Pharmacie sans ordonnance livraison rapide 24h: acheter viagra en ligne en France.

No job name

Bioconjugate Chem. 2002, 13, 518−524
Methotrexate Conjugate with Branched Polypeptide Influences
Leishmania donovani
Infection in Vitro and in Experimental
Animals†

Gyo¨rgy Ko´cza´n,‡ Asoke C. Ghose,§ Ananda Mookerjee,§ and Ferenc Hudecz*,‡ Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eo¨tvo¨s L. University,Budapest 112, POB 32, Hungary, H-1518 and Department of Microbiology, Bose Institute, P-1/12 C. I. T.
Scheme 7M, Calcutta, 700054, India. Received July 18, 2001 Methotrexate (MTX) has been coupled to various structurally related, polycationic (poly[Lys(DL-Alam)](AK), poly[Lys(Seri-DL-Alam)] (SAK), poly[Lys(DL-Alam-Leui)] (ALK)), or amphoteric (poly[Lys(Glui-DL-Alam)] (EAK)) synthetic branched polypeptides containing poly[L-Lys] backbone by the aid of BOPreagent. The average degree of MTX incorporation was found to be dependent on the charge propertiesof the polymer. Under the experimental conditions used, the molar substitution ratio achieved washigher for polycations (25%) than for the amphoteric polypeptide (10%). We have studied the effect ofpolycationic polypeptides on Leishmania donovani infection. Results demonstrated that MTXconjugates in which the drug is covalently attached to carrier have pronounced leishmanicid activity.
In this communication we showed that (a) a branched polypeptide-methotrexate conjugate with apolycationic carrier (ALK) increases the effect of MTX against Leishmania donovani infection in mice;(b) the covalent bond between the carrier and methotrexate is essential for both in vivo and in vitroactivity; and (c) the number of Leishmania donovani parasites in infected macrophages are markedlyreduced in conjugate treated animals. In vitro observation might also indicate that the MTX conjugateexhibits an effect through an uptake by macrophages which is different from that of the free drug.
glucosylated-BSA (7, 8), mannosylated-HSA (9), or ma-leylated-BSA (10), for delivery into cells containing L. Methotrexate (MTX,1 L-4-amino-N10-methylpteroyl- donovani parasites or to polylysine for studying the glutamic acid), a folate antimetabolite, has been in mechanism of action of cellular uptake by pinocytosis in clinical use for more than 35 years. It is a potent mice and human tumor cell lines (11-14).
anticancer agent of proven benefit in the treatment ofacute leukemia, osteogenic sarcoma (1), and of rheuma- The methods of conjugation of methotrexate to these tological disorders (2-4). Recently its inhibitory potential carriers have involved predominantly the glutamic acid has been demonstrated against a group of intracellular moiety. Activation of R- and γ-carboxyl groups has been parasites (Leishmania) of macrophages (5). Visceral achieved in situ in the presence of the carrier using water leishmaniasis or kala-azar in man is caused by the soluble carbodiimide like EDC (15) or by ester formation protozoan parasite Leishmania donovani, which prolifer- with N-hydroxysuccinimide (11). (None of the above ates intracellularly within the mononuclear phagocytes methods reported have been used for selective derivati- zation of R- or γ-carboxyl groups of MTX (15).) Methotrexate has been linked to various macromol- MTX was also one of the first antitumor drug attached ecules such as BSA, mannosylated-, galactosylated-, or covalently to high molecular weight carriers to improvethe therapeutic index by site-specific targeting and/or bychanging the pharmacological properties of MTX to * To whom correspondence should be addressed: Dr. Ferenc provide controlled release. It has been demonstrated that Hudecz, Research Group of Peptide Chemistry, Hungarian the biodistribution of branched polypeptide attached Academy of Sciences, Pa´zma´ny P. se´ta´ny 1A, Budapest, Hun- drugs (e.g., MTX, daunomycin, amylorid) can be strongly gary, H-1117, Fax: 36 1 372 2620, Tel: 36 1 372 2828, e-mail: modified by the charge and side chain structure of the † A preliminary account of this work was read at 10th carrier resulting in elevated and more prolonged blood International Congress of Immunology, New-Delhi, India, 1998.
levels, slower drug excretion, and a longer half-life (16, For the investigation of the conjugate’s leishmanicid 1 Abbreviations (in order of appearance in the text): MTX, activity we have prepared a new set of methotrexate- methotrexate; EAK, poly[Lys(Glui-DL-Alam)]; SAK, poly[Lys(Seri- branched polypeptide conjugates (18) (Figure 1). In these DL-Alam)]; ALK, poly[Lys(DL-Alam-Leui)]; AK, poly[Lys(DL-Alam)]; compounds MTX molecules are attached to polycationic DP , number average of the degree of polymerization; PBS, poly[Lys(DL-Alam)] (AK), poly[Lys(Seri-DL-Alam)] (SAK), phosphate-buffered saline, pH 7.3; DS, average degree of poly[Lys(DL-Alam-Leui)] (ALK) or amphoteric poly[Lys- substitution; MTX-SAK, poly[Lys(MTXj-Seri-DL-Alam)]; MTX- (Glui-DL-Alam)] (EAK) polypeptide carrier by amide bonds.
