Synthesis and In Vitro Evaluation of 8-Pyridinyl-Substituted Benzo[e]imidazo[2,1-c][1,2,4]triazines as Phosphodiesterase 2A Inhibitors

Phosphodiesterase 2A (PDE2A) is highly expressed in distinct areas of the brain, which are known to be related to neuropsychiatric diseases. The development of suitable PDE2A tracers for Positron Emission Tomography (PET) would permit the in vivo imaging of the PDE2A and evaluation of disease-mediated alterations of its expression. A series of novel fluorinated PDE2A inhibitors on the basis of a Benzoimidazotriazine (BIT) scaffold was prepared leading to a prospective inhibitor for further development of a PDE2A PET imaging agent. BIT derivatives (BIT1–9) were obtained by a seven-step synthesis route, and their inhibitory potency towards PDE2A and selectivity over other PDEs were evaluated. BIT1 demonstrated much higher inhibition than other BIT derivatives (82.9% inhibition of PDE2A at 10 nM). BIT1 displayed an IC50 for PDE2A of 3.33 nM with 16-fold selectivity over PDE10A. This finding revealed that a derivative bearing both a 2-fluoro-pyridin-4-yl and 2-chloro-5-methoxy-phenyl unit at the 8- and 1-position, respectively, appeared to be the most potent inhibitor. In vitro studies of BIT1 using mouse liver microsomes (MLM) disclosed BIT1 as a suitable ligand for 18F-labeling. Nevertheless, future in vivo metabolism studies are required

Preparation of (177Lu-DOTA)n-PAMAM-[Nimotuzumab-F(abâ)2] as a Therapeutic Radioimmunoconjugate for EGFR Overexpressed Cancers Treatment

Intact monoclonal antibodies with a high molecular weight tend to have a poor pharmacokinetic profile and tumor penetration, and potential for eliciting host antibody responses. F(abâ)2 fragments smaller than intact monoclonal antibodies that still maintain antigen binding could solve this problem. The objective of this study was to optimize the digestion process of nimotuzumab, an anti-EGFR monoclonal antibody, into its F(abâ)2 fragment and investigate its potential as a therapeutic radioimmunoconjugate. Optimal conditions for digestion of nimotuzumab to its F(abâ)2 fragment were found to be 6 hours of digestion time with a pH of 3.5 and 1:100 mol ratio of pepsin to nimotuzumab. The purity of the F(abâ)2-nimotuzumab was confirmed by SDS-PAGE and HPLC analysis. Prior to its labeling with lutetium-177 radionuclide, the nimotuzumab-F(abâ)2 was conjugated to DOTA-PAMAM dendrimer [DOTA denotes 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, PAMAM denotes poly(amidoamine)] to form conjugate of (DOTA)n-PAMAM-[nimotuzumab-F(abâ)2]. Radiolabeling of DOTA-PAMAM-[nimotuzumab-F(abâ)2] conjugate with 177Lu resulted in (177Lu-DOTA)n-PAMAM-[nimotuzumab-F(abâ)2] with radiochemical purity > 99% after purification with a PD-10 column. Further studies still need to be performed in order to confirm the potential of this radioimmunoconjugate as a radioimmunotherapeutic agent for EGFR overexpressed cancers

Preparation of 99mtc-tricine-edda-hynic-folate, a Potential Radiopharmaceutical for Radiodiagnosis of Folate Receptors Over Expressed Cancer

PREPARATION OF 99mTc-TRICINE-EDDA-HYNIC-FOLATE, A POTENTIAL RADIOPHARMACEUTICAL FOR RADIODIAGNOSIS OF FOLATE RECEPTORS OVER EXPRESSED CANCER. Folate receptors (FRs) have been reported to be over expressed on various types of cancers. Therefore, it would be possible for its ligand in this case folic acid, also known as vitamin B9, to be used as delivery agent for diagnosis and therapy of FRs over expressed cancers. The aim of this projectwas to prepare 99mTc radiolabeled folic acid via 6-hydrazinonicotinamido-hydrazido (HYNIC) in the form of 99mTc-tricine-ethylenediamine diacetate-HYNIC-folate (99mTc-tricine-EDDA-HYNIC-folate), which was expected to be potential for radiodiagnosis of the FRs over expressed cancers. Preparation of 99mTc-tricine-EDDA-HYNIC-folate was initiated by preparation of HYNIC-folate by reacting of folate-γ- hydrazide with 6-chloronicotinic acid NHS ester which was then followed by addition of hydrazine-hydrate. The HYNIC-folate was recovered in its HCl salt-form which was then formulated to form a freeze dried kit which consisted of HYNIC-folate, tricine and EDDA (co-ligands) and Sn(II) as reducing agent. The formation of 99mTc-tricine-EDDA-HYNIC-folate was carried out by addition of 99mTc into tricine-EDDA-HYNIC-folate freeze dried kit which resulted in 99mTc-tricine-EDDA-HYNIC-folate with radiochemical purity of 97.0 ± 1.8% met with the requirement of a good radiopharmaceutical (≥ 90%). The stability test showed that the 99mTc-tricine-EDDA-HYNIC-folate was still intact (radiochemical purity ~ 95%) when stored at 37 °C for four hours

