An activatable theranostic for targeted cancer therapy and imaging

Article abstract of DOI:10.1002/anie.201311133

A new theranostic strategy is described. It is based on the use of an all in one prodrug, namely the biotinylated piperazine-rhodol conjugate 4 a. This conjugate, which incorporates the anticancer drug SN-38, undergoes self-immolative cleavage when exposed to biological thiols. This leads to the tumor-targeted release of the active SN-38 payload along with fluorophore 1 a. This release is made selective as the result of the biotin functionality. Fluorophore 1 a is 32-fold more fluorescent than prodrug 4 a. It permits the delivery and release of the SN-38 payload to be monitored easily in?vitro and in?vivo, as inferred from cell studies and ex?vivo analyses of mice xenografts derived from HeLa cells, respectively. Prodrug 4 a also displays anticancer activity in the HeLa cell murine xenograft tumor model. On the basis of these findings we suggest that the present strategy, which combines within a single agent the key functions of targeting, release, imaging, and treatment, may have a role to play in cancer diagnosis and therapy. All in one: A new theranostic prodrug was developed containing a biotinylated piperazine-rhodol conjugate linked to the drug SN-38 through a self-immolative disulfide spacer. When exposed to cellular thiols in cancer cells, it is able to release the active chemotherapeutic, SN38, along with a diagnostic fluorophore. This theranostic framework permits the targeted delivery, release of an active agent (SN-38), and its facile monitoring in vitro and in vivo.

Full text of DOI:10.1002/anie.201311133

Angewandte
Chemie
DOI: 10.1002/anie.201311133
Drug Delivery
An Activatable Theranostic for Targeted Cancer Therapy and
Imaging**
Sankarprasad Bhuniya, Sukhendu Maiti, Eun-Joong Kim, Hyunseung Lee, Jonathan L. Sessler,*
Kwan Soo Hong,* and Jong Seung Kim*
Abstract: A new theranostic strategy is described. It is based on
the use of an “all in one” prodrug, namely the biotinylated
piperazine-rhodol conjugate 4a. This conjugate, which incor-
porates the anticancer drug SN-38, undergoes self-immolative
cleavage when exposed to biological thiols. This leads to the
tumor-targeted release of the active SN-38 payload along with
fluorophore 1a. This release is made selective as the result of
the biotin functionality. Fluorophore 1a is 32-fold more
fluorescent than prodrug 4a. It permits the delivery and
release of the SN-38 payload to be monitored easily in vitro
and in vivo, as inferred from cell studies and ex vivo analyses
of mice xenografts derived from HeLa cells, respectively.
Prodrug 4a also displays anticancer activity in the HeLa cell
murine xenograft tumor model. On the basis of these findings
we suggest that the present strategy, which combines within
a single agent the key functions of targeting, release, imaging,
and treatment, may have a role to play in cancer diagnosis and
therapy.
all” concept associated with most current drug development
efforts, theranostic approaches could help move the field of
medicine toward an era of more effective and personalized
treatment.[1] For this promise to be realized, advances in
imaging, targeting, and therapy must be made and effective
targeting and release strategies must be developed. Herein,
we report an activatable prodrug strategy wherein a biotiny-
lated piperazine-rhodol is combined with the topoisomerase I
inhibitor, SN-38. This combination permits targeted imaging
and cytotoxicity in cellular cancer models. It also allows for
tumor-selective localization and antitumor activity in mouse
xenografts.
As we conceive it, a number of elements need to be
optimized to create a functioning theranostic that would be
attractive for use in cancer treatment protocols. These include
the imaging modality, the choice of active therapeutic agent,
and tumor targeting. A scission strategy that permits the
concurrent release of the signaling and therapeutic agent
would also be beneficial from an operational perspective.
Such an approach is especially attractive for cancer treatment,
as a number of enzymes overexpressed in malignant cells[2]
have a cleavage activity that could be conveniently used to
activate a prodrug via bond scission. When complemented by
the use of ligands selective for a specific cell type, a combi-
nation of targeting and drug release should be possible that is
highly effective.[3] If fluorophores[4] are included within the
theranostic design, real-time monitoring by fluorescence
imaging should be possible. Based on this strategy, we
recently developed a theranostic prodrug system based on
Gemcitabine where a biotin subunit was incorporated as
a potential localizer and coumarin was used as the fluoro-
phore.[5] While this system permitted in vitro imaging, tumor
localization was not demonstrated in vivo.
