CHAPTER 5 *4
m..4
Prodrugs and Drug Latentiation
FORREST T. SMITH AND C. RANDALL CLARK
HISTORY
In 1958. Albert initially coined the term prodrug and used
it to refer to a pharmacologically inactive compound that is
transformed by the mammalian system into an active sub-
stance by either chemical or metabolic means.' This included
both compounds that are designed to undergo a transforma-
tion to yield an active substance and those that were discov-
ered by serendipity to do so. These two situations were dis-
tinguished by Harper. who in 1959 introduced the term drug
Iatentiat,o,i to refer to drugs that were specifically designed
to require bioactivation.2
These ideas led to the development of a number of cur-
rently used drugs that have advantages over their nonprodrug
counterparts. The type of prodrug to be produced depends
on the specific aspect of the drug's action that requires im-
provement and the type of functionality that is present in
the active drug. Generally. prodrug approaches are under-
taken to improve patient acceptability of the agent (i.e., re-
duce pain associated with administration), alter absorption.
alter distribution, alter metabolism, or alter elimination. The
chemical nature of the prodrugs that can be prepared is some-
what limited, however, by the chemical nature of the active
species.
Recently, the terms hard drugs and soft drugs were intro-
duced.3 Hard drugs arc compounds that are designed to
contain the structural characteristics necessary for pharma-
cological activity but in a form that is not susceptible to
metabolic or chemical transformation. In this way, the pro-
duction of any toxic metabolite is avoided, and there is in-
creased efficiency of action. Since the drug is not inactivated
by metabolism, it may be less readily eliminated. On the
other hand, soft drugs are active compounds that after exert-
ing their desired pharmacological effect arc designed to
undergo metabolic inactivation to give a nontoxic product.
Thus soft drugs are considered to be the opposite of prod-
rugs.
BASIC CONCEPTS
A prodrug by definition is inactive and must be converted
into an active species within the biological system. There
are a variety of mechanisms by which this conversion may
be accomplished. Generally, the conversion to an active form
is most often carried out by metabolizing enzymes within
the body. Conversion to an active form may be accomplished
by chemical means (e.g.. hydrolysis or decarboxylation). al-
though this is less common. Chemical transformation does
not depend on the presence or relative amounts of memaboliz-
142
ing enzymes, and therefore, less interpatient variability in
activation is since such compounds are chemically un-
stable. however, storage of these compounds may present a
problem.
Prodrugs can be conveniently grouped into carrier-linked
prodrugs and bioprccursor prodrugs.5 Carrier-linked pro-
drugs are drugs that have been attached through a metaboli-
cally labile linkage to another molecule, the so-called pro-
moiety, which is not necessary for activity but may impart
some desirable property to the drug, such as increased lipid
or water solubility or site-directed delivery. Several advan-
tages may be gained by generating a prodrug: increased ab-
sorption. alleviation of pain at the site of injection if the
agent is given parenterally. elimination of an unpleasant taste
associated with the drug, decreased toxicity, decreased meta-
bolic inactivation, increased chemical stability, and pro-
longed or shortened action, whichever is desired in a par-
ticular agent. An example of such a prodrug form of
chloramphenicol is provided below (Scheme 5-I
Administration of a drug parenserally may cause pain at
the site of injection, especially if the drug begins to precipi-
tate Out of solution and damage the surrounding tissue. This
situation can be remedied by preparing a drug with increased
solubility in the administered solvent. Since chlorampheni-
col has low water solubility, the succinale ester was prepan.'d
to increase the water solubility of the agent and facilitate
parenteral administration. The succinate ester usd1 is inac-
tive as an antibacterial agent, so it must be converted to
chloramphenicol for this agent to be effective. This occurs
in the plasma to give the active drug and succinate. The ester
hydrolysis reaction can be catalyzed by esterases present in
large amounts in the plasma. The ability to prepare ester-
type prodrugs depends, of course, on the presence of either
a hydroxyl group or a carboxyl moiety in the drug molecule.
The promoiety should be easily and completely removed
after it has served its function and should be nontoxic. as is
indeed the case with succinate. The selection of the appropri-
ate promoiety depends on which properties are sought br
the agent. If it is desirable to increase water solubility. then
a promoiety containing an ionizable function or numerous
polar functional groups is used. If. on the other hand, the goal
is to increase lipid solubilimy or decrease water solubility. a
nonpolar promoiety is appropriate.
A slight variation on the currier-iinked prodrug approach
is seen with mutual prodrugs in which the carrier also has
activity. The antineoplastic agent estramustinc, which is
used in the treatment of prostatic cancer, provides an exam-
ple of such an approach (Scheme Estramustine is com-
posed of a phosphorylated steroid ( 17a-estradiol) linked toa
nomiustard IHN(CFI2CI-l2CI)21 through acarbamate linkage.
The steroid portion of the molecule helps to concentrate the

OH
Cl2
H 20
Scheme 5—1 • Hydrolysis of chioramphenicol succinate.
drug in the prostate, where hydrolysis occurs to give the
norniustard and The normustard then acts as an alkylat-
Hg agent and etcits a cytotoxic effect. The I 7a-estradiol
has an antiandrogcnic effect on the prostate and.
hcrehy, slows the growth of the cancer cells. Since both
he stcmid and the mustard possess activity. estramustine is
coned a mama! pradrug. Note that phosphorylation of the
cart be used to increase the water solubility, which
jho constitutes a prodrug modification. Both types of esters
earbantates and phosphates) are hydrolyzed by chemical or
encymatic means.
In contrast to carrier-linked prodrugs. bioprecursor pro-
drugs contain no promoiety but rather rely on metabolism
introduce the functionality necessary to create an active
For example, the nonsteroidal anti-inflammatory
drug (NSAIDt sulindric is inactive as the sulfoxide and must
reduced metabolically to the active sulfide (Scheme
OPO3Na2
Scheme 5—2 • Activation of estramustine.
Chapter 5 • Prnulruox and Drug Lizesniazion 143
OH
H C '2
H
a
Sodium Sucdnate
53)5 Sulindac is administered orally, absorbed in the small
intestine, and subsequently reduced to the active species.
Administration of the inactive form has the benefit of reduc-
ing the gastrointestinal (CII) irritation associated with the
sulfide. This example also illustrates one of the problems
associated with this approach, namely, participation of alter-
nate metabolic paths that may inactivate the compound. In
this case, after absorption of sulindac, irreversible metabolic
oxidation of the sulfoxide to the sulfone can also occur to
give an inactive compound.
Although seen less frequently, some prodrugs rely on
chemical mechanisms for conversion of the prodrug to its
active form. For example. hetacillin is a prodrug form of
ampieillin in which the amide nitrogen and a-amino func-
tionalities have been allowed to react with acetone to give
an imidazolidinone ring system (Scheme This de-
creases the basicity of the a-amino group and reduces pro-
H0
lTa-abadIol
H + + + 2Na
 144 Wilson and Gisvolsi',c Textbook of Organic Medicinal and Pharmaceutical Che,nistrt

