Aruna Korde

Solid lipid nanoparticles and nanosuspension formulation of Saquinavir: preparation, characterization, pharmacokinetics and biodistribution studies

Solid lipid nanoparticles (SLNs) and nanosuspensions (NSs) have shown great promise for improving bioavailability of poorly water-soluble drugs. This study was aimed to develop SLNs and NS of Saquinavir (SQ) for improvement in bioavailability. These formulations were characterized and their pharmacokinetics and biodistribution in mice were evaluated. Saquinavir-loaded SLNs (SQSLNs) showed particle size 215 ± 9 nm and entrapment efficiency 79.24 ± 1.53%, while solid-state studies (differential scanning calorimetry and X-ray diffraction) indicated entrapment of the drug in SLNs. Saquinavir NS (SNS) showed particle size 344 ± 16 nm with fourfold increase in saturation solubility and its solid-state studies showed reduction in crystallinity. Pharmacokinetics and biodistribution studies of orally administered SQSLN and SNS in mice exhibited higher plasma level concentration compared to saquinavir microsuspension (SMS). The relative bioavailabilities for SNS and SQSLN were 37.39% and 66.53%, respectively, compared to 18.87% bioavailability obtained after administration of SMS, indicating suitability of nanoparticulate formulations for improving bioavailability.

Solid lipid nanoparticles and nanosuspension of adefovir dipivoxil for bioavailability improvement: formulation, characterization, pharmacokinetic and biodistribution studies

The present study was aimed at developing colloidal formulations like solid lipid nanoparticles (SLN) and nanosuspension (NS) for improving bioavailability of adefovir dipivoxil (AD), a nucleoside reverse transcriptase inhibitor which displays poor oral bioavailability. SLNs were prepared by solvent injection method while NS was prepared by pearl milling method. The prepared formulations were characterized for physicochemical parameters such as particle size, ζ potential, drug content, X-ray Diffraction (XRD), Differential Scanning Calorimetry (DSC). Pharmacokinetic and biodistribution studies were performed in mice to evaluate in vivo fate of the formulations. The SLNs showed particle size of 267 ± 18 nm and entrapment efficiency of 73.5 ± 2.12%. The particle size obtained for NS was 393 ± 13 nm against 710 ± 70 μm for bulk drug, which led to significant improvement in saturation solubility. DSC and XRD studies of NS and SLN showed reduction in crystallinity while in vitro studies showed improved dissolution rate in both cases. Pharmacokinetics studies of orally administered formulations in mice exhibited higher plasma concentration compared to plain drug. Biodistribution studies showed higher accumulation of drug in liver, kidneys, intestine and stomach. The higher concentration of AD in liver after 24 hr highlights its potential advantage for effective treatment of chronic hepatitis infection. The relative bioavailability for adefovir NS and SLN were 52.46% and 78.23% respectively compared to 34.34% bioavailability obtained after administration of adefovir micro suspension (AMS), indicating suitability of both nanoparticulate formulations for improving bioavailability. SLNs were found to performed better as compared to NS for improving the bioavailability of AD.

Preparation and preliminary bioevaluation of 99mTc(CO)3-11β-progesterone derivative prepared via click chemistry route

INTRODUCTION Progesterone receptors (PRs) overexpressed in breast cancers serve as potential targets for developing radiotracers for use in nuclear medicine. Hence, suitably derivatized progesterone can be envisaged as a potential vector for targeting overexpression of receptors in breast cancer. In the present article, we report the preparation of a (99m)Tc(CO)(3)-progesterone triazole using the Cu(I)-catalyzed novel click chemistry route. Preliminary evaluation of the radiolabeled derivative has been carried out in binding studies with MCF 7 cell lines. METHODS 11-Hydroxyprogesterone has been synthetically derivatized to 11-azidoprogesterone. Subsequently, the cycloaddition reaction between progesterone azide and propargyl glycine was carried out to prepare 1,4-bifunctionalized progesterone triazole analogue. The clicked progesterone triazole derivative was radiolabeled with (99m)Tc and characterized by HPLC. The chemical characterization of (99m)Tc(CO)(3)-progesterone triazole has been carried out by preparing its corresponding rhenium complex using the [NEt(4)](2)[Re(CO)(3)Br(3)] precursor. While in vitro studies were carried out in MCF7 cell lines, in vivo distribution studies were performed in female Swiss mice. RESULTS The radiolabeled complex could be prepared in >95% radiochemical yield as determined by HPLC. In vitro studies of (99m)Tc(CO)(3)-progesterone complex in MCF7 cell lines overexpressing receptors for breast cancer showed binding up to 30%. In vivo distribution studies in female Swiss mice have shown uterine uptake of 0.41 (0.06) % ID/g at 3 h postinjection (pi) and retention therein till 24 h pi. CONCLUSION The present study demonstrates a novel and facile route for preparation of (99m)Tc-labeled progesterone complex using click chemistry. This strategy can be further extended towards preparation of radiolabeled complexes of other steroidal derivatives.