T
heranostics refers to an appealing new approach to drug
development wherein therapeutic modalities are combined
with those associated with diagnostic imaging. If successfully
implemented, theranostic strategies could permit the con-
current monitoring and treatment of a particular disease,
while permitting increased insights into drug delivery kinetics,
local response, and overall efficacy. Ultimately, theranostics
could allow treatment protocols to be adjusted to the needs of
individual patients in a way that thus far has not been
generally possible. By going beyond the current “one size fits
[*] Dr. S. Bhuniya,[+] Dr. E. J. Kim,[+] H. Lee, Dr. K. S. Hong
Division of MR Research, Korea Basic Science Institute
Cheongwon 363-883 (Korea)
Here, we describe a prodrug (4a in Scheme 1) that is
based on the topoisomerase I inhibitor, SN-38. This inhibitor
is the active ingredient in CPT11 (camptothecin), a therapeu-
tic agent used to treat various carcinomas,[6] and is one of the
most efficient drugs in this class.[7] The use of a biotin subunit
provides for tumor localization,[8] whereas a piperazine-
rhodol permits fluorescent-based monitoring both in vitro
and in vivo. A further key design feature of the present
theranostic is the use of a labile, self-immolative linker based
on disulfide bonds that permits release of both the active SN-
38 therapeutic agent and the N-biotinylated piperazine-
rhodol fluorophore in the presence of intracellular thiols. To
the best of our knowledge, this is the first system where drug
targeting, release, and fluorescence-based monitoring have
been demonstrated in an animal model.
E-mail: kshong@kbsi.re.kr
Dr. S. Maiti,[+] Prof. J. S. Kim
Department of Chemistry, Korea University
Seoul 136-701 (Korea)
E-mail: jongskim@korea.ac.kr
Prof. J. L. Sessler
Department of Chemistry, University of Texas at Austin
Austin, TX 78712-1224 (USA)
E-mail: sessler@cm.utexas.edu
[+] These authors contributed equally to this work.
[**] This work was supported by the Creative Research Initiative
program (No. 2009-0081566) of National Research Foundation of
Korea, the CAP (PBC066) funded by the Korea Research Council of
Fundamental Science and Technology, and by a grant from the
Korea Basic Science Institute (D33400).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201311133.
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in intensity by ca. 25-fold upon the addition of GSH (5.0 mm).
The fluorescence emission signal corresponding to 4a (5.0 mm
in PBS containing 25% DMSO) at lmax = 544 nm (excitation
at lmax = 510 nm) was also enhanced by ca. 32-fold following
exposure to GSH (5.0 mm) as shown in Figure 1b. As
À
expected for a system undergoing S S bond cleavage and
release of an active fluorophore, the emission spectrum of the
solution of 4a + GSH proved identical to that for the
reference fluorescent system 1a recorded at the same
Scheme 1. Synthesis of theranostic prodrugs 4a,b. Reagent and reac-
tion conditions: a) 1-(3-hydroxyphenyl)-piperazine, TFA, pressure tube,
958C, 3 h; b) RCO2H, EDCI, DMAP, DMF, RT, 12 h; c) mono-O-
dimethoxytrityl-2-hydroxyethyl disulfide, 4-nitrophenyl chloroformate,
DIPEA, DCM, 08C–RT, 3 h then 1a,b, TEA, DMF, RT, 24 h; d) DCM,
AcOH, RT, 24 h; e) 4-nitrophenyl chloroformate, DIPEA, DCM, 08C–RT,
3 h then SN-38, TEA, DMF, RT, 24 h. TFA=trifluoroacetic acid;
EDCI=N’-(3-dimethylaminopropyl)-N-ethylcarbodiimide; DMAP=
4-dimethylaminopyridine; DIPEA=diisopropylethylamine; DCM=
dichloromethane; TEA=triethylamine.
