Biphalin (Tyr-D-Ala-Gly-Phe-NH-NH<-Phe<-Gly<-D-Ala<-Tyr) is an opioid octapeptide with a dimeric
structure based on two identical pharmacophore portions, derived from enkephalins, joined “tail to tail” by a hydrazide
bridge. This particular structure enhances the antinociceptive activity of the native enkephalins with an unknown
mechanism, probably based on a cooperative binding and improved enzymatic stability. Biphalin has excellent binding
affinity for μ and δ receptors and it is a highly potent analgesic, as potent as or more than ethorphine. A definitive
explanation of the extraordinary in vivo potency shown by this compound, which has pronounced efficacy in pain
modulation, is still not available; it has been suggested, however, that the high agonist activity may be related to its
binding mode at both μ and δ opioid receptors. Biphalin has significantly higher potency than other analgesics with novel
biological profiles; in particular, most recent data show that biphalin is unlikely to produce dependency in chronic use. In
the past 20 years, there have been many attempts to modify its structure to obtain products unaffected by the action of
enkephalinases, to enhance its antinociceptive activity and to modify the BBB penetration. In addition, structure-activity
relationship studies (SAR) were performed in order to understand the elements responsible for biphalin’s high activity.
The aim of the studies reported in this review was to clarify: i) the role of the hydrazide bridge, ii) the role of residues in
position 4, 4’ and 3, 3’, iii) the consequences of molecular simplifications (truncation, delection), iv) the consequences of
cyclization through a disulfide bridge, v) conjugation with PEG and fluorescet residues, and vi) radiolabeling on Tyr1.