Autio et al. EJNMMI Research 2011, 1:10
`http://www.ejnmmires.com/content/1/1/10
`
`O RIG IN AL R ES EA RC H
`Mini-PEG spacering of VAP-1-targeting
`68Ga-DOTAVAP-P1 peptide improves PET imaging
`of inflammation
`Anu Autio1, Tiina Henttinen2, Henri J Sipilä1, Sirpa Jalkanen3 and Anne Roivainen1,4*
`
`Open Access
`
`Abstract
`
`Background: Vascular adhesion protein-1 (VAP-1) is an adhesion molecule that plays a key role in recruiting
`leucocytes into sites of inflammation. We have previously shown that 68Gallium-labelled VAP-1-targeting peptide
`(68Ga-DOTAVAP-P1) is a positron emission tomography (PET) imaging agent, capable of visualising inflammation in
`rats, but disadvantaged by its short metabolic half-life and rapid clearance. We hypothesised that prolonging the
`metabolic half-life of 68Ga-DOTAVAP-P1 could further improve its imaging characteristics. In this study, we
`evaluated a new analogue of 68Ga-DOTAVAP-P1 modified with a mini-polyethylene glycol (PEG) spacer (68Ga-
`DOTAVAP-PEG-P1) for in vivo imaging of inflammation.
`Methods: Whole-body distribution kinetics and visualisation of inflammation in a rat model by the peptides 68Ga-
`DOTAVAP-P1 and 68Ga-DOTAVAP-PEG-P1 were evaluated in vivo by dynamic PET imaging and ex vivo by measuring
`the radioactivity of excised tissues. In addition, plasma samples were analysed by radio-HPLC for the in vivo stability
`of the peptides.
`Results: The peptide with the mini-PEG spacer showed slower renal excretion but similar liver uptake as the
`original peptide. At 60 min after injection, the standardised uptake value of the inflammation site was 0.33 ± 0.07
`for 68Ga-DOTAVAP-P1 and 0.53 ± 0.01 for 68Ga-DOTAVAP-PEG-P1 by PET. In addition, inflammation-to-muscle ratios
`were 6.7 ± 1.3 and 7.3 ± 2.1 for 68Ga-DOTAVAP-P1 and 68Ga-DOTAVAP-PEG-P1, respectively. The proportion of
`unchanged peptide in circulation at 60 min after injection was significantly higher for 68Ga-DOTAVAP-PEG-P1 (76%)
`than for 68Ga-DOTAVAP-P1 (19%).
`Conclusion: The eight-carbon mini-PEG spacer prolonged the metabolic half-life of the 68Ga-DOTAVAP-P1 peptide,
`leading to higher target-to-background ratios and improved in vivo PET imaging of inflammation.
`Keywords: gallium-68, inflammation imaging, mini-PEG spacer, positron emission tomography, vascular adhesion
`protein-1
`
`Background
`In vivo imaging of inflammation is a demanding task,
`and novel molecular imaging targets are called for. The
`gold standard in nuclear medicine is the radiolabelling
`of white blood cells, which is both time consuming and
`potentially hazardous for the technical personnel.
`Vascular adhesion protein-1 (VAP-1) is an inflamma-
`tion-inducible endothelial adhesion protein involved in
`
`* Correspondence: anne.roivainen@utu.fi
`1Turku PET Centre, University of Turku and Turku University Hospital, Turku,
`Finland
`Full list of author information is available at the end of the article
`
`the leucocyte trafficking from the blood stream into the
`tissues. VAP-1 is stored in intracellular granules within
`endothelial cells. However, upon inflammation, it is
`rapidly translocated to the endothelial cell surface, for
`example, in the synovial tissue in rheumatoid arthritis
`and at the site of ischemic reperfusion injury [1,2].
`Therefore, VAP-1 is both an optimal candidate for anti-
`inflammatory therapy and a potential target for in vivo
`imaging of inflammation. This approach may open new
`opportunities for diagnosing, therapy planning and mon-
`itoring of the treatment efficacy, as well as for the drug
`discovery and development processes [3-6].
`
`© 2011 Autio et al; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution
`License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium,
`provided the original work is properly cited.
