Oral estrogen receptor PROTAC® vepdegestrant (ARV-471) is highly efficacious as monotherapy and in combination with CDK4/6 or PI3K/mTOR pathway inhibitors in preclinical ER+ breast cancer models.

PURPOSE: Estrogen Receptor (ER) alpha signaling is a known driver of ER-positive (ER+)/human epidermal growth factor receptor 2 negative (HER2-) breast cancer. Combining endocrine therapy (ET) such as fulvestrant with CDK4/6, mTOR or PI3K inhibitors is now a central strategy for the treatment of ER+ advanced breast cancer. However, suboptimal ER inhibition and resistance resulting from ESR1 mutation dictates that new therapies are needed. EXPERIMENTAL DESIGN: A medicinal chemistry campaign identified vepdegestrant (ARV-471), a selective, orally bioavailable, potent small molecule PROteolysis-TArgeting Chimera (PROTAC®) degrader of ER. We used biochemical and intracellular target engagement assays to demonstrate the mechanism of action of vepdegestrant, and ESR1 wild-type and mutant ER+ preclinical breast cancer models to demonstrate ER degradation-mediated tumor growth inhibition. RESULTS: Vepdegestrant induced ≥90% degradation of wild-type (WT) and mutant ER, inhibited ER-dependent breast cancer cell line proliferation in-vitro and achieved significant tumor growth inhibition (TGI) (87-123%) in MCF7 orthotopic xenograft models, better than the ET agent fulvestrant (31-80% TGI). In the hormone-independent ER Y537S patient derived xenograft (PDX) breast cancer model ST941/HI, vepdegestrant achieved tumor regressions and was similarly efficacious in the ST941/HI/PBR palbociclib-resistant model (102% TGI). Vepdegestrant induced robust tumor regressions in combination with each of the CDK4/6 inhibitors palbociclib, abemaciclib, and ribociclib, the mTOR inhibitor everolimus, and the PI3K inhibitors alpelisib and inavolisib. CONCLUSIONS: Vepdegestrant achieved greater ER degradation in-vivo compared to fulvestrant, which correlated with improved tumor growth inhibition, suggesting vepdegestrant could be a more effective backbone ET for patients with ER+/HER2- breast cancer.

Dosing, collection, and quality control issues in cerebrospinal fluid research using animal models.

Cerebrospinal fluid (CSF) is a complex fluid filling the ventricular system and surrounding the brain and spinal cord. Although the bulk of CSF is created by the choroid plexus, a significant fraction derives from the interstitial fluid in the brain and spinal cord parenchyma. For this reason, CSF can often be used as a source of pharmacodynamic and prognostic biomarkers to reflect biochemical changes occurring within the brain. For instance, CSF biomarkers can be used to diagnose and track progression of disease as well as understand pharmacokinetic and pharmacodynamic relationships in clinical trials. To facilitate the use of these biomarkers in humans, studies in preclinical species are often valuable. This review summarizes methods for preclinical CSF collection for biomarkers from mice, rats, and nonhuman primates. In addition, dosing directly into CSF is increasingly being used to improve drug levels in the brain. Therefore, this review also summarizes the state of the art in CSF dosing in these preclinical species.

Passive immunization with phospho-tau antibodies reduces tau pathology and functional deficits in two distinct mouse tauopathy models.

In Alzheimer's disease (AD), an extensive accumulation of extracellular amyloid plaques and intraneuronal tau tangles, along with neuronal loss, is evident in distinct brain regions. Staging of tau pathology by postmortem analysis of AD subjects suggests a sequence of initiation and subsequent spread of neurofibrillary tau tangles along defined brain anatomical pathways. Further, the severity of cognitive deficits correlates with the degree and extent of tau pathology. In this study, we demonstrate that phospho-tau (p-tau) antibodies, PHF6 and PHF13, can prevent the induction of tau pathology in primary neuron cultures. The impact of passive immunotherapy on the formation and spread of tau pathology, as well as functional deficits, was subsequently evaluated with these antibodies in two distinct transgenic mouse tauopathy models. The rTg4510 transgenic mouse is characterized by inducible over-expression of P301L mutant tau, and exhibits robust age-dependent brain tau pathology. Systemic treatment with PHF6 and PHF13 from 3 to 6 months of age led to a significant decline in brain and CSF p-tau levels. In a second model, injection of preformed tau fibrils (PFFs) comprised of recombinant tau protein encompassing the microtubule-repeat domains into the cortex and hippocampus of young P301S mutant tau over-expressing mice (PS19) led to robust tau pathology on the ipsilateral side with evidence of spread to distant sites, including the contralateral hippocampus and bilateral entorhinal cortex 4 weeks post-injection. Systemic treatment with PHF13 led to a significant decline in the spread of tau pathology in this model. The reduction in tau species after p-tau antibody treatment was associated with an improvement in novel-object recognition memory test in both models. These studies provide evidence supporting the use of tau immunotherapy as a potential treatment option for AD and other tauopathies.

