Ozanimod (RPC1063) is a potent sphingosine-1-phosphate receptor-1 (S1P1 ) and receptor-5 (S1P5 ) agonist with autoimmune disease-modifying activity.

BACKGROUND AND PURPOSE: Sphingosine1-phosphate (S1P) receptors mediate multiple events including lymphocyte trafficking, cardiac function, and endothelial barrier integrity. Stimulation of S1P1 receptors sequesters lymphocyte subsets in peripheral lymphoid organs, preventing their trafficking to inflamed tissue sites, modulating immunity. Targeting S1P receptors for treating autoimmune disease has been established in clinical studies with the non-selective S1P modulator, FTY720 (fingolimod, Gilenya™). The purpose of this study was to assess RPC1063 for its therapeutic utility in autoimmune diseases. EXPERIMENTAL APPROACH: The specificity and potency of RPC1063 (ozanimod) was evaluated for all five S1P receptors, and its effect on cell surface S1P1 receptor expression, was characterized in vitro. The oral pharmacokinetic (PK) parameters and pharmacodynamic effects were established in rodents, and its activity in three models of autoimmune disease (experimental autoimmune encephalitis, 2,4,6-trinitrobenzenesulfonic acid colitis and CD4(+) CD45RB(hi) T cell adoptive transfer colitis) was assessed. KEY RESULTS: RPC1063 was specific for S1P1 and S1P5 receptors, induced S1P1 receptor internalization and induced a reversible reduction in circulating B and CCR7(+) T lymphocytes in vivo. RPC1063 showed high oral bioavailability and volume of distribution, and a circulatory half-life that supports once daily dosing. Oral RPC1063 reduced inflammation and disease parameters in all three autoimmune disease models. CONCLUSIONS AND IMPLICATIONS: S1P receptor selectivity, favourable PK properties and efficacy in three distinct disease models supports the clinical development of RPC1063 for the treatment of relapsing multiple sclerosis and inflammatory bowel disease, differentiates RPC1063 from other S1P receptor agonists, and could result in improved safety outcomes in the clinic.

Pharmacokinetics, pharmacodynamics, allometry, and dose selection of rPSGL-Ig for phase I trial.

rPSGL-Ig is a recombinant, soluble, and chimeric form of P-selectin glycoprotein ligand-1, which is developed as an antagonist to P-selectin. Allometric and pharmacokinetic/pharmacodynamic modeling was used to select doses for human clinical trials. Pharmacokinetic parameters of rPSGL-Ig such as clearance (CL), volume of distribution (Vc), and t(1/2) across animal species are well described by power functions with body weight as an independent variable. The power functions for CL, Vc, and t(1/2) were CL = 0.37. W(0.93) ml/h (r(2) = 0.94), Vc = 45.0.W(1.064) ml (r(2) = 0.988), and t(1/2) = 190.W(0.159) h (r(2) = 0.75), respectively. These functions provide a means to predict pharmacokinetics of rPSGL-Ig in humans. For a 70-kg human, the values of CL, Vc, and t(1/2) are predicted to be 19.9 ml/h, 4138 ml, and 15.5 days, respectively. The predicted pharmacokinetics in humans is used in conjunction with pharmacological data to estimate appropriate doses for clinical trials. The doses that may provide potential effects in humans range from 0.13 to 4.7 mg/kg. The predicted doses produce concentrations above those that are associated with efficacy in animal disease models and, maintain concentrations above the EC(50) of in vitro binding between rPSGL-Ig and stimulated human platelets. Hence, rPSGL-Ig in clinical trials may provide therapeutic activities for P-selectin-mediated diseases.

Implantation of recombinant human bone morphogenetic proteins with biomaterial carriers: A correlation between protein pharmacokinetics and osteoinduction in the rat ectopic model.

