HPLC method for the determination of oxytocin in pharmaceutical dosage form and comparison with biological method.

Conditions have been established for the determination of oxytocin by the HPLC method; the method has been validated. The results of HPLC determinations are compared with those obtained by the biological method.

Capillary electrophoretic separation of N-acetylcysteine and its impurities as a method for quality control of pharmaceuticals.

Capillary electrophoresis has been applied to separate and determine N-acetylcysteine (NAC) and related impurities. Determination conditions were found to be optimum with 100 mmol/l borate as the buffer, pH 8.40. The limit of detection was established for each substance examined. The method has been validated by examining linearity ranges, precision and repeatability. The method was used to determine the content of NAC in, and purity of, pharmaceutical preparations. The major impurities (N,N-diacetylcystine, N,S-diacetylcysteine and cystine) were determined at levels of 0.1%.

New renin inhibitors with hydrophilic C-terminus.

Four new peptide-based renin inhibitors, Boc-Phe(4-OMe)-MePhe-AHPPA-epsilon Ahx-EA (11), Boc-Phe(4-OMe)-MeLeu-AHP-PA-epsilon Ahx-EA (15), Boc-Phe(4-OMe)-MePhe-Sta-epsilon Ahx-EA (20) and Boc-Phe(4-OMe)-MeLeu-Sta-epsilon Ahx-EA (21) have been synthesized in search of structures with improved biological properties. They were designed as compounds with moderate hydrophobicity (5.28, 4.79, 4.79 and 4.30), respectively. All synthesized inhibitors were resistant to chymotrypsin activity, all were poorly soluble in buffers pH 2.0 and pH 7.4. The inhibitory potency of renin activity in vitro of 11, 15, 20 and 21 expressed as IC50 was 7.0 x 10(-4), 7.5 x 10(-5), 6.0 x 10(-4) and 2.5 x 10(-4) M/l, respectively.

Determination of protamine sulphate in drug formulations using high performance liquid chromatography.

Protamine sulphate, which has the property of neutralising heparin, is determined in pharmaceutical formulations using spectrophotometric (BP 1995) or biochemical methods (USP XXIII 1995). Accuracy of these methods is not very high. We applied the HPLC technique for the assay of protamine sulphate in a gel formulation. The assay was carried out on a diol-type column with a mobile phase containing 8% acetonitrile in 0.15% trifluoroacetic acid at pH 2.5. The flow rate was 1.0 ml min(-1). The results obtained show that HPLC can be used for the determination of protamine sulphate in pharmaceutical preparations. The method is rapid and more accurate than those described until now.

Renin inhibitors containing nicotinic or isonicotinic acid at the N-terminus. Part V.

Four new compounds: Nic-Phe/4-OMe/-MePhe-Sta-epsilonAhx-OMe/23/,Nic-Phe/4-OMe/-MePhe- Sta-epsilonAhx-Iaa/24/,iNic-Phe/4-OMe/-MeLeu-Sta-ep silonAhx-OMe/29/ and iNic-Phe/4-OMe/-MeLeu-Sta-epsilonAhx-Iaa/30/ have been synthesized in search after renin inhibitors of improved biological properties. Their stability against chymotrypsin activity, solubility in water at pH 7.4, 6.9 and 2.0, partition coefficient and activity in vitro were determined. All synthesized inhibitors are resistant to enzymatic degradation, all are very good soluble in water at pH 2.0, poorly soluble at pH 6.9 and insoluble at pH 7.4. Partition coefficients go up together with increase of pH worth of buffer. IC50 of obtained inhibitors 23,24,29 and 30 is 3 x 10(-4),7.5 x 10(-4),4 x 10(5) and 4 x 10(-3)M/1 respectively.

Enzymatically stable renin inhibitors containing statine and 6 aminohexanoic acid. Part IV.

