Preclinical studies of BB-1701, a HER2-targeting eribulin-containing ADC with potent bystander effect and ICD activity.

BACKGROUND: Several HER2-targeting antibody-drug conjugates (ADC) have gained market approval for the treatment of HER2-expressing metastasis. Promising responses have been reported with the new generation of ADCs in patients who do not respond well to other HER2-targeting therapeutics. However, these ADCs still face challenges of resistance and/or severe adverse effects associated with their particular payload toxins. Eribulin, a therapeutic agent for the treatment of metastatic breast cancer and liposarcoma, is a new choice of ADC payload with a distinct mechanism of action and safety profile. METHODS: We've generated a novel HER2-tageting eribulin-containing ADC, BB-1701. The potency of BB-1701 was tested in vitro and in vivo against cancer cells where HER2-expressing levels vary in a large range. Bystander killing effect and toxin-induced immunogenic cell death (ICD) of BB-1701 were also tested. RESULTS: In comparison with HER2-targeting ADCs with DM1 and Dxd payload, eribulin-containing ADC demonstrated higher in vitro cytotoxicity in HER2-low cancer cell lines. BB-1701 also effectively suppressed tumors in models resistant to DM1 or Dxd containing ADCs. Mode of action studies showed that BB-1701 had a significant bystander effect on HER2-null cells adjacent to HER2-high cells. In addition, BB-1701 treatment induced ICD. Repeated doses of BB-1701 in nonhuman primates showed favorable pharmacokinetics and safety profiles at the intended clinical dosage, route of administration, and schedule. CONCLUSIONS: The preclinical data support the test of BB-1701 in patients with various HER2-expressing cancers, including those resistant to other HER2-targeting ADCs. A phase I clinical trial of BB-1701 (NCT04257110) in patients is currently underway.

Antibody-drug conjugate MORAb-202 exhibits long-lasting antitumor efficacy in TNBC PDx models.

The antibody-drug conjugate (ADC) MORAb-202, consisting of farletuzumab paired with a cathepsin B-cleavable linker and eribulin, targets folate receptor alpha (FRA), which is frequently overexpressed in various tumor types. MORAb-202 was highly cytotoxic to FRA-positive cells in vitro, with limited off-target killing of FRA-negative cells. Furthermore, MORAb-202 showed a clear in vitro bystander cytotoxic effect in coculture with FRA-positive/negative cells. In vivo antitumor efficacy studies of MORAb-202 were conducted with a single administration of MORAb-202 in triple-negative breast cancer (TNBC) patient-derived xenograft (PDx) models expressing low and high levels of FRA. MORAb-202 exhibited durable efficacy proportional to tumor FRA expression. Toxicology studies (Q3Wx2) in nonhuman primates suggested that the major observed toxicity of MORAb-202 is hematologic toxicity. Overall, these findings support the concept that MORAb-202 represents a promising investigational ADC for the treatment of TNBC patients.

A randomized, placebo-controlled trial of canakinumab in patients with peripheral artery disease.

Extensive atherosclerotic plaque burden in the lower extremities often leads to symptomatic peripheral artery disease (PAD) including impaired walking performance and claudication. Interleukin-1ß (IL-1ß) may play an important pro-inflammatory role in the pathogenesis of this disease. Interruption of IL-1ß signaling was hypothesized to decrease plaque progression in the leg macrovasculature and improve the mobility of patients with PAD with intermittent claudication. Thirty-eight patients (mean age 65 years; 71% male) with symptomatic PAD (confirmed by ankle-brachial index) were randomized 1:1 to receive canakinumab (150 mg subcutaneously) or placebo monthly for up to 12 months. The mean vessel wall area (by 3.0 T black-blood magnetic resonance imaging (MRI)) of the superficial femoral artery (SFA) was used to measure plaque volume. Mobility was assessed using the 6-minute walk test. Canakinumab was safe and well tolerated. Markers of systemic inflammation (interleukin-6 and high-sensitivity C-reactive protein) fell as early as 1 month after treatment. MRI (32 patients at 3 months; 21 patients at 12 months) showed no evidence of plaque progression in the SFA in either placebo-treated or canakinumab-treated patients. Although an exploratory endpoint, placebo-adjusted maximum and pain-free walking distance (58 m) improved as early as 3 months after treatment with canakinumab when compared with placebo. Although canakinumab did not alter plaque progression in the SFA, there is an early signal that it may improve maximum and pain-free walking distance in patients with symptomatic PAD. Larger studies aimed at this endpoint will be required to definitively demonstrate this. ClinicalTrials.gov Identifier: NCT01731990.

