Surface Layer Modification of Poly(d,l-lactic- co-glycolic acid) Nanoparticles with Targeting Peptide: A Convenient Synthetic Route for Pluronic F127-Tuftsin Conjugate.

Nanoparticles consisting of biodegradable poly(d,l-lactic- co-glycolic acid) (PLGA) are promising carriers for drug molecules to improve the treatment of tuberculosis. Surface modifiers, such as Pluronic F127, are essential for biocompatibility and for the protection against particle aggregation. This study demonstrates a successful approach to conjugate Pluronic F127 coated PLGA nanoparticles with Tuftsin, which has been reported as a macrophage-targeting peptide. Transformation of Pluronic F127 hydroxyl groups-which have limited reactivity-into aldehyde groups provide a convenient way to bind aminooxy-peptide derivatives in a one-step reaction. We have also investigated that this change has no effect on the physicochemical properties of the nanoparticles. Our data showed that coating nanoparticles with Pluronic-Tuftsin conjugate markedly increased the internalization rate and the intracellular activity of the encapsulated drug candidate against Mycobacterium tuberculosis. By employing this approach, a large variety of peptide targeted PLGA nanoparticles can be designed for drug delivery.

Successful modulation of murine lupus nephritis with tuftsin-phosphorylcholine.

In areas where helminths infections are common, autoimmune diseases are rare. Treatment with helminths and ova from helminths, improved clinical findings of inflammatory bowel disease, multiple-sclerosis and rheumatoid-arthritis. The immunomodulatory functions of some helminths were attributed to the phosphorylcholine (PC) moiety. We aimed to decipher the tolerogenic potential of Tuftsin-PC (TPC) compound in mice genetically prone to develop lupus. Lupus prone NZBXW/F1 mice received subcutaneously TPC (5 µg/1 ml), 3 times a week starting at 14 weeks age. Autoantibodies were tested by ELISA, T-regulatory-cells by FACS, cytokines profile by RT-PCR and cytokines protein levels by DuoSet ELISA. Glomerulonephritis was addressed by detection of proteinuria, and immunoglobulin complex deposition in the mesangium of the kidneys of the mice by immunofluorescence. Our results show that TPC attenuated the development of glomerulonephritis in lupus prone mice, in particular, it ameliorated proteinuria (p < 0.02), and reduced immunoglobulin deposition in the kidney mesangium. TPC also enhanced the expression of TGFß and IL-10 (p < 0.001), and inhibited the production of IFNγ and IL-17 (p < 0.03). TPC Significantly enhanced the expansion of CD4+CD25+FOXP3+ T-regulatory cells (Tregs) phenotype in the treated mice. These data indicate that TPC hampered lupus development in genetically lupus prone mice which was exemplified by moderate glomerulonephritis, attenuation of pro-inflammatory cytokines and enhancement of anti-inflammatory cytokines expression, as well as Tregs expansion. Our results propose harnessing novel natural therapy for lupus patients.

Synthesis and immunomodulatory activity of [60]fullerene-tuftsin conjugates.

Immunomodulating peptide tuftsin (Thr-Lys-Pro-Arg) was covalently conjugated to fullerene C(60) by two different ways to prepare NH(2)-tuftsin-C(60) and C(60)-tuftsin-COOH. The two new compounds were intensively characterized. The synthetic C(60)-tuftsin conjugates were assayed for their stability against leucine aminopeptidase degradation. And the immunostimulating activities to murine peritoneal macrophages were investigated in vitro. Compared with the natural tuftsin, significant enhancement of phagocytosis, chemotaxis activities and major histocompatibility complex class II (MHC II) molecule expression were observed in macrophages stimulated by both of the conjugates. The two conjugates also exhibit complete resistance to enzymatic hydrolysis, and they are non-toxic to macrophages in the tested concentrations. On all accounts, these results suggest that the C(60)-tuftsin conjugates can be used as potential candidates of immunomodulators and vaccine adjuvants.

Solid phase synthesis and biological activity of tuftsin conjugates.

