Preliminary Evaluation of Iron Oxide Nanoparticles Radiolabeled with 68Ga and 177Lu as Potential Theranostic Agents.

Theranostic radioisotope pairs such as Gallium-68 (68Ga) for Positron Emission Tomography (PET) and Lutetium-177 (177Lu) for radioisotopic therapy, in conjunction with nanoparticles (NPs), are an emerging field in the treatment of cancer. The present work aims to demonstrate the ability of condensed colloidal nanocrystal clusters (co-CNCs) comprised of iron oxide nanoparticles, coated with alginic acid (MA) and stabilized by a layer of polyethylene glycol (MAPEG) to be directly radiolabeled with 68Ga and its therapeutic analog 177Lu. 68Ga/177Lu- MA and MAPEG were investigated for their in vitro stability. The biocompatibility of the non-radiolabeled nanoparticles, as well as the cytotoxicity of MA, MAPEG, and [177Lu]Lu-MAPEG were assessed on 4T1 cells. Finally, the ex vivo biodistribution of the 68Ga-labeled NPs as well as [177Lu]Lu-MAPEG was investigated in normal mice. Radiolabeling with both radioisotopes took place via a simple and direct labelling method without further purification. Hemocompatibility was verified for both NPs, while MTT studies demonstrated the non-cytotoxic profile of the nanocarriers and the dose-dependent toxicity for [177Lu]Lu-MAPEG. The radiolabeled nanoparticles mainly accumulated in RES organs. Based on our preliminary results, we conclude that MAPEG could be further investigated as a theranostic agent for PET diagnosis and therapy of cancer.

Synthesis and in vitro proof-of-concept studies on bispecific iron oxide magnetic nanoparticles targeting PSMA and GRP receptors for PET/MR imaging of prostate cancer.

Prostate cancer (PCa) is the most common malignancy worldwide in men. This is a proof-of-concept study describing the development of 68Ga-magnetic iron oxide nanoparticles (mNP) targeting prostate specific membrane antigen (PSMA) and gastrin releasing peptide (GRPR) receptors as potential tools for diagnosis of PCa with PET/MRI. Two pharmacophores targeting PSMA, 1, and GRPR, 2, were coupled to mNPs carrying -SH (mNP-S1/2) or -NH2 (mNP-N1/2) groups. The mNP-S1/2 and mNP-N1/2 were characterized for their size, zeta potential, structure, and efficiency of functionalization using dynamic light scattering (DLS), FT-IR and RP-HPLC. A direct 68Ga-labelling procedure was followed, where 68Ga-mNP-N1/2 proved superior to 68Ga-mNP-S1/2 regarding radiolabelling efficiency, and thus were further evaluated in vitro. Toxicity studies in PCa cells (LNCaP, PC-3) showed low toxicity, and minimal hemolysis of red blood cells. In vitro assays in cells expressing PSMA (LNCaP), and GRPR (PC-3), showed specific time-dependent binding (40 min to plateau), high avidity (PC-3: Kd = 28.27 nM, LNCaP: Kd = 11.49 nM) and high internalization rates for 68Ga-mNP-N1/2 in both cell lines.

Investigation of Tumor Cells and Receptor-Ligand Simulation Models for the Development of PET Imaging Probes Targeting PSMA and GRPR and a Possible Crosstalk between the Two Receptors.

Prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPR) have both been used in nuclear medicine as targets for molecular imaging and therapy of prostate (PCa) and breast cancer (BCa). Three bioconjugate probes, the PSMA specific: [68Ga]Ga-1, ((HBED-CC)-Ahx-Lys-NH-CO-NH Glu or PSMA-11), the GRPR specific: [68Ga]Ga-2, ((HBED-CC)-4-amino-1-carboxymethyl piperidine-[D-Phe6, Sta13]BN(6-14), a bombesin (BN) analogue), and 3 (the BN analogue: 4-amino-1-carboxymethyl piperidine-[(R)-Phe6, Sta13]BN(6-14) connected with the fluorescent dye, BDP-FL), were synthesized and tested in vitro with PCa and BCa cell lines, more specifically, with PCa cells, PC-3 and LNCaP, with BCa cells, T47D, MDA-MB-231, and with the in-house created PSMA-overexpressing PC-3(PSMA), T47D(PSMA), and MDA-MB-231(PSMA). In addition, biomolecular simulations were conducted on the association of 1 and 2 with PSMA and GRPR. The PSMA overexpression resulted in an increase of cell-bound radioligand [68Ga]Ga-1 (PSMA) for PCa and BCa cells and also of [68Ga]Ga-2 (GRPR), especially in those cell lines already expressing GRPR. The results were confirmed by fluorescence-activated cell sorting with a PE-labeled PSMA-specific antibody and the fluorescence tracer 3. The docking calculations and molecular dynamics simulations showed how 1 enters the PSMA funnel region and how pharmacophore Glu-urea-Lys interacts with the arginine patch, the S1', and S1 subpockets by forming hydrogen and van der Waals bonds. The chelating moiety of 1, that is, HBED-CC, forms additional stabilizing hydrogen bonding and van der Waals interactions in the arene-binding site. Ligand 2 is diving into the GRPR transmembrane (TM) helical cavity, thereby forming hydrogen bonds through its amidated end, water-mediated hydrogen bonds, and π-π interactions. Our results provide valuable information regarding the molecular mechanisms involved in the interactions of 1 and 2 with PSMA and GRPR, which might be useful for the diagnostic imaging and therapy of PCa and BCa.

Radiolabeled PSMA Inhibitors.

PSMA has shown to be a promising target for diagnosis and therapy (theranostics) of prostate cancer. We have reviewed developments in the field of radio- and fluorescence-guided surgery and targeted photodynamic therapy as well as multitargeting PSMA inhibitors also addressing albumin, GRPr and integrin αvß3. An overview of the regulatory status of PSMA-targeting radiopharmaceuticals in the USA and Europe is also provided. Technical and quality aspects of PSMA-targeting radiopharmaceuticals are described and new emerging radiolabeling strategies are discussed. Furthermore, insights are given into the production, application and potential of alternatives beyond the commonly used radionuclides for radiolabeling PSMA inhibitors. An additional refinement of radiopharmaceuticals is required in order to further improve dose-limiting factors, such as nephrotoxicity and salivary gland uptake during endoradiotherapy. The improvement of patient treatment achieved by the advantageous combination of radionuclide therapy with alternative therapies is also a special focus of this review.

PET Diagnostic Molecules Utilizing Multimeric Cyclic RGD Peptide Analogs for Imaging Integrin αvß3 Receptors.

Multimeric ligands consisting of multiple pharmacophores connected to a single backbone have been widely investigated for diagnostic and therapeutic applications. In this review, we summarize recent developments regarding multimeric radioligands targeting integrin αvß3 receptors on cancer cells for molecular imaging and diagnostic applications using positron emission tomography (PET). Integrin αvß3 receptors are glycoproteins expressed on the cell surface, which have a significant role in tumor angiogenesis. They act as receptors for several extracellular matrix proteins exposing the tripeptide sequence arginine-glycine-aspartic (RGD). Cyclic RDG peptidic ligands c(RGD) have been developed for integrin αvß3 tumor-targeting positron emission tomography (PET) diagnosis. Several c(RGD) pharmacophores, connected with the linker and conjugated to a chelator or precursor for radiolabeling with different PET radionuclides (18F, 64Cu, and 68Ga), have resulted in multimeric ligands superior to c(RGD) monomers. The binding avidity, pharmacodynamic, and PET imaging properties of these multimeric c(RGD) radioligands, in relation to their structural characteristics are analyzed and discussed. Furthermore, specific examples from preclinical studies and clinical investigations are included.

Synthesis, characterization and evaluation of 68Ga labelled monomeric and dimeric quinazoline derivatives of the HBED-CC chelator targeting the epidermal growth factor receptor.

