Detection of HER2 expression using 99mTc-NM-02 nanobody in patients with breast cancer: a non-randomized, non-blinded clinical trial.

BACKGROUND: 99mTc radiolabeled nanobody NM-02 (99mTc-NM-02) is a novel single photon emission computed tomography (SPECT) probe with a high affinity and specificity for human epidermal growth factor receptor 2 (HER2). In this study, a clinical imaging trial was conducted to investigate the relationship between 99mTc-NM-02 uptake and HER2 expression in patients with breast cancer. METHODS: Thirty patients with pathologically confirmed breast cancer were recruited and imaged with both 99mTc-NM-02 SPECT/computed tomography (CT) and 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET)/CT. According to the treatment conditions before recruitment, patients were divided into two groups, the newly diagnosed group (n = 24) and the treated group (n = 6). The maximal standard uptake value (SUVmax) of 18F-FDG and SUVmax and mean SUV (SUVmean) of 99mTc-NM-02 in the lesions were determined to analyze the relationship with HER2 expression. RESULTS: No meaningful relationship was observed between 18F-FDG uptake and HER2 expression in 30 patients with breast cancer. 99mTc-NM-02 uptake was positively correlated with HER2 expression in the newly diagnosed group, but no correlation was observed in the treated group. 99mTc-NM-02 uptake in HER2-positive lesions was lower in those with effective HER2-targeted therapy compared with the newly diagnosed group. 99mTc-NM-02 SPECT/CT detected brain and bone metastases of breast cancer with a different imaging pattern from 18F-FDG PET/CT. 99mTc-NM-02 showed no non-specific uptake in inflamed tissues and revealed intra- and intertumoral HER2 heterogeneity by SPECT/CT imaging in 9 of the 30 patients with breast cancer. CONCLUSIONS: 99mTc-NM-02 SPECT/CT has the potential for visualizing whole-body HER2 overexpression in untreated patients, making it a promising method for HER2 assessment in patients with breast cancer. TRIAL REGISTRATION: NCT04674722, Date of registration: December 19, 2020.

Drug-loaded hybrid hydrogels for sonodynamic-chemodyanmic therapy and tumor metastasis suppression.

Introduction: Although various therapies have been adopted to treat cancer, metastasis of tumor cells still is a big challenge that compromises therapeutic benefits. Methods: We herein report an injectable drug-loaded hybrid hydrogel that can achieve sonodynamic therapy (SDT) and chemodyanmic therapy (CDT) combined action and suppression of tumor metastasis. This alginate (ALG)-based hydrogel (termed as AMPS) contains manganese dioxide (MnO2) nanoparticles as the CDT agents, an organic polymer as the sonosensitizer, and a SIS3 drug as metastasis inhibitor. Results: AMPS is formed via the chelation of ALG by Ca2+ in tumor microenvironment, in which MnO2 nanoparticles mediate CDT via Fenton-like reaction and the organic polymers enable SDT under ultrasound (US) irradiation by generating singlet oxygen (1O2), allowing for combinational action of CDT and SDT. In addition, SIS3 is released from AMPS hydrogels to inhibit the metastasis of tumor cells. As such, the AMPS enables a combinational action of SDT and CDT to greatly inhibit the growths of subcutaneous tumors in living mice and also completely suppress the tumor metastasis in lungs and livers. Conclusion: This study thus offers a hybrid hydrogel platform for combinational therapy and metastasis suppression simultaneously.

[Diagnostic and Prognostic Value of 18F-FDG PET/CT in Bone Marrow Infiltration of Newly Diagnosed Diffuse Large B-Cell Lymphoma].

