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Biochemistry and Biophysics |
-Melanocyte-stimulating Hormone Peptide Analogues1
Departments of Biochemistry [JQ. C., T. P. Q.] and Chemistry [Z. C., S. S. J.], University of Missouri-Columbia, Columbia, Missouri 65211, and Harry S. Truman Memorial Veterans Hospital, Columbia, Missouri 65201 [T. J. H.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-MSH3–13 (CCMSH)
exhibits high tumor uptake and retention values in a murine melanoma
mouse model. In this report, the tumor targeting mechanism of
99mTc-CCMSH was studied and compared with four other
radiolabeled -melanocyte stimulating hormone (-MSH) peptide
analogues: 125I-(Tyr2)-[Nle4,
D-Phe7]-MSH
[125I-(Tyr2)-NDP];
99mTc-CGCG-NDP; 99mTc-Gly11-CCMSH;
and 99mTc-Nle11-CCMSH. In vitro
receptor binding, internalization, and cellular retention of
radiolabeled -MSH analogues in B16/F1 murine cell line demonstrated
that >70% of the receptor-bound radiolabeled analogues were
internalized together with the receptor. Ninety % of the internalized
125I-(Tyr2)-NDP, whereas only 36% of
internalized 99mTc-CCMSH, was released from the cells into
the medium during a 4-h incubation at 37°C. Two mouse models, C57
mice and severe combined immunodeficient (Scid) mice, inoculated s.c.
with B16/F1 murine and TXM-13 human melanoma cells were used for the
in vivo studies. Tumor uptake values of 11.32 and 2.39
[% injected dose (ID)/g] for 99mTc-CCMSH at 4 h
after injection, resulted in an uptake ratio of tumor:blood of 39.0 and
11.5 in murine melanoma-C57 and human melanoma-Scid mouse models,
respectively. Two strategies for decreasing the nonspecific kidney
uptake of 99mTc-CCMSH, substitution of Lys11 in
CCMSH with Gly11 or Nle11, and lysine
coinjection, were evaluated. The biodistribution data for the modified
peptides showed that Lys11 replacement dramatically
decreased the kidney uptake, whereas the tumor uptakes of
99mTc-Nle11- and
99mTc-Gly11-CCMSH were significantly lower than
that of 99mTc-CCMSH. Lysine coinjection significantly
decreased the kidney uptake (e.g., from 14.6% ID/g to
4.5% ID/g at 4 h after injection in murine melanoma-C57 mice)
without significantly changing the value of tumor uptake of
99mTc-CCMSH. In conclusion, the compact cyclic structure of
99mTc-CCMSH, its resistance to degradation, and its
enhanced intracellular retention are the major contributing factors to
the superior in vivo tumor targeting properties of
99mTc-CCMSH. Lys11 residue in
99mTc-CCMSH is critical to the tumor targeting in
vivo, and lysine coinjection rather than lysine replacement can
significantly decrease the nonspecific renal radioactivity accumulation
without impeding the high melanoma-targeting properties of
99mTc-CCMSH. The metal-cyclized CCMSH molecule displays
excellent potential for the development of melanoma-specific diagnostic
and therapeutic agents. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An alternative approach for tumor targeting is the use of lower
molecular weight, high-affinity receptor binding ligands, such as
peptides, for tumor-selective delivery of radionuclides (13
, 14)
. For instance, the receptor for
-MSH3
is found on murine (15)
and human melanoma cells
(16)
. It has been reported that >80% of human melanoma
tumor samples obtained from patients with metastatic melanoma bear
-MSH receptors (17)
, enabling the use of radiolabeled
-MSH peptide analogues as specific melanoma diagnostic and
therapeutic agents. Wild-type -MSH
(Ac-Ser1-Tyr2-Ser3
-Met4-Glu5-His6-Phe7-Arg8-Trp9-Gly10-Lys11-Pro12-Val13-NH2)
is a tridecapeptide that is primarily responsible for the regulation of
skin pigmentation (18)
. Several superpotent -MSH
analogues have been synthesized that possess increased receptor
affinity and stability (19
, 20)
. For example, substitution
of Met4 with Nle4 and
Phe7 with
D-Phe7 yields the NDP analogue, which
is one of the most cited -MSH analogues because of its subnanomolar
receptor affinity and resistance to enzymatic degradation (16
, 19)
. It has also been reported that -MSH peptides cyclized
via disulfide bond [Cys (4
, 10)
,
D-Phe7]-MSH (20)
or
lactam bond formation [Asp5,
D-Phe7,
Lys11-MSH (21
, 22)
display
increased receptor binding affinity and resistance to proteolysis. High
receptor affinity and stability have made these -MSH analogues
attractive as site-specific delivery vehicles for radionuclides,
toxins, and chemotherapeutic molecules (23
, 24)
.
