Preclinical and clinical in vitro in vivo correlation of an hGH dextran microsphere formulation

Abstract

Purpose: To investigate the in vitro in vivo correlation of a sustained release formulation for human growth hormone (hGH) based on hydroxyethyl methacrylated dextran (dex-HEMA) microspheres in Pit-1 deficient Snell dwarf mice and in healthy human volunteers.

Materials and methods: A hGH-loaded microsphere formulation was developed and tested in Snell dwarf mice (pharmacodynamic study) and in healthy human volunteers (pharmacokinetic study).

Results: Single subcutaneous administration of the microspheres in mice resulted in a good correlation between hGH released in vitro and in vivo effects for the hGH-loaded microsphere formulation similar to daily injected hGH indicating a retained bioactivity. Testing the microspheres in healthy volunteers showed an increase (over 7-8 days) in hGH serum concentrations (peak concentrations: 1-2.5 ng/ml). A good in vitro in vivo correlation was obtained between the measured and calculated (from in vitro release data) hGH serum concentrations. Moreover, an increased serum concentration of biomarkers (insulin-like growth factor-I (IGF-I), IGF binding protein-3 (IGFBP-3) was found again indicating that bioactive hGH was released from the microspheres.

Conclusions: Good in vitro in vivo correlations were obtained for hGH-loaded dex-HEMA microspheres, which is an important advantage in predicting the effect of the controlled drug delivery product in a clinical situations.

Fig. 1

Schematic overview of model used to predict the in vivo serum concentration of hGH.

Fig. 2

Cumulative release of hGH from DS 12 (closed squares, open squares), and 20 (closed triangles, open triangles) microspheres. The initial water content was 50% and the protein load was 3% (closed symbols) and 20% (open symbols). The values are the average of two independent measurements and deviated less than 10%.

Fig. 3

Cumulative release of hGH from dex-HEMA microsphere (DS 16, initial water content 50%, initial protein loading 10% w/w) batches prepared without homogenization (ATM1, Table II; closed squares) and with homogenization of the emulsion prior to polymerization (ATM2, Table II; closed triangles). ATM1 and ATM2 were used in the animal study. The values are the average of two independent measurements and deviated less than 15%.

Fig. 4

Cumulative release of hGH from dex-HEMA microspheres (DS 16, initial water content 50%, initial protein loading) used in the clinical study (CTM, Table II). The values are the average of two independent measurements and deviated less than 5%.

Fig. 5

Cumulative % increase in body length of dwarf mice during 4 weeks after sc injection of saline (open squares), hGH 8.3 μg/per day for 28 days (open triangles), ATM1 microspheres (closed squares) and ATM2 microspheres (closed triangles). Mean ± S.E. (n = 5). Curves are significant different (P < 0.05).

Fig. 6

aIn vitro in vivo correlation between cumulatively administered/released hGH (open symbols) and cumulative increase in body length (closed symbols) for hGH 8.3 μg/per day (open triangles, closed triangles), microspheres of ATM1 (open squares, closed squares) and microspheres ATM2 (open circles, closed circles) and b cumulatively administered/released hGH vs cumulative increase in body length for hGH 8.3 μg/per day (closed triangles), microspheres of ATM1 (closed squares) and microspheres ATM2 (closed circles).

Fig. 7

Binding of ¹²⁵I-hGH to hGH-antibodies present in sera of Snell dwarf mice having received a single injection microspheres of ATM1 (closed squares) and microspheres of ATM2 (closed triangles) and daily injection of saline (open squares) or daily injection of a solution containing 8.3 μg hGH-190 (open triangles).

Fig. 8

Measured hGH concentration in serum of human volunteers after administration of CTM microspheres, (closed triangles) and hGH concentration in serum (dotted line) calculated from the in vitro release of hGH using the model as described in the materials and methods. Mean ± S.E. (n = 10).

Fig. 9

Serum concentration IGF-1 after administration of CTM microspheres. Mean ± S.E. (n = 10).

Fig. 10

Serum concentration IGFBP-3 after administration of CTM microspheres. Mean ± S.E. (n = 10).