2.1.

Synthesis Cy5.5-c(KRGDf)]-Conjugate

c(KRGDf) was synthesized on linker-PL-DMA resin using Fmoc solid phase chemistry as described previously.¹⁵ Briefly, peptide was cleaved from the support with 1% trifluoroacetic acid (TFA) with all sidechain protecting groups intact. The head-to-tail cyclization was then carried out in DMF using benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexaflurophosphate, 1-hydroxybenzotriazole, and N,N-diisopropylethylamine (DIPEA) as coupling agents. After removal of all sidechain protecting groups the cyclic peptide was purified by reverse phase HPLC eluted with H ₂ O /acetonitrile containing 0.1% TFA. For conjugation with the fluorescence dye, a solution of Cy5.5-NHS [Eq. (1)] and c(KRGDf)] [Eq. (1.3)] in DMF/DIPEA (10/1,v/v) was stirred at room temperature overnight. After all solvents and by-products were removed under vacuum, the compound was purified by reverse phase HPLC eluted with a 0.01 M solution of NH ₄ OAc in water/methanol. The Cy5.5-c(KRGDf) product was validated by analytic HPLC and MALDI mass spectrometry.

2.2.

Cell Adhesion Assay

KS1767 cells were seeded in DMEM/F12 culture medium supplemented with 10% fetal bovine serum for 24 h. Cells (1×10⁵) with different concentrations of c(KRGDf) or Cy5.5-c(KRGDf) were added to vitronectin-coated microtiter wells under serum-free conditions and incubated at 37 °C for 1 h. After washing steps, the bound cells were stained with 5% crystal violet, followed by addition of 0.1 M HCl to each well. The concentrations of crystal violet were determined by ultraviolet/visible absorption at 627 nm. IC ₅₀ values were estimated from the dose-activity curves.

2.3.

Animal Model

Six-week-old female athymic nude mice (nu/nu; 18–22 g) were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN) and housed five per cage and fed sterilized pelleted food (Harlan Sprague Dawley Inc., Indianapolis, IN) and sterilized water. Animals were maintained in a specific pathogen-free mouse colony in the Department of Veterinary Medicine (The University of Texas M. D. Anderson Cancer Center, Houston, TX). The facility is accredited by the American Association for Laboratory Animal Care and all experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee.

Tumor cells to be implanted into mice were harvested near confluence by incubation with 0.05% trypsin-EDTA. Cells were pelleted by centrifugation at 450×g for 5 min and resuspended in sterile PBS. Cells (2–3×10⁶ /animal) were implanted subcutaneous into the thigh region of mice.

Animals were subdivided into four test groups based on the contrast agent administered. Animals in the first group (N=2) received the free Cy5.5 dye (6 nmol/mouse), while animals in the remaining three groups were administered the Cy5.5-c(KRGDf) conjugate (6 nmol/mouse). Of those animals receiving the conjugate dye, one-third received the conjugate dye alone (N=3); one-third received the conjugate one hour after the intravenous injection of unconjugated c(KRGDf) peptide (N=3); and the remaining third received the conjugate 24 h after the intravenous injection of the unconjugated c(KRGDf) peptide (N=3). The unconjugated c(KRGDf) peptide was administered at 200 times the dose of the Cy5.5-c(KRGDf) conjugate.

2.4.

Experimental Imaging Method

Before fluorescence imaging, mice were anesthetized by intraperitoneal injection of 50 mg (kg bw)⁻¹ Nembutal (Sigma, St. Louis, MO) and a catheter was positioned into the tail vein to facilitate the subsequent delivery of the fluorescent dye. Fluorescence images were obtained every 7 s for a period of approximately 18 min following the injection of contrast agent. Excitation light provided by a laser diode (660 nm, 35 mW) whose beam was expanded via a planoconvex lens and a holographic diffuser to provide a uniform excitation field of approximately 12 cm in diameter over the surface of the animal. An intensified CCD, comprised of a 16-bit CCD camera (Photometrics, model CH350/L, Tucson, AZ) coupled to an image intensifier (model FS9910C, ITT Night Vision, Roanoke, VA) via a 105 mm lens, functioned as an area detector of re-emitted fluorescence light.¹⁶ A holographic notch-plus band rejection filter (660 nm center wavelength; Kaiser Optical Systems, Inc., Ann Arbor, MI) and a bandpass filter (710 nm center wavelength; CVI Laser, Albuquerque, NM) were positioned prior to a 50 mm lens to reject backscattered and reflected excitation light and isolate only re-emitted fluorescence light. For the acquisition of white-light images, a low-power lamp provided a white-light source and the filters were removed. Image acquisition was controlled by a personal computer equipped with V++ imaging software (Digital Optics, Auckland, New Zealand). The first five images were acquired prior to the bolus administration of the fluorescent probe, and thus, served as base line or background images.

