Friederike M. Mansfeld

Synthesis and biological evaluation of an orally active glycosylated endomorphin-1

The endogenous opioid peptide endomorphin-1 (1) was modified by attachment of lactose to the N-terminus via a succinamic acid spacer to produce compound 2. The carbohydrate modification significantly improved the metabolic stability and membrane permeability of 2 while retaining μ-opioid receptor binding affinity and agonist activity. Analogue 2 produced dose-dependent antinociceptive activity following intravenous administration in a chronic constriction injury (CCI) rat model of neuropathic pain with an ED(50) of 8.3 (± 0.8) μmol/kg. The corresponding ED(50) for morphine was 2.6 (± 1.4) μmol/kg. Importantly, compound 2 produced dose-dependent pain relief after oral administration in CCI rats (ED(50) = 19.6 (± 1.2) μmol/kg), which was comparable with that of morphine (ED(50) = 20.7 (±3.6) μmol/kg). Antineuropathic effects of analogue 2 were significantly attenuated by pretreatment of animals with the opioid antagonist naloxone, confirming opioid receptor-mediated analgesia. In contrast to morphine, no significant constipation was produced by compound 2 after oral administration.

Lipo-endomorphin-1 derivatives with systemic activity against neuropathic pain without producing constipation.

To enhance the drug-like properties of the endogenous opioid peptide endomorphin-1 (1 = Tyr-Pro-Trp-Phe-NH2), the N-terminus of the peptide was modified with 2-aminodecanoic acid, resulting in compound 3. Tyr in compound 1 was replaced with 2,6-dimethyltyrosine yielding compound 2. Derivative 2 was also substituted with 2-aminodecanoic acid producing compound, 4. Lipoamino acid-modified derivatives showed improved metabolic stability and membrane permeability while maintaining high μ-opioid (MOP) receptor binding affinity and acting as a potent agonist. In vivo studies showed dose-dependent antinociceptive activity following intravenous (i.v.) administration of compounds 3 and 4 in a chronic constriction injury (CCI)-rat model of neuropathic pain with ED50 values of 1.22 (±0.93) and 0.99 (±0.89) µmol/kg, respectively. Pre-treatment of animals with naloxone hydrochloride significantly attenuated the anti-neuropathic effects of compound 3, confirming the key role of opioid receptors in mediating antinociception. In contrast to morphine, no significant constipation was produced following i.v. administration of compound 3 at 16 µmol/kg. Furthermore, following chronic administration of equi-potent doses of compound 3 and morphine to rats, there was less antinociceptive tolerance for compound 3 compared with morphine.

Synthesis and in vitro evaluation of glycosyl derivatives of luteinizing hormone-releasing hormone (LHRH)

Luteinizing hormone-releasing hormone (LHRH) analogues are used extensively for the treatment of various hormone-dependent diseases. However, none of the currently marketed derivatives can be administered orally. Modification of peptide sequences by attachment of carbohydrate moieties is a promising strategy that may increase the metabolic stability of the target peptide and enhance its transport across cell membranes, subsequently improving peptide bioavailability. In this study, either the N- or C-terminus of the LHRH peptide was altered by attachment of carbohydrate moieties. Caco-2 cells were chosen as an in vitro model to investigate both the permeability and stability of the new LHRH analogues. Our findings show that conjugating sugar moieties to the N-terminus of the LHRH peptide significantly increased both permeability and metabolic stability of most of the modified LHRH derivatives.

Peripherally acting novel lipo-endomorphin-1 peptides in neuropathic pain without producing constipation

We previously described two novel analogues of endomorphin-1 (Tyr-Pro-Trp-Phe-NH2, 1), modified with an 8-carbon lipoamino acid (C8LAA) with or without replacement of Tyr(1) with 2,6-dimethyltyrosine (Dmt) at the N-terminus of the peptide (compounds 3 and 4, respectively). They were shown to be more stable and permeable, and acted as potent μ-opioid receptor agonists. In this study we report that the C8LAA modification resulted in successful systemic delivery of both analogues. They produced potent dose-dependent pain relief in a chronic constriction injury-rat model of neuropathic pain after intravenous administration with ED50 values obtained at 6.58 (±1.22) μmol/kg for 3 and 6.18 (±1.17) μmol/kg for 4. Using two different rat models of constipation that assess the effects of μ-opioid receptor agonists on stool hydration and gastro-intestinal motility, compound 3 produced insignificant constipation at 16 μmol/kg, whereas morphine elicited significant constipation at 2 μmol/kg. Compound 3 in contrast to morphine, did not attenuate the hypercapnic ventilatory response at 5 μmol/kg, a dose that fully alleviated hindpaw sensitivity at the time of peak effect in CCI-rats. This finding revealed the lack of respiratory depression effect at antinociceptive dose.

Active immunisation of mice with GnRH lipopeptide vaccine candidates: Importance of T helper or multi-dimer GnRH epitope.

Active immunisation against gonadotropin releasing hormone (GnRH) is a potential alternative to surgical castration. This study focused on the development of a GnRH subunit lipopeptide vaccine. A library of vaccine candidates that contained one or more (up to eight) copies of monomeric or dimeric GnRH peptide antigen, an adjuvanting lipidic moiety based on lipoamino acids, and an additional T helper epitope, was synthesised by solid phase peptide synthesis. The candidates were evaluated in vivo in order to determine the minimal components of this vaccine necessary to induce a systemic immune response. BALB/c mice were immunised with GnRH lipopeptide conjugates, co-administered with or without Complete Freunds Adjuvant, followed by two additional immunisations. Significant GnRH-specific IgG titres were detected in sera obtained from mice immunised with four of the seven lipopeptides tested, with an increase in titres observed after successive immunisations. This study highlights the importance of for epitope optimisation and delivery system design when producing anti-hapten antibodies in vivo. The results of this study also contribute to the development of future clinical and veterinary immunocontraceptives.

