Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates

The reactive thiol in cysteine is used for coupling maleimide linkers in the generation of antibody conjugates. To assess the impact of the conjugation site, we engineered cysteines into a therapeutic HER2/neu antibody at three sites differing in solvent accessibility and local charge. The highly solvent-accessible site rapidly lost conjugated thiol-reactive linkers in plasma owing to maleimide exchange with reactive thiols in albumin, free cysteine or glutathione. In contrast, a partially accessible site with a positively charged environment promoted hydrolysis of the succinimide ring in the linker, thereby preventing this exchange reaction. The site with partial solvent-accessibility and neutral charge displayed both properties. In a mouse mammary tumor model, the stability and therapeutic activity of the antibody conjugate were affected positively by succinimide ring hydrolysis and negatively by maleimide exchange with thiol-reactive constituents in plasma. Thus, the chemical and structural dynamics of the conjugation site can influence antibody conjugate performance by modulating the stability of the antibody-linker interface.     

Determination of 1-methoxy-2-propanol and its metabolite 1,2-propanediol in rat and mouse plasma by gas chromatography

A method utilizing capillary GC and flame ionization detection was developed for the simultaneous determination of 1-methoxy-2-propanol (propylene glycol monomethyl ether; PGME) and its metabolite 1,2-propanediol (propylene glycol; PG) in rat and mouse plasma. The calibration graphs for rat and mouse plasma were linear with correlation coefficients at>0.997 over the range 2–700 μg/ml. The limit of quantification was ca. 2 μg/ml (2 ng on-column) for both compounds in plasma of each species. The ranges of the precision and accuracy for PGME were 2.8–8.8% and 3.2–13%, respectively, and for PG were 11–26% and 10–25%, respectively. The recovery of PGME from rat and mouse plasma was ca. 73% and for PG it was ca. 65 and 31% from rat and mouse plasma, respectively. The method was used to study the oral absorption and metabolism of PGME in mice. PGME was readily absorbed and metabolized to PG following oral gavage administration at 90 mg/kg. The maximum concentrations of PGME and PG in plasma were attained at 20 and 30 min following dosing, respectively.     

Influence of hypoxanthine and other purines on the lecithin:cholesterol acyltransferase reaction in the plasma of rat and other species.

1. Esterification of radiolabelled cholesterol in the plasma of rat, mouse, pig, ox and, to a lesser extent, guinea pig was partially inhibited by hypoxanthine, xanthine and guanine; esterification in human plasma and in plasma from 12 other vertebrate species was unaffected by purines. 2. Esterification of endogenous cholesterol and the formation of lysolecithin in rat plasma were decreased in the presence of purines indicating that it was the lecithin:cholesterol acyltransferase (LCAT) reaction that was inhibited rather than the isotopic equilibration of labelled cholesterol with the endogenous substrate lipoproteins. 3. Maximum inhibition of the LCAT reaction in rat plasma occurred at 1.4 mM hypoxanthine or xanthine; inhibition was not dependent upon the concentration of LCAT or plasma lipoproteins but increased with the amount of lipoprotein depleted rat plasma (LDRP) present in the incubation mixture. 4. Partial inhibition of the LCAT reaction in rat or mouse plasma by purines had no significant effect on the fatty acyl composition of the cholesteryl esters (CE) formed by LCAT. 5. In the presence of heated rat plasma, LDRP or, to a lesser extent, rat high density lipoproteins (HDL) prepared from heated plasma, the LCAT reaction in human plasma was inhibited by hypoxanthine. 6. Rat HDL and LDRP prepared from plasma pre-incubated at 37 degrees C for 4 hr before heating increased and decreased, respectively, the inhibitory effect of hypoxanthine on human plasma LCAT compared with HDL and LDRP prepared from unincubated rat plasma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Validated hydrophilic interaction LC-MS/MS method for determination of N-methyl-2-pyrrolidinone residue: Applied to a depletion study of N-methyl-2-pyrrolidinone in swine liver following intramuscular administration of drug N-methyl-2-pyrrolidinone formulation