PREPARATION OF 99mTc-TRICINE-EDDA-HYNIC-FOLATE, A POTENTIAL RADIOPHARMACEUTICAL FOR RADIODIAGNOSIS OF FOLATE RECEPTORS OVER EXPRESSED CANCER

PREPARATION OF 99mTc-TRICINE-EDDA-HYNIC-FOLATE, A POTENTIAL RADIOPHARMACEUTICAL FOR RADIODIAGNOSIS OF FOLATE RECEPTORS OVER EXPRESSED CANCER. Folate receptors (FRs) have been reported to be over expressed on various types of cancers. Therefore, it would be possible for its ligand in this case folic acid, also known as vitamin B9, to be used as delivery agent for diagnosis and therapy of FRs over expressed cancers. The aim of this projectwas to prepare 99mTc radiolabeled folic acid via 6-hydrazinonicotinamido-hydrazido (HYNIC) in the form of 99mTc-tricine-ethylenediamine diacetate-HYNIC-folate (99mTc-tricine-EDDA-HYNIC-folate), which was expected to be potential for radiodiagnosis of the FRs over expressed cancers. Preparation of 99mTc-tricine-EDDA-HYNIC-folate was initiated by preparation of HYNIC-folate by reacting of folate-γ- hydrazide with 6-chloronicotinic acid NHS ester which was then followed by addition of hydrazine-hydrate. The HYNIC-folate was recovered in its HCl salt-form which was then formulated to form a freeze dried kit which consisted of HYNIC-folate, tricine and EDDA (co-ligands) and Sn(II) as reducing agent. The formation of 99mTc-tricine-EDDA-HYNIC-folate was carried out by addition of 99mTc into tricine-EDDA-HYNIC-folate freeze dried kit which resulted in 99mTc-tricine-EDDA-HYNIC-folate with radiochemical purity of 97.0 ± 1.8% met with the requirement of a good radiopharmaceutical (≥ 90%). The stability test showed that the 99mTc-tricine-EDDA-HYNIC-folate was still intact (radiochemical purity ~ 95%) when stored at 37 °C for four hours

Uji Preklinis 177Lu-DOTA-Trastuzumab: Radiofarmaseutika Potensial untuk Terapi Kanker Payudara HER-2 Positif

Radiopharmaceutical 177Lu-(1,4,7,10-tetraazacyclododecane-N,N’,N”,N”’-tetra acetic acid)-trastuzumab (177Lu-DOTA-trastuzumab), based on antihuman epithelial receptor type 2 (HER-2) monoclonal antibody which is expected to be potential for diagnostic and therapeutic agent of breast cancer positive HER-2, had been successfully prepared with radiochemical purity of > 99%. Preclinical studies aimed in providing basic data for clinical trial and particularly in finding out the effectiveness of 177Lu-DOTA-trastuzumab in killing cancer cells which over express HER-2, have been performed. The data included urine and faeces clearance tests, imaging with gamma camera and cytotoxicity test. The results showed that the excretion of radioactivity post injection of 177Lu-DOTA-trastuzumab in normal rats were more rapid through urine as compared to the excretion through faeces. The gamma camera image on normal rat 144 hours post injection of 177Lu-DOTA-trastuzumab showed that there was remaining a trace of radioactivity in hepatic area. The residue of radioactivity (< 5%, quantified by biodistribution test) was found to be relatively lower than reported for 111In-NSL-trastuzumab. However, this residue of radioactivity has to be seriously considered when 177Lu-DOTA-trastuzumab is going to be applied for treatment of cancer. Cytotoxicity test showed that 177Lu-DOTA-trastuzumab was far more effective in killing cancer cells positive HER-2 (SKOV-3 cell lines) than that of trastuzumab.Radiofarmaka 177Lu-(1,4,7,10-tetraazacyclododecane-N,N’,N”,N”’-tetra acetic acid)-trastuzumab (177Lu-DOTA-trastuzumab), berbasis antihuman epithelial receptor type 2 (HER-2) antibodi monoklonal, yang diharapkan potensial untuk diagnosis dan terapi kanker payudara positif HER-2, dengan kemurnian radiokimia besar dari 99% telah berhasil dipreparasi. Uji preklinis yang dimaksudkan untuk mendapatkan data dasar sebelum pelaksanaan uji klinis dan khususnya untuk melihat efektifitas 177Lu-DOTA-trastuzumab dalam membunuh sel kanker yang mengekspresikan HER-2, telah berhasil dilakukan. Data dasar tersebut diantaranya adalah data clearance, citra dengan gamma kamera dan data hasil uji sitoksisitas. Hasil uji clearance memperlihatkan bahwa ekskresi radioaktif setelah pemberian 177Lu-DOTA-trastuzumab pada tikus sehat lebih cepat melalui urin dibandingkan dengan eksresi melalui feses. Hasil pencitraan dengan kamera gamma pada tikus normal 144 jam setelah pemberian 177Lu-DOTA-trastuzumab memperlihat adanya residu radioaktif di daerah hati (< 5%, berdasarkan hasil uji biodistribusi). Residu ini lebih rendah jika dibandingkan dengan residu radioaktif pada hati yang dilaporkan untuk 111In-NSL-trastuzumab. Walaupun residu radioaktif ini relatif rendah, tetapi harus tetap menjadi perhatian yang serius pada saat 177Lu-DOTA-trastuzumab akan digunakan pada pasien penderita kanker. Uji sitotoksisitas memperlihatkan 177Lu-DOTA-trastuzumab jauh lebih efektif dalam membunuh sel kanker positif HER-2 (SKOV-3 cell lines) dibandingkan dengan trastuzumab tidak bertanda