New tuftsin/retro-tuftsin conjugates were designed and synthesized using a classical fluorenylmethoxycarbonyl (Fmoc) solid phase procedure. All the peptide conjugates were divided into three series: 1,4-dihydroxyanthraquinone (type A), 1-nitroacridine (type B), and 4-carboxyacridone (type C) derivatives. In type A conjugates, the N-terminal group of the peptide chain is directly connected to the anthraquinone ring at C1 (Scheme 1), whereas types B and C conjugates possess an amide bond formed between the carboxyl group of heterocyclic molecule and the N-termini of the tuftsin chain. The in vitro cytotoxic activity of the tuftsin conjugates and their precursors using two human tumor cell lines (lung adenocarcinoma (A549) and myeloblastic leukemia (HL-60)) was investigated. The analogues from groups A and C exhibited low cytotoxic activity, whereas several compounds of type B showed a potent and selective cytotoxic activity against tested tumor cell lines. None of the examined tuftsin conjugates demonstrated any significant effect on the catalytic activity of types I and II DNA topoisomerases.

Design, synthesis, and in vitro activity of novel drug delivery systems containing tuftsin derivatives and methotrexate.

During the past decade, biodegradable polymers or oligopeptides recognized by cell-surface receptors have been shown to increase drug specificity, lowering systemic drug toxicity in contrast to small-size fast-acting drugs. The goal of the present study was to develop anticancer bioconjugates based on chemotactic drug targeting (CDT). These constructs are composed of methotrexate (Mtx) attached to a tuftsin-like peptide carrier through an enzyme-labile pentapeptide spacer (GFLGC) and several copies of a chemotactic targeting moiety (H-TKPR, For-TKPR, H-TKPKG, and Ac-TKPKG). Carriers with targeting moieties in the branches were prepared by solid-phase synthesis using mixed Boc and Fmoc strategies. The drug molecule connected to an enzyme-labile spacer was attached to the branched oligopeptide in solution. In vitro chemotaxis, cellular uptake, and cytotoxicity assays were carried out on the MonoMac6 cell line. The most effective conjugates with H-TKPR or Ac-TKPKG targeting moieties in the branches, which have the most advantageous chemotactic properties, can be internalized rapidly, and these conjugates trigger higher toxic effect than the free drug (Mtx). The results suggest that our tuftsin-based drug delivery systems might be potential candidates for targeting cancer chemotherapy.

Synthesis of oligotuftsin-based branched oligopeptide conjugates for chemotactic drug targeting.

The synthesis and chemotactic properties of a new class of branched oligopeptide-based conjugates are described. Tetratuftsin derivatives containing chemotactic formyl tripeptides (For-MLF, For-NleLF or For-MMM) in branches were prepared by stepwise solid-phase peptide synthesis. The influence of the composition and ionic charge of the carrier-branched oligopeptide on the chemotactic behaviour of the conjugate was studied in Tetrahymena pyriformis. Conjugates with methotrexate (Mtx) as a drug component was also prepared. For this, a GFLGC spacer, cleavable by cathepsin B, was used. The spacer with N-terminal methotrexate was coupled to the chloroacetylated chemotactic carrier molecule by thioether bond formation. The chemotactic activity and cytotoxity of Mtx conjugates were also studied.

Synthesis of linear, branched, and cyclic peptide chimera.

Chimeric peptides are unnatural constructs consisting of bioactive compounds from at least two different peptide(s) and/or protein(s) or two sequences from different parts of the same protein. Such multifunctional peptide combinations are prepared to enhance the biological activity or selectivity of their components. New biological effects can also be achieved with the chimera. In this chapter the synthesis of three different types of chimeric peptides will be described. In a linear chimera, two peptide epitopes from different parts of glycoprotein D (gD) of herpes simplex virus (HSV) are combined. A branched chimera, built from linear peptides, consists of tuftsin oligomers with immunostimulatory activity and an epitope peptide of HSV gD. The third compound is a cyclic chimeric molecule, where alpha-conotoxin GI as a host peptide is modified by the incorporation of a core epitope from HSV gD as a guest sequence.

Synthesis and biological activity of tuftsin, its analogue and conjugates containing muramyl dipeptides or nor-muramyl dipeptides.

