Novel prodrug approach to amprenavir-based HIV-1 protease inhibitors via O-->N acyloxy migration of P1 moiety

We have developed a new approach to prodrugs, which utilizes a pH-induced intramolecular O-->N migration of an acyloxy group in carbonate moiety to a free amino moiety at neutral pH. This method is exemplified by facile rearrangement of highly water-soluble prodrug 3 to carbamate 4, a close analogue of HIV-1 protease inhibitor Amprenavir. The O-->N acyloxy migration is unprecedented in the context of prodrugs and it enables a high atom economy due to recycling of the 'pro' moiety.     

Effect of the acyl groups on O-->N acyl migration in the water-soluble prodrugs of HIV-1 protease inhibitor

To improve the low water-solubility of HIV-1 protease inhibitors KNI-272, -279 and -727, we previously reported the water-soluble prodrugs of these inhibitors based on O-->N intramolecular acyl migration reaction. These prodrugs were rapidly converted to the corresponding parent drugs under physiological conditions. To understand the steric and electrostatic effects of O-acyl moiety on the migration rate, we examined several types of prodrug. A remarkably slow migration was observed in the benzoyl-type prodrugs, and Hammett plot of migration rate constants of p-substituted benzoyl-type prodrugs gave a linear free energy relationship.     

Water-soluble prodrugs of dipeptide HIV protease inhibitors based on O-->N intramolecular acyl migration: Design, synthesis and kinetic study

To improve the low water-solubility of HIV protease inhibitors, we synthesized water-soluble prodrugs of KNI-727, a potent small-sized dipeptide-type HIV-1 protease inhibitor consisting of an Apns-Dmt core (Apns; allophenylnorstatine, Dmt; (R)-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid) as inhibitory machinery. These prodrugs contained an O-acyl peptidomimetic structure with an ionized amino group leading to an increase in water-solubility, and were designed to regenerate the corresponding parent drugs based on the O-->N intramolecular acyl migration reaction via a five-membered ring intermediate at the alpha-hydroxy-beta-amino acid residue, that is Apns. The synthetic prodrug 3a improved the water-solubility (13 mg/mL) more than 8000-fold in comparison with the parent compound, which is the practically acceptable value as water-soluble drug. Furthermore, to understand the structural effects of the O-acyl moiety on the migration rate, we evaluated several phenylacetyl-type and benzoyl-type prodrugs. These prodrugs were stable as an HCl salt and in a strongly acidic solution corresponding to gastric juice (pH 2.0), and could be converted to the parent compounds promptly under aqueous conditions from slightly acidic to basic pH at 37 degrees C.     

New water-soluble prodrugs of HIV protease inhibitors based on O-->N intramolecular acyl migration

To improve the low water-solubility of HIV protease inhibitors, we synthesized water-soluble prodrugs of KNI-272 and KNI-279 which are potent HIV-1 protease inhibitors consisting of an Apns–Thz core structure (Apns; allophenylnorstatine, Thz; thiazolidine-4-carboxylic acid) as an inhibitory machinery. The prodrugs, which contained an O-acyl peptidomimetic structure with an ionized amino group leading to the increase of water-solubility, were designed to regenerate the corresponding parent drugs based on the O→N intramolecular acyl migration reaction at the -hydroxy-β-amino acid residue, that is allophenylnorstatine. The synthetic prodrugs 3, 4, 6, and 7 improved the water-solubility (>300 mg/mL) more than 4000-fold in comparison with the parent compounds, which is the practically acceptable value as water-soluble drugs. These prodrugs were stable as an HCl salt and in a strongly acidic solution corresponding to gastric juice (pH 2.0), and could be converted to the parent compounds promptly in the aqueous condition from slightly acidic to basic pH at 37 °C, with the suitable migration rate, via a five-membered ring intermediate. Using a similar method, we synthesized a prodrug (12) of ritonavir, a clinically useful HIV-1 protease inhibitor as an anti-AIDS drug. In contrast to the prodrugs 3, 4, 6, and 7, the prodrug 12 was very slowly converted to ritonavir probably through a six-membered ring intermediate, with the t_1/2 value of 32 h that may not be suitable for practical use.     