Open-label Extension Phase of a Chronic Diabetic Foot Ulcer Multicenter, Controlled, Randomized Clinical Trial Using Cryopreserved Placental Membrane.

OBJECTIVE: The results of the single-arm, open-label extension phase of the Grafix (cryopreserved placental membrane; CPM; Osiris Therapeutics, Inc, Columbia, MD) multicenter, blinded, randomized, controlled clinical trial for chronic diabetic foot ulcers (DFUs) is reported. MATERIALS AND METHODS: Twenty-six patients in the standard wound care (SWC) arm whose DFUs did not close in the blinded phase chose to receive weekly applications of the CPM in an open-label extension phase. RESULTS: In the extension phase, 17 (65.4%) patients closed their wounds in a median of 34 days and 3 visits. There were fewer total adverse events (AEs) (24 CPM vs. 52 SWC) and index wound-related infections (5 CPM vs. 12 SWC) during the CPM application compared with the number of AEs for the same patients during the SWC treatment in the blinded phase of the trial. CONCLUSIONS: These results corroborate the benefits of this CPM combined with SWC over SWC alone for chronic DFUs previously reported for the blinded randomized phase of the trial, which directly relate to lower health care costs.

Targeting endosialin/CD248 through antibody-mediated internalization results in impaired pericyte maturation and dysfunctional tumor microvasculature.

Over-expression of endosialin/CD248 (herein referred to as CD248) has been associated with increased tumor microvasculature in various tissue origins which makes it an attractive anti-angiogenic target. In an effort to target CD248, we have generated a human CD248 knock-in mouse line and MORAb-004, the humanized version of the mouse anti-human CD248 antibody Fb5. Here, we report that MORAb-004 treatment significantly impacted syngeneic tumor growth and tumor metastasis in the human CD248 knock-in mice. In comparison with untreated tumors, MORAb-004 treated tumors displayed overall shortened and distorted blood vessels. Immunofluorescent staining of tumor sections revealed drastically more small and dysfunctional vessels in the treated tumors. The CD248 levels on cell surfaces of neovasculature pericytes were significantly reduced due to its internalization. This reduction of CD248 was also accompanied by reduced α-SMA expression, depolarization of pericytes and endothelium, and ultimately dysfunctional microvessels. These results suggest that MORAb-004 reduced CD248 on pericytes, impaired tumor microvasculature maturation and ultimately suppressed tumor development.

The efficacy and safety of Grafix(®) for the treatment of chronic diabetic foot ulcers: results of a multi-centre, controlled, randomised, blinded, clinical trial.

In a randomised, controlled study, we compared the efficacy of Grafix(®) , a human viable wound matrix (hVWM) (N = 50), to standard wound care (n = 47) to heal diabetic foot ulcers (DFUs). The primary endpoint was the proportion of patients with complete wound closure by 12 weeks. Secondary endpoints included the time to wound closure, adverse events and wound closure in the crossover phase. The proportion of patients who achieved complete wound closure was significantly higher in patients who received Grafix (62%) compared with controls (21%, P = 0·0001). The median time to healing was 42 days in Grafix patients compared with 69·5 days in controls (P = 0·019). There were fewer Grafix patients with adverse events (44% versus 66%, P = 0·031) and fewer Grafix patients with wound-related infections (18% versus 36·2%, P = 0·044). Among the study subjects that healed, ulcers remained closed in 82·1% of patients (23 of 28 patients) in the Grafix group versus 70% (7 of 10 patients) in the control group (P = 0·419). Treatment with Grafix significantly improved DFU healing compared with standard wound therapy. Importantly, Grafix also reduced DFU-related complications. The results of this well-controlled study showed that Grafix is a safe and more effective therapy for treating DFUs than standard wound therapy.

