Gelatin-methotrexate conjugate microspheres as a potential drug delivery system

Gelatin-methotrexate microspheres for intra-tumor administration have possibilities for minimizing systemic toxicities of methotrexate (MTX) and overcoming its resistance. Gelatin-MTX conjugates prepared by a carbodiimide reaction were crosslinked with glutaraldehyde to form microspheres (MTX:gelatin molar ratios of 2:1, 15:1, and 21:1). Microspheres were evaluated under in vitro tumor conditions at pH 6.5 and 37 degrees C with and without Cathepsin B (Cat B). Some microspheres were capped with an ethanolamine/cyanoborohydride procedure. SEM of broken microspheres revealed a hollow shell structure. Superficial Cat B degradation influenced some free MTX release but produced no conjugate fragment release. HPLC measured release of fragments (<10 kDa) was very little and release of free MTX was small. However, higher drug load microspheres released less free MTX than lower drug load, a substantial lag phase of free MTX release from capped microspheres changed to an initial rapid release in uncapped microspheres, and fragments were only released from uncapped microspheres. Opened unstable Schiff base crosslinks in uncapped microspheres may allow enzyme to produce conjugate fragments not observed in capped microspheres. Free MTX release may occur from dissolved uncrosslinked conjugate within the hollow microspheres. Important relationships and observations are described that will be useful for gelatin and perhaps other proteinaceous microspheres.     

Characterization and in vitro release of methotrexate from gelatin/methotrexate conjugates formed using different preparation variables

The purpose of this study was to evaluate effects of preparation variables on the composition of gelatin-methotrexate conjugates, and to evaluate their in vitro stability. Conjugation variables of pH, amount of conjugating agent 1-ethyl-3-(diaminopropyl)carbodiimide HCl (EDC), and methotrexate (MTX), with unfractionated gelatin were examined. Conjugate composition was determined spectrophotometrically. The molar ratios of MTX to gelatin in the conjugates ranged from 5.9 to 64.9. Molar ratios increased with molecular weight (MW) of gelatin in the conjugate, but the weight ratio was constant. This common conjugating procedure, however, produces by-product crosslinking and produces a mix of covalent MTX binding to carboxyl and amino groups of the gelatin. For release studies, gelatin was fractionated by size exclusion spectra (SEC) into MW of 21, 91, and 195 kDa prior to conjugation. MTX release from conjugates in dialysis cassettes at 25, 37, and 50 degrees C, in isotonic pH 7.4, buffer over 72 h was assayed by high performance liquid chromatography (HPLC). There was no effect of gelatin MW on MTX release. MTX release was approximately linear and attained 2.3, 7.2, and 13% by 72 h at 25, 37, and 50 degrees C, respectively, for the 91 kDa conjugates. First-order release rate constants were 0.23 x 10(-3), 0.95 x 10(-3), and 1.8 x 10(-3) x h(-1), respectively. The calculated activation energy for MTX release was 15.8 kcal/mol. Rate constants and the activation energy for MTX release are consistent with hydrolysis of a peptide bond. Non-degraded MTX species were found in the release medium at amounts similar to free MTX and were attributed to MTX polymers and MTX/gelatin fragments < 10 kDa.     

Characterization and In Vitro Methotrexate Release from Methotrexate/Gelatin Conjugates of Opposite Conjugate Bond Polarity

Purpose. Our laboratory has previously prepared gelatin/methotrexate (MTX) conjugates containing mixed conjugation sites and by-product crosslinking, both of which may alter conjugate effectiveness. In this study, we prepared and evaluated gelatin/MTX conjugates having specific conjugate bond sites and minimal by-product crosslinking. Methods. Opposite polarity conjugates were produced by coupling gelatin having blocked amino groups with MTX (G-MTX) and by coupling MTX having blocked amino groups with gelatin (M-GEL) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl. Amino groups were blocked using citraconic anhydride and deblocked under acidic conditions. Gelatin and MTX contents were determined spectrophotometrically. The stability of each conjugate was determined by evaluating their in vitro release of MTX in isotonic buffer at pH 7.4 and 37°C for 7 days. Results. The G-MTX and M-GEL conjugates contained 21 and 1.2 mole MTX/mole gelatin and released 12 and 17% MTX by 7 days resulting in pseudo-first order release rate constants of 0.76×10^–3 and 1.0×10^–3 hr^–1, respectively. Alternate MTX species ( 10%) were detected during the release study and were attributed to low molecular weight gelatin/MTX fragments and MTX polymers. Conclusions. Gelatin/MTX conjugates having opposite conjugate bond polarities and minimal by-product crosslinking have been produced and slowly released MTX by hydrolytic cleavage indicating good stability for future cell culture studies.     