Tyrosine kinase (TK) receptors including epidermal growth factor receptors (EGFRs) are known to be overexpressed in a wide variety of solid tumors associated with poor prognosis. The HBED-CC chelator N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid 1 was coupled via one or both its propionic acid moieties with the quinazoline EGFR-TK inhibiting pharmacophore 4-amino-N-(4-((3-bromophenyl)amino)quinazolin-6-yl)butanamide 3 resulting in either a monomeric 4 or a dimeric 5 species. Ligands 4 and 5 reacted with Ga3+ generating the corresponding complexes Ga4 and Ga5. Both ligands and complexes were characterized with mass spectrometry and NMR spectroscopy and evaluated in vitro with MTT assays in A431 cells, where they showed IC50 values in the range 51.6 to 68.8 µM. Labeling of ligands 4 and 5 with the PET radionuclide 68Ga was quantitative and resulted in tracers [68Ga]Ga4 and [68Ga]Ga5 with radiochemical purities greater than 98%, which were also characterised by comparative RP-HPLC studies with Ga4 and Ga5 respectively. Radiotracers [68Ga]Ga4 and [68Ga]Ga5 were stable (intact tracer over 98%) in the reaction mixture (120 min) and in human serum (30 min). Both tracers were evaluated in vivo with biodistribution experiments in SCID mice bearing A431 tumors presenting tumor uptake of 1.34 for [68Ga]Ga4 and 1.01 %ID/g for [68Ga]Ga5 at 5 min, which was slightly decreased at 60 min p.i. and then remained stable until 120 min p.i. To the best of our knowledge, this is the first report of monomeric and dimeric quinazoline conjugates with the chelator HBED-CC, which can serve as a basis for further development of EGFR-TKI targeting tracers.

A theranostic PSMA ligand for PET imaging and retargeting of T cells expressing the universal chimeric antigen receptor UniCAR.

Chimeric antigen receptor (CAR) T cells have shown impressive therapeutic potential. Due to the lack of direct control mechanisms, therapy-related adverse reactions including cytokine release- and tumor lysis syndrome can even become life-threatening. In case of target antigen expression on non-malignant cells, CAR T cells can also attack healthy tissues. To overcome such side effects, we have established a modular CAR platform termed UniCAR: UniCAR T cells per se are inert as they recognize a peptide epitope (UniCAR epitope) that is not accessible on the surface of living cells. Bifunctional adapter molecules termed target modules (TM) can cross-link UniCAR T cells with target cells. In the absence of TMs, UniCAR T cells automatically turn off. Until now, all UniCAR TMs were constructed by fusion of the UniCAR epitope to an antibody domain. To open up the wide field of low-molecular-weight compounds for retargeting of UniCAR T cells to tumor cells, and to follow in parallel the progress of UniCAR T cell therapy by PET imaging we challenged the idea to convert a PET tracer into a UniCAR-TM. For proof of concept, we selected the clinically used PET tracer PSMA-11, which binds to the prostate-specific membrane antigen overexpressed in prostate carcinoma. Here we show that fusion of the UniCAR epitope to PSMA-11 results in a low-molecular-weight theranostic compound that can be used for both retargeting of UniCAR T cells to tumor cells, and for non-invasive PET imaging and thus represents a member of a novel class of theranostics.

Bispecific radioligands targeting prostate-specific membrane antigen and gastrin-releasing peptide receptors on the surface of prostate cancer cells.

Metastases of prostate cancer usually show highly heterogeneous or partly lost prostate-specific membrane antigen (PSMA) expression. In order to image and treat both PSMA positive and negative tissues, the extension of PSMA tracer specificity to other receptors, also highly expressed on the surface of prostate cancer cells, has been suggested. Prostate cancer cells usually express both PSMA and gastrin-releasing peptide (GRP) receptors; thus, bispecific heterodimeric molecules, addressing both targets at the same time, may significantly improve prostate cancer imaging and therapy. This article summarizes preclinical data regarding low-molecular weight molecules targeting PSMA and GRP receptors.