A hydrophilic interaction high-performance liquid chromatography coupled with tandem mass spectrometry method for determination of N-methyl-2-pyrrolidinone in swine liver was developed and validated. After the fortification of N-methyl-d(3)-2-pyrrolidinone-d(6) as the deuterium-labeled internal standard, N-methyl-2-pyrrolidinone in swine liver was extracted by acetonitrile and the supernatant was led through a C18+WAX mixed-mode SPE cartridge for removal of the matrix interferences. The final eluate was acidified by formic acid and then injected onto a 3μm 15cm×2.1mm TX column for hydrophilic interaction chromatographic analysis. Mass spectrometry detection was carried on a PE Sciex API 4000 triple quadrupole mass spectrometer using positive turbo-ion spray ionization mode. The MRM transitions were 100→58 for N-methyl-2-pyrrolidinone and 109→62 for N-methyl-d(3)-2-pyrrolidinone-d(6). Solvent calibration standards could be readily used for quantitative analysis of N-methyl-2-pyrrolidinone with excellent precision and accuracy, although there are endogenous levels of N-methyl-2-pyrrolidinone in many blank matrices. The true recovery was nearly 100% and the MRM signal of N-methyl-2-pyrrolidinone was suppressed about 30% because of the matrix effect. Nevertheless, N-methyl-d(3)-2-pyrrolidinone-d(6) completely compensated the ion-suppression effect and the injection-to-injection variation. The detection limit was 5ngg(-1) swine liver. The validated method was applied to a depletion study of N-methyl-2-pyrrolidinone in swine liver following intramuscular administration of a drug N-methyl-2-pyrrolidinone formulation.     

The origin of plasma alkaline phosphatase activity in mice and rats

Swiss albino mice displayed the highest activity of alkaline phosphatase at 4-6 weeks with a precipitous decline by 18 weeks of age to a value seen in the mature animal. Circulating activity of alkaline phosphatase was significantly higher in the rat than the mouse in the fed state. With fasting, enzyme activity declined in the rat yet increased in the mouse. The net result was significantly higher plasma alkaline phosphatase activity in the mouse than the rat after the 48 hr fast. L-Phenylalanine inhibition of plasma alkaline phosphatase was greater in plasma from the rat than the mouse in the fed state. Yet in the fed condition, L-homoarginine and L-p-bromotetramisole inhibited alkaline phosphatase activity in plasma from mice to a greater extent than in rats. Heat inactivation as well as urea denaturation of alkaline phosphatase was significantly faster with plasma of the mouse than the rat in the fed state. Thus, it appears that the alkaline phosphatase isoenzyme of skeletal origin contributes a greater proportion of the circulating activity in the fed Swiss albino mouse than occurs in the Sprague-Dawley rat in which the intestinal isoenzyme plays a greater role in the fed condition.     

Detection of a cell surface antigen common to a mouse myeloma and a population of mouse thymocytes.

A new antigen termed PTA (plasmocytoma thymus antigen) is described which occurs on 70--80% of mouse thymocytes of all mouse strains tested and on an IgG 2b producing BALB/c plasma cell tumor. PTA is detectable on mouse spleen and lymph node cells. It is absent from liver, brain, bone marrow, and antibody plaque forming cells. PTA is not related to theta antigen and is not detectable on rat thymocytes.     

Differences in deoxycytidine metabolism in mouse and rat.

Bone-marrow macrophages from both rat and mouse release deoxycytidine derived from phagocytosed nuclei. Mouse plasma contains no detectable deoxycytidine (less than 0.1 microM), whereas the concentration in rat plasma is 18 microM. Enzyme assays of tissue extracts show that both mouse and rat spleen contain high deoxycytidine kinase activity. Mouse organs, including kidney, liver and lung, also have deoxycytidine deaminase activity. In contrast, rat tissues have virtually no deoxycytidine deaminase activity. Lack of deaminase provides an explanation for the presence of deoxycytidine in rat plasma. Cytotoxicity assays show that cultured mouse lymphoid cells grown in undialysed rat serum are more resistant to cytotoxic effects of deoxyadenosine than are those cells grown in dialysed rat serum. The results suggest that a major difference in deoxycytidine metabolism between mouse and rat may account for discrepancies in the pharmacological response of the two animals to certain nucleoside compounds.     

Effects of alpha-macroglobulins and murinoglobulins on the hemagglutination by influenza C virus

Rat alpha-1- and alpha-2-macroglobulins as well as rat murinoglobulins I and II were shown to inhibit hemagglutination by influenza C virus. In marked contrast, neither alpha-macroglobulins nor murinoglobulins from mouse or guinea pig plasma had the inhibitory activity. These results suggest that the hemagglutination-inhibiting activity of rat alpha-macroglobulins or murinoglobulins is not related to their protease-binding capacity.     

