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.     

Preparation and evaluation of temperature sensitive liposomes containing adriamycin and cytarabine

Temperature sensitive liposomes (TSL) containing adriamycin (ADM) and cytarabine (Ara-C) were prepared. ADM and Ara-C were selected as model compounds of amphiphilic and hydrophilic drug, respectively. Encapsulation efficiency of ADM entrapped into TSL was about twice greater than that of Ara-C. It might be due to different polarity of the drugs. Lipid compositions of TSL had no effect on the encapsulation efficiency of drugs. Thermal behavior of TSL using a differential scanning calorimetry (DSC) was also investigated. Phase transition temperature (T_c) of TSL was dependent on the lipid compositions of TSL.ADM broadened thermogram of TSL but Ara-C did not. However, T_c of TSL was not changed by any drug. Release rate of drugs was highly dependent on temperature. The release profile of ADM was similar to that of Ara-C. The maximum release rate of drugs from TSL was occurred at the near T_c and observed at 39–41°C for DPPC (Dipalmitoylphosphatidylcholine) only, 52–54°C for DSPC (Distearoylphosphatidylcholine) only, 41–43°C for DPPC and DSPC (3∶1), and 43–45°C for DPPC and DSPC (1∶1), respectively. Effect of human serum albumin (HSA) on the release rate of ADM was investigated. HSA had no significant effect on the release of ADM below T_c. However, ADM release from TSL was increased at the near and above T_c. The HSA-induced leakage of drug may result from the interaction of liposomal constituents with HSA structure at the near T_c. From the fact that the release profiles of ADM from freshly prepared TSL and stored TSL for 1 week at 4°C was not changed, the TSL was considered to be stable for at least 1 week at 4°C. Based on these findings, TSL may be useful to deliver drugs to preheated target sites due to its thermal behaviors.     

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.     

Effect of bovine serum albumin on the stability of methotrexate-encapsulated liposomes

The effect of bovine serum albumin (BSA) on the encapsulation efficiency and stability of liposomes containing methotrexate (MTX) having different surface charges and cholesterol contents were investigated. The encapsulation efficiency of MTX was lower and the release of MTX was faster by the addition of BSA. The leaking of MTX from lipid bilayer depends upon the BSA concentrations. These results may be derived from the interaction of BSA with lipid bilayers. The dynamic structural changes of BSA were monitored indirectly using circular dichroism spectra. Observed dynamic structural changes of BSA with liposomes are presumed to reflect the interaction of BSA with liposomes. Negatively charged liposomes have more strong interaction with BSA than neutral and positively charged liposomes. BSA attacks lipid bilayers whether it is at the inner or at the outer phase of lipid bilayer and induces leakage of entrapped MTX. Especially, negatively charged liposomes are more sensitive than others. The inclusion of cholesterol in the lipid layers inhibits the interaction of BSA with liposomes and shows protective effect against BSA-induced leakage of MTX. To endure the attacking of BSA, liposomes as drug carriers should be made using cholesterol.     

The in vivo antitumor activity of LHRH targeted methotrexate-human serum albumin nanoparticles in 4T1 tumor-bearing Balb/c mice

The use of targeted drug delivery systems is a growing trend in cancer treatment to decrease the adverse effect of anti-cancer drugs. In this study, we sought to conjugate methotrexate-human serum albumin nanoparticles (MTX-HSA NPs) with luteinizing-hormone releasing hormone (LHRH). The LHRH was intended to target LHRH receptors overexpressed on the several types of tumors. The expression of LHRH receptors on the 4T1 breast cancer cells was confirmed by FITC conjugated LHRH receptor antibody using fluorescence microscopy. Female Balb/c mice bearing 4T1 breast cancer tumor were treated with a single i.v. injection of free MTX, non-targeted MTX-HSA NPs and LHRH targeted MTX-HSA NPs. LHRH targeted MTX-HSA nanoparticles showed stronger anti-tumor activity in vivo. By 7 days after treatment, average tumor volume in the LHRH targeted MTX-HSA NPs treated group decreased to 8.67% of the initial tumor volume when the number of attached LHRH molecules on MTX-HSA NPs was the highest, while the average tumor volume in non-targeted MTX-HSA NPs treated mice grew rapidly and reached 250.7% of the initial tumor volume 7 days after the treatment. LHRH targeted MTX-HSA NPs could significantly extend the survival time of tumor bearing mice compared with the non-targeted MTX-HSA NPs and free MTX formulations.     

