Population Pharmacokinetics and Pharmacodynamics of Lampalizumab Administered Intravitreally to Patients With Geographic Atrophy.

Intravitreally administered lampalizumab is an investigational complement inhibitor directed against complement factor D (CFD) for the treatment of geographic atrophy (GA) secondary to age-related macular degeneration. We sought to develop an integrated ocular and systemic pharmacokinetic/pharmacodynamic model for lampalizumab in patients with GA using the data from the clinical phase I and II studies. The kinetics of lampalizumab and CFD disposition were well described by the combined ocular/serum target-mediated drug disposition model using a quasi-steady-state approximation. This model takes into account the drug, target, and drug-target complex clearance, their transfer rates between ocular and serum compartments, and turnover kinetics of CFD. The constructed model provided a prediction of target occupancy in ocular tissues and supported that the two dosing regimens (10 mg q4w and 10 mg q6w) selected for the phase III studies are expected to be efficacious and able to achieve near-complete target engagement in the vitreous humor.

Soluble P-selectin glycoprotein ligand 1 inhibits ocular inflammation in a murine model of allergy.

PURPOSE: To assess the anti-inflammatory modality of a soluble extracellular form of P-selectin glycoprotein ligand 1 (sPSGL-1) in a mouse model of ocular allergic response. METHODS: Potential anti-inflammatory effects of sPSGL-1 were investigated in SWR/J mice sensitized by topical application of short ragweed pollen to the nasal mucosa followed by a challenge of the ocular mucosa with the same allergen. Five experimental groups were included in these studies: A, mice neither sensitized nor challenged with pollen (control group 1); B, animals sensitized but not challenged (control group 2); C, animals not sensitized but challenged (control group 3); D, animals sensitized and challenged; and E, sensitized animals treated with sPSGL-1 before pollen challenge. All experimental groups were evaluated for gross morphologic ocular changes, and histologic assessments were made to determine the onset/progression of inflammatory reactions and to look for evidence of eosinophil infiltration. RESULTS: Mice sensitized and challenged with pollen developed clinical signs consistent with human allergic conjunctivitis. These signs correlate with histologic changes in the conjunctival epithelium and stroma (e.g., edema and extensive eosinophil infiltration). Moreover, the ocular changes also correlated with evidence of eosinophil degranulation. However, sensitized and challenged mice concurrently treated with sPSGL-1 displayed no inflammatory ocular changes associated with a ragweed-induced type-1 hypersensitivity reaction. The lack of ocular changes included the absence of histologic late-phase inflammatory changes of the conjunctiva and a 97% reduction in the induced eosinophil infiltrate. CONCLUSIONS: The antagonistic intervention of cell- cell interactions through the blockade of selectin-dependent leukocyte adhesion may offer novel therapeutic strategies to modulate inflammatory responses. The potent inhibitory effects on eosinophil recruitment and late-phase inflammation suggest a role for sPSGL-1 in the treatment of ocular allergic diseases.

Guanine-adenine ligation-mediated polymerase chain reaction in vivo footprinting.

The analysis of functional DNA regulatory sequences involved in transcriptional control is critical to establishing which proteins mediate cell-specific gene expression. The organization of erythroid LCRs is complex, consisting of multiple, interdigested cis elements. As in situ binding to these sites is determined by the accessibility of these regulatory regions in native chromatin and the availability of relevent cell-specific and ubiquitous factors, in vivo footprinting was used to define protein DNA interactions in human globin LCRs. To further enhance the detection of protein contacts with this technique, we have modified the dimethyl sulfate-based ligation-mediated PCR in vivo footprinting procedure to permit the assessment of protein binding at guanine and adenine resides, rather than exclusively at guanines. This modification, termed GA-LMPCR in vivo footprinting, was essential for the analysis of GATA-1 motifs in the alpha-LCR and HS-3 of the beta-LCR. Moreover, GA-LMPCR in vivo footprinting provided high-resolution analysis of AP-1/NF-E2 elements and revealed protein contacts at sequences that are not coincident with previously described regulatory motifs. A comprehensive discussion of this modification and sample illustrations from our studies have been presented to demonstrate the enhanced detection and resolution obtained with this procedure.

Guanine-adenine ligation-mediated PCR in vivo footprinting.

The identification of relevant DNA regulatory sequences involved in transcriptional control is critical to establishing which proteins mediate cell-specific gene expression. As an approach to investigating the mechanisms of gene regulation, in vivo footprinting studies reveal protein-DNA interactions as they actually occur in situ. We have used in vivo footprinting to complement in vitro studies of the human globin locus control regions (LCRs) in erythroid cells. To further enhance the detection of protein contacts with this technique, we have modified the dimethyl sulfate-based ligation-mediated PCR (LMPCR) in vivo footprinting procedure to permit the assessment of protein binding at guanine and adenine residues, rather than exclusively at guanines. This modification, termed GA-LMPCR in vivo footprinting, was essential for the analysis of GATA-1 motifs in the alpha-LCR and HS-3 of the beta-LCR. Moreover, GA-LMPCR in vivo footprinting provided high-resolution analysis of AP-1/NF-E2 elements and revealed protein contacts at sequences that are not coincident with previously described regulatory motifs. A comprehensive discussion of the GA-LMPCR in vivo footprinting methodology and representative analyses from our studies, including GATA-1 and AP-1/NF-E2 motifs, are presented to illustrate the modified technique.

In vivo protein-DNA interactions at hypersensitive site 3 of the human beta-globin locus control region.

