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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5371_Библиотеки_им_академика_М_И_Перельмана
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Chemistry and Pharmacology of Drug Discovery
antigens on the host tissues. In April 2012, CTI BioPharma Corp. (formerly known as
Cell Therapeutics Inc., CTI hereafter) and S*BIO Pte. Ltd. entered into an asset purchase
agreement under which CTI would acquire worldwide rights to pacritinib from S*BIO.
CTI completed the acquisition of pacritinib (1) in June 2012. CTI would have sole
responsibility for developing and commercializing pacritinib (1) worldwide.
Pacritinib (1) received its first approval on February 28, 2022, in the United
States for the treatment of adults with intermediate- or high-risk primary or secondary
(post-polycythemia vera or post-essential thrombocythemia) myelofibrosis with a platelet
count below 50 109/L. This accelerated approval is based on the demonstration of
×
spleen volume reduction in pacritinib (1) recipients in phase III PERSIST-2 trial.
However, the continued approval of pacritinib (1) is conditional upon an ongoing trial
(PACIFICA) being completed to confirm the clinical benefit of this spleen volume
1
reduction.
Pacritinib (1) is available on the market as 100 mg capsules for oral use. The
recommended dosage of pacritinib (1) is 200 mg administered twice daily, with or
without food. Pacritinib (1) is currently under investigation in phase II trial for prostate
cancer and phase I/II in graft-vs-host disease and breast cancer. Clinical trials of
pacritinib (1) in other indications previously started (e.g., colorectal cancer, acute
myeloid leukemia, and lymphoma) has now been discontinued.
After the pioneer work of Meydan et al.2 in 1996 to develop the small molecular
inhibitor of JAK inhibitor with antileukemic activity, great efforts have been made to
develop potent and selective JAK inhibitors in the last two decades.
potent, orally available, selective inhibitor of both JAK1 and JAK2 of the JAK-STAT
signaling pathway developed by Incyte Corp and Novartis. Ruxolitinib (2) was the first
JAK inhibitor approved by the US Food and Drug Administration (FDA) for the
treatment of patients with intermediate or high-risk myelofibrosis, including primary
Ruxolitinib (2) is a
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Chapter 8. Pacritinib (Vonjo)
myelofibrosis, post-polycythemia Vera myelofibrosis, and post-essential
thrombocythemia myelofibrosis in November 2011. Tofacitinib (3) is an orally active
small molecule inhibitor of JAK1, 2, and 3 developed by Pfizer, for the treatment of
immunological disorders. It was approved for the treatment of rheumatoid arthritis by
3
FDA in 2012.
Baricitinib (4) is an orally active small-molecule inhibitor of JAK1/2
developed by Eli Lilly. It was approved by the European Medicine Agency (EMA) in
2017 for the treatment of rheumatoid arthritis. Baricitinib (4) was approved by the FDA
4
for the treatment of moderate-to-severe rheumatoid arthritis in adults in June 2018.
Upadacitinib (5) an orally administered JAK-1 inhibitor, is being developed by
5
AbbVie for the treatment of rheumatoid arthritis was approved by FDA in 2019.
In the
same year, FDA approved fedratinib (6) developed by Impact Biomedicines, Inc. for
adults with intermediate-2 or high-risk primary or secondary (post-polycythemia vera or
6
post-essential thrombocythemia) myelofibrosis.
Abrocitinib (7) is another oral smallmolecule inhibitor of JAK-1 being developed by Pfizer for the treatment of moderate-tosevere atopic dermatitis (AD) got FDA approval in September 2020.7
2 Pharmacology
The JAK/STAT signaling pathway plays a crucial role in many cellular functions,
including cell proliferation, stem cell maintenance and differentiation as well as in
inflammation and autoimmune diseases.
more complex. The basic and simple version of molecular mechanism underlying
cytokine signaling through the JAK/STAT pathway is shown in Figure 1.9 Cytokines
bind to the cytokine receptors and form activated and phosphorylated homo- or
heteropolymers complex with their JAK partners. Then the cytosolic DNA-binding
STAT proteins bind to the receptor-JAK complex and are themselves phosphorylated by
JAK family members. Upon phosphorylation, phosphorylated STAT proteins form
homo- and heteropolymers which then enter the nucleus. Phosphorylated STAT dimer
8
In the reality JAK/STAT signaling is much
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Chemistry and Pharmacology of Drug Discovery
transcription factors then bind specific DNA binding sites regulating gene transcription
and cellular function.
