A mutated cathepsin-D devoid of its catalytic activity stimulates the growth of cancer cells.

Cathepsin-D, a lysosomal aspartyl proteinase, is highly secreted by breast cancer cells and its over-expression by transfection stimulates cancer cell proliferation. The mechanism by which this protease affects proliferation remains, however, unknown. In order to determine whether proteolytic activity is necessary, we abolished its enzymatic activity using site-directed mutagenesis followed by stable transfection in 3Y1-Ad12 cancer cells. Substitution of the aspartic acid residue 231 by an asparagine residue in its catalytic site abrogated the cathepsin-D proteolytic activity but did not affect its expression level, processing or secretion. However, like wild-type cathepsin-D, this mutated catalytically-inactive cathepsin-D retained its capacity to stimulate proliferation of cells embedded in Matrigel or collagen I matrices, colony formation in soft agar and tumor growth in athymic nude mice. Addition on the mock-transfected cells, of either conditioned media containing the wild-type or the mutated pro-cathepsin-D, or of the purified mutated pro-cathepsin-D, partially mimicked the mitogenic activity of the transfected cathepsin-D, indicating a role of the secreted pro-enzyme. Moreover, addition of two anti-cathepsin-D antibodies on the cathepsin-D transfected cells inhibited their proliferation, suggesting an action of the secreted pro-cathepsin-D via an autocrine loop. A synthetic peptide containing the 27-44 residue moiety of the cathepsin-D pro-fragment was, however, not mitogenic suggesting that a receptor for the pro-fragment was not involved. Furthermore, the cathepsin-D mitogenicity was not blocked by inhibiting the interaction of pro-cathepsin-D with the mannose-6-phosphate receptors. Our results altogether demonstrate that a mutated cathepsin-D devoid of catalytic activity is still mitogenic and suggest that it is acting extra-cellularly by triggering directly or indirectly a yet unidentified cell surface receptor.

Switchability of neoral and equoral according to Food and Drug Administration rules and regulations.

According to the US Food and Drug Administration (FDA), if a drug product contains a drug substance that is chemically identical and is delivered to the site of action at the same rate and extent as another drug product, then it is equivalent and can be substituted (switchable) for that drug product. Methods used to define bioequivalence as stated by the FDA rules (FDA 21 CFR 320, 24) are (1) pharmacokinetic (PK) studies in healthy volunteers, (2) comparative clinical trials, and (3) pharmacodynamic (PD) studies (bioactivity). We evaluated the switchability of Equoral (IVAX-USA) with Neoral (Novartis Switzerland using all FDA rules. In a single oral dose, we undertook a comparative bioavailability study of Equoral (IVAX, USA) Neoral (Novartis, USA), and Neoral (Novartis UK). The pharmacokinetics of Equoral and Neoral were determined with blood levels at 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 10, 12, 16, 24, 30, 36, 42, and 48 hours. The area under curve (AUC), AUC extrapolated to infinity (AUC0-inf), rate of absorption (Tmax), extent of absorption (Cmax), half time (t1/2) of Equoral and Neoral were all within the 90% confidence interval of 80% to 125% boundaries. A comparative multinational multicenter clinical trial in stable renal transplant patients included 70 patients (22 women and 48 men) of mean age of 33 years (range, 26 to 43) was performed in Turkey, Lebanon, and Pakistan. In this study the ratios of LSM and the 90% confidence intervals for the Nontransformed/Parameters (AUC0-t, AUCinf, Tmax, and Cmax) of Equoral and Neoral SGC were 98% and 95%, respectively, which are within the 80% to 125% FDA acceptance range. For immunosuppressive drugs, the site of action is the lymphocyte and the measurable response is the decrease in lymphocyte count caused by the relative concentration of the drug in the lymphocyte. In a controlled switch, fixed-dose study, both Equoral and Neoral achieved the same concentration in the lymphocytes and caused the same degree of lymphocyte count reduction. The results of the testing (bioavailability-bioequivalence, clinical studies, and pharmacodynamic-bioactivity) required by FDA for interchangeability ("switchability") of immunosuppressive agents suggests that Neoral and Equoral are switchable.

Biosynthesis of destruxins.