ALK, poly[Lys(MTXj-DL-Alam-Leui)]; MTX-AK, poly[Lys(MTXj- Branched polypeptides with the general formula poly- DL-Alam)]; MTX-EAK, poly[Lys(MTXj-Glui-DL-Alam)]; BOP ben- zotriazol-1-yl-oxytris(dimethylamino)phosphoniumhexafluorophosphate; i-DL-Alam)] (XAK) or poly[Lys(DL-Alam-Xi)] (AXK), DIEA, N,N-diisopropylethylamine; DMF, dimethylformamide; where i < 1 and m ∼ 3, have been used as biodegradable DMSO, dimethyl sulfoxide; HOBt, 1-hydroxybenzotriazole.
(19) macromolecular carriers for targeting antitumor Antileishmanial MTX-Branched Polypeptide Conjugate Bioconjugate Chem., Vol. 13, No. 3, 2002 519
Table 1. Characteristics of Branched Polypeptides
poly[Lys(DL-Alam -Leui)] ALK a Code of branched polypeptides is based on one letter symbol of amino acids. b Determined by amino acid analysis after hy-drolysis in 6 M HCl at 105 °C for 24 h. c Calculated from thenumber average degree of polymerization of polylysine (DP )60 ( 2 or DP ) 90 ( 2*) and from the amino acid composition of laboratories as described earlier (19-21). The character-istics of these compounds are summarized in Table 1.
Polylysine with DP ) 60 ( 2 or DP )90 ( 2 (EAK) and M /M )1.65 ( 0.15 was used.
Animals. Inbred mice (BALB/c strain) were obtained
from the National Institute of Nutrition, Hyderabad,India. Female mice, 6-8 weeks old and of 20-25 g bodyweight were used in these experiments.
Parasites. The Leishmania donovani strain (BI2302)
was isolated from the bone marrow material of a kala-azar patient. The strain was maintained in vivo throughserial passages in hamsters (35).
Coupling of Methotrexate to Branched Polypep-
tides. Coupling was performed with BOP in the presence
of 1-hydroxybenzotriazole (HOBt) and DIEA in water-
DMF (1:9, v/v) mixture. Briefly: Branched polypeptide
ALK (10 mg, 0.24 µmol) was dissolved in 0.5 mL of
Figure 1. Schematic representation of branched polypeptide-
deionized water, and the clean and viscous solution was MTX conjugates: poly[Lys(MTXj-DL-Alam-Leui)] (MTX-ALK).
diluted with 4.5 mL of DMF (c ) 2 mg/mL). Driedmethotrexate (5.62 mg, 11.0 µmol) (0.5 mol calculated for agents (16-25), radioligands (26, 27), immunomodulatory amino group of side chains of the polymer), 4.88 mg (11.0 peptides (28), or constructing synthetic antigens with T µmol) of BOP reagent, 1.7 mg (11.0 µmol) of HOBt, and or B cell epitopes (29-31).
2.75 µL (22 µmol) of N,N-diisopropylethylamine (DIEA) In this paper we describe the improved synthesis of were dissolved in 2 mL of DMF and the reaction MTX conjugates with selected polycationic and ampho- proceeded for 10 min at 4 °C. The solution with preac- teric polypeptides and the results of comparative studies tivated MTX was added to the solution of branched against Leishmania donovani infection using these com- polypeptide. The mixture was stirred at room tempera- pounds. These conjugates have been tested and compared ture and allowed to react for 12 h at RT in the dark. The with that of the free drug and of free carrier in the control final concentration of polypeptide was 1.53 mg/mL. The of L. donovani infection in BALB/c mice. We have also input molar ratios of MTX to polypeptide were 0.1:1, 0.3: investigated the mechanism of leishmanicid activity in 1, and 0.5:1. Since the relative amounts of R- and mouse macrophages in vitro following their treatment γ-coupled material are unknown, the error introduced by with MTX, MTX + branched polypeptide mixture, and assuming no effect of coupling, although probably small, MTX-branched polypeptide conjugates.
is not known accurately. After conjugation, the solventwas removed in vacuo, and the resulting oil was tritu- rated with 5 mL of diethyl ether three times. The product Abbreviations used in this paper follow the rules of the was dissolved in 1 mL of glacial acetic acid and diluted IUPAC/IUB Commission of Biochemical Nomenclature with 3 mL of water. The solution was placed in Visking (32) in accord with the recommended nomenclature of tubing (molecular mass cutoff 12000-14000 Da, Fisons, Loughborough, UK) and dialyzed extensively against Materials. Methotrexate was obtained from Lederle
1.0% acetic acid for 48 h. The average degree of molar Laboratories (Gosport, U.K.); N,N-diisopropylethylamine substitution (DS) was estimated by amino acid analysis (DIEA), 1-hydroxybenzotriazole (HOBt), and trifluoro- and spectrophotometrically at λ ) 373 nm assuming that acetic acid (TFA) were Fluka products (Buchs, Switzer- the extinction coefficient of methotrexate at λ ) 373 nm land), while benzotriazol-1-yloxytris(dimethylamino)- is 7800 M-1 cm-1. Analytical data on the conjugates are phosphonium hexafluorophosphate (BOP) was purchased from Bachem (Bubendorf, Switzerland). Hydroxylamin, Analysis of Ester Bound Methotrexate to Poly-
glacial acetic acid, diethyl ether, and dimethylformamide [Lys(Seri-DL-Alam)]. Ser-containing conjugate poly[Lys-
(DMF) were purchased from REANAL (Budapest, Hun- (MTXj-Seri-DL-Alam)] (MTX-SAK) was treated with 1.1 M gary). DMF was stored over molecular sieve. Branched hydroxylamine (0.1 M final concentration) in 0.1 M polypeptides used in these studies were prepared in our carbonate buffer (pH 9.0) at 37 °C overnight, similarly 520 Bioconjugate Chem., Vol. 13, No. 3, 2002
Table 2. Chemical Characterization of MTX-Branched
with five doses of MTX covalently coupled to different Polypeptide Conjugates
branched polypeptides (poly[Lys(MTXj-DL-Alam)] (MTX- AK), MTX-SAK and poly[Lys(MTXj-DL-Alam-Leui)] (MTX- ALK)) so that the administered compounds contained equivalent amounts of MTX (100 µg/injection/animal).
