Design, synthesis, and biological evaluation of novel cRGD-paclitaxel conjugates for integrin-assisted drug delivery.

The efficacy of taxane-based antitumor therapy is limited by several drawbacks which result in a poor therapeutic index. Thus, the development of approaches that favor selective delivery of taxane drugs (e.g., paclitaxel, PTX) to the disease area represents a truly challenging goal. On the basis of the strategic role of integrins in tumor cell survival and tumor progression, as well as on integrin expression in tumors, novel molecular conjugates were prepared where PTX is covalently attached to either cyclic AbaRGD (Azabicycloalkane-RGD) or AmproRGD (Aminoproline-RGD) integrin-recognizing matrices via structurally diverse connections. Receptor-binding assays indicated satisfactory-to-excellent α(V)ß(3) binding capabilities for most conjugates, while in vitro growth inhibition assays on a panel of human tumor cell lines revealed outstanding cell sensitivity values. Among the nine conjugate ensemble, derivative 21, bearing a robust triazole ring connected to ethylene glycol units by an amide function and showing excellent cell sensitivity properties, was selected for in vivo studies in an ovarian carcinoma model xenografted in immunodeficient mice. Remarkable antitumor activity was attained, superior to that of PTX itself, which was associated with a marked induction of aberrant mitoses, consistent with the mechanism of action of spindle poisons. Overall, the novel cRGD-PTX conjugates disclosed here represent promising candidates for further advancement in the domain of targeted antitumor therapy.

Homo- and heterodimeric Smac mimetics/IAP inhibitors as in vivo-active pro-apoptotic agents. Part I: Synthesis.

Novel pro-apoptotic, homo- and heterodimeric Smac mimetics/IAPs inhibitors based on the N-AVPI-like 4-substituted 1-aza-2-oxobicyclo[5.3.0]decane scaffold were prepared from monomeric structures connected through a head-head (8), tail-tail (9) or head-tail (10) linker. The selection of appropriate decorating functions for the scaffolds, and of rigid and flexible linkers connecting them, is described. The synthesis, purification and analytical characterization of each prepared dimer 8-10 is thoroughly described.

Dimeric Smac mimetics/IAP inhibitors as in vivo-active pro-apoptotic agents. Part II: Structural and biological characterization.

Novel pro-apoptotic, homodimeric and heterodimeric Smac mimetics/IAPs inhibitors connected through head-head (8), tail-tail (9) or head-tail linkers (10), were biologically and structurally characterized. In vitro characterization (binding to BIR3 and linker-BIR2-BIR3 domains from XIAP and cIAP1, cytotoxicity assays) identified early leads from each dimer family. Computational models and structural studies (crystallography, NMR, gel filtration) partially rationalized the observed properties for each dimer class. Tail-tail dimer 9a was shown to be active in a breast and in an ovary tumor model, highlighting the potential of dimeric Smac mimetics/IAP inhibitors based on the N-AVPI-like 4-substituted 1-aza-2-oxobicyclo[5.3.0]decane scaffold as potential antineoplastic agents.

Cyclic RGD functionalized gold nanoparticles for tumor targeting.

Integrin α(v)ß(3) is an adhesion molecule involved in physiological and pathological angiogenesis as well as tumor invasion and metastasis. Therefore, it is considered an important target for molecular imaging and delivery of therapeutics for cancer, and there is a strong interest in developing novel agents interacting with this protein. Nevertheless, the interaction of individual ligands is often still weak for efficient tumor targeting, and many research groups have synthesized multivalent displays in order to overcome this problem. Gold nanoparticles can be considered a smart platform for polyvalent presentation on account of their globular shape, tunable size, facile surface chemistry, and biocompatibility. Moreover, their unique physical properties render gold nanoparticles ideal candidates for tumor diagnosis and therapy. Here, we report the synthesis and characterization of gold nanoparticles functionalized with cRGD integrin ligand and their employment for targeting human cancer cells expressing α(v)ß(3) integrin.

