A comparison of the pharmacokinetics and safety of enzalutamide in subjects with hepatic impairment and matched healthy subjects.

WHAT IS KNOWN AND OBJECTIVE: Enzalutamide is an androgen receptor inhibitor approved for treatment of metastatic castration-resistant prostate cancer. Enzalutamide is highly protein bound and eliminated primarily by hepatic metabolism; therefore, it is important to understand whether enzalutamide pharmacokinetics is altered by hepatic impairment. METHODS: Pharmacokinetic data were obtained from two non-randomized, open-label, single-dose, phase 1 studies conducted in patients with mild (Child-Pugh class A, n = 6) or moderate (Child-Pugh class B, n = 8) hepatic impairment (NCT01901133) or severe (Child-Pugh class C, n = 8) hepatic impairment (NCT02138162) and their corresponding matched healthy controls; data from both studies are presented here. Subjects with hepatic impairment had liver cirrhosis (n = 19) or chronic hepatitis (n = 3). All subjects received a single oral dose of 160 mg enzalutamide under fasting conditions, with blood samples collected predose and up to 49 days post-dose. RESULTS AND DISCUSSION: Exposure to enzalutamide active moieties, based on the area under the curve of the sum of enzalutamide and N-desmethyl enzalutamide (an active metabolite with similar potency to enzalutamide), increased by 13%, 18% and 4% in subjects with mild, moderate and severe hepatic impairment, respectively, relative to matched controls. Compared with healthy controls, the mean maximum plasma concentration for enzalutamide active moieties was 24% higher in subjects with mild hepatic impairment and 11% and 41% lower in subjects with moderate and severe hepatic impairment, respectively. Enzalutamide was generally well tolerated, with no clinically significant trends in abnormal laboratory findings, vital signs or electrocardiograms. WHAT IS NEW AND CONCLUSIONS: No major differences in single-dose pharmacokinetics were observed in subjects with hepatic impairment vs. matched healthy controls. Therefore, these studies indicate that no initial dose adjustment is necessary when administering enzalutamide to patients with hepatic impairment.

Treatment of mildly to moderately active ulcerative colitis with a tryptase inhibitor (APC 2059): an open-label pilot study.

BACKGROUND: Mast cells isolated from the colonic mucosa in active ulcerative colitis appear to be partially degranulated, suggesting the release of tryptase. AIM: To investigate the safety and activity of APC 2059, a highly specific tryptase inhibitor, in the treatment of ulcerative colitis. METHODS: This was an open-label, Phase 2, multicentre pilot study in patients with mildly to moderately active ulcerative colitis, with a disease activity index of 6-9 on a 12-point scale. Fifty-six adults received 20 mg APC 2059 subcutaneously twice daily and 53 completed 28 days of treatment. The primary end-point was response, defined as a final disease activity index of < or = 3. Supplementary analyses were also performed. RESULTS: Sixteen (29%) of 56 patients responded. Five (9%) showed complete remission (disease activity index=0). Twenty-seven (49%) improved, with a final disease activity index of < or = 3 or a four-point reduction. Improvement or normalization in each category of the disease activity index was as follows: stool frequency, 64%; bleeding, 64%; endoscopy, 50%; physicians' rating, 63%. There were no significant relationships between outcome and pharmacokinetics. The most common adverse events were related to the injection site (32.1%). CONCLUSIONS: In this pilot study, the tryptase inhibitor APC 2059 was safe and there was evidence of activity in the treatment of ulcerative colitis.

Intraocular pharmacokinetics and safety of a humanized monoclonal antibody in rabbits after intravitreal administration of a solution or a PLGA microsphere formulation.