Figure 1. JAK/STAT pathway. Step 1: Cytokine binding, complex formation, activation,
and phosphorylation of JAK. Step 2: Recruitment and phosphorylation of STAT. Step 3:
Phosphorylated STAT (p-STAT) dimerization. Step 4: Nuclear translocation and DNA
binding of p-STAT dimer. Step 5: Gene transcription. Source: Bryan and Rajapaksa
9
/with permission of American Chemical Society
The JAK family includes four members JAK1, JAK2, JAK3, and TY2K. JAKs
have seven homology domains, JH1–JH7 (Figure
2). Starting from the C-terminal, JH1 is
the first domain, also known as the kinase domain. JH1 has the kinase activity and
phosphorylates a substrate. JH2 is a pseudokinase domain that is structurally similar to
the kinase domain but has no kinase activity. It regulates the activity of the kinase
domain. JH3 and one-half of JH4 constitutes the Src-homology SH2 domain, while the
combination of one-half of JH4, JH5, JH6, and JH7 constitutes the FERM domain. These
two domains, SH2 and FERM mainly regulate the binding of JAK and cytokine-receptor
10
membrane-proximal box1/2 regions.
Among these kinases, JAK1 plays a crucial role in allergic rhinitis, pruritic
dermatitis, inflammatory bowel disease, and asthma. Small molecule inhibitors of JAK1
11–13
have proved efficacy in the treatment of these diseases.
Furthermore, several small
molecules with JAK1 and JAK2 inhibitory activity have also provided therapeutic
benefits in the treatment of psoriasis, rheumatoid arthritis, and pruritis. Besides this,
several JAK3 selective inhibitors have been evaluated for their efficacy in the treatment
of rheumatoid arthritis. Moreover, selective inhibitors of TYK2 may also be useful in the
14
treatment of autoimmune diseases.
Excessive activation of JAKs has also been reported
in different types of cancer. The JAK/STAT3 pathway plays an important role in the
proliferation and angiogenesis of solid tumors.
15
Pacritinib (1)
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Chapter 8. Pacritinib (Vonjo)
Figure 2. Domain structure of JAKs
Pacritinib (1) is thought to exert clinical efficacy by inhibiting two distinct
pathways,
JAK/STAT and TLR/Myddosome/IRAK1, leading to suppression of NFκB
and downstream inflammatory cytokine cascade, reduction in splenomegaly, and
myelofibrosis symptom control.
inhibitor with high specificity for JAK2 (IC
Relative to JAK2, pacritinib (1) is 2-fold less potent against TYK2 (IC
fold less potent against JAK3 (IC
= 1280 nM). Pacritinib (1) also a potent inhibitor of FLT3 (IC50 = 22 nM) and its
(IC
50
D835Y
mutant FLT3
(IC50 = 6 nM), which impact in myelofibrosis is unknown (Table 1).17
16
Pacritinib (1) is a potent competitive reversible kinase
= 23 nM), JAK2
50
= 520 nM) and 56-fold less potent against JAK1
50
V617F
(IC50 = 19 nM).
= 50 nM), 23-
50
Pacritinib (1) is significantly more effective than best available therapy (BAT), including
ruxolitinib (2), for reducing splenomegaly and symptoms in patients with myelofibrosis
and thrombocytopenia.
Pacritinib (1) is orally administered and readily absorbed regardless of food
intake, with a mean terminal elimination half-life of 27.7 h. It is predominantly
metabolized by the CYP3A4 isozyme and biliary excretion. Pacritinib (1) achieves
maximum plasma concentration (C
) at approximately 4–5 h post dose and has an
max
apparent volume of distribution of 229 L with 98.8% plasma protein binding (PBB).
Pacritinib (1) is generally metabolized via CYP3A4, which will be discussed in detail
18
later.
Compound Kinase
Table 1. In vitro kinase spectrum of pacritinib (1)
IC
(nM) Selectivity vs JAK2
50
JAK1 1280 56
JAK2 23 1.0
V617F
JAK2
19 0.8
JAK3 520 23
TYK2 50 2.2
FLT3 22 1.0
B835Y
FLT3
6 0.3
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Chemistry and Pharmacology of Drug Discovery
3 Structure–Activity Relationship (SAR)
From in-house library screening, S*BIO Pte Ltd’s19 identified compound 8 that showed
broad kinase inhibition with reasonable activity in cell lines tested. To avoid any
intellectual property contradiction, they connected the open ends of 8, to form
macrocycle 9. Interestingly, they found that the binding mode to the kinase hinge region
is not compromised. A study of various linkers with and without R1 groups demonstrated
that these templates inhibited JAK2/FLT3 with selectivity against JAK1/3 is shown in
2. They found that the most potent JAK2 linkers were 8 atoms in length with
Table
symmetrical dibenzylic linkers, as employed in compounds 10e and 10f, were quite
potent against JAK2 with good selectivity over JAK1 and 3. 10e inhibit JAK2 with IC
of 70 nM and is 27- and 17-fold selective against JAK1 and JAK3, respectively. They
decided to focus on the progression of the most potent dibenzylic compound 10e and 10f.
