Peficitinib, a JAK Inhibitor, in the Treatment of Moderate-to-Severe Rheumatoid Arthritis in Patients With an Inadequate Response to Methotrexate.

OBJECTIVE: To evaluate the efficacy and safety of orally administered once-daily peficitinib in combination with methotrexate (MTX) in patients with moderate-to-severe rheumatoid arthritis (RA) who had an inadequate response to MTX. METHODS: In this multinational, phase IIb, randomized, double-blind, placebo-controlled, dose-ranging trial, patients with RA (n = 378) were treated with peficitinib 25 mg, 50 mg, 100 mg, or 150 mg plus MTX, or matching placebo plus MTX once daily for 12 weeks. The primary end point was the percentage of patients who met the American College of Rheumatology 20% improvement criteria (achieved an ACR20 response) at week 12. RESULTS: ACR20 response rates at week 12 were 43.9%, 61.5% (P < 0.05 versus placebo), 46.4%, 57.7%, and 44.4% in the peficitinib 25 mg, 50 mg, 100 mg, 150 mg, and placebo groups, respectively. Significant decreases from baseline in the Disease Activity Score in 28 joints using the C-reactive protein level were seen in the peficitinib 50 mg (P < 0.05) and 150 mg (P < 0.01) groups compared with placebo at week 12. Overall, the incidence of adverse events (AEs) was similar between peficitinib and placebo. The most common AEs were urinary tract infection (n = 22 [6%]), upper respiratory tract infection (n = 16 [4%]), and diarrhea (n = 16 [4%]). There were 3 cases of herpes zoster infection (2 in the peficitinib 100 mg group and 1 in the 150 mg group) and 2 cases of serious infection (viral infection in the peficitinib 100 mg group and erysipelas in the 150 mg group). CONCLUSION: The ACR20 response rate in the group receiving peficitinib 50 mg plus MTX was significantly different compared with the rate in patients receiving placebo, but there were no apparent dose-dependent responses, and the placebo response rate was high. Peficitinib plus MTX in patients with moderate-to-severe RA was well tolerated, with limited safety signals emerging.

Removal of viruses from human intravenous immune globulin by 35 nm nanofiltration.

Viral safety is an important prerequisite for clinical immunoglobulin preparations. A common manufacturing practice is to utilize several virus removal/inactivation process steps to ensure the safety of human intravenous immunoglobulin (IVIg). In this regard, we examined the use of Planova 35 nm filters to reduce potential loads of both non-enveloped and enveloped viruses prior to end-stage solvent detergent treatment. The nanofiltration process was validated for removal of a variety of enveloped and non-enveloped viruses ranging in size from 70 nm to 18 nm including: Sindbis virus, Simian Virus 40 (SV40), Bovine Viral Diarrhoea virus (BVDV), Feline Calicivirus, Encephalomyocarditis virus (EMC), Hepatitis A virus (HAV), Bovine Parvovirus (BPV) and Porcine Parvovirus (PPV). The filtration procedure was carried out by first spiking a 7% solution of IVIg with < 10(8) virus. The spiked IVIg solution was then filtered through a 75 nm Planova filter followed by two Planova 35 nm filters in series (75/35/35). The 75 nm prefilter is incorporated into this process to increase the capacity of the 35 nm viral removal filters. As a result of the inclusion of the 75 nm pre-filtration step it was possible to assess the removal of virus by the 35 nm filters independent of possible aggregation of the initial viral spiking material. Samples were collected at each step and immediately titred by viral plaque assay. A process control sample of the spiked load solution was held at the same conditions for the duration of the filtration process and then titred to determine the extent to which antibody neutralization may have contributed to overall viral reduction. Control assays of spiked IVIg were performed to establish the degree of toxicity of the IVIg solution to the indicator cell lines and the extent to which the IVIg interfered with plaque formation in the assay system. This combined data was used to establish assay sensitivity for the calculation of log removal by the filtration process. It was noted that toxicity/interference effects could have a significant effect upon apparent log reductions, and these effects could vary greatly, even within viruses of the same family. The results of these studies indicate that 35 nm filtration is very effective for removing substantial quantities of both non-enveloped and enveloped viruses from IVIg. Complete clearance (to the limits of detection of the assay) was obtained for all viruses larger than 35 nm. Interestingly, viruses reported to have mean diameters of less than 35 nm (EMC and HAV) were at least partially removed by the filtration (4.3 and > 4.7 logs removal, respectively). Even small viruses such as PPV were to some extent removed from the IVIg solution by the filters (2.6 logs removal). Reduction of BPV would not be assessed due to extensive neutralization and interference with plaque formation by the IVIg. Sindbis and SV40 also were subject to neutralization and assay interference due to the IVIg, though to a lesser extent. We conclude from these studies that the 35 nm mean pore size is functionally efficient in removal of smaller size viruses from spiked IVIg concentrates.

