Effects of Monoclonal Antibodies against Nerve Growth Factor on Healthy Bone and Joint Tissues in Mice, Rats, and Monkeys: Histopathologic, Biomarker, and Microcomputed Tomographic Assessments.

Tanezumab, an anti-nerve growth factor (NGF) antibody, is in development for management of chronic pain. During clinical trials of anti-NGF antibodies, some patients reported unexpected adverse events requiring total joint replacements, resulting in a partial clinical hold on all NGF inhibitors. Three nonclinical toxicology studies were conducted to evaluate the effects of tanezumab or the murine precursor muMab911 on selected bone and joint endpoints and biomarkers in cynomolgus monkeys, Sprague-Dawley rats, and C57BL/6 mice. Joint and bone endpoints included histology, immunohistochemistry, microcomputed tomography (mCT) imaging, and serum biomarkers of bone physiology. Responses of bone endpoints to tanezumab were evaluated in monkeys at 4 to 30 mg/kg/week for 26 weeks and in rats at 0.2 to 10 mg/kg twice weekly for 28 days. The effects of muMab911 at 10 mg/kg/week for 12 weeks on selected bone endpoints were determined in mice. Tanezumab and muMab911 had no adverse effects on any bone or joint parameter. There were no test article-related effects on bone or joint histology, immunohistochemistry, or structure. Reversible, higher osteocalcin concentrations occurred only in the rat study. No deleterious effects were observed in joints or bones in monkeys, rats, or mice administered high doses of tanezumab or muMab911.

Nerve growth factor inhibition with tanezumab influences weight-bearing and subsequent cartilage damage in the rat medial meniscal tear model.

OBJECTIVE: To investigate whether the effects of nerve growth factor (NGF) inhibition with tanezumab on rats with medial meniscal tear (MMT) effectively model rapidly progressive osteoarthritis (RPOA) observed in clinical trials. METHODS: Male Lewis rats underwent MMT surgery and were treated weekly with tanezumab (0.1, 1 or 10 mg/kg), isotype control or vehicle for 7, 14 or 28 days. Gait deficiency was measured to assess weight-bearing on the operated limb. Joint damage was assessed via histopathology. A second arm, delayed onset of treatment (starting 3-8 weeks after MMT surgery) was used to control for analgesia early in the disease process. A third arm, mid-tibial amputation, evaluated the dependency of the model on weight-bearing. RESULTS: Gait deficiency in untreated rats was present 3-7 days after MMT surgery, with a return to normal weight-bearing by days 14-28. Prophylactic treatment with tanezumab prevented gait deficiency and resulted in more severe cartilage damage. When onset of treatment with tanezumab was delayed to 3-8 weeks after MMT surgery, there was no increase in cartilage damage. Mid-tibial amputation completely prevented cartilage damage in untreated MMT rats. CONCLUSIONS: These data suggest that analgesia due to NGF inhibition during the acute injury phase is responsible for increased voluntary weight-bearing and subsequent cartilage damage in the rat MMT model. This model failed to replicate the hypotrophic bone response observed in tanezumab-treated patients with RPOA.

Biosimilars: Practical Considerations for Pharmacists.

OBJECTIVE: To review the scientific and regulatory aspects of biosimilar development and practical considerations for the use of biosimilars that are relevant to pharmacists. DATA SOURCES: Literature searches of PubMed and congress abstracts for publications pertaining to biosimilars were conducted from January 2016 to January 2017. Individual drug company web pages and governmental, regulatory, and other agency websites were also reviewed. STUDY SELECTION/DATA EXTRACTION: Published articles, regulatory guidelines, and other sources covering biologic/biosimilar development and approval, reporting results of biosimilar studies or survey research, and/or identifying biosimilars in development or approved for use in Europe or the United States were reviewed and included. DATA SYNTHESIS: Biologic therapies have revolutionized the treatment of serious diseases, including hematological or autoimmune disorders and cancers. A biosimilar is highly similar to a licensed biologic (ie, reference or originator) and has no clinically meaningful differences in safety, purity, and potency. Unlike small-molecule drugs, biologics are large, complex proteins that cannot be exactly replicated, so the concept of a generic equivalent cannot be applied to biologics. Regulatory agencies have provided a framework for biosimilar approval, but there are many practical considerations for pharmacists, including interchangeability, substitution, naming, indication extrapolation, product labeling, therapeutic drug monitoring, manufacturer attributes, logistics of product use, and reimbursement. CONCLUSIONS: Pharmacists will play a key role in managing the introduction of biosimilars into health care systems. Understanding the principles of biosimilar development and evolving regulatory guidelines relevant to their use will allow pharmacists to make informed decisions regarding formulary inclusion and educate patients and other health care providers about biosimilars.

