TransCon CNP, a Sustained-Release C-Type Natriuretic Peptide Prodrug, a Potentially Safe and Efficacious New Therapeutic Modality for the Treatment of Comorbidities Associated with Fibroblast Growth Factor Receptor 3–Related Skeletal Dysplasias

Vibeke Miller Breinholt; Caroline E. Rasmussen; Per Holse Mygind; Mads Kjelgaard-Hansen; Frank Faltinger; Ana Bernhard; Joachim Zettler; Ulrich Hersel

doi:10.1124/jpet.119.258251

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Fig. 1.
MAP following a single subcutaneous administration of either vehicle, CNP-38 (800 µg CNP/kg), or TransCon CNP (800 µg CNP/kg) to mice. Footnote: MAP recorded up to 48 hours postdose. Means (±S.E.M.). *Significant difference from vehicle P < 0.05. **Significant difference from vehicle P < 0.01. William’s test comparing treatment groups with vehicle control at each time point.
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Fig. 2.
SAP following a single subcutaneous administration of TransCon CNP and daily CNP-39 molecule to cynomolgus monkeys compared with vehicle control. Footnote: SAP recorded up to 48 hours postdose. (A) Means. (B) Baseline-adjusted means (±S.E.M.).
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Fig. 3.
Growth from baseline of body, tail, tibia, and ulna following weekly administration of TransCon CNP to monkeys (n = 4/group) compared with the daily CNP-39 molecule (positive control) and vehicle treatment (negative control); (A) for duration of study (26 weeks), and (B) comparing relative change from baseline (±95% CI) at week 26. Footnote: Estimates and test are derived from an analysis of covariance model, including treatment as a fixed effect and baseline value as a covariate. *Significant difference in absolute growth from negative control, P < 0.05. Neg.Ctrl, negative control; Pos.Ctrl, positive control.
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Fig. 4.
Histomorphometry imaging of the proximal tibial physis following weekly administration of (A) vehicle control compared with (B) TransCon CNP (100 µg CNP/kg per week) for 26 weeks to monkeys. Footnote: PZ, proliferative zone; HZ, hypertrophic zone; PZi, increased cellularity and width of proliferative zone; HZi, increased cellularity and width of hypertrophic zone.

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TABLE 1

CNP-38 conjugates used for in vivo and in vitro studies

CNP-38 Conjugate (Linker Type, Linker Attachment Site)	Description
5-kDa PEG-linker CNP-38 (permanent, Lys26)	Linear 5-kDa PEG carrier conjugated to the Lys26 side chain of CNP-38 via a permanent linker
5-kDa PEG-linker CNP-38 (permanent, N-terminus)	Linear 5-kDa PEG carrier conjugated to the N-terminus of CNP-38 via a permanent linker
4 × 10-kDa PEG-linker CNP-38 (permanent, Lys26)	4-Arm–branched 4 × 10-kDa PEG carrier conjugated to the Lys26 side chain of CNP-38 via a permanent linker^{^a}
2 × 20-kDa PEG-linker CNP-38 (transient, Lys26)	2-Arm–branched 2 × 20-kDa PEG carrier conjugated to the Lys26 side chain of CNP-38 via a TransCon linker^{^a}
4 × 10-kDa PEG-linker CNP-38 (transient, Lys26), TransCon CNP	4-Arm–branched 4 × 10-kDa PEG carrier conjugated to the Lys26 side chain of CNP-38 via a TransCon linker^{^a}
C^{^b}(10-kDa PEG-linker CNP-38)₄ (permanent, Lys26)	Four CNP-38 molecules conjugated to a 4-arm–branched 4 × 10-kDa PEG; one CNP-38 is attached via its Lys26 side chain and via a permanent linker to the distal end of each of the four PEG arms

↵^a The linker attachment site to the PEG carrier is close to the branching site of the PEG arms.
↵^b C represents the central carbon atom, which is the branching point of the four PEG arms.