`
`MPI EXHIBIT 1095 PAGE 1
`
`MPI EXHIBIT 1095 PAGE 1
`
`

`

`Autio et al. EJNMMI Research 2011, 1:10
`http://www.ejnmmires.com/content/1/1/10
`
`Page 2 of 7
`
`Peptide-based imaging agents are small molecules that
`possess favourable properties such as rapid diffusion in
`target tissue, rapid clearance from the blood circulation
`and non-target tissues, easy and low-cost synthesis, and
`low toxicity and immunogenicity. We are particularly
`interested in developing radiolabelled peptides for VAP-
`1 targeting for the purposes of in vivo imaging of leuco-
`cyte trafficking. The linear peptide, VAP-P1, has been
`characterised by Yegutkin et al. and proven to bind the
`enzymatic groove of VAP-1 and dose-dependently inhi-
`bit VAP-1-dependent lymphocyte rolling and firm adhe-
`sion to primary endothelial cells [7]. We have previously
`shown that 68Ga-labelled DOTA-conjugated VAP-P1
`peptide (68Ga-DOTAVAP-P1) is able to delineate
`inflammation in rats by a VAP-1-specific way using
`positron emission tomography (PET) [8-10]. Disadvanta-
`geously, the 68Ga-DOTAVAP-P1 peptide has relatively
`short plasma half-life and very rapid clearance by the
`kidneys to the urine.
`PEGylation, the process by which polyethylene glycol
`(PEG) chains or its derivatives, e.g., mini-PEGs are
`attached to a peptide, has been used for modifying the
`properties of radiolabelled compounds, such as antibo-
`dies and peptides, in order to improve their imaging
`characteristics. The goal of PEGylation is mainly to
`improve the tracer’s kinetics and distribution pattern by
`increasing its metabolic half-life and by lowering its
`non-specific binding. By increasing the molecular mass
`of the peptide and by shielding it from proteolytic
`enzymes, PEGylation may modify its biodistribution and
`pharmacokinetics [11]. Thus, the method could over-
`come the above mentioned shortcomings. However,
`because PEGylation may also have unfavourable effects,
`such as inhibition of receptor binding and reduction of
`target-to-background ratio, its impact must be carefully
`evaluated for a new peptide.
`We hypothesised that prolonging the metabolic half-
`life of 68Ga-DOTAVAP-P1 would further improve its
`potential for in vivo imaging of inflammation. In this
`study, we evaluated a new mini-PEG spacered analogue
`of 68Ga-DOTAVAP-P1 (68Ga-DOTAVAP-PEG-P1) for
`in vivo PET imaging of inflammation.
`
`Methods
`68Ga-DOTA-peptides
`The DOTA-conjugated peptides were purchased from
`Almac Sciences (By Gladsmuir, Scotland, UK), ABX
`advanced biochemical compounds GmbH (Radeberg,
`Germany) and NeoMPS (Strasbourg, France).
`Linear 9-amino acid DOTA-chelated peptide
`(GGGGKGGGG) with and without a PEG linker (8-
`amino-3,6-diooxaoctanoyl, PEG derivative, MW 145.16
`Da) between the DOTA and the N terminal amino acid
`was labelled with 68Ga as previously described [8], and
`
`named as 68Ga-DOTAVAP-P1 and 68Ga-DOTAVAP-
`PEG-P1. Briefly, 68Ga was obtained in the form of
`68GaCl3 from a 68Ge/68Ga generator (Cyclotron Co.,
`Obninsk, Russia) by elution with 0.1 M HCl. The
`68GaCl3 eluate (500 μl) was mixed with sodium acetate
`(18 mg; Sigma-Aldrich, Seelze, Germany) to give a pH
`of approximately 5.5. Then, DOTA-peptide (35 nmol)
`was added and the mixture was incubated at 100°C for
`20 min. No further purification was needed.
`The radiochemical purity was determined by reversed-
`phase HPLC (μBondapak C18, 7.8 × 300 mm2, 125 Å,
`10 μm; Waters Corporation, Milford, MA, USA). The
`HPLC conditions for 68Ga-DOTAVAP-P1 have been
`described previously [9]. The HPLC conditions for 68Ga-
`DOTAVAP-PEG-P1 were slightly different and as fol-
`lows: flow rate = 4 ml/min, l = 215 nm, A = 2.5 mM
`trifluoroacetic acid, B = acetonitrile and C = 50 mM
`phosphoric acid. Linear A/B/C gradient was 100/0/0 for
`0 to 3 min, 40/60/0 for 3 to 9 min, and 0/0/100 for 9 to
`16 min. The radio-HPLC system consisted of LaChrom
`instruments (Hitachi; Merck, Darmstadt, Germany):
`pump L7100, UV detector L-7400 and interface D-7000;
`an on-line radioisotope detector (Radiomatic 150 TR,
`Packard, Meriden, CT, USA); and a computerised data
`acquisition system.
`
`In vitro stability and solubility
`The in vitro stability of the 68Ga-labelled peptides was
`evaluated in human and rat plasma. Several samples
`were taken during the 4-h incubation period at 37°C.
`Proteins from plasma samples were precipitated with
`10% sulphosalicylic acid (1:1 v/v), centrifuged at 3,900 ×
`g for 3 min at 4°C, and filtered through 0.45-μm Minis-
`pike filter (Waters Corporation). The filtrate was ana-
`lysed by radio-HPLC.
