A new strategy for attachment of antibodies to sterically stabilized liposomes resulting in efficient targeting to cancer cells.

The development of long-circulating formulations of liposomes (S-liposomes), sterically stabilized with lipid derivatives of poly(ethylene glycol) (PEG), has increased the likelihood that these liposomes, coupled to targeting ligands such as antibodies, could be used as drug carriers to deliver therapeutic drugs to specific target cell populations in vivo. We have developed a new methodology for attaching monoclonal antibodies to the terminus of PEG on S-liposomes. A new end-group functionalized PEG-lipid derivative pyridylthiopropionoylamino-PEG- distearoylphosphatidylethanolamine (PDP-PEG-DSPE) was synthesized for this purpose. Incorporation of PDP-PEG-DSPE into S-liposomes followed by mild thiolysis of the PDP groups resulted in formation of reactive thiol groups at the periphery of the lipid vesicles. Efficient attachment of maleimide-derivatized antibodies took place under mild conditions even when the content of the functionalized PEG-lipid in S-liposomes was below 1% of total lipid. The resulting S-immunoliposomes showed efficient drug remote loading, slow drug release rates and increased survival times in circulation compared to liposomes lacking PEG. When antibodies recognizing several different tumor-associated antigens were coupled to the PEG terminus of S-liposomes a significant increase in the in vitro binding of liposomes to the target cells was observed. The binding of S-immunoliposomes containing entrapped doxorubicin to their target cell population resulted in increased cytotoxicity compared to liposomes lacking the targeting antibody.

Long circulating, cationic liposomes containing amino-PEG-phosphatidylethanolamine.

Ligand attachment to polyethylene glycol (PEG) grafted, long circulating liposomes at the polymer terminus is of interest for targeting but the effect of positively charged groups is unknown. Amino-polyethylene glycol-phosphatidylethanolamine (AminoPEG-PE), prepared in four steps from alpha-amino-omega-hydroxy-PEG, was tested for influence on liposome interactions in vivo: blood circulation and biodistribution. Despite surface amines on each liposome conferring cationic behavior, in vivo properties are comparable to those obtained with methoxy-PEG-PE. The consequences are profound for targeting and possibly systemic delivery of cationic lipidic-polynucleotide complexes.

An investigation of angiotensin II agonist and antagonist analogues with 5,5-dimethylthiazolidine-4-carboxylic acid and other constrained amino acids.

To probe the receptor-bound conformational requirements of angiotensin II (ANG II) octapeptide agonists and antagonists, the synthesis and biological activities of [Sar1]ANG II agonist and [Sar1,X8]ANG II antagonist analogues (X8 = Ile, D-Phe, or Aib) bearing conformational constraints in positions 3, 5, and 7 were investigated and compared with previous literature efforts. The conformational constraints that were examined include Pro, Dtc (5,5-dimethylthiazolidine-4-carboxylic acid), Aib, Cle, (NMe)Ala, (NMe)Ile, and the lactam modification, L,L-lactam-Phe, previously described by Freidinger et al. (J. Org. Chem. 1982, 47, 104-109). Both [Sar1,(NMe)Ala3 and Pro3]ANG II retained agonist activity, while only [Sar1,(NMe)Ala3,Ile8]ANG II retained antagonist activity. [Sar1,Dtc5]ANG II displayed superior agonist activity, while both [Sar1,Dtc5 and Cle5,Ile8] ANG II displayed superior antagonist activity. In contrast to position 5, Dtc7 substitution for Pro7 of either [Sar1]ANG II or [Sar1,Ile8]ANG II gave analogues with reduced activities. These results are consistent with the hypothesis that conformations of [Sar1]ANG II and [Sar1,Ile8]ANG II containing a C7 conformation in position 7 are preferred for both ANG II agonist and antagonist activity. Incorporation of the L,L-lactam-Phe modification into [Sar1]ANG II gives a pure ANG II antagonist (pA2 8.3), comparable to saralasin (pA2 8.6). In positions 3, 5, and 7 the conformational requirements for the ANG II agonist [Sar1]ANG II and the ANG II antagonist [Sar1,Ile8]ANG II may be different. Individual substitution of (NMe)Ala3, Dtc5, D-Phe8 and Aib8 [[Sar1,Aib8]ANG II: Khosla et al. J. Med. Chem. 1977, 20, 1051-1055] into [Sar1,Ile8]ANG II gives analogues that retain antagonist activity. Multiple substitutions of these types of residues into [Sar1,Ile8]ANG II gives analogue 45 [Sar1,(NMe)Ala3,Dtc5,Aib8]ANG II, 46 [Sar1(NMe)Ala3,D-Phe8]AII, and 47 [Sar1,Dtc5,D-Phe8]AII, which display considerably reduced antagonist activity. In ANG II antagonists the construction of highly constrained analogues may not be possible by the additive substitution of "preferred" constrained amino acids into a single analogue.

The role of position 4 in angiotensin II antagonism: a structure-activity study.

