Clinical pharmacology and clinical trials in Japan.

Clinical pharmacology is the pursuit of rational therapeutics by following the scientific principles of medicine and pharmacology. In Japan the roles for clinical pharmacology and clinical pharmacologists have been evolving since the discipline appeared in the 1950s. Clinical pharmacology and clinical trials for drug development depend on each other, and clinical pharmacologists play an important role in drug development in Japan. As the discipline becomes more important and complicated, many issues regarding drug therapeutics and clinical trials in Japan have been raised, and several points of view have been expressed. The following suggestions have been made to improve clinical pharmacology in Japan: (a) Medical education in the field of clinical pharmacology must be improved by creating or improving clinical pharmacology programs in medical schools. (b) The appropriate infrastructure for clinical trials must be established so that the physicians' workload is reduced, and patients' participation in clinical trials becomes much easier. (c) Scientific and ethical standards of the pharmaceutical industry must be improved, and the effort should be made to produce drugs with new mechanisms of action or with significant expected benefits. (d) The regulatory agency must provide stronger support, encompassing all the various points of view of academic institutes and the pharmaceutical industry. In light of the enthusiasm demonstrated by the government, physicians, and pharmaceutical industry in Japan for continued progress in clinical pharmacology, it seems likely that all its challenges will be overcome in the near future. Hence, despite the various problems discussed here the future seems promising for the continued development of clinical pharmacology.

Fate of recombinant human renin administered exogenously to anesthetized monkeys.

Highly purified recombinant human renin (rh-renin), synthesized by Chinese hamster ovary cells, was labeled with iodine-125 and was given intravenously to pentobarbital-anesthetized common marmosets (Callithrix jacchus) to study the fate of the circulating renin. Specific anti-rh-renin antiserum was used to identify the 125I-rh-renin. Plasma disappearance of the exogenously administered 125I-rh-renin in marmosets (n = 6) showed two exponential components, with a half-life of 12.1 +/- 1.9 minutes for the rapid component and 120.3 +/- 16.4 minutes for the slow component. The metabolic clearance rate was 1.17 +/- 0.26 ml/min/kg. Thirty minutes after the injection of 125I-rh-renin, 43.1 +/- 0.9 and 3.5 +/- 0.5% of the injected dose had distributed to the liver and the kidneys, respectively. With time, the accumulated 125I-rh-renin in the liver and kidneys decreased. The accumulation of 125I-rh-renin was less than 1% of the dose injected in other organs such as lungs, heart, spleen, adrenal glands, testes, and ovaries. Analysis of liver and kidney extracts by high performance liquid chromatography at 30 and 120 minutes indicated that immunoreactive 125I-rh-renin decreased with time and was accompanied by an increase in nonimmunoreactive degradation products of a low molecular weight. The incubation of 125I-rh-renin with monkey or human plasma at 37 degrees C did not degrade the labeled renin. Therefore, rh-renin was rapidly cleared from the circulation by the liver and the kidney.

Urinary excretion of renin and its biochemical properties in dogs.

The amount and biochemical properties of renin excreted by anesthetized dogs were investigated to elucidate the significance of urinary excretion in the metabolism of renin. Mean arterial blood pressure was 127 +/- 4 mm Hg, renal blood flow was 170 +/- 8 ml/min, glomerular filtration rate, 38.6 +/- 2.3 ml/min, and urine flow rate, 0.37 +/- 0.09 ml/min (n = 11). Urinary renin concentration (URC) was 9.2 +/- 2.1 ng angiotensin I (ANG I)/ml X hr (n = 11), as determined by radioimmunoassay for ANG I generated by incubation with semipurified homologous renin substrate. The ANG I-producing activity was inhibited by more than 90% by a specific antibody to dog kidney renin. The renin secretion rate from the left kidney into the renal vein was 76.4 +/- 13.3 ng ANG I/ml X hr per min (n = 11), and the simultaneous urinary excretion rate of renin was 2.3 +/- 0.4 ng ANG I/ml X hr per min (n = 11). Molecular weight of the urinary renin was 40,000 daltons. The pH dependent curves of the angiotensin-forming capacity of renin showed an optimum between pH 5.5 to 6.0, and the estimated Michaelis constant was 0.42 microM. These biochemical parameters were similar to the findings in the case of renin in the plasma and the kidney. Moreover, neither acid nor trypsin treatment altered the renin activity in the urine. Thus, the active form of renin with a molecular weight 40,000 was excreted into the urine in dogs. Urinary excretion of renin was a small percentage of the renin secretion rate, thereby indicating the minor role of urinary excretion in the metabolism of renin.