Eight new peptide renin inhibitors: Boc-Phe/4-OMe/His-Sta-epsilonAhx-Iaa(13), Boc-Phe/4-OMe/-His-Sta-episilonAhx-OMe,(21),Boc-Phe/4-OMe/-MePhe-S ta-epsilonAhx-Iaa(27),Boc-Phe/4-OMe/-MePhe-Sta-epsilonAhx-++ +epsilonAhx-Iaa(32),Boc-Phe/4-OMe/-MePhe-Sta-Val-epsilonAhx- OMe (38),Boc-Phe/4-OMe/-MeVal-Sta-Val-Iaa(48),Boc-Phe/4-OMe/-Me Val-Sta-Iaa(51), Boc-Phe/4-OMe/-MeLeu-Sta-epsilonAhx-Iaa (57) have been synthesized in search after compounds of improved biological properties. All peptides were obtained by carbodimide method in solution by stepwise elongation of the peptide chain or by fragment condensation. Their potency was assayed in vitro by a spectrofluorometric method/assay of Leu-Val-Tyr-Ser released from N-acetyltetradecapeptide substrate by renin in the presence of an inhibitor/. Their resistance to enzymatic degradation was assayed by determination of stability to chymotrypsin activity. The most potent inhibitor was (13):IC50 = 7 x 10(-8)M/1. All inhibitors were stable to chymotrypsin.

Renin inhibitors containing statine and 6-aminohexanoic acid. Part III.

Five peptide renin inhibitors containing the sequence: Phe-His-Sta-epsilon Ahx (Sta = 4(S)-amino-3(S)-hydroxy-6-methylheptanoic acid, epsilon Ahx = 6-aminohexanoic acid) were synthesized and their potency was assayed in vitro by a spectrofluorometric method (assay of Leu-Val-Tyr-Ser released from N-acetyltetradecapeptide substrate by renin in the presence of an inhibitor). Their stability was tested by assay of Phe and Pro-Phe released after incubation with chymotrypsin. The most potent inhibitor was Boc-Phe-His-Sta-epsilon Ahx-OMe (IC50 = 5 x 10(-9) M/l), the most stable--Boc-Pro-Phe-His-Sta-epsilon Ahx-OMe (resistant to incubation with chymotrypsin for 4 h).

[Comparative determination of lipase activity in pharmaceutical preparations]. / Porównawcza ocena aktywnosci lipazy w preparatach farmaceutycznych.

Lipase activity in various pharmaceuticals has been assayed with different methods expressing the results in different units. Conversion factors enabling comparison of the international and Wallersteins units in FIP units which is current way of pancreatin enzyme activity expression have been calculated. Lipase activity conversion factors are diversified and depend on the type of pharmaceutical preparation containing pancreatic extract. Conversion factors facilitate comparison of lipase activity in different Polish and foreign-made pharmaceutical preparations and proper dosage.

Synthesis and antienzymic activity of two new angiotensin converting enzyme inhibitors.

Two new analogs of captopril were synthesized. Inhibition of angiotensin converting enzyme (ACE) by these compounds in vitro and in vivo was determined using fluorimetric method. The obtained compounds were much weaker ACE inhibitors than captopril.

Methionine dependence of virus-infected cells.

Mouse embryo cells nonproductively infected with human cytomegalovirus differed from noninfected cells by the impaired ability to grow in the medium containing homocysteine instead of methionine. Virus infection of mouse embryo cells grown in both kinds of media resulted in the increase of protein synthesis. In the infected cells grown on homocysteine this increase was followed by a quick decrease. The effects of homocysteine substitution could be abolished by the addition of low amounts of methionine (0.1 mM). Methionine uptake in the infected cells grown on homocysteine for 48 h was significantly higher than that in the noninfected cells.

Effects of retinoic acid on plasminogen activator activity and cellular proliferation.

This paper reports effects of retinoic acid (RA) on the expression of plasminogen activator (PA) activity and their relation to the effects of the vitamin on cellular proliferation. RA at the concentrations of 10 microM ml and 1 microM/ml did not affect PA activity in the cells of human melanoma cell line 10-135 but produced a transient decrease of PA activity as well in two other human melanoma cell lines as in RK 13 and IAR 6-7 cells. Unlike 10-135 cells which were resistant to retinoic acid all the remaining cell lines were susceptible to inhibition of the growth by the vitamin. Replacement of the medium with RA by standard medium produced a reversal of the inhibitory effects of the vitamin on PA activity and cell proliferation.

[Measurement of angiotensin I convertase activity in the serum of the rat]. / Pomiar aktywnosci konwertazy angiotensyny I w surowicy krwi szczura.

A fluorimetric method has been developed for the determination of activity of angiotensin I convertase in rat blood serum. The method proved highly sensitive. It enables determination of the activity of angiotensin I converting enzyme in 0.01 cm3 of blood serum. The method may be applied to in vitro and in vivo testing of inhibitors of angiotensin I convertase.