Poly(lactic-co-glycolic) acid (PLGA) bioresorbable microspheres are used for controlled-release drug delivery and are particularly promising for ocular indications. The objective of the current study was to evaluate the pharmacokinetics and safety of a recombinant human monoclonal antibody (rhuMAb HER2) in rabbits after bolus intravitreal administration of a solution or a PLGA-microsphere formulation. On Day 0, forty-eight male New Zealand white rabbits (2.3-2.6 kg) were immobilized with intramuscular ketamine/xylazine, and the test materials were injected directly into the vitreous compartment. Group 1 animals received rhuMAb HER2 in 50:50 lactide: glycolide PLGA microspheres; Group 2 animals received rhuMAb HER2 in solution (n = 24/group). The dose for each eye was 25 microg (50 microl). After dosing, animals were sacrificed at 2 min, and on 1, 2, 4, 7, 14, 23, 29, 37, 44, 50, and 56 days (n = 2/timepoint/group). Safety assessment included direct ophthalmoscopy, clinical observations, body weight, and hematology and clinical chemistry panels. At necropsy, vitreous and plasma were collected for pharmacokinetics and analysis for antibodies to rhuMAb HER2, and the vitreal pellet (Group 1) was prepared for histologic evaluation. All animals completed the study per protocol-both treatments were well tolerated, and no suppurative or mixed inflammatory cell reaction was observed in the vitreal samples (Group 1) at any of the time points examined. Antibodies to rhuMAb HER2 were detected in plasma samples by Day 7 in both treatment groups, but infrequently in vitreous samples. There were no safety implications associated with this immune response. The in vitro characterization of the PLGA microspheres provided reasonable projections of the in vivo rhuMAb HER2 release kinetics (Group 1). The total amount of antibody that was released was similar in vitro (25.9%) and in vivo (32.4%). RhuMAb HER2 (Group 2) was cleared slowly from the vitreous compartment, with initial and terminal half-lives of 0.9 and 5.6 days, respectively. The volume of distribution approximated the vitreous volume in a rabbit eye.

Comparisons of the intraocular tissue distribution, pharmacokinetics, and safety of 125I-labeled full-length and Fab antibodies in rhesus monkeys following intravitreal administration.

Access of recombinant proteins to the retina following intravitreal administration is poorly understood. A study was conducted in male Rhesus monkeys (15 to 28 mo of age; 2.8-3.3 kg) in order to compare the intraocular tissue distribution, pharmacokinetics, and safety of 125Iodine (I)-labeled full-length humanized rhuMAb HER2 antibody (148 kD) and of 125I-labeled humanized rhuMAb vascular endothelial growth factor Fab antibody (48.3 kD) following bilateral bolus intravitreal injection on day 0 (5 animals/group). The dose administered to each eye was 25 microg (9-10 microCi) in 50 microl. Animals were euthanatized on day 0 (1 hr postdose) and on days 1, 4, 7, and 14. Safety assessment included direct ophthalmoscopy, intraocular pressure measurements, clinical observations, body weight, and hematology and clinical chemistry panels. Blood and vitreous samples were collected daily (blood only) and at necropsy for pharmacokinetics and analysis for antibodies to the test materials; the ocular tissue distribution of the test material was evaluated by microautoradiography. All animals completed the study. Microautoradiography demonstrated that the full-length antibody did not penetrate the inner limiting membrane of the retina at any of the time points examined. In contrast, the Fab antibody fragment diffused through the neural retina to the retinal pigment epithelial layer at the 1-hr time point and persisted in this location for up to 7 days. Systemic exposure to test material was low but variable: the highest plasma concentration of the full-length antibody was 20.3 ng/ml, whereas plasma concentrations for the Fab antibody remained below the limit of quantitation (i.e., <7.8 ng/ml). An immune response to the test material was not evident in either treatment group. The half-life in vitreous was 5.6 days for the full-length antibody and 3.2 days for the Fab antibody. The shorter intravitreal half-life of the Fab antibody is related to its smaller size and its significant diffusion through the retinal layers. The differences in pharmacokinetics and tissue distribution that are noted between the full-length and Fab antibodies in this study identify potential therapeutic approaches that may be exploited in specific disease conditions.