Robust changes in expression of brain-derived neurotrophic factor (BDNF) mRNA and protein across the brain do not translate to detectable changes in BDNF levels in CSF or plasma.

Adult rats were treated acutely with peripheral kainic acid (KA), and changes in brain-derived neurotrophic factor (BDNF) mRNA and protein were tracked over time across multiple brain regions. Despite robust elevation in both mRNA and protein in multiple brain regions, plasma BDNF was unchanged and cerebrospinal fluid (CSF) BDNF levels remained undetectable. Primary neurons were then treated with KA. BDNF was similarly elevated within neurons, but was undetectable in neuronal media. Thus, while deficits in BDNF signaling have been implicated in a number of diseases, these data suggest that extracellular concentrations of BDNF may not be a facile biomarker for changes in neurons.

Hyperdynamic microtubules, cognitive deficits, and pathology are improved in tau transgenic mice with low doses of the microtubule-stabilizing agent BMS-241027.

Tau is a microtubule (MT)-stabilizing protein that is altered in Alzheimer's disease (AD) and other tauopathies. It is hypothesized that the hyperphosphorylated, conformationally altered, and multimeric forms of tau lead to a disruption of MT stability; however, direct evidence is lacking in vivo. In this study, an in vivo stable isotope-mass spectrometric technique was used to measure the turnover, or dynamicity, of MTs in brains of living animals. We demonstrated an age-dependent increase in MT dynamics in two different tau transgenic mouse models, 3xTg and rTg4510. MT hyperdynamicity was dependent on tau expression, since a reduction of transgene expression with doxycycline reversed the MT changes. Treatment of rTg4510 mice with the epothilone, BMS-241027, also restored MT dynamics to baseline levels. In addition, MT stabilization with BMS-241027 had beneficial effects on Morris water maze deficits, tau pathology, and neurodegeneration. Interestingly, pathological and functional benefits of BMS-241027 were observed at doses that only partially reversed MT hyperdynamicity. Together, these data suggest that tau-mediated loss of MT stability may contribute to disease progression and that very low doses of BMS-241027 may be useful in the treatment of AD and other tauopathies.

Tau transgenic mice as models for cerebrospinal fluid tau biomarkers.

Levels of tau in cerebrospinal fluid (CSF) are elevated in Alzheimer's disease (AD) patients. It is believed this elevation is related to the tau pathology and neurodegeneration observed in AD, but not all tauopathies have increased CSF tau. There has been little pre-clinical work to investigate mechanisms of increased CSF tau due to the difficulty in collecting CSF samples from mice, the most commonly used pre-clinical models. We developed methods to collect CSF from mice without contamination from tau in brain tissue, which is approximately 50,000 fold more abundant in brain than CSF. Using these methods, we measured CSF tau from 3xTg, Tg4510, and Tau Alone transgenic mice. All three lines of mice showed age-dependent increases in CSF tau. They varied in phenotype from undetectable to severe tau pathology and neurodegeneration, suggesting that degenerating neurons are unlikely to be the only source of pathologic CSF tau. Overall, CSF tau levels mirrored expression levels and changes of tau in the brain, but they did not always correlate exactly. CSF tau was often more sensitive to changes in brain transgene expression and pathology. In addition, we also developed ELISA assays specific to different regions of the tau protein. We used these assays to provide evidence that CSF tau exists as fragments, with little intact C-terminus and partial loss of the N-terminus. Taken together, these assays and mouse models may be used to facilitate a deeper understanding of CSF tau in neurodegenerative disease.

A liver-specific nitric oxide donor improves the intra-hepatic vascular response to both portal blood flow increase and methoxamine in cirrhotic rats.

