Recent advances in chromatographic and electrophoretic methods for the study of drug-protein interactions

Drug-protein binding is an important process in determining the activity and fate of a pharmaceutical agent once it has entered the body. This review examines various chromatographic and electrophoretic methods that have been developed to study such interactions. An overview of each technique is presented along with a discussion of its strengths, weaknesses and potential applications. Formats that are discussed include the use of both soluble and immobilized drugs or proteins, and approaches based on zonal elution, frontal analysis or vacancy peak measurements. Furthermore, examples are provided that illustrate the use of these methods in determining the overall extent of drug-protein binding, in examining the displacement of a drug by other agents and in measuring the equilibrium or rate constants for drug-protein interactions. Examples are also given demonstrating how the same methods, particularly when used in high-performance liquid chromatography or capillary electrophoresis systems, can be employed as rapid screening tools for investigating the binding of different forms of a chiral drug to a protein or the binding of different proteins and peptides to a given pharmaceutical agent.     

Development of a trichloroacetic acid precipitation assay for covalent adducts of thymidylate synthase

The use of trichloroacetic acid as a protein precipitant and denaturant in the quantitative measurement of covalent complexes of thymidylate synthase is described. Enzyme inactivated with N[3H]ethylmaleimide and inhibitory ternary complex (formed with native enzyme, 5-[6-3H]fluoro-2'-deoxyuridylate, and methylenetetrahydrofolate) served as reagents which were used to establish the conditions under which trichloroacetic acid precipitation, washing, and solubilization steps provided quantitative results. The ternary complex formed by dihydrofolate reductase with [3H]methotrexate and NADPH was used as a control to assess whether tight, but noncovalent, enzyme:ligand complexes survived trichloroacetic acid precipitation. The fact that no counts above background were detected in the pellet of precipitated protein demonstrated that the noncovalent complexes were completely dissociated by this treatment. The dynamic range of linear response for the inhibitory ternary complex of thymidylate synthase spanned five orders of magnitude, and the assay detected levels of enzyme as low as 10 fmol, a value which was essentially limited by the specific radioactivity of 5-[6-3H]fluoro-2'-deoxyuridylate. The ability of the enzyme to bind 5-[6-3H]fluoro-2'-deoxyuridylate specifically, as measured by the trichloroacetic acid assay, generated a specific binding value of 13.4 nmol of enzyme/mg protein (assuming a binding ratio of 1.5 for the inhibitory ternary complex). Specific binding values were compared to specific activity values (obtained from the spectrophotometric assay) at each stage of purification of the enzyme from Lactobacillus casei and were found to give parallel results. The characteristics of the trichloracetic acid assay procedure, which exclusively detects covalent enzyme-ligand adducts, are compared to those for other ligand binding assays for thymidylate synthase.     

Quantification of formaldehyde-mediated covalent adducts of adriamycin with DNA.

Duplex DNA incubated with adriamycin, dithiothreitol (DTT), and Fe3+ under aerobic, aqueous conditions yields double-stranded (DS) DNA bands by denaturing polyacrylamide gel electrophoresis (DPAGE) analysis, characteristic of DNAs which are interstrand cross-linked. Another laboratory has provided evidence that formaldehyde produced under these conditions promotes the covalent linkage of adriamycin to one strand of DNA and suggested that this complex results in the anomalous DPAGE behavior. We provide herein strong support for this interpretation. We show: (a) that mixtures of DNA and adriamycin incubated with DTT/Fe3+, H2O2, or formaldehyde all show DS DNA bands on DPAGE, (b) that the DS DNA bands and the formaldehyde-mediated lesion (detected by an indirect, GC-MS analysis) form with similar time courses, and in similar amounts, and (c) that the DNA in the DS DNA bands contains approximately one such lesion per DNA, whereas the single-stranded DNA is devoid of it. These results further support the interpretation that adriamycin does not create interstrand cross-links in DNA, and that the DS DNA observed in DPAGE experiments derives from the formaldehyde-mediated monoadduct.     

Separation procedures capable of revealing DNA adducts

Detection and quantification of DNA adducts are very important in relation to diseases such as cancer. Both high sensitivity and high selectivity are required for the detection of DNA adducts because the content of adducts in DNA is very small compared with those of normal bases and only small amounts of DNA samples are available for analysis in general cases. In this paper are described separation procedures such as liquid chromatography, gas chromatography and capillary electrophoresis combined with a detection and identification method such as 32P-postlabeling, mass spectrometry, electrochemical detection, fluorescence detection and immunoassay. The merits and demerits of the procedures are also discussed.     