Clathrate Ba8Au16P30: the "gold standard" for lattice thermal conductivity.

A novel clathrate phase, Ba8Au16P30, was synthesized from its elements. High-resolution powder X-ray diffraction and transmission electron microscopy were used to establish the crystal structure of the new compound. Ba8Au16P30 crystallizes in an orthorhombic superstructure of clathrate-I featuring a complete separation of gold and phosphorus atoms over different crystallographic positions, similar to the Cu-containing analogue, Ba8Cu16P30. Barium cations are trapped inside the large polyhedral cages of the gold-phosphorus tetrahedral framework. X-ray diffraction indicated that one out of 15 crystallographically independent phosphorus atoms appears to be three-coordinate. Probing the local structure and chemical bonding of phosphorus atoms with (31)P solid-state NMR spectroscopy confirmed the three-coordinate nature of one of the phosphorus atomic positions. High-resolution high-angle annular dark-field scanning transmission electron microscopy indicated that the clathrate Ba8Au16P30 is well-ordered on the atomic scale, although numerous twinning and intergrowth defects as well as antiphase boundaries were detected. The presence of such defects results in the pseudo-body-centered-cubic diffraction patterns observed in single-crystal X-ray diffraction experiments. NMR and resistivity characterization of Ba8Au16P30 indicated paramagnetic metallic properties with a room-temperature resistivity of 1.7 mΩ cm. Ba8Au16P30 exhibits a low total thermal conductivity (0.62 W m(-1) K(-1)) and an unprecedentedly low lattice thermal conductivity (0.18 W m(-1) K(-1)) at room temperature. The values of the thermal conductivity for Ba8Au16P30 are significantly lower than the typical values reported for solid crystalline compounds. We attribute such low thermal conductivity values to the presence of a large number of heavy atoms (Au) in the framework and the formation of multiple twinning interfaces and antiphase defects, which are effective scatterers of heat-carrying phonons.

BaAu2P4: layered zintl polyphosphide with infinite ∞(1)(P­) chains.

Barium gold polyphosphide BaAu2P4 was synthesized from elements and structurally characterized by single crystal X-ray diffraction. BaAu2P4 crystallizes in a new structure type, in the orthorhombic space group Fddd (No. 70) with a = 6.517(1) Å, b = 8.867(2) Å, c = 21.844(5) Å. The crystal structure of BaAu2P4 consists of Au­P layers separated by layers of Ba atoms. Each Au­P layer is composed of infinite ∞(1)(P­) chains of unique topology linked together by almost linearly coordinated Au atoms. According to Zintl­Klemm formalism, this compound is charge balanced assuming closed shell d10 configuration for Au: Ba2+(Au+)2(P­)4. Magnetic and solid state NMR measurements together with quantum-chemical calculations reveal diamagnetic and semiconducting behavior for the investigated polyphosphide, which is as expected for the charged balanced Zintl phase. Electron localization function and crystal orbital Hamilton population analyses reveal strong P­P and Au­P bonding and almost nonbonding Au­Au interactions in BaAu2P4.

Child and family factors associated with teacher-reported behavior problems in young children of substance abusers.

BACKGROUND: We examined child and family factors associated with teacher-reported behavior problems in 79 children of substance abusers (COSAs). METHOD: Using regression models, we examined the impact of four family risk factors, cumulatively and individually, on children's behavior and explored children's engagement of adults as a protective factor. RESULTS: More than half (55%) of children had clinically elevated behavior problems. Cumulative family risks were associated with increased problems, whereas the presence of a father in the home and the child's ability to engage adults were protective. CONCLUSIONS: These findings may help explain the variation in behavioral outcomes of COSAs.

Abdominal apoplexy: two unusual cases of hemoperitoneum.