Renal metabolism in mice of exogenously administered 125I labelled renin.

125I labelled mouse submaxillary renin was administered intravenously to anaesthetized male Institute of Cancer Research, USA (ICR) mice in an attempt to determine the organ-related degradation of circulating renin. A specific antirenin antiserum was used for identification. The disappearance rate of labelled renin from plasma was estimated on the basis of a two-compartmental model. The mean half-time of clearance of labelled renin from plasma in intact control and 70% hepatectomized mice was 13.3 +/- 0.8 and 12.0 +/- 0.9 min respectively; these values were not significantly different. The mean half-time of clearance of labelled renin from plasma in nephrectomized mice was 40.0 +/- 2.4 min; this was significantly longer than intact controls and 70% hepatectomized mice. Fifty per cent of the administered renin accumulated in the kidney and 9% in the liver. A small amount of 125I labelled renin was identified in urine, and a large amount of 125I liberated from labelled renin was detected. Since the high performance liquid chromatography profiles showed that plasma renin rapidly moves into the kidney, clearance of circulating renin may take place mainly in this organ.

Whole body autoradiographic distribution of exogenously administered renin in mice.

The distribution of exogenously administered renin was investigated using whole body autoradiography. Purified renin from mouse submaxillary gland (SR) was labeled with radioactive iodine (125I). This labeled renin (125I-SR) and Na125I were administered into the tail vein of male ddY mice, in doses of 10.2 and 16.4 mu Ci/30 g body weight, respectively. Mice were killed by an overdose of ether, and autoradiography was performed on whole body sections. To separate free 125I liberated from 125I-SR, sections were treated with perchloric acid. A major accumulation of 125I-SR, acid-insoluble, was evident in the renal cortex, whereas the hepatic accumulation of 125I-SR was minor. Radioactivity in the thyroid and submaxillary glands, in the stomach, and in urine was also apparent, but disappeared after acid treatment, except in the thyroid glands. Radioactivity in the brain, intestinal content, spleen, and adrenal glands was nil. These autoradiograms provide the first evidence that exogenously administered renin is mainly distributed in the renal cortex.

Whole-body autoradiographic distribution of exogenously administered renal renin in rats.

We studied, by whole-body autoradiography, the distribution of exogenously administered renal renin in rat. Rat renal renin was completely purified and labeled with 125I ([125I]-renin) and was then injected into the tail veins of conscious rats at a dose of 30 microCi, 430 ng. After various intervals, rats were killed by an overdose of ether, the whole body rapidly frozen in acetone-dry ice, and autoradiography performed on sagittal whole-body sections. To remove breakdown products ([125I]-tyrosine and free 125I) from [125I]-renin, sections were treated with perchloric acid solution. The main accumulation of [125I]-renin acid-insoluble radioactivity was observed in liver and renal cortex. The accumulation in these organs was already evident 2 min after the injection, reached a maximum level by 15 min, then gradually decreased. A small amount of [125I]-renin was also evident in spleen, bone marrow, and adrenal gland. Thirty min after the injection, radioactivity began to appear in the thyroid gland, stomach, and small intestine, but disappeared with acid treatment, except in the thyroid. Radioactivity was negligible in other organs including brain, submaxillary gland, lung, heart, and testis. These autoradiographs clearly demonstrate that exogenously administered renal renin is distributed mainly in the liver and renal cortex.