Several conjugates of muramyl dipeptide (MDP) or nor-muramyl dipeptide (nor-MDP) with tuftsin were synthesized. Conjugates 8a-f were prepared by acylation of protected tuftsin with the isoglutamine carboxyl group of MDP or nor-MDP 2a-f. Also tuftsin analogue 6 (H-Thr-Lys-Pro-Arg(NO2)-OH) was obtained. All synthesized compounds were investigated at the Medical University of Gdansk. The biological activity of the examined compounds was estimated using in vitro cultures of human monocytes and lymphocytes. The substances displayed cytotoxic effects, as was revealed in the viability tests performed. The effects were most probably mediated by the induction of an oxidative burst in monocytes and the stimulation of redox enzymes in lymphocytes. In addition, the analogues turned out to be efficient stimulators of TNFalpha and IL6 secretion by monocytes and lymphocytes. Nevertheless, the secretion of cytokines did not affect the viability of the leukocyte population used in the experiments.The beneficial properties of the compounds examined (mainly 6, 3, 8a and 8c), which implies their usefulness as potential therapeutic agents, are connected with their rapid start of action and more efficient effects compared with tuftsin alone. An in vivo assay on animal models will be performed.

Tuftsin-AZT conjugate: potential macrophage targeting for AIDS therapy.

The IgG-derived immunomodulating peptide tuftsin, Thr-Lys-Pro-Arg, is recognized by specific receptors on phagocytic cells, notably macrophages, and is capable of targeting proteins and peptides to these sites. Aiming to target 3'-azido-3'-deoxythymidine (AZT) to HIV-infected macrophages, a conjugate of AZT with tuftsin was synthesized. The AZT-tuftsin chimera possesses the characteristic capacities of its two components. Thus, like AZT, it inhibits reverse transcriptase activity and HIV-antigen expression, and similarly to tuftsin, it stimulates IL-1 release from mouse macrophages and augments the immunogenic function of the cells. Importantly, the conjugate is not cytotoxic to T-cells. The results suggest that the AZT-tuftsin conjugate might have potential use in AIDS therapy.

Increase in the immunogenicity of HIV peptide antigens by chemical linkage to polytuftsin (TKPR40).

The use of synthetic peptide antigens in human prophylaxis still suffers from the very important problem of finding suitable carriers devoid of side effects. A desirable carrier for use in humans would be poorly immunogenic by itself, yet it would enhance the immune response to the peptide antigen. In the study reported herein, we examined the role of polytuftsin (TKPR40), a synthetic polymer of the natural immunomodulator tuftsin, as a carrier for synthetic peptides of HIV derived from the gp41 and gp120 proteins. Chimeric immunogens were constructed by chemical linkage between synthetic peptides of HIV and polytuftsin. These were employed for immunization of mice of different MHC haplotypes, and the humoral and cellular immune responses developed against the peptides were assessed by measuring total IgG, IgG, subclasses, T-cell proliferation, and in vitro cytokine release. A significantly stronger immune response was observed in mice immunized with the peptide-polytuftsin conjugates than in mice receiving the peptide dimers (peptide-peptide). Peptide-polytuftsin conjugates induced IgG2a and IgG2b isotype switching after both primary and secondary immunization. In addition, there was a positive correlation between the amounts of cytokines and the shift in the IgG isotypes. These data suggest that the use of polytuftsin as a carrier may increase the immune response against poorly immunogenic synthetic peptides.

Tuftsin-THF-gamma 2 chimeric peptides: potential novel immunomodulators.

Synthesis of two chimeric peptides composed of tuftsin and thymic humoral factor-gamma 2 (THF-gamma 2) conjugates was accomplished. Our goal was the generation of novel immunomodulators. Initially, we demonstrate an IL-6 inducing activity of the phagocytic cells stimulant, tuftsin, on murine macrophages. This activity was documented only in the presence of antigen, either KLH or lysozyme. The augmentation was dose dependent, with optimal activity at a concentration of 200 and 20 nM, respectively. The chimeric peptides, either H2N-tuftsin-THF-gamma 2-OH or H2N-THF-gamma 2-tuftsin-OH, were also evaluated in the IL-6 system in the presence of the more potent antigen, KLH. The IL-6 inducing effect was maintained, although maximal activity appeared only at a concentration an order of magnitude greater than that of tuftsin. The chimeric peptides were further tested in an assay evaluating enhancement in murine bone marrow myeloid colony formation, a system in which THF-gamma 2, a T cell stimulant, has an established beneficial effect. The compounds were found to be inactive at the 25-200 ng/ml (14-112 nM) concentration range evaluated. Finally, the chimeric peptides were tested in a combined macrophages-T cells assay, i.e. antigen presentation, in which H2N-tuftsin-THF-gamma 2-OH was found to be more active than either parent peptide, thus representing a possible therapeutic agent.

Synthesis and immunological evaluation of the conjugates composed from a muramyl peptide GMDP and tuftsin.