Characterization of in-vitro drug release and biological activity of methotrexate-bovine serum albumin conjugates

Two series of methotrexate (MTX)-bovine serum albumin (BSA) conjugates have been prepared containing either 96 +/- 16 mg (mean +/- s.d.) or 32 +/- 13 mg of MTX per gram of conjugate. The conjugates released MTX in-vitro in a biphasic manner, the release rate being dependent on the quantity of MTX in the conjugate and on the pH of the release medium. An initial rapid release over 6 h appears to be due to physically adsorbed MTX with the slower secondary release due to covalently bound drug. The conjugates retain a degree of antineoplastic activity in-vitro, but this might be related to the small fraction of MTX that is tightly physically bound.     

The Effect of Molecular Weight,Drug Load,and Charge of Gelatin–MTX Conjugates on Growth Inhibition of HL-60 Leukemia Cells

Purpose  Gelatin–methotrexate conjugates (G-MTX) with known molecular weight (MW), drug load, and charge were prepared and evaluated for growth inhibition on leukemia cells. Methods  Gelatin (34 to 171 kDa) was reacted with a carbodiimide to prepare G-MTX with high (G-MTX-H) and low (G-MTX-L) drug loads. Cationic conjugates were prepared by ethylenediamine modification. MTX:gelatin molar ratios were determined spectrophotometricaly. Isoelectric focusing electrophoresis (IEF) and turbidity were used to measure isoelectric points (IEP). Growth inhibition profiles and IC₅₀ values were determined on HL-60 cells using a modified MTT assay. Results  IC₅₀ values of anionic G-MTX-L (drug loads 0.5:1 to 2.2:1) increased linearly from 46 to 180 nM with MW. But, IC₅₀ values for anionic G-MTX-H (drug loads 7.4:1 to 25:1) showed little, if any, MW dependence and were about two times higher. IC₅₀ values for cationic G-MTX-L ranged from 770 to 2,900 nM and the relationship with MW was non-linear. Conclusions  The growth inhibition ranking was MTX > anionic G-MTX-L > anionic G-MTX-H > cationic G-MTX-L. High drug load may hinder lysosomal enzyme degradation and drug release and contribute to suppression of the MW effect observed with G-MTX-L. A mechanism change is suggested as the cationic conjugates increase to the highest MW.     

Macrophage activation for antitumour function by muramyl dipeptide-protein conjugates

A muramyl dipeptide (MDP) has been conjugated directly with various proteins by means of a water-soluble carbodiimide. The enhancement of the antitumour activity of mouse peritoneal macrophages by the MDP-protein conjugates has been investigated to assess the ability of the proteins for targeting MDP to the macrophages. These were activated to inhibit the in-vitro growth of tumour cells much more effectively, when immunoglobulin (IgG), fibronectin (FN), and gelatin conjugates were used than when MDP was used alone. The minimum amount of MDP in both the MDP-gelatin and the MDP-IgG conjugates necessary for macrophage activation was approximately 2000 times lower than the amount of free MDP needed. The macrophages activated by the conjugates exhibited growth inhibitory activity against phenotypically diverse tumour cells. The activity induced by the MDP-gelatin conjugate was higher than that of the MDP-IgG conjugate over the range of MDP concentrations, regardless of the isoelectric point of the gelatin used. When MDP was conjugated with bovine serum albumin (BSA), the antitumour activity of macrophages was reduced as the amount of BSA conjugated increased. With both free MDP and MDP-protein conjugates, the macrophages were more strongly activated, the longer they were pretreated. However, less pretreatment time was needed to potentiate macrophage activation by the MDP-gelatin conjugate than by free MDP. Also, the macrophages pretreated with the MDP-gelatin conjugate could maintain their activated state for longer than those pretreated with free MDP. It is concluded that gelatin is an effective carrier protein for the targeting MDP to macrophages, resulting in their activation.     