poly[Lys(MTXj-Seri-DL-Alam)] Whenever required, groups of infected mice were also poly[Lys(MTXj-Glui-DL-Alam)] treated with unconjugated branched polypeptides or with poly[Lys(MTXj -DL-Alam-Leui)] MTX-ALK a mixture of MTX and branched polypeptides using a a Code of conjugates is composed of the abbreviation of branched similar protocol. The control group of infected mice polypeptide and of methotrexate attached. b Average degree of received only normal saline (0.9% NaCl) injections. All substitution determined from the amino acid analysis after mice (treated or control) were sacrificed on day 28, and hydrolysis in 6 M HCl at 105 °C for 24 h. c Calculated from the parasitemia in their liver was determined microscopically number average degree of polymerization of polylysine (DP ( 2 or DP ) 90 ( 2*) and from the amino acid composition of In Vitro Treatment of L. donovani Infected Mouse
Macrophages with MTX Alone or in Combination
to the method of Endo et al. (34) with some modifications with Branched Polypeptides. Antileishmanial activi-
(39). The samples were run on RP-HPLC as described ties of MTX or branched polypeptide ALK or their combinations were determined in vitro by using mouse RP-HPLC Analysis of MTX-Branched Polypep-
macrophages infected with leishmania parasites (37). For tide Conjugates. The HPLC system consisted of one
this, peritoneal exudate cells were collected from mice Model 600 ternary gradient pump and controller, a Model pretreated with 2% (w/v) thioglycolate solution. Washed 490E programmable multi-wavelength UV-visible detec- cells were resuspended in RPMI-1640 medium containing tor, a Model 717 autosampler, and an in-line degasser 10% heat inactivated fetal calf serum (RPMI-FCS) and (all from Waters, Milford, MA). All injections were made overlaid on glass cover slips taken in 35 mm diameter by the autosampler. Data were recorded and processed plastic Petri dishes. Dishes were kept at 37 °C in air with Millenium manager software. A 15 cm × 4.6 mm containing 5% CO2 for 4 h to allow attachment of cells.
column with a spherical 5 µm silica (300 Å pore size) with Following washing, cells remaining attached to the cover a C18 hydrophobic bonded phase were used (Ultrasphere, slips were further incubated overnight with RPMI-FCS.
Beckman, CA). The mobile phase consisted of 0.1% (v/v) Next, L. donovani promastigotes freshly harvested from TFA in HPLC grade water (eluent A), and 0.1% (v/v) TFA a log phase culture of the parasite grown in a liquid in 80% (v/v) acetonitrile-water mixture (eluent B).
culture medium (38) were added to the Petri dishes in a Gradient elution was used at 1 mL/min flow rate; the B cell-to-parasite ratio of about 1:10. After incubation for content of the eluent was increased from 5% to 60% 6 h at 37 °C, free parasites were removed by washing between 5 and 35 min. All samples were dissolved in and the Petri dishes were incubated with RPMI-FCS distilled water and were filtered before analysis using containing appropriate doses of MTX (25 µg/mL of 0.45 µm Spartan 13 (Schleicher and Schuell, Dassel, incubation medium) or ALK (108 µg/mL) or their mixture Germany) filters. UV absorbance was monitored at λ ) or poly[Lys(MTXj-DL-Alam-Leui)] (MTX-ALK) conjugate.
373 nm for MTX containing samples and at λ ) 214 nm The control dish (with parasitized macrophages in cover for free polypeptide samples. The injected volume was slips) contained neither drug nor conjugates. Following selected so that the absorbance value at the peak incubation of the dishes for a period of 72 h, cover slips maximum was below 1.0 absorbance unit. Quantitative containing parasitized macrophages were taken out, analysis of conjugates was performed using peak-area fixed, stained with Giemsa, and examined microscopically measurement calibrated with appropriate standards. All to determine the number of intracellular parasites per analyses were carried out at ambient temperature.
macrophage. At least, 100 macrophages were screened Amino Acid Analysis. The amino acid composition
of branched polypeptides and their MTX conjugates wasdetermined by amino acid analysis using a Beckman (Fullerton, CA) model 6300 amino acid analyzer. Prior Synthesis and Chemical Characterization of MTX-
to the analysis, samples were hydrolyzed in 6 M HCl in Branched Polypeptide Conjugates. The coupling of
sealed and evacuated tubes at 110 °C for 24 h.
