LC-MS/MS-Based Monitoring of In Vivo Protein Biotransformation: Quantitative Determination of Trastuzumab and Its Deamidation Products in Human Plasma.

An LC-MS/MS-based method is described for quantitatively monitoring the in vivo deamidation of the biopharmaceutical monoclonal antibody trastuzumab at a crucial position in its complementarity determining region (CDR). The multiplexed LC-MS/MS assay using selected reaction monitoring (SRM) allows simultaneous quantitation of five molecular species derived from trastuzumab after tryptic digestion: a stable signature peptide (FTISADTSK), a deamidation-sensitive signature peptide (IYPTNGYTR), its deamidated products (IYPTDGYTR and IYPTisoDGYTR), and a succinimide intermediate (IYPTsuccGYTR). Digestion of a 50 µL plasma sample is performed at pH 7 for 3 h at 37 °C, which combines a reasonable (>80%) digestion efficiency with a minimal (<1%) formation of deamidation products during digestion. Rapid in vitro deamidation was observed at higher pH, leading to a (large) overestimation of the concentrations of deamidation products in the original plasma sample. The LC-MS/MS method was validated in accordance with international bioanalytical guidelines over the clinically relevant range of 0.5 to 500 µg/mL with bias and CV values well below 15%. Deamidation of trastuzumab was observed in plasma both in a 56 day in vitro forced degradation study (up to 37% of the total drug concentration) and in samples obtained from breast cancer patients after treatment with the drug for several months (up to 25%). Comparison with a validated ELISA method for trastuzumab showed that deamidation of the drug at the CDR leads to a loss of recognition by the antibodies used in the ELISA assay.

Symptoms and course of intoxication with mesuximide--a case report.

We report on a 31-year-old, female patient who presented with somnolence due to an intoxication by the antiepileptic drug, mesuximide (MSM). The serum concentration of its metabolite n-desmethyl-mesuximide (85.7 mg/L) was above the so-called therapeutic range (10-40 mg/L) but below the concentration range that led to an impairment of consciousness in previous cases according to the German SPC (>150 mg/L). The symptoms remitted quickly under hemodialysis. In somnolent patients treated with MSM, the treating physicians should be aware of drug intoxication as a possible etiology. Therefore, the serum concentration should be measured early. Due to the, often, long latency until the results are available, treatment initiation may be necessary based on suspicion.

CFSE flow cytometric quantification of lymphocytic proliferation in extracorporeal photopheresis: use for quality control.

Quality control is essential to validate extracorporeal photopheresis (ECP) processes. There is just one protocol based on PHA-induced proliferation. Since it involves the use of radioactive thymidine, we developed another technique using CFSE labeling. We compared the two tests in a paired series including 18 procedures. The thymidine test was valid. Once proliferation was obtained (10 patients out of 13), the CFSE test was in close agreement with it. In particular, two cases of residual proliferation after ECP were simultaneously detected by both techniques. Only the CFSE test allows targeted analysis of lymphocytes, thus identifying a surviving lymphocytic sub-population.

Erythrocyte survival time in Greyhounds as assessed by use of in vivo biotinylation.

OBJECTIVE: To determine erythrocyte survival time in Greyhounds. ANIMALS: 6 Greyhounds used as blood donors and 3 privately owned non-Greyhound dogs. PROCEDURES: In vivo biotinylation of erythrocytes was performed by infusion of biotin-N-hydroxysuccinimide into each dog via a jugular vein catheter. Blood samples were collected 12 hours later and then at weekly intervals and were used to determine the percentage of biotin-labeled erythrocytes at each time point. Erythrocytes were washed, incubated with avidin-fluorescein isothiocyanate, and washed again before the percentage of biotinylated erythrocytes was measured by use of flow cytometry. Survival curves for the percentage of biotinylated erythrocytes were generated, and erythrocyte survival time was defined as the x-intercept of a least squares best-fit line for the linear portion of each curve. RESULTS: The R2 for survival curves ranged from 0.93 to 0.99 during the first 10 weeks after infusion of erythrocytes. Erythrocyte survival time for the 3 non-Greyhound dogs was 94, 98, and 116 days, respectively, which was consistent with previously reported values. Erythrocyte survival time for the 6 Greyhounds ranged from 83 to 110 days (mean, 93 days; median, 88 days). As determined by use of in vivo biotinylation, erythrocyte survival times in Greyhounds were similar to those determined for non-Greyhound dogs and did not differ significantly from erythrocyte survival times reported previously for non-Greyhound dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Erythrocyte survival time was similar in Greyhounds and non-Greyhound dogs. Greyhounds can be used as erythrocyte donors without concerns about inherently shorter erythrocyte survival time.