In vitro Drug release characteristics of methotrexate-human serum albumin and 5-fluorouracil-acetic acid-human serum albumin conjugates

The release rates of methotrexate (MTX) from MTX-human serum albumin (HSA) conjugate, and 5-fluorouracil (5-FU) from 5-FU acetic acid (AA)-HSA conjugate were determined after incubation of the conjugates in various conditions. The concentrations of 5-FU released from the conjugate increased monoexponentially, however those of MTX increased biexponentially in all studies. It indicated that there are two distinct types of MTX-HSA linkage, weakly and tightly bound linkages. The release rates of 5-FU were lower than those of MTX in all studies indicating that the bond of 5-FU-AA-HSA conjugate is very stable, which is supported by the higher value of activation energy (39.9 vs 10.7 Kcal/mole) using Arrhenius equation. The release rates of MTX and 5-FU from the conjugates increased with incubation temperatures. Proteolytic enzyme and liver homogenates accelerated significantly the release rates of MTX and 5-FU. Approximately 1.30 and 22.0% of MTX were released after 12 hours of incubation in the absence and presence of protease, respectively. The corresponding values for 5-FU were 1.0 and 17.0% Approximately 10.3 and 11.9% of 5-FU and MTX, respectively were released after 12 hours of incubation with rat liver homogenates which were diluted 6 times with phosphate buffer of pH 6.0. The MTX-HSA and 5-FU-AA-HSA conjugates were very stable in rat plasma.     

Synthesis and in vitro characterization of novel dextran-methylprednisolone conjugates with peptide linkers: effects of linker length on hydrolytic and enzymatic release of methylprednisolone and its peptidyl intermediates

To control the rate of release of methylprednisolone (MP) in lysosomes, new dextran-MP conjugates with peptide linkers were synthesized and characterized. Methylprednisolone succinate (MPS) was attached to dextran 25 kDa using linkers with 1-5 Gly residues. The release characteristics of the conjugates in pH 4.0 and 7.4 buffers, blood, liver lysosomes, and various lysosomal proteinases were determined using a size-exclusion and/or a newly developed reversed-phase HPLC method capable of simultaneous quantitation of MP, MPS, and all five possible MPS-peptidyl intermediates. We synthesized conjugates with >or=90% purity and 6.9-9.5% (w/w) degree of MP substitution. The conjugates were stable at pH 4.0, but released MP and intact MPS-peptidyl intermediates in the pH 7.4 buffer and rat blood, with faster degradation rates for longer linkers. Rat lysosomal fractions degraded the conjugates to MP and all the possible intermediates also at a rate directly proportional to the length of the peptide. Whereas the degradation of the conjugates by cysteine peptidases (papain or cathepsin B) was relatively substantial, no degradation was observed in the presence of aspartic (cathepsin D) or serine (trypsin) proteinases, which do not cleave peptide bonds with Gly. These newly developed dextran conjugates of MP show promise for controlled delivery of MP in lysosomes.     

Doxorubicin-loaded microgels composed of cinnamic acid–gelatin conjugate and cinnamic acid–Pluronic F127 conjugate

Microgels were prepared by cinnamic acid–gelatin (type B) conjugate (CA-GelB) and cinnamic acid–Pluronic F127 conjugate (CA-Plur). ¹H NMR confirmed that CA was conjugated to gelatin and the gelatin to CA residue molar ratio was estimated to be 1:4.7 by a colorimetric method. CA-Plur of which the CA residue to Plur molar ratio was 1.2:1 was used as a thermo-sensitive polymer. The CA residues of CA-Plur/CA-GelB mixture were readily photo-dimerized to form microgels by UV irradiation. The isoelectric point of the microgel was found to be pH 5.8 and the hydrodynamic diameter decreased when the suspension temperature increased. The microgel could hardly retard the release of doxorubicin (DOX) at pH 3.0 and pH 5.0, but it could suppress and control the release at pH 7.4 possibly due to electrostatic attraction. Meanwhile, the release of DOX at pH 7.4 was less suppressed when the medium temperature was higher, possibly because of thermal thinning of Pluronic chain layer.     