Comparative nonclinical assessments of the proposed biosimilar PF-05280586 and rituximab (MabThera®).

Comparative nonclinical studies were conducted with the proposed biosimilar PF-05280586 and rituximab-EU (MabThera®). In side-by-side analyses, peptide maps and complement-dependent cytotoxicity assay results were similar. Sexually-mature cynomolgus monkeys were administered PF-05280586 or rituximab-EU as a single dose of 0, 2, 10, or 20 mg/kg on day 1 and observed for 92 days (single-dose study) or as 5 weekly injections of 0 or 20 mg/kg and necropsied on day 30, the day after the 5th dose, or on day 121 (repeat-dose study). The pharmacokinetic and pharmacodynamic profiles for both molecules were similar. Marked depletion of peripheral blood B cells 4 days after dosing was followed by near or complete repletion (single-dose study) or partial repletion (repeat-dose study). In the single-dose study, anti-drug antibodies (ADA) were detected by day 29 in all animals administered PF-05280586 or rituximab-EU and persisted through day 85, the last day tested. In the repeat-dose study, ADA were detected on day 121 in 50% of animals administered PF-05280586 or rituximab-EU. Both molecules were well tolerated at all doses. In all endpoints evaluated, PF-05280586 exhibited similarity to rituximab-EU.

Quantitative prediction of renal transporter-mediated clinical drug-drug interactions.

Kidney plays a critical role in the elimination of xenobiotics. Drug-drug interactions (DDIs) via inhibition of renal organic anion (OAT) and organic cation (OCT) transporters have been observed in the clinic. This study examined the quantitative predictability of renal transporter-mediated clinical DDIs based on basic and mechanistic models. In vitro transport and clinical pharmacokinetics parameters were used to quantitatively predict DDIs of victim drugs when coadministrated with OAT or OCT inhibitors, probenecid and cimetidine, respectively. The predicted changes in renal clearance (CLr) and area under the plasma concentration-time curve (AUC) were comparable to that observed in clinical studies. With probenecid, basic modeling predicted 61% cases within 25% and 94% cases within 50% of the observed CLr changes in clinic. With cimetidine, basic modeling predicted 61% cases within 25% and 92% cases within 50% of the observed CLr changes in clinic. Additionally, the mechanistic model predicted 54% cases within 25% and 92% cases within 50% of the observed AUC changes with probenecid. Notably, the magnitude of AUC changes attributable to the renal DDIs is generally less than 2-fold, unlike the DDIs associated with inhibition of CYPs and/or hepatic uptake transporters. The models were further used to evaluate the renal DDIs of Pfizer clinical candidates/drugs, and the overall predictability demonstrates their utility in the drug discovery and development settings.

Analysis of ß-nerve growth factor and its precursor during human pregnancy by immunoaffinity-liquid chromatography tandem mass spectrometry.

ß-Nerve growth factor (NGF) is a neurotrophin that plays a critical role in fetal development during gestation. ProNGF is the precursor form of NGF with a distinct biological profile. In order to investigate the role of NGF and proNGF in pregnant human females, a sensitive and selective immunoaffinity liquid chromatography-tandem mass spectrometry assay was developed and qualified to simultaneously measure the levels of total NGF (tNGF; sum of mature and proNGF) and proNGF using full and relative quantification strategies, respectively. The assay was used to determine serum tNGF and proNGF levels in the three gestational trimesters of pregnancy and in non-pregnant female controls. Mean tNGF ± SD were 44.6 ± 12.3, 42.6 ± 9.3, 65.4 ± 17.6 and 77.0 ± 17.8 pg/mL for non-pregnant, first, second, and third trimesters, respectively, demonstrating no significant increase in circulating tNGF between the control and the first trimester, and a moderate yet significant 1.7-fold increase through gestation. proNGF levels during the first trimester were unchanged compared to control. In contrast to tNGF, however, proNGF levels during gestation remained stable without significant changes. The development of this sensitive, novel immunoaffinity duplexed assay for both tNGF and proNGF is expected to enable further elucidation of the roles these neurotrophins play in human pregnancy as well as other models.

