Anticancer activity of the liposome-encapsulated cyclic dipeptides, cyclo(His-Gly) and cyclo(His-Ala).

Cyclic dipeptides have been well characterized for their biological activity, including antimicrobial and anticancer activities. Cyclo(His-Gly) and cyclo(His-Ala) have also recently demonstrated significant anticancer activity against a range of cell lines, however, as a result of their physicochemistry, namely high solubility and low lipophilicity, it can be predicted that cellular permeability would be low, making them ideal candidates for liposome drug delivery. Liposomes were composed of phosphatidylcholine, hydrogenated soy phosphatidylcholine (HSPC), stearylamine, alpha-tocopherol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (PEG-DSPE) or folate-polyethylene glycol-cholesteryl hemisuccinate (F-PEG-CHEMS) using the thin-film hydration method and characterized for size and encapsulation. The cytotoxic activity of the encapsulated cyclic dipeptides was tested against HeLa, low folate HeLa and MCF-7 cells and found to have limited improvement in activity. However, modification of the polyethylene glycol with folic acid to target folate receptors significantly decreased the IC50 values recorded in all cells lines tested, particularly HeLa cells cultured in media containing physiological concentrations of folic acid with the lowest IC50 being recorded as 0.0962 mM for folate-targeted cyclo(His-Ala). Therefore, hydrophilic cyclic dipeptides are ideal candidates for inclusion into targeted drug delivery systems such as liposomes.

Antimicrobial activity of liposome encapsulated cyclo(L-tyrosyl-L-prolyl).

Various studies have shown the potentially beneficial biological activities of cyclic dipeptides and in particular, cyclo(L-tyrosyl-L-prolyl) (cyclo(Tyr-Pro)) has shown fair antibacterial activity in vitro. This study aimed to determine if liposome encapsulation would have any significant effects on the antibacterial activity of this compound. The thin-film hydration method with extrusion was used to produce small unilamellar vesicles containing cyclo(Tyr-Pro) that were shown to have an average encapsulation of 9.4% with a mean particle size of 160.4 nm. Minimum inhibitory concentrations tested against Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Bacillus subtillis were shown to be lower in liposome encapsulated cyclo(Tyr-Pro) than for the free form, while no antimicrobial activity was noted for either encapsulated nor non-encapsulated drug against the fungus Candida albicans or two methicillin-resistant strains of Staphylococcus aureus (MRSA). A positive control of liposome encapsulated amoxicillin was shown to be extremely active against both MRSA strains. The results confirm that liposome encapsulation has the potential to enhance activity as well as to overcome bacterial resistance towards current antibacterial agents.

The synthesis and anticancer activity of selected diketopiperazines.

Six selected diketopiperazines, cyclo(Gly-Val), cyclo(Gly-D-Val), cyclo(Gly-Leu), cyclo(Gly-Ile), cyclo(Phe-Cys) and cyclo(Tyr-Cys), were synthesized via various synthetic routes. Their potential to inhibit cancer cell growth in HT-29, HeLa and MCF-7 cells was determined. Cyclo(Tyr-Cys) caused the greatest inhibition in cervical carcinoma cells with near equivalent activity against HT-29 and MCF-7 cells. The other cyclic dipeptides tested were effective in the inhibition of colon, cervical and breast carcinoma cells, respectively, but the percentage inhibition was lower than for cyclo(Tyr-Cys).

The biological activity of the histidine-containing diketopiperazines cyclo(His-Ala) and cyclo(His-Gly).

