drug Discovery 163 Introns | j E Pre-mRNA ranslation Proteins Modification Posttranslationally modified protein Path from DNA to pro- e proteins can also direct the three-dimen- gene into a transmissible vector that can be transcribed, am- n. Posttranslational modifications occur in plified, and propagated by à host cell's biochemical machin- ery; transferring it to that host cell, and facilitating the tran- the protein is transported out of the cell. seription into mRNA and translation into proteins. Cloned jonal modifications occur only in higher DNA can also be removed or altered by using an appropriate ie notin bacteria. The three-base genetic codon sys- restriction endonuclecase. Since genes encode the language El Enown and has been conserved among all organ- of proteins, in theory it is possible to creale any protein if allows TDNA procedures to work and facilitates one can obtain a copy of the corresponding gene. rDNA of a model for the amino acid sequence methods require:” e An efficient method for cleaving and rejoining phosphodiester bonds on fragmenta of DNA (genes) derived from an array of different sources DNA (rFDMA) Technology * Suitable veciors or carmers capable of replicating both them- selves and the foreign DNA linked to them techniques involved in working with rDNA e A means of introducing the DNA into a bacterial, yeast, plant, E OF COpying à gene; inserting the precise or mammalian cell 164 Wilson and Gisvold's Texthook of Organic Medicinal and Pharmaceutical Chemistry * Procedures for sercening and selecting a clone of cells thai has sequired the FONA molecule from a large population of cells There are two primary methods for cloning DNA“ using genomic and cDNA libraries as the primary sources of DNA tragments, which, respectively, represent either the chromo- somal DNA of a particular organism or the cDNA prepared from mRNA present in a given cell, tissue, or organ. In the first method, a library of DNA fragments is created from a cell's genome, which represents all of the genes present. The library is then sereened against special DNA probes. Lysing the genomic contents to generate fragments of different sizes and compositions, some of which should contain the genetic sequences thai encode the specific activity that one is seek ing, creates the library. With knowledge of the protein se- quence that the gene specifies, DNA probes can be synthe- sized that should hybridize with corresponding fragments in the library. By labeling the probes with fluorescent or radioactive tags, probe molecules that hybridize and form double-helical DNA can be identified and isolated electro- phoretically. The DNA from the library can then be ampli- ficd by a technique such as the polymerase chain reaction (PCR), inserted into a vector, and transferred into a host cell. À comparison of these methods is given in Table 6-1 The second major method for cloning DNA represents only genes that are being expressed” at à given time and involves first the isolation of the mRNA that encodes the amino acid sequence of the protein of interest. Treating the mRNA with the viral enzyme reverse transcriptase in the presence of nuclcoside triphosphates (NTPs) causes a strand of DNA to be synthesized complementary to the mRNA matrix, affording a RNA-DNA hybrid. The RNA strand is broken down in alkaline conditions, yielding a single- stranded molecule of DNA. The DNA polymerase reaction atfords a complementary or copy strand of DNA (cDNA), which on fusion of a promotor sequence can be attached to a transport vector. Figure 6-5 depiets these reactions, LÊ the amino acid sequence of a protein (and, hence, the codon sequence) 1s known. automated synthesis of DNA through chemical or enzymatic means represents a third way lhat genes can be engincered. This method is useful only for TABLE 6-1 Characteristics of Genomic Versus cDNA Libraries Characteristic Genomic cDNA Source of genetic miterial Genômic DNA Coll cw tissue mRNA Complexity (independent > [O LONIO S 000 20,000 recombinanis | Size range of recormbimants bp”) 1 ODO-SA,D00 30. HO 000 Presence of introns Yes No Presence of regulatory elements Yes Mayhe Suitable for heterologous Maybe Yes Expression bp. huse pair mRNA + Rivera Transcripiago DNA Synthesis, mRNA as Matrix RNA-DNA Hybrid , Elmo (bene comer RNA) , —————————— Difyá, DMA Potymoraso , cODNA Fuse in promotor ragmon ration of cODNA from a mRNA Figure 6-5 = Method for preí transcript relatively small proteins. In principle, for the preparation of a genomic library the cellular origin of the DNA is not an issue, whereas the cellular origin of mRNA is central to the preparation of a cDNA library. Therefore, genomic libraries vary from species to species but not from tissue to tissue withun that species, CDNA varies with tissue and the devel opmental stages of cells, tissues, or species. Another impor tant distinction is that the fragment of DNA from cukaryotie chromosomes will contain exons tprotem coding segments) and introns (noncoding segments between exons), whereas in cDNA the introns are spliced out Restriction Endonucleases"* º5 The restriction endonuclease (or restricion enzyme) is prob: ably best described as a set of “molecular seissors” in ma ture. Restriction endonuclcases aré bacterial enzymes that, as the name implies, cleave internal phosphodiester bonds of a DNA molecule. The cleavage site on a segment of DNA lies within a specific nucleotide sequence of about six to eight base pairs. More than 500 restriction endonucleases have been discovered, and these react with more than 00 differem cleavage sites. The chemical reaction of the restric tion endonuclease releases the 3º end of one base as an alco- hol and the 5º end as a monophosphate. The general reaction is shown in Figure 6-6 The recognition sites for restriction endonuclcases arc specific palindromic sequences o! DNA“ not more lhan À bp long. A number of these palindromes are listed in Table 6-2. A palindrome is à sequence of letters that reads the same way forward and backward, for instance: “A man, à plan, a canal; Panama!” ““DNA-land,'* “Did Hannah see bees? Hannah did” Restriction endonuclcases cleave DNA at palindromic sites to yield several types of cuts: l SCCCTAGG Y -— — SºCCTAG 3º Y GGATCC 5º Y GATCE 5 The above cut yields an overhanging C/C *stcky” end that Figure 6-1 tion IS Felativel: DNA mole 5 CCiGo 3 3 GGICE 5 Frequires my DNA. Palir ton enzym cleavage si Eroup of em; ing in the fix to their use make their « DNA Lig: When the ge DNA by the vector DNA endonucleas ent DNA ma the first step stranded DN is added to th fatos pairing, ter bonds are e TABLE 6-2 eee Alul AGLCT EcoRW GATLATO Mihel AGATO 166 Wilsen and Gisvold's Texthook of Organic Medicinal and Pharmaceutical Chemistry EcoRI Restriction Site Left Arm CE km Right Arm EE à DNA 48,502 base pairs Antiblotic Resistance Gene Bactariophage À EcoRl Restrictlon Site E. coli plasmid pBR322 bacteriophage A, and when the virus infects, inserted into cells. Hybridization is then detected by screening with DNA probes.” In addition, there are special vectors called phagemids, vaccinia and adenovirus for cloning into mam- malian cells, and yeast artificial chromosomes (YACs) that facilitate cloning in yeasts“* Differences among these vec- tors concerm the size of the insert that they will accept, the methods used in the selection of the clones, and the proce- dures for propagation Once the passenger DNA has been created and the plasmid vector cul (both with the same