Mechanisms of the biphasic responses to endothelin-3 in dog coronary arteries.

1. Endothelin-3 (ET-3) elicited relaxations at low concentrations (up to 10(-8) M) and contractions at higher concentrations in dog isolated coronary arteries precontracted with prostaglandin F2 alpha (PGF2 alpha). The relaxation by ET-3 was not affected by endothelium denudation nor treatment with NG-nitro-L-arginine, but was abolished or reversed to a contraction by treatment with indomethacin and markedly suppressed by tranylcypromine, a PGI2 synthetase inhibitor, or diphloretin phosphate, a prostaglandin receptor antagonist. ET-1 produced only concentration-dependent contractions. 2. BQ-123, a new selective ETA receptor antagonist, caused relaxation of the strips contracted with ET-3 in a dose-dependent manner and prevented the ET-3-induced contraction but did not affect the contraction produced by PGF2 alpha. The relaxation caused by ET-3 was enhanced by treatment with BQ-123. 3. It is concluded that the relaxations elicited by ET-3 in dog coronary arteries are mediated via liberation of PGI2 by activation of non-ETA receptors, located in subendothelial tissues, possibly smooth muscle cells, whereas the peptide-induced contractions are mediated via ETA receptors.

Acute effects of E-3174, a human active metabolite of losartan, on the cardiovascular system in tachycardia-induced canine heart failure.

The aim of this study was to evaluate the acute effects of E-3174, a human active metabolite of the AT1 receptor antagonist, losartan, on hemodynamic functions in dogs with severe heart failure (HF). In dogs, insignificant plasma levels of E-3174 are present following administration of losartan, and therefore, the effects of these two drugs can be studied independently in the dog. HF was established by rapid pacing of the right ventricle (250-270 beats/min) for 4 weeks. We examined changes in cardiovascular functions after acute intravenous administration of losartan (1 mg/kg) and E-3174 (0.3 and 1 mg/kg), as well as an ACE inhibitor, enalapril (0.3 and 1 mg/kg), under condition of HF. The HF before treatment was characterized by increases in pre- and after-load of the left ventricle (LV), consequent low cardiac output, and LV dilatation. E-3174 at 0.3 and 1 mg/kg reduced pulmonary artery pressure (-13+/-6% and -22+/-3% from baseline, respectively, p<0.05), pulmonary capillary wedge pressure (-18+/-4% and -36+/-10%, p<0.05) and mean arterial pressure (-24+/-2% and -36+/-7%, p<0.05), increased stroke volume (SV: +12+/-7% p>0.05; +36 +/-19%, p<0.05), and reduced peripheral resistance (-23+/-5% and -41+/-9%, p<0.05), but had no effect on the first derivative of left ventricular pressure (dP/dt/P) or the time constant for relaxation. Effects of losartan at 1 mg/kg were similar to those of 0.3 mg/kg of E-3174. Enalapril at 1 mg/kg caused changes comparable to those seen after E-3174 administration (1 mg/kg), except that the increase in SV (+16+/-8%, p<0.05) with enalapril was not as great as that with E-3174. Both losartan at 1 mg/kg and E-3174 at 0.3 and 1 mg/kg increased fractional shortening to a similar extent (FS: +52+/-12%, +47+/-8% and +56+/-8%), while enalapril at 0.3 and 1 mg/kg had no significant effects on FS. Reflex elevation of plasma renin activity induced by 1 mg/kg of E-3174 was similar to that caused by 1 mg/kg of enalapril, suggesting that the two drugs achieved similar inhibition of the endogenous renin angiotensin system. Our study demonstrated that acute blockade of the AT1 receptor with E-3174 reduced elevated pre- and after-load and consequently increased stroke volume in a canine HF model. With the exception of changes in stroke volume, these effects of E-3174 were comparable to those produced by enalapril, and were 3 times stronger than those by losartan.