L-[Me-13C]-Methionine, sodium [2-13C]acetate and sodium [1,2-13C]acetate were incorporated into destruxins A, B and E by the entomopathogenic fungus Metarhizium anisopliae. NMR analysis established that methionine is involved in the N-methylation of L-valine and L-alanine, and that one intact acetate unit participates in the biosynthesis of the -CH(OH)-COOH fragment of the hydroxy acid moiety. The presence of two acetate units in proline and isoleucine agrees with the accepted biochemical schemes.

Destruxin Ed1 a cyclopeptide from the fungus Metarhizium anisopliae.

A new destruxin Ed1 has been isolated from the entomopathogenic fungus Metarhizium anisopliae. Its structure was deduced from the NMR and mass spectral data.

Beauverolides L and La from Beauveria tenella and Paecilomyces fumosoroseus.

New beauverolides L and La were isolated and identified from the entomopathogenic fungi, Beauveria tenella and Paecilomyces fumosoroseus. Their structures, cyclo-[3-hydroxy-4-methyldecanoyl-L-phenylalanyl-L-alanyl-D-leucyl ], and cyclo-[3-hydroxy-4-methyldecanoyl-L-phenylalanyl-L-alanyl-D-allo-i soleucyl] were deduced from HPLC and GC-mass spectrometric analyses of their hydrolysates and NMR and mass spectral data.

Cyclosporins from Tolypocladium terricola.

New natural cyclosporins were isolated from the mycelium of surface cultivated fungus Tolypocladium terricola. The chemical structures of [Leu4] CS and [MeLeu1] CS = cyclosporin-J, were deduced from the NMR and mass spectral data. Biological activity of new cyclosporins is reported based on the proliferative mitogen stimulation test.

Detection of a P-glycoprotein related pump in Chironomus larvae and its inhibition by verapamil and cyclosporin A.

A membrane associated ATP-dependent efflux pump, similar in function to mammalian P-glycoprotein, was detected in anal papillae of Chironomus riparius larvae. Immunohistochemical analysis of larval tissues, using monoclonal antibodies against P-glycoprotein, was supplemented by functional in vivo and in vitro assays which confirmed the existence of a mechanism for transporting xenobiotic substances. The in vitro ATPase activity of homogenate fractions increased in the presence of typical P-glycoprotein substrates (vinblastine, actinomycin D or ivermectin). This increase was unaffected by inhibitors of other membrane ATPases (sodium azide, EGTA, ouabain), but sensitive to vanadate, cyclosporin A and verapamil which inhibit mammalian P-glycoprotein mediated ATP-consumption. Sublethal concentrations of specific P-glycoprotein-inhibitors such as verapamil or cyclosporin A synergistically enhanced the mortality of C. riparius towards ivermectin. Although cyclosporin A originates from entomopathogenic fungi, its mode of action in insects and its function during infection are not understood. Our results lend some credit to the hypothesis that this compound is possibly released to promote poisoning of the infected host by xenobiotics which are normally removed by a P-glycoprotein related pump. The putative role of insect P-glycoprotein homologues in the context of multiple resistance towards insecticides in discussed.

Morphological alterations accompanying the effect of peptaibiotics, alpha-aminoisobutyric acid-rich secondary metabolites of filamentous fungi, on Culex pipiens larvae.

The effect of different representatives of the group of peptaibiotics, alpha-amino-isobutyric acid rich secondary metabolites of filamentous fungi, on Culex pipiens larvae was studied. Light and transmission electron microscopy techniques were used to localize the intracellular damage and to determine the target organells for the mode of action of peptaibols in mosquito larvae. Though different in insecticidal activity, all tested compounds induced the same type of tissue damage, which was characterized by heavy challenge of mitochondria followed by partial swelling, crystaeolysis and destruction of mitochondrial walls. It is concluded that the mode of action of peptaibols in mosquito larvae is mediated through the damage of mitochondria. The structure-mosquitocidal effect of these compounds, their potential mode of action and role in the natural fungal entomopathogenic process are briefly discussed.

The role of generics in transplantation: TM-MMF versus Cellcept in healthy volunteers.