Recently, several radiolabeled -MSH peptide analogues have been
investigated for melanoma-specific targeting. Direct halogenation of
NDP with 125I at Tyr2
resulted in a radioiodinated -MSH analogue with excellent
cell-binding characteristics in vitro (25)
, but
it was subject to dehalogenation in vivo. Dehalogenation has
been largely overcome by labeling NDP with succinimidyl 3- or
4-(125I or 18F) benzoate,
yielding molecules that display significant improvements in stability
and clearance from normal tissues (26
, 27)
.
111In-labeled -MSH derivatives containing two
NDP sequences linked together via a single DTPA molecule have been
examined for their abilities to image the melanoma lesions in patients
(14)
. Although the 111In-labeled
DTPA-bis-NDP conjugates were able to image melanoma tumors in
vivo, routine clinical use appears limited because of high
nonspecific radioactivity accumulation in the liver and kidneys
(28)
. To decrease the background in the liver and kidneys,
111In-labeled DTPA-mono-NDP has also been
investigated (29)
. However, the tumor uptake of
111In-labeled DTPA-mono-NDP was significantly
lower than that of 111In-labeled DTPA-bis-NDP.
Our previous results with
99mTc/188Re-NDP, labeled
with either the CGCG chelating tetrapeptide (30)
or
MAG2 chelate (tetrafluorophenyl
mercapto-acetylglycylglycyl-
-aminobutyrate), illustrated that
tumor-targeted radioactivity was rapidly washed out of the tumor tissue
(31)
.
Significantly higher tumor uptake values were obtained for
99mTc/188Re-labeled CCMSH,
an 11-amino acid -MSH peptide analogue cyclized via metal
coordination with three Cys3,4,10 sulfhydryls and
one Cys4 amide nitrogen positioned in the
sequence of the peptide (23
, 32)
. The
99mTc/188Re-CCMSH exhibited
excellent tumor radioactivity retention and fast whole body clearance
via the kidneys into the urine. However, significant kidney
radioactivity accumulation was observed, which could limit the
therapeutic index if a ß- or -emitting radionuclide is coordinated
to the peptide for therapeutic purposes.
In this study, the mechanism of 99mTc-CCMSH tumor
uptake was studied by characterizing the receptor binding,
internalization, and cellular retention of the radiolabeled complex in
B16/F1 murine melanoma cells in vitro and compared with
125I-(Tyr2)-NDP and
99mTc-CGCG-NDP. Self-regulation of the -MSH
receptor by NDP and receptor recovery was also investigated. The
in vivo tumor targeting capacity of
99mTc-CCMSH was evaluated in both C57 BL/6 and
Fox Chase ICR Scid mouse melanoma models, inoculated s.c. with B16/F1
murine and TXM-13 human melanoma cells and compared with that of
125I-(Tyr2)-NDP and
99mTc-CGCG-NDP. The specificity of
99mTc-CCMSH tumor uptakes was determined in
vivo by a coinjection of 2 µg of nonradiolabeled NDP. In
addition, two strategies of blocking nonspecific kidney uptake of
99mTc-CCMSH were examined. One of the strategies
involved the replacement of Lys11 in CCMSH with
Nle11 or Gly11. The
in vitro receptor binding affinity
(IC50) and in vivo biodistribution of
the modified peptides,
99mTc-Nle11-CCMSH and
99mTc-Gly11-CCMSH, were
tested and compared with those of 99mTc-CCMSH.
The other strategy investigated for reducing nonspecific renal uptake
was lysine coinjection. Blocking the nonspecific kidney uptake with two
different quantities and times of lysine injection was examined in
healthy C57 mice and then in B16/F1 melanoma-bearing mice. Tumor
imaging was performed at 1 and 8 h after the administration of
99mTc-CCMSH in murine melanoma-bearing C57 mice.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preparation of Radiolabeled Complexes.
125I-(Tyr2)-NDP (2000
Ci/mmol) was obtained from Advanced ChemTech (Louisville, KY).
99mTc-labeled CCMSH,
Gly11-CCMSH, Nle11-CCMSH,
and CGCG-NDP were prepared by using stannous chloride as a reducing
agent and glucoheptonate as a transfer ligand. Briefly, 30 µl of 2
mg/ml SnCl2 in 0.2 M glucoheptonate
were added into 200 µl of fresh
99mTcO4-
eluate from a 99 Mo/99mTc
generator (1–4 mCi). After the formation of
99mTc-glucoheptonate and the adjustment of the pH
to 9 with 0.1 M NaOH, 10 µg of the peptide were added,
and the reaction mixture was incubated at 75°C for 30 min. The
radiolabeled complex was purified by HPLC using a C-18 RP column. A
20-min gradient of 17–22% acetonitrile/0.1% TFA versus
H2O/0.1% TFA was used for the purification of
99mTc-CCMSH, and a 20-min gradient of 20–25% or
24–29% acetonitrile/0.1% TFA versus
H2O/0.1% TFA was used for the purification of
99mTc-Gly11-CCMSH,
99mTc-Nle11-CCMSH, or
99mTc-CGCG-MSH, respectively. Purified
preparations were flushed with nitrogen gas to remove the acetonitrile,
and the pH was adjusted to 7 by addition of 0.2 M
sodium phosphate (pH 8.0)/150 mM NaCl. The
stability of the radiolabeled complexes was evaluated in 0.01
M, pH 7.4 PBS.