Designing tracers for PET imaging of the urokinase-type plasminogen activator receptor from a cyclic uPA-derived peptide: first in vitro evaluations.

The treatment of cancer remains a major challenge, especially after tumour cell dissemination and metastases formation. Expression of the urokinase-type plasminogen activation system including urokinase (uPA) and its receptor (uPAR) has been associated with the complex process of cell migration, a tumour's invasive potential as well as a reduced overall and disease-free survival of patients with solid cancers and haematological disorders. A cyclic peptide cyclo[21,29][d-Cys21 ,Cys29 ]-uPA21-30 was designed from the growth factor-like domain (GFD) of urokinase whose binding to uPAR was found to inhibit tumour growth and spread of human ovarian cancer cells in mice. With the aim of visualising uPAR expression using PET imaging to attempt an estimate on the tumour's aggressiveness, the cyclic peptide was modified with an either C- or N-terminally attached variable spacer and chelator. The free ligands were evaluated for their binding affinities to the isolated human uPAR and labelled with 68 Ga and 177 Lu to assess their lipophilicities and stabilities in human serum. Although retaining the full binding potential displayed by cyclo[21,29][d-Cys21 ,Cys29 ]-uPA21-30 to its target was found to be a challenging task upon both C- and N-terminal modification, chelator-bearing ligands were identified that can serve as promising starting points in the development of uPAR-addressing PET tracers.

Monomeric and Dimeric 68Ga-Labeled Bombesin Analogues for Positron Emission Tomography (PET) Imaging of Tumors Expressing Gastrin-Releasing Peptide Receptors (GRPrs).

The GRPr, highly expressed in prostate PCa and breast cancer BCa, is a promising target for the development of new PET radiotracers. The chelator HBED-CC ( N, N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine- N, N'-diacetic acid) was coupled to the bombesin peptides: HBED-C-BN(2-14) 1, HBED-CC-PEG2-[d-Tyr6,ß-Ala11,Thi13,Nle14]-BN(6-14) 2, HBED-CC-Y-[d-Phe6,Sta13,Leu14]-BN(6-14) (Y = 4-amino-1-carboxymethylpiperidine) 3, and HBED-CC-{PEG2-Y-[d-Phe6,Sta13,Leu14]-BN(6-14)}2 4 (homodimer). Compounds 1-4 presented high binding affinities for GRPr (T47D, 0.56-3.51 nM; PC-3, 2.12-4.68 nM). In PC-3 and T47D cells, agonists [68Ga]1 and [68Ga]2 were mainly internalized while antagonists [68Ga]3 and [68Ga]4 were surface bound. Cell-related radioactivity reached a maximum after 45 min, while tracer levels followed GRPr expression (PC-3 > T47D > LNCaP > MDA-MB-231). [68Ga]4 showed the highest cell-bound radioactivity (PC-3 and T47D). In vivo, tumor (PC-3) targeting for [68Ga]3 and [68Ga]4 increased over time, with dynamic µPET showing clearer tumors images at later time points. [68Ga]3 and [68Ga]4 can be considered suitable PET tracers for imaging PCa and BCa expressing GRPr.

Improving the Imaging Contrast of 68Ga-PSMA-11 by Targeted Linker Design: Charged Spacer Moieties Enhance the Pharmacokinetic Properties.