Rat atrial natriuretic factor stimulates renin release from renal cortical slices.

We investigated the effect of synthetic rat atrial natriuretic factor (ANF) on renin release from rat renal cortical slices. Rat ANF (10(-6) M) increased renin release from the slices with a concomitant increase in the levels of cGMP contents. The increase in cGMP was also prominent in the case of incubation with 10(-4) M sodium nitroprusside but was not accompanied by an enhanced release renin. 8-Bromo-cGMP did not stimulate renin release. We propose that the stimulation of renin release from rat renal cortical slices is not related to an increase in endogenous cGMP.

Antihypertensive effects of BQ-123, a selective endothelin ETA receptor antagonist, in spontaneously hypertensive rats treated with DOCA-salt.

We determined the antihypertensive effects of BQ-123 (cyclo(D-Trp-D-Asp-L-Pro-D-Val-L-Leu-), sodium salt), a selective endothelin ETA receptor antagonist, in spontaneously hypertensive rats treated with deoxycorticosterone acetate-salt (DOCA-salt SHR). BQ-123 (1-30 mg/kg/h) decreased blood pressure in DOCA-salt SHR in a dose-dependent manner, although plasma immunoreactive endothelin-1 did not significantly increase and the maximal contractile response to endothelin-1 in the aorta significantly decreased as compared with values observed in age-matched SHR. These results suggest that endogenous endothelin-1 is involved in the maintenance of hypertension in DOCA-salt SHR, and that circulating endothelin-1 is not sufficient to reflect the physiological role of endothelin-1.

The role of endothelin and nitric oxide in modulation of normal and spastic cerebral vascular tone in the dog.

To investigate the roles of endothelin and nitric oxide (NO) in the regulation of cerebral vascular tone under basal conditions and in cerebral vasospasm following subarachnoid hemorrhage in dogs, we used BQ-123 (cyclo(-D-Trp-D-Asp-L-Pro-D-Val-L-Leu-) sodium salt), an endothelin ETA receptor antagonist, L-arginine, a substrate for the formation of NO, and NG-nitro-L-arginine methyl ester, an NO synthesis inhibitor, and measured the angiographic diameter of the basilar artery in vivo. In normal dogs, intracisternal (i.c.) injection of BQ-123 (0.6 mg/kg) produced a 29.4 +/- 6.11% (P < 0.01) increase in the basal diameter 24 h after injection. NG-nitro-L-arginine methyl ester (0.6 mg/kg i.c.) produced a 19.3 +/- 2.93% (P < 0.05) decrease in the basal diameter 2 h after injection. This decrease was significantly attenuated by both BQ-123 (0.06-0.6 mg/kg i.c.) and L-arginine (6 mg/kg i.c.), but not by D-arginine. In the two-hemorrhage canine model, BQ-123 significantly inhibited the development of cerebral vasospasm (36.9 +/- 4.11% decrease on day 5 and 42.0 +/- 4.54% decrease on day 6 in controls vs 21.7 +/- 4.75% decrease (P < 0.05) on day 5 and 20.8 +/- 4.14% decrease (P < 0.05) on day 6 for 0.6 mg/kg i.c.) significantly attenuated the cerebral vasospasm on day 4 from a mg/kg i.c.). Furthermore, in this model, L-arginine (6 30.9 +/- 5.78% decrease (before)) to a 12.6 +/- 5.99% decrease (after). The immunoreactive endothelin-1 levels in the endothelial layer and the adventitia of the basilar artery were much higher on days 3 and 7 after the injection of autologous blood than on day 0 before blood injection. These results suggest that endogenous endothelin and NO both participate in regulating the basal tone of cerebral arteries, and, therefore, the development of cerebral vasospasm following subarachnoid hemorrhage may be at least partially attributed to an impairment of the balanced action of endothelin and NO. Furthermore, endothelin ETA antagonists or NO products may be useful in the treatment of cerebral vasospasm following subarachnoid hemorrhage.

