Novel hyaluronic acid–methotrexate conjugate suppresses joint inflammation in the rat knee: efficacy and safety evaluation in two rat arthritis models

Table 1 Release of MTX and MTX-α peptides from DK226

Treatment	pH	Released concentration (ng/mL) MTX MTX-α-Phe MTX-α-Phe-Phe			% of released MTX	% peak area of MTX-α-Phe after 24-h incubation
Synovial tissue homogenate	4.0	8.82	BLQ^b	BLQ^c	1.2	0^e
Synovial tissue homogenate	7.4	BLQ^a	BLQ^b	BLQ^c	0	100
Synovial fluid	4.0	1.14	2.02 (1.53)^d	1.29 (0.783)^d	0.5	80.1
Synovial fluid	7.4	BLQ^a	BLQ^a	BLQ^a	0	100

DK226 was incubated in rabbit synovial tissue homogenate or synovial fluid at pH 4.0 or pH 7.4 for 24 h at 37 °C. Release of MTX and MTX-α peptides was measured by LC/Q-TOF-MS. MTX-α-Phe was used as a reference compound to assess the metabolic stability, and the percent peak area of intact MTX-α-Phe and MTX released from MTX-α-Phe were monitored by LC-UV
MTX methotrexate, Phe phenylalanine, BLQ below lower limit of quantification
^aBLQ (below LLOQ); <1 ng/mL
^bBLQ; <5 ng/mL
^cBLQ; <10 ng/mL
^dMTX-equivalent concentration
^ePercent peak area of MTX-α-Phe after 3-h incubation

ISSN: 1478-6362