比较三种新型生物制剂治疗中重度活动性类风湿关节炎的疗效与安全性

目的：观察利妥昔单抗、托珠单抗及阿达木单抗联合慢作用抗风湿药物（DMARDs）治疗中重度活动性类风湿关节炎24周后的疗效和安全性。方法：共入组87例DAS28＞3.2的活动性类风湿关节炎患者,给予利妥昔单抗或托珠单抗或阿达木单抗同时联合DMARDs,或仅用DMARDs药物治疗,主要疗效指标为达到ACR20的患者比例,次要疗效指标为达到ACR50、ACR70的比例及DAS28-CRP的改善情况。结果：在24周时,利妥昔单抗、托珠单抗及阿达木单抗达到ACR20的比例分别为66.67%、94.44%和73.08%,而单用DMARDs组则为53.57%,同时各治疗组患者DAS28-CRP亦较基线及仅用DMARDs组显著下降,且托珠单抗及阿达木单抗组优于利妥昔单抗组。最常见的不良事件为上呼吸道感染,其次是淋巴细胞下降及肝功能异常。结论：利妥昔单抗、托珠单抗及阿达木单抗对类风湿关节炎均具有显著的疗效和良好的安全性,阿达木单抗及托珠单抗的疗效优于利妥昔单抗。     

Integration of efficacy, pharmacokinetic and safety assessment of interleukin-1 receptor antagonist in a preclinical model of arthritis

Pharmacokinetic properties and safety profile of a drug are likely influenced by the disease state of a patient. In this study, we investigated the influence of arthritic processes on pharmacokinetics and immunotoxicity of interleukin-1 receptor antagonist (Anakinra) in the rat adjuvant arthritis model. Anakinra dose-dependently suppressed joint inflammation and degradation as demonstrated by reduced clinical arthritis score, paw thickness, synovial infiltration and bone degradation. In addition, plasma levels of chemokines MCP-1 and GRO/KC were reduced. Pharmacokinetic behaviour of Anakinra was influenced by disease state of the rats as judged from a decrease in C(max) and an increase of the MRT as the disease progressed at a dose of 24 and 72 mg Anakinra/kg body weight. The pharmacokinetic parameters increased dose-dependently, but non-proportionally with increasing dose. Low level anti-Anakinra antibody formation was observed at prolonged exposure to the biologic. Safety parameters, including haematology, splenic lymphocyte subset analysis, ex vivo stimulation of spleen cells and histopathology of immune system organs were affected by the disease itself to such extent that no additional effects of Anakinra could be observed. In conclusion, we demonstrated that pharmacokinetic behaviour of Anakinra was influenced by the arthritis background of the rats resulting in decreased internal exposure.     

Importance of cynomolgus monkeys in development of monoclonal antibody drugs

Animal species used in the preclinical studies for development of monoclonal antibody (mAb) drugs are surveyed in this review. Relevant animal species for preclinical studies of mAb candidates are those express desired epitope of mAb candidates. Cynomolgus monkeys cross-react with mAb drugs much higher than other animal species commonly used in preclinical studies such as absorption, distribution, metabolism and excretion (ADME), efficacy, and toxicity studies, for development of new drugs. Moreover, plasma exposure of the mAb drugs in humans is predicted well from the exposure in the monkeys, and the placental transfer of immunoglobulin G (IgG, all the mAb drugs contain IgG) from mother to fetus is similar between humans and the monkeys from a viewpoint of time course and plasma level of IgG transferred. These observed findings indicate that the monkeys are the most suitable animal species used in the ADME and toxicity studies for development of new mAb drugs.     

Nonclinical safety assessment of Efinaconazole Solution (10%) for onychomycosis treatment

Efinaconazole is a triazole developed as a 10% solution for topical treatment of onychomycosis, a common fungal nail infection. Efinaconazole solution and topical formulation vehicle administered dermally to mice (13 weeks), rats (6 months) and minipigs (9 months) produced transient erythema, minimal to modest hyperkeratosis, and mild microscopic skin inflammation. The liver was the target organ of systemic toxicity; reversible, minimal to moderate vacuolated changes were noted in the rat dermal study at 15 and 50 mg/kg/day. No systemic toxicity was observed in mice and minipigs, at approximate high dermal doses of 930 and 170 mg/kg/day, respectively. Daily subcutaneous injection of propylene glycol vehicle or efinaconazole to rats for 6 months produced severe local inflammation and systemic spread, evidenced by peritoneal adhesions, spinal cord necrosis and urinary tract disease. Mortalities occurred in all groups but were increased at the high dose (30 or 40 mg/kg/day), suggesting that vehicle effects were exacerbated by efinaconazole. Efinaconazole was not carcinogenic in a 2-year mouse dermal study and was not genotoxic. Exposure-based safety margins at the NOAEL were 70–698 relative to onychomycosis patients. In conclusion, efinaconazole demonstrated low/moderate toxicity, consistent with other azole antifungals, and high safety margins for topical onychomycosis therapy.     

