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•Chlorambucil was linked to HYNIC to form HYNIC-chlorambucil conjugate.•HYNIC-chlorambucil labeled with 99mTcTc, with > 95 % radiochemical purity.•Appreciable tumor uptake with ...gradual clearance with time observed.•Increasing tumor-to-blood ratio with time suggests tumor selective retention.•Biological behaviour of 99mTcTc-chlorambucil is influenced by the BFCA.
Chlorambucil is an alkylating drug that finds application towards chemotherapy of different types of cancers. In order to explore the possibility of utilization of this drug as an imaging agent for early diagnosis of solid tumors, attempt was made to synthesize a 99mTc complex of chlorambucil and evaluate its potential in tumor bearing small animal model. HYNIC-chlorambucil was synthesized by conjugation of HYNIC with chlorambucil via an ethylenediamine linker. All the intermediates and final product were purified and characterized by standard spectroscopic techniques viz. FT-IR, 1H/13C-NMR as well as by mass spectrometry. HYNIC-chlorambucil conjugate was radiolabeled with 99mTcTc and found to be formed with > 95 % radiochemical purity via RP-HPLC studies. The partition coefficient (Log10Po/w) of the synthesized complex was found to be −0.78 ± 0.25 which indicated the moderate hydrophilic nature for the complex. Biological behaviour of 99mTcTc-HYNIC-chlorambucil, studied in fibrosarcoma bearing Swiss mice, revealed a tumor uptake of about 4.16 ± 1.52 %IA/g at 30 min post-administration, which declined to 1.91 ± 0.13 % IA/g and 1.42 ± 0.14 %IA/g at 1 h and 2 h post-administration, respectively. A comparison of different 99mTcTc-chlorambucil derivatives (reported in the contemporary literature) formulated using different methodologies revealed that tumor uptake and pharmacokinetics exhibited by these agents strongly depend on the lipophilicity/hydrophilicity of such agents, which in turn is dependent on the bifunctional chelators used for formulating the radiolabeled chlorambucils.
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•natCu/64CuCu-labeled porphyrin complex was envisaged as a dual modality agent.•Radiolabeling of porphyrin using carrier added 64CuCu was demonstrated.•Copper metalation in the ...porphyrin core led to quenching of fluorescence intensity.•Copper metalated porphyrin exhibited reduced singlet oxygen generation ability.•natCu-porphyrin complex exhibited lower photocytotoxicity than free base porphyrin.
An unsymmetrically substituted porphyrin derivative namely, 5-carboxymethyleneoxyphenyl-10,15,20-tris(4-methoxyphenyl)porphyrin (UTriMA) was synthesized with an aim to explore the utility of non-radioactive copper complex of UTriMA (natCu-UTRiMA) towards possible application in photodynamic therapy (PDT). Efforts were also directed to prepare the corresponding radioactive analog i.e. 64CuCu-UTriMA in order to exemplify the utility of UTriMA as a dual modality agent. UTriMA was synthesized via a three-step reaction procedure. All the intermediates and final product were purified and characterized by standard spectroscopic techniques viz. UV–Vis, FT-IR, 1H/13C NMR and mass spectrometry. Copper complex of UTriMA (natCu-UTriMA) was synthesized using non-radioactive copper and was characterized by UV–Vis spectroscopy and mass spectrometry. Both UTriMA and natCu-UTriMA were evaluated for their differential ability, if any, towards generation of singlet oxygen using di-phenylisobenzofuran (DPBF) as well as for light dependent toxicity by carrying out in-vitro studies in A549 cancer cells (lung adenocarcinoma) and WI26 (lung fibroblasts). UTriMA was radiolabeled with 64Cu, produced in-house employing (n,γ) reaction and the percentage radiolabeling yield was determined using RP-HPLC. Post complexation with non-radioactive copper, singlet oxygen generation ability of UTriMA was observed to be compromised. During in-vitro experiments, a reduction in photocytotoxicity of natCu-UTriMA, relative to free base porphyrin, was also observed. Copper-64-labeled UTriMA could be prepared with a radiochemical purity of ∼90 % under the optimized reaction conditions. The results of the present study demonstrated significant reduction in photocytotoxic potential of porphyrin derivative upon complexation with copper. Though successful radio-complexation of the same porphyrin with 64Cu was demonstrated, the lowered photocytotoxic potential of the non-radioactive analog weakened its envisaged candidature for dual modality applications.
Good's buffer or HEPES has advantages over other buffers commonly used in radiopharmaceutical preparation as it exhibits significantly lower complexation tendency with metal ions. However, use of ...HEPES buffer for radiolabeling reactions, meant for clinical applications, has been underrated due to the non-availability of sufficient toxicity data. The objective of the present study is to find the evidences towards safety of intravenous administration of HEPES through systemic toxicological studies in small animal model to support its safe application for clinical exploitation.
A pilot study was performed to investigate the lethal dose of HEPES in female Sprague Dawley rats by administering seven different doses of HEPES solution (150 to 2000 mg/kg), through intravenous pathway. Similarly, for determining maximum tolerated dose (MTD), gradually increasing doses of HEPES (50 to 950 mg/kg) were administered in the same species via similar pathway. Various hematological and clinical pathological investigations were carried out in order to find out the safe administration dose of HEPES in rats.
No mortality was observed up to 2000 mg/kg doses of HEPES. The doses beyond 300 mg/kg resulted few temporary adverse effects, though these were found to disappear within 4–5 days of dosing.
