Characterizing the Suture Pullout Force for Human Small Bowel.

Performing a small bowel anastomosis, or reconnecting small bowel segments, remains a core competency and critical step for the successful surgical management of numerous bowel and urinary conditions. As surgical education and technology moves toward improving patient outcomes through automation and increasing training opportunities, a detailed characterization of the interventional biomechanical properties of the human bowel is important. This is especially true due to the prevalence of anastomotic leakage as a frequent (3.02%) postoperative complication of small bowel anastomoses. This study aims to characterize the forces required for a suture to tear through human small bowel (suture pullout force, SPOF), while analyzing how these forces are affected by tissue orientation, suture material, suture size, and donor demographics. 803 tests were performed on 35 human small bowel specimens. A uni-axial test frame was used to tension sutures looped through 10 × 20 mm rectangular bowel samples to tissue failure. The mean SPOF of the small bowel was 4.62±1.40 N. We found no significant effect of tissue orientation (p = 0.083), suture material (p = 0.681), suture size (p = 0.131), age (p = 0.158), sex (p = .083), or body mass index (BMI) (p = 0.100) on SPOF. To our knowledge, this is the first study reporting human small bowel SPOF. Little research has been published about procedure-specific data on human small bowel. Filling this gap in research will inform the design of more accurate human bowel synthetic models and provide an accurate baseline for training and clinical applications.

Shackling in the Hospital.

Hospitalized incarcerated patients are commonly shackled throughout their duration of treatment in community medical centers to prevent escape or harm to others. In the absence of overarching policies guiding the shackling of non-pregnant, incarcerated patients, clinicians rarely unshackle patients during routine care. We provide a medical-legal lens through which to examine inpatient shackling, review the limited evidence supporting the practice, and highlight harms associated with shackling in the hospital. We conclude by offering guidance to advance evidence-based shackling practices that prevent physical harm, reduce prejudice towards incarcerated patients, and relinquish reliance on shackles in favor of tailored security measures.

A Case Study Comparing Heterogeneous Lysine- and Site-Specific Cysteine-Conjugated Maytansinoid Antibody-Drug Conjugates (ADCs) Illustrates the Benefits of Lysine Conjugation.

Antibody-drug conjugates are an emerging class of cancer therapeutics constructed from monoclonal antibodies conjugated with small molecule effectors. First-generation molecules of this class often employed heterogeneous conjugation chemistry, but many site-specifically conjugated ADCs have been described recently. Here, we undertake a systematic comparison of ADCs made with the same antibody and the same macrocyclic maytansinoid effector but conjugated either heterogeneously at lysine residues or site-specifically at cysteine residues. Characterization of these ADCs in vitro reveals generally similar properties, including a similar catabolite profile, a key element in making a meaningful comparison of conjugation chemistries. In a mouse model of cervical cancer, the lysine-conjugated ADC affords greater efficacy on a molar payload basis. Rather than making general conclusions about ADCs conjugated by a particular chemistry, we interpret these results as highlighting the complexity of ADCs and the interplay between payload class, linker chemistry, target antigen, and other variables that determine efficacy in a given setting.

FRET Reagent Reveals the Intracellular Processing of Peptide-Linked Antibody-Drug Conjugates.

Despite the recent success of antibody-drug conjugates (ADCs) in cancer therapy, a detailed understanding of their entry, trafficking, and metabolism in cancer cells is limited. To gain further insight into the activation mechanism of ADCs, we incorporated fluorescence resonance energy transfer (FRET) reporter groups into the linker connecting the antibody to the drug and studied various aspects of intracellular ADC processing mechanisms. When comparing the trafficking of the antibody-FRET drug conjugates in various different model cells, we found that the cellular background plays an important role in how the antigen-mediated antibody is processed. Certain tumor cells showed limited cytosolic transport of the payload despite efficient linker cleavage. Our FRET assay provides a facile and robust assessment of intracellular ADC activation that may have significant implications for the future development of ADCs.

High-Throughput Cysteine Scanning To Identify Stable Antibody Conjugation Sites for Maleimide- and Disulfide-Based Linkers.