Developmental toxicity assessment of tanezumab,an anti-nerve growth factor monoclonal antibody,in cynomolgus monkeys (Macaca fascicularis)

Two intravenous studies with tanezumab, an anti-nerve growth factor monoclonal antibody, were conducted in pregnant cynomolgus monkeys to assess potential effects on pregnancy and pre- and postnatal development. Study 1 evaluated infants up to 12 months of age following weekly maternal dosing (0, 0.5, 4 or 30 mg/kg; 18 per group) from gestation day (GD) 20 through parturition. Study 2 evaluated infants 2 months postnatally following weekly maternal dosing (0, 0.5 or 30 mg/kg; 20–21 per group) from GD 20 through 48. In the absence of maternal toxicity, tanezumab increased stillbirth and post-birth infant mortality/morbidity, decreased infant growth and resulted in microscopic changes in the peripheral sympathetic and sensory nervous system of the infants at all doses. Decreased primary antibody responses and increased incidences in skin changes in infants were also observed. The no-observed-adverse-effect-level for maternal toxicity was 30 mg/kg and <0.5 mg/kg for developmental toxicity.     

Drug safety evaluation through biomarker analysis--a toxicity study in the cynomolgus monkey using an antibody-cytotoxic conjugate against ovarian cancer

Antibody-cytotoxin conjugates are complex novel therapeutic agents whose toxicological properties are not presently well understood. The objective of this study was to identify serum biomarkers that correlate with MLN8866 (an Antibody-Cytotoxic Conjugate, mAb8866-CT) pathological events in monkeys and to predict the maximal tolerated dose (MTD) level using biomarkers. Cynomolgus monkeys were administered a single dose MLN8666 (5, 15 or 30 mg/kg) by intravenous infusion and evaluated over a 7-day period. Exposure levels were determined by quantifying MLN8866 levels (Cmax and AUC(0-96 h)) in serum. The increase in MLN8866 Cmax and AUC(0-96 h) was approximately dose proportional. Two biomarkers in serum (m/z 316 and m/z 368) were identified to be correlated with MLN8866 toxicological outcomes. The predicted MTD, 11.4 mg/kg, was within the MTD range set by pathology results (5-15 mg/kg). Administration of MLN8866 at 15 mg/kg and 30 mg/kg dose levels resulted in changes in hematology parameters associated with impaired hematopoiesis and bone marrow toxicity. The projected MLN8866 MTD exposure level was integrated with toxicokinetic analysis and showed Cmax=236 microg/mL and AUC(0-96 h)=7246 h mg/mL. The safety of three different MLN8866 dosing regimens with three dosing schedules was explored with pharmacokinetic modeling.     

Assessment of ixekizumab,an interleukin-17A monoclonal antibody,for potential effects on reproduction and development,including immune system function,in cynomolgus monkeys

The reproductive and developmental toxicity of ixekizumab, a selective inhibitor of interleukin-17A (IL-17A), was assessed in the following studies in cynomolgus monkeys: fertility (3-month dosing), embryo-fetal development (EFD; dosing from gestation day (GD) 20 through 139), and pre-postnatal development (PPND; dosing from GD 20 through parturition). Because IL-17A has functional roles in innate and humoral immunity, immune system modulation was evaluated in the EFD and PPND studies; immunological evaluations in infants comprised peripheral blood immunophenotyping, Natural Killer cell cytolytic activity, and T-cell-dependent antibody (IgG and IgM) primary and secondary responses to antigen challenge. Ixekizumab exposure was sustained during the dosing periods in most adult monkeys. Fetal exposure at Cesarean section (GD 140–142; EFD study) was 18–25% of maternal exposure and ixekizumab was present in infants for up to 29 weeks postpartum. There were no adverse effects attributed to ixekizumab in any study. Importantly, immune system development and maturation were unaffected.     