The injected dose of Cy5.5-c(KRGDf) and the image exposure time were equivalent to 6 nmol (in 0.3 mL volume) and 800 ms, respectively, and were constant for all the imaging studies conducted. Fluorescence images were obtained at 24 h postinitial injection and again every 24 h thereafter until little or no fluorescence signal was detectable from the tumor region of interest, as determined qualitatively, for up to 168 h.

2.5.

Analysis of Pharmacokinetics from Dynamic Imaging

Upon completion of the imaging routine, a background image was subtracted from each of the acquired fluorescence images. The white light image was then used to define two regions of interest (ROI); one was located entirely within the circumference of the tumor and the other, approximately equivalent in size, defined a region of normal tissue located symmetrically opposite the tumor site. Utilizing Matlab software (The MathWorks, Inc., Natick, MA) the mean of the fluorescence intensity within each ROI was computed for every fluorescence image acquired. Plots of mean fluorescence intensity versus time were then generated for each of the imaging studies performed. The mean fluorescence intensity as a function of time after agent administration within each ROI was fitted via a nonlinear Levenberg–Marquardt regression algorithm to the PK equation described in the following section.

3.1.

Derivation of Three-Compartment Pharmacokinetic Model

In this section we develop a pharmacokinetic model in order to describe the time-dependent distribution of the fluorescing dye-peptide conjugate throughout the animal. The model, depicted schematically in Fig. 1, is comprised of three compartments in which Cy5.5 resides: (i) the blood plasma compartment, (ii) the extravascular, extracellular compartment, and (iii) the tissue compartment representing the “bound” Cy5.5 associated to the integrin receptor.

Figure 1

Schematic depicting the three-compartment pharmacokinetic model employed.

The dye is administered rapidly into the blood plasma compartment via an intravenous bolus injection. From this central blood compartment, the dye diffuses across the vascular endothelium, and partitions into the extravascular, extracellular space (EES) represented by the second compartment. The endothelial transfer coefficient or the rate constant for the distribution of dye from the blood to the EES is given by k_p while k_r is the rate constant describing the reflux from the EES to the blood. A measure of vascular permeability can be obtained from an analysis conducted on first pass circulation data, or data obtained immediately following the bolus injection of the agent. In the past, Cuccia etal. employed a pharmacokinetic analysis to quantify physiologic parameters related to capillary permeability following the administration of the nonspecific dye indocyanine green.¹⁷

In addition to extravasation into the EES, the dye is eliminated from the blood compartment via other routes such as kidney filtration and liver metabolism, described collectively by the rate constant k_el. For nonspecific dyes, that is, dyes that do not exhibit cellular specificity, the blood and EES compartments may be sufficient to describe their distribution.^{17 18} However, for dyes that exhibit molecular specificity, a third compartment is included. This third compartment represents the association of the dye at a molecular level, whether it is through surface receptor binding or cellular uptake. The rate at which the dye accumulates in the cellular compartment, whether through association with cellular membrane receptors or internalization, is denoted by R.

If the rate of accumulation of dye in the cellular compartment, R, is constant, determining the time-dependent dye concentration in each of the compartments is relatively straightforward. However, in general, the association between the dye and the cell is a saturable process. Therefore, the rate of dye uptake by the cell may be expressed as

The detected fluorescence intensity at the tissue surface is mediated by a number of factors including the tissue optical properties of scattering and absorption and the depth and concentration of the embedded fluorophore within the tissue. For purposes of monitoring the temporal fluorescence intensity we can assume optical properties of the tissue are constant and can express the detected fluorescence intensity as a weighted sum of the concentration of the dye in each compartment

