Development of a long acting human growth hormone analog suitable for once a week dosing

Article abstract of DOI:10.1016/j.bmcl.2012.11.104

Human growth hormone was conjugated to a carrier aldolase antibody, using a novel linker by connecting a disulphide bond in growth hormone to a lysine-94 amine located on the Fab arm of the antibody. The resulting CovX body showed reduced affinity towards human growth hormone receptor, reduced cell-based activity, but improved pharmacodynamic properties. We have demonstrated that this CovX-body, given once a week, showed comparable activity as growth hormone given daily in an in vivo hypophysectomized rat model.

Full text of DOI:10.1016/j.bmcl.2012.11.104

Bioorganic & Medicinal Chemistry Letters 23 (2013) 402–406
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of a long acting human growth hormone analog suitable
for once a week dosing
⇑
Moorthy S. S. Palanki , Abhijit Bhat, Ben Bolanos, Florence Brunel, Joselyn Del Rosario, Danielle Dettling,
Mark Horn, Rodney Lappe, Ryan Preston, Annette Sievers, Nebojsa Stankovic, Gary Woodnut, Gang Chen
CovX, Pfizer Worldwide Research and Development, 9381 Judicial Drive, Suite 200, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Human growth hormone was conjugated to a carrier aldolase antibody, using a novel linker by connect-
ing a disulphide bond in growth hormone to a lysine-94 amine located on the Fab arm of the antibody.
The resulting CovX body showed reduced affinity towards human growth hormone receptor, reduced
cell-based activity, but improved pharmacodynamic properties. We have demonstrated that this
CovX-body, given once a week, showed comparable activity as growth hormone given daily in an
in vivo hypophysectomized rat model.
Received 12 September 2012
Revised 21 November 2012
Accepted 26 November 2012
Available online 5 December 2012
Keywords:
Human growth hormone
Antibody
Ó 2012 Elsevier Ltd. All rights reserved.
CovX body
Disulphide linker
Somatotroph cells located in the anterior pituitary gland inter-
mittently releases growth hormone (hGH), which has many physio-
logical functions including growth promotion, protein synthesis,
lipolysis and regulation of metabolic processes.1 Although growth
hormone has many biological functions, its major target organs
are bone and muscle, promoting the growth of the bone and increas-
ing the mass of the muscles.2 GH deficiency in children results in a
condition called pituitary dwarfism, which is characterized by
slowed long bone growth, younger facial features and sometimes a
chubby body build. Puberty may come late or not at all in older
children. It is estimated that 10,000–15,000 children in the United
States have growth failure due to growth hormone deficiency.3
Recombinant human growth hormone (GenotropinÒ), a 191
amino acid polypeptide chain, is widely used to treat diseases
due to growth hormone deficiency both in children and adults.4
GH is given as a daily subcutaneous injection due to its short
half-life of 20–30 min. Rapid proteolysis and ligand–receptor inter-
nalization result in a short half-life. A long acting growth hormone
receptor (GHR) agonist with sufficient stability to be dosed weekly
is therefore a highly desirable alternative especially for young
children. A long acting N-terminally PEGylated growth hormone
was advanced to phase II clinical trials but subsequently the
studies were terminated due to injection site lipoatrophy.5,6
CovX-Bodies represent a novel class of biotherapeutic agents
created by the fusion of a therapeutic with an antibody, which
serves as a carrier protein scaffold. The fused payload determines
the targeting property of a CovX-Body whereas the antibody scaf-
fold provides an antibody like pharmacokinetic and distribution
profile which prevents it from crossing the blood–brain barrier.
CovX-Bodies are formed by the conjugation of a pharmacophore
functionalized with an azetidinone (AZD) linker with a specific ly-
sine residue in the Fab region of a humanized antibody that serves
as the carrier scaffold. We have successfully applied this technol-
ogy to peptide based therapeutic agents and we envisioned the
application to the protein therapeutic space such as GH.7–9 We
developed a long-acting hGH analogue conjugated to an aldolase
carrier antibody engineered in such a way that human growth hor-
mone was irreversibly linked to one of the Fab arms. The resulting
conjugated molecule, known as GH-CovX-Body, has the pharmaco-
logical properties of GH with the benefit of extended therapeutic
half-life.10 To the best of our knowledge, this is the first demonstra-
tion that half-life of human growth hormone can be extended by
conjugating to a carrier antibody that has no biological function.