A novel ligand, [125I]BQ-3020, reveals the localization of endothelin ETB receptors.

The precise localization of an endothelin (ET) receptor subtype, the ETB receptor, in porcine lung was elucidated by in vitro microautoradiography using a novel ETB-selective radioligand, [125I]BQ-3020 ([125I-Tyr]-N-acetyl-Leu-Met-Asp-Lys-Glu-Ala-Val-Tyr-Phe-Ala-His-Leu-Asp -Ile-Ile-Trp). Of the labeled native ET isopeptides, [125I]ET-3 is selective for ETB receptors. However, [125I]ET-3 was not suitable for autoradiography due to its high degree of non-specific binding. On the other hand, [125I]BQ-3020 showed extremely low non-specific binding on autoradiography. The distribution of [125I]BQ-3020 binding in porcine lung was clearly different from that of [125I]ET-1, which showed more widespread binding than [125I]BQ-3020 due to a high affinity to both ETA and ETB receptors. [125I]BQ-3020 was found to bind to parenchyma, parasympathetic ganglia, pulmonary and submucosal plexuses, but bound only slightly to circular smooth muscle layers and the epithelium of airway tracts. Although [125I]ET-1 bound to the smooth muscle layer of all blood vessels, the binding of [125I]BQ-3020 differed among blood vessels. [125I]BQ-3020 binding in blood vessels paralleled acetylcholinesterase activity, suggesting that ETB receptors in blood vessels are located on parasympathetic nerves. Thus, the radioligand [125I]BQ-3020 is very useful for studying the precise localization of ETB receptors.

Protective effect of a selective endothelin receptor antagonist, BQ-123, in ischemic acute renal failure in rats.

To elucidate the pathophysiological role of endogenous endothelin (ET), we examined the effects of the newly synthesized ETA receptor-selective antagonist, BQ-123, on ischemic acute renal failure induced by bilateral clamping of renal artery and vein followed by reperfusion in rats. BQ-123, when given by i.v. infusion of 0.5 mg/kg per min for 2.5 h during the pre- and post-ischemic period, was found to prevent the decrease in creatinine clearance and increases in blood urea nitrogen, plasma creatinine and the fractional excretion of sodium. Morphological observation also showed an effect of BQ-123, i.e. prevention of proximal tubular (S3 segment) necrosis. At 2 h after the start of reperfusion, the ET-1 content in the kidney increased to its maximal level. At this time, the Ca2+ content in the mitochondrial fraction of the renal cortex increased, with a concomitant increase in blood urea nitrogen. However, these increases were limited by treatment with BQ-123. Thus, BQ-123 was effective to both prevent mitochondrial Ca2+ accumulation in the early phase of ischemic acute renal failure and protect proximal tubular cells from post-ischemic degeneration. We conclude that ET may be at least partially involved in the pathogenesis of tubular cell injury in this acute renal failure model.

Disproportional release of differently glycosylated forms of human renin by furosemide.

Concanavalin A (con A) chromatography of human plasma revealed the presence of three differently glycosylated forms of active renin(AR) and prorenin(PR), including the con A unbound forms(AR-I and PR-I), the loosely-bound forms (AR-II and PR-II), and the tightly-bound forms (AR-III and PR-III). These three forms of AR and PR were observed in human renal extracts. Normal male volunteers were intravenously given the diuretic furosemide (20 mg), kept standing for one hr and the effect on each form of renin was examined. These treatments elevated the plasma concentrations of AR-I, II and III by 2.1 +/- 0.2, 2.6 +/- 0.5, and 6.3 +/- 1.1-fold, respectively (n = 12), thereby indicating that the increase in AR-III was significantly larger than that in the other two forms (P less than 0.01). This disproportional increase was accompanied by a significant increase in the relative percent of AR-III in plasma from 21.6 +/- 2.5 to 42.2 +/- 3.0% (P less than 0.01). On the other hand, increase in the plasma levels of PR-I, II, and III was small (1.4 +/- 0.1, 1.0 +/- 0.1, and 1.1 +/- 0.2-fold, respectively). These results provide evidence for the presence of differently glycosylated forms of AR and PR in human plasma and suggest the preferential release of AR-III with the acute stimulation of renin, by furosemide.