To obtain the time-dependent dye concentration in each of the compartments, the system of differential equations given by Eqs. (3 4 5) may be solved simultaneously subject to the initial condition that at the time of i.v. bolus injection of the dye, t=0, C_B=C _Bo , C _EES =0, and C_c=0. The results may then be applied to Eq. (6) to obtain the following expression for the time-dependent observed fluorescence intensity:

An examination of Eqs. (11) and (12) reveals that the complex rate constant α must be greater than β. As a result, at early time points the expression for the observed fluorescence intensity is more strongly influenced by the term containing α. However, the exponential term containing α approaches zero more rapidly than does the term containing β. Consequently, at later time points the expression for the observed fluorescence intensity becomes a function of β alone. Therefore, the early time portion of the fluorescence intensity profile is referred to as the α phase whereas the later portion is known as the β phase.

Furthermore, it is evident that the values of α and β are intimately related to the values of all other rate constants. It is particularly interesting to examine how α and β vary with the magnitude of the elimination rate constant, k _el . For example, if the value of k _el is varied while all other rate constants remained fixed, the root function that appears in the expression for both α and β would asymptotically approach k_B−k _EES for large values of k _el and k _EES −k_B for small values. As a result, α approaches k_B while β approaches k _EES when the intrinsic elimination is very rapid. The trend is reversed when elimination is slow; α approaches k _EES while β approaches k_B. Finally, and more importantly, because α and β differ by only the sign in front of the root function, the sum of α and β yields the sum of all rate constants between the various compartments of the model, α+β=k _el +k_p+k_r+k_c. One may exploit this result to determine the magnitude of the rate constant for specific uptake of contrast agent within the cellular compartment, k_c as follows: for a given specific contrast agent administered in an animal bearing a tumor model in which the specific cellular uptake mechanism has been compromised while the remaining mechanisms of dye uptake remain unchanged, the net decrease in α+β reflects the magnitude of the rate constant for specific cellular uptake of the contrast agent.

As a final note, from Eq. (7) it is evident that I₀ represents the fluorescence intensity at the time of contrast agent administration or background fluorescence intensity level. Thus, when one applies a background subtraction technique, the mathematical equivalent of setting I₀ equal to zero, the number of parameters to be fit is reduced such that the time-dependent observed fluorescence intensity may be expressed as

In summary, the four parameter, double-exponential equation represents the analytical solution to a three-compartment pharmacokinetic model with first order distribution between (i) the vascular, (ii) the extravascular extracellular space, and that associated with (iii) molecularly specific binding. The parameters α and β are complex rate constants governing uptake into each of the three compartments and its sum, (α+β) represents the sum of first order rate constants.

4.1.

In vitro Binding Studies

To test the antiadhesive activity of Cy5.5-c(KRGDf) and c(KRGDf) peptides, attachment of KS1767 cells (1×10⁵) to vitronectin-coated microtiter wells were studied using different concentrations of each peptide. Both Cy5.5-c(KRGDf) and c(KRGDf) behaved similarly, blocking the attachment of KS1767 cells to microtiter wells in a dose-dependent manner with estimated IC ₅₀ of 2.5 and 1.2 μM, respectively. Figure 2 shows the dose-dependent inhibition of KS1767 cell adhesion as indicated by the diminution of crystal blue dye with increasing concentrations of both Cy5.5-c(KRGDf) and c(KRGDf). Cell attachment was inhibited because the peptides compete for ligand binding sites of the cell surface integrins. These in vitro results confirm the molecular specificity of Cy5.5-c(KRGDf) and c(KRGDf) to the cell surface integrins. Furthermore, using fluorescence microscopy we have confirmed the inhibition of Cy5.5-c(KRGDf) binding to KS1767 cells by preincubation with c(KRGDf).¹⁵

Figure 2

Dose-dependent inhibition of adhesion of KS1767 cells to vitronectin-coated microplate wells by c(KRGDf) and Cy5.5-c(KRGDf).

4.2.

Pharmacokinetic Analysis

Figure 3 represents typical dynamic fluorescence intensity profiles in the (a) tumor ROI and (b) normal ROI and the corresponding least-squares fit obtained from one representative animal of each of the groups that received an injection of the Cy5.5-c(KRGDf) conjugate. The coefficient of determination R² for the regression was 0.99 or greater in all cases. In both normal and tumor ROIs, uptake was (i) greatest when the animal received Cy5.5-c(KRGDf) conjugate alone, (ii) reduced when the animal received the Cy5.5-c(KRGDf) conjugate 1 h after administration of free c(KRGDf) as a blocking agent, and (iii) recovered when the animal received the Cy5.5-c(KRGDf) conjugate 24 h after administration of free c(KRGDf) as a blocking agent. In addition, normal tissue experiences a diminished reduction of update with administration of competitive binding of c(KRGDf) than seen in the Kaposi’s sarcoma. This is presumably due to the lower integrin receptor density found in normal tissues as compared to Kaposi’s sarcoma.