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TABLE 2
Evaluation analysis parameters for XtremeCT data
Evaluation Ulna Growth Plate Evaluation Radius Metaphysis Evaluation Radius Diaphysis
Sigma 0.8 0.8 0.8
Support 1 1 1
Lower threshold 95 150 (total/cortical) and 110 (trabecular) 150
Upper threshold 1000 1000 1000

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TABLE 3

In vitro NEP resistance, activity, and NPR-C affinity of CNP-38 conjugates

CNP-38 Conjugate	NEP Resistance	Activity^{^a}	NPR-C Affinity^{^b}^,^{^c}
(Linker Type, Attachment Site)	Half-Life [h]	[%]	[%]
CNP-38	12.6 ± 3.9^{^d}	100	100
5-kDa PEG-linker CNP-38 (permanent, N-terminus)	Stable^{^e}	14	199 ± 12
5-kDa PEG-linker CNP-38 (permanent, Lys26)	65.4	<1^{^f}	83 ± 7
2 × 20-kDa PEG-linker CNP-38^{^g} (transient, Lys26)	ND^{^h}	<1^{^f}	11 ± 4
4 × 10-kDa PEG-linker CNP-38^ⁱ (permanent, Lys26)	Stable^{^e}	<1^{^f}	<2^{^f}
4 × 10-kDa PEG-linker CNP-38^ⁱ (transient, Lys26), TransCon CNP	ND	<1^{^f}	<1^{^f}^,^{^j}
C(10-kDa PEG-linker CNP-38)₄^{^k} (permanent, Lys26)	ND	ND	68 ± 11

↵^a Activity to elicit intracellular cGMP response in a NIH3T3 cell assay, relative to CNP-38.
↵^b n = 2 to 3.
↵^c Ratio IC₅₀ CNP-38/IC₅₀ CNP-38 conjugate.
↵^d n = 4.
↵^e Almost no degradation after 96 h.
↵^f Limitation due to assay range.
↵^g Branched 40-kDa PEG carrier having the CNP attachment site near the branching site of two 20-kDa arms.
↵^h ND, not determined.
↵ⁱ Branched 40-kDa PEG carrier having the CNP attachment site near the branching site of four 10-kDa arms.
↵^j Calculated from a 25× concentrated sample.
↵^k Branched 4 × 10-kDa PEG carrier having a total of four CNPs attached. The CNP attachment site is not close to the branching site of the four 10-kDa arms, but at the distal end of each of the arms.

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TABLE 4
Mean pharmacokinetic parameters of CNP (after first dose) released from TransCon CNP following administration of TransCon CNP compared with the daily CNP-39 molecule in monkeys
Group Dose (µg CNP/kg) T_max^{^a} (h) C_max (pmol/l) AUC_(0-t) (h × pmol/l) T_1/2 (h)
Daily CNP-39 molecule 20/day 0.25 3850 2560 0.35
TransCon CNP 40/wk 48 16.5 1748 92.6
TransCon CNP 100/wk 24 39.4 4186 86.7
AUC, area under the curve; T_1/2, half-life; T_max, time to maximum plasma concentration.
↵^a Median T_max is presented.

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TABLE 5

Bone length [millimeters [95% confidence interval] (% change from control)] determined by X-ray after 5 wk of daily bolus injection or continuous infusion of CNP-38 or vehicle control in FVB mice

	Subcutaneous Bolus Injection		Subcutaneous Continuous Infusion
Measurement	Vehicle Control	CNP-38 203 µg/kg Per Day	Vehicle Control	CNP-38 203 µg/kg Per Day
Femur length^{^a}	14.2 [13.7; 14.8]	15.0 [14.5; 15.6] (5.5%)^*	13.6 [13.0; 14.1]	14.6 [14.1; 15.2] (7.1%)^*
Tibia length^{^b}	17.9 [17.7; 18.1]	18.7 [18.5; 18.9] (4.0%)^*	17.6 [17.4; 17.8]	19.8 [19.6; 20.0] (12.2%)*^,#
Spine length^{^c}	53.8 [52.4; 55.2]	59.8 [58.5; 61.2] (11.3%)^*	53.6 [52.2; 55.0]	67.0 [65.6; 68.4] (25.0%)*^,#

↵^a Right femur.
↵^b Right tibia.
↵^c Lateral view.
↵^* Different from vehicle control; ^#different from growth under subcutaneous bolus injection (one-factor ANOVA model on logarithmic transformed data); P < 0.05.

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TABLE 6
Percent increase in bone formation markers BAP and PINP following weekly TransCon CNP or daily CNP-39 molecule administration for 26 wk to cynomolgus monkeys
TransCon CNP 40 µg CNP/kg Per Week TransCon CNP 100 µg CNP/kg Per Week Daily CNP-39 Molecule 20 µg/kg Per Day
No. animals per group 4 4 4
BAP — 14^{^a} 51^{^a}
PINP — 53^{^a} 144^{^a}
↵^a Significantly different from control group; P < 0.05, based on statistical analysis of group means.