Influence of retinoic acid on protein synthesis and transport of L-methionine in cultured L cells.

The effects of retinoic acid (RA) on cell proliferation, activity of acid phosphatase, protein synthesis and methionine uptake were studied in transformed murine LPA cells. Early inhibition of protein synthesis was demonstrated under experimental conditions in which the rate of cell proliferation was diminished and non-specific effects of vitamin action could be excluded. Measurements of L-methionine uptake revealed a decrease to approximately one-half of that in control cultures after treatment with RA at the concentrations of 5 X 10(-5) M and 10(-4) M.

Methionine regulation of N-5-methyltetrahydrofolate: homocysteine methyltransferase and its influence on the growth and protein synthesis in normal, neoplastic, and transformed cells in culture.

Normal human embryonic fibroblasts (CLV-58) and normal embryonic rat fibroblasts (REF) revealed equal growth in media containing 0.2 mM DL-homocysteine thiolactone (HOM) or in methionine (METH) enriched with 1.5 microM cyanocobalamin and 0.1 mM folic acid, whereas L 5178Y lymphoblasts and transformed rat fibroblasts (REF-S) showed more or less acute need for exogenous METH. In normal cells general protein synthesis remained unimpaired during 24 hours after substitution of METH by HOM. In L 5178Y and REF-S cells, however, protein synthesis was significantly inhibited. Measurements of N-5-methyltetrahydrofolate:homocysteine methyltransferase revealed a two- to threefold increase of enzyme activity in normal cells grown on HOM and in L 5178Y and REF-S cells grown on HOM with added METH. HeLa cells showed variability in growth pattern, in general protein synthesis response, and in inducibility of methyltransferase upon substitution of METH by HOM.

Transport of methionine by normal and transformed rat embryo fibroblasts.

Methionine uptake was 2-2.3-fold higher in the transformed rat embryo fibroblasts as compared with the non-transformed cells. This was associated with a 2-fold greater Vmax, with no detectable changes in Km values. Methionine starvation caused an increase in the rate of transport, the increase being higher in the transformed cells. Addition of methionine abolished the starvation effect, although to a smaller extent in the transformed cells. It is suggested that differences in methionine uptake might be due to a quicker depletion of intracellular methionine, and not to changes in the transport system of the transformed cells.

[Effect of lack of exogenous methionine on protein synthesis in normal and leukemic leukocytes cultured in vitro]. / Wplyw braku metioniny egzogennej na synteze bialka w prawidlowych i bialaczkowych krwinkach bialych w hodowli in vitro.

Experiments were performed on lymphocytes and leucocytes isolated from peripheral blood of healthy donors and on leucocytes from patients with acute myeloblastic and acute lymphoblastic leukaemia. The influence on protein synthesis of methionine depletion in the medium or replacement of methionine by homocysteine was measured in labelling experiments with tritiated valine. The rate of protein synthesis in lymphocytes and leucocytes from healthy donors cultivated in homocysteine--containing medium and stimulated with PHA did not differ significantly in the course of 5 days from the rate of protein synthesis in the basic medium. In leukaemic leucocytes cultivated in vitro in the time period of 34 h in the rate of protein synthesis in homocysteine--containing medium was similar to the rate of protein synthesis in the medium devoid of methionine.

Effect of excess methionine on methotrexate cell-killing action in vitro.

Methionine at the concentrations 10, 15 and 20 times higher than in complete Fischer's medium produced a transient impairment or a transient block in the growth of L 5178 Y cells in culture without influencing their viability. Autoradiographic data revealed that methionine-induced inhibition of DNA synthesis was followed by a partial synchronization of cells in the phase of DNA synthesis. Methionine at a high concentration (1.5 mg/ml) enhanced the cell-killing action of methotrexate under conditions of delayed drug addition and did not diminish it when added with the drug simultaneously.

Dependence on exogenous methionine of rat sarcoma and murine leukemia cells in culture.

A comparative study was performed on methionine auxotrophy of rat sarcoma and murine leukemia cells taken directly from the organism and grown in culture in media lacking methionine or in which methionine was substituted by homocysteine. Methionine auxotrophy was observed in both kinds of cells. At low levels of methionine in the media containing homocysteine rat sarcoma cells showed an increase in growth. Addition of homocysteine to the media with low levels of methionine did not influence the survival of murine leukemia cells.