OBJECTIVE: To explore the diagnostic value of 18F-FDG PET/CT in bone marrow infiltration (BMI) of newly diagnosed diffuse large B-cell lymphoma (DLBCL), compared with the results of bone marrow biopsy (BMB) and investigate whether the BMI diagnosed by 18F-FDG PET/CT and other factors have independent prognostic values. METHODS: Ninety-four newly diagnosed DLBCL patients who underwent PET/CT in Clinical Medical College of Shanghai General Hospital of Nanjing Medical University were included. BMB was performed within 2 weeks before or after PET/CT, and standardized treatment was performed after PET/CT. The manifestations of bone marrow (BM) FDG uptake were recorded. The diagnostic criteria of BMI were BMB positive or focal BM FDG uptake confirmed by imaging follow-up. The relationship between clinical features and BM FDG uptake and the values of PET/CT and BMB in the diagnosis of BMI was analyzed. The progression-free survival (PFS) was analyzed by Kaplan-Meier survival curves, log-rank test was used to compare PFS rate, and Cox regression model was used to analyze the independent risk factors affecting PFS. RESULTS: Among 94 DLBCL patients, 34 patients showed focal BM uptake (fPET), 7 patients showed super BM uptake (sBMU), 11 patients showed diffuse homogenous uptake higher than liver (dPET), and the other 42 patients had normal BM uptake (nPET) (lower than liver). BMB positive was found in all sBMU patients, in 20.6%(7/34) of fPET patients, and in 27.3% (3/11) of dPET patients. All nPET patients had negative BMB results. dPET patients were associated with lower hemoglobin level and leukocyte count compared with nPET group (P < 0.001, P =0.026). Compared with fPET patients, sBMU patients were more likely to have B symptoms and elevated lactate dehydrogenase (LDH). A total of 44 patients were diagnosed BMI, including 17 cases with BMB+. The sensitivity and specificity of BMB in the diagnosis of BMI was 38.6% (17/44) and 100% (50/50), respectively. Using fPET and sBMU as criteria of PET BMI, the diagnostic sensitivity and specificity of PET/CT was 93.2% (41/44) and 100% (50/50), respectively. Kaplan-Meier analysis showed that there was no significant difference in 2-year PFS rate between nPET and dPET patients (P >0.05), while sBMU patients had lower 2-year PFS rate compared with fPET patients (P < 0.001). Multivariate analysis showed that higher Ann Arbor stage (HR=9.010, P =0.04) and sBMU (HR=3.964, P =0.002) were independent risk factors affecting PFS. CONCLUSIONS: Increased BM FDG uptake of DLBCL can be manifested as dPET, fPET and sBMU. fPET and sBMU can replace BMB to diagnose BMI. Although dPET cannot completely exclude the possibility of BMI, it does not affect the prognosis, so it can be diagnosed as PET BMI negative. sBMU is an independent prognostic risk factor.

131I-Labeled Anti-HER2 Nanobody for Targeted Radionuclide Therapy of HER2-Positive Breast Cancer.

Purpose: The unique structure of nanobodies is advantageous for the development of radiopharmaceuticals for nuclear medicine. Nanobodies targeted to human epidermal growth factor receptor 2 (HER2) can be used as tools for the imaging and therapy of HER2-overexpressing tumors. In this study, we aimed to describe the generation of a 131I-labeled anti-HER2 nanobody as a targeted radionuclide therapy (TRNT) agent for HER2-positive breast cancer. Methods: The anti-HER2 nanobody NM-02 was labeled with 131I using the iodogen method, and its radiochemical purity and stability in vitro were assessed. The pharmacokinetic profile of 131I-NM-02 was investigated in normal mice. Tumor accumulation, biodistribution, and therapeutic potential of 131I-NM-02 were evaluated in HER2-positive SKBR3 xenografts; HER2-negative MB-MDA-231 xenografts were used as the control group. Results: 131I-NM-02 could be readily prepared with satisfactory radiochemical purity and stability in vitro. Apparent tumor uptake was observed in HER2-positive tumor-bearing mice with rapid blood clearance and favorable biodistribution. 131I-NM-02 could significantly inhibit tumor growth and extend the life of these mice with good organ compatibility. Negligible tumor accumulation and inhibitory effects of 131I-NM-02 were observed in the negative control group. Conclusion: 131I-NM-02 has the potential to be explored as a novel tool for TRNT of HER2-positive breast cancer.

Second near-infrared photoactivatable nanomedicines for enhanced photothermal-chemodynamic therapy of cancer.

Nanomedicines have been widely used for cancer therapy, while controlling their activity for effective and safe treatment remains a big challenge. Herein, we report the development of a second near-infrared (NIR-II) photoactivatable enzyme-loaded nanomedicine for enhanced cancer therapy. Such a hybrid nanomedicine contains a thermoresponsive liposome shell loaded with copper sulfide nanoparticles (CuS NPs) and glucose oxidase (GOx). The CuS nanoparticles mediate the generation of local heat under 1064 nm laser irradiation, which not only can be used for NIR-II photothermal therapy (PTT), but also leads to the destruction of the thermal-responsive liposome shell to achieve the on-demand release of CuS nanoparticles and GOx. In a tumor microenvironment, GOx oxidizes glucose to produce hydrogen peroxide (H2O2) that acts as a medium to promote the efficacy of chemodynamic therapy (CDT) by CuS nanoparticles. This hybrid nanomedicine enables the synergetic action of NIR-II PTT and CDT to obviously improve efficacy without remarkable side effects via NIR-II photoactivatable release of therapeutic agents. Such a hybrid nanomedicine-mediated treatment can achieve complete ablation of tumors in mouse models. This study provides a promising nanomedicine with photoactivatable activity for effective and safe cancer therapy.