BACKGROUND/AIMS: A decreased intra-hepatic nitric oxide (NO) production participates on the pathogenesis of portal hypertension in cirrhosis. We tested the hemodynamic effects of a liver-specific NO donor (NCX-1000) derived from ursodeoxycholic acid in portal hypertensive cirrhotic rats. METHODS: After a 14-day treatment with ursodeoxycholic acid or NCX-1000 by gavage, ascitic cirrhotic rats (CCl4-induced) were used in two studies: (1) in vivo mean arterial pressure (MAP), portal pressure (PP) and superior mesenteric artery (SMA) blood flow measurements before and during progressive blood volume expansion (blood infusion); and (2) in situ liver perfusion to obtain dose/response curves to methoxamine (alpha1-adrenergic agonist) and flow/pressure curves. RESULTS: Basal heart rate, MAP, and PP were similar in both groups. During blood infusion, similar MAP and SMA flow increases were observed in both groups; however, PP increase observed in control rats was blunted in NCX-1000 treated rats (P=0.015). In liver perfusions, flow/pressure curves were similar in both groups; however, NCX-1000-treated livers showed a lower response to methoxamine (P=0.016). cGMP concentration in NCX-1000-treated livers was higher (P=0.015) than in controls. CONCLUSIONS: Treatment with a liver-specific NO donor improves the portal system adaptability to portal blood flow increase and ameliorates the intra-hepatic response to methoxamine in cirrhotic rats.

Bacterial translocation up-regulates GTP-cyclohydrolase I in mesenteric vasculature of cirrhotic rats.

In cirrhosis, arterial vasodilation and the associated hemodynamic disturbances are most prominent in the mesenteric circulation, and its severity has been linked to bacterial translocation (BT) and endotoxemia. Synthesis of nitric oxide (NO), the main vasodilator implicated, is dependent on the essential cofactor tetrahydrobiopterin (BH(4)). The key enzyme involved in BH(4) synthesis is GTP-cyclohydrolase I (GTPCH-I), which is stimulated by endotoxin. Therefore, we investigated GTPCH-I activity and BH(4) biosynthesis in the mesenteric vasculature of cirrhotic rats with ascites, as well as their relationship with BT and endotoxemia, serum NO, and mean arterial pressure (MAP). GTPCH-I activity and BH(4) content in mesenteric vasculature was determined by high-performance liquid chromatography. BT was assessed by standard bacteriologic culture of mesenteric lymph nodes (MLNs). Serum endotoxin was measured by a kinetic turbidimetric limulus amebocyte lysate assay, and serum NO metabolite (NOx) concentrations were assessed by chemiluminescence. BT was associated with local lymphatic and systemic appearance of endotoxin and was accompanied by increases in serum NOx levels. GTPCH-I activity and BH(4) content in mesenteric vasculature were both increased in animals with BT and correlated significantly (r = 0.69, P <.01). Both GTPCH-I activity and BH(4) levels significantly correlated with serum endotoxin and NOx levels (r = 0.69 and 0.54, 0.81 and 0.53, P <.05). MAP (a marker of systemic vasodilatation) correlated with endotoxemia (r = 0.58, P <.03) and with GTPCH-I activity (r = 0.69, P <.01). In conclusion, in cirrhotic animals BT appears to lead to endotoxemia, stimulation of GTPCH-I, increased BH(4) synthesis, and further enhancement of vascular NO production that leads to aggravation of vasodilatation.

Mesenteric vasoconstriction triggers nitric oxide overproduction in the superior mesenteric artery of portal hypertensive rats.

BACKGROUND & AIMS: Vasoconstriction of the superior mesenteric artery (SMA) is the earliest hemodynamic event occurring after partial portal vein ligation (PVL). We tested the hypothesis that this early vasoconstriction of the SMA may initiate eNOS up-regulation in PVL. METHODS: Portal hypertension with or without mesenteric vasoconstriction was induced by differentially calibrated stenosis of the portal vein (PVL-20G and PVL-18G, respectively). In a separate group of rats, mesenteric vasoconstriction was achieved by renal artery ligation. Sham-operated rats were used as controls. Effects of vasoconstriction of the SMA in PVL and RAL rats were evaluated by measuring perfusion pressure changes in isolated SMA beds in response to methoxamine, nitric oxide synthase activity, and eNOS protein expression. Mean arterial pressure, portal pressure, and SMA blood flow were measured by catheterization and Doppler flowmetry. SMA vascular resistance was calculated from arterial pressure, portal pressure, and SMA flow. RESULTS: There was a significant increase in SMA vascular resistance in PVL-20G (2.33 +/- 0.13 vs. 1.22 +/- 0.03 mm Hg/% flow; P < 0.05) and RAL (2.32 +/- 0.18 vs. 1.18 +/- 0.02 mm Hg/% flow; P < 0.05) but not in PVL-18G, showing mesenteric vasoconstriction in both PVL-20G and RAL groups. The mesenteric vasculature of PVL-20G and RAL animals showed hyporeactivity to methoxamine (P < 0.01). Whereas both PVL groups were portal hypertensive (P < 0.01), RAL rats were not. The SMA hyporeactivity of PVL-20G and RAL rats was corrected by N(G)()-monomethyl-L-arginine, and nitric oxide synthase enzyme activity was significantly higher in PVL-20G and RAL rats (P < 0.05). CONCLUSIONS: Mesenteric arterial vasoconstriction plays a triggering role in up-regulation of eNOS catalytic activity in the SMA of portal hypertensive rats.

