Large-scale synthesis of peptides.

Recent advances in the areas of formulation and delivery have rekindled the interest of the pharmaceutical community in peptides as drug candidates, which, in turn, has provided a challenge to the peptide industry to develop efficient methods for the manufacture of relatively complex peptides on scales of up to metric tons per year. This article focuses on chemical synthesis approaches for peptides, and presents an overview of the methods available and in use currently, together with a discussion of scale-up strategies. Examples of the different methods are discussed, together with solutions to some specific problems encountered during scale-up development. Finally, an overview is presented of issues common to all manufacturing methods, i.e., methods used for the large-scale purification and isolation of final bulk products and regulatory considerations to be addressed during scale-up of processes to commercial levels.

An assessment of lower back pain in young adults: implications for college health education.

A convenience sample of 243 undergraduates completed a 36-item questionnaire on their knowledge about back care and exercise patterns before they attended lectures and a workshop on back mechanics. At the workshop, the students were individually evaluated for posture, hamstring flexibility, hip flexor flexibility, back and abdominal strength, and lifting technique. Twenty-nine percent of the students reported that they experienced no back pain; 71% experienced lower back pain 1 to 5 days a week. The majority were neglectful of their posture, lifting and carrying techniques, and scored fair-to-poor on the hamstring flexibility test, possibly foreshadowing back problems in later life. The majority of respondents were unsure of what exercises to do for back care. Within the subgroup of students who claimed they were knowledgeable about exercise, more than 50% were performing ineffective and potentially harmful exercises. The results underscored the potential worth of health education on back care offered through didactic instruction and experiential workshops.

T-cell-dependent immunobiological activity of a desmuramyl analog of muramyl dipeptide, adamantylamide dipeptide (AdDP), and D-isoglutamine.

The present experiments demonstrate that, similar to immunomodulatory muramyl dipeptide, its desmuramyl analog adamantylamide dipeptide is able to induce mild and fully reversible paw edema in mice. This effect is an immune-related phenomenon depending on the activation of T-cell/macrophage interactions and on production of prostaglandins. Possible involvement of certain immunoregulatory/inflammatory cytokines (e.g. IL-1, IL-2) has been suggested. The most probable intrinsic moiety of the adamantylamide dipeptide molecule responsible for triggering the edema formation is obviously D-isoglutamine.

Pharmacokinetic profile of the immunomodulating compound adamantylamide dipeptide (AdDP), a muramyl dipeptide derivative in mice.

A pharmacokinetic profile of 14C-AdDP with uniformly labelled alanine was investigated. It was shown that the distribution phase after an i.v. administration is very short with a half-life of 2.1 min. The half-life of elimination phase after the i.v. administration is about 2.85 hours, that is longer than those of MDP and its derivatives. The total body clearance (30 ml/min/kg) is caused predominantly by metabolism of the compound. All the radioactivity found in urine in a 48 hours interval after a s.c. administration represents only 3.1% of the administered dose. Only a smaller part of the excreted radioactivity is formed by unmetabolised AdDP. The concentration curve after a s.c. administration is characterized by a very fast absorption with a half-life shorter than 1 minute. The distribution and elimination phases are prolonged (20 min, 11 hours respectively) in comparison with an i.v. injection. The decreased absolute bioavailability after a s.c. administration (65%) is probably not biologically significant because of a slower release of the compound from the site of the s.c. administration. A relatively very high radioactivity was found in liver, kidney, thymus, spleen and brain very soon which suggest a very good penetration into tissues. It is an agreement with the high apparent distribution volume of peripheral compartment and higher lipophilicity of AdDP as compared to MDP.

Synthesis of inhibitors of the meso-diaminopimelate-adding enzyme from Escherichia coli.

In order to obtain inhibitors of the meso-diaminopimelate-adding enzyme, which participates in the biosynthesis of bacterial peptidoglycan, several N alpha-propionyl-dipeptides of the general formula Pr-L-Ala-ambo-Xaa-OH were synthesized. Xaa represented methionine S,S-dioxide, methionine S-oxide, methionine sulfoximine, and 2-amino-4-phosphonobutyric acid, i.e. transition state analogs of glutamine synthetase and gamma-glutamyl-cysteine synthetase, which catalyze the same type of reaction as our target enzyme. After synthesis, the diastereoisomers were separated by preparative HPLC or t.l.c.; those containing methionine derivatives could be identified thanks to previously synthesized reference compounds. After preincubation with the meso-diaminopimelate-adding activity from Escherichia coli, the LD diastereoisomers displayed moderate inhibitory effects, whereas the LL ones were inefficient. The best inhibition was obtained with one diastereoisomer of Pr-L-Ala-zeta-2-amino-4-phosphonobutyrate, presumably the LD one. A chloromethylketone derivative Pr-L-Ala-D-Glu(CH2Cl)-OH, potential affinity labeler of the meso-diaminopimelate-adding enzyme, was also synthesized. In the assay with preincubation, this compound behaved as the best inhibitor.

Solid-phase synthesis of a nonadecapeptide coded for by the v-myb oncogene.