restriction enzyme), the insert is Hgated into the plasmid along with a promoter (a shom DNA sequence that enhances the transeription of the adija- cent gene). Often, a gene imparting antibiotic resistance linked to the desired gene is inserted as à selection tool, The idea behind this is that if the gene is inserted in the proper location, lhe bacterial cell will grow on à medium containing the antibiotic. Bacteria that do not contain the resistance gene and, hence, lack the required gene will not grow. This makes the task of sercening for integration of the desired gene casier. After the molecule is ligated, the vector is finally a TDNA molecule that can be inserted into à host cell Host cells can be bacteria (e.g., E. coli), cukaryotic veast (Saccharomyces cerevisige), or mammalian cell lines includ ing Chinese hamster ovary (CHO), African green monkey kidney (VERO). and baby hamster kidney (BHK). It is casy to grow high concentrations of bacteria and yeast cells in fermenters to yield high protein concentrations. Mammalian cell culture systems typically give poorer protein yields, but sometimes this is acceptable, especially when the product demands the key posttranslational modifications that do not occur in bacteria. Host cells containing the vector are grown 4,361 base pairs Origin of Replication Figure 6-7 a Types of cloning qe amd à vectors: a bacterópha plasmid in small-scale cultures and screened for the desired gene” When the clone providing the best protein yield 1s located, the organism is grown under carefully controlled conditions and used to inoculate pilot-scale fermentations. Parameters such as production medium composition, pH, aeration, agita tion, and temperature are investigated at this stage to opti mize the fermentation. The hosteells divide and the plasmids in them replicate, producing the desired “new” protein. The fermentation is scaled up into larger bioreactors for large scale isolation of the recombinant protein. Obviously, the cultures secrete their own natural proteins along with the cloned protemm. Purification steps are required before the re- combinant protein is suitable for testing as a new, genetically engineered pharmaceutical agent, Once the host cell line expressing the recombinant gene is isolated, it is essêntial to maintain selection pressure on itso that it does not sponta neously lose the plasmid. Typically, this pressure is applied by maintaining the cells on medium containing am antibione to which they bear a resistance gene. SOME TYPES OF CLONING A listing of some types of cloning is given in Table 6-3, Functional Expression Cloning”? Functional expression cloning focuses on obtaining a spe cific cDNA of known function. There are many variations on this approach, but they all rely on the ability to search for and isolate cDNAs based on some functional actrvily 167 Chapter 6 = Biotechnologv and Drug Discovery “TABLE 6-3 Cloning Strategies Strategy Advantages Disadvantages Example Pesitional Provides information underiying the 23x 10" bp, difficult to use with diseases Cystle fibrosis gene product genetic basis of known disenses Vields genetic information encoding proteins of known structure and function purification Yields genetic information encoding proteins of known structure amd Function Yiclds genetio information encoding a functionally nctive protein; does ot require protein purification Identification of related gomes or gene families; relatively simple Expression based High ihroughput: idemification of novel cDNAs Knowledge of total genome; Identification of all potemtin] gene proclucis sequence tapging genomic soquencing caused by multiple interacting alleles Protein purification, especially for Ba-Adrenergio receptor lovw-nbundance proteins, is exacting: availability of appropriate libraries, incompleto coding sequenoes Envolves protein purification, unrecognized cross-reactivity, incompleto coding sequentes Vitamin DD receptor Function must be compatible with existing tibrary-soreening technology Substanoe K receptor Deperds on precxisting gene sequenço, can yield incomplete genes or genes of unknown function Muscarinio receptor Incomplete coding sequences or genes of unem function 33» 10"bp, labor intensive; genes of unknown function Hacimephilius inflnende dioning lhatcan be measured, e.g., the electrophystological measure- ogv-based cloning, takes advamage of the fact that nucleo e and a ment of jon conductances following expression of cDNAs tide sequences encoding important functional domains of da frog oocytes. By incrementally subdividing the cDNAs proteins tend to be conserved during the process of evolu- imo pools and following the activity, it is possible to obtain ton. Thus, nucleotide sequences encoding regions involved mo single cDNA clone that encodes the functionality. The with ligand binding or enzymatic activity can be used as Eca antage of functional expression eloning is Uhat it does probes that will hybridize to complementary nucleotide se- ocated, Hot rely on knowledge of the primary amino acid sequence. quences that may be present on other genes that bind similar iditiong This às q definite advantage when atempting to clone pro- ligands or have similar enzymatic activity, This approach trees feims of low abundance. can be combined with PCR?! “2 to amplify the DNA se- | agias quences. The use of homology-based cloning has the advan- bo cpa Positional Cloning?º tages that it can be used to identify families of related genes, lasmids a - : does not rely on the punification or functional activity of a dn. The Positional cloning can be used to localize fragments of DNA given protein, and can provide novel targets for drug discov- rasa depesenting genes prior to isolating the DNA. An example cry, Its usefulness is offset by the possibility that the isolated “ly, the Bl lhe use of positional cloning is the cloning of the gene fragment may not encode a complete or functional protein cith the Eponsible for cystic fibrosis (CF). By studying the patterns or tha in spite of knowledge of the shared sequence, lhe pad e of ção e e at dio Ana with actual function of the clone may be difficult to identify. etc; : mv chromosormal markers Chinkage analysis), MU was pos- . > ell line Pablo, without knowing the function of the gene, to locate the ssential Rene on human chromosome 7. Then, by using à technique sponta- own as chromosome walking, lhe gene was localized to applied BG DNA sequence that encodes a protein now known as the EXPRESSION OF CLONED DNA tibiotie Bic fibrosis transmembrane conductiance regulator K” Loo, HORTR). This protein, previously unknown, was shown to Once cloned, there are many different possibilities for the edefective in CF patients and could account for many of the pa und manipulation of DNA sequences, As tl con- ptoms of the disease. Like functional cloning, positional cerns the use of cloned genes in the process of drug discovery | : ng has the advantage that specific knowledge of the and development, there are many obvious ways in which the p ? is not required. It is also directly relevant to the expression of DNA sequençces can be applied. One of the ding of human disease, and it can provide impor- most obvious is in replacement of older technologies that :63, new biological targets for drug development and the involve the purification of proteins for human use from either es OÍ discasc animal sources or human by-products, such as blood. An example of this is factor VHI, a clotting cascade