Effect of Polymer Blending on the Release of Ganciclovir from PLGA Microspheres

Purpose The aim of the study is to investigate the effect of polymer blending on entrapment and release of ganciclovir (GCV) from poly(d,l-lactide-co-glycolide) (PLGA) microspheres using a set of empirical equations. Methods Two grades of PLGA, PLGA 7525 [d,l-lactide:glycolide(75:25), MW 90,000–126,000 Da] and Resomer RG 502H [d,l-lactide:glycolide(50:50), MW 8000 Da], were employed in the preparation of PLGA microspheres. Five sets of microsphere batches were prepared with two pure polymers and their 1:3, 1:1, and 3:1 blends. Drug entrapment, surface morphology, particle size analysis, drug release, and differential scanning calorimetric studies were performed. In vitro drug-release data were fitted to a set of empirical sigmoidal equations by nonlinear regression analysis that could effectively predict various parameters that characterize both diffusion and degradation cum diffusion-controlled release phases of GCV. Results Entrapment efficiencies of GCV ranged from 47 to 73%. Higher amounts of GCV were entrapped in polymer blend microspheres relative to individual polymers. Triphasic GCV release profiles were observed, which consisted of both diffusion and degradation cum diffusion-controlled phases. In vitro GCV release was shortest for Resomer RG 502H microsphere (10 days) and longest for PLGA 7525 microspheres (90 days). Upon blending, the duration of release gradually decreased as the content of Resomer RG 502H in the matrix was raised. Equations effectively estimated the drug-release rate constants during both the phases with high R² values (>0.990). GCV release was slower from the blend microsphere during the initial diffusion phase. Majority of entrapped drug (70–95%) was released during the matrix degradation cum diffusion phase. Conclusions Drug entrapment and release parameters estimated by the equations indicate more efficient matrix packing between PLGA 7525 and Resomer RG 502H in polymer-blended microspheres. The overall duration of drug release diminishes with rising content of Resomer RG 502H in the matrix. Differential scanning calorimetry studies indicate stronger binding between the polymers in the PLGA 7525/Resomer RG 502H∷ 3:1 blend. Polymer blending can effectively alter drug-release rates of controlled delivery systems in the absence of any additives.     

Reversible binding of tolmetin,zomepirac, and their glucuronide conjugates to human serum albumin and plasma

Acyl glucuronides of drugs and bilirubin have been shown in the past decade to be reactive metabolites undergoing acyl migration and irreversible binding. The latter reaction has been hypothesized to be facilitated by or to proceed through the formation of a reversible complex. Furthermore, it has been suggested that the decreased binding seen in patients with compromised excretory function may be due to competition by elevated plasma concentrations of the glucuronides. In these reversible binding studies, we characterized the extent and the “site” of binding of tolmetin, zomepirac, their glucuronides and isomeric conjugates. We also examined the displacement between the parent drugs and their glucuronide conjugates using a rapid ultrafiltration method. Tolmetin exhibited three classes of binding sites with a primary association constant of 1.7×10⁶ M⁻¹ (K_dl=0.60 μM). The primary association constant of zomepirac (1.16×10⁶ M⁻¹, K_dl=0.86 μM) is similar to that of tolmetin. The β 1 and α/β3 glucuronides of both compounds bind to a lesser extent than their parent aglycones. The isomeric glucuronide conjugates of both compounds showed much stronger binding than the β/1 conjugates. Of the four glucuronides investigated, tolmetin glucuronide-α/β3 isomer was bound by fatty acid free human serum albumin with the highest affinity (4.6×10⁵ M⁻¹, K_d=2.22 μM). Protein binding of the parent drugs and conjugates were decreased significantly at pH 5.0. In displacement studies, except for salicylate and acetylsalicylate, drugs known to bind to Sites I and II as well as the digitoxin and tamoxifen binding sites had little inhibitory effect on the binding of tolmetin, zomepirac, and their glucuronide conjugates. Supported in part by Grant GM 36633 from the National Institute of General Medical Sciences.     

