Biorecognition of HPMA Copolymer-Adriamycin Conjugates by Lymphocytes Mediated by Synthetic Receptor Binding Epitopes

Purpose. The EDPGFFNVE nonapeptide (NP) was recognized as the CD21 (CR2) binding epitope of the Epstein-Barr virus (EBV) gp350/ 220 envelope glycoprotein which mediates the virus attachment to human B lymphocytes (Nemerow et al., Cell 56:369-377, 1989). Here we evaluated the targeting potential of a synthetic receptor binding epitope (NP) covalently attached to a water-soluble polymeric drug carrier. In particular, the biorecognition of N-(2-hydroxypropyl) metha-crylamide (HPMA) copolymer-NP conjugates by B- and T-cells and the cytotoxicity of HPMA copolymer-NP-adriamycin (ADR) conjugates toward B-cells, T-cells, and peripheral blood lymphocytes (PBL) were evaluated. Methods. HPMA copolymer-NP and optionally ADR conjugates varying in the NP density and mode of NP attachment were incubated with Raji B-cells (human Burkitt's lymphoma), CCRF-CEM T-cells (acute human lymphoblastic leukemia), and CCRF-HSB-2 T-cells (human lymphoblastic leukemia). The kinetics of binding was studied, the Langmuir adsorption isotherms analyzed, binding constants calculated, and IC₅₀ doses determined. Results. Flow cytometry studies revealed that binding was homogeneous to both cell types. The apparent binding constants to T-cells were about two times higher when compared to B-cells. The binding and cytotoxicity increased with increased amount of epitopes per polymer chain. Attachment of the NP via a GFLG spacer resulted in increased biorecognition when compared with conjugates containing NP bound via a GG spacer. HPMA copolymer-NP-ADR conjugates possessed specific cytotoxicity to T- and B-malignant cells. Concentrations, which were lethal to the latter, were not toxic for PBL. Conclusions. The data obtained seem to indicate the potential of the HPMA copolymer-NP conjugates as polymer anticancer drug carriers targetable to immunocompetent cells.     

Targeted Delivery of Doxorubicin by HPMA Copolymer-Hyaluronan Bioconjugates

Purpose. Overexpression of hyaluronan (HA) receptors on cancer cells results in enhanced endocytotic uptake of the drug conjugate. An N-(2-hydroxypropyl)methacrylamide (HPMA)-HA polymeric drug delivery system was used for targeted delivery of doxorubicin to cancer cells. Methods. HA-doxorubicin (DOX) bioconjugates (HA-DOX), and HPMA copolymer-DOX conjugates containing HA as a side chain (HPMA-HA-DOX) were synthesized. The cytotoxicity of the polymer-drug conjugate was evaluated via in vitro cell culture. The internalization of the conjugate was visualized by fluorescence microscopy. Results. Cytotoxicity of HPMA-HA-DOX targeted bioconjugate was higher against human breast cancer (HBL-100), ovarian cancer (SKOV-3), and colon cancer (HCT-116) cells when compared to the non-targeted HPMA-DOX conjugate. Fluorescence confocal microscopy revealed that the targeted HPMA-HA-DOX conjugates were internalized more efficiently by cancer cells relative to the non-targeted HPMA-DOX conjugate. Both HPMA-DOX and HPMA-HA-DOX showed minimal cytotoxicity toward mouse fibroblast NIH 3T3 cells. The internalization of polymer conjugates was correlated with their cytotoxicity. Conclusions. Selective delivery of anti-cancer agents to cancer cells was achieved by biochemical targeting. The HA-modified HPMA copolymer showed improved toxicity due to receptor-mediated uptake of the macromolecular drug.     