Ocular Safety and Toxicokinetics of Bevacizumab-bvzr (Zirabev), a Bevacizumab Biosimilar, Administered to Cynomolgus Monkeys by Intravitreal Injection.

Purpose: Bevacizumab-bvzr (Zirabev®), a recombinant humanized monoclonal antibody targeting vascular endothelial growth factor and a biosimilar to bevacizumab, is approved for intravenous administration for various indications worldwide. The objectives of this study were to evaluate the ocular toxicity, systemic tolerability, and toxicokinetics (TKs) of bevacizumab-bvzr following repeat intravitreal (IVT) injection to cynomolgus monkeys. Methods: Male monkeys were administered saline, vehicle, or bevacizumab-bvzr at 1.25 mg/eye/dose once every 2 weeks (3 doses total) for 1 month by bilateral IVT injection, followed by a 4-week recovery phase to evaluate the reversibility of any findings. Local and systemic safety was assessed. Ocular safety assessments included in-life ophthalmic examinations, tonometry (intraocular pressure, IOP), electroretinograms (ERGs), and histopathology. In addition, concentrations of bevacizumab-bvzr were measured in serum and in ocular tissues (vitreous humor, retina, and choroid/retinal pigment epithelium) and ocular concentration-time profiles and serum TKs were evaluated. Results: Bevacizumab-bvzr was tolerated locally and systemically, with an ocular safety profile comparable to the saline or vehicle control group. Bevacizumab-bvzr was observed in both serum and in the evaluated ocular tissues. There were no bevacizumab-bvzr-related microscopic changes or effects on IOP or ERGs. Bevacizumab-bvzr-related trace pigment or cells in vitreous humor (in 4 of 12 animals; commonly associated with IVT injection) and transient, nonadverse, mild ocular inflammation (in 1 of 12 animals) were noted upon ophthalmic examination and fully reversed during the recovery phase. Conclusions: Bevacizumab-bvzr was well tolerated via biweekly IVT administration in healthy monkeys, with an ocular safety profile comparable to saline or its vehicle control.

How current understanding of clearance mechanisms and pharmacodynamics of therapeutic proteins can be applied for evaluation of their drug-drug interaction potential.

Increasing use of therapeutic proteins (TPs) in polypharmacy settings calls for more in-depth understanding of the biological interactions that can lead to increased toxicity or loss of pharmacological effect. Factors such as patient population, medications that are likely to be coadministered in that population, clearance mechanisms of a TP, and concomitant drugs have to be taken into account to determine the potential for drug-drug interactions (DDIs). The most well documented TP DDI mechanism involves cytokine-mediated changes in drug-metabolizing enzymes. Because of the limitations of the current preclinical models for addressing this type of DDI, clinical evaluation is currently the most reliable approach. Other DDI mechanisms need to be addressed on a case-by-case basis. These include altered clearance of TPs resulting from the changes in the target protein levels by the concomitant medication, displacement of TPs from binding proteins, modulation of Fcγ receptor expression, and others. The purpose of this review is to introduce the approach used by Pfizer scientists for evaluation of the DDI potential of novel TP products during drug discovery and development.

A multiple-dose toxicity study of tanezumab in cynomolgus monkeys.

Nerve growth factor (NGF) is an important mediator of pain and hyperalgesia and has become a target of novel analgesic therapeutics. Tanezumab is a humanized IgG(2) antibody that binds NGF with high affinity and specificity. In a study to assess the toxicity and pharmacokinetic properties of tanezumab in adult, male and female, cynomolgus monkeys following weekly intravenous administration of 1, 10, or 30 mg/kg for up to 26 weeks (followed by an 8-week recovery period), tanezumab was well tolerated with no macroscopic or microscopic effects on those brain, spinal cord, nerve, or ganglia sections evaluated. One fifth of tanezumab-treated monkeys developed an antibody response to tanezumab that prevented maintenance of tanezumab exposure between dosing. In the antibody-negative animals, accumulation of tanezumab was observed; steady state was achieved approximately 8 weeks after the first dose of study drug, and exposure to tanezumab was approximately dose proportional with no observed difference between male and female animals. One monkey died during the study; this monkey had findings suggestive of hypersensitivity reaction. The favorable toxicity and pharmacokinetic profile of tanezumab seen in this study supports its further evaluation for the treatment of pain in clinical practice.