68Ga-Glu-urea-Lys-(Ahx)-HBED-CC (68Ga-PSMA-11) represents a successful radiopharmaceutical for PET/CT imaging of prostate cancer. Further optimization of the tumor-to-background contrast might significantly enhance the sensitivity of PET/CT imaging and the probability of detecting recurrent prostate cancer at low PSA values. This study describes the advantage of histidine (H)/glutamic acid (E) and tryptophan (W)/glutamic acid (E) containing linkers on the pharmacokinetic properties of 68Ga-PSMA-11. The tracers were obtained by a combination of standard Fmoc-based solid-phase synthesis and copper(I)-catalyzed azide-alkyne cycloaddition. Their 68Ga complexes were compared to the clinical reference 68Ga-PSMA-11 with respect to cell binding, effective internalization, and tumor targeting properties in LNCaP-bearing balb/c nu/nu mice. The introduction of (HE)i (i = 1-3) or (WE)i (i = 1-3) into PSMA-11 resulted in a significantly changed biodistribution profile. The uptake values in kidneys, spleen, liver, and other background organs were reduced for (HE)3 while the tumor uptake was not affected. For (HE)1 the tumor uptake was significantly increased. The introduction of tryptophan-containing linkers also modulated the organ distribution profile. The results clearly indicate that histidine is of essential impact in order to improve the tumor-to-organ contrast. Hence, the histidine/glutamic acid linker modifications considerably improved the pharmacokinetic properties of 68Ga-PSMA-11 leading to a reduced uptake in dose limiting organs and a significantly enhanced tumor-to-background contrast. Glu-urea-Lys-(HE)3-HBED-CC represents a promising 68Ga complex ligand for PET/CT-imaging of prostate cancer.

In vivo biodistribution and imaging studies with a 99mTc-radiolabeled derivative of the C-terminus of prothymosin alpha in mice bearing experimentally-induced inflammation.

Prothymosin alpha (ProTα) is a highly conserved mammalian polypeptide (109 amino acids in man) exerting in vitro and in vivo immunoenhancing activities. Recently, our team has developed a 99mTc-radiolabeled derivative of the C-terminal bioactive decapeptide of ProTα ([99mTc]C1) and employed it in in vitro studies, the results of which support the existence of binding sites on human neutrophils that recognize [99mTc]C1, intact ProTα as well as the C-terminal decapeptide of ProTα and presumably involve Toll-like receptor 4. In the present work, [99mTc]C1 was administered to Swiss albino mice with experimentally-induced inflammation for in vivo biodistribution and imaging studies, in parallel with a suitable negative control, which differs from [99mTc]C1 only in bearing a scrambled version of the ProTα decapeptide. The biodistribution data obtained with [99mTc]C1 demonstrated fast clearance of radioactivity from blood, heart, lungs, normal muscle, and predominantly urinary excretion. Most importantly, slow clearance of radioactivity from the inflammation focus was observed, resulting in a high ratio of inflamed/normal muscle tissue (9.15 at 30min post injection, which remained practically stable up to 2h). The inflammation-targeting capacity of [99mTc]C1 was confirmed by imaging studies and might be attributed to neutrophils, which are recruited at the inflamed areas and bear binding sites for [99mTc]C1. In this respect, apart from being a valuable tool for further studies on ProTα in in vitro and in vivo systems, [99mTc]C1 merits further evaluation as a radiopharmaceutical for specific imaging of inflammation foci.

Co-administration of succinylated gelatine with a (99m)Tc-bombesin analogue, effects on pharmacokinetics and tumor uptake.

The bombesin analogue, [(99m)Tc-GGC]-(Ornithine)3-BN(2-14), (99m)Tc-BN-O, targeting gastrin releasing peptide receptors (GRPrs) on the surface of tumors, was pre-clinically investigated as potential imaging agent for single photon emission computed tomography (SPECT). In addition, the improvement of its pharmacokinetic profile (PK) was investigated through the co-administration of a succinylated gelatin plasma expander (Gelofusine), aiming to reduce its kidney accumulation and enhance its tumor-to-normal tissue contrast ratios. Biodistribution data were collected from normal mice and rats, and PC-3 tumor bearing mice, in reference to its PK, metabolism and tumor uptake. Imaging data were also collected from PC-3 tumor bearing mice. Biodistribution and imaging experiments showed that (99m)Tc-BN-O was able to efficiently localize the tumor (5.23 and 7.00% ID/g at 30 and 60min post injection, respectively), while at the same time it was rapidly cleared from the circulation through the kidneys. HPLC analysis of kidney samples, collected at 60min p.i. from normal mice and rats, showed that the majority of radioactivity detected was due to intact peptide i.e. 56% for mice and 73% for rats. Co-administration of (99m)Tc-BN-O with Gelo resulted in the reduction of kidney uptake in both animal models. The integrated area under the curve (AUC30-60 min) from the concentration-time plots of kidneys was decreased in both mice and rats by 25 and 50%, respectively. In PC-3 tumor bearing mice, an increase of tumor uptake (AUCtumor increased by 69%) was also observed with Gelo. An improvement in tumor-to-blood and tumor-to-normal tissue ratios was noted in all cases with the exception of the pancreas, which normally expresses GRPr. The results of this preclinical study may also be extended to other similar peptides, which are utilized in prostate cancer imaging and present similar PK profile.