酸敏感靶向的阿霉素聚酰胺-胺型树枝状高分子的抗肿瘤研究(英文)

聚酰胺-胺型树枝状高分子是近年来新出现的一种表面有多官能团的药物载体。本研究中,顺-4,7,10,13,16,19-二十二碳六烯酸(DHA)和阿霉素(DOX)共价连接到2.5代的PAMAM分子上。通过这种设计,载体结构可以优化药物在血液中的循环,药物和载体之间的酸敏感键可以优化药物的释放。体外实验结果表明,pH4.5条件下阿霉素的12小时累积释放量约为pH7.4条件下的两倍。这种酸敏感的药物释放可以提高药效。药动学研究表明,DHA-PAMAM-DOX能提高药物浓度-时间曲线下面积。在荷B16黑色素瘤小鼠模型中,实验构建的DHA-PAMAM-DOX抑瘤效果优于同等剂量(5 mg/kg)的阿霉素溶液。研究表明DHA-PAMAM-DOX在肿瘤药物靶向给药方面有很好的发展前景。     

Methotrexate Esters of Poly(Ethylene Oxide)-Block-Poly(2-Hydroxyethyl-L-Aspartamide). Part I: Effects of the Level of Methotrexate Conjugation on the Stability of Micelles and on Drug Release

Purpose. To study the effects of hydrophobicity of the micelle-formingblock copolymeric drug conjugate, methotrexate (MTX) esters ofpoly-(ethylene oxide)-block-poly(2-hydroxyethyl-L-aspartamide) (MTXesters of PEO-b-PHEA), on the stability of micelles and on drug release. Methods. MTX esters of PEO-b-PHEA with three levels of MTXconjugation were synthesized. Size distribution of the micelles wasmeasured by dynamic light scattering (DLS). The critical micelleconcentration (CMC) was determined by a light scattering study. Sizeexclusion high performance liquid chromatography (SEC-HPLC) wasused to study the equilibrium between unimers and micelles, and releaseof MTX at pH 7.4. Results. MTX esters of PEO-b-PHEA with MTX substitution of 7.4%,22%, and 54% were prepared. The conjugates formed micelles basedon DLS. The stability of the micelles correlated with the level of MTXconjugation. The conjugate with 54% MTX had a lower CMC (0.019mg/mL) than the conjugates with 22% MTX (0.081 mg/mL) or 7.4%MTX (0.14 mg/mL). Micelle dissociation was significantly slower forthe conjugate with 54% MTX than that with 22% and 7.4% MTX.Slower release of MTX from the micelles was also observed for theconjugate with the higher MTX attachment. Conclusions. MTX esters of PEO-b-PHEA can be structurallymodulated by varying the degree of MTX substitution, which in turn changesthe hydrophobicity of the conjugate, thereby modifying micelle stabilityand controlling drug release.     

HPMA Copolymer-1,5-Diazaanthraquinone Conjugates as Novel Anticancer Therapeutics

1,5-Diazaanthraquinones (DAQs) are promising anticancer drugs, however, their clinical potential is limited due to poor solubility. Conjugation of anticancer agents to hydrophilic water-soluble polymers can overcome this problem and has already been used to generate conjugates with demonstrated clinical benefit. Here a library of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates containing a novel amino-functionalised 1,5-diazaanthraquinone derivative (amino-DAQ) have been synthesised. The conjugates were fully characterised by UV, HPLC, SEC, FT-Raman and NMR spectroscopy. Conjugation to HPMA copolymers improved amino-DAQ aqueous solubility (>7-fold). The HPMA copolymer-amino-DAQ conjugates were slightly less haemolytic than the parent compound (2% Hb released in 1 h for conjugate HPMA copolymer-GFLG (5 mol%)-amino-DAQ conjugate compared to 13% obtained with amino-DAQ). When conjugates were incubated with isolated rat liver lysosomal enzymes (Tritosomes) the rate of amino-DAQ release was influenced by both drug loading and the composition of the peptidyl side chain used to link the drug to the carrier. The higher the drug loading the lower the rate of drug release. Whereas the GG linker did not release amino-DAQ, up to 26% of the amino-DAQ was released from a GFLG linker over 24 h. The in vitro cytotoxicity of these conjugates was evaluated against two different cell lines, B16F10 murine melanoma and MCF-7 human breast cancer cells. HPMA copolymer-amino-DAQ conjugates, which are internalised by cells by the endocytic pathway, showed much lower in vitro cytotoxicity (IC₅₀ for HPMA copolymer-GFLG (5 mol%)-amino-DAQ conjugate > 397 μM drug-equiv.) than the free drug (the IC₅₀ for amino-DAQ was 12.6 and 2.8 μM against the B16F10 murine melanoma and the MCF-7 breast cancer cell line, respectively). Nonetheless, the observed lysosomal activation of the HPMA copolymer-GFLG-amino-DAQ conjugates, suggests that evaluation of the antitumour potential in vivo is warranted.     

