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. • Design of Renin Inhibitors
. Fig. 24.1 Catalytic mechanism of aspartic proteases. Awater mol-
ecule, placed at the apex between the two catalytically active aspartates, is polarized by the deprotonated carboxylic acid. As aresult, it
acts as anucleophile to attack the carbonyl carbon atom of the amide
bond to be cleaved. The second, less acidic and protonated aspartate
. Fig. 24.2 Possible transition
state isosteres for the design
of aspartic protease inhibitors.
Hydroxyl groups are particularly
well suited. Statin, anonproteinogenic amino acid, is found in
many inhibitor structures
where hydrogen bonds are formed to the peptide backbone of the substrate. Asimilar pattern is found on the
upper half of the tunnel, which is not shown in the image
because it has been clipped off. The hydrogen-bonding
pattern along the tunnel is common to all aspartic proteases. Individual binding pockets to the left and right of
the cleavage site are responsible for the selective recognition of substrates. They accommodate the side chains
of the residues of the substrate molecules. It is striking
that, in contrast to serine proteases, the pockets are well
forms an H-bond to the carbonyl oxygen of this amide bond. This
increases the electrophilicity of the carbonyl carbon(a). With the intermediate formation of atetrahedral coordinated diol and the simultaneous cleavage of the former amide bond(b), the original peptide
chain decomposes into the cleaved chain products(c)
Hamao Umezawa isolated one of the rst potent
and specic aspartic protease inhibitors, pepstatin, from
a culture of Streptomyces sp. This peptide, Iva–Val–
Val–Sta–Ala–Sta–OH, 24.1 (. Fig.24.4) is agood- to
high-afnity inhibitor of many members of the aspartic
protease family. It contains the nonproteinogenic amino
acid statin with ahydroxyethyl group. The 3D structure
of the pepsin–pepstatin complex shows that statin actually binds as atransition state mimicry to the catalytic
aspartic acids.
established on both sides of the cleavage site. This observation is explained by the reaction mechanism. Unlike
serine proteases, no intermediate is formed that is co-
24.2 Design of Renin Inhibitors
valently bound to the enzyme. Aspartic proteases often
cleave between hydrophobic amino acids (. Table24.1).
Since strong and directional interactions cannot form
with such residues, recognition and xation of substrate
molecules over several positions to the left and right of
the cleavage site is important. This explains the distinct
character of the pockets on either side. Therefore, to
design inhibitors, one must rst nd groups that mimic
good interactions with the binding pockets S3, S2, S1 and
,
,
. One of the groups shown in . Fig.24.2, which
is an analogue of the transition state, is placed directly
at the cleavage site.
Renin is an aspartic protease that is composed of 340
amino acids. It plays apivotal role in endogenous blood
pressure regulation and in electrolyte and water homeo-
stasis. The enzyme cleaves the peptide angiotensinogen to
form the decapeptide angiotensinI (. Fig.24.5). This is
subsequently cleaved by angiotensin-converting enzyme
(ACE, Sect.25.4), ametalloprotease, to give the octa-
peptide angiotensinII, which increases blood pressure.
Inhibition of the enzyme renin leads to adecrease in the
concentration of angiotensinI and, as aconsequence, of
angiotensinII. Renin inhibition, therefore, has ahypo-
tensive effect. Because of the great therapeutic success of
24
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Chapter • Aspartic Protease Inhibitors
cde
. Fig. 24.3 Sectional view of the binding pockets of ve aspartic
proteases: aHIV protease, bendothiapepsin, ccathepsinD, dplasme-
psin, and erenin. The catalytic center extends as atunnel through the
proteases (top left, schematic view from the side into the tunnel). In the
gures, the proteins are cut in such away that the clipping plane passes
through the center of the tunnels (right part of the schematic drawing,
the cut protease has to be rotated by 90°). When looking from the side
ACE inhibitors, many pharmaceutical companies began
searching for selective renin inhibitors in the early 1980s.
Renin has an unusually high specicity. Angiotensino-
gen is the only known natural substrate of this enzyme.
Therefore, it should be possible to nd ahighly specic
renin inhibitor that does not block other enzymes and
does not cause side effects, which is not the case with
many other antihypertensive agents.
