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Article 111

Use of In-Hexavalent Lactoside for Liver Reserve Estimation in Rodents with Thioacetamide-Induced Hepatic Fibrosis Mei-Hui Wang, Chuan-Yi Chien, Hung-Man Yu, Ping-Yen Wang, and Wuu-Jyh Lin Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00326 • Publication Date (Web): 13 Aug 2018 Downloaded from http://pubs.acs.org on August 14, 2018

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Molecular Pharmaceutics

Use of

111

In-Hexavalent Lactoside for Liver Reserve Estimation in Rodents with

Thioacetamide-Induced Hepatic Fibrosis Mei-Hui Wang*, Chuan-Yi Chien, Hung-Man Yu, Ping-Yen Wang, Wuu-Jyh Lin* . Institute of Nuclear Energy Research, Taoyuan 325, Taiwan

Corresponding authors: * Mei-Hui Wang, PhD, Isotope Application Division, Institute of Nuclear Energy Research, P.O. Box 3-27, Longtan, Taoyuan 325, Taiwan. Tel: 886-3-4711400 ext 7162. Fax: 886-3-4711416. Email: [email protected] *Wuu-Jyh Lin, PhD, Institute of Nuclear Energy Research, 1000 Rd Wenhua, Longtan, Taoyuan 32546, Taiwan. Tel: 886-3-4711400 ext 2761. Fax: 886-3-4713960. Email: [email protected]

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Abstract Many biochemical tests detecting the presence of liver disease are not liver-specific and may be abnormal in non-hepatic conditions. Asialoglycoprotein receptor (ASGPR) is a hepatocyte-specific receptor for Gal/GalNAc-terminated glycopeptide or glycoprotein. The number of these receptors decreases in patients with chronic liver diseases. Here, we aimed to evaluate the use of 111In-hexavalent lactoside, a known ASGPR imaging biomarker, as a more sensitive probe to detect small changes in liver reserve in animal models of chronic liver injury. Thioacetamide (TAA) treatment via intraperitoneal injection every two days in BALB/c mice continued for 1, 2, 3, or 4 months. The liver fibrosis stages were determined by Sirius Red staining and were based on the METAVIR classification method. Serum transaminase enzymes (alanine transaminase (ALT) and aspartate transaminase (AST)), alkaline phosphatase, albumin, and bilirubin were measured using a FUJI FDC3500 i/s analyzer. The ASGPR staining was performed by immunohistocytochemical stain. The percentages of fibrosis and ASGPR were calculated using ImageJ software after collagen staining and anti-ASGPR staining, respectively. A nanoSPECT/CT was used for molecular imaging and liver uptake measurement. We observed fibrosis grades of F0-F1 in mice treated with TAA for 1 month, F2 in mice treated for 2 months, F3-F4 in mice treated for 3 months, and F4 in mice treated for 4 months. The levels of ALT and albumin were not significantly different in the TAA groups from those in the controls. Although the average levels of AST, alkaline phosphatase, and bilirubin in the TAA groups were different from those in the control group, there was little difference between TAA groups. More sensitive distinctions among TAA groups were detected in 111

In-hexavalent lactoside uptake of ASGPR, ASGPR staining, and fibrosis % than when

using the conventional AST, ALT, albumin, alkaline phosphatase, and bilirubin tests. The


2

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absorption and distribution of

111

In-hexavalent lactoside were lower in the chronic hepatitis

models than the normal controls. The liver reserves measured by

111

In-hexavalent lactoside

uptake were 71.7±7.5% and 50.9±5.6% after 1 and 2 months, respectively, of TAA treatment. As an ASGPR biomarker,

111

In-hexavalent lactoside has higher sensitivity than

traditional liver function tests and collagen stain to provide more objective data for evaluating compensated cirrhosis or the changes of the liver damage. ASGPR staining can reflect the regenerated hepatocytes, but the need for a biopsy limits its use.

111


measurement is comparable with ASGPR staining, which suggests that

111

In-hexavalent

lactoside measurement will be more useful as a practical, noninvasive test of chronic liver injury.

Keywords: asialoglycoprotein receptor immunostaining, asialoglycoprotein receptor imaging, compensated cirrhosis, I11In-hexavalent lactoside scintigraphy, liver fibrosis.


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Introduction 111

In-hexavalent lactoside is a promising imaging biomarker with high specificity and

accuracy for functional liver mass.1, 2 As methods for determining liver reserve in patients with cirrhosis are urgently needed, we investigated 111In-hexavalent lactoside absorption in a thioacetamide (TAA)-induced fibrosis model to evaluate if

111


scintigraphy could measure the change in liver reserves in an animal model of chronic liver injury. Viral hepatitis has been well controlled in Taiwan since a successful vaccine policy was instituted; however, drug-induced hepatitis remains a serious problem. In particular, the severity of this disease is difficult to determine using a simple blood screen because aspartate transaminase (AST) and alanine transaminase (ALT) levels are often irrelevant, even in the presence of drug-induced hepatitis. Patients with drug-induced fulminant hepatitis often face the prospect of liver transplantation. People with sufficient liver reserves can survive through supportive therapy, but individuals with little or no liver reserve may die without successful liver transplantation. In addition, liver transplant patients must take immunosuppressant drugs for the remainder of their lives, thereby increasing the risk of infection from bacteria and viruses. Therefore, clinicians must determine which patients require urgent liver transplantation due to low liver reserves. Doctors require a sensitive technique to evaluate liver function and determine the appropriate medical treatment. Bile duct ligation and TAA treatments have been shown to induce liver fibrosis. The former causes bile juice obstruction and then fibrosis. The latter uses a hepatotoxin to induce chronic liver injury.3 TAA is oxidized in the liver by the cytochrome P450 system to produce acetamide and TAA sulfoxide and is then transformed further into TAA sulfodioxide, a highly reactive compound that binds to cellular macromolecules to cause hepatocyte damage and oxidative stress.4, 5 Because the fibrosis pattern induced by this method is similar to that


4

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observed in humans,6 increasing numbers of drug development studies use this method to establish a liver fibrosis model.7, 8 Currently, drug-induced liver fibrosis remains a problematic disease. Patients who are diagnosed early with fibrosis may recover completely through appropriate therapy. Thus far, biopsy remains the gold standard, but it has some disadvantages, including human error in sampling, observer deviation and possible complications.9 In addition, the invasiveness of the procedure can deter patients from undergoing necessary checkups. In the clinic, MRI and ultrasound imaging are two powerful and widely used non-invasive methods for the detection of liver stiffness. However, while they provide good diagnosis for advanced fibrosis, it is still difficult to detect very early fibrosis or small changes.10, 11 Molecular targeting probes would be better for detection and therapeutic drug delivery. The

99m

Tc-sulfur colloid liver scan is

FDA-approved, but it is used for Kupffer cell scans and lacks liver-specific characteristics. In a previous paper, we revealed the liver-targeting and low-background properties of 111

In-hexavalent lactoside scintigraphy.1 Here, we further evaluate whether asialoglycoprotein

receptor (ASGPR) imaging using

111

In-hexavalent lactoside can sensitively detect earlier

changes and determine the degree of severity of drug-induced fibrosis in a rodent model.