Cell Culture.
B16/F1 murine melanoma cells were obtained from American Type Tissue
Culture Collection, and human TXM-13 melanoma cells were supplied by
Dr. Isaiah J. Fidler from the Cell Biology Department, University of
Texas M. D. Anderson Cancer Center. The human melanoma line TXM-13JQ
was derived from the parent cell line TXM-13 after successive passages
in vitro. Melanoma cells were cultured in RPMI 1640
containing NaHCO3 (2 g/l), which was supplemented
with 10% heat-inactivated FCS, 2 mM
L-glutamine, and 48 mg of gentamicin. The cells
were expanded in 75-cm2 tissue culture flasks and
kept in a humidified atmosphere of 5% CO2 at
37°C. The media were changed every other day. A confluent monolayer
was detached with 0.02% EDTA in Ca2+ and
Mg2+ free, pH 7.4, 0.01 M
PBS and dissociated into a single-cell suspension for further cell
culture.
In Vitro Cell Assays.
Receptor binding affinity, internalization and cellular retention
assays were performed on B16/F1 murine cells. Cell binding experiments
were performed as follows. Cells were seeded at a density of 0.2
million/well in 24-well tissue culture plates and allowed to attach
overnight. After washing once with the binding medium [MEM with 25
mM HEPES, 0.2% BSA, and 0.3 mM
1,10-phenanthroline (Sigma, St. Louis, MO)], the cells were incubated
at 25°C for 3 h with 
50,000 cpm of
125I-(Tyr2)-NDP or 200,000
cpm of the 99mTc-labeled analogues in 0.5 ml of
binding medium. The nonspecific binding was determined by coincubation
with nonradiolabeled NDP at a final concentration of 10
µM. The cells were rinsed twice with pH 7.4, 0.01
M PBS/0.2% BSA and lysed in 0.5 ml of 1 M NaOH
for 5 min, and their radioactivity was measured.
Internalization and cellular retention of
99mTc-CCMSH was evaluated and compared with
99mTc-CGCG-NDP and
125I-(Tyr2)-NDP in B16/F1
murine melanoma cells. The cells were prepared as described above in
24-well tissue culture plates and incubated at 25°C for a period of 5
min to 4 h in 0.5 ml of binding media with 50,000 cpm of
125I-labeled or 200,000 cpm of the
99mTc-labeled complexes. Internalization of the
radiolabeled complex was assessed by washing the cells with acid buffer
[40 mM sodium acetate (pH 4.5) containing 0.9% NaCl and
0.2% BSA] to remove the membrane bound radiocomplex, and the
remaining internalized radioactivity in the cells was measured. The
cellular retention properties of the internalized
125I- or 99mTc-labeled
-MSH analogues were determined by incubating B16/F1 cells with the
radiolabeled analogues for 3 h, removing the membrane-bound
radioactivity with acid buffer wash, and monitoring radioactivity
release into the media at 25°C or 37°C. At different time points
over a 4-h incubation period, the radioactivity in the medium and in
the cells were separately collected and counted. The radioactive moiety
in the medium was analyzed using a C-18 Sep-Pak Cartridge (Water
Corporation, MA). After equilibrating the cartridge with 10%
acetonitrile/H2O, 200 µl of the sample were
loaded onto the column, which was then rinsed with 3 ml of 10%
acetonitrile/H2O, followed by 3 ml of 60%
acetonitrile/H2O. Free
99mTcO4-,
99mTc-labeled analogues, and
125I-(Tyr2)-NDP were used
as controls.
Self-regulation of -MSH receptors by NDP was examined in B16/F1
cells. Cells were treated for 24 h in RPMI 1640 supplemented with
10% dialyzed FCS (10,000 molecular weight cutoff dialyzed FCF; Sigma
Chemical Co., St. Louis, MO) or in 10% normal FCS-supplemented RPMI
1640 containing NDP at a concentration of 20 or 50
nM. After removal of the membrane-bound NDP by washing the
cells with acid buffer, the cells were further cultured in medium. At
different time points within a period of 96 h, the binding
capacity of the treated cells for
125I-(Tyr2)-NDP was
determined.
The IC50, the concentration of competitor required to inhibit 50% of radioligand binding, of CCMSH, ReCCMSH, Gly11-CCMSH, and Nle11-CCMSH was determined in competitive binding assays with 125I-(Tyr2)-NDP over a 10-14–10-6 (M) concentration range. The Bmax of B16/F1 and TXM-13 cells was determined by incubating a fixed number of cells with serial concentrations of 125I-(Tyr2)-NDP under the receptor binding conditions described above. Meanwhile, the binding fraction (1/r) of 125I-(Tyr2)-NDP to B16/F1 cells was assayed according to a method that was developed to determine the immunoreactive fraction of radiolabeled antibodies (33) . Briefly, serially diluted B16/F1 cells suspensions (0.125–16 million) in 0.5 ml of binding medium were incubated with a certain concentration of 125I-(Tyr2)-NDP at 25°C for 3 h. Total applied radioactivity over specific binding (total/specific binding) was used as a function of the reverse cell concentration (ml/million), and r was obtained by linear extrapolation to the ordinate.