Growth hormone has two binding sites (site 1 and 2) and binds
to two growth hormone receptors in 1:2 complex and initiate
intracellular signaling event through receptor homo-dimerization,
which is critical for signaling.11,12 After the signaling event, the en-
tire receptor–ligand complex is internalized resulting in signal ter-
mination.13 By preventing the internalization one could keep the
GH in circulation longer. PEGylated growth hormone, which
showed reduced affinity and improved half-life, was suitable for
once a week dosing.14 However the development of PEGylated
growth hormone was discontinued due to injection site lipoatro-
phy. Our approach for developing long-acting growth hormone
was to reduce the affinity in a way that maintains the required
⇑
Corresponding author. Tel.: +1 858 964 2053.
E-mail address: Moorthy.palanki@pfizer.com (M.S.S. Palanki).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.104

M. S. S. Palanki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 402–406
403
potency but likely reduces the internalization rate (or efficiency)
and then use such a modified molecule with CovX-Body conjuga-
tion to give the desired pharmacokinetic profile. We reasoned that
by lowering the affinity of GH towards GHr one could keep the GH
in circulation longer since the lower affinity could result in reduced
internalization. Several mutants of growth hormone have been
shown to have reduced affinity towards GHr, which have the po-
tential to stay in the circulation longer.15 These studies showed
that the affinity towards the receptor can be reduced by as much
as 30-fold without affecting the cellular EC50. Our internal studies
have shown that by modulating the affinity towards its receptor,
hGH can affect the internalization without affecting cellular activ-
ity. The traditional approach to modulate the protein affinity to-
wards its receptor is to mutate appropriate amino acids on the
surface of the protein. One of the mutated amino acids is then used
to attach a linker which could be connected to an antibody. We
decided to work with wt-GH for generating an agonist with re-
duced affinity to reduce the risk of formation of an antibody to
the drug molecule.
N-hydroxysuccinamide18 with an amine of a lysine for linker con-
nection. However, this approach resulted in a mixture of products
due to natural abundance of lysines on the surface of GH. Our next
approach involved inserting a maleimide based linker in between a
C-terminal disulphide bond (Cys182–Cys189) and connect GH to
the aldolase antibody. This approach offered several advantages.
The maleimide group was small and could be inserted between a
disulphide bond with high selectivity and efficiency in buffered
solutions at room temperature.19 The examination of crystal struc-
ture revealed that both cysteine residues are buried inside and not
easily accessible. Hence we reasoned that even if the linker is bro-
ken, the cysteine disulphide modification should not elicite any
meaningful immunogenic response. The growth hormone by itself
has a very short half-life and it is removed from the circulation
through receptor internalization if it is cleaved. By introducing a
linker at the above location, we anticipated a change in conforma-
tion of the C-terminal end which would lower the affinity of GH to-
wards its receptor. It was anticipated that these conformational
changes would allow the GH analog to bind to the receptor long
enough to trigger the signaling event but not long enough to inter-
nalize immediately. We also anticipated by connecting the GH to
the aldolase antibody, the GH was protected from proteolysis
and stays in circulation longer. On one end of the linker we intro-
duced a diphenylthiomaleimide group which reacts with a thiol
group of the protein and on the other end an electrophile to con-
nect to the antibody. The aldolase antibody has a lysine at position
93 of the heavy chain and located deep in the hydrophobic binding
pocket on each of the two Fab arms.20 We have developed phenyl-
propanoylazetidin-2-one to react selectively with Lys-93 located
deep in the hydrophobic pocket as described earlier.21,22 We pro-
posed linker 1 which would be useful for conjugating either pep-
tides or proteins to the aldolase antibody, provided the peptide
or protein has at least one disulphide bond.