Risk and safety assessment of exogenous human brain natriuretic peptide in cynomolgus monkeys (Macaca fascicularis)—A subchronic study

Safety evaluation of synthetic human brain natriuretic peptide (shBNP) was carried out in cynomolgus monkeys (Macaca fascicularis) by 2-week intravenous toxicity studies. System exposure was also assessed throughout the whole administration. Three test groups received doses of 432, 1440 and 4320 μg/kg/day of shBNP, with a high infusion rate of 36 mL/kg/hr for 30 min compared to the clinical protocol of continuous infusion over 24 h. Commercially available recombinant human brain natriuretic peptide (rhBNP) of 1440 μg/kg/day was used as positive control. The 2-week repeated intravenous doses of shBNP resulted in reversible increased serum LDH and CPK in monkeys receiving 1440 and 4320 μg/kg/day dose with no pertinent histopathological changes. Some changes related to the pharmacologic effects of BNP including hypotension was observed after administration. No treatment-related mortalities or renal dysfunction were found. Controversy about the safety issue of BNP as an exogenous hormone concerning ventricular remodeling and myocardial cell apoptosis, coupled with our results, were also discussed. The no-observed-adverse-effect level (NOAEL) was considered to be 432 μg/kg /day, which is about 20 times higher than the commonly used clinical dose. The results of the present study advocate the safety of shBNP in cynomolgus monkeys at levels used in the study.     

Combination of cassette-dosing and microsampling for reduced animal usage for antibody pharmacokinetics in cynomolgus monkeys,wild-type mice,and human FcRn transgenic mice

Pharmaceutical Research - The aim of this study was to develop a useful antibody PK evaluation tool using a combination of cassette-dosing and microsampling in mice and monkeys in order to reduce...     

RF、CRP和抗CCP抗体联合检测对类风湿关节炎的诊断意义

目的：通过分析类风湿关节炎的临床资料及进行类风湿因子（RF）、C反应蛋白（CRF）及抗环瓜氨酸肽抗体（抗CCP抗体）的检查,探讨RF、CRP及抗CCP抗体联合检测在诊断类风湿关节炎患者的临床应用价值。方法对本院的115例类风湿关节炎的患者及131例非类风湿性患者的血清,用酶联免疫吸附试验检测抗CCP抗体、免疫比浊法对RF、CRP进行定量检测,然后对RF、CRP及抗CCP抗体检查结果进行分析。结果类风湿关节炎组RF、CRP及抗CCP抗体检测的阳性率显著高于非类风湿关节炎组,抗CCP抗体对类风湿关节炎的敏感性为64.5%, CRP对类风湿关节炎的敏感性为69.9%, RF对类风湿关节炎的敏感性为60.5%,略低于抗CCP抗体和CRP;RF对类风湿关节炎的特异性为79.3%,抗CCP抗体的特异性最高94.0%, CRP较低为55%;抗CCP抗体和RF、CRP联合检测时对RA诊断的敏感性为73.5%,特异性为98.9%。经统计学检验, RF、CRP及抗CCP抗体联合检测的灵敏性与单独应用时差异无统计学意义;RF、CRP及抗CCP抗体联合检测特异性比抗CCP抗体、RF、CRP高,差异有统计学意义。结论临床单独将RF、CRP或抗CCP抗体的检查用于类风湿关节炎的诊断敏感性和特异性一般,为提高类风湿关节炎诊断的敏感性和特异性,应该将RF、CRP和抗CCP抗体检测进行联合应用。     

国内萘丁美酮治疗类风湿关节炎的疗效及安全性的Meta分析

目的:系统评价国内萘丁美酮治疗类风湿关节炎的临床疗效和安全性。方法:对有关萘丁美酮治疗类风湿关节炎的疗效和不良反应研究文献进行Meta分析。结果:共有9项研究符合纳入标准。萘丁美酮治疗类风湿关节炎疗效总体优于对照组[RR=1.11,95%C(I1.02,1.20),P=0.01],不良反应发生率低于对照组[RR=0.71,95%C(I0.41,1.22),P=0.007]。结论:现有研究显示,萘丁美酮治疗类风湿关节炎疗效优于对照组,且安全性较高。但由于本研究中纳入文献在质量及数量上的局限,上述结论尚需大样本、长期随访的高质量随机对照试验进一步证实。     

Elevated levels of soluble Endothelial protein C receptor in rheumatoid arthritis and block the therapeutic effect of protein C in collagen-induced arthritis