Chemoattraction of infective larvae of Ancylostoma braziliense to rodent plasmas and to salts

Infective larvae of Anyclostoma braziliense were tested for orientational response to rat plasma, to mouse plasma, to rat plasma fractions, and to salts. A high percentage of larvae accumulated at sources of rat plasma, mouse plasma, rat plasma diffusate, concentrated rat plasma dialysate, and some salts, notably sodium chloride. Because sodium chloride is present at an effective concentration in mammalian plasma, and because this salt may form a gradient between the blood and the skin surface, sodium chloride from the blood may direct the penetrating larvae through host's skin. Preliminary tracking of the larvae in gradients of rat plasma and of sodium chloride suggests that orientation to sources of these attractants was via a taxis, possibly a klinotaxis, whereas accumulation at these sources was via a klinokinesis.     

Phospholipase D2 localizes to the plasma membrane and regulates angiotensin II receptor endocytosis

Phospholipase D (PLD) is a key facilitator of multiple types of membrane vesicle trafficking events. Two PLD isoforms, PLD1 and PLD2, exist in mammals. Initial studies based on overexpression studies suggested that in resting cells, human PLD1 localized primarily to the Golgi and perinuclear vesicles in multiple cell types. In contrast, overexpressed mouse PLD2 was observed to localize primarily to the plasma membrane, although internalization on membrane vesicles was observed subsequent to serum stimulation. A recent report has suggested that the assignment of PLD2 to the plasma membrane is in error, because the endogenous isoform in rat secretory cells was imaged and found to be present primarily in the Golgi apparatus. We have reexamined this issue by using a monoclonal antibody specific for mouse PLD2, and find, as reported initially using overexpression studies, that endogenous mouse PLD2 is detected most readily at the plasma membrane in multiple cell types. In addition, we report that mouse, rat, and human PLD2 when overexpressed all similarly localize to the plasma membrane in cell lines from all three species. Finally, studies conducted using overexpression of wild-type active or dominant-negative isoforms of PLD2 and RNA interference-mediated targeting of PLD2 suggest that PLD2 functions at the plasma membrane to facilitate endocytosis of the angiotensin II type 1 receptor.     

Determination of lecithin:cholesterol acyltransfer in mouse plasma and the influence of mercaptoethanol and sulphydryl blocking agents on its activity.

1. The cholesterol esterifying activity in mouse plasma has been identified as lecithin:cholesterol acyltransferase (LCAT) on the basis of stoichiometric data, predominant transfer of polyunsaturated fatty acids, wide pH optimum and inhibition of esterification by phospholipase A2 and sulphydryl blocking agents. The esterifying activity differed from that present in plasma of man, rat and other species since it was partially inhibited by mercaptoethanol and other thiols. 2. Stoichiometric correlations between unesterified cholesterol, lecithin and lysolecithin were not exact, suggesting possible involvement of other enzymes in the overall esterification process during in vitro incubation of mouse plasma. 3. The initial rate of cholesterol esterification was determined by in vitro incubation of mouse plasma, whose cholesterol had been labelled by prior in vivo injection of 3H-mevalonic acid. The mean rate was 281 +/- 74 nmol/ml/hr (mean +/- S.D., n = 12) and correlated with unesterified cholesterol concentration (r = 0.73, P less than 0.01).     

Adrenal glands of mouse and rat do not synthesize androgens.

Human adrenal glands produce considerable amounts of the C-19 steroids dehydroepiandrosterone (DHEA) and androstenedione. To investigate the capability of rodent adrenals to produce these steroids, cell suspensions of mouse and rat adrenal glands were incubated in the absence and presence of adrenocorticotropic hormone (ACTH). Corticosterone levels in the incubation medium increased dramatically in the presence of ACTH, but no significant amounts of 17-hydroxyprogesterone or androstenedione could be detected. This indicates that the adrenals of rat and mouse lack the enzyme 17 alpha-hydroxylase. Absence of plasma cortisol in the presence of high levels of corticosterone confirmed these data. Plasma levels of androstenedione were significantly decreased in castrated male rats as compared to levels observed in intact males, showing the contribution of the testes to the plasma content of androstenedione. Very low levels of androstenedione were observed in female, male and castrated male mice. Plasma concentrations of DHEA were not detectable in intact and castrated male mice and rats. It is concluded that rat and mouse lack the enzyme necessary to synthesize adrenal C-19 steroids and that the adrenals in these animals, therefore, do not contribute to plasma levels of androstenedione and DHEA.     