protein used B 1 Cloni for the treatment of the genetically linked bleeding disorder a spe- E ng hemophilia. Until recently, the only source of purified factor riations ré ah . À genes to guide identification and cloning of eve many reluted genes. This approach. referred to as hor Another cloning strategy involves the use of previously VIH was human blood, and tragically, before the impact of AIDS was fully appreciated, stocks of factor VII had be- come contaminated with HIV-=1, resulting in the infection qu tod ligand-binding domams at rransmambrane domains eemecirimi Dc. intracelular sgnaling mat, domaimes Receptor A Receptor B É Rieceptors re: Figure 6-8 = Chimeric receptors. ss illustrated in Figure 6-8 for two receptors labeled A UH, Each receptor has functional domains that are respon- for ligand binding. integration into lhe plasma mem- e, and activation of intracellular signaling pathways. TONA techniques, one can exchange these funcional ns bo create chimeric receptors that, for example, con- Win he ligand-binding domain of receptor B but the trans- nbrane and intracelular signaling domains of receptor ; The application of the Lusion protein strategy is discussed ither in connection with the human growth hormone recep- under the heading, Novel Drug-Screening Strategies) pd with denileukim difiitox. Another reason for combining elements of two proteins into One recombinant protein às to facilitate its expression purficaton. For example, recombinant glutathione S- sferase 1GST), cloned from the parasitic worm Schisto- Ra aponicim, is strongly expressed in E. coli and has a indine site for glutathione. Heterologous seguences encod- he the functional domains from other proteins can be fused, qto the carboxy terminus of GST, and the resulting protein is often expressed at the same levels as GST “Jo addition, the resulting fusion protein still retaims . ab to bind glutathione, which means that affinity immtography, using glutathione that has been covalently led to agarose, cam be used for a single-step purification lhe fusion protein. The functional activity of the heterolo- oimains that have been fused to GST can then be stud- Eras par of the fusion protein or separately following of the fusion protein with specific proteases that e the function berween GST and the heterologous à Puried fusion proteins can also be used to generate dies to the heterologous domains and for other bio- pal studies. Sometimes, fusion proteins are made to a recombinant protein that cam be casily identified. ple of this is a technique called epitope ragging, in well-characterized antibody recognition sites ure lh recombinant proteins. The resulting recombinant Chapter 6 » Biotechnology and Drug Discovere 169 protein can then be identified by immunofluorescence or can be purified with antibodies that recognize the epitope. NEW BIOLOGICAL TARGETS FOR DRUG DEVELOPMENT One of the outcomes of the progress that has been made in the identification and cloning of genes is that many proteins encoded by these genes represent entirely new targets for drug development. In some cases, the genes themselves may represent the ultimate target for the treatment of à discase in the form of gene therapy. The cloning of the cystic fibrosis gene is an example of both à new drug target and a gene that could potentially be used to treat the disease. The protein encoded by this gene, CFTR, is a previously unknown inte- gral membrane protein that functions as a channel for chlo- ride ions. Mutations in CFTR underlie the pathophysiology of cystic fibrosis, and in principle, replacement or coexpres- sion of the defective gene with the healthy, nonmutated gene would cure the disease, lt is also possible, however, that by understanding te structure and function of the healthy CFTR, drugs could be designed to interact with the mutated CFTR and improve its function. An important outgrowth of the study of new drug targets is the recognition that many traditional targets, such as en- zymes und receptors, are considerably more heterogençous than previously thought, Thus, instead of one enzyme or receptor, there may be several closely related subtypes, or isoforms, each with the potential of representing a separate drug target. This can be ilustrated with the enzyme cyeloox- vgenase (COX), which is pivotal to the formation of prosta- elandins und which is the target of aspirin and the nonsteroi- dal anti-inflammatory agents (NSAIDs). Until recently, COX was considered to be a single enzyme, but pharmaco- logical and gene cloning studies have revealed that there are at least three enzyme forms, named COX-1, COX-2, and COX-3. Imerestingly, they are differentially regulated. COX-1 is expressed constitutively in many tissues, whereas the expression of COX-2 is induced by inflammatory pro- cesses. Thus, the development of COX-2 selective agents can yield NSATDs with the same efficacy as existing (nonse- lecuve) agents bui with fewer side elfects, such as those om the gastric mucosa. The elucidation of the Family of adrenergic receptors is another example in which molecular cloning studies have revealed previously unknown heterogeneity, with the conse- quence of providing new targets for drug development, The adrenergic receptors mediate the physiological effects of the catecholamines epinephrine and norepinephrine. They are also the targets for many drugs used in the treatment of such conditions as congestive heart failure, asthma, hypertension, glaucoma, and benign prostatic hypertrophy. Prior to the molecular cloning and purification of adrenergic receptors, the pharmacological classification of this family of receptors consisted of four subtypes: ay, 05. By and 8 The initial cloning of the a-adrenergic receptor in 1986 and subsequent gene cloning studies revealed at least nine subtypes: &. Bs. Ba tras Cs Cp, Conv Con, and ese (Chapter 16). The evidence that there are nine subtypes of adrenergic receptors is very important in terms of understanding the 170 Wilson and Gisvold's Texthook of Organic Medicinal and Pharmaceutical Chemistry TABLE 6-4 Selected Examples of Receptor Subtype Heterogeneity Receptor Superfamily Original Subtypes Present Subtypes C-protein-coupled Adrenergio Pi, Ps tap ts Bi. Bs Bs ias tum ri aÃ, 58, 40, Dopamine Do. D; D,, Dj, Ds. Du, Ds Prostugiandin Es EP, EPs, EPs EP, EPs RP", EPs Receptor tyrosine kinase netrotrophins DNA binding Estrogen Thyrold hormone Remnoic acid Ligand-activated channels Glycine GABAs Estrogen receptor Nerve growth factor receptor Thyroid hormone receptor Retimic acid receptor Glycine and/or strychmine receptor GABA andior bengodinzenine receptor TRA, TrkB, TR ERRI, ERRZ TRa, TRÊ RARa, RARB drgs traços (oultisabunit” Epa ao jo Ra, Oie to (riam Abermative mRNA splicing creates additional receptor heterogencity Omby the heeterogemetoy of the Dngamd- boncirg suando do bintoad; arrasto mamas siructuro comibinco math the heterogeneity of the other sabunits crostes a very barge mumbser of por Mental subery ques. physiology of the adrenergic receptors and of developing drugs that can selectively interact with these subtypes. For example, in the case of the ay-agonist p-aminoclonidine, an agent used to lower intraocular pressure (TOP) in the treat- ment of glaucoma, it may now be possible to explain some of the drug's pharmacological side effects (e.g., bradyeardia and sedation) by invoking interactions with the additional as-adrenergic receptor subtypes. Of considerable interest is the possibility that these