Radiosynthesis and Biological Investigation of a Novel Fluorine-18 Labeled Benzoimidazotriazine-Based Radioligand for the Imaging of Phosphodiesterase 2A with Positron Emission Tomography

A specific radioligand for the imaging of cyclic nucleotide phosphodiesterase 2A (PDE2A) via positron emission tomography (PET) would be helpful for research on the physiology and disease-related changes in the expression of this enzyme in the brain. In this report, the radiosynthesis of a novel PDE2A radioligand and the subsequent biological evaluation were described. Our prospective compound 1-(2-chloro-5-methoxy phenyl)-8-(2-fluoropyridin-4-yl)-3- methylbenzo[e]imidazo[5,1-c][1,2,4]triazine, benzoimidazotriazine (BIT1) (IC50 PDE2A = 3.33 nM; 16-fold selectivity over PDE10A) was fluorine-18 labeled via aromatic nucleophilic substitution of the corresponding nitro precursor using the K[18F]F-K2.2.2-carbonate complex system. The new radioligand [18F]BIT1 was obtained with a high radiochemical yield (54 ± 2%, n = 3), a high radiochemical purity (≥99%), and high molar activities (155–175 GBq/μmol, n = 3). In vitro autoradiography on pig brain cryosections exhibited a heterogeneous spatial distribution of [18F]BIT1 corresponding to the known pattern of expression of PDE2A. The investigation of in vivo metabolism of [18F]BIT1 in a mouse revealed sufficient metabolic stability. PET studies in mouse exhibited a moderate brain uptake of [18F]BIT1 with a maximum standardized uptake value of ~0.7 at 5 min p.i. However, in vivo blocking studies revealed a non-target specific binding of [18F]BIT1. Therefore, further structural modifications are needed to improve target selectivity

Synthesis and In Vitro Evaluation of 8-Pyridinyl-Substituted Benzo[e]imidazo[2,1-c][1,2,4]triazines as Phosphodiesterase 2A Inhibitors

Phosphodiesterase 2A (PDE2A) is highly expressed in distinct areas of the brain, which are known to be related to neuropsychiatric diseases. The development of suitable PDE2A tracers for Positron Emission Tomography (PET) would permit the in vivo imaging of the PDE2A and evaluation of disease-mediated alterations of its expression. A series of novel fluorinated PDE2A inhibitors on the basis of a Benzoimidazotriazine (BIT) scaffold was prepared leading to a prospective inhibitor for further development of a PDE2A PET imaging agent. BIT derivatives (BIT1–9) were obtained by a seven-step synthesis route, and their inhibitory potency towards PDE2A and selectivity over other PDEs were evaluated. BIT1 demonstrated much higher inhibition than other BIT derivatives (82.9% inhibition of PDE2A at 10 nM). BIT1 displayed an IC50 for PDE2A of 3.33 nM with 16-fold selectivity over PDE10A. This finding revealed that a derivative bearing both a 2-fluoro-pyridin-4-yl and 2-chloro-5-methoxy-phenyl unit at the 8- and 1-position, respectively, appeared to be the most potent inhibitor. In vitro studies of BIT1 using mouse liver microsomes (MLM) disclosed BIT1 as a suitable ligand for 18F-labeling. Nevertheless, future in vivo metabolism studies are required

