2.1.

Synthesis and Characterization of Fluorogenic PSA FAST Agents

A nine amino acid oligopeptide substrate based on a known PSA-cleavable tetrapeptide sequence, Chg-Gln-Ser-Ser,⁸ was purchased as the trifluoroacetic acid salt from Tufts Core Facility, Boston, MA. VivoTag-S 750 was obtained from PerkinElmer, Inc. (Waltham, MA). All chemicals were used as purchased without further purification. To generate a fluorophore quenched PSA substrate sequence, the N-Ac oligopeptide ($\sim 10\mu \mathrm{mol}$) was dissolved in 1 mL $N,N$-dimethyl formamide (DMF) containing 10 μL of triethylamine. A solution of VivoTag-S 750 ($\sim 25\mu \mathrm{mol}$) in 2 mL of anhydrous DMF was added and stirred for 30 min at ambient temperature in the dark. Purification was carried out by preparative high-performance liquid chromatography (HPLC) using a Varian system and a Phenomenex Jupiter Phenylhexyl column (10 μm, $100\AA$, $250\times 21.2\mathrm{mm}$) to give the fluorophore labeled oligopeptide. The incorporation of two fluorophores per oligopeptide was confirmed by electrospray mass spectrometry on a single quadrupole Waters ZQ: calculated: 2274; found: 1138 1/2 $(\mathrm{M}+2\mathrm{H})/2$.

As a labeled oligopeptide alone would not have the desired pharmacokinetic properties in vivo, a chemical modification was made to increase circulation half-life. Briefly, the labeled oligopeptide ($\sim 8\mu \mathrm{mol}$) was dissolved in 6 mL of DMF containing the pharmacokinetic modifier (PKM) amine (40 kDa polyethylene glycol, 12 μmol), $N$-hydroxybenzotriazole (13 μmol), and $N$-methylmorpholine (14 μmol). To the solution was added 1-ethyl 3-[(3-dimethylamino) propyl] carbodiimide hydrochloride (13 μmol) and the solution was stirred for 40 min at ambient temperature in the dark. The product was purified by an anion exchange column (Q-Sepharose Fast Flow anion exchange gel, 10 mL bed volume) and the desired fractions were pooled, lyophilized, and stored at $-20°\mathrm{C}$. UV–visible absorbance and fluorescence emission spectra of the native and enzyme activated agent were recorded on Cary 50 and Cary Eclipse spectrophotometers, respectively, in $1\times$ PBS using 740 nm for fluorescence excitation. For this, 5 μM of PSA750 was cleaved in the presence of 1 μM activated PSA for 6 h in a final volume of 250 μL 50 mM Tris, pH 8, and 1 M NaCl. PSA was preactivated by Thermolysin ($1\mu \mathrm{g}/\mathrm{mL}$ for 5 min in 50 mM Tris, 10 mM ${\mathrm{CaCl}}_2$, 150 mM NaCl, 0.05% Brij-35, pH 7.5) and the reaction stopped by the addition of 1,10-Phenanthroline (20 mM final). The proteolysis of the oligopeptide substrate at the anticipated cleavage site was confirmed by electrospray mass spectrometry on a single quadrupole Waters ZQ: calculated mass of fragment A: 1607.6 Da; found: 803 Da $(\mathrm{M}-2\mathrm{H})/2$.

2.2.

Cell Lines and Reagents

Human prostate carcinoma LNCaP (${\mathrm{PSA}}^{+}$) and human grade IV prostate adenocarcinoma prostate cancer 3 (PC3) (${\mathrm{PSA}}^{-}$) cells (ATCC, Manassas, VA) were grown in RPMI-1640 medium containing 10% heat-inactivated fetal calf serum at 37°C in a humidified atmosphere containing 5% ${\mathrm{CO}}_2$.

2.3.