Circulating Hormones and Mammographic Density in Premenopausal Women.

Prior research suggests that several endogenous hormones in premenopausal women are associated with breast cancer risk; however, few studies have evaluated associations of endogenous hormones with mammographic density (MD) in premenopausal women. We conducted a cross-sectional study of plasma hormone levels in relation to MD among 634 cancer-free premenopausal women in the Nurses' Health Study II. We measured percent MD from screening mammograms using a computer-assisted method. We assayed estradiol, estrone, and estrone sulfate in blood samples timed in early follicular and mid-luteal phases of the menstrual cycle as well as testosterone, androstenedione, progesterone, dehydroepiandrosterone (DHEA), DHEA sulfate, sex hormone-binding globulin (SHBG), and anti-Müllerian hormone in luteal or untimed samples. We used multivariable linear regression to quantify the association of %MD with quartiles of each hormone, adjusting for age, body mass index, and breast cancer risk factors. Women in the highest quartile of follicular estradiol levels had significantly greater %MD compared to those in the lowest quartile [difference, 6.7 percentage points; 95% confidence interval (CI) 2.2, 11.3; p-trend < 0.001]. Similar associations were observed for follicular free estradiol but not luteal-phase estradiol. Also, women in the top (vs. bottom) quartile of free testosterone had significantly lower %MD (difference, - 4.7; 95% CI - 8.7, - 0.8; p-trend = 0.04). Higher SHBG was significantly associated with higher percent MD (difference, 4.8; 95% CI 1.1, 8.6; p-trend = 0.002). Percent MD was not strongly associated with other measured hormones. Results were similar in analyses that excluded women with anovulatory cycles. Our findings suggest that follicular estradiol and SHBG may play an important role in premenopausal percent MD.

Affinity labeled somatomedin-C-binding proteins in rat sera.

We have developed an affinity labeling technique that uses disuccinimidyl suberate to covalently cross-link [125I]somatomedin-C (Sm-C) to specific binding proteins in rat serum. Normal rat serum contains four major classes of intensely labeled [125I]Sm-C-binding protein complexes which are sensitive to competition with unlabeled Sm-C with relative molecular masses of 95, 49, 36-33, and 26-23 K. In addition, less intensely labeled complexes are observed migrating between 175 and 115 K. Of the Sm-C binding complexes observed in normal serum, hypophysectomized (hypox) serum contains only an intensely labeled 36-33-K complex and a faint 49-K complex. Chronic administration of ovine (100 micrograms, ip, daily) to hypox rats induces the 95-K complex and possibly complexes between 175-115 K. With increasing duration of treatment, these complexes as well as the 49-K complex appear to increase in intensity. Binding proteins in both hypox and normal sera do not appear to distinguish between Sms, since both unlabeled Sm-C and multiplication-stimulating activity were equally potent in competing with [125I]Sm-C for binding. This affinity labeling technique appears to be a useful investigative tool to study the physiology and structure of Sm-binding proteins.

Tracking of leukocyte recruitment into tissues of mice by in situ labeling of blood cells with the fluorescent dye CFDA SE.

Models of inflammatory and immune diseases are extensively studied in mice with engineered genetic mutations, and tracking the recruitment of blood leukocytes into tissues is an important component of these studies. A direct in situ method for labeling the total pool of blood cells in mice by a single intravenous injection of the fluorescent dye CFDA SE (CFSE) is described. The fluorescence intensity of labeled cells initially declines, but remains stable after 4 h, enabling detection weeks after labeling. Labeled leukocytes can be tracked as they accumulate in lymphoid tissues and sites of inflammation and then be immunophenotyped for analysis by flow cytometry. This method is rapid, reproducible and simple to perform.

Methsuximide for intractable childhood seizures.

Methsuximide was added to the therapeutic regimens of 25 children with intractable epilepsy. In 15 patients the drug was well tolerated and resulted in a 50% or greater reduction in seizure frequency. No serious or irreversible adverse effects were seen. Methsuximide is frequently overlooked and may be an effective adjunctive antiepileptic for children with intractable seizures.

Measurement of methsuximide and N-desmethylmethsuximide using solid-phase extraction and wide-bore capillary gas chromatography.

