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A novel strategy to develop site-activated multifunctional chelators for targeting multiple etiologies of Alzheimer's disease is reported. The novel prochelator HLA20A with improved cytotoxicity shows little affinity for metal ions until it is activated by binding and inhibiting acetylcholinesterase (AChE), releasing an active chelator HLA20 that modulates amyloid precursor protein (APP) regulation and beta-amyloid (Abeta) reduction, suppresses oxidative stress, and passivates excess metal ions (Fe, Cu, and Zn) in the brain.

Affecting an estimated 5% of adults over 65 years of age, Parkinson's disease and Alzheimer's disease are the most common neurodegenerative disorders. Accumulating evidence suggests that oxidative stress induced by the breakdown of iron homeostasis is a major contributor to the neuronal loss observed in neurodegeneration. Thus, brain-permeable iron chelators may present potential therapeutic benefits. In the present study, iron-chelating hydroxamate groups were introduced into the NAP (NAPVSIPQ) peptide, whose neuroprotective qualities have been widely demonstrated. Our experiments revealed that the novel dihydroxamate peptide 3 is capable of inhibiting iron-catalyzed hydroxyl radical formation and lipid peroxidation, abilities that are not part of the repertoire of its parent peptide. In addition, peptide 3 was superior to native NAP in protecting human neuroblastoma cell cultures against the toxicity of hydrogen peroxide. These results suggest that NAP-based iron chelators deserve further investigation in the search for drug candidates for neurodegeneration.

Polymyxin B nonapeptide (PMBN), a cationic cyclic peptide derived by enzymatic processing from the naturally occurring peptide polymyxin B, is able to increase the permeability of the outer membrane of Gram-negative bacteria toward hydrophobic antibiotics probably by binding to the bacterial lipopolysaccharide (LPS). We have synthesized 11 cyclic analogues of PMBN and evaluated their activities compared to that of PMBN. The synthetic peptides were much less potent than PMBN in their capacity to sensitize Escherichia coli and Klebsiella pneumoniae toward novobiocin and to displace dansyl-PMBN from Escherichia coli LPS. Moreover, unlike PMBN, none of the analogues were able to inhibit the growth of Pseudomonas aeruginosa. The structural-functional features of PMBN were characterized and identified with regard to the ring size, the distance between positive charges and peptide backbone, the chirality of the DPhe-Leu domain, and the nature of the charged groups. Apparently, the structure of PMBN is highly specific for efficient perturbation of the outer membrane of Gram-negative bacteria as well as for LPS binding. The present study further increases our understanding of the complex PMBN-LPS and may, potentially, enable the design of compounds having enhanced permeabilization potency of the Gram-negative outer membrane.

Four novel 2,4-methano amino acids (MAAs, 1-aminocyclobutane-1-carboxylic acids) were synthesized. These include the basic MAA analogs of lysine (16), ornithine (5), and arginine (6) and the neutral methanovaline (22), related to proline. The above MAAs, as well as the MAA analog of homothreonine (7), were incorporated into the peptide chain of the immunomodulatory peptide tuftsin, Thr-Lys-Pro-Arg, known to enhance several biological activities mediated by phagocytic cells. The synthetic methano tuftsin analogs were assayed for their ability to stimulate interleukin-6 (IL-6) secretion by mouse peritoneal macrophages and for their stability in human serum toward enzymatic degradation. It was found that, at 2 x 10(-7) M, [MThr1]tuftsin (24) and an isomer of [MVal3]tuftsin (27a) were considerably more active than the parent peptide in augmentation of cytokine release. [MOrn2]Tuftsin (25) was equally potent. The analogs [MThr1]tuftsin (24) and [MOrn2]tuftsin (25), both pertaining to the proteolytically sensitive Thr-Lys bond of tuftsin, exhibited high resistance to enzymatic hydrolysis as compared to tuftsin. Using specific rabbit anti-tuftsin antibodies in a competitive enzyme-linked immunosorbent assay (ELISA) revealed that none of the MAA analogs can cross-react with tuftsin. It may indicate that the peptides assume global structures different than that of tuftsin.

Pegylation is a powerful technology to prolong the action of proteins in vivo, but it is impractical for low-molecular-weight (LMW) drugs, which are usually inactivated upon such modification. Here, we have applied a recently developed strategy of reversible pegylation to gentamicin, a LMW antibiotic. Variable length polyethyleneglycol (PEG-SH) chains were covalently linked to gentamicin using two heterobifunctional agents, each containing a spontaneously hydrolyzable bond. The inactive derivatives regained full antibacterial potency upon incubation under physiological conditions in vitro, and following systemic administration to rats, they released native active gentamicin with half-lives 7- to 15-fold greater than those of systemically administered nonderivatized gentamicin. In conclusion, reversibly pegylated prodrug derivatives of gentamicin were found to be capable of releasing gentamicin for prolonged periods in vivo. Most importantly, the major drawback of conventional pegylation, namely, the loss of pharmacological potency following irreversible derivatization, has been overcome.