High-molecular-weight renin converting enzyme from dog kidney: detection and fractionation.

Renal cortical high-molecular-weight renin (Mw:60,000) of the dog is a complex of renin (low-molecular-weight renin; Mw:40,000) and a renin binding protein. We detected an enzyme-like substance that catalyzes the conversion from high- into low-molecular-weight renin. When the renal cortical extract was added to the high-molecular-weight renin and the preparation incubated at 37 degrees C for 30 min, the high-molecular-weight renin was converted into the low-molecular-weight form. No such conversion occurred in the case of renal medullary extract. This converting substance was fractionated using concanavalin A Sepharose, 70% ammonium sulfate saturation and DEAE-cellulose chromatography. The converting activity was inhibited by potassium tetrathionate, N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid). These events suggest that this substance is an enzyme possessing sulfhydryl moieties. However, a cathepsin B inhibitor leupeptin did not affect the activity. Accordingly, the high-molecular-weight renin converting enzyme, which is sensitive to sulfhydryl oxidation, may explain the mechanism of interconversion between high- and low-molecular-weight renin involving the oxidation-reduction of tissue sulfhydryl groups.

Intrarenal localization of renin binding substance in rats.

Renin binding substance is a protein that reacts with renin (Mw:40,000) to form a high-molecular-weight renin (Mw:60,000). There is evidence that this substance is present in the renal cortex. However, the exact localization has not been determined. We now report that when glomeruli and tubular segments were isolated from the rat kidney cortex and were frozen and thawed to extract proteins, the high-molecular-weight renin was detected by high performance liquid chromatography, when renin was mixed with an extract of tubular segments, but was not detected with an extract of the glomeruli. Thus, the renin binding substance was demonstrated in the cortical tubular cells but not in the glomeruli. Thus, the renin binding substance was demonstrated in the cortical tubular cells but not in the glomeruli, and the renin binding substance probably does not contribute to the process of biosynthesis of renin in juxtaglomerular cells. Rather, this substance may play a role in tubular functions in the kidney.

Antihypertensive effect of a newly synthesized endothelin antagonist, BQ-123, in a genetic hypertensive model.

A newly synthesized ET(A)-selective antagonist, BQ-123, was examined with respect to its anti-endothelin(ET) action in vitro and in vivo and its effect on blood pressure in Wistar Kyoto rats (WKY), spontaneously hypertensive rats (SHR) and stroke-prone spontaneously hypertensive rats (SHRSP). In isolated porcine coronary arteries, BQ-123 (0.07 microM to 6.0 microM) shifted the concentration-response curve for ET-1 to the right without affecting the maximal response of ET-1, its pA2 value being 7.35. Intravenous infusion of BQ-123 at a rate of 1.2 and 30 mg/kg/hr produced a significant decrease in blood pressure in 20- to 29-week-old SHRSP, but did not alter blood pressure in 13- to 16-week-old WKY or in 18- to 19-week-old and 40-week-old SHR. The hypotensive effect of BQ-123 depended on the pretreatment blood pressure level. These results suggest that ET-1 is involved in part in the maintenance of high blood pressure in malignant hypertension, as exemplified by SHRSP.

Analysis of responses to endothelins in isolated porcine blood vessels by using a novel endothelin antagonist, BQ-153.