F r2COOH
.CH2COOH

CH3

Sulfide Sulindac
C— (Inactive) (Inadive)

Scheme 5—3 • Metabolism of sulindac.

tonation in the small intestine so that the agent is more lipo-
philic. In this manner, the absorption of the drug from the
small intestine is increased after oral dosing, and chemical
hydrolysis after absorption regenerates ampicillin. In such
an approach. the added moiety, or promoiety. in this case
acetone, must be nontoxic and easily removed after it has
performed its function.
PRODRUGS OF FUNCTIONAL GROUPS
Carboxyllc Acids and Alcohols
Prodrugs of agents that contain carboxylic acid or alcohol
functionalities can often be prepared by conversion to an
ester. This is the most common type of prodrug because of
the ease with which the ester can be hydrolyzed to give
the active drug. Hydrolysis is normally accomplished by
estera.se enzymes present in plasma and other tissues that
are capable of hydrolyzing a wide variety of ester linkage.s
(Scheme Included below are a numberoithe different
types of esterases that prodrugs may use:

As mentioned above, there are a variety of different types of Ester hydrulase

prodrugs. and a comprehensive discussion of each individual Lipase
agent is beyond the scope of this chapter. The major types Cholesterol ester,tsc
of prodrugs (grouped according to functional group) and Acetylchotinestera.se
bioprecursor drugs (grouped according to type of metabolic Carboxypeptidase
activation), however, are discussed briefly below. Cholinesterase

NH2

0 COOH
H 20
Miplclhs

COOH +

CH H3

Scheme 5—4 • Hydrolysis of hetacuilin.


0 0
Drug—C—O—Promolety
or
Drug—O —"—Pro moiety
Scheme 5—5 • Activation of ester prodrugs,
In addition to these agents, microflora present within the
gut produce a wide variety of enzymes that can hydrolyze
esters. Chemical hydrolysis of the ester function may also
occur to some extent. An additional factor that has contrib-
uted to the popularity of esters as prodrugs is the ease with
which they can be formed, lithe drug molecule contains
either an alcohol or carboxylic acid functionality, an ester
prodrug may be synthesized easily. The carboxylic or alco-
hol pronlolety can be chosen to provide a wide range of
lipaphilic or hydrophilic properties to the drug, depending
ott what is desired. Manipulation of the scene and electronic
properties of the prontolety allows control of the rate and
extent of hydrolysis. This can be an important consideration
when the active drug must be revealed at the correct point
in its movement through the biological system.
When ills desired to decrease water solubility. a nonpolar
alcohol or carboxylic acid is chosen as the prodrug moiety.
Decreasing the hydrophilicity of the compound may yield a
number of benelits, including increased absorption. de-
creased dissolution in the aqueous environment of the stom-
ach, and a longer duration of action. An example of increased
absorption by the addition of a nonpolar carboxylic acid is
seen with dipivefrin HCI (Scheme 5-6). This is a prodrug
fonu of epinephnine in which the catechol hydroxyl groups
have been used in the formation of an ester linkage with
pivalic acid." The agent is used in the treatment of open-
angle glaucoma. The increased lipophilicity relative to epi-
CH3
CH34,..CH3
OH CHn
cie
Dlptvetrin HCI
Scheme 5—6 • Hydrolysis of d,p,vefrin HCI.
Chapter 5 • Prodrz,ç'.t anti I)rug Lan'nnaiwn 145
Drug—C—OH + HO —Promolety
or
Drug—OH + HO—11---Promolety
nephnine allows the agent, when applied. to move across the
membrane of the eye easily and achieve higher intraocular
concentrations. Hydrolysis of the ester functions then occurs
in the cornea. conjunctiva. and aqueous humor to generate
the active form. epinephrine. Using pivalic acid as the pro-
moiety increases the stenic bulk around the scissile ester
bond, which slows the ester hydrolysis relative to less bulky
groups. yet still allows this reaction to proceed after the drug
has crossed the membrane harriers of the eye. In addition
to this benefit, the catechol system is somewhat susceptible
to oxidation, and protecting the cacechol as the die.sler pre-
vents this oxidation and the resulting drug inactivation.
Decreasing the water soluhility ola drug by the formation
of a prodrug may have additional benefits beyond simply
increasing absorption. A number of agents have an unpleas-
ant taste when given orally. This results when the drug be-
gins to dissolve in the mouth and then is capable of interact-
ing with taste receptors. This can present a significant
problem, especially in pediatric patients.. and may lead to
low compliance. A prodrug with reduced water solubility
does not dissolve to any appreciable extent in the tnouth
and, therefore, does not interact with taste receptors. This
approach has been used in the case of the antibacterial chlor-
amphenicol. which produces a bitter taste when given as the
parent drug (Scheme 5-7). The hydrophobic palmitate ester
does not dissolve to any appreciable extent in the mouth, so
there is little chance for interaction with taste receptors.'7
OH
P'1H2
HO
C
HO
Epinephilne
+
C H
CH,
Ptvaøc Add
 _____

146 WiI.con and Gisvojd'.c Textbook of Or,ianic Medicinal and Pharmaceutical

OH

H 1(CH C 12

.1(CHCI2
02N OH

02N Chcot
H3
+

Chioramphenicot Paimitate
0
CH3(CH2)14 OH

Scheme 5—7 • Hydrolysis of chloramphenicol palmitate.

