Where Did the Linker-Payload Go? A Quantitative Investigation on the Destination of the Released Linker-Payload from an Antibody-Drug Conjugate with a Maleimide Linker in Plasma.

The reactive thiol of cysteine is often used for coupling maleimide-containing linker-payloads to antibodies resulting in the generation of antibody drug conjugates (ADCs). Currently, a numbers of ADCs in drug development are made by coupling a linker-payload to native or engineered cysteine residues on the antibody. An ADC conjugated via hinge-cysteines to an auristatin payload was used as a model in this study to understand the impact of the maleimide linkers on ADC stability. The payload was conjugated to trastuzumab by a protease-cleavable linker, maleimido-caproyl-valine-citruline-p-amino-benzyloxy carbonyl (mcVC-PABC). In plasma stability assays, when the ADC (Trastuzumab-mcVC-PABC-Auristatin-0101) was incubated with plasma over a 144-h time-course, a discrepancy was observed between the measured released free payload concentration and the measured loss of drug-to-antibody ratio (DAR), as measured by liquid chromatography-mass spectrometry (LC-MS). We found that an enzymatic release of payload from ADC-depleted human plasma at 144 h was able to account for almost 100% of the DAR loss. Intact protein mass analysis showed that at the 144 h time point, the mass of the major protein in ADC-depleted human plasma had an additional 1347 Da over the native albumin extracted from human plasma, exactly matching the mass of the linker-payload. In addition, protein gel electrophoresis showed that there was only one enriched protein in the 144 h ADC-depleted and antipayload immunoprecipitated plasma sample, as compared to the 0 h plasma immunoprecipitated sample, and the mass of this enriched protein was slightly heavier than the mass of serum albumin. Furthermore, the albumin adduct was also identified in 96 h and 168 h postdose in vivo cynomolgus monkey plasma. These results strongly suggest that the majority of the deconjugated mc-VC-PABC-auristatin ultimately is transferred to serum albumin, forming a long-lived albumin-linker-payload adduct. To our knowledge, this is the first report quantitatively characterizing the extent of linker-payload transfer to serum albumin and the first clear example of in vivo formation of an albumin-linker-payload adduct.

Effect of attachment site on stability of cleavable antibody drug conjugates.

The systemic stability of the antibody-drug linker is crucial for delivery of an intact antibody-drug conjugate (ADC) to target-expressing tumors. Linkers stable in circulation but readily processed in the target cell are necessary for both safety and potency of the delivered conjugate. Here, we report a range of stabilities for an auristatin-based payload site-specifically attached through a cleavable valine-citrulline-p-aminobenzylcarbamate (VC-PABC) linker across various sites on an antibody. We demonstrate that the conjugation site plays an important role in determining VC-PABC linker stability in mouse plasma, and that the stability of the linker positively correlates with ADC cytotoxic potency both in vitro and in vivo. Furthermore, we show that the VC-PABC cleavage in mouse plasma is not mediated by Cathepsin B, the protease thought to be primarily responsible for linker processing in the lysosomal degradation pathway. Although the VC-PABC cleavage is not detected in primate plasma in vitro, linker stabilization in the mouse is an essential prerequisite for designing successful efficacy and safety studies in rodents during preclinical stages of ADC programs. The divergence of linker metabolism in mouse plasma and its intracellular cleavage offers an opportunity for linker optimization in the circulation without compromising its efficient payload release in the target cell.

Evaluation of two forms of N-benzoyl-L-tyrosyl p-aminobenzoic acid in pancreatic function testing of dogs.

Plasma para-aminobenzoic acid (PABA) concentrations were compared in 12 dogs after oral administration of either a powdered suspension or a solution of N-benzoyl-L-tyrosyl-PABA. Peak PABA plasma concentrations were significantly higher at 30, 60 and 90 minutes after administration of the solution (P less than 0.05). As the solution may now be used as a clinical test, interpretation of the results by comparison with normal absorption curves obtained after administration of the suspension could contribute to a failure to diagnose canine exocrine pancreatic insufficiency.

Diagnostic value of plasma PABA level in pancreatic diseases.