`The octanol-water distribution coefficient, logD, of
`the 68Ga-DOTA-peptides was determined using the
`following procedure. Approximately 5 kBq of 68Ga-
`labelled peptide in 500 μl of phosphate-buffered saline
`(PBS, pH 7.4) was added to 500 μl of 1-octanol. After
`the mixture had been vortexed for 3 min, it was centri-
`fuged at 12,000 × g for 6 min, and 100-μl aliquots of
`both layers were counted in a gamma counter (1480
`Wizard 3″ Gamma Counter; EG&G Wallac, Turku,
`Finland). The test was repeated three times. The logD
`was calculated as = log10 (counts in octanol/counts in
`PBS).
`
`Animals
`All animal experiments were approved by the Lab-Ani-
`mal Care & Use Committee of the State Provincial
`Office of Southern Finland and carried out in compli-
`ance with the Finnish laws relating to the conduct of
`animal experimentation.
`
`MPI EXHIBIT 1095 PAGE 2
`
`MPI EXHIBIT 1095 PAGE 2
`
`

`

`Autio et al. EJNMMI Research 2011, 1:10
`http://www.ejnmmires.com/content/1/1/10
`
`Page 3 of 7
`
`Male Sprague-Dawley rats (n = 14) were purchased
`from Harlan, Horst, The Netherlands. Twenty-four
`hours before the PET studies, turpentine oil (Sigma-
`Aldrich; 0.05 ml per rat) was injected subcutaneously
`into their neck area in order to induce a sterile inflam-
`mation [10]. Six rats were PET imaged and additional
`eight animals were used for in vivo metabolite analyses.
`
`PET imaging and ex vivo biodistribution
`The whole-body distribution and kinetics of 68Ga-
`DOTAVAP-P1 (n = 3) and 68Ga-DOTAVAP-PEG-P1 (n
`= 3) in rats harbouring a sterile inflammation were stu-
`died with a high-resolution research tomograph (Sie-
`mens Medical Solutions, Knoxville, TN, USA). The rats
`were anaesthetised with isoflurane (induction 3%, main-
`tenance 2.2%). Two rats were imaged at the same time,
`and they were kept on a warm pallet during the imaging
`procedure. Following a 6-min transmission for attenua-
`tion correction, the rats were intravenously (i.v.) injected
`with 68Ga-DOTAVAP-P1 (15.8 ± 3.0 MBq, 19.4 ± 0.0
`μg, 19.6 ± 0.0 nmol) or with 68Ga-DOTAVAP-PEG-P1
`(17.7 ± 1.6 MBq, 21.0 ± 1.3 μg, 18.5 ± 1.1 nmol) as a
`bolus via a tail vein using a 24-gauge cannula (BD Neo-
`flon, Becton Dickinson Infusion Therapy AB, Helsing-
`borg, Sweden). Dynamic imaging lasting for 60 min
`started at the time of injection. The data acquired in list
`mode were iteratively reconstructed with a 3-D ordered
`subsets expectation maximisation algorithm with 8 itera-
`tions, 16 subsets and a 2-mm full-width at half-maxi-
`mum post-filter into 5 × 60 s and 11 × 300 s frames.
`Quantitative analyses were performed by drawing
`regions of interest (ROI) on the inflammatory foci, mus-
`cle (hind leg), heart, kidney, liver and urinary bladder.
`Time-activity curves (TACs) were extracted from the
`corresponding dynamic images (Vinci software, version
`2.37; Max Planck Institute for Neurological Research,
`Cologne, Germany). The average radioactivity concen-
`trations in the ROIs (kilobecquerels per millilitre) were
`used for further analyses. The uptake was reported as
`standardised uptake value (SUV), which was calculated
`as the radioactivity of the ROI divided by the relative
`injected radioactivity expressed per animal body weight.
`The radioactivity remaining in the tail was compensated.
`After the PET imaging, the animals were sacrificed.
`Samples of blood, urine and various organs were col-
`lected, weighed and measured for radioactivity using the
`gamma counter (Wizard, EG&G Wallac). The results
`were expressed as SUVs.
`
`Blood analyses
`Blood samples (0.2 ml of each) were drawn at 5, 10, 15,
`30, 45, 60 and 120 min after injection of 68Ga-DOTA-
`peptides into heparinised tubes (Microvette 100; Sar-
`stedt, Nümbrecht, Germany). Radioactivity of whole
`
`blood was measured with the gamma counter (Wizard,
`EG&G Wallac). Plasma was separated by centrifugation
`(2,200 × g for 5 min at 4°C), and plasma radioactivity
`was measured. The ratio of radioactivity in blood versus
`plasma was calculated. To determine plasma protein
`binding, proteins were precipitated with 10% sulphosa-
`licylic acid, and the radioactivity in protein precipitate
`and supernatant was measured. The plasma supernatant
`was further analysed by radio-HPLC in order to evaluate
`the in vivo stability of the 68Ga-labelled peptides.
