Modification of curcumin with polyethylene glycol enhances the delivery of curcumin in preadipocytes and its antiadipogenic property.

Conjugation of curcumin (CCM) by polyethylene glycol (PEG) has been previously developed to improve water solubility of the natural form of CCM and its antiproliferative role in some human cancer cell lines. This study examined the cellular uptake kinetics of the natural form of CCM and CCM-PEG. Their cytotoxic effect in proliferating preadipocytes and antiadipogenic property in differentiating preadipocytes had also been investigated. CCM and CCM-PEG were found to be differently absorbed in 3T3-L1 preadipocytes and adipocytes with a limited amount of CCM-PEG absorption in the cell. The improved water solubility of CCM-PEG was correlated with increased cellular retention of CCM in 3T3-L1 cells, particularly in preadipocytes. Consequently, CCM-PEG treatment sensitized proliferating preadipocytes to CCM-induced cell toxicity. Furthermore, incubation of differentiating 3T3-L1 cells with CCM-PEG resulted in improvement of the inhibitory role of CCM in adipocyte differentiation with no toxic effect. These results suggest that pegylation-improved water solubility and cellular retention of CCM may be uniquely useful for improving the delivery of CCM in preadipocytes and its antiadipogenic ability.

Synthesis and preliminary biological evaluation of high-drug-load paclitaxel-antibody conjugates for tumor-targeted chemotherapy.

The goal of this study was to design paclitaxel (PTX)-monoclonal antibody (mAb) prodrug conjugates (PTXMAbs) with the ability to deliver therapeutically significant doses of the drug to the tumor while avoiding the previously observed solubility limitations of conjugates with PTX:mAb molar ratios of >3. New PTX conjugates were synthesized using the discrete poly(ethylene glycol) (dPEG) as linkers. These compounds, PTX-L-Lys[(dPEG12)(3)-dPEG4]-dPEG6-NHS (9a and 9b, for L = GL or SX, respectively), were then conjugated to the antiepidermal growth factor receptor mAb, C225 at increasing PTX:C225 ratios, producing completely soluble conjugates. Unlike the earlier PTXMAbs, buffered solutions of these conjugates remained homogeneous for extended periods of time. Fluorescence-activated cell sorting (FACS) analysis indicated preserved immunogenicity of the conjugates at all four substitution ratios, while cytotoxicity studies in MDA-MB-468 breast cancer cells indicated preservation of drug cytotoxicity. These conjugates may have potential in the development of high-drug-load tumor-targeting taxanes.

Recent developments in taxane drug delivery.

Paclitaxel (taxol, 1a) and docetaxel (taxotere, 1b) have established themselves as an important class of antitumor drugs currently available to the oncologist. While the great contribution of these drugs to the management of the disease and their effect on the improvement of the patient quality of life could not be overemphasized, a great deal of research has been ongoing to improve two key pharmacologic factors, antitumor activity and systemic toxicity. Both physical and chemical means have been employed towards the enhancement of antitumor activity and at the same time, lowering the inherent toxicity and side effects of these drugs. This mini-review compiles the recent reported works on the design and development of taxane delivery systems through tumor cell surface receptor-targeted delivery mechanisms such as small-molecule peptides and monoclonal antibodies, as well as those on non-targeted procedures such as liposomes, nanostructures, and natural and synthetic polymers.

Design and development of water-soluble curcumin conjugates as potential anticancer agents.

Conjugates of curcumin to two differently sized poly(ethylene glycol) molecules were synthesized in an attempt to overcome the low aqueous solubility of this natural product with cytotoxic activity against some human cancer cell lines. The soluble conjugates exhibited enhanced cytotoxicity as compared to that of the parent drug. Synthesis, analyses of the rate of drug release, and cytotoxicity studies are herein reported. The water-soluble conjugates may provide information useful for the development of injectable curcumin conjugates.

Assessment of indocyanine green-labeled cetuximab to detect xenografted head and neck cancer cell lines.