Two cyclic dipeptides, cyclo(His-Ala) and cyclo(His-Gly,) were synthesized from their linear counterparts and their structures elucidated using standard elucidation techniques. Molecular modeling and predictive NMR results indicated that the majority of energetically favourable conformers adopted a boat conformation with respect to the diketopiperazine ring. Cyclo(His-Ala), at concentrations of 100 microM inhibited the growth, in vitro, of various cancer cell lines, including HT-29, MCF-7 and HeLa carcinoma cells while cyclo(His-Gly) inhibited the growth of MCF-7 cells. While the antibacterial potential of these two compounds was limited, both cyclic dipeptides significantly inhibited the growth of C. albicans. Both compounds at a concentration of 100 microM resulted in a decrease in heart rate, coronary flow rate and left ventricular systolic pressure in the isolated rat heart. Inhibition of thrombin, amounting to a 63.3% and 36.7% reduction in the rate of fibrin formation, was noted for cyclo(His-Ala) and cyclo(His-Gly), respectively. While cyclo(His-Ala) showed no notable effects on platelet aggregation, cyclo(His-Gly) significantly inhibited both pathways tested with greatest effects on thrombin-induced platelet aggregation, yielding an IC(50) of 0.0662 mM (R(2)=0.989). The results of the anticancer and hematological studies indicate that histidine-containing diketopiperazines have potential as a novel group of cytotoxic agents with antithrombotic effects.

Biological activity of selected tyrosine-containing 2,5-diketopiperazines.

The study investigates two cyclic dipeptides, cyclo(Tyr-Tyr) (cTT) and cyclo(Phe-Tyr) (cPT) with respect to their biological activity. Investigations using the whole-cell patch-clamp technique testing the effects of 100 microM cyclic dipeptide on ion channels, revealed reversible voltage-dependant blockade for cTT, while cPT exhibited irreversible time-dependant blockade of L-type calcium channels. The isolated retrogradely-perfused rat heart was used to determine the effects of 100 microM of either cTT or cPT on heart rate (HR), coronary flow rate (CFR), left ventricular systolic pressure (LVSP) and cardiac conduction speed. Results indicated opposing effects for the two compounds, where cTT increased HR and CF while cPT decreased HR, CF, LVSP as well as conduction speed. Other biological investigations included opioid binding and anti-neoplastic assays. Competitive binding curves, using tritiated DAMGO, revealed significant binding to micro-opioid receptors with IC50 values for cTT and cPT being 0.82 microM and 69.7 microM respectively. Anti-neoplastic activity was tested using three cultured cell lines: HT-29, MCF-7 and HeLa which were exposed to 2.5 mM cyclic dipeptide or 0.1 mM melphalan as a positive control. While cTT showed little activity against these lines, cPT resulted in as much as a 75.6% growth inhibition of MCF-7 cells, while also being active against HeLa (73.4% inhibition) and HT-29 (60.6%). The results indicate potential biological activity, showing a need for more investigation into tyrosine containing cyclic dipeptides and their analogues as potential bioactive compounds.

Interstitial laser hyperthermia model development for minimally invasive therapy of breast carcinoma.

BACKGROUND: This investigation describes the preclinical development of a laser fiberoptic interstitial delivery system for the thermal destruction of small breast cancers. We propose adaptation of this technology to stereotactic mammographic instrumentation currently employed for diagnostic core biopsy to thermally ablate a site of disease with maximal treatment efficacy, minimal observable superficial change, reduced patient trauma, and lowered overall treatment costs. STUDY DESIGN: Laser hyperthermia is a clinical modality that seeks to achieve tumor destruction through controlled tissue heating. The advantage of laser-induced hyperthermia over traditionally used heat sources such as ultrasound, microwave, or radiowave radiation lies in the ability to focus heat localization to the specific tumor tissue site. Neodymium:yttrium aluminum garnet (Nd:YAG) laser light transmitted through a fiberoptic cable to a diffusing quartz tip can induce such temperature increases leading to localized tissue destruction. Because breast cancer occurs with greatest frequency in the mature woman whose breast tissue has undergone glandular involution with fatty replacement, this study concentrates on determining the resultant laser energy heat distribution within fat and fibrofatty tissue. This investigation studied the time-temperature responses of ex vivo human breast and porcine fibrofatty tissue, which led to an in vivo subcutaneous porcine model for the practical demonstration of a laser hyperthermia treatment of small volumes of porcine mammary chain tissue. RESULTS: Spatial recordings of the resultant temperature fields through time exhibited similar, reproducible thermal profiles in both ex vivo human breast and subcutaneous porcine fat. In vivo laser-produced temperature fields in porcine subcutaneous fat were comparable to those in the ex vivo analyses, and showed a histologically, sharply defined, and controllable volume of necrosis with no injury to adjacent tissues or to overlying skin. CONCLUSIONS: Interstitially placed, fiberoptically delivered Nd:YAG laser energy is capable of controlled tissue denaturation to a defined volume for the treatment of small breast cancers. It is hoped that this minimally invasive approach, with further investigation and refinement, may lead to the effective treatment of small, well-defined breast cancers that are commonly diagnosed through stereographic mammography and stereotactic core biopsy. The juxtaposition of such a localized treatment modality with these increasingly used diagnostic tools is of considerable promise.