Alchemical Free Energy Calculations and Isothermal Titration Calorimetry Measurements of Aminoadamantanes Bound to the Closed State of Influenza A/M2TM.

Adamantane derivatives, such as amantadine and rimantadine, have been reported to block the transmembrane domain (TM) of the M2 protein of influenza A virus (A/M2) but their clinical use has been discontinued due to evolved resistance in humans. Although experiments and simulations have provided adequate information about the binding interaction of amantadine or rimantadine to the M2 protein, methods for predicting binding affinities of whole series of M2 inhibitors have so far been scarcely applied. Such methods could assist in the development of novel potent inhibitors that overcome A/M2 resistance. Here we show that alchemical free energy calculations of ligand binding using the Bennett acceptance ratio (BAR) method are valuable for determining the relative binding potency of A/M2 inhibitors of the aminoadamantane type covering a binding affinity range of only ∼2 kcal mol(-1). Their binding affinities measured by isothermal titration calorimetry (ITC) against the A/M2TM tetramer from the Udorn strain in its closed form at pH 8 were used as experimental probes. The binding constants of rimantadine enantiomers against M2TMUdorn were measured for the first time and found to be equal. Two series of alchemical free energy calculations were performed using 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipids to mimic the membrane environment. A fair correlation was found for DPPC that was significantly improved using DMPC, which resembles more closely the DPC lipids used in the ITC experiments. This demonstrates that binding free energy calculations by the BAR approach can be used to predict relative binding affinities of aminoadamantane derivatives toward M2TM with good accuracy.

Specific in vitro binding of a new (99m)Tc-radiolabeled derivative of the C-terminal decapeptide of prothymosin alpha on human neutrophils.

Prothymosin alpha (ProTα) is a conserved mammalian polypeptide with intracellular functions associated with cell proliferation and apoptosis and an extracellular role associated with immunopotentiation. The N-terminal fragment [1-28], which is identical with the immunostimulating peptide thymosin α1 (Tα1), was earlier considered as the immunoactive region of the polypeptide; however, recent data suggest that ProTα may exert a discrete immunomodulating action through its central or C-terminal region, via targeting Toll-like receptor- 4 (TLR4). In this work, a derivative of the C-terminal fragment ProTα[100-109] (ProTα-D1) that can be radiolabeled with (99m)Tc was developed. The biological activity of the non-radioactive (185/187)rhenium-complex of this derivative ([(185/187)Re]ProTα-D1, structurally similar with [(99m)Tc]ProTα-D1) was verified through suitable in vitro bioassays on human neutrophils. Subsequent cell-binding studies revealed specific, time-dependent and saturable binding of [(99m)Tc]ProTα-D1 on neutrophils, which was inhibited by intact ProTα and ProTα[100-109], as well as by a "prototype" TLR4-ligand (lipopolysaccharide from Escherichia coli). Overall, our results support the existence of ProTα-binding sites on human neutrophils, recognizing [(99m)Tc]ProTα-D1, which might involve TLR4. [(99m)Tc]ProTα-D1 may be a useful tool for conducting further in vitro and in vivo studies, aiming to elucidate the extracellular mode of action of ProTα and, eventually, develop ProTα-based immunotherapeutics.