A nonadecapeptide comprising a predicted B-cell determinant from the v-myb oncoprotein was synthesized by Merrifield's solid-phase method. Hydrogen chloride in dichloromethane was used for protective t-butyloxycarbonyl group removal; the deprotection was monitored using a new qualitative deprotection test. The nonadecapeptide coupled to a carrier elicited a high titre of protein-reactive antipeptide antibodies.

[Comparison of the effects of tercleucine and tryptophan superanalogs of LH-RH on gonadotropin secretion in heifers]. / Porovnání úcinku tercleucinového a tryptofanového superanalogu LH-RH na sekreci gonadotropinu u jalovic.

In six heifers without sexual organs the effects were compared of the superanalogues LH-RH [(D-Tle6) and (D-Trp6)] on the secretion of luteinizing hormone (LH) and follicles of stimulating hormone (FSH). The superanalogues were given i. m. at dosages of 10, 50 and 100 micrograms pro toto. Dosages of 50 and 100 micrograms of both superanalogues had a greater effect on the secretion of LH than the dosage of 100 micrograms. The highest average concentration of LH during the time of application (D-Tle6) was recorded in the dosage of 10 micrograms (7.84 ng.ml-1). The average concentrations of LH after application (D-Trp6) were within the range of 5.46 to 9.29 ng.ml-1. Because of the great variability of the concentrations of LH no significant differences were ascertained due to the influence of dosage. From a comparison of the concentrations of LH with the same dosage it emerged that (D-Trp6) after an application of 50 micrograms significantly increased the concentration of LH from the 60th to the 240th minute and thus had a more protracted effect. With dosages of 10 and 100 micrograms of the superanalogues no statistically significant differences were recorded after two hours from application. With (D-Trp6) the higher stimulative effect on the secretion of LH was statistically confirmed. The highest concentrations of follicles of stimulation hormone (FSH) (204.6 and 228.6 ng.ml-1) were found from the 40th to the 100th minute after the application of dosages of 50 and 100 micrograms (D-Tle6). The protracted effect was greatest with a dosage of 100 micrograms (270 mins). (D-Trp6) at a dosage of 100 micrograms caused the greatest effect on the secretion of FSH (226.8 ng.ml-1 for a period of 180 mins). The lower dosages of analogues scarcely differed in response. The dosage of superanalogues has an influence on the concentration of FSH in peripheral blood and on the duration of the protracted effect.

The immunomodulatory property of a novel synthetic compound adamantylamide dipeptide.

The adjuvant effect of a novel synthetic compound adamantylamide dipeptide (AdDP) was tested in a model of delayed hypersensitivity to ovalbumin in guinea pigs, and of immunostimulatory activity in the experiments with 3H thymidine where DNA biosynthesis was measured in several immunocompetent organs after administration of adamantylamide dipeptide. In both tests, the compound proved to be active since delayed hypersensitivity was potentiated and a marked increase in the utilization of 3H thymidine in liver, thymus and spleen has been observed. The novel compound appeared to be devoid of other effects of MDP such as pyrogenicity and arthritogenicity.

Protracted milk-ejecting effect of some oxytocin analogues in rats.

Protracted effect of [2-0-methyltyrosine]-deamino-1-carba-oxytocin, [2-0-methyltyrosine]-oxytocin and its dimeric form was studied on the mammary gland of lactating rat in vivo. Biphasic course of biological response found for monomers and slow onset of response observed for dimer led to the assumption that the substances behaved like hormonogens.

Circular-dichroic spectra of vasopressin analogues and their cyclic fragments.

The circular dichroic spectra of [Arg8]vasopressin, [Mpr1, Arg8]vasopressin, [Mpr1, D-Arg8]-vasopressin, pressinamide, deaminopressinamide, tocinamide, deaminotocinamide, [Leu4, D-Arg8]-vasotocin, [Mpr1, Leu4, D-Arg8]vasotocin and [Phe2, Lys8]vasopressin have been studied. All these substances showed a characteristic positive dichroic band at about 225 nm due to the presence of tyrosine in sequence position 2. The intensity of this band was affected by interactions between the tyrosine side-chain and other structural elements in the molecule, such as the Na-amino group, the side-chain of phenylalanine in position 3 and the linear C-terminal peptide. Analysis of the response of this band to structural modifications of the molecule and change in the solvent (particularly comparing neutral aqueous solutions with hexafluoroacetone solutions) allowed some conformational conclusions. The linear C-terminal tripeptide is probably situated over the cyclic portion of the molecule both in vasopressin and oxytocin substances. Its steric interaction with the tyrosine side-chain seems to be particularly efficient in molecules containing D-arginine in position 8. In the vasopressin series the stacking interaction of neighbouring aromatic amino acid residues furthermore limits the conformational freedom of the tyrosine side-chain and also probably distorts the dihedral angles of residues 1-3 in comparison with oxytocin. The interactions of phenylalanine and arginine with tyrosine relatively decrease the conformational effects of the primary amino group. Consequently the local conformation of vasopressin in the region of the tyrosine residue is more rigid and less sensitive to changes in medium than that of oxytocin. The circular dichroic spectra did not show any basic conformational differences in the backbone peptide chain of oxytocin and vasopressin substances. A weak negative disulphide band at about 290 nm could be observed in the spectra of both series of substances.