Efficacy and concentration-response of murine anti-VEGF monoclonal antibody in tumor-bearing mice and extrapolation to humans.

The development of a neovascular supply (angiogenesis) is a major aspect of tumorigenesis. Recent work has indicated that vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis. In vitro and in vivo studies have demonstrated that an anti-VEGF antibody is capable of suppressing the growth of human tumor cell lines. The following study was conducted in tumor-bearing nude mice to evaluate the concentration-response relationship of murine anti-VEGF monoclonal antibody (muMAb VEGF) so that an efficacious plasma concentration of the recombinant humanized form (rhuMAb VEGF) in cancer patients could be estimated. (This study was included in our Investigational New Drug application to support the clinical dosing regimen and projected human safety factors for the toxicology program.) Additionally, the growth dynamics of the tumors were evaluated as a function of dose to explore whether a mechanismic interpretation of tumor growth inhibition by muMAb VEGF is possible. On day 1, A673 human rhabdomyosarcoma cells (2 x 10(6) cells/mouse) were injected subcutaneously in 188 beige nude mice (16-24 g). Treatment with muMAb VEGF (0.05-5.0 mg/kg; n = 24/group), phosphate-buffered saline (n = 10), or anti-gp120 isotype-matched control antibody (5.0 mg/kg; n = 10) began 24 hr later. Each animal received intraperitoneal injections of test material twice weekly for 4 wk. Immediately prior to each dose, 2 mice from each muMAb VEGF group were selected randomly, and plasma was collected for pharmacokinetic evaluation; at the end of the study, samples were collected from all animals for pharmacokinetic evaluation. Tumor dimensions were recorded weekly, and at the end of the study, tumor weight and dimensions were recorded. Satisfactory tumor suppression in nude mice was achieved at muMAb VEGF doses of > or =2.5 mg/kg, where the average trough muMAb VEGF plasma concentration was 30 microg/ml (concentrations in individual animals >10 microg/ml). Assuming the pharmacokinetics of rhuMAb VEGF in patients will resemble the pharmacokinetics of a similar humanized anticancer monoclonal antibodies, a clinical dosing regimen was designed to maintain the rhuMAb VEGF plasma concentration in this efficacious range. This study shows an approach that can be used to estimate a human dosing regimen from preclinical pharmacokinetic/pharmacodynamic data. Because we have just initiated clinical trials with rhuMAb VEGF we cannot judge clinical outcome in relation to these preclinical predictions; nonetheless, it is hoped that by sharing our approach and thought processes with other investigators we can assist the discovery and development of anticancer therapeutics.

Design of biological equivalence programs for therapeutic biotechnology products in clinical development: a perspective.

The determination of biological equivalence requires that studies are conducted to establish that two molecules, two formulations, of two dosing regimens, for example, are indistinguishable with respect to safety and efficacy profiles that have been previously established. The criteria that are used to establish biological equivalence will depend on the nature of the change (e.g., molecular, process, formulation), the stage of the development program, the duration of treatment, and the intended clinical indications. Key components of an equivalence program include chemical characterization, in vitro and in vivo bioactivity against reference material, pharmacokinetics, and safety. Special considerations for patient populations, endogenous concentrations, environmental factors, immunogenicity, assay methodology, biochemical identity, pharmacodynamic equivalence, and statistical methodology are discussed. In addition, the role of preclinical in vivo assessments is addressed. Specific case studies provide insight into the varied nature of approaches that are currently employed.

Pharmacokinetics, tissue distribution, and expression efficiency of plasmid [33P]DNA following intravenous administration of DNA/cationic lipid complexes in mice: use of a novel radionuclide approach.