Designing Smac-mimetics as antagonists of XIAP, cIAP1, and cIAP2.

Inhibitor of apoptosis proteins (IAPs) such as XIAP, cIAP1, and cIAP2 are upregulated in many cancer cells. Several compounds targeting IAPs and inducing cell death in cancer cells have been developed. Some of these are synthesized mimicking the N-terminal tetrapeptide sequence of Smac/DIABLO, the natural endogenous IAPs inhibitor. Starting from such conceptual design, we generated a library of 4-substituted azabicyclo[5.3.0]alkane Smac-mimetics. Here we report the crystal structure of the BIR3 domain from XIAP in complex with Smac037, a compound designed according to structural principles emerging from our previously analyzed XIAP BIR3/Smac-mimetic complexes. In parallel, we present an in silico docking analysis of three Smac-mimetics to the BIR3 domain of cIAP1, providing general considerations for the development of high affinity lead compounds targeting three members of the IAP family.

Functionalized cyclic RGD peptidomimetics: conjugable ligands for αvß3 receptor imaging.

The α(v)ß(3) integrin is an adhesion molecule involved in physiological and pathological angiogenesis as well as in tumor invasion and metastasis, and therefore, there is a strong interest in developing novel agents interacting with this molecule. We report the synthesis and characterization of fluorescent α(v)ß(3) integrin probes and their use to visualize integrin α(v)ß(3) expression on human normal and cancer cells. The fluorescent probes we describe here may be of use for noninvasive imaging of α(V)ß(3) integrin expression also in vivo.

4-Aminoproline-based arginine-glycine-aspartate integrin binders with exposed ligation points: practical in-solution synthesis, conjugation and binding affinity evaluation.

An expedient and practical in-solution synthesis of three new 4-aminoproline-based arginine-glycine-aspartate integrin binders--compounds 15, 17 and 19--is presented. Two candidates carrying exposed azide and amine functional points were further advanced to trimeric platform 21 as well as fluorescein- and DOTA-conjugates 23 and 25. The new compounds were assayed for their binding affinity towards human alpha(V)beta3 and alpha(V)beta5 integrin receptors. Both monomeric candidates and covalent conjugates revealed potent ligand competence for the alpha(V)beta3 receptor in the one-digit nanomolar range (IC50 alpha(V)beta3 = 0.2-8.0 nM; IC50 alpha(V)beta5 = 5.0-1621 nM), thus demonstrating that conjugation does not impair the exquisite binding profile of this new generation of integrin ligands.

Rational design, synthesis and characterization of potent, non-peptidic Smac mimics/XIAP inhibitors as proapoptotic agents for cancer therapy.

Novel proapoptotic Smac mimics/IAPs inhibitors have been designed, synthesized and characterized. Computational models and structural studies (crystallography, NMR) have elucidated the SAR of this class of inhibitors, and have permitted further optimization of their properties. In vitro characterization (XIAP BIR3 and linker-BIR2-BIR3 binding, cytotox assays, early ADMET profiling) of the compounds has been performed, identifying one lead for further in vitro and in vivo evaluation.

Structure revision of the lantibiotic 97518.

The lantibiotic 97518, produced by a Planomonospora sp., was reported as a 2194 Da polypeptide comprising 24 amino acid residues with five thioether bridges. It was assigned to the mersacidin subgroup of type B lantibiotics by Castiglione et al. (Biochemistry 2007, 46, 5884-5897) and named planosporicin. New analytical, chemical, and genetic data and reinterpretation of the published NMR chemical shifts enable structure revision of 97518. The resulting revision of the 97518 structure involves both a shift of two amino acids and a reorganization of two thioether bridges. With this revision, the lantibiotic 97518 becomes a clear member of the nisin subgroup of compounds.

Impact of occult hepatitis B virus infection in HIV patients naive for antiretroviral therapy.