Figure 1. a) Absorption spectra of 4a (15.0 mm in PBS, 25% DMSO)
recorded in the presence and absence of GSH (5.0 mm). b) Fluores-
cence spectra of 4a (5.0 mm) recorded in the presence and absence of
GSH (5.0 mm). c) Fluorescence changes of 4a (5.0 mm) after treatment
with increasing concentrations of GSH (0–80 equiv). d) Fluorescence
spectra of 4a (5.0 mm) recorded in the presence of GSH (5.0 mm) as
a function of pH. Excitation was effected at 510 nm with the excitation
and emission slit widths both set at 3 nm.
The theranostic prodrug 4a was synthesized as outlined in
Scheme 1. The piperazine-rhodol was prepared as previously
described.[9]
The biotin-containing fluorescent precursor 1a was
obtained by reaction of biotin with the crude piperazine-
rhodol, followed by amide coupling. Next, mono-O-DMTr-2-
hydroxyethyl disulfide was reacted with 4-nitrophenyl chlor-
oformate, followed by reaction with 1a; this gave compound
2a in moderate yield. Removal of the O-DMTr protecting
group gave 3a in 60% yield. Compound 3a was then reacted
with 4-nitrophenyl chloroformate to give a reactive inter-
mediate, which was subsequently reacted with SN-38 to give
the prodrug target 4a. The non-targeted prodrug 4b was also
synthesized as a reference compound so as to judge the cell
specificity of 4a. Detailed synthetic procedures, including
spectroscopic data, are provided in the experimental section.
Before proceeding to cell imaging experiments, we
explored whether a biologically relevant thiol was able to
induce cleavage of the disulfide bond in prodrug 4a. Towards
this end conjugate 4a was treated with glutathione (GSH)
under physiological conditions. The changes in the optical
features were then monitored using UV/Vis and fluorescence
spectroscopy. As prepared, prodrug 4a (15.0 mm in PBS
(phosphate-buffered saline) containing 25% DMSO
(dimethyl sulfoxide)) exhibits a strong absorption peak at
lmax = 510 nm in its absorption spectrum. This peak increases
concentration (Figure S1 in the Supporting Information).
We also observed that in the presence of 80 equiv GSH, the
fluorescence intensity reached saturation (Figure 1c). These
results are fully consistent with the design expectation that the
disulfide bond of 4a is readily cleaved by GSH.
Since GSH and other reduced thiols are present in cancer
cells,[10] the bond scission seen under the conditions of the
above analyses is expected to translate into an increase in the
intracellular concentration of SN-38 while also providing an
easy-to-monitor fluorescent marker.
We also analyzed the pH-dependent changes in the
intensity of the fluorescence emission (lmax = 544 nm) of 4a
(5.0 mm) in the absence and presence of 5.0 mm GSH. As
shown in Figure 1d and S2, in the absence of GSH, no
immediate change in the fluorescence features of 4a are seen
over the pH range of 3.2 to 8.6. In the presence of GSH,
however, a significant increase in the fluorescence intensity is
seen over a pH range of 5 to 8.6 within minutes of mixing (see
below for detailed time-course studies). These observations
provide further support for the suggestion that prodrug 4a
contains a cleavable linker.
2
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To assess whether other biologically relevant analytes
might interfere with the proposed GSH-induced cleavage
process in vitro or in vivo, the interaction of 4a with various
metal ions and thiols, thiol-free amino acids were investigated
under the same reaction conditions used to test the effect of
GSH (Figure 2a and b).
Figure 2. Fluorescence spectra of 4a (5.0 mm in PBS, 25% DMSO)
recorded in the presence and absence of various metal cations
(1.0 mm) and GSH, Cys, Hcy (5.0 mm), respectively. Excitation was
effected at 510 nm with the excitation and emission slit widths both
set at 3 nm.
Addition of cysteine (Cys) or homocysteine (Hcy) to 4a
caused changes in the emission similar to those elicited by
GSH. On the other hand, no significant spectroscopic changes
were detected upon exposure to thiol-free amino acids or
biologically relevant metal cations, including Na+, K+, Ca2+,
Mg2+, Zn2+, Fe2+, Fe3+, Cu2+ respectively. On this basis, we
conclude that the disulfide bond in 4a will undergo specific
thiol-mediated cleavage without substantial interference from
other chemical entities that would be expected to be
prevalent in a biological environment.
