Healthy volunteers for bioequivalence trials: predictive factors for enrollment failures--a case-control study.

OBJECTIVE: To identify social predictors for enrollment failures of healthy volunteers (hv) in bioequivalence trials. METHODS: Retrospective case-control study. Data was collected from clinical files of hv recruited in 13 bioequivalence trials approved by an independent IRB and local regulatory authority carried out between January and December 2009 at a Pharmacokinetic Unit in Buenos Aires, Argentina. All hv signed the Inform Consent Form. Only subjects who fulfilled all inclusion criteria required by the protocols were studied. Cases (enrollment failures): hv who fulfilled the protocols eligibility criteria but were not enrolled in the trials by their own decision. CONTROLS: hv who fulfilled all the protocols eligibility criteria and were enrolled. Cases and controls were matched by demographic/ physical data and compared in relation to database contact, unemployment, alcoholic/ drug family environment, history of alcohol/ drug abuse, and other social variables. Chi2-test and t-test were used to compare data; variables presenting statistical difference were included in a logistic regression model. RESULTS: A sample of 375 hv. was analyzed. cases: 81/375(21.60%). Controls: 294/375 (78.40%). Cases did not differ from controls in relation to nationality, educational level, length of study and history of alcohol abuse. Statistical differences between cases and controls were found in non-database contact, unemployment, alcoholic environment, drug abuse environment and personal history of drug abuse. In a multivariate analysis only unemployment, (OR: 4.20, p < 0.001), non-database contact, (OR: 2.35, p = 0.004) and alcoholic environment, (OR: 1.94, p = 0.045) remained as predictive factors. CONCLUSION: In bioequivalence trials, an unemployment condition, and an alcoholic family environment were identified as negative predictors for effective enrollment in new healthy volunteers.

Differential effects of acetylcholine and bradykinin on prostanoid release from the rat mesenteric bed: role of endothelium and of nitric oxide.

The roles of nitric oxide and of endothelium in the effects of the vasorelaxing agents acetylcholine and bradykinin on the production of prostanoids was studied in the isolated and perfused mesenteric vascular bed of the rat. Prostanoids were measured in the perfusate by high-performance liquid chromatography (HPLC). In the intact vascular bed, 1 microM bradykinin increased the release of 6-keto-prostaglandinF(1alpha) (stable metabolite of prostacyclin) and of prostaglandin E2 and 10 microM acetylcholine stimulated the efflux of prostacyclin only. In the de-endothelialized vascular bed, bradykinin increased the release of prostacyclin whereas acetylcholine increased the efflux of thromboxane. The inhibition of nitric oxide synthesis with 100 microM N(G)-nitro-L-arginine methyl ester prevented the effect of bradykinin but did not modify the effects of acetylcholine on prostanoid release. In addition, 100 microM L-arginine reversed the inhibitory effect of N(G)-nitro L-arginine methyl ester on bradykinin-stimulated prostaglandin production. It is concluded that acetylcholine and bradykinin stimulate prostanoid release in the rat mesenteric vascular bed with different patterns and through different mechanisms.

Time-course of the alterations in prostanoid production and in contractile responses of mesenteric beds isolated from streptozotocin diabetic rats.

The prostanoid production and the effect of indomethacin on the noradrenaline-induced contractions were studied in the mesenteric bed of rats at different times (1-8 weeks) after the administration of streptozotocin (STZ). The production of prostacyclin (measured as 6-keto-PGF1alpha) and prostaglandin (PG) E2 was unchanged one week after STZ, but it was reduced to 50% of control values 4 weeks after STZ without further changes 8 weeks after the treatment. The release of thromboxane (TX) A2 (measured as TXB2) and PGF2alpha, increased by 100% one week after STZ and returned to basal values at 3 weeks. TX release was below control values 8 weeks after STZ. The ratio 6-keto-PGF1alpha/TXB2 was reduced one week after STZ, recovered to control values at 4 weeks and augmented at 8 weeks. Indomethacin (10 microM) reduced the contractile responses to noradrenaline in the controls, whereas in STZ-treated rats this effect was observed solely 8 weeks after the treatment. Since this recovery coincided with an increase of the vasodilator/vasoconstrictor prostanoid ratio, a time-dependent compensation of the vascular alterations caused by STZ can be proposed from the present results.

Endothelium-mediated and N omega-nitro-L-arginine methyl ester-sensitive responses to cromakalim and diazoxide in the rat mesenteric bed.

The effects of two 'K+ channel openers', (+/-)-6-cyano-3,4-dihydro-2,2-dimethyl-trans-4-(2-oxo-1-pyrrolidyl )-2 H-benzo[b]-pyran-3-ol (cromakalim) and 7-chloro-3-methyl-2 H-1,2,4-benzothiadiazine 1,1-dioxide (diazoxide), were studied on the rat isolated mesenteric bed. Differences in the perfusion pressure were measured as a parameter of vascular resistance. Cromakalim (0.1-700 microM) and diazoxide (1 microM-1 mM) reduced to 60% the contractions elicited by 10 microM noradrenaline and to 30% those evoked by 100 mM KCl. The relaxant effects of cromakalim and diazoxide on the noradrenaline-induced contractions were reduced by the K(+)-ATP channel blocker, 5-chloro-N-[2-[4-[[[(cyclohexylamino) carbonyl]amino]-sulfonyl]phenyl]ethyl]-2-methoxybenzamide (glibenclamide, 0.01-0.3 microM), endothelium removal with 0.1% saponin and pretreatment with the nitric oxide synthesis inhibitor, S(+/-)-N5-[imino(nitroamino)methyl]-L-ornithine methyl ester hydrochloride (L-NAME, 500 microM). Reductions in the relaxant responses after endothelium removal or L-NAME pretreatment were observed with 1-100 microM cromakalim and with 30 microM diazoxide but not with 100 and 300 microM diazoxide. Pretreatment with the inactive stereoisomer D-NAME as well as with the prostanoid synthesis inhibitor, 1-[p-chlorobenzoyl]-5-methoxy-2-methylindole-3-acetic acid (indomethacin, 10 microM), did not affect the reductions in contractile responses to noradrenaline caused by either cromakalim or diazoxide. It is concluded that the relaxant effects of cromakalim and diazoxide in the rat mesenteric bed are endothelium-mediated and L-NAME-sensitive and could at least partially involve the participation of nitric oxide.