Abdominal apoplexy, or idiopathic spontaneous intraperitoneal hemorrhage, is a rare and often fatal condition resulting from a variety of disease processes affecting the arterial and venous abdominal vasculature. Preoperative and intraoperative diagnosis and treatment of abdominal apoplexy are challenging. The source of bleeding may remain elusive even after careful autopsy dissection given the absence of intravascular pressure. Despite these challenges, early diagnosis and rapid treatment remain central to a successful outcome, as nonsurgical mortality has approached 100%. Presented here are two fatal cases of abdominal apoplexy, one involving a patient with arterial dissection of the gastroduodenal artery and one involving rupture of the superior mesenteric-portal venous system with perivascular pseudoaneurysm formation.

Biomechanical and biologic effects of meniscus stabilization using triglycidyl amine.

The susceptibility of meniscus allografts to enzymatic degradation may be reduced through tissue stabilization. We have previously reported on an epoxide-based crosslinker, triglycidyl amine (TGA), which can be used alone or with a bisphosphonate (MABP) to stabilize heterograft heart valves and reduce their pathologic calcification. Our objective was to evaluate the effects of TGA and TGA-MABP pretreatment on an orthopedic allograft involving meniscus crosslinking, degradation, calcification, and compressive properties. Ovine menisci treated with TGA or TGA-MABP for up to seven days and glutaraldehyde crosslinked controls were examined in vitro for degree of crosslinking, resistance to degradation by collagenase, and material property changes. Likewise treated menisci were implanted in rats for eight weeks and examined for calcium content and biomechanical changes. TGA treatment for three days significantly reduced collagen loss by 88% and increased thermal denaturation temperatures (Ts) above 80 degrees C versus Ts of 70 degrees C or less for non-crosslinked meniscus. In vitro, TGA and TGA-MABP significantly increased aggregate modulus by 19% and 32% compared to native controls, respectively. TGA decreased permeability by 53% while TGA-MABP increased it by 303%. In vivo, TGA significantly reduced explant calcification by 42% compared to glutaraldehyde, and including MABP reduced it by 90%. Analyses revealed that TGA and TGA-MABP stabilized menisci had significantly lower modulus and permeability values than glutaraldehyde controls by at least 28% and 86%, respectively. It is concluded that TGA crosslinking of meniscus increases resistance to both collagenase degradation and pathologic calcification, while demonstrating comparable or improved biomechanical properties versus glutaraldehyde controls.

Fenfluramine disrupts the mitral valve interstitial cell response to serotonin.

Serotonin (5HT) receptor signaling and 5HT-related agents, such as the anorexogen fenfluramine (Fen), have been associated with heart valve disease. We investigated the hypothesis that Fen may disrupt mitral valve interstitial cell (MVIC) homeostasis through its effects on mitogenesis and extracellular matrix biosynthesis. Normal and myxomatous mitral valves, both human and canine, were harvested, and primary MVIC cultures were established. 5HT caused increased phosphorylation of extracellular signal-related kinase in MVIC; Fen alone did not. However, Fen combined with 5HT increased the level of MVIC extracellular signal-related kinase, when compared with 5HT alone. In addition, MVIC mitogenesis per (3)H-thymidine ((3)HTdR) demonstrated a 5HT dose-dependent increase, with no effect of Fen alone. In contrast, Fen combined with 5HT inhibited the MVIC (3)HTdR response when compared with 5HT alone. Furthermore, fluoxetine, a 5HT transporter inhibitor, while having no effect alone, suppressed Fen-5HT (3)HTdR inhibition when administered with Fen plus 5HT. Finally, MVIC incorporations of (3)H-proline and (3)H-glucosamine, measures of extracellular matrix collagen and glycosaminoglycan respectively, were increased with 5HT alone; however, Fen did not affect MVIC glycosaminoglycan or collagen either alone or in combination with 5HT. Taken together, the ratios of (3)H-proline or (3)H-glycosaminoglycan to (3)HTdR in MVIC, normalized to 5HT alone, demonstrated a significant imbalance of extracellular matrix production versus proliferation in MVIC cultures with Fen plus 5HT exposure. This imbalance may explain in part the pathophysiology of Fen-related mitral valve disease.

Foxl1-Cre BAC transgenic mice: a new tool for gene ablation in the gastrointestinal mesenchyme.