This study was carried out to determine the effect of recombinant human bone morphogenetic protein (rhBMP) pharmacokinetics (PK) on rhBMP-induced osteoinductive activity. It was our working hypothesis that the PK of a rhBMP significantly affects its osteoinductive activity. The PK of various rhBMPs (rhBMP-2, rhBMP-4, rhBMP-6, and chemically modified rhBMP-2) implanted with four biomaterial carries (Helistat, hDBM, Osteograf/N, and Dexon) was determined using (125)I-labeled proteins in the rat ectopic assay. A select combination of rhBMP and carriers then was evaluated in the rat ectopic assay for osteoinductive activity using a semi-quantitative histologic scoring system. The results indicate that initial protein retention is dependent on protein isoelectric point (pI); proteins with a higher pI yielded a higher implant retention. Subsequent PK was not strongly dependent on the pI or on the carrier. Because of the difference in early retention, the rhBMP-carrier combinations exhibited a >100-fold difference in implant-retained protein dose. When rhBMP-2 and rhBMP-4 were implanted with the carriers, more rhBMP-2 was retained in an implant, and the osteoinductive potency of rhBMP-2 typically was higher than rhBMP-4 at low implantation doses. We conclude that protein pI plays a significant role in the local retention of implanted rhBMP and that higher retention yields a higher osteoinductive activity.

rhBMP-collagen sponges as osteoinductive devices: effects of in vitro sponge characteristics and protein pI on in vivo rhBMP pharmacokinetics.

Osteoinductive devices, comprised of biodegradable collagen scaffolds and recombinant human Bone Morphogenetic Proteins (rhBMPs), are being currently pursued for local bone induction. To better understand the biological performance of such devices, we have carried out a series of studies to investigate the effects of sponge properties and protein structural features on the pharmacokinetics of implanted rhBMPs. The results indicated little dependence of the rhBMP-2 pharmacokinetics on the in vitro determined sponge properties. The protein isoelectric point (pI), on the other hand, was found to significantly affect the initial implant retention of rhBMPs, but not the subsequent pharmacokinetics. A 100-fold difference in the implant-retained dose could be observed depending on the type of rhBMP implanted. We conclude that protein structural features are important variables controlling in vivo pharmacokinetics of rhBMPs, and possibly the osteoinductive potency of the devices.

Characterization of rhBMP-2 pharmacokinetics implanted with biomaterial carriers in the rat ectopic model.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) is a member of the bone morphogenetic protein family involved in de novo bone induction. Successful use of rhBMP-2 requires implantation with a biomaterial which can act as a scaffold for cell invasion for osteoinduction and retains rhBMP-2 at a site of implantation. This study was carried out to characterize rhBMP-2 pharmacokinetics from a variety of biomaterial carriers in a rat ectopic model. Retention of rhBMP-2 within carriers after 3 h was variable among the carriers (range, 75-10%), with collagenous sponges retaining the highest fraction of implanted dose. A gradual loss of rhBMP-2 was subsequently observed, the kinetics of which was strongly dependent on the implanted carrier. Collagenous carriers were observed to lose rhBMP-2 gradually from the implant site, whereas some of the mineral-based carriers retained a fraction of implanted rhBMP-2 within the implants. These differences in protein pharmacokinetics among carriers, in addition to their physicochemical nature, are expected to affect the biological activity of implanted rhBMP-2.

Antiproliferative effects of recombinant human bone morphogenetic protein-2 on human tumor colony-forming units.

Bone morphogenetic protein-2 (BMP-2) is a differentiation factor for normal osteoblasts. BMP-2 is structurally related to transforming growth factor-beta which inhibits cell proliferation and enhances apoptosis. A recent study has shown the presence of BMP-2 receptors on several cancer cell lines. In this study, we attempted to determine if recombinant human BMP-2 (rhBMP-2) can modulate the proliferation of human tumor colony-forming units taken from 113 patients. Tumor cells were cultured in soft agar and continuously exposed to three concentrations of rhBMP-2 (10, 100 and 1000 ng/ml) for 14 days in the capillary cloning system. There were 65 evaluable specimens, including 17 breast cancers, 15 ovarian cancers, 14 non-small cell lung cancers and five prostate cancers. Importantly, rhBMP-2 did not stimulate the tumor cell proliferation. A significant inhibition (50% or less survival of tumor colony-forming units) was seen in 16 of 65 specimens (24.6%) at 1000 ng/ml, including five of 14 non-small cell lung cancers, five of 17 breast tumors and two of 15 ovarian tumors. A concentration-response relationship was observed (p<0.001 by Mantel-extension test). The results of this study encourage further evaluation of the antiproliferative effects of rhBMP-2 against human cancers.