The Influence of a Colonic Microbiota on HPMA Copolymer Lectin Conjugates Binding in Rodent Intestine

Germ-free (GF) animals lack a colonic microflora like that seen in conventional (CV) animals. Bacterial presence plays a role in the development of glycoproteins in the gastrointestinal (GI) tract; the absence of a microbiota has been seen to suppress the production of certain glycoproteins and glycolipids. Binding patterns of lectins are modified when glycoprotein structures are altered (e.g., during development or disease). Little information on lectin binding patterns in mature GF animals is available. We examined the binding of free and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-conjugated fluorescein isothiocyanate (FITC)-labeled wheat germ agglutinin (WGA) [P(HPMA)-(WGA-FITC)] and FITC-labeled peanut agglutinin (PNA) [P(HPMA)-(PNA-FITC] in CV and GF mouse colon with and without neuraminidase pretreatment. Anti-Thomsen-Friedenreich (TF) antigen (a development and disease-related glycoprotein) antibody binding was also examined in these tissues. Subtle differences were seen in the binding patterns between CV and GF animals. CV animals showed strong P(HPMA)-(WGA-FITC) binding in goblet cells, but minimal P(HPMA)-(PNA-FITC) binding was visible. In GF animals, luminal surface binding of P(HPMA)-(WGA-FITC) was visible, and goblet cell binding of P(HPMA)-(PNA-FITC) was seen. These subtle changes suggest that altered glycoprotein expression occurred under GF conditions.     

HPMA Copolymer-Anticancer Drug-OV-TL-TL16 Antibody Conjugates. 1. Influence of the Method of Synthesis on the Biding Affinity to OVCAR-3 Ovarian Carcinoma Cells In Vitro

The influence of different methods of binding the OV-TL16 antibody and its Fab′ fragment to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer—drug (adriamycin {ADR} or meso chlorin e₆ mono(N-2-aminoethylamide) {Mce₆} conjugates on the affinity of conjugates to an ovarian carcinoma (OVCAR-3) cell associated antigen was investigated. The binding of the antibody to HPMA copolymer—drug (ADR or Mce₆) conjugates via amino groups resulted in conjugates which were heterogeneous in their antigen binding. Coupling the HPMA copolymer—Mce₆ conjugate to the carbohydrate region of the antibody resulted in conjugates with a more homogeneous distribution of affinity constants than conjugates prepared by linking the antibody to the polymer via amino groups. However, both methods resulted in a decrease in the affinity constant compared to the native antibody. Conjugates prepared with the Fab′ fragment of the OV-TL16 antibody demonstrated a more homogeneous affinity than either conjugate prepared with the whole antibody. To verify the hypothesis that the changes in the binding affinity and homogeneity are a consequence of conformational changes in the antibody structure, a series of physicochemical methods where employed to characterize the conjugates. The excitation energy transfer between OV-TL16 antibody and drugs (ADR and Mce₆) and the spectral properties of Mce₆ were used to monitor the interactions between the antibody and drugs. The quenching of the intrinsic fluorescence of the antibody was also employed to study its conformational changes. An attempt has been made to correlate the biorecognition at the cellular surface with the interactions of drug with the antibody molecule and with changes in antibody conformation.     

Comparison of active and passive targeting of doxorubicin for somatostatin receptor 2 positive tumor models by octreotide-modified HPMA copolymer-doxorubicin conjugates

Somatostatin receptor 2 (SSTR2), specifically over-expressed on many tumor cells, is a potential receipt for active targeting in cancer therapy. In the present study, octreotide (Oct), which had high affinity to SSTR2, was attached to N-(2-hydroxypropyl) methacrylamide (HPMA) polymeric system to enhance the antitumor efficiency of the anticancer drug doxorubicin (DOX). Two kinds of cell lines (HepG2 and A549), which overexpress SSTR2, were chosen as cell models. Compared with non-modified conjugates, Oct-modified conjugates exhibited superior cytotoxicity and intracellular uptake on both HepG2 and A549 cell lines. This might be due to the mechanism of receptor-mediated endocytosis. Subsequently, the in vivo biodistribution and antitumor activity evaluations showed that Oct modification significantly improved the tumor accumulation and antitumor efficacy of HPMA copolymer conjugates in SSTR2 over-expressed Kunming mice bearing H₂₂ tumor xenografts. In summary, Oct-modified HPMA polymer-DOX conjugates might be a promising system for the treatment of SSTR2 over-expressed cancers.     