Syntheses, structures and properties of novel 3D lanthanide metal-organic frameworks with paddle-wheel building blocks

Three new complexes Ln(BDC)_1.5(DEF) [Ln = La(1), Ce(2), Nd(3); H₂BDC = 1,4-benzenedicarboxylic acid; DEF = N,N-diethylformamide] have been solvothermally synthesized and characterized by single-crystal X-ray diffraction. Complexes 1–3 are isostructural and crystallize in triclinic space group . They display three-dimensional frameworks and 18.7986 Å × 11.3230 Å rhombic channels along the a axis without interpenetration. Two eight-coordinated metal centers are connected with two dimonodentate BDC groups and two chelating/bridging bidentate BDC groups to give a paddle-wheel building block. The building blocks are connected through two carboxyl groups to lead to a one-dimensional infinite inorganic chain (–M–O–C–O–M–) along the [1 0 0] direction. Thermogravimetric analyses (TGA) and X-ray powder diffraction (XPRD) were determined for the three complexes. An observed intense blue luminescence with an emission band peaking at 372 nm for them may result from ligand-to-metal charge transfer (LMCT). Variable-temperature magnetic susceptibility of 2 and 3 was investigated and found deviated from the Curie–Weiss law.     

Acid-activatable prodrug nanogels for efficient intracellular doxorubicin release

Endosomal pH-activatable doxorubicin (DOX) prodrug nanogels were designed, prepared, and investigated for triggered intracellular drug release in cancer cells. DOX prodrugs with drug grafting contents of 3.9, 5.7, and 11.7 wt % (denoted as prodrugs 1, 2, and 3, respectively) were conveniently obtained by sequential treatment of poly(ethylene glycol)-b-poly(2-hydroxyethyl methacrylate-co-ethyl glycinate methacrylamide) (PEG-b-P(HEMA-co-EGMA)) copolymers with hydrazine and doxorubicin hydrochloride. Notably, prodrugs 1, 2, and 3 formed monodispersed nanogels with average sizes of 114.4, 75.3, and 66.3 nm, respectively, in phosphate buffer (PB, 10 mM, pH 7.4). The in vitro release results showed that DOX was released rapidly and nearly quantitatively from DOX prodrug nanogels at endosomal pH and 37 °C in 48 h, whereas only a minor amount (ca. 20% or less) of drug was released at pH 7.4 under otherwise the same conditions. Confocal laser scanning microscope (CLSM) observations revealed that DOX prodrug nanogels delivered and released DOX into the cytosols as well as cell nuclei of RAW 264.7 cells following 24 h incubation. MTT assays demonstrated that prodrug 3 had pronounced cytotoxic effects to tumor cells following 72 h incubation with IC(50) data determined to be 2.0 and 3.4 μg DOX equiv/mL for RAW 264.7 and MCF-7 tumor cells, respectively. The corresponding polymer carrier, PEG-b-P(HEMA-co-GMA-hydrazide), was shown to be nontoxic up to a tested concentration of 1.32 mg/mL. These endosomal pH-activatable DOX prodrug nanogels uniquely combining features of water-soluble macromolecular prodrugs and nanogels offer a promising platform for targeted cancer therapy.     

On the two forms of bacteriorhodopsin

In our previous work [(1993) FEBS Lett. 313, 248-250; (1993) Biochem. Int. 30,461-469] M-intermediate formation of wild-type bacteriorhodopsin was shown to involve two components differing in time constants (τ₁ = 60–70 μs and τ₂ = 220–250 μs), which were suggested to reflect two independent pathways of M-intermediate formation. The contribution of the fast M was 4-times higher than the slow one. Our present research on M-intermediate formation in the D115N bacteriorhodopsin mutant revealed the same components but at a contribution ratio of 1:1. Upon lowering the pH, the slow phase of M-formation vanished at a pK of 6.2, and in the pH region 3.0–5.5 only the M-intermediate with a rise time of 60 μs was present. A 5–6 h incubation of D115N bacteriorhodopsin at pH 10.6 resulted in the irreversible transformation of 50% of the protein into a form with a difference absorbance maximum at 460 nm. This form was stable at pH 7.5 and had no photocycle, including M-intermediate formation. The remaining bacteriorhodopsin contained 100% fast M-intermediate. The disappearance of the 250-μs phase concomitant with bR460 formation indicates that at neutral pH bacteriorhodopsin exists as two spectroscopically indistinguishable forms.     