A number of [Sar1,(pX)Phe4]-ANG II and [Sar1,(pX)Phe4,Ile8]-ANG II analogues were prepared. A good correlation between pX structure in [Sar1,(pX)Phe4]-ANG II and antagonist activity could not be found. However, the data suggest a general trend: Position 4 para substituents that are hydrophilic and capable of donating a hydrogen atom in a hydrogen bond promote agonist activity, while para substituents that are hydrophobic and incapable of donating a hydrogen atom promote antagonist activity. These properties were found to be optimal in the p-chloro substituent. The resulting analogue [Sar1,(pCl)Phe4]-ANG II is a potent ANG II antagonist in vivo. The pX substituents that promote antagonist activity in the [Sar1,(pX)Phe4]-ANG II series were unfavorable in [Sar1,(pX)Phe4,Ile8]-ANG II analogues. ANG II analogues that are antagonists by virtue of an alteration in position 8 require a position 4 agonist side chain. Concurrent modifications of positions 4 and 8 do not give rise to potent antagonists with reduced partial agonist activity.

Potent angiotensin II antagonists with non-beta-branched amino acids in position 5.

Amino acids with lipophilic side chains that contain more than one functional group on the beta-carbon, i.e. a beta-branched hydrocarbon moiety, are required in position 5 of angiotensin II (AII) analogue with potent agonist activity. This requirement for agonist activity does not follow for AII analogues with potent antagonist activity. Straight-chain amino acids may be substituted into position 5 of [Sar1,X5,Ile8]AII with retention or enhancement of antagonist activity, e.g. (X5,pA2 rabbit aorta) Phe, 9.15; Tyr, 9.6; His, 9.0; Glu,9.0; Nle, 8.85, compared to Ile, 9.1. beta-Branched side chains can still enhance the antagonist activities of [Sar1,X5,Ile8]AII analogues, e.g. X5 = (beta Me)Phe, pA2 = 9.3. An X-ray crystal structure of the Boc-(beta Me)Phe DCHA salt, prepared for the synthesis of [Sar1,-(beta Me)Phe5, Ile8]AII, revealed an S,S configuration of alpha- and beta-carbon atoms. Contrary to previous literature reports, chemical nonequivalence of the deta-protons of Pro was observed in the 1H NMR spectra of [Sar1,X5,Ile8]AII analogues bearing both beta-branched X5 side chains (X5 = Ile) and non-beta-branched X5 side chains (X5 = Ala, His).

The importance of residues 2 (arginine) and 6 (histidine) in high-affinity angiotensin II antagonists.

The structure-antagonist activity relationship is described for analogues of [Sar1,Ile8]angiotensin II substituted in position 2 (arginine) and position 6 (histidine). An extreme sensitivity of potency to alterations in these positions was observed, suggesting that both residues are important for binding. Evidence is presented suggesting that the position 6 histidine side chain in angiotensin II (AII) is not involved in receptor stimulation. The structure-activity relationship is also explored for both [des-Asp1] AII (AIII) and [des-Asp1,Ile8]AII analogues substituted in position 2 (arginine). The substitution of D-N-methylalanine, D-(NMe)Ala, into position 2 of both [des-Asp1]AII and [des-Asp1,Ile8]AII gives analogues 39 and 40 that appear to be more potent than the native [Arg2]peptides and that are the most potent AIII agonists and antagonists described to date.

Triamcinolone impairs the synthesis of collagen and noncollagen proteins in condylar cartilage of newborn mice.

The effects of triamcinolone hexacetonide (TH) on the synthesis of collagen and noncollagen proteins were tested in mandibular condylar cartilage of newborn mice. Four-day-old ICR mice received a single i.p. injection of TH at doses ranging from 0.4 to 4.0 mg/kg body weight. Hydrocortisone, deoxycorticosterone, dexamethasone, and progesterone were administered at a dose of 4.0 mg/kg. Test animals and nontreated and vehicle-treated controls were sacrificed after 24, 48, and 72 hours and were processed for electron microscopy. Additional animals were injected with 5 microCi of 3H-proline 2 hours before sacrifice. The specimens were extracted with 5% TCA containing 1 mM proline followed by 5% TCA, acetone, and ether, homogenized and digested with purified bacterial collagenase, and the amounts of radioactivity in collagenase digestible (CDP) and noncollagen proteins (NCP) were determined. The present results revealed that triamcinolone led to a significant dose-dependent decrease in the protein content of the tissue that lasted for 3 days (12-14% at the dose of 4 mg/kg). The incorporation of 3H-proline into CDP was reduced by 39, 57, and 42% at 24, 48, and 72 hours, respectively whereas the incorporation into NCP was reduced by 20, 35, and 23%, respectively. When compared with other steroids, dexamethasone revealed a similar inhibitory effect, whereas hydrocortisone and deoxycorticosterone had no significant effect. Progesterone, on the other hand, showed a transient (24 hours) stimulatory effect on the synthesis of collagen synthesis (21%, P less than 0.05). Electron microscopy showed an atypical arrangement of collagen fibers and accumulation of large aggregates of collagen that filled the entire matrical space between cartilage cells.