Genotoxicity testing of an aqueous mistletoe extract in vitro.

An aqueous mistletoe extract (AME, the active principle of Lektinol) was investigated for its genotoxic potential in a wide range of in vitro studies, according to the actual international standard guidelines. Gene mutation tests on bacteria and mammalian cells in vitro all gave negative results. From the findings of cytogenetic studies, there was no indication of a clastogenic activity in human lymphocytes. A cell transformation assay revealed no malignant changes of Syrian hamster embryo cell cultures. In summary, the results suggest that AME has no genotoxic potential in vitro.

The Fanconi anemia complementation group C gene (FAC) suppresses transformation of mutant fibroblasts by the SV40 virus.

Fanconi anemia (FA) is a heterogeneous genetic syndrome manifested by bone marrow failure and consisting of at least five complementation groups (A, B, C, D, E). Mutations in a gene termed FAC are responsible for the C complementation group, but the function of the FAC protein remains obscure. FA patients are also highly cancer-prone; the molecular basis for this susceptibility is unclear but has led to the hypothesis that the wild-type FA gene may act as a tumor suppressor. In vitro, mutant FA primary fibroblasts are 3- to 50-fold more sensitive than normal fibroblasts to transformation in culture by the SV40 virus. We confirmed this marked susceptibility to transformation of a FAC-mutant primary fibroblast cell line, GM449. We then introduced a copy of the wild-type FAC cDNA into GM449 cells using a recombinant adeno-associated virus (rAAV) vector. We found that GM449 cells transduced with a copy of the normal FAC cDNA by a FAC-rAAV vector were at least 10-fold less prone to form transformed foci. Diminished transformation potential of transduced cells was a specific effect of the FAC cDNA since GM449 cells transduced with a rAAV vector not containing FAC retained marked susceptibility to SV40 transformation.

A comparison of methods for the estimation of retroviral burden.

The presence of retroviral contamination is of vital concern in the manufacture of cell culture-derived biopharmaceuticals. These cell lines usually have A- or C-type retrovirus-like particles which are visible by transmission electron microscopy (TEM) even when infectivity (IF) or reverse transcriptase activity (RTA) cannot be demonstrated. The supernatant of the post-production cell cultures, therefore, also needs to be evaluated by TEM for viral burden. A major question, however, is how to establish a quantitative viral load estimate for the evaluation of a purification process. The FDA recommends that a purification process for viral contaminants remove or inactivate 3-5 logs over the estimated viral burden. Viral particles are difficult to identify and quantify, however, by conventional negative staining. We present a comparison of infectivity assay, reverse transcriptase assay, negative staining, and thin sectioned TEM. These assays were performed on four samples. Ultracentrifuged sediments of cleared cell-culture media were measured, fixed and processed. Thin sections were evaluated by TEM and the number of viral particles estimated by morphometric derived quantification. Retrovirus particles were easily identified and quantified when examined by TEM as compared to negative staining and correlated with the other viral assays (IF, RTA). These results demonstrate that the TEM thin section method was a superior technique to negative staining for estimating viral particle load in cell-culture supernatant. To validate further the plastic embedding with thin sectioning, we evaluated cell culture supernatants (pellets) for retroviral burden at various dilutions, from two cell lines. Morphometric determinations were made of the number of viral particles present per unit volume and compared to results obtained by infectivity assay. Since the morphometric calculation for viral density assumes even distribution of viral particles, we also evaluated and calculated viral counts on multiple thin sections taken throughout selected pellets.

Virus removal studies using nanofiltration membranes.