Structural modifications of 99mTc-labelled bombesin-like peptides for optimizing pharmacokinetics in prostate tumor targeting.

PURPOSE: The main goal of the present study was to investigate the importance of the addition of a positively charged aa in the naturally occurring bombesin (BN) peptide for its utilization as radiodiagnostic agent, taking into consideration the biodistribution profile, the pharmacokinetic characteristics and the tumor targeting ability. METHODS: Two BN-derivatives of the general structure [M-chelator]-(spacer)-BN(2-14)-NH(2), where M: (99m)Tc or (185/187)Re, chelator: Gly-Gly-Cys-, spacer: -(arginine)(3)-, M-BN-A; spacer: -(ornithine)(3)-, M-BN-O; have been prepared and evaluated as tumor imaging agents. RESULTS: The peptides under study presented high radiolabelling efficiency (>98%), significant stability in human plasma (>60% intact radiolabelled peptide after 1h incubation) and comparable receptor binding affinity with the standard [(125)I-Tyr(4)]-BN. Their internalization rates in the prostate cancer PC-3 cells differed, although the amount of internalized peptide was the same. The biodistribution and the dynamic γ-camera imaging studies in normal and PC-3 tumor-bearing SCID mice have shown significant tumor uptake, combined with fast blood clearance, through the urinary pathway. CONCLUSION: The addition of the charged aa spacer in the BN structure was advantageous for biodistribution, pharmacokinetics and tumor targeting ability, because it reduced the upper abdominal radioactivity levels and increased tumor/normal tissue contrast ratios.

Dittany of Crete: a botanical and ethnopharmacological review.

Origanum dictamnus (Lamiaceae family), an endemic plant of the Greek island of Crete, is widely used as a traditional medicine since antiquity, all over Europe. The aim of the present review is to present comprehensive information of the plant's botanical taxonomy and morphology, as well as of the chemical constituents, biological and pharmacological research on O. dictamnus, which will be presented and critically evaluated. The paper also highlights particularly interesting aspects and common medicinal uses not previously described in the specific ethnobotanical literature. An increasing number of chemical and pharmacological studies have been reported recently, some of which strongly support its traditional medicinal uses against various illnesses such as sore throat, cough and gastric ulcer. A variety of compounds, including flavonoids, lipids and terpenoids (mainly carvacrol and thymol) have been identified from the plant. Current studies have showed that the extracts, the essential oil, as well as their active principles possess several pharmacological properties, like antimicrobial, antioxidant and anti-ulcer ones. The recent scientific data and the rich historical evidence of its medicinal uses could support further research as well as its use as a safe herbal medicinal product.

Chemical composition and antimicrobial activity of the essential oil of Algerian Phlomis bovei De Noé subsp. bovei.

The chemical composition of essential oil obtained by steam distillation of dried aerial parts of Phlomis bovei De Noé subsp. bovei collected from Algeria, was analyzed by GC and GC/MS. Seventy five constituents (corresponding to 86.37% of the total weight) were identified. The main components were: germacrene D, beta-caryophyllene, beta-bournonene, thymol and hexahydrofarnesyl acetone. Furthermore, the antimicrobial activity of the oil was evaluated against six gram (+/-) bacteria and three pathogenic fungi, using the agar dilution technique. It was found that the oil exhibited strong antimicrobial activity against most of the tested microorganisms.

Cytotoxic activity of Origanum dictamnus.

Several extracts of Origanum dictamnus, an endemic plant of Greece growing only in the island of Crete and the bioassay-directed isolated ursolic acid, were tested in vitro against the P388 (murine leukemia) and the human bronchial epidermoid cancer NSCLC-N6 (non small cell lung cancer) cell lines. Both the initial dichloromethane extract and the isolated from it ursolic acid exhibited cytotoxic activity. Ursolic acid was also tested in vivo, on murine ascite leukemia P388, where it exhibited at a dose of 50 mg/kg a marginal antileukemic activity.