Synthesis and In Vitro Evaluation of 8-Pyridinyl-Substituted Benzo[e]imidazo[2,1-c][1,2,4]triazines as Phosphodiesterase 2A Inhibitors

Phosphodiesterase 2A (PDE2A) is highly expressed in distinct areas of the brain, which are known to be related to neuropsychiatric diseases. The development of suitable PDE2A tracers for Positron Emission Tomography (PET) would permit the in vivo imaging of the PDE2A and evaluation of disease-mediated alterations of its expression. A series of novel fluorinated PDE2A inhibitors on the basis of a Benzoimidazotriazine (BIT) scaffold was prepared leading to a prospective inhibitor for further development of a PDE2A PET imaging agent. BIT derivatives (BIT1–9) were obtained by a seven-step synthesis route, and their inhibitory potency towards PDE2A and selectivity over other PDEs were evaluated. BIT1 demonstrated much higher inhibition than other BIT derivatives (82.9% inhibition of PDE2A at 10 nM). BIT1 displayed an IC50 for PDE2A of 3.33 nM with 16-fold selectivity over PDE10A. This finding revealed that a derivative bearing both a 2-fluoro-pyridin-4-yl and 2-chloro-5-methoxy-phenyl unit at the 8- and 1-position, respectively, appeared to be the most potent inhibitor. In vitro studies of BIT1 using mouse liver microsomes (MLM) disclosed BIT1 as a suitable ligand for 18F-labeling. Nevertheless, future in vivo metabolism studies are required

Synthesis and In Vitro Evaluation of 8-Pyridinyl-Substituted Benzo[e]imidazo[2,1-c][1,2,4]triazines as Phosphodiesterase 2A Inhibitors

Phosphodiesterase 2A (PDE2A) is highly expressed in distinct areas of the brain, which are known to be related to neuropsychiatric diseases. The development of suitable PDE2A tracers for Positron Emission Tomography (PET) would permit the in vivo imaging of the PDE2A and evaluation of disease-mediated alterations of its expression. A series of novel fluorinated PDE2A inhibitors on the basis of a Benzoimidazotriazine (BIT) scaffold was prepared leading to a prospective inhibitor for further development of a PDE2A PET imaging agent. BIT derivatives (BIT1–9) were obtained by a seven-step synthesis route, and their inhibitory potency towards PDE2A and selectivity over other PDEs were evaluated. BIT1 demonstrated much higher inhibition than other BIT derivatives (82.9% inhibition of PDE2A at 10 nM). BIT1 displayed an IC50 for PDE2A of 3.33 nM with 16-fold selectivity over PDE10A. This finding revealed that a derivative bearing both a 2-fluoro-pyridin-4-yl and 2-chloro-5-methoxy-phenyl unit at the 8- and 1-position, respectively, appeared to be the most potent inhibitor. In vitro studies of BIT1 using mouse liver microsomes (MLM) disclosed BIT1 as a suitable ligand for 18F-labeling. Nevertheless, future in vivo metabolism studies are required

Preparation of (177Lu-DOTA)n-PAMAM-[Nimotuzumab-F(ab’)2] as a Therapeutic Radioimmunoconjugate for EGFR Overexpressed Cancers Treatment

Intact monoclonal antibodies with a high molecular weight tend to have a poor pharmacokinetic profile and tumor penetration, and potential for eliciting host antibody responses. F(ab’)2 fragments smaller than intact monoclonal antibodies that still maintain antigen binding could solve this problem. The objective of this study was to optimize the digestion process of nimotuzumab, an anti-EGFR monoclonal antibody, into its F(ab’)2 fragment and investigate its potential as a therapeutic radioimmunoconjugate. Optimal conditions for digestion of nimotuzumab to its F(ab’)2 fragment were found to be 6 hours of digestion time with a pH of 3.5 and 1:100 mol ratio of pepsin to nimotuzumab. The purity of the F(ab’)2-nimotuzumab was confirmed by SDS-PAGE and HPLC analysis. Prior to its labeling with lutetium-177 radionuclide, the nimotuzumab-F(ab’)2 was conjugated to DOTA-PAMAM dendrimer [DOTA denotes 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, PAMAM denotes poly(amidoamine)] to form conjugate of (DOTA)n-PAMAM-[nimotuzumab-F(ab’)2]. Radiolabeling of DOTA-PAMAM-[nimotuzumab-F(ab’)2] conjugate with 177Lu resulted in (177Lu-DOTA)n-PAMAM-[nimotuzumab-F(ab’)2] with radiochemical purity > 99% after purification with a PD-10 column. Further studies still need to be performed in order to confirm the potential of this radioimmunoconjugate as a radioimmunotherapeutic agent for EGFR overexpressed cancers