The crystal structure studies have shown that GH is a four helix
bundle with two disulphide bonds which are located towards the
C-terminal end of GH (Fig. 1).16 Several residues of the
a-helix on
the C-terminal side located in site 1 of GH interact with GH recep-
tor.17 Our approach to develop a growth hormone with lower affin-
ity was to introduce small conformational changes towards the C-
terminal end which would disrupt some of the interactions with
the receptor. Initially we examined reaction of linkers containing
The synthesis of a disulphide linker 1 is shown in Scheme 1. 3-(4-
Nitrophenyl)propionyl chloride was prepared from 2 as described.23
The carboxylic acid group in 2 was converted to propionyl acid chlo-
ride using thionyl chloride. An amide proton in azetidin-2-one was
deprotonated with n-butyl lithium in tetrahydrofuran and reacted
with the propionyl chloride to give 3. Hydrogenation over palladium
on carbon gave 4 that was treated with 1,4-dioxane-2,6-dione to
give an intermediate acid and the acid group was converted to pen-
tafluorophenyl ester 5 by treating with diisopropylcarbodiimide and
pentafluorophenol. The treatment of pentafluorophenyl ester 5 with
2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethanol (7) resulted in
compound 8 that was converted to 1 as described.24
The preparation of the GH-aldolase antibody conjugate CovX-
Body) is shown in Scheme 2. Wt-hGH (10 mg/mL in 5 mM sodium
phosphate and 34 mM glycine at pH 7.5) was treated with Tris[2-
carboxyethyl] phosphine (TCEP, 0.287 mg in 36
phosphate at pH 7.0) for 20 min at room temperature. Compound 1
(1.8 mg in 150 L of dimethylsulfoxide) was added and shaken
lL of 0.1 M sodium
l
very gently overnight at room temperature. The protein was puri-
fied using a PD-10 column (PBS buffer, pH 7.4, Mediatech Cat. No.
21-040-CM) and the excess linker and TCEP were removed. Accu-
rate measurement of the intact mass of GH + compound 1 using
a QTof mass spectrometer confirmed the addition of one linker
onto the GH protein. About 95% of the total GH protein was
observed containing 1 linker, with only trace levels of underiv-
atized protein or protein with two (2) or greater linkers attached.25
Further analysis by MALDI-TOF In-Source Decay mass spectros-
copy methodology was used to establish that the linker was at-
tached selectively between the disulphide bond of Cys-182 and
Cys-189 in GH.25 GH + 1 protein was desalted and mixed 2:1 with
the matrix ‘super’ dihydroxybenzoic acid (sDHB, Bruker Daltonics)
and spotted on a stainless steel target. In-Source Decay MS gener-
ates protein fragment ions sequentially from the N- and C-termini
Figure 1. X-ray crystal structure of hGH (PDB ID: 1hgu). Both disulphide bonds are
towards the C-terminal end. The disulphide bond between Cys182–Cys189 is more
easily accessible for the introduction of a linker. The crystal structure of wild-type
growth hormone at 2.5 Å resolution (PDB 1D: 1hgu).

404
M. S. S. Palanki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 402–406
O
O
O
O
O
O
O
N
d
HO
a
N
N
c
F
F
F
F
O
O
e
b
O
2
3
4
N
H
O
F
NO2
NO2
NH2
5
O
O
O
O
O
f
g
H2N
O
O
OH
O
N
H
O
OH
6
7
O
h
N
O
O
O
O
O
N
H
N
O
OH
H
8
O
O
PhS
PhS
Br
Br
NH
NH
i
9
10 O
O
O
O
N
O
O
O
O
O
O
N
N
H
O
N
SPh
SPh
H
1
O
Scheme 1. Reagents and conditions: (a) SOCl2, toluene, rt; (b) azetidin-2-one, n-BuLi, THF, À78 °C to rt, 57%; (c) H2, 1 N HCl, Pd/C, methanol, 35 °C, 65%; (d) 1,4-dioxane-2,6-
dione, N,N-diisopropylethylamine, dichloromethane, rt, 88%; (e) diisopropylcarbodiimide, pentafluorophenol, THF, rt, 81%; (f) trifluoroacetic acid, dichloromethane, rt, 88%;
(g) 7, DMF, rt, 77%; (h) thiophenol, sodium bicarbonate, methanol, rt, 99%; (i) diisopropyl azodicarboxylate, triphenyl phosphine, neopentyl alcohol, dichloromethane, THF,
À78 °C to rt, 69%.