Recombinant human factor IX: replacement therapy, prophylaxis, and pharmacokinetics in canine hemophilia B.

Recombinant human factor IX (rFIX) has been expressed in transduced cultured cell systems since 1985. Because there has been limited in vivo testing of rFIX in hemophilia B subjects, this study was undertaken using the severe hemophilia B canines of the Chapel Hill strain. Three groups of hemophilic dogs received either 50, 100, or 200 IU/kg of rFIX. As a control, a fourth group of hemophilic dogs received 50 IU/kg of a high purity, plasma-derived human FIX (pdFIX). The coagulant and hemostatic effects of rFIX and pdFIX were similar with all comparative dosing regimens. Based on activity data, the elimination half-life of rFIX was 18.9 +/- 2.3 hours and pdFIX was 17.9 +/- 2.1 hours. A prophylactic regimen administering rFIX daily resulted in a continuous therapeutic level of plasma FIX and was accompanied by a two-fold increase in recovery levels by day 5, compared to that observed with administration of a single bolus. The mechanisms of the high to complete recovery of FIX with the prophylactic regimen could depend not only on the degree of saturation of the vascular endothelial binding sites but also on the altered dynamics of the balance of FIX distribution between the intravascular and extravascular compartments. The pharmacokinetic (PK) parameters for rFIX and pdFIX were similar. However, the relative PK values for V1 and V5s of both products on day 5 differed greatly from day 1 and may reflect the changing equilibrium of FIX between compartments with elevated levels of plasma FIX. Neutralizing antihuman FIX antibodies resulting from human FIX antigen being administered to FIX deficient dogs were observed beginning at 14 days. The antigenicity of rFIX and pdFIX appeared to be comparable. Despite the very different procedures used for production of rFIX and pdFIX products, in vivo testing in hemophilia B dogs showed the functional behavior of these products is similar; they are highly effective for replacement therapy and for prophylaxis.

The importance of N- and O-linked oligosaccharides for the biosynthesis and in vitro and in vivo biologic activities of erythropoietin.

Erythropoietin (EPO) plays a critical role in stimulating the proliferation and differentiation of erythroid precursor cells. EPO is heavily glycosylated with three asparagine (N)-linked tetraantennary oligosaccharides that may contain N-acetyl-lactosamine repeats and a single serine (O)-linked oligosaccharide. EPO expressed in Chinese hamster ovary cells exhibits biologic properties and amino acid and carbohydrate composition similar to natural urinary EPO. The importance of the complex N-linked and the O-linked carbohydrate was studied by expressing EPO in cells that are deficient in UDP-galactose/UDP-N-acetylgalactosamine 4-epimerase activity. In these cells, the ability to add galactose and N-acetylgalactosamine to glycoproteins can be controlled by the addition of these sugars to the culture medium. The results demonstrate that a block in O-linked glycosylation and/or the ability to process N-linked carbohydrate to completion does not alter EPO secretion. EPO produced without O-linked carbohydrate exhibits normal in vitro and in vivo biologic activity and in vivo clearance. However, EPO produced with incompletely processed N-linked oligosaccharides exhibits normal in vitro activity but is at least 500-fold less effective in stimulating erythropoiesis in vivo. Studies on the survival of bioactive EPO remaining in the circulation demonstrated that EPO with incomplete N-linked oligosaccharides exhibits a sevenfold increased rate of clearance. However, this increased clearance may not fully account for the 500-fold loss of in vivo activity. These results suggest a potentially important unique requirement for appropriate complex N-linked oligosaccharides for the intrinsic biologic activity of EPO in vivo.

Protein engineering of novel plasminogen activators with increased thrombolytic potency in rabbits relative to activase.