MTX to branched polypeptide was achieved by the BOP In Vivo Treatment of L. donovani Infected BALB/c
reagent-based activation method in which one carboxyl Mice with MTX Alone or in Combination with MTX
group of the molecule was linked to the R amino group Conjugates. Groups of four to five mice were infected
of the side chains of branched polypeptide AK, SAK, ALK, with L. donovani amastigotes (about 2 × 107 parasites/ or EAK to provide covalent R-amide type bonding (Figure 0.1 mL/animal) via the intracardial (ic) route. Different 1). The carboxyl groups of MTX were activated by groups of animals received different types of treatment equimolar BOP and HOBt in situ using a tertiary amine as per the following protocol. For initial experiments (DIEA). It should be noted that no precipitate was groups of animals were treated with MTX (50, 100, or observed during the synthesis of the conjugate under 200 µg/injection/animal) by the intraperitoneal (ip) route these conditions. The MTX-XAK/AXK preparations were on every alternate day starting from day 10 following the triturated followed by dialysis, and free MTX content was infection. Five such injections were given and animals assessed by reversed-phase HPLC. The amino acid were sacrificed on day 28 following the infection, i.e., 10 composition of purified conjugates was determined by days after the last injection. The degree of parasitaemia amino acid analysis. Considering the amino acid compo- in the sacrificed animals was determined by microscopic sition of the free carrier and of the Glu content of MTX counting of the number of amastigotes in their liverimpression smears stained with Giemsa. At least 500 the average degree of molar substitution (DS) was nucleated cells were screened for the purpose and results calculated. Characteristic values of the conjugates (MTX are expressed as the number of amastigotes/100 cells (36).
content, M ) are summarized in Table 2. The results Other groups of infected animals were similarly treated presented in this table indicate that the amount of MTX Antileishmanial MTX-Branched Polypeptide Conjugate Bioconjugate Chem., Vol. 13, No. 3, 2002 521
incorporation into the polypeptide conjugate dependsmainly on the charge properties of the terminal aminoacid residue of the side chain. The average molarsubstitution ratio was in the range of 10.2-26.3%expressed as % of modified side chains in the conjugates.
Interestingly enough no marked differences have beenobserved among the respective DS values of polyca-tionic polypeptides (DS for MTX-AK 26.0%, for MTX-SAK 25.1%, and for MTX-ALK 25.0%), while in case ofMTX conjugate with amphoteric EAK only 10.2% of theside chains were substituted at 0.5:1 input MTX:EAKmolar ratio. These values indicate that 10-26% of thefree amino groups of branched polypeptide was modifiedby MTX. Consequently, all MTX conjugates studied stillpreserved the polycationic or amphoteric character of thebranched polypeptide used.
Under these conditions the active ester derivative of MTX could react with the hydroxyl group of the Serresidue producing an ester linkage between MTX andbranched polypeptide SAK. The ester bond can be cleavedby hydroxylamine (34, 39). To verify the absence of ester-linked MTX two sets of MTX-SAK conjugate sampleswere investigated. Conjugates before and after hydroxyl-amine treatment were analyzed by RP-HPLC using two Figure 2. RP-HPLC chromatograms of a mixture of poly[Lys-
wavelengths to access free drug in the MTX conjugate (MTXj-Seri-DL-Alam)] (MTX-SAK) conjugate and MTX (A) and preparations. The retention time for MTX, absorbing at λ ) 373 nm, was found to be 10.1 min. Free SAK wasdetected at λ ) 214 nm at the void volume, whereas the Initially, different groups of L. donovani infected mice were treated with MTX branched polypeptide conjugates R value for MTX-SAK was 14.5 min at both λ ) 214 and 373 nm, indicating the presence of covalently bound (MTX-AK, MTX-SAK, and MTX-ALK). It should be noted MTX. Quantitative analysis, performed on a reversed- that due to the high tendency of gel formation under phase HPLC column (pore size of 300 Å, gradient elution), experimental conditions MTX-EAK conjugate was not indicated that less than 0.01% of free MTX besides the investigated. All three preparations showed significant total MTX content in the conjugate samples can be levels of antileishmanial activity (data not shown).
detected using peak-area measurement calibrated with However, the conjugate MTX-ALK produced most en- appropriate standards. It should be noted that no free couraging data (>90% reduction in the number of para-site). Therefore, detailed studies were undertaken with MTX was detected in the MTX-SAK samples before this conjugate and with the corresponding branched hydroxylamine treatment. These data are in accord with our earlier observation that conditions for the preparationof conjugates highly influence the extent of ester bound Table 3 shows the effect of treatment of leishmania formation between OH group and active ester derivative infected mice with free MTX as well as in its conjugated of MTX. The lack of free MTX in MTX-SAK samples form with ALK. About 42% reduction in the liver parasite suggests that the formation of the amide linkage between burden was noted in the MTX treated group of animals MTX and branched polypeptide is favored in a DMF- as compared to those of untreated control group, and the difference was statistically significant (0.01 < P <0.05).
Treatment of animals with MTX-ALK conjugate, how- After analyzing the purity of freshly synthesized ever, led to more marked reduction (∼95%) of their liver compounds, their stability was investigated. Typical parasite burden as compared to those in the control group chromatograms of free MTX and of MTX-SAK conjugate and the difference was statistically highly significant (P containing free MTX as a control are shown in Figure 2.