The use of Cre-loxP technology for the purpose of cell type-specific gene ablation has revolutionized developmental biology and biomedicine. Several transgenic mouse lines have been developed for the analysis of gene function in the gastrointestinal tract, but in all of these the expression of Cre is limited to the epithelial cell layer. No Cre- expressing transgenic mouse lines ("Cre lines") exist for the deletion of loxP-flanked genes specifically in gut mesoderm. To address this deficiency, we have derived a bacterial artificial chromosome based transgenic mouse line in which the Cre gene is controlled by the Foxl1 promoter and enhancer elements. X-Gal staining of Foxl1-Cre; Rosa26R bi-transgenic lines confirm that Foxl1-Cre results in recombination specifically in the gastrointestinal mesenchyme. The Foxl1-Cre line will facilitate the dissection of mesenchymal to epithelial signaling that is known to play a major role in the patterning and function of the gastrointestinal tract.

Biological stability of polyurethane modified with covalent attachment of di-tert-butyl-phenol.

Polyurethane cardiovascular implants are subject to oxidation initiated surface degradation, which is mediated by monocyte-derived macrophages (MDM); this often leads to surface cracking and device failure. The present studies examined the hypothesis that covalently attaching antioxidant, di-tert-butylphenol (DBP), to the urethane nitrogens of a polyether polyurethane (PU) via bromo-alkylation reactions could prevent this problem. PU was configured with two dosages of DBP, 0.14 mM DBP/g PU of DBP (PU-DBP) and a more highly modified (HM) 0.40 mM DBP/g PU (PU-DBP-HM). THP-1 cells, a human MDM cell line, stimulated with phorbol ester and seeded on PU, PU-DBP, and PU-DBP-HM films were assessed for reactive oxygen species (ROS) production via a fluorescent based dihydrorhodamine-123 assay. Results from these studies showed a significant dose-dependent reduction of ROS levels for THP-1 cells seeded on PU-DBP versus unmodified PU. PU, PU-DBP, or PU-DBP-HM films were implanted into subdermal pouches of Sprague-Dawley rats. Films were explanted after 10 weeks and assessed for oxidative degradation via light and scanning electron microscopy (SEM) and Fourier transformation infrared spectroscopy (FTIR). Light microscopy showed extensive surface cracking, which was confirmed via SEM, on unmodified PU surfaces that was absent in both PU-DBP and PU-DBP-HM explanted films. FTIR analysis showed reduction in oxidation-induced ether crosslinking that was directly related to DBP dosages. It is concluded that modifying PU with the covalent attachment of an antioxidant confers biodegradation resistance in vivo in a dose dependent manner; this effect is likely due to quenching of the ROS generated by the adherent macrophages.

Mechanisms of the in vivo inhibition of calcification of bioprosthetic porcine aortic valve cusps and aortic wall with triglycidylamine/mercapto bisphosphonate.

Heart valve replacements fabricated from glutaraldehyde (Glut)-crosslinked heterograft materials, porcine aortic valves or bovine pericardium, have been widely used in cardiac surgery to treat heart valve disease. However, these bioprosthetic heart valves often fail in long-term clinical implants due to pathologic calcification of the bioprosthetic leaflets, and for stentless porcine aortic valve bioprostheses, bioprosthetic aortic wall calcification also typically occurs. Previous use of the epoxide-based crosslinker, triglycidyl amine (TGA), on cardiac bioprosthetic valve materials demonstrated superior biocompatibility, mechanics, and calcification resistance for porcine aortic valve cusps (but not porcine aortic wall) and bovine pericardium, vs. Glut-prepared controls. However, TGA preparation did not completely prevent long-term calcification of cusps or pericardium. Herein we report further mechanistic investigations of an added therapeutic component to this system, 2-mercaptoethylidene-1,1-bisphosphonic acid (MABP), a custom synthesized thiol bisphosphonate, which has previously been shown in a preliminary report to prevent bioprosthetic heterograft biomaterial calcification when used in combination with initial TGA crosslinking for 7 days. In the present studies, we have further investigated the effectiveness of MABP in experiments that examined: (1) The use of MABP after optimal TGA crosslinking, in order to avoid any competitive interference of MABP-reactions with TGA during crosslinking; (2) Furthermore, recognizing the importance of alkaline phosphatase (ALP) in the formation of dystrophic calcific nodules, we have investigated the hypothesis that the mechanism by which MABP primarily functions is through the reduction of ALP activity. Results from cell-free model systems, cell culture studies, and rat subcutaneous implants, show that materials functionalized with MABP after TGA crosslinking have reduced ALP activity, and in vivo have no significant calcification in long-term implant studies. It is concluded that bioprosthetic heart valves prepared in this fashion are compelling alternatives for Glut-prepared bioprostheses.

