Electrochemiluminescence from tunicate, tunichrome-metal complexes and other biological samples.

Low level intrinsic electrochemiluminescence (ECL) was induced from body fluids and homogenized tissues of oysters and several species of tunicates. No significant ECL was detected in human blood cell lysates, or bovine haematin, but minor ECL was observed in avian blood cell lysates. Both terrestrial grass and seagrass exhibited ECL, which is probably attributable to chlorophyll, since dead (brown) grass did not demonstrate ECL. It was postulated that organic-metal complexes in marine invertebrates were, at least in part, responsible for the intrinsic ECL, since such animals are known to be rich in organically bound metals. However, alternative biochemical mechanisms for the observed ECL, which do not involve metal chelates, are possible. Various metal ions were added to the invertebrate preparations to determine whether exogenous metals could enhance or inhibit the ECL reactions. Strongly oxidizing metal ions such as Ag+, Au+, Cu2+, Hg2+ and Sb2+ at > or = 100 ppm severely inhibited the intrinsic ECL response. No statistically significant ECL enhancement due to addition of metal ions was noted. ECL "profiles' were generated which demonstrated differences in the ECL responses of individual tunicate preparations to the presence of various exogenous metal ions. Differences in ECL profiles may represent differences in types or levels of endogenous metal chelates or other biochemical constituents. In addition, synthetic tunichromes (tunicate pigments) were analysed for ECL in the presence and absence of various added metal ions. One synthetic tunichrome isomer demonstrated a specific ECL interaction with Hg2+, while the other demonstrated broader ECL activity with several metal ions.

Development of a sensitive activity assay for high-volume evaluation of human renin inhibitory peptides in rat serum: results with U-71,038.

A sensitive activity assay for high volume evaluation of human renin inhibitory peptides (RIPs) in rat sera (range 2-80 ng/ml) was developed based on the low affinity of RIPs to rat renin and their high affinity to human renin. The utility of this activity assay was tested by measuring concentrations of a human RIP, U-71,038 (BOC-Pro-Phe-N-MeHis-Leu psi [CHOHCH2]Val-Ile-Amp), in rat sera, determined by the activity assay, by a sensitive radioimmunoassay (RIA), and by tracking tritiated drug. Rats were given radiolabeled drug as an intravenous bolus, and blood samples were collected at various times after dosing. The serum level of U-71,038 equivalents was determined by the three techniques. Whole blood was also counted for total radioactivity to evaluate the potential for U-71,038 incorporation into red blood cells. Results from the three serum assays indicate good agreement between the calculated U-71,038 equivalents for the 30 min and 1 hr collection times. The 2 and 4 hr collection times show excellent agreement for the activity assay and RIA; [3H]-U-71,038 determinations gave substantially higher values. Serum levels for U-71,038 determined 30 min after dosing averaged less than 300 ng equivalents/ml suggesting that less than 1% of the administered dose was in the systemic circulation at that time. Thus, U-71,038 was rapidly cleared. At the 4 hr collection time, the level of U-71,038 equivalents, as determined by activity assay and RIA, was ten times the in vitro IC50 for the renin inhibitory activity of U-71,038.(ABSTRACT TRUNCATED AT 250 WORDS)

A primate bioassay for the determination of renin inhibitory peptides in serum.

The study of renin inhibitory peptides (RIPs) in rodents and primates requires the establishment of a simple, high volume method for determining the concentration of RIPs in serum after intravenous or oral dosing. The human renin inhibition assay useful for rodents is not directly applicable to primates due to inherent production of angiotensin I from the primate serum angiotensinogen and added recombinant human renin. Therefore, a novel approach to analyze the serum concentrations of RIPs in primates is described based on in vitro studies with monkey serum. The procedure involves the inactivation of monkey angiotensinogen and monkey renin by thermal denaturation prior to analysis. Application of this assay was demonstrated by analyzing serum samples from an in vivo study in monkeys using ditekiren (U-71,038), a renin inhibitory peptide, and by validation of the assay and results using a tritium-based radioimmunoassay (RIA) for ditekiren. The minimum detectable limit of ditekiren for both the RIA and the bioassay for primates was 10ng/ml serum. The reported bioassay should be of value for monitoring serum levels of thermostable RIPs from pharmacokinetic, bioavailability, and pharmacodynamic studies in primates as well as in humans.

Substrate specificity and inhibitor structure-activity relationships of recombinant human renin: implications in the in vivo evaluation of renin inhibitors.