The ester moiety is subsequently hydrolyzed in the GI tract. Not all carboxylic esters are easily hydrolyzed in vivo.
and the agent is absorbed as chloramphenicol. Stcric inhibition around the ester in some cases prevents the
Listed below are a number of other agents that have been prodrug from being hydrolyzed. This is seen in the $-lac-
converted into ester prodrugs and other types of prodrugs to turns, in which it is often desirjblc to increase the hydropho-
overcome an unpleasant taste: bicity of the agent to improve absorption or prevent dissolu-
tion in the stomach where acid-catalyzed decomposition may
Chloramphenicol palmitate occur. Simple esters 01 the carboxylic acid moiety, however,
N-Acetyl sulfisoxazole
are not hydrolyzed in vivo to the active carboxylate (Scheme
N-Acetyl sulfamethoxypyridazine
Erythmmycin e,stolate 5-8).
Clindamycin palmitate A solution to this problem was to use the so-called double-
Troleandomycin ester approach, in which an additional ester or carbonate

I
Penic8in Esters

H
R1 NH
\_L__s CH3
No ReactIon

CO OR2

R2 Propyl. But)l, Ptienyt

— Esters
H
H

No ReactIon

Ethyl, Propyl, Butyt. Phemyl

Scheme 5—8 • Simple esters of fl-lactams with resistance to enzymatic hydrolysis.


(unction is incorporated into the R2 substitueni further re—
ninved from the helerocyclic nucleus.'5 " Hydrolysis of
such a function occurred readily, and the moiety was selected
that chemical hydrolysis of the second ester occurred
quickly. This is seen in the cephalosporin celpodoxime pro-
wheN a carbonate function was used (Scheme 5_9)20
The carbonate is also susceptible to the action of esterase
cnLyines. aiid the unstable product undergoes further reac-
jun to give the active carboxylate. This approach is fre-
quently used to improve absorption or prevent dissolution
in the stomach and the subsequent acid-catalyzed decompo-
of aminopenicillins and second- and third-generation
C H3
N
H 2N __
2)7...JL1N H
—
H3
H
H
Scheme 5—9 • Hydrolysis mechanism of cefpodoxime proxetil.
Chapter 5 • Prodrug.c and Drug La,enria,ion 147
cephalosporins (celpodoxime proxetil has been classilied as
both a second- and a third-generation agent) so that these
agents can be administered orally (see Scheme 5-10 for sev-
eral examples).
To increase the hydrophilicity of an agent, several differ-
ent types of ester prodrugs have been used, including succi-
nates. phosphates. and sulfonates. Alt are ionized at physio-
logical pH and, therefore, increase the water solubilily of
the agents, making them more suitable for parenteral or oral
administration when high water solubility is desirable
(Scheme 5-I I).
Succinate esters containing an ionizable carhoxylate are
H
, Co2 + H —O
H H3
148 Wj150,l and Gjcvoldx of Organic Medicinal and Phannaci'aaieul Chenii,srr

C H3

H
H2N

CH3
II o—(CH3

Ce

C H3

0 —CH—0 CH3

CH3

H2N

0
OCH2CH3
CH3

Scheme 5—10 • Some examples of double esters of $-lactarns.


Dnig—O—ll-CH2-CH2-—'I—O"N a
Sucdnates
0
a
OH
Scheme 5—11 • Succinate and phosphate esters
useful when rapid in viva hydrolysis of the ester functional-
ly The rapid hydrolysis is related to the intramo-
keular attack of the carboxylate on the ester linkage, which
not require the participation of enzymes (Scheme
As a result, these agents may be somewhat unstable
in and should he dissolved immediately prior to
admInistration.
Phosphate esters of alcohols offer another method of in-
cressing the water solubility of an agent. The phosphates
are completely ionized at physiological p1-I and generally
hydrolyzed rapidly in vivo by phosphatase enzymes. Ioniza-
tion of the phosphate function imparts high stability to these
densativcs in solution, and solutions for administration can
he stored for long periods of time without hydrolysis of the
phosphate. Such an approach has been used to produce din-
phosphate, which produces less pain at the injection
site Ihan clindamycin itself (Scheme 5-13). Pain after paren-
icral adniinistration is associated with local irritation caused
by low aqueous solubility or highly acidic or basic solutions.
With chindamycin phosphate, the reduction in pain is attrib-
uted to the increased water solubility of the agent.
Dedvatization of amines to give amides has not been widely
used us a prodrug strategy because of the high chemical
stability of the amide linkage and the lack of amidase en-
I.ymcs necessary for hydrolysis. There have been efforts at
incorporating amines into peptide linkages in which the pep-
Succinate Pmdnig
Scheme 5-12 • Intramolecular cleavage of succinate esters.
Chapter 5 a I'rodrug.s wul Drug La:ensiatiw: 149
Drug—OH +
0
Drug—OH +
OH
tide serves to increase cellular uptake by use alan amino acid
transporter. The amino acids are then cleaved by specific
peptidase enzymes. A more common approach has been to
use Mannich bases as a prodrug form of the amines. Mannieh
bases result from the reaction of two amines with an aIde-
hyde or ketonc. As seen with hetacillin (see Scheme 5-4), the
effect of fornting the Mannich base is to tower the basicity of
the amine and, thereby, increase lipophilicity and absorp-
tion.
When nitrogen is present in an amide linkage, it is some-
times desirable to use the amide nitrogen as one of the
amines necessary to form a Monnich base. This approach
was used with the antibiotic tetracycline—the amide nitro-
gen was allowed to react with formaldehyde and pyrrolidine
to give the Mannich base rolitetracycline (Scheme
In this case, addition of the basic pyrrolidine nitrogen intro-
duces an additional ionizable functionality and increases the
water solubility of the parent drug. The Mannich base hydro-
lyzes completely and rapidly in aqueous media to give the
active tetracycline.
Azo Unkage
Amines have occasionally been incorporated into an azo
linkage to produce a prodrug. In fact. ii was an azo dye.
prontosil. that led to the discovery of the sulfonamides as the
first antibacterials to be used to treat systemic infections.22
Although prontosil itself was inactive in vitro, it was active
Drug—OH
Succinic Mhydrlde
150 Wi/so,, and Gisvolds Textbook of Organic Medicinal and P!iarn,aceutical Che,nis:rv

