Steven E. Hall

Purification of the human blood platelet thromboxane A2/prostaglandin H2 receptor protein

The human platelet thromboxane A2/prostaglandin H2 receptor has been purified 6100-fold to apparent homogeneity by a three-step chromatographic procedure with an overall yield of 6%. A 6-fold purification of the receptor was first achieved by chromatography of 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate (CHAPS)-solubilized membrane proteins from human platelets on a diethylaminoethyl (DEAE)-Sepharose column. The DEAE eluate fractions containing receptor activity were then applied to a newly developed affinity column using the cyclohexyl derivative of SQ30,741 (SQ31,491) as the immobilized ligand. Elution of the receptor from the affinity column with BM13.177 yielded a further purification of 1700-fold. An additional 4-fold receptor purification from the affinity column eluate was achieved by HPLC using GPC 500 and GPC 100 columns connected in tandem. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the HPLC eluate containing purified receptor revealed a single, distinct band with a molecular weight of 55,000. The receptor binding activity was detected with [3H]SQ29,548 using a newly developed binding assay which involved immobilization of the receptor on polyethyleneimine-treated glass fiber filters. The binding of [3H]SQ29,548 to the purified receptor was time dependent, saturable, reversible and highly specific. Unlabeled SQ29,548, BM13.505, and U46619 (but not thromboxane B2 or 6-keto prostaglandin F1 alpha) competed for [3H]SQ29,548 binding to the purified receptor in a concentration-dependent manner. Scatchard analysis of [3H]SQ29,548 binding to the purified receptor revealed the presence of a single class of high-affinity binding sites, with a Kd of 4 nM and a Bmax of 17 nmol/mg protein.

Development of Glatopa® (Glatiramer Acetate): The First FDA-Approved Generic Disease-Modifying Therapy for Relapsing Forms of Multiple Sclerosis

The multiple sclerosis (MS) treatment landscape in the United States has changed dramatically over the past decade. While many disease-modifying therapies (DMTs) have been approved by the US Food and Drug Administration (FDA) for the treatment of relapsing forms of MS, DMT costs continue to rise. The availability of generics and biosimilars in the MS-treatment landscape is unlikely to have a major impact on clinical benefit. However, their availability will provide alternative treatment options and potentially lower costs through competition, thus increasing the affordability of and access to these drugs. In April 2015, the first generic version of the complex drug glatiramer acetate (Glatopa® 20 mg/mL) injection was approved in the United States as a fully substitutable generic for all approved indications of the 20 mg/mL branded glatiramer acetate (Copaxone®) dosage form. Despite glatiramer acetate’s complex nature—being a chemically synthesized (ie, nonbiologic) mixture of peptides—the approval occurred without conducting any clinical trials. Rather, extensive structural and functional characterization was performed to demonstrate therapeutic equivalence to the innovator drug. The approval of Glatopa signifies an important milestone in the US MS-treatment landscape, with the hope that the introduction of generic DMTs and eventually biosimilar DMTs will lead to future improvements in the affordability and access of these much-needed treatments for MS.

Inhibition of prostaglandin biosynthesis by SQ 28,852, A 7-oxabicyclo-[2.2.1]heptane analog

7-Oxabicyclo[2.2.1]heptane analogs of prostaglandin (PG) H2 can act as thromboxane (Tx) A2 receptor antagonists or agonists, PGI2 and/or PGD2 receptor agonists, or exhibit a mixture of the above activities. SQ 28,852, a new analog with a hexyloxymethyl omega side chain, is a potent inhibitor of PG synthesis. SQ 28,852 inhibited collagen and arachidonic acid (AA)-induced platelet aggregation and TxB2 and PGE2 formation, but did not block platelet aggregation induced by ADP or the TxA2 mimics, 9,11-azo PGH2, SQ 26,655, and U-46,619. It also blocked conversion of AA to TxB2, PGE2, and 6-keto PGF1 alpha by microsomal preparations of human platelets, bovine seminal vesicles, and bovine aortas, respectively, but did not inhibit the conversion of PGH2 to TxA2 by the platelet microsomal preparation. SQ 28,852 (p.o.) protected mice against the lethal effects of AA (75 mg/kg, i.v.). The I50 values for SQ 28,852, indomethacin and aspirin were 0.025, 0.05 and 15 mg/kg, respectively. Neither SQ 28,852 nor indomethacin protected mice from death caused by 9,11-azo PGH2. SQ 28,852 (0.01 to 1 mg/kg, i.v.) inhibited AA-induced bronchoconstriction in anesthetized guinea pigs for at least 60 min. As an inhibitor of AA-induced bronchoconstriction, SQ 28,852 was 16- and 45-times more potent than indomethacin at 3 and 60 min after i.v. administration, respectively. SQ 28,852 did not inhibit bronchoconstriction induced by histamine or 9,11-azo PGH2, indicating its specificity of action in vivo. SQ 28,852 is the first example of a new class of cyclooxygenase inhibitors whose structure is similar to that of the naturally occurring endoperoxide, PGH2.