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.     

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.     

Controlled release of dexamethasone from PLGA microspheres embedded within polyacid-containing PVA hydrogels

The development of zero-order release systems capable of delivering drug(s) over extended periods of time is deemed necessary for a variety of biomedical applications. We hereby describe a simple, yet versatile, delivery platform based on physically cross-linked poly(vinyl alcohol) (PVA) microgels (cross-linked via repetitive freeze/thaw cycling) containing entrapped dexamethasone-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres for controlled delivery over a 1-month period. The incorporation of polyacids, such as humic acids, Nafion, and poly(acrylic acid), was found to be crucial for attaining approximately zero-order release kinetics, releasing 60% to 75% of dexamethasone within 1 month. Microspheres alone entrapped in the PVA hydrogel resulted in negligible drug release during the 1-month period of investigation. On the basis of a comprehensive evaluation of the structure-property relationships of these hydrogel/microsphere composites, in conjunction with their in vitro release performance, it was concluded that these polyacids segregate on the PLGA microsphere surfaces and thereby result in localized acidity. These surface-associated polyacids appear to cause acid-assisted hydrolysis to occur from the surface inwards. Such systems show potential for a variety of localized controlled drug delivery applications such as coatings for implantable devices.     

Neurosteroids may differentially affect the function of two native GABAA receptor subtypes in the rat brain

Hippocampal noradrenergic and cerebellar glutamatergic axon terminals are known to possess GABA_A receptors mediating, respectively, enhancement of noradrenaline (NA) and glutamate release. It has been recently found that the hippocampal receptor is benzodiazepine-sensitive, whereas the cerebellar receptor is insensitive to benzodiazepine agonists. We here tested the effects of neurosteroids on these two native GABA_A receptors using superfused rat hippocampal and cerebellar synaptosomes. Allopregnanolone (3α,5α-P), at nanomolar concentrations, potentiated the GABA-induced [³H]-NA release from superfused hippocampal synaptosomes; in the absence of GABA, the steroid was ineffective up to 10μM. The enhancement by GABA of the K⁺-evoked [³H]-D-aspartate release from cerebellar synaptosomes also was potentiated by nanomolar 3α,5α-P; in addition, at 1–10μM, the steroid increased [³H]-D-aspartate release in the absence of GABA. Both in hippocampus and cerebellum the potentiations of the GABA effects produced by nanomolar 3α,5α-P were abolished by dehydroepiandrosterone sulphate (DHEAS). Added up to 10μM, DHEAS could not inhibit the effects of GABA alone. The enhancement of [³H]-D-aspartate release elicited by 3μM 3α,5α-P in the absence of added GABA was antagonized completely by bicuculline and picrotoxin and halved by DHEAS. To conclude, 3α,5α-P, at nanomolar concentrations, behaves as a positive allosteric GABA modulator at both the GABA_A receptors under study. Low micromolar 3α,5α-P can directly activate the cerebellar receptor, whereas the hippocampal GABA_A receptor is insensitive to the neurosteroid alone. DHEAS appears to be a pure antagonist at the neurosteroid allosteric sites. Along with the previously observed differential sensitivity to benzodiazepines, the present data strengthen the idea that the two receptors investigated represent native subtypes of the GABA_A receptor having distinct pharmacology, neuronal localization and function. Received: 13 October 1997 / Accepted: 30 December 1997     

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.     

Hydrolytic Degradation and Drug Release of Ricinoleic Acid–Lactic Acid Copolyesters

A systematic study on the degradation and drug release from l-lactic acid and ricinoleic-acid-based copolyesters is reported. These copolyesters were synthesized by ring opening polymerization (ROP), melt condensation (COND) and transesterification (TRANS) of high molecular weight poly(lactic acid) (PLA) with ricinoleic acid (PLA-RA), and repolymerization by condensation to yield random and block copolymers of weight average molecular weights (Mw) between 3000 and 13,000. All polymers showed an almost zero-order weight loss, with a 20–40% loss after 60 days of incubation. Lactic acid release to the degradation solution is proportional to weight loss of the polymer samples. The main decrease in molecular weight was observed during the first 20 days, followed by a slow degradation phase, which kept the number average molecular weight (Mn) at 4000–2000 for another 40 days. Water-soluble 5FU was released from ricinoleic-acid-based polymers faster than slightly water-soluble triamcinolone. Drug release into phosphate-buffered saline (pH 7.4, 0.1 M) at 37°C from P(LA-RA) 60:40 prepared by condensation of the acids was faster than from pasty P(PLA-RA) 60:40 synthesized by transesterification for both drugs.     