Scheme 2. Preparation of GH-CovX body.
of proteins. After linker derivatization of GH, extensive terminal se-
quence coverage was observed, which terminated only at residue
53 on the N-terminus and residue 165 on the C-terminus, which
suggests the presence of an intact disulfide bridge between these
residues. It was further determined that fragment ions containing
Cys residues 182 and 189 were modified with a mass correspond-
ing to the linker 1.25 The protein with the linker was added to the
antibody (20 mg/mL, 10 mM Histidine, 130 mM Glycine, 130 mM
Sucrose, pH 6.5, 1:1 molar ratio) and very gently shaken over night
at room temperature. The reaction mixture was purified using size
exclusion column (CM column, buffer A: 20 mM 2-(N-morpho-
lino)ethanesulfonic acid, pH 6.0; buffer B: 20 mM 2-(N-morpho-
lino)ethanesulfonic acid, 1 M sodium chloride, pH 6.0) to isolate
11 (34%) and 12 (32%). Compounds 11 and 12 were isolated in
>95% pure based on mass spectral analysis. Compound 11 has
one GH per antibody and compound 12 has two GHs per antibody.

M. S. S. Palanki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 402–406
405
These two compounds were evaluated using NB2-11 cells which
80
60
40
20
0
Cumulative BW Change
proliferate in response to lactogenic hormones such as GH. These
cells were maintained in Fisher’s Medium supplemented with
10% fetal bovine serum and 10% Lactogen deficient horse serum.
Prior to GH exposure, cells were serum starved overnight in Lacto-
gen deficient media (Fisher’s Medium, 1% Fetal Bovine Serum, 10%
Horse Serum, 0.05 mM 2-mercaptoethanol). Culture plates were
prepared with starved NB2 cells and stimulated with GH for
48 h. Following GH stimulation, the amount of NB2-11 cell prolif-
eration was determined and found to show identical biological
activity in in vitro assays (Table 1).26 Since there is no advantage
for having more than one growth hormone molecule per antibody,
we have chosen 11 to study further.27
PBS (non-HYPOX)
PEG-hGH 1.8 mpk
50 mg/kg
5 mg/kg
***
***
***
*
*
1.5 mg/kg
PBS (Hypox rats)
-20
0
5
10
15
Day
* P<0.05, ***P<0.001 vs PBS, One-Way ANOVA, Dunnett
Figure 2. Mean cumulative body weight gain for hypophysectomized rats treated
with 11 s.c. using various dosing regimens (n = 5 per treatment group).
Compound 11 was evaluated in vivo in hypophysectomized
rats. PEGylated growth hormone (PEG-hGH) was included in this
study.28 Pituitary glands in young female Sprague Dawley rats
were surgically removed and the rats were placed randomly in four
different groups. Three groups of rats were subcutaneously dosed
once a week with compound 11 at three different doses (1.5, 5,
50 mg/kg/week). Two control groups (hypophysectomized rats
and non- hypophysectomized (normal) rats) were given weekly
s.c. doses of PBS. Compound 11, PBS and PEG-hGH were adminis-
tered at days 0 and 7 and monitored for the duration of the study.
The weights of the rats were measured and the change in body
weight can be seen in Figure 2. Animals dosed with compound
11 showed a significant body mass gain in all three dose groups.
The group that received 11 at 50 mg/kg/week dose showed compa-
rable weight gain as a group of hypophysectomized rats that re-
in cell activity. However, after a single injection, the compound
showed a long duration of action as evidenced by the continued
growth of the animals. Following day 5 the weight of the animals
were stabilized. When a second dose was given at day 7, the ani-
mals continued their weight gain. The compound is currently in
pre-clinical stage and additional information will be disclosed in
due time.
Acknowledgments
We would like to thank Anna Russell Tempczyk for molecular
modeling pictures and Kathy Ogilvie for critically reading the
manuscript.
ceived GH at 5 l
g/day, s.c.29 The group that received PEG-hGH
gained comparable weight as the group that received 50 mpk of
compound 11 during the first week. However, during the 2nd
week, the group that received PEG-hGH gained more weight com-
pared to the group that received 50 mpk of compound 11.
References and notes
1. Strobl, J. S.; Thomas, M. J. Pharmacol. Rev. 1994, 46, 1.
2. Rosen, T.; Bengtsson, B. A. Lancet 1990, 336, 285.
3. http://www.hgfound.org/pub_growth.html.