`In vivo stability data were used in order to generate
`metabolite-corrected plasma TACs for 68Ga-DOTA-
`VAP-P1 and 68Ga-DOTAVAP-PEG-P1, which were
`further used for the calculation of pharmacokinetic
`parameters. The area under curve (AUC) of the plasma
`TAC from 0 to infinity was calculated using a non-com-
`partmental analysis employing the trapezoidal rule. The
`clearance (CL) of the 68Ga-labelled peptides after a sin-
`gle intravenous bolus dose was calculated by dividing
`the injected dose by the AUC. The plot of the natural
`logarithm of parent tracer concentration against time
`after bolus injection became linear in the end phase, as
`the tracer was eliminated according to the laws of first-
`order reaction kinetics. The elimination rate constant
`(kel) was calculated as the negative slope of the linear
`part of the plot. The plasma elimination half-life (t1/2)
`was calculated as t1/2 = ln(2)/kel. The metabolic half-
`lives of the 68Ga-DOTA-peptides were calculated
`according to the results of radio-HPLC, i.e. the time
`point when 50% of the total radioactivity is still bound
`to the intact peptide.
`
`Statistical analyses
`All the results are expressed as means ± standard devia-
`tion (SD) and range. The correlations between PET ima-
`ging and ex vivo measurement values were evaluated
`using linear regression analysis. Inter-group comparisons
`were made using an unpaired t test. Statistical analyses
`were conducted using Origin 7.5 software (Microcal,
`Northampton, MA, USA). A P value less than 0.05 was
`considered as statistically significant.
`
`Results
`In vitro studies
`The radiochemical purities of 68Ga-DOTAVAP-P1 and
`68Ga-DOTAVAP-PEG-P1 were 97 ± 1% and 99 ± 1%,
`and specific radioactivities 2.27 ± 0.47 and 2.55 ± 0.45
`MBq/nmol, respectively. The retention times for 68Ga-
`DOTAVAP-P1 and 68Ga-DOTAVAP-PEG-P1 were 6.6
`± 0.1 and 6.7 ± 0.1 min, respectively. The retention time
`for free gallium was approximately 12 min, and it eluted
`only with phosphoric acid. The in vitro stabilities of
`68Ga-DOTAVAP-P1 and 68Ga-DOTAVAP-PEG-P1 were
`very similar. The amounts of unchanged peptide after
`
`MPI EXHIBIT 1095 PAGE 3
`
`MPI EXHIBIT 1095 PAGE 3
`
`

`

`Autio et al. EJNMMI Research 2011, 1:10
`http://www.ejnmmires.com/content/1/1/10
`
`Page 4 of 7
`
`the 4-h incubation in human or rat plasma were 88 ±
`3% and 82 ± 11% for 68Ga-DOTAVAP-P1 and 89 ± 8%
`and 90 ± 6% for 68Ga-DOTAVAP-PEG-P1, respectively.
`Both peptides were highly hydrophilic; logD was -3.30
`for 68Ga-DOTAVAP-P1 and -3.50 for 68Ga-DOTAVAP-
`PEG-P1.
`
`PET studies with rat model of inflammation
`Both peptides were capable of visualising inflammatory
`foci from surrounding tissues by PET imaging (Figure
`1a). The inflammation uptakes expressed as SUVs were
`0.33 ± 0.07 (range, 0.26 to 0.40) and 0.53 ± 0.01 (range,
`0.42 to 0.60) for 68Ga-DOTAVAP-P1 and 68Ga-DOTA-
`VAP-PEG-P1, respectively, at 60 min after injection.
`Inflammation-to-muscle ratios at 60 min after injection
`were 6.7 ± 1.3 (range, 5.2 to 7.5) and 7.3 ± 2.1 (range,
`5.6 to 9.7) for 68Ga-DOTAVAP-P1 and 68Ga-DOTA-
`VAP-PEG-P1, respectively. The kinetics of 68Ga-DOTA-
`VAP-P1 and 68Ga-DOTAVAP-PEG-P1 in inflammatory
`foci were quite fast, and the peak radioactivity was
`reached within 20 min for both peptides. On the aver-
`age, the inflammation uptake of 68Ga-DOTAVAP-PEG-
`P1 was 59% higher than that of 68Ga-DOTAVAP-P1,
`and the difference was statistically significant (P =
`0.047). According to the whole-body dynamic PET ima-
`ging, 68Ga-DOTAVAP-PEG-P1 showed slower renal
`excretion to urine but otherwise rather
`similar
`
`distribution kinetics as the original peptide 68Ga-DOTA-
`VAP-P1 (Figure 1b, c, d, e).
