Of mice and men: an open-label pilot study for treatment of immune thrombocytopenic purpura by an inhibitor of Syk

Anna Podolanczuk, Alan H. Lazarus, Andrew R. Crow, Elliot Grossbard and James B. Bussel

Data supplements

  • 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) -
      (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.

Article Figures & Data


  • Figure 1

    Inhibition of Syk prevents murine antibody-mediated cytopenias. (A) CD1 mice were pretreated with nothing (Nil), vehicle, R788 (at the dose indicated), or IVIg on day 1. Mice in the vehicle and R788 groups were injected again (8 hours apart) on day 2. All mice except for the unmanipulated group were injected with antiplatelet antibody on day 2. Mice were bled on day 3 for platelet counting as detailed in “Methods.” n = 6 mice per group. (B) Mice were treated as in panel A, except that anemia was induced by injection of antierythrocyte (TER119) antibody. All data are expressed as mean plus or minus SEM (n = 6 mice per group).

  • Figure 2

    Study flowchart and patient response. (A) Study flowchart, with all patients enrolled in study as of January 31, 2008. Patient numbers correspond to panel B. (B) Graphical representation of response for each study patient at each study visit. At each visit every patient was classified as either responder (Y, black) or nonresponder (N, gray). Response was defined as platelet count increased by at least 20 × 109/L (20 000/mm3) to greater than 30 × 109/L (30 000/mm3) with no IVIg treatment in the preceding 2 weeks. Blank space indicates no study visit in a given week.

  • Figure 3

    Baseline, maximal, and median platelet counts for maintained responders, nonmaintained responders, nonresponders, and all patients in study. Maintained response is defined as platelet count increased by at least 20 × 109/L (20 000/mm3) to greater than 30 × 109/L (30 000/mm3) on at least 66% of the study visits. Nonmaintained response is defined as platelet counts increased by at least 20 × 109/L (20 000/mm3) to greater than 30 × 109/L (30 000/mm3) on less than 66% of the study visits. Nonresponders did not achieve any increases in platelet counts as per the above definition. Baseline platelet counts represent the lowest count within 1 month of study start. Maximum and average platelet counts were calculated as the median of peak and average counts, respectively, achieved by each patient in a given group.

  • Figure 4

    Sample platelet responses. Sample platelet response for maintained responder (A), nonmaintained responder (B), and nonresponder (C). No significant difference was observed in the baseline characteristics between the 3 groups. Embedded Image indicates dose increase; and Embedded Image, rescue treatment.


  • Table 1

    Baseline characteristics for 16 enrolled patients

    Baseline characteristicValue
    Median age, y (range)66 (31-81)
    Sex, n, male/female6/10
    Race, n
        Hispanic or Latino1
    Median duration of ITP, y (range)9 (1 to ≥ 29)
        n, median3
        At least 3 comorbidities, n (%)8 (50)
        History of ≥ 3 prior treatments, n (%)15 (94)
    Prior treatments, n (%)
        Steroids16 (100)
        IVIg15 (94)
        Rituximab14 (88)
        Splenectomy11 (69)
        Anti-D9 (56)
        Danazol8 (50)
        Azathioprine6 (38)
        Thrombopoietic agents5 (31)
        Vincristine5 (31)
        GMA1613 (19)
        Anti-CD40L2 (13)
        Helicobacter pylori1 (6)
    Median baseline platelet count, n × 109/L, (range)16 (2-28)
    • Baseline platelet count is defined as lowest count within 1 month of starting treatment. The most common comorbidities included hypertension (n = 5), dyslipidemia (n = 3), arthritis (n = 3), and diabetes (n = 3).

  • Table 2

    Platelet response of enrolled patients

    Patient IDVisits as responder, %*Visits with plt count > 30 × 109/L, %Visits with plt count > 50 × 109/L, %Visits IVIg given, %Baseline plt count, ×109/LMax plt count, ×109/LMed plt count, ×109/L§No. of dose changesSteroids tapered
    Maintained response
    Nonmaintained response#
    No response**
    • Subjects 1-3, 5, 6, 8-10, and 12-14 had undergone splenectomy; subjects 6, 8, 9, 13, and 16 had failed a thrombopoietic agent.

    • Plt indicates platelet; max, maximum; and med, median.

    • * Responder was defined as increase in platelet count by more than 20 × 109/L to greater than 30 × 109/L.

    • Baseline value is within 1 month before first dose of study drug.

    • Max value is while on study drug with no IVIg treatment in preceding 2 weeks.

    • § Median value is while on study drug with no IVIg treatment in preceding 2 weeks.

    • Steroids include prednisone, methylprednisolone, and danazol.

    • Platelets increased by greater than 20 × 109/L to greater than 30 × 109/L for at least 66% of visits.

    • # Platelets increased by greater than 20 × 109/L to greater than 30 × 109/L for less than 66% of study visits.

    • ** Platelets did not increase by greater than 20 × 109/L to greater than 30 × 109/L.

  • Table 3

    All adverse reactions reported by patients on study

    Adverse reactionPatients reporting reaction, nReaction probably related to R788
    SBP increased by > 10 mm Hg5Yes
    Weight gain > 5 kg3Yes
    Abdominal pain3Yes
    Shortness of breath3No
    ALT > 2× ULN2Yes
    Chest tightness1No
    Blurry vision1No
    Lung hemangioma1No
    • SBP indicates systolic blood pressure; ALT, alanine aminotransferase; ULN, upper limit of normal; UTI, urinary tract infection; DVT, deep vein thrombosis; and LAD, lymphadenopathy.