Release of peptides from sustained delivery systems (microcapsules and microparticles) in vivo

Sustained delivery systems (microcapsules, microparticles, or implants) developed for once a month administration of peptides are efficacious and convenient. Long acting formulations of several bioactive peptides are based on microcapsules of a biodegradable polymer poly(dl -lactide-co-glycolide) (PLG), but a better understanding is required of the mechanism of the peptide release from the microcapsules, which is assumed to be primarily by diffusion through pores. In order to clarify this mechanism, microcapsules and microparticles of the agonist [d -Trp⁶]-LHRH and microcapsules of the LHRH antagonist SB-75 were given i.m. to rats 2 h and 1, 2, 4, 7, 14 and 21 days before histological and immunohistochemical investigation. Signs of biodegradation of the PLG matrix could be seen the first day after the injection, in a form of vacuole development in the interior of the particles and connected with the presence of macrophages within the matrix. The microcapsules showed excellent tissue-compatibility, and no significant foreign body reaction was detected. Immunohistochemical study on the microcapsules revealed no visible decrease in peptide concentration in the remnants of the matrix even 2 weeks after the injection. Evaluation of serum [d -TrP⁶]-LHRH showed that after an initial burst, both microcapsules and microparticles maintained elevated serum [d -Trp⁶]-LHRH levels for more than 3 weeks. Our results suggest that the previously proposed mechanisms do not reflect the experimental findings, particularly for the insoluble peptides. The peptide release from the PLG microcapsules or microparticles appears to be controlled mostly by the speed of the biodegradation of the polymer matrix and the diffusion of the peptides from the PGL is negligible.     

New antagonists of LHRH II. Inhibition and potentiation of LHRH by closely related analogues

Modifications of the previously described LHRH antagonists, [Ac-d -Nal(2)¹, d -Phe(4Cl)², d -Trp³, d -Cit⁶, d -Ala¹⁰]LHRH and the corresponding d -Hci⁶ analogue, have been made to alter the hydrophobicity of the N-terminal acetyl-tripeptide portion. Substitution of d -Trp³ with the less hydrophobic d -Pal(3) had only marginal effects on the antagonistic activities and receptor binding potencies of the d -Cit/d -Hci⁶ analogues, but it appeared to further improve the toxicity lowering effect of d -Cit/d -Hci⁶ substitution. Antagonists containing d -Pal(3)³ and d -Cit/d -Hci⁶ residues, i.e. [Ac-d -Nal(2)¹, d -Phe(4Cl)², d -Pal(3)³d -Cit⁶, d -Ala¹⁰]LHRH (SB-75) and [Ac-d -Nal(2)¹, d -Phe(4Cl)², d -Pal(3)³, d -Hci⁶, d -Ala¹⁰]LHRH (SB-88), were completely free of the toxic effects, such as cyanosis and respiratory depression leading to death, which have been observed in rats with the d -Trp³, d -Arg⁶ antagonist and related antagonists. Replacement of the N-acetyl group with the hydrophilic carbamoyl group caused a slight decrease in antagonistic activities, particularly in vitro. Introduction of urethane type acyl group such as methoxycarbonyl (Moc) or t-butoxycarbonyl (Boc) led to analogues that showed LHRH-potentiating effect. The increase in potency induced by these analogues, e.g. [Moc-d -Nal(2)¹, d -Phe(4Cl)², d -Trp³, d -Cit⁶, d -Ala¹⁰]LHRH and [Boc-d -Phe¹, d -Phe(4Cl)², d -Pal(3)³, d -Cit⁶, d -Ala¹⁰]LHRH, was 170-260% and persisted for more than 2 h when studied in a superfused rat pituitary system.     