The starting point for the work was the peptide sequence of the substrate angiotensinogen. Renin cleaves
angiotensinogen between Leu and Val. First, asuitable
surrogate for the Leu–Val unit was sought that would
allow the retention of the amino acids in the positions
to
P
(. Table24.3). The octapeptide His–Pro–Phe–
5
His–Leu–Val–Ile–His is cleaved as arenin substrate. Replacement of the Leu–Val amide bond that is cleaved by
the enzyme with the stable, isosteric groups –CH2NH–
(in the direction of the arrow), only the back side of the tunnels can
be seen. The protein surfaces are cut in the upper and lower part and
show the course of the polypeptide chain (red ribbons). The blue areas
on the backside of the tunnel refer to H-bond donor and acceptor sites
beneath the protein surface to which a bound substrate is attached
along its peptide backbone
. Fig. 24.4 Pepstatin 24.1 is an inhibitor for alarge number of dif-
ferent aspartic proteases. (Iva isovaleric acid, Sta statin)
or –COCH2– resulted in modestly effective inhibitors.
The isostere with the hydroxyethylene group, –CH(OH)
CH2–, was better suited as atransition state analogue,
and afforded astrong inhibitor (IC50 = 3 nM). The incorporation of the nonproteinogenic amino acid statin
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. • Design of Renin Inhibitors
. Fig. 24.5 The renin–angiotensin system. The conversion of angio-
tensinogen to angiotensinII (ATII), which increases blood pressure, is
accomplished in two steps. Degradation by an Asp–aminopeptidase,
(see . Fig.24.4) produced astrongly binding inhibitor.
As adipeptide isostere, statin replaces the P1–
unit in
the Leu–Val segment of the substrate.
The next step was the optimization of the P1 moiety.
Different groups were investigated as areplacement for
the leucine side chain. The results of such astructural
variation of 24.2 are listed in . Table24.4. Replacing the
iso-butyl group with alarger cyclohexylmethylene group
increased the afnity by afactor of20. An adamantylmethylene group is obviously too large for the pocket,
as the corresponding derivative only weakly inhibits the
enzyme. Next, the P2 moiety was investigated. Replacing the histidine with another group did not signicantly
improve the binding afnity. Nevertheless, the substitution of the basic histidine in the P2 position was amajor
advance in renin research because it allowed the discovery that glycols are potent renin inhibitors. Afew com-
pounds from the 24.3 class are listed in . Table24.5. The
introduction of asecond hydroxyl group in the correct
conguration increased the afnity by afactor of 10–200
depending on the chosen P1 side chain. Accordingly, it
was possible to nd tripeptide analogues with binding
constants of about 1 nM. Several companies developed
renin inhibitors to the point of clinical trials. Examples
are A-64662 24.4 (. Fig.24.6) from Abbott and Ro 455892 24.5 from Roche.
However, the desired goal had not yet been achieved.
The compounds had short half-lives and were not orally
available. It turned out that the amide bond between
the P3 residue Phe and the P2 residue His was rapidly
angiotensinaseA, leads to angiotensinIII (ATIII), which is still biologically active. Different angiotensinases (aminopeptidases, carboxypeptidases) degrade these two peptides into inactive fragments
. Table 24.3 The replacement of the cleavable amide bond
in Leu–Val (highlighted in bold) by astable isostere leads to
potent renin inhibitors. The Leu–Val group is replaced by
agroup in the inhibitors that the enzyme cannot cleave
Substrate/Inhibitor IC50
(nM)
His–Pro–Phe–His–Leu–Val–Ile–His 300,000
His–Pro–Phe–His–Leu[COCH2]–Val–Ile–His 500
His–Pro–Phe–His–Leu[CH2NH]–Val–Ile–His 200
His–Pro–Phe–His–Statin–Ile–His 20
His–Pro–Phe–His–Leu[CHOHCH2]–Val–Ile–His
a
Substrate, KM value
a
3
cleaved by the digestive enzyme chymotrypsin. The high
molecular weight of the compounds, which led to rapid
biliary excretion, was also aproblem. Further work focused on nding asuitable replacement for the P2 and
P3 side chains.
Inhibitor stability against chymotrypsin was achieved
by modication of the P3 group, phenylalanine. The stabilities for some of these modied renin inhibitors 24.6
are summarized in . Table24.6. One compound that
was no longer cleaved by chymotrypsin was obtained
by using β,β-dimethylphenylalanine. This is because, in
contrast to phenylalanine, the very bulky side chain no
longer ts into the specicity pocket of chymotrypsin.