Properties Influencing the Relative Binding Affinity of Pteroate Derivatives and Drug Conjugates Thereof to the Folate Receptor

Purpose  Using in vitro competition assays, determine salient chemical features of pteroates and pteroate-drug conjugates which afford high affinity to the folate receptor. Materials and Methods  Both folate binding protein-coated polystyrene plates and adherent human cell-based assays were used to evaluate the effects of assay temperature and buffer composition on pteroate/pteroate-drug conjugate binding affinity. Following assay selection and optimization, the relative binding affinities of ten vitamers and derivatives as well as seven pteroate-drug conjugates were evaluated. Results  Compared to polystyrene plates containing immobilized folate binding protein, adherent KB cells were determined to be an equally effective, more desirable source of folate receptor for such analyses. Using the latter method, we discovered that a charged group positioned in close proximity to the pteroate’s aryl moiety is critical for retaining high binding affinity. We also found that a diverse set of bioactive small molecule agents can be attached to folic acid in a manner that does not appreciably disturb this vitamin’s intrinsic high affinity for the folate receptor. However, conjugation of lipophilic, high protein-binding agents to folate was sometimes found to dramatically reduce affinity, which is a finding that best exemplifies the need for having a reliable in vitro assay for determining a compound’s RA. Conclusion  Molecules which bind best to the human folate receptor are those that contain hydrophilic regions distal to the ligand’s aryl group, and for drug conjugates, an extended hydrophilic spacer placed in-between the pteroate and drug cargo moieties.     

Synthesis and in vitro anti-tumor activity of novel HPMA copolymer-drug conjugates with potential cell surface targeting property for carcinoma cells

In several groups of malignant tumors including head and neck tumors, a protein named Hsp47/CBP2 leaked from the cell was expressed on the tumor cell surface. Several synthetic peptides have been identified as effective ligands for binding to Hsp47/CBP2. This study has focused on the synthesis and in vitro characterization of a targeting delivery system of 5-fluorouracil (5-FU) to human head and neck squamous cell carcinoma (HNSCC) in order to improve anti-cancer efficacy and reduce dose-limiting toxicity of 5-FU. An N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer, with Hsp47/CBP2 binding peptide sequence (namely WHYPWFQNWAMA) as a targeting ligand, was synthesized by a novel and simplified synthetic route. Under the controlled synthetic conditions, 1,3-dimethylol-5-FU, derived from 5-FU, was attached to the HPMA copolymer backbone via the lysosomally degradable GFLG linker, while the WHYPWFQNWAMA was conjugated via a non-degradable Gly-Gly (GG) linker. A control polymer without targeting moiety was also synthesized (P-FU). The in vitro cytotoxicity, internalization and apoptosis assays of the polymeric conjugates were evaluated. The characteristic apoptotic morphological changes were also assessed. Compared to 5-FU and P-FU, the HPMA copolymer containing the Hsp47/CBP2 binding peptide (P-FU-peptide) exhibited the highest cytotoxic efficacy to cell line of human head and neck squamous cell carcinoma (p < 0.05) and was internalized much faster than P-FU, especially after being incubated for 30 min. Both of the morphology and apoptosis analyses demonstrated that the treatment of P-FU-peptide resulted in more apoptotic and necrotic induction of tumor cells than P-FU. Meanwhile, the rate of apoptosis induced by P-FU-peptide was higher than that of necrosis. In summary, the HPMA copolymer-Hsp47/CBP2 binding peptide conjugates showed a promising future for the treatment of HNSCC with improved efficacy.     