OBJECTIVE: To study the impact of occult hepatitis B virus (HBV) infection in 115 consecutive anti-HIV-positive, hepatitis B surface antigen-negative patients, naive for antiretroviral treatment. METHODS: Of these 115, 86 patients were followed for at least 6 months (range 6-36) with serial determinations of HIV RNA and HBV DNA by polymerase chain reaction and other laboratory tests. RESULTS: Of the 86 patients having a follow-up, plasma HBV DNA was detected in 17 (19.8%), 13 on admission and four during follow-up. HBV DNA was more frequently found in patients with isolated anti-hepatitis B core (HBc; 35.5% of 31 cases) than in those lacking anti-HBc and anti-hepatitis B surface (8.8% of 41, P < 0.005), or showing both (21.4% of 14). Twenty-eight patients (32.5%) experienced a hepatic flare during the follow-up; this event was more frequent in the 17 HBV-DNA-positive patients than in the 69 negative (64.7% versus 24.6%, P < 0.005). Of the 13 HBV-DNA-positive patients on admission, 11 receiving HAART containing lamivudine became HBV-DNA negative, but two of these again became positive and experienced a hepatic flare during treatment and two both during and after lamivudine treatment. A hepatic flare also occurred under lamivudine treatment in two of the four patients in whom HBV DNA became detectable during follow-up. The role of immune reconstitution inflammatory syndrome and HAART in inducing a hepatic flare was found to be marginal in 49 patients with no HBV or hepatitis C virus marker. CONCLUSION: The study suggests that HBV occult infection, relatively frequent in anti-HIV-positive patients, is associated with hepatic flares.

Clinical and virological improvement of hepatitis B virus-related or hepatitis C virus-related chronic hepatitis with concomitant hepatitis A virus infection.

BACKGROUND: We evaluated the clinical and virological characteristics of hepatitis A virus infection in persons concomitantly infected with hepatitis B virus (HBV) or hepatitis C virus (HCV). METHODS: We enrolled 21 patients with acute hepatitis A and chronic hepatitis with no sign of liver cirrhosis, 13 patients who were positive for hepatitis B surface antigen (case B group), 8 patients who were anti-HCV positive (case C group), and 21 patients with acute hepatitis A without a preexisting liver disease (control A group). Two control groups of patients with chronic hepatitis B (control B group) or C (control C group) were also chosen. All control groups were pair-matched by age and sex with the corresponding case group. RESULTS: Fulminant hepatitis A was never observed, and hepatitis A had a severe course in 1 patient in the case B group and in 1 patient in the control A group. Both patients recovered. On admission, HBV DNA was detected in 1 patient in the case B group (7.7%) and in 13 patients (50%) in the control B group; HCV RNA was found in no patient in the case C group and in 16 patients (81.2%) in the control C group. Of 9 patients in the case B group who were followed up for 6 months, 3 became negative for hepatitis B surface antigen and positive for hepatitis B surface antibody, 2 remained positive for hepatitis B surface antigen and negative for HBV DNA, and 4 became positive for HBV DNA with a low viral load [corrected] Of 6 patients in the case C group who were followed up for 6 months, 3 remained negative for HCV RNA, and 3 had persistently low viral loads. CONCLUSION: Concomitant hepatitis A was always self-limited, associated with a marked inhibition of HBV and HCV genomes, and possibly had a good prognosis for the underlying chronic hepatitis.

Hepatitis C virus superinfection in hepatitis B virus chronic carriers: a reciprocal viral interaction and a variable clinical course.