The conjugates of a muramyl dipeptide analog GMDP (N-acetylglucosaminyl beta 1-->4 N-acetylmuramyl-L-alanyl-D-isoglutamine) and tuftsin (Thr-Lys-Pro-Arg) were synthesized from unprotected GMDP by mixed anhydride procedure. The identity of the conjugates was confirmed by high resolution NMR and their immunomodulating properties were determined in various tests. It was found that the conjugate in which the GMDP carboxyl group forms an amide bond with the epsilon-amino group of tuftsin lysyl residue exceeds GMDP in all the activities determined. Synergism of GMDP and tuftsin was found in phagocytosis stimulation assay.

Enhanced phagocytosis activity of cyclic analogs of tuftsin.

Cyclic analogs of the physiological immunostimulating peptide tuftsin (Thr-Lys-Pro-Arg), cyclo(Thr-Lys-Pro-Arg-Gly) (ctuf-G) and cyclo(Thr-Lys-Pro-Arg-Asp) (ctuf-D), were synthesized based on molecular modeling studies, and assayed for the ability to stimulate phagocytosis by human polymorphonuclear leukocytes. As predicted, the synthesis of ctuf-D resulted in two isomers with the correct molecular mass and amino acid composition. In phagocytosis assays, tuftsin, ctuf-G and two isomers of ctuf-D showed the usual bell-shaped activity profiles. The optimum concentration of ctuf-G was 50-fold less than that of tuftsin, whereas the degree of stimulation was similar. One isomer of ctuf-D was almost inactive, and the other ctuf-D exhibited the same degree of phagocytosis as tuftsin but its optimum concentration was 5-fold lower. The enhanced potency of ctuf-G and one isomer of ctuf-D may be due to conformational effects and/or to the possibility that these cyclic peptides are resistant to proteolytic degradation.

Single-step bulk purification of synthetic tetrapeptide tuftsin by high performance liquid chromatography.

A protocol on reversed phase high performance liquid chromatogrpahy (RP-HPLC) using water-acetonitrile gradients under lesser stringent conditions, has been devised to obtain highly purified tuftsin in bulk amount. The protocol was also tested for two different preparations of tuftsin to yield identical quality of peptide.

Synthesis and biological activity of [L-hydroxyproline]3-tuftsin analogue and its alpha- or beta-O-D-glucosylated derivatives.

Syntheses are described of the Hyp3-tuftsin analogue and of its derivatives alpha- or beta-O-glycosylated at the side chain function of the hydroxyproline residue. The carbohydrate-free tetrapeptide was prepared by reacting Z-Thr-Lys(Z)-OH with H-Hyp-Arg(NO2)-OBzl by the mixed anhydride procedure. In the synthesis of the alpha-glycosylated analogue the O-glycosyl amino acid was incorporated by reacting Boc-(Glc alpha+beta)Hyp-OH with H-Arg(NO2)-OBzl through the same procedure. The alpha-glucosylated dipeptide was isolated from the diastereomeric mixture, selectively deblocked, and acylated with Z-Thr-Lys(Z)-OH by the mixed anhydride procedure. In the preparation of the beta-glucosylated analogue the BOP procedure was used for reacting Boc-[Glc(Ac)4 beta]Hyp-OH with H-Arg(NO)2-OBzl was well as for the final coupling to tetrapeptide. Removal of protecting groups from crude tetrapeptides was achieved by catalytic hydrogenation. Deacetylation of the sugar moiety of the beta-glucosylated tetrapeptide was achieved by treatment with sodium methoxide in methanol. The synthetic compounds were isolated by ion exchange chromatography, and characterized by elemental analysis, amino acid analysis, optical rotation and proton NMR. Their capacity to evoke the release of interleukin 1 from mouse peritoneal macrophages and to modulate immunogenic activity of antigen-fed cells was evaluated, in comparison with tuftsin and rigin. All of the analogues were found to possess tuftsin-like activity.

Immunostimulation by a partially modified retro-inverso-tuftsin analogue containing Thr1 psi[NHCO](R,S)Lys2 modification.