HPMA copolymer-1,5-diazaanthraquinone conjugates as novel anticancer therapeutics

1,5-diazaanthraquinones (DAQs) are promising anticancer drugs, however, their clinical potential is limited due to poor solubility. Conjugation of anticancer agents to hydrophilic water-soluble polymers can overcome this problem and has already been used to generate conjugates with demonstrated clinical benefit. Here a library of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates containing a novel amino-functionalised 1,5-diazaanthraquinone derivative (amino-DAQ) have been synthesised. The conjugates were fully characterised by UV, HPLC, SEC, FT-Raman and NMR spectroscopy. Conjugation to HPMA copolymers improved amino-DAQ aqueous solubility (>7-fold). The HPMA copolymer-amino-DAQ conjugates were slightly less haemolytic than the parent compound (2% Hb released in 1 h for conjugate HPMA copolymer-GFLG (5 mol%)-amino-DAQ conjugate compared to 13% obtained with amino-DAQ). When conjugates were incubated with isolated rat liver lysosomal enzymes (Tritosomes) the rate of amino-DAQ release was influenced by both drug loading and the composition of the peptidyl side chain used to link the drug to the carrier. The higher the drug loading the lower the rate of drug release. Whereas the GG linker did not release amino-DAQ, up to 26% of the amino-DAQ was released from a GFLG linker over 24 h. The in vitro cytotoxicity of these conjugates was evaluated against two different cell lines, B16F10 murine melanoma and MCF-7 human breast cancer cells. HPMA copolymer-amino-DAQ conjugates, which are internalised by cells by the endocytic pathway, showed much lower in vitro cytotoxicity (IC50 for HPMA copolymer-GFLG (5 mol%)-amino-DAQ conjugate>397 microM drug-equiv.) than the free drug (the IC50 for amino-DAQ was 12.6 and 2.8 microM against the B16F10 murine melanoma and the MCF-7 breast cancer cell line, respectively). Nonetheless, the observed lysosomal activation of the HPMA copolymer-GFLG-amino-DAQ conjugates, suggests that evaluation of the antitumour potential in vivo is warranted.     

E-PHA介导的化疗药物对离体胃癌细胞的抑制效应

本文报道了用E-PHA作载体,偶联三种化疗药物(ADM、MMC和MTX),对离体培养的胃癌(SGC)细胞的抑制和杀伤作用进行了观察。结果表明:偶联药物对胃癌细胞表现出一定的抑制和杀伤作用,但效果没有游离药物明显,另外偶联药对正常细胞也显示了一定的保护作用。并就相关问题进行了讨论。     

Development of novel polymeric micellar drug conjugates and nano-containers with hydrolyzable core structure for doxorubicin delivery.

Novel micelle-forming poly(ethylene oxide)-block-poly(epsilon-caprolactone) (PEO-b-PCL) block copolymers bearing doxorubicin (DOX) side groups (PEO-b-P(CL-DOX)) on the PCL block were synthesized. Prepared block copolymers were characterized, assembled to polymeric micellar drug conjugates and assessed for the level of DOX release at pH 7.4 and pH 5.0 using a dialysis membrane to separate released and conjugated drug. The possibility for the degradation of PCL backbone for PEO-b-P(CL-DOX) micelles was investigated using gel permeation chromatography. Micelle-forming DOX conjugate did not show any signs of DOX release at 37 degrees C within 72h of incubation at both pHs, but revealed signs of poly(ester) core degradation at pH 5.0. In further studies, PEO-b-PCL micelles bearing benzyl, carboxyl or DOX groups in the core were also used as micellar nano-containers for the physical encapsulation of DOX, where maximum level of drug-loading and control over the rate of DOX release was achieved by polymeric micelles containing benzyl groups in their core, i.e., PEO-b-poly(alpha-benzylcarboxylate-epsilon-caprolactone) (PEO-b-PBCL) micelles. The in vitro cytotoxicity of chemically conjugated DOX as part of PEO-b-P(CL-DOX) and physically encapsulated DOX in PEO-b-PBCL against B16F10 murine melanoma cells was assessed and compared to that of free DOX. Consistent with the results of in vitro release study, cytotoxicity of micellar PEO-b-P(CL-DOX) conjugate (IC(50) of 3.65mug/mL) was lower than that of free and physically encapsulated DOX in PEO-b-PBCL (IC(50) of 0.09 and 3.07mug/mL, respectively) after 24h of incubation. After 48h of incubation, the cytotoxicity of conjugated DOX (IC(50) of 0.50mug/mL) was still lower than the cytotoxicity of free DOX (IC(50) of 0.03mug/mL), but surpassed that of physically encapsulated DOX in PEO-b-PBCL (IC(50) of 1.54mug/mL). The results point to a potential for PEO-b-P(CL-DOX) and PEO-b-PBCL as novel polymeric micellar drug conjugates and nano-containers bearing hydrolyzable cores for DOX delivery.     