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Experimental Section Animals Male BALB/c mice (age, 5-6 w; weight, 22±2 g) were purchased from the National Animal Center, Taipei, Taiwan. Male Wistar rats (age, 6-8 w; weight, 200±20 g) were purchased from the National Taiwan University Animal Center, Taipei, Taiwan. All animals were bred in a specific pathogen-free animal room maintained at a constant temperature (23±3°C) and relative humidity (50±20%), with periodic air changes and 12 h of light per day. Food and tap water were supplied ad libitum during the experimental periods. All animal experiments were approved by the Institutional Animal Care and Use Committee at the Institute of Nuclear Energy Research (Taoyuan, Taiwan).

Animal model BALB/c mice (5-6 w old, n=4) were treated with 200 mg/kg of TAA via intraperitoneal injection every 2 days for 1-4 months to induce liver fibrosis.12 The ASGPR content was examined using anti-ASGPR staining. The stage and degree of liver fibrosis were determined using Sirius Red staining13 according to the METAVIR classification. The fibrosis and ASGPR contents were quantified using Photoshop CS3 (Adobe Systems, San Jose, CA, USA) and ImageJ software (http://imagej.nih.gov/ij/).14 Serum AST, ALT, alkaline phosphatase (ALP), albumin, and bilirubin were measured using a FUJIFILM Dry-Chem 3500 i/s analyzer. ASGPR biomarker imaging was performed and measured for 15 min using a nano-single-photon-emission computed tomography (SPECT)/computed tomography (CT) scanner (Mediso Ltd., Budapest, Hungary) after an intravenous injection of 18.5 MBq of 111

In-hexavalent lactoside in each mouse. The position of the liver was selected to measure

the image intensity.


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Pathological staining and clinical biochemical assay Liver tissue was removed and fixed using a 10% (v/v) buffered formalin solution and embedded in paraffin wax for conventional histological examination. Briefly, 5-7-µm-thick sections were cut, stained with hematoxylin and eosin and observed under a microscope to assess the morphological changes. For fibrosis staining, the liver tissue was dyed with 0.1% Sirius Red (in saturated picric acid) to determine the severity of fibrosis/cirrhosis.15 The fibrosis stage was determined according to the METAVIR classification: F0=no fibrosis, F1=portal fibrosis without septa, F2=portal fibrosis with few septa, F3=numerous septa without cirrhosis and F4=cirrhosis. The fibrosis percentage was measured by determining the fibrosis color, converting the image to grayscale with Adobe Photoshop CS3, and then statistically analyzing the grayscale area fraction relative to the total area with ImageJ software. The ASGPR immunostaining was carried out on Vantana BenchMark autostainer (Vantana BenchMark Systems, USA). Sections were stained with an anti-ASGPR antibody (1:2000, Santa Cruz Biotechnology, Inc.) at 4°C for 12 h, and then washed twice with phosphate-buffered saline (PBS), treated with goat anti-mouse IgG biotin-labeled secondary antibody (1:500; Vector Laboratories) at room temperature for 1 h and developed with an ABC kit (Vector Laboratories) and counterstained with hematoxylin according to the manufacturer’s instructions. The slides were then examined under a microscope. The ASGPR-stained region was selected, and the image selected was converted to grayscale with Adobe Photoshop CS3. ImageJ software was used to statistically analyze the grayscale area fraction of the total area.15 The ASGPR area fraction (%) was measured by dividing the ASGPR-stained area by the total area. A blood biochemistry assay was used to measure liver-related biochemical indicators, including AST activity, ALT activity, ALP activity, bilirubin level and albumin level, using a


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FUJIFILM Dry-Chem 3500 i/s automatic analyzer (Fujifilm Corporation, Japan).

Radiosynthesis of 111In-hexavalent lactoside Radiosynthesis

was

performed

by

a

quantitative

reaction

of

111

InCl

and

DTPA-hexavalent lactoside with a molar ratio 1 to 20, which was performed by adding 185 MBq (i.e., 1.15 x 10-10 moles) of sterile and apyrogenic

111

InCl (pH 2) to the lyophilized

INER HL kit, a patented formulation containing 8 µg (i.e., 2.2 x 10-9 moles) of DTPA-hexavalent lactoside, 5.5 mg of citric acid, 6.2 mg of trisodium citrate, and 10 mg of mannitol (the final pH was 4), followed by incubation for 10-15 min at room temperature.2 DTPA-hexavalent lactoside was synthesized using the method reported by Lee and colleagues.1, 16 Briefly, a “nitrile-triacetic acid” (NTA) linker group with an α-amino group was used to attach three lactose glycoside residues to each carboxyl group of NTA. Then, another linker, the amino-protected hexanoyl-L-aspartic acid, was used to dimerize two trivalent-lactoside residues to form hexavalent lactoside, and the ε-amino group was unmasked and modified with DTPA dianhydride for radiochemical purity of

111

111

In-chelating purposes. The

In-hexavalent lactoside was determined using a radio-instant

thin-layer chromatography (radio-ITLC) method with 10 mM sodium citrate (about pH 5) as the

developing

solvent2

and

was

validated

with

radio-high-performance

liquid

chromatography (radio-HPLC) using an Atlantis T3 C18 column (250 x 4.6 mm, 5 µm). The ITLC analysis used ITLC-SG film (Gelman Sciences Corp., USA) as the stationary phase. The position approximately 2 cm from the bottom of an ITLC-SG film was marked with a pencil as the origin, while the position approximately 8 cm from the bottom was marked as the end. A few samples (0.2 µL) were dropped at the origin by a micropipette. Then, the ITLC-SG film was developed in 10 mM sodium citrate buffer (about pH 5). The film was taken out to dry in the fume cabinet while the developing solvent reached the end.


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The film was scanned with a radio-TLC scanner (Bioscan AR-2000, USA) to collect the chromatogram for 1 min. The retention factor (Rf) was measured as distance traveled by the sample divided by distance traveled by the developing solvent. The radio-ITLC results showed an Rf of

111

In-hexavalent lactoside = 0–0.1 and an Rf of

(Supplementary Fig. S1A-B).

