The chemical and topological diversity of peptides offer tremendous possibilities to identify new diagnostic imaging compounds. Peptides have been widely used to target an imaging probe to a specific protein or receptor and thereby provide greater specificity. Typically an imaging reporting moiety (e.g. positron emitter, gamma emitter, paramagnetic ion, near infrared fluorophore) is conjugated to the peptide. The site of conjugation, the linker, and the type of imaging reporter all play a role in determining biological activity and pharmacokinetics.1, 2
For peptide-based magnetic resonance imaging (MRI) contrast agents, an additional factor is detection sensitivity of the imaging probe.3
Multiple copies of the MR active reporter, typically a gadolinium complex, are required to provide robust image contrast.
An additional major challenge to creating new drugs from peptides is peptide degradation by endogenous peptidases. There are numerous medicinal chemistry approaches to improve peptide stability, biological activity, and/or bioavailability that increase in complexity from modified peptides to pseudopeptides to small molecule peptidomimetics.4, 5
In this report, we explore the potential of using the imaging reporter to block peptidase activity.
have been interested in developing gadolinium-based peptide-targeted MR imaging agents. Compared to other modalities, MRI provides a favorable combination of high spatial resolution, depth penetration, and lack of ionizing radiation. Unlike nanoparticles, these relatively small molecules can rapidly reach targets in extravascular spaces and can be readily excreted through the kidneys to reduce/avoid long-term gadolinium retention and toxicity. On the other hand, extravasation into the kidneys and liver exposes these compounds to a range of peptidases.
There is some flexibility as to where and how the gadolinium chelates are conjugated to the peptide. Conjugation is possible at the N- or C-terminus and/or within the peptide structure.6
We recently reported some fibrin-specific peptides conjugated with one or four [Gd(DTPA)]2−
The construct with highest affinity had two peptides linked via their N-terminus to a GdDTPA tetramer, i.e. PepN
Pep, termed EP-1084 (Cmpd 1
in ). 1
showed good fibrin affinity and specificity, high relaxivity, and positive uptake in an in vivo venous thrombus model. However, subsequent in vivo studies revealed that this compound was being metabolized over the course of the study, and that the Gd-containing metabolites no longer bound fibrin. This communication describes the effort to improve the metabolic stability of this class of compounds while maintaining their biological and relaxometric activity. We demonstrate that the Gd-chelate moieties can be used to block peptidase activity in addition to their role in MRI signal generation. Parts of this work have been communicated previously in conference abstracts.15, 16
shows the compounds described in this communication which used 3 similar peptides that differ only at the C-terminus. The L-Asp to D-Asp substitution did not affect fibrin affinity, but D-amino acids are known to sometimes provide metabolic stability.4
The Leu to biphenylalanine (Bip) substitution resulted in higher fibrin affinity8
and it was hoped that the unnatural amino acid would improve stability. Peptides are linked to the Gd-chelates via an oxime or amide bond.
A typical in vitro assay for metabolism is to incubate the compound with tissue homogenate or liver microsomes. Rat liver homogenate consists of a mixture of proteases and other enzymes and represents a harsh challenge for compound stability. The half-life of 1 in liver homogenate at 37 °C was 10.8 min, which was consistent with observed instability in vivo. The peptides alone, with or without the Bip or D-Asp substitutions, were completely metabolized within the time taken for measurement, t1/2 < 2 min. The bulky hydrophilic GdDTPA tetramer at the N-terminus appeared to block metabolism. This was supported by studies with the single peptide analog 2 (Gd4-NPep). The half-life for this compound was similar to 1 (10.0 min, ) suggesting that blockade of the C-terminus might be required for enhanced stability. Compound 3 (Bpc-NPep-Gd4) was synthesized with the Gd tetramer conjugated directly to the C-terminus and the N-terminus capped with biphenyl carboxamide (Bpc), a group used to block exopeptidase activity. However this compound also had a similar half-life in rat liver homogenate (9 min). With compound 4, we took this strategy further and capped the C- and N-termini with a GdDTPA dimeric unit (Gd2A-NPep-Gd2A) resulting in a 6 to 7-fold increase in metabolic stability. This suggests that while the D-amino acid and unnatural Bip in 2 and the Bpc in 3 may increase stability, these substitutions are not as effective as using the metal chelate with this specific peptide.
Effect of peptide-chelate architecture on metabolic stability, as assessed by half-life (t1/2) in rat liver homogenate, and affinity to DD(E) (soluble fibrin fragment).
While metabolic stability was improved with compound 4
, unfortunately its fibrin affinity was significantly reduced. also shows inhibition constants, Ki
, for each compound to inhibit the binding of a fluorescent peptide to the soluble fibrin fragment DD(E). A lower Ki
value indicates higher fibrin affinity. The Ki
values for 1
are comparable to the binding dissociation constants to insoluble fibrin reported previously (for 1
(DD(E)) = 1.1 μM, Kd
(fibrin) = 1.9 μM). To improve the fibrin affinity of metabolically stable 4, we made the Leu to Bip substitution used in 1
to give compound 5
(Gd2B-Pep-Gd2B), which resulted in a 2–3 fold increase in fibrin affinity, while maintaining the metabolic stability. The other modifications from 4
were practical: the m
-bis-(aminomethyl)-benzene linker is much less expensive than the para
analog; the bis(GdDTPA) moiety Gd2B prepared from diethylene triamine gave better yields than Gd2A prepared from diaminopropionic acid. Although the affinity of 5
was still less than 1
, it was a simpler and more cost-effective molecule that only utilized one peptide per compound. Compound 5
(aka EP-1242) subsequently showed efficacy in a guinea pig model of arterial thrombosis.17
Based on its favorable fibrin affinity and metatabolic stability properties, we sought to further characterize the MR properties of 5. Nuclear magnetic relaxation dispersion (NMRD) profiles of 5 in either pH 7.4 Tris buffered saline (TBS), 30 μM human fibrinogen/TBS solution, human plasma, or 30 μM gelled human fibrin in TBS are shown in . The large enhancement in relaxivity going from TBS to fibrin and the peak in relaxivity at ca. 30 MHz are consistent with binding to fibrin and a reduction in the rotational correlation time (τR). There was little relaxivity enhancement in fibrinogen solution suggesting 5 does not bind/binds weakly to fibrinogen. Some enhanced relaxivity in plasma suggests some weak binding to plasma proteins. Relaxivity in is plotted on a per molecule basis to demonstrate the increased detection sensitivity of 5 compared to commercial [Gd(DTPA)]2−.