In Vivo Biodistribution, Imaging, and Tumor Tissue
Histopathological Studies.
C57 BL/6 and Fox Chase ICR Scid female mice (Harlan Sprague Dawley,
Indianapolis, IN) were inoculated s.c. in the right flank with
1 x 106 cultured B16/F1 murine
and TXM-13JQ human melanoma cells, respectively. Ten days after the
inoculation, tumors reached a weight of 500 mg. It took 4 weeks for
the TXM-13 human melanoma tumors to reach a weight of 500 mg,
induced by s.c. inoculation with 3 x 106 cells in Scid mice. Two to four µCi of
99mTc- or 125I-labeled
peptide (72,000 Ci/mmol or 2,000 Ci/mmol, with a corresponding peptide
amount of 0.065 or 2.6 ng, respectively) was injected into each mouse
through the tail vein for biodistribution and in vivo tumor
targeting studies. About 20 µCi of
99mTc-peptide was administrated into each mouse
for the 24-h postinjection group. After injection, the mice were housed
separately, and their urine and feces were collected. Groups of five
mice were sacrificed at different time points after injection. Tumor
and normal tissues of interest were dissected, and the blood on the
samples was sponged off with gauze. The contents in the GI were not
removed. Tissue samples and the carcass were weighed, and their
radioactivity was measured in a gamma counter, together with the
collected urine and feces. The total blood value was counted as 6.5%
of the whole body weight. The radioactivity uptake in the tumor and
normal tissues of interest was expressed as a percentage of the injected radioactivity dose per gram of tissue (% ID/g) or percentage
of the injected dose (% ID). The specificity of the in vivo
tumor uptake of 99mTc-CCMSH was determined by a
coinjection with 2 µg of nonradiolabeled NDP, a 30,000 fold molar
excess to 99mTc-CCMSH.
The effect of lysine coinjection on the reduction of nonspecific kidney uptake of 99mTc-CCMSH was determined in healthy C57 BL/6 mice. Group 1 was i.p. injected with 20 mg of lysine (L-lysine monohydrochloride; Fisher Biotech, Houston, TX), and 5 min later injected with a mixture of 99mTc-CCMSH and 25 mg of lysine through the tail vein. Group 2 was i.v. injected with a mixture of 99mTc-CCMSH and 25 mg of lysine only. A single injection of 99mTc-CCMSH with 30 mg of lysine was used for further studies in the tumor model to check whether the lysine coinjection would interfere with the tumor targeting property of 99mTc-CCMSH.
In the imaging studies, mice were injected with 100 µCi of
99mTc-CCMSH through the tail vein. Mouse images
were acquired at 1 and 8 h after injection by a Siemens LEM + Mobile camera equipped with a low energy parallel hole
collimator. The images were collected on a 512 matrix with x1.5
magnification and 8-bit depth.
For histopathological examination, the tumor tissues were dissected and then immediately fixed in buffered 10% formalin. The fixed tumor tissue was sliced in a thickness of 5 µm and stained in H&E staining. Histopathological studies were performed by the Research Animal Diagnostic and Investigative Laboratory (University of Missouri, Columbia, MO).
All of the animal studies were carried out in compliance with Federal and local institutional rules for the conduct of animal experimentation. Statistical analysis was performed using the Student’s t test for unpaired data.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-MSH analogues, except
125I-(Tyr2)-NDP, were
synthesized by solid-phase Fmoc synthesis, purified by RP-HPLC and
characterized by FAB mass spectrometry. Table 1
lists the sequences used in this study and the calculated and measured
molecular weights of the -MSH analogues. The
99mTc-labeled peptides were completely separated
from their nonradiolabeled counterparts by RP-HPLC because of an
increase in hydrophobicity. The structure of metal-cyclized
99mTc-CCMSH is shown in Fig. 1
. In addition to mass spectrometry, the identity of the
99mTc-CCMSH was confirmed by analytical HPLC
analysis. A Re-CCMSH nuclear magnetic resonance standard sample was
coinjected with a 99mTc-CCMSH radiolabeling
reaction mixture. The major product of the
99mTc-CCMSH reaction comigrated with the Re-CCMSH
standard (Fig. 2)
. A similar analytic HPLC analysis, using a Re-CGCG-NDP synthetic
standard (30)
, was used to confirm the identity of the
99mTc-CGCG-NDP (data not shown). The
radiochemical stability of all of the
99mTc-labeled analogues was evaluated in pH 7.4,
PBS. Over a 24-h period of incubation at 25°C in PBS, only
radiolabeled peptide and no detectable free radioactivity was observed
by RP-HPLC.