H H ,.C H3
CH3CH3CH2 CH3

H H

HO H

HO

C-
cn — +

H3P04
Scheme 5—13 • Clindamycin activation by phosphate hydrolysis.

in vivo and was converted by aio reductase enzymes in the
gut to sutfanilamide. the active species (Scheme 5-15).
Although prontosil is no longer used as an antibacterial.
this type of linkage appears in sulfasalazine. which is used
in the treatment of ulcerative colitis. The azo linkage is bro-
ken in the gut by the action of azo reductases produced by
microflora. This releases the active agent, aminosalicylic
acid, which has an anti-inflammatory effect on the colon, and
sulfapyridine (Scheme 5-16). The advantage of this prodrug
approach is that the combination of cleavage of the azo link-
age and generation of aminosalicylic acid prior to absorption
prevents the systemic absorption of the agent and helps con-
centrate the active agent at the site of action.
Carbonyl Compounds
A number of different functionalities have been evaluated
as prodrug derivatives of carbonyls (e.g., aldehydes and
tones), although this approach has not found wide clinical
use. These have generally involved derivatives in which the

CH3 OH N(CH3)2 N(CH3)2

CH3 OH

4120
IOH
H 20 o CO N H —C 2
OH 0 OH 0

Rolft*ecycflne

C H2=O

Fo,makMltyde

HNG

Scheme 5—14 • Rolitetracycline synthesis and activation.

 Chapter 5 a and I)rug Latenlia,ian 151

H2N =N Azo
Dwg)

NH2
+

H2N H2

NH2
Scheme 5—15 • Azo cleavage of prontosil

HO H2

HO OC

Add
HO =N H —rjj Azo Reductase

HO OC

Sulfasabzlne
H

Suttapyddine
Scheme 5—16 • Action of azo reductase on 5ulfasalazine.

hybridiied carbonyl carbon is converted to an sp3 hybri-
carbon attached to two heteroatoms. such as oxygen,
nitrogeil, or sulilir. Under hydrolysis conditions, these firne-
tionalities are reconvened to the carbonyl compounds. An
of this approach is methenaminc. shown below
Methenainine releases formaldehyde in the
urinc. which acts u.s an antibacterial agent by reacting with
nuckophiles present iii bacteria. The agent is administered
in enteric-coated capsules to protect it 1mm premature hy-
drolysis in the acidic environment of the stomach. After dis-
solution of the enteric-coated capsules in the intestine, the
agent is absorbed and moves into the bloodstream, eventu-
ally ending up in the urine, where the acidic pH catalyzes
the chemical hydmlysis to give lbrmaldehyde. Use of this

H
6CH20 + 4NH3

Formaldehyde Ammonia

Methenamine
Scheme 5—17 • Methenamune hydrolysis.
152 Wilson and GisI'(,ld'.s Textbook of Organic Medicinal and Plsam,aceu:ical Chemistry
prodrug approach prevents the systemic relea.sc of formalde-
hyde and reduces toxicity.
Other prodrug approaches have involved the use of ox-
imes, imines, and enol esters, although these types of com-
pounds have not been used clinically. A number of agents
contain imine and oxime linkages, such as many of the third-
generation cephalosporins (e.g.. cefotaxime, ceftizoxime).
but these are not prodrugs.
BIOPRECURSOR PRODRUGS
As indicated above, bioprecursor prodrugs do not contain a
carrier or promoiety but rather contain a latent functionality
that is metabolically or chemically transformed to the active
drug molecule. The types of activation often involve oxida-
tive activation, reductive activation, phosphorylation, and
in some cases chemical activation. Of these, oxidation is
commonly seen, since a number of endogenous enzymes can
carry out these transformations. Phosphorylation has been
widely exploited in the development of antiviral agents. and
many currently available agents depend on this type of acti-
vation.
The abundance of oxidizing enzymes in the body has
made this type of bioactivation a popular route. Isozymes
of cytochrome P450 can oxidize a wide variety of function-
alities. generally to produce more polar compounds that can
be excreted directly or undergo phase 2 conjugation reac-
tions and subsequently undergo elimination. This occurs in
a fairly predictable manner and, therefore, has been success-
fully exploited in prodrug approaches.
A good example of a prodrug that requires oxidative acti-
vation is the NSAID nabumetone (Relafen) (Scheme
NSAIDs produce stomach irritation, which in pa-
tients with preexisting conditions or patients taking large
amounts of NSAIDs for extended periods may be severe.
This irritation is associated in part with the presence of an
acidic functionality in these agents. The carboxylic acid
functionality commonly found in these agents is un-ionized
in the highly acidic environment of the stomach. As a result,
these agents are more lipophilic in nature and may pass into
the cells of the gastric mucosa. The intracellular pH of these
cells is more basic than that of the stomach lumen, and the
NSAID becomes ionized. This results in backflow of
from the lumen into these cells, with concomitant cellular
damage. This type of damage could be prevented if the car-
Nabumetone
(Pmdrug)
Scheme 5—18 • Oxidative activation of nabumetone.
boxylic acid function could be eliminated from these agents:
this functional group is required for activity, however.
Nahumetonc contains no acidic functionality and passes
through the stomach without producing the irritation nor-
mally associated with this class of agents. Subsequent ab-
sorption occurs in the intestine, and metabolism in the liver
produces the active compound as shown in Scheme 5-18.
This approach, however, did not completely eliminate the
gastric irritation associated with nabumetone. since it is due
only in part to a direct effect on the stomach. Inhibition of
the target enzyme, cyclooxygenase. while having an anti-
inflammatory effect, also results in the increased release of
gastric acid, which irritates the stomach. So, while nabumet-
one induces less gastric irritation than other NSAIDs. this
undesirable eFfect was not completely eliminated by a pro-
drug approach. Such an effect was also seen above with the
NSAID sulindac (see Scheme 5-3). whose Gl irritation was
reduced hut not completely eliminated.
Reductive activation is occasionally seen as a method of
prodrug activation but, because there are fewer reducing en-
zymes, is generally less common than oxidative activation.
One of the best known examples of reductive activation is
for the antineoplastic agent mitomycin C. which is used in
the treatment of bladder and lung cancer (Scheme 5-I
Mitomycin C contains a quinone functionality that under.
goes reduction to give a hydroquinone. This is important
because of the differential effect of the quinone and hydro-
quinone on the electron pair of the nitrogen. Whereas the
quinone has an electron-withdrawing effect on this electron
pair, the hydroquinone has an electron-releasing effect.
which allows these electrons to participate in the expulsion
of methoxide and the subsequent loss of the carbamate to
generate a reactive species that can alkylate DNA.
The cascade of events that leads to an alkylating active
drug species is initiated by the reduction of the quinone func-
tionality in mitomycin C. The selectivity of mitomycin for
hypoxic cells is minimal, however. The selectivity is deter-
mined in part by the reduction potential of the quinone.
which can be influenced by the substituents attached to the
ring. In an effort to modify the reduction potential of mito-
mycin C. various analogues have been prepared and tested
for antineoplastic activity. It was hoped that the reduction
potential could be altered so that the analogues would only
be activated in hypoxic conditions, such as those found in
slow-growing solid tumors that are poorly vascularized. In
the.se tissues with a low oxygen Content it was thought that
reductive metabolism might be more prevalent than in nor-
Adive Fonn
 Chapter 5 • Pradruga and Drug Latt',t:ia:ion 153