Human tissue-type plasminogen activator (t-PA) is a glycoprotein used currently in thrombolytic therapy for patients with acute myocardial infarction. Due to its rapid rate of clearance from the circulation, continuous intravenous administration of approximately 100 mg over 3 h is recommended. We have previously characterized novel thrombolytic variant forms of t-PA which offer the potential of administration by bolus injection and reduced dosage due to their slower rates of clearance, relative to t-PA. This study was undertaken to quantitatively compare the pharmacokinetics, thrombolytic activity, and hemostatic effects of two of these variant forms, called delta FE1X and delta FE3X plasminogen activator (PA), with commercially available recombinant t-PA (Activase). These evaluations were performed in rabbits after bolus intravenous injection of the proteins. Following injection of 0.25 mg of protein/kg of body weight, the rates of clearance for delta FE3X and delta FE1X PA antigen were decreased approximately 9- and 18-fold, respectively, relative to Activase. Plasma plasminogen activator activity was also measured and the rates of clearance of delta FE3X and delta FE1X PA activity were similarly decreased by approximately 9- and 22-fold, respectively, relative to Activase. To quantitate thrombolytic activity we used the rabbit jugular vein thrombosis model and demonstrated that approximately 50% thrombolysis was achieved with delta FE1X and delta FE3X PA at approximately an 8.6- and 3-fold lower dose than Activase, respectively. No major differences in fibrinogen and alpha 2-antiplasmin depletion were observed among the agents at doses required to produce 50% thrombolysis, indicating similarities in fibrin specificities among these agents. These results demonstrate a reciprocal relationship between thrombolysis and rate of clearance for these thrombolytic proteins. The 8.6-fold increase in potency of delta FE1X PA relative to Activase supports the future clinical testing of this novel engineered protein as a thrombolytic agent.

Replacement of finger and growth factor domains of tissue plasminogen activator with plasminogen kringle 1. Biochemical and pharmacological characterization of a novel chimera containing a high affinity fibrin-binding domain linked to a heterologous protein.

A novel triple-kringle plasminogen activator protein, PK1 delta FE1X, has been produced which is a genetic chimera between the fibrin binding kringle 1 domain of plasminogen and the two kringles and serine protease domains of naturally occurring wild-type tissue plasminogen activator (wt t-PA). This chimera also contains a modification to prevent high mannose type N-linked glycosylation on kringle 1 of t-PA. PK1 delta FE1X is biochemically and fibrinolytically similar to wt t-PA in vitro but retains the decreased plasma clearance rate characteristic of other t-PA variants which lack fibronectin finger-like and epidermal growth factor domains. The serine protease domain of PK1 delta FE1X exhibits the amidolytic activity characteristic of wt t-PA. In an indirect coupled plasminogen activator assay, the specific activity of PK1 delta FE1X is approximately 1.4 times greater than that of wt t-PA. In a fibrin film-binding assay, greater binding to untreated fibrin is observed with wt t-PA than with PK1 delta FE1X. However, following limited plasmin digestion of the fibrin film, PK1 delta FE1X binding increases to the level observed with wt t-PA. The incremental binding to plasmin-digested fibrin observed with PK1 delta FE1X is eliminated if plasmin digestion of the fibrin film is followed by carboxypeptidase B treatment. This result suggests that plasminogen kringle 1 binds plasmin-digested fibrin even after recombination with a heterologous protein. The fibrinolytic activity of PK1 delta FE1X in human plasma clot lysis assays was similar to that of wt t-PA at activator concentrations of approximately 1 microgram/ml. At substantially lower concentrations, approximately 0.1 microgram/ml, PK1 delta FE1X was only slightly less active than wt t-PA. Pharmacokinetic analysis showed that wt t-PA activity is cleared approximately 15 times as rapidly as PK1 delta FE1X following intravenous bolus injection. In a rabbit jugular vein clot lysis model, intravenous bolus injection of 0.06 mg/kg of PK1 delta FE1X showed greater thrombolytic potency than a similar administration of 0.5 mg/kg of wt t-PA. Thus it appears that in vitro exon shuffling techniques can be used to generate novel fibrinolytic agents which biochemically and pharmacologically represent the combination of individual domains of naturally occurring proteins.

Site-directed mutagenesis in human tissue-plasminogen activator. Distinguishing sites in the amino-terminal region required for full fibrinolytic activity and rapid clearance from the circulation.