Homogeneous, active recombinant human renin obtained from Chinese hamster ovary (CHO) cells was characterized in vitro by (i) determination of its relative rates of hydrolysis of plasma angiotensinogens (ANGs) from human, monkey, baboon, rat, pig, rabbit, hamster, and dog and (ii) analysis of several synthetic ANG-based, inhibitors ranging in IC50 from 10(-10) to 10(-6) M. Comparison of the recombinant human renin with human kidney renin showed that these enzymes were indistinguishable from each other in terms of their plasma ANG specificities and inhibition by synthetic renin inhibitors. Porcine kidney renin was also characterized and shown to display plasma ANG hydrolysis profiles and inhibitor potencies that were markedly different from those of human renins. Finally, the results using the above plasma ANGs extend previous studies showing that the substrate specificity of human renin may be influenced by the amino acid residues at P2 (i.e., Ile, Val, or Tyr) and P3 (i.e., His or Tyr) sites. The relevance of these data to in vivo evaluation of renin inhibitors in animal models is discussed.

Effect of angiotensinogen concentration on renin activity following nephrectomy and adrenalectomy: implications in the activation of plasma prorenin.

We have studied the effects of bilateral nephrectomy and adrenalectomy on angiotensinogen concentration, plasma renin activity, and total plasma renin activity obtained after trypsin activation in rats and compared with controls. Our results show that the plasma angiotensinogen concentration of nephrectomized (NX) rats is 5-fold higher than that of adrenalectomized (AX) rats. On the other hand, plasma angiotensinogen concentration of AX rats was about 2.5-fold lower than that of control rats. While NX rat plasma possess no renin activity, its mixing (1:1, v/v) with AX plasma results in 2-fold increase in renin activity over that observed for AX plasma alone. These results suggest that the apparent increase in renin activity upon mixing these two plasmas is at least partly due to an overall increase in angiotensinogen concentration in the mixture. To show that it is not due to activation of NX plasma prorenin by a convertase from AX plasma, prorenin-free NX rat plasma was prepared by using an anti-renin immunoaffinity column. When this prorenin-free NX plasma was mixed (1:1, v/v) with AX, again a 2-fold increase in renin activity was observed which is attributed to the overall increase in plasma angiotensinogen in the mixture. It is concluded that rat plasma prorenin is probably not activated within the circulation by a prorenin convertase from the rat kidney.

Recombinant human interleukin-1 alpha and recombinant human interleukin-1 beta stimulate cartilage matrix degradation and inhibit glycosaminoglycan synthesis.

Recombinant human interleukin-1 alpha (rhIL-1 alpha) and recombinant human interleukin 1 beta (rhIL-1 beta) stimulated the time- and concentration-dependent release of glycosaminoglycan (GAG) from bovine nasal cartilage explants. Maximum GAG release occurred during six to eight days of cartilage exposure to either species of rhIL-1; and rhIL-1 alpha was consistently more potent than rhIL-1 beta. In addition to inducing cartilage matrix resorption, rhIL-1 alpha and rhIL-1 beta also inhibited the incorporation of [35SO4]sulfate into cartilage, which is a reflection of the suppression of GAG synthesis. IL-1 had no capacity to stimulate GAG relase from or inhibit GAG synthesis by dead cartilage. Cycloheximide, an inhibitor of protein synthesis, and 1, 10-phenanthroline, a metalloproteinase inhibitor, suppressed rhIL-1-stimulated cartilage matrix resorption. Polyclonal antisera to rhIL-1 alpha and rhIL-1 beta specifically neutralized the respective cytokines.

Preparation and application of polyclonal antibodies to recombinant human interleukin-1 alpha and interleukin-1 beta.

We report the preparation and application of polyclonal antisera to the analysis and quantitation of human interleukin-1 alpha (IL-1 alpha) and human interleukin-1 beta (IL-1 beta). The anti IL-1 alpha antibodies specifically react with the alpha form of IL-1 and do not cross react (less than 0.1%) with the beta form of IL-1 and vice versa. Data reported here demonstrate that detection of human IL-1 alpha or beta by a radioimmunoassay technique is sensitive enough to measure picogram levels of these lymphokines. The practical application of using these highly specific antisera for radioimmunoassays was established by measuring exogenously added IL-1 alpha or IL-1 beta to human plasma. Potential benefits of these reagents and the radioimmunoassay procedures described herein are discussed in relation to the biological assays which cannot distinguish between human IL-1 alpha and human IL-1 beta.

Renin activity determination using human plasma as a substrate.

