Sapropterin dihydrochloride, (6R
)-L-erythro-5,6,7,8-tetrahydrobiopterin dihydrochloride (BH4
·2HCl), referred also as sapropterin, is a synthetic version of the naturally occurring 6R
, which is, among others, a cofactor of phenylalanine-, tyrosine-, and tryptophan hydroxylases. Sapropterin is the active ingredient of the drug Kuvan®
, a medicine approved by the Food and Drug Administration (FDA) in 2007 and the European Medicines Agency (EMA) in 2008, for the treatment of hyperphenylalaninaemia (HPA), a rare disease caused by defects in the phenylalanine hydroxylase (PAH) [1
] or by a defect in the biosynthesis or recycling of BH4
. Mutations in the phenylalanine hydroxylase gene (PAH) lead to phenylketonuria (PKU) which, depending on the seriousness of the mutations, can be mild (Phe levels 300–600 µM), moderate (Phe levels 600–1200 µM), or severe (Phe levels >1200 µM). On another side, defects in BH4
synthesis or recycling lead to BH4
deficiency. The latter occurs in only around 2% of the HPA patient population [2
]. In Europe, patients who are found to respond to a seven-day BH4
loading test (30% decrease of phenylalanine level in the bloodstream), could benefit from a life-long treatment with Kuvan®
, at a daily dose of between 5 and 20 mg/kg [3
Sapropterin is a relatively complex and unstable molecule, whose synthesis is made difficult by the presence of three adjacent asymmetric centers. Until 1999, sapropterin was obtained as a mixture of 6R
diastereomers in a 69:31 ratio. However, only the 6R
isomer is biologically active, the 6S
isomer has been reported to cause an irreversible inactivation of rat liver PAH [4
]. HPA commonly requires life-long treatment; therefore, the medicine must be of the highest quality and stability in order to avoid the ingestion and accumulation of potentially dangerous impurities over the patient’s life. Over the years, some papers have raised doubts about the quality of some generic drugs, which are indicated to be of lower quality compared to their branded counterparts [5
]. Such papers prompted FDA Commissioner, Dr Scott Gottlieb, and the Center for Drug Evaluation and Research (CDER) Director, Dr Janet Woodcock, to publish a statement to deny any alleged lower safety and/or effectiveness of FDA approved generics versus their corresponding branded drugs [6
]. Notwithstanding this initiative, the feeling that branded drugs cannot indiscriminately be exchanged by their corresponding generics, is widely spread among practitioners and some patient communities.
The aim of the present study was to experimentally verify this perception by investigating the composition and stability of the life-long treatment sapropterin-containing drug Kuvan® and its generic sapropterin Dipharma (Diterin®). Furthermore, our data, based on HPLC-UV and LC-high resolution mass spectrometry (LC-HRMS) analysis, moved a step forward in providing the most detailed profile available to date of the impurities present in sapropterin-containing products, leading to the identification of nine by-products.
Sapropterin is an effective agent in lowering blood hyperphenylalaninaemia (HPA) in patients with phenylketonuria (PKU) or BH4 deficiency, and, to date, no other natural or synthetic agent has demonstrated the same efficacy. Sapropterin is formulated for oral administration in tablets (or powder in sachets) that are dissolved in water prior to administration. The active pharmaceutical ingredient (API) is obtained synthetically through a somewhat complex route, with the main difficulty residing in controlling the configuration of three asymmetric centers. The processes giving rise to the API present in Kuvan® and Diterin®, enable the obtention of the 6R form with high purity, although very small amounts of the undesired 6S stereoisomer may be present. It is to be noted that the 6S form is not only inactive, but it may cause inactivation of phenylalanine hydroxylase.
Sapropterin is a relatively unstable compound and its degradation chemistry is very complex, mostly ascribable to the sensitivity of side chain and ring moiety to oxidation. Tautomeric equilibria on the oxidized products render even more complex a comprehensive understanding of the exact formation and quantification of all the impurities. It has been estimated that in a sample of sapropterin left in the open, traces of biopterins 3
will start to appear after only 15 min incubation, and after 30 min, the amount of sapropterin is halved [11
]. To minimize the impact of these reactions, sapropterin-based tablets contain the antioxidant ascorbic acid.
