Cellular Internalization and Inhibition Capacity of New Anti-Glioma Peptide Conjugates: Physicochemical Characterization and Evaluation on Various Monolayer- and 3D-Spheroid-Based in Vitro Platforms.

Most therapeutic agents used for treating brain malignancies face hindered transport through the blood-brain barrier (BBB) and poor tissue penetration. To overcome these problems, we developed peptide conjugates of conventional and experimental anticancer agents. SynB3 cell-penetrating peptide derivatives were applied that can cross the BBB. Tuftsin derivatives were used to target the neuropilin-1 transport system for selectivity and better tumor penetration. Moreover, SynB3-tuftsin tandem compounds were synthesized to combine the beneficial properties of these peptides. Most of the conjugates showed high and selective efficacy against glioblastoma cells. SynB3 and tandem derivatives demonstrated superior cellular internalization. The penetration profile of the conjugates was determined on a lipid monolayer and Transwell co-culture system with noncontact HUVEC-U87 monolayers as simple ex vivo and in vitro BBB models. Importantly, in 3D spheroids, daunomycin-peptide conjugates possessed a better tumor penetration ability than daunomycin. These conjugates are promising tools for the delivery systems with tunable features.

In vitro biological evaluation of new antimycobacterial salicylanilide-tuftsin conjugates.

Tuberculosis is caused by Mycobacterium tuberculosis, an intracellular pathogen that can survive in host cells, mainly in macrophages. An increase of multidrug-resistant tuberculosis qualifies this infectious disease as a major public health problem worldwide. The cellular uptake of the antimycobacterial agents by infected host cells is limited. Our approach is to enhance the cellular uptake of the antituberculars by target cell-directed delivery using drug-peptide conjugates to achieve an increased intracellular efficacy. In this study, salicylanilide derivatives (2-hydroxy-N-phenylbenzamides) with remarkable antimycobacterial activity were conjugated to macrophage receptor specific tuftsin based peptide carriers through oxime bond directly or by insertion of a GFLG tetrapeptide spacer. We have found that the in vitro antimycobacterial activity of the salicylanilides against M. tuberculosis H37Rv is preserved in the conjugates. While the free drug was ineffective on infected macrophage model, the conjugates were active against the intracellular bacteria. The fluorescently labelled peptide carriers that were modified with different fatty acid side chains showed outstanding cellular uptake rate to the macrophage model cells. The conjugation of the salicylanilides to tuftsin based carriers reduced or abolished the in vitro cytostatic activity of the free drugs with the exception of the palmitoylated conjugates. The conjugates degraded in the presence of rat liver lysosomal homogenate leading to the formation of an oxime bond-linked salicylanilide-amino acid fragment as the smallest active metabolite.

Pharmacokinetics and bioavailability of tuftsin-derived T peptide, a promising antitumor agent, in beagles.

Tuftsin, a natural phagocytosis-stimulating tetrapeptide, had aroused much interest in tumor immunotherapy, but the poor pharmacokinetics hampered its clinical developments, for that it was extremely susceptible to degradation by enzymolysis in vivo. T Peptide (TP) was a newly designed tuftsin derivative aimed to enhance stability and was proved to have significant antitumor activity. In this study, the pharmacokinetics and bioavailability of TP was first clarified in beagles with subcutaneous administration, by using a simple and robust competitive ELISA method. Dose-dependency and non-linear dynamics of TP after single-dose (2, 6 and 18 mg kg(-1), respectively) were found, and the half-time of TP was proved to reach 1.3-2.8 h. Multiple dosing of 6 mg kg(-1) once a day for 7 days resulted in a slight accumulation (accumulation index was 1.92 ± 0.43), indicating that the dosing interval in the following clinical trial needs to be extended. The absolute bioavailability of TP was 31.1 ± 6.2% after subcutaneous administration. These results first demonstrated the pharmacokinetics and bioavailability data of TP in vivo, which illustrated the potential druggability of TP and provided useful information for the dosage regimen design in the following clinical trials, as well as a simple and feasible analytical method for clinical sample analysis.

Safety, efficacy and pharmacokinetics of tuftsin-loaded nystatin liposomes in murine model.