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shows the cell binding and internalization of
125I-(Tyr2)-NDP (Fig. 3A)
, 99mTc-CCMSH (Fig. 3B)
,
and 99mTc-CGCG-NDP (Fig. 3C)
on B16/F1
cells at 25°C over a 4-h incubation period. Four to 8% cell binding
was obtained for all three radiolabeled analogues with 3 h
incubation. The internalization was assessed after removal of free and
membrane-bound radioactivity by washing the cells with pH 4.5, 40
mM sodium acetate acid buffer and collecting the
remaining cellular radioactivity. Approximately 70% of the
receptor-bound radiolabeled complexes was internalized as early as 5
min after incubation for the
125I-(Tyr2)-NDP in B16/F1
cells. Although receptor binding increased over the incubation period,
the percentages of the radioactivity internalized versus
total binding did not significantly change. Similar results were
observed among the three 99mTc- and
125I-labeled analogues.
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illustrates the cellular radioactive retention of
99mTc-CCMSH,
99mTc-CGCG-NDP, and
125I-(Tyr2)-NDP in B16/F1
cells over time at different temperatures. Approximately 36% of the
internalized radioactivity of 99mTc-CCMSH and
99mTc-CGCG-NDP and 90% of the internalized
125I-(Tyr2)-NDP were
released from the cells into the medium during a 4-h incubation at
37°C (Fig. 4A)
. Greater than 75% of the internalized
125I-(Tyr2)-NDP activity
was released from the cells within the first 2 h. However, no
significant difference in cellular radioactive retention was found
among these three radiolabeled analogues at 25°C incubation
temperature (Fig. 4B)
. The radioactive moiety in the medium
released from the cells was analyzed on a C-18 Sep-Pak column. Free
99mTcO4-
was rinsed off the column at 10% acetonitrile/H2O, whereas
125I- and 99mTc-labeled
peptides were eluted by 60% acetonitrile/H2O. In
media samples isolated from B16/F1 cells treated with
125I-(Tyr2)-NDP or
99mTc-labeled analogues, 80% of the
radioactivity was eluted with 10%
acetonitrile/H2O, suggesting that the majority of
the radioactivity released from the cells into the media consisted of
low molecular weight forms.
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-MSH receptor regulation and turnover was examined in
B16/F1 cells. B16/F1 cells were incubated for 24 h with NDP (20 or
50 nM) or with dialyzed FCS. The binding capacity of the
treated cells for
125I-(Tyr2)-NDP
versus incubation time at 25°C is shown in Fig. 5A
. Compared with the control group, cell binding increased by
20% for the cells cultured with media supplemented with 10%
dialyzed FCS. The percentage of cell binding for the hormone-treated
cells, both at 20 and 50 nM, was low at all time
points over the 3-h incubation period. The binding capacity of the 50
nM NDP-treated cells for
125I-(Tyr2)-NDP did not
completely recover until 96 h of incubation in normal media (Fig. 5B)
.
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-MSH analogues were examined in healthy and tumor-bearing mouse
models. Fig. 6
illustrates a comparison of the biodistribution properties among
99mTc-CCMSH,
99mTc-CGCG-NDP, and
125I-(Tyr2)-NDP in B16/F1
murine melanoma-bearing C57 mice at 4 h after injection. The tumor
uptake for 99mTc-CCMSH was significantly higher
than that of 99mTc-CGCG-NDP and
125I-(Tyr2)-NDP
(P < 0.001 and 0.001 < P < 0.01, respectively). The percentage of
radioactivity in the blood of mice injected with
99mTc-CCMSH and
99mTc-CGCG-NDP was significantly lower than that
of 125I-(Tyr2)-NDP
(P < 0.001). The majority of the
radioactivity for 99mTc-CCMSH and
125I-(Tyr2)-NDP was
eliminated via the kidneys into the urine, whereas the radioactivity of
99mTc-CGCG-NDP was cleared from both urine and
the GI tract. Because of the high tumor uptake and fast clearance, the
tumor:blood uptake ratio for 99mTc-CCMSH was 6.3-
and 21.5-fold higher than that of 99mTc-CGCG-NDP
and 125I-(Tyr2)-NDP,
respectively. However, the kidney uptake of
99mTc-CCMSH was 14.6 ± 1.9 (%
ID/g), which was 5–12 times higher than that of
99mTc-CGCG-NDP and
125I-(Tyr2)-NDP.