The timing of drug release from its prodrug form can have
a critical impact on its efficacy.[11] To investigate the time-
dependent release of the active drug SN-38 and fluorophore
components present in 4a, the extent of fluorescence signal
recovery at lmax = 544 nm was recorded as a function of time
at different GSH concentrations (Figure S3). It was found
that the fluorescence intensity reached a maximum and
plateaued within 10 min upon exposure to 5 mm GSH. This
GSH concentration mimics the average intracellular gluta-
thione level. Thus, this finding leads us to suggest that the
active components (fluorophore and drug) will be released
shortly after prodrug 4a enters a cell.
To confirm whether the fluorescence enhancement
observed when 4a is exposed to GSH reflects SN-38 release
(as opposed to just the active fluorophore) prodrug 4a was
treated with 5.0 mm GSH at 378C for 2 h. An aliquot was then
subjected to mass spectrometric (MS) analysis. Molecular
mass peaks corresponding to SN-38 (MW= 392.4) and 1a
(MW= 626.3) were observed (Figure S34). This is taken as
evidence that the disulfide bond present in 4a is readily
cleaved by GSH and that this cleavage leads to release of free
SN-38 along with the fluorescent probe as shown in Scheme 2.
Based on the products observed, we suggest this release
process involves intramolecular cyclization and concomitant
scission of a neighboring carbonate bond.
Scheme 2. a) Thiol-induced cleavage of prodrug 4a and b) schematic
representation of its proposed intracellular processing.
To gain insight into whether the cellular uptake of 4a
could be targeted, studies were carried out using biotin
receptor-enriched tumor cells. Control studies were con-
ducted using normal NIH3T3 cells that express a low level of
the biotin receptor.[12] The precursor 3a and prodrug 4a were
incubated at various concentrations with HeLa cells over-
expressing the biotin receptor[13] for 2 h, and the efficiency of
cellular uptake was measured using flow cytometry (Fig-
ure 3a).
In the case of both 3a and 4a cellular uptake proved
concentration-dependent, with plateaus being reached at
similar concentration levels for both compounds of 10 mm.
Since 4a contains a potent anti-cancer drug, SN-38, which
could induce cytotoxicity and affect the background cellular
fluorescence, we used precursor 3a, rather than 4a, to follow
internalization. As shown in Figure 3b, the biotin receptor-
positive HeLa and A549 cancer cell lines exhibited strong
fluorescence within 2 h of incubation with 3a (10 mm). In
contrast, a very weak fluorescence signal was observed in the
NIH3T3 cells. Such a difference in emission among these cell
lines is consistent with the suggestion that the selective uptake
of 3a by HeLa and A549 cells can be attributed to receptor-
mediated endocytosis.
Furthermore, as assessed by fluorescence-activated cell
sorting (FACS), the uptake of 3a proved dose-dependent in
both the biotin receptor-positive HeLa and receptor-negative
NIH3T3 cells, with the uptake in the HeLa cells being
routinely more than 3-fold higher than that in the NIH3T3
cells (Figure S4). However, the non-biotinylated precursor 3b
gives rise to random cellular uptake (Figure S5) irrespective
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Figure 4. a) Optical images of the biotin receptor-positive HeLa and
A549 cells pretreated with biotin (100 mm) for 1 h, followed by
incubation with 3a (10 mm) in PBS for 2 h. b) The reduced cellular
uptake efficiency was further demonstrated by FACS analysis. Nuclear
counter-staining using DRAQ5 (red). Scale bar: 10 mm. Images were
obtained using excitation wavelengths of 510 nm and 630 nm, with the
emission being monitored over the 540–600 and 680–750 nm spectral
regions for the green and red signals, respectively.
Figure 3. a) Cellular uptake of 3a and 4a by HeLa cells as assessed by
FACS analysis performed after 2 h incubation. b) Fluorescence images
of HeLa, A549, and NIH3T3 cells treated with 10 mm of 3a in PBS for
2 h. Nuclear counter-staining using DRAQ5 (red). Scale bar, 10 mm.
Images were obtained using excitation wavelengths of 510 nm and
630 nm, with the emission being monitored over the 540–600 and
680–750 nm spectral regions for the green and red signals, respec-
tively.