The initiation of liver development is dependent on Foxa transcription factors.

The specification of the vertebrate liver is thought to occur in a two-step process, beginning with the establishment of competence within the foregut endoderm for responding to organ-specific signals, followed by the induction of liver-specific genes. On the basis of expression and in vitro studies, it has been proposed that the Foxa transcription factors establish competence by opening compacted chromatin structures within liver-specific target genes. Here we show that Foxa1 and Foxa2 (forkhead box proteins A1 and A2) are required in concert for hepatic specification in mouse. In embryos deficient for both genes in the foregut endoderm, no liver bud is evident and expression of the hepatoblast marker alpha-fetoprotein (Afp) is lost. Furthermore, Foxa1/Foxa2-deficient endoderm cultured in the presence of exogenous fibroblast growth factor 2 (FGF2) fails to initiate expression of the liver markers albumin and transthyretin. Thus, Foxa1 and Foxa2 are required for the establishment of competence within the foregut endoderm and the onset of hepatogenesis.

The MODY1 gene HNF-4alpha regulates selected genes involved in insulin secretion.

Mutations in the gene encoding hepatocyte nuclear factor-4alpha (HNF-4alpha) result in maturity-onset diabetes of the young (MODY). To determine the contribution of HNF-4alpha to the maintenance of glucose homeostasis by the beta cell in vivo, we derived a conditional knockout of HNF-4alpha using the Cre-loxP system. Surprisingly, deletion of HNF-4alpha in beta cells resulted in hyperinsulinemia in fasted and fed mice but paradoxically also in impaired glucose tolerance. Islet perifusion and calcium-imaging studies showed abnormal responses of the mutant beta cells to stimulation by glucose and sulfonylureas. These phenotypes can be explained in part by a 60% reduction in expression of the potassium channel subunit Kir6.2. We demonstrate using cotransfection assays that the Kir6.2 gene is a transcriptional target of HNF-4alpha. Our data provide genetic evidence that HNF-4alpha is required in the pancreatic beta cell for regulation of the pathway of insulin secretion dependent on the ATP-dependent potassium channel.

Mild nephrogenic diabetes insipidus caused by Foxa1 deficiency.

Foxa1 is a member of the winged helix family of transcription factors and is expressed in the collecting ducts of the kidney. We investigated its potential contribution to renal physiology in Foxa1-deficient mice on a defined genetic background. Foxa1(-/-) mice are dehydrated and exhibit electrolyte imbalance as evidenced by elevated hematocrit and plasma urea levels, hypernatremia, and hyperkalemia. This phenotype is the consequence of decreased urine osmolality secondary to renal vasopressin resistance. Mutations of the human genes encoding the vasopressin 2 receptor and aquaporin 2 cause nephrogenic diabetes insipidus; however, expression of these genes is maintained or increased, respectively, in Foxa1(-/-) mice. Likewise, expression of the genes encoding the Na-K-2Cl cotransporter (NKCC2), the potassium channel ROMK, the chloride channel CLCNKB, barttin (BSND), and the calcium-sensing receptor (CASR), each of which is important in sodium reabsorption in the loop of Henle, is maintained or even increased in Foxa1-deficient mice. Thus, we have shown that Foxa1(-/-) mice represent a new model of nephrogenic diabetes insipidus with unique molecular etiology, and we have identified the first transcription factor whose mutation leads to a defect in renal water homeostasis in vivo.