THIOMAB antibody technology utilizes cysteine residues engineered onto an antibody to allow for site-specific conjugation. The technology has enabled the exploration of different attachment sites on the antibody in combination with small molecules, peptides, or proteins to yield antibody conjugates with unique properties. As reported previously ( Shen , B. Q. , et al. ( 2012 ) Nat. Biotechnol. 30 , 184 - 189 ; Pillow , T. H. , et al. ( 2017 ) Chem. Sci. 8 , 366 - 370 ), the specific location of the site of conjugation on an antibody can impact the stability of the linkage to the engineered cysteine for both thio-succinimide and disulfide bonds. High stability of the linkage is usually desired to maximize the delivery of the cargo to the intended target. In the current study, cysteines were individually substituted into every position of the anti-HER2 antibody (trastuzumab), and the stabilities of drug conjugations at those sites were evaluated. We screened a total of 648 THIOMAB antibody-drug conjugates, each generated from a trastuzamab prepared by sequentially mutating non-cysteine amino acids in the light and heavy chains to cysteine. Each THIOMAB antibody variant was conjugated to either maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E (MC-vc-PAB-MMAE) or pyridyl disulfide monomethyl auristatin E (PDS-MMAE) using a high-throughput, on-bead conjugation and purification method. Greater than 50% of the THIOMAB antibody variants were successfully conjugated to both MMAE derivatives with a drug to antibody ratio (DAR) of >0.5 and <50% aggregation. The relative in vitro plasma stabilities for approximately 750 conjugates were assessed using enzyme-linked immunosorbent assays, and stable sites were confirmed with affinity-capture LC/MS-based detection methods. Highly stable conjugation sites for the two types of MMAE derivatives were identified on both the heavy and light chains. Although the stabilities of maleimide conjugates were shown to be greater than those of the disulfide conjugates, many sites were identified that were stable for both. Furthermore, in vitro stabilities of selected stable sites translated across different cytotoxic payloads and different target antibodies as well as to in vivo stability.

Attachment Site Cysteine Thiol pKa Is a Key Driver for Site-Dependent Stability of THIOMAB Antibody-Drug Conjugates.

The incorporation of cysteines into antibodies by mutagenesis allows for the direct conjugation of small molecules to specific sites on the antibody via disulfide bonds. The stability of the disulfide bond linkage between the small molecule and the antibody is highly dependent on the location of the engineered cysteine in either the heavy chain (HC) or the light chain (LC) of the antibody. Here, we explore the basis for this site-dependent stability. We evaluated the in vivo efficacy and pharmacokinetics of five different cysteine mutants of trastuzumab conjugated to a pyrrolobenzodiazepine (PBD) via disulfide bonds. A significant correlation was observed between disulfide stability and efficacy for the conjugates. We hypothesized that the observed site-dependent stability of the disulfide-linked conjugates could be due to differences in the attachment site cysteine thiol pKa. We measured the cysteine thiol pKa using isothermal titration calorimetry (ITC) and found that the variants with the highest thiol pKa (LC K149C and HC A140C) were found to yield the conjugates with the greatest in vivo stability. Guided by homology modeling, we identified several mutations adjacent to LC K149C that reduced the cysteine thiol pKa and, thus, decreased the in vivo stability of the disulfide-linked PBD conjugated to LC K149C. We also present results suggesting that the high thiol pKa of LC K149C is responsible for the sustained circulation stability of LC K149C TDCs utilizing a maleimide-based linker. Taken together, our results provide evidence that the site-dependent stability of cys-engineered antibody-drug conjugates may be explained by interactions between the engineered cysteine and the local protein environment that serves to modulate the side-chain thiol pKa. The influence of cysteine thiol pKa on stability and efficacy offers a new parameter for the optimization of ADCs that utilize cysteine engineering.

Effects of Drug-Antibody Ratio on Pharmacokinetics, Biodistribution, Efficacy, and Tolerability of Antibody-Maytansinoid Conjugates.

