Relative Oral Bioavailability and Food Effects of Two Sepiapterin Formulations in Healthy Participants.

Sepiapterin is an orally administered drug in development for the treatment of phenylketonuria, an inborn error of metabolism characterized by the deficiency of the phenylalanine-metabolizing enzyme phenylalanine hydroxylase. This study characterized the pharmacokinetics, safety, and tolerability of 2 clinical sepiapterin formulations (Phase 1/2, Phase 3) and the effects of food on the pharmacokinetics of the Phase 3 formulation in healthy participants. In Part A, 18 participants were randomized to one of 2 treatment sequences, each with 4 dosing periods comprising a single dose (20 or 60 mg/kg) of the Phase 1/2 or the Phase 3 formulation with a low-fat diet. In Part B, 14 participants were randomized to one of 2 sequences, each comprising 4 dosing periods of a single dose (20 or 60 mg/kg) of the Phase 3 formulation under fed (high-fat) or fasted conditions. Following oral administration, sepiapterin was quickly absorbed and rapidly and extensively converted to tetrahydrobiopterin (BH4). BH4 was the major circulating active moiety. Under low-fat conditions, the Phase 3 formulation was bioequivalent to the Phase 1/2 formulation at 20 mg/kg, while slightly lower BH4 exposure (approximately 0.81×) for the Phase 3 formulation was observed at 60 mg/kg. BH4 exposure increased to approximately 1.7× under the low-fat condition and approximately 2.8× under the high-fat condition at a dose of either 20 or 60 mg/kg for the Phase 3 formulation, compared with the fasted condition. Both sepiapterin formulations were well tolerated, with no serious or severe adverse events reported. All treatment-emergent adverse events were mild or moderate in severity.

Phase I clinical evaluation of CNSA-001 (sepiapterin), a novel pharmacological treatment for phenylketonuria and tetrahydrobiopterin deficiencies, in healthy volunteers.

Tetrahydrobiopterin (BH4) is the natural cofactor of aromatic amino acid hydroxylases and essential for degradation of phenylalanine and synthesis of catecholamines and serotonin. It can be synthesized either de novo from GTP or through the salvage pathway from sepiapterin. Sepiapterin, a natural precursor of BH4, is a more stable molecule and is transported more efficiently across cellular membranes, thus having potentially significant advantage over BH4 as a pharmacological agent for diseases associated with BH4-deficient conditions. We report the results of a first-in-humans, randomized, double-blind, placebo-controlled, dose-ranging, Phase I clinical trial in 83 healthy volunteers of CNSA-001, a novel formulation of sepiapterin. Single oral doses of 2.5-80 mg/kg CNSA-001 caused dose-related increases in plasma sepiapterin (mean Cmax 0.58-2.92 ng/mL) and BH4 (mean Cmax 57-312 ng/mL). Maximum plasma concentrations were achieved in about 1-2 h (sepiapterin) or about 4 h (BH4) after CNSA-001 oral intake. Increases in plasma BH4 were substantially larger in absolute terms and on a dose-for-dose basis following treatment with CNSA-001 vs. sapropterin dihydrochloride, a synthetic form of BH4. The pharmacokinetics of plasma sepiapterin and BH4 were similar before and after seven days of repeat daily dosing with CNSA-001 at 5, 20 or 60 mg/kg indicating little or no drug accumulation. Oral administration of CNSA-001 resulted in higher concentrations of sepiapterin in fasted vs. fed subjects, but overall BH4 plasma exposure following CNSA-001 intake increased by 1.7-1.8-fold in fed subjects. CNSA-001 was well tolerated, with no clear dose-relationship for adverse events (AE), no serious AE and no study discontinuations for AE. These data indicate that CNSA-001 is rapidly and efficiently converted to BH4 in humans supporting further clinical evaluation of CNSA-001 for the management of PKU, primary BH4 deficiencies and other diseases associated with deficient BH4 metabolism.

Targeting the tumor-associated folate receptor with an 111In-DTPA conjugate of pteroic acid.