Present study was performed to evaluate the efficacy, toxicity and pharmacokinetics of antifungal drug nystatin incorporated in immunomodulator tuftsin-bearing liposomes. In vitro toxicity of free nystatin and nystatin incorporated in tuftsin-free or tuftsin-loaded liposomes was assessed by incubation of nystatin formulations with human erythrocytes. The toxicity profile of free nystatin and liposomal formulations of nystatin with or without tuftsin was also analyzed by monitoring the level of blood urea nitrogen (BUN) and serum creatinine in the treated BALB/c mice. The results of the present work showed that tuftsin-loaded nystatin liposomes like conventional nystatin liposomes exerted less toxicity to human erythrocytes as compared with free nystatin. Moreover, mice treated with tuftsin-loaded nystatin liposomes showed insignificant elevation in the biochemical values of serum creatinine and blood urea. The stability of nystatin liposomes upon incorporation of tuftsin was evaluated by monitoring the leakage of the entrapped drug in human serum. Tuftsin-loaded liposomes held nystatin for longer duration in the presence of serum than identical nystatin liposomes without tuftsin. Pharmacokinetics of the both tuftsin-free or tuftsin-loaded liposomal formulations nystatin was analyzed by determining the level of nystatin in the systemic circulation of mice at different time points. Mice injected with tuftsin-loaded nystatin liposomes showed higher level of the drug in the systemic circulation compared with those treated with conventional nystatin liposomes. The efficacy of tuftsin-loaded nystatin liposomes against A. fumigatus was evaluated by assessing the fungal burden in the lungs of treated mice. Treatment with tuftsin-loaded nystatin liposomes was most effective in eliminating fungal burden from lung tissues of infected mice compared to those treated with free nystatin or nystatin liposomes without tuftsin. The immunopotentiating activity, increased stability and less toxicity of tuftsin-incorporated nystatin liposomes, supports the idea for its prophylactic and therapeutic use in the clinical setting.

Toxicity, stability and pharmacokinetics of amphotericin B in immunomodulator tuftsin-bearing liposomes in a murine model.

OBJECTIVES: In the present study we evaluated the pharmacokinetics and toxicity of amphotericin B in immunomodulator tuftsin-loaded liposomes in a murine model. METHODS: Stability of amphotericin B liposomes was tested by incubating one volume of liposomal formulations of amphotericin B with nine volumes of serum. The pharmacokinetics of amphotericin B in Candida albicans-infected mice treated with conventional and tuftsin-loaded amphotericin B liposomes was evaluated over a period of 24 h. In vitro toxicity of amphotericin B deoxycholate, as well as amphotericin B liposomes, was tested by incubation with human erythrocytes for 1 h at 37 degrees C. To assess amphotericin B-induced in vivo toxicity, BALB/c mice were injected with three doses of amphotericin B deoxycholate, as well as amphotericin B liposomal formulations on days 1, 2 and 3 post C. albicans infection. Blood from treated mice was taken by retro-orbital puncture to test renal function parameters such as serum creatinine and urea. RESULTS: In vitro stability studies revealed that tuftsin-bearing amphotericin B liposomes released only 11% of the total liposomal amphotericin B in the serum, while it was found to be 19% from identical tuftsin-free amphotericin B liposomes. Both tuftsin-loaded as well as tuftsin-free liposomal formulations of amphotericin B induced approximately 20% haemolysis of erythrocytes at a dose of 40 mg/L, while the same amount of drug in amphotericin B deoxycholate caused 100% lysis of the erythrocytes. Pharmacokinetic studies revealed that subsequent to administration of various formulations of amphotericin B, there was 32 mg/L amphotericin B in the systemic circulation of mice treated with tuftsin-bearing amphotericin B liposomes, while it was 25 mg/L for amphotericin B liposomes, 4 h post drug administration. In vivo toxicity studies demonstrated that the amphotericin B deoxycholate formulation induced elevations in serum creatinine (approximately 300% of control) and blood urea (approximately 380% of control) values, while these values were substantially less (blood urea approximately 150% of control and serum creatinine approximately 210% of control) in the animals treated with the tuftsin-loaded amphotericin B liposomal formulation. Further, the administration of amphotericin B deoxycholate (1 mg/kg) in BALB/c mice at a dose of 1 mg/kg body weight led to the accumulation of 18.6 +/- 5.25 g/kg (of amphotericin B) in kidneys. On the other hand, administration of liposomal amphotericin B and tuftsin-bearing liposomal amphotericin B at a dose of 5 mg/kg body weight resulted in accumulation of 8.8 +/- 2.0 and 4.0 +/- 1.6 g/kg of amphotericin B, respectively, in the kidneys of treated animals. CONCLUSIONS: Co-administration of immunomodulator tuftsin along with liposomal formulations of amphotericin B successfully minimizes toxicity, as well as other side effects of the drug. Interestingly, tuftsin also increased the stability of liposomal amphotericin B. Superior efficacy, reliable safety and favourable pharmacodynamics of tuftsin-loaded amphotericin B liposomes suggest their potential therapeutic value in the management of fungal infections.

[The pharmacokinetics and metabolism of heptapeptide--a prospective synthetic analog of tuftsin with psychostimulating action in rats]. / Farmakokinetika i metabolizm geptapeptida--perspektivnogo sinteticheskogo analoga taftsina s psikhostimuliruiushchim deistviem u krys.

The pharmacokinetics of a new heptapeptide with psychotropic activity was studied in rats in experiments in vitro and in vivo by means of high-performance liquid chromatography. The heptapeptide was detected in its natural form in rat blood plasma for 7-10 min; its T1/2 exceeded that of other oligopeptides of similar structure. The structure of the main pharmacologically active heptapeptide metabolite was identified by high performance liquid chromatography; it proved to be tetrapeptide Thr-Lys-Pro-Arg (taphtsin). The heptapeptide was distributed in well vascularized organs of rats: liver > kidneys > heart.