Recombinant variants of tissue plasminogen activator (t-PA) containing either substitutions or deletions of amino acids within the fibronectin finger-like domain (residues 6-50) were found to exhibit widely varying in vivo clearance profiles in rats and fibrinolytic activity in 125I-fibrin clot lysis assays. Clearance was not significantly affected by changes in the densely charged region of amino acid residues 7-10. Deletions or substitutions of amino acids in the region 14-32 decreased both fibrinolytic activity and the clearance of the enzyme. Modifications within the predicted omega loop of residues 37-41 affected clearance only to a small degree, whereas amino acid alterations in the region of residues 42-49 resulted in as much as a 6-fold decrease in the rate of clearance with only relatively minor decreases in the fibrinolytic activity of the variants. The cumulative results distinguish discrete sections of the NH2-terminal region of the enzyme as determinants of in vivo clearance and fibrinolytic activity of t-PA. In addition, the fibrinolytic activity of a variant containing the substitutions Gln42----Asn, His44----Glu, and Asn117----Gln, when compared with wild-type t-PA in an in vivo rabbit venous clot lysis model, was found to have similar lytic efficacy at approximately one-fourth the dose. We conclude that decreases in the in vivo clearance of t-PA can result in more potent thrombolytic agents in vivo, even though the in vitro fibrinolytic activity of the enzyme may be somewhat impaired.

The disposition of quinine in the rat isolated perfused liver: effect of dose size.

We have investigated the pharmacokinetics of both free and total quinine in the rat isolated perfused liver at three doses, 6.25, 12.5 and 25 mg. The plasma concentrations of free and total quinine decayed biexponentially over 4 h. However, on increasing dose, the terminal half-life of free and total quinine showed marked increases ranging from 12.4 +/- 3.7 min at 6.25 mg to 176.0 +/- 153 min at 25 mg (total quinine). Quinine clearance was reduced approximately by half as the dose was doubled. At 10 min post dosage, quinine extraction at the 6.25 mg dose (56 +/- 16.3%) was more than twice that of the highest dose (25 mg, 25.0 +/- 6.5%). Free quinine at the 6.25 mg dose was cleared at approximately 100% of perfusate flow, whereas at 25 mg, clearance was less than one fifth of that value. Unchanged quinine elimination in bile was low, with less than 1% of the parent drug being detected at the 12.5 and 25 mg doses. Relatively little parent drug was recovered from the liver at 4 h. At the 25 mg dose, less than or equal to 6% was recovered as parent drug. HPLC analysis revealed some polar metabolites of quinine in the bile and in the liver homogenates. Dose dependent kinetics of quinine were demonstrated in this study, as hepatic extraction of quinine decreased with increasing dose and input concentration.

The disposition of ethiofos (WR-2721) in the isolated perfused rat liver.

We have investigated the disposition of ethiofos (20 mg, 4 microCi [14C]ethiofos) in the isolated perfused rat liver preparation to determine the hepatic contribution to the poor oral bioavailability of the drug. Ethiofos clearance (10.6 +/- 3.3 ml h-1) was only a small fraction (1.2 +/- 0.03%) of the perfusate flow rate. The elimination half-life was calculated at 7.1 +/- 1.9 h. The area under curve, AUC0-4 h, for ethiofos (2858 +/- 314 nM h ml-1) was not significantly different from that of 14C (3038 +/- 692 nM h ml-1) or total material convertible to WR-1065 (total WR-1065, 3324 +/- 612 nM h ml-1), indicating a low level of metabolism. The AUC0-4 h for free WR-1065 (37.5 +/- 23.3 nM h ml-1) was less than 2% of ethiofos. Biliary elimination of ethiofos, WR-1065, and 14C was below 1%. At 4 h postdose, 7.9 +/- 1.9% of the dose of radioactivity remained in the liver. Less than 1.5% could be identified as ethiofos (0.12 +/- 0.09%) or total WR-1065 (1.09 +/- 0.05%). Ethiofos, 14C, and total WR-1065 were approximately evenly distributed between the 10,000-g pellet and supernatant. However, significantly more ethiofos, WR-1065, and 14C were recovered from the 105,000-g supernatant compared with the pellet. In summary, both the metabolism and biliary elimination of ethiofos and its derivatives were sparing. Hence it is likely that in the rat, the contribution of the liver to the presystemic biotransformation and poor bioavailability of ethiofos is relatively minor.