We examined the effects of a novel ETA-selective endothelin (ET) antagonist, BQ-153, on vascular responses to ET-1 and ET-3 in isolated porcine coronary and pulmonary blood vessels, to clarify the roles of ET receptor subtypes in the regulation of vascular smooth muscle tension. With endothelium-denuded vascular tissues, the concentration-contraction curve (CCC) for ET-1 appeared as a single sigmoidal shape for all types of tissue. The CCC for ET-1 was antagonized by BQ-153 (2 and 10 microM) in all tissues, but part of the contraction was resistant. The CCC for ET-3 usually consisted of two different phases with higher (first phase) and lower (second phase) sensitivities to the peptide. Only the second phase of CCC for ET-3 was completely inhibited by BQ-153 (2 microM) in all tissues, while the first phase was resistant. The BQ-153-resistant contractile phases of ET-1 and ET-3-induced vasoconstriction appeared to have similar sensitivity in all tissues, and the contractile activity varied with each type of tissue. With endothelium-intact materials, the potencies of ET-1 and ET-3 for endothelium-dependent vasorelaxation in pulmonary artery were almost equivalent. BQ-153 (10 microM) did not inhibit ET-induced vasorelaxation. These results indicate that ET-induced vasoconstriction is mediated not only through ETA but also through ETnonA (probably ETB), and that the relative proportions of the ET-receptor subtypes mediating contractions vary in different vascular areas. In addition, results showed that ET-induced endothelium-dependent vasorelaxation is mediated through ETB.

Down regulation of epidermal growth factor receptors in rat hepatocytes treated with clofibric acid.

The effect of clofibric acid (CA), a peroxisome proliferator and a non-genotoxic hepatocarcinogen was investigated on epidermal growth factor (EGF) receptors in hepatocytes of female Sprague-Dawley rats treated at a dose of 9000 ppm in a diet for up to 13 weeks. Hepatocyte plasma membranes were isolated in Weeks 1 and 13, and assayed with [125I]EGF. The binding of EGF to the hepatocyte plasma membranes was reduced in Week 1 as a result of decreased number of low-affinity receptors. The fall of binding capacity was further evident in Week 13, which was associated with decreased numbers of both high- and low-affinity receptors. The equilibrium dissociation constant remained unchanged either in Week 1 or 13. These results were in agreement with previous observations of a decreased hepatocyte response to mitogens after prolonged treatment with CA. This suggested that the CA-associated liver tumor promoting effect is related to its ability to decrease the number of EGF receptors and the resultant aberrant growth environment.

Experimental methods for immunization and challenge in antigenicity studies in guinea pigs.

Optimal experimental methods for antigenicity studies in guinea pigs were investigated on: (1) the effects of different immunizing methods using complete or incomplete Freund's adjuvants (CFA or IFA), and various injection sites, the number of immunizations, the immunizing doses, and the immunizing periods, (2) the relationship between the severity of active systemic anaphylaxis (ASA) reactions and passive cutaneous anaphylaxis (PCA) titers, (3) positive control for oral administration, and (4) the effects of incubation mixture of drug and serum protein as the challenge for the ASA assay. The following results provided useful information for designing more appropriate methods for antigenicity studies: (1) The optimal immunization method for benzylpenicillin (PcG), cephaloridine, 2,4,6-trinitrobenzene sulfonic acid and adriamycin, which were selected as positive controls for low molecular medicines in this experiment, involved subcutaneous administration of 1 ml of a test substance in CFA (1st immunization) or IFA (2nd and 3rd immunizations) at two doses, 1 and 10 mg/animal, 3 times at 2-week intervals on the back of a guinea pig. Blood collection for PCA assay was needed 2 weeks after the last immunization, and ASA assay, 1 or 2 days after the blood collection. (2) The insensitivity of ASA reactions in bovine serum albumin-immunized animals with very high PCA titers was overcome by increasing the challenge antigen dose from 1 to 10 mg/animal. (3) Most animals administered lysozyme at 0.1, 1 or 10 mg/animal by gavage for 2 weeks or more showed ASA and PCA reactions. (4) Incubation of a mixture of 20 mg/ml of PcG and 2 mg/ml of guinea pig serum albumin for 4 hr was the most effective as challenge for the induction of ASA reaction in PcG-immunized guinea pigs.