Automated detection of covalent adducts to human serum albumin by immunoaffinity chromatography, on-line solution phase digestion and liquid chromatography-mass spectrometry

A generic method for the detection of covalent adducts to the cysteine-34 residue of human serum albumin (HSA) has been developed, based on an on-line combination of immunoaffinity chromatography for selective sample pre-treatment, solution phase digestion, liquid chromatography and tandem mass spectrometry. Selective anti-HSA antibodies immobilized on agarose were used for sample pre-concentration and purification of albumin from the chemically produced alkylated HSA. After elution, HSA and HSA adducts are mixed with pronase and directed to a reaction capillary kept at a digestion temperature of 70 degrees C. The digestion products were trapped on-line on a C18 SPE cartridge. The peptides were separated on a reversed-phase column using a gradient of organic modifier and subsequently detected using tandem mass spectrometry. Modified albumin samples consisted of synthetically alkylated HSA by the reactive metabolite of acetaminophen, N-acetyl-p-benzoquinoneimine (NAPQI), and using the alkylating agent 1-chloro-2,4-dinitrobenzene (CDNB) as reference. The resulting mixture of alkylated versus non-modified albumin has been applied to the on-line system, and alkylation of HSA is revealed by the detection of the modified marker tetra-peptide glutamine-cysteine-proline-phenylalanine (QCPF) adducts NAPQI-QCPF and CDNB-QCPF. Detection of alkylated species was enabled by the use of data comparison algorithms to distinguish between unmodified and modified HSA samples. The in-solution digestion proved to be a useful tool for enabling fast (less than 2 min) and reproducible on-line digestion of HSA. A detection limit of 1.5 micromol/L of modified HSA could be obtained by applying 10 microL of NAPQI-HSA sample.     

Reversible covalent enzyme immobilization methods for reuse of carriers

Reversible immobilization techniques which allow for multiple use of the carrier are relevant for applications, such as enzymatic microreactors, biosensors with specific setups and for expensive carriers such as superparamagnetic particles. The activity of immobilized enzymes reduces with time, so that the introduction of fresh immobilized enzyme becomes necessary. Thus, methods for reversible immobilization and multiple carrier reuse can help to reduce purchase costs and facilitate reactor construction. In this work, we present a method that makes use of the reduction and oxidation of cystamine, a cleavable linker with disulfide bond and amine functionality. For a proof of principle, α-chymotrypsin was immobilized on polyethylene glycol with terminal epoxy groups using cystamine as a crosslinker. The enzyme was highly active and could be used in repeated cycles. After the enzymatic reaction was demonstrated, α-chymotrypsin was cleaved off the particle by reducing agents. The resulting thiols on the particle surface were oxidized to disulfides by means of cysteamine, the reduction product of cystamine. This way, an almost complete oxidation of surface thiols with cysteamine was possible, restoring amine functionalization for further reactions. Reduction and oxidation were repeated several times without a decrease in the extent of amine coupling. Finally, immobilization of α-chymotrypsin could be repeated with results comparable to first run.     

ABTS radical-driven oxidation of polyphenols: isolation and structural elucidation of covalent adducts

The formation of covalent adducts obtained from the reaction of the polyphenols, trans-3,3',4',5,7-pentahydroxyflavan (catechin) and 1,3,5-trihydroxybenzene (phloroglucinol), with ABTS radicals is reported. Two adducts derived from (+)-catechin and three adducts from phloroglucinol were isolated and identified using reversed-phase high performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS). The molecular masses of the (+)-catechin-derived adducts (I(c) and II(c)) were found to be 802 and 559 Da, respectively, whereas the masses of phloroglucinol-derived adducts (I(p), II(p), and III(p)) were 638, 395, and 381 Da, respectively. The initially formed adducts (I(c), I(p)) were unstable and degraded to secondary adducts (II(c), II(p), and III(p)) releasing part of the ABTS molecule. The structures of these adducts were elucidated by interpreting the results of MS/MS analysis of prominent ions generated by both positive and negative ion ESI-MS. The adducts were found to scavenge ABTS radicals, an observation that could explain the complex kinetic behaviour manifested by the reactions of ABTS radicals with polyphenols. A mechanism, which accounts for both the formation of the adducts and the degradation products of ABTS radicals, is proposed.     