The effects of creatine on the retrogradely perfused isolated rat heart.

Although the role of creatine in muscle metabolism is well understood, there is still uncertainty as to its effects at supplemented levels. With this in mind, this study was designed to investigate the direct effects of commercially available creatine on the isolated rat heart, retrogradely perfused and infused with varying concentrations of creatine (1.25, 2.5, 5 and 10 mM) to determine its effects on heart rate, coronary flow and ventricular pressure. Furthermore, tissue from these hearts was used to investigate the cardiotoxic potential of supplemented levels of creatine. Results indicate that creatine directly improves the functioning of the heart under normal conditions with respect to heart rate and ventricular pressure, but may be detrimental to the functioning of energy-deprived hearts. It also showed no cardiotoxic properties since it increased the baseline levels of adenosine triphosphate (ATP) and decreased the levels of isocitrate dehydrogenase (lCD), indicating a decrease in cellular death compared with non-supplemented control hearts.

Cyclic dipeptides in the induction of maturation for cancer therapy.

Studies have suggested a possible form of therapy based on the use of maturation-inducing compounds to induce differentiation of neoplastic cells and stimulate faster recovery of the normal cell population. The study of the effects of nine cyclic dipeptides on biochemical markers of differentiation implicated their potential to induce differentiation. Studies were undertaken to determine the specificity of these agents for HT-29 cell cultures as well as the identification of the signal transduction pathways affected by these agents inducing the differential gene expression observed in the cells. The cyclic dipeptides studied showed a high degree of specificity, having no significant effect on Caco-2 cells (P > 0.05), representing the normal gastrointestinal mucosa. All inducers administered were shown to affect the total energy state of HT-29 cells, an effect which increased the probability of HT-29 cell differentiation. Results indicated that those agents which induced differential gene expression acted at different steps in the isolated signal transduction pathway. Cyclo(Trp-Trp) and cyclo(Phe-Pro) induced a high degree of acetylation of histones (P < 0.05), while the remaining cyclic dipeptides induced a high degree of phosphorylation of histones (P < 0.05) (cyclo(Trp-Trp) induced a moderate degree of histone phosphorylation). The results from histone phosphorylation and acetylation and cyclic AMP responsive element binding protein phosphorylation studies suggest that the cyclic dipeptides activate a chromatin switch, which leads to the increase in accessibility of lineage-specific genes for transcription.

The biological activity of selected cyclic dipeptides.