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. The receptor binding affinity of the modified analogues was about 10
times lower than that of NDP, with IC50s of 1.9,
3.8, and 0.21 nM for Nle11-CCMSH,
Gly11-CCMSH, and NDP, respectively. However, the
receptor binding affinity of the modified peptides did not change
significantly compared with that of CCMSH (Table 2)
.
|
. Although the kidney uptake of
99mTc-Nle-11-CCMSH and
99mTc-Gly11-CCMSH was
dramatically decreased, their tumor uptake was also significantly lower
than that of 99mTc-CCMSH (P < 0.001). There were also differences between these two modified
peptides, with the tumor and kidney uptakes of
99mTc-Nle11-CCMSH being
significantly higher than those of
99mTc-Gly11-CCMSH. In
addition, the replacement of the Lys11 in CCMSH
with Nle11 or Gly11
partially changed the path of the clearance from urine to GI. Greater
than 50% ID of the radioactivity of
99mTc-Nle11-CCMSH and
99mTc-Gly11-CCMSH was
cleared through the GI at 1 h after injection, whereas only about
20% ID was eliminated in urine (Fig. 7)
.
|
shows the results of the lysine coinjection for blocking the
nonspecific kidney uptake of 99mTc-CCMSH in two
different groups of healthy C57 mice at 30 min and 1 h after
injection. Group 1 was first i.p. injected with 20 mg of lysine and
then i.v. injected with a mixture of 99mTc-CCMSH
and 25 mg of lysine, and group 2 was i.v. injected with a mixture of
99mTc-CCMSH and 25 mg of lysine only. There was
no significant difference in the efficiency of reducing the kidney
uptake between these two groups (Table 3)
. Hence, a single coinjection
of 99mTc-CCMSH with 30 mg of lysine was performed
in melanoma-bearing C57 mice to determine whether the lysine
coinjection would interfere with the tumor uptake. Table 4
lists the biodistribution data of 99mTc-CCMSH
with or without a 30-mg lysine coinjection in murine melanoma-bearing
mice at 0.5, 1, 4, and 24 h after injection. Except for the
kidney, the uptake values of 99mTc-CCMSH in both
tumor and normal tissues of interest and the clearance kinetics were
not significantly changed by the lysine coinjection. Lysine coinjection
was successful in reducing nonspecific kidney uptake by 48, 55, and
70% at 0.5, 1, and 4 h after injection, respectively (Table 4)
.
|
|
.
|
. Although the radioactivity accumulation in the bladder is very high,
the tumor spot is identifiable 1 h after injection image (Fig. 9
,
left). After the mouse voids its bladder contents, the
extremely high tumor uptake and low background in the normal tissues
are observed as illustrated in the image obtained 8 h after
injection of radioactivity (Fig. 9
, right).
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shows the biodistribution, tumor uptake, and tumor:tissue uptake ratios
of 99mTc-CCMSH in TXM-13 human melanoma bearing
Scid mice at 30 min, 1, 4, and 24 h after injection with or
without lysine coinjection. The biodistribution of the
99mTcCCMSH in normal tissues of interest was
comparable between the two mouse models (e.g., TXM-13 human
melanoma-bearing Scid mice and B16/F1 murine melanoma-bearing C57 mice;
Tables 4
and 5
). Compared with murine melanoma, the tumor uptake of
99mTc-CCMSH in human melanoma was lower, which
may be attributable to its lower receptor density and differences in
in vivo behavior. However, a tumor uptake of 2.39 ± 0.42% ID/g was obtained at 4 h after injection in TXM-13
melanoma-bearing Scid mice, resulting in an uptake ratio of
tumor:normal tissue of 11.5 and 50.9 for the blood and the muscle,
respectively (Table 5)
. Biodistribution results of
99mTc-CCMSH in Scid mice bearing the melanonic
TXM-13JQ human melanoma at 30 min and 4 h after injection are
illustrated in Fig. 10
. The tumor uptake at both 30 min and 4 h after injection was
(7.64 ± 2.12 and 6.55 ± 1.31,
respectively), significantly higher than that in Scid mice bearing the
amelanonic solid TXM-13 human melanoma (0.001 < P < 0.01 and P < 0.001, respectively).
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. All of the three pictures show unencapsulated masses composed of round
cells with distinct borders, moderate basophilic cytoplasm, often
containing brown to black intracytoplasmic pigment granules. The cells
are also marked by anisokaryosis of the round nucleoli, which are
consistent with malignant melanoma. Morphologically, the B16/F1- and
TXM-13JQ-induced tumors formed melanonic gelatinous masses, whereas
TXM-13 induced an amelanonic solid mass tumor. Moreover, severe
necrosis was observed in the amelanonic solid TXM-13 human melanoma
tumor (Fig. 11C)
, whereas necrosis was much less prevalent
in the TXM-13JQ tumor tissue and rarely seen in the B16 tumor (Fig. 11, A and B)
.
|
|
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this investigation, the data illustrated that 70% of the
membrane-bound radiolabeled -MSH analogues were internalized in both
B16/F1 murine (Fig. 3)
and TXM-13 human melanoma cell lines (data not
shown). Although there was no difference in cellular retention between
these three radiolabeled analogues at 25°C, the cellular retention of
the 99mTc-labeled analogues was 2.5-fold higher
than that of the radioiodinated analogue at 37°C (Fig. 4)
. C-18
Sep-Pak column analysis of the radioactive moiety in the medium showed
that the majority of the radioactivity released from the cells into the
media had been metabolized into low molecular weight forms. No
differences in the cellular retention of
99mTc-CCMSH and
99mTc-CGCG-NDP were found in vitro,
contradicting the results observed in vivo (Fig. 6)
, which
might be attributable to differences in metabolism of the radiolabeled
complexes in vitro and in vivo.