Binding characteristics of (-)- and (+)-nicotine to the rat brain P2 fraction

Saturation studies employing (-)- and (+)-[3H]nicotine indicate that the isomers bind to different very high and high affinity sites since the binding density for (-)-[3H]nicotine is 10 times that for (+)-[3H]nicotine. Both isomers also bind to a low affinity site (KDS = approximately 10(-5) to 10(-4) M). Competition studies employing unlabelled (-)- and (+)-nicotine reveal greater complexities. The isomers also appear to bind to a separate site which enhances binding at the (-)- and (+)-nicotine high affinity sites. (+)-Nicotine is more effective in increasing the binding of (-)-[3H]nicotine at its high affinity site than (-)-nicotine. Further, (+)-nicotine has a greater specificity for enhancing binding than (-)-nicotine in that it enhances (-)-[3H]nicotine binding at lower concentrations and inhibits binding at higher concentrations than (-)-nicotine.     

Binding of β-Carbolines and Tetrahydroisoquinolines by Opiate Receptors of the δ-Type

Abstract: Effects of various β-carbolines (BC's) and two tetrahydroisoquinolines (TIQ's) on the specific binding of a natural opiate δ-receptor ligand, leucine enkephalin, have been studied in rat synaptosomal membranes, and compared with the effects on the binding of mu-receptor ligands dihydromorphine and naloxone. Harmaline (7-MeO-1-Me-dihydro-BC) was the most potent compound studied (Ki value 3.5 μM), while the two TIQ's (salsolinol and salsolidine) were less potent than BC's (Ki> 100 μM) in inhibiting the binding of δ-receptors. In general, BC's showed more affinity for δ-receptors than for μ-receptors; salsolinol was more potent against the binding of μ-receptors. Inhibition of binding was generally of the competitive type: Kd values increased and Bmax values were not altered. The Na dependence suggests that BC's and salsolinol are antagonists or partial agonists of opioids. Since the binding affinity of BC's and TIQ's was on the micromolar level only, the opiate receptors do not appear to be the major sites of action for BC's or TIQ's.     

Binding of certain acidic drugs to human albumin: Theoretical and practical estimation of fundamental parameters

Summary The binding to human albumin of four acidic drugs, acenocoumarin, sodium chlorophenoxyisobutyrate, phenylbutazone and warfarin, has been measured by equilibrium dialysis and ultrafiltration at 37°C and pH 7.4. The affinity constants and numbers of binding sites for each drug were then calculated according to the law of mass action. With the aid of these values, it was possible to estimate the percentage binding of each drug after a therapeutic dose and assuming an albumin concentration of 40 g/l. The results obtained were identical to those previously measuredin vivo.     

The Determination of Naproxen by Spectrofluorometry and its Binding to Serum Proteins

Abstract A fluorometric method for the determination of naproxen in serum, albumin solutions and protein free buffer solutions is described. The detection limit is about 10 ng/ml. Furosemide, thiopental and salicylic acid did not show any disturbing fluorescence while phenprocoumon did. The in vitro binding of naproxen in albumin solutions and serum was studied by equilibrium dialysis. A small but significant increase was found in the percentual binding in albumin solutions as compared to serum. The percentual binding was not affected by changes in the pH from 5-8. By fitting the binding data to a model assuming two classes of binding sites, association constants and binding capacities were determined. A very high affinity and a high capacity were found. The association constant for the first class of binding sites was higher for human serum albumin than for Serum (8.5 versus 5.3 · 10⁶ M^-1). The difference in the protein binding to the first class of binding sites in human serum albumin and serum can be explained by the existence of a competitive inhibitor in serum.     

Effect of FSH Receptor‐Binding Inhibitor‐8 on FSH‐Mediated Granulosa Cell Signaling and Proliferation