聚氧乙烯骨架缓释片的处方及体外释药机制研究

目的以聚氧乙烯（PEO）为亲水凝胶骨架制备缓释片剂,并考察其体外释药机制。方法基于两种规格PEO的用量比与释药速率之间的关系,优化缓释片处方。通过考察片剂的体外释放度和溶蚀比探讨其释药机制,并对不同溶解度药物的体外释放行为进行比较。结果缓释片体外释药速率与PEO用量比呈线性关系,所得优化处方在12h内以接近恒速释药,其体外释药与溶蚀过程基本同步,且在所考察用量范围内不同溶解度药物的体外释放度相近。结论PEO制成的亲水凝胶骨架片缓释性能良好,其体外释药是药物扩散和骨架溶蚀协同作用的结果。     

Controlled Delivery of Pilocarpine. 1. In Vitro Characterization of Gelfoam® Matrices

The potential of Gelfoam absorbable gelatin sponge as a carrier for ophthalmic delivery of pilocarpine was examined. Prolonged in vitro release of pilocarpine was achieved through pharmaceutical modification of the device by embedding a retardant in the pores. The device embedded with cetyl ester wax released pilocarpine in a zero-order pattern (release exponent = 0.93 ± 0.04) for up to 5 hr. This result corresponded well with a linear penetrant uptake by this device. The device impregnated with polyethylene glycol 400 mono-stearate exhibited anomalous drug transport with a release exponent of 0.63 ± 0.02. The absorption of water by this retardant and the formation of a gel layer on the surface slowed the penetration of the release medium into the deeper sections of the matrix, as well as the rapid outward diffusion of drug, resulting in a prolonged release of pilocarpine.     

Preparation and characterization of methotrexate-loaded microcapsules

Abstract

Methotrexate (MTX) has toxic effect to healthy tissues. Microencapsulation coats particles with a functional coat to optimize storage stability and to modulate release. In the present study, a new MTX encapsulated microcapsules were synthesized for controlling MTX release. Controlled drug release provides releasing of efficient dose and prevent drug side effect to tissues and also protects MTX from oxygen, pH and other interactions. MTX was encapsulated through biocompatible hyaluronic acid (HA) and sodium alginate (SA) with an encapsulation system to reduce its toxicity and for controlled release. The microcapsules prepared by vibrating nozzle were cross-linked with SA, HA and calcium chloride. Nozzle diameter and MTX concentration were changed according to loaded MTX and encapsulation efficiency were determined using HPLC. For the reliability of the data, validation studies of the HPLC method were performed. The precision of the method was demonstrated using intra- and inter-day assay relative standard deviation (RSD) values which are less than 2% in all instances. For the characterization of microcapsules, particle size, drug loading and in vitro drug release studies were performed. Diameters of MTX-loaded microcapsules were acquired approximately 160, 400 and 800?µm. Surface morphology of encapsulated microcapsules were displayed with light microscope. Eighty-nine percent MTX encapsulation efficiencies were achieved. Encapsulated MTX microcapsules showed controlled release when compared to pure MTX. While powder MTX dissolved completely in 10?min in the dissolution medium, MTX release from encapsulated MTX microcapsules became 40?h in 0.1?M PBS pH 7.4, including NaCl. MTX release from MTX-loaded microcapsules was reached to 79%. Moreover, drug efficiency was examined in vitro cell culture tests. Viability of 5RP7 cells were decreased to 88.5% for 96?h. When MTX was given directly to 5RP7 cells, viability of 5RP7 cells was decreased to 49.7% for 96?h. Flow cytometry studies also showed that, MTX microcapsules induced apoptosis. The goal of this study is to provide controlled release of MTX and to reduce the toxic effect of MTX.     