Figure 1 NMRD showing field- and medium-dependent relaxivity of 5 at 35 °C in TBS buffer (open triangles), 30 μM human fibringogen/TBS (open circles), human plasma (filled triangles), and 30 μM fibrin gel (filled circles). NMRD of [Gd(DTPA)] (more ...)
shows the dual effect of fibrin targeting and relaxation enhancement on thrombus imaging. Two 500 μL solutions were prepared, each containing 30 μM compound 5, in fibrinogen enriched (10 mg/mL) human plasma. One sample was clotted by addition of 4 μL of a 2 M CaCl2 solution and 2 μL of human thrombin (0.6 units). After clotting, the clot was allowed to retract by gentle agitation with an Eppendorf pipet tip. Relative to 5 in plasma (), the signal intensity in the clot () is higher because of increased concentration of 5 due to binding and also because of higher relaxivity. The concentration of 5 in the serum is depleted resulting in lower signal intensity.
T1-weighted MR images of 5 in unclotted (A) and clotted (B) human plasma from a 1.5T clinical scanner (General Electric) with a 6 cm surface coil, using a spoiled gradient echo sequence (TE/TR/α: 3/39/40°)
Additional NMRD at 5, 15, 25, and 35 °C for 5
in TBS or in fibrin gel, and variable temperature O-17 solvent relaxation studies (T1
) for 5
in TBS were performed to better understand the underlying dynamics influencing relaxation. shows the results of these studies with solid lines as fits to the data. The high field NMRD and O-17 data were analyzed as described previously18
using the usual Solomon Bloembergen Morgan equations with two-site exchange.19
The data were well described with an isotropic model of rotation, and τR
of this peptide-gadolinium tetramer is substantially increased compared to [Gd(DTPA)]2−
(390 ps vs 44 ps20
at 37 °C) accounting for the increased relaxivity of 5
compared to [Gd(DTPA)]2−
. Not surprisingly, the water exchange rate and parameters for electronic relaxation at the backbone modified Gd(DTPA)(H2
O) moiety in 5
are very similar to those of [Gd(DTPA)]2−
and related derivatives.18, 20
Figure 3 Relaxometric analysis of 5: A) VT O-17 NMR (1/T1 open squares, 1/T2 filled squares) at 7.05T in TBS. VT NMRD in TBS (B) and fibrin gel (C) at 5° (open circles), 15° (filled circles), 25° (open triangles), and 35°C (filled (more ...)
The lack of temperature dependence on the relaxivity of fibrin-bound 5
implies that there is substantial internal motion limiting relaxivity. If fibrin binding caused true immobilization, then relaxivity should decrease with decreasing temperature because relaxation would be limited by slow water exchange. The NMRD data of the fibrin-bound 5
was modeled with the Lipari-Szabo formalism where two correlation times describe rotational diffusion: a slow, global correlation time (τR
) for the protein-bound compound and a shorter local correlation time for (τl
) for internal motion.18, 21
These are weighted by an order parameter, 1 ≥ F2
≥ 0 where F2
=1 represents isotropic global motion and F2
=0 represents local motion decoupled from the slow global motion. The NMRD data was well described by adjusting the rotational parameters without changing the electronic relaxation and water exchange parameters from those determined in TBS. Best fits were obtained when the global τR
was very long, >20 ns. However F2
was quite small (0.08) indicating that relaxivity of fibrin-bound 5
was limited by internal. As temperature is decreased, internal motion is reduced but this positive effect on relaxivity is offset by a decrease in water exchange rate. The overall effect is a near temperature independence on relaxivity.
The NMRD analysis indicates that relaxivity at 37 °C is not likely to be improved by changing the Gd chelate to one with faster water exchange kinetics since relaxivity at 37 °C is limited by fast internal motion and not water exchange. However reducing internal motion should have a major impact on increasing relaxivity at 1.5T (the field at which the vast majority of clinical MRI scanners operate). Nonetheless, the relaxivity observed for 5 is still quite high: 18 mM−1s−1 per Gd and 72 per molecule bound to fibrin at 1.5T. This is 4–5 fold higher than GdDTPA on a per Gd basis and 18 fold higher per molecule, providing the sensitivity to detect thrombi in vivo.
A similar Gd2-Pep-Gd2 design was used in EP-2104R,9, 22
which has been used to detect thrombus by MRI in animal models and in human clinical trials. 22
EP-2104R uses a different peptide than the compounds in this report, but its metabolic stability may arise in part because of blocking both the C- and N- termini with Gd chelates.
In summary, the metabolic stability of a fibrin-targeted peptide is greatly increased when the Gd chelates are positioned at both the C- and N-termini to block exopeptidase activity. Using multiple Gd chelates also results in increased molecular relaxivity, although NMRD analysis suggests that relaxivity can be even further increased by reducing internal motion. Using more than one site of chelate conjugation may represent a general strategy to increase metabolic stability of peptide-targeted imaging agents.