Supramolecular complexes of polyethylene glycol-alpha-Chymotrypsin covalent and noncovalent adducts with polyoxyethylene-modified cyclo dextrins

Complexes of covalent and noncovalent adducts of polyethylene glycol (PEG) and alpha-chymotrypsin (ChT), PEG-ChT, were generated in the presence of beta-cyclodextrin derivatives of polyoxyethylene (beta CD-PEO), and their thermal stability was studied. The covalent [PEG-ChT]c conjugates were obtained by chemical modification of the protein amino groups with the monoaldehyde derivatives of monomethoxypolyethylene glycol. The noncovalent [PEG-ChT]n complexes were obtained by the treatment of ChT-PEG mixtures with increasing pressure (1.1-400 MPa). Supramolecular structures resulting from complex formation between PEG chains of the PEG-ChT adducts (PEGad) and beta CD-PEO were studied. The decrease in the rate constant of the slow stage of ChT thermal inactivation in PEG-ChT adducts (k2) can serve as confirmation of complex formation between beta CD-PEO and PEGad. The stoichiometric composition of our supramolecular structures was determined from the k2 dependence on the molar ratio of beta CD-PEO to PEGad. It was shown that each polymeric chain in the [PEG-ChT]c conjugates forms an inclusion complex with beta CD-PEO, whereas only half of the PEGad polymeric chains participate in the formation of supramolecular structures in the case of [PEG-ChT]n complexes. Although covalent and noncovalent PEG-ChT adducts of the same composition significantly differ in their thermal stability, the maximal values of the k2 rate constants for [PEG-ChT]c and [PEG-ChT]n adducts in the triple system attainable at the (beta CD-PEO) to (PEGad) ratio corresponding to the stoichiometry of the resulting ternary systems are practically the same (k2 = 0.007 c-1 at 45 degrees C in 0.02 M Tris-HCl buffer solution, pH 8.0). Structures for the supramolecular dendrite-like ensembles formed upon the interaction of covalent and noncovalent PEG-ChT adducts with beta CD-PEO were suggested.     

Separation methods for camptothecin and related compounds

This paper reviews working procedures for the analytical determination of camptothecin and analogues. We give an overview of aspects such as the chemistry, structure–activity relationships, stability and mechanism of action of these antitumor compounds. The main body of the review describes separation techniques. Sample treatment and factors influencing high-performance liquid chromatography development are delineated. Published high-performance liquid chromatographic methods are summarized to demonstrate the variability and versatility of separation techniques and a critical evaluation of separation efficiency, detection sensitivity and specificity of these methods is reported.     

Separation and identification methods for metalloproteinase inhibitors

Metalloproteinase inhibitors are being explored for the treatment of a wide variety of human diseases including cancers, arthritis, cardiovascular disorders, human immunodeficiency virus infection, and central nervous system illnesses. This review provides an overview of various analytical sample preparation, separation, detection, and identification techniques employed for the quantitative and qualitative determination of these inhibitor compounds. Special emphasis is placed on biological sample preparation by automated solid-phase extraction, liquid–liquid extraction, and protein precipitation by centrifugation or filtration. Other sample preparation methodologies are also evaluated. Applications of high-performance liquid chromatography, gas chromatography, and capillary electrophoresis to the quantitative determination of metalloproteinase inhibitors are described. Examples of qualitative analysis of metalloproteinase inhibitors by hyphenated liquid chromatography with mass spectrometry and nuclear magnetic resonance are also presented. The advantages and limitations of these separation and identification methodologies as well as other less frequently employed techniques are assessed and discussed.     

Bile duct ligation promotes covalent drug-protein adduct formation in plasma but not in liver of rats given zomepirac