Chondroitin sulfate-based anti-inflammatory macromolecular prodrugs.

Macromolecular prodrugs of three non-steroidal anti-inflammatory drugs (NSAIDs), ibuprofen, ketoprofen, and naproxen, were prepared by the covalent attachment of the drugs onto chondroitin sulfate (ChS) using PEG 1000 as a spacer. Drug-PEG adducts were synthesized using 1,1'-carbonyl diimidazole as a coupling agent in dimethyl sulfoxide, followed by the reaction with ChS in highly dilute aqueous solution at pH 6.8 via N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC) as a conjugation agent. The drug-ChS conjugates were confirmed by FTIR, 1H NMR and 13C NMR and the molar percent of drug substitution onto ChS was characterized by 1H NMR using the peak areas of the three protons of -PhiCHCH3 on the drugs to those of -NHCOCH3 on ChS. All drug-ChS conjugates are water-soluble. The release amounts of the free drugs from their corresponding drug-ChS conjugates were evaluated in the presence or absence of either esterase or chondroitinase, and the both enzymes in pH 7.4 Tris-buffer solutions at 37 degrees C by high performance liquid chromatography (HPLC). Keto-ChS conjugates released approximately 100% ketoprofen within 12h in the presence of esterase, but the combination with chondroitinase did not accelerate the release rate. The degradation of Keto-ChS conjugates by chondroitinase was confirmed by gel permeation chromatography (GPC). The Keto-ChS conjugates still retained the enzymatic recognition even at the substitution of ketoprofen as high as 56 mol%. The inhibition percent of carrageenan-induced edema of Keto-ChS-56 was comparable to that of a simple blend of ChS and ketoprofen, suggesting that biologically active ChS and ketoprofen could be liberated from the conjugate.     

Role of hepatic lysosomes in the degradation of metallothionein.

The degradation of metallothionein (MT) by rat liver was examined. Degradation of MT by liver homogenate was greater than by cytosol. In addition, MT degradation by the homogenate at pH 5.5 was more than that at pH 7.2. Because lysosomal proteases function at acidic pH, these findings suggest the importance of lysosomes in MT degradation. The degradation by the lysosomal fraction was about 400-fold greater than that by the cytosol. Because cathepsins are the principal lysosomal proteases, we used cathepsin-specific inhibitors, such as leupeptin, E-64 and pepstatin, to determine the relative importance of different cathepsins in degrading MT. The study reveals that cathepsin B and/or L is (are) probably the most important enzyme(s) in degrading hepatic MT, because leupeptin, which blocks cathepsin B and L activity, inhibited the degradation of apo-MT by about 80%. Cathepsin D appears to be of least importance in MT degradation, because inhibition of this enzyme by pepstatin reduced degradation by only 20%. Studies on the degradation of apo-MT, ZnMT, and CdMT indicated that apo-MT is about 1500-fold more sensitive to degradation than ZnMT and CdMT. These data suggest that metals protect MT from degradation. This is further supported by a reconstitution experiment, which shows that with a progressive decrease of MT: metal ratio following titration of apo-MT by metals, there is a concomitant reduction in degradation. At a lysosomal pH of around 4.7, about 60% of Zn and 20% of Cd are displaced from MT, thereby making it susceptible to degradation. We propose, therefore, that lysosomes are probably important for MT degradation in vivo and that metal release is a prerequisite for degradation. With the release of metals, MT becomes susceptible to degradation, which is probably accomplished by the lysosomal cathepsins, in particular cathepsins B and L.     