Cyclic dipeptides are widely used as models for larger peptides because of their simplicity and limited conformational freedom. Some cyclic dipeptides have been shown to be antiviral, antibiotic and anti-tumour. The aim of this study was to determine the biological activity of four cyclic dipeptides synthesized in this laboratory: cyclo(L-phenylalanyl-L-prolyl), cyclo(L-tyrosyl-L-prolyl), cyclo(L-tryptophanyl-L-prolyl) and cyclo(L-tryptophanyl-L-tryptophanyl). The enhancement or inhibition of calcium channels in ventricular myocytes from rats and delayed-rectifier potassium channels in ventricular myocytes from guinea-pigs were determined by use of the whole-cell patch-clamp technique. The induction of differentiation in HT-29 cells was assessed by assaying for an increase in the expression of alkaline phosphatase. Antibiotic properties against Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Bacillus subtilus and Streptococcus sp. were determined by use of the Kirby-Bauer disc-diffusion assay. Results from these assays indicate that the cyclic dipeptides have biological activity in both prokaryotes and eukaryotes. Three of the dipeptides block cation channels in ventricular myocytes and all increase the expression of alkaline phosphatase. All the dipeptides have concentration-dependent antibacterial properties. These results suggest that with increased solubility the cyclic dipeptides might have potential as muscle relaxants, anti-tumour compounds and antibiotics.

An investigation into the biological activity of the selected histidine-containing diketopiperazines cyclo(His-Phe) and cyclo(His-Tyr).

Although cyclic diketopiperazines have been known since the beginning of the century, only now have they attracted considerable interest with respect to their biological activity. The aim of this study was to determine if the diketopiperazines cyclo(L-histidyl-L-phenylalanyl) (cyclo(His-Phe)) and cyclo(L-histidyl-L-tyrosyl) (cyclo(His-Tyr)) have significant biological activity relevant to the treatment of cardiovascular-related disease states, cancer and infectious diseases. Haematological studies were performed, including thrombin substrate binding, blood clotting time, platelet adhesion, platelet aggregation and fibrinolysis assays. A cytotoxicity screening utilizing a tetrazolium-based assay on the cell lines HeLa, WHCO3, and MCF-7 was performed. The whole-cell patch-clamp technique was used to investigate ion-channel activity in ventricular myocytes of rats, and isolated rat heart studies were performed to investigate the cardiac effects involving heart rate and coronary flow rate. Cyclo(His-Tyr) produced a significant prolongation of blood clotting time, slowing of clot lysis and inhibition of ADP-induced platelet adhesion and aggregation (P < 0.05). Cyclo(His-Phe) showed significant (P < 0.05) anti-tumour activity, causing greatest reduction of cell viability in cervical carcinoma cells. Preliminary results from patch-clamp studies indicate that both diketopiperazines caused blocking of sodium and calcium ion channels, but opening of inward rectifying potassium ion channels. In the rat isolated heart studies, cyclo(His-Phe) caused a gradual reduction in heart rate (P = 0.0027) and a decrease in coronary flow rate (P = 0.0017). Cyclo(His-Tyr) significantly increased the heart rate (P = 0.0016) but did not cause any significant change of coronary flow rate (P > 0.05). Cyclo(His-Tyr) showed notable (P < 0.05) antibacterial activity and both diketopiperazines showed excellent antifungal activity (P < 0.05). These observations reveal diketopiperazines to be ideal lead compounds for the rational design of an agent capable of preventing metastasis, inhibiting tumour growth, and as potential chemotherapeutic, antiarrhythmic and antihypertensive agents, as well as potential antibacterial and antifungal agents.

Hepatotoxicity of the isomers of cyclo(Trp-Pro).