Deficit in nitric oxide production in cirrhotic rat livers is located in the sinusoidal and postsinusoidal areas.

Intrahepatic nitric oxide (NO) production is decreased in cirrhotic livers. Our objective was to identify, in cirrhotic rat livers, intrahepatic vascular segments where the deficit of NO facilitates the effect of vasoconstrictors. By using a modified rat liver perfusion system with measurement of both the perfusion and sinusoidal (wedged hepatic vein) pressures, we studied the effect of the NO synthase blocker N(omega)-nitro-l-arginine (l-NNA) on the response to methoxamine (alpha(1)-adrenoreceptor agonist) in different segments of the intrahepatic circulation of normal and cirrhotic rat livers. l-NNA enhanced the presinusoidal, sinusoidal, and postsinusoidal responses to methoxamine in normal livers as well as the presinusoidal response in cirrhotic livers. However, l-NNA did not change the already enhanced sinusoidal/postsinusoidal response to methoxamine in cirrhotic livers. The postsinusoidal response to methoxamine was higher in cirrhotic rats with ascites than in those without ascites. We concluded that NO modulates the presinusoidal, sinusoidal, and postsinusoidal vascular tone in normal livers. NO production in cirrhotic rat livers is severely impaired in the sinusoidal and postsinusoidal areas but is preserved in the presinusoidal area, as evidenced by its normal response to l-NNA. We speculate that an increased postsinusoidal response to catecholamines may participate in the genesis of ascites in cirrhosis.

Mice with targeted deletion of eNOS develop hyperdynamic circulation associated with portal hypertension.

Systemic vasodilation is the initiating event of the hyperdynamic circulatory state, being most likely triggered by increased levels of vasodilators, primarily nitric oxide (NO). Endothelial NO synthase (eNOS) is responsible for this event. We tested the hypothesis that gene deletion of eNOS and inducible NOS (iNOS) may inhibit the development of the hyperdynamic circulatory state in portal hypertensive animals. To test this hypothesis, we used mice lacking eNOS (eNOS-/-) or eNOS/iNOS (eNOS/iNOS-/-) genes. A partial portal vein ligation (PVL) was used to induce portal hypertension. Sham-operated animals were used as a control. Hemodynamic characteristics were tested 2 wk after surgery. As opposed to our hypothesis, PVL also caused significant reduction in peripheral resistance in eNOS-/- compared with sham animals (0.33 +/- 0.02 vs. 0.41 +/- 0.03 mmHg. min x kg body wt x ml(-1); P = 0.04) and in eNOS/iNOS-/- animals with PVL compared with that of the sham-operated group (0.44 +/- 0.02 vs. 0.54 +/- 0.04; P = 0.03). This demonstrates that, despite gene deletion of eNOS, the knockout mice developed hyperdynamic circulation. Compensatory vasodilator molecule(s) are upregulated in place of NO in the systemic and splanchnic circulation in portal hypertensive animals.

Bioactivation of nitroglycerin and vasomotor response to nitric oxide are impaired in cirrhotic rat livers.

Nitroglycerin (NTG), a nitric oxide (NO) donor, has been shown to reduce portal pressure in cirrhotic patients. Using the in situ perfusion of normal and cirrhotic rat livers, we compared the vascular relaxation induced by either NTG or the spontaneous nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP). Normal and cirrhotic livers were perfused (40 mL/min, 37 degrees C) with Krebs' solution in a recirculating system. After preconstriction with methoxamine (10(-4) mol/L), a dose-response study was performed using 6 cumulative doses of NTG or SNAP (10(-7) to 3 x 10(-5) mol/L). NO(x) (NO(-)(2) + NO(-)(2) production in the perfusate was measured by chemiluminescence. Cirrhotic livers exhibited lower vasorelaxant responses, compared with normal livers, to both NTG (P <.0001) and SNAP (P =.0020). In normal livers, NTG and SNAP induced similar vasorelaxant responses (P =.44). In cirrhotic livers, NTG induced less vasorelaxation than SNAP (P <.0001). In the presence of NTG (P =.0045), but not SNAP (P =.99), NO(x) production in experiments with cirrhotic livers was lower than in experiments with normal livers. In conclusion, in cirrhotic rat livers, the vasorelaxant response to NTG is impaired owing to both a decreased metabolism of this NO donor and an inability of the hepatic vasculature to respond to NO.