Nuc
H2N
H2N \t=O

H2N,
H2N, -OCH
Red

Mtomydn C

H2N H2N

OH OH __O
H
H2N -OCOWH 2

CH3 N
CH3 IH
OH

Further

IH

Scheme 5—19 • Mechanism of activation of mitomycin C.

mat Iisaues. so the agents would be selectively activated and,
selectively toxic.
therefore,
Although mitomycin was the first agent used clinically to
he recogniaed as requiring rcductivc activation, it is only
modestly selective for hypoxic cells. A much more selective
agent. is currently undergoing phase ill clini-
cal trials.- Tirapazamine is reported to he 100 to 2(X) times
more selective for hypoxic cells than for normal cells. The
mechanism of activation involves a one-electron reduction
that is catalyied by a number of enzymes, including cyto-
chrome P.450 and cytochromc P-450 reductase to give a
radical species (Scheme 5-20). This species, which is shown
as a carbon-centered radical, can initiate breaks in the DNA
chain under hypoxic conditions. Under aerobic conditions,
hydroside radical is formed, which can initiate chain breaks.
Phosphorylation is a common metabolic function of' the
body, which is used to produce high-energy phosphodiester
bonds such as those present in ATP and GTP. The body
then typically uses these molecules to phosphorylate other
molecules and, in the process of doing so, activates these
molecules. The type of activation achieved depends on the
molecule phosphorylated. but in many cases. phosphoryla-
Lion introduces a leaving group, which can be displaced by
an incoming nucleophile. This is seen, for example. in the
synthesis of DNA and RNA. in which nucleotides are added
to the 3' end of a growing chain of DNA or RNA (Scheme
5-21).
Phosphorylation is commonly required for the binactiva-
Lion of antiviral agents. These agents are commonly nucleo-
sides, which must be converted to the nucleotides to have
154 Wilsi,,, und Gjsvuhls Te,aho(;k of Organic MedEcinul and Pharmaceutical Chemist,

0 N

&. H

°2 02
[ • OH
Tirapazamine
DNA

Double-Strand
Scheme 5—20 • Reductive activa- Breaks
tion of tirapazamine. Oxidized DNA

activity. Most often. arniviral agents disrupt the synthesis or
function of DNA or RNA. which is generally accomplished
by conversion to the triphosphate. Since normal cells are
also involved in the synthesis of DNA and RNA. compounds
have been sought that would be converted to the triphos-
phates. the active form, in greater amounts in infected cells
than in normal cells. Therefore. nucleosides that have higher
affinity for the viral kinase enzymes than the mammalian
kinases are desirable and have greater selective toxicity.
This can be seen in the prodrug idoxuridiiie. which was
the first agent to show clinical effectiveness against viruses
(Scheme 5-22))° The nucleoside enters the cell, where it is
phosphorylated. In virally infected cells, this phosphoiyla-
tion is accomplished preferentially by viral thymidine ki-
nase, because the idoxuridine is a better substrate for the
viral enzyme than for the corresponding mammalian en-
zyine. Therefore, the drug is activated to a greater extent in
the virally infected cells and achieves some selective toxic.
ity. although this selectivity is rather low, and there is signifi-
cant toxicity to normal cells. Once the drug has been phos.
phoiylated to the triphosphate stage, it can inhibit DNA
synthesis in a number of ways, including inhibition of viral
DNA polymerase and incorporation into DNA. which results
in incorrect base pairing that disrupts the ability of DNA to
function as a template for DNA and RNA synthesis.
In addition to the selective toxicity mentioned, the prodrug
approach offers the additional advantage of increased cell
penetration. The prodrug can easily enter the cell via active

DNA chak'm

0-

DNA Polyrnerase

thymkie

0 0
-o —Lo
Scheme 5—21 • DNA synthesis.