In conclusion, we have developed a long acting human growth
hormone analog using CovX technology and demonstrated its effi-
cacy in an in vivo model. The introduction of a cysteine mutant of-
fers a thiol nucleophile which provides a convenient handle to
introduce selective modifications. However the above method suf-
fers from the fact that the introduction of cysteine mutant in a pro-
tein containing disulphide bonds can potentially cause misfolding.
Consequently one has to make several different cysteine mutants
to identify a suitable one for development. The reduction of native
disulphide bond offers two thiol nucleophiles and a convenient
way to introduce a linker. The use of maleimide linker offers sev-
eral advantages including small size, rapid reaction and re-con-
struction of bridge to mimic the role of natural disulfide bond.
We have developed a linker that targets surface exposed disulfide
bonds for attaching a linker. These type of protein modifications
using the above linker can be accomplished under mild conditions
and have potential applications in developing modified proteins for
therapeutics. The approach was based on the hypothesis that: (a)
one could keep a protein in circulation longer by lowering its affin-
ity towards the receptor thus reducing its internalization rate and;
(b) by attaching a protein to an antibody, the protein stays in cir-
culation longer and is protected from proteolysis. By conjugating
GH to the CovX antibody, we saw an approximately 45-fold drop
4. Blethen, S. L.; Baptista, J.; Kuntze, J.; Foley, T.; LaFranchi, S.; Johanson, A. J. Clin.
Endocrinol. Metab. 1997, 82, 418.
5. Nemirovskiy, O.; Zheng, Yi-J.; Tung, D.; Korniski, B.; Settle, S.; Skepner, A.;
Yates, M.; Aggarwal, P.; Sunyer, T.; Aguiar, D. J. Xenobiotica 2010, 40, 586.
6. Touraine, P.; D’Souza, G. A.; Kourides, I.; Abs, R.; Barclay, P.; Xie, R.; Pico, A.;
Torres-Vela, E.; Ekman, B. Eur. J. Endocrinol. 2009, 161, 533.
7. Doppalapudi, V. R.; Tryder, N.; Li, L.; Aja, T.; Griffith, D.; Liao, F. F.; Roxas, G.;
Ramprasad, M. P.; Bradshaw, C.; Barbas, C. F. Bioorg. Med. Chem. Lett. 2007, 17,
501.
8. Bower, K. E.; Lam, S. N.; Oates, B. D.; del Rosario, J. R.; Corner, E.; Osothprarop, T.
F.; Kinhikar, A. G.; Hoye, J. A.; Preston, R. R.; Murphy, R. E.; Campbell, L. A.;
Huang, H.; Jimenez, J.; Cao, X.; Chen, G.; Ainekulu, Z. W.; Datt, A. B.; Levin, N. J.;
Doppalapudi, V. R.; Pirie-Shepherd, S. R.; Bradshaw, C.; Woodnutt, G.; Lappe, R.
W. J. Med. Chem. 2011, 54, 1256.
9. Doppalapudi, V. R.; Huang, J.; Liu, D.; Jin, P.; Liu, B.; Li, L.; Desharnais, J.; Hagen,
C.; Levin, N. J.; Shields, M. J.; Parish, M.; Murphy, R. E.; Del Rosario, J.; Oates, B.
D.; Lai, J.; Matin, M. J.; Ainekulu, Z.; Bhat, A.; Bradshaw, C. W.; Woodnutt, G.;
Lerner, R. A.; Lappe, R. W. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 22611.
10. Del Rosario, J.; Palanki, M. S. S.; Ainekulu, Z. Bacica, M.; Campbell, L.; Dettling,
D.; Gropp, K.; Horn, M.; Sievers, A.; Stankovic, N.; Wilkie, D.; Bhat, A.; Lappe, R.;
Woodnutt, G.; Chen, G. Endo 2012, Houston, TX, June 23–26, 2012, Poster No.
MON-684.
11. Ilondo, M. M.; Damholt, A. B.; Cunningham, B. A.; Wells, J. A.; De Meyts, P.;
Shymko, R. M. Endocrinology 1994, 134, 2397.
12. Pearce, K. H., Jr.; Cunningham, B. C.; Fuh, G.; Teeri, T.; Wells, J. A. Biochemistry
1999, 38, 81.
13. Cunningham, B. C.; Ultsch, M.; de Vos, A. M.; Mulkerrin, M. G.; Clauser, K. R.;
Wells, J. A. Science 1991, 254, 821.