The hepatotoxicity of the novel cyclic dipeptide cyclo(Trp-Pro), which has shown potential usage in various pharmacological fields, had not been assessed. Further studies on the isomers of this cyclic dipeptide (cyclo(L-Trp-L-Pro), cyclo-(L-Trp-D-Pro), cyclo(D-Trp-L-Pro) and cyclo(D-Trp-D-Pro)) revealed further biological activities. The assessment of hepatotoxicity of these isomers was thus warranted. In vitro screens were performed on primary isolated rat hepatocytes, the Chang liver and N-2-alpha cell lines. In vivo screening involved the assessment of serum levels of lactate dehydrogenase, aspartate transaminase, ATP, Ca2+ and albumin after intraperitoneal injection over a 1 and 5 day period in the rat model. Liver samples were also obtained for the assessment of lipid peroxidation. It was found that only cyclo(D-Trp-L-Pro) was hepato-specific in its action, while the other isomers were not. The greatest effect on any biochemical or physiological parameter was noted after 5 days. LDH secretion was greatly increased in the presence of cyclo(L-Trp-L-Pro) and cyclo-(L-Trp-D-Pro) (p < 0.05). Significantly increased levels of lipid peroxidation were observed in all the isomer-treated samples (p < 0.05), while Ca2+ concentrations were decreased at day 5. Decreased protein synthesis was noted in the presence of all the isomers at day 1. These results indicate the potential harm involved in the administration of the isomers, which may limit their potential usage in the treatment of various diseases.

The influence of acetoacetate and butyrate on calcium influx and ATP concentrations in HT-29 cells.

The effects of acetoacetate and butyrate on Ca(2+)-influx in HT-29 cells were unknown. Extracellular signals can be transferred to the intracellular environment of the cell via changes in the Ca(2+)-concentration. Extracellular Ca2+ may enter the cell via Ca(2+)-channels in the plasma membrane. Physiological processes occurring within the cell are dependent on Ca(2+)-concentration, including enzyme activity. Intracellular Ca(2+)-concentrations were measured using Fura-2/AM, a fluorescent intracellular Ca(2+)-probe. Ca(2+)-concentrations were measured immediately on application of the inducers to the cells, as well as after a 9 day incubation period. The effect of these inducers on the L-type voltage-operated Ca(2+)-channels were determined using the whole-cell patch-clamp technique. To validate these results for the intestinal epithelial model, membrane current studies were performed on HT-29 cells grown on a polycarbonate membrane. ATP concentrations were measured, and the theoretical effect of the inducers on PDE 4 activity was determined. It was found that both acetoacetate and butyrate blocked Ca(2+)-influx through the L-type voltage-operated Ca(2+)-channels, resulting in the initial low Ca(2+)-concentration (p < 0.05). The blockage effect is short-lived as after a 9 day incubation period in the presence of the inducers, Ca(2+)-concentrations were higher than that of the HT-29 control sample (p < 0.05). ATP concentrations of the cells were decreased in the presence of the inducers (p < 0.05), whilst it was suggested that no interaction between the catalytic site of PDE 4 and the inducers existed.

In vitro anti-HIV activity of five selected South African medicinal plant extracts.

AIM OF THE STUDY: Five South African medicinal plants, Bulbine alooides (L.) Willd. (Asphodelaceae), Crinummacowani Baker (Amaryllidaceae), Hypoxis sobolifera var. sobolifera (Jacq.) Nel (Hypoxidaceae), Leonotisleonurus (L.) R.Br. (Lamiaceae) and Tulbaghiaviolacea Harv (Liliaceae) used for the treatment of various ailments, including infectious diseases, were screened for activity against human immunodeficiency virus (HIV). MATERIALS AND METHODS: Aqueous and ethanol extracts were tested for inhibitory activity in HIV-1 infected CEM.NK(R)-CCR5 cells, and against HIV-1 reverse transcriptase (RT) and HIV-1 protease (PR). RESULTS: In CEM.NK(R)-CCR5 cells, ethanol extracts of Leonotisleonurus inhibited HIV-1 significantly (33% reduction in HIV-1 p24, P<0.05). HIV-1 RT inhibition (> or =50%) was shown for extracts of Bulbine alooides (aqueous and ethanol), Hypoxis sobolifera (aqueous and ethanol) and Leonotisleonurus (aqueous), but inhibitory activity was lost upon dereplication for removal of non-specific tannins/polysaccharides. HIV-1 PR inhibition was observed for extracts of Hypoxis sobolifera (aqueous), Bulbine alooides (aqueous and ethanol) and Leonotisleonurus (ethanol). Only ethanolic extracts of Bulbine alooides and Leonotisleonurus retained HIV-1 PR inhibition after dereplication with IC50 of 94 microg/ml and 120 microg/ml, respectively. CONCLUSION: The dereplicated ethanolic extracts of Leonotisleonurus and Bulbine alooides showed the greatest anti-HIV potential in this study through inhibition of HIV-1 PR.