Lymphocyte Cytotoxicity Assay for Predicting Preterm Delivery of Small for Date Babies

ABSTRACT: Cytotoxic activity of lymphocytes, obtained from 84 pregnant women was tested, using human embryonic fibroblast cells as target cells. Lymphocytes of 8 women who delivered small for date babies before term and those of 3 women who had spontaneous abortions were significantly more cytotoxic (p < 0.001) than lymphocytes of women who delivered small for date babies at term, appropriately grown babies before term, or appropriately grown babies at term. Data suggest that a higher level of in vitro lymphocyte cytotoxic activity of pregnant women forecasts either the preterm delivery of small for date babies or the occurrence of spontaneous abortions.     

Membrane fluidity of trophoblast cells and susceptibility to natural cytotoxicity

This study examines the relationship between membrane lipid microviscosity and susceptibility of villous trophoblast to lysis by natural cytotoxic cells. Trophoblast-enriched cell suspensions prepared from term human placentae were treated with cholesteryl hemisuccinate (CHS)--a modulator of membrane lipid microviscosity. CHS-treated cells were more susceptible targets for natural lymphocyte cytotoxicity than were untreated controls. In binding experiments, increased binding of lymphocytes to CHS-treated target cells was found. Preincubation with progesterone prevented membrane rigidification by CHS. Progesterone, cortisol, and estriol restored the impaired resistance of CHS-treated trophoblast cells to lysis. We determined microviscosity and progesterone concentration in villous surface membranes, prepared from placentae from idiopathic spontaneous abortions and normal first-trimester pregnancies. An inverse relationship was found between progesterone content and microviscosity of the membranes. Microviscosity of the membranes from abortion placentae was significantly higher (P less than .01) and progesterone concentration was significantly lower (P less than .001) than those in the membranes of normal first trimester placentae.     

Progesterone-Prostaglandin Balance Influences Lymphocyte Function in Relation to Pregnancy

ABSTRACT: The effect of progesterone and prostglandin F₂α on cytotoxic activity of lymphocytes was investigated. Thirty-three pregnant women with a clinical diagnosis of threatened premature labour were treated with the prostaglandin synthesis inhibitor acetylsalicylic acid (2.7 gm/day) for 1 week. Cytotoxic activity of the lymphocytes significantly decreased while their progesterone binding capacity increased by the end of the treatment. Ten healthy pregnant women received a single 15 mg dose of prostaglandin F₂α for pregnancy termination. One day after prostaglandin administration lymphocytes showed significantly higher cytotoxic activity and lower progesterone binding capacity than before. Lymphocytes of 44 healthy nonpregnant volunteers were preincubated with prostaglandin F₂α and/or progesterone. Prostaglandin F₂α (8 nmole) significantly decreased cytotoxic activity of 4 × 10⁶ lymphocytes. In the presence of progesterone prostaglandin F₂α failed to stimulate lymphocytes.     

Increased NK Activity Is Responsible for Higher Cytotoxicity to HEF Cells by Lymphocytes of Women With Threatened Preterm Delivery

ABSTRACT: Recently we have shown that lymphocytes of pregnant women with threatened preterm delivery (risk group) exerted significantly higher cytotoxic activity to human embryonic fibroblast (HEF) cells than those of healthy pregnant women. The purpose of this study was to get information on the mechanism of this cytotoxicity. The possibility of prior sensitization to embryonic antigen was excluded, since no difference could be demonstrated between cytotoxic activity of lymphocytes obtained from women with two or more previous pregnancies and that of lymphocytes from never-pregnant women. For determining the effector cell type responsible for cytotoxicity, lymphocytes of 50 healthy pregnant women and those of 50 risk patients were tested in different cytotoxicity tests using HEF and K-.562 target cells. The proportion of NK cells among lymphocytes was determined by counting large granular lymphocytes (LGL), IgG Fc receptor bearing cells, and cells positively stained by NK specific monoclonal antibody. Though no difference in the proportion of NK cells between the two groups was found, risk patients' lymphocytes were significantly more cytotoxic to K-562 target cells than those of healthy pregnant women. Investigations at the single-cell level made it obvious that this higher cytotoxic activity originated from increased target cell lysing ability of their lymphocytes, while their conjugating capacity did not differ significantly from that of lymphocytes obtained from healthy pregnant women. Staining conjugates with monoclonal antibodies showed that NK cells were concentrated on the surface of both HEF and K-.562 cells, so it seems likely that NK activity is responsible for the cytotoxic activity of pregnancy lymphocytes to HEV cells.     