pharmacological effects (Le, lower- ing of OP, bradycardia, and sedation) are cach medisted by one of the three different ay-receptor subtypes. If this is true, it might be possible to develop a subrype-selective a- agonist that lowers TOP bui does not cause bradycardia or sedation. Likewise, il might even be possible to take advan- tage of the pharmacology and develop es-adrenergic agents that selectively lower heart rate or produce sedation, The discovery of subtypes of receptors and enzymes by molecular cloning studies seems to be the rule rather than the exception and is offering a plethora of potential new drug targets (Table 6-4). To note just a few: 5 dopamine receptor subtypes have been cloned, replacing 2 defined pharmacologically (Chapter 15), 7 serotonin receptor sub- tvpes have been cloned, replacing 3; 4 genes encoding recep- tors for prostaglandin E, have been isolated, including 12 addinonal alternative mRNA splice variants; and 3 receptors for nerve growth factor have been cloned, replacing |, NOVEL DRUG-SCREENING STRATEGIES The combination of the heterologous expression of cloned DNA, the molecular cloning of new biological targets, and the ability to manipulate gene sequences has created power- ful new tools that can be applied to the process of drug discovery and development. In its most straightforward ap- plication, the ability to simply express newly identified re- ceptor protein targets offers a novel means of obtaining in- formation that may be difficult, or even impossible, to obtain from more complex native biological systems. There is a reason for this. À newly identified protein can be expressed in isolation. Even for closely related ensyme or receptor subtypes, heterologous expression of the individual subtype can potentially provide data that are specific for the subtype being expressed, whereas lhe data from native biological systems will reflect the summation of the individual subrypes that may be present. The potential advantage of heterologous expression is ih lustrated in Figure 6-9 for the interaction of a drug will multiple binding sites. In panel A, which can represent he data obtained from a native biological system, the data are complex, and the curve reflects interactions of the drug wiih two populations of receptors; one with high afinity, repre- senting 50% of the total receptor population, and one wilh low affinity, representing the remaining 509%. The individual contributions of these two populations of receptors are indi- cated in panel B, which could also reflect the data obtained if FDNA encoding these two receptors were expressed indi vidually in a heterologous expression system, Although iz some cases the data, as in panel A, can be analyzed will success, frequently they cannot, especially 1f more tham (wo subtypes are present or if any one subiype makes up les than 109% of the total receptor population or if the affinite | of the drug for the two receptor populations differ by less than TO-fold. Another important reason for imegrating heterologom expression into drug-screening strategies is that data can us ally be obtained for the human target protein rather than am animal substitute, This does not mean that organ prepare tions or animal models will be totally replaced. For the pur poses of the identification of lead compounds and the optim sation of selectivity, affinity, etc, however, the use dl recombinant expression systems provides some obvious ab vantages, By combining heterologous expression with novel fume tional assays, il is possible to increase both specificity amd throughput (the number of compounds that can be screen! 172 B-galactosidase, the activity of the reporter will be increased as the number of cells increase as à consequente of receptor activation. Initially, a limitation of this assay was that dt only worked with receptors that normally coupled to cellular proliferation; by making a mutation in one of the second- messenger proteins involved with the proliferative response, however, il was possible to get additional receptors to work in this assay. This second-messenger protein, G,. Was cloned, and à recombinant chimera was made that included part of another second messenger known as G, In native cells, receptors thal acuvate G, are not known for their stimu- lation of cell proliferation, but when such receptors aré coex- pressed in the r-SAT assay with the chimeric G,. their activ- ity can be measured. A similar strategy involving chimeric proteins has been used for receptors whose second-messenger signaling path- ways are not clearly understood. For example, the develop- ment of potential (herapeutic agents acting on the human growth hormone receptor has been difficult because of a lack of a good signaling assay. The functional activity of other receptors that are structurally and functionally related to the growih hormone receptor can be measured, however, in a cell proliferation assay. One such receptor that has been cloned is the murine receptor for granulocyte colony-stimu- Iming facior (G-CSF). By making a recombinant chimeric receptor containing the ligand-binding domain of the human growth hormone receptor with the second-messenger-coup- ling domain of the murein G-CSF receptor, it was possible to stimulate cellular proliferation with human growth hormone. In addition to providing a useful pharmacological sereen for human growth hormone analogues. the construction of wis chimeric recepror provides considerable insight into the mechanism of agonist-induced growth hormone receptor ac- tivation. The growil hormone-binding domain is clearly localized to the extracellular amino terminus of the receptor, while the transmembrane and intracelular domains are im- plicated in the signal transduction process. ll was also deter- mined that successful signal transduction required receptor dimerization by the agonist (L.e., simultaneous interaction of wo receptor molecules wii one molecule of growth hor- mone), On the basis of this information, a mechanism-based strategy was used for the design of potential antagonists. Thus. human growth hormone analogues were prepared that were incapable of producing receptor dimerization and were found to be potent antagonists, PROCESSING OF THE RECOMBINANT PROTEIN Processing the fermentation contents to isolate a recombi- nam protein is often a difficult operation, reguiring as much art as science. In the fermentation broth are whole bacterial cells, Iysed cells, cellular fragments, nucleotides, normal bacterial proteins, the recombinant protein, and particulate medium components. [fa Gram-negative bactenum such as E. coli has been used, lipopolysaccharide endotóxins (pyro- gens) may be present. When animal cell cultures are used, it is commonly assumed that virus panticles may be present. Viruses can also be introduced by the culture nutrients, gen- Wilson cmd Gisvoldl's Texthook of Organte Medicinal and Pharmacentical Chemistry erated by an infected cell line, or introduced by animal serum, Purification of a FDNA protein while maintaining the factors that keep it in its active three-dimensional conformas ton from this mixture may be difficult because cach step must be designed to ensure that the protein remains intact and pharmacologically active. Assays must be designed that allow the activity of the protein to be assessed at cach purifi- cation step. Consequently, the structure and activity of the recombinant protein must be considered at all stages of puri- fication, and assays