Non-clinical studies in the process of new drug development - Part II: Good laboratory practice, metabolism, pharmacokinetics, safety and dose translation to clinical studies

The process of drug development involves non-clinical and clinical studies. Non-clinical studies are conducted using different protocols including animal studies, which mostly follow the Good Laboratory Practice (GLP) regulations. During the early pre-clinical development process, also known as Go/No-Go decision, a drug candidate needs to pass through several steps, such as determination of drug availability (studies on pharmacokinetics), absorption, distribution, metabolism and elimination (ADME) and preliminary studies that aim to investigate the candidate safety including genotoxicity, mutagenicity, safety pharmacology and general toxicology. These preliminary studies generally do not need to comply with GLP regulations. These studies aim at investigating the drug safety to obtain the first information about its tolerability in different systems that are relevant for further decisions. There are, however, other studies that should be performed according to GLP standards and are mandatory for the safe exposure to humans, such as repeated dose toxicity, genotoxicity and safety pharmacology. These studies must be conducted before the Investigational New Drug (IND) application. The package of non-clinical studies should cover all information needed for the safe transposition of drugs from animals to humans, generally based on the non-observed adverse effect level (NOAEL) obtained from general toxicity studies. After IND approval, other GLP experiments for the evaluation of chronic toxicity, reproductive and developmental toxicity, carcinogenicity and genotoxicity, are carried out during the clinical phase of development. However, the necessity of performing such studies depends on the new drug clinical application purpose.

The mechanism of gentisic acid-induced relaxation of the guinea pig isolated trachea: the role of potassium channels and vasoactive intestinal peptide receptors

We examined some of the mechanisms by which the aspirin metabolite and the naturally occurring metabolite gentisic acid induced relaxation of the guinea pig trachea in vitro. In preparations with or without epithelium and contracted by histamine, gentisic acid caused concentration-dependent and reproducible relaxation, with mean EC50 values of 18 æM and Emax of 100 percent (N = 10) or 20 æM and Emax of 92 percent (N = 10), respectively. The relaxation caused by gentisic acid was of slow onset in comparison to that caused by norepinephrine, theophylline or vasoactive intestinal peptide (VIP). The relative rank order of potency was: salbutamol 7.9 > VIP 7.0 > gentisic acid 4.7 > theophylline 3.7. Gentisic acid-induced relaxation was markedly reduced (24 + or - 7.0, 43 + or - 3.9 and 78 + or - 5.6 percent) in preparations with elevated potassium concentration in the medium (20, 40 or 80 mM, respectively). Tetraethylammonium (100 æM), a nonselective blocker of the potassium channels, partially inhibited the relaxation response to gentisic acid, while 4-AP (10 æM), a blocker of the voltage potassium channel, inhibited gentisic acid-induced relaxation by 41 + or - 12 percent. Glibenclamide (1 or 3 æM), at a concentration which markedly inhibited the relaxation induced by the opener of ATP-sensitive K+ channels, levcromakalim, had no effect on the relaxation induced by gentisic acid. Charybdotoxin (0.1 or 0.3 æM), a selective blocker of the large-conductance Ca2+-activated K+ channels, caused rightward shifts (6- and 7-fold) of the gentisic acid concentration-relaxation curve. L-N G-nitroarginine (100 æM), a NO synthase inhibitor, had no effect on the relaxant effect of gentisic acid, and caused a slight displacement to the right in the relaxant effect of the gentisic acid curve at 300 æM, while methylene blue (10 or 30 æM) or ODQ (1 æM), the inhibitors of soluble guanylate cyclase, all failed to affect gentisic acid-induced relaxation. D-P-Cl-Phe6,Leu17[VIP] (0.1 æM), a VIP receptor antagonist, significantly inhibited (37 + or - 7 percent) relaxation induced by gentisic acid, whereas CGRP (8-37) (0.1 æM), a CGRP antagonist, only slightly enhanced the action of gentisic acid.

Efficacy, safety, quality control, marketing and regulatory guidelines for herbal medicines (phytotherapeutic agents)

This review highlights the current advances in knowledge about the safety, efficacy, quality control, marketing and regulatory aspects of botanical medicines. Phytotherapeutic agents are standardized herbal preparations consisting of complex mixtures of one or more plants which contain as active ingredients plant parts or plant material in the crude or processed state. A marked growth in the worldwide phytotherapeutic market has occurred over the last 15 years. For the European and USA markets alone, this will reach about $7 billion and $5 billion per annum, respectively, in 1999, and has thus attracted the interest of most large pharmaceutical companies. Insufficient data exist for most plants to guarantee their quality, efficacy and safety. The idea that herbal drugs are safe and free from side effects is false. Plants contain hundreds of constituents and some of them are very toxic, such as the most cytotoxic anti-cancer plant-derived drugs, digitalis and the pyrrolizidine alkaloids, etc. However, the adverse effects of phytotherapeutic agents are less frequent compared with synthetic drugs, but well-controlled clinical trials have now confirmed that such effects really exist. Several regulatory models for herbal medicines are currently available including prescription drugs, over-the-counter substances, traditional medicines and dietary supplements. Harmonization and improvement in the processes of regulation is needed, and the general tendency is to perpetuate the German Commission E experience, which combines scientific studies and traditional knowledge (monographs). Finally, the trend in the domestication, production and biotechnological studies and genetic improvement of medicinal plants, instead of the use of plants harvested in the wild, will offer great advantages, since it will be possible to obtain uniform and high quality raw materials which are fundamental to the efficacy and safety of herbal drugs