Many peptide and protein drugs have a short circulatory half-life in vivo. The covalent attachment of polyethylene glycol (PEG) chains (PEGylation) can overcome this deficiency, but pegylated peptides and proteins are often inactive. In this study, we present a novel PEG-IFNalpha2 conjugate, PEG(40)-FMS-IFNalpha2, capable of regenerating native interferon alpha2 (IFNalpha2) at a slow rate under physiological conditions. A 2-sulfo-9-fluorenylmethoxycarbonyl (FMS) containing bifunctional reagent, MAL-FMS-NHS, has been synthesized, enabling the linkage of a 40 kDa PEG-SH to IFNalpha2 through a slowly hydrolyzable bond. By use of a BIAcore binding assay, the in vitro rate of regeneration of native interferon was estimated to have a half-life of 65 h. Following subcutaneous administration to rats and monitoring circulating antiviral activity, active IFNalpha2 levels peaked at 50 h, with substantial levels still being detected 200 h after administration. This value contrasts with a half-life of about 1 h measured for unmodified interferon. The concentration of active IFNalpha2 scaled linearly with the quantity injected. Comparing subcutaneous to intravenous administration of PEG(40)-FMS-IFNalpha2, we found that the long circulatory lifetime of IFNalpha2 was affected both by the slow rate of absorption of the PEGylated protein from the subcutaneous volume and by the slow rate of discharge from the PEG in circulation. A numerical simulation of the results was in good agreement with the results observed in vivo. The pharmacokinetic profile of this novel IFNalpha2 conjugate combines a prolonged maintenance in vivo with the regeneration of active-native IFNalpha2, ensuring ready access to peripheral tissues and thus an overall advantage over currently used formulations.

Photodynamic therapy uses a combination of light, oxygen, and a photosensitizer to induce the death of malignant cells. To improve the selectivity of a photosensitizer toward cancerous cells that express gonadotropin-releasing hormone (GnRH) receptors, protoporphyrin IX (PpIX) was conjugated to a GnRH agonist, [d-Lys6]GnRH, or to a GnRH antagonist, [d-pGlu1, d-Phe2, d-Trp3, d-Lys6]GnRH. The condensation of the peptide with PpIX was carried out in a homogeneous solution using benzotriazole-1-yloxytris(pyrrolidinophosphonium) hexafluorophosphate as a coupling reagent. Although these conjugates had lower binding affinity to rat pituitary GnRH receptors than their parent analogues, they fully preserved their agonistic or antagonistic activity in vitro and in vivo. The GnRH agonist conjugate proved to be long-acting in vivo. Thus, 24 h after its administration to rats (2 nmol/rat), serum LH concentrations were significantly higher than in rats treated with the same amount of the parent peptide. The conjugates, notably the agonist, were more phototoxic toward pituitary gonadotrope alphaT3-1 cell line than was unconjugated PpIX. In contrast to PpIX, the phototoxicity of the conjugates toward alphaT3-1 cells or to human breast cancer cells (MCF-7 cells that were transfected with human GnRH receptors) was alleviated by co-incubation with the parent peptide, indicating that phototoxicity is receptor-mediated. The selectivity of the GnRH antagonist conjugate to gonadotrope cells in a primary pituitary culture was approximately 10 times higher than that of the unconjugated PpIX. Thus, GnRH-based conjugates may affect cancer cells not only by acting as classic GnRH analogues to reduce the plasma levels of steroids by desensitization of the pituitary gland but also by selective photodamage of cells that express GnRH receptors.