Antibody-drug conjugates (ADCs) are being actively pursued as a treatment option for cancer following the regulatory approval of brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla). ADCs consist of a cytotoxic agent conjugated to a targeting antibody through a linker. The two approved ADCs (and most ADCs now in the clinic that use a microtubule disrupting agent as the payload) are heterogeneous conjugates with an average drug-to-antibody ratio (DAR) of 3-4 (potentially ranging from 0 to 8 for individual species). Ado-trastuzumab emtansine employs DM1, a semisynthetic cytotoxic payload of the maytansinoid class, which is conjugated via lysine residues of the antibody to an average DAR of 3.5. To understand the effect of DAR on the preclinical properties of ADCs using maytansinoid cytotoxic agents, we prepared a series of conjugates with a cleavable linker (M9346A-sulfo-SPDB-DM4 targeting folate receptor α (FRα)) or an uncleavable linker (J2898A-SMCC-DM1 targeting the epidermal growth factor receptor (EGFR)) with varying DAR and evaluated their biochemical characteristics, in vivo stability, efficacy, and tolerability. For both formats, a series of ADCs with DARs ranging from low (average of ∼2 and range of 0-4) to very high (average of 10 and range of 7-14) were prepared in good yield with high monomer content and low levels of free cytotoxic agent. The in vitro potency consistently increased with increasing DAR at a constant antibody concentration. We then characterized the in vivo disposition of these ADCs. Pharmacokinetic analysis showed that conjugates with an average DAR below ∼6 had comparable clearance rates, but for those with an average DAR of ∼9-10, rapid clearance was observed. Biodistribution studies in mice showed that these 9-10 DAR ADCs rapidly accumulate in the liver, with maximum localization for this organ at 24-28% percentage injected dose per gram (%ID/g) compared with 7-10% for lower-DAR conjugates (all at 2-6 h post-injection). Our preclinical findings on tolerability and efficacy suggest that maytansinoid conjugates with DAR ranging from 2 to 6 have a better therapeutic index than conjugates with very high DAR (∼9-10). These very high DAR ADCs suffer from decreased efficacy, likely due to faster clearance. These results support the use of DAR 3-4 for maytansinoid ADCs but suggest that the exploration of lower or higher DAR may be warranted depending on the biology of the target antigen.

Understanding How the Stability of the Thiol-Maleimide Linkage Impacts the Pharmacokinetics of Lysine-Linked Antibody-Maytansinoid Conjugates.

Antibody-drug conjugates (ADCs) have become a widely investigated modality for cancer therapy, in part due to the clinical findings with ado-trastuzumab emtansine (Kadcyla). Ado-trastuzumab emtansine utilizes the Ab-SMCC-DM1 format, in which the thiol-functionalized maytansinoid cytotoxic agent, DM1, is linked to the antibody (Ab) via the maleimide moiety of the heterobifunctional SMCC linker. The pharmacokinetic (PK) data for ado-trastuzumab emtansine point to a faster clearance for the ADC than for total antibody. Cytotoxic agent release in plasma has been reported with nonmaytansinoid, cysteine-linked ADCs via thiol-maleimide exchange, for example, brentuximab vedotin. For Ab-SMCC-DM1 ADCs, however, the main catabolite reported is lysine-SMCC-DM1, the expected product of intracellular antibody proteolysis. To understand these observations better, we conducted a series of studies to examine the stability of the thiol-maleimide linkage, utilizing the EGFR-targeting conjugate, J2898A-SMCC-DM1, and comparing it with a control ADC made with a noncleavable linker that lacked a thiol-maleimide adduct (J2898A-(CH2)3-DM). We employed radiolabeled ADCs to directly measure both the antibody and the ADC components in plasma. The PK properties of the conjugated antibody moiety of the two conjugates, J2898A-SMCC-DM1 and J2898A-(CH2)3-DM (each with an average of 3.0 to 3.4 maytansinoid molecules per antibody), appear to be similar to that of the unconjugated antibody. Clearance values of the intact conjugates were slightly faster than those of the Ab components. Furthermore, J2898A-SMCC-DM1 clears slightly faster than J2898A-(CH2)3-DM, suggesting that there is a fraction of maytansinoid loss from the SMCC-DM1 ADC, possibly through a thiol-maleimide dependent mechanism. Experiments on ex vivo stability confirm that some loss of maytansinoid from Ab-SMCC-DM1 conjugates can occur via thiol elimination, but at a slower rate than the corresponding rate of loss reported for thiol-maleimide links formed at thiols derived by reduction of endogenous cysteine residues in antibodies, consistent with expected differences in thiol-maleimide stability related to thiol pKa. These findings inform the design strategy for future ADCs.