A method for the determination of the anti-epileptic drug methsuximide (MSM) and its active metabolite N-desmethylmethsuximide (NDM) is presented. 5-Methyl-5-phenylhydantoin is used as the internal standard. A simple solid-phase extraction procedure utilizing disposable reversed-phase C18 columns is described. Samples are analyzed by gas chromatography with flame ionization detection using a wide-bore capillary column with a permanently bonded, non-polar stationary phase. The MSM assay possesses linearity to 6.0 micrograms/mL, sensitivity to 0.5 microgram/mL, recovery ranging from 93 to 110%, and precision reflected by a SD of +/- 0.37 microgram/mL. The NDM assay displays linearity up to 80.0 micrograms/mL, sensitivity to 5.0 micrograms/mL, recovery of 90 to 100%, and precision reflected by a SD +/- 0.90 microgram/dL. Lack interference is documented for 6 commonly prescribed anti-epileptic drugs and 4 drugs with similar retention times on this stationary phase; only guaifenesin was found to potentially interfere with the determination of methsuximide. We conclude that the method reported here is ideally suited for monitoring therapeutic and toxic levels of this anti-epileptic drug.

Renal damage, metabolism and covalent binding following administration of the nephrotoxicant N-(3,5-dichlorophenyl)succinimide (NDPS) to male Fischer 344 rats.

In vivo metabolism, nephrotoxicity and covalent binding to proteins were evaluated in male Fischer 344 rats that received [2,3-14C]-N-(3,5-dichlorophenyl)succinimide (14C-NDPS). Some animals were pretreated with the enzyme inducer phenobarbital (PB, 80 mg/kg per day, for 3 days, i.p. in saline) prior to receiving a non-nephrotoxic dose of 14C-NDPS (0.2 mmol/kg, i.p. in corn oil). Other rats were pretreated with the cytochrome P450 inhibitor 1-aminobenzotriazole (ABT, 100 mg/kg, 1 h prior to NDPS, i.p. in saline) before administration of a non-toxic or a toxic dose (0.2 or 0.6 mmol/kg, respectively, i.p. in corn oil) of 14C-NDPS. Non-pretreated animals received either dose of 14C-NDPS, but did not receive PB or ABT. All rats were sacrificed 6 h after administration of 14C-NDPS. Nephrotoxicity was monitored by measuring urine volume, urine protein concentrations, blood urea nitrogen levels, and kidney weights. The NDPS metabolic profile in tissue, blood, and urine was analyzed by HPLC. Covalent binding of 14C-NDPS-derived radioactivity to tissue proteins was also measured. Compared with non-pretreated rats, PB-pretreatment potentiated the toxicity of the non-toxic dose of 14C-NDPS. In contrast, ABT-pretreatment protected the rats against NDPS nephrotoxicity. The amount of N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA), an oxidative, nephrotoxic metabolite of NDPS, was elevated in kidney homogenates and urine by PB-pretreatment (0.2 mmol/mg NDPS). ABT pretreatment inhibited NDPS metabolism at both doses. Covalent binding of 14C-NDPS (0.2 mmol/kg)-derived radioactivity to renal and plasma proteins was higher in the PB-pretreated rats than in the non-pretreated animals. In contrast, ABT-pretreatment partially inhibited covalent binding at both doses of 14C-NDPS. Our results suggest that there is a relationship between oxidative metabolism of NDPS, covalent binding of an NDPS metabolite to renal proteins, and NDPS-induced nephrotoxicity in rats.

2-Pyrrolidinone and succinimide endogenously present in several mammalian species.

2-Pyrrolidinone and succinimide were identified in blood plasma of man, rat, and mouse. Dog plasma contained only traces of 2-pyrrolidinone not exceeding significantly the detection limit of our GCMS-method. Succinimide but not 2-pyrrolidinone could also be found in the brains of rat and mouse. Evidence is presented for a metabolic pathway leading from 2-pyrrolidinone to succinimide, with 5-hydroxy-2-pyrrolidinone as an intermediate.

Serum protein binding of desmethyl-methsuximide.

Serum protein binding of desmethyl-methsuximide (DM-MSM) in serum from 23 patients on polytherapy were determined using ultrafiltration and high-performance liquid chromatography. Desmethyl-methsuximide, The active metabolite of methsuximide (MSM), was found to have a moderate protein binding ranging between 45% and 60%.

Pharmacokinetics of methsuximide and a major metabolite in dogs.

The pharmacokinetics of methsuximide and its major metabolite 2-methyl-2-phenylsuccinimide were studied in dogs after single intravenous doses. Plasma methsuximide levels were described by a two-compartment open model, and those of the metabolite were described by a one-compartment open model. An expression was derived that describes both methsuximide and metabolite plasma levels after methsuximide administration. Excellent fits were obtained between observed data and those predicted from the model. The metabolite accounted for 40% of the overall elimination of methsuximide, and the half-life of the metabolite (15 hr) was much greater than that of the parent drug (1--3.5 hr). The results suggest that pharmacological effects after methsuximide administration may be due primarily to the metabolite, which may accumulate in the body during repeated doses.

alpha,alpha-Diphenylsuccinimide: evaluation of anticonvulsant and hydrophobic properties.