Follicle‐stimulating hormone is important for mammalian reproduction. It acts through specific receptors located on the plasma membrane of granulosa cells in ovaries and Sertoli cells in testes. The binding of follicle‐stimulating hormone to its receptor activates intracytoplasmic signaling pathways leading to steroidogenesis. These steroids in turn regulate the follicle‐stimulating hormone action from the anterior pituitary through exerting negative feedback effect. In addition to steroids, non‐steroidal factors secreted by the ovaries are believed to modulate follicle‐stimulating hormone action through autocrine/paracrine mode. One such low molecular weight peptide referred to as follicle‐stimulating hormone receptor‐binding inhibitor‐8 purified from human follicular fluid has been extensively studied. Follicle‐stimulating hormone receptor‐binding inhibitor‐8 has been shown to inhibit binding of follicle‐stimulating hormone to its receptor. The present article describes the effect of follicle‐stimulating hormone receptor‐binding inhibitor‐8 on follicle‐stimulating hormone‐induced signaling in rat granulosa cells. Follicle‐stimulating hormone receptor‐binding inhibitor‐8 inhibited the follicle‐stimulating hormone‐induced cAMP, and the effect was observed to be mediated through the protein kinase A. Further, an inhibitory effect of follicle‐stimulating hormone receptor‐binding inhibitor‐8 on the granulosa cell proliferation was evaluated using COV434 cell line which is derived from the human granulosa cell tumor. The effect of the peptide on the cell cycle analysis showed an increase in apoptotic population and the arrest of G1 phase. These findings suggest that follicle‐stimulating hormone receptor‐binding inhibitor‐8 acts as a follicle‐stimulating hormone antagonist and affects the follicle‐stimulating hormone‐mediated signaling and proliferation in the granulosa cells.     

芳烷酮哌嗪类化合物SCP-1和SCP-2与5-HT1,2受体的结合实验

目的研究芳烷酮哌嗪类化合物SCP-1和SCP-2与5-HT1和5-HT2受体体外是否有结合作用.方法5-HT1受体取材于大鼠大脑皮质,5-HT2受体取材于大鼠海马.采用受体-配体结合测定法测定10-3mol·L-1的SCP-1和SCP-2对5-HT1,5-HT2受体的最大结合率、半数抑制浓度(IC50)和Hill系数.结果10-3mol·L-1的SCP-1和SCP-2与5-HT1受体的最大结合率分别为75%和63%.SCP-1和SCP-2与5-HT1受体结合的IC50分别为1.584和5.495μmol·L-1,二者与5-HT1受体结合的Hill系数分别为0.98和1.02.10-3mol·L-1的SCP-1和SCP-2与5-HT2受体的最大结合率分别为92%和87%.SCP-1和SCP-2与5-HT2受体结合的IC50分别为1.0和2.512μmol·L-1,二者与5-HT2受体结合的Hill系数分别为0.86和0.88.结论SCP-1和SCP-2与5-HT1受体的结合方式是与单一受体位点结合,并且这种结合服从质量作用定律(Hill系数接近于1).SCP-1和SCP-2与5-HT2受体呈现不规则性结合,也可能有负相互作用或多种结合位点(Hill系数偏离1).     

Differences in the Intracellular Distribution of Acid-Sensitive Doxorubicin-Protein Conjugates in Comparison to Free and Liposomal Formulated Doxorubicin as Shown by Confocal Microscopy

Purpose. To investigate differences in the cellular uptake and intracellular distribution of protein-bound doxorubicin in comparison to free doxorubicin and a liposomal formulation (CAELYX®) Methods. LXFL 529 lung carcinoma cells were incubated with an acid-sensitive transferrin and albumin conjugate of doxorubicin, a stable albumin doxorubicin conjugate, and free and liposomal doxorubicin for up to 24 h. The uptake of doxorubicin was detected with confocal laser scanning microscopy (CLSM). To investigate the intracellular localization of the anticancer drug, lysosomes, Golgi apparatus, and mitochondria were also stained by various organelle-specific fluorescent markers. In vitro efficacy of the doxorubicin derivatives was examined with the BrdU incorporation assay. Results. The acid-sensitive albumin and transferrin doxorubicin conjugates showed enhanced cytotoxicity in comparison to liposomal doxorubicin, whereas the stable albumin-doxorubicin conjugate showed only marginal activity. Of all compounds tested, doxorubicin showed the highest cytotoxicity. CLSM studies with specific markers for lysosomes, mitochondria, and the Golgi apparatus demonstrated that protein-bound doxorubicin or liberated doxorubicin was accumulated in the mitochondria and Golgi compartments, but not in the lysosomes after 24 h. Free doxorubicin showed a time-dependent intracellular shift from the nucleus to the mitochondria and Golgi apparatus. Fluorescence resulting from incubation with CAELYX was primarily detected in the nucleus. Conclusions. Our results indicate that other organelles in addition to the cell nucleus are important sites of accumulation and interaction for protein-bound doxorubicin or intracellularly released doxorubicin as well as for free doxorubicin.     

