Antiulcer agents. 4-substituted 2-guanidinothiazoles: reversible, competitive, and selective inhibitors of gastric H+,K(+)-ATPase.

A series of 4-substituted 2-guanidinothiazoles has been found to inhibit the gastric proton-pump enzyme H+,K(+)-ATPase. In general, these compounds were reversible inhibitors of canine gastric H+,K(+)-ATPase, competitive at the K+ site, and selective relative to canine renal Na+,K(+)-ATPase. Structure-activity relationship (SAR) studies on this series revealed no general replacement for the guanidinothiazole. On the other hand, use of pyrrolyl, phenyl, and indolyl groups as the C-4 substituent yielded active compounds. Extensive studies of substitution patterns on these 4-aryl groups led to more active compounds, but no consistent SAR became apparent. Monosubstitution of the guanidine and substitution of the thiazole at C-5 both often led to increased activity, but combining these changes generated compounds less active than the parents. Despite 100-fold improvement in in vitro inhibitory potency, only a 3-fold increase in gastric antisecretory activity in rats was observed for these agents.

Discovery of the hemifumarate and (alpha-L-alanyloxy)methyl ether as prodrugs of an antirheumatic oxindole: prodrugs for the enolic OH group.

Ether, ester, and carbonate derivatives of the antirheumatic oxindole 1 were prepared and screened as potential prodrugs of 1. This effort led to the discovery of the (alpha-L-alanyloxy)-methyl ether and hemifumarate derivatives of 1 which deliver the drug efficiently into the circulation of test animals, are stable in the solid state, and possess good stability in solution at low pH as required to ensure gastric stability. Success in achieving acceptable bioavailabilities of 1 across species (rats, dogs, and monkeys) followed the inclusion of ionizable functionality within the promoiety to compensate for masking the polar enolic OH group of the free drug. However, the introduction of ionizable functionality was often associated with decreased stability, as demonstrated by the hemisuccinate, hemiadipate, hemisuberate, and alpha-amino ester derivatives of 1 which could not be isolated. A clear exception was the hemifumarate derivative of 1 which was not only isolable but actually more stable at neutral pH than the nonionizable ester analogues. The solution and solid state stability of the hemifumarate, together with its activity as a prodrug of 1, suggests that hemifumarate be considered as an alternative to hemisuccinate as a prodrug derivative for alcohols, particularly in situations where solution state stability is an issue.

Inhibition of the human eyeblink reflex: an evaluation of the sensitivity of the Wendt-Yerkes method for threshold detection.

In two experiments, human subjects showed a reduced eyewink reflex to an airpuff (S2) if it was preceded by a light flash or a noise burst (S1). The first (N=6) showed that a light inhibited the blink maximally at a 100-msec lag. In the second (N=8), thresholds for noise pips were established with the method of limits. Then the pips were presented in multiples of threshold values as S1 100 msec before S2. Sujbects pressed a button whenever they heard the sound, on S1-S2 pairs, on S1 alone, or on S2 alone trials. Eyeblinks to S2 declined in amplitude and increased in latency as S1 intensity increased. The threshold S1 (approximately 2 dB over a 50-dB background) produced reliable inhibition in 7 of 8 subjects, and a weaker S1 which never evoked a button press was sufficient to produce reliable inhibition for the group as a whole. Inhibition in any particular trial was unrelated to button pressing. The sensitivity of the reflex technique and its relative invariance to task demands suggest its usefulness as a behavioral psychophysical procedure.

Vitamin B-6 content of selected foods served in dining halls.

Roast beef and green beans lost significant quantities of vitamin B-6 during institutional cooking and holding procedures; some was leached into juice and pot liquor. Whole baking potatoes lost smaller quantities of the vitamin during these procedures. A mean of 60% of the vitamin that was present in the foods before cooking was actually served. More vitamin B-6 will be retained if cooking and holding times are held to a minimum.

Vitamin B-12 and folate intakes and plasma levels of black adolescent females.

The vitamin B-12 and folate intakes and plasma levels of 39 healthy black female adolescents were evaluated. Estimated dietary folate intakes of 97% of the subjects were less than 2/3 of the RDA, and 74% had plasma folate levels indicative of marginal (3.0 to 5.9 ng/ml [less than 6 nmol/:]), nmol/L]), and 3%, deficient (below 3.0 ng/ml [less than 6 nmol/L]) folate status. Thirteen percent of the subjects had estimated dietary intakes below 2/3 of the RDA for vitamin B-12; all subjects had plasma levels indicative of adequate vitamin B-12 status.

Reflex modulation: a new method of measuring hearing in children.

The term reflex modulation (RM) as used here refers to an auditorially induced modification in the magnitude of a cutaneous reflex. the audiometric potential of RM was examined in two children, while they enjoyed viewing silent cartoons. Eyeblink reflexes elicited by an airpuff were reduced in amplitude or "inhibited" by a variety of audible tones presented 100 msec prior to reflex elicitation. The amount of relative blink inhibition was directly related to the intensity of the tones. The weakest tone intensities which produced reliable blink-inhibition were usually about 10 dB above voluntary threshold. Implications for testing the difficult-to-test are discussed.