The expression of beta-globin genes in developing erythroid cells is dependent on distant, upstream regulatory sequences, known as the locus control region (LCR), which are marked in chromatin by DNase I hypersensitive sites (HS-1 to HS-4). Linkage of the beta-globin gene complex LCR or fragments surrounding core regions of 200-300 base pairs to the human beta-globin gene permits consistent, high-level expression of the transgene in mice. To define the array of nuclear factors interacting with beta-LCR HS-3, we have performed in vivo dimethyl sulfate footprinting of the active HS-3 core in erythroid cells by a modified procedure that permits assessment of protein-DNA contacts at adenine, as well as guanine, residues. In vivo protein occupancy differs considerably from that predicted from previous in vitro binding analyses. In vivo footprinting detects protein binding at four sites recognized by the erythroid transcription factor GATA-1, at two CACC/GT motifs, and at a single AP-1/NF-E2 site. The regulatory elements occupied in vivo in HS-3 appear similar to those described previously in globin gene promoters and 3' enhancers. These findings suggest that the distinctive properties of the HS-3 region may be attributable to the organization of these occupied motifs and the consequent protein interactions, rather than to the binding of unique LCR regulatory factors.

In vivo footprinting of the human alpha-globin locus upstream regulatory element by guanine and adenine ligation-mediated polymerase chain reaction.

A major regulatory element required for expression of the human alpha-globin genes is located 40 kb upstream of the embryonic zeta-globin gene. To understand how this and other locus control region (LCR) elements contribute to high-level expression in erythroid cells, we have performed high-resolution, in vivo dimethyl sulfate footprinting. In addition, we have modified the dimethyl sulfate-based ligation-mediated polymerase chain reaction in vivo footprinting procedure to permit the assessment of interactions at guanine and adenine residues, rather than guanines alone. In vivo footprinting of the human alpha-LCR element carried on chromosome 16 in a mouse erythroleukemia cell environment revealed protein occupancy at GATA-1, AP-1/NF-E2, and CACC/GGTGG motifs, specific differences compared with in vitro protein binding, and distinct changes in one region upon dimethyl sulfoxide-induced cellular maturation. No protein contacts were detected in nonexpressing hepatoma cells. In addition, we have demonstrated that two AP-1 motifs in the alpha-LCR element which are occupied in vivo bind purified mouse NF-E2 protein in vitro. Our data suggest that three proteins, GATA-1, NF-E2, and unknown CACC/GGTGG factors, are minimally required as DNA-binding proteins for the function of LCR-like elements. The juxtaposition and interaction of these factors with each other, and with accessory proteins not directly in contact with DNA, are likely to account for the relative position independence of the upstream globin regulatory elements.

CCAAT displacement protein as a repressor of the myelomonocytic-specific gp91-phox gene promoter.

The cytochrome b heavy chain (gp91-phox) is expressed exclusively in terminally differentiating myelomonocytic cells. The human gp91-phox gene spans approximately 30 kilobases, and is divided into 13 exons. A ubiquitous factor that is indistinguishable from the CCAAT-binding factor CP1 interacts in vitro with the distal gp91-phox promoter CCAAT box motif. CP1 binding is prevented, however, by a CCAAT displacement protein (CDP) that binds to the region surrounding the CCAAT box. CDP DNA-binding activity is found in nuclear extracts prepared from cells in which the endogenous gp91-phox gene is transcriptionally inactive, but is absent or reduced in expressing cells, consistent with CDP functioning as a repressor of gp91-phox transcription. Introduction of gp91-phox promoter/reporter constructs into nonexpressing cells yields significantly less expression than that produced by the parental reporter vector alone. The reduction in expression is relieved when the CDP/CP1-binding site is removed from the gp91-phox promoter, confirming that it is a target for repression. No derepression is observed if the CP1-binding site is selectively mutated. Derepression of expression exhibited upon deletion of the CDP/CP1-binding site suggests that, in addition to blocking the interaction of the CCAAT-binding factor with the gp91-phox promoter, CDP may also repress transcription mediated through a distinct cis-element(s). We propose that down-regulation of CDP DNA-binding activity is a necessary step in the induction of myelomonocytic-specific expression of the gp91-phox gene.

Analysis of a human V beta gene subfamily.

We have isolated and sequenced five germline V beta gene segments that are homologous to the V region of the YT35 cDNA encoding the beta chain of the T cell antigen receptor from the tumor MOLT-3. One of these gene segments is identical to the YT35 V segment, and therefore is the corresponding germline V beta gene segment encoding the YT35 cDNA. The other four V beta members exhibit 77-98% homology to the YT35 V gene segment. Two of these V beta gene segments are pseudogenes. Analyses of the coding region sequences reveal that, although the V beta segments are very diverse, they are mutating at a rate comparable to that observed in most eukaryotic genes. Analyses of the genomic clones show that the spacing distance between germline V beta gene segments ranges from 3 kb to greater than 30 kb, and the entire V beta 8 subfamily appears to be linked by a total of no more than 110 kb of DNA.

Specific-primer-directed DNA sequencing.

A simple and rapid strategy for DNA sequence analysis based on the Sanger chain-termination method is described. This procedure utilizes full-sized inserts of 1 to 4 kb of DNA cloned into M13 bacteriophage vectors. After the sequence of the first 600-650 bp of the insert DNA has been determined with the commercially available universal vector primer, a specific oligonucleotide is synthesized utilizing the sequence data obtained from the 3' end of the sequence and used as a primer to extend the sequence analysis for another 600-650 nucleotides. Additional primers are synthesized in a similar manner until the nucleotide sequence of the entire insert DNA has been determined. General guidelines for the selection of oligonucleotide length and composition and the use of unpurified primers are discussed. The use of the specific-primer-directed approach to dideoxynucleotide sequence analysis, in association with highly purified single-stranded template DNA, reduces considerably the time required for the analysis of large segments of DNA.