`The PET imaging results were verified by ex vivo mea-
`surements (Figure 2). Linear regression analysis showed
`reasonable correlation between in vivo PET and ex vivo
`tissue samples (R = 0.58, P = 0.023 for 68Ga-DOTA-
`VAP-P1 and R = 0.80, P < 0.001 for 68Ga-DOTAVAP-
`PEG-P1). When the tissue uptakes of 68Ga-DOTAVAP-
`P1 and 68Ga-DOTAVAP-PEG-P1 were compared, the
`inflammation, lung, small intestine, skin and urinary
`bladder radioactivities were significantly different.
`Although the PET and ex vivo methods correlate well,
`there are some discrepancies between the results. For
`example, in the PET image analysis, the urine and blood
`of kidney are included in the “kidney” ROI, whereas for
`ex vivo measurement, the excised tissue samples are
`dotted dry on a paper. Since the radioactivity of urine is
`extremely high, the in vivo kidney SUV is higher than
`that of ex vivo.
`The blood-plasma ratios and the plasma free fractions
`(fp), i.e. the fraction of total radioactivity in plasma that
`is unbound to plasma proteins, were 1.3 ± 0.1 and 0.84
`± 0.04 for 68Ga-DOTAVAP-P1 and 1.3 ± 0.1 and 0.86 ±
`0.02 for 68Ga-DOTAVAP-PEG-P1, respectively. The in
`vivo stability of 68Ga-DOTAVAP-PEG-P1 was better
`than that of 68Ga-DOTAVAP-P1. The proportions of
`unchanged peptides in rat plasma at 60 and 120 min
`
`Figure 1 PET images and time-activity curves. (a) Representative coronal PET images of Sprague-Dawley rats with sterile turpentine oil-
`induced inflammation as a sum image of 10 to 60 min after i.v. injection of 68Ga-DOTAVAP-P1 (13.8 MBq) or 68Ga-DOTAVAP-PEG-P1 (17.5 MBq).
`Time-activity curves of (b) inflammation and muscle, (c) kidney, (d) liver and (e) urinary bladder for 68Ga-DOTAVAP-P1 and 68Ga-DOTAVAP-PEG-
`P1.
`
`MPI EXHIBIT 1095 PAGE 4
`
`MPI EXHIBIT 1095 PAGE 4
`
`

`

`Autio et al. EJNMMI Research 2011, 1:10
`http://www.ejnmmires.com/content/1/1/10
`
`Page 5 of 7
`
`Figure 2 Biodistribution of the 68Ga-DOTA-peptides at 60 min after injection. Results are given as the mean ± SD of three experiments.
`Asterisks indicate statistically significant differences between the peptides. *P < 0.05; **P < 0.01.
`
`after injection were 19 ± 4% and 4 ± 1% for 68Ga-
`DOTAVAP-P1 and 76 ± 18% and 49 ± 6% for 68Ga-
`DOTAVAP-PEG-P1, respectively (Figure 3). The meta-
`bolic half-lives of 68Ga-DOTAVAP-P1 and 68Ga-DOTA-
`VAP-PEG-P1 were 24 and 125 min, respectively. Based
`on in vivo plasma measurements, 68Ga-DOTAVAP-
`PEG-P1 showed significantly slower kel and total CL and
`larger AUC values. In addition, 68Ga-DOTAVAP-PEG-
`P1 had a longer elimination t1/2 than the original 68Ga-
`DOTAVAP-P1, although the difference was not statisti-
`cally significant (Table 1).
`
`Figure 3 In vivo stability of the i.v. administered 68Ga-DOTA-
`peptides in blood circulation of the rat. Results are given as the
`means of three to seven experiments. NS, not significant; **P < 0.01;
`***P < 0.001.
`
`Discussion
`Previously, we have reported the feasibility of the VAP-
`1-targeting peptide, 68Ga-DOTAVAP-P1, for PET ima-
`ging of inflammation in different rat models [8-10].
`However, as a limitation, 68Ga-DOTAVAP-P1 is cleared
`very rapidly from circulation and its in vivo stability
`against degradation by enzymes is only moderate. In
`this study, we showed that the incorporation of a mini-
`PEG spacer in 68Ga-DOTAVAP-P1 enhanced its in vivo
`stability
`and
`improved
`the PET imaging
`of
`inflammation.
`The animal model used in our experiments involves
`turpentine oil injection-induced subcutaneous inflamma-
`tion as described previously [10]. In that study, we were
`able to show that the H & E staining of the inflamed
`site demonstrated infiltration of leucocytes and macro-
`phages at the site of inflammation. The abscess centre
`with few cells, including residual injected oil, exudates
`and cell debris, was surrounded by an abscess wall. The
`dermis also appeared to be inflamed. In the present
`study, inflammation was evaluated in every animal by
`visually observing the pale colour of inflamed subcuta-
`neous tissue. We performed in vitro, ex vivo and in vivo
`experiments to evaluate the VAP-1 targeting, inflamma-
`tion imaging efficacy and pharmacokinetics of 68Ga-
`DOTAVAP-PEG-P1 in comparison to the original 68Ga-
`DOTAVAP-P1. The incorporation of a mini-PEG spacer
`had no apparent effect on the in vitro properties of the
`VAP-1 binding peptide; both peptides were stable in
`plasma incubations and their solubility was very similar.