Because several studies having revealed a relation between early graft rejection and long-term graft survival, potential benefits have been attributed to MMF. The cost of the drug is, however, prohibitive, which renders its long-term use in countries with limited income. We thus compared the pharmacokinetic profiles of a new MMF generic formulation (MM-Cept developed by Ivax CR) with those of Cellcept (Hoffman La Roche) in healthy volunteers. This open label, balanced randomized, two-treatment, two-period, two-sequence, single-dose, crossover, comparative oral bioavailability study was conducted in non-smoking adult male healthy volunteers between the ages of 18 and 45 years. The study was performed in accordance with the basic principles defined in the US 21 CFR Part 312.20 and the principles enunciated in the Declaration of Helsinki (World Medical Association Declaration of Helsinki). The subjects were given a single oral 1 g dose with a washout period of 10 days. Pharmacokinetic profiles included blood levels at 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 4, 6, 10, 12, 16, 20, 24, 30, 36, and 48 hours following each dose. The formulations were MMCept 500 mg tablets and Cellcept 500 mg tablets. Subjects were fasted overnight and for 4 hours postdosing. Mycophenolic acid (MPA) concentrations were determined using HPLC. Physical examinations, hematology, urinalysis, and serum chemistry tests including liver enzymes were performed at screening and at the end of the study. Subjects were monitored for safety and adverse events throughout the study. Both products showed similar bioavailability. The LSM were within the limits for FDA approval (80 to 125), suggesting that the two products are equivalent and switchable.

The pharmacokinetics of equoral versus neoral in stable renal transplant patients: a multinational multicenter study.

We studied the pharmacokinetics (PKs) of the new generic cyclosporine formulation, Equoral capsules, after the switch from original formulation Neoral capsules in stable renal transplant patients. The study was carried out in accordance with the basic principles defined in the US 21 CFR Part 312.20 and the principles of the Declaration of Helsinki. The study included clinically stable first renal transplant patients maintained on cyclosporine with no rejection episode during the past 6 months. Hematology, biochemistry, and urine chemistry were determined on day 7, and day 21. The patients were all switched to Neoral (lot number 416MFD0601) on day 0 when the first sparse sampling PK was performed. On day 14 a 12-hour PK profile included predose, 30 minutes; 1 hour; 1 hour 30 minutes; 2 hours; 3 hours; 4 hours; 5 hours; 6 hours; 8 hours; 10-hours and 12-hour samples. Cyclosporine levels were determined using a CYA kit (Abbott TDx). On day 15 the patients were switched from Neoral capsules to Equoral capsules (lot 5T111014) at an equivalent dosage (mg/mg). The second sparse sampling PK was performed on day 21 and a 12-hour PK was performed on day 28. On the morning of day 29 patients were switched from Equoral capsules to Neoral capsules at an equivalent dosage (mg/mg). Additional concentrations were measured on days -7, 18, and 35. Safety parameters were monitored at each visit. The pharmacokinetics of both formulations were equivalent. The mean AUC for Neoral and Equoral was 2856 and 2892, respectively. The ratios of LSM and the 90% confidence intervals for the in-transformed parameters (AUC o-t, AUC inf, and Cmax) of Equoral and Neoral SGC were 98% and 95%, respectively, suggesting that Equoral and Neoral SGC are bioequivalent.

Effects of destruxins, cyclic depsipeptide mycotoxins, on calcium balance and phosphorylation of intracellular proteins in lepidopteran cell lines.

The influence of the destruxin E, cyclodepsipeptidic mycotoxin produced by the filamentous entomopathogenic fungus Metarhizium anisopliae, on calcium fluxes and protein phosphorylation of in vitro cultivated lepidopteran cell lines have been studied. The use of the fluorescent calcium indicator Fura 2 AM did not show a fast increase in the level of cytosolic calcium in lepidopteran and human cell lines treated with dtx E. In contrast, 45Ca+2 assays detected a late but significant calcium influx in insect cells exposed to dtx E and A for at least 30 min. This effect was not inhibited by calcium channel blockers with the exception of nifedipine 25 microM. A viral treatment potentiated the effect of dtx E on calcium balance. Dtx E also induced a strong, fast (10 min), transient phosphorylation of high molecular weight (250 kDa) cellular proteins. The activation of phosphorylation was only partially inhibited by EDTA. This study provides the first direct evidence of dtx E influence on calcium fluxes and protein phosphorylation.

Effects of beauverolide L and cyclosporin A on humoral and cellular immune response of the greater wax moth, Galleria mellonella.