The r1 relaxivity and T1 imaging properties of dendrimer-based manganese and gadolinium chelators in magnetic resonance imaging.

We report the preparation and characterization of gadolinium (Gd)- or manganese (Mn)-loaded dendrimers and Gd-loaded dendrimer-entrapped gold nanoparticles (Gd-Au DENPs) to examine the relationship between the number of metal ion chelators and r1 relaxivity. In this study, amine-terminated fifth-generation poly(amidoamine) dendrimers (G5.NH2) modified with different numbers of DOTA-NHS chelators were used to chelate Gd and Mn ions. The remaining amine groups were then acetylated completely, followed by the use of materials with better r1 relaxivities and T1-weighted imaging performances as templates to synthesize Gd-Au DENPs. The Gd and Mn chelators as well as Gd-Au DENPs were characterized via different techniques. We show that the r1 relaxivity and T1 imaging performance increase with loading of greater numbers of Gd and Mn ions on the G5.NH2 and that the acetylation process affects the relaxivity and imaging properties to a certain extent. After entrapment with Au NPs, the r1 relaxivity and T1-weighted imaging performance of Gd-Au DENPs decrease with greater loading of Au NPs. This systematic study of the relaxivities and T1-weighted imaging performances of Gd, Mn, and Gd-Au DENP chelators are expected to be a theoretical basis for developing multifunctional dual-mode contrast agents.

Prodrug and Glucose Oxidase Coloaded Photodynamic Hydrogels for Combinational Therapy of Melanoma.

With the advantages of high safety and selectivity, photodynamic therapy (PDT) has been widely used for cancer treatments, while the anticancer efficacy is often limited because of its relying on oxygen concentrations. Therefore, sole PDT fails to achieve the desired therapeutic effect for hypoxic tumors. To address this issue, we herein report the construction of prodrug and glucose oxidase (GOx) coloaded alginate (ALG) hydrogels for PDT-combined chemotherapy of melanoma. The hydrogels are in situ formed in tumor sites after injection of ALG solution containing semiconducting polymer nanoparticles, hypoxia-responsive prodrug tirapazamine (TPZ), and GOx, which is based on chelation of ALG by endogenous Ca2+. Due to the presence of semiconducting polymer nanoparticles acting as photosensitizers, the hydrogels mediate PDT to produce singlet oxygen (1O2) for directly killing tumor cells, in which oxygen is consumed to create a more hypoxic tumor microenvironment. Moreover, the loaded GOx within hydrogels can deplete oxygen to further aggravate tumor hypoxia. As such, TPZ is effectively activated by hypoxia to cause cancer cell death via chemotherapy. Thus, the hydrogels with laser irradiation achieve a combinational action of PDT with chemotherapy to almost completely eradicate tumors, leading to a much higher therapeutic efficacy relative to sole PDT. This study will provide a promising injectable hydrogel platform for effective treatments of cancer.

Albumin-Stabilized Manganese Oxide/Semiconducting Polymer Nanocomposites for Photothermal-Chemodynamic Therapy of Hepatic Carcinoma.

Hepatic carcinoma is one of the most common cancers worldwide, while its treatment remains a great challenge. Traditional therapeutic methods often have disadvantages such as limited therapeutic efficacy and potential side effects. In this study, we report the construction of bovine serum albumin (BSA)-stabilized manganese oxide (MnO2)/semiconducting polymer (SP) nanocomposites to combine photothermal therapy (PTT) and chemodynamic therapy (CDT) for treatment of hepatic carcinoma in living mouse models. Such nanocomposites are composed of BSA, SP, and MnO2 as the stabilizer, PTT, and CDT agent, respectively. SP produced local heat under near-infrared (NIR) laser irradiation for PTT, and MnO2 nanoparticles mediated CDT in the tumor microenvironment, leading to apoptosis of cancer cells. Such nanocomposite-mediated combinational therapy showed a much higher efficacy in inhibiting growth of subcutaneous HepG2 tumors in nude mice than sole treatment. This study thus provides a multifunctional nanoplatform for safe and effective treatment of hepatic carcinoma.

Development of a 99mTc-Labeled Single-Domain Antibody for SPECT/CT Assessment of HER2 Expression in Breast Cancer.