Preparation and In-Vivo Evaluation of 188Re(CO)3-Colchicine Complex for Use as Tumor-Targeting Agent

The Re(I)-tricarbonyl synthon, [(188)Re(H(2)O)(3)(CO)(3)](+), was prepared by using carbon monoxide gas and amine-borane as the reducing agent. Colchicine, a naturally occurring cytotoxic alkaloid, was derivatized to iminodiacetic acid, the required array for the tridentate ligand system for coordination to the Re(I)-tricarbonyl core. (188)Re(CO)(3)-colchicine iminodiacetic acid (IDA) complex could be prepared in >95% radiochemical purity, as determined by high-performance liquid chromatography. The chemical characterization of (188)Re(CO)(3)-colchicine-IDA complex has been carried out by preparing the corresponding cold Re(CO)(3)-complex. The radiolabeled complex was stable at room temperature, even after 48 hours postpreparation, as well as against histidine and cysteine ligand exchange studies. Biodistribution studies were carried out in the murine fibrosarcoma tumor model. Tumor uptake of 1.7 +/- 0.03 percent injected dose per g (%ID/g) was observed at 3 hours postinjection (h.p.i.), which increased to 4.1 +/- 1.3 %ID/g at 24 h.p.i. Tumor-blood and tumor-muscle ratios were 0.14 and 1 at 1 h.p.i. that increased to 0.95 and 4 at 24 h.p.i., respectively. Retention of the complex in tumor for more than one half-life of (188)Re(t(1/2) = 17 hours) indicates its potentiality for tumor therapy.

Radiosynthesis and Biological Evaluation of 68Ga-Labeled Colchicine Conjugates

OBJECTIVE Colchicine, a plant-derived alkaloid, is a known substrate for P-glycoprotein (Pgp), which confers multidrug resistance (MDR) to cancer cells and tumors, through enhanced efflux of chemotherapeutic drugs. Hence, radiolabeled colchicine can be a suitable probe for imaging of activity of Pgp transport in vivo and early diagnosis of MDR. METHODS In the present study, colchicine was hydrolyzed to desacetylcolchiceine for conjugation with p-SCN-Bn-DOTA and p-SCN-Bn-NOTA. The resulting conjugates, DOTA-desacetylcolchiceine and NOTA-desacetylcolchiceine, were radiolabeled with 68Ga. The radiotracers 68Ga-DOTA-desacetylcolchiceine (68Ga-1) and 68Ga-NOTA-desacetylcolchiceine (68Ga-2) were evaluated in vitro (MCF-7 and T47D breast cancer cell lines) and in vivo (biodistribution studies, Swiss mice bearing fibrosarcoma tumors). RESULTS The radiotracers prepared in >97% radiochemical yield showed good in vitro binding and significant inhibition with 100-fold cold colchicine (p<0.05). In vivo the tumor uptake reached maximum at 120 minutes postinjection (68Ga-1: 2.35%±0.39% injected dose per gram [ID/g]; 68Ga-2: 1.5%±0.31% ID/g). Of the two radiotracers 68Ga-2 cleared faster from blood (p<0.05) with lower uptake in nontargeted organs as compared with 68Ga-1. CONCLUSIONS The radiotracer 68Ga-2 has shown improved pharmacokinetic features over 68Ga-1 and the previously reported 99mTc(CO)3-colchicine radiotracer. The preliminary studies with 68Ga-2 indicate its potential for in vivo targeting of tumor. However, the efficacy of the radiotracer for imaging of multidrug-resistant states will be ascertained in future.