The effects of beauverolide L and cyclosporin A, cyclic peptidic metabolites, produced by several genera of entomopathogenic fungi on immune responses of last instar larvae of the greater wax moth Galleria mellonella have been examined. Intrahemocoelic injection of either metabolite-coated silica particles or dissolved metabolites in a concentrations ranging between 10 and 30 micrograms per larvae caused no mortality but activated humoral responses in G. mellonella larvae. The challenge induced a significant release of lysozyme and cecropin-like activity into the hemolymph, suggesting stimulatory activity on humoral immune responses. Injected metabolite-coated particles were rapidly surrounded by hemocytes which subsequently accomplished formation of melanized nodules, which increased in size and number compared with controls. In vitro assays with dissolved metabolites indicated no adverse effects of beauverolide L or cyclosporin A on attachment or spreading of isolated plasmatocytes but dose-dependent inhibition of their phagocytic activity. Isolated plasmatocytes incubated with cyclosporin A or beauverolide L exhibited cytoskeleton alterations that differed from those observed in plasmatocytes from infected G. mellonella larvae or reported from other fungal secondary metabolites. The experiments provided further data to elucidate the role of fungal secondary metabolites in development of mycoses in insects.

Detection of antigens common to salivary glands and other tissues of tsetse fly, Glossina palpalis palpalis (Diptera: Glossinidae).

The study demonstrates the common antigens to salivary gland, fat body, mesenteron, thorax muscle, native whole body, and dried whole body homogenates of tsetse flies, G. palpalis palpalis. The possibilities of their origin and the role in hypersensitivity induction and its propagation are discussed.

The effect of tolypin in Tolypocladium niveum crude extract against mosquito and blackfly larvae in the laboratory.

The efficacy of tolypin against mosquito and blackfly larvae was studied under laboratory conditions. It was tested against Culex molestus, Aedes aegypti, Anopheles maculipennis, Simulium noelleri and Odagmia ornata. A concentration 0.1 mg/ml caused 100% mortality in all species tested and a concentration 0.001 mg/ml caused 100% mortality only in the two species of blackflies used within 24 hours.

Passive transfer of humoral resistance against adults of the tsetse fly, Glossina palpalis palpalis (Diptera, Glossinidae), in rabbits.

The possibility of passive transfer of rabbit humoral immunity against tsetse fly bites was investigated for the first time. Partial immunity of recipient animals was achieved after two intravenous injections of 15 ml of serum from immunized (donor) rabbits during 48 hrs. This treatment induced an apparent increase of resistance in the passively immunized group of rabbits expressed as direct mortality ("killing effect") of sucking flies within the following 72 hr period. The immunological state of immune sera of both donors and recipients was examined by ELISA, using the water-soluble proteins of tsetse salivary glands as antigen. No direct correlation between the titre of antibodies and the killing of Glossina was detected. These results indicate that these antibodies were not the only humoral factor responsible for tsetse mortality since their titre did not substantially change in the course of 7 days while the "killing effect" had disappeared from the recipient's blood within 72 hrs.

Antigenic characterization of rat louse Polyplax spinulosa.

Number and relative molecular weights of proteins in whole body homogenates of the louse, Polyplax spinulosa were determined using SDS-PAGE. 13 protein components from the total of 28 bands were defined as Con A binding glycoproteins. In immunoblotting sera of lice infested laboratory rats of BN strain specifically recognized 3 major protein components with r.m.w. of 77,000, 105,000 and 230,000 Da and from 6 to 8 minor protein components with r.m.w. of 31,000-180,000 Da.

Tolypocladium terricola sp. N., a new mosquito-killing species of the genus Tolypocladium Gams (Hyphomycetes).

A new entomopathogenic species of the genus Tolypocladium, T. terricola is described from a soil sample from Finalnd. From other known Tolypocladium species, T. terricola differs in morphology, production of secondary metabolites and possession of relatively strong mosquitocidal activity. The fungus is characterized by broad oval conidia (2.5 x 2 microns) with one pointed end which are produced in grape-like clusters, and are not firmly adherent. When treated with T. terricola, mosquito larvae show typical features of intoxication characterized by the concentration of larvae in the centre of cup, hanging by their siphons on the surface.