The Effect of Neonatal Vasectomy on Testicular Function

The effect of unilateral or bilateral vasectomy on testicular weight and basal testosterone production in vitro was studied in neonatal intact and hemicastrated rats. Five days after right-side vasectomy ipsilateral to vasectomy testicular weight increased, and basal testosterone production decreased. Ten days postvasectomy the changes were opposite, and affected both testes. In hemicastrated animals hemivasectomy did not interfere with compensatory hypertrophy but induced a significant decrease in basal testosterone production. The data of the present study suggest that in neonatal animals intact ductus deferens bundle(s) is also required for the full control of testicular weight and basal testosterone secretion.     

Synthesis and in vitro and in vivo activity of analogs of growth hormone-releasing hormone (GH-RH) with C-terminal agmatine

In the search for more active analogs of human growth hormone-releasing hormone (GH-RH), 37 new compounds were synthesized by solid phase methodology, purified, and tested biologically. Most of the analogs contained a sequence of 27 amino acids and N-terminal desaminotyrosine (Dat) and C-terminal agmatine (Agm), which are not amino acids. In addition to Dat in position 1 and Agm in position 29, the majority of the analogs had Ala¹⁵ and Nle²⁷ substitutions and one or more additional L- or D-amino acid modifications. [Dat¹, Ala¹⁵, Nle²⁷]GH-RH(1-28)Agm (MZ-2-51) was the most active analog. Its in vitro GH-releasing potency was 10.5 times higher than that of GH-RH(1-29)NH₂ and in the i.v. in vivo assay, MZ-2-51 was 4-5 times more active than the standard. After s.c. administration to rats, MZ-2-51 showed an activity 34 times higher at 15min and 179 times greater at 30min than GH-RH(1-29)NH₂ and also displayed a prolonged activity. D-Tyr¹⁰, D-Lys¹², and D-Lys¹² homologs of MZ-2-51 also showed enhanced activities. Thus, [Dat¹, D-Tyr¹⁰, Ala¹⁵, Nle²⁷]GH-RH(1-28)Agm (MZ-2-159), [Dat¹, D-Lys¹², Ala¹⁵, Nle²⁷]GH-RH(1-28)AGM (MZ-2-57), and [Dat¹, Ala¹⁵, D-Lys²¹, Nle²⁷]GH-RH(1-28)Agm (MZ-2-75) were 4-6 times more active in vitro than GH-RH(1-29)NH₂. In vivo, after i.v. administration, analog MZ-2-75 was equipotent and analogs MZ-2-159 and MZ-2-57 about twice as potent as the standard. After S.C. administration, the potencies of MZ-2-57 and MZ-2-75 were 10-14 times higher than the standard at 15 min and 45-89 times greater when determined at 30 min. Most of the analogs containing two or more D-amino acid substitutions were less active than GH-RH(1-29)NH₂ or inactive. Our studies indicate that very potent GH-RH analogs can result from the combination of agmatine in position 29 with other substitutions.     

Naloxone has a Local Effect on the Testis of Immature Rats

Local effect of naloxone on testicular weight, compensatory hypertrophy, and differentiation of the testis and serum testosterone level has been studied in immature rats. A single injection of naloxone in a dose of 2 micrograms/testis under the testicular capsule significantly increased the testicular weight, promoted testicular differentiation and enhanced the rate of compensatory hypertrophy of the remaining testis in unilaterally orchidectomized animals. Data suggest that naloxone has a direct effect on the testis of immature rats.