2, 17

111

InCl3 = 0.9–1.0

The radiochemical purity was calculated by the peak area

of 111In-hexavalent lactoside divided by the total area of all peaks. The radio-HPLC method used an Atlantis T3 C18 column (250 x 4.6 mm, 5 µm). Mobile phase A was 0.1% TFA in acetonitrile, and 0.1% TFA in water was mobile phase B, and the flow rate was 1.0 mL/min. The gradient profile was A/B from 0/100 to 100/0 for 0–30 min. The radio-HPLC results showed retention time of 16.40 min for 111In-hexavalent lactoside.1 The radio-ITLC method was validated to have good correlation with the radio-HPLC method. Briefly,

111

In-hexavalent lactoside with different radiochemical purities made by

mixing different proportions of 111In-hexavalent lactoside and 111InCl3 were analyzed using methods radio-ITLC and radio-HPLC. The radiochemical purity analyzed by method radio-ITLC was compared with that of radio-HPLC. The correlation linear relationship was Y=0.9606X+3.170, R2=0.9924 (Supplementary Fig. S1C). Here, X refers to radiochemical purity determined via radio-ITLC, and Y refers to radiochemical purity determined via radio-HPLC. The radiolabeled compound in its final formulation was stable with higher than 96±1% radiochemical purity for more than 24 h at 4°C (Supplementary Fig. S2A).2 To mimic the stability of 111

111

In-hexavalent lactoside under in vivo physiological conditions, aliquots of

In-hexavalent lactoside (about 50 µL) were added into PBS (1 mL) or mouse serum (1 mL)

and incubated at 37°C for 24 h. At 0.25, 1, 4, and 24 h, aliquots of samples (about 0.2 µL) were analyzed for radiochemical purity using the radio-ITLC method.


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Page 10 of 39

Biodistribution assay 111

In-hexavalent lactoside (148 kBq) was injected into each mouse via the tail vein (n=4),

and mice were sacrificed at 15 min post-injection by cervical dislocation. Organs and fluids, including whole blood, brain, muscle (thigh), bone, stomach, spleen, pancreas, lung, heart, kidney, and liver, were collected as biological samples. The samples were weighed, placed in a measuring tube and then measured with a Gamma counter. The percentage injected dose per gram (% ID/g) was measured to determine the biodistribution of radioactivity in each organ.

111

In-hexavalent lactoside scintigraphy 111

In-hexavalent lactoside (18.5 MBq) was intravenously injected into the tail vein of

each mouse for

111

In-hexavalent lactoside scintigraphy. Imaging data were acquired

immediately after the injection. SPECT/CT imaging was performed using a pinhole collimator nanoSPECT/CT scanner (Mediso, Budapest, Hungary) and standard animal scan procedures. The mice were anesthetized with 1.5% isoflurane during the imaging period. The 3D image is the congregation of each 2D slice image. After imaging, nanoSPECT/CT image reconstruction and fusion, volumes of interest (VOIs) were selected around the liver using PMOD (Pmod Technology LLC, Zürich, Switzerland) software. The values of liver VOIs (counts) were converted to radioactivity using a known amount of radioactivity (10-50 µCi 111

In) as a reference, which was scanned with mice. The percentage of injection dose (%ID)

was calculated by dividing the radioactivity of the whole liver by the total injected radioactivity. The association between the percentage fibrosis in the liver slice and the percentage of the injected dose in the liver VOI quantified using InVivo Scope software was analyzed using GraphPad Prism (GraphPad Software, San Diego, CA, USA) software.


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Statistical analysis The data are expressed as means ± standard deviation and were analyzed using a two-tailed unpaired t-test. Correlations (R2 value) were calculated using the Pearson correlation coefficient. Statistical analyses indicating a significant difference were labeled with *, **, and *** when the p-values were less than 0.05, 0.01, and 0.001, respectively. All analyses were performed with GraphPad Prism software version 6.03 for Windows.


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Page 12 of 39

Results Pathological staining for fibrosis and related biochemical tests The TAA-induced fibrosis pattern in mice was similar to the pattern observed in humans (Fig. 1) and was classified according to the METAVIR method. The METAVIR fibrosis severities were F0-F1, F2, F3-F4, and F4 for the mice treated with TAA for 1, 2, 3 and 4 months, respectively (Fig. 1). The fibrotic areas represented 1.17±0.46%, 5.37±0.84%, 9.57±1.37%, 10.73±3.23% and 15.76±1.83% of the liver tissues in mice that underwent TAA treatment for 0, 1, 2, 3 and 4 months, respectively (Fig. 2). Generally, the fibrosis severity was consistent with the duration of TAA treatment (Figs. 1 and 2). The statistical analyses indicated a significant increase in fibrosis after treatment with TAA for 1, 2, 3, and 4 months (P < 0.001) (Fig. 2). The serum levels of ALT and albumin were not significantly different between the untreated mice and the TAA groups treated for 1, 2, 3, and 4 months (P > 0.05; Fig. 3A-B), indicating that serum ALT and albumin were statistically insensitive or irrelevant to TAA-induced fibrosis. The serum levels of AST, ALP and bilirubin in the TAA groups were abnormal compared to those in the untreated mice, but there were no significant differences between the TAA-treated groups (P > 0.05, Fig. 3A, 3C, 3D), which indicates that these tests are less correlated with the fibrosis severity.

Immunohistochemical staining of hepatocytes from healthy mice and mice with TAA-induced fibrosis using an anti-ASGPR antibody Pathological staining with an anti-ASGPR antibody revealed that the expression of ASGPR was decreased in mice with TAA-induced fibrosis (Fig. 4). As shown in Fig. 4A, ASGPR was only detected on the surface membranes of hepatocytes, and the hepatocytes


12

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were arranged surrounding the central vein by radiating cords. Figs. 4B-C show that ASGPR expression was decreased in the TAA-induced hepatocytes as the TAA induction duration increased. In the 3-month TAA treatment group, some increased abnormal immunostaining was present (Fig. 4D), and in the 4-month TAA treatment group, we observed many irregular, smaller hepatocytes regenerating around the central vein (Fig. 4E). The ASGPR area fractions in Fig. 4A-E were 7.6%, 3.3%, 2.3%, 4.9%, and 3.9%, respectively. The slightly increased ASGPR area fractions in the TAA 3-month treatment (Fig. 4D) and TAA 4-month treatment (4E) were mostly due to the production of new ASGPR.

Radiochemistry and stability measurements 111

In-hexavalent lactoside was prepared by reacting

111

InCl3 with DTPA-hexavalent

lactoside in a 1:20 molar ratio in 0.1 M sodium citrate buffer (pH 4.0). Radiolabeling was completed at room temperature for 15 min. Since there is no further purification, the radiolabeling efficiency is the same as the radiochemical purity. Both the radiolabeling efficiency and radiochemical purity assayed by radio-ITLC and radio-HPLC were greater than 98±1%, and the specific activity reached 82.7±8.4 MBq/nmol (i.e. 22.6±2.3 GBq/mg). Table 1 shows the stability of 111In-hexavalent lactoside in PBS and mouse serum. The 111

In-hexavalent lactoside was incubated in either PBS or mouse serum at 37°C for 24 h. The

results measured by radio-ITLC showed that

111

In-hexavalent lactoside exhibited a good

stability with over 94% radiochemical purity in either PBS or mouse serum at 0.25–4 h. After 24 h of incubation at 37°C, the radiochemical purity levels of 111In-hexavalent lactoside were 89.3±0.1% in PBS and 91.9±0.6% in serum.