Chapter • Aspartic Protease Inhibitors
24
. Table 24.4 Optimization of the P1 side chainR. The bind-
ing pocket is lipophilic and obviously has just the right size for
acyclohexylmethylene group
R IC50 (nM)
Isobutyl 81
Cyclohexylmethylene 4
Cyclohexyl 150
Adamantylmethylene 2500
Benzyl 15
Boc=tert-butoxycarbonyl protecting group
. Table 24.5 The introduction of asecond hydroxyl group
in the R2 position leads to asignicant increase in the binding
afnity
R
1
Isobutyl H 1500
Cyclohexylmethyl H 10
Boc=tert-butoxycarbonyl protecting group
R
2
OH 11
OH 1.5
IC50 (nM)
The extended search for possible replacements of
Phe–His as the P3–P2 moiety led to aplethora of new nonpeptidic renin inhibitors with high afnity. The introduction of aterminal basic group was highly effective, even
though adequate oral bioavailability was not achieved.
Typical examples are 24.8 and 24.9 (. Fig.24.7).
Despite enormous efforts, renin research stagnated
worldwide because no compound could achieve the required oral bioavailability. All compounds contained at
least one amide bond and their molecular weights were
too high. In addition, the 3D structure of renin did not
become available until the late 1980s in the laboratory
of Michael James in Edmonton, Alberta, Canada. By
then, it had been recognized that renin had acertain,
albeit modest, sequence homology of 20–30% with aspartic proteases from fungi, for which 3D structures were
known. This was the starting point for homology model-
. Fig. 24.6 Enalkiren 24.4 and remikiren 24.5 were the rst renin
inhibitors to be tested in clinical trials
ing in several laboratories. The rst model was published
by Tom Blundell’s group in 1984. They used the crystal
structure of endothiapepsin as areference. First, the renin sequence was compared with that of other aspartic
proteases to nd structurally conserved regions. They
then modeled the interior of the protein by replacing
residues in endothiapepsin with those in renin. Deletions and insertions in the polypeptide chain had to be
taken into account. The ap region was of particular
importance. It opens to allow the ligand to enter and
form hydrogen bonds with the protein. Its structural architecture is, therefore, important for ligand binding. Unfortunately, the renin sequence differed from that of the
fungal enzymes in the ap region. Acomparison of the
renin model with the later determined crystal structure
of renin showed good agreement, especially in the lower
part of the binding pocket near the two aspartic acids.
However, signicant differences were found in the loops
of the ap region. In the context of the overall protein
architecture, these were less important. In the context
of drug design, however, they were critical! Errors in the
structural model had to inevitably lead to incorrect suggestions for inhibitor design.
The structure of renin complexed with the inhibitor
CGP-38560 24.10 determined by Markus Grütter and
John Priestle at Ciba in Basel, Switzerland, is shown in
. Fig.24.8. It was on the basis of this structure that
the researchers at Ciba, now Novartis, made their breakthrough. Looking at the arrangement in the binding
pocket of renin, it is apparent that the S1 and S3 pockets
merge into one large hydrophobic cavity. The substituents in the P1 and P3 positions, acyclohexylmethylene
and abenzyl group, come spatially close together. Instead of spanning the molecule with its side chains on
apeptidic backbone, the scientists broke the chain at the
amide bond. This created anew polar moiety, aterminal
charged amino group. In place of the former backbone,
. • Design of Renin Inhibitors
. Table 24.6 By modifying the P3 substituentR, the stability to chymotrypsin is improved
R IC50 (nM) Hydrolysis by chymotrypsin t
0.35 2.2
0.76 727
0.58 Stable
. Fig. 24.7 Structures 24.7–24.9 are afew renin inhibitors
with moderate oral availability. All have in common adiol
unit and acyclohexylmethylene side chain that binds in the
pocket
P
1
1/2
(min)
24
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Chapter • Aspartic Protease Inhibitors
. Fig. 24.8 Superposition of the crystal structures of renin complex-
es with the inhibitors CGP-38560 24.10 (gray carbon atoms) and aliskiren 24.12 (light-green carbon atoms). The inhibitors bind with their
peptide-like architecture in an extended, pleated-sheet-like conformation. Compound 24.10 orients its benzyl and cyclohexylmethyl groups
in the broad S3/S1 pocket. To design aliskiren, the two hydrophobic
side chains of 24.10 were chemically linked (pink arrow). The newly
the linkage of the molecule was redirected to the closely
adjacent hydrophobic substituents in the large S3/S1
pocket. The result was acompletely new dipeptide-like
scaffold (24.11, . Figs.24.8 and24.9). It had an IC50 of
6 nM. Finally, several steps of side chain optimization
were performed on the aromatic ring and the amide linkage. The methoxypropoxy side chain occupies aslightly
different pocket than the corresponding groups in CGP-
38560. It results in asignicant increase in binding afnity. The optimized substituent in
has little effect
on in vitro afnity, but is critical for duration of action.