Accurate determination of human epidermal growth factor receptor 2 (HER2) expression is essential for HER2-targeted therapy in patients with cancer. HER2 expression in a complex environment, such as in a heterogeneous tumor, makes the precise assessment of the HER2 status difficult using current methods. In this study, we developed a novel 99mTc-labeled anti-HER2 single-domain antibody (99mTc-NM-02) as a molecular imaging tracer for the noninvasive detection of HER2 expression and investigated its safety, radiation dosimetry, biodistribution, and tumor-targeting potential in 10 patients with breast cancer. Our data showed that no drug-related adverse reactions occurred. The tracer mainly accumulated in the kidneys and liver with mild uptake in the spleen, intestines, and thyroid; however, only background tracer levels were observed in other organs where primary tumors and metastases typically occurred. The mean effective dose was 6.56 × 10-3 mSv/MBq, and tracer uptake was visually observed in the primary tumors and metastases. A maximal standard uptake value of 1.5 was determined as a reasonable cutoff for identifying HER2 positivity using SPECT/CT imaging. Our 99mTc-NM-02 tracer is safe for use in breast cancer imaging, with reasonable radiation doses, favorable biodistribution, and imaging characteristics. 99mTc-NM-02 SPECT imaging may be an accurate and noninvasive method to detect the HER2 status in patients with breast cancer.

LyP-1-Modified Multifunctional Dendrimers for Targeted Antitumor and Antimetastasis Therapy.

We designed and synthesized 131I-labeled dendrimers modified with the LyP-1 peptide as a multifunctional platform for single-photon emission computed tomography (SPECT) imaging, radionuclide therapy, and antimetastasis therapy of cancer. The multifunctional platform was constructed by modifying amine-terminated generation 5 poly(amidoamine) dendrimers with 33.1 LyP-1 peptide and 9.2 3-(4'-hydroxyphenyl)propionic acid-OSu (HPAO), followed by acetylation of the remaining dendrimer terminal amines and radiolabeling with 131I via the HPAO moieties. The LyP-1-modified dendrimers showed favorable cytocompatibility in the studied concentration range of 0.1-10 µM for 24 h and could be labeled by 131I with satisfactory radiochemical purity (>99%) and stability (>90% even at 16 h). The 131I-labeled LyP-1-modified dendrimers were capable of being utilized as a diagnostic probe for SPECT imaging and as a therapeutic agent for radionuclide therapy and antimetastasis of cancer cells in vitro and in a subcutaneous tumor model in vivo. Based on analyses of the tumor microenvironment, the antitumor and antimetastasis effects could be because of the reduced levels of the molecular markers associated with proliferation and metastasis, improved local hypoxia, and increased apoptosis rate. The developed 131I-labeled dendrimeric nanodevice may hold great promise to be used as a nanotheranostic platform for cancer diagnosis and therapy.

Anti-metastatic effect of 131I-labeled Buthus martensii Karsch chlorotoxin in gliomas.

The present study investigated the underlying molecular mechanism by which Buthus martensii Karsch chlorotoxin (BmK CT) inhibits the invasion and metastasis of glioma cells and the possibility of 131I­labeled BmK CT (131I­BmK CT) as a novel targeted agent for the treatment of glioma. The impact of BmK CT with and without 131I radiolabeling on the invasion and metastasis of glioma cells in vitro was studied. Cell viability was assessed using Cell Counting Kit­8 and plate colony formation assays in order to confirm the cytotoxicity of BmK CT and 131I­BmK CT at different concentrations. Transwell invasion and wound­healing assays were conducted in order to investigate the inhibitory effects BmK CT and 131I­BmK CT on cell migration and invasion. Furthermore, western blotting, ELISA immunofluorescence and a gelatin zymography assay were performed to evaluate changes in the protein expression levels of glioma cells following treatment with BmK CT or 131I­BmK CT. The results indicated that BmK CT inhibits the invasion and metastasis of glioma cells via regulation of tissue inhibitor of metalloproteinase­2 expression and that 131I­BmK CT has the potential to be a novel targeted therapeutic drug for glioma.

SPECT imaging and radionuclide therapy of glioma using 131I labeled Buthus martensii Karsch chlorotoxin.