Chemical Structure and Concentration of Intratumor Catabolites Determine Efficacy of Antibody Drug Conjugates.

Despite recent technological advances in quantifying antibody drug conjugate (ADC) species, such as total antibody, conjugated antibody, conjugated drug, and payload drug in circulation, the correlation of their exposures with the efficacy of ADC outcomes in vivo remains challenging. Here, the chemical structures and concentrations of intratumor catabolites were investigated to better understand the drivers of ADC in vivo efficacy. Anti-CD22 disulfide-linked pyrrolobenzodiazepine (PBD-dimer) conjugates containing methyl- and cyclobutyl-substituted disulfide linkers exhibited strong efficacy in a WSU-DLCL2 xenograft mouse model, whereas an ADC derived from a cyclopropyl linker was inactive. Total ADC antibody concentrations and drug-to-antibody ratios (DAR) in circulation were similar between the cyclobutyl-containing ADC and the cyclopropyl-containing ADC; however, the former afforded the release of the PBD-dimer payload in the tumor, but the latter only generated a nonimmolating thiol-containing catabolite that did not bind to DNA. These results suggest that intratumor catabolite analysis rather than systemic pharmacokinetic analysis may be used to better explain and predict ADC in vivo efficacy. These are good examples to demonstrate that the chemical nature and concentration of intratumor catabolites depend on the linker type used for drug conjugation, and the potency of the released drug moiety ultimately determines the ADC in vivo efficacy.

Antibody Drug Conjugates Differentiate Uptake and DNA Alkylation of Pyrrolobenzodiazepines in Tumors from Organs of Xenograft Mice.

Pyrrolobenzodiazepine (PBD)-dimer is a DNA minor groove alkylator, and its CD22 THIOMAB antibody drug conjugate (ADC) demonstrated, through a disulfide linker, an efficacy in tumor reduction for more than 7 weeks with minimal body weight loss in xenograft mice after a single 0.5-1 mg/kg i.v. dose. The DNA alkylation was investigated here in tumors and healthy organs of mice to understand the sustained efficacy and tolerability. The experimental procedures included the collection of tumors and organ tissues of xenograft mice treated with the ADC followed by DNA isolation/hydrolysis/quantitation and payload recovery from reversible DNA alkylation. PBD-dimer formed a considerable amount of adducts with tissue DNA, representing approximately 98% (at 24 hours), and 99% (at 96 hours) of the total PBD-dimer in tumors, and 78-89% in liver and lung tissues, suggesting highly efficient covalent binding of the released PBD-dimer to tissue DNA. The amount of PBD-DNA adducts in tumor tissues was approximately 24-fold (at 24 hours) and 70-fold (at 96 hours) greater than the corresponding amount of adducts in liver and lung tissues. In addition, the DNA alkylation levels increased 3-fold to 4-fold from 24 to 96 hours in tumors [41/106 base pairs (bp) at 96 hours] but remained at the same level (1/106 bp) in livers and lungs. These results support the typical target-mediated cumulative uptake of ADC into tumors and payload release that offers an explanation for its sustained antitumor efficacy. In addition, the low level of DNA alkylation in normal tissues is consistent with the tolerability observed in mice.

A novel anti-CD37 antibody-drug conjugate with multiple anti-tumor mechanisms for the treatment of B-cell malignancies.

CD37 has gathered renewed interest as a therapeutic target in non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL); however, CD37-directed antibody-drug conjugates (ADCs) have not been explored. Here, we identified a novel anti-CD37 antibody, K7153A, with potent in vitro activity against B-cell lines through multiple mechanisms including apoptosis induction, antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis, and complement-dependent cytotoxicity. The antibody was conjugated to the maytansinoid, DM1, a potent antimicrotubule agent, via the thioether linker, N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and the resulting ADC, IMGN529, retained the intrinsic antibody activities and showed enhanced cytotoxic activity from targeted payload delivery. In lymphoma cell lines, IMGN529 induced G2/M cell cycle arrest after internalization and lysosomal processing to lysine-N(ε)-SMCC-DM1 as the sole intracellular maytansinoid metabolite. IMGN529 was highly active against subcutaneous B-cell tumor xenografts in severe combined immunodeficient mice with comparable or better activity than rituximab, a combination of cyclophosphamide, vincristine, and prednisone, or bendamustine. In human blood cells, CD37 is expressed in B cells at similar levels as CD20, and IMGN529 resulted in potent and specific depletion of normal and CLL B cells. These results support evaluation of the CD37-targeted ADC, IMGN529, in clinical trials in patients with B-cell malignancies including NHL and CLL.