As a test of whether thiol-induced disulfide bond cleavage
and the concurrent increase in fluorescence is mediated by
thiols within cells, the fluorescence behavior of precursor 3a
was analyzed in HeLa and A549 cells pretreated with N-
ethylmaleimide (NEM). NEM reacts with thiols and was
expected to reduce the effective concentration of GSH (and
of the cell line chosen for study. This observation leads us to
conclude that biotin functionality present in 3a plays an
important role in terms of mediating the cellular uptake of 3a
(or 4a) depending on the level of overexpressed biotin
receptors present in the cancer cells. It is envisioned that 4a
may be a possible tool for the targeted delivery of chemo-
therapeutic and diagnostic imaging agents to tumor sites.
To provide further confirmation for the proposed endo-
cytosis-mediated uptake mechanism, the biotin receptor-
positive HeLa and A549 cells were pre-treated with an
excess of biotin prior to incubating with 3a for 2 h. This
treatment was expected to block the receptors and prevent or
reduce competitive binding by 3a. The results of the
fluorescence imaging analyses revealed that, in accord with
expectations, the number of fluorescent cells was significantly
reduced (Figure 4a). Further, the overall fluorescence inten-
sity was significantly decreased (Figure 4b) in the HeLa and
A549 cells pretreated with biotin. These findings are thus fully
consistent with the suggestion that cellular uptake of pre-
cursor 3a in receptor-positive cells occurs by receptor-
mediated endocytosis.[14]
À
other free thiols), thus inhibiting the critical S S scission
process.[15] In fact, the intracellular fluorescence ascribed to
3a was substantially reduced in the presence of 50 mm NEM
(Figure S6). On this basis, we conclude that the fluorescence
enhancement seen for 3a in vitro is triggered, at least for the
most part, by reaction with intracellular thiols (Scheme 2). It
is inferred that the same mode of action holds for 4a, although
because of concerns involving cytotoxicity and artifacts (see
above) this was not tested directly.
To determine whether the enhancement in long-wave-
length fluorescence intensity observed for prodrug 4a under
conditions of bond scission (cf. Figure 1) can be correlated
with intracellular release of SN-38, an effort was made to
detect SN-38 directly.[16] Towards this end, lysates of HeLa
cells were treated with 4a (10 mm) and subjected to fluoro-
metric analysis with monitoring effected at 370 nm. This
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wavelength corresponds to the UV absorption maximum of
SN-38 (Figure S7). In fact, the intensity of the 370 nm and
548 nm emission signals (the latter ascribable to 1a) were
À
both seen to increase under conditions where S S bond
scission occurs. As a result, we conclude that intracellular
release of SN-38 from prodrug 4a could be monitored by
following the increase in the intensity of the easier-to-
visualize 548 nm fluorescence emission.
The anti-cancer efficacy of prodrug 4a and 4b were
evaluated in three cell lines characterized by different
expression levels of the biotin receptor.
This was done using standard cell viability protocols.
Briefly, cells were treated separately with 4a and 4b,
incubated for an additional 48 h, and analyzed using a stan-
dard MTT assay. As shown in Figure 6 prodrug 4a exhibited
significantly enhanced dose-dependent antiproliferative
activity against HeLa and A549 cells with high biotin receptor
levels.[13] In conjunction with the results shown in Figure 5 and
S8, these data also serve to confirm that the biotin moiety can
guide prodrug 4a to biotin receptor-positive HeLa and A549
Figure 5. Cell viability of HeLa, A549, and NIH3T3 cells pretreated with
various concentrations of prodrug 4a in PBS. After treatment, the cells
were incubated for 48 h. Cell viability was assessed using a standard
MTT assay.
À
tumor cells. Following S S bond scission, this gives rise to the
observed anti-proliferative activity as the result of releasing
SN-38 within the cell. In contrast, the non-biotinylated
prodrug 4b did not show such specific antiproliferative
activity; nor, was a depend-
conditions, the non-biotinylated prodrug 4b did not give
rise to a tumor specific biodistribution of wither the chemo-
therapeutic or imaging agents (Figure 6a, right side).
ence on biotin-receptor levels
in
different
cell
lines
observed (Figure S9).