Ageminal substitution with two methyl groups and aterminal carboxamide group proved to be optimal for the
position. The resulting inhibitor was launched in 2006 as
aliskiren (24.12), the rst orally available renin inhibitor.
Despite being so well optimized, the compound does not
have ideal bioavailability. As aresult, it must be administered at relatively high doses. However, aliskiren shows
virtually no binding to other aspartic proteases such as
cathepsinD or pepsin.
Roche achieved another success with their work
on renin, which later proved to be stimulating for the
formed bond allowed the peptide chain to be cleaved and anew, polar
N-terminus could be formed. (7 https://sn.pub/BGi2k5)
entire eld of research. With remikiren 24.5, the company had apotent inhibitor that unfortunately lacked
the desired oral bioavailability. The company, therefore, initiated anew comprehensive screening program.
Chlorophenylmethoxybenzyloxypiperidine 24.13 was
discovered (. Fig.24.10) with an IC50 value of 50 μM.
This structure was surprising because it did not have the
typical group mimicking the transition state. The crystal structure of avery similar derivative showed that the
protonated nitrogen on the piperidine ring binds between
the two catalytic aspartic acids. The lipophilic chlorophenyl moiety aligns with the broad S1/S3 pocket normally
occupied by the leucine and phenylalanine residues of
the angiotensinogen substrate. Since the available space
in this pocket was not yet fully occupied, the Roche researchers initially focused on structural variations in the
para-position of the aromatic ring as asurrogate for the
chlorine atom. The introduction of aromatic groups with
variable chain lengths yielded derivatives with up to 100fold improved activity. It appeared to be critical that only
hydrophobic groups could be placed in this position. The
best results were obtained with apropylenedioxybenzyl
. • Design of Substrate Analogue HIV Protease Inhibitors
. Fig. 24.10 A piperidine derivative 24.13 that was found in
ascreening campaign for renin inhibitors at Roche. Acrystal structure
was determined with the optimized compound24.14
family in more conformations than the closed-ap conformation. The open conformer can also be stabilized by
an inhibitor. These exemplary studies on renin provided
important information for novel work on the aspartic
proteases (Sect.24.6).
. Fig. 24.9 For the further development of 24.10 to aliskiren 24.12
as an orally available renin inhibitor the benzyl and cyclohexylmethyl
side chain groups in 24.10 were tethered together to yield 24.11. Then
the peptide chain could be cleaved after the nitrogen atom, and anew
polar group could be formed that binds to the catalytic center. The
analogous molecular parts in both inhibitors are highlighted in red
side chain. This moved the compounds into the subnanomolar inhibition range. Acrystal structure determination was performed on derivative 24.14, which showed
acompletely unexpected binding mode (. Fig.24.11).
The protonated nitrogen of the piperidine ring is still
between the two aspartic acid residues, but the lipophilic
naphthyl group is oriented in the broad S1/S3 pocket. The
long hydrophobic side chain of the 4′-substituted phenyl group opens anew pocket in renin. Like all aspartic
proteases, renin has aexible ap region that collapses
over the binding pocket after substrate binding. In this
case, the ap is pushed outwards by the inhibitor. The
enzyme adopts ageometry that is more consistent with
an open-ap conformation. Ahydrogen bond between
Trp39 and Tyr75, which closes the ap, is ruptured. At
the same time, the 4-phenyl group of the inhibitor occupies aregion where the aromatic ring of Tyr75 would be
located if the ap were closed. This structure provided
the researchers with two important pieces of information: (i)anitrogen-containing heterocycle is an interesting peptidomimetic that binds to the catalytic aspartic
acids, and (ii)inhibitors can bind to the aspartic protease
24.3 Design of Substrate Analogue HIV
Protease Inhibitors
AIDS (Acquired Immune Deciency Syndrome) is an
infectious disease caused by the human immunode-
ciency virus, HIV, which was isolated and identied in
1983 by the French virologists Luc Antoine Montagnier and Françoise Barré-Sinoussi. Many years later, in
2008, they were awarded the Nobel Prize for their work.