OBJECTIVE: The aim of this study is to determine the efficacy of indocyanine green (ICG) conjugated to antiepidermal growth factor receptor antibody (cetuximab) to image head and neck cancer. STUDY DESIGN: Mice (n = 3) were injected with unconjugated ICG and imaged at 100-second intervals for a total of 1000 seconds to assess imaging characteristics. Mice (n = 10) xenografted with SCC-1 cells were then systemically injected with cetuximab conjugated to indocyanine green and imaged over a 72-hour period. To assess the sensitivity and specificity, xenografted tumors underwent subtotal resections and then were assessed for residual disease by fluorescence stereomicroscopy and confirmed by histology. RESULTS: Tumors demonstrated excellent fluorescence 24 hours after injection of cetuximab-ICG. There was a direct relationship between fluorescence and the given dose of cetuximab-ICG. Following subtotal resection, we found fluorescence correlated with a sensitivity of 78.4% and specificity of 96%. CONCLUSIONS: This study provides evidence that supports further preclinical investigation of cetuximab in the evaluation of surgical margins, but linkage to ICG lacks the sensitivity for use in a clinical setting.

Single-drug multiligand conjugates: synthesis and preliminary cytotoxicity evaluation of a paclitaxel-dipeptide "scorpion" molecule.

To improve the targeting properties of receptor-directed drug-peptide conjugates, a multiligand approach was proposed and a model "scorpion" conjugate (6, Figure 1), consisting of two peptide "claws" and a paclitaxel (PTX) "tail", was synthesized. The cell surface receptor-directed peptide used in this single-drug multiligand (SDML) model was a segment of the amphibian peptide bombesin (BBN) which had the Y6Q7W8A9V10G11H12L13M14-NH2 sequence, designated here as BBN[6-14] (2, Figure 2). Due to the lipophilic nature of both PTX and BBN[6-14], compound 6 had a low water solubility. To enhance the solubility, PEG derivatives of this conjugate were prepared with the polymer inserted either in the claws or in the tail regions. In a preliminary random screening, conjugate 6 showed superior cytotoxic activity in several GRPR-positive human cancer cell lines as compared to free PTX and two single-drug single-ligand (SDSL) conjugates. In a receptor blocking experiment, addition of excess unconjugated BBN[6-14] ligand reduced the cytotoxicity of conjugate 6, indicating the receptor-mediated mechanism of drug delivery. The PEG-derived conjugates showed activities which were intermediate between SDSL and the SDML congeners. Also, an increase in the number of the PEG segments lowered cytotoxicity, possibly due to steric hindrance against ligand-receptor binding. Taken together, these results demonstrate the potential of the multiligand approach in the design of receptor-targeting conjugates for tumor-specific drug delivery.

Synthesis and biological evaluation of a paclitaxel immunoconjugate.

Targeted cancer therapy is a promising strategy for the treatment of this disease. In this approach, a cytotoxic agent (CA), such as a drug or a radionuclide, is attached, usually covalently, to a "targeting" vehicle (TV), which in turn is capable of recognizing specific receptor motifs on the surface of the tumor cells. Once administered systemically, the construct would localize on the tumor through the TV moiety and would release the CA cargo, resulting in the destruction of the malignant tissue. Small-molecule peptides as well as monoclonal antibodies have been used as TVs. The synthesis, antigen binding, and cytotoxicity of a covalent conjugate of the anticancer drug paclitaxel (taxol) to the anti-epidermal growth factor receptor (EGFR) monoclonal antibody C225 (IMC-C225; Erbitux, ImClone Systems, Somerville, NJ) are described in this chapter to illustrate the methods used for the construction and in vitro evaluation of these conjugates.

Site-specifically traced drug release and biodistribution of a paclitaxel-antibody conjugate toward improvement of the linker structure.

Tumor-directed drug delivery is a promising strategy in cancer treatment, and in this field, monoclonal antibodies constitute an important class of targeting vehicles. A critical issue in the design of targeting conjugates is the timing of the release of the cytotoxic payload, with the ideal situation being the release at the maximum tumor uptake of the targeting molecule. A site-specific radiolabeling technique was used to elucidate the biodistribution and in vivo drug release pattern of an antibody conjugate of paclitaxel (PTX, 1, Figure 1) in which the drug and the antibody moieties were connected by a succinate (SX) linker. In this new method, a metabolite of PTX, 3'-(4-hydroxyphenyl)paclitaxel (3'-OH-PTX, 2, Figure 1) was used as a tyrosine mimic for the synthesis of the drug site-labeled conjugate (DSL, [(125)I]-3'-OH-PTXSXC225). This was achieved by iodogen (125)I-labeling of 3'-OH-PTXSX and subsequent conjugation to C225. The antibody site-labeled conjugate (ASL, PTXSX-[(125)I]-C225) was prepared by direct radioiodination of PTXSXC225. Biodistribution of these compounds was studied in Balb/c nude mice bearing DU-145 human prostate carcinoma xenografts. While the 4 and 24 h tumor uptake (in percent injected dose per gram of tissue, %ID/g) for [(125)I]-3'-OH-PTXSXC225 were 3.3 +/- 1.5 and 1.7 +/- 0.6%ID/g, the PTXSX-[(125)I]-C225 showed tumor uptake values of 3.8 +/- 4.2 and 14.8 +/- 4.2%ID/g at these time points. This difference in the tumor uptake over time indicates an early cleavage of the drug with respect to the antibody tumor localization. This was further confirmed by an in vitro drug release kinetics study leading to a half-life of about 2 h for PTXSXC225 under physiological conditions. To increase the stability of the PTX-MAb bond, a new conjugate (PTXGLC225) with glutaric acid (GL) as the linker was synthesized. Under the same conditions, the PTXGLC225 showed a 16-fold increase in the half-life (t(1/2)) of the drug release. The effect of the increased t(1/2) of this compound on the antitumor activity of the conjugate was tested in a DU-145 human prostate tumor-implanted mouse model. In comparison to a previous similar experiment with PTXSXC225, better antitumor activity was observed for the PTXGLC225 conjugate as compared to controls. These results demonstrated the first time use of radioiodinated 3'-OH-PTX for in vivo tracing of a paclitaxel conjugate and application of the resulting information to the design of a therapeutically more useful PTX-MAb linker.