Acyl glucuronides are reactive electrophilic metabolites of carboxylate drugs, capable of undergoing hydrolysis, rearrangement and covalent binding reactions with proteins in vivo. Such covalent drug-protein adducts may be prerequisites for certain idiosyncratic immune and toxic responses in susceptible individuals. The present study examined the effect of experimental cholestasis on the extent and pattern of formation of protein adducts in plasma and liver of rats given the non-steroidal antiinflammatory drug (NSAID) zomepirac (ZP). Groups of intact, bile-exteriorized and bile duct-ligated rats given a 50 mg/kg i.v. dose of ZP were studied for 24 hr. In intact rats, only 1.4% of the dose was recovered as the sum of ZP, ZP acyl glucuronide (ZAG) and its rearrangement isomers (iso-ZAG) in urine in 24 hr. In bile-exteriorized animals, 0.5% of the dose was recovered in urine in 24 hr, with 31.6% of the dose being recovered in bile (2.7% as ZP, 20.0% as ZAG and 8.9% as iso-ZAG). In the bile duct-ligated group, recovery of dose in 24 hr urine totalled 17.5% (1.7% as ZP, 6.7% as ZAG and 9.1% as iso-ZAG). ZAG and iso-ZAG were measurable in plasma only in the bile duct-ligated group, and covalent binding of ZP to plasma proteins was much higher (5-6 fold) than in intact or bile-exteriorized rats. Total adduct concentrations in liver were not significantly different among the three groups. Immunoblotting using a polyclonal ZP antiserum confirmed that serum albumin was a major target protein in plasma. The major ZP-modified bands in the livers of intact and bile-exteriorized rats were at about 110, 140 and 200 kDa. However, the bands at 110 and 140 kDa were much lower in the livers of bile duct-ligated rats. The results show that about 30% of ZP doses are normally excreted as ZAG and its isomers in bile, with only minor excretion in urine. Bile duct ligation shunts the glucuronide into blood (and urine), strongly promoting adduct formation with plasma proteins, and alters the pattern but not the total quantity of drug-modified proteins formed in the liver.     

Separation methods for tricyclic antiviral drugs

A review of the published analytical methodology for the tricyclic antiviral (TAV) drugs is presented. While amantadine and rimantadine are the only two approved drugs for the prophylaxis and treatment of the influenza A virus, amantadine has also been approved for the treatment of Parkinson’s disease. In addition, a few structurally related compounds are finding important clinical applications in other central nervous system-related disorders. To effectively evaluate the pharmacokinetics, biotransformations, stability, and other critical parameters that are necessary for pre-clinical and clinical studies, analytical methodology that conforms to the rigors of regulatory requirements must be developed and made available. This review discusses the analytical methods used in the determination of amantadine, rimantadine, tromantadine and memantine and the pre-clinical and clinical application of these techniques.     

Separation methods for pharmacologically active xanthones

Xanthones, as a kind of polyphenolic natural products with many strong bioactivities, are attractive for separation scientists due to the similarity and diversity of their structures resulting in difficult separation by chromatographic methods. High performance liquid chromatography (HPLC) and thin layer chromatography (TLC) are traditional methods to separate xanthones. Recently, capillary electrophoresis (CE), as a micro-column technique driven by electroosmotic flow (EOF), with its high efficiency and high-speed separation, has been employed to separate xanthones and determine their physicochemical properties such as binding constants with cyclodextrin (CD) and ionization constants. Since xanthones have been used in clinic treatment, the development of chromatographic and CE methods for the separation and determination of xanthones plays an essential role in the quality control of some herbal medicines containing xanthones. This article reviewed the separation of xanthones by HPLC, TLC and CE, citing 72 literatures. This review focused on the CE separation for xanthones due to its unique advantages compared to chromatographic methods. The comparison of separation selectivity of different CE modes including capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), microemulsion electrokinetic capillary chromatography (MEEKC) and capillary electrochromatography (CEC) was discussed. Compared with traditional chromatographic methods such as HPLC and TLC, CE has higher separation efficiency, faster separation, lower cost and more flexible modes. However, because of low sensitivity of UV detector and low contents of xanthones in herbal medicines, CE methods have seldom been applied to the analysis of real samples although CE showed great potential for xanthone separation. The determination of xanthones in herbal medicines has been often achieved by HPLC. Hence, how to enhance CE detection sensitivity for real sample analysis, e.g. by on-line preconcentration and CE-MS, would be a key to achieve the quantitation of xanthones.     

Separation methods for antiviral phosphorus-containing drugs

Among antiviral drugs, phosphorus-containing compounds, foscarnet and cidofovir, present adverse effects including renal toxicity. Since their main therapeutic target is the treatment of CMV retinitis, which needs lifelong maintenance therapy, accurate analytical methods are required for drug monitoring. According to the high hydrophilic property of the two compounds, ion pair reversed-phase HPLC methods were proposed for their separation in drug formulations and biological samples. Their lack of UV absorption at wavelengths above 205 nm does not allow the use of this detection technique for biological fluids. Electrochemical detection methods (coulometry and amperometry) led to a quantification limit of 15 μM for foscarnet. Fluorescent derivatives obtained by modification of cidofovir cytosine nucleus with α-haloketones offered advantage over UV detection and allowed to reach a detection limit of 5 ng/ml, making possible investigations on the drug time-course in biological fluids.     