The results of NDP-induced down-regulation of -MSH receptors on
B16/F1 cells (Fig. 5)
were consistent with previous reports (38
, 39)
, which suggested that -MSH peptides are internalized
together with their receptors upon binding. The use of dialyzed FCS
eliminated trace levels of -MSH hormone in the FCS supplemented
media, resulting in a slight increase in B16/F1 cell receptor binding
of 125I-(Tyr2)-NDP.
However, the treatment of the B16/F1 cells with NDP at a concentration
of as low as 20 nM blocked -MSH receptor-mediated
binding of the iodinated NDP analogue, illustrating sensitivity of the
receptor for its cognate ligand. Receptor internalization and slow
recovery highlight the importance of developing a -MSH receptor
targeting agent that not only possess high affinity but also an equally
high degree of cellular retention.
In this study, a cell binding value of 4–8% was obtained for all
three radiolabeled analogues, which is significantly lower than the
percentage of cell binding values reported for radiolabeled antibodies
(40)
. The difference in percentage of cell binding values
between 99mTc-CCMSH and radiolabeled antibodies
is primarily attributable to the concentration of receptor or antigen.
To illustrate the effect of receptor concentration on the binding
capacity, the binding fraction (1/r) of
125I-(Tyr2)-NDP to B16/F1
cells was tested by an immunoreactivity assay, commonly used to
determine the bioactivity of a radiolabeled antibody (33)
.
A binding fraction (1/r) of 0.37 was obtained for the
radioiodinated NDP (2,000 Ci/mmol) to B16/F1 cells. Because the
receptor density on B16/F1 cells was 7,000/cell, a cell concentration
of 32 million cells/ml represented a receptor concentration of 0.37
nM, which was approximately the Ki of
NDP. Hence, the low percentage of cell binding for
125I-(Tyr2)-NDP observed in
this investigation (Fig. 3)
was attributable to the low receptor
concentration on the cells (e.g., 0.2 million cells/0.5 ml
only represented a receptor concentration of 4.6
pM, which is 45 times lower than the
Ki of NDP). This result demonstrates that the
immunoreactivity assay is difficult to apply when receptor
concentration is low.
The in vivo results showed that the tumor uptake of
99mTc-CCMSH was significantly higher than that of
99mTc-labeled and radioiodinated linear NDP
analogues in the murine melanoma mouse model. The tumor:blood uptake
ratio of 99mTc-CCMSH at 4 h after the
injection time point was 6.3- and 21.5-fold higher than that of
99mTc-CGCG-NDP and
125I-(Tyr2)-NDP,
respectively (Fig. 6)
. This result was consistent with our previous
report that the radiolabeled linear -MSH analogues were rapidly
washed out of the tumor tissue (31)
. In comparison with
the linear analogues, 99mTc-CCMSH is cyclized via
the site-specific metal coordination by three
Cys3,4,10 sulfhydryls and a
Cys4 amide nitrogen (23
, 32)
. The
cyclized and compacted structure of 99mTc-CCMSH
appears to enhance the resistance of the molecule to proteolysis and
contributes significantly to the high in vivo tumor uptake
and retention. The high tumor uptake of
99mTc-CCMSH was shown to be specific, because
84% of the tumor uptake was identically blocked by coinjection of 2
µg of nonradiolabeled NDP (Fig. 8)
. The extremely high tumor uptake
and retention of 99mTc-CCMSH is also identified
in the 8-h postinjection imaging (Fig. 9)
.
The nonspecific radioactivity accumulation in the kidneys is often
associated with the in vivo application of radiolabeled
peptides and antibody fragments (23
, 28
, 34)
. It was
hypothesized that the positive charge of the lysine residue in the
99mTc-CCMSH sequence contributed significantly to
the nonspecific radioactivity kidney retention. Substitution of
Lys11 with Nle11 or
Gly11 in the 99mTc-CCMSH
sequence yielded analogues with reduced kidney uptake (Fig. 7)
, but the
tumor uptake of the two analogues was also significantly lower compared
with that of 99mTc-CCMSH. Although the
Nle11-CCMSH and Gly11-CCMSH
analogues showed similar receptor binding affinities to CCMSH in
vitro (Table 2)
, the loss of the lysine residue at position 11
adversely affected tumor uptake in vivo (Fig. 7)
. These
results demonstrate that the lysine residue in
99mTc-CCMSH is critical for effective melanoma
targeting in vivo. Moreover, the Lys11
replacement in 99mTc-CCMSH altered the pathway of
clearance from urine to the GI tract, which slowed the clearance
kinetics of the 99mTc-labeled analogues from the
whole body. However, nonspecific kidney retention of
99mTc-CCMSH was effectively reduced by lysine
coinjection. A single injection of 30 mg of lysine together with the
99mTc-CCMSH decreased the kidney uptake by 48,
55, and 70% at 30 min and 1 and 4 h after injection without
altering tumor uptake. Lysine coinjection showed more efficient
blocking at 4 h after injection, suggesting that part of the
radioactivity in the kidney at earlier time points was attributable to
radioactivity clearance from the kidney into the urine. Kidney uptake
with the lysine coinjection was lower, but without statistical
difference (P > 0.05), than that of the
control group at 24 h after radiocomplex administration (Table 4)
.