BACKGROUND: The virological and clinical impact of hepatitis C virus (HCV) superinfection in chronic hepatitis B virus (HBV) carriers has been poorly characterized. OBJECTIVE: To evaluate the viral interaction, clinical presentation and course of the disease in four HBsAg/HBV-DNA positive chronic hepatitis patients who developed acute HCV infection. STUDY DESIGN: To evaluate clinical, virological and laboratory data for at least 6 months from the onset of acute HCV infection in patients with chronic HBV infection. RESULTS: Three patients with acute HCV infection had a normal clinical course, but the remaining patient had severe disease with ascites and a marked decrease in prothrombin activity. In all cases, plasma HBV-DNA, which had been detectable prior to the HCV infection, was no longer detectable when the acute HCV infection occurred. The inhibition exerted by HCV on HBV-DNA persisted throughout the follow-up period in three patients, but was temporary in the one patient who experienced an acute exacerbation of chronic HBV infection. HCV-RNA became persistently undetectable in two patients and reduced to low levels in the other two. CONCLUSIONS: Acute HCV infection in the four HBV chronic carriers was characterized by a reciprocal inhibition of HBV-HCV genomes and, in one case, by a severe course of disease.

Biological and molecular properties of a new alpha(v)beta3/alpha(v)beta5 integrin antagonist.

The aim of the present study was to identify specific alpha(v)beta3/alpha(v)beta5 integrin antagonists active on tumor-induced angiogenesis. To this purpose, in vitro integrin-binding assays were used to screen a library of conformationally constrained bicyclic lactam Arg-Gly-Asp-containing pseudopeptides. The results identified ST1646 as a high-affinity specific ligand for alpha(v)beta3 and alpha(v)beta5 integrins with negligible interacting with alpha5beta1 integrin. In all the assays, ST1646 was equipotent to or more potent than the well-characterized integrin antagonists c(RGDfV) and cyclo(Arg-Gly-Asp-d-Phe-[NMe]Val) (EMD121974). In the chorioallantoic membrane assay, topical administration of ST1646 was able to prevent the angiogenic responses elicited by recombinant fibroblast growth factor-2 or vascular endothelial growth factor. In addition, systemic administration of ST1646 in mice exerted a significant antiangiogenic activity on neovascularization triggered by mammary carcinoma MDA-MB435 cells implanted s.c. in a dorsal air sac via a (Millipore Filter Corporation, Bedford, MA) chamber. Moreover, ST1646 delivery via an osmotic pump inhibited the growth and vascularization of tumor xenografts originating from the injection of alpha(v)beta3/alpha(v)beta5-expressing human ovarian carcinoma cells in nude mice. In agreement with the biochemical and pharmacologic studies, Monte Carlo/Stochastic Dynamics simulation showed that the bicyclic scaffold in ST1646 forced the compound to assume a preferred conformation superimposable to the X-ray conformation of alpha(v)beta3-bound EMD121974. Accordingly, computer-docking studies indicated that the ST1646-alpha(v)beta3 integrin complex maintains the ligand-receptor distances and interactions observed in the crystalline EMD121974-alpha(v)beta3 integrin complex. Taken together, these observations indicate that ST1646 represents a dual alpha(v)beta3/alpha(v)beta5 integrin antagonist with interesting biochemical and biological features to be tested in cancer therapy.

[An attempt to improve classification of HCV-correlated chronic hepatitis]. / Un tentativo di migliorare la classificazione delle epatiti croniche HCV-correlate.

To verify the clinical efficacy of the Desmet classification of chronic hepatitis C we reviewed 801 liver biopsies from patients with HCV-chronic hepatitis (CH). The diagnosis of chronic hepatitis was assessed according to the Desmet classification based on the Knodell Histological Activity Index (HAI) (minimal CH=score 1-3; mild CH= 4-8; moderate CH= 9-12; severe CH= 13-18). Liver fibrosis was assessed according to the Scheuer scoring system. One hundred forty-eight patients had cirrhosis and 653 CH. Of these 653, according to the Desmet classification 145 patients showed minimal, 424 mild, 73 moderate and 11 severe chronic hepatitis. Since the classification underestimated the moderate and severe forms of HCV-related chronic hepatitis, we evaluated the possibility of improving the Desmet classification of chronic hepatitis C using our classification: minimal CH= score 1-3; mild CH= 4-6; moderate CH= 7-8; severe CH= 9-18. According to our classification 145 showed minimal CH, 363 mild CH, 61 moderate CH and 84 severe CH. All the 61 patients who crossed over from mild CH under the Desmet to moderate CH under our classification showed a periportal inflammation of grade 3, and all the 73 patients but 8 who crossed over from moderate to severe showed a grade of periportal inflammation higher than 3. The Desmet classification of HCV-related chronic hepatitis underestimated the severe forms of HCV-CH, while our classification seems to be suitable also for chronic hepatitis C.