Separation methods for anthraquinone related anti-cancer drugs

The quinoid anthracycline-related anti-cancer agents represent an important group of anti-tumour drugs with a wide spectrum of activity. We review here some of the separation techniques used for the analysis of anthracyclines and related compounds. In this review we have covered a range of compounds from the early anthracycline antibiotics such as doxorubicin to the more recent anthracenediones and anthrapyrazoles such as mitoxantrone and losoxantrone, respectively. We also include novel compounds such as AQ4N and C1311, both awaiting clinical trial. Separations of the anthraquinone related anti-cancer agents are predominantly by HPLC. These separation techniques have been used for a variety of applications including drug stability, protein binding and therapeutic drug monitoring as well as detailed pharmacokinetic and metabolic studies. Pharmacokinetics, and therefore drug analysis, plays a central role in both the development of new agents and also leads to a better understanding of clinically established agents in this class. Sample preparation and extraction methods including solid-phase and liquid–liquid extraction have also been highlighted. Many anthraquinone related compounds are highly coloured and fluoresce. They are suitable for a range of detection methods including UV–Vis, electrochemical and fluorescence. The methods described are used for sometimes complex separations that are needed for the evaluation of such compounds in biological samples.     

Stereoselective pharmacokinetics of flurbiprofen and formation of covalent adducts with plasma protein in adjuvant-induced arthritic rats

To determine the effect of arthritis on the disposition of flurbiprofen (FP) and its acyl glucuronide (FPG) as well as formation of covalent adducts with plasma protein, a pharmacokinetic study was carried out in adjuvant-induced arthritic (AA) rats. In control animals the pharmacokinetics of FP were stereoselective following intravenous bolus injection of rac-FP: (-)-(R)-FP showed higher plasma clearance (CL(tot)) and shorter mean residence time (MRT) compared to (+)-(S)-FP. The CL(tot) and clearance for the glucuronide formation (CL(glu)) of both enantiomers in AA rats were extremely increased compared to those in control rats. Increased total clearance in AA rats was due, at least in part, to a remarkable increase in the plasma unbound fraction of FP, consistent with a decrease in the plasma albumin level. The yield of covalent binding of FP to plasma protein in AA rats was less than that in controls, being consistent with the decrease in the plasma acyl glucuronide level.     

Computational methods for exon detection

Computer methods for the complete and accurate detection of genes in vertebrate genomic sequences are still a long way to perfection. The intermediate task of identifying the coding moiety of genes (coding exons) is now reasonably well achieved using a combination of methods. After reviewing the intrinsic difficulties in interpreting vertebrate genomic sequences, this article presents the state-of-the-art, with an emphasis on similarity search methods and the resources available through Internet.     

Neocarzinostatin chromophore-DNA adducts: evidence for a covalent linkage to the oxidized C-5'' of deoxyribose.

The nonprotein chromophore of neocarzinostatin forms a variety of adducts with DNA. The predominant adduct recovered from nuclease digests of chromophore-treated poly(dA-dT). poly(dA-dT) is a compound with structure chromophore-d(TpApT). Mild acid hydrolysis of this compound released free adenine, while snake venom exonuclease (pH 6.5) released 5'-dTMP leaving in both cases adducts of slightly altered chromatographic mobility. These results eliminate adenine and 5'-dTMP as possible sites of covalent chromophore attachment. Electrophoresis data suggest that the adduct is not a phosphotriester. At pH 8.6, chromophore-d(TpApT) spontaneously hydrolyzed, releasing chromophore and 3'-dTMP, leaving a modified d(ApT) which contained deoxyadenosine-5'-aldehyde. Deoxyadenosine-5'-aldehyde was released from the modified d(ApT) by snake venom exonuclease, and identified by a series of derivatizations including 1) mild oxidation to deoxyadenosine-5'-carboxylic acid, 2) NaBH4 reduction to deoxyadenosine, and 3) formation of a hydrazone with phenylhydrazine. Since deoxyadenosine-5'-aldehyde cannot exist as such in the chromophore-d(TpApT) adduct, we suggest that the chromophore may be covalently attached to the C-5' of deoxyadenosine as a phosphorylacetal or similar structure. Hydrolysis of the chromophore-acetal bond at pH 8.6 would leave a phosphorylhemiacetal on C-5', which would be expected to spontaneously decompose to yield the observed 3'-phosphate and 5'-aldehyde groups.