GM2 gangliosidosis (GM2), (MIM 230700), is an inherited neurodegenerative disorder characterized by progressive deterioration of motor, cerebral and spinocerebellar function caused by deficiency of lysosomal β-hexosaminidase A (Hex A). Normal human tissues contain two major β-hexosaminidase (Hex) isozymes, Hex A and Hex B. Hex A is a heterodimer made up of non-identical α and β subunits, encoded by two evolutionarily related genes, HEXA
, respectively. Hex B is a homodimer made up of two identical β-subunits. A third minor, unstable Hex isozyme, Hex S, is a dimer of α-subunits and is only detected unequivocally in tissues from patients with the Sandhoff disease variant (SD) (MIM 268800) of GM2. SD results from HEXB
mutations producing a deficiency of functional β-subunits and both Hex A (αβ) and Hex B (ββ) activities. On the other hand, Tay–Sachs disease (TSD; MIM 272800) is caused by HEXA
mutations resulting in a deficiency of α-subunits, and Hex A (αβ) activity, but normal levels of Hex B (reviewed in [1
]). Deficiency of the non-catalytic GM2 activator protein, a substrate-specific cofactor for Hex A, results in the third very rare AB-variant form of GM2.
GM2 is characterized by a marked clinical heterogeneity. The most severe and devastating forms are the infantile or acute variants of TSD and SD, associated with <0.5% of normal Hex A activity. It is characterized by rapid neurodegeneration, culminating in death in infancy. By contrast, the late-onset variants, which are subdivided into juvenile or sub-acute and adult or chronic forms, are associated with residual Hex A activities 2–4% of normal. Asymptomatic individuals have been described with as low as 10% of normal Hex A activity, i.e. pseudo-deficiencies. Patients with juvenile GM2 usually present with evidence of neurodeterioration starting after one year of age, and experience a slower rate of progression than patients with the infantile forms. Patients with adult-onset disease may present with spinocerebellar, psychiatric and/or peripheral neuropathies, which do not significantly affect longevity in some cases (reviewed in [1
]). The rate of disease progression and severity has been found to correlate roughly with the level of residual Hex A activity: generally, clinical disease does not develop unless residual Hex A activity drops below a critical threshold of 5–10% of normal, as measured in patient fibroblasts. Thus, only a low level of residual Hex A activity is apparently needed to prevent or reverse substrate-storage in these conditions [3
], i.e. 1.5- to 3-fold enhancement of Hex A levels in late onset patients.
Pharmacological chaperones (PC) are small molecules which are often also competitive inhibitors of their target enzyme. Thus PCs bind to and stabilize the “native” folded conformation of the protein. This results in a functional, active enzyme, unless the mutation affects a functional residue in the enzyme; e.g. αR178H [4
] associated with the B1-variant of TSD [5
]. Arg178 in the α-subunit is directly involved in first binding the substrate and then in stabilizing the reaction intermediate [6
]. Once formed into its native structure, the enzyme ceases to be a substrate for the endoplasmic reticulum (ER)-associated degradation pathway (ERAD) and is transported to lysosomes [7
]. In lysosomes it is believed that the stored substrate will displace the PC and continue to stabilize the enzyme. However, some PCs, like pyrimethamine (PYR), have the added advantage of inhibiting their target enzyme better at the neutral pH of the ER than at the acidic pH of lysosomes. Additional stability is gained once a lysosomal enzyme is folded correctly in the ER, because disulfide bonds are then formed [8
], and the mutant subunit assembles with a wild-type subunit to form the Hex A heterodimer. Dimerization involves an extensive subunit-subunit interface, ~2700 Å2
, which occurs between the catalytic domains of the two subunits, with several residues from one subunit structurally completing and stabilizing active site residues of the other [6
]. Stability is further increased upon lysosomal compartmentalization, because Hex A, like most lysosomal enzymes; e.g., glucocerebrosidase [9
], is more stable at acidic pH than at the neutral pH of the ER, i.e. the melting temperature of wild-type Hex A increases from 52 °C at pH 7.0 to 59 °C at pH 4.3 (Tropak et al., unpublished data). The PC approach has been shown to enhance the enzyme levels of five different mutant lysosomal enzymes causing chronic forms of the lysosomal storage diseases, GM2 gangliosidosis [4
], Fabry [11
], Gaucher [9
], and GM1 gangliosidosis [15
By screening a 1040-compound library of FDA approved drugs obtained from the National Institute of Neurological Diseases and Stroke (NINDS) for Hex inhibitors, we identified PYR as a μM competitive inhibitor of Hex and thus a potential PC [4
]. Pyrimethamine is a selective inhibitor of parasitic dihydrofolate reductase (folate antagonist) [http://www.rxlist.com/daraprim-drug.htm
]. It is approved for the treatment of chloroquine-resistant malaria and malaria prophylaxis and toxoplasmosis treatment. It is not generally recommended alone for the treatment of acute malaria, but is used for malaria prophylaxis in situations where the parasite is not resistant to the drug. It is used as a first-line treatment of toxoplasmosis, along with sulfonamides, particularly in immunocompromised individuals. The drug is used in dosages up to 75 mg per day in adults. The toxic side effects relate primarily to hypersensitivity reactions and the folate antagonist properties of the drug and are preventable by concomitant treatment with folinic acid.
