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Blood, Vol. 113, Issue 14, 3154-3160, April 2, 2009
Of mice and men: an open-label pilot study for treatment of immune thrombocytopenic purpura by an inhibitor of Syk
Blood Podolanczuk et al.
113: 3154
Supplemental materials for: Podolanczuk et al
Syk Inhibition Assay
A Basotest assay (Orpegen, Germany) was used to assess levels of basophil degranulation as a functional marker of Syk inhibition. The assay was performed as previously described.9 Briefly, blood samples were drawn before a scheduled dose (trough) and the findings with the assay compared to those obtained at peak blood levels (1–2 hours after scheduled dose). Basophil degranulation, a Syk-dependent process, was measured as IgE+CD63+ cells by flow cytometry, as a marker of Syk activity. Subjects were run in duplicate and average counts taken whenever possible. To calculate the percent reduction in syk inhibition, % CD63+ cells after treatment were subtracted from % CD63+ cells before treatment, all divided by % CD63+ cells before treatment. Syk inhibition assay
The Syk inhibition assay only became available late in the course of the study. In order to assess the role of Syk inhibition in relation to platelet responses, all 9 of the 12 responders on treatment when the assay became available were tested using the basotest assay (Orpegen, Germany) for Syk inhibition. Non-responders and those discontinuing treatment because of toxicity were no longer on study drug at the time the assay was available and could not be tested. Fig. 5a shows the mean percentage reduction from immediately prior to dosing (presumed trough level of Syk inhibition) compared to peak levels of drug and therefore presumed peak degree of inhibition after dosing (peak) for sustained responders and non-sustained responders. Sustained responders had significantly less change from pre- to post-R788 dose (p<0.05), suggesting a more prolonged inhibition of Syk based on q12 hour dosing. Fig. 5b shows a weak correlation (r = −0.46) between median platelet count on study drug and reduction in Syk activity after a dose of R788 for the same patients. Two patients were re-assayed after a drug holiday of 3 days (Fig. 5c). The sustained responder had a 15% inhibition of Syk activity after this period of time, while the non-sustained responder only had a 3% reduction in Syk activity, suggesting a weak effect of the drug in this patient and that a parameter of response might be the degree of inhibition Syk, which was variable across patients but related to the platelet response. This is further corroborated by pk data for these patients. While the sustained responder had a 73% increase in pk levels post R788 dose with repeated twice daily dosing, after a 3-day drug holiday pk levels went up 792% one hour after taking R788 (figure 5c). The nonsustained responder had a slightly lower change in pk levels with repeated twice daily dosing (64%), and failed to show a significant increase in pk levels after the 3-day drug holiday, suggesting that individual mechanism of drug metabolism may be play a role in determining response in different patients.
Files in this Data Supplement:
- Table S1. Pre and post counts represent average counts for the 4 weeks preceding study start and 4 weeks after starting R788 (PDF, 11.8 KB)
- Figure S1. Syk activity (JPG, 74.6 KB)
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(A) Average percent reduction in Syk inhibition, as measured by percent CD63 expression, 1 hour after taking R788 as compared to immediately before ingesting R788 for sustained responders (n = 6) and non-sustained responders (n = 3); p<0.05. Percent reduction, reflecting the change in inhibition of Syk, was calculated using basotest assay for basophil degranulation where percent of IgE+CD63+ cells was measured by flow cytometry before and after R788dose. (B) Relationship between percent reduction in Syk activity and median platelet count on study for each patient assayed; n = 9; r = −0.47 showing that higher median platelet counts were seen in patients with less change in inhibition of Syk. The dotted line separates the two groups at changes more or less than 10% inhibition. (C) Subjects 1 and 9, representing one patient from each group (sustained responder and non-sustained responder, respectively), were assayed after stopping the study drug for 3 days. Values represent percent Syk activity, as measured by IgE+CD63+ cells, on study and after a 3 day drug holiday. Pharmacokinetic (PK) levels of the drug (in ng/mL) for each of the two patients were measured by Quest Pharmaceuticals can be found in the table below. A single dose of R788, following the drug holiday, achieved a degree of inhibition of Syk only in the sustained responder but was not nearly at the same level as the degree of inhibition seen with repeated twice daily dosing.
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