14. Choa, C.; Danielb, T.; Buechlera, Y. J.; Litzingerc, D. C.; Maioa, Z.; Putnama, A. H.;
Kraynova, V. S.; Sim, B.; Bussella, S.; Javahishvilia, T.; Kaphlea, S.; Viramontesa,
G.; Onga, M.; Chua, S.; GCa, B.; Lieud, R.; Knudsena, N.; Castiglionib, P.;
Normana, T. C.; Axelroda, D. W.; Hoffmane, A. R.; Schultzf, P. G.; Dimarchi, R.
D.; Kimmel, B. E. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 9060.
15. Pearce, K. H., Jr.; Cunningham, B. C.; Fuh, G.; Teeri, T.; Wells, J. A. Biochemistry
1999, 38, 81.
16. Chantalat, L.; Jones, N. D.; Korber, F.; Navaza, J.; Pavlovsky, A. G. Protein Pept.
Lett. 1995, 2, 333.
17. De Vos, A. M.; Ultsch, M.; Kossiakoff, A. A. Science 1992, 255, 306.
18. Solulink Technology, http://store.solulink.com.
Table 1
In vitro efficacy of human growth hormone and its analogs were evaluated
in MTS proliferation bioassay (Cell titer-glo kit from Promega) using
prolactin-dependent Nb2 Tumor Cell Lines (Sigma–Aldrich)
Compound
NB2-11 cell assay, EC50, pM
Wt-GH
11
12
25
882
899
19. (a) Tedaldi, L. M.; Smith, M. E. B.; Nathani, R. I.; Baker, J. R. Chem. Commun.
2009, 6583; (b) Smith, M. E. B.; Schumacher, F. F.; Ryan, C. P.; Tedaldi, L. M.;
Papaioannou, D.; Waksman, G.; Caddick, S.; Baker, J. R. J. Am. Chem. Soc. 2010,

406
M. S. S. Palanki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 402–406
132, 1960; (c) Jones, M. W.; Strickland, R. A.; Schumacher, F. F.; Caddick, S.;
Baker, J. R.; Gibson, M. I.; Haddleton, D. M. J. Am. Chem. Soc. 2012, 134, 1847.
20. Martin, A. C. Proteins 1996, 25, 130.
21. Bradshaw, C.; Sakamuri, S.; Fu, Y.; Oates, B.; Desharnais, J.; Tumelty, D. WO
2008081418, 2008; Chem. Abstr. 2008, 149, 168435.
25. Bolanos, B.; Preston, R.; Palanki, M. MALDI-TOF In-Source Decay (ISD) for
Characterization of Biotherapeutic Protein Conjugates. 60th American Society for
Mass Spectrometry Conference, May 20–24, 2012, Vancouver, BC, Canada.
26. Ishikawa, M.; Nimura, A.; Horikawa, R.; Katsumata, N.; Arisaka, O.; Wada, M.;
Honjo, M.; Tanaka, T. J. Clin. Endocrinol. Metab. 2000, 85, 4274.
22. Palanki, M. S. S.; Bhat, A.; Lappe, R. W.; Liu, B.; Oates, B.; Rizzo, J.; Stankovic, N.;
Bradshaw, C. Bioorg. Med. Chem. Lett. 2012, 22, 4249.
23. Maitra, U.; Balasubramanian, S. J. Chem Soc., Perkin Trans. 1 1995, 83.
24. Schumacher, F. F.; Nobles, M.; Ryan, C. P.; Smith, M. E. B.; Tinker, A.; Caddick, S.;
Baker, J. R. Bioconjug. Chem. 2011, 22, 132.
27. Complete details on assay conditions and results will be published separately.
28. Nemirovskiy, O.; Zheng, Yi-J.; Tung, D.; Korniski, B.; Settle, S.; Skepner, A.;
Yates, M.; Aggarwal, P.; Sunyer, T.; Aguiar, D. J. Xenobiotica 2010, 40, 586.
29. Osborn, B. L.; Sekut, L.; Corcoran, M.; Poortman, C.; Sturm, B.; Chen, G.; Mather,
D.; Lin, H. L.; Parry, T. J. Eur. J. Pharmacol. 2002, 456, 149.