< 0.001). The MTX-ALK conjugate treated group had also The stability data of conjugate samples obtained after significantly (0.001 < P < 0.01) less number of liver 4-180 days storage at 4 °C. No free MTX was detected parasites than that of the MTX alone treated group.
Further experiments were carried out to determine the Treatment of L. donovani Infected Mice with
antileishmanial effect of ALK treatment alone or in the MTX Alone or in Combination with Branched
form of a mixture with MTX. Results presented in Table Polypeptides. In preliminary experiments, different
3 demonstrate that treatment of L. donovani infected groups of L. donovani infected mice were treated with mice with free ALK did not induce any significant (P > different doses (50-200 µg/injection/animal) of MTX, and 0.1) level of reduction of their liver parasite burden. On their liver parasite load was estimated. About 20%, 36%, the other hand, treatment of animals with the MTX+ALK and 47% reduction of their liver parasite load was mixture produced about 35% reduction in the parasitemia demonstrable following treatment with 50 µg, 100 µg, and as compared to that of the control group and the decrease 200 µg/injection/animal, respectively (five injections ad- was statistically significant (0.001 < P < 0.01). However, ministered as per the protocol described in the Materials the MTX+ALK mixture treated group failed to show any and Methods section). A dose of 100 µg of MTX was significant difference (P > 0.1) in their liver parasitemia subsequently chosen for all subsequent experiments for as compared to that of the MTX alone treatment group.
the determination of any additional benefit, which may Treatment of L. donovani Infected Mouse Mac-
be derived from the combination therapy of MTX and rophages in Vitro with MTX Alone or in Combina-
tion with the Branched Polypeptide ALK. The effect
522 Bioconjugate Chem., Vol. 13, No. 3, 2002
Table 3. Reduction of Parasite Burden in the Liver of L.
donovani
Infected Mice Following Treatment with MTX
(alone) or in Conjugation with ALK

66.2 ( 14.6 0.01 < P* < 0.05 Figure 3. Effect of treatment of L. donovani-infected mouse
macrophages in vitro with MTX (B), MTX + poly[Lys-(DL-Alam- Leui)] (ALK) mixture (C) and poly[Lys-(MTXj-DL-Alam-Leui)] (MTX-ALK) conjugate (D). (A) show infected macrophages without any treatment (control). Microscopic magnification: increased and selective uptake of the drug and thus more beneficial therapeutic effect. For the construction of macromolecule-drug conjugates it is important to pro- vide rational basis to the selection of the proper carrier.
n ) number of animals. b Animals were treated with MTX (100 To this end we have previously reported data on the µg/injection/animal), with ALK (432 µg/injection/animal) orALK+MTX (a mixture of 100 µg of MTX and 432 µg of ALK/ synthesis, biodistribution and in vitro cytotoxicity of the injection/animal) or ALK-MTX (100 µg of MTX conjugated to 432 antimetabolite drug methotrexate-branched polypeptide µg of ALK/injection/animal). c *Compared to group I. **Compared conjugates using osteogenic sarcoma cell line (16). In this contribution we extended our studies by investigation ofantileishmanial effect of MTX conjugates in experimental of in vitro treatment of parasite infected mouse mac- animals. For this a new set of branched polypeptide- rophages with MTX alone or in combination with ALK based conjugates was prepared by an improved synthetic was studied, and results are presented in Table 4 and procedure. After covalent coupling of MTX to the R-amino Figure 3. It may be seen that treatment of macrophages groups of AK, SAK, or ALK conjugates with a similar with MTX (alone) (Figure 3B) or in the form of a mixture average degree of molar substitution the overall charge with ALK (Figure 3C) induced only marginally significant (0.05 < P <0.1) reduction in the intracellular parasite It is believed that positive charge density is required level while ALK (alone) failed to show any reduction of for uptake of MTX-linear poly(R-amino acid) (e.g., polyly- this kind (P > 0.1). On the other hand, considerable sine, polyornitine) by various mouse or human tumor (about 54%) decrease of the parasite load was noted cells (11, 13). A mode of action of the conjugates involves following MTX-ALK conjugate treatment (Figure 3D vs binding to the cell-surface, subsequent endocytic inter- Figure 3A), and the reduction was significant (0.001 < P nalization, localization in the lysosomal system, and < 0.01) as compared to that of the control. The MTX- degradation by lysosomal enzymes to liberate the drug ALK conjugate was also found to be more efficient than (12, 13, 40). This hypothesis was supported by experi- MTX + ALK mixture or MTX alone (0.01 < P <0.05) in mental results using polylysine-coupled MTX and L929 reducing the number of amastigotes within the macro- mouse fibroblast (12) and mouse mammary tumor MM46 cells (40) in vitro. MTX attached to poly(D-lysine) directlyor through a Leu-Ala-Leu-Ala tetrapeptide spacer be- tween the drug and the -amino groups of poly(D-lysine) Synthetic effort with soluble carriers to discover alter- was also investigated. No effect was observed with MTX- nate means to introduce MTX into cells could lead poly(D-lysine), while the conjugate containing MTX at the Table 4. Effect of Treatment of L. donovani Infected Mouse Macrophages in Vitro with MTX, ALK, and Their
Combinations

number of parasites/macrophage (after 72 h of treatment) 0.001 < P* < 0.010.01 < P** < 0.050.01 < P*** < 0.05 a Macrophages were treated with MTX (25 µg/mL of culture), or ALK (108 µg/mL) or ALK+MTX mixture (25 µg/mL of MTX and 108 µg of ALK/mL) or ALK-MTX (c) (25 µg of MTX conjugated to 108 µg of ALK/mL). b n.d. ) not determined. c *Compared to Group I.