As with any other medicine, sapropterin tablets can contain minor impurities deriving from their synthetic procedure as well as products formed upon degradation of the API itself. The aims of this work were: (a) to identify a separation method able to efficiently separate and quantify as many impurities as possible; (b) to apply this method to evaluate the impurities of commercially available sapropterin-containing tablets; and (c) to compare the impurity profile of generic and branded sapropterin-containing tablets, and finally evaluate them over time. To our knowledge, the method reported above, based on the use of an ion-exchange HPLC column, is one of the most efficient reported to date, as it enables the efficient separation and detection of nine impurities/degradation products from sapropterin-containing tablets.
Data reported in Table 2
, evidence that the tablets contain an impurity percentage concentration ranging from 0.3% to 0.7%. If we had also taken into account the stabilizer ascorbic acid and the other excipients (mainly mannitol), the impurity percentage concentration would have been obviously further lowered. Figure 3
schematically depicts the trend of impurity concentration for the examined products, when stored at 40 °C and 75% RH. As expected, an increase in the impurity level is observed over time for both tested commercial products, which can be clearly ascribed to an increase in the concentration of degradation products. The detailed trend of impurities is complex, and its full rationalization is extremely difficult; however, a more detailed analysis of the behavior of some selected impurities can better clarify this point.
The concentration of compound 1 is practically constant over the time and this could be explained by its origin as a synthetic impurity of the active principle sapropterin. The assignment of this peak fully supports this hypothesis since it has been assigned as biopterin, a key intermediate in the syntheses of sapropterin. The average level of this impurity is in the same range for both Kuvan® and Diterin®.
reports a likely, although necessarily simplified, conversion scheme that could help to rationalize the formation of the other compounds. Biolumazines (6
) are the products of hydrolysis of sapropterin (or of its epimer), resulting in the substitution of the amino group on the pyrimidine ring with a hydroxy group and subsequent tautomerism. Dihydrobiopterins 3
are regioisomeric oxidation products of sapropterin [14
], and the trend of their concentrations is interesting. The concentration of 3
decreased over the time, probably because this compound was further oxidized to sepiapterin (2
), thus concurring to the marked increase at T
= 6 of 2
. In our experiments, at T
= 6, sepiapterin 2
was the major impurity in both products, accounting for 0.169% in Diterin®
and 0.221% in Kuvan®
. In Diterin®
sepiapterin concentration actually increased more than 4-fold, meanwhile 7,8-dihydrobiopterin (3
) decreased 3-fold. It was interesting to note that the same trend, even if little bit more accentuated, was found in Kuvan®
. On the other hand, direct oxidation of sapropterin to 4
caused an increase of this degradation product at T
= 6 (2-fold more intense as in Kuvan®
), although, in turn, the oxidation of compound 4
could provide a further source of pterin (5
) and biopterin (1
). Impurity 5
showed a marked increase at T
= 6 in Kuvan®
, while only a small increase was noticed in Diterin®
. In Kuvan®
, a parallel decrease of biopterin (1
) at T
= 6 was observed, therefore a conversion of 1
may be hypothesized in this sample.
A highly undesired impurity is (6S
)-sapropterin epimer (8
), which is capable of irreversibly inactivating the PAH enzyme [4
]. Although both products contained a very little amount of this undesired side-product, after six months of storage in accelerated conditions, Diterin®
demonstrated a roughly 50% inferior amount of 8
with respect to Kuvan®
. Finally, 5,6,7,8-tetrahydropterin (9
) could derive from a direct cleavage of the side chain of sapropterin and this impurity is present at T
= 6 in higher concentrations in Diterin®
than in Kuvan®
. Grouping together all the impurities, we can observe that Kuvan®
ranges from 0.389% (t
= 0) to 0.694% (t
= 6) (78% increase), while Diterin®
ranges from 0.285% (t
= 0) to 0.395% (t
= 6) (39% increase).
In conclusion, data from an efficient HPLC-based analysis, able to separate and detect nine impurities/degradation products, showed an overall lower level of impurities/degradation products and higher stability of the sapropterin-containing generic tablets Diterin® formulation, which mostly relate to a lower increase of impurities 2, 4 and 6, compared to Kuvan®. The lower increase of the efficacy-reducing impurity 8, is also worthy of being mentioned, because although it is numerically less significant, it is potentially clinically significant.
We are well aware that a detailed comparison between the two commercial products would require further investigation; it is, nevertheless, undisputable that both Kuvan® and Diterin® completely conform to the standard quality of the major regulatory authorities, even after six months storage in accelerated stability conditions.