The characterization of the relevant PC properties of the drug was performed using fibroblast cell-lines from TSD- or SD-variant patients with subacute (juvenile) and late-onset (adult) GM2. Each of the mutant cell lines from our patients’ responded differently to PYR [4
]. Recently an increased turn-over rate, comparable to the enhancement levels of Hex A, of a fluorescent derivative of GM2 ganglioside loaded into the lysosomes of adult Tay–Sachs fibroblasts treated with 12 μM (~3 mg/L) PYR has been demonstrated [17
]. However one potential problem is that PYR contains two primary amines that could, if high intra-lysosomal concentrations were reached, decrease lysosomal pH leading to increased secretion of lysosomal enzymes through inhibition of mannose-6-phosphate receptor recycling, i.e. a lysosomotropic effect. In tissue culture the level of PYR needed to produce such an effect was ~100 μM (~25 mg/L) [4
The present study was undertaken to examine the potential clinical benefit of the treatment of late-onset forms of both TSD- and SD-variants of GM2 gangliosidosis with doses of PYR similar to those used for the treatment of parasitic diseases, such as malaria. The protocol was designed as a Phase I/II clinical trial with a primary focus on the establishment of the tolerability of the treatment and indications of efficacy based on measurements of leukocyte Hex A activity, and the levels of PYR in patients’ plasma. Clinical assessments were done frequently, primarily to identify early evidence of adverse drug-related reactions. The clinical assessments were not sufficiently sensitive to identify any subtle clinical improvements in the patients during this short-term study.
All the subjects, both males and females, were 18 years of age or older. All had biochemically and genetically confirmed GM2 gangliosidosis caused by known HEXA or HEXB mutations, with clinical characteristics consistent with juvenile- or adult-onset disease. Each provided formal, signed consent for participation in the study, with the consent forms and protocol approved by the Institutional Review Boards of the Hospital for Sick Children (Toronto) and the New York University Medical Center (New York).
- biochemically and genetically confirmed diagnosis of GM2 gangliosidosis caused by β-hexosaminidase deficiency resulting from mutations in the HEXA or HEXB genes;
- having HEXA or HEXB mutations shown to be responsive to PYR in vitro;
- 18 years of age or over at the time of study initiation;
- able to understand and cooperate with the requirements of the study protocol;
- mentally competent, have the ability to understand and willingness to sign the informed consent form;
- able to travel to one of the two participating study sites;
- women of child-bearing potential must use accepted contraceptive methods and must have a negative serum or urine pregnancy test within one week prior to treatment initiation;
- fertile men must practice effective contraceptive methods during the study period, unless documentation of infertility exists;
- laboratory values≤2 weeks prior to beginning the trial must show adequate hematologic, hepatic, renal, and coagulation function; and body weight >40 kg.
- serious medical illness, significant cardiac disease or severe debilitating pulmonary disease;
- any hematologic abnormality, especially megaloblastic anemia, leukopenia, thrombocytopenia, pancytopenia;
- any active uncontrolled bleeding or any bleeding diathesis (e.g., active peptic ulcer disease);
- possible folate deficiency, and those receiving therapy (such as phenytoin) affecting folate levels;
- any complex disease that may confound treatment assessment;
- pregnant women or women of child-bearing potential not using reliable means of contraception;
- lactating females;
- fertile men unwilling to practice contraceptive methods during the study period;
- unwilling or unable to follow protocol requirements;
- known hypersensitivity reactions, intolerance or adverse reactions to PYR;
- evidence of active infection, or serious infection within the past month;
- HIV infection;
- a history of cancer of any type;
- receiving any other standard or investigational treatment for any indication within the past 4 weeks prior to initiation of PYR treatment;
- receiving immunotherapy of any type within the past 4 weeks prior to initiation of PYR treatment; or any condition or abnormality which may, in the opinion of the investigator, compromise the safety of patients.
The study was a two-center, open-label, study of 10 patients with late-onset forms of GM2 gangliosidosis, examining the effect of escalating doses of PYR to a maximum of 100 mg per day administered orally. All subjects received folinic acid, 5 mg per day, throughout the study. Tolerability was assessed by regular clinical assessments, as well as frequent measurements of a wide range of hematologic and biochemical parameters. Efficacy was evaluated by weekly measurements of leukocyte Hex A activity normalized to β-glucuronidase (Glcr) activity to compensate for variations in the quality of the leukocyte pellet preparation and to monitor for any non-specific lysosomotropic effects of the treatment. Additionally, levels of PYR and Glcr were measured in plasma samples in order to roughly assess the drug’s pharmacokinetics and as a secondary method of detecting any lysosomotropic effects of the treatment, respectively.
The clinical trial protocol is summarized in .
Outline of clinical trial protocol.