L. R. Roberts et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4173–4178
4177
Table 2
In summary, a series of programmed CovX-Bodies have been
made derivatizing a small kappa opioid peptide by attaching link-
ers to the C-terminus of various lengths and conformational rigid-
ity. The most interesting of which from a potency perspective was
compound 20. Evidence from compound 13 that the terminal half-
life is greatly extended over the small peptide further shows the
utility of combining small peptides with an antibody.
Rat pharmacokinetics of 13 dosed at 5 and 0.3 mg/kg iv
5 mg/kg
0.3 mg/kg
AUC (h ng/mL)
Alpha T1/2 (h)
Beta T1/2 (h)
Cmax (ng/mL)
74,95,027
8.6
142
56,016
5.7
118
183,204
2598
References and notes
1. Kivell, B.; Prisinzano, T. E. Psychopharmacology 2010, 210, 109.
2. Riviere, P. J.-M. Br. J. Pharmacol. 2004, 141, 1331.
3. Bhat, A.; Laurent, O.; Lappe, R. In Fusion protein technologies for
biopharmaceuticals: Applications and Challenges Chapter 10: CovX Bodies, S.R.
Schmidt (Ed.); Wiley-Blackwell: Hoboken, NJ, USA; in press.
4. Huang, H.-H.; Lai, J.-Y.; Do, J.; Liu, D.-G.; Li, L.-N.; Del Rosario, J.; Doppalapudi, V.
R.; Pirie-Shepherd, S.; Levin, N.; Bradshaw, C.; Woodnutt, G.; Lappe, R.; Bhat, A.
Clin. Cancer Res. 2011, 17, 1001.
5. 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, R.
D.; Lai, J.-Y.; 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.
6. Dooley, C. T.; Ny, P.; Bidlack, J. M.; Houghten, R. A. J. Biol. Chem. 1998, 273,
18848.
7. Arendt-Nielsen, L.; Olesen, A. E.; Staahl, C.; Menzaghi, F.; Kell, S.; Wong, G. Y.;
Drewes, A. M. Anesthesiology 2009, 111, 616.
8. For synthesis of AZD linkers 4 and 5 see Bradshaw, C.; Sakamuri, S.; Fu, Y.;
Oates, B.; Desharnais, J.; Tumelty, D. PCT Int. Appl. WO2008081418.
9. Peptide pharmacophores with AZD linkers were mixed at a 3:1 molar ratio
with CVX-2000, a humanized IgG1 monoclonal aldolase antibody, at 20 mg/mL
in a buffer of 10 mmol/L histidine, 10 mmol/L glycine, 2% sucrose, pH 6.5. After
incubating at room temperature overnight, free peptide was removed by size
exclusion chromatography, using a Superdex 200 10/300 GL column on an
AKTAPurifier (GE healthcare). The buffer was 0.5 mol/L sodium chloride,
0.1 mol/L sodium phosphate, pH 6.5. CovX-Bodies were exchanged into PBS
using centrifugal filters and their valency was confirmed by LC–MS. See Backes,
B. J.; Virgilio, A. A.; Ellman, J. A. J. Am. Chem. Soc. 1996, 118, 3055.
10. Backes, B. J.; Ellman, J. A. J. Org. Chem. 1999, 64, 2322.
Figure 3. Graphpad plot of 13 at 5 and 0.3 mg/kg iv.
in antibody engineering as a flexible hinge region. We avoided
attaching a small PEG group directly onto our C-terminus as dialk-
oxy ethylene groups energetically prefer a gauche conformation
and we did not want steric clashes of the PEG chain against the side
of our receptor before exiting.16 However, we did add an optional
small PEG group between the maleimide ring and the AZD as an
additional spacer.