Development of stereotactically guided laser interstitial thermotherapy of breast cancer: in situ measurement and analysis of the temperature field in ex vivo and in vivo adipose tissue.

BACKGROUND AND OBJECTIVE: The size (0.5-1.0 cm) of early nonpalpable breast tumors currently detected by mammography and confirmed by stereotactic core biopsy is of the order of the penetration depth of near infrared photons in breast tissue. In principle, stereotactically biopsied tumors, therefore, could be safely and efficiently treated with laser thermotherapy. The aim of the current study is to confirm the controlled heating produced by clinically relevant power levels delivered with an interstitial laser fiber optic probe adapted for use with stereotactic mammography and biopsy procedures. STUDY DESIGN/MATERIALS AND METHODS: Temperature increases and the resultant thermal field produced by the irradiation of ex vivo (porcine and human) and in vivo (porcine) tissue models appropriate to the treatment of human breast tissue by using cw Nd:YAG laser radiation delivered with a interstitial fiber optic probe with a quartz diffusing tip, were recorded with an array of fifteen 23-gauge needle thermocouple probes connected to a laboratory computer-based data acquisition system. RESULTS: By using a stepwise decreasing power cycle to avoid tissue charring, acceptably symmetric thermal fields of repeatable volumetric dimensions were obtained. Reproducible thermal gradients and predictable tissue necrosis without carbonization could be induced in a 3-cm-diameter region around the fiber probe during a single treatment lasting only 3 minutes. The time-dependences of the temperature rise of the thermocouples surrounding the LITT probe were quantitatively modeled with simple linear functions during the applied laser heating cycles. CONCLUSION: Analysis of our experimental results show that reproducible, symmetric and predictable volumetric temperature increases in time can be reliably produced by interstitial laser thermotherapy.

In situ temperature measurements with thermocouple probes during laser interstitial thermotherapy (LITT): quantification and correction of a measurement artifact.

BACKGROUND AND OBJECTIVE: The purpose of this work was to quantify the magnitude of an artifact induced by stainless steel thermocouple probes in temperature measurements made in situ during experimental laser interstitial thermo-therapy (LITT). A procedure for correction of this observational error is outlined. STUDY DESIGN/MATERIALS AND METHODS: A CW Nd:YAG laser system emitting 20W for 25-30 s delivered through a fiber-optic probe was used to create localized heating. The temperature field around the fiber-optic probe during laser irradiation was measured every 0.3 s in air, water, 0.4% intralipid solution, and fatty cadaver pig tissue, with a field of up to fifteen needle thermocouple probes. RESULTS: Direct absorption of Nd:YAG laser radiation by the thermocouple probes induced an overestimation of the temperature, ranging from 1.8 degrees C to 118.6 degrees C in air, 2.2 degrees C to 9.9 degrees C in water, 0.7 C to 4.7 C in intralipid and 0.3 C to 17.9 C in porcine tissue after irradiation at 20W for 30 s and depending on the thermocouple location. The artifact in porcine tissue was removed by applying exponential and linear fits to the measured temperature curves. CONCLUSION: Light absorption by thermocouple probes can induce a significant artifact in the measurement of laser-induced temperature increases. When the time constant of the thermocouple effect is much smaller than the thermal relaxation time of the surrounding tissue, the artifact can be accurately quantified. During LITT experiments where temperature differences of a few degrees are significant, the thermocouple artifact must be removed in order to be able accurately to predict the treatment outcome.