Effects of gabapentin and pregabalin on K<Superscript>+</Superscript>-evoked <Superscript>3</Superscript>H-GABA and <Superscript>3</Superscript>H-glutamate release from human neocortical synaptosomes

One site of action of the anticonvulsant, analgesic, and anxiolytic drugs gabapentin and pregabalin is the α₂δ-subunit of voltage-sensitive Ca²⁺ channels (VSCC). We therefore analyzed the effects of gabapentin and pregabalin on K⁺-evoked release of ³H-γ-aminobutyric acid (GABA) and ³H-glutamate from superfused human neocortical synaptosomes. These neurotransmitters are released by Ca²⁺-dependent exocytosis and by Ca²⁺-independent uptake reversal. When a GABA transport inhibitor was present throughout superfusion to isolate exocytotic conditions, gabapentin and pregabalin (100 μM each) reduced K⁺-evoked ³H-GABA release by 39% and 47%, respectively. These effects were antagonized by the α₂δ-ligand l-isoleucine (1 μM) suggesting the α₂δ-subunit of terminal VSCC to mediate the reduction of exocytosis. Both drugs had no effect on exocytotic ³H-glutamate release and also failed to modulate the release of ³H-GABA and ³H-glutamate caused by reversed uptake in the absence of external Ca²⁺. Thus, an inhibition of glutamate release by gabapentin and pregabalin as main anticonvulsant principle is not supported by our experiments. An anticonvulsant mode of action of both drugs may be the reduction of a proconvulsant exocytotic GABA release.     

Methotrexate: a detailed review on drug delivery and clinical aspects

Introduction: Uses of methotrexate (MTX) are well established for the treatment of various types of malignancy, psoriasis, rheumatological diseases and the medical termination of pregnancy. Formulation and targeting approaches for MTX with controlled release carriers, multiparticulate systems, prodrug and drug conjugates have been found to improve bioavailability, reduce adverse effects and maximize clinical efficacy, compared with conventional methods.

Areas covered: This exhaustive literature survey on different electronic databases covers drug delivery and clinical trials on MTX. This review deals with the challenges and achievements of controlled release, multiparticulate, prodrug and drug conjugate systems of MTX.

Expert opinion: Therapeutic drug monitoring of MTX is crucial to attain a good efficacy. In spite of the advantages of multiparticulate, prodrug and drug conjugates, clinical applications of such formulations of MTX are still under infancy. These drug delivery systems require the special attention of medical experts for its wider clinical usage, and pharmaceutical experts for its scale-up. The combination of MTX with other antineoplastic and immunosuppressants should also be subjected to clinical trials, such as the combination of misoprostol with MTX in abortion.     

Growth inhibition, drug load, and degradation studies of gelatin/methotrexate conjugates

Macromolecular gelatin-methotrexate conjugates have potential therapeutic advantages over the free drug. Conjugates with MTX:gelatin molar ratios (MR) ranging from 1:1 to 27:1 were examined for cell growth inhibition, stability, degradation, and methotrexate (MTX) release. Conjugate growth inhibition was less than that of free MTX whose IC(50) value of 1.3 x 10(-8) M was about 10-fold less. Cell uptake of fluorescein labeled gelatin (145 kD) was observed by 24-30 h. Higher MR conjugates produced less growth inhibition, measurably greater stability at pH 7.4 based on MTX release, and had less gelatin degradation in the conjugate by the lysosomal enzyme Cathepsin B (Cat B) compared to low MR conjugates. Cat B conjugate degradation was greater at the in vitro lysosomal pH of 4.8 than the intra-tumor pH of 6.5. The presence of Cat B did not meaningfully affect MTX release, but less MTX was released at pH 4.8 than pH 6.5. The maximum MTX release was a relatively low 7% after 72 h at pH 6.5 for the low MR conjugate. Low molecular weight conjugate fragments were also produced and were also influenced by pH and MR. Reduced growth inhibition by high MR conjugates may be due to a hindered enzymatic degradation in the lysosomes. A strong peptide conjugate bond at lysosomal pH and a 24-30 h delayed gelatin uptake may contribute to reduced growth inhibition of the conjugate compared to free MTX. MTX release under these in vitro conditions occurs by aqueous hydrolysis, not by Cat B cleavage of the conjugate bond.