The pharmacokinetics, tissue distribution, and efficacy of a systemic gene transfer method were examined in male BALB/c mice (6-8 weeks old) using 33P-labeled plasmid DNA for luciferase. The DNA was delivered via tail vein injection in saline ([33P]DNA) or in a cationic lipid formulation ([33P]DNA/lipid). One group of mice received approximately equal to 1-3 microCi (45 micrograms of DNA) of either formulation, and mice were euthanized at 2 and 20 min, and 1 and 24 h postdose (2 mice/time point). Blood and plasma radioactivity were quantified, and whole body autoradiographic (WBAR) images were obtained from 20-microns whole body sections. A tissue distribution (TD) study was conducted in a second group of mice, which received approximately equal to 4-6 microCi (45-60 micrograms of DNA) of [33P]DNA/lipid. Mice were euthanized at 1.5 h (1 mouse; [33P]DNA/lipid) or 24 h (2 mice/ group), and organ radioactivity and luciferase expression were measured in lung, liver, kidney, spleen thymus, and parotid salivary gland by direct quantitation methods. Microautoradiography (MAR) was performed on a third group of mice (n = 2), which received 3 microCi (45 micrograms of DNA) of [33P]DNA/lipid and were euthanized at 24 h postdose. For WBAR, the [33P]DNA/lipid tissue distribution (% dose equiv/g) at 2 min was lung >> liver > spleen (red pulp) > kidney (cortex); at 24 h the ranking was spleen (red pulp) > liver > lung, kidney (cortex). The [33P]DNA organ distribution observed at 2 min was liver >> spleen (red pulp) > lung, blood > kidney (cortex); at 24 h the ranking was liver, spleen (red pulp) > kidney (cortex) > lung, blood. High levels of radioactivity in bone (cortical, marrow, growth plate) in both groups may represent uptake of the 33P-labeled test articles by the cellular component of the bone marrow, particularly macrophages, as well as deposition of [33P]phosphate in the bone matrix following metabolism of the [33P]DNA. In the luciferase component of the study, no expression was observed in the [33P]DNA group at 24 h. The [33P]- DNA/lip group exhibited expression as early as 1.5 h in the lung; at 24 h, expression was seen in all the organs examined. Microautoradiography of 24-h tissue samples revealed radioactivity in hepatic Kupffer cells, reticuloendothelial system cells in the marginal zone of the spleen, and diffusely along alveolar septae with scattered accumulations in alveolar macrophages. The results of the WBAR, TD, MAR, and luciferase assay show that the use of cationic lipids significantly altered the biodistribution and resulting expression of the DNA plasmid. Further, 33P (0.25 MeV beta, half-life = 25 days) was shown to be an excellent radionuclide for quantitative WBA and MAR, providing sharp images with less personal hazard and greater ease of handling than 32P (1.71 MeV beta, half-life = 14.3 days).

Pharmacokinetics and interspecies scaling of recombinant human factor VIII.

Recombinant human (rh) factor VIII is a glycoprotein consisting of multiple polypeptides with relative mobilities (M(r)) ranging from 80,000 to 210,000. It is produced in mammalian cells. Single-dose intravenous pharmacokinetic studies were conducted with rh factor VIII (Kogenate rh Antihemophilic Factor, Miles, Inc.) in male mice (21.0-25.8 g) and rats (252.0-254.2 g). Each species received 400 IU/kg, and blood was collected up to 12 hr (mice) or 32.5 hr (rats) post-dose. Immunoreactive factor VIII concentrations in plasma were quantified by a sensitive and specific ELISA. In both species, the disposition profiles were described by the sum of two exponentials. The pharmacokinetics of rh factor VIII in mouse were as follows: clearance, 27.7 ml/hr/kg; initial volume of distribution, 72 ml/kg; steady-state volume of distribution, 148 ml/kg; and terminal half-life, 4.1 hr. In rat, the mean estimates were as follows: clearance, 16.0 ml/hr/kg; initial volume of distribution, 41 ml/kg; steady-state volume of distribution, 125 ml/kg; and terminal half-life, 5.5 hr. These pharmacokinetic parameters for rh factor VIII in animals and human rh factor VIII pharmacokinetic parameters from the literature were evaluated to determine if the parameters can be represented by the allometric relationship, Y = aWb, where Y is the pharmacokinetic parameter, and W is body weight. The following allometric relations were obtained for rh factor VIII: clearance (ml/hr) = 10.4W0.69, half-life (hr) = 7.5 W0.18, initial volume of distribution (ml) = 43.6 W1.04, and steady-state volume of distribution (ml) = 99.1 W0.84. The allometric exponents for each parameter conformed to theory and were within the range of values commonly observed for xenobiotics and therapeutic proteins. These studies suggest that the pharmacokinetics of rh factor VIII in laboratory animals are predictive of the disposition in humans despite the complex nature of its biological interactions and the chemical diversity of the purified material.