HIV protease, which is required for viral replication, is
encoded as alarge proprotein in the viral genome. The
function of HIV protease is to cleave the initial polypeptide chain produced in the life cycle of the virus into
smaller functional proteins. Inhibitors of HIV protease
should, therefore, be able to suppress HIV replication.
The existence of HIV protease was postulated in 1985
and experimentally conrmed in 1988.
In 1989, the rst 3D structure of the enzyme as well
as afew enzyme–inhibitor complexes were determined.
HIV protease is ahomodimer made up of two identical
chains. One catalytic aspartic acid comes from each chain
of the homodimer. The dimeric structure of HIV protease with its twofold symmetry is shown in . Fig.24.12.
It was soon discovered that HIV protease is also
inhibited by pepstatin. This was the starting point for
the search for HIV protease inhibitors. Many companies already active in the renin eld tested compounds
generated in these programs for possible HIV protease
inhibition. Starting with the nonproteinogenic amino
acid statin known from the renin work, anumber of active HIV protease inhibitors were discovered. As with
renin, the hydroxyethylene isostere proved to be apartic-
ularly suitable building block. For example, H-261 24.15
(. Fig.24.13) is apotent HIV protease inhibitor with
Ki = 5 nM.
24
0
1
Chapter • Aspartic Protease Inhibitors
. Fig. 24.11 The crystallographically determined binding mode
of the piperidine lead structure 24.14 with renin. The basic nitrogen
of the inhibitor binds between the two aspartic acids of the catalytic
dyad. The lipophilic side chain lies in anewly opened binding pocket.
It was formed by breaking ahydrogen bond that was originally present
between Trp39 and Tyr75 in the uncomplexed protein. Both residues
Heptapeptides have been identied as aminimal sub-
strate for HIV protease. Ser–Leu–Asn–Phe–Pro–Ile–Val
is such asubstrate. Cleavage of the amide bond occurs
between the amino acids Phe and Pro. Replacement of
the cleavable amide bond with ahydrolytically stable hydroxyethylamino group –CHOH–CH2–NH– led to 24.16
(JG 365, . Fig.24.13), ahigh-afnity HIV protease inhibitor (Ki = 0.66 nM). This compound was, however,
inactive in cell culture assays. It is unable to penetrate the
cell membrane to exert its antiviral effects.
Chemists at Roche have demonstrated that the design
of an HIV protease substrate analogue can lead to an effective drug. Proline is often found in the
position (e.g.,
24.17, . Fig.24.14). Therefore, isosteres of analogues of
the dipeptide Phe–Pro were investigated as HIV protease
inhibitors. Replacement of proline by homoproline 24.18
or decahydroisoquinoline 24.19 resulted in asignicant
increase in potency. In addition, 24.19 showed marked
selectivity towards the other aspartic proteases renin,
adopt anew position with larger distance between one another after
binding of 24.14. (7 https://sn.pub/lrGrOq)
pepsin, cathepsinD, and cathepsinE. More importantly,
the compound was active in acellular assay. It has the
ability to penetrate the cell membrane. In enzyme assays,
24.19 inhibited HIV protease with Ki < 0.12 nM. Viral
replication is inhibited in cell culture with EC50 values
of 1–10 nM. The activity in cells is, therefore, on the
same order of magnitude as the pure enzyme inhibition.
Saquinavir 24.19 was the rst HIV protease inhibitor to
complete all phases of clinical testing and receive marketing approval in November 1995. In the years since, other
pharmaceutical companies have succeeded in bringing
substrate-like HIV protease inhibitors to market. As aresult, our drug arsenal now includes eight approved drugs
(24.19–24.27) with peptide-like scaffolds (. Fig.24.15).