The cell membrane folate receptor is a potential molecular target for tumor-selective drug delivery. To probe structural requirements for folate receptor targeting with low molecular weight radiometal chelates, specifically the role of the amino acid fragment of folic acid (pteroylglutamic acid) in mediating targeting selectivity, the amide-linked conjugate pteroyl-NHCH(2)CH(2)OCH(2)CH(2)OCH(2)CH(2)NH-DTPA was prepared by a three-step procedure from pteroic acid, 2,2'-(ethylenedioxy)-bis(ethylamine), and t-Bu-protected DTPA. This conjugate, 1-{2-[2-[(2-(biscarboxymethyl-amino)ethyl)-carboxymethyl-amino]ethyl]-carboxymethyl-amino}-acetylamino-3,6-dioxa-8-pteroylamino-octane (1), was employed for synthesis of the corresponding (111)In(III) radiopharmaceutical. Following intravenous administration to athymic mice, the (111)In complex of 1 was found to selectively localize in folate receptor-positive human KB tumor xenografts and to afford prolonged tumor retention of the (111)In radiolabel (5.4 +/- 0.8, 5.6 +/- 1.1, and 3.6 +/- 0.6% of the injected dose per gram of tumor at 1, 4, and 24 h, respectively). The observed tumor localization was effectively blocked by co-administration of folic acid with the (111)In-1 complex, consistent with a folate receptor-mediated targeting process. In control studies, tumor targeting with this pteroic acid conjugate appears as effective as that seen using (111)In-DTPA-folate, a radiopharmaceutical that has progressed to clinical trials for detection of folate receptor-expressing gynecological tumors.

Chronic oral supplementation with sepiapterin prevents endothelial dysfunction and oxidative stress in small mesenteric arteries from diabetic (db/db) mice.

We previously reported that acute incubation with tetrahydrobiopterin (BH4) or sepiapterin, a cofactor for endothelial nitric oxide synthase and a stable precursor of BH4, respectively, enhanced the acetylcholine (Ach)-induced relaxation of isolated small mesenteric arteries (SMA) from diabetic (db/db) mice. In this study, we investigated the effect of chronic oral supplementation of sepiapterin (10 mg x kg-1 x day-1) to db/db mice on endothelium function, biopterin levels and lipid peroxidation in SMA. Oral dietary supplementation with sepiapterin had no effect on glucose, triglyceride, cholesterol levels and body weight. SMA from db/db mice showed enhanced vascular reactivity to phenylephrine, which was corrected with sepiapterin supplementation. Furthermore, Ach, but not sodium nitroprusside-induced relaxation, was improved with sepiapterin supplementation in db/db mice. BH4 levels and guanosine triphosphate cyclohydrolase I activity in SMA were similar in db/+ and db/db mice. Sepiapterin treatment had no effects on BH4 or guanosine triphosphate cyclohydrolase I activity. However, the level of dihydrobiopterin+biopterin was higher in SMA from db/db mice, which was corrected following sepiapterin treatment. Thiobarbituric acid reactive substance, malondialdehyde, a marker of lipid peroxidation, was higher in SMA from db/db mice, and was normalized by sepiapterin treatment. These results indicate that sepiapterin improves endothelial dysfunction in SMA from db/db mice by reducing oxidative stress. Furthermore, these results suggest that decreased biosynthesis of BH4 may not be the basis for endothelial dysfunction in SMA from db/db mice.

The effect of side-chain, para-aminobenzoyl region, and B-ring modifications on dihydrofolate reductase binding, influx via the reduced folate carrier, and cytotoxicity of the potent nonpolyglutamatable antifolate N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L- ornithine.