Safety, pharmacokinetics, and antiviral response of CD4-immunoglobulin G by intravenous bolus in AIDS and AIDS-related complex.

To assess the safety, pharmacokinetics, and antiviral effects of intravenous recombinant CD4 immunoglobulin G (CD4-IgG), a 12-week Phase One study with an optional maintenance phase was performed. Twenty-two subjects with advanced human immunodeficiency virus (HIV) infection were enrolled; 15 subjects completed the initial 12 weeks. CD4-IgG doses were 30, 100, or 300 micrograms/kg weekly; 1,000 micrograms/kg once, twice, or three times per week; or 3,000 micrograms/kg twice weekly. Serum concentrations of CD4-IgG increased linearly with dose, with average peak serum concentrations of 22 micrograms/ml with 1,000 micrograms/kg. CD4-IgG was well tolerated; one patient had self-limited tachycardia and flushing associated with CD4-IgG therapy. No changes were seen in CD4 cell counts, hematologic or coagulation studies, serum chemistries, HIV p24 antigen titers, or plasma HIV titers. No subject developed anti-CD4 antibodies. HIV isolates from five patients had IC90 values that were higher than the peak concentrations of CD4-IgG achieved in those patients. Additional studies that achieve higher CD4-IgG concentrations are necessary to evaluate the antiviral activity of this compound.

Characterization of the MN gp120 HIV-1 vaccine: antigen binding to alum.

PURPOSE: The characterization of recombinant MN gp120/alum vaccine requires the study of the gp120-alum interaction for the successful formulation of an alum-based HIV-1 vaccine. METHODS: Several observations suggest that the gp120-alum interaction is weak, wherein buffer counterions such as phosphate, sulfate, bicarbonate may cause the desorption of gp120 from alum. Comparison of gp120 with other proteins using particle mobility measurements shows that the weak binding of gp120 to alum is not an anomaly. Serum and plasma also cause desorption of gp120 from alum with a half-life of only a few minutes, wherein this half-life may be faster than the in-vivo recruitment of antigen presenting cells to the site of immunization. RESULTS: Immunization of guinea pigs, rabbits and baboons with gp120 formulated in alum or saline demonstrated that alum provides adjuvant activity for gp120, particularly after early immunizations, but the adjuvant effect is attenuated after several boosts. CONCLUSIONS: These observations indicate that both the antigen and the adjuvant require optimization together.

Engineering linear F(ab')2 fragments for efficient production in Escherichia coli and enhanced antiproliferative activity.

We developed a novel bivalent antibody fragment, the linear (L-) F(ab')2, comprising tandem repeats of a heavy chain fragment VH-CH1-VH-CH1 cosecreted with a light chain. Functional humanized L-F(ab')2 directed against p185HER2 was secreted from Escherichia coli at high titer (> or = 100 mg/l) and purified to homogeneity. The L-F(ab')2 binds two equivalents of antigen with an apparent affinity (Kd = 0.46 nM) that is within 3-fold of the corresponding thioether-linked F(ab')2 fragment. The N-terminal site binds antigen with an affinity (Kd = 1.2 nM) that is approximately 4-fold greater than that for the C-terminal site, as shown by the comparison of L-F(ab')2 variants containing a single functional binding site. L-F(ab')2 has greater antiproliferative activity than the thioether-linked F(ab')2 against the p185HER2-overexpressing tumor cell line BT474. Linear and thioether-linked F(ab')2 have very similar pharmacokinetic properties in normal mice, and their serum permanence times are respectively 7- and 8-fold longer than the corresponding Fab fragment. L-F(ab')2 offers a facile route to bivalent antibody fragments that are potentially suitable for clinical applications, and that may have improved biological activity compared with thioether-linked F(ab')2 fragments.