Design of Coltuximab Ravtansine, a CD19-Targeting Antibody-Drug Conjugate (ADC) for the Treatment of B-Cell Malignancies: Structure-Activity Relationships and Preclinical Evaluation.

Coltuximab ravtansine (SAR3419) is an antibody-drug conjugate (ADC) targeting CD19 created by conjugating a derivative of the potent microtubule-acting cytotoxic agent, maytansine, to a version of the anti-CD19 antibody, anti-B4, that was humanized as an IgG1 by variable domain resurfacing. Four different linker-maytansinoid constructs were synthesized (average ∼3.5 maytansinoids/antibody for each) to evaluate the impact of linker-payload design on the activity of the maytansinoid-ADCs targeting CD19. The ADC composed of DM4 (N(2')-deacetyl-N(2')-[4-mercapto-4-methyl-1-oxopentyl]maytansine) conjugated to antibody via the N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) linker was selected for development as SAR3419. A molar ratio for DM4/antibody of between 3 and 5 was selected for the final design of SAR3419. Evaluation of SAR3419 in Ramos tumor xenograft models showed that the minimal effective single dose was about 50 µg/kg conjugated DM4 (∼2.5 mg/kg conjugated antibody), while twice this dose gave complete regressions in 100% of the mice. SAR3419 arrests cells in the G2/M phase of the cell cycle, ultimately leading to apoptosis after about 24 h. The results of in vitro and in vivo studies with SAR3419 made with DM4 that was [(3)H]-labeled at the C20 methoxy group of the maytansinoid suggest a mechanism of internalization and intracellular trafficking of SAR3419, ultimately to lysosomes, in which the antibody is fully degraded, releasing lysine-N(ε)-SPDB-DM4 as the initial metabolite. Subsequent intracellular reduction of the disulfide bond between linker and DM4 generates the free thiol species, which is then converted to S-methyl DM4 by cellular methyl transferase activity. We provide evidence to suggest that generation of S-methyl DM4 in tumor cells may contribute to in vivo tumor eradication via bystander killing of neighboring tumor cells. Furthermore, we show that S-methyl DM4 is converted to the sulfoxide and sulfone derivatives in the liver, suggesting that hepatic catabolism of the payload to less cytotoxic maytansinoid species contributes to the overall therapeutic window of SAR3419. This compound is currently in phase II clinical evaluation for the treatment of diffuse large B cell lymphoma.

Relationships of self-reported and objective measures of sleep, sleepiness, and sleep quality in Parkinson's disease.

INTRODUCTION: Disrupted sleep and excessive daytime sleepiness (EDS) are common and disabling symptoms of Parkinson's disease (PD). The relationships between subjective and objective assessments of sleep and sleepiness in PD are not well established. We aimed to examine the relationships between self-reported (subjective) and objective assessments of sleep and sleepiness in PD. METHODS: Epworth Sleepiness Scale (ESS), Pittsburg Sleep Quality Index (PSQI), Parkinson's Disease Sleep Scale (PDSS), sleep diaries, and overnight polysomnography (PSG) with next-morning multiple sleep latency testing (MSLT) were collected from 27 individuals with PD and EDS who participated in a clinical trial of light therapy for EDS in PD. RESULTS: No significant correlations were found between measures of EDS and night-time sleep quality and quantity. PDSS was correlated with PSQI. PDSS and PSQI had significant relationships with multiple metrics derived from sleep diaries, including sleep latency, quality, and ease of falling asleep. Several PSG-derived sleep metrics correlated well with sleep diary metrics. CONCLUSIONS: There is a poor correlation between metrics used to assess sleep and sleepiness in PD. A sleep diary may be a valuable tool for this purpose. Accurate clinical and research assessment and monitoring require refinement of existing and development of novel methods for measuring sleep and sleepiness in PD.