Substantial tumor uptake of 99mTc-CCMSH was also
demonstrated in the TXM-13 and TXM-13JQ human melanoma-bearing Scid
mice models (Table 5
; Fig. 10
). The biodistribution of
99mTc-CCMSH in normal tissue in TXM-13 human
melanoma-bearing Scid mice was similar to that in B16/F1 murine
melanoma-bearing C57 mice (Tables 4
and 5)
. It appeared that the lower
-MSH receptor density and tumor morphology contributed to the
reduced tumor uptake of 99mTcCCMSH in the
TXM-13 human melanoma xenografts compared with that in the B16/F1
murine melanoma in the C57 mouse model. There was a significant
difference in uptake of 99mTc-CCMSH between
TXM-13- and TXM-13JQ-induced human melanoma tumors. Tumor uptake of
99mTc-CCMSH at 4 h after injection increased
from 2.39 ± 0.41 (% ID/g) in TXM-13 to 6.55 ± 1.31 (% ID/g) in TXM-13JQ. The TXM-13JQ cell line was derived
from the original TXM-13 cell line by successive passaging in
vitro. In vivo TXM-13JQ formed uniform melanonic gelatinous tumors
with limited necrotic centers in contrast to TXM-13, which formed
amelanonic solid tumors with extensive necrotic centers (Fig. 11)
.
Clearly, the extent of necrosis within the TXM-13 human melanoma
affects the tumor uptake. However, even in the solid TXM-13 human
melanoma Scid mouse model, the uptake ratio of tumor:blood or
tumor:muscle of 11.5 or 50.9, respectively, was obtained for
99mTc-CCMSH at 4 h after injection because
of the high tumor uptake and low background in the normal tissues
(Table 5)
.
In conclusion, the cyclic -MSH analogue
99mTc-CCMSH showed superior tumor uptake and
retention and fast whole body clearance in both murine and human
melanoma mouse models, compared with any other radiolabeled -MSH
analogue (14
, 25, 26, 27, 28, 29, 30, 31)
. The compacted cyclic structure of
99mTc-CCMSH (23
, 32)
exhibits
resistance to degradation and enhanced intracellular retention,
resulting in the high in vivo tumor-targeting properties of
the molecule. Lysine coinjection rather than peptide modification of
the Lys11 residue in
99mTcCCMSH was shown to significantly
decrease the nonspecific radioactivity accumulation in the kidneys
without significantly interfering with the high melanoma-targeting
properties of 99mTc- CCMSH. The metal-cyclized
CCMSH molecule displays high potential for the development of
melanoma-specific diagnostic and therapeutic agents.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported by Grant ER61661 from the Department
of Energy (to T. P. Q.) and a grant from the University of Missouri
Molecular Biology Postdoctoral Fellowship (to JQ. C.). 
2 To whom requests for reprints should be
addressed, at Department of Biochemistry, 117 Schweitzer Hall,
University of Missouri-Columbia, Columbia, MO 65211. Phone:
(573) 882-6099; Fax: (573) 882-5635; E-mail: quinnt{at}missouri.edu
3 The abbreviations used are: -MSH,
-melanocyte stimulating hormone; DTPA, diethylenetriaminepentaacetic
acid; CGCG, Ac-Cys-Gly-Cys-Gly; Scid, severe combined immunodeficient;
HPLC, high-performance liquid chromatography; RP, reverse phase; TFA,
trifluoroacetic acid; GI, gastrointestinal tract; ID, injected dose;
FAB, fast atom bombardment; NDP, [Nle4,
D-Phe7]-MSH; CCMSH,
[Cys3,4,10,
D-Phe7]-MSH3–13;
Gly11-CCMSH, [Cys3,4,10,
D-Phe7,
Gly11]-MSH3–13; Nle11-CCMSH,
[Cys3,4,10, D-Phe7,
Nle11]-MSH3–13.
Received 3/ 7/00. Accepted 8/17/00.
|
REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and internalized activity (
) are
expressed as a percentage of applied activity.
) radioactivity in B16/F1
cells over time at 37°C (A) or 25°C
(B).
) or 50 nM NDP (
) or with 10%
dialyzed FCS (
), for 125I-(Tyr2)-NDP. Bars,
SD. B, recovery of
125I-(Tyr2)-NDP binding capacity in B16/F1
cells treated with 50 nM NDP (