The efficacy of Bacillus thuringiensis var. israelensis against larvae of the blackfly Odagmia ornata (Meig.) (Simuliidae) at low temperatures.

The effect of the suspension of Bacillus thuringiensis var. israelensis spores on larvae of the blackfly Odagmia ornata was studied in the laboratory and under field conditions of a natural biotope in southern Bohemia. The preparation Moskitur was used and its effect was tested in laboratory at temperatures 0.1-2.9 degrees C and 17-19 degrees C. Although O. ornata larvae were able to filter feed on a lethal dose of the preparation even at a lower temperature than 2.9 degrees C, no marked manifestation of mortality was observed at low temperatures in comparison with a control sample.

Antibody-mediated response of pigeons to Argas polonicus larval feeding and characterization of larval antigen.

Circulating antibodies to larval Argas polonicus antigen detected in the blood of pigeons by means of ELISA reach their highest level 3-6 days post-tick attachment. During 6-8 days post infestation when most larvae detach from their host, there is an abrupt drop of the antibody level in blood followed by second peak at day 10-15. During the secondary and subsequent infestations the dynamics of the antibody production is analogous, but the maximum absorbance values found are higher with each following infestation. This is in direct correlation with the growth of immune resistance of hosts. The transfer of immunoglobulins of resistant pigeons produces in naive hosts a partial resistance in a statistically significant (P less than 0.01) reduction of the number of engorged larvae, in the shortening of larval feeding period and in the decrease of their mean weight after feeding. However this resistance was significantly (P less than 0.01) less expressed than in naturally resistant pigeons during secondary infestation. The protracted effect on the duration of premoulting period and the percentage of moulted larvae manifested in larvae after secondary infestation was not apparent in molecular weight of approximately 19, 21, 23, 27, 45 and 165 kilodaltons, were recognized by serum of resistant pigeons.

Prograf five milligrams versus Tacrolimus medis in healthy volunteers: a bioequivalence study.

For FDA approval, bioequivalence of a generic version of Tacrolimus must be demonstrated in a randomized, two-treatments, two-periods, two-sequences, single-dose crossover study in healthy adult volunteers. Currently there are at least 3 differents generic equivalent for Tacrolimus, that are approved by the EMA and the FDA, with a USA market share of nearly 50%. However, the market share of generic immunosuppressive drugs in the Middle East region is still very low due to the reluctance of the physician to accept Tacrolimus generics, considered to be a narrow therapeutic window drug, that are approved using the standard bioequivalence criteria of 80% to 125%. Herein we present a bioequivalence study of a new Tacrolimus generic, Tacrolimus Medis 5 mg developed by Medis Tunisia batch number 12G3003 compared with Prograf® 5 mg batch number 7202 manufactured by Astellas Toyama Co., Ltd. Japan and HIKMA Pharmaceuticals, Amman-Jordan in healthy adult volunteers using the 90%-111% criteria recommended for drugs with narrow therapeutic window. The study was, balanced, randomized, two-treatments, two-periods, two-sequences, single dose, crossover, comparative oral bioavailability study in healthy adult human volunteers. The study was carried out in accordance with the Basic Principles defined in the U.S. 21 CFR Part 312.20, the principles enunciated in the Declaration of Helsinki (World Medical Association Declaration of Helsinki). Thirty six non-smoking healthy, as determined by medical history, volunteers, 18 years and older, were included. Following randomization using a computer software (pharma solution) the volunteers were given a single oral dose of 5 milligrams following a 12 hour fast with a wash out period of 7 days. Pharmacokinetics profile with blood levels at: 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, and 24 hours were performed following each dose. Tacrolimus plasma level was determined using an HPLC validated method (Transmedical For Life S.A.R.L. Beirut Lebanon), for accuracy, suitability, reproducibility, precision , long-term stability and robustness. Physical examinations, hematology, urine analysis and serum chemistry tests were performed at screening and before dosing in each period and at end of the study. Volunteers were monitored for safety and adverse events throughout the study. Both products were bioequivalent at the entire pharmacokinetic parameters tested. The LSM were 95.31%-101.21% for AUC, 94.65%-101.11% for AUC0-inf, 97.15%-100.02% for Cmax and 91.54%-103.75% for Half-life. Respectively all of which are within the EU and FDA approval limits (90-111%) indicating that the 2 products are equivalent and switchable.