In vitro and in vivo evaluation of a 64Cu-labeled polyethylene glycol-bombesin conjugate.

The goal of this study was to synthesize and evaluate a novel bombesin (BN) analogue containing a polyethylene glycol (PEG) linker that can be radiolabeled with 64Cu through the DOTA bifunctional chelate. It is hypothesized that PEG linkers would improve the pharmacokinetics of radiolabeled bombesin analogues to optimize their tumor-to-normal tissue ratios for radiotherapy applications. The formation of this conjugate (DOTA-PEG-BN(7-14)) was confirmed by MALDI-TOF mass spectrometry and was radiolabeled with 64Cu at a specific activity of 2.7 MBq/nmol. DOTA-PEG-BN(7-14) bound specifically to gastrin-releasing peptide receptor (GRPR)-positive PC-3 cells with an IC50 value of 3.9 microM for displacing 125I-Tyr4-BN. Internalization of 64Cu-DOTA-PEG-BN(7-14) into PC-3 cells showed that 5.7%, 13.4%, and 21.0% was internalized at 0.5, 2, and 4 hours, respectively. Biodistribution of 64Cu-DOTA-PEGBN(7-14) was evaluated in normal, athymic nude mice 2, 4, and 24 hours after i.v. injection. This showed that most of the tissues had a similar uptake and clearance of 64Cu-DOTA-PEG-BN(7-14) compared to a control peptide with an alkyl linker (DOTA-Aoc-BN(7-14)) at the given time points. There was uptake of 10.8% ID/g of 64Cu-DOTA-PEG-BN(7-14) 4 hours after i.v. injection in the GRPR-positive pancreas that was inhibited to 2.4% upon injection of an excess of Tyr4-BN. These studies demonstrate that BN analogues can be conjugated with PEG linkers, radiolabeled with 64Cu, and bind to GRPR. Future studies will attempt to increase the affinity of these analogues for GRPR and alter the pharmacokinetics of the 64Cu-labeled conjugates through the use of various sized PEG linkers.

Synthesis and biological evaluation of paclitaxel-C225 conjugate as a model for targeted drug delivery.