The tuftsin retro-inverso analogue H-Thr psi[NHCO](R,S)Lys-Pro-Arg-OH was synthesized through a novel procedure for the high-yield incorporation of isolated retro-inverso bonds into peptide chains and the use of the new Meldrum's acid derivative (CH3)2C(OCO)2CH(CH2)4NHCOCF3 followed by its efficient coupling in solution to trimethylsilylated H-D-Thr(t-Bu)NH2. Closely related peptide impurities were eliminated both from the crude final peptide and the fully protected tetrapeptide amide precursor via ion-exchange and reversed-phase displacement chromatography, respectively. The tuftsin retro-inverso analogue proved to be completely resistant to enzymatic degradation in vitro, either against isolated aminopeptidases or human plasma proteolytic enzymes. When administered either orally or intravenously, it was significantly more active than normal tuftsin in increasing the number of specific antibody secreting cells in spleen of mice immunized with sheep erythrocytes. Furthermore, the analogue exerted an enhanced stimulatory effect on the cytotoxic activity of splenocytes against YAC-1 tumor cells. Finally, retro-inverso-tuftsin was about 10-fold more potent than the native peptide in reducing rat adjuvant arthritis. The resistance of the retro-inverso analogue to peptidases might explain the increased in vivo activities and allows its further immunopharmacological characterization.

Approaches to some biochemical mechanisms of action of tuftsin and analogues.

Tuftsin, T-K-P-R, is a phagocytosis-stimulating peptide described as a natural immunostimulant. Four analogues of this peptide were synthesized. These compounds were assayed for their ability to compete with [3H]tuftsin for its specific receptor from thioglycollate-elicited mouse peritoneal macrophages. They were also tested for their ability to change level in intracellular cGMP and to stimulate phagocytosis through the nitroblue tetrazolium reduction measurement. Surprisingly, all the analogues were poor competitors of [3H]tuftsin binding but possess potent tuftsin-like activities.

An active pseudopeptide analog of the leucokinin insect neuropeptide family.

A pseudopeptide analog of the active core of the leucokinin insect neuropeptide family was synthesized and found to retain myotropic activity. No reports of active pseudopeptide analogs of an insect or other invertebrate neuropeptide have previously appeared in the literature. The pseudopeptide (Phe psi [CH2-NH] Phe-Ser-Trp-Gly-NH2) contains a reduced-amide linkage between the two N-terminal Phe residues. Unlike its amide-bond containing counterpart, the activity of the pseudopeptide was not destroyed upon exposure to aminopeptidase M.

Synthesis of glycosylated tuftsins and tuftsin-containing IgG fragment undecapeptide.

Syntheses are described of two new tuftsin derivatives containing a 2-acetamido-2-deoxy-D-galactopyranosyl unit alpha- or beta-glycosidically linked to the threonine's hydroxy side chain function and of the glycosylated undecapeptide corresponding to the tuftsin region of the heavy chain of IgG (amino acid sequence 289-299). The glycosylated tuftsins were synthesized by the solution procedure. Fmoc-[Gal NAc(Ac)3 alpha]Thr-OH and Fmoc-[GalNAc(Ac)3 beta]Thr-OH were allowed to react with H-Lys(Z)-Pro-Arg(NO2)-OBzl by the mixed anhydride procedure and the resulting glycosylated tetrapeptides were fully deblocked by catalytic hydrogenation followed by treatment with potassium cyanide, purified by ion exchange chromatography and characterized by analytical HPLC, elemental and amino acid analyses, optical rotation, and proton NMR spectroscopy. Synthesis of the glycosylated undecapeptide was achieved by the continuous flow solid phase procedure on 4-hydroxymethylphenoxyacetyl-norleucyl derivatized Kieselguhr-supported resin. Fmoc-amino acid symmetrical anhydrides or pentafluorophenyl esters, in the presence of N-hydroxybenzotriazole, were used as the acylating agents. To mimic the native sequence of the tuftsin region at the Fc-domain of immunoglobulin G a 2-acetamido-2-deoxy-beta-D-glucopyranosyl unit was N-glycosidically linked to the amide side chain of Asn 297. The glycosylated asparagine residue was introduced as N2-fluorenylmethyloxycarbonyl-N4-(2-acetamido-3,4,6-tri-O-acetyl-2 -deoxy-beta-D - glucopyranosyl)-asparagine pentafluorophenyl ester. After cleavage from the resin the glycopeptide was deprotected, purified by ion exchange chromatography, and characterized by analytical HPLC, amino acid analysis, high voltage electrophoresis, and proton NMR. The conformational features of the glyco-undecapeptide were determined by circular dichroism measurements both in water and in 98% trifluoroethanol. Results of biological assays will be published elsewhere.