Acute hepatitis B and C virus coinfection: a virological and clinical study of 3 cases.

We report the virological interaction in, clinical presentation of, and course of disease observed in 3 male injection drug users with acute hepatitis B virus (HBV) and hepatitis C virus (HCV) coinfection. In all 3 cases, HBV infection presented first and quickly resolved. Diagnosis of acute HBV/HCV coinfection requires a long follow-up period with careful observation.

Uselessness of liver biopsy in patients with hepatitis C virus chronic infection and persistently normal aminotransferase levels.

This case-control study evaluated the real need for liver biopsy in subjects with persistently normal aminotransferase values over a long period by comparing the histological features of these subjects with those of patients with abnormal aminotransferase values. We considered as "Cases" all 32 consecutive anti-HCV/HCV-RNA positive subjects with at least eight normal serum ALT values during the last twelve months; for each "Case", we selected as a "Control" one anti-HCV/HCV-RNA positive patient with at least two abnormal serum ALT values during the last twelve months. The Cases and Controls were matched for age ( 5 years) and sex. In the Case group, 1 subject showed normal liver tissue, 18 minimal chronic hepatitis (CH) and 13 mild CH. In the Control group, 7 subjects showed minimal CH, 19 mild CH, 3 moderate CH, 1 severe CH and 2 cirrhosis. The subjects in the Control group showed a significantly higher HAI score (5.39+2.81) than those in the Case group (2.96+1.62, p < 0.001). The subjects in the Control group more frequently showed a fibrosis score greater than 1 (28.1%) compared to the Case group (9.4%; p<0.05). Finally, steatosis was more frequent and more severe in the Control group than in the Case group (respectively, 78.1% vs 50%, p < 0.05; and 1.47+1.16 vs 0.6+0.71, p < 0.001). The HAI and fibrosis scores did not correlate with the ALT value, HCV genotype or HCV viral load in either the Case or Control group. Our findings showed that the subjects with a persistently normal serum ALT value had minimal or mild chronic hepatitis, thus demonstrating that a liver biopsy is not indicated for these subjects.

Hepatitis viruses and HIV infection in the Naples area.

In 189 anti-HIV positive subjects (130 males and 59 females; median age 32 years, range 17-57) we evaluated the prevalence of patients with hepatitis infections, the role of parenteral and sexual risk factors on the acquisition of these infections and the reciprocal influence between HIV and HCV infections. HCV infection was detected in 53.9% of cases and HBV infection in 8.4%. In only 32% of our patients no marker of hepatitis virus infection was detected. The presence of a hepatitis virus infection was associated to drug addiction; indeed in 91 drug abusers HIV/HCV co-infection was present in 80% of cases and HIV infection alone in 7.7%, p<0.0001. On the other hand, the association between unsafe sexual activity, whether homosexual or heterosexual, and sexual activity with a steady anti-HIV positive partner with HCV infection was less evident, although the high prevalence of anti-HCV in these cases (10.4%, 15.4% and 26.4% respectively) clearly suggests that HIV infection may improve the sexual transmission of HCV. No substantial differences in the level of immunodeficiency, nor in the HIV viral load nor in the frequency of AIDS cases were observed between patients with HIV infection alone and those with HIV/HCV co-infection. In fact, the percentage of patients with AIDS was similar in these two groups. However, we observed a statistically significant association between an advanced HIV clinical stage and the presence of HIV/HCV co-infection (p<0.005), since subjects with co-infection more frequently than with HIV infection alone were in the CDC-B clinical stage. The presence of a more severe liver disease was linked to a multiple hepatitis virus infection, regardless of the degree of immunodeficiency.