Comparison of different algorithms for predicting clinical drug-drug interactions, based on the use of CYP3A4 in vitro data: predictions of compounds as precipitants of interaction.

Cytochrome P450 3A4 (CYP3A4) is the most important enzyme in drug metabolism and because it is the most frequent target for pharmacokinetic drug-drug interactions (DDIs) it is highly desirable to be able to predict CYP3A4-based DDIs from in vitro data. In this study, the prediction of clinical DDIs for 30 drugs on the pharmacokinetics of midazolam, a probe substrate for CYP3A4, was done using in vitro inhibition, inactivation, and induction data. Two DDI prediction approaches were used, which account for effects at both the liver and intestine. The first was a model that simultaneously combines reversible inhibition, time-dependent inactivation, and induction data with static estimates of relevant in vivo concentrations of the precipitant drug to provide point estimates of the average magnitude of change in midazolam exposure. This model yielded a success rate of 88% in discerning DDIs with a mean -fold error of 1.74. The second model was a computational physiologically based pharmacokinetic model that uses dynamic estimates of in vivo concentrations of the precipitant drug and accounts for interindividual variability among the population (Simcyp). This model yielded success rates of 88 and 90% (for "steady-state" and "time-based" approaches, respectively) and mean -fold errors of 1.59 and 1.47. From these findings it can be concluded that in vivo DDIs for CYP3A4 can be predicted from in vitro data, even when more than one biochemical phenomenon occurs simultaneously.

Assessment of three human in vitro systems in the generation of major human excretory and circulating metabolites.

An early understanding of key metabolites of drugs is crucial in drug discovery and development. As a result, several in vitro models typically derived from liver are frequently used to study drug metabolism. It is presumed that these in vitro systems provide an accurate view of the potential in vivo metabolites and metabolic pathways. However, no formal analysis has been conducted to validate their use. The goal of the present study was to conduct a comprehensive analysis to assess if the three commonly used in vitro systems, pooled human liver microsomes, liver S-9 fraction, and hepatocytes, adequately predict in vivo metabolic profiles for drugs. The second objective was to compare the overall capabilities of these three systems to generate in vivo metabolic profiles. Twenty-seven compounds in the Pfizer database and 21 additional commercially available compounds of diverse structure and routes of metabolism for which the human ADME data was available were analyzed in this study to assess the performance of the in vitro systems. The results suggested that all three systems reliably predicted human excretory and circulating metabolite profiles. Furthermore, the success in predicting primary metabolites and metabolic pathways was high (>70%), but the predictability of secondary metabolites was less reliable in the three systems. Thus, the analysis provides sufficient confidence in using in vitro systems to reliably produce primary in vivo human metabolites and supports their application in early discovery to identify metabolic spots for optimization of metabolic liabilities anticipated in humans in vivo. However, the in vitro systems cannot solely mitigate the risk of disproportionate circulating metabolites in humans and may need to be supplemented with metabolic profiling of plasma samples from first-in-human studies or early human radiolabeled studies.

Synthesis and SAR of tolylamine 5-HT6 antagonists.

The synthesis and SAR of tolylamines with 5-HT(6) receptor antagonist activity is presented. The amine, core aromatic, peripheral aromatic, and ether linker moieties of HTS hit 1 were modulated and the effect on potency at 5-HT(6) examined. Tolylpiperidine ether 9h was found to possess desirable pharmacokinetic (PK) properties, and was also shown to enhance cognition in the rat novel object recognition paradigm.

Bioanalytical assays in support of tanezumab developmental and reproductive toxicity studies: challenges and learnings.