In Vitro Enzymatic Activation

In vitro activation of PSA750 was carried out in a final concentration of 0.1 μM of each enzyme and 0.5 μM PSA750 at the optimal buffer and pH conditions for each individual enzyme. Recombinant human PSA, Kallikrein I, Kallikrein II, u-plasminogen activator/urokinase and cathepsin B (CatB) were purchased from R&D Systems (Minneapolis, MN), and MMP-9, MMP-12, MMP-13, human plasma alpha-1 ACT, chymotrypsin, and thrombin were purchased from Calbiochem. Enzymatic reactions were carried out in buffers recommended by manufacturers, at room temperature in 250 μl in 96-well plates with black sides and bottom in recommended buffers. PSA was preactivated by Thermolysin (R&D Systems) and 1,10-Phenanthroline (Sigma, St. Louis, MO) was used to stop the Thermolysin activity. All the reactions were monitored at various time points at excitation/emission wavelengths of $750/770\mathrm{nm}$ using a fluorescence plate reader (Molecular Devices, San Leandro, CA). The released fluorescence is shown after subtracting background fluorescence of the agent without enzyme.

2.4.

Pharmacokinetics

Twenty-four female retired breeder BALB/c mice (age 12 to 16 weeks, Charles River Laboratories, Wilmington, MA) were injected intravenously (i.v.) with 2 nmol PSA750 in PBS. Terminal blood samples ($n=2\mathrm{mice}/\text{time point}$) were collected by cardiac puncture from each mouse (following carbon dioxide asphyxiation) in ethylenediamine tetra-acetic acid-containing tubes and plasma was obtained by centrifugation (15,000 rpm for 10 min at 4°C). Fifty microliters of each plasma sample was mixed with 150 μL cold methanol, centrifuged (12,000 rpm, 4°C, 10 min), and supernatants were analyzed by HPLC.

2.5.

Agent Characterization by HPLC

HPLC analyses were performed on a Waters model 2695 (Waters Corporation, Milford, MA). The PDA, Waters model 2998, was set to scan from 225 to 800 nm. The wavelength corresponding to the absorbance maximum of the fluorophore, 750 nm, was extracted from the PDA trace. Samples were analyzed on a C4, $300\AA$, 5 μm, $150\times 4.6\mathrm{mm}$ HPLC column (Phenomenex, Torrance, CA). The aqueous mobile phase contained 25 mM ammonium formate. Samples were eluted with acetonitrile at a flow rate of $1\mathrm{mL}/\min$. An organic phase of 0.1% formic acid in methanol was used to elute native plasma proteins during the run. A gradient of 15% to 75% organic provided sufficient resolution. Standards were prepared with PSA750 (0 to 1 μM) in mouse plasma. Standard curves had correlation coefficients $>0.99$.

2.6.

Prostate Tumor Models

Two tumor models were used in these studies: PSA positive LNCaP and PSA negative PC3 human prostate carcinoma xenografts. Male Nu/Nu mice (age 6 to 8 weeks, Charles River Laboratories, Wilmington, MA) were injected subcutaneously (s.c.) in the upper chest region with ${5\times 10}^6$ tumor cells. Mice were housed in environmentally controlled specific-pathogen free conditions with water and low-fluorescence mouse chow (Harlan Teklad, Madison, WI). All animal experimental procedures were approved by PerkinElmer’s Institutional Animal Care and Use Committee and in accordance with veterinarian requirements. When tumors reached the desired volume, as measured by calipers (${\sim 200\mathrm{mm}}^3$), mice were injected i.v. with 2 nmol of PSA750 and imaged by FMT (FMT™ 4000, PerkinElmer Inc., Waltham, MA) at various time points.

2.7.

PSA750 Activation in Tumor Sections

To validate the specificity of PSA750 activation by active PSA present in tumors, LNCaP tumor-bearing mice were euthanized by ${\mathrm{CO}}_2$ inhalation and tumors were removed and snap frozen in OCT. Five micrometer tumor sections were incubated with 5 μM PSA750 in the absence or presence of ACT (5 μM) at 37°C in a humidified incubator for 30 min. Slides were viewed on a Zeiss Axioskop2 $\mathrm{MOT}+$ fluorescence microscope with a Hammamatsu Orca RC monochromatic camera using PerkinElmer’s Volocity software. Cell nuclei were visualized using a DAPI nuclear counterstain.

2.8.