Wal ThdWie
Scheme 5—22 • Idoxuridine activation.
mechanisms, whereas the active nucleotides are
unable to use this process and arc too polar to cross the
iiiembrane via passive diffusion.
A good example of chemical activation is seen with the
pngnn pump inhibitors such as omeprazole. In this case,
chemical activation is provided by the highly acidic environ-
neSt in and around the parietal cell of the stomach (Scheme
5.23i. This allows protonation of nitrogen on the benzimid-
awte flog followed by attachment of the pyridine nitrogen.
Ring opening then gives the sulfenic acid that subsequently
cydiies with the loss of water. Attachment by a sulfliydryl
group present on the proton pump of the parietal cell then
occurs and inactivates this enzyme, preventing further re-
lease of H' into the GI tract, which is useful in treating
gastric ulceration.
CHEMICAL DEUVERY SYSTEMS
The knowledge gained front drug metabolism and prodrug
studies may be used to target a drug to it.c site of action.
Site'specifie chemical delivery systems take advantage of
Chapter 5 • Prodrs,gs and Drug LsUe,usar,on 155
0
HN-I
o_J_o O=<—>_I
OH
DNA —
DNA
higher levels of activity in a metabolic or chemical pathway
at the target site. A prodrug form of the active drug is de-
signed to serve as a substrate in that specific pathway, thus
yielding a high concentration of active drug at the target
site. Site-specific chemical delivery requires that the prodrug
reaches the target site and that the enzymatic or chemical
process exists at the target site for conversion of the prodrug
to the active drug. Many factors are involved in the relative
success of site-specilic drug delivery, including extent of
target organ perfusion. rate of conversion of prodrug to ac-
tive drug in both target and nontarget sites, and input/output
rates of prodrug and drug from the target sites.
Site-specific chemical delivery systems represent but otte
approach to the selective delivery of drug molecules to their
site of action for increased therapeutic effectiveness and lim-
ited side effects. Other than chemical drug delivery, many
carrier systems have been evaluated lir drug delivery, in-
cluding proteins. polysaccharides. liposomes. emulsions.
cellular carriers (erythrocytes and leukocytes). magnetic
control targeting, and implanted mechanical As thc
fate of drugs in the human body has become more clearly
understood, research activity to improve the delivery of ac-
tive drug to the target site has increased. The basic goal
156 Wilso,, and Gisyold's Textbook of Organic Medicinal and Pharmacewical Chemistry
@ __<s=O
x
Scheme 5—23 • Mechanism of activation of proton pump inhibitors.
of these efforts is to protect the drug from the nonspecific
biological environment and to protect the nonspecific bio-
environment fiom the drug to achieve some site-specific
drug delivery. Site-specific drug delivery has been evaluated
extensively for drugs with narrow therapeutic windows, such
as many of the anticancer drugs.
The site-specific delivery of the active drug via its prodrug
counterpart requires that the prodrug be readily transported
to the site of action and rapidly absorbed at the site. On
arrival at the target site, the prodrug should be selectively
converted to drug relative to its rate of conversion at nontar-
get sites. Since high metabolic activity occurs in highly per-
fused tissues such as liver and kidney, delivery to these or-
gans has a natural advantage. Unfortunately. prodrug
delivery of active drug to other organs or tissues is disadvan-
taged for the same reasons.
Furthermore, it is highly desirable to have the active drug.
once formed, migrate from the target site at a slow rate.
On the basis of all these requirements, clearly site-specific
delivery of drug to the target by a prodrug chemical delivery
system is a far more complex undertaking than designing a
prodrug to improve one aspect of its overall properties. Yet
there are several excellent examples of site-specific chemical
delivery systems in use in modem drug therapy. The target
sites include cancer cells, GI tract, kidney and urinary tract,
bacterial cells, viral material, ocular tissue, and the
blood—brain barrier.
The prodrug methenamine, described above in this chapter
(Scheme 5-li), can be considered a site-specific chemical
delivery system for the urinary tract antiseptic agent formal-
dehyde.3' The low pH of the urine promotes the hydrolysis
H-N NH
of methenamine to formaldehyde, the active antibacterial
agent. The rate of hydrolysis increases with increased acidity
(decreased pH). and this can be promoted by administration
of urinary pH-lowering agents or by diet. The pH of the
plasma is buffered to about 7.4. and the rate of hydrolysis
is low, preventing systemic toxicity from formaldehyde. As
mentioned abovc, this compound is administered in enteric-
coated tablets that prevent dissolution and, therefore, prema-
ture hydrolysis in the highly acidic environment of the
stomach.
A number of prodrugs for cancer chemotherapy have been
designed for selective delivery o active drug to tumor tissue,
based on higher levels of activating enzyme in the tumor
cell than in normal Many enzymatic systems show
higher activity in tumor cells than in normal tissue because
of the higher growth rates associated with tumor tissue. Pep-
tidases and proteolytic enzymes are among those systems
showing higher activity in and near tumor cells. Thus, one
means of attempting to produce higher rates of drug incorpo-
ration into tumors than in surrounding normal tissue involves
deriving a drug molecule with an amino acid or peptide frag-
ment.
Capecitabine is an example of a prodrug chemical delivery
system that requires a series of enzymatic steps for conver-
sion to the active antitumor drug species. 5-Iluorouracil
(Scheme Tumors located in tissues with high levels
of the required enzymes should respond best to treatment
with capecitabine. Esterase activity occurs primarily in the
liver, allowing the intact ester capecitabine to be the ab-
sorbed species following oral administration. The ester hy-
drolysis product itself shows some specific toxicity toward