Nanofiltration of immunoglobulin products was investigated as a virus removal step using Planova-controlled pore size membranes. For these studies, purified preparations of an IgG and an IgM were tested with 15 nm and 35 nm Planovace nanofiltration membranes, respectively. The results of spiking studies with four model viruses indicated that both the Planova 15 and 35 membranes removed 6-7 log10 plaque or focus forming units of murine xenotropic retrovirus, simian virus 40 and pseudorabies virus. Although two tandem Planova 35 membranes were tested for clearance of poliovirus, only the Planova 15 membrane removed this virus. Nanofiltration experiments were carried out with protein concentrations up to 12 mg/ml for the IgG and 1-2 mg/ml for the IgM, and protein recovery was excellent.

Tenidap in rheumatoid arthritis. A 24-week double-blind comparison with hydroxychloroquine-plus-piroxicam, and piroxicam alone.

OBJECTIVE: To compare the clinical efficacy, effect on serum C-reactive protein (CRP), serum amyloid A (SAA), and plasma interleukin-6 (IL-6) levels, and safety of tenidap with a combination of hydroxychloroquine-plus-piroxicam, and piroxicam alone, in the treatment of rheumatoid arthritis (RA) patients. METHODS: A double-blind, randomized, multicenter study in which patients with active RA were treated with tenidap 120 mg/day, hydroxychloroquine 400 mg/day and piroxicam 20 mg/day, or piroxicam alone 20 mg/day, for 24 weeks. RESULTS: At weeks 12 and 24, tenidap produced greater improvements than piroxicam based on 5 primary efficacy parameters; this improvement showed statistical significance in 4 of the 5 measures at week 12, and in 3 of the 5 measures at week 24. Clinical improvements in the hydroxychloroquine-plus-piroxicam-treated with tenidap. Compared with piroxicam, tenidap was associated with significantly greater reductions in serum CRP concentrations at 4, 12, and 24 weeks, and significantly greater reductions in SAA concentrations at weeks 12 and 24. The decrease in SAA concentrations was also significantly greater at weeks 4 and 24 in the tenidap-treated group than in the hydroxychloroquine-plus-piroxicam-treated group. Significant reductions in plasma IL-6 levels were observed at weeks 4, 12, and 24 within the tenidap group, and at week 24 within the hydroxychloroquine-plus-piroxicam-treated group. The overall occurrence of side effects, including gastrointestinal side effects, was similar in all 3 treatment groups. A small proportion of tenidap-treated patients (6.4%) manifested mild, nonprogressive, reversible proteinuria of presumed renal proximal tubular origin, and 3-4% of patients had elevated transaminase levels. CONCLUSION: In the treatment of patients with RA, tenidap is as effective as the combination of hydroxychloroquine-plus-piroxicam, and is more effective than piroxicam alone; moreover, tenidap's safety profile is comparable to that observed with piroxicam alone, and with hydroxychloroquine-plus-piroxicam. The clinical response observed in this study, as well as the prompt decreases in acute-phase protein levels of CRP and SAA, and in plasma IL-6 levels, suggest that tenidap represents a new type of antiarthritic medication, with properties similar to, but not identical to, a therapeutic combination of a nonsteroidal antiinflammatory drug with disease-modifying antirheumatic drugs.

Industrial experience with the detection of retroviruses.

An important aspect of the virological assessment of cell substrates used to produce biologicals is their retroviral status. In this presentation we will attempt to document some of the practical experiences of industry when testing cell substrates for retroviruses using infectivity tests, reverse transcriptase and electron microscopy. We will also explore some important aspects of the experimental design of these retrovirus assay systems and review some results from the application of these methods in model, experimental systems.

Toxicity of Echinacea purpurea. Acute, subacute and genotoxicity studies.

Single oral or intravenous doses of the expressed juice of Echinacea purpurea (EP) proved virtually non-toxic to rats and mice. After 4 weeks of oral administration in doses amounting to many times the human therapeutic dose laboratory tests and necropsy findings gave no evidence of any toxic effects in rats. Tests for mutagenicity carried out in microorganisms and mammalian cells in vitro and in mice all gave negative results. In an in vitro carcinogenicity study EP did not produce malignant transformation in hamster embryo cells.