Figure 3

Fluorescence intensity versus time profiles obtained from one representative animal of each of the groups that received an injection of the RDG-Cy5.5 conjugate, acquired from the (a) tumor ROI and (b) normal ROI. The symbols denote experimental measurements while the solid line denotes the corresponding least-squares fit. The squares (□) denote data from an animal receiving the Cy5.5-c(KRGDf) conjugate alone, while the circles (○) and diamonds (⋄) represent data obtained from an animal receiving the conjugate 1 h and 24 h, respectively, after the injection of RGD peptide.

Figure 4 shows that the sum of the pharmacokinetic rate constants, α+β, does not appear to differ between the normal ROIs of all animal groups; a series of homoscedastic pooled t tests confirmed that the mean estimates of α+β are not significantly different, at a 95% significance level. For animals injected with free Cy5.5 and animals pretreated with c(KRGDf) peptide one hour prior to the injection of the Cy5.5-c(KRGDf) conjugate, the values of α+β in tumor ROIs are statistically similar at a 95% level of significance to each other and to the mean values of α+β determined from the normal ROIs. These results suggest that the free Cy5.5 dye exhibits similar targeting, or lack thereof, in KS1767 tumor tissue and in normal tissue. In contrast, the value of α+β was found to be three to fourfold greater for the tumor ROIs of the animals which received Cy5.5-c(KRGDf) conjugate (mean α+β=0.21) than for those values (i) from the normal tissue ROIs in animals injected with Cy5.5 or Cy5.5-c(KRGDf) (mean α+β=0.049 or 0.068) and (ii) from the tumor ROIs in animals injected with Cy5.5 (mean α+β=0.055). Statistical analysis revealed that the estimates for α+β from within the tumor ROIs of the test groups administered Cy5.5-c(KRGDf) alone or the conjugate dye 24 h after treatment with free c(KRGDf) peptide were significantly different from the values of the remaining test groups.

Figure 4

Results of the nonlinear least-squares regression in determining the sum of the pharmacokinetic complex rate constants α+β. The column height represents the mean value of the test group listed along the abscissa while the error bars represent the standard deviation. The time in parenthesis represents the time between injection of the RGD peptide and the Cy5.5-c(KRGDf) conjugate.

The conjugate Cy5.5-c(KRGDf) exhibited a lack of tumor targeting when the administration of the dye is preceded by the injection of free c(KRGDf) peptide by 1 h, as evidenced by the fact that no statistical difference in α+β value was found between the tumor ROIs of animals receiving Cy5.5 dye and that of mice receiving c(KRGDf) followed by Cy5.5-c(KRGDf) 1 h later. When the c(KRGDf) peptide preceded injection of the conjugate by 24 h, the value of α+β in tumor ROIs was recovered and became once again significantly different from the α+β values of the normal tissue ROIs and from the tumor ROIs of mice injected with Cy5.5 and mice injected with c(KRGDf) followed by Cy5.5-c(KRGDf) with a 1 h interval.

From the studies involving the administration of the RGD peptide prior to the administration of the Cy5.5-c(KRGDf) conjugate, one may draw the following conclusions. First, because the net effect of administering the RGD peptide one hour prior to the Cy5.5-c(KRGDf) conjugate is the reduction of the tumor uptake and since the α_vβ₃ integrin receptor overexpressed on KS1767 binds the RGD peptide, the pharmacokinetic analyses from dynamic imaging verify the specific binding of the Cy5.5-c(KRGDf) conjugate. Second, because the value of α+β obtained from the tumor ROIs of animals injected with the conjugate dye alone or the conjugate dye 24 h following the pretreatment with the RGD peptide are statistically similar, one may speculate that the turnover of the α_vβ₃ integrin receptor expressed on the cell surface of KS1767 occurs within 24 h. Given experimental dynamic in vivo fluorescence data obtained subsequent to the injection of the Cy5.5-c(KRGDf) conjugate at various intervals following the administration of the c(KRGDf) peptide, one may be able to experimentally determine receptor turnover time. Finally, since α+β is equivalent to the sum of all the rate constants of the compartmental pharmacokinetic model, and since the α_vβ₃ integrin receptors bind the c(KRGDf) peptide such that pretreatment with the c(KRGDf) peptide results in the elimination of the cellular uptake of the dye conjugate, the difference in the value of α+β between animals injected with the conjugate dye alone and animals injected with the conjugate 1 h after the pretreatment with the RGD peptide may indicate the molecularly specific cellular uptake of the conjugate. Using this rationale, we found the rate constant for the specific cellular uptake of Cy5.5-c(KRGDf) to have a mean value of k_c=0.16 s ⁻¹.