Detecting and remediating external meatal collapse during audiologic assessment.

We present a case study in which the jaw/mouth-open procedure during air-conduction testing is employed to both detect and remediate external auditory meatal collapse. A prospective investigation was also conducted to compare the efficacy of the proposed jaw/mouth-open procedure with that of the traditional insert receivers in remediating meatal collapse during audiologic assessment. Meatal collapse was present in 6 of the 136 subjects evaluated. In these 6 subjects, the air-conduction thresholds under supra-aural headphones in the jaw/mouth-open condition were similar to those under insert receivers. We discuss the implications of this finding in clinical settings where insert receivers are not available.

Peptidic p-nitroanilide substrates of interleukin-1 beta-converting enzyme.

Peptidic p-nitroanilides are useful colorimetric substrates for enzymes. With the aim of developing a convenient, quantitative assay for inhibitors of interleukin-1 beta-converting enzyme (ICE), we have explored three approaches to the synthesis of peptidic p-nitroanilides relevant to this enzyme. The first approach involved a late stage oxidation of a p-aminoanilide such as CbzValAlaAsp(beta- tert-butyl)-p-(t-Boc-amino)anilide. The second and third approaches used the preformed amino acid p-nitroanilides HAsp-p-nitroanilide hydrochloride and HAsp(beta-tert-butyl)-p-nitroanilide which were coupled iteratively with preactivated amino acid derivatives or with an appropriate peptide, respectively. While each approach had it merits and limitations, all three produced p-nitroanilides that were substrates for ICE.

Experiments re: clinical application of reflex modulation audiometry.

Two experiments were conducted on normal hearing adults to assess the audiometric test potential of reflex modulation (RM) when administered according to the descending method of limits. RM describes the inhibitory effect of a tone upon a subsequently occurring eyeblink reflex. Although RM conducted according to the method of constant stimuli has already been shown to produce RM results which agree well with voluntary audiometry in normal and hearing-impaired adults and children, the present concern is with the potential clinical application of RM, and in this regard the descending method may offer certain distinct advantages. In study one, a 2-kHz tone was presented at several descending intensities, whereas in study two, four different frequencies (0.5, 1.2, and 4 kHz) were presented in concurrent fashion, but again, the intensity of the complete set was decreased in successive steps. Sensitivity in both cases was 10 to 15 dB SL. Implications for the clinical application of RM audiometry are discussed.

Interleukin-1 beta converting enzyme. Synthesis of hydroxyethyl dipeptide surrogate-containing compounds as potential ICE inhibitors.

A series of compounds containing a hydroxyethyl-based dipeptide surrogate have been prepared as probes to evaluate the possibility of ICE being an aspartic protease. The aldehyde t-BocAsp(beta-t-butyl)H reacted with the organochromium species derived from phenethyl bromide and CrCl2 to give the expected addition product. Lactonization, reprotection of the amine and oxidation with RuCl3 gave the two protected dipeptide surrogates 7a and 7b. These were incorporated into tetra-, penta- and hexapeptide-like molecules and evaluated as inhibitors of the enzyme. The failure of these compounds to inhibit ICE indicated that this enzyme was very unlikely to be an aspartic protease.

Reflex modulation and loudness recruitment.

Eyeblink reflexes to airpuffs to the orbit are inhibited by preliminary tones even of low intensity, and the amount of inhibition or "reflex modulation" (RM) is correlated with tone loudness. Brief pure tones were presented 100 msec prior to an airpuff to the eye and eyeblink amplitude was recorded in 7 normal-hearing adults. Tones of several levels were either in quiet or in a white noise of 73.5 dbC. As tones increased in intensity, RM increased faster to stimulation in the masked than in the unmasked ear, simulating the classical "pseudo-recruitment" in the noise-masked ear. In addition, very rapid growth of RM was found at 4 kc/s in a patient with sensorineural impairment of 70 db at that frequency as compared with the RM from a frequency with normal HTL. These data confirm the potential of RM in the objective quantification of loudness recruitment.

Reflex modulation: a hearing test for the difficult-to-test.

This study was designed to determine the effectiveness of reflex modulation (RM) audiometry in assessing the hearing sensitivity of difficult-to-test subjects. RM describes the inhibitory effect of a tone upon a subsequently elicited eyeblink reflex. Five ENT subjects were referred to the University of Kansas audiology clinic because of medicolegal problems or doubts regarding their hearing status. After conventional pure tone and speech audiometry had produced equivocal results, the subjects were tested by RM. The study indicates that RM is effective as an objective test of hearing for these subjects. Since RM sensitivity is not frequently dependent, it has a potential role as an adjunct to the brainstem evoked response (BSER) for testing low frequencies.

Difluoroketones as inhibitors of matrix metalloprotease-13.

Substrate-like difluoroketones have been prepared as potential inhibitors of MMP-13. Weak inhibition was seen with the key target 2. This and the more potent activity of intermediate 7b illustrates that hydrated ketones can be used to inhibit MMP-13 and perhaps other members of this class of enzymes.