`However, when i.v. administered, 68Ga-DOTAVAP-
`PEG-P1 showed significantly longer metabolic and
`
`MPI EXHIBIT 1095 PAGE 5
`
`MPI EXHIBIT 1095 PAGE 5
`
`

`

`Autio et al. EJNMMI Research 2011, 1:10
`http://www.ejnmmires.com/content/1/1/10
`
`Page 6 of 7
`
`Table 1 Pharmacokinetic parameters of the VAP-1-targeting 68Ga-DOTA-peptides
`68Ga-DOTAVAP-P1
`68Ga-DOTAVAP-PEG-P1
`Elimination t1/2 (min)
`13.4 ± 1.8
`28.0 ± 10.7
`0.05 ± 0.01
`0.02 ± 0.01
`kel (1/min)
`950 ± 170
`2,400 ± 68
`AUC (min*kBq/ml)
`0.017 ± 0.001
`0.008 ± 0.002
`CL (ml/min)
`kel, elimination rate constant; AUC, area under curve; CL, clearance; NS, not significant.
`
`P value
`NS (0.079)
`0.018
`0.022
`0.006
`
`elimination half-lives and slower total clearance com-
`pared to 68Ga-DOTAVAP-P1. Furthermore, our results
`revealed that while both peptides were able to visualise
`experimental inflammation by PET imaging, 68Ga-
`DOTAVAP-PEG-P1 showed a higher inflammation-to-
`muscle ratio than the original 68Ga-DOTAVAP-P1. As
`regards 68Ga-DOTAVAP-P1, the results of this study
`are in line with our previous publications [8-10]. The
`renal excretion of 68Ga-DOTAVAP-PEG-P1 was slower,
`resulting in a significantly lower urinary bladder radioac-
`tivity in comparison to 68Ga-DOTAVAP-P1. The liver
`uptake was rather high for both peptides, which is, at
`least in part, due to the high number of VAP-1 recep-
`tors in the sinusoidal endothelial cells in the liver [12].
`Some degradation products of 68Ga-DOTA-peptides,
`such as free 68Ga, also tend to accumulate in the liver
`[13]. Although modification with a mini-PEG spacer
`generally decreases liver uptake, the two peptides
`behaved quite similarly in our study, suggesting a VAP-
`1-specific binding in this tissue.
`PEGylation has widely been used for improving the in
`vivo kinetics of pharmaceuticals. However, the results of
`such modifications depend much on the nature of the lead
`compound and the choice of PEG linker [14-20]. In most
`cases, PEGylation of radiopeptides has advantageous
`effects, such as increased metabolic half-life, decreased
`kidney uptake, and improved targeting and subsequent
`improved targeting for high-quality imaging. However, dis-
`advantageous results have also been reported, e.g. the
`insertion of a long PEG chain may induce a higher liver
`uptake and reduce receptor binding [16].
`In this study, we incorporated an eight-carbon mini-
`PEG spacer between the DOTA and the VAP-P1 pep-
`tide in order to prolong its biological activity. The 8-
`amino-3,6-dioxaoctanoic acid contains the shortest ether
`structure possible of PEG with two ethylene oxide units.
`A similar spacer has previously been used in imaging
`agents by Burtea et al. [21], Ke et al. [22] and Silvola et
`al. [23].
`Modification with a mini-PEG spacer increased meta-
`bolic stability of VAP-1-targeting DOTA-peptide. In
`addition, it also improved in vivo imaging of inflamma-
`tion suggesting that PEGylation had other highly pro-
`nounced in vivo effects beyond modification of
`pharmacokinetics. Although the modification with a
`
`mini-PEG spacer increased the target-to-background
`ratio, the SUV values in the inflamed area were still very
`low. Thus,
`further improvement of
`the tracer is
`warranted.
`
`Conclusion
`The incorporation of a mini-PEG spacer enhanced the
`in vivo stability and pharmacokinetics of the VAP-1-tar-
`geting peptide, thus leading to higher target-to-back-
`ground ratios and improved in vivo PET imaging of
`experimental inflammation. 68Ga-DOTAVAP-PEG-P1
`warrants further investigations for its feasibility in PET
`imaging of inflammation.
`
`Abbreviations
`HPLC, high performance liquid chromatography; H & E, haematoxylin and
`eosin; MW, molecular weight; PEG, polyethylene glycol; PET, positron
`emission tomography; VAP-1, vascular adhesion protein-1.