Preparation,characterization and in vitro activity of a docetaxel–albumin conjugate

Docetaxel is one of the most effective anticancer drugs. However, the current formulation of docetaxel contains Tween 80 and ethanol as the solvent, which can cause severe side effects. Consequently, the development of new type of formulation of docetaxel with high efficiency and low side effects is a very important issue. In this study, we explored the covalent linking of docetaxel and albumin via one organic linker. 6-Maleimidocaproic acid was applied to link the C2′ hydroxyl group of docetaxel with the cysteine-34 of albumin to obtain 1:1 docetaxel–albumin conjugate. The synthesized conjugate can control the release of docetaxel in the bovine serum. Furthermore, in vitro cell cytotoxicity experiments indicated that the docetaxel–albumin conjugate have high activities for human prostate cancer cell line PC3 and human breast cancer cell line MCF-7. The present study provides a valuable strategy for further development of a new type of docetaxel–albumin prodrug.     

Reductive activation of 5-fluorodeoxyuridine prodrug possessing azide methyl group by hypoxic X-irradiation

We prepared a 5-fluorodeoxyuridine (5-FdUrd) derivative possessing azide methyl group (N(3)-FdUrd) as a novel radiation-activated prodrug. The parent antitumor agent, 5-FdUrd, was released efficiently from N(3)-FdUrd by hypoxic X-irradiation. On the other hand, the activation of N(3)-FdUrd was suppressed upon X-irradiation under aerobic conditions. A biological assay using A549 cells revealed that the cytotoxicity of N(3)-FdUrd was significantly enhanced by hypoxic X-irradiation.     

Caprine luteinizing hormone isoforms during the follicular phase and anestrus

The relative proportion of the circulating luteinizing hormone isoforms in goats during follicular phase (pre-ovulatory peak; F) and anestrus (A) was investigated. Estrus was synchronized in six goats with a prostaglandin analogue. After estrus was detected, blood samples were taken at 1 h intervals for 24 h. Four anestrous goats received 100 μg i.v. of GnRH and blood samples were collected every 15 min for 5 h. Samples with the greatest LH concentration in follicular phase and after GnRH administration (anestrus) were analyzed by chromatofocusing and eluted with a pH gradient from 10.5 to 3.5. For quantification purposes eluted LH was grouped into basic (pH ≥ 7.5), neutral (pH 7.4–6.5) and acidic isoforms (pH ≤ 6.4) as well as by pH unit. In both physiological conditions (PC), basic and acidic isoforms were greater than the neutral. With this grouping criteria, there was an interaction between PC and pH group, with the proportion of neutral isoforms being greater (p < 0.05) in A (12.0 ± 0.8%) as compared with F (5 ± 2%). Analysis by pH unit showed a very basic group of eluted isoforms (pH ≥ 10), which amounted to a percentage of 6.0 ± 0.4% of the total observed during A, and 3 ± 1% during F (p < 0.05). Predominant isoforms in A eluted in the pH range 9.99–9.0 (42 ± 3%) as compared to 7 ± 3% (p < 0.01) in that pH range in F. In contrast, the predominant isoforms in F eluted in the pH range 8.99–8.0, representing 55 ± 8%, while in A the proportion was 11 ± 2% (p < 0.01). Isoforms eluted at the pH range 7.9–7 represented a significantly greater proportion during A (5.0 ± 0.6%) as compared with F (3 ± 1%). This is the first report on goat LH circulating isoforms. During A the LH isoforms secreted by the pituitary are more basic than during F.     

Synthetic approaches to a mononucleotide prodrug of cytarabine

Synthetic pathways to a mononucleotide prodrug of cytarabine (Ara-C) bearing S-pivaloyl-2-thioethyl (tBuSATE) groups, as biolabile phosphate protections, are reported. Using a common phosphoramidite approach, two different kinds of nucleoside protecting groups have been investigated. During this study, we observed an intermolecular migration of the Boc protecting group in the course of the tert-butyldimethylsilyl ether cleavage using tetrabutyl ammonium fluoride.     