The amount of HEPES to be administered during clinical intervention is usually much lower (typically 1–2.5 mg per kg of body weight of healthy adult) than the MTD determined in rat model during present report. Hence, the utilization of this buffer for preparation of radiolabeled drugs for human investigation may be safe. However, further detailed investigations may be warranted for supporting the candidature of Good's buffer for regular clinical exploitation.
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Trastuzumab is a US‐FDA‐approved humanized monoclonal antibody used for the treatment of human epidermal growth factor receptor 2 (HER2)‐positive breast cancer. The aim of the present work is to ...optimize a freeze‐dried formulation of DOTA‐Trastuzumab conjugate for the preparation of patient doses of 177LuLu‐Trastuzumab for radioimmunotherapy of breast cancer. The formulation of 177LuLu‐Trastuzumab usually takes a long time, and thus, such a process is not suitable for the routine preparation of this agent in hospital radiopharmacies. To circumvent this, a pre‐synthesized DOTA‐Trastuzumab conjugate as a freeze‐dried formulation is proposed. In the present work, DOTA‐Trastuzumab conjugate was subjected to a freeze‐drying process after the addition of optimized amounts of radioprotectant and cryoprotectant. 177LuLu‐DOTA‐Trastuzumab was prepared by incubating the lyophilized powder of the kit vial with medium‐specific activity 177LuCl3. The final radiochemical purity of 177LuLu‐DOTA‐Trastuzumab, prepared using freeze‐dried kit, was determined to be >95%. To ascertain the reproducibility of the procedure, six consecutive batches of the freeze‐dried formulation were prepared, radiolabeled, and evaluated by carrying out both in vitro and ex vivo studies. The consistency of the results of all the six consecutive batches confirmed the robustness and utility of the in‐house optimized freeze‐dried formulation for the preparation of patient doses of 177LuLu‐Trastuzumab at hospital radiopharmacies.
Trastuzumab is a US‐FDA approved humanized monoclonal antibody used for immunotherapy of human epidermal growth factor receptor 2 (HER2) positive breast cancer. Radioimmunotherapy of ca breast employing 177LuLu‐Trastuzumab is currently undergoing clinical investigations. In the present work, formulation of patient dose of 177LuLu‐Trastuzumab using an in‐house developed freeze‐dried kit of DOTA‐Trastuzumab has been demonstrated. The efficacy of 177LuLu‐Trastuzumab towards targeting HER2 receptors was showcased by detailed in vitro and ex vivo studies in cancer cell lines and small animal model, respectively.
The objective of the present work was to evaluate the potential of a nuclear localization signal (NLS) toward facilitating intracellular delivery and enhancement in the therapeutic efficacy of the ...molecular cargo. Toward this, an in-house synthesized porphyrin derivative, namely, 5-carboxymethyelene-oxyphenyl-10,15,20-
(4-methoxyphenyl) porphyrin (UTriMA), was utilized for conjugation with the NLS sequence PKKKRKV. The three compounds synthesized during the course of the present work, namely DOTA-Lys-NLS, DOTA-UTriMA-Lys-NLS, and DOTA-Lys-UTriMA, were evaluated for cellular toxicity in cancer cell lines (HT1080), wherein all exhibited minimal dark toxicity. However, during photocytotoxicity studies with DOTA-Lys-UTriMA and DOTA-UTriMA-Lys-NLS conjugates in the same cell line, the latter exhibited significantly higher light-dependent toxicity compared to the former. Furthermore, the photocytotoxicity for DOTA-UTriMA-Lys-NLS in a healthy cell line (WI26VA4) was found to be significantly lower than that observed in the cancer cells. Fluorescence cell imaging studies carried out in HT1080 cancer cells revealed intracellular accumulation for the NLS-conjugated porphyrin (DOTA-UTriMA-Lys-NLS), whereas unconjugated porphyrin (DOTA-Lys-UTriMA) failed to do so. To evaluate the radiotherapeutic effects of the synthesized conjugates, all three compounds were radiolabeled with
Lu, a well-known therapeutic radionuclide with high radiochemical purity (>95%). During in vitro studies, the
LuLu-DOTA-UTriMA-Lys-NLS complex exhibited the highest cell binding as well as internalization among the three radiolabeled complexes. Biological distribution studies for the radiolabeled compounds were performed in a fibrosarcoma-bearing small animal model, wherein significantly higher accumulation and prolonged retention of
LuLu-DOTA-UTriMA-Lys-NLS (9.32 ± 1.27% IA/g at 24 h p.i.) in the tumorous lesion compared to
LuLu-UTriMA-Lys-DOTA (2.3 ± 0.13% IA/g at 24 h p.i.) and
LuLu-DOTA-Lys-NLS complexes (0.26 ± 0.17% IA/g at 24 h p.i.) were observed. The results of the biodistribution studies were further corroborated by recording serial SPECT-CT images of fibrosarcoma-bearing Swiss mice administered with
LuLu-DOTA-UTriMA-Lys-NLS at different time points. Tumor regression studies performed with
LuLu-DOTA-UTriMA-Lys-NLS in the same animal model with two different doses 250 μCi (9.25 MBq) and 500 μCi (18.5 MBq) resulted in a significant reduction in tumor mass in the treated group of animals. The above results revealed a definite enhancement in the targeting ability of molecular cargo upon conjugation with NLS and hence indicated that this strategy may be helpful for the preparation of drug-NLS conjugates as multimodal agents.
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