In Vivo Fluorescence Molecular Tomography

Mice imaged by FMT (FMT^® 4000, PerkinElmer, Inc., Waltham, MA) were first anesthetized by gas anesthesia (isoflurane/oxygen mixture), placed in the FMT 4000 system imaging chamber (equipped with gas anesthesia), one at a time, and imaged. Images were analyzed using TrueQuant software (PerkinElmer Inc., Boston, MA) by drawing three-dimensional (3-D) regions of interests (ROIs) around tumor regions and an adjoining background area, and using a threshold equal to 30% of the mean value of fluorescence in the background. The total amount (in pmol) of fluorochrome was automatically calculated relative to internal standards generated with known concentrations of the appropriate dye (VivoTag-S 750). Quantification accuracy of the FMT system has been described previously.^9,10

2.9.

Organ Distribution

Immediately following imaging, a cohort of mice was sacrificed ($n=2$, experiment repeated twice), organs excised, and imaged in epifluorescence mode using the FMT 4000. The mean fluorescence intensity was measured after drawing an ROI around each organ. No threshold was applied. Data are expressed as mean fluorescence (in counts/energy).

2.10.

Tissue Localization

LNCaP tumor-bearing mice were injected i.v. with 10 nmol of agent. Twenty four hours later, mice were injected with $25\mathrm{mg}/\mathrm{kg}$ Hoechst 33342 (Life Technologies, Grand Island, NY). Five minutes later, mice were sacrificed, and tumors were excised, and snap frozen in OCT. Ten micron sections was prepared and slides were viewed on a Zeiss Axioskop2 $\mathrm{MOT}+\text{fluorescence}$ microscope with a Hammamatsu Orca RC monochromatic camera using PerkinElmer’s Volocity software.

2.11.

Statistical Analysis

Data are presented as $\text{mean}\pm \mathrm{SEM}$. Significance analysis of differences between groups was conducted using a two-tailed unpaired Student’s $t$ test. Probability values of $<5\%$ were considered significant.

3.1.

Synthesis and Characterization of PSA750

The fluorogenic agent PSA750 consists of an oligopeptide substrate sequence comprising seven amino acids containing a Chg-Gln-Ser-Ser tetrapeptide moiety, and flanked by two lysines at both the C-terminus and the N-terminus. Each of the two lysines was linked at the amino group on the side chain to VivoTag-S 750, an NIR fluorochrome, resulting in a self-quenched agent. To optimize the plasma half-life of the agent for in vivo imaging applications, the substrate was further conjugated to a 40 kDa polyethylene glycol PKM at the C-terminus at a ratio of one substrate per polymer molecule [Fig. 1(a)]. The product has a MW of ${\sim \mathrm{42,500}\mathrm{gmol}}^{-1}$ and was characterized by reversed phase HPLC to be $>95\%$ pure at 750 nm. Confirmation of the cleavage site between Q and S amino acid residues was achieved by mass spectral analysis of PSA-cleaved fragments, as described in Sec. 2: calculated mass of fragment A: 1607.6 Da; found: 803 Da $(\mathrm{M}-2\mathrm{H})/2$.

Fig. 1

Design and optical characteristics of PSA750. (a) The fluorogenic peptide substrate containing a Gln-Ser cleavage site is conjugated to a pharmacokinetic modifier (PKM) and flanked by two NIR fluorophores. Upon cleavage of the peptide by active PSA, the fluorophores become fluorescent. (b) Absorbance spectra of the PSA-activated fluorescent form (gray line) shows a bathochromic shift in the absorbance maximum relative to the native autoquenched state (black line). (c) The fluorescence emission is increased more than 18-fold upon proteolytic activation with a maximum at 770 nm.