A method for human renin activity determination using human plasma as the substrate is described. Angiotensin I is generated by incubating renin with human plasma, which is available along with the commercial radioimmunoassay kit for angiotensin I. The method obviates the need to isolate and purify the substrate, human angiotensinogen, from human plasma. In addition, the assay is highly renin specific, sensitive, and convenient to use for the routine determination of active human renin during its isolation and purification from tissue extracts or from genetically engineered bacterial and nonbacterial expression systems.

Folding and activation of recombinant human prorenin.

In vitro folding of mature renin, prorenin, and fused prorenin, all produced in denatured form in inclusion bodies in recombinant Escherichia coli, has been studied in order to evaluate the importance of prosequence in the folding of human renin. These studies have been compared with the in vivo folding and subsequent in vitro activation of recombinant human prorenin secreted by a nonbacterial expression system, namely Chinese hamster ovary (CHO) cells grown in serum-free medium. It is concluded that prosequence is essential in the folding of human renin and, therefore, the DNA coding for this sequence cannot be removed without affecting the recovery of active human renin from recombinant bacterial and nonbacterial systems.

Secretion of enzymatically active human renin from mammalian cells using an avian retroviral vector.

Recombinant plasmid-based retroviral expression vectors were constructed using a modified spleen necrosis virus (SNV) containing the Herpes simplex virus thymidine kinase gene promoter controlling the expression of the Tn5 neomycin phosphotransferase II gene (NPTII gene). The human renin (HRn) gene (hrn) was inserted into the 5' end of the SNV sequences such that in concatemeric plasmid DNA its expression was controlled by the strong promoter in the SNV long terminal repeat (LTR). Dog cells transfected with the concatemeric plasmid DNA secreted a small amount of a HRn-like 43-kDa protein. After cotransfection of chicken cells with concatemeric plasmid DNA and proviral DNA of reticuloendotheliosis virus strain A, infectious stocks of viruses were recovered. Cells infected with the virus carrying the viral LTR-hrn gene oriented for expression secreted the 43-kDa HRn-like protein at about 100-fold higher levels than the cells transfected with the plasmid DNAs. Biological activity of secreted HRn was determined by measuring levels of angiotensin I generated by incubating culture media with either a porcine or human angiotensinogen substrate. Infected dog cells produce about 40 ng of enzymatically active HRn per 10(6) cells per 24 h. These data indicate that retroviral expression vectors provide a good system for obtaining the secretion of high levels of enzymatically active heterologous proteins from mammalian cells.

Steroid-induced urogenital tract changes and urine retention in laboratory rodents.

Since previous literature suggested that estrogen-treated male mice are models for human benign prostatic hypertrophy, a series of studies was designed to examine urine retention and urogenital tract changes in rodents given chronic estradiol-17 beta (E) and dihydrotestosterone (DHT) treatments. In Study 1, intact and castrate male mice received E, DHT or E plus DHT for four weeks via subcutaneous Silastic capsules. Bladder urine volume increased in the groups given E and this effect was not altered by castration, DHT or removal of E capsules two weeks before necropsy. Estrogen treatment also increased mortality. In Study 2, intact male, intact female, adrenalectomized (Adx) male and sham Adx male mice received 16 weeks of steroid treatments. Bladder urine volume increased in all E treated groups regardless of sex or Adx. Hydronephrosis, hydroureter and increased mortality were found in the E treated mice of both sexes. Estrogen induced epithelial changes and edema of the prostate, vas deferens and the utriculus prostaticus. In further studies male rats, hamsters and guinea pigs were given several different dosages of E but no evidence of urine retention or increased mortality was found. Taken together these studies suggest that E-induced urine retention is unique to mice. Although urine retention and hydronephrosis found in the mice were similar to those in humans with BPH, the lesion that results in the urine obstruction is not similar.

Synthesis and biological activity of prostaglandin lactones.

Most of the primary prostaglandins and several biologically important prostaglandin analogues were converted to 1,9-, 1,11- or 1,15-lactones, in order to investigate the biological profiles of these internal esters and to assess their potential as prodrugs for the corresponding open-chain hydroxy acids. In each case, the key lactonization step was done using Corey's "double activation" procedure (cyclization of omega-hydroxy-2-pyridinethiol esters). In general, the 1,9-lactones exhibited less than 1% of the biological activity of the parent hydroxy acids in the standard prostaglandin test systems. The 1,11- and 1,15-lactones, on the other hand, were essentially equal to the parent hydroxy acids as antifertility agents (a 4-day assay which would allow time for in vivo enzymatic lactone hydrolysis). The 1,11- and 1,15-lactones exhibited very low activity in acute or in vitro screens (e.g., rat blood pressure and gerbil colon stimulation), assays which more closely reflect the intrinsic activity of the lactones themselves. These results are consistent with the observed relative ease of enzymatic hydrolysis of the prostaglandin lactones (1,15 greater than or equal to 1,11 much greater than 1,9). Several of the lactones whose parent hydroxy acids are resistant to metabolic inactivation (e.g., 15-methyl, 16-phenoxy, and 17-phenyl) exhibited potent abortifacient activity in the hamster. These lactones, with greatly diminished activity in the blood pressure and smooth muscle assays (indicators of potential side effects), represent a therapeutically useful class of antifertility agents.