`
`Acknowledgements
`The study was conducted within the Finnish Centre of Excellence in
`Molecular Imaging in Cardiovascular and Metabolic Research supported by
`the Academy of Finland, the University of Turku, the Turku University
`Hospital and the Åbo Akademi University. The study was further supported
`by grants from the Turku University Hospital (EVO grants, A.R. and A.A) and
`from the Academy of Finland (grant no. 119048, A.R). Anu Autio is a PhD
`student supported by the Drug Discovery Graduate School. Erja Mäntysalo is
`thanked for excellent assistance with animal experiments.
`
`Author details
`1Turku PET Centre, University of Turku and Turku University Hospital, Turku,
`Finland 2Department of Biology, Division of Genetics and Physiology,
`University of Turku, Turku, Finland 3MediCity Research Laboratory, University
`of Turku, Turku, Finland 4Turku Center for Disease Modeling, University of
`Turku, Turku, Finland
`
`Authors’ contributions
`AA participated in the design of the study, carried out the in vitro and in
`vivo PET studies and drafted the manuscript. TH participated in the design
`of the study and drafted the manuscript. HJS performed the labelling
`chemistry and participated in in vitro studies. AR and SJ conceived the study,
`participated in its design and coordination and critically revised the
`manuscript. All authors read and approved the final manuscript.
`
`Competing interests
`The authors declare that they have no competing interests.
`
`Received: 11 May 2011 Accepted: 26 July 2011 Published: 26 July 2011
`
`References
`Salmi M, Jalkanen S: A 90-kilodalton endothelial cell molecule mediating
`1.
`lymphocyte binding in humans. Science 1992, 257:1407-1409.
`Jaakkola K, Jalkanen S, Kaunismäki K, Vänttinen E, Saukko P, Alanen K,
`Kallajoki M, Voipio-Pulkki LM, Salmi M: Vascular adhesion protein-1,
`
`2.
`
`MPI EXHIBIT 1095 PAGE 6
`
`MPI EXHIBIT 1095 PAGE 6
`
`

`

`Autio et al. EJNMMI Research 2011, 1:10
`http://www.ejnmmires.com/content/1/1/10
`
`Page 7 of 7
`
`3.
`
`4.
`
`5.
`
`6.
`
`7.
`
`8.
`
`9.
`
`12.
`
`intercellular adhesion molecule-1 and P-selectin mediate leukocyte
`binding to ischemic heart in humans. J Am Coll Cardiol 2000, 36:122-129.
`Smith DJ, Salmi M, Bono P, Hellman J, Leu T, Jalkanen S: Cloning of
`vascular adhesion protein 1 reveals a novel multifunctional adhesion
`molecule. J Exp Med 1998, 188:17-27.
`Salmi M, Jalkanen S: VAP-1: an adhesin and an enzyme. Trends Immunol
`2001, 22:211-216.
`Tohka S, Laukkanen M-L, Jalkanen S, Salmi M: Vascular adhesion protein 1
`(VAP-1) functions as a molecular brake during granulocyte rolling and
`mediates recruitment in vivo. FASEB J 2001, 15:373-382.
`Jalkanen S, Salmi M: VAP-1 and CD73, endothelial cell surface enzymes in
`leukocyte extravasation. Arterioscler Thromb Vasc Biol 2008, 28:18-26.
`Yegutkin GG, Salminen T, Koskinen K, Kurtis C, McPherson MJ, Jalkanen S,
`Salmi M: A peptide inhibitor of vascular adhesion protein-1 (VAP-1)
`blocks leukocyte-endothelium interactions under shear stress. Eur J
`Immunol 2004, 34:2276-2285.
`Lankinen P, Mäkinen TJ, Pöyhönen TA, Virsu P, Salomäki S, Hakanen AJ,
`Jalkanen S, Aro HT, Roivainen A: (68)Ga-DOTAVAP-P1 PET imaging
`capable of demonstrating the phase of inflammation in healing bones
`and the progress of infection in osteomyelitic bones. Eur J Nucl Med Mol
`Imaging 2008, 35:352-364.
`Ujula T, Salomäki S, Virsu P, Lankinen P, Mäkinen TJ, Autio A, Yegutkin GG,
`Knuuti J, Jalkanen S, Roivainen A: Synthesis, 68Ga labeling and
`preliminary evaluation of DOTA peptide binding vascular adhesion
`protein-1: a potential PET imaging agent for diagnosing. osteomyelitis.
`Nucl Med Biol 2009, 36:631-641.
`10. Autio A, Ujula T, Luoto P, Salomäki S, Jalkanen S, Roivainen A: PET imaging
`of inflammation and adenocarcinoma xenografts using vascular
`adhesion protein 1 targeting peptide (68)Ga-DOTAVAP-P1: comparison
`with (18)F-FDG. Eur J Nucl Med Mol Imaging 2010, 37:1918-1925.