Response to 2009 pandemic influenza A (H1N1) vaccine in HIV-infected patients and the influence of prior seasonal influenza vaccination

Background

The immunogenicity of 2009 pandemic influenza A(H1N1) (pH1N1) vaccines and the effect of previous influenza vaccination is a matter of current interest and debate. We measured the immune response to pH1N1 vaccine in HIV-infected patients and in healthy controls. In addition we tested whether recent vaccination with seasonal trivalent inactivated vaccine (TIV) induced cross-reactive antibodies to pH1N1. (clinicaltrials.gov Identifier:{"type":"clinical-trial","attrs":{"text":"NCT01066169","term_id":"NCT01066169"}}NCT01066169)

Methods and Findings

In this single-center prospective cohort study MF59-adjuvanted pH1N1 vaccine (Focetria®, Novartis) was administered twice to 58 adult HIV-infected patients and 44 healthy controls in November 2009 (day 0 and day 21). Antibody responses were measured at baseline, day 21 and day 56 with hemagglutination-inhibition (HI) assay. The seroprotection rate (defined as HI titers ≥1∶40) for HIV-infected patients was 88% after the first and 91% after the second vaccination. These rates were comparable to those in healthy controls. Post-vaccination GMT, a sensitive marker of the immune competence of a group, was lower in HIV-infected patients. We found a high seroprotection rate at baseline (31%). Seroprotective titers at baseline were much more common in those who had received 2009–2010 seasonal TIV three weeks prior to the first dose of pH1N1 vaccine. Using stored serum samples of 51 HIV-infected participants we measured the pH1N1 specific response to 2009–2010 seasonal TIV. The seroprotection rate to pH1N1 increased from 22% to 49% after vaccination with 2009–2010 seasonal TIV. Seasonal TIV induced higher levels of antibodies to pH1N1 in older than in younger subjects.

Conclusion

In HIV-infected patients on combination antiretroviral therapy, with a median CD4+ T-lymphocyte count above 500 cells/mm³, one dose of MF59-adjuvanted pH1N1 vaccine induced a high seroprotection rate comparable to that in healthy controls. A second dose had a modest additional effect. Furthermore, seasonal TIV induced cross-reactive antibodies to pH1N1 and this effect was more pronounced in older subjects.     

Synthetic Approaches to a Mononucleotide Prodrug of Cytarabine

Synthetic pathways to a mononucleotide prodrug of cytarabine (Ara-C) bearing S-pivaloyl-2-thioethyl (tBuSATE) groups, as biolabile phosphate protections, are reported. Using a common phosphoramidite approach, two different kinds of nucleoside protecting groups have been investigated. During this study, we observed an intermolecular migration of the Boc protecting group in the course of the tert-butyldimethylsilyl ether cleavage using tetrabutyl ammonium fluoride.     

Solution conformation of brazzein by H nuclear magnetic resonance: resonance assignment and secondary structure

Brazzein is a sweet-tasting protein isolated from the fruit of the West African plant Pentadiplandra brazzeana Baillon. It is the smallest and the most water-soluble sweet protein discovered so far, it is also highly thermostable. The proton NMR study of brazzein at 600 MHz (pH 3.5, 300K) is presented. Complete sequence specific assignment of the individual backbone and sidechain proton resonances were achieved using through-bond and through-space connectivities obtained from standard two-dimensional NMR techniques. The secondary structure of brazzein contains one -helix (residues 21–29), one short 3₁₀-helix (residues 14–17), two strands of antiparallel β-sheet (residues 34–39, 44–50) and probably a third strand (residues 5–7) near the N-terminus.     

Properties of a glucose oxidase covalently immobilized on amorphous AlPO4 support

Glucose oxidase (GOD) was covalently immobilized on amorphous AlPO₄ as well as on an AlPO₄/clay mineral Sepiolite system. Immobilization of the enzyme was carried out through the -amino group of lysine residues through an aromatic Schiff's-base. Activation of the support was obtained after reaction of appropriate molecules with support surface –OH groups. The enzymatic activities of native, and different immobilized GOD systems and filtrates, were followed by the amount of liberated -gluconic acid obtained in the enzymatic β- -glucose oxidation with the aid of an automatic titrator. The kinetic properties of native and immobilized GOD were obtained for glucose concentrations in the range of physiological conditions and at different working conditions such as reaction temperature, reaction pH, and enzyme concentration.