Most low-molecular-weight drugs are short-lived species in the circulatory system, being rapidly eliminated by glomerular filtration in the kidney. However, binding to human serum albumin (HSA) can slow clearance and prolong lifetime profile in vivo. In this study, we have engineered a gentamicin derivative with affinity to albumin by linking three (2-sulfo)-9-fluorenylmethoxycarbonyl (FMS) to three amino groups of gentamicin C(1). FMS(3)-gentamicin associates with HSA with a K(a) value of (1.31 +/- 0.2) x 10(5) M(-1). It has less than 1% the antibacterial potency of native gentamicin. Upon incubation at pH 8.5 and 37 degrees C, the FMS moieties from FMS(3)-gentamicin undergo slow hydrolysis (t(1/2) = 8.0 +/- 0.2 h), leading to a linear regeneration of the antibacterial potency with a t(1/2) value of 11 +/- 0.7 h. FMS(3)-gentamicin is a long-lived species in the rat circulatory system. Following a single subcutaneous or intravenous administration, it maintains a prolonged pharmacokinetic profile with a peak and a "through" concentration of immuno/antibacterial active gentamicin exceeding 4-5 times the duration obtained by administered native gentamicin. To sum up, an approach aimed at elongating the lifetime of low-molecular-weight drugs in vivo has been examined here with gentamicin. Two to three FMS per mole of compound are to be introduced to obtain an albumin associating affinity of K(d) = 7.6-9.2 microM and, hence, to significantly extend the drug's lifetime in situ following administration. By use of this technology, the loss of pharmacological potency with derivatization is of no consequence, since FMS moieties are hydrolyzed and activity is generated at physiological conditions.

The design, synthesis, and biological evaluation of a gonadotropin-releasing hormone (GnRH) agonist, [D-Lys(6)(1,3,8-trihydroxy-6-carboxyanthraquinone)]GnRH ([D-Lys(6)(Emo)]GnRH), is described. Synthesis of this analogue was carried out in a homogeneous solution as well as on a polymer support. [D-Lys(6)(Emo)]GnRH was found to bind to rat pituitary GnRH receptors (IC(50) = 0.25 nM), to induce luteinizing hormone (LH) release (ED(50) = 27 pM), and to be devoid of any toxicity. This analogue also proved to be a very potent agonist in vivo and exhibited a prolonged bioactivity. Six hours after its administration to rats, LH levels were substantially higher than those of rats treated with a 10-fold higher dose of the parent peptide. Moreover, chronic treatment of adult male rats with [D-Lys(6)(Emo)]GnRH (0.1 nmol/rat) for one week resulted in a further decrease of the weight of the testes and prostate as compared to those of rats that were treated with a higher dose of [D-Lys(6)]GnRH (1 nmol/rat). The prolonged activity of [D-Lys(6)(Emo)]GnRH may be attributed to its emodic acid moiety, which enhances the binding affinity of the analogue to human serum albumin. Indeed, we found that emodic acid binds to human serum albumin almost completely at the examined range of concentrations.

We have previously determined that Ac-D-Trp-Leu-Asp-Ile-Ile-Trp (peptide I), an endothelin antagonist, binds specifically (Ki = 1.9 microM) to the rat pituitary gonadotropin-releasing hormone (GnRH) receptor. Moreover, peptide I exhibits a GnRH agonistic activity, mediated directly by the GnRH receptor. We now report structure-activity studies of peptide I in respect to its interactions with the GnRH receptor. Our studies suggest that the bioactive conformation of peptide I, recognized by the GnRH receptor, is of a cyclic nature. Thus cyclic analogues of peptide I exhibit higher affinity to the GnRH receptor and increased agonistic potencies as compared to peptide I itself. A linear peptide, Ile-Ile-Trp-D-Trp-Leu-Asp, which presumably forms a similar cyclic conformation, was also shown to be a GnRH agonist. Intraperitoneal administration of Ac-Ile-Ile-Trp-D-Trp-Leu-Cys-OH (Ki = 0.32 microM), one of the cyclic hexapeptides that we have synthesized, to rats induces secretion of luteinizing hormone (LH) with a potency which is only 1 order of magnitude less than that of GnRH itself. Moreover, plasma levels of LH remained elevated for a longer period of time following the administration of the cyclic hexapeptide. This novel class of GnRH agonists may prove useful in the development of new therapeutics.

A novel strategy for designing reduced-size analogues of the decapeptide gonadotropin-releasing hormone (GnRH) was developed. As opposed to previous attempts to delete residues from either of the peptide's termini, our approach is based upon the known importance of both C- and N-terminals of GnRH analogues for receptor recognition, whereas the central part of the molecule is replaced by a short spacer. The present truncation strategy was successful for generation of reduced-size hexapeptide and heptapeptide antagonists possessing potent antagonistic capacity. The same methodology was not suitable for the generation of reduced-size agonists, suggesting different conformational characteristics for GnRH agonists and antagonists. A heptapeptide antagonist designed by this method was shown to inhibit serum levels of luteinizing hormone in castrated rats in vivo. Structure-activity studies suggested that the structural preferences for GnRH receptor recognition are similar to those reported for decapeptide antagonists. Our studies resulted in a heptapeptide GnRH antagonist (Ac-D-Nal2-D-Cpa-D-Pal-Gly-Arg-Pro-D-Ala-NH2) with high receptor binding affinity (IC50 = 7 nM), as compared to that of GnRH itself (IC50 = 2 nM). The highest affinity of a hexapeptide antagonist that we have synthesized was somewhat lower (IC50 = 45 nM).