Compared to the absorbance maximum of VivoTag-S 750, a hypsochromic shift was observed in the absorbance maximum of the nonactivated agent (${\lambda }_{\mathrm{Max}}$ at 684 nm and an ${\epsilon =\mathrm{170,000}\mathrm{cm}}^{-1}{\mathrm{M}}^{-1}$ in $1\times \mathrm{PBS}$) reflecting static, groundstate quenching of the two fluorochromes [Fig. 1(b)]. Measurement of fluorescence at 770 nm indicated $>95\%$ quenching in the nonactivated form. Upon cleavage with active PSA, an 18-fold increase in fluorescence was detected while the absorption ${\lambda }_{\mathrm{Max}}$ shifted to 750 nm, $\epsilon {=\mathrm{240,000}\mathrm{cm}}^{-1}{\mathrm{M}}^{-1}$, similar to that of the starting fluorophore [Fig. 1(c)]. PSA750 was found to be stable in the presence of mouse plasma over a period of 24 h, as assessed by HPLC (data not shown).

3.2.

In Vitro Enzyme Assays

The main objective of developing a PSA-activatable agent was to target enzymatically active PSA over inactive or bound PSA as well as other disease-relevant enzymes. The in vitro specificity of PSA750 toward PSA is illustrated in Fig. 2. Activation of PSA750 by PSA was blocked in vitro by prior complexing with alpha-1 ACT, a known natural inhibitor of PSA, indicating the specificity of the agent. PSA750 is activated more effectively when compared to other cancer- and inflammation-relevant enzymes such as MMPs 12 ($p<0.001$ at 24 h, $<0.0001$ at all other time points) and 13 ($p<0.001$ at 24 h, $p<0.0001$ at all other time points), chymotrypsin (comparable values at 1 min $p<001$ at 24 h, $p<0.001$ at other times), kallikreins, and thrombin (comparable values at 1 min, $p<001$ at 24 h, $p<0.001$ at other times). The kinetics of PSA750 activation reveal that a fluorescent signal is released as early as 1 min after addition of the enzyme, with peak fluorescence seen at around 6 h. The very low-level signal seen with MMP-12 and chymotrypsin required 24 h for effective measurement.

Fig. 2

PSA750 is activated several-fold by active PSA when compared to other disease-relevant enzymes. The agent (0.5 μM) was activated in vitro by a panel of enzymes (0.1 μM) in optimized buffers and pH for each enzyme and the fluorescence monitored up to 24 h in a fluorescence microplate reader. Released fluorescence was obtained by subtracting the fluorescence of agent only from that of the PSA750 in the presence of enzymes. PSA complexed with it natural inhibitor, ACT, does not cleave the agent.

3.3.

Ex Vivo Tissue Activation

Activation of PSA750 was tested in tumor tissue samples excised from tumor-bearing nude mice (Fig. 3). PSA positive LNCaP tumor slices (5 μm) displayed activation of PSA750 after incubation in a 5 μM solution of the agent. PSA750-associated fluorescence was also inhibited when adjacent tumor slices were incubated with an equal concentration of ACT.

Fig. 3

Effect of ACT on the activation of PSA750 ex vivo in tumor sections. LNCaP tumor-bearing mice were sacrificed and tumors excised and snap frozen. PSA activity was assessed in situ by incubating tumor sections (5 μm thick) with 5 μM PSA750 at 37°C for 30 min in the absence or presence of the PSA inhibitor ACT (5 μM). Fluorescent microscopy images were captured with a microscope equipped with a xenon light source and Cy7 filters. Shown are representative images at a final $400\times$ magnification. In light gray, DAPI nuclear stain; bright stain, activated PSA750.

3.4.

Pharmacokinetics and Organ Distribution

To determine the optimal in vivo imaging time point, kinetic imaging, and blood pharmacokinetic studies were performed. In a preliminary imaging study, five tumor-bearing mice were imaged at 6 and 24 h post-agent injection. The mean fluorescence of the tumors and background area were quantified. At 6 h, there was no statistically significant difference between the tumor and background signals ($p=0.066$), although there was a 3.12 tumor to background ratio. At 24 h, however, the ratio increased to 7.54 and there was a statistically significant difference between tumor and background signals ($p=0.025$). An additional study [Fig. 4(a)] assessing tumor fluorescence over time, from 24 to 168 h, allowed determination of the clearance kinetics. Mouse plasma levels of PSA750 were measured by HPLC [Fig. 4(a)], which detects the intact parent molecule. The plasma half-life was determined to be 2 h, with $<10\%$ remaining in plasma at 24 h, confirming this as the earliest optimal time point for in vivo imaging. Tissue clearance [Fig. 4(a)] and organ distribution of fluorescence [Fig. 4(b)] were also measured in ${\mathrm{PSA}}^{+}$ LNCaP tumor-bearing mice at 24 h. This assessment provides useful data with regard to understanding the PSA750 tumor versus other potential sites of interfering signal at the time of optimal tumor imaging. High fluorescence in the bladder indicates mostly renal clearance. From in vivo and ex vivo tumor fluorescence measurements, it was determined that the tissue half-life of PSA750 was approximately 2 days, with full clearance by day 7.