Copper deficient rats and mice both develop anemia but only rats have lower plasma and brain iron levels

Iron homeostasis depends on adequate dietary copper but the mechanisms are unknown. Mice (Mus musculus) and rat (Rattus norvegicus) offspring were compared to determine the effect of dietary copper deficiency (Cu-) on iron status of plasma, liver, brain and intestine. Holtzman rat and Hsd:ICR (CD-1) outbred albino mouse dams were fed a Cu- diet and drank deionized water or Cu supplemented water. Offspring were sampled at time points between postnatal ages 13 and 32. Cu- rat and mouse pups were both anemic, but only rat pups had lower plasma and brain iron levels. Plasma iron was lower throughout the suckling period in Cu- rats but not Cu- mice. Cu- mice derived from dams restricted of Cu only during lactation were also severely anemic without hypoferremia. Intestinal metal analysis confirmed that Cu- pups had major reductions in intestinal concentration of Cu, increased Fe, and normal Zn. However, whole mouse (less the intestine) analysis demonstrated normal content of Fe indicating that the limitation in iron transport by intestinal hephaestin had no consequence to total iron reserves of the mouse. Further research will be needed to determine the reason Cu- mice were anemic since the "ferroxidase" hypothesis does not explain this phenotype.     

9-Cis-retinoyl-β-glucuronide is a major metabolite of 9-Cis-retinoic acid

The in vivo metabolism of 9-cis-retinoic acid (9-c-RA), an endogenous ligand of retinoid X receptors (RXRs), which can also bind to retinoic acid receptors (RARs), was examined in pregnant mice and rats following a single oral dose of 100 mg 9-cis- retinaldehyde (9-c-RAL) / kg body weight. 9-Cis-retinoyl-β-glucuronide (9-c-RAG), a metabolite not found in vivo before, was a major metabolite of 9-c-RA in mouse plasma and was also present in all mouse tissues examined as well as in rat plasma. In both species putative oxidation products of retinoic acids and high levels of retinyl esters were found. Concentrations of retinoic acid isomers and retinoyl-β-D-glucuronides in the mouse plasma greatly exceeded those of the rat plasma. The finding of high levels of 9-c-RAG underlines the importance of glucuronidation in the metabolism of retinoids.     

Rat plasma murinoglobulin: isolation, characterization, and comparison with rat alpha-1- and alpha-2-macroglobulins

Murinoglobulin, a newly identified mouse plasma protein with trypsin-protein esterase activity (Saito, A. & Sinohara, H. (1985) J. Biol. Chem. 260, 775-781), was also found in rat plasma and purified to apparent homogeneity. The serum level of rat murinoglobulin was 14.1 mg/ml, amounting to 1/3 of the total serum globulin fraction. Rat murinoglobulin was a monomeric glycoprotein (Mr = 210,000) containing 12% carbohydrate. Rat plasma contained two isoforms of murinoglobulin, termed I and II, which showed complete immunological identity on double diffusion analysis using rabbit antiserum raised against isoform I or II. These antisera also showed partial cross-reactivity towards mouse murinoglobulin and rat alpha-1-macroglobulin but not towards rat or human alpha-2-macroglobulin. The chemical compositions, peptide mapping patterns and electrophoretic mobilities of the two isoforms resembled each other but clearly differed from those of rat alpha-1- or alpha-2-macroglobulin. Rat murinoglobulin inhibited the proteolytic activity of trypsin towards casein and remazol brilliant blue hide powder. The inhibition as to the latter substrate was greater than that as to the former. When molar ratios of inhibitor to trypsin were low, murinoglobulin and the two alpha-macroglobulins stimulated the amidolytic activity of trypsin towards a synthetic substrate. At higher ratios, however, murinoglobulin, but not the alpha-macroglobulins, inhibited the same activity. The trypsin-protein esterase activity of murinoglobulin and the two alpha-macroglobulins was impaired by a molar excess of soybean trypsin inhibitor. Murinoglobulin and the two alpha-macroglobulins were inactivated by methylamine with a concomitant unmasking of the thiol group. Murinoglobulin was much more sensitive to soybean trypsin inhibitor and methylamine than the two alpha-macroglobulins.     