Methods for the detection of adventitious viruses in cell cultures used in the production of biotechnology products.

The possibility for viral contamination exists in established cultures as well as in primary cultures. The use of established genetically engineered cultures in the production of biologicals for human use requires that these cultures be monitored for adventitious viral agents. Among the methods used for this purpose are animal inoculation and in vitro assays which provide a broad-spectrum screen for viral agents.

Intra-articular orgotein in osteoarthritis of the knee: a placebo-controlled efficacy, safety, and dosage comparison.

PURPOSE: Superoxide dismutase (orgotein for injection) has been used in managing osteoarthritis for more than seven years in Europe; however, well-controlled studies to establish an optimum dosage regimen have not been conducted. In this study, three orgotein dose/regimens were compared with placebo in terms of efficacy, safety, and duration of effect in patients with active osteoarthritis of the knee. PATIENTS AND METHODS: A total of 139 patients with osteoarthritis of the knee were enrolled in the study. Nonsteroidal anti-inflammatory agents were withdrawn to induce a flare of disease activity. Patients were then randomly assigned to receive one intra-articular injection of either placebo or orgotein (8 mg to 32 mg) each week for three weeks. Both investigators and patients evaluated disease activity and adverse experiences at a series of follow-up visits for three months. RESULTS: Orgotein was effective in reducing symptoms of osteoarthritis for up to three months after treatment; 16 mg given twice was the most effective and most best-tolerated regimen. Discomfort at the injection site was drug related, although this effect also occurred occasionally after injection of placebo. CONCLUSION: The long-lasting effects of intra-articular superoxide dismutase contribute to a favorable risk-benefit ratio and support the importance of the free-radical anion, superoxide (O2-), in the biochemical pathology of osteoarthritis.

An interlaboratory comparison of enhanced morphological transformation of Syrian hamster embryo cells cultured under conditions of reduced bicarbonate concentration and pH.

Initial studies performed in our laboratory indicated that early passage Syrian hamster embryo (SHE) cells exhibit optimal clonal proliferation when cultured in medium with a sodium bicarbonate concentration of 8.9 mM and pH of 6.70 instead of 44 mM and pH 7.35 as used previously by others. Subsequent studies indicated that morphological transformation frequency induced by benzo[a]pyrene (BP) was also enhanced at pH 6.70 compared to 7.35 and the level of enhancement was affected by cell density and duration of culture. With optimal conditions identified, the carcinogens BP, 3-methylcholanthrene, N-methyl-N'-nitro-N-nitrosoguanidine, 2-acetylaminofluorene and the non-carcinogen anthracene were tested at pH 6.70 and 7.35 in our laboratory and at Microbiological Assoc. Inc. under code. Additionally, the non-carcinogens 4-acetylaminofluorene, and caprolactam were tested in our laboratory. Results from these studies indicate that all carcinogens tested caused a significant increase in morphological transformation frequency compared to controls at pH 6.70. In contrast, only BP caused a significant increase in the morphological transformation frequency at pH 7.35. The non-carcinogens did not significantly increase the morphological transformation frequency compared to controls. Interlaboratory comparisons were in qualitative agreement despite the fact that different lots of serum and hamster cell isolates were used by the two laboratories. However, different dose-response curves for the various chemicals were observed between the two labs. It was also demonstrated that the enhanced morphological transformation frequency is not due to a decrease in culture medium osmolality or Na concentration, a condition which accompanies media with a reduced bicarbonate concentration and pH. These results demonstrate that the chemicals tested, low pH transformation of SHE cells agrees with carcinogenic potential and that assay variability is minimized. The implications of these results regarding use of the SHE cell assay as a short-term test for predicting the carcinogenic potential of chemicals are discussed.

Ketoprofen versus indomethacin in patients with rheumatoid arthritis: a multicenter double blind comparative study.

Oral ketoprofen (200-300 mg/day) and indomethacin (100-150 mg/day) were compared in a 12-week double blind study involving 140 patients with rheumatoid arthritis. The treatments were generally equally effective in most assessments, producing highly significant (p less than 0.01) improvements from baseline values within one week. Only isolated statistically significant differences (p less than 0.05) were detected between the 2 treatments: ketoprofen had a more pronounced effect than indomethacin in functional class (Weeks 1 and 12), swollen joint score (Week 1), and patients' global assessments (Week 12); indomethacin was significantly superior in improving grip strength at Week 4. The clinical significance of these statistically established differences may be questioned. The incidence of side effects, primarily gastrointestinal and neurologic, was also comparable in the 2 treatments.