Enhanced Cytotoxicity to Cancer Cells by Codelivery and Controlled Release of Paclitaxel‐loaded Sirolimus‐conjugated Albumin Nanoparticles

Recently, it is suggested that mTOR signaling pathway is an important mediator in many cancers especially breast cancer. Therefore, effects of sirolimus as a mTOR inhibitor in breast cancer have been studied in combination with paclitaxel with or without controlled release effect. In this work, we prepared a water‐soluble formulation of sirolimus‐conjugated albumin nanoparticles loaded with paclitaxel, to study the effects of sirolimus concentration when it releases more later than paclitaxel in comparison with sirolimus–paclitaxel‐loaded albumin nanoparticles. Also effects of paclitaxel loading on cytotoxic properties of nanoparticles were studied. Sirolimus was succinylated at 42‐OH with enzymatic reaction of Candida antarctica lipase B, and then its carboxylic group was activated with EDC/NHS and conjugated to the lysine residues of albumin. Paclitaxel was loaded on albumin surface by nab technique in concentration range of 0–10 μg/mL. Sirolimus‐conjugated nanoparticles with 0.01 μg/mL paclitaxel showed lowest cell viability of 44% while it was 53% for non‐conjugated nanoparticles in MDA‐MB‐468 cell lines after 48 h (p‐value = 0.003). In MCF‐7 cell lines, sirolimus‐conjugated nanoparticles with 0.1 μg/mL paclitaxel showed lowest cell viability of 35.69% while it was 48% for non‐conjugated nanoparticles after 48 h (p‐value = 0.03). We guess that when cancer cell lines arrest in G2‐M by anticancer drugs like paclitaxel, Akt activates mTOR to make cells continue living, then inhibiting mTOR can enhance anticancer effects.     

Fatty acid and drug binding to a low-affinity component of human serum albumin,purified by affinity chromatography

Binding equilibria for decanoate to a defatted, commercially available human serum albumin preparation were investigated by dialysis exchange rate determinations. The binding isotherm could not be fitted by the general binding equation. It was necessary to assume that the preparation was a mixture of two albumin components about 40% of the albumin having high affinity and about 60% having low affinity. By affinity chromatography we succeeded in purifying the low-affinity component from the mixture. The high-affinity component, however, could not be isolated. We further analyzed the fatty acid and drug binding abilities of the low-affinity component. The fatty acids decanoate, laurate, myristate and palmitate were bound with higher affinity to the mixture than to the low-affinity component. Diazepam was bound with nearly the same affinity to the low-affinity component as to the albumin mixture, whereas warfarin was not bound at all to the low-affinity component.     

Binding of cations to individual gamma-carboxyglutamate residues of conantokin-G and conantokin-T

Abstract: Conantokin-G (con-G) and conantokin-T (con-T) are naturally occurring γ-carboxyglutamate (Gla)-containing peptides that interact with multivalent cations in functionally relevant manners. Selective ¹³C-enrichment of C^γ and C^δ in each of the Gla residues has allowed metal binding affinities to be measured at individual side chains. Con-T possesses two metal binding sites, one with high affinity at Gla¹⁰/Gla¹⁴ and another with weak binding at Gla³/Gla⁴. Con-G contains two sites of comparable low affinity for Ca²⁺. Analysis of the ¹³C line-widths of con-G in the presence of Mg²⁺ allowed the order of metal binding to be determined, with Gla¹⁰/Gla¹⁴ loading before the Gla³/Gla⁴/Gla⁷ cluster. While the variant peptide, apo-con-T[Lys⁷Gla], was shown to have a very low α-helical content, this peptide binds a second metal with much greater affinity than wild-type con-T. This provides additional evidence that Gla⁷ in con-G is primarily responsible for destabilizing the apo-form, but is an important ligand for metal chelation. The residue-specific α-helical stabilities of con-G and con-T in their metal-free and metal-loaded states were estimated by determining rates of proton exchange from backbone peptide bond amides with deuterium atoms from ²H₂O-containing solvents. For both peptides, the lifetimes of protons on several peptide bond amides increased as metals of higher affinity were bound to the peptides, with the longest half-lives found in the region of the α-helical turn stabilized by the Gla¹⁰/Gla¹⁴ metal coordination site. We propose that Gla¹⁰ and Gla¹⁴ constitute the primary tight metal ion binding site in both peptides. This detailed analysis with physiologically relevant metal cations is crucial for deciphering the roles of critical amino acids in the bioactivity of the conantokin peptides.     