N(alpha)-(4-Amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-o rnithine (PT523) is an unusually tight-binding dihydrofolate reductase (DHFR) inhibitor and is efficiently taken up into cells via the reduced folate carrier (RFC). Unlike classical DHFR inhibitors with a glutamate side chain, such as methotrexate and aminopterin, PT523 cannot form polyglutamates. Thus, it resembles lipophilic antifolates such as trimetrexate in not requiring metabolic activation by folylpolyglutamate synthetase in order to produce its antifolate effect. However, in contrast to trimetrexate, PT523 retains growth inhibitory activity in cells with the multidrug resistance phenotype. As part of the preclinical development of this drug, we have performed systematic modification of several regions of the PT523 molecule, with the aim of defining the optimal structural features for DHFR binding, influx into cells via the RFC, and the ability to inhibit cell growth. The following structure-activity correlations have emerged from this ongoing investigation, and are discussed: (1) the hemiphthaloylornithine side chain has the optimal length; (2) the preferred location of the aromatic carboxyl group is the ortho position; and (3) replacement of the phenyl ring of the para-aminobenzoic acid moiety by naphthalene, of nitrogen at the 10-position of the bridge by carbon, and of nitrogen at the 5- and/or 8-position of the B-ring by carbon are all well tolerated. Several of the second generation analogs of PT523 are more potent DHFR inhibitors and better RFC substrates than PT523 itself, and are more potent inhibitors of tumor cell growth in culture.

Pharmacokinetics, antifolate activity and tissue distribution of PT523 in SCC VII tumor-bearing mice.

PURPOSE: To monitor the pharmacokinetics of PT523 and methotrexate in C3H mice with transplanted SCC VII tumors; to compare the impact of PT523 and methotrexate on tumor and normal host 5,10-methylenetetrahydrofolate levels; and to synthesize [14C]PT523 and determine its time-dependent tissue distribution in tumor and host tissues. METHODS: C3H mice bearing SCC VII tumors were given i.p. PT523 or methotrexate. Plasma drug levels and tumor, gut and marrow 5,10-methylenetetrahydrofolate were assayed. [14C]PT523 was synthesized and administered i.v. to tumor-bearing mice for tissue distribution analysis. RESULTS: Areas under the curve, mean residence times, whole body clearances, apparent distribution volumes, and plasma protein binding of PT523 vs methotrexate were, respectively, 4311 vs 6472 microM x min(-1); 20 vs 16 min; 0.56 vs 0.36 ml min(-1); 532 vs 325 ml x kg(-1); and 70% vs 30%. Both PT523 and methotrexate caused time-dependent declines in 5,10-methylenetetrahydrofolate in tumor and marrow, but not in gut mucosa [corrected]. Gut levels began to recover within 4 h in the PT523-treated group only. [14C]PT523 distributed mainly into the liver, duodenum, kidneys, lungs, tumor, pancreas and muscle; less into the spleen, blood cells, heart, brain and testicles; and very little into gut [corrected. Only 35% of the dose was excreted, and 2.9-fold more in feces than urine. CONCLUSIONS: Despite its more rapid clearance, accumulation of PT523 in extravascular tissues was greater than that of methotrexate. Consequently, less PT523 was recovered in feces and urine and its apparent volume of distribution was greater. PT523 selectively depleted 5,10-methylenetetrahydrofolate pools in tumor and, less persistently, in marrow, but spared the gut mucosa [corrected]. [14C]PT523 tissue distribution correlated with organ mass and blood supply.

Neurochemical effects following peripheral administration of tetrahydropterin derivatives to the hph-1 mouse.

The hph-1 mouse which displays tetrahydrobiopterin deficiency and impaired dopamine and serotonin turnover, has been used to study cofactor replacement therapy for disorders causing brain tetrahydrobiopterin deficiency. Subcutaneous administration of 100 mumol/kg (30 mg/kg) of tetrahydrobiopterin resulted in a twofold increase in brain cofactor concentration 1 h after administration. Concentrations remained above the endogenous level for at least 4 h but returned to normal by 24 h. The lipophilic tetrahydrobiopterin analogue 6-methyltetrahydropterin entered the brain five times more efficiently than tetrahydrobiopterin but was cleared at a faster rate. Tetrahydropterins linked to the lipoidal carrier N-benzyl-1, 4-dihydronicotinoyl did not result in a detectable increase in levels of brain pterins over the period of the study (1-4 h). Stimulation of monoamine turnover was not observed at any time point with either natural cofactor or the methyl analogue. Increasing the amount of tetrahydrobiopterin to 1,000 mumol/kg resulted in elevation of cofactor concentrations, a brief increase in the activity of tyrosine and tryptophan hydroxylase 1 h postadministration, and increased turnover of dopamine and serotonin metabolites lasting 24 h. However, 2 of 12 (17%) mice died following administration of this dose of cofactor. Our findings suggest that acute peripheral tetrahydrobiopterin administration is unlikely to stimulate brain monoamine turnover directly unless very large and potentially toxic doses of cofactor are used.