CP-66,948: an antisecretory histamine H2-receptor antagonist with mucosal protective properties.

CP-66,948 is a histamine H2-receptor antagonist with gastric antisecretory activity and mucosal protective properties. The affinity of CP-66,948 for the guinea pig atria histamine H2-receptor is 15 times greater than that of cimetidine and seven times greater than that of ranitidine. In vivo, the ED50 value for inhibition of gastric acid secretion in pylorus-ligated rats is 2 mg/kg intraduodenally, and in histamine or pentagastrin-stimulated Heidenhain pouch dogs the antisecretory ED50 values are 0.3 mg/kg per os and 1.0 mg/kg per os, respectively. CP-66,948 also inhibits ethanol-induced gastric hemorrhagic lesions in rats following either oral or systemic administration (ED50 values of 12 mg/kg per os and 6 mg/kg subcutaneously). In addition, the mucosal protective activity is independent of prostaglandin synthesis. CP-66,948 inhibits gastric acid secretion in man, and its mucosal protective activity may provide additional benefits in peptic ulcer therapy.

trans-3-Benzyl-4-hydroxy-7-chromanylbenzoic acid derivatives as antagonists of the leukotriene B4 (LTB4) receptor.

The SAR of a series of 2-(7-chromanyl)benzoic acids has been investigated with the aim of identifying potent and selective LTB4 receptor antagonists that maintain potency in complex biological fluids. We found optimal activity in derivatives with electron-withdrawing groups in the benzoic acid ring and with an unsubstituted C-3 benzyl group on the chromanol nucleus. While compounds containing a 3-(4-phenyl)benzyl chromanol substituent were potent LTB4 receptor antagonists, the increased lipophilicity imparted by the additional phenyl substituent led to decreased potency in the presence of plasma proteins. From among the potent compounds identified, CP-195543, the 5'-trifluoromethyl 3-benzyl chromanol, was selected for development.

The preclinical pharmacological profile of the potent and selective leukotriene B4 antagonist CP-195543.

CP-195543 [(+)-2-(3-benzyl-4-hydroxy-chroman-7-yl)-4-trifluoromethyl-benzoic acid] is a structurally novel, selective and potent leukotriene B4 (LTB4) receptor antagonist. In vitro CP-195543 inhibited [3H]LTB4 binding to high-affinity LTB4 receptors on human neutrophils (HN) and murine spleen membranes with IC50 values of 6.8 nM (Ki = 4.9 nM) and 37.0 nM (Ki = 26.9 nM), respectively. CP-195543 inhibited human and mouse neutrophil chemotaxis mediated by LTB4 with IC50 values of 2.4 nM and 7.5 nM, respectively. Evidence of noncompetitive antagonist effects on the HN high-affinity LTB4 receptor was obtained by Scatchard analysis of [3H]LTB4 binding to and chemotaxis of HN to LTB4. Scatchard analyses of [3H]LTB4 binding to low-affinity receptors on HN indicated that CP-195543 acted as a competitive antagonist at this receptor, and inhibition of LTB4-mediated CD11b up-regulation on HN was inhibited competitively by CP-195543 (pA2 = 7.66). In whole blood, CP-195543 also blocked CD11b up-regulation on HN (pA2 = 7.12) and murine neutrophils (pA2 = 7.06) with a similar potency. LTB4-mediated CD11b up-regulation on human monocytes and eosinophils in whole blood were inhibited by CP-195543 with IC50 values of 270 nM and 420 nM, respectively. CP-195543 at 10 microM failed to inhibit HN chemotaxis and CD11b up-regulation mediated through alternative (i.e., complement fragment 5a, interleukin-8, platelet-activating factor) G-protein-coupled chemotactic factor receptors. In vivo, after oral administration, CP-195543 blocked LTB4-mediated neutrophil infiltration in guinea pig and murine skin with ED50 values of 0.1 mg/kg and 2.8 mg/kg p.o., respectively. When administered in osmotic pumps, CP-195543 reduced the clinical symptoms and attendant weight loss in an IL-1-exacerbated murine model of collagen-induced arthritis with half-maximal effects associated with plasma drug levels of 0.4 to 0.5 microg/ml. Collectively these data provide evidence of the in vitro potency and in vivo efficacy of a novel LTB4 antagonist and support its clinical evaluation in a variety of inflammatory diseases in man.

Inhibition of MMP-1 and MMP-13 with phosphinic acids that exploit binding in the S2 pocket.

Through the use of empirical and computational methods, phosphinate-based inhibitors of MMP-1 and MMP-13 that bind into the S2 pocket of these enzymes were designed. The synthesis and testing of 2 suggested that binding was occurring as hypothesized. Structure determination of a co-crystal of 2 bound to the catalytic domain of MMP-1 confirmed the binding mode. Substituents binding into S2, S1', S2' and S3', were optimized yielding compounds with low double-digit nM IC50's against these enzymes.