Preparation andIn vitro release characteristics of hydrophilic albumin microspheres containing methotrexate and methotrexate-human serum albumin conjugates

Release characteristics of five different types of hydrophilic albumin microspheres (HAM) containing different ratios of methotrexate-albumin (MTX-HSA) conjugates to free MTX; 1∶0 (HAMC), 3∶1 (HAMC3F), 1∶1 (HAMCF), 1∶3 (HAMCF3) and 0∶1 (HAMF) were investigated in the absence or presence of protease using dissolution tester. In all the HAMs studied except HAMC, the MTX was released bi-exponentially in the absence of protease; an initial fast release period up to approximately 6 h, and thereafter the release rate was very much slower. The fast release of MTX from the HAMs (such as HAMC3F, HAMCF, HAMCF3 and HAMF) at the initial phase is probably due to the release of “physically associated” MTX on the surface and/or entrapped in the near inner surface of HAMs and the slow release at the second phase is due to the release of entrapped free MTX from the core of the HAMs. The initial rate constants were 7.2, 8.7, 8.5 and 5.9 times greater than the second rate constants for HAMF, HAMCF3, HAMCF and HAMC3F, respectively. MTX release from HAMC was very slow and mono-phasic. It was at most 2.2% of the total entrapped amount by 24 h. The protease accelerated the release of MTX from the HAMs. The percentages of MTX released from HAMs up to 24 h were 100, 89.0, 75.0, 66.0 and 61.0% for HAMF, HAMCF3, HAMCF, HAMC3F and HAMC, respectively in the presence of protease and the corresponding values in the absence of protease were 30.2, 19.0, 10.0, 6.5 and 2.2%, respectively.In vitro release of MTX in the presence of protease varied according to the ratios of MTX-HSA conjugates to MTX; the data set from HAMF, HAMCF3 and HAMCF fits better to monophasic first-order profile more adequately than to zero-order profile, that of HAMC3 F to mono-phasic zero-order, and that of HAMC to bi-phasic zero-order. Above results suggested that zero-order release rate can be achieved by adjusting the ratio of MTX-HSA conjugates to MTX in the preparation of HAMs such as HAMC3F.     

Pharmacokinetics of methotrexate after intramuscular injection of methotrexate-polylysine conjugate in rabbits

Methotrexate (MTX)-poly-L-lysine (PLL) conjugate was relatively stable in phosphate buffer of pH 7.4 and in plasma. However, liver homogenate accelerated the release of MTX from the conjugate. Pharmacokinetics and tissue distribution of MTX were compared after intramuscular injection of MTX (treatment I) and MTX-PLL conjugate (treatment II), 10 mg/kg as free MTX to rabbits. The peak concentrations of MTX in treatment II were significantly lower than those in treatment I. The amount of MTX excreted in 24-hr urine was significantly reduced in treatment II and it suggested that MTX be more metabolized in treatment II than in treatment I. The amounts of MTX remaining in each organ after 24-hr of intramuscular injection were not significantly different in both treatments.     

Lipophilic methotrexate conjugates with antitumor activity.

Lipophilic methotrexate (MTX)-lipoamino acid conjugates coupled with amide or ester linkages (1a-1r) were synthesised. The inhibitory activity of the conjugates was evaluated on bovine liver DHFR. The in vitro growth inhibitory effect against MTX-sensitive human lymphoblastoid CCRF-CEM cells and an MTX-resistant sub-line (CEM/MTX), which displays defective intracellular transport of MTX, was determined under short-term and continuous (120-h incubation) exposure conditions. The alpha, gamma, or alpha,gamma amide conjugates showed different activity in inhibiting the growth of parent cells. CEM/MTX cells were much less susceptible than CCRF-CEM cells to inhibition by alpha or alpha,gamma-substituted lipoamino acid conjugates, whereas both cell lines were almost equally sensitive to the MTX-gamma conjugates. Although less potent than MTX, they could partially circumvent the impaired transport system. These findings confirm that lipophilic MTX conjugates may be good lead compounds on the drug development for the treatment of some MTX-resistant tumors. Ester-type conjugates displayed an interesting activity against parent CCRF-CEM cells, although they were less potent against the transport-resistant sub-line. Stability studies on these molecules indicated that they are not degraded into MTX in the culture medium, thus suggesting that they are not able to over-cross cell resistance despite of their lipophilicity.