However, nelnavir 24.24 was withdrawn from the European market in 2007. It was noticed that tablets containing this substance had an unusual smell. Subsequent
analysis revealed that the drug was contaminated with
ethyl mesylate from synthesis. Because saquinavir has
0
1
. • Structure-Based Design of Nonpeptidic HIV Protease Inhibitors
. Fig. 24.12 The 3D Structure of HIV protease complexed with the
peptide substrate Arg–Pro–Gly–Asn–Phe–Leu–Gln–Ser–Arg–Pro.
The structure with the substrate could be obtained with acatalytically
inactive enzyme variant because both acidic aspartic acids of the catalytic dyad had been mutated to asparagines. The protease exists as
aC2-symmetric homodimer. The peptide chains are shown in green
and red, respectively. (7 https://sn.pub/r7ffVB)
unsatisfactory bioavailability (3–5%), it is administered
in combination with ritonavir 24.20, apotent CYP3A4
inhibitor (Ki = 17 nM; Sect. 27.6). This signicantly
minimizes the rst pass effect when coadministered with
saquinavir. Ritonavir has established itself as abooster
for other drugs and is increasingly used in combinations
with other drug molecules. For example, it is used in
combination with the recently approved COVID-19 inhibitor nirmatrelvir (Paxlovid®, Sect.23.9). Amprenavir
24.23 was withdrawn in 2004 because it was replaced by
the more soluble prodrug fosamprenavir (Lexiva®).
24.4 Structure-Based Design of
Nonpeptidic HIV Protease Inhibitors
The relationship to the parent substrate is clearly seen in
the inhibitors introduced in the last section. The compounds are still essentially peptides. The crystal structures of the peptidic HIV protease inhibitors complexed
with the enzyme all show that the inhibitors form essentially the same H-bonding pattern in the immediate
vicinity of the catalytically active aspartic acid residues
(. Fig.24.16). One water molecule is of particular interest because it is present in all crystal structures. This
water molecule forms hydrogen bonds with both the inhibitor and the enzyme. Inhibitors designed to displace
this water molecule were hoped to increase the binding
afnity by the entropically favorable release of this water (Sect.4.6). The release of water molecules is often
associated with an entropic gain. Note, however, that at
best this gain is not fully compensated by acomparable
loss of enthalpy, leaving anet contribution to binding
afnity. Moreover, it was expected that such an approach
would also increase selectivity, since awater molecule
with asimilar function is not known to exist in the other
aspartic proteases of similar pharmaceutical relevance.
At Dupont–Merck, a3D database was searched for
new scaffolds for HIV protease inhibitors. Apharmacophore pattern was derived from the crystal structure
of the enzyme. The occupancy of the S1 and
pockets
was considered essential for binding and interaction with
the catalytic aspartates. Two lipophilic groups, separated
by 8.5–12 Å and also 3.5–6.5 Å from ahydrogen bond
24
Chapter • Aspartic Protease Inhibitors
. Fig. 24.13 The peptidic HIV protease inhibitors
H-261 24.15 and JG365 24.16 are potent inhibitors
in the enzyme assay. They are inactive in cell culture
. Fig. 24.14 The stepwise optimization of the
substrate analogue inhibitor 24.17 led to the highly
potent HIV protease inhibitor Ro 31-8959 24.19
via 24.18. This compound was the rst protease
inhibitor to pass clinical trials and is marketed with
the name saquinavir
acceptor or donor, were sought (. Fig.24.16, right). In
addition, there should be afunctional group between
the two lipophilic groups that can displace the structurally conserved water molecule from the binding pocket.
Asearch in the Cambridge database (Sect.17.11) yielded
amolecular scaffold derived from asubstituted phenol
(24.28). This led to the idea of using 4-hydroxycyclohexanone as the scaffold (. Fig.24.16). Modeling studies and
intensive discussions with synthetic chemists eventually
led to acyclic urea (24.29) as the scaffold for the new
inhibitors 24.30–24.33 (. Fig.24.17). The rst result of
this development was DMP-323 24.32, asmall-molecule
HIV protease inhibitor. The 3D structure of 24.31 complexed with the protease is shown in . Fig.24.18. It conrms the hypothesis that the carbonyl group displaces the
structural water molecule and the two hydroxyl groups
bind to the catalytic aspartate residues. As promising as
the design of the cyclic urea as an HIV protease inhibitor seemed, no compound has yet survived all stages of
clinical testing to gain regulatory approval.