Dihydrofolate reductase binding and cellular uptake of nonpolyglutamatable antifolates: correlates of cytotoxicity toward methotrexate-sensitive and -resistant human head and neck squamous carcinoma cells.

Several mechanisms have been demonstrated to be independently involved in methotrexate (MTX) resistance, including increased dihydrofolate reductase (DHFR) activity, decreased membrane transport, and decreased conversion to noneffluxing polyglutamates by folylpolyglutamate synthetase. We conducted the present study to test the hypothesis that nonpolyglutamatable antifolates with an N delta-hemiphthaloyl-L-ornithine side chain could be more potent than MTX against MTX-sensitive and -resistant human carcinoma cells via tighter DHFR binding, more efficient cellular uptake, the ability to bypass defective polyglutamation, or a combination. Two nonpolyglutamatable antifolates, N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine (PT523) and the new B-ring analogue N alpha-[4-[N-(2,4-diamino-5-chloroquinazolin-6-yl)methyl]amino] benzoyl-N delta-hemiphthaloyl-L-ornithine (PT619), were tested as inhibitors of purified recombinant human DHFR and were found to bind somewhat better to the enzyme than MTX as determined by competitive radioligand binding assay. PT523 and PT619 were 9- and 14-fold, respectively, more active than MTX as inhibitors of parental SCC25 human and neck squamous carcinoma cell growth in 72-hr cultures. Moreover, there was an even greater increase in relative potency against two previously described MTX-resistant cell lines with an increased DHFR content and a decreased ability to convert MTX to polyglutamates: SCC25/R1 (selected with MTX) and SCC25/CP (selected with cisplatin but collaterally resistant to MTX). Both PT523 and PT619 very efficiently inhibited [3H]MTX uptake by SCC25 cells in a 1-hr assay, with PT523 being 11-fold more potent and PT619 being 17-fold more potent than MTX. Greater inhibition of [3H]MTX uptake with PT523 and PT619 than with MTX was also observed in SCC25/R1 and SCC/CP cells. However, the increase in activity of PT523 and PT619 relative to MTX in uptake experiments was less than that in growth-inhibition assays, especially for SCC25/CP cells. This suggested that additional cytotoxicity determinants may exist in these resistant cells.

The transport of pteridines in CCRF-CEM human lymphoblastic cells.

The transport routes used by CCRF-CEM human lymphoblastoid cells for the influx and efflux of unconjugated pteridines were analyzed using [3H]6-hydroxymethylpterin as a model compound. Influx proceeds by a mechanism that exhibits a Km of 66.7 microM and a Vmax of 0.077 nmol/min per mg cellular protein. The process is somewhat sensitive to metabolic inhibitors, particularly uncouplers of oxidative phosphorylation, and is significantly affected by the presence of other pteridines in the extracellular medium. The results suggest that pterins with either no 6-substituent (pterin) or those with methyl, hydroxyl, or formyl groups in this position, which exhibit Ki values between 25 and 77 microM, may share the same pathway for uptake. 6-Carboxypterin exhibits low affinity for the system (Ki greater than 500 microM), as do 7-substituted and 6,7-di-substituted derivatives and compounds with larger groups at the 6-position, such as neopterin and biopterin (Ki = 250-300 microM). Efflux of [3H]6-hydroxymethylpterin occurs rapidly and can proceed by at least two routes. The first, comprising approximately 50% of total efflux, is inhibited by extracellular pterins and exhibits similar properties to the uptake system in both its pattern of sensitivity to metabolic inhibitors and its specificity for pteridine structure. The route by which the remaining efflux occurs is relatively insensitive to metabolic inhibition. Adenine significantly inhibits 6-hydroxymethylpterin influx and efflux (Ki = 10.6 microM for uptake) but does not appear to share the same transport system. Similarly, methotrexate and folic acid exhibit little affinity for the unconjugated pteridine transport routes.