Transport of recombinant human CD4-immunoglobulin G across the human placenta: pharmacokinetics and safety in six mother-infant pairs in AIDS clinical trial group protocol 146.

Recombinant CD4-immunoglobulin G (rCD4-IgG) is a 98-kDa human immunoglobulin-like protein that is produced by fusing the gp120 binding domain of CD4 to the Fc portion of the human IgG1 heavy chain. This hybrid molecule was given to human immunodeficiency virus (HIV)-infected pregnant women at the onset of labor by intravenous bolus at 1 mg/kg of body weight (group A; n = 3) and 1 week prior to and at the onset of labor by the same route and at the same dose (group B; n = 3). In addition to pharmacokinetic studies, safety in the mothers and infants was determined through routine chemistries, hematology, and urinalysis; immunologic and HIV infection statuses in the infants were assessed through lymphocyte cultures, p24 antigen level determination, culture of HIV from plasma, PCR, lymphocyte subset enumeration, quantitative immunoglobulin analysis, and lymphocyte proliferation. Thirty minutes after the rCD4-IgG injection, concentrations in maternal serum were 12 to 23 micrograms/ml. These concentrations declined slowly, with initial and terminal half-lives (mean +/- standard deviation) of 9.95 +/- 3.23 and 47.6 +/- 22.3 h, respectively. Infants were born 2.6 to 46.5 h after rCD4-IgG administration; concentrations of rCD4-IgG in cord blood ranged from 28 to 107 ng/ml. The half-life of rCD4-IgG in infants ranged from 5 to 29 h. These data demonstrate that the transfer of rCD4-IgG from the mother to the fetus is rapid and that newborns do not appear to have any difficulty eliminating rCD4-IgG. No safety concerns in mothers or infants were encountered. Although the study did not address the question of efficacy, none of the infants was HIV type 1 infected 36 months later. In summary, these findings document that bifunctional immune molecules can be transported across the placenta, and this general approach may be used in the future to block vertical transmission of HIV type 1.

Expression of exposure in negative carcinogenicity studies: dose/body weight, dose/body surface area, or plasma concentrations?

Evaluation of positive findings in a rodent carcinogenicity study and the subsequent extrapolation to humans is based on chemical structure, mutagenicity, pharmacology, hormone changes, chronic toxicity, and the nature of the tumors induced. For negative studies, adequacy of exposure may become an issue. The use of plasma concentrations as a metric for exposure assumes that each species responds in a similar manner to a given concentration; data are now available that demonstrate that this is not generally true for carcinogenicity. The use of the body surface area metric (i.e., mg/m2) is a special case of interspecies allometric scaling (i.e., W0.67). For a chemical to be amenable to such scaling in toxicology, it must satisfy 3 criteria: (a) the concentration-time profile of the putative toxicant at the site of action must be governed by a scalable pharmacokinetic process (e.g., glomerular filtration); (b) the mechanism of action and the susceptibility of each species to a given systemic exposure must be the same and, for example, be independent of lifespan, cellular repair mechanism/rate, and so forth; and (c) the biological response must depend only on size (e.g., not on race, strain, gender, age, or parity). Carcinogens rarely, if ever, meet these criteria. An empirical analysis of carcinogenic potency data in rodents and in humans shows that, in general, exposure is best expressed in terms of mg/kg body weight.