**Compared to Group II. ***Compared to Group IV.
Antileishmanial MTX-Branched Polypeptide Conjugate Bioconjugate Chem., Vol. 13, No. 3, 2002 523
N-terminal R-amino group of the Leu-Ala-Leu-Ala unit exhibited potent cytotoxicity, indicating that the spaceris cleaved in the secondary lysosomes (40). In addition, These studies were supported by grants from the the cytotoxicity of a terner conjugate in which MTX-Leu- Hungarian Research Fund (OTKA T-038038), from the Ala-Leu-Ala was coupled to monoclonal antibody specific Hungarian-Indian Intergovernmental Program (4/1996), for MM46 cells was not inhibited by TPP, an inhibitor of from the Hungarian Ministry of Health (T-12/2000), the membrane active transport system for MTX (40).
Budapest, and from Hungarian Ministry of Education(MediChem, 047/2001) Hungary.
Taken together these data suggest that the transportpathways of MTX and of MTX-carrier conjugates aredifferent and independent (12). Trouet et al. (41) has demonstrated that a daunorubicin conjugate with Ala- (1) Embleton, M. J., and Garnett, M. C. (1985) Antibody Leu-Ala-Leu spacer and succinylated BSA carrier was targeting of anticancer agents. Monoclonal antibodies for stable in serum but was cleaved by lysosomal hydrolases.
cancer detection and therapy (R. W. Baldwin, and V. S. Byers, These findings could be adopted for the speculation on Eds.) pp 317-344, Academic Press, London.
the mechanism of action of MTX-ALK conjugate with (2) Furst, D. E., and Kremer, J. M. (1988) Methotrexate in marked antileishmania activity observed in vivo and in rheumatoid arthritis. Arthritis Rheum. 31, 305-314.
(3) Gardner-Medwin, J. M., and Powell, R. J. (1996) One patient, two unusual conditions and three basic lessons. Ann. It has been described that MTX attached to mannose- BSA strongly inhibits the growth of Leishmania donovani (4) Vardy, D. A., Cohen, A., Kachko, L., Zvulunov, A., and inside macrophages. This conjugate was 100 times more Frankenburg, S. (1999) Relapse of cutaneous leishmaniasis active than the free MTX. In contrast MTX conjugated in a patient with an infected subcutaneous rheumatoid to BSA or other nonspecific neoglycoproteins such as nodule. Br. J. Dermatol. 141, 914-917.
galactose-BSA and glucose-BSA have leishmanicidal (5) Mukhopadhyay, A., Chaudhuri, G., Arora, S. K., Sehgal, S., effects comparable to that of the free MTX (7). Further and Basu, S. K. (1989) Receptor mediated drug delivery to results indicated that mannose-BSA based MTX conju- macrophages in chemotherapy of leishmaniasis. Science 244, gate binds specifically to mannose-specific receptor of macrophages and is internalized and degraded in lyso- (6) Bryceson, A. D. M. (1966) Manson’s Tropical Diseases (G.
somes, releasing the active drug to act on Leishmania C. Cook, Ed.) pp 1213-1245, W. B. Saunders, London.
parasites (8, 9, 42). These data suggest that the uptake (7) Chakraborty, P., Bhaduri, A. N., and Das, P. K. (1990) Sugar mechanism of mannose-containing conjugates is related receptor mediated drug delivery to macrophages in thetherapy of experimental visceral leishmaniasis. Biochem. to the presence of mannose receptor of infected macro- Biophys. Res. Commun. 166, 404-410.
phages. On the other hand, our findings clearly indicate (8) Chakraborty, P., Bhaduri, A. N, and Das, P. K. (1990) that MTX conjugates without a mannose moiety could Neoglycoproteins as carriers for receptor-mediated drug also be introduced into macrophages.
targeting in the treatment of experimental visceral leishma- Recently it has been documented that Leishmania and niasis. J. Protozool. 37, 358-364.
other trypanosomatid protozoa require reduced pteridines (9) Sett, R., Sarkar, H. S., and Das, P. K. (1993) Pharmacoki- (pterins and folates) for growth, suggesting that inhibi- netics and biodistribution of methotrexate conjugated to tion of these pathways could be targeted for effective mannosyl human serum albumin. J. Antimicrob. Chemother.
31,
151-159.
chemotherapy (43). Findings suggest that successful (10) Chaudhuri, G., Mukhopadhyay, A., and Basu, S. K. (1989) antifolate chemotherapy in Leishmania will have to Selective delivery of drugs to macrophages through a highly target simultaneously both dihydrofolate reductase specific receptor. An efficient chemotherapeutic approach (DHFR) and pteridine reductase 1 (PTR1). It is attractive against leishmaniasis. Biochem. Pharmacol. 38, 2995-3002.
to speculate on the mechanism of action of branched (11) Ryser, H. J. P., and Shen, W. C. (1978) Conjugation of polypeptide attached MTX. On the basis of published methotrexate to poly(L-Lysine) increases drug transport and data outlined above and our own observations reported overcomes drug resistance in cultured cells. Proc. Natl. Acad. in this communication, it is likely that MTX released from MTX-branched polypeptide conjugate might act as (12) Shen, W. C., and Ryser, H. J. P. (1981) Selective protection an inhibitor of enzymes involved in the unusual pteridine against the cytotoxicity of methotrexate and methotrexate- poly(lysine) by thiamine pyrophosphate, heparin and leu-civorin Life Sci. 28, 1209-1214.