Bioanalytical challenges were encountered during developmental and reproductive toxicity studies of tanezumab in cynomolgus monkeys. Possible changes in breast milk composition over the postpartum period potentially complicated assessment of tanezumab concentration in this matrix, requiring validation of the quantification assay across different time intervals. Immunogenicity assessment in maternal serum was complicated by apparent increases in the incidence of antidrug antibody-positive results in treatment-naive samples as pregnancy progressed that were due to changes in the concentration of nerve growth factor, tanezumab's target protein. This was overcome by employing gestational day-specific cut points throughout pregnancy. Researchers should recognize potential challenges associated with dynamic matrices/physiological conditions and anticipate that assays developed under normal conditions may require adaptation for specialized situations.

Application of CYP3A4 in vitro data to predict clinical drug-drug interactions; predictions of compounds as objects of interaction.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: Numerous retrospective analyses have shown the utility of in vitro systems for predicting potential drug-drug interactions (DDIs). Prediction of DDIs from in vitro data is commonly obtained using estimates of enzyme K(i), inhibitor and substrate concentrations and absorption rate for substrate and inhibitor. WHAT THIS STUDY ADDS: Using a generic approach for all test compounds, the findings from the current study showed the use of recombinant P450s provide a more robust in vitro measure of P450 contribution (fraction metabolized, f(m)) than that achieved when using chemical inhibitors in combination with human liver microsomes, for the prediction of potential CYP3A4 drug-drug interactions prior to clinical investigation. The current study supported the use of SIMCYP(R), a modelling and simulation software in utilizing the in vitro measures in the prediction of potential drug-drug interactions. AIMS: The aim of this study was to explore and optimize the in vitro and in silico approaches used for predicting clinical DDIs. A data set containing clinical information on the interaction of 20 Pfizer compounds with ketoconazole was used to assess the success of the techniques. METHODS: The study calculated the fraction and the rate of metabolism of 20 Pfizer compounds via each cytochrome P450. Two approaches were used to determine fraction metabolized (f(m)); 1) by measuring substrate loss in human liver microsomes (HLM) in the presence and absence of specific chemical inhibitors and 2) by measuring substrate loss in individual cDNA expressed P450s (also referred to as recombinant P450s (rhCYP)) The fractions metabolized via each CYP were used to predict the drug-drug interaction due to CYP3A4 inhibition by ketoconazole using the modelling and simulation software SIMCYP. RESULTS: When in vitro data were generated using Gentest supersomes, 85% of predictions were within two-fold of the observed clinical interaction. Using PanVera baculosomes, 70% of predictions were predicted within two-fold. In contrast using chemical inhibitors the accuracy was lower, predicting only 37% of compounds within two-fold of the clinical value. Poorly predicted compounds were found to either be metabolically stable and/or have high microsomal protein binding. The use of equilibrium dialysis to generate accurate protein binding measurements was especially important for highly bound drugs. CONCLUSIONS: The current study demonstrated that the use of rhCYPs with SIMCYP provides a robust in vitro system for predicting the likelihood and magnitude of changes in clinical exposure of compounds as a consequence of CYP3A4 inhibition by a concomitantly administered drug.

Impact of physiological, physicochemical and biopharmaceutical factors in absorption and metabolism mechanisms on the drug oral bioavailability of rats and humans.

The onset, intensity and duration of therapeutic response to a compound depend on the intrinsic pharmacological activity of the drug and pharmacokinetic factors related to its absorption, distribution, metabolism and elimination that are inherent to the biological system. The process of drug transfer from the site of administration to the systemic circulation and the interspecies factors that impact this process are the scope of this review. In general, the factors that influence oral drug bioavailability via absorption and metabolism can be divided into physicochemical/biopharmaceutical and physiological factors. Physicochemical and biopharmaceutical factors that influence permeability and solubility tend to be species independent. Although there are significant differences in the anatomy and physiology of the gastrointestinal tract, these are not associated with significant differences in the rate and extent of drug absorption between rats and humans. However, species differences in drug metabolism in rats and humans did result in significant species differences in bioavailability. Overall, this review provides a better understanding of the interplay between drug physicochemical/biopharmaceutical factors and species differences/similarities in the absorption and metabolism mechanisms that affect oral bioavailability in rats and humans. This will enable a more rational approach to perform projection of oral bioavailability in human using available rat in vivo data.

Innovative solutions: a plurality of vision--integrating the chaplain into the critical care unit.