BackgroundEndothelial protein C receptor (EPCR) is a membranous protein that can be combined with a variety of ligands and plays important roles in anticoagulant and anti-inflammation. Recent reports have shown that surface EPCR expression on T cells is negatively associated with Th17 differentiation and is co-expressed with other immunosuppressive molecules, such as The programmed cell death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4). Hence, we hypothesized that EPCR may play a critical role in rheumatoid arthritis (RA) disease progression that is mediated by Th17 differentiation. In order to explore the role of EPCR on RA disease pathogenesis, we detected membranous EPCR (mEPCR) expression in CD4⁺ T cells and soluble EPCR (sEPCR) expression in the sera of RA patients.MethodsThe proportion of CD4⁺/EPCR⁺ T cells in the peripheral blood of RA patients was detected by flow cytometry, and the expression of sEPCR in the sera of RA patients was detected by enzyme-linked immunosorbent assay (ELISA). For in vitro experiments, protein C (PC) and EPCR recombinant proteins were used to block peripheral blood mononuclear cell (PBMC) activation and to detect Th17 differentiation. For in vivo experiments in DBA/1 mice with collagen-induced arthritis (CIA), we administered PC and EPCR recombinant proteins, monitored disease progression, and evaluated the role of EPCR in disease progression.ResultsThe proportion of CD4⁺/EPCR⁺ T cells in the peripheral blood of RA patients was lower than that of osteoarthritis (OA) patients, while the expression level of sEPCR in the sera of RA patients was concomitantly higher than that in OA patients. Subsequent analysis revealed that sEPCR expression was positively correlated with rheumatoid factors (RF) and other inflammatory indicators in RA patients. Further studies confirmed that sEPCR administration alleviated the progression of collagen-induced arthritis and partially blocked the therapeutic effect of PC in CIA mice.ConclusionSoluble EPCR is associated with RA disease progression and induces disease remission in CIA mice by inhibiting Th17 differentiation.     

Cetuximab: an IgG1 monoclonal antibody for the treatment of epidermal growth factor receptor-expressing tumours

The epidermal growth factor receptor (EGFR) plays an essential role in normal cell growth and differentiation and in the survival of healthy and cancerous cells. EGFR expression is a common feature of non-haematological malignancies and is associated with poor clinical prognosis. Cetuximab is an IgG₁ monoclonal antibody that blocks EGFR activation. It has efficacy alone, and in combination with irinotecan, in the treatment of metastatic colorectal cancer that has progressed on irinotecan-containing therapy. It has been approved for use in combination with irinotecan in both Switzerland and the US and as monotherapy in the US. Cetuximab also has efficacy in cancers of the head and neck and non-small cell lung cancer. Cetuximab is well-tolerated and does not exacerbate the side effects of co-administered cytotoxic chemotherapy.     

Repeated injection of KMRC011, a medical countermeasure for radiation,can cause adverse health effects in cynomolgus monkeys

High‐dose radiation‐induced tissue damage is a major limiting factor in the medical application of nuclear technology. Herein, we tested 28‐day repeated‐dose toxicity of KMRC011, an agonist of toll‐like receptor (TLR) 5, which is being developed as a medical countermeasure for radiation, using cynomolgus monkeys. KMRC011 (0.01, 0.02 or 0.04 mg/kg/day) was intramuscularly injected once daily for 4 weeks, and each two monkeys in both control and 0.04 mg/kg/day group were observed for an additional 2‐week recovery period. There were no dose‐related toxicological changes in mortality, clinical observations, body weight, food consumption, ophthalmological findings, electrocardiographs, coagulation, serum chemistry, organ weights, or urinalysis and urine chemistry. Although treatment‐related changes, such as increased white blood cells, increased absolute and relative neutrophils, decreased relative lymphocytes and inflammatory lesions, were noted in the maximum dose group, these findings were not observed after the 2‐week recovery period. Further, we considered that the kidneys and heart may be target organs of TLR5 agonists, as well as the spleen, and that autophagic signals can be triggered in tissue damage and the repair process. Importantly, accumulation of p62 protein, an indicator of autophagy, and a decrease of caveolin‐1 protein, a regulator of TLR5 protein half‐life, were found in both tissues from the highest dose group. Therefore, we conclude that the no‐observed‐adverse‐effect level for KMRC011 may be greater than 0.04 mg/kg/day in male and female monkeys. Additionally, we propose that further studies are needed to identify the molecular signals, which are related to KMRC011‐induced adverse effects.     