In an effort to assess
whether drug release from
4a would occur in tumor-
affected tissues, a mouse xen-
ograft model was used to
evaluate tumor targeting and
organ distribution. In these
studies, HeLa tumor-bearing
mice were treated with 4a by
intravenous
injection
(2.5 mgkgÀ1). Ex vivo optical
and fluorescence imaging was
then carried out. A strong
fluorescent signal was seen
for
the
tumor-affected
regions after treatment with
4a. In contrast, only weak or
negligible signals were seen
in the case of other organs,
including the liver, kidney,
lung, and spleen (Figure 6a).
Also
tumor-selective
enhancements in fluores-
cence were seen for cryo-
Figure 6. Ex vivo biodistribution of prodrugs (4a, 4b) and in vivo therapeutic efficacy of prodrug 4a.
a) Fluorescent images of tumor region and major organs obtained from tumor-bearing nude mice after
systemic injection of the animals with prodrug 4a and 4b in PBS. b) Fluorescent images of tumor tissues
excised from mice treated with saline and prodrug 4a (green, prodrug 4a; blue, nuclei). Scale bar: 50 mm.
Images were obtained using excitation wavelengths of 360 nm and 510 nm, and recording the output over
the 440–500 nm and 540–600 nm spectral regions, for the blue and green signals, respectively. c) Time
course of solid tumor volumes for HeLa cell mice xenografts. Prodrug 4a was injected into the tail vein in
5 doses of 125 mgkgÀ1 each every other day starting day 10 after inoculation to induce the xenograft.
d) Photographic images of the solid tumors excised from each group 35 days post initial inoculation of
tumor cells.
sectioned
tumor
tissues
obtained from mice injected
with 4a (Figure 6b). An
appreciable
fluorescence
signal was seen only in the
tissues bearing the HeLa
tumor cells. Under similar
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Based on the above studies the tumor specific in vitro
cellular uptake, imaging, antiproliferative activity, and bio-
distribution of 4a (but not control 4b) was considered
confirmed. Therefore, the antitumor efficacy of 4a was
evaluated in vivo. This was done using a xenograft murine
model created by subcutaneous inoculation with HeLa cells
and measuring the extent of tumor growth (Figure 6c and d).
In these studies, prodrug 4a was administered every other day
for a total of 5 doses of 125 mgkgÀ1 each via tail vein injection
starting at day 10 post-inoculation. Saline was used as
a control. In the mice treated with 4a, the tumor volume
was significantly reduced compared with control group (p <
0.05 at day 35 post-inoculation). We take this as an indication
that the biotinylated prodrug 4a works according to design
expectations in vivo as well as in vitro. Specifically, based on
our findings we suggest that this cleavable theranostic
1) displays tumor-targeting features both in vitro and
in vivo, 2) is able to deliver SN-38 into tumor cells, 3) allows
visualization of cancerous lesions, and 4) exhibits antiproli-
ferative activity in xenograft tumor models.
xenografts served to confirm an increase in fluorescence. A
statistically significant anticancer effect was also seen in these
tumor models. On this basis, we conclude that prodrug 4a and
analogues represent a potentially useful new approach to
theranostic design and represent species that could provide
new tools for tumor-targeted drug delivery and concomitant
monitoring of uptake and function by fluorescent imaging. To
our knowledge, this is the first time these design principles
have been successfully demonstrated both in vitro and in vivo.
Received: December 22, 2013
Revised: February 17, 2014
Published online: && &&, &&&&
Keywords: biotin · cellular imaging · glutathione · SN-38 ·
.
theranostic
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www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 1 – 7
These are not the final page numbers!

Angewandte
Chemie
Communications
Drug Delivery
All in one: A new theranostic prodrug was
developed containing a biotinylated
piperazine-rhodol conjugate linked to the
drug SN-38 through a self-immolative
disulfide spacer. When exposed to cellular
thiols in cancer cells, it is able to release
the active chemotherapeutic, SN38, along
with a diagnostic fluorophore. This
theranostic framework permits the tar-
geted delivery, release of an active agent
(SN-38), and its facile monitoring in vitro
and in vivo.
S. Bhuniya, S. Maiti, E. J. Kim, H. Lee,
J. L. Sessler,* K. S. Hong,*
J. S. Kim*
&&&& — &&&&
An Activatable Theranostic for Targeted
Cancer Therapy and Imaging
Angew. Chem. Int. Ed. 2014, 53, 1 – 7
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
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These are not the final page numbers!