Tracheal responsiveness to histamine, PAF-acether and acetylcholine in normal and actively ovalbumin-sensitized guinea pigs

The contractile effects of histamine (His), PAF-acether (PAF) and acetylcholine (Ach) were analyzed in tracheal rings from normal and ovalbumin-sensitized (OAS) guinea pigs. Although maximal responses to all agonists were enhanced in preparations from OAS guinea pigs, only the EC50 of Ach was significantly decreased. Previous treatment with 1 micronM indomethacin (IND) or 10 micronM quinacrine (QUIN) markedly displaced the dose-response curves for Ach on OAS preparations to he left, whereas the EC50 of His was only increased by QUIN. PAF-responses, however, were completely abolished by IND in preparations from both senstized and unsensitized animals. These results indicate that ovalbumin sensitization significantly affects the tracheal response to Ach, His and PAF, an effect that appears to be mediated by arachidonic acd metabolites

Effect of PAF-acether on the reactivity of the isolated rat myometrium

In non-pregnant rat isolated uterine strips PAF-acether (1-1000 nM) produced contractile effects on 74% of the preparations tested which were concentration-dependent in 44% of the cases (EC50 = 28 nM). All the preparations tested exhibited contractile responses to either acetylcholine or potassium. PAF-acether was less potent on myometrial strips from pregnant animals (EC50 = 0.3 micronM) and was only effective on 24% of the preparations tested. A second contractile concentration-response curve was reproducible in only 14% of the preparations from non-pregnant rats (EC50 = 13 nM), whereas all strips from pregnant animals were completely refractory to a second challenge with PAF-acether. These results indicate that PAF-acether induces contraction of the isolated rat myometrium within the Ssame range of concentration at which it is active in other tissues

Demonstration of anti-bradykinin compounds in callus cultures of Mandevilla velutina

Calluses from stems and leaves of Mandevilla velutina were grown in culture for 30,45 and 60 days. Thin-layer chromatography of ethyl acetate extracts of calluses from stems of M. velutina revealed the presence of 6 compounds. Two of them had RF values identical to those of the anti-bradykinin (BK) compounds MV8609 and MV8610 previously isolated from the natural plant. The ethyl acetate extract (20 to 80 microng/ml) from stem callus culture antagonized in a concentration-dependent and reversible manner contractions caused by BK (0.1-100 nM) in the isolated rat uterus. The extracts obtained from calluses cultured for 30,45 and 60 days were about 79-, 63-and 31-fold more potent, respectively, in antagonizing BK than the crude extract obtained from the rhizome of the plant

Gossypol affects the responsiveness of isolated rat myometrium and vas deferens

The effects of gossypol on responsiveness of both rat myometrium and vas deferens were analyzed. In myometrial strips, gossypol (1-30 micronM) produced rightward displacemtns of the cumulative concentration-response curves to acetylcholine, bradykinin and oxytocin, accompanied by reductions in maximal responses. Gossypol (30 micronM) also completely abolished the contractions induced by field stimulation of the rat vas deferens. The IC50 values for gossypol against agonist-uinduced myometrial contractions and field-stimulated vas deferens contractions were similar, ranging between 13 and 18 micronM. These results provide additional evidence that gossypol exerts a direct and irreversible inhibition of the contractility of both male and female reproductive organs

Blockade of the bradykinin-evoked diphasic response of isolated rat duodenum by the crude extract of and compounds obtained from Mandevilla velutina

The influence of an aqueous/ethanolic extract of M. velutina (CE) and of two compound (MV 8608 and MV 8612) purified from the plant on BK-, Ad- and K+ -induced responses of the rat duodenum was analyzed in vitro. In preparations precontracted with Ca2+, the CE (1 and 2 mg/ml) markedly inhibited BK -induced relaxation in a dose-dependent manner but was less effective against Ad-induced relaxation. The CE (0.5 mg/ml) also antagonized BK -induced relaxation and contraction of strips bathed in normal medium. The two compounds from M. velutina (10 and 20 microng/ml) also promoted a dose-dependent blockade of both responses to BK, only slightly depressing the response to K+

Bone formation in the clitoris of neonatallu testosterone treated female dogs.

O tratamento precose de cachorrinhas no 1o. e 3o. dias de vida com propianato de testosterona provoca crescimento do clitoris que se apresenta com aspecto penisoide Por outro lado a castracao pos-natal de cachorrinha impede a evolucao da estrutura ossea peniana. Estes resultados falam a favor da hipotese de que tanto o clitoris como o penis caninos apresentam a mesma potencialidade de resposta ao estimulo androgenico