Banner Good Samaritan Medical Center is a 650-bed quarternary care facility located in the Southwestern United States. It contains 12 intensive care units (ICUs) and experience a high patient acuity as a result of being a referral center for Arizona. The palliative care nurse practitioner and ICU clinical nurse specialist collaborated with the chaplain to entrance his visibility in the ICUs and to incorporate the philosophy of spiritual care assessments in the ICU.

From the Cover: Evaluation of the Effects of Tanezumab, a Monoclonal Antibody Against Nerve Growth Factor, on the Sympathetic Nervous System in Adult Cynomolgus Monkeys (Macaca fascicularis): A Stereologic, Histomorphologic, and Cardiofunctional Assessment.

Tanezumab, a humanized monoclonal antibody against nerve growth factor is in development for treatment of chronic pain. Three nonclinical studies assessed effects of clinically relevant and supratherapeutic doses of tanezumab on the sympathetic nervous system (SNS) of adult nonhuman primates. Study 1 evaluated potential effects of subcutaneous (SC) tanezumab (1.2 mg/kg every 8 weeks [Q8W]) on SNS in cynomolgus monkeys for 3 or 6 months and reversibility or persistence of any effects through a nondosing/recovery period. Study 2 evaluated whether neuronal cell death occurs shortly after a single SC tanezumab injection (1.2 mg/kg). Assessments for these two studies included evaluations of superior cervical and cervicothoracic ganglia for neuronal cell death and morphology. Study 3 evaluated effects of SC tanezumab (1.2 mg/kg Q8W and 30 mg/kg/week) over 6 months on sympathetic control of cardiovascular function. Tanezumab exposure was associated with stereologic changes in sympathetic ganglia, including smaller ganglion volume, and smaller average neuron size/area beginning at 2 weeks and reaching maximal levels by 1 month with no further progression through 6 months. These changes were not associated with clinical signs, completely reversed upon tanezumab withdrawal, and were not considered adverse. Tanezumab had no adverse effects on sympathetic control of cardiovascular function. These data support the conclusion that tanezumab administration for up to 6 months has no adverse effects on SNS morphology or function and does not cause neuronal cell death in adult nonhuman primates.

Innovative solutions: pharmacy and nursing collaboration on a high-risk medication project.

Banner Good Samaritan Medical Center is a 650-bed tertiary care facility in the Southwestern United States. The pharmacy and nursing departments collaborated on a multifocal project to address the educational and operational needs of the facility regarding the new JCAHO Medication Management Review Standards for high-risk and high-alert medications. The project encompassed policy creation, nursing education, identification and evaluation of HRHA medications, and facility-wide distribution of information related to high-risk and high-alert medications.

Emerging Agricultural Biotechnologies for Sustainable Agriculture and Food Security.

As global populations continue to increase, agricultural productivity will be challenged to keep pace without overtaxing important environmental resources. A dynamic and integrated approach will be required to solve global food insecurity and position agriculture on a trajectory toward sustainability. Genetically modified (GM) crops enhanced through modern biotechnology represent an important set of tools that can promote sustainable agriculture and improve food security. Several emerging biotechnology approaches were discussed in a recent symposium organized at the 13th IUPAC International Congress of Pesticide Chemistry meeting in San Francisco, CA, USA. This paper summarizes the innovative research and several of the new and emerging technologies within the field of agricultural biotechnology that were presented during the symposium. This discussion highlights how agricultural biotechnology fits within the context of sustainable agriculture and improved food security and can be used in support of further development and adoption of beneficial GM crops.

In vitro ADME phenotyping in drug discovery: current challenges and future solutions.

Drug-metabolizing enzymes and drug transporters are key regulators of drug disposition and pharmacodynamics, which are closely linked to drug efficacy and safety. In this article, current challenges and future solutions to predicting their influence on pharmacokinetics and inter-organ distribution in humans, from data generated during the drug discovery decision-making process, are presented. In vitro phenotyping strategies for drug metabolizing enzymes (eg, CYP3A4, UGT1A1) and transporters (eg, OATP1B1) are offered, including perspectives on a selection of in vitro systems, novel in vitro phenotyping reagents and remaining technology gaps, challenges in extrapolating in vitro data to the in vivo situation, in silico models for the prediction of whether compounds are enzyme or transporter substrates, and the impact of pharmacogenomics.