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TABLE 7

Estimated relative effects of CNP treatment on growth of body, tail, tibia, and ulna (relative increase in growth compared with vehicle treatment [% (95% confidence interval) in monkeys]

	TransCon CNP 40 µg CNP/kg Per Week	TransCon CNP 100 µg CNP/kg Per Week	Daily CNP-39 Molecule 20 µg CNP/kg Per Day
Body	1.0 (−2.3; 4.4)	4.8 (1.1; 8.5)^*	3.3 (0.0; 6.6)^*
Tail	1.4 (−2.7; 5.5)	8.6 (3.7; 13.4)^*	2.8 (−1.3; 6.9)
Tibia	1.7 (−2.0; 5.4)	6.0 (2.1; 9.9)^*	2.9 (−1.0; 6.8)
Ulna	0.9 (−2.8; 4.7)	2.7 (−1.1; 6.5)	0.8 (−2.9; 4.5)

↵^* P < 0.05.

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TABLE 8

Summary of XtremeCT values (mean ± S.D.) as percentage change from acclimation period (n = 4) after weekly subcutaneous administration of TransCon CNP or daily CNP-39 molecule for 26 wk

Treatment		Control	Daily CNP-39 Molecule	TransCon CNP
CNP Dose		Vehicle	20 µg/kg Per Day	40 µg/kg Per Week	100 µg/kg Per Week
Right Radius, Distal Metaphysis—Trabecular Region
Trabecular region vBMD	mg HA/cm3	19.8 ± 10.3	18.2 ± 14.9	27.0 ± 17.9	16.5 ± 10.7
Trabecular tissue BMD	mg HA/cm3	−0.27 ± 2.1	0.45 ± 1.8	−0.12 ± 1.1	1.7 ± 0.83
Trabecular BV/TV	%	18.2 ± 8.2	18.7 ± 15.0	29.9 ± 19.2	18.2 ± 12.4
Trabecular number	1/mm	13.2 ± 11.6	5.8 ± 6.0	12.9 ± 8.8	8.9 ± 5.3
Trabecular thickness	mm	4.6 ± 3.7	8.9 ± 6.7	16.2 ± 14.5	14.0 ± 11.8
Trabecular separation	mm	−14.6 ± 8.4	−5.9 ± 7.0	−12.0 ± 9.0	−10.5 ± 5.0
Right Radius, Distal Metaphysis—Cortical Region
Cortical region vBMD	mg HA/cm3	−5.2 ± 1.2	−7.0 ± 2.2	−8.7 ± 2.9	−2.7 ± 2.3
Cortical tissue BMD	mg HA/cm3	−3.8 ± 1.4	−5.9 ± 1.6	−5.9 ± 1.2	−2.4 ± 1.8
Cortical thickness	mm	−3.6 ± 16.0	−9.4 ± 11.2	−16.7 ± 11.5	4.5 ± 10.7
Right Radius, Distal Diaphysis—Cortical Region
Cortical region vBMD	mg HA/cm3	−1.8 ± 1.6	−3.3 ± 1.6	−7.8 ± 9.8	−2.4 ± 2.3
Cortical tissue BMD	mg HA/cm3	−4.8 ± 1.9	−6.7 ± 1.4	−6.3 ± 3.6	−3.7 ± 1.6
Cortical thickness	Mm	7.0 ± 3.0	8.2 ± 5.0	1.1 ± 8.4	8.9 ± 4.7

BMD, bone mineral density; BV, bone volume; TV, tissue volume.

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TABLE 9

Histomorphometry data of the proximal tibial growth plate following weekly administration of TransCon CNP compared with the daily CNP-39 molecule for 26 wk to monkeys (% change from vehicle control)

		Treatment
	TransCon CNP 40 µg CNP/kg Per Week	TransCon CNP 100 µg CNP/kg Per Week	Daily CNP-39 Molecule 20 µg/kg Per Day
No. animals per group	4	4	4
Bone, tibia (no. measured)	4	4	4
Proliferative zone width	−2	37^{^a}	16^{^a}
Hypertrophic zone width	7	38^{^a}	39^{^a}
Epiphyseal plate thickness	−4	16^{^a}	7