Gliomas, the most prevalent type of brain tumor in adults, are associated with high rates of morbidity and mortality. Recent studies on 131I labeled scorpion toxins suggest they can be developed as tumor-specific agents for glioma diagnosis and treatment. This study investigated the potential of 131I labeled Buthus martensii Karsch chlorotoxin (131I-BmK CT) as a new approach for targeted imaging and therapy of glioma. The results showed that 131I can be successfully linked to BmK CT with satisfactory radiochemical purity and stability and that 131I-BmK CT markedly inhibited glioma cell growth in a dose and time dependent manner, with significant accumulation in glioma cells in vitro. Persistent intratumoral radioiodine retention and specific accumulation of 131I-BmK CT were observed in C6 glioma tumor, which was clearly visualized by SPECT imaging. Both intratumoral and intravenous injections of 131I-BmK CT could result in significant tumor inhibition efficacy and prolonging the lifetime of tumor-bearing mice. Based on these promising results, it is concluded that 131I-BmK CT has the potential to be explored as a novel tool for SPECT imaging and radionuclide therapy of glioma.

A promising dual mode SPECT/CT imaging platform based on 99mTc-labeled multifunctional dendrimer-entrapped gold nanoparticles.

Multifunctional 99mTc-labeled dendrimer-entrapped gold nanoparticles (99mTc-Au DENPs) were designed and synthesized. Our results show that the type of surface groups (acetyl or hydroxyl) significantly impact the biodistribution profile of the 99mTc-Au DENPs, thereby allowing for preferential SPECT/CT imaging of different organs.

LAPONITE-Polyethylenimine Based Theranostic Nanoplatform for Tumor-Targeting CT Imaging and Chemotherapy.

In this study, laponite (LAP) nanodisks and polyethylenimine (PEI) were used to build a hybrid theranostic nanoplatform for targeted computed tomography (CT) imaging and chemotherapy of cancer cells overexpressing CD44 receptors. First, amphiphilic copolymer poly(lactic acid)-poly(ethylene glycol) (PLA-PEG-COOH) were assembled on the surface of LAP nanodisks via hydrophobic interaction, and then PEI were conjugated by the formation of amide groups via1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) coupling chemistry. The developed LAP-PLA-PEG-PEI nanoparticles were used as templates for the embedding of gold nanoparticles (Au NPs), followed by modification with hyaluronic acid (HA) as a targeting ligand for cancer cells overexpressing CD44 receptors. Finally, anticancer drug doxorubicin (DOX) was loaded. The formed LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes display good stability, a high drug loading efficiency as 91.0 ± 1.8%, and sustained drug release profile with a pH-sensitive manner. In vitro cell viability assay, flow cytometric analysis, and laser scanning confocal microscopy observation demonstrate that the formed nanocomplexes can specifically deliver and inhibit cancer cells overexpressing CD44 receptors. In vivo experiments illustrate that LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes can not only significantly inhibit the growth of tumors and decrease the side-effect of DOX, but also be used as a targeted contrast agent for CT imaging of tumors. Therefore, the developed LAP-PLA-PEG-PEI-(Au0)50-HA/DOX nanocomplexes can be used as a promising theranostic platform for targeted imaging and chemotherapy of CD44-overexpressed tumors.

[(18)F-FDG PET/CT for extranodular natural killer/T-cell lymphoma nasal type: imaging findings and clinical value].

OBJECTIVE: To explore the value of (18)F-FDG PET/CT in the diagnosis and treatment evaluation in patients with pretreatment or recurrent extranodular natural killer/T-cell lymphoma nasal type (ENTCL). METHODS: (18)F-FDG PET/CT images and clinical records of 35 cases (67 scans) of pathologically confirmed ENTCL treated in our hospital within the last 9 years were analyzed. The imaging characteristics of the upper aerodigestive tract (UAT) and the non-aerodigestive tract (NUAT) lesions were analyzed. Lesion distribution, clinical stages, SUVmax and patient survival data were compared between pretreatment and recurrent cases. RESULTS: s All the ENTCL lesions were hypermetabolic. The UAT lesions involved mainly the nasal cavity and pharynx, while the NUAT lesions may involve the lymph nodes and all the organs. UAT lesions were more common in pretreatment cases while NUAT lesions tended to increase in recurrent cases. The SUVmax of pretreatment and recurrent lesions were 10.4∓4.4 and 9.6∓5.2, and showed no significant difference among patients with different lesion distribution patterns, clinical stages, or treatment history. The tumor remission rate evaluated by PET/CT were higher in cases with an initial diagnosis than in those with recurrence [(89.5% (17/19) vs 33.3% (5/15), P<0.005)]. Cox regression analysis revealed no significant differences in the survival rates among patients with different treatment history, clinical stages, lesion distribution patterns, or SUVmax levels (P>0.05). CONCLUSION: (18)F-FDG PET/CT can sensitively detect the pretreatment or recurrent lesions in ENTCL patients and helps in accurate tumor staging and curative effect evaluation.