Gratifying clinical experience with an indigenously formulated single-vial lyophilized HYNIC-TOC kit at the radiopharmaceutical division of BARC: a pivotal boost for building up a peptide receptor radionuclide therapy programme in an Indian setting

Dear Sir, Peptide receptor radionuclide therapy (PRRT) with Lubased somatostatin receptor analogues has seen a rapid expansion and has generated significant interest over recent years amongst the nuclear medicine fraternity. In India, this has received a major boost in recent years. The primary impetus for this could be ascribed to two developments as a part of radiopharmaceutical research in the country’s premier atomic energy establishment Bhabha Atomic Research Centre (BARC): (1) the availability of Lu-LuCl3 at a much lower cost due to indigenous production (less than one-third of commercially available material) and (2) indigenous production of a single-vial kit for the formulation of Tc-HYNIC-TOC, which has played an important role in centres that do not have access to a germanium/gallium generator. Both these developments at the Radiopharmaceutical Division of BARC could be considered major societal contributions to radiopharmaceutical research in this country in recent years that has benefited a large number of patients with neuroendocrine tumours of various subtypes, both from a diagnostic and a therapeutic standpoint, in multiple clinical centres active in this domain. A single-vial kit formulation for the preparation of TcHYNIC-TOC has been described byGuggenberg et al. [1]. This kit allows a simple method for the preparation of up to two patient doses (10 – 15 mCi, 370 – 555 MBq, per adult patient) of Tc-HYNIC-TOC. Considering a higher cancer patient population density per centre in India, a single vial kit capable of providing up to four patient doses was thought to be more appropriate and was therefore formulated. The lyophilized kit developed in BARC contained 33 μg of HYNIC-TOC, 10 mg of ethylenediaminodiacetic acid, 20 mg of tricine, 40 μg SnCl2, 4.5 mg of sodium phosphate dibasic and 1 mg of sodium phosphate monobasic as ingredients. A significant modification in the present kit was the inclusion of the appropriate buffer which takes care of the final pH of the formulation. This avoids the need for the addition of buffer solution to the kit vial by the user, as is necessary with some of the commercial kits. No deterioration in radiochemical purity (RCP) of Tc-HYNICTOC was observed due to this modification. This kit also allowed relaxation in the maximum volume of Tcpertechnetate that can be added to the kit vial which is restricted to 1 ml in commercial kits. The volume restriction makes commercial kits economically less tenable when the activity per millilitre of the generator eluate goes below the recommended maximum activity for the kit formulation. BARC kits can be reconstituted with 1 – 3 ml of Tc-pertechnetate solution containing a maximum of 80 mCi (2,960 MBq) without compromising the RCP (>90 %) of Tc-HYNIC-TOC. This kit can thus be used for the preparation of multipatient doses evenwhen the activity per millilitre in the Mo/Tc generator eluate is as low as 20 – 25 mCi/ml (740 – 925 MBq/ml). At the Radiation Medicine Centre in Mumbai, which deals with patients from all walks of life including many of lower S. Basu (*) : P. Kand :H. Shimpi Radiation Medicine Centre (B.A.R.C), Tata Memorial Centre Annexe, Jerbai Wadia RoadParel Mumbai 400012, India e-mail: drsanb@yahoo.com

Synthesis and Preliminary Bioevaluation of 99mTc(CO)3-17α-Triazolylandrost-4-Ene-3-One Derivative Prepared via Click Chemistry Route