Radioactivity uptake in mice with various severities of chronic hepatitis Significantly less

111

In-hexavalent lactoside was absorbed in the livers of the


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Page 14 of 39

TAA-induced chronic hepatitis group than in the normal group (P < 0.001 for the TAA treatment groups compared with the normal group), according to the results of the biodistribution assay. The liver uptake of

111

In-hexavalent lactoside presented as %ID/g in

liver decreased as the duration of TAA induction increased (Fig. 5). The image of Figure 6A is a 2D image indicating one slice image of 3D volume and the quantification of %ID is calculated from 3D liver volume. The gray-white color of the livers from 2D images of the 3-month and 4-month TAA treatment groups indicates cirrhosis. Because 2D image did not represent the total uptake of liver, for quantitation, the %ID from total liver volume should be more reliable. In Figure 6B, vertical axis in the left shows the real percentage liver uptake of normal and treatment groups, while the vertical axis in the right refers to the relative percentage liver uptake (RPLU). The ASGPR imaging showed that the percentage of

111

In-hexavalent

lactoside injected dose in the liver was 53±6%, 38±4%, 27±3%, 34±2%, and 31±2% after 0, 1, 2, 3, and 4 months of TAA treatment, respectively, according to the total liver uptake of 111

In-hexavalent lactoside calculated in scintigraphy experiment (Fig. 6A). RPLU is obtained

by dividing the liver uptake (%ID) of each treatment group by that of normal group, which demonstrates the relative liver uptake of disease groups compared to normal group. Hence, the trend of RPLU for the disease group was 71.7±7.5%, 50.9±5.6%, 64.1±3.8%, and 58.4±3.7% after 1, 2, 3, and 4 months of TAA treatment, respectively (Fig. 6B). A linear correlation between the fibrosis intensity and the radioactivity uptake in the liver was observed at 0, 1, and 2 months of TAA treatment (correlation efficiency=0.9925; Fig. 6B). In the advanced fibrosis, increase abnormal immunostaining was present (Fig. 4D) and new hepatocytes were generated (Fig. 4E), and ASGPR trend in TAA-treatment course was slightly increased in 3-month and 4-month group (Fig. 6). The trend of ASGPR imaging was in accordance with that of ASGPR immunostaining among animal groups (Fig 7).


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Discussion This is the first report in which ASGPR imaging was in accordance with ASGPR staining, confirming that ASGPR imaging can correlate to the changes of the chronic liver damage. In our study, the longer the TAA treated, the higher degree of fibrosis produced (Fig. 1). However, the fibrosis percentage cannot reflect regenerated hepatocytes in advanced fibrosis (i.e. compensated cirrhosis), whereas ASGPR immunocytochemical staining can indicate such conpensation by showing the increased abnormal immunostaining (Fig. 4D) and proliferation of many small hepatocytes (Fig. 4E). Since ASGPR plays a vital role in maintaining serum glycoprotein hemostasis by recognizing galactose residue termini of asialoglycoprotein and performing receptor-mediated endocytosis,18, 19 ASGPR molecular imaging with 111In-hexavalent lactoside is more reliable to measure liver reserve because of its anticipated role in most liver functions, including hepatocyte-specific adsorption, metabolism and excretion. Moreover, immunocytochemistry requires a biopsy, which limits its development; in contrast, ASGPR scintigraphy uptake provides more objective information, such as liver compensation leading to slightly increased ASGPR. The paper previously published involved using wild-type mice and mouse models of hepatoma, partial hepatectomy and acute hepatitis to evaluate the threshold of liver reserve for survival when utilizing 111In-hexavalent lactoside for ASGPR imaging.1 The present report emphasizes that small changes (such as small hepatocyte regeneration) in chronic liver injuries are difficult to detect via traditional liver function tests but can be revealed using ASGPR immunostaining and scintigraphy. The present work could potentially introduce a sensitive imaging tool for monitoring the changes of the liver damage or compensated cirrhosis, and would be valuable for designing theranostic strategies for chronic liver diseases. Furthermore, this study provides a series of systemic assessments of a fibrosis model,


15


Page 16 of 39

including fibrosis staining, ASGPR immunostaining, ASGPR imaging and traditional liver function examinations, that will improve understanding of liver fibrosis. Although the fibrotic degree increased over the course of TAA treatment (Fig. 2), the increased abnormal immunostaining and regeneration of hepatocyte were observed in the pathology slices of 3-month (Fig. 4D) and 4-month (Fig. 4E) TAA-treated group. Generally, the ASGPR uptake negatively correlated with the severity of liver fibrosis (Fig. 6). There is a slightly higher uptake observed in the 3-month and 4-month TAA-treated mice. The phenomenon of increased abnormal immunostaining and liver regeneration may be the interpretation of the increase of liver uptake of

111

In-hexavalent lactoside tracer in the

3-month and 4-month treated group. Since the severity of liver fibrosis cannot predict the outcome of chronic liver disease, fibrosis percentage measured using Adobe Photoshop CS3 and ImageJ software on the Sirius Red-stained fibrotic liver slice or fibrosis scan with ultrasound or MRI techniques would not be a good application for prognosis of chronic liver disease; whereas,

111

In-hexalavent lactoside which correlates to the changes of the liver

damage or the liver compensation may have the potential to be an indicator for prognosis. Our study revealed that ASGPR imaging is in accordance with ASGPR immunostaining (Fig 7); therefore, the ASGPR uptake measurement will be more applicable in the clinic. More than 96±1% radiochemical purity in the final formulation was maintained up to 120 h at 4°C (Supplementary Fig. S2A),

1, 2

while

111

In-hexavalent lactoside in PBS at 37°C

or in mouse serum at 37°C had slight demetalation over periods > 24 hrs (Table 1, Supplementary Fig. S2B). The major differences between incubation environments are the incubation pH and temperature. The

111

In radiolabeling of DTPA-hexavalent lactoside

requires acidic conditions (pH 2-4, Supplementary Fig. S3).2 In PBS and serum, pH value is around 7, In(III) chelates will converse to monohydroxo forms; as a result, 111In chelates may become labile.20 Price et al mentioned that the acyclic chelator DTPA has a greater extent of


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demetalation than macrocyclic chelators such as DOTA in physiological condition.21 However, the radiochemical purity remained higher than 94% within 4 h in PBS or mouse serum, indicating that the kinetics of demetalation of acyclic chelate is very slow. Since 111