The binding percentage of enzymes was in the 50–80% range, with residual and specific activities in the 65–80% and 90–150% ranges, respectively. No change in the pH optimum and only slight changes in the V_max and K_M kinetic parameters with respect to native GOD were observed, so that not only was little deactivation of enzyme obtained throughout the immobilization process but also that the stability of the covalently bound enzyme in the two supports appeared to have increased with respect to the soluble enzyme. GOD immobilization also increased its efficiency and operational stability in repeated uses on increasing the amount of immobilized enzyme.     

A xylose-tolerant beta-xylosidase from Paecilomyces thermophila: characterization and its co-action with the endogenous xylanase

An extracellular β-xylosidase from the thermophilic fungus Paecilomyces thermophila J18 was purified 31.9-fold to homogeneity with a recovery yield of 2.27% from the cell-free culture supernatant. It appeared as a single protein band on SDS–PAGE with a molecular mass of approx 53.5 kDa. The molecular mass of β-xylosidase was 51.8 kDa determined by Superdex 75 gel filtration. The enzyme was a glycoprotein with a carbohydrate content of 61.5%. It exhibited an optimal activity at 55 °C and pH 6.5, respectively. The enzyme was stable in the range of pH 6.0–9.0 and at 55 °C. The purified enzyme hydrolyzed xylobiose and higher xylooligosaccharides but was inactive against xylan substrates. It released xylose from xylooligosaccharides with a degree of polymerization ranging between 2 and 5. The rate of xylose released from xylooligosaccharides by the purified enzyme increased with increasing chain length. It had a K_m of 4.3 mM for p-nitrophenol-β-d-xylopyranoside and was competitively inhibited by xylose with a K_i value of 139 mM. Release of reducing sugars from xylans by a purified xylanase produced by the same organism increased markedly in the presence of β-xylosidase. During 24-hour hydrolysis, the amounts of reducing sugar released in the presence of added β-xylosidase were about 1.5–1.73 times that of the reaction employing the xylanase alone. This is the first report on the purification and characterization of a β-xylosidase from Paecilomyces thermophila.     

Development of novel water-soluble photocleavable protective group and its application for design of photoresponsive paclitaxel prodrugs

A novel coumarin-based highly water-soluble photocleavable protective group was designed and synthesized, and then this photosensitive protecting group was used to design paclitaxel prodrugs. These novel paclitaxel conjugates demonstrated excellent water solubility, over 100mgmL(-1). Thus, the use of a detergent in the formulation can be omitted completely, even at high doses. Phototaxel 11 released the parent drug, paclitaxel, quickly and efficiently by minimal tissue-damaging 365nm UV light irradiation at low power, while laser activation at 355nm led to extensive decomposition of the prodrug. The carbamate-type prodrug, phototaxel 11, was stable in the dark prior to activation, whereas carbonate-type phototaxel 9 demonstrated poor stability under aqueous conditions. For such prodrugs, tumor-tissue targeting after administration could be achieved by selective light delivery, similar to that used in photodynamic therapy. In addition, newly designed coumarin derivative 8 can be applied in organic chemistry as a photosensitive protective group and for the design of caged compounds.     

Gelation properties of extruded lemon cell walls and their water-soluble pectins

The amount of water-soluble pectins was largely increased after extrusion-cooking of lemon fibres. These pectins showed the ability to form a gel in the presence of sucrose and at acidic pH. The gels obtained with the water-extracted pectins after extrusion-cooking and with pectins acid-extracted on a laboratory scale were softer than those prepared with commercial citrus pectins. The water-extracted pectins after extrusion-cooking and the pectins acid-extracted on a laboratory scale contained long neutral side-chains and required a higher sucrose concentration to gel than the commercial citrus pectins. The extruded lemon fibres showed the ability to form gels in the presence of sucrose and at acidic pH. The gels obtained with the extruded fibres containing some water-solube pectins of high molecular weight were stronger than those obtained with the extruded fibres containing higher amounts of more depolymerised water-soluble pectins. The extruded fibres containing 12.5–14.9% of water-soluble pectins of high molecular weight (intrinsic viscosity: 413–504 mL/g) were those showing the better gelation properties.     