In this study we designed, prepared, and analyzed a water-soluble, long-acting insulin derivative whose protracted action in vivo is based on a new principle rather than on slower absorption rates of suspended insulin formulations. To this end, we have prepared (9-fluorenylmethoxycarbonyl-SO(3)H)(3)-insulin ((FMS)(3)-insulin), a derivative having three 9-fluorenylmethoxycarbonyl-SO(3)H (FMS) moieties covalently linked to the three amino side chains of insulin. (FMS)(3)-insulin is soluble in aqueous buffers at neutral pH, at a concentration range of 0.15-0.60 mM, and has about 1% of both the biological potency and the receptor-binding affinity of the native hormone. Upon incubation at pH 7.4 and 37 degrees C, it undergoes a slow hydrolysis with linear regeneration of insulin possessing full biological potency. A single subcutaneous administration of (FMS)(3)-insulin to streptozocin-treated rats lowered circulating glucose levels for a prolonged period (t(1/2) = 30 h). Similarly, intraperitoneal administration of (FMS)(3)-insulin to healthy rats had a prolonged glucose-lowering effect. In this experimental system, recovery from hypoglycemia proceeded with a t(1/2) value of 14 +/- 1 h, as compared with t(1/2) = 8.0 +/- 1 h for native insulin and t(1/2) = 10.0 +/- 1 h for NPH-insulin. (FMS)(3)-insulin is more resistant to proteolysis and appears to be nonimmunogenic. On the whole, (FMS)(3)(-)insulin represents a prototype version of a water-soluble, long-acting preparation of insulin. It is nearly inactive at the time of administration, and therefore can be administered, at high dose, with no concern for hypoglycemia. Because of its decreased receptor-binding affinity, (FMS)(3)-insulin evades receptor-mediated endocytosis and degradation and, hence, persists for a long period in the circulation. The insulin constituent of the (FMS)(3)-insulin conjugate undergoes a slow, spontaneous activation in the circulatory system, manifesting a prolonged glucose-lowering action in vivo. According to the data presented here, (FMS)(3)-insulin represents a typical prodrug: a compound which by itself shows only marginal activity but over time it is chemically hydrolyzed to the fully active hormone.

The synthesis of 11 peptides, ranging in composition from 9 to 17 amino acid residues, by solid-phase methodology was accomplished with the purpose of studying how the amphiphilic and hydrophobic character, the size of the molecule, and the charge distribution modulate the antibacterial activity. It was found that peptides composed of 16 and 17 amino acid residues, with high hydrophobic (mainly due to Trp or Phe) and hydrophilic (due to Lys) character distributed along opposite amphiphilic faces, showed considerable antibacterial activity against clinically isolated bacteria together with Gram positive and Gram negative ATCC bacterial strains. However, the hemolytic capacity of the peptides was also significant. Decreasing the hydrophobic character of the molecule by replacing Trp or Phe with Leu residues while maintaining the basic contribution of Lys drastically reduced the hemolytic activity and only slightly decreased the bioactivity. Peptides composed of 9-10 amino acid residues with high hydrophobic and basic nature possess antibacterial activity but, in general, are less active than the larger counterpart peptides. By replacing all Trp residues of a short peptide by Leu residues, the activity was considerably reduced. Circular dichroism studies and antibacterial assays showed that shorter peptides with very low helical content, and thus deprived of amphiphilic character, still have appreciable bioactivity. This observation, coupled with the fact that due to their small size they cannot span the bacterial outer lipid bilayer, may suggest different mechanisms of action for long-chain vis-a-vis short-chain peptides.

Thirteen analogues of the natural macrophage activator peptide tuftsin, ten of which are novel, were synthesized with the aim of exploring the relation between their biological potency and their capacity to attach specifically to cellular tuftsin's receptors. The analogues representing modifications and chain extensions at various parts of the parent tuftsin molecule can be classified as N-terminal analogues, C-terminal analogues, "within-chain" derivatives, or dimers of tuftsin and retrotuftsin. The various synthetic routes employed to prepare the analogues are described. A direct correlation was found between the ability of analogues to inhibit [3H-Arg4]tuftsin specific binding to mice peritoneal macrophages and their capacity to enhance phagocytosis or to inhibit tuftsin-mediated phagocytosis by the cells and to potentiate the cell's immune response.