`11. Harris JM, Chess RB: Effect of pegylation on pharmaceuticals. Nat Rev
`Drug Discov 2002, 3:214-221.
`Lalor PF, Edwards S, McNab G, Salmi M, Jalkanen S, Adams D: Vascular
`adhesion protein-1 mediates adhesion and transmigration of
`lymphocytes on human hepatic endothelial cells. J Immunol 2002,
`169:983-992.
`13. Ujula T, Salomäki S, Autio A, Luoto P, Tolvanen T, Lehikoinen P, Viljanen T,
`Sipilä H, Härkönen P, Roivainen A: 68Ga-chloride PET reveals human
`pancreatic adenocarcinoma xenografts in rats–comparison with FDG.
`Mol Imaging Biol 2010, 12:259-268.
`14. Chen X, Hou Y, Tohme M, Park R, Khankaldyyan V, Gonzales-Gomez I,
`Bading JR, Laug WE, Conti PS: Pegylated Arg-Gly-Asp peptide: 64Cu
`labeling and PET imaging of brain tumor alphavbeta3-integrin
`expression. J Nucl Med 2004, 45:1776-1783.
`15. Wen X, Wu Q, Ke S, Ellis L, Charnsangavej C, Delpassand AS, Wallace S, Li C:
`Conjugation with (111)In-DTPA-poly(ethylene glycol) improves imaging
`of anti-EGF receptor antibody C225. J Nucl Med 2001, 42:1530-1537.
`16. DeNardo SJ, Liu R, Albrecht H, Natarajan A, Sutcliffe JL, Anderson C, Peng L,
`Ferdani R, Cherry SR, Lam KS: 111In-LLP2A-DOTA polyethylene glycol-
`targeting a4b1 integrin: comparative pharmacokinetics for imaging and
`therapy of lymphoid malignancies. J Nucl Med 2009, 50:625-634.
`17. Wu Z, Li ZB, Cai W, He L, Chin FT, Li F, Chen X: 18F-labeled mini-PEG
`spacered RGD dimer (18F-FPRGD2): synthesis and microPET imaging of
`αvβ3 integrin expression. Eur J Nucl Med Mol Imaging 2007, 34:1823-1831.
`Liu Z, Niu G, Shi J, Liu S, Wang F, Liu S, Chen X: 68Ga-labeled cyclic RGD
`dimers with Gly3 and PEG4 linkers: promising agents for tumor integrin
`αvβ3 PET imaging. Eur J Nucl Med Mol Imaging 2009, 36:947-957.
`Liu Z, Liu S, Wang F, Liu S, Chen X: Noninvasive imaging of tumor
`integrin expression using 18F-labeled RGD dimer peptide with PEG4
`linkers. Eur J Nucl Med Mol Imaging 2009, 36:1296-1307.
`Ke S, Wen X, Wu QP, Wallace S, Charnsangavej C, Stachowiak AM,
`Stephens CL, Abbruzzese JL, Podoloff DA, Li C: Imaging taxane-induced
`tumor apoptosis using PEGylated, 111In-labeled annexin V. J Nucl Med
`2004, 45:108-115.
`21. Burtea C, Laurent S, Lancelot E, Ballet S, Murariu O, Rousseaux O, Port M,
`Vander Elst L, Corot C, Muller RN: Peptidic targeting of phosphatidylserine
`for the MRI detection of apoptosis in atherosclerotic plaques. Mol Pharm
`2009, 6:1903-1919.
`Ke T, Jeong EK, Wang X, Feng Y, Parker DL, Lu ZR: RGD targeted poly(l-
`glutamic acid)-cystamine-(Gd-DO3A) conjugate for detecting
`
`18.
`
`19.
`
`20.
`
`22.
`
`23.
`
`angiogenesis biomarker αvβ3 integrin with MRTI mapping. Int J
`Nanomedicine 2007, 2:191-199.
`Silvola J, Autio A, Luoto P, Jalkanen S, Roivainen A: Preliminary evaluation
`of novel 68Ga-DOTAVAP-PEG-P2 peptide targeting vascular adhesion
`protein-1. Clin Physiol Funct Imaging 2010, 30:75-78.
`
`doi:10.1186/2191-219X-1-10
`Cite this article as: Autio et al.: Mini-PEG spacering of VAP-1-targeting
`68Ga-DOTAVAP-P1 peptide improves PET imaging of inflammation.
`EJNMMI Research 2011 1:10.
`
`Submit your manuscript to a
`journal and benefi t from:
`7 Convenient online submission
`7 Rigorous peer review
`7 Immediate publication on acceptance
`7 Open access: articles freely available online
`7 High visibility within the fi eld
`7 Retaining the copyright to your article
`
` Submit your next manuscript at 7 springeropen.com
`
`MPI EXHIBIT 1095 PAGE 7
`
`MPI EXHIBIT 1095 PAGE 7
`
`