The anticonvulsant potencies (ED50) of alpha,alpha-diphenylsuccinimide, phenytoin, and phenobarbital were evaluated in mice by a standard maximal electroshock technique. Potencies were expressed in terms of intraperitoneal dosage and blood and brain concentrations. Overt neurotoxicity (TD50) was assessed by the rotorod method. These data were compared with relative hydrophobicities for the above compounds and three others [carbamazepine, cyheptamide, and (diphenylacetyl)urea] taken from the literature. An approximate parabolic dependence of anticonvulsant potency on hydrophobicity was observed regardless of the means of expressing potency (intraperitoneal dosage, blood concentration, or brain concentration); approximate optimal hydrophobicities were in the range of 2.18-2.23 (log P). Calculated therapeutic indices (TD50/ED50) also displayed a parabolic dependence on hydrophobicity, while toxic potency (TD50) displayed a linear dependence (within the limited range of log P values studied). Implications of the parabolic dependence of anticonvulsant potency and linear dependence of toxic potency on hydrophobicity are discussed with respect to the possible mechanisms involved.

The determination of anticonvulsants in biological samples by use of high-pressure liquid chromatography.

The procedure for anticonvulsant analysis represent use of a relatively new technology. The procedures compare favorably in accuracy and precision with accepted methodology using gas chromatography [14]. It is possible to determine a greater number of anticonvulsants simultaneously with liquid chromatography than with reported gas-chromatographic procedures. This is because gas chromatography necessitates manipulation of the sample to be analyzed, the various components of which may have completely different manipulation requirements. Liquid chromatography is able to effect the analysis without extensive manipulation of the sample. The columns recommended in Sect. III are currently available standard columns. Column technology is developing at an accelerating rate. It is now anticipated that new developments in microparticulate packings and improvements in packing techniques will give the analyst greater column efficiency. This may be used either to shorten overall analysis time or to include more drugs in a simultaneous analysis. There is not yet available to the analyst a variety of different detectors suitable for routine use. This situation will change. In the case of the successful use of ultraviolet spectrophotometric detectors, the technique is not being fully exploited. Ultraviolet spectrophotometry is firmly established in practice, but as yet it is not routine to apply dual-wavelength spectroscopy to the column effluent. Application of this technique may be expected to improve selectivity and quantitation besides permitting optical resolution of some components that are not separated by the column. In the procedures given in this chapter there was no detailed discussion on gradient elution. This technique is potentially of great utility for simultaneous analyses of many compounds. It should be used only where isocratic elution is out of the question because it calls for somewhat stricter requirements of equipment in control of chromatography conditions. An additional time for equilibration between analyses may be considered too high a price to pay for any advantage gradient elution may give the analyst. The problem of fully automatic operation of liquid chromatographs for the type of analysis discussed here remains to be solved. Automatic operation would advance the use of liquid chromatography in the clinical laboratory probably more than any other single improvement. However, regardless of refinements of this type, liquid chromatography in its modern manifestation has versatility and sensitivity in abundance for analyzing compounds of toxicological and clinical interest. It will therefore assume greater importance in these areas.

Comparison of the in vitro and in vivo stability of a succinimide intermediate observed on a therapeutic IgG1 molecule.

Deamidation of asparagine residues, a post-translational modification observed in proteins, is a common degradation pathway in monoclonal antibodies (mAbs). The kinetics of deamidation is influenced by primary sequence as well as secondary and tertiary folding. Analytical hydrophobic interaction chromatography (HIC) is used to evaluate hydrophobicity of candidate mAbs and uncover post-translational modifications. Using HIC, we discovered atypical heterogeneity in a highly hydrophobic molecule (mAb-1). Characterization of the different HIC fractions using LC/MS/MS revealed a stable succinimide intermediate species localized to an asparagine-glycine motif in the heavy chain binding region. The succinimide intermediate was stable in vitro at pH 7 and below and increased on storage at 25°C and 40°C. Biacore evaluation showed a decrease in binding affinity of the succinimide intermediate compared with the native asparagine molecule. In vivo studies of mAb-1 recovered from a pharmacokinetic study in cynomolgus monkeys revealed an unstable succinimide species and rapid conversion to aspartic/iso-aspartic acid. Mutation from asparagine to aspartic acid led to little loss in affinity. This study illustrates the importance of evaluating modifications of therapeutic mAbs both in vitro and in serum, the intended environment of the molecule. Potential mechanisms that stabilize the succinimide intermediate in vitro are discussed.