must be conducied to measure the amount of purified, intact protein. A general scheme for purification of a TDNA protein is as follows: º Particulate removal. Particulutes may be removed by centrifu- gation, filtration, ultrafiltration, and tangential Flow filtration Virus particles may be inactivated by heating if the rDNA peptide cam tolerate lhe procedure * Concentration. The volume of the mixture às reduced, which increases lhe concentration of the contents. Often, concentra tion is achievable by the filtration step, espectally if ultrafilivas ton is used e Initial purification, The initial purification of the mixture às sometimes secomplished by precipitation of the proteins, using a slow, stepwise incrense of the tonic strength MR solution (salting out). Ammontum sulfate is a typical salt Uhal can be used in cold, aqueous solutions. Water-miscible organi solvents such as tnchloroacetic acid and polveihylene glyool change the diclectric constant of the solution and also effeci precipitation of proteins o Intermediate purificanion. In this stage, lhe proteins may be diulyzed against water to remove salts thal were used in the precipitation step. lon exchange chromatography is used to effect a somewhat crude separation of the proteins based oa their behavior in a pH or salt gradient on the resin, Another step that may be taken às size exclusion (gel filtration) chroma tography. Gels of appropriate molecular weight cutoffs cam vield a somewhat low-resolution separation of proteins of p desired molecular weight 1 a mutive bacterial protein that hus been carried this (ar is nearly the same molecular weight as the FDNA protein, no separatiom will occur e Final purification; Final purification usually mvolves the use of high-resolution chromatography. twpically Ingh-perior mance liquid chromatography. An abundance of commercial stationary phases allows various types of adsorpton chroma tography (normal and reversed phase), jon exchange chromia: tography, immunoaffinity chromatography, hydrophobie in tersetion chromatography, and size excluston chm- matography. The protein fractions are simply collected wlwen they elute from the column and are concentrated and assayól for metivaty. o Sterilizarion and formulation, This step can be nccomplished by ultrafiltration to remove pyrogens or by heating if the pros tein cam withstand this. Formulation might involve reconstitu- ton into stable solutions for administration or determining the optimum conditions for stability when submitting for clinical trials. Complicating factors include (a) proteins unfolding into an inactive conformation during processing (it may not be possible to refold the protein comecty) and (6) proteases tal are commonly produced by bacterial, yeast. and mammalian cells, which may partially degrade the protein. ACEUTICS OF RECOMBINANT DNA RODUCED AGENTS IA methods have facilitated the production of very pure, » useful proteins. The physicochemical and ceutical properties of these agents are those of pro- , veich means that pharmacists must understand the tand the chemistry of instability) of proteins to handle, dispense, reconstitute, and administer these pdmgs. Instabilítios among proteins may be physical cul. In the former case, the protein might stick to às 01 Hocculate, altering the dose that the patient ivo. In the latter case, chemical reactions taking om te protein may alter the type or stercochemistry amino acids, change the position of disulfide bonds, e the peptide chains themselves, and alter the charge of the protein. Any of these can cause unfolding l Job the protein and loss of activity, rendering qule useless as q drug. Chemical instability can be m during the purification stages of a protein, when Ecule might be subjected to acids or bases, bul insta- peu occur at the point of administration when, for &, a Iyophilized protein is reconstituted. The pharma- is understand a few concepts of the chemical and iestability of proteins to predict and handle potential Instability of Proteins"” 61 yr Eifdmlyis. Hydrolyic reactions of the peptide bonds con Rea (ho polymer chain. Asparinie residues hydrolyze 100 Es faster im dilute acids than do other amino acids under same conditions. As a general mile of peptide hydrolysis, Pro > Asp-X or X-Asp bonds. This property of Asp is ly due to am autocatalytic function of the Asp side chain hor y! group. Asn, Asp. Gln, and Glu hydrolyze exceprion- V emily dE they occur nestto Gly, Ser, Ala, und Pro, Within groupings, Asn and Gln aceelerato hydrolysis more at e pl wlile Asp and Glu hydrolyze most readily at high Dwben the side chain carboxy! groups are jonized. ntdasen. Glncand Asm undergo hydrolytic reactions that Wmidate (heir side chains. These reactions convert neutral acid residues into charged ones. Gln 15 converted to and Asm to Asp: The amino acid type is changed, but E chain às not clesved. This process is, cffectively, primary quence jomerization, and may influence biological active The desmidutios resction of Asn residues is sccelerated Er beutral or alkaline pH conditions. A five-membered de imide intermediate formed by intramolecular attack of mtrogen atom on the carbonyl carbon of the Asn side chain he accelerant. The cyclic imide spontaneously hydrolyzes ugive a mixiure of residues—the asparty! peptide and an iso foution. Buse-calilyzed racemization redctions cam In any of the amino acids except glycine, which is Racemizations yield proteins with mixtures of L- and | acid configurations The reaction occurs following lhe abstracton ofihe a-hydrogen from the amino acid to form bamon. As should be expected, the stability of the carban- controls the rate of the reaction. Asp, which undergues nation via a cyclic imide intermediate, racemizes 105 Es faster than free Asn. By comparison, other amino acids Chapter 6 » Biotechnology and Drug Discovery 173 in a protem mcemize about 2 to 4 times faster than Uheir free counterparts e Eliminar. Proteins containing Cys, Ser, The, Phe, and Lys undergo facile P-elimination in alkuline conditions that facilite formation of an a carbanion + Ouidation. Oxidation can occur at the sulfur-contuning amino acids Met and Cys and at the aromatic amino acids His, Trp, and Tyr. These reactions can occur during protein processing as well as in storage. Methionine (CH,-S-Ri is oxidizable at low pH by hydrogen peroside or molecular oxygen to yield a sulfoxide (R-S0-CH ) and a sulfone (R-S0,-CH;), The thiol group of Cys (R-SH) can undergo successive oxidation to the corresponding sulfeme acid (R-SOH), disulfide (R-5-S- Ri, sulfimo acid (R-SO,H), and sulfonic acid (R-S0,M. À number of factors, including pH, influence these reactions. Free -SH groups can be converted into disulfide bonds (-S- S-) und vice versa. In the phenomenon of disulfide exchange, disulfide bonds break and reform in different positions, caus- ing incorrect folding of the protein. Major changes in (he three- dimensional structure of the peptide can abolish activity, Oxi dation of the aromatic rings of His, Trp, and Tyr residues |s believed to occur with a variety of oxidizing enzymes. Physical Instability of Proteins? Chemical alterations are not the only source of protein insta- bility. À protein is a large. globular polymer that exists im some specific forms of secondary, tertiary, and quaternary structure. A protein is not à fixed, rigid structure. The mole- cule is in dynamic motion, and the structure samples an array of three-dimenstonal space, During this motion, noncovalent