Nabumetone versus naproxen in the treatment of osteoarthritis. A six-month trial.

The efficacy and safety of nabumetone (1,000 mg at bedtime) and naproxen (250 mg twice daily) were compared in a six-month, randomized, double-blind study of patients with osteoarthritis. Forty patients were entered in the study and completed a washout phase, and 37 were evaluable for efficacy. Of these, 18 patients received nabumetone and 19 received naproxen. Both treatment groups had significant improvement from baseline in all of the efficacy variables measured. All 40 patients were evaluated for safety. Fifty percent of the patients treated with nabumetone and 65 percent of the patients treated with naproxen had at least one drug-related or unknown-related adverse experience. Fifteen percent of patients in both groups had treatment-related adverse experiences that were moderate or severe. There were no statistically significant differences in the two treatment groups with regard to efficacy or adverse experiences.

Six-month multi-center study comparing nabumetone with naproxen in the treatment of osteoarthritis.

This six-month, double-blind, controlled, randomized, parallel study at 13 medical centers compared the safety and efficacy of nabumetone (1,000 mg taken at bedtime) with that of naproxen (250 mg twice daily) in the treatment of osteoarthritis in symptomatic adult outpatients. Five efficacy parameters were measured: patients' assessment of overall osteoarthritis activity and pain, physicians' assessment of overall osteoarthritis activity and pain, and physicians' assessment of pain with respect to a declined activity. All 489 patients who took medication were included in the evaluation of safety, and 455 patients (227 in the nabumetone group and 228 in the naproxen group) were evaluated for efficacy. Significant improvement in all five efficacy parameters occurred in both groups. No significant differences were found between the two groups at the end of the study in any of the five efficacy parameters. Twenty-three percent of nabumetone and 17 percent of naproxen patients withdrew from the study for lack of efficacy. At least one possible or probable treatment-related adverse experience was reported for 45 percent of nabumetone-treated patients and 42 percent of those given naproxen, and in 19 percent of the nabumetone-treated and 18 percent of the naproxen-treated patients these experiences were moderate or severe. However, only 7 percent of patients in each group withdrew from the study due to adverse experiences. Nabumetone and naproxen have comparable safety and efficacy, suggesting that a single, nighttime dose of nabumetone is a convenient, effective, and safe treatment for osteoarthritis.

A method for accelerating progression to a malignant phenotype in rodent cells.

This report describes a system of suspension culturing that enhances the expression of transformed cells in carcinogen-treated rodent cells and decreases the time required to observe clear evidence of the neoplastic or malignant phenotype by 2-8 wk or more. Retrovirus-infected Fischer rat embryo cells, uninfected Balb/c 3T3 mouse cells and Syrian hamster embryo cells in monolayer culture were treated with the chemical carcinogens, dimethylbenzanthracene, benzo[a]pyrene or N-methyl-N'-nitro-N-nitrosoguanidine, following a protocol appropriate to each cell type. The cultures were divided into two groups, one seeded directly onto a plastic surface, and the other suspended in liquid medium over agar before seeding onto a plastic surface. In all three cell types incluson of the suspension phase accelerated chemically induced transformation as indicated by clonogenicity in soft agarose (rat, mouse and hamster cells) and by morphological transformation and formation of tumours in athymic nude mice (hamster cells).

A selection procedure for the rapid expression of carcinogen-induced malignant transformation of retrovirus-infected Fischer rat embryo cells.