Design of antibody-maytansinoid conjugates allows for efficient detoxification via liver metabolism.

Antibody-maytansinoid conjugates (AMCs) are targeted chemotherapeutic agents consisting of a potent microtubule-depolymerizing maytansinoid (DM1 or DM4) attached to lysine residues of a monoclonal antibody (mAb) using an uncleavable thioether linker or a stable disulfide linker. Most of the administered dose of an antibody-based therapeutic is slowly catabolized by the liver and other tissues of the reticuloendothelial system. Maytansinoids released from an AMC during this catabolic process could potentially be a source of toxicity. To investigate this, we isolated and identified liver metabolites in mice for three different [(3)H]AMCs with structures similar to those currently undergoing evaluation in the clinic. We then synthesized each metabolite to confirm the identification and assessed their cytotoxic potencies when added extracellularly. We found that the uncleavable mAb-SMCC-[(3)H]DM1 conjugate was degraded to a single major maytansinoid metabolite, lysine-SMCC-[(3)H]DM1, that was nearly 50-fold less cytotoxic than maytansine. The two disulfide-linked conjugates, mAb-SPP-[(3)H]DM1 and mAb-SPDB-[(3)H]DM4, were also found to be catabolized to the analogous lysine-linked maytansinoid metabolites. However, subsequent reduction, S-methylation, and NADPH-dependent oxidation steps in the liver yielded the corresponding S-methyl sulfoxide and S-methyl sulfone derivatives. The cytotoxic potencies of the oxidized maytansinoids toward several human carcinoma cell lines were found to be 5- to 50-fold less potent than maytansine. Our results suggest that liver plays an important role in the detoxification of both cleavable and uncleavable AMCs.

Calicheamicin Antibody-Drug Conjugates with Improved Properties.

Calicheamicin antibody-drug conjugates (ADCs) are effective therapeutics for leukemias with two recently approved in the United States: Mylotarg (gemtuzumab ozogamicin) targeting CD33 for acute myeloid leukemia and Besponsa (inotuzumab ozogamicin) targeting CD22 for acute lymphocytic leukemia. Both of these calicheamicin ADCs are heterogeneous, aggregation-prone, and have a shortened half-life due to the instability of the acid-sensitive hydrazone linker in circulation. We hypothesized that we could improve upon the heterogeneity, aggregation, and circulation stability of calicheamicin ADCs by directly attaching the thiol of a reduced calicheamicin to an engineered cysteine on the antibody via a disulfide bond to generate a linkerless and traceless conjugate. We report herein that the resulting homogeneous conjugates possess minimal aggregation and display high in vivo stability with 50% of the drug remaining conjugated to the antibody after 21 days. Furthermore, these calicheamicin ADCs are highly efficacious in mouse models of both solid tumor (HER2+ breast cancer) and hematologic malignancies (CD22+ non-Hodgkin lymphoma). Safety studies in rats with this novel calicheamicin ADC revealed an increased tolerability compared with that reported for Mylotarg. Overall, we demonstrate that applying novel linker chemistry with site-specific conjugation affords an improved, next-generation calicheamicin ADC.

Tumor delivery and in vivo processing of disulfide-linked and thioether-linked antibody-maytansinoid conjugates.

