The Use of Preclinical Models to Improve the Treatment of Retinoblastoma

Rodent models play an essential role in the development of new chemotherapeutics and dosing regimes. It is often difficult to carryout a clinical study for pediatric cancers due to the small patient population. Retinoblastoma, a pediatric cancer of the eye, is one example of a pediatric cancer that can benefit from preclinical studies. Over the years various retinoblastoma rodent models have been developed used to test various combination of broad-spectrum systemic chemotherapy. It was found form these studies that the combination of topotecan and carboplatin was effective. However both drugs cause myelosuppression and therefore administrating both of these drugs systemically is not possible. An alternative effective therapy in the clinic was the use of a subconjunctival administration. We thought if we could administer both drugs, one by systemic and one by a subconjunctival injection, perhaps we could decrease the systemic exposure with good tumor response. Detailed pharmacokinetic studies were conducted to understand the subconjunctival injections of topotecan and carboplatin. It was found that both drugs could successfully penetrate the eye and increase drug exposure. In addition, in the presence of a tumor, drug exposure to the vitreous was greater. Additionally comparative pharmacodynamic studies combining topotecan subconjunctival injection with carboplatin intraperitoneal or carboplatin subconjunctival injection with topotecan intraperitoneal were conducted. The tumor response, systemic toxicity and local toxicity were studied. There was tumor response in both combinations and no ocular toxicity was seen with a single eye subconjunctival injection for either drug. However, rats that received the combination with topotecan subconjunctival injection and carboplatin intraperitoneal experienced great toxicity and morbidity. The data and observations suggest the death is due to dehydration. Therefore it was concluded that the alternative combination was better. The above data suggested an appropriate drug combination and schedule for a preclinical study. However, the noninvasive methods to follow tumor progression and choosing the correct genetic model needed to be determined. This was essential to ensure the preclinical study could be easily translated for future clinical studies. A characterization study of five modalities, retina camera, optomotry, tonometer, ultrasound and MRI, was done with retinoblastoma mice. We determined the feasibility of each technique. It was found that the retina camera could detect the tumor the earliest in a high throughput manner. Additionally, the tonometer and optomotry machines could assess ocular health. While the ultrasound and MRI could image the eye and tumor in one field of view, MRI could capture the posterior chamber in more detail along with the extraocular space. With different software programs, the tumor to eye ratio volume measurement were determined and compared to the gold standard of enucleation, embedding, serial sectioning and hand tracing. It was found that there was a better correlation between the ultrasound and hand tracing histological sections. Concurrently, the tumor progression of six different genotypes was assessed. The tumor progression depended on the number and different genes deleted. Additionally, based on genotypes, it was determined there was not a strong genotypic trend in the increase in IOP or the loss of vision. From the studies of tumor progression we have learn more about the influence of genes on tumor progression, which will benefit additional genetic studies in mouse model systems and human tumors

Coexpression of Normally Incompatible Developmental Pathways in Retinoblastoma Genesis

It is widely believed that the molecular and cellular features of a tumor reflect its cell of origin and can thus provide clues about treatment targets. The retinoblastoma cell of origin has been debated for over a century. Here, we report that human and mouse retinoblastomas have molecular, cellular, and neurochemical features of multiple cell classes, principally amacrine/horizontal interneurons, retinal progenitor cells, and photoreceptors. Importantly, single-cell gene expression array analysis showed that these multiple cell type-specific developmental programs are coexpressed in individual retinoblastoma cells, which creates a progenitor/neuronal hybrid cell. Furthermore, neurotransmitter receptors, transporters, and biosynthetic enzymes are expressed in human retinoblastoma, and targeted disruption of these pathways reduces retinoblastoma growth in vivo and in vitro

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

MDM2-A, a common Mdm2 splice variant, causes perinatal lethality, reduced longevity and enhanced senescence

MDM2 is the predominant negative regulator of p53 that functions to maintain the appropriate level of expression and activity of this central tumor suppressor. Mdm2-a is a commonly identified splice variant of Mdm2; however, its physiological function is unclear. To gain insight into the activity of MDM2-A and its potential impact on p53, an Mdm2-a transgenic mouse model was generated. Mdm2-a transgenic mice displayed a homozygous-lethal phenotype that could be rescued by a reduction in p53 expression, demonstrating a dependence upon p53. Mdm2-a hemizygous mice exhibited reduced longevity, and enhanced senescence was observed in their salivary glands. In addition, the transgenic mice lacked typical, accelerated aging phenotypes. Growth of transgenic mouse embryonic fibroblasts (MEFs) was inhibited relative to wild-type MEFs, and MDM2-A was shown to bind to full-length MDM2 in an interaction that could increase p53 activity via reduced MDM2 inhibition. Evidence of p53 activation was shown in the Mdm2-a transgenic MEFs, including p53-dependent growth inhibition and elevated expression of the p53 target protein p21. In addition, MDM2-A increased senescence in a p21-independent manner. In conclusion, unexpected roles for MDM2-A in longevity and senescence were identified in a transgenic mouse model, suggesting that Mdm2 splice variants might be determinants of these phenotypes in vivo

Coexpression of Normally Incompatible Developmental Pathways in Retinoblastoma Genesis

It is widely believed that the molecular and cellular features of a tumor reflect its cell of origin and can thus provide clues about treatment targets. The retinoblastoma cell of origin has been debated for over a century. Here, we report that human and mouse retinoblastomas have molecular, cellular, and neurochemical features of multiple cell classes, principally amacrine/horizontal interneurons, retinal progenitor cells, and photoreceptors. Importantly, single-cell gene expression array analysis showed that these multiple cell type-specific developmental programs are coexpressed in individual retinoblastoma cells, which creates a progenitor/neuronal hybrid cell. Furthermore, neurotransmitter receptors, transporters, and biosynthetic enzymes are expressed in human retinoblastoma, and targeted disruption of these pathways reduces retinoblastoma growth in vivo and in vitro.This is a manuscript of an article from Cancer Cell 20 (2011): 260, doi: 10.1016/j.ccr.2011.07.005. Posted with permission.</p

Whole-Body Physiologically Based Pharmacokinetic Model for Nutlin-3a in Mice after Intravenous and Oral AdministrationS⃞

Nutlin-3a is an MDM2 inhibitor that is under investigation in preclinical models for a variety of pediatric malignancies, including retinoblastoma, rhabdomyosarcoma, neuroblastoma, and leukemia. We used physiologically based pharmacokinetic (PBPK) modeling to characterize the disposition of nutlin-3a in the mouse. Plasma protein binding and blood partitioning were assessed by in vitro studies. After intravenous (10 and 20 mg/kg) and oral (50, 100, and 200 mg/kg) dosing, tissue concentrations of nutlin-3a were determined in plasma, liver, spleen, intestine, muscle, lung, adipose, bone marrow, adrenal gland, brain, retina, and vitreous fluid. The PBPK model was simultaneously fit to all pharmacokinetic data using NONMEM. Nutlin-3a exhibited nonlinear binding to murine plasma proteins, with the unbound fraction ranging from 0.7 to 11.8%. Nutlin-3a disposition was characterized by rapid absorption with peak plasma concentrations at approximately 2 h and biphasic elimination consistent with a saturable clearance process. The final PBPK model successfully described the plasma and tissue disposition of nutlin-3a. Simulations suggested high bioavailability, rapid attainment of steady state, and little accumulation when administered once or twice daily at dosages up to 400 mg/kg. The final model was used to perform simulations of unbound tissue concentrations to determine which dosing regimens are appropriate for preclinical models of several pediatric malignancies

Pneumonic Plague Pathogenesis and Immunity in Brown Norway Rats

The Brown Norway rat was recently described as a bubonic plague model that closely mimics human disease. We therefore evaluated the Brown Norway rat as an alternative small animal model for pneumonic plague and characterized both the efficacy and potency of vaccine candidates. When infected by intranasal instillation, these rats rapidly developed fatal pneumonic plague within 2 to 4 days of infection. Plague disease was characterized by severe alveolar edema and vascular hemorrhage in the lung in addition to fulminant necrotizing pneumonia caused by massive bacterial replication and inflammation. Twenty-four hours before death, animals developed systemic disease with an apparent delayed inflammatory response. We evaluated the ability of the protective antigen, LcrV, and a mutant derivative, V10, to protect these rats from pneumonic plague. Both were highly effective vaccines because complete protection was observed at challenge doses of 7500 LD50. Antibody analyses suggested stronger potency of V10 immune sera compared with LcrV in the passive transfer of immunity to bubonic plague, with multiple neutralizing epitopes in LcrV. Taken together, these data demonstrate the effectiveness of inhibiting type III secretion in the prevention of pneumonic plague in rats and reveal critical contributions from both the cellular and humoral immune systems. Thus, the Brown Norway rat is an appealing alternative small animal model for the study of pneumonic plague pathogenesis and immunity