We observed a significant loss in potency between the non-con-
jugated payload and the corresponding CovX-Bodies in the thiol-
maleimide series, for example 6 versus 7. Another observation
looking across the thiol-maleimide series of compounds is that
before bioconjugation, there is only a relatively small difference
in potency depending on linker length, that is 6 versus 12. How-
ever, after conjugation there is less of a drop-off in potency with
the longer linker lengths, that is 13 is more potent than 7. It is
likely that with a larger tether the pharmacophore is further away
from the CovX body and able to interact more efficiently with the
Kappa receptor. The discrepancy in potency between the payload
and CovX-Body was significantly reduced when a directing linker
such as a piperidine was used at the C-terminus of the peptide
when comparing examples 17 versus 18 and 19 versus 20. At least
in these examples, it looks like a rigid directional linker helped im-
prove the potency of the compounds.
One of the key reasons for conjugating these peptides to a CovX
body is to improve the overall terminal half-life of the peptide. The
pharmacokinetics of 13 was determined in rat at two doses (Table
2 and Fig. 3).17 The terminal half-life at 5 and 0.3 mg/kg was 142
and 118 h, respectively which is significantly longer than the small
peptide. This has to be put into context, however, in that the anal-
ysis and detection of the PK samples only looks at the CVX-2000
antibody not the combined CovX body which also includes the lin-
ker and pharmacophore. To compensate for this, a FACS pharmaco-
kinetic binding assay was also performed this time in mouse, on
the same compound. However, the sensitivity of this assay is less
than the immunoassay, so extrapolation for determination of a ter-
minal half-life was not possible.18 We can say that the distribution
profile obtained from the FACS experiment mirrors that observed
in the rat pharmacokinetic experiment over the first three hours,
indicating that at least for these points, the molecule is sufficiently
stable.
11. Synthesis of 16
Pd/C
H2, HCl
O
O
O
O
N
N
H2N
O2N
22
95%
21
+
HBTU
DIEA
DMF
O
O
O
OH
O
N
N3
N
H
16
N3
68%
To a mixture of 218 (0.01 mol, 2.5 g) in methanol (0.1 M, 100 ml) was added
concentrated hydrochloric acid (12 M, 1.3 mL) followed by addition of
palladium on carbon (50% water, 10% w/w, 880 mg). The stirred mixture was
purged with nitrogen then hydrogenated (1 atm) overnight. The mixture was
filtered through Celite™ and washed with methanol (100 mL), filtrates
combined and concentrated to dryness. The crude material was dissolved in
methanol (30 mL), followed by drop-wise addition of ethyl acetate (270 mL),
resulting in product precipitation. After 3 h, the product was isolated by
filtration and dried under high-vacuum over night to give 228 (2.4 g, 95%). A
solution of 22 (100 mg, 0.39 mmol), 4-azidobenzoic acid (63 mg, 0.39 mmol),
HBTU (140 mg, 0.39 mmol) and N,N-dimethylformamide (0.2 M, 1.95 mL) was
treated with DIEA (1.56 mmol, 0.27 mL) and the mixture stirred at room
temperature for 4 h. Reaction mixture was extracted into ethyl acetate (25 mL)
and washed with water/brine mixture (1:1, v/v, 25 mL). Extraction repeated
two more times, organic layers combined and concentrated to dryness. Crude
product material was then re-suspended in ethyl acetate/hexanes mixture
(1:1, v/v, 5 mL) and stirred for 2 h. Product was then filtered off, cake washed
with hexanes (3 mL) and dried under high-vacuum over night to give 16
(97 mg, 68% yield). 1H NMR (400 MHz, acetone-d6) d 8.10 (2H, d), 7.50 (2H, d),
7.25–7.15 (4H, m), 3.60–3.50 (2H, m), 3.10–3.05 (2H, m) and 2.95–2.90 (4H, m)
ESCI-MS: m/z: [M+H]+ 364 (100%).
12. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem., Int. Ed.
2002, 41, 2596.
13. KOR b-arrestin recruitment assay methods: U2OS cells recombinantly expressing
the
j-opioid receptor were linked to the DiscoveRx ProLink tag and the b-
arrestin/EA complex. They were maintained in modified Eagle’s medium
(MEM) supplemented with 10% (v/v) heat inactivated foetal calf serum (HI-
FCS), 2 mM L-Glutamine, 20 lg/mL geneticin and 10 lg/mL Hygromycin, in