Synthesis, biological activity and structure–activity relationship of endomorphin-1/substance P derivatives

Endomorphins have been shown to produce potent analgesia in various rodent models of pain. However, their central administration led to the development of tolerance and physical dependence. Conjugation of C-terminal substance P (SP) fragments to opioids and opioid peptides was previously shown to produce hybrid peptides with strong analgesic activity, with low or no propensity to develop tolerance. In this study, four peptides (2-5) comprised of endomorphin-1 (1) and C-terminal fragments of SP (four or five amino acids, SP(8-11) (2) or SP(7-11) (4), respectively), with an overlapping Phe residue, were synthesized. To overcome low metabolic stability and poor membrane permeability of the peptide, the N-terminus of 2 and 4 was further modified with a C10-carbon lipoamino acid (C10LAA) achieving 3 and 5, respectively. LAA-modification of the hybrid peptides resulted in a significant increase in metabolic stability and membrane permeability compared to peptides 1, 2 and 4. Compound 5 showed potent μ-opioid receptor binding affinity (K(iμ)=3.87 ± 0.51 nM) with dose-dependent agonist activity in the nanomolar range (IC(50)=45 ± 13 nM). In silico modeling was used to investigate the binding modes and affinities of compounds 1-5 in the active site of μ-opioid receptors. The docking scores were in agreement with the K(iμ) values obtained in the receptor binding affinity studies. The more active LAA-modified hybrid peptide showed a lower total interaction energy and higher negative value of MolDock score.

Lipidated analogues of luteinizing hormone-releasing hormone (LHRH) reduce serum levels of follicle-stimulating hormone (FSH) after oral administration

A diverse range of diseases involving the reproductive system are treated with luteinizing hormone-releasing hormone (LHRH) agonists which must be administered daily. Currently, an efficient oral delivery system is not available. Here, we show the facile inclusion of lipoamino acids into the peptide sequence of LHRH, rendering it more stable towards enzymatic degradation, as well as enhancing permeability across Caco-2 cell monolayers. Selected LHRH derivatives were tested in vivo by daily oral administration to rats. The size and weight of the sex organs remained unchanged and the levels of LH were stable over the course of the experiment. However, some of the lipidic peptides (3, 8 and 9) were able to reduce serum levels of follicle-stimulating hormone (FSH), an important finding towards the development of orally available LHRH agonists.

A study on liposomal encapsulation of a lipophilic prodrug of LHRH

Abstract This study aimed at evaluating whether derivatization of luteinizing hormone-releasing hormone (LHRH) peptide with an amphiphilic lipoamino acid moiety could allow, along with other technological and/or pharmacokinetic advantages, to improve its encapsulation in liposomes, potentially driving its further body distribution and cellular uptake. Experimental data confirmed that a lipophilic derivative of LHRH was efficiently incorporated in various liposomal systems, differing in lipid composition and surface charge, and obtained using different methods of production. Incubation of liposomes, loaded with a fluorescent derivative of the LHRH prodrug, with NCTC keratinocytes or Caco-2 cell cultures showed that the carriers can be rapidly internalized. Conversely, the internalization of the free prodrug occurred only at very high concentrations.

Investigation of structure-activity relationships of synthetic anti-gonadotropin releasing hormone vaccine candidates.

The immunoneutralization of gonadotropin‐releasing hormone (GnRH) can be used for the treatment of human hormone‐dependent male and female cancers or as immunocontraceptives in animals. Vaccine candidates 1 [Th(K‐LP)GnRH], 2 [GnRH(K‐LP)Th], 3 [GnRH(K‐Th)LP], and 4 [Th(K‐GnRH)LP] (for which K=lysine, LP=lipopeptide Ser‐Ser‐C16‐C16, and Th=T helper cell epitope influenza HA2), were synthesized by assembling a CD4+ T helper cell epitope (Th), GnRH, and an adjuvanting lipid moiety (LP) in various spatial arrangements. All compounds were efficiently taken up by antigen‐presenting cells with significant immunogenicity without an external adjuvant. Compounds 2, 3, and 4, in which GnRH is conjugated through its C terminus, produced higher GnRH‐specific antibody responses than construct 1, in which the GnRH moiety is conjugated through its N terminus. All four constructs induced a significant antiproliferative effect (up to 55 %) on GnRH‐receptor‐positive LNCaP cells, but showed weaker activity in the GnRH‐receptor‐negative SKOV‐3 cell line. Marked degenerative changes were observed in morphology and follicular development in the ovaries of immunized mice, with approximately 30 % higher degenerative antral and atretic follicles.

Calorimetry and Langmuir-Blodgett studies on the interaction of a lipophilic prodrug of LHRH with biomembrane models

The interaction between an amphiphilic luteinizing hormone-releasing hormone (LHRH) prodrug that incorporated a lipoamino acid moiety (C12-LAA) with biological membrane models that consisted of multilamellar liposomes (MLVs) and phospholipid monolayers, was studied using Differential Scanning Calorimetry (DSC) and Langmuir-Blodgett film techniques. The effect of the prodrug C12[Q1]LHRH on the lipid layers was compared with the results obtained with the pure precursors, LHRH and C12-LAA. Conjugation of LHRH with a LAA promoiety showed to improve the peptide interaction with biomembrane models. Basing on the calorimetric findings, the LAA moiety aided the transfer of the prodrug from an aqueous solution to the biomembrane model.