One concern of this high cLogP compounds was the very low solubility. They found the
solubility of 10b and 10d less than 10 μg/mL in PBS buffered at pH 7.0. Therefore, they
search for locations to install a solubilizing group in this template to improve solubility.
50
1900
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Chapter 8. Pacritinib (Vonjo)
Table 2. Search for a suitable linker for selectivity toward JAK2
Compd JAK2
IC50 (nM)
JAK1 IC
(nM)
50
JAK3
IC
50
(nM)
TYK2 IC
(nM)
50
FLT3
IC
50
cLogP
(nM)
10a 1,200 >10,000 >10,000 5200 1100 4.7
10b 230 >10,000 >10,000 70 880 4.3
10c 130 3000 ND 150 310 4.6
10d 260 5800 ND 120 78 4.0
10e 70
1200 210 190 4.1
10f 53 400 1100 170 170 3.4
Figure 3. Compound 10e docked into the ATP-binding site of JAK2. Source: William et
19
/with permission of American Chemical Society
al.
Docking of 10e into the JAK2 ATP binding pocket (Figure 3) showed that the
macrocycle structure fills out the available space in the binding site quite well with the
opportunities for building additional interactions with the protein from various sites. The
docking pose of 10e showed that the R
to the solvent that may offer potential for installation of a basic center to increase the
solubility.
substituent points directly toward a channel out
1
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Chemistry and Pharmacology of Drug Discovery
Based on their docking results, they explored both the R1 and R2 positions on the
benzylic ring. They found that a range of N and O-substituents were very well tolerated.
Though sterically large groups like morpholine and piperazines were potent and
selective for JAK2, but sparingly soluble in aqueous media. They used side chain like
hydroxyethyl piperazine, or less sterically open chain analogues and amide (structures not
mentioned here). However, improved solubility with good potency was achieved with
oxygen-linked substituents. For example, compounds 1 and 11a,b, containing an
aminoethyl ether side-chain, were found uniformly potent against JAK2 and selective
against JAK1/3. The direct R
analogue of 1, 11c, was also potent against JAK2 but with
2
sub-micromolar CDK2 activity. Morpholine analogue 11d, however, was less CDK2
potent but suffered a 5-fold reduction in JAK2 potency over the most potent R
compounds (Table 3).
Table 3. SAR of optimization solubility
1
Compd JAK2
IC
(nM)
11a 24 3500 620 79 29 4700 147
1 23 1280 520 50 22 3900 >150
11b 15 950 750 84 34 2700 ND
11c 48 2100 830 80 22 570 145
11d 96 2400 3300 81 19 2700 40
In search of better drug candidate compound, they further explored the effect of
small groups substitution on the aromatic rings (Table
pyrimidine 12b exhibited 4-fold higher potency for JAK2 (IC
JAK1
50
IC
50
(nM)
JAK3
IC
50
(nM)
TYK2
IC
50
(nM)
FLT3
IC
50
(nM)
SDK2
IC
50
(nM)
Solubility
(μg/mL)
3). Indeed 5′-methyl substituted
6 nM) as compared to 1
50
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Chapter 8. Pacritinib (Vonjo)
with good solubility. However, this compound was compromised somewhat by its
selectivity against JAK3 which had decreased by nearly15-fold. When 5-methyl group
was replaced by electron-withdrawing fluoro 12c, it reduced JAK2 activity by 3-fold,
which confirming that hydrophobic groups are ultimately preferred. Substitution at R
12d with methoxy is unfavorable for JAK family activity. On the other hand, substitution
12e with electron-withdrawing fluoro is well tolerated but reduces selectivity over
at R
5
JAK1 and solubility. Substituting methoxy at the R
potency and selectivity for JAK2 over JAK1 (Table
position in 12f resulted in excellent
6
4). Though the effects of adding
JAK1 activity to a JAK2 inhibitor are poorly understood. Therefore, they prioritized more
selective compounds in the expectation that they would have reduced off-target toxicity.