Hypophysectomized male rats treated with polydimethylsiloxane capsules containing testosterone: effects on spermatogenesis, fertility, and reproductive tract concentrations of androgens.

Spermatogenesis, reproductive luminal contents and androgen concentrations were examined in hypophysectomized male rats treated with 1 of 3 testosterone (T) dosages for 60-64 days and in sham-operated controls. Hypophysectomized rats treated with 2 cm long T implants showed normal mating but reduced fertility, while normal fertility was maintained in animals given 8 or 3 x 8 cm T. Spermatogenesis in the hypophysectomized groups treated with 2 cm T for 10 days was generally arrested at the spermatocyte stage, while in the hypophysectomized animals treated with 2 cm T for 64 days spermatogenesis was halted at the spermatid stage. The 8 and 3 x 8 T-treated hypophysectomized animals had normal spermatogenesis with only minimal focal areas of degenerating seminiferous epithelium. Serum T concentrations were reduced by hypophysectomy, maintained at control levels by the 2 cm T dosage and increased to pharmacological levels in a dose-dependent manner by 8 and 3 x 8 cm T treatments. Testicular T concentrations also responded in a dose-dependent manner but the 3 x 8 cm T dose was not sufficient to keep testicular T at control levels. Dihydrotestosterone (DHT) in the caput epididymidis was maintained at control levels by the 8, reduced by the 2 and elevated by the 3 x 8 cm T treatment. Without gonadotropins, higher than normal levels of serum T are required to maintain normal fertility, although this normal reproductive capacity is possible even with greatly reduced testicular T concentrations.

Influence of testosterone, estradiol-17 beta and dihydrotestosterone on circulating LH and FSH in castrate male guinea pigs.

The influence of testosterone, dihydrotestosterone, estradiol-17 beta or combinations of these steroids on gonadotropin regulation was examined in castrate male guinea pigs. Physiological replacement therapy with testosterone prevented the postcastration rise in LH. Treatment with dihydrotestosterone and the combination of estradiol-17 beta plus testosterone was also effective. In contrast, these treatments reduced but did not prevent the postcastration rise of FSH. The negative feedback effects of these steroid treatments was not altered when 3, 6 or more than 8 weeks elapsed between castration and the initiation of steroid replacement therapy. Our data indicate that the guinea pig testosterone and dihydrotestosterone are effective regulators of LH but that other hormones may be important in the regulation of FSH.

Plasma concentrations of 9-deoxo-16-dimethyl-9-methylene-PGE2 in rhesus monkeys after administration by various routes.

A method is described for the estimation of 9-deoxo-16, 16-dimethyl-9-methylene-PGE2 by double antibody radioimmunoassay. Plasma samples obtained from animals treated with 9-methylene-16, 16-dimethyl-PGE2, 1-adamantanamic salt were extracted with diethyl ether to recover the prostaglandin. The validation of sample preparation and assay procedure are presented. Rhesus females were treated by several routes of administration and the samples assayed for drug content. Maximum blood levels were probably reached 30 minutes following subcutaneous injection and within 30 seconds of an intravenous injection. Results of the acute intravenous injection indicate an initial half-life of approximately one minute in peripheral circulation. Continuous intravenous infusion at 3 increasing doses of this compound resulted in a stepwise increase in plasma drug concentrations. Vaginal administration of 9-methylene-16, 16-dimethyl-PGE2, 1-adamantanamine salt in suppositories produced a dose dependent increase in plasma drug concentration. Higher plasma drug concentrations were produced when the prostaglandin was delivered in H-15 base suppositories than in E-76 base suppositories.

Prostaglandin prodrugs III: Synthesis and biological properties of C9- and C15-monoesters of dinoprost (prostaglandin F2 alpha).