Fig. 4

Plasma half-life and bio-distribution of PSA750. (a) LNCaP tumor-bearing mice were injected with 2 nmol of PSA750 and imaged at different times thereafter by FMT. CD-1 mice were injected i.v. with 2 nmol PSA750 and blood was collected at different times. Plasma was prepared and analyzed by HPLC (inset). Pharmacokinetic and pharmacodynamics profiles of PSA750 show a plasma half-life of 2 h, while in vivo tumor imaging reveals a the tissue fluorescence half-life of PSA750 is approximately 2 days, with full clearance by 7 days. This allows weekly repeat imaging. (b) LNCaP tumor-bearing mice were injected with 2 nmol of PSA750 different tissues excised 24 h later. Organ fluorescence was determined using FMT in epifluorescence mode. Regions of interest were drawn around each organ using the FMT software and the mean fluorescence normalized to the geometric mean of the ROI (${[\text{counts}/\text{energy}]/\mathrm{m}}^3$) determined. Shown are data from two representative mice (the experiment was repeated twice with similar results). PSA750 selectively distributes to tumor tissue compared to other vital organs. Inset shows an image of the fluorescence detected in different organs of an LNCaP tumor-bearing mouse.

3.5.

In Vivo Fluorescence Molecular Tomography

PSA750 was used to visualize both ${\mathrm{PSA}}^{+}$ LNCaP and ${\mathrm{PSA}}^{-}$ negative PC3 tumors in vivo using 3-D fluorescence molecular tomography [Fig. 5(a)]. Tomographic images showed a higher PSA750 signal in the LNCaP tumors, which was confirmed quantitatively, exhibiting a significant increase ($\sim 20\times$) in fluorescence concentration in the PSA positive cells when compared to PSA negative PC3 tumors [Fig. 5(b)]. In vivo tumor fluorescence was also found to correlate well ($r^2=0.90$) with tumor volume [Fig. 5(c)].

Fig. 5

In vivo tumor imaging and quantification. LNCaP and PC3 tumor-bearing mice were injected with 2 nmol PSA750 and imaged 24 h later by FMT. (a) FMT representation of PSA750 activity in PSA positive (LNCaP) and negative (PC3) tumor-bearing mice. LNCaP tumors show more tumor definition and brighter signal, which is quantified in (b), the total amount of fluorescence (pmol) was quantified in specific ROIs encompassing each tumor. LNCaP tumors have significantly higher PSA-associated signal as compared to adjoining muscle (left) or PC3 tumors (right). (c) The total fluorescence of PSA750 also correlates well ($r^2=90$) with tumor volume.

3.6.

Tumor Localization

PSA750 localization was determined in LNCaP tumor sections excised from nude mice that had been injected with both PSA750 and Hoechst 33342 to visualize tumor vasculature. Fluorescence microscopy shows that PSA750-associated fluorescence is seen in nonvascularized tissue (Fig. 6), indicating that PSA750 is activated by PSA in the tissue as expected. Perfused regions allow plasma ACT to ablate PSA activity.

Fig. 6

Localization of activated PSA750 ex vivo. Tumors were snap frozen in OCT for fluorescence microscopy. The distribution of an NIR fluorescence was determined using fluorescence microscopy. Digital images were captured using appropriate filters for Hoechst (blue), and the near-infrared agent PSA750 (red). Final magnification $400\times$. Each panel from a different tumor section shows activated PSA-associated fluorescence in nonvascularized areas as expected. (PSA is immediately inactivated by inhibitors in plasma, active PSA is found in the tumor matrix.)