intramolecular bonds can break, reform, and break again, but the overall shape remains centered around an energy minimum that represents the most likely (and pharmacologi- cally active) conformer of the molecule. Any major change in the conformation can abolish the activity of the protein, Small drug molecules do not demonstrate this problem. A globular protein normally folds so that the hydrophobie groups are directed to the inside and the hydrophilic groups are directed to the outside. This arrangement facilitates the water solubility of the protein, IH the normal protein unfolds, it cam refold to yield changes in hydrogen bonding, charge, and hydrophobic effects. The protein loses its globular struc- ture, and the hydrophobic groups can be repositioned to the outside, The unfolded protein can subsequently undergo fur- ther physical interactions. The loss of the globular structure of a protein is referred to as denaturation. Denaturation is, by far, the most widely studied aspect of protein instability. In the process, the three-dimensional folding of the native molecule is disrupted at the tertiary and, possibly, the secondary structure level. When a protein denatures, physical structure rather than chemical composi- tion changes. The normally globular protein unfolds, expos- ing hydrophobic residues and abolishing the native three- dimensional structure. Factors that affect the denaturation of proteins are temperature, pH, jonie strength of the medium, inclusion of organic solutes (urea. guanidine salts, acetam- ide, and formamide), and the presence of organic solvents such as alcohols or acetone. Denaturation can be reversible or imeversible. [f the denatured protein can regain its native form when the denaturant is removed by dialysis, reversible denaturation will occur. Denatured proteins are generally insoluble in water, lack biological activity, and become sus- Die to cnzymatic bydrolysis. The air-water interface ts a hydrophobic surface that can facilitate protein mon. Interfaces like these are commonly encoun- im dmg delivery devices and intravenous (IV) bags e ulsorption of proteins is characterized by adhe- DE ihe protein tó surfaces, such as the walls of the con- Rs olihe dosage form and drug delivery devices, ampuls, tubing. Proteins can adhere to glass, plasties, rubber, «and polyvinylehloride. This phenomenon is Hed feras Mocentarion. The internal surfaces of intrave- Nery pumps and IV delivery bags pose particular is of this kind. Flocculated proteins cannot be dosed LO Eregution results when protein molecules, in aqueous nn selhassociate to form dimers, trimers, tetramers, E und large macromolecular aggregates. Self-asso- ai depends on the pH of the medium as well as solvent bn, tonic strength, and dielectric properties. Mod- bunts of denaturants (below the concentration that À cause denaturation) may also cause protein aggrega- lally unfolded intermediates have a tendency to ate. Concentrated protein solutions, such as an immu- lin for injection, may aggregate with storage time Il, The presence of particulates in the preparation is ist's clue that the antibody solution is defective. ipitation usually occurs along with denaturation. De- nvestigations have been conducted with insulin, forms a finely divided precipitate on the walls of an om device or its dosage form container, It is believed Him undergoes denaturation at the air-water inter- Aihtuting lhe precipitation process. The concentration e jon, pH, and the presence of adjuvants such as prot- E also affect the precipitation reaction of insulin hylheir very nature aré antigens. A human protein, Dus al its typical physiological concentration, may completely different immunogenic properties when ed in the higher concentration that would be used ig Unless a biotechnology-derived protein is engi- do be 100% complementary to the human form, it Er among several major epitopes. The protein may pdifications of its amino acid sequence (substitutions E amino acid for another). There may be additions or ns Of amino acids, N-terminal methionyl groups, in- orabnormal folding patterns, or oxidation of a sulfur- E side chain of a methionine or a cysteine. Addi- When à protein has been produced by using a bacte- Or, a finite amount of immunoreactive material may the final product. All of these listed items contribute Ngenicity of a biotechnologically produced protein. Div administered to a human patient, the host's im- dem will react to the protein just as it would to a Bilack and neutralize it. This is why research has enaken to create 1009% human protein drugs, such which patients will need to take for a long time. om, some 0f the most promising biotechnology prod- he monocional antibodies, are produced in mice by use manized genes to avoid human reaction to the mouse Chapter 6 8 Biotechnology and Drug Discovery 175 E DELIVERY AND PHARMACOKINETICS OF BIOTECHNOLOGY PRODUCTS“ As with any drug class, the medicinal chemist and pharma cist must be concerned with the absorption, distribution, me- tabolism, and excretion (ADME) parameters of protein drugs. Biotechnology -produced drugs add complexities thai are not encountered with “traditional” Jow-molecular- weight drug molecules, ADME parameters are necessary to compute pharmacokinetic and pharmacodynamic param- eters for a given protein. As for any drug. these parameters are essential in calculating the optimum dose for a given response, determining how often to administer the drug to obtain a steady state, and adjusting the dose to obtain (he best possible residence time at the receptor (pharmacodynamic parameters). Delivery of drugs with the molecular weighis and proper- ties OF proteins into the human body is a complex task. The oral route cannot be used with a protein because the acidity of the stomach will catalyze its hydrolysis unless the drug is enteric conted, Peptide bonds are chemically labile, and proteolytic enzymes that are present throughout the body can attack and destroy protein drugs. Hydrolysis and peptidase decomposition also occur during membrane transport through the vascular endothelium, at the site of administra- tion, and al sites of reaction in the liver, blood, kidneys, and most tissues and fluids of the body. It is possible to circum- vent these enzymes by saturating them with high concentra- tions of drug or by coadministering peptidase inhibitors, Ox- idative metabolism of aromatic rings and sulfur oxidation can also occur. Proteins typical decompose into small frag- ments that are readily hydrolyzed, and the individual amino acids are assimilated into new peptides. A potentially serious hindrance to a pharmacokinetic profile is the tendency of proteins administered as drugs to bind to plasma proteins such as serum albumin. [f this happens, they enter a new biodistribution compartment from which they may slowly exit. Presently, the routes of administration that are available for protein drugs are largely subcutaneous and intramuscu- lar. Much ongoing research is targeted at making peptide drugs more bioavailable. An example of this is conjugation of interleukin-2 with polyethylene glycol (PEG). These so- called pegylated proteins tend to have a slower elimination clearance and a longer 1, than interleukin? alone. Another strategy being used is lhe installation of a prosthetic sugar moiety onto the peptide. The sugar moiety will adjust the partition coefficient of the drug, probably making it more water soluble. a RECOMBINANT DRUG PRODUCTS Hormones