(13) McGuire, J. J., and Russell, C. A. (1990) A human leukemia cell culture system for testing new antifols: differentialsenstivity of lymphoid and nonlymphoid cell lines to uncon- These studies have indicated that synthetic branched jugated and methotrexate-conjugated polymers of basic amino polypeptides can be considered as potential candidates for constructing suitable conjugates for methotrexate (14) Galivan, J., Balinska, M., and Whiteley, J. M. (1982) delivery into Leishmania donovani infected macrophages.
Interaction of methotrexate-poly(L-Lys) with transformed In this communication we showed that (a) a branched hepatic cells in culture. Arch. Biochem. Biophys. 216, 544- polypeptide-methotrexate conjugate with a polycationic carrier (ALK) increase the leishmanicid activity of MTX (15) Upeslacis, J., and Hinman, L. (1988) Chemical modification in mice; (b) the covalent bond between the carrier and of antibodies for cancer chemotherapy Annual Reports in methotrexate is essential for the effect; and (c) the Medicinal Chemistry (N. Saltzman, Ed.) pp 151-160, Acad.
Press, New York.
number of L. donovani parasites in infected macrophages (16) Hudecz, F., Clegg, J. A., Kajta´r, J., Embleton, M. J., Pimm, are markedly reduced in conjugate treated animals. The M. V., Szekerke, M., and Baldwin, R. W. (1993) Influence of latter observation might indicate that the MTX conjugate carrier on biodistribution and in vitro cytotoxicity of meth- exhibits its effect throught an uptake by macrophages otrexate-branched polypeptide conjugates. Bioconjugate Chem.
which is different from that of the free drug. Further comparative studies are in progress to clarify the mech- (17) Hudecz, F., Clegg, J. A., Kajta´r, J., Embleton, M. J., Szekerke, M., and Baldwin, R. W. (1992) Synthesis, confor- 524 Bioconjugate Chem., Vol. 13, No. 3, 2002
mation, biodistribution and in vitro cytotoxicity of daunomy- (30) Wilkinson, K. A., Hudecz, F., Vordermeier, H. M., Ivanyi, cin-branched polypeptide conjugates. Bioconjugate Chem. 3, J., and Wilkinson R. J. (1999) Enhancement of the T cell response to a mycobacterial peptide by conjugation to syn- (18) Ko´cza´n, Gy., Ghosh, A. K, Mookherjee, A., Ghose, A. C., thetic branched polypeptide Eur. J. Immunol. 29, 2788-2796.
and Hudecz, F. (1998) Application of branched chain poly- (31) Mezo¨, G., Mihala, N, Andreu, D., and Hudecz, F. (2000) meric polypeptides for methotrexate targeting to macrophages Conjugation of epitope peptides to branched chain polypep- in Leishmania donovani infection. The Immunologist S1, 610.
tides via Cys(Npys). Bioconjugate Chem. 11, 484-491.
(19) Hudecz, F. (1995) Design of synthetic branched-chain (32) IUPAC-IUB Commission on Biochemical Nomenclature.
polypeptides as carriers for bioactive molecules. Anti-Cancer (1972) Biochem. J. 127, 753-756.
(20) Mezo¨, G., Kajta´r, J., Hudecz, F., and Szekerke, M. (1993) (33) IUPAC-IUB Commission on Biochemical Nomenclature.
Carrier design: conformational studies of amino acid (X) and (1984) Eur. J. Biochem. 138, 9-37.
oligopeptide (X-DL-Alam) substituted poly[L-lysine]. Biopoly- (34) Endo, N., Takeda, Y., Umemoto, N., Kishida, K., Watanabe, K., Saito, M., Kato, Y., and Hara,T. (1988) Nature of linkage (21) Hudecz, F., Pimm, M. V., Rajnavo¨lgyi, E and mode of action of methotrexate conjugated with antitu- A., Gaa´l, D., Kova´cs, A. L., Horva´th, A., and Szekerke, M.
mor antibodies: implications for future preparation of con- (1999) Carrier design: New generation of polycationic branched jugates. Cancer Res. 48, 3330-3335.
polypeptides containing OH groups with prolonged blood (35) Dasgupta, S., Mookerjee, A., Chowdhury, S. K., and Ghose, survival and diminished in vitro cytotoxicity. Bioconjugate A. C. (1999) Immunosuppression in hamsters with progres- sive visceral leishmaniasis: an evaluation of the role of nitric (22) Gaa´l, D., and Hudecz, F. (1998) Low toxicity and high oxide towards the impairment of lymphoproliferative re- antitumour activity of daunomycin by conjugation to immu- sponse. Parasitol. Res. 85, 594-596.
npotential amphoteric branched polypeptide. Eur. J. Cancer (36) Stauber, L. A. (1958) Host resistance to the khartoum strain of Leishmania donovani. Rice Inst. Pamph. 45, 80- (23) Mezo¨, G., Sa´rmay, G., Hudecz, F., Kajta´r, J., Nagy, Zs., Gergely, J., and Szekerke, M. (1996) Synthesis and charac-terization of p-borono-Phe - branched polypeptide - mono- (37) Ghose, A. C., Mookerjee, A., Sengupta, K., Ghosh, A. K., clonal antibody ternary systems for potential use in boron Dasgupta, S., and Ray, P. K. (1999) Therapeutic and prophyl- neutron capture therapy (BNCT) J. Bioact. Compat. Polym. actic uses of the immunomodulator Protein A in the control of Leishmania donovani infection in experimental animals.