While the sum of α and β can be used to provide model-dependent information about molecular binding as described earlier, the regression values for the PK complex rate constants, α and β as well as the PK pre-exponential factors A and B are individually presented in Fig. 5. As shown in Figs. 5(a) and 5(b), the complex rate constant, α, is greater than β in these early time-course studies and shows significant differences between normal and tumor tissues when Cy5.5-c(KRGDf) is administered. The differences disappear when Cy5.5-c(KRGDf) is administered 1 h after c(KRGDf) is administered as a blocking ligand, but reappear again when administered 24 h after c(KRGDf) is administered. The complex rate constant, β, does not show significant differences between these groups in the early time courses investigated herein. Further study at longer times after administration of Cy5.5-c(KRGDf) need to be conducted in order to effectively address possible differences. The pre-exponential factors illustrated in the histograms of Figs. 5(c) and 5(d) are truly arbitrary factors which could vary with external factors such as variation in illumination power on the animal surface. Nonetheless, with the exception of the free Cy5.5 dye, the tumor regions possess greater values of the pre-exponential factors indicative of perhaps of the increased vascularity. Clearly, owing to the large number of parameters that constitute α, β, A, and B individually, it is not possible to extract biophysical information in the same manner as one can for the sum of α+β.

Figure 5

Results of nonlinear least-squares regression for determining the PK complex rate constants (a) α and (b) β as well as the PK pre-exponential factors (a) A and (b) B from Eq. (13). The column height represents the mean value of the test group listed along the abscissa while the error bars represent the standard deviation. The time in parentheses represents the time between injection for the c(KRGDf) peptide and the Cy5.5-c(KRGDf) conjugate. Figure 5(b) is similar to Fig. 4 owing to the small values of β.

Fluorescence images of all animals studied were also obtained at 24 h intervals following the administration of the contrast agent. Figure 6 displays the images of raw fluorescence (i.e., no background subtraction) overlaid with white light images and obtained 24 and 48 h following the initial injection of the contrast agent for a representative animal of each of the four test groups studied. From the images of Fig. 6(a) it is clear that the KS1767 tumor was not visible from the fluorescence images at any time following the injection of the free Cy5.5 dye. In this case and the other cases as well, the visible fluorescence intensity originating from the animal gut is due to animals’ diet. The KS1767 tumor was clearly visible 24 and 48 h after the administration of the Cy5.5-c(KRGDf) peptide, as evidenced in Figs. 6(b)–6(d), and returned to base line after about 168 h. At any given time, the fluorescence intensity originating from the tumor was highest for the animal that was administered the Cy5.5-c(KRGDf) conjugate alone, Fig. 6(b); slightly diminished for the animal injected with the RGD peptide 24 h prior to the administration of the conjugate dye, Fig. 6(d); and lower still for the animal that received the RGD peptide 1 h prior to the administration of the Cy5.5-c(KRGDf) conjugate, Fig. 6(c). These results further substantiate the specific in vivo cellular binding of the Cy5.5-c(KRGDf) conjugate to KS1767.

Figure 6

Raw fluorescence images (no background subtraction) obtained 24 h (first column) and 48 h (second column) after the administration of (a) Cy5.5, (b) Cy5.5-c(KRGDf) conjugate alone, (c) Cy5.5-c(KRGDf) conjugate 1 h after the injection of RGD peptide, and (d) Cy5.5-c(KRGDf) conjugate 24 h after injection of the RGD peptide. The white arrows indicate the location of the xenografted Kaposi’s sarcoma tumor.