NHCOO(CH2)4CH3
NH7
H F
N H.
-iC
0
HO OH
0 ated with this active species.
0 specific chemical delivery system that delivers the dntg do-
5-Fluorouracil
Scheme 5—24 • Metabolic conversion of capecitabine.
01 Llact tissue, which prevents this molecule from serving
as an effective prodnig delivery form of 5-fluorouracil. The
other two enzymes involved in the formation of 5-fluoroura-
cii occur in high concentrations in target tissues such as
cers'iv, breast. kidney, and colon.
There is considerable current interest in the general con-
cept of tumor-activated prodrugs. and a number of strategies
have been proposed for drug targeting in tumor One
of the more interesting approaches is linking an exogenous
nonhuman) enzyme to a tumor-specific antibody. Based on
immunological response, the antibody would carry the
enzyme to the tumor surface, where it would be
available tbr prodrug activation. Prodrugs activated by this
cxopenoin enzyme would be converted to the active species
only at the tumor site. Since the activating exogenous en-
zyune is not normally found in human tissue, maximum accu-
racy in drug targeting should be achieved in this antibody-
directed enzyme prodrug therapy.
The antiviral drugs, such as idoxuridine (Scheme 5-22),
arc an interesting example of site-specific chemical dcliv-
These drugs serve as substrates for phosphorylating
cneymes found in viruses, and the phosphorylated species
is the active antiviral agent. The active phosphorylated spe-
vies is incorporated into viral DNA, disrupting viral replica-
non and, thus, producing the antiviral effect. These drugs
do not undergo phosphorylation by mammalian cells, so the
HO
NH2
Scheme 5—25 • Decarboxylation of v-dopa in the CNS to yield the active drug dopamine.
Chapter S • Pradrugs and Drug Latrn:ia:ian 157
prodrug is specific for those sites where it serves as a sub-
strate for phosphorylation enzymes. One of the requirements
for site-specific chemical delivery discussed above was the
proper input/output ratios for prodrug and active drug spe-
cies at the target. The relative physicochemical properties
of prodrug and its phosphorylated derivative suggest an ap-
propriate input/output ratio for site specificity. The prodrug
can readily penetrate the virus, and the increased polarity of
the phosphorylated derivative would serve to retain that ac-
tive species inside the virus. The combination of increased
polarity and viral retention of the active phosphorylated spe-
cies likely reduces any human toxicity that might he associ-
The amino acid drug L-dopa can be considered a site-
pamine to the brain. The brain has an active transport system
that operates to incorporate L amino acids into the central
nervous system (CNS). and L-dopa is transported into the
brain in this manner. Once across the blood—brain barrier.
L-dopa undergoes decarboxylation, as shown in Scheme
5-25, to yield the active metabolite, dopamine. Direct sys-
temic administration of dopamine does not produce signiti-
cant levels of the drug in the brain because of its high polarity
and poor membrane permeability as well as its facile meta-
bolic degradation by oxidative deamination. Dopamine
formed on the inside of the blood—brain barrier is held there.
however, because of the poor membrane permeability of this
drug. Although some specificity for brain tissue is achieved
by this delivery method, peripheral side effects of t.-dopa
are the direct result of decarboxylation to dopamine in other
organ systems. In this case, the enzyme activating system is
not localized at the target site, and its presence in other tis-
sues and organs leads to undesirable side effects.
Another example of the chemical delivery of a drug to
the brain and CNS is the prodrug form of 2-PAM (pro-2-
PAM), an important antidote for the phosphate and carha-
mate acetylcholinesterase inhibitors used in insecticides and
nerve gases.3° The polar properties of 2-PAM. a permanent
cationic species, prevent this drug from being absorbed fol-
lowing oral administration and restrict the drug from access
to the brain, even after IV administration. Pro-2-PAM is
a dihydropyridine derivative that undergoes metabolic and
chemical oxidation to yield the active drug 2-PAM (Scheme
5-26). The nonionic pro-2-PAM can easily cross the
blood—brain barrier, and oxidation to 2-PAM within the
brain essentially traps the active cationic drug species inside
the brain. Oxidation of the dihydropyridine ring of pro-2-
PAM occurs throughout the mammalian system, not just in
the brain, and the levels of the resulting 2-PAM arc approxi-
mately the same in peripheral tissue as in the brain. IV ad-
HO
158 WiLson and Giss'old'x Textbook of Medicinal and Pharmaceutical Chenii,qrv
I II Oxidation
CH=N014 CH=NOH
CH,
Pro-2.PAM 2-PAM
Scheme 5—26 • Oxidation of pro-2-PAM.
ministration of pro-2-PAM, however, yields brain levels of
2-PAM thai are approximately 10 times higher than those
achieved by IV administration of the parent drug.
The delivery of drugs across the blood—brain barrier has
been a significant issue in the design of many therapeutic
compounds. Only very lipophilic drugs can cross into the
brain without the aid of some active uptake process. such
as the one that operates to incorporate essential amino acids
into the CNS. The facile oxidation of the dihydropyridine
ring system has been extensively investigated as a general
process for chemical delivery of a number of drugs to the
CNS. The approach has been described as a chemical deliv-
ery system. not just a prodrug designed to penetrate the
blood—brain This process is a multistep procedure
involving delivery of the drug—dihydropyridine derivative to
(he brain via facile diffusion across the blood—brain barrier,
followed by oxidation to the quaternary pyridine cation,
which is trapped in the brain. The drug is then released from
the pyridine cation by a second metabolic/chemical event.
A number of functional groups can be added to the dihydro-
pyridine to facilitate the derivatization of various functional
groups found in CNS drugs. Since many CNS drugs are
amines. amides of dihydropyridinc carhoxylic acids are often
prepared and used to deliver the drugs across the
blood—brain barrier into the brain. Additionally, these amide
derivatives often serve to protect the amines from metabolic
degradation before they reach the target site. Primary amines
such as dopamine and norepinephrine and ninny others are
readily tuetabolized and degraded by oxidative deamination
before reaching the CNS. The dihydropyridinc derivative of
a doparnine ester, shown in Scheme 5-27. has access to the
OCOR
C H 2C H
Dopamine
H
Scheme 5—27 • Dihydropyridine-based drug delivery system for dopamine.
CNS via passive absorption of the tertiary amine, which on
oxidation restricts the resulting pyridinium amnide to the
brain. Amide hydrolysis then delivers the active form of the
drug at or near its site of action. The amide hydrolysis step
may be slower than the dihydropyridine oxidation step, and
thus a reservoir of pyridinium antide precurmr may be avail.
able for conversion to the active drug species.
The use of prodrug concepts has been very successful in
the delivery of active drug species to the human eye follow-
ing local application. Lipophilic esters of epinephrine. such
u.s the dipivaloyl ester described above tsee Scheme 5-6).
show better corneal penetration following direct application
to the eye than the more polar parent drug epinephrine.°
The esterases necessary for the hydrolysis of thc prodnig
are readily available in the eye and skin. The more polar drug
species. epinephrine. is then localized within the lipophilic
membrane barriers of the eye, and the drug remains available
at the target site to produce its antiglaucoma The
local application of the prodrug species to the skin or eye
allows metabolic processes to activate the drug without con-
cern forcompetitive reactions at other tissues or sites of loss.
The delivery of drugs to the colon and lower GI tract has
taken advantage of the unique enzymatic processes found
in colon bacteria. The glucosidase activity of these bacteria
allows hydrolysis of glucosidc derivatives of drugs in the
colon and provides higher concentrations of active drug.32
A number of steroid drugs Schenie 5-28) demonstrate in-
creased effectiveness in the lower GI tract following admnin-
istration as their glucoside derivatives. The polar glucoside
derivatives of the steroids are not well absorbed into the
bloodstream from the GI tract and remain available to serve
as substrates for the bacteria that are found primarily in the
human colon,
The prodrug approach for the delivery of anticancer drugs
to the site of action has been used in a number of cases in
an effort to increa.se effectiveness and lower side effects.
Several enzyme systems that show higher activity in and
near the cancer cells have been evaluated for their ability to
activate the prodrug species. In most cases, the enzyme
hy level is simply higher near the faster growing cancer cells,
OCOR
x Pyridtnium Ion Intermediate
 Chapter 5 • Prodrugx too! Drug L.a:emianio,s 159