Phase IV study comparing diurnal glycemic profile following the administration of 2 NPH plus regular human DNA recombinant insulin regimens in type 1 diabetes mellitus (T1DM) adult patients.

Intensive insulin therapy (IIT) based on multiple daily injections of long plus rapid-acting insulin has been demonstrated to reduce mortality and morbidity associated with chronic hyperglycemia in T1DM patients. The objective of this study was to assess and compare the postprandial glycemic profile over a diurnal 12 h-period produced by the administration of a new NPH plus regular human DNA recombinant IIT (test regimen) relative to the reference IIT in T1DM patients. A phase IV, single-center, open-label, randomized, multiple-dose, balanced, cross-over study in 12 T1DM patients was conducted. Patients were assigned to receive either the test (Densulin® N (NPH) plus Densulin® R (regular),100 UI/ml, Denver Farma, Argentina) followed by the reference (InsulatardHM® (NPH) plus ActrapidHM®,100 UI/ml, Novo Nordisk Pharma Argentina) regimens or viceversa, according to a random sequence. Each treatment regimen consisted of 2 phases of an ambulatory run-in period of 7 days followed by 12 h confinement period. Blood glucose levels were measured. Glycemic profile was evaluated through glycemic plasma-concentration time curves, area under the time-concentration glycemic curves from basal to 2 h (GlyAUC0-2) and to 12 h (GlyAUC0-12) postprandial, and maximum glycemic postprandial concentration (GlyCmax). 12 hour glycemic concentration-time curves were similar for both test and reference regimens. Geometric least square means ratios Test/ref regimens and their 90% confidence interval for GlyAUC0-2, GlyAUC0-12 and GlyCmax were 94.33 (81.13-125.09), 107.75 (94.05-123.45) and 105 (92.89-118.68), respectively. Both regimens presented similar safety profile. This study demonstrated that the new human DNA recombinant NPH and regular insulin is equally effective to the reference regimen for postprandial diurnal glycemic profile.

Effects of the chronic in vivo administration of L-NAME on the contractile responses of the rat perfused mesenteric bed.

The effects of the chronic in vivo inhibition of nitric oxide synthase (NOS) with N(omega)-nitro-L-arginine methyl ester (L-NAME) on vascular contractility were studied in the rat perfused mesenteric bed. The chronic treatment with L-NAME during 4 weeks induced a rise in systolic blood pressure (basal: 115.1 +/- 6.5 mmHg; chronic L-NAME treatment: 171.7 +/- 7.7 mmHg, n = 16, P < 0.05). After the chronic NOS inhibition, the potentiation of the maximal vasoconstrictor responses to noradrenaline, phenylephrine and KCl was to the same extent as that observed after the in vitro exposure to 100 microM L-NAME. No further potentiation of the contractile responses was achieved when the mesenteric beds isolated from L-NAME treated rats were incubated in vitro with 100 microM L-NAME. The endothelium removal but not the inhibition of prostanoid synthesis with either 10 microM indomethacin or 10 microM 17-octadecynoic acid potentiated the contractions to noradrenaline and to KCl both under control conditions as well as after the chronic in vivo administration of L-NAME. These observations taken together suggest that after chronic L-NAME maximum inhibition of nitric oxide synthase was achieved and no compensatory mechanisms able to counterbalance the increase in contractile responses were developed. Further studies are necessary to elucidate the nature of the factors, other than nitric oxide, that contribute to the potentiation of contractile responses observed when the endothelium is removed after L-NAME treatment.

Effects of aging on ATP sensitive K(+) channels and on prostanoid production in the rat mesenteric bed.

The aim of the present work was to evaluate, in the rat isolated mesenteric bed, whether increasing age is associated with alterations in the ATP sensitive K(+) channels functionality. Moreover, studies were performed in order to evaluate the effects of aging on the synthesis of vascular prostanoids as well as on its possible contribution to the pressor responses of this vascular bed. Male Wistar rats of 3 month (adults) and 24 month (aged) were used. Although no differences were found among adult and aged rats in pressor responses to 2-30 nmol noradrenaline and to 40-160 nmol KCl, the relaxant responses to the K(+) channel opener, 10(-6) M cromakalim, were significantly diminished in the aged group compared to the adults. On the other hand, whereas PGF2α and 6-keto PGF1α production was not modified with age, the thromboxane B2 and prostaglandin E2 production in the mesenteric bed from 24 month old rats was significantly increased compared to the adult group. Furthermore, the cyclooxigenase synthesis inhibitor, 10(-5) M indomethacin reduced the pressor responses induced by noradrenaline in the mesenteric beds from adults but not from aged rats. It is concluded that there is an age related reduction in the functionality of the ATP sensitive K(+) channels in the rat mesenteric bed. In addition, aging produces an increase in the release of vasoconstrictor as well as of vasodilator prostanoids, whose contribution to noradrenaline induced pressor responses appears to be less relevant in the older animals.