Highly specific,quantitative polymerase chain reaction probe for the quantification of human cells in cynomolgus monkeys

Quantification of human cells may be performed using quantitative polymerase chain reaction (qPCR). In preclinical studies, the human Alu sequence is widely used as biomarker for human DNA. However, because the Alu gene is shared by primates, its use is limited to non-primate studies. The biodistribution of human cells in primates is also necessary for translational studies. Therefore, we aimed to design a novel, human-specific primer/probe that enables the quantification of human cells in primates and other animal models. A novel primer/probe set was successfully designed based on highly repetitive LINE1 sequences. qPCR efficiency (94.95–99.21%) and linearity of calibration curves (r² = 0.996–0.999) were confirmed in tissue homogenates of cynomolgus monkey. The lower limit of detection was 10 cells per 15-mg tissue sample, a sensitivity that is equivalent to existing Alu primers/probes. The set was also effective in other animal models such as mice, rabbits, pigs, and common marmosets.To our knowledge, this is the first study describing the successful design of a human-specific qPCR primer/probe for human cell quantification in various animals, including non-human primates, using LINE1 sequence. The excellent selectivity, sensitivity, and versatility of the LINE1 primers/probes make it a promising quantification tool in preclinical biodistribution studies.     

Involvement of the peripheral benzodiazepine receptor in the development of rheumatoid arthritis in Mrl/lpr mice

In this study, the effects of different peripheral benzodiazapine receptor ligands: PK 11195 [1-(2-chloro-phenyl)-N-methyl-N-(1-methylpropyl)-1-isoquinoline carboxamide], Ro5-4864 [7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one] and the newly described SSR 180575 (7-chloro-N,N,5-trimethyl-4-oxo-3-phenyl-3,5-dihydro-4H-pyridozine[4,5-b] indole-1-acetamide) were analysed on the progression and severity of rheumatoid arthritis in vivo in the Mrl/lpr mice model, following chronic treatment (at 3 mg/kg, i.p. for 30 days). We found that peripheral benzodiazepine receptor ligands have significant beneficial therapeutic action on the development of spontaneous rheumatoid arthritis-like signs. Concomitantly, we mapped immunoreactive peripheral benzodiazepine receptor in inflamed tissues, and we observed that in addition to the infiltrated leukocytes, peripheral benzodiazepine receptor was expressed in synovial membranes, at the cartilage pannus junction and in chondrocytes. Interestingly, we observed that peripheral benzodiazepine receptor expression in chondrocytes was reduced when Mrl/lpr mice developed the pathology and restored upon peripheral benzodiazepine receptor ligand treatment. Altogether, our data provide further evidence of a role played by peripheral benzodiazepine receptor in the regulation of inflammation processes and support new therapeutic applications for specific potent peripheral benzodiazepine receptor ligands.     

Development of a human antibody fragment directed against the alpha folate receptor as a promising molecule for targeted application

Alpha folate receptor (FRα) is currently under investigation as a target for the treatment of patients with non-small-cell lung cancer (NSCLC), since it is highly expressed in tumor cells but is largely absent in normal tissue. In this study, a novel human variable domain of a heavy-chain (VH) antibody fragment specific to FRα was enriched and selected by phage bio-planning. The positive phage clone (3A102 VH) specifically bound to FRα and also cross-reacted with FRβ, as tested by ELISA. Clone 3A102 VH was then successfully expressed as a soluble protein in an E. coli shuffle strain. The obtained soluble 3A102 VH demonstrated a high affinity for FRα with affinity constants (K_aff) values around 7.77 ± 0.25 × 10⁷ M⁻¹, with specific binding against both FRα expressing NSCLC cells and NSCLC patient-derived primary cancer cells, as tested by cell ELISA. In addition, soluble 3A102 VH showed the potential desired property of a targeting molecule by being internalized into FRα-expressing cells, as observed by confocal microscopy. This study inspires the use of phage display to develop human VH antibody (Ab) fragments that might be well suited for drug targeted therapy of NSCLC and other FRα-positive cancer cells.     

MORAb-003, a fully humanized monoclonal antibody against the folate receptor alpha, for the potential treatment of epithelial ovarian cancer

Morphotek Inc is developing MORAb-003, a humanized monoclonal antibody directed against folate receptor alpha, for the potential treatment of ovarian cancer. Phase II clinical trialsof MORAb-003, either alone or in combination with platinum/taxane chemotherapy, are underway in ovarian cancer patients experiencing their first disease recurrence.     

Infant toxicology: State of the science and considerations in evaluation of safety

Differences in the physiology and biological susceptibilities of adults and infants have led to growing interest in safety evaluation methods for exposures from infant formula packaging. In addition to potential physiological differences, infants aged 0–6 months may consume a sole source of food, infant formula or breast milk, and consume higher amounts of food relative to body weight compared to adults. While the duration of the exposure is short compared to the expected lifespan of the individual, it occurs during a period of important developmental processes. The purpose of this document is to (1) review key biological and exposure elements that may impact the evaluation of safety for food contact products intended for use by infants, (2) summarize the current reproductive and developmental toxicity testing protocols available, and (3) identify potential data gaps concerning this period of development.