Tumor-targeted drug delivery is an attractive strategy in cancer treatment. We have previously reported a paclitaxel model conjugate using a bombesin receptor-recognizing peptide in which the drug cytotoxicity against H1299 human nonsmall cell lung cancer was enhanced compared to unconjugated taxol. In an effort to expand the development of tumor-recognizing taxanes, paclitaxel (PTX, taxol) was conjugated to the anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody (MAb) Erbitux (C225) to serve as a model MAb-mediated drug delivery compound. Thus, paclitaxel was derivatized at its 2'-hydroxy function by introduction of a succinate linker, and the carboxyl group of the latter was covalently attached to C225 through amide bond formation. The final product conjugate (PTXC225) was analyzed mass spectrometrically for assessment of the drug-to-antibody ratios. Cytotoxicity screening of the drug-antibody conjugate against A431, UM-SCC-1, and UM-SCC-6 cells indicated an enhancement in cytocidal effect of paclitaxel as compared to those of the free drug, the intact antibody, and a physical mixture of the two (the controls). In A431 cells, the conjugate showed 25.2% +/- 2.2% of apoptosis induction as compared to little or no apoptosis caused by the controls. Biodistribution analysis of the PTXC225 in tumor-implanted nude mice and a tyrosine-kinase assay showed that conjugation of the drug did not interfere with the immunoreactivity of the antibody. The 24-h tumor uptake of C225 and PTXC225 were 11.7% +/- 6.0% and 7.1% +/- 3.6% of the injected dose per gram of tissue (%ID/g), respectively, which were not significantly different. Also, in A431-implanted nude mice, the conjugate and C225 showed tumor growth inhibition effects of 57.2% and 41.2%, respectively, against a saline-treated control, which were not significantly different from each other. This lack of difference in the in vivo antitumor activity of the MAb-delivered drug and free PTX may be due to either a relatively low dose of the antibody-delivered drug (346 microg/kg), or an untimely release of it, or both. The tumor growth inhibition pattern of the conjugate, however, was identical to that of C225, indicating that the attachment of PTX did not affect the antigen-binding and growth inhibitory features of the MAb. These preliminary results demonstrate the potential of tumor-targeted delivery of taxol as a promising strategy in cancer treatment and warrant further work to develop more suitable drug-MAb linkers as well as improved dosage and treatment protocols.

De novo synthesis of a new diethylenetriaminepentaacetic acid (DTPA) bifunctional chelating agent.

Diethylene triamine pentaacetic acid (DTPA) has been in extensive use as a metal chelator in the development of radiopharmaceuticals and contrast agents. The former application uses DTPA mostly as a bifunctional chelating agent (BCA) conjugated to tumor-targeting vehicles (TTVs) such as monoclonal antibodies (MAbs) and receptor-directed peptides. A new bifunctional DTPA derivative was synthesized by a fully organic scheme. This compound, N(4),N(alpha),N(alpha),N(epsilon),N(epsilon)-[pentakis(carboxymethyl)]-N(4)-(carboxymethyl)-2,6-diamino-4-azahexanoic hydrazide (20) was prepared by a convergent synthesis strategy using N(alpha)-benzyloxycarbonyl-2,3-diaminopropionic acid as the starting compound. This commercially available material was used to build a functionalized triamine which served as the molecular core template for assembling the target molecule. To evaluate the conjugation and radiolabeling capabilities of this new molecule, it was covalently attached to the anti-TAG-72 MAb, Delta CH2HuCC49, and the conjugate was radiolabeled in near-quantitative yields with yttrium-90 ((90)Y) and lutetium-177 ((177)Lu). Biodistribution of the (177)Lu-labeled DTPA-Delta CH2HuCC49 in tumor-bearing nude mice demonstrated preservation of the immunoreactivity of the MAb as indicated by high tumor uptake. In addition to the introduction of a new bifunctional DTPA, this work reports on a novel synthetic approach for preparation of this useful metal chelator and introduces a new conjugation protocol.

Synthesis of the first diethylenetriaminepentahydroxamic acid (DTPH) bifunctional chelating agent.

Synthesis of a new pentahydroxamic acid bifunctional chelating agent (BCA), constructed on the aminoazaalkyl core of diethylenetriaminepentaacetic acid (DTPA), is reported. Rational modifications in the structure of DTPA, which could result in an enhancement of its chelation properties, add to the collection of diagnostic and therapeutic metals bound by this chelator, and might implement significant improvements in the in vivo behavior of this compound, are described. Further improvements in the stability of the ligand-metal complexes of DTPA may improve both diagnostic and therapeutic outcomes such as tumor-to-normal tissue ratios and target-delivered radioactivity. A combination of hydroxamate functions with the azaalkyl backbone of DTPA might be a suitable approach to generate such higher stabilities. This rationale may be justified by the well-known affinity of hydroxamates against different transition metals and favorable properties of DTPA as a versatile chelator. Thus, the N(4),N(alpha),N(alpha),N(epsilon),N(epsilon)-pentakis[[((N-hydroxy-N-methyl]carbonyl)methyl]-2, 6-diamino-4-azahexanoic hydrazide (5, DTPH) was designed and synthesized through a convergent synthesis and in 40.7% overall yield. Conjugation of this compound to the monoclonal antibody (MAb) Delta Ch2HuCC49, used as a model protein, was carried out to evaluate the efficiency of this molecule as a BCA. Radiolabeling of the DTPH-Delta CH2HuCC49 conjugate with lutetium-177 ((177)Lu) and biodistribution of the labeled conjugate in athymic nude mice, bearing LS174T human colon carcinoma xenografts, are reported.