Methods are described for the synthesis of dinoprost C9- and C15-monoesters using protective groups. Esters at C9 were synthesized by acylation of dinoprost 11,15-bis(tetrahydropyran-2-yl)ether followed by acid-catalyzed protective group removal. Esters at C15 were synthesized by initial formation of the protected intermediate, dinoprost 9,11-n-butylboronate, followed by acylation and hydrolytic protective group removal. Many esters were active in vivo in the hamster antifertility screen. Plasma hydrolysis studies showed that the C15-esters were more readily cleaved than the C9-esters. In vivo studies in the rat showed that both the C9- and C15-esters resulted in urinary excretion of 5 alpha, 7 alpha-dihydroxy-11-ketotetranorprosta-1,16-dioic acid in amounts comparable to those obtained after dosing with dinoprost, indicating that ester hydrolysis occurred in vivo.

2-(Aminomethyl)-2-decarboxyprostaglandin F2 alpha type analogs.

The 2-(aminomethyl)-2-decarboxy analogs of prostaglandin F2 alpha (PGF2 alpha), (15S)-15-methyl-PGF2 alpha, 16-phenoxy-omega-tetranor-PGF2 alpha and 16,16-dimethyl-PGF2 alpha were synthesized. The amino analogs closely resemble the parent PGF2 alpha compounds as antifertility agents in the hamster.

Measurement of (15S)-15-methyl prostaglandin F2alpha by radioimmunoassay.

Antibodies against (15s)-15-methyl prostaglandin F2alpha (15-MF) were produced in rabbits immunized with a 15-MF bovine serum albumin conjugate. Tritium labeled 15-MF was prepared from (15S)-15-methyl prostaglandin E2 by reduction with tritiated sodium borohydride. Antiserum specificity and label specific activity (12 Ci/mM) were sfficient to enable development of a sensitive, accurate and relatively specific radioimmunoassay for this prostaglandin analog. The assay can detect as little as five picograms of 15-MF in 100 mul of unextracted blood plasma. Endogenous concentrations of PGF2alpha should not cause a significant blank using current assay conditions, as cross reactivity with this molecule was less than one percent. There is, however, significant affinity (45%) for the C-1 methyl ester of 15-MF. This makes the assay also applicable for measuring this molecule, although no distinction can be made between it and the free acid when neat plasma is assayed. Plasma samples from human and monkey studies were assayed for 15-MF before and after recipients were treated with pharmacologically active doses of this compound. Blank values (0 Hour) of less than 0.1 nanograms per ml were found for all pre-treatment samples while post-treatment samples contained detectable levels. Results of the human study was compared and found to be in excellent agreement with values that were obtained using a gas-liquid chromatographic - mass spectrographic assay.

Preparation and quantitation of urinary metabolites of prostaglandin F2alpha by radioimmunoassay.

Radioimmunoassay systems are described which have been developed to quantitate two principle urinary metabolites of PGF2alpha; 9alpha,11alpha-dihydroxy-15-oxo-2,3,4,5-tetranorprostanoic acid (I) and 9alpha,11alpha-dihydroxy-15-oxo-2,3,4,5-tetranorprosta-1,20-dioic acid (II). Preparation of the required metabolites was achieved by total synthesis (I) or by bioconversion (isolation from urine of animals treated with 15-keto-PGF2alpha, II). These metabolites were used to prepare conjugates for immunization. Labelled metabolites, suitable as binding markers, were prepared by metabolism of 3-H-PGF2alpha in vitro (I) or in vivo (II). Specificity of the resulting antibodies was compared to an antibody to PGF2alpha and to 13,14-dihydro-15-keto PGF2alpha. Antisera of II had little or no affinity for 20-carbon precursors (PGF2alpha or 13,14-dihydro-15-keto PGF2alpha), but had nearly equal affinity for metabolite I. Antisera of I, however, had little or no affinity for antigen of II. Therefore, analysis of samples by both assay systems enables quantitation of these excretion products of PGF2alpha. Other assay parameters (binding, affinity, recovery, precision and the repeatability of the assays) were similar to those previously described for other RIA systems, and were considered satisfactory for quantitation of compounds in biological fluids. Quantitation of 24 hour urinary excretion of di-acid metabolite in humans was in close agreement with previously published values determined by physical-chemical means. Greater quantity of di-acid metabolite was excreted by human males (42.0 mug/24 hr) than by females sampled either during the follicular (20.0) or luteal phase (21.2) of the menstrual cycle. The total quantity of C-16 metabolites (as approximated by system II) excreted/kg body weight by the rhesus monkey was similar to that excreted by the human. However, the ratio of di-acid to monoacid was much nearer unity in the monkey than the human.