In-hexavalent lactoside has excellent liver-targeting characteristics, and this imaging can be

performed in 15 min, radiochemical purity maintained more than 94% for 4h is sufficient for imaging. Therefore, the gradual demetalation after 24 h in physiological conditions is not a problem in liver reserve measurement using 111In-hexavalent lactoside. In this study,

111

In-hexavalent lactoside was validated in a TAA-treated mouse model

(Figs. 5 & 6) and was compared with the existing tools. The existing tools included fibrosis collagen staining (Figs. 1 & 2), liver-related routine laboratory examinations (Fig. 3), and ASGPR staining (Fig. 4). In the present study, ASGPR was measured in TAA-treated mice (Figs. 4-6). ASGPR level was validated to be negatively correlated with the extent of liver severity using anti-ASGPR pathological staining (Fig. 4), a biodistribution assay (Fig. 5) and the ASGPR imaging technique (Fig. 6). Reduced anti-ASGPR staining was observed in the fibrotic livers, which indicated that ASGPR was reduced in the TAA-induced fibrotic livers. ASGPR expression can reflect the liver severity; therefore, the use of ASGPR imaging to predict small changes in functional liver reserves is reasonable. The biomarker uptake was negatively correlated with the extent of liver severity, suggesting that it might be a sensitive, noninvasive determination of liver reserve. Due to the similar pathogenesis of chronic liver disease in mice and humans, ASGPR expression levels in rodents and humans are expected to reflect the levels of liver severity in these organisms. The TAA-induced fibrosis pattern was more similar to the pattern observed in humans, and the METAVIR classification was applicable, making this system a good liver fibrosis model for drug development (i.e., pre-clinical development and translation into the clinic). When we compared this experiment to our bile duct ligation experiment (Supplementary Fig.


17


Page 18 of 39

S4), the fibrosis stage was difficult to classify in the bile-duct-ligated mice, even though they had ascites. Our study showed there was no good discriminator between two TAA treatment points in the biochemical tests, including AST, ALT, albumin, bilirubin, and alkaline phosphatase. Based on the

111

In-hexavalent lactoside scintigraphy results,

111


scintigraphy was a more sensitive method for detecting changes in functional liver reserve than biochemical tests, including ALT, AST, ALP, bilirubin, and albumin levels, because ASGPR imaging can distinguish differences in fibrosis between the TAA treatment groups, but general biochemical tests cannot. Aminotransferases (both ALT and AST) are soluble cytoplasmic enzymes that are produced as a result of hepatocellular damage.22 They are good indicators of acute hepatitis; however, they are not very reliable for detecting chronic liver change. Albumin is synthesized exclusively by the liver; thus, the concentration of albumin can be used to reflect the liver synthesis function.22 In liver disease, albumin should be theoretically reduced. However, our study indicated that albumin was insensitive or statistically irrelevant in the detection of the severity of liver fibrosis. Alkaline phosphatase is a membrane-associated enzyme and is anchored in biliary canaliculus.22 The circumstances related to ALP release from the membrane have not been fully understood until now. Both the proteolytic and detergent actions of bile acid are possible mechanisms for this release.22 Generally, ALP is released into serum in small amounts following hepatocyte apoptosis because it is anchored in the membrane. It is generally increased during cholestasis. In our study, ALP was decreased in the 2-, 3-, and 4-month TAA-treated groups. To our knowledge, this phenomenon has not been reported before. Though the explanation is not yet clear, based on the release mechanism, we hypothesize that this phenomenon is due to a reduction in the proteolytic action.


18

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Bilirubin is insoluble in water and is bound to albumin in serum. In the liver, bilirubin is taken up by hepatocytes through an organic anionic transporter

23

and is conjugated with

glucuronic acid by the enzyme glucuronyl transferase to a relatively hydrophilic molecule. The conjugated bilirubin can be secreted by the liver through bile ducts. Elevated total bilirubin in serum may be related to fewer organic anionic transporters due to hepatocyte damage. However, the plasma bilirubin concentration is also influenced by non-hepatic factors, such as increased production due to hemolysis. In our study, it was difficult for bilirubin tests to distinguish the chronic liver disease severity between TAA-treated groups (Fig. 3D). Above all, conventional biochemical tests cannot reflect the degree of chronic liver injury, nor the liver reserve. Compared with pathological staining combined with the degree of fibrosis or METAVIR staging, ASGPR imaging biomarkers can more accurately and sensitively reflect small changes in liver status. First, ASGPR imaging and staining have good correlations in chronic liver injuries. Second, the method of analyzing liver reserves through imaging is more convenient and noninvasive than conventional biopsy and pathological methods. Fibrosis is usually unevenly distributed in fibrotic livers; therefore, pathological staining requires a large sample size. The substantial effort required to perform pathological staining also limits its applicability. In addition, an accurate determination of liver reserves using the simple METAVIR classification is difficult. Furthermore, ASGPR imaging measures the overall ASGPR uptake, which is more objectively related to the residual liver reserve. ASGPR residing on hepatocyte membranes is responsible for maintaining the balance of physiological proteins via endocytosis and for degrading old serum proteins targeted via receptor-mediated endocytosis for routine turnover.19, 24 Notably, patients with early liver fibrosis have a greater opportunity for recovery; thus, earlier detection of functional liver


19


Page 20 of 39

reserve changes will be beneficial to providing proper treatment to prevent liver deterioration. Since ASGPR abundance would be one indicator of liver function, it is reasonable that 111

In-hexavalent lactoside could be an imaging biomarker for residual liver function (i.e.,

liver reserve). The ASGPR biomarker, via either in vitro immunocytochemical staining or in vivo ASGPR scintigraphy, exhibits higher sensitivity than traditional liver function tests and provides more objective information for evaluating functional liver changes. Thus, ASGPR may be a promising ligand for measuring functional liver reserve and monitoring compensated cirrhosis. In livers at METAVIR stage 4, the unanticipated increased total overall ASGPR staining and

111

In-hexavalent lactoside imaging absorption might be

attributable to regenerated hepatocytes. One limitation is that although ASGPR imaging can detect the severity of liver disease based on the relative liver reserve measurement, the differentiation of fibrosis and hepatitis is difficult because both subjects with fibrosis and subjects with hepatitis might exhibit the same liver reserve according to the ASGPR imaging technique. However, survival depends on the liver reserve; thus, it is most important to control the liver reserve. In our study, 111In-hexavalent lactoside uptake was significantly different between normal mice and mice with fibrotic livers. Recently, ASGPR imaging has been reported to exhibit potential for measuring functional liver reserves in a chronic hepatitis model. Kao et al. (2013) applied monovalent galactoside (18F-FBHGal) as an ASGPR imaging probe in a dimethylnitrosamine (DMN)-induced mouse model and revealed that

18

F-FBHGal exhibits

reduced uptake in fibrotic livers and prolonged systemic circulation in a fibrotic model. The authors reported a significant difference in ASGPR uptake (P < 0.01) between mice with normal (20.50±1.51% ID) and fibrotic nodules caused by fibrous septa (14.84±1.10% ID).25 However, the difference was too small and the sensitivity was insufficient for the early