Development of a humanized disulfide-stabilized anti-p185HER2 Fv-beta-lactamase fusion protein for activation of a cephalosporin doxorubicin prodrug.

The humanized anti-p185HER2 antibody, humAb4D5-8, has completed Phase II clinical trials for p185HER2-overexpressing breast cancer. Here, this antibody is used as a building block to engineer a disulfide-linked Fv (dsFv) beta-lactamase fusion protein for use in antibody-dependent enzyme-mediated prodrug therapy using cephalosporin-based prodrugs. Three Fv variants were designed with an interchain disulfide bond buried at the VL/VH interface and secreted from Escherichia coli. One variant, dsFv3 (VL L46C VH D101C0, has similar affinity for antigen (Kd = 0.7 nM) as the wild-type Fv and was used to construct a fusion protein in which beta-lactamase, RTEM-1, is joined to the carboxy terminus of VH. The dsFv3-beta-lactamase fusion protein secreted from E. coli efficiently activates a cephalothin doxorubicin prodrug (PRODOX, kcat/km = 1.5 x 10(5) s-1 M-1). PRODOX is approximately 20-fold less toxic than free doxorubicin against breast tumor cell lines SK-BR-3 and MCF7, which express p185HER2 at elevated and normal levels, respectively. Prebinding the dsFv3-beta-lactamase fusion protein specifically enhances the toxicity level of PRODOX to that of doxorubicin against SK-BR-3 but not MCF7 cells. The fusion protein retains both antigen-binding plus kinetic activity in murine serum and is cleared rapidly as judged by pharmacokinetic analysis in nude mice (initial and terminal half-lives of 0.23 and 1.27 h, respectively). Development and characterization of the dsFv3-beta-lactamase fusion protein is an important step toward targeted prodrug therapy of p185HER2-overexpressing tumors.

Molecular and biological properties of an interleukin-1 receptor immunoadhesin.

Overproduction of the cytokine interleukin 1 (IL-1) is an important factor in the pathogenesis of several autoimmune and inflammatory disease. To develop a recombinant inhibitor of IL-1 with an extended pharmacologic half-life, we constructed an IL-1 receptor immunoadhesin (IL-1R-IgG), by fusing the extracellular domain of the type IL-1 receptor with the hinge and Fc regions of human IgG1 heavy chain. Transfected human 293 cells express IL-1R-IgG as a secreted, disulfide-bonded homodimer. The secreted protein contains an intact antibody Fc region, as indicated by immunoblotting, and a functional IL-1 receptor region, as indicated by ligand-blotting. Saturation binding analysis indicates an equilibrium dissociation constant (KD) of 350 pM for the binding of IL-1R-IgG to its ligand, IL-1 beta. Kinetic analysis of the binding reveals an off rate of 0.1 min-1 and an on rate of 1.5 x 10(8) min-1 M-1, yielding a calculated KD of 770 pM. These binding properties are similar to those of cell-surface type I IL-1 receptor. IL-1R-IgG is capable of inhibiting the biological activity of IL-1 beta in vitro, as evidenced in a thymocyte proliferation assay. Pharmacokinetic analysis in mice indicates that IL-1R-IgG has a terminal half-life of 91 hr in the blood circulation. This half-life is markedly longer than the values reported for other recombinant inhibitors of IL-1 such as the IL-1 receptor antagonist or soluble IL-1 receptor. Thus, IL-1R-IgG may be useful for investigating the interaction of IL-1 with its receptor and the role of IL-1 in disease, as well as for potential intervention in pathological situations involving overproduction of IL-1.

Modification of CD4 immunoadhesin with monomethoxypoly(ethylene glycol) aldehyde via reductive alkylation.