Azolyl steroids are known to manifest antiprostate cancer and antiandrogenic activities. These azolyl steroids have been shown to express affinity toward androgen receptors (ARs) overexpressed on LNCaP (human prostate adenocarcinoma) cell line. Hence, suitably derivatized azolyl steroids can be envisaged as potential vectors for targeting overexpression of ARs in prostate cancer. In the present study, testosterone has been derivatized to 17α-azidoandrost-4-ene-3-one using microwave-mediated azidation of the mesylate. Subsequently, a facile one-pot Cu(I)-catalyzed Click reaction was carried out to synthesize (99m)Tc(CO)(3)-labeled 17α-triazolylandrost-4-ene-3-one, which was characterized by HPLC. The chemical characterization of (99m)Tc(CO)(3)-17α-triazolylandrost-4-ene-3-one was carried out by preparing its corresponding rhenium complex using [NEt(4)](2)[Re(CO)(3)Br(3)] precursor. The radiolabeled complex could be prepared in >95% radiochemical yield as determined by HPLC. In vitro studies of (99m)Tc(CO)(3)-17α-triazolylandrost-4-ene-3-one complex in LNCaP cell lines overexpressing ARs showed binding of 4.95%±1.2%, with inhibition of 8%±0.9%. In vivo biodistribution studies in male Wistar rats have shown uptake in the prostate to the extent of 0.48%±0.19% injected dose/g at 1 hpi and retention therein till 3 hpi. The present study demonstrates a novel and facile one-pot reaction for preparation of (99m)Tc-labeled 17α-triazolylandrost-4-ene-3-one complex using Click chemistry. The corresponding Re-analog has been prepared for purpose of comparative characterization with the (99m)Tc-labeled complex. The radiosynthetic strategy described in this article can be further extended toward preparation of radiolabeled complexes of other triazolyl steroidal derivatives.

Cellular Toxicity and Apoptosis Studies in Osteocarcinoma Cells, a Comparison of 177Lu-EDTMP and Lu-EDTMP

OBJECTIVE The most promising bone pain palliative agent such as 177Lu-EDTMP emerges as a newer radiopharmaceutical for cancer management. Thus, it was of interest to study the cell uptake of this agent in osteocarcinoma cell line and investigate the underlying mechanism of cellular toxicity. METHODS The cell binding studies of 177Lu-EDTMP were carried out in osteocarcinoma cells (MG63) after induction of bone mineralization. Cellular toxicity studies were carried out with varying amounts of 177Lu-EDTMP and compared with equivalent amount of cold Lu-EDTMP. Cell viability was assessed by trypan blue, LDH and MTT assay. Different studies such as DNA fragmentation and Western blotting for apoptosis related proteins were carried out to elucidate the mechanism of cell death. RESULTS Maximum cell binding of 177Lu-EDTMP, observed with mineralized MG63 cells was 19 ± 0.122%. Nearly 12% cell death was observed in MG63 cells treated with 37 MBq of 177Lu-EDTMP as compared to controls. Apoptosis studies were carried out by ELISA to estimate DNA fragmentation and it was found that DNA enrichment factor was 1.8, compared to the corresponding control. Down regulation of anti-apoptotic protein, bcl-2 and cleavage of PARP protein was evident by Western blot results. CONCLUSION These studies indicate that the 177Lu-EDTMP binds to mineralized bone cells and induces apoptotic cell death in MG63 cells.

Stereoselective synthesis of an iodinated resveratrol analog: preliminary bioevaluation studies of the radioiodinated species.

Stereoselective synthesis of an E-hydroxystilbene has been carried out using the McMurry reaction. Synthesis of a monoiodinated hydroxystilbene has been carried out by a McMurry cross-coupling reaction. For the purpose of biological evaluation, the facile electrophilic substitution route has been attempted to radioiodinate it with (125)I. The HPLC pattern of the radioiodinated hydroxystilbene, which could be obtained in >90% radiochemical purity, was found to be identical to that of its non-radioactive analog that has been independently prepared using the McMurry cross-coupling route. In vitro cell uptake studies were carried out in breast cancer cells MCF7, overexpressing estrogen receptors. In vivo biodistribution studies in female Swiss mice show a uterine uptake of 0.85±0.4% ID/g at 3h.p.i. with a uterus to muscle ratio of 2.83. Uptake in the thyroid was insignificant indicating good in vivo stability of the radioiodinated hydroxystilbene.

Preparation and Evaluation of 125I-Aflatoxin B1

Aflatoxin B1 (AfB1), present in fungus infested crops is highly carcinogenic and is measured by immunoassays. 125I labeled aflatoxin B1 is a key reagent for development of radioimmunoassay (RIA) which exhibits less interference and better sensitivity than other immunoassays. Since AfB1 lacks suitable functional groups for radiolabeling, an oxime derivative of AfB1 was synthesised and evaluated by UV-spectrophotometry and 1H NMR spectroscopy. 125I-histamine was conjugated to AfB1 oxime by mixed anhydride method and purified by solvent extraction followed by TLC. The tracer obtained was immunoreactive, stable as ethanolic solution and could be used in RIA.