20

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detection of liver reserve changes. Chang et al. (2013) later developed

99m

Tc-labeled

mono-galactoside and di-galactoside for ASGPR imaging of DMN-induced mice. The half-life of mono-galactoside is too short to reveal any difference between normal mice and DMN-induced mice, and the imaging signal of di-galactoside is not sufficiently stable for detection because a significant difference in ASGPR imaging uptake occurred only between normal and DMN-induced mice at 5 min post-injection,26 a short detection period that represents a considerable limitation in routine work. In addition, many organs were shown to exhibit

radioactivity

interference

in

both

Kao’s

mono-galactoside

and

Chang’s

mono-galactoside and di-galactoside methods, and the high background noise and background radiation will limit the development of these methods due to their low sensitivity. Because Gal-terminated triantennary structures exhibit a 106-fold stronger affinity than monovalent component structures,27 we used hexa-lactoside, which is a multivalent galactoside. According to our results, 111In-hexavalent lactoside scintigraphy is more sensitive in distinguishing the difference between mice treated with TAA for one month and normal mice, as well as between TAA-treated groups. Ultrasound (US)-based technology is the most widely used tool for liver fibrosis staging and possesses good diagnostic performance, providing an alternative to liver biopsy. However, there are still some drawbacks. Because of US attenuation, the measurement is unreliable in deeper regions of the livers of obese individuals10. Additionally, ascites will also interfere with the transmission of US waves. MRI-based technology also presents good accuracy regarding liver fibrosis, but some procedures still have limitations, such as requiring additional hardware and post-processing software that is not installed in every clinical center; in addition, some types of diagnostic performance are not yet well validated, and some are sensitive to motion.28 Moreover, there are reports in the literature that hepatic steatosis, inflammation, cholestasis, or right heart failure can interfere with the evaluation of liver


21


Page 22 of 39

fibrosis in both US- and MRI-based diagnostic methods10. Furthermore, our experiments showed fibrosis intensity cannot reveal compensated cirrhosis. However,

111

In-hexavalent

lactoside is a receptor-specific agent; therefore, theoretically, it has fewer biological confounders than US- and MRI-based technologies. Moreover, 111In-hexavalent lactoside can provide information about residual liver function that is not provided by US- and MRI-based technologies. Hence, the development of

111

In-hexavalent lactoside will apparently benefit

liver diagnosis and prognosis in the future. The maintenance of sufficient ASGPR content on the membrane of hepatocytes is extremely important for survival. Liver reserve measurement is often used in pre-surgery assessments. The published paper has shown ASGPR imaging exhibits considerable potential as an indicator of liver reserve.1 Because liver fibrosis is related to liver reserve and early detection of fibrosis improves recovery, the early detection of changes in liver reserve is important for the maintenance of liver health. In addition,

111

In-hexavalent lactoside was

liver-specific and can be uptaken by ASGPR even in the context of chronic liver injuries. Thus,

111

In-hexavalent lactoside not only has diagnostic value but also possesses

liver-targeting properties that render it suitable for use in drug delivery systems for chronic liver disease therapy. Furthermore, ASGPR imaging provides a more sensitive method for detecting compensated cirrhosis and may serve as a good prognosis predictor of liver reserve with advanced fibrosis in routine healthcare. In conclusion,

111

In-hexavalent lactoside imaging is in accordance with ASGPR

immunostaining. ASGPR level is a good indicator that correlates to the changes of the liver damage or liver compensation. ASGPR imaging using

111

In-hexavalent lactoside shows

promise as a sensitive imaging tool for use as a diagnostic or prognostic biomarker of the functional liver mass because it exhibits a specific liver-targeting behavior and has the potential to sensitively reflect changes in liver reserve associated with liver disease.


22

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Acknowledgments This study was supported by the Atomic Energy Council of Taiwan (98-2001-01-D-10, 99-2001-01-D-07, and 100-2001-01-D-12).

Supporting Information Fig. S1: Correlation of radiochemical purity measurement by radio-ITLC and radio-HPLC techniques. Fig. S2: Stability of 111In-hexavalent lactoside in final formulation, PBS or mouse serum. Fig. S3: The effect of pH on radiochemical yield. Fig. S4: Molecular imaging of a normal Wistar rat and one with bile duct ligation.


23


Page 24 of 39

References Reference (1) Wang, M. H.; Chien, C. Y.; Wang, P. Y.; Yu, H. M.; Lee, H. S.; Lin, W. J.

The

specificity and accuracy of (111)In-hexavalent lactoside in estimating liver reserve and its threshold value for mortality in mice. J Hepatol 2015, 63, (2), 370-7. (2) Wang, M. H.; Lin, W. J.; Yu, H. M.; Chien, C. Y.; Wang, P. Y. Liver-receptor imaging injection, dispensing method and pharmaceutical composition thereof. US9040017B2. 2015. (3) Muller, A.; Machnik, F.; Zimmermann, T.; Schubert, H.

Thioacetamide-induced

cirrhosis-like liver lesions in rats--usefulness and reliability of this animal model. Exp Pathol 1988, 34, (4), 229-36. (4) Bruck, R.; Aeed, H.; Shirin, H.; Matas, Z.; Zaidel, L.; Avni, Y.; Halpern, Z.

The

hydroxyl radical scavengers dimethylsulfoxide and dimethylthiourea protect rats against thioacetamide-induced fulminant hepatic failure. J Hepatol 1999, 31, (1), 27-38. (5) Porter, W. R.; Gudzinowicz, M. J.; Neal, R. A.

Thioacetamide-induced hepatic

necrosis. II. Pharmacokinetics of thioacetamide and thioacetamide-S-oxide in the rat. J Pharmacol Exp Ther 1979, 208, (3), 386-91. (6) Moreira, E.; Fontana, L.; Periago, J. L.; Sanchez De Medina, F.; Gil, A.

Changes in

fatty acid composition of plasma, liver microsomes, and erythrocytes in liver cirrhosis induced by oral intake of thioacetamide in rats. Hepatology 1995, 21, (1), 199-206. (7) Honda, H.; Ikejima, K.; Hirose, M.; Yoshikawa, M.; Lang, T.; Enomoto, N.; Kitamura, T.; Takei, Y.; Sato, N. Leptin is required for fibrogenic responses induced by thioacetamide in the murine liver. Hepatology 2002, 36, (1), 12-21. (8) Kornek, M.; Raskopf, E.; Guetgemann, I.; Ocker, M.; Gerceker, S.; Gonzalez-Carmona, M. A.; Rabe, C.; Sauerbruch, T.; Schmitz, V.