(131)I-labeled multifunctional dendrimers modified with BmK CT for targeted SPECT imaging and radiotherapy of gliomas.

AIM: The poly(amidoamine) dendrimers modified with Buthus martensii Karsch chlorotoxin (BmK CT) were developed as a (131)I delivery system for glioma-targeted imaging and therapy. MATERIALS & METHODS: Dendrimers before and after labeling (131)I were synthetized and their physicochemical properties were tested. The targeting and therapeutic efficacy of (131)I-G5.NHAc-HPAO-(PEG-BmK CT)-(mPEG) dendrimer against glioma was evaluated in vitro and in vivo. RESULTS: All the dendrimers were stable under different conditions. BmK CT modification increased the cellular uptake of dendrimers in C6 glioma cells, but not in the normal RLE-6TN cells. (131)I-G5.NHAc-HPAO-(PEG-BmK CT)-(mPEG) dendrimer was radiochemically pure and could be applied in glioma-targeting single-photon emission CT (SPECT) imaging and radiotherapy. CONCLUSION: (131)I-G5.NHAc-HPAO-(PEG-BmK CT)-(mPEG) complex is a promising multifunctional nanoplatform for glioma-specific nuclear imaging and radiotherapy.

Ultrastable polyethyleneimine-stabilized gold nanoparticles modified with polyethylene glycol for blood pool, lymph node and tumor CT imaging.

Development of new long-circulating contrast agents for computed tomography (CT) imaging of different biological systems still remains a great challenge. Here, we report the design and synthesis of branched polyethyleneimine (PEI)-stabilized gold nanoparticles (Au PSNPs) modified with polyethylene glycol (PEG) for blood pool, lymph node, and tumor CT imaging. In this study, thiolated PEI was first synthesized and used as a stabilizing agent to form AuNPs. The formed Au PSNPs were then grafted with PEG monomethyl ether via PEI amine-enabled conjugation chemistry, followed by acetylation of the remaining PEI surface amines. The formed PEGylated Au PSNPs were characterized via different methods. We show that the PEGylated Au PSNPs with an Au core size of 5.1 nm have a relatively long half-decay time (7.8 h), and display a better X-ray attenuation property than conventionally used iodine-based CT contrast agents (e.g., Omnipaque), and are hemocompatible and cytocompatible in a given concentration range. These properties of the Au PSNPs afford their uses as a contrast agent for effective CT imaging of the blood pool and major organs of rats, lymph node of rabbits, and the xenografted tumor model of mice. Importantly, the PEGylated Au PSNPs could be excreted out of the body with time and also showed excellent in vivo stability. These findings suggest that the formed PEGylated Au PSNPs may be used as a promising contrast agent for CT imaging of different biological systems.

Structure-function analysis of porcine cytochrome P450 3A29 in the hydroxylation of T-2 toxin as revealed by docking and mutagenesis studies.

T-2 toxin, one of the type A trichothecenes, presents a potential hazard to human and animal health. Our previous work demonstrated that porcine cytochrome P450 3A29 (CYP3A29) played an important role in the hydroxylation of T-2 toxin. To identify amino acids involved in this metabolic process, T-2 toxin was docked into a homology model of CYP3A29 based on a crystal structure of CYP3A4 using AutoDock 4.0. Nine residues of CYP3A29, Arg105, Arg106, Phe108, Ser119, Lys212, Phe213, Phe215, Arg372 and Glu374, which were found within 5 Å around T-2 toxin were subjected to site-directed mutagenesis. In the oxidation of nifedipine, the CLint value of R106A was increased by nearly two-folds compared with the wild-type CYP3A29, while the substrate affinities and CLint values of S119A and K212A were significantly reduced. In the hydroxylation of T-2 toxin, the generation of 3'-OH-T-2 by R105A, S119A and K212A was significantly less than that by the wild-type, whereas R106A slightly increased the generation of 3'-OH-T-2. These results were further confirmed by isothermal titration calorimetry analysis, suggesting that these four residues are important in the hydroxylation of T-2 toxin and Arg105 may be a specific recognition site for the toxin. Our study suggests a possible structure-function relationship of CYP3A29 in the hydroxylation of T-2 toxin, providing with new insights into the mechanism of CYP3A enzymes in the biotransformation of T-2 toxin.