Human insulin, Recombinant,'90-102 Human insulin was the first pharmacologically active biological macromol ecule to be produced through genetic engineering. The FDA approved the drug in 1982 for the treatment of type | (insulin- dependent) diabetes (see Chapter 25). The insulin protein is a two-chain polypeptide containing 51 amino acid residues. Chain A is composed of 21 amino acids, and chain B con- 176 Wilson and Gisvold's Texthook of Organic Medicinal and Pharmaceutical Chemistry tains 30. The human insulin molecule has three disulfide linkages, CysÃ; to CysBo, CysÃoo to CysB jo, and an in- trachain linkage, CysÃa to CysÃ. Insulin is secreted by the f-cells of the pancreatio islets of Langerhans, initially as à single peptide chain called proinsulin. Enzymatic cleav- age of the propeptide releases the insulin. Historically, insulin was isolated from bovine or porcine sources. Using these agents was not without difficulty. Both porcine and bovine insulin differ in amino acid sequence, with Ala replacing Thr at the C terminus of the human B chain (Bs). Bovine insulin also differs in sequence from human insulin, with Ala substituting for Thr at Ag and Val substituting for isoleucine at Ap. These differences, small though they may seem, result in immunological reactions in some patients, Adjustments to the formulation of bovine and porcine insulin led to products that differed in time of onset, time to peak reduction in glucose, and duration of action. These parameters were vaned by addition of protamine and zine (which vielded a particulate insulin with a longer dura- tion of action), and adjustment of the pH to neutrality, which stabilized the preparation, Insulins were characterized as reg- ular (short-duration Iletin, 4 to 12 hours), semilente (ultra- short duration), lente (intermedinte [1 to 3 hours to peak, 24 hours duration |), and ultralente (extended duration). An addnvonal form of insulin was NPH (neutral protamine Hagedorn), which had an intermediate time of onset and time to peak (1 to 3 hours) and a long duration of action (16 to 24 hours). Producing a recombinant insulin that is chemically and physically indistinguishable from the human pancreatic hor- mone was à major accomplishment. The problem with im- munoreactivity has been eliminated, the pyrogen content of the FDNA product is nil, the insulin is not contaminated with other peptides, and the hormone can be biosynthesized in larger quantities. Human insulin (FDNA) is available as Hu- mulin, Novolin, and a number of analogues that differ in their pharmacokinetic profiles. Humulin is produced by using recombinant E. coli; Novolin is prepared by using recombinant 8. cerevisiae, a yeast. There have been modifi- cations in the production procedure since the inítial success- ful biosynthesis. Prior to 1986, Humulin was produced by creating two different vectors, one for the A chain and one for the B chain, and inserting them into E. coli. The A cham and the B chain would be secreted into the medium, and the two were joined chemically to form rDNA insúlin. Today, the entire proinsuhn gene is used to creme à recombinant organism, and the connecting peptide in proinsulin is cleaved by two enzymes (an endopeptidase and à carboxypeptidase B), vielding insulin (for details sec Chapter 25). Insulin FDNA is available in several!” forms, Insulin lis- pro (Humalog) has a more rapid (15 to 30 minutes) onsel and a shorter duration (3 to 6.5 hours) of action than regular human insulin (onset 30 to 60 minutes, duration 6 to TO hours). Tt is effective when administered 15 minutes before a meal, unlike regular insulin, which must be inected 30 minutes before a meal. In lispro, the B-chain amino acids BssPro and BayLys are exchanged. Insulin aspart (Novolog), onset 15 to 30 minutes, duration à to 6,5 hours, with a single amino acid substitution of Asp for Pro at Bas, is effective when administered 5 to 10 minutes before a meal. The ultra- long-acting agent insulin glargine has the Asp at Az, re- placed by Gky and has two Arg residues added at the € terminus of the B chain, Insulin glargine, administered sub cutancously (SC), has à duration of action of 24 to 48 hours The alteration in basicity of this agent causes il to precipitate at neutral pH, creating a depot effect. Insulin FDNA has been very successful, The only problem has arisen in patients who have been using porcine or bovine insulin for a long time. Some patients who are switched to DNA human insulin report difficulty in “feeling their ghu- cose level,” and these patients require extra counseling in the use of the recombinant hormone, Glucagon.'* The hormone glucagon (GlucaGen) |) biosynthesized in the pancreas as a high-molecular-weigh! protein from which the active macromolecule is released by proteolytic cleavage. Glucagon is a single chain of 29 amino acids and generally opposes the actions of insulin. Bovindl and porcine glucagons, which possess structures identical with human glucagon, have been in use for years. The rDNA form has been approved by the FDA for use in severe hypos glycemia and as a radiological diagnostic aid. Glucagon 1 laxes smooth muscles in lhe gastrointestinal (GT) tract, de creasing Gl motility and improving the quality df radiological examinations. In the treatment of severe hype! glycemia in insulin-dependent diabetes, GlucaGen cause the tiver to convem glycogen to glucose, Left untreated, vere hypoglycemia (low-blood-sugar reactions) can cus prolonged loss of consciousness and may be fatal, The TDMA drug has the benefit that there is no chance of nequinm bovine spongiform encephalopathy from glucagon therapo This condition, also known as mad cow disease, is cousalhe a prion that was suspected to infect animal pancreas tis Human Growth Hormone, Recombinant'S Human growth hormone (hGH) is a protein that is esse for normal growth and development in humans. hGH aflos many aspects of human development and metabolism & cluding longitudinal growth, regulation (increase) of profs synthesis and lipolysis, and regulation (decrease) of glum metabolism. hGH has been used as a drug since the 198 and ithas been extremely successful in the treatment ofd sic growth hormone deficiency. chronic renal insufficia Turner's syndrome, failure to lactate in women, and Pra Will syndrome. In its long history the hormone has hu remarkably successful and free of side effects. The primary form of hGH in the circulation is a 22 nonglycosylated protein produced in the anterior piu and composed of 191 amino acid residues linked by disulã bridges in two peptide loops. The structure of hGH is ghê lar, with four antiparallel a-helical regions, gen hGH is composed of about 85% of the 22-kKDa mono to 10% of a 20-kDa monomer, and 59% of a mixture fam fide-linked dimers, oligomers, and other modified fá From the late 1950s, hGH was isolated from pituitaçã tracts of cadavers. À prion associated with the prepir was suspected to cause Creutzfeldi-Jakob disease, degenerative neurological disorder. The first use of recombinant hGH (hGH) was rea in 1982. hGH preparations were first produced in These preparations contained a terminal methionine amino acids. Natural sequence rhGH has since b duced in mammalian (mouse) cell culture, 178 Pluripotent Stem Cell Myelold Stem a o x Ze E IL-9 q e, Ol OM IL-3 SCF SCF GM-CSF y IL-3 , IL-9 EPO GM-CSF IL-3 | / | GM-CSF / EPO ” ORRO IL-1, SCF a 4 a Wales and Gisvold's Tedbook of Organic Medicinal and Pharmaceutical Chemistry Qulescent Daughter Cell Predá