Combination of systemic thioacetamide (TAA)

injections and ethanol feeding accelerates hepatic fibrosis in C3H/He mice and is associated with intrahepatic up regulation of MMP-2, VEGF and ICAM-1. J Hepatol 2006, 45, (3), 370-6. (9) Sumida, Y.; Nakajima, A.; Itoh, Y.

Limitations of liver biopsy and non-invasive

diagnostic tests for the diagnosis of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. World J Gastroenterol 2014, 20, (2), 475-85. (10) Tang, A.; Cloutier, G.; Szeverenyi, N. M.; Sirlin, C. B.

Ultrasound Elastography and

MR Elastography for Assessing Liver Fibrosis: Part 2, Diagnostic Performance, Confounders, and Future Directions. American Journal of Roentgenology 2015, 205, (1), 33-40. (11) Pomper, M. G.; Lee, S.

Molecularly Targeted MR Imaging Agent in Liver Fibrosis:

High Sensitivity and Low Gadolinium Mean High Translational Potential. Radiology 2018, 287, (2), 590-591.


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(12) Hessien, M. H.; El-Sharkawi, I. M.; El-Barbary, A. A.; El-Beltagy, D. M.; Snyder, N. Non-invasive index of liver fibrosis induced by alcohol, thioacetamide and Schistosomal infection in mice. BMC Gastroenterol 2010, 10, 53. (13) Kisseleva, T.; Cong, M.; Paik, Y.; Scholten, D.; Jiang, C.; Benner, C.; Iwaisako, K.; Moore-Morris, T.; Scott, B.; Tsukamoto, H.; Evans, S. M.; Dillmann, W.; Glass, C. K.; Brenner, D. A.

Myofibroblasts revert to an inactive phenotype during regression of liver

fibrosis. Proc Natl Acad Sci U S A 2012, 109, (24), 9448-53. (14) Tang, X. N.; Berman, A. E.; Swanson, R. A.; Yenari, M. A.

Digitally quantifying

cerebral hemorrhage using Photoshop and Image J. J Neurosci Methods 2010, 190, (2), 240-3. (15) Jonker, A. M.; Dijkhuis, F. W.; Boes, A.; Hardonk, M. J.; Grond, J. Immunohistochemical study of extracellular matrix in acute galactosamine hepatitis in rats. Hepatology 1992, 15, (3), 423-31. (16) Lee, R. T.; Wang, M. H.; Lin, W. J.; Lee, Y. C.

New and more efficient multivalent

glyco-ligands for asialoglycoprotein receptor of mammalian hepatocytes. Bioorg Med Chem 2011, 19, (8), 2494-500. (17) Yu, H. M.; Wang, M. H.; Chen, J. T.; Lin, W. J.

Labelling of Peptide Derivative with

In-111 for Receptor Imaging. J Label Compd Radiopharm 2010, 53 406-489. (18) Ashwell, G.; Harford, J.

Carbohydrate-Specific Receptors of the Liver. Annual Review

of Biochemistry 1982, 51, (1), 531-554. (19) Ashwell, G.; Morell, A. G.

The role of surface carbohydrates in the hepatic recognition

and transport of circulating glycoproteins. Adv Enzymol Relat Areas Mol Biol 1974, 41, (0), 99-128. (20) Clarke, E. T.; Martell, A. E. Stabilities of the Fe(III), Ga(III) and In(III) chelates of N,N ′ ,N ″ -triazacyclononanetriacetic acid. Inorganica Chimica Acta 1991, 181, (2), 273-280. (21) Price, E. W.; Cawthray, J. F.; Bailey, G. A.; Ferreira, C. L.; Boros, E.; Adam, M. J.; Orvig, C.

H4octapa: an acyclic chelator for 111In radiopharmaceuticals. J Am Chem Soc 2012, 134,

(20), 8670-83. (22) Whitby, L. G.; Smith, A. F.; Beckett, G. J.; Walker, S. W., Lecture notes on clinical biochemistry. Blackwell Scientific publications: 1993; p 105-123. (23) Yh, C.; Koenig, J.; Leier, I.; Buchholz, U.; Keppler, D., Hepatic Uptake of Bilirubin and Its Conjugates by the Human Organic Anion Transporter SLC21A6. 2001; Vol. 276, p 9626-30. (24) Morell, A. G.; Irvine, R. A.; Sternlieb, I.; Scheinberg, I. H.; Ashwell, G.

Physical and

chemical studies on ceruloplasmin. V. Metabolic studies on sialic acid-free ceruloplasmin in vivo. J Biol Chem 1968, 243, (1), 155-9. (25) Kao, H. W.; Chen, C. L.; Chang, W. Y.; Chen, J. T.; Lin, W. J.; Liu, R. S.; Wang, H. E.


25


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(18)F-FBHGal for asialoglycoprotein receptor imaging in a hepatic fibrosis mouse model. Bioorg Med Chem 2013, 21, (4), 912-21. (26) Chang, W. Y.; Kao, H. W.; Wang, H. E.; Chen, J. T.; Lin, W. J.; Wang, S. J.; Chen, C. L. Synthesis and biological evaluation of technetium-99m labeled galactose derivatives as potential asialoglycoprotein receptor probes in a hepatic fibrosis mouse model. Bioorg Med Chem Lett 2013, 23, (23), 6486-91. (27) Lee, Y. C.; Townsend, R. R.; Hardy, M. R.; Lonngren, J.; Arnarp, J.; Haraldsson, M.; Lonn, H.

Binding of synthetic oligosaccharides to the hepatic Gal/GalNAc lectin.

Dependence on fine structural features. J Biol Chem 1983, 258, (1), 199-202. (28) Petitclerc, L.; Sebastiani, G.; Gilbert, G.; Cloutier, G.; Tang, A.

Liver fibrosis: Review

of current imaging and MRI quantification techniques. J Magn Reson Imaging 2017, 45, (5), 1276-1295.


26

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Table 1 Stability of 111In-hexavalent lactoside in PBS (37°°) or mouse serum (37°°C) Radiochemical purity (%) 15 min

1h

4h

24 h

PBS

97.8± ±0.7

96.8± ±0.2

95.5± ±0.7

89.3± ±0.1

Mouse serum

97.0± ±0.1

96.7± ±0.1

94.3± ±0.4

91.9± ±0.6

In vitro stability of 111In-hexavalent lactoside in PBS and mouse serum at 37°C over 24 h. Values are the mean ± SD (n=3) for each time point.

Figure legends

Fig. 1. Sirius Red staining of mouse liver tissues (5-7 µm). Panel A: Image of liver tissues from normal mice. Panels B, C, D, and E: Images of liver tissues from mice treated with thioacetamide for 1, 2, 3, and 4 months, respectively. The bar scale indicates 200 µm.