Antibody-drug conjugates (ADCs) are designed to eradicate cancer cells that express the target antigen on their cell surface. A key component of an ADC is the linker that covalently connects the cytotoxic agent to the antibody. Several antibody-maytansinoid conjugates prepared with disulfide-based linkers such as those targeting the CanAg antigen have been shown to display more activity in preclinical mouse xenograft models than corresponding conjugates prepared with uncleavable thioether-based linkers. To investigate how the linker influences delivery and activation of antibody-maytansinoid conjugates, we isolated and characterized the [(3)H]maytansinoids from CanAg-positive tumor tissues following a single intravenous administration of 300 microg/kg (based on maytansinoid dose) of anti-CanAg antibody (huC242)-(3)H-maytansinoid conjugates prepared with cleavable disulfide linkers and an uncleavable thioether linker. We identified three target-dependent tumor metabolites of the disulfide-linked huC242-SPDB-DM4, namely, lysine-N(epsilon)-SPDB-DM4, DM4, and S-methyl-DM4. We found similar metabolites for the less hindered disulfide-linked huC242-SPP-DM1 conjugate with the exception that no S-methyl-DM1 was detected. The sole metabolite of the uncleavable thioether-linked huC242-SMCC-DM1 was lysine-N(epsilon)-SMCC-DM1. The AUC for the metabolites of huC242-SMCC-DM1 at the tumor over 7 d was about 2-fold greater than the corresponding AUC for the metabolites of the disulfide-linked conjugates. The lipophilic metabolites of the disulfide-linked conjugates were found to be nearly 1000 times more cytotoxic than the more hydrophilic lysine-N(epsilon)-linker-maytansinoids in cell-based viability assays when added extracellularly. The cell killing properties associated with the lipophilic metabolites of the disulfide-linked conjugates (DM4 and S-methyl-DM4, and DM1) provide an explanation for the superior in vivo efficacy that is often observed with antibody-maytansinoid conjugates prepared with disulfide-based linkers in xenograft mouse models.

Enantioselective inhibition of squalene synthase by aziridine analogues of presqualene diphosphate.

Squalene synthase catalyzes the conversion of two molecules of (E,E)-farnesyl diphosphate to squalene via the cyclopropylcarbinyl intermediate, presqualene diphosphate (PSPP). Since this novel reaction constitutes the first committed step in sterol biosynthesis, there has been considerable interest and research on the stereochemistry and mechanism of the process and in the design of selective inhibitors of the enzyme. This paper reports the synthesis and characterization of five racemic and two enantiopure aziridine analogues of PSPP and the evaluation of their potencies as inhibitors of recombinant yeast squalene synthase. The key aziridine-2-methanol intermediates (6-OH, 7-OH, and 8-OH) were obtained by N-alkylations or by an N-acylation-reduction sequence of (+/-)-, (2R,3S)-, and (2S,3R)-2,3-aziridinofarnesol (9-OH) protected as tert-butyldimethylsilyl ethers. S(N)2 displacements of the corresponding methanesulfonates with pyrophosphate and methanediphosphonate anions afforded aziridine 2-methyl diphosphates and methanediphosphonates bearing N-undecyl, N-bishomogeranyl, and N-(alpha-methylene)bishomogeranyl substituents as mimics for the 2,6,10-trimethylundeca-2,5,9-trienyl side chain of PSPP. The 2R,3S diphosphate enantiomer bearing the N-bishomogeranyl substituent corresponding in absolute stereochemistry to PSPP proved to be the most potent inhibitor (IC(50) 1.17 +/- 0.08 muM in the presence of inorganic pyrophosphate), a value 4-fold less than that of its 2S,3R stereoisomer. The other aziridine analogues bearing the N-(alpha-methylene)bishomogeranyl and N-undecyl substituents, and the related methanediphosphonates, exhibited lower affinities for recombinant squalene synthase.

Antibody-cytotoxic agent conjugates: preparation and characterization.

Conjugates of antibodies with cytotoxic agents offer a targeted therapeutic strategy against cancer cells expressing target antigens. Several antibodies against various cancer cell-surface antigens have been conjugated with different cytotoxic agents that inhibit essential cellular targets such as microtubules or DNA. Antibody-cytotoxic agent conjugates (ACCs) against several types of cancer are currently in advanced stages of clinical trials and one, gemtuzumab ozogamicin (Mylotarg), is approved for the treatment of acute myeloid leukemia. The linker group connecting the antibody to the cytotoxic agent is an important feature of the ACC, modulating the release of the active cytotoxic agent in the targeted cell. Several linker strategies employed for ACCs in current clinical trials include cleavable linkers with disulfide, hydrazone, lysosomal protease-substrate groups, and non-cleavable linkers. This chapter describes the methods of preparation of conjugates of antibodies with small-molecule cytotoxic agents (maytansinoids, calicheamicin, and auristatins) bearing different linkers. Methods to evaluate the in vitro cytotoxicity and in vivo anti-tumor efficacy of ACC are described in brief. Analytical methods are described to evaluate the mechanism of cellular processing of the ACCs with different linkers and the generation of the active metabolites.