HOC H2 Chemistry ul Drug Design and Drug Action York. Academic

Press. 1992. Chap. 8, pp. 352—41)1.
O O—R (,. Glaako. A. J.. Carnes. H. F... A.. ci at.: Antihuot Anna. 792:
Glucosidaso 752—802. 1957.
OH R —OH 7. Riley. T. N.: .1. Chem. Edur. 65:947-953. 198)4.
8. Duggan. D. F..: Drug Melab. Rev. 12:725-337. 19111.
4)0 DrUg 9. Hurdcasitc. (I. A.. Johnson. D. A.. Panetla. C. A.. ci al.: .1. Org. (item
OH 31:897—899. 1966.
10. Tsujj, A.. and Yamana. T.: Chem. Pharm, huh. 22:2434-2443. 1974.
Diug.Glucoslde II. Jusko, W. i. and Lewis. P.: J. Ptiarm. Sri. 62:69—76. 913
12. Jusko. W. J.. Lewis, G. P.. and Schmitt. (. W.: Clin. Pharun ilmer. 14
Stheme 5—28 • Activation of drug-glucoside by bacterial glu- 1973.
(Os!dase. 13. Schwartz. MA., and Hayton. W. 1.: 3. Ptiann. Sri. 61:91)6—9(13. 1q72.
14. Bungared. H.: Ada Pharni. Succ. 13:9-26. 1976.
IS. Sinkula. A. A.. and Yalkowsky. S. H.: 3. Phiurm. Sri. (v4:181. 1975.
16. Wci. C. P. Anderson. 1. A.. and Leumputld. I.: Ophlhalunuul. Vis.
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This briel discussion of site-specific drug delivery shows 18. Jan.sen. A. B. A.. and Russell. 1. 3.: .1. Client. Soc.. 965, 2127.

hal in some cases the prodrug was in use before its mecha-
nt delivery and specificity was discovered. Thus, some
cenipounds were discovered to represent site-specific drug
dclivcry well after they were placed into therapeutic use. An
emaluahion of the properties of these agents has produced the
Itamework for the design of other prodrug.c with target sites
in specific tissues. This process is really no different from
the general drug discovery process in which a unique sub-
stance is observed to have desirable pharmacological effects,
and studies of its properties lead to the design of better drugs.
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CHAPTER 6
Biotechnology and Drug Discovery
JOHN M. BEALE, JR.
BIOTECHNOLOGY: AN OVERVIEW
Developments in biotechnology in recent 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. A grxxl reflection of the impact of biotechnology
is the GenBank database. GenBank is an electronic reposi-
tory of gene sequence information, specifically the nuclea-
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.' 2 The growth of the GenBank database has been
rapid, and it has been increasing steadily since about
Figures 6-I and 6-2 graphically depict these growth rates.
In October 2002, the Pharmaceutical Research and Manu-
facturers Association (PhRMA) 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-
eases. 26 are new drugs for autoimmune diseases. 22 are
new drugs for neurological disorders, and 21 are new drugs
for human immumxleliciency virus (HIV) and acquired im-
munodeitciency syndrome (AIl)S) and related conditions.4
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
sclerosis, leukemia, hepatitis, rheumatoid arthritis, breast
cancer. diabetes, congestive heart failure. lymphoma. renal
cancer, cystic fibrosis, and other diseases. The number of
approvals of biotechnology drugs per year has been increas-
ing steadily. These data are shown in Figure 6-3.
The Human Gcnomc Project, an international etlort to ob-
tain complete genetic maps. including nucleotide sequences.
of each of the 24 human chromosomes, has spawned much
new knowledge and technology. It is awesome to consider
that in the mere 30 years since 1972. the science has reached
the stage of attempting genetic cures for some diseases, such
as cystic fibrosis and immune deficiency disorders.
BIOTECHNOLOGY AND PHARMACEUTICAL
CARE
As it affects 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 (rDNA)-driven drug
discovery process is beginning to yield new avenues for the
preparation of some old drugs. For example, insulin, once
prepared by isolation 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 pituitaxy glands of the deceased, can now
be prepared in pure form. Recombinant systems such as
these provide high-yielding, reproducible hatches 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 costs of
biotechnology drugs.232" implementation in a practice set-
regulatory issues.'34" product evaluation and for-
mulation.4748 patient compliance.49 and finding informs-
tion.5153 Additionally. there are a number of general
review and a general resource refer-
ence eatalogue."• 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 cloning of
novel receptors can provide access to tremendous tools for
the testing of drugs (e.g.. the adrenergic receptors), while
ctoning of a novel growth factor might potentially provide
a 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.
teifls 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 diseases such as cystic fibrosis