(24) Mezo¨, G., Mezo¨, I., Sepro˜di, A., Tepla´n, I., Kova´cs, M., Vincze, B., Pa´lyi, I., Kajta´r, J., Szekerke, M., and Hudecz, F.
(38) Ghosh, A. K., Dasgupta, S., and Ghose, A. C. (1995) (1996) Synthesis, conformation, biodistribution and hormon Immunoglobulin G subclass specific antileishmanial antibody related in vitro antitumor effect of a GnRH antagonist- responses in Indian kala-azar and post kala-azar dermal branched polypeptide conjugate. Bioconjugate Chem. 7, 642- leishmaniasis. Clin. Diagn. Lab. Immunol. 2, 291-296.
(39) Hudecz, F., Garnett, M. C., Khan, T., and Baldwin, R. W.
(25) Pato´, J., Ulbrich, K., Baker, P., Mezo¨, G., and Hudecz, F.
(1992) The influence of synthetic conditions on the stability (1999) Synthesis of macromolecular conjugates of a urokinase of methotrexate-monoclonal antibody conjugates determined inhibitor. J. Bioact. Compat. Polym. 14, 99-121.
by reversed phase high performance liquid chromatography.
(26) Pimm, M. V., and Hudecz, F.(1996) Biodistribution in Biomed. Chromatogr. 6, 128-132.
tumour-bearing mice of polycationic, amphoteric and poly- (40) Umemoto, N., Kato, Y., Endo, N., Takeda, Y., and Hara, anionic branched polypeptides with poly(L-lysine) backbone T. (1989) Preparation and in vitro toxicity of a MTX-anti- labeled with 125I and 111In: Tumour accumulation less than MM46 monoclonal antibody conjugate via an oligopeptide that of labeled serum proteins. J. Cancer Res. Clin. Oncol. spacer. Int. J. Cancer. 43, 677-684.
(27) Perkins, A. C., Frier, M., Pimm, M. V., and Hudecz, F.
(41) Trouet, A., Masquelier, M., Baurain, R., and Deprez-De (1998) Tc99m-branched chain polypeptide (BCP): a potential Campeneere, D. (1982) A covalent linkage between dauno- synthetic radiopharmaceutical. J. Labelled Compds 41, 631- rubicin and proteins that is stable in serum and reversible by lysosomal hydrolases, as required for a lysosomotropic (28) Mezo¨, G., Kajta´r, J., Szo´ka´n, Gy., Sa´rmay, G., Gergely, J., drug-carrier conjugate: in vitro and in vivo studies. Proc. and Szekerke, M. (1991) Branched polypeptides as carriers Natl. Acad. Sci. U.S.A. 79, 2626-2629.
of tuftsin analogues: synthesis, structure and immunostimu- (42) Basu, N., Sett, R., and Das, P. K (1991) Down-regulation latory activity. Peptides 1990 (E. Giralt, and D. Andreu, Eds.) of mannose receptors on macrophages after infection with Leishmania donovani. Biochem. J. 277, 451-456.
(29) Wilkinson, K. A., Vordermeier, M. H., Wilkinson, R., (43) Nare, B., Luba, J., Hardy, L. W., and Beverley, S. (1997) Iva´nyi, J., and Hudecz, F. (1998) Synthesis and in vitro T New approaches to Leishmania chemotherapy: pteridine cell immunogenicity of conjugates with dual specificities: reductase 1 (PTR1) as a target and modulator of antifolate attachment of epitope peptides of 16 kDa and 38 kDa proteins sensitivity. Parasitology 114 Suppl. S101-110.
from M. tuberculosis to branched polypeptide. BioconjugateChem. 9, 539-547.

Source: http://www.chemotaxis.sote.hu/CHTXhpg/Leishmania/DT/Koczan-G-BioconjChem-2002.pdf

appcb.ap.nic.in

Environmental Impact Assessment of Proposed 420 TPA Bulk Drugs & Intermediates Manufacturing with R&D facility at APIIC Industrial Park, Annarugudem Village, Khammam District, Andhra Pradesh Executive Summary Sponsor : M/s. Varun Laboratories Private Limited, Hyderabad EIA Consultant: KKB Envirocare Consultants Pvt. Ltd., Tarun Plaza,

drthchowdary.net

Conduct of seminars, discussions, round-tables and workshops devoted to the boardaspects of telecommunications policy, organisation, performance, technology re-search, consumer protection, telecom laws, etc., Involving providers and consumersof service, economists, intellectuals and policy makers; ICTs AND SOCIETY Involving consumer associations and providers of service in discussions; Editor

Copyright © 2010-2014 Sedative Dosing Pdf