Binding to opioid receptors of enkephalin derivatives taking α-helical conformation and its dimer

The opioid receptor binding of [Leu]enkephalin derivatives with extended address segment to the C-terminal was studied. The extension peptide is designed to take an amphiphilic helical structure in order to evaluate effects of helical conformation and membrane affinity of enkephalin moiety of the derivatives on receptor binding. In the σ-receptor-selective binding assay, Tyr-Gly-Gly-Phe-Leu-Lys-Aib-Leu-Aib-OH (1) showed the same affinity as enkephalinamide, whereas in the μ-receptor-selective binding assay, a 7-fold reduction in affinity was observed. On the other hand, Tyr-GIy-Gly-Phe-Leu-(Lys-Aib-Leu-Aib)₂-OH (2) showed 51-and 96-fold decreases in affinities for δ- and μ-receptors, respectively, compared with enkephalinamide. The low receptor affinity of derivative 2 is considered due to α-helical conformation, which might not be compatible with topological requirements of δ- and μ-receptors. A dimer, Tyr-Gly-GIy-Leu-Phe-(Lys-Aib-Leu-Aib)₂-Lys(X)-Aib-OCH₃ (X = Tyr-Gly-Gly-Phe-Leu-, (4)), showed 2.5- and 3.0-fold increases in affinities respectively for δ- and μ-receptors compared with the monovalent derivative 2, possibly due to cross-linking of neighboring receptors. The Hill plot of the binding of the dimer to bovine brain membranes was composed of two phases, although such a heterogeneity of receptors was not observed in the presence of naloxone or in the binding to NG108-15 cell membranes. These findings indicate the presence of the bivalent-ligand-induced interactions between δ- and μ-receptors in bovine brain membranes.     

The Binding of Thiopental to Serum Proteins Determined by Ultrafiltration and Equilibrium Dialysis

Abstract: An ultra filtration method is described by which it is possible to estimate the protein bound fraction of a drug at its original concentration in a serum sample. The determination is carried out at 37° and pH 7.4. For thiopental no difference was found between the values of protein binding whether they were determined by the ultrafiltration method or by a less time consuming equilibrium dialysis against an equal volume of phosphate buffered isotonic sodium chloride solution. The equilibrium dialysis was used to measure the concentration of bound and unbound thiopental molecules; and the binding parameters in a two class binding model were determined. No evidence was found for binding to other proteins than albumin. About one binding site belonging to the first class was found per 1000 albumin molecules whereas an average of about 5 secondary sites were found for each albumin molecule. The association constants for the primary class of binding sites were 3.4×10⁶M^?1 for albumin and 13.3×10⁶M^?1 for serum while the values for the secondary class were 2.2 and 1.2×10³M^?1 for albumin and serum respectively. The estimates of association constants and number of binding sites were based on experiments with total thiopental concentrations ranging from 0.4 to 80 μg/ml. In a phosphate buffered albumin solution with 2 g albumin per 100 ml, a pH increase from 5 to 8 caused an increase in protein binding from 36 to 76%. A Scatchard plot using data from experiments with increasing albumin concentrations resulted in a “plot” with positive slope. The use of a curve like this is discussed and it is concluded that the binding parameters for thiopental are influenced by the albumin concentration.