Assignment of three rat genes coding for plasma proteins, transferrin, third component of complement, and beta-fibrinogen to rat chromosomes 8, 9, and 2

The assignment of three rat genes coding for plasma proteins has been deduced from the analysis of rat x mouse cell hybrids. The transferrin and the third component of complement genes were localized on rat chromosomes 8 and 9 respectively, and the beta-fibrinogen gene on chromosome 2.     

A protein associated with the mouse and rat hepatocyte junctional complex

Summary In this study we have examined a protein associated with bile canaliculi of mouse and rat hepatocytes that is detected by monoclonal antibody BG9.1. The protein is seen by indirect-immunofluorescence microscopy as 2 discrete parallel lines at the lateral borders of adjacent hepatocytes. This pattern is present during development in the day 13 fetal mouse liver. Electron microscopy with immuno-gold labeling indicated that the protein is associated with the cytoplasmic surface of junctional complexes located on either side of bile canaliculi. BG9.1 reacts with a protein of 192000 apparent molecular weight on immunoblots of plasma membrane isolated from mouse and rat hepatocytes. It has been reported that unlike most cellular components, tight junctions are not soluble in sodium deoxycholate. Extraction of isolated hepatic plasma membrane sheets with deoxycholate and other reagents did not eliminate the pattern seen by indirect-immunofluorescence microscopy and enhanced the intensity of reactions on immunoblots. BG9.1 also binds to the junctional-complex region in other epithelial cell types. These results indicate that BG9.1 detects a deoxycholate-insoluble protein associated with junctional complexes and suggests that the protein is a component of tight junctions.     

Characterization of rodent liver and kidney AVP receptors: pharmacologic evidence for species differences.

Radioligand binding studies with [3H]vasopressin (AVP) were used to determine the affinities of AVP receptor agonists and antagonists for mouse liver and kidney plasma membrane preparations. Both membrane preparations exhibited one class of high-affinity binding site. AVP ligand binding inhibition studies confirmed that mouse liver binding sites belong to the V1A subtype while kidney binding sites belong to the V2 receptor subtype. The affinity of each ligand for mouse V1A receptors was very similar to that for rat V1A receptors, showing differences in Ki values of less than 3-fold. In contrast, several peptide (d(CH2)5Tyr(Me)AVP) and nonpeptide (OPC-21268 and SR 49059) ligands had different affinities for mouse and rat kidney V2 receptors, with differences in Ki values ranging from 14- to 17-fold. These results indicate that mouse and rat kidney V2 receptors show significant pharmacologic differences.     

Studies on plasma membranes

Summary Plasma membranes were isolated from rat and mouse livers, one rat hepatoma (and its subline) and two mouse hepatomas, and their lipid class compositions were determined. Lipids accounted for 30 to 35% of the dry weight of the membranes of livers and mouse hepatomas, and for 45% in the case of rat hepatoma-subline. Of the total lipids of rat-liver plasma membranes, 60% consisted of phospholipids, the corresponding values for mouse-liver and rat-hepatoma plasma membranes amounting to 55% and for both mouse-hepatoma plasma membranes to about 50%. The free cholesterol and cholesteryl ester contents of all hepatoma plasma membranes were significantly increased as compared with normal. Evidence is presented that the increase of free cholesterol was not a preparative artefact. The major phospholipid classes in all plasma membranes were phosphatidyl choline, sphingomyelin, phosphatidyl ethanolamine and phosphatidyl serine. The relative proportions in each plasma membrane species could differ appreciably, the mouse- and rat-liver membranes showing the closest resemblance. Possible reasons for (a) the higher level of phosphatidyl serine as compared with published values, and (b) the wide divergencies which may be found among the phospholipid profiles of rat-liver plasma membranes reported by other authors, are presented. Cardiolipin was absent from liver plasma membranes, but some could be found in the hepatoma membranes due to mitochondrial contamination. No consistent phospholipid profile characterized hepatoma as distinct from liver plasma membranes, nor did the hepatoma data-including plasmalogens-resemble the few available data on other hepatomas.