Drug Development

This process starts with the synthesis ofnovel chemical compounds. Substanceswith complex structures may be obtained from various sources, e.g., plants(cardiac glycosides), animal tissues(heparin), microbial cultures (penicillinG), or human cells (urokinase), or bymeans of gene technology (human insulin). As more insight is gained into structureactivity relationships, the searchfor new agents becomes more clearlyfocused.Preclinical testing yields information on the biological effects of new substances. Initial screening may employ biochemical pharmacological investigations (e.g., receptorbinding assaysp. 56) or experiments on cell cultures,isolated cells, and isolated organs. Sincethese models invariably fall short ofreplicating complex biological processes in the intact organism, any potentialdrug must be tested in the whole animal. Only animal experiments can reveal whether the desired effects will actually occur at dosages that produce little or no toxicity. Toxicological investigations serve to evaluate the potential for:(1) toxicity associated with acute orchronic administration; (2) geneticdamage (genotoxicity, mutagenicity);(3) production of tumors (onco or carcinogenicity); and (4) causation of birthdefects (teratogenicity). In animals, compounds under investigation alsohave to be studied with respect to theirabsorption, distribution, metabolism,and elimination (pharmacokinetics).Even at the level of preclinical testing,only a very small fraction of new compounds will prove potentially fit for usein humans. Pharmaceutical technology provides the methods for drug formulation.Clinical testing starts with Phase Istudies on healthy subjects and seeks todetermine whether effects observed inanimal experiments also occur in humans. Doseresponse relationships are determined. In Phase II, potential drugsare first tested on selected patients for therapeutic efficacy in those diseasestates for which they are intended.Should a beneficial action be evidentand the incidence of adverse effects beacceptably small, Phase III is entered, involving a larger group of patients inwhom the new drug will be comparedwith standard treatments in terms oftherapeutic outcome. As a form of human experimentation, these clinicaltrials are subject to review and approvalby institutional ethics committees according to international codes of conduct (Declarations of Helsinki, Tokyo,and Venice). During clinical testing, many drugs are revealed to be unusable.Ultimately, only one new drug remainsfrom approximately 10,000 newly synthesized substances.The decision to approve a newdrug is made by a national regulatorybody (Food & Drug Administration inthe U.S.A., the Health Protection BranchDrugs Directorate in Canada, UK, Europe, Australia) to which manufