Discovery and development of COVID-19 vaccines and therapeutics: nonclinical perspectives.

The development and regulatory review of BNT162b2, a COVID-19 vaccine, and PaxlovidTM (nirmatrelvir tablets/ritonavir tablets), a COVID-19 therapeutic, are benchmarks for accelerated innovation during a global pandemic. Rapid choice of the SARS-CoV-2 spike protein and main protease (Mpro) as targets for the vaccine and therapeutic, respectively, leveraged the available knowledge of the biology of SARS-CoV-2 and related viruses. The nonclinical immunogenicity and safety of BNT162b2 was rigorously assessed. Likewise, a comprehensive nonclinical safety assessment was conducted for the therapeutic candidates, lufotrelvir (PF-07304814) and nirmatrelvir (PF-07321332). The development and regulatory review of BNT162b2 and Paxlovid was enabled through close collaboration of the pharmaceutical industry with regulatory agencies and public health organizations. This experience highlights approaches that could be adopted for pandemic preparedness including risk-based investment strategies, conduct of activities in parallel that normally are conducted sequentially, quick kill decisions, simultaneous evaluation of multiple candidates, and use of flexible, established vaccine platforms.

Translation of nonclinical to clinical safety findings for 27 biotherapeutics.

Human adverse drug reactions (ADRs), and in vivo nonclinical adverse and nonadverse findings, were identified in 27 biotherapeutic programs and placed into organ categories to determine translation. The sensitivity of detecting human ADRs was 30.8% with a positive predictive value (PPV) of 53.3% for nonclinical adverse findings; sensitivity increased to 67.3% and PPV fell to 35.0% when including nonadverse findings. Nonclinical findings were associated with a greater likelihood of a human ADR in that organ category, especially for adverse findings [positive likelihood ratio (LR+) >10 (lower 95% confidence interval [CI] of >5)]. The specificity and negative predictive value (NPV) were very high (>85%). A lack of nonclinical findings in an organ category was associated with a lower likelihood of a human ADR in that organ category. About 40-50% of human ADRs and nonclinical adverse findings, and about 30% of nonclinical nonadverse findings, were attributed to pharmacology. Slightly more than half of the human ADRs with a translating nonclinical finding had findings in animals that could be considered very similar. Overall, 38% of nonclinical findings translated to a human ADR at the organ category level. When nonclinical findings did not translate to humans, the cause was usually higher exposures or longer dosing in animals. All programs with human ADRs attributed to immunogenicity also had nonclinical adverse or nonadverse findings related to immunogenicity. Overall, nonclinical adverse and nonadverse findings were useful in predicting human ADRs, especially at an organ category level, and the majority of human ADRs were predicted by nonclinical toxicity studies.

Toxicological Assessments of a Pandemic COVID-19 Vaccine-Demonstrating the Suitability of a Platform Approach for mRNA Vaccines.

The emergence of SARS-CoV-2 at the end of 2019 required the swift development of a vaccine to address the pandemic. Nonclinical GLP-compliant studies in Wistar Han rats were initiated to assess the local tolerance, systemic toxicity, and immune response to four mRNA vaccine candidates encoding immunogens derived from the spike (S) glycoprotein of SARS-CoV-2, encapsulated in lipid nanoparticles (LNPs). Vaccine candidates were administered intramuscularly once weekly for three doses at 30 and/or 100 µg followed by a 3-week recovery period. Clinical pathology findings included higher white blood cell counts and acute phase reactant concentrations, lower platelet and reticulocyte counts, and lower RBC parameters. Microscopically, there was increased cellularity (lymphocytes) in the lymph nodes and spleen, increased hematopoiesis in the bone marrow and spleen, acute inflammation and edema at the injection site, and minimal hepatocellular vacuolation. These findings were generally attributed to the anticipated immune and inflammatory responses to the vaccines, except for hepatocyte vacuolation, which was interpreted to reflect hepatocyte LNP lipid uptake, was similar between candidates and resolved or partially recovered at the end of the recovery phase. These studies demonstrated safety and tolerability in rats, supporting SARS-CoV-2 mRNA-LNP vaccine clinical development.

COVID-19 Therapeutics and Vaccines: A Race to Save Lives.

COVID-19 (Coronavirus Disease 2019), the disease caused by SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) is an ongoing global public health emergency. As understanding of the health effects of COVID-19 has improved, companies and agencies worldwide have worked together to identify therapeutic approaches, fast-track clinical trials and pathways for emergency use, and approve therapies for patients. This work has resulted in therapies that not only improve survival, reduce time of hospitalization, and time to recovery, but also include preventative measures, such as vaccines. This manuscript discusses development programs for 3 products that are approved or authorized for emergency use at the time of writing: VEKLURY (remdesivir, direct-acting antiviral from Gilead Sciences, Inc.), REGEN-COV (casirivimab and imdevimab antibody cocktail from Regeneron Pharmaceuticals Inc.), and Comirnaty (Pfizer-BioNTech COVID-19 Vaccine [Pfizer, Inc.-BioNTech]), and perspectives from the U.S. Food and Drug Administration.

Maternal immunization with Group B Streptococcus six-valent polysaccharide conjugate vaccine supported by lack of toxicity in rat and rabbit fertility and developmental toxicity studies.

A maternal Group B Streptococcus (GBS) six-valent polysaccharide conjugate vaccine (GBS6) is being developed to protect neonates and infants up to 3 months of age through passive transfer of antibodies from the mother to the infant. Fertility and developmental toxicity studies were conducted in female Sprague Dawley rats and New Zealand White rabbits with GBS6 (20 µg capsular polysaccharide/serotype formulated with or without AlPO4 , the highest clinical dose). Females were administered the full human dose of the GBS6 formulation intramuscularly twice prior to mating and twice during gestation, to ensure that high antibody levels were maintained throughout gestation and lactation. Approximately, half of the rats and rabbits were evaluated at the end of gestation, and the remainder were evaluated at the end of lactation. Maternal blood for GBS6 serology, to measure antibody titers to the GBS6 antigens, was collected prior to the first dose, prior to mating, and at each necropsy. Blood for serology was also collected from offspring at the end of gestation and lactation. In both species, there was no evidence of vaccine-related effects on fertility, embryo-fetal development, or postnatal development of the offspring, supporting regulatory guidance that single-species evaluation would have been sufficient. Functional serum antibodies to all six serotypes in the vaccine were confirmed in maternal animals and functional serum antibodies to one or more of the six serotypes was also confirmed in some rat offspring and most of the rabbit offspring. The results of these studies supported the safety of GBS6 vaccine administration to pregnant women.

Lack of effects on female fertility and prenatal and postnatal offspring development in rats with BNT162b2, a mRNA-based COVID-19 vaccine.

BNT162b2 is a vaccine developed to prevent coronavirus disease 2019 (COVID-19). BNT162b2 is a lipid nanoparticle formulated nucleoside-modified messenger RNA (mRNA) encoding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein locked in its prefusion conformation. A developmental and reproductive toxicity study was conducted in rats according to international regulatory guidelines. The full human BNT162b2 dose of 30 µg mRNA/dose (>300 times the human dose on a mg/kg basis) was administered intramuscularly to 44 female rats 21 and 14 days prior to mating and on gestation days 9 and 20. Half of the rats were subject to cesarean section and full fetal examination at the end of gestation, and the other half were allowed to deliver and were monitored to the end of lactation. A robust neutralizing antibody response was confirmed prior to mating and at the end of gestation and lactation. The presence of neutralizing antibodies was also confirmed in fetuses and offspring. Nonadverse effects, related to the local injection site reaction, were noted in dams as expected from other animal studies and consistent with observations in humans. There were no effects of BNT162b2 on female mating performance, fertility, or any ovarian or uterine parameters nor on embryo-fetal or postnatal survival, growth, physical development or neurofunctional development in the offspring through the end of lactation. Together with the safety profile in nonpregnant people, this ICH-compliant nonclinical safety data supports study of BNT162b2 in women of childbearing potential and pregnant and lactating women.

PF-06804103, A Site-specific Anti-HER2 Antibody-Drug Conjugate for the Treatment of HER2-expressing Breast, Gastric, and Lung Cancers.

The approval of ado-trastuzumab emtansine (T-DM1) in HER2+ metastatic breast cancer validated HER2 as a target for HER2-specific antibody-drug conjugates (ADC). Despite its demonstrated clinical efficacy, certain inherent properties within T-DM1 hamper this compound from achieving the full potential of targeting HER2-expressing solid tumors with ADCs. Here, we detail the discovery of PF-06804103, an anti-HER2 ADC designed to have a widened therapeutic window compared with T-DM1. We utilized an empirical conjugation site screening campaign to identify the engineered ĸkK183C and K290C residues as those that maximized in vivo ADC stability, efficacy, and safety for a four drug-antibody ratio (DAR) ADC with this linker-payload combination. PF-06804103 incorporates the following novel design elements: (i) a new auristatin payload with optimized pharmacodynamic properties, (ii) a cleavable linker for optimized payload release and enhanced antitumor efficacy, and (iii) an engineered cysteine site-specific conjugation approach that overcomes the traditional safety liabilities of conventional conjugates and generates a homogenous drug product with a DAR of 4. PF-06804103 shows (i) an enhanced efficacy against low HER2-expressing breast, gastric, and lung tumor models, (ii) overcomes in vitro- and in vivo-acquired T-DM1 resistance, and (iii) an improved safety profile by enhancing ADC stability, pharmacokinetic parameters, and reducing off-target toxicities. Herein, we showcase our platform approach in optimizing ADC design, resulting in the generation of the anti-HER2 ADC, PF-06804103. The design elements of identifying novel sites of conjugation employed in this study serve as a platform for developing optimized ADCs against other tumor-specific targets.

A Novel GUCY2C-CD3 T-Cell Engaging Bispecific Construct (PF-07062119) for the Treatment of Gastrointestinal Cancers.

PURPOSE: Gastrointestinal cancers remain areas of high unmet need despite advances in targeted and immunotherapies. Here, we demonstrate potent, tumor-selective efficacy with PF-07062119, a T-cell engaging CD3 bispecific targeting tumors expressing Guanylyl Cyclase C (GUCY2C), which is expressed widely across colorectal cancer and other gastrointestinal malignancies. In addition, to address immune evasion mechanisms, we explore combinations with immune checkpoint blockade agents and with antiangiogenesis therapy. EXPERIMENTAL DESIGN: PF-07062119 activity was evaluated in vitro in multiple tumor cell lines, and in vivo in established subcutaneous and orthotopic human colorectal cancer xenograft tumors with adoptive transfer of human T cells. Efficacy was also evaluated in mouse syngeneic tumors using human CD3ε transgenic mice. IHC and mass cytometry were performed to demonstrate drug biodistribution, recruitment of activated T cells, and to identify markers of immune evasion. Combination studies were performed with anti-PD-1/PD-L1 and anti-VEGF antibodies. Toxicity and pharmacokinetic studies were done in cynomolgus macaque. RESULTS: We demonstrate that GUCY2C-positive tumors can be targeted with an anti-GUCY2C/anti-CD3ε bispecific, with selective drug biodistribution to tumors. PF-07062119 showed potent T-cell-mediated in vitro activity and in vivo efficacy in multiple colorectal cancer human xenograft tumor models, including KRAS- and BRAF-mutant tumors, as well as in the immunocompetent mouse syngeneic tumor model. PF-07062119 activity was further enhanced when combined with anti-PD-1/PD-L1 treatment or in combination with antiangiogenic therapy. Toxicity studies in cynomolgus indicated a monitorable and manageable toxicity profile. CONCLUSIONS: These data highlight the potential for PF-07062119 to demonstrate efficacy and improve patient outcomes in colorectal cancer and other gastrointestinal malignancies.

Inflammatory Cell Findings in the Female Rabbit Heart and Stress-associated Exacerbation with Handling and Procedures Used in Nonclinical Studies.

Despite the use of rabbits in biomedical research, including regulatory toxicology and cardiovascular studies, little data exist on heart findings in this species. This study was designed to document myocardial findings in female rabbits and the impact of study-related procedures typical for vaccine toxicology studies. One hundred and forty 6- to 8-month-old female New Zealand White rabbits were divided equally into 2 groups, high and low study procedure groups (group 1 and group 2, respectively). All animals received intramuscular (IM) injections of sterile saline every 2 weeks for 5 times and were necropsied 2 days after the final IM injection. Clinical chemistry, hematology, and urinalysis were evaluated. Blood for stress biomarkers (norepinephrine, epinephrine, cortisol, and corticosterone), C-reactive protein, cardiac troponin I, and creatine kinase were collected at time 0 (just before dose administration) and then at 4, 24, and 48 hr after dose administration in group 1 only. Hearts were assessed histologically. Focal to multifocal minimal inflammatory cell infiltrates were common (∼80%), particularly in the left ventricle and interventricular septum, and were similar to the types of infiltrates identified in other laboratory animal species. Additionally, study-related procedures elevated serum stress biomarkers and exacerbated the frequency and severity of myocardial inflammatory cell infiltrates.

Mechanistic investigations of test article-induced pancreatic toxicity at the endocrine-exocrine interface in the rat.

Pancreatic toxicity commonly affects the endocrine or exocrine pancreas. However, it can also occur at the endocrine-exocrine interface (EEI), where the capillary network of the islet merges with the capillaries of the surrounding acinar tissue, that is, the insulo-acinar portal system. The goal of this article is to describe a novel, test article-induced pancreatic toxicity that originated at the EEI and to summarize investigations into the mechanistic basis of the injury. This injury was initially characterized by light microscopy in 7/14 day-toxicity studies in Sprague-Dawley (Crl: CD®[SD]) rats with undisclosed test articles. Microvascular injury at the interface resulted in peri-islet serum exudation, fibrin deposition, hemorrhage, inflammation, and secondary degeneration/necrosis of surrounding exocrine tissue. More chronic injury presented as islet fibrosis and lobular atrophy. Direct cytotoxicity affecting the capillary endothelium at the EEI was confirmed ultrastructurally on day 4. Endothelial microparticle and blood flow studies further confirmed endothelial involvement. Similar lesions occurred less frequently in 2 other rat strains and not in the mouse, dog, or cynomolgus macaque. In summary, in vivo and investigative study data confirmed primary endothelial cytotoxicity in the pathogenesis of this lesion and suggested that the lesion may be rat/rat strain-specific and of uncertain relevance for human safety risk assessment.

Activation of TrkB with TAM-163 results in opposite effects on body weight in rodents and non-human primates.

Strong genetic data link the Tyrosine kinase receptor B (TrkB) and its major endogenous ligand brain-derived neurotrophic factor (BDNF) to the regulation of energy homeostasis, with loss-of-function mutations in either gene causing severe obesity in both mice and humans. It has previously been reported that peripheral administration of the endogenous TrkB agonist ligand neurotrophin-4 (NT-4) profoundly decreases food intake and body weight in rodents, while paradoxically increasing these same parameters in monkeys. We generated a humanized TrkB agonist antibody, TAM-163, and characterized its therapeutic potential in several models of type 2 diabetes and obesity. In vitro, TAM-163 bound to human and rodent TrkB with high affinity, activated all aspects of the TrkB signaling cascade and induced TrkB internalization and degradation in a manner similar to BDNF. In vivo, peripheral administration of TAM-163 decreased food intake and/or body weight in mice, rats, hamsters, and dogs, but increased food intake and body weight in monkeys. The magnitude of weight change was similar in rodents and non-human primates, occurred at doses where there was no appreciable penetration into deep structures of the brain, and could not be explained by differences in exposures between species. Rather, peripherally administered TAM-163 localized to areas in the hypothalamus and the brain stem located outside the blood-brain barrier in a similar manner between rodents and non-human primates, suggesting differences in neuroanatomy across species. Our data demonstrate that a TrkB agonist antibody, administered peripherally, causes species-dependent effects on body weight similar to the endogenous TrkB ligand NT-4. The possible clinical utility of TrkB agonism in treating weight regulatory disorder, such as obesity or cachexia, will require evaluation in man.

Agonistic TAM-163 antibody targeting tyrosine kinase receptor-B: applying mechanistic modeling to enable preclinical to clinical translation and guide clinical trial design.

TAM-163, an agonist monoclonal antibody targeting tyrosine receptor kinase-B (TrkB), is currently being investigated as a potential body weight modulatory agent in humans. To support the selection of the dose range for the first-in-human (FIH) trial of TAM-163, we conducted a mechanistic analysis of the pharmacokinetic (PK) and pharmacodynamic (PD) data (e.g., body weight gain) obtained in lean cynomolgus and obese rhesus monkeys following single doses ranging from 0.3 to 60 mg/kg. A target-mediated drug disposition (TMDD) model was used to describe the observed nonlinear PK and Emax approach was used to describe the observed dose-dependent PD effect. The TMDD model development was supported by the experimental determination of the binding affinity constant (9.4 nM) and internalization rate of the drug-target complex (2.08 h(-1)). These mechanistic analyses enabled linking of exposure, target (TrkB) coverage, and pharmacological activity (e.g., PD) in monkeys, and indicated that ≥ 38% target coverage (time-average) was required to achieve significant body weight gain in monkeys. Based on the scaling of the TMDD model from monkeys to humans and assuming similar relationship between the target coverage and pharmacological activity between monkey and humans, subcutaneous (SC) doses of 1 and 15 mg/kg in humans were projected to be the minimally and the fully pharmacologically active doses, respectively. Based on the minimal anticipated biological effect level (MABEL) approach for starting dose selection, the dose of 0.05 mg/kg (3 mg for a 60 kg human) SC was recommended as the starting dose for FIH trials, because at this dose level<10% target coverage was projected at Cmax (and all other time points). This study illustrates a rational mechanistic approach for the selection of FIH dose range for a therapeutic protein with a complex model of action.

"Candidatus Mycoplasma haemomacaque" and Bartonella quintana bacteremia in cynomolgus monkeys.

Here, we report latent infections with Bartonella quintana and a hemotropic Mycoplasma sp. in a research colony of cynomolgus monkeys (Macaca fascicularis). Sequence alignments, evolutionary analysis, and signature nucleotide sequence motifs of the hemotropic Mycoplasma 16S rRNA and RNase P genes indicate the presence of a novel organism.

Ultra performance liquid chromatography-mass spectrometry profiling of bile acid metabolites in biofluids: application to experimental toxicology studies.

We have developed an ultra performance liquid chromatography-mass spectrometry (UPLC-MS(E)) method to measure bile acids (BAs) reproducibly and reliably in biological fluids and have applied this approach for indications of hepatic damage in experimental toxicity studies. BAs were extracted from serum using methanol, and an Acquity HSS column coupled to a Q-ToF mass spectrometer was used to separate and identify 25 individual BAs within 5 min. Employing a gradient elution of water and acetonitrile over 21 min enabled the detection of a wide range of endogenous metabolites, including the BAs. The utilization of MS(E) allowed for characteristic fragmentation information to be obtained in a single analytical run, easily distinguishing glycine and taurine BA conjugates. The proportions of these conjugates were altered markedly in an experimental toxic state induced by galactosamine exposure in rats. Principally, taurine-conjugated BAs were greatly elevated ( approximately 50-fold from control levels), and were highly correlated to liver damage severity as assessed by histopathological scoring (r = 0.83), indicating their potential as a sensitive measure of hepatic damage. The UPLC-MS approach to BA analysis offers a sensitive and reproducible tool that will be of great value in exploring both markers and mechanisms of hepatotoxicity and can readily be extended to clinical studies of liver damage.

The use of probiotics in the prevention and treatment of antibiotic-associated diarrhea with special interest in Clostridium difficile-associated diarrhea.

Antimicrobials are effective agents used to combat virulent bacterial, yeast, and fungal infections that may otherwise cause rampant disease leading to skyrocketing social/economic costs and possible epidemic morbidity and mortality rates. Antibiotics are designed to attack specific bacterial pathogens but, in the process, indiscreetly reduce the number of beneficial human microbiota that is part of the gut-associated lymphatic tissue. Broad-spectrum antibiotics can upset this uniquely balanced gut ratio, allowing pathogens to propagate in a largely unrestrained environment, which may result in antibiotic-induced diarrhea. Critical illness, age, immunosuppression, exposure to nosocomial microorganisms, and the length of hospitalization are additional factors that contribute to the overgrowth of opportunistic pathogens. In mild to moderate cases of diarrhea, absorptive impairment may occur, thereby reducing micro/macronutrient assimilation, resulting in malnutrition and growth issues in children. In severe cases, infectious diarrhea can have devastating complications. Of particular interest is the bacterium Clostridium difficile, which has the potential to cause a host of symptoms ranging from mild diarrhea to severe life-threatening conditions. C. difficile infection can increase mortality rates by 10%-30%. Probiotic supplementation may prevent and treat antibiotic-associated diarrhea. Specific probiotics may modulate the intestinal mucosa by antagonizing pathogens through the production of antimicrobial compounds and chemicals, thereby reducing the rate of nosocomial infection and recurrence of C. difficile.

Metabonomic evaluation of metabolic dysregulation in rats induced by PF 376304, a novel inhibitor of phosphoinositide 3-kinase.

Phosphoinositide 3-kinase (PI3K) is an enzyme fundamental to the regulation of various metabolic processes. Metabonomic studies were undertaken in order to gain mechanistic insight into significant, yet unexplained, toxicity issues associated with PF 376304, a nonspecific PI3K inhibitor under development for anti-inflammatory indications. Two experiments were conducted in which rats were given daily doses of up to 1000 mg of PF 376304/kg for as long as 7 days. Mortality rapidly ensued (within 72 h) at doses of >or=300 mg/kg. Doses of >or=100 mg/kg were associated with a profound but transient glucosuria. Despite the magnitude of this effect, within 72 h urinary glucose excretion in surviving animals returned to control levels even with continued dosing. Other metabolic effects associated with drug treatment included increased urinary beta-hydroxybutyrate and creatine and decreased citrate. A time-course study revealed elevated serum glucose within 1 h, followed by increases in serum insulin and decreases in serum triglycerides. Serum corticosterone was also significantly elevated within 1 h of treatment. All metabolic effects were largely reversed within 24 h of administration of the third daily dose and remained that way through day 7. The likely explanation for the onset of effects involves the role of PI3K in regulation of glucose at multiple points, but the reversal of the effects in the presence of continued exposure to the drug has not been explained. Finally, the data demonstrate the power of metabonomics technology in mechanistic toxicology investigations.

Metabonomic evaluation of Schaedler altered microflora rats.

Previously, we identified two distinct metabonomic phenotypes in Sprague-Dawley rats sourced from two different rooms (colonies) in the Charles River, Raleigh facility [Robosky, L. C., Wells, D. F., Egnash, L. A., Manning, M. L., Reily, M. D., and Robertson, D. G. (2005) Metabonomic identification of two distinct phenotypes in Sprague-Dawley (Crl:CD(SD)) rats. Toxicol. Sci. 87, 277-284]. On the basis of literature reports and cohabitation experiments, we concluded that the differing phenotypes were due to different gut flora populations. One hypothesis explaining this phenomenon was attributed to the practice of initiating new colonies with rats derived from foundation colonies that had limited gut floral populations, the Charles River altered Schaedler flora (CRASF) rats. We hypothesized that the lack of differentiation of CRASF rats to the full complement of microflora was responsible for the altered phenotype characterized by increased urinary chlorogenic acid metabolites and decreased hippurate (CA rats) as opposed to the prevalent phenotype characterized by the inverse ratio of these metabolites (HIP rats). Upon receipt, it was confirmed that the CRASF rats exhibited a metabonomic profile similar to CA rats that remained constant while animals were housed individually in a dedicated animal room. However, exposure of CRASF rats to HIP rats, or their bedding, led to a relatively rapid but variable rate of reversion to the historic HIP type metabolic profile. On the basis of the results, we conclude that CRASF rats have a unique metabolic profile due to their limited gut flora constitution. If rigorous isolation procedures are not employed, the CRASF phenotype will eventually differentiate into the more typical HIP phenotype with a time course that may be quite variable. Given the marked metabolic heterogeneity between the phenotypes, this work highlights the importance of monitoring rat metabolic profiles.

All-trans retinoic acid antagonizes the action of calciferol and its active metabolite, 1,25-dihydroxycholecalciferol, in rats.

An antagonistic interaction between retinol and calciferol has been established. However, the mechanism by which this antagonism occurs is unclear. One possibility is that retinol affects the metabolism of calciferol. To investigate this hypothesis, retinol- and calciferol-depleted rats were given various amounts of ergocalciferol, cholecalciferol, 1alpha,25-dihydroxycholecalciferol [1,25(OH)2D3], or 24,24-difluoro-1alpha,25-dihydroxycholecalciferol [24-F2-1,25(OH)2D3] in combination with various amounts of retinyl acetate or all-trans retinoic acid (ATRA) in a series of studies. Rats administered 1720 or 3440 microg retinyl acetate once every 3 d for 33 d in combination with 25.8 ng ergocalciferol or 25 ng cholecalciferol every 3 d had lower serum calcium and greater serum phosphorus concentrations than rats fed 0 or 11.4 mug retinyl acetate every 3 d. In addition, rats fed 400 microg ATRA/d in combination with 25.8 ng ergocalciferol every 3 d, 25 ng cholecalciferol every 3 d, 2-5 ng 1,25(OH)2D3/d, or 0.5-1 ng 24-F2-1,25(OH)2D3/d had significantly lower serum calcium and higher serum phosphorus concentrations than rats not given ATRA in the diet. Therefore, both retinyl acetate and ATRA are able to antagonize the action of ergocalciferol and cholecalciferol in vivo. Additionally, ATRA antagonizes the in vivo action of 1,25(OH)2D3 and an analog, 24-F2-1,25(OH)2D3, that cannot be 24-hydroxylated. Together, these results suggest that retinol does not antagonize the action of calciferol by altering the metabolism of calciferol or 1,25(OH)2D3, but does so by another mechanism.

A juxtamembrane tyrosine in the colony stimulating factor-1 receptor regulates ligand-induced Src association, receptor kinase function, and down-regulation.

Recent literature implicates a regulatory function of the juxtamembrane domain (JMD) in receptor tyrosine kinases. Mutations in the JMD of c-Kit and Flt3 are associated with gastrointestinal stromal tumors and acute myeloid leukemias, respectively. Additionally, autophosphorylated Tyr559 in the JMD of the colony stimulating factor-1 (CSF-1) receptor (CSF-1R) binds to Src family kinases (SFKs). To investigate SFK function in CSF-1 signaling we established stable 32D myeloid cell lines expressing CSF-1Rs with mutated SFK binding sites (Tyr559-TFI). Whereas binding to I562S was not significantly perturbed, Y559F and Y559D exhibited markedly decreased CSF-1-dependent SFK association. All JMD mutants retained intrinsic kinase activity, but Y559F, and less so Y559D, showed dramatically reduced CSF-1-induced autophosphorylation. CSF-1-mediated wild-type (WT)-CSF-1R phosphorylation was not markedly affected by SFK inhibition, indicating that lack of SFK binding is not responsible for diminished Y559F phosphorylation. Unexpectedly, cells expressing Y559F were hyperproliferative in response to CSF-1. Hyperproliferation correlated with prolonged activation of Akt, ERK, and Stat5 in the Y559F mutant. Consistent with a defect in receptor negative regulation, c-Cbl tyrosine phosphorylation and CSF-1R/c-Cbl co-association were almost undetectable in the Y559F mutant. Furthermore, Y559F underwent reduced multiubiquitination and delayed receptor internalization and degradation. In conclusion, we propose that Tyr559 is a switch residue that functions in kinase regulation, signal transduction and, indirectly, receptor down-regulation. These findings may have implications for the oncogenic conversion of c-Kit and Flt3 with JMD mutations.

Bone resorption activity of all-trans retinoic acid is independent of vitamin D in rats.

The mechanism by which all-trans retinoic acid (ATRA) induces bone resorption is unknown. However, an interaction between vitamin A and vitamin D has been established. In fact, although the mechanism is still unclear, vitamin A has been shown to be a weak antagonist of the actions of vitamin D. Taking into account this interaction and the influence of vitamin D on other calcitropic hormones, such as parathyroid hormone, the effect of vitamin D on ATRA-induced bone resorption was investigated. Vitamin D-deficient rats were fed diets containing 0 or 150 micro g of ATRA/g of diet. The rats then were orally administered 0 or 625 ng of cholecalciferol (vitamin D(3)) daily. Various bone parameters were measured after 3-8 wk. Regardless of the presence or absence of vitamin D(3), ATRA was able to cause bone resorption. In addition to examining the effect of vitamin D on ATRA-induced bone resorption under normal conditions, this effect also was studied under conditions that inhibit bone mineralization or growth by altering dietary calcium (Ca) and phosphorus (P) levels. Changes in dietary levels of Ca and P did not affect the ability of ATRA to cause bone resorption. Interestingly, despite its ability to stimulate bone resorption, ATRA did not affect serum calcium or phosphorus levels. Overall, the ability of ATRA to cause bone resorption is not dependent on vitamin D(3), dietary Ca or dietary P.