SCF IL-3 U-5 e GM-CSF eita EPO E Do Lymphold Stem Cell So O) IL=7 O ONO GM-CSF IL-3 IL-3 IL-2 IL-6 IL-6 IL=7 GM-CSF | GM-CSF EPO TPO | mese | IL-3 | G-CSF Y Y r Y 4 20 - fe OR NO NCMONMO | Ertncoyina Platotota Macrophages Neutrophils Eosinophits Basophiis H Lymphosyties T Lymphocytus Figure 6-12 = Cytokine-mediated cascade leading to different blood cell types. EPO, erythropoietin, &- CSF gra colony-stimulating | IE-X, interleukins, M-CSF loevte LNOCYTE the kidney, and 1t activates the proliferation and differentia- tion of specially committed erythroid progenttors in the bone murrow, Epoetin alfa (Epogen) is a 165-amino acid glyco- protein that às manufactured in mammalian cells by rDNA technology. The protein is heavily glycosylated and has a molecular mass of approximately 30,400 Da. Erythropoietin Is composed of four antiparallel o helices. The «DNA protein has the same amino acid sequence as natural erythropoietin. Epoetin is indicated to treat anemia of chronic renal failure patients, anemia in zidovudine-treated HIV-infected pa- tents, and in cancer patients taking chemotherapy. The re- sults in these cases have been dramatic; most patients re- spond with a clinically significant increase in hematocrit tia, tis Filgrastim. Filgrastim, granuloeyte colony-stim- ulating factor (G-CSF), Neupogen, stimulates the prolifera- or; GM-CSF macrophage cdi etimalatina factor; SCF granulocyte- -macrophag 3e colony-stimulating factor stem cell factor; TPO, thrombopotetin tion of granulocytes (especially neutrophils) by mobilink hematopoietic stem cells in the bone marrow. Endogem G-CSF is à glycoprotein produced by monocytes, fik blasts, and endothelial cells. G-CSF is a protein of 174 ami acids, with a molecular mass of approximately 18,800 1 The native protein is glycosylated Filgrastim selectively stimulates proliferation and dia entiation of neutrophil precursors in the bone marrow leads to the release of mature neutrophils into the circulal from the bone marrow. Filgrastim also affects mature phils by enhancing phagocytic activity, priming the cel metabolic pathways associated with the respiratory enhancing antibody-dependent killing. and increasing expression of some functions associated with cell 3 antigens. do patients receiving chemotherapy with drugs such as Elophosphamide, doxorubicin, and etoposide, the inci- v! neutropenia accompanied by fever is rather high. histration of G-CSF reduces the time of neutrophil re- avery and duration of fever in adults with acute myeloge- nous Jeukemia, The number of infections, days that antibiot- Es re required, and duration of hospitalization are also Pilgrim O is identical wilh G-CSF in its amino acid ve, except that it contains an N-terminal methionine ds necessary for expression of the vector in E, coli. The ds not glycosylated. Filgrastim is supplied in a 0,0] dum acemte buffer comtaining 5% sorbitol and 0,004% orbate SO, It should be stored at 2 to 8º€ without freez- E Under these conditions, the shelf life is 24 months. shaking when reconstituting; although the foaming not harm the product, it may alter the amount of drug at às drawn into a syringe. : Sargr: aos cara Sargrumostim, granulocyte- emophage colony-stimulating factor (GM-CSF), Leukine, ulvcoprotein of 127 amino acids, consisting of three ular subunits of 19,500, 16,800, and 15,500 Da. The senous form of GM-CSF is produced by T Iympho- endothelial fibroblasts, and macrophages. Recombi- GM-CSF, produced in 8. cerevisiae, differs from native 1OM-CSF only by substitution of a leucine for an at position 23. This substitution facilitates expres- Bo Of lhe gene in the yeast. The site of glycosylation in lhe recombinant molecule may possibly differ from that of he nútive protein. Sargramostim binds to specific receptors on target cells induces proliferation, activation, and maturation, Ad- inistration to patients causes a dose-related increase in the enpheral white blood cell count. Unlike G-CSF. GM-CSF multilincage hematoporetic growth factor thal induces ly comantted progenitor cells to proliferate and differ- ente along the granulocyte and the macrophage pathways. ] so enhances the function of mature granulocytes and prophages/monocytes. GM-CSF increases the chemotac- e antifungal, and antiparasitic activities of granulocytes monoeytes. lt also increases the cytotoxicity of mono- es poward neoplastic cell lines and activates polymorpho- muclear leukocytes to inhibit the growth of tumor cells. Sargramostim is used to reconstitute lhe myeloid tissue her autologous bone marrow transplant and following olherapy in acute myelogenous leukemia. The prepara- indecreases the incidence of infection, decreases the num- DE days that antibiotics are required, and decreases the pol hospital stays. SOU D mumostim is supplied as à solution or powder (for f à Both forms should be stored at 2 to 8ºC wilhout ng. The liquid and powder have expiration dates of 24 OW. minis. The reconstituted powder and the aqueous solution | not be shaken. nO: HS Becaplermin, Regranex Gel, an en- nous polvpeptide that is released from cells that are bd im the healing process, is a recombinant human etdenved growth factor (r-hPDGF-BB). The “BB” that becaplermin is the homodimer of the B chain. Chapter 6 = Biotechnology and Drug Discovery 179 King —— + di, Intorteron vs y E (om Maturál Killar Infociod Host Neighboring Cell Cell Ceil Figure 6-13 = Anliviral mechanism of action of the inter- ferons Becaplermin is produced by a recombinant strain of $. cere- visiae comaining the gene for the B chain of PDGF. The protein has a molecular mass of approximately 25 kDa and is a homodimer composed of two identical polypeptide chains that are linked by disulfide bonds. Itis a growth factor that activates cell proliferation, differentiation, and function, and it is released from cells involved in the healing process. Becaplermin is formulated as a gel recommended for topi- cal use in the treatment of ulcerations of the skin secondary to diabetes, Interferons The interferons are a family of small proteins or glycopro- teins of molecular masses ranging from 15,000 to 25,000 Da and 145 to 166 amino acids long. Eukaryotic cells secreto interferons in response to viral infection. Their mechanism of action is bimodal, The immediate effect is the recruitment of natural killer (NK) cells to kill the host cell harboring the virus (Fig. 6-13). Interferons then induce a state of viral resistance in cells in the immediate vicinity, preventing spread of the virus. Additionallv, interferons induce a cas- cade of antiviral proteins from the target cell, one of which is 2,5oligoadenylate synthetase. This enzyme catalyzes the conversion of ATP into 2º,5-oligoadenylate, which activates ribonuclease R. hydrolyzing viral RNA. Interferons can be defined as cytokines that mediate antiviral, antiproliferative, and immunomodulatory activities. Three classes of interferon (TFN) have been characterized: a (alpha), 8 (beta), and y (gamma) (see Table 6-6). a-Inter- ferons are glycoproteins derived from human leukocytes. &- Imerferons are glycoproteins derived from fibroblasts and macrophages. They share à receptor with a-interferons. + Interferons are glycoproteins derived from human T lympho- TABLE 6-6 Interferons Used Therapeutically Interferon Endogenous Available Type Source Drug Products Alpha Leukocytes Interferon alfa-2a Interferon alfa-2h Interferon alfa-2e Beta Fibroblasts, macrophages Beta-la Beta-lb Gamma TLymphocytes, natural CGiamma-Ib killer cetis 