Addition of an additional substituent to the right-hand side aniline ring of 1 resulted in
electron rich 12a which did not compromise JAK2 activity or selectivity and showed
improved FLT3 activity as compared with 1.
Table 4. SAR Exploration of aromatic ring substitutions with small groups
4
Compd JAK2,
IC
(nM)
12a 36 1700 ND 170 6 3400 154
12b 7 1000 89 57 19 >10 178
12c 17 830 1000 140 15 1550 ND
12d 330 4900 7200 620 12 >10 ND
12e 24 380 ND 36 8 2000 2.6
12f
12g
For further in vitro study, they chosed compounds 1, 11a, 11b, 12b, and 12f. 1
inhibited the JAK2-mediated production of p-STAT5 and p-STAT3 dose dependently in
Ba/F3 cells. Although, 12b exhibiting desirable single digit nanomolar potency toward
JAK2 but inhibit CYP3A4 with IC
of 0.37 μM. On the other hand, 1 and 12f were much less active against CYP3A4
IC
50
50
JAK1,
IC
50
(nM)
JAK3,
IC
50
(nM)
TYK2,
IC
50
(nM)
FLT3,
IC
50
(nM)
SDK2,
IC
50
(nM)
Solubility
μg/mL
19 >10 890 180 92 >10 60.8
25 4600 720 ND 40 3250 ND
of 2.5 μM. Similarly 12b also inhibit CYP3A4 with
50
Pacritinib (1)
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Chemistry and Pharmacology of Drug Discovery
and both compounds had good selectivity and microsomal stability. Although, compound
12f has excellent enzyme selectivity, was less active against FLT3. Compound 1 shows
an overall balanced profile meeting all target criteria (Table 5).19 It was also active
against the V617F mutant of JAK2 with IC
with IC
= 6 nM. Its protein kinase selectivity was evaluated by testing against more
50
= 19 nM and the D835Y mutant of FLT3
50
than 50 other protein kinases covering all major families of the human protein kinome.
Thus, they selected 1 was their drug candidate compound.
Table 5. ADME profile of pacritinib (1)
Properties Values
HLM (t
DLM (t
RLM (t
MLM (t
, min) >60
1/2
, min) 41
1/2
, min) 18
1/2
, min) 22
1/2
Human CYP inhibition IC50 (μM) >5
PPB (%) in human 99.88
PPB (%) in dog 99.63
PPB (%) in mouse 99.41
Permeability (
˟10–6 cm/s) 16
Papp, A-B,
4 Pharmacokinetics and Drug Metabolism
It has been noticed that increases in pacritinib (1) exposure are less than doseproportional over a dose range of 100–600 mg in patients with advanced myeloid
malignancies.
myelofibrosis, the mean peak concentration (C
8.4 mg/L. Generally, pacritinib (1)’s C
Administration of pacritinib (1) with food does not significantly impact its
pharmacokinetics. A steady state is achieved within a week of commencing twice-daily
administration of pacritinib (1) and its accumulation is 386%. At steady state, the median
apparent volume of distribution of pacritinib (1) is 229 L. As mentioned in Table 4,
pacritinib (1) is highly bound to plasma proteins (~ 99%). Pacritinib (1) pharmacokinetics
are not significantly affected by age, sex, race, or body weight.
CYP3A4. The metabolism of pacritinib (1), investigated by Jayaraman et al. revealed the
formation of four metabolites by liver microsomes in both humans and mice (Figure 4).18
The identified metabolites include two oxidized metabolites formed by oxidation of the
pyrrole ring (M1) and pyrrole nitrogen (M3). While the third metabolite (M2) formed by
O-dealkylation of the pyrrole-bearing side chain and the fourth metabolite (M4) was
20
When pacritinib (1) 200 mg is administered twice daily in patients with
) of pacritinib (1) at steady state is
max
is reached after ~ 4–5 h dose administration.
max
1
The metabolic study of pacritinib (1) revealed that it is mostly metabolized by
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formed by reduction of the double bond of the linker of pacritinib (1). Pacritinib (1) has a
mean apparent steady-state clearance of 2.09 L/h and a mean effective half-life of 27.7 h.
When a single dose of radiolabeled pacritinib (1) 400 mg was orally administered in
healthy adults, the radioactivity was mostly recovered in feces (87%, none as unchanged
drug). While a small proportion of the drug administered was recovered in urine (6%,
with 0.12% as unchanged drug).
Chapter 8. Pacritinib (Vonjo)
Figure 4. Schematic representation of molecular ionic structures of pacritinib (1) and its
metabolites
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