NBT-PABA test was performed in 72 subjects with simultaneous determination of the plasma PABA time-concentration curve. The curve in 25 controls after oral administration of N-benzoyl-L-tyrosyl-P-aminobenzoic acid (NBT-PABA) peaked (3.88 +/- 0.78 micrograms/ml, M +/- SD) at the third hour. In patients with chronic pancreatitis (24) and pancreatic carcinoma (6), the curves were flattened, and peaked at the fifth hour (2.7 +/- 1.4 micrograms/ml, M +/- SD), and fourth hour (3.14 +/- 2.26 micrograms/ml, M +/- SD), respectively. The discrimination between the controls and patients with chronic pancreatitis was most significant in the third-hour plasma PABA concentration (P less than 0.0001). This study shows that determination of the third-hour plasma PABA concentration is better in sensitivity and specificity than the 6-hour urinary excretion of PABA, suggesting that the third-hour plasma PABA concentration after oral administration of NBT-PABA might be a sensitive index of exocrine pancreatic function test.

Effectiveness of the anaesthetic MS-222 in gilthead seabream, Sparus aurata: effect of feeding time and day-night variations in plasma MS-222 concentration and GST activity.

Feeding time is a potent zeitgeber capable of synchronising behavioural and physiological daily rhythms in fish. However, the effect of feeding time on the daily rhythm of drugs toxicity and/or effectiveness remains unexplored to date. In this paper we investigated the day/night variations in the effectiveness of an anaesthetic commonly used in fish (Tricaine, MS-222) in a teleost of great chronobiological and aquaculture interest (gilthead seabream). To this end, fish were kept under LD 12:12 and fed at mid-light (ML), mid-darkness (MD) or random times (RD). The time needed to induce anaesthesia (reduction of locomotor activity) during MS-222 exposure (65 mg/L) as well as the recovery period were investigated at ML and MD in the three experimental groups using specialised video tracking software. In addition, daily rhythms of GST activity in the liver (as an indicator of detoxification processes) and plasma MS-222 concentration (related to uptake) were determined. The results revealed that MS-222 effectiveness in the ML group was higher during the day than at night (significant reduction of activity after 3 min vs. 5 min) whereas in the MD group, the daily variation of MS-222 effectiveness was inverted (significant reduction of activity after 7 min at ML vs. 2 min at MD), suggesting that feeding time can shift the day-night variations in the effectiveness of MS-222. Hepatic GST also seemed to be affected by feeding time: in fish fed at MD or RD this enzyme activity showed significant differences during the day, and the highest levels were found at different times of the day in each group. Plasma MS-222 concentrations were higher at ML (142.4±12.8 ng/ml) than at MD (96.3±10.9 ng/ml) (t-Student, p<0.05). These results suggest that the daily variation in MS-222 concentration following exposure might be involved, among other factors, in the existence of day-night variations in the effectiveness of this anaesthetic. Furthermore, manipulation of the feeding schedule can be used to modify the daily variations in MS-222 effectiveness, which has basic as well applied implications for optimising anaesthesia protocols in fish aquaculture.

[Specificity and sensitivity of the 4-N-acetyl-L-tyrosyl-p- aminobenzoic acid test in childhood with serum p-aminobenzoic acid determination]. / Spezifität und Sensitivität des ALTAB-Testes im Kindesalter bei Serum-Paba-Bestimmung.

Specificity and sensitivity of ALTAB test have been estimated in 40 healthy children and 13 cystic fibrosis patients. The test has been carried out in the modification as 2-hour-serum PABA-test. The found specificity was 90%, the sensitivity 100% respectively. Therefore this test is suitable for evaluation of exocrine pancreas function.

Liquid-chromatographic measurement of p-aminobenzoic acid and its metabolites in serum.

This is a high-performance liquid-chromatographic method for measuring p-aminobenzoic acid (PABA) and its metabolites in plasma or serum. Samples are deproteinized, then extracted with organic solvents before chromatography. For quantification, the peak height of the individual compound is compared with that of the internal standard. Analytical recoveries ranged from 41% to 100%, depending on the compound studied. Comparison of patients' samples after oral administration of either N-benzoyl-L-tyrosyl-p-aminobenzoic acid or free PABA revealed that PABA is extensively metabolized and conjugated to either p-acetamidobenzoic acid, p-aminohippuric acid, or p-acetamidohippuric acid. PABA concentrations in serum as measured with the Bratton-Marshall ultraviolet spectrophotometric procedure would appear predominantly to reflect measurements of metabolites, with only a minor contribution from PABA itself.

Pancreatic function testing: serum PABA measurement is a reliable and accurate measurement of exocrine function.

A comparison between the NBT-PABA/14C-PABA test (NBT-PABA, n-benzoyl-tyrosyl para-aminobenzoic acid) using the PABA excretion index (PEI) and serum PABA estimation at 90 minutes has been made in 42 consecutive subjects attending for investigation of possible pancreatic disease to a District General Hospital (DGH). The PEI was unobtainable or incorrect on 38% of occasions compared with 9% for the serum test. Sensitivity, specificity, and efficiency for the PEI (n = 33 valid results) were 71%, 88%, and 79% respectively and for the serum PABA (n = 41 valid results), 95%, 90%, and 93% respectively. These results confirm that measurement of serum PABA is a simpler, more reliable, and a more accurate method of assessing pancreatic function.