Targeted enzyme prodrug therapy for metastatic prostate cancer – a comparative study of L-methioninase,purine nucleoside phosphorylase,and cytosine deaminase

Background

Enzyme prodrug therapy shows promise for the treatment of solid tumors, but current approaches lack effective/safe delivery strategies. To address this, we previously developed three enzyme-containing fusion proteins targeted via annexin V to phosphatidylserine exposed on the tumor vasculature and tumor cells, using the enzymes L-methioninase, purine nucleoside phosphorylase, or cytosine deaminase. In enzyme prodrug therapy, the fusion protein is allowed to bind to the tumor before a nontoxic drug precursor, a prodrug, is introduced. Upon interaction of the prodrug with the bound enzyme, an anticancer compound is formed, but only in the direct vicinity of the tumor, thereby mitigating the risk of side effects while creating high intratumoral drug concentrations. The applicability of these enzyme prodrug systems to treating prostate cancer has remained unexplored. Additionally, target availability may increase with the addition of low dose docetaxel treatment to the enzyme prodrug treatment, but this effect has not been previously investigated. To this end, we examined the binding strength and the cytotoxic efficacy (with and without docetaxel treatment) of these enzyme prodrug systems on the human prostate cancer cell line PC-3.

Results

All three fusion proteins exhibited strong binding; dissociation constants were 0.572 nM for L-methioninase-annexin V (MT-AV), 0.406 nM for purine nucleoside phosphorylase-annexin V (PNP-AV), and 0.061 nM for cytosine deaminase-annexin V (CD-AV). MT-AV produced up to 99% cell death (p < 0.001) with limited cytotoxicity of the prodrug alone. PNP-AV with docetaxel created up to 78% cell death (p < 0.001) with no cytotoxicity of the prodrug alone. CD-AV with docetaxel displayed up to 60% cell death (p < 0.001) with no cytotoxicity of the prodrug alone. Docetaxel treatment created significant increases in cytotoxicity for PNP-AV and CD-AV.

Conclusions

Strong binding of fusion proteins to the prostate cancer cells and effective cell killing suggest that the enzyme prodrug systems with MT-AV and PNP-AV may be effective treatment options. Additionally, low-dose docetaxel treatment was found to increase the cytotoxic effect of the annexin V-targeted therapeutics for the PNP-AV and CD-AV systems.     

Characteristics of human types 1, 2 and 3 17β-hydroxysteroid dehydrogenase activities: Oxidation/reduction and inhibition

Following transfection of types 1, 2 and 3 17β-hydroxysteroid dehydrogenase (17β-HSD) cDNAs into transformed embryonal kidney (293) cells, we have characterized the selective directional and inhibitory characteristics of these activities. While homogenates of transfected cells could catalyze interconversion of the substrate and product, in agreement with the general belief on the activity of these enzymes, the same activities measured in intact cells, in order to better reflect the physiological conditions, showed an unidirectional reaction. Types 1 and 3 17β-HSD catalyzed the reduction of estrone to estradiol and 4-androstenedione to testosterone, respectively, while type 2 17β-HSD catalyzed the oxidative transformation of both testosterone and 17β-estradiol to 4-androstenedione and estrone, respectively. In addition, types 1, 2 and 3 17β-HSD activities showed different pH optima. While types 1 and 3 showed pH optimum values centered at around 5 and 6, respectively, type 2 17β-HSD activity, which preferentially catalyzes the oxidation reaction, has higher activity at an alkaline pH (8–10). Differences in the optimum incubation temperatures were also observed: type 1 17β-HSD shows a relatively high temperature tolerance (55°C). In contrast, type 2 and 3 functioned best at 37°C. Types 1, 2 and 3 17β-HSD activities could be also differentiated by their sensitivity toward various specific inhibitors: type 1 was potently inhibited by an estradiol derivative containing a bromo/or iodopropyl group at position 16. On the other hand a derivative of estrone containing a spiro-γ-lactone at position 17 showed a potent inhibitory effect on type 2 17β-HSD, whereas type 3 was strongly inhibited by 1,4-androstadiene-1,6,17-trione.