Fig. 2. Fibrosis percentage in normal and fibrotic mice. The mice were intraperitoneally injected with 200 mg/kg of TAA every other day for 1, 2, 3, or 4 months to induce fibrosis. Fibrotic tissues were stained with Sirius Red, and the fibrosis fraction was measured using Adobe Photoshop CS3 and ImageJ software. TAA: thioacetamide.

Fig. 3. Biochemical tests in normal and TAA-treated mice. Panel A: Serum transaminase levels in normal mice and mice treated with thioacetamide for 1, 2, 3, and 4 months. Panel B: Albumin. Panel C: Alkaline phosphatase. Panel D: Total bilirubin. TAA, thioacetamide; ALT, alanine transaminase; AST, aspartic transaminase.

Fig. 4. Immunostaining of mouse liver tissues (5-7 µm) with an anti-asialoglycoprotein


27


Page 28 of 39

receptor antibody. Panel A: Images of normal BALB/c mouse liver tissue. Panels B-E: Images of liver tissues from BALB/c mice treated with thioacetamide for 1, 2, 3, and 4 months, respectively. The black arrow in panel D shows that some abnormal immunostaining increased around the central vein in mice treated with thioacetamide for 3 months. The red circle in panel E shows many irregular, smaller hepatocytes regenerating around the central vein. The bar scale indicates 200 µm.

Fig. 5. Comparison of the biodistribution data between the normal and TAA-treated groups. The radioactivity uptake in the organs was measured by collecting tissues from normal mice and mice that had been treated with TAA for 1-4 months and sacrificed at 15 min after injection of 148 kBq of 111In-hexavalent lactoside. TAA, thioacetamide.

Fig. 6. Molecular images of normal and fibrotic livers from mice treated with TAA. The normal mice and the mice that had been treated with TAA for 1, 2, 3, and 4 months were injected with

111

In-hexavalent lactoside, and ASGPR imaging was analyzed using

nanoSPECT/CT (Mediso, Hungary). Panel A: NanoSPECT/CT 2D imaging and 3D volume of liver uptake quantitation (mean ± standard deviation) were performed immediately after intravenous injection of

111

In-hexavalent lactoside (18.5 MBq); scanning was performed for

15 min. The red color in the bar represents a higher uptake, and blue represents a lower uptake. The mice with TAA-induced fibrosis for 1 or 2 months exhibited significantly lower ASGPR uptake than the normal group (P < 0.01). The gray-white color in 2D image of the livers from the 3-month and 4-month TAA treatment groups indicates cirrhosis. Panel B: A comparison of the radioactivity uptake by ASGPR between normal and fibrotic mice. The percentage of injection dose (%ID) in the liver volume of interest was quantified using InVivo Scope and PMOD software and compared to the liver slice fibrosis %; the liver


28

Page 29 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


section was stained with Sirius Red, and the fibrotic area was measured using Photoshop 6.0 and ImageJ softwares. A relationship figure for the % radioactivity uptake in the liver and fibrosis percentage in normal and TAA-induced mice was generated according to the above measurement method. The vertical axis in the left shows the real percentage liver uptake of normal and treatment groups, while the vertical axis in the right refers to the relative percentage liver uptake (RPLU). RPLU is obtained by dividing the liver uptake of each treatment group by normal group, which demonstrates the relative liver uptake of disease groups compared to normal group. TAA, thioacetamide.

Fig. 7. ASGPR measured by immunocytochemical staining and SPECT/CT scintigraphy of normal and fibrotic livers from mice treated with TAA. From normal mice and mice that had been treated with TAA for 1, 2, 3, and 4 months to induce fibrosis, fibrotic tissues were stained with anti-ASGPR. The anti-ASGPR staining fraction was measured using Adobe Photoshop CS3 and ImageJ software and compared with the liver slice fibrotic percentage measured using Adobe Photoshop CS3 and ImageJ software on the Sirius Red-stained fibrotic liver slice. The ASGPR imaging was analyzed using

111

In-hexavalent

lactoside nanoSPECT/CT (Mediso, Hungary) and compared with the liver slice fibrotic percentage calculated using ImageJ software after collagen staining. TAA, thioacetamide.


29


ASGP-Receptor imaging (% of injected dose)

Table of Contents Graphic

60

20

ASGP-Receptor imaging (% of injected dose) ASGP-Receptor staining (%)

40

15 10

20 5 0

0 0

5

10

15

ASGP-Receptor staining %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 39

20

Liver fibrosis %


30

Page 31 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56


A

B

C

D

E

Fig 1



20

***

15.761.83%

15

*** ***

10

10.733.23%

9.571.37%

***

5

5.370.84%

on th s

TA A

4

m

on th s m 3

TA A

2 TA A

TA A

1

m

m

on th s

al

on th

1.170.46%

0

no rm

Fibrosis fraction (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56

Page 32 of 39

Fig 2


Page 33 of 39

A 150 125

U/L

100

**

75

**

**

**

ALT

50

AST

25 0 0

B

5

10

15

20

fibrosis % 3.5

Albumin (g/dL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56


3.0 2.5 2.0 1.5 0

5

10

fibrosis %

Fig 3A-B


15

20


C Alkaline phophastase (U/L)

800 700 600

*

** ***

500 400 0

5

10

15

20

fibrosis (%)

D 1.5

Total bilirubin (mg/dL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56

Page 34 of 39

** 1.0

**

**

*

5

10

0.5

0.0 0

fibrosis (%)

Fig 3C-D


15

20

Page 35 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56


A

B

C

D

E

Fig 4


50 40 30

Page 36 of 39

Normal TAA 1 month TAA 2 month TAA 3 month TAA 4 month

20 10 0

B M rain us cl e St Bo om ne a Sp c h Pa l e nc en re a Li s v B er lo od Lu n H g e K ar id t ne Te y st is

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56

Percentage of Injected Dose per gram organ


Fig 5


Page 37 of 39

B 60

% of injected dose

53%

100%

50

**

40

30 first three points

*** ***

71.7%

*** 50.9%

20 Y = -3.085X + 55.96 R2 =0.9925

10

all points Y=-1.481X+49.24 R 2 =0.6709

0 0

2

4

6

8

10 12 14 16 18 20

% of fibrosis

Fig 6


110 100 90 80 70 60 50 40 30 20 10 0

relative % of injected dose

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56



ASGPR uptake (% of injected dose)

60

20

ASGPR uptake (% of ID) ASGPR stain (%)

15

40 10 20 5 0

0 0

5

10

15

fibrosis %

Fig 7


20

ASGPR stain %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56

Page 38 of 39

ASGP-Receptor imaging (% of injected dose) ASGP-Receptor staining (%)

60

40

20 15 10

20 5 0

0 0

5

10

15

ASGP-Receptor staining %

1 2 3 4 5 6

ASGP-Receptor imaging (% of injected dose)

Page 39 of 39 Molecular Pharmaceutics

20

Liver fibrosis %


Use of In-Hexavalent Lactoside for Liver Reserve ... - ACS Publications

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