Colorimetric plasma assay for the bentiromide test (BT-PABA) for exocrine pancreatic insufficiency.

Bentiromide is a synthetic peptide, N-benzoyl-L-tyrosyl-p-aminobenzoic acid, which has been used as a test for exocrine pancreatic function. Following oral administration, bentiromide is hydrolyzed by chymotrypsin to yield free p-aminobenzoic acid (PABA) which is absorbed, conjugated and excreted in the urine. The PABA conjugates reach their peak levels in blood in 90-120 min. Healthy individuals have higher levels of PABA than patients with pancreatic insufficiency. A simple, accurate, and precise method for the determination of PABA in blood has been developed and validated. The plasma (1 ml) is deproteinized by perchloric acid. The conjugates are hydrolyzed and the total PABA is determined colorimetrically by the Bratton-Marshall test. The standard curve in plasma is linear up to 8 micrograms/ml of PABA. A similar semimicro method using 200 microliter of plasma suitable for pediatric samples shows comparable results. Average analytical recovery is 97% and precision studies of pooled within-run and total between-run showed CV% of 5.0 and 5.7%, respectively.

N-acetylation of drugs. Pharmacogenetic studies in rabbits selected for their acetylator characteristics.

Studies on acetylation of sulfadiazine, isoniazid, and p-aminobenzoic acid in selected lines of slow and rapid acetylator rabbits are described. Pedigree analysis of rabbits classified as slow or rapid sulfadiazine acetylators confirmed previous studies that the rate of sulfadiazine elimination (acetylation) is genetically controlled, with rapid elimination dominant over slow elimination of the drug. Pharmacokinetic studies in rabbits of specified sulfadiazine acetylator genotypes with isoniazid and p-aminobenzoic acid show that the rate of isoniazid elimination is under the same genetic control as is sulfadiazine, whereas the rate of p-aminobenzoic acid elimination is not. A new drug acetylation polymorphism, which controls the rate of enzymatic acetylation of p-aminobenzoic acid in peripheral blood cells and which is related to the sulfadiazine acetylation polymorphism, is described.

[Serum paraaminobenzoic acid following the use of N-benzoyl-L-tyrosyl-paraaminobenzoic acid in the diagnosis of pancreatic insufficiency]. / Paraaminobenzoesäurespiegel im Serum nach Gabe von N-Benzoyl-L-tyrosyl-Paraaminobenzoesäure als Suchtest der exkretorischen Pankreasfunktion.

In 25 patients with chronic exocrine pancreatic insufficiency and 37 controls, the PABA test was compared with the concentration of chymotrypsin in stool. In additional 16 patients the test could not be evaluated (rate 21%). By determination of the PABA levels in serum before and one hour after ingestion of N-benzoyl-L-tyrosyl-PABA the test can be shortened and simplified: 1 hour serum levels in 9 patients with exocrine pancreatic insufficiency were 0.5 +/- 0.5 nMol/ml compared with 8 controls with 6.4 +/- 2.0 nMol/ml. PABA serum levels correlated significantly with chymotrypsin in stool. Compared with the conventional PABA test, since it is easily applicable in outpatients and the results are not influenced by medication or food.

Value of serum PABA as a pancreatic function test.

In a total of 71 subjects (19 controls, 24 patients with non-pancreatic gastrointestinal disease, and 27 patients with pancreatic disease) an oral pancreatic function test using N-benzoyl-L-tyrosyl-PABA (BT-PABA) was performed with simultaneous determination of the serum para-aminobenzoic acid (PABA). Urinary excretion of PABA was significantly less (p less than 0.001) in patients with chronic pancreatitis (n = 12) and pancreatic carcinoma (n = 10) than in controls and in patients with non-pancreatic disease. The serum concentration curve in patients with chronic pancreatitis was significantly flattened (p less than 0.001) compared with that of the control group and the patients with non-pancreatic gastrointestinal disease. The discrimination between the controls and the patients with chronic pancreatitis was best at 120 minutes after administration of BT-PABA (lower limit of normal: 2.8 micrograms/ml). The results of our study show that determination of PABA serum concentration two hours after administration of BT-PABA is as valuable an index of pancreatic function as the urinary excretion of PABA.