This study evaluates a procedure that accelerates the expression of the transformation of retrovirus-infected Fischer rat embryo cells. The endpoints used were anchorage-independent growth (formation of colonies in soft agarose) and formation of foci on a contact-inhibited monolayer. The cells were treated in monolayer with chemical, solvent (negative control) or medium alone for 3 days; then the chemical was removed and the cultures re-fed with medium alone for an additional 3 days. Cells in monolayer were disaggregated, suspended in liquid medium over solid agar for 4 days, disaggregated again and seeded into monolayer. After 2-4 wk without additional subculturing, cells from monolayer were seeded in soft agarose. Suspension of retrovirus-infected rat embryo cells above agar after chemical treatment resulted in rapid expression of neoplastic phenotypes. Cells treated in monolayer with the carcinogens, benzidine dichloride, dimethyl benzanthracene, 4,4-oxydianiline, 4-nitroquinoline-N-oxide and N-methyl-N'-nitro-N-nitrosoguanidine demonstrated colony formation in soft agarose or morphological transformation within 2-4 wk after being held in suspension for 4 days. In addition two carcinogens, benzo[a]pyrene and N-2-acetylaminofluorene and two noncarcinogens, benzo[e]pyrene and N-4-acetylaminofluorene did not induce neoplastic changes in this time period. The suspension technique may be a useful modification of this assay because it selectively amplifies neoplastic transformation after treatment with a number of chemicals.

Effect of donor age on the levels of activity of rat, hamster and human liver S9 preparations in the Salmonella mutagenicity assay.

Liver S9 fractions were prepared from male and female Syrian Golden hamsters and Sprague-Dawley rats, 1, 3, 6 and 12 months of age, which were either uninduced (corn-oil treated) or induced with Aroclor 1254 suspended in corn oil. These preparations were compared at varying protein levels for their ability to metabolize polycyclic aromatic hydrocarbons (benzo(a)pyrene, 3-methylcholanthrene, 7,12-dimethylbenzanthracene), aromatic amines (N-2-acetyl-aminofluorene, beta-naphthylamine, benzidine), and nitroso compounds (N-nitroso-diethylamine, nitrosopyrrolidine, nitrosodiethylmethylurea) to products mutagenic to Salmonella typhimurium. With 3-methylcholanthrene or benzo(a)pyrene in the presence of S9 preparations from Aroclor-treated male rats, the numbers of revertant colonies decreased with increasing age of the animals. Mutagenicity of aromatic amines was not affected by the age of the donor animals from which the S9 was prepared. The use of liver S9 from 1-month-old hamsters produced the highest number of revertant colonies with nitrosodiethylamine. This number decreased with preparations from animals of increasing age. The greatest number of revertant colonies with nitrosopyrrolidine occurred with preparations from male hamsters. A decrease in numbers of revertant colonies with increasing age was observed with the S9 preparation from Arcolor-treated male rats. Nitroso-diethylmethylurea was mutagenic only in the presence of S9 from male or female Aroclor-treated hamsters and the metabolic activity of the S9 preparations did not change with age. S9 preparations from livers of 50-70-year-old humans were compared for their ability to produce mutagenic metabolites at a number of protein levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased carcinogen metabolism and survival of retrovirus-infected Fischer rat embryo cells following repetitive carcinogen treatment.

The carcinogens N-2-acetylaminofluorene (AAF) and diethylnitrosamine (DEN) often give negative results when tested in the Fischer rat embryo cell survival assay with the standard single 72-hour regimen, whereas another carcinogen, benzo(a)pyrene [B(a)P], may yield varied results between different laboratories and may require relatively high concentrations (compared with other polycyclic aromatic hydrocarbons) for a positive result to occur. Enhanced survivals (compared with controls) were 56% or less with these carcinogens. In place of the standard single 72-hour treatment with test chemical, the cells were exposed to three consecutive 24-hour treatments. The amount of B(a)P metabolized during the last of the three 24-hour treatment periods was 3.2 times greater than that during the first 24-hour period, indicating that an induction effect occurred. Furthermore, the total amount of metabolites of B(a)P formed with repetitive treatments was 2.1 times greater than with a single 72-hour treatment. The total amount of AAF metabolites formed with repeated treatments was 1.6 times greater than with the single treatment regimen, although no induction effect was observed between treatment periods. Survival enhancement with the repetitive regimen increased to 181% with B(a)P, 172% with AAF, and 188% with DEN. With benzo(e)pyrene, anthracene, and pyrene, enhanced survival was 14% or less following the single treatment regimen and did not increase following repetitive treatments. When the carcinogen cinnamyl anthranilate was tested using repetitive treatments, survival enhancement was more than 100% at three of six doses, versus less than 0% when the standard single treatment regimen was used.