Helicobacter spp. and liver diseases.

To test whether Helicobacter species play a role in the enhancement of liver necro-inflammation and fibrosis and in the development of hepatocellular carcinoma (HCC), we sought DNA sequences of Helicobacter species in liver specimens from patients with viral-related chronic hepatitis, HCC or metastatic liver carcinoma. We enrolled 28 consecutive patients with ultrasound evidence of hepatic nodule(s) on their first liver biopsy: 21 had histological evidence of HCC (Group I) and 7 of metastatic liver carcinoma (Group II). In the same period we observed 27 consecutive patients with chronic hepatitis on their first liver biopsy (Group III). Helicobacter sequences were sought by PCR using primers for the 16S rDNA of Helicobacter spp, designed to amplify a 400 base-pair fragment, and detected by 2% agarose gel and hybridization with a specific biotinylated probe. We used, as positive controls for the DNA extraction from liver tissue, hepatic biopsy sections in which HBV infection was confirmed by HBcAg positivity and in which we amplified HBV-DNA by specific primers; positive controls for the amplification of Helicobacter spp were obtained from gastric biopsy sections in which Helicobacter pylori infection was confirmed by biochemical and histochemical tests. HBV-DNA was found in all five HBcAg positive liver biopsies. Helicobacter spp 16S rDNA was detected in all five biopsy specimens of gastric mucosa and in none of liver specimens from patients in any group. Our data suggest that Helicobacter species were not involved in the pathogenesis of virus-related HCC, chronic hepatitis or liver carcinoma metastasis.

Synthesis of Gd and (68)Ga complexes in conjugation with a conformationally optimized RGD sequence as potential MRI and PET tumor-imaging probes.

We report the synthesis of novel chelates of Gd and (68)Ga with DPTA, DOTA, HP-DOA3, as well as with AAZTA, a novel chelating agent developed by our research group. These chelating agents were appropriately conjugated, prior to metal complexation, with DB58, an RGD peptidomimetic, conformationally constrained on an azabicycloalkane scaffold and endowed with high affinity for integrin α(ν)ß(3) . Because α(ν)ß(3) is involved in neo-angiogenesis in solid tumors and is also directly expressed in cancer cells (e.g. glioblastomas, melanomas) and ovarian, breast, and prostate cancers, these constructs could prove useful as molecular imaging probes in cancer diagnosis by MRI or PET techniques. Molecular modeling, integrin binding assays, and relaxivity assessments allowed the selection of compounds suitable for multiple expression on dendrimeric or nanoparticulate structures. These results also led us to an exploratory investigation of (68)Ga complexation for the promising (68)Ga-PET technique; the AAZTA complex 15((68)Ga) exhibited uptake in a xenograft model of glioblastoma, suggesting potentially useful developments with new probes with improved affinity.

Recognition of Smac-mimetic compounds by the BIR domain of cIAP1.

Inhibitor of apoptosis proteins (IAPs) are negative regulators of apoptosis. As IAPs are overexpressed in many tumors, where they confer chemoresistance, small molecules inactivating IAPs have been proposed as anticancer agents. Accordingly, a number of IAP-binding pro-apoptotic compounds that mimic the sequence corresponding to the N-terminal tetrapeptide of Smac/DIABLO, the natural endogenous IAPs inhibitor, have been developed. Here, we report the crystal structures of the BIR3 domain of cIAP1 in complex with Smac037, a Smac-mimetic known to bind potently to the XIAP-BIR3 domain and to induce degradation of cIAP1, and in complex with the novel Smac-mimetic compound Smac066. Thermal stability and fluorescence polarization assays show the stabilizing effect and the high affinity of both Smac037 and Smac066 for cIAP1- and cIAP2-BIR3 domains.