CD4 immunoadhesin (CD4-IgG) is a chimeric glycoprotein molecule comprised of the gp120-binding portion of human CD4 fused to the hinge and Fc portions of human IgG. As a candidate for human therapeutic use, CD4-IgG represents an important advance over soluble CD4, insofar as the systemic clearance in humans of CD4-IgG is significantly slower. In an effort to prolong its in vivo residence time even further, we have modified CD4-IgG chemically by attaching monomethoxypoly(ethylene glycol) (MePEG) moieties to lysine residues via reductive alkylation. We synthesized MePEG aldehyde and investigated reaction conditions for adding a range of MePEG moieties per protein molecule. At neutral pH in the presence of sodium cyanoborohydride, the reaction was sufficiently slow to allow for significant control over the extent of MePEGylation. Addition of 7.7 or 14.4 MePEG moieties to CD4-IgG resulted in an approximately 4- or 5-fold increase, respectively, in the persistence of the protein in rats, as compared with unmodified CD4-IgG. These results suggest that the therapeutic utility of a human receptor IgG chimera can be improved by MePEGylation technology, provided that the modified immunoadhesin retains its biological activity in vivo. Such modification can lead to a significant additional increase in the in vivo residence time of the protein.

Pharmacokinetics and tissue distribution of recombinant human transforming growth factor beta 1 after topical and intravenous administration in male rats.

Recombinant human transforming growth factor beta (rhTGF-beta 1) enhances the healing process after topical application to various animal wound models. A detailed pharmacokinetic and tissue distribution study was performed to support the clinical development of rhTGF-beta 1 for wound healing indications. Rats received radioiodinated or unlabeled rhTGF-beta 1 as an intravenous (iv) bolus or as a topical formulation applied to a full thickness wound. Plasma concentrations of TGF-beta 1 were estimated from TCA-precipitable radioactivity or were measured by ELISA. Following iv administration, the initial half-life was rapid (< 11 min), regardless of whether radiolabeled or unlabeled rhTGF-beta 1 was used. The terminal half-life was long (163 min) when the test material was radioiodinated and administered as a trace dose and relatively short (< or = 61 min) when given at high doses and assayed by ELISA. Analysis of plasma radioactivity by SDS-PAGE revealed a time-dependent clearance of the 25-kDa parent molecule without a significant appearance of lower molecular weight radiolabeled metabolites. The majority of the radioactivity was associated with highly perfused organs, known iodide elimination pathways, and the thyroid at 1 and 8 hr after iv injection. After topical administration of a high dose (0.8 mg/kg), no immunoreactive TGF-beta 1 was detectable in plasma samples taken over a 48-hr period. However, trace amounts (< or = 0.05 ng/mL) of acid-precipitable radioactivity were detected in plasma after topical application of [125I]rhTGF-beta 1 (1 microgram/kg, 126 microCi/kg).(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of TNF by a TNF receptor immunoadhesin. Comparison to an anti-TNF monoclonal antibody.

TNF is an important mediator of inflammation, which can have deleterious effects when produced inappropriately. We have described a recombinant inhibitor of TNF, termed TNFR-IgG, or TNFR immunoadhesin, composed of the extracellular portion of the type 1 (p55) TNF receptor (TNFR) linked to the hinge and Fc regions of IgG heavy chain. This bivalent, Ab-like molecule is a potent inhibitor of TNF, exhibiting significantly higher affinity for the cytokine than soluble TNFR. Here, we compare the TNF-neutralizing capacity of TNFR-IgG to that of an anti-TNF mAb. In vitro, TNFR-IgG was 10- to 50-fold more potent than anti-TNF mAb at blocking the cytotoxic effect of exogenous TNF on actinomycin D-treated murine L-M cells. In vivo, the plasma half-life of TNFR-IgG in mice was approximately 6 days, similar to that reported for the anti-TNF mAb. However, the immunoadhesin was approximately 10-fold more effective than the Ab at neutralizing the activity of endogenous TNF, as assessed in a model for murine listeriosis. These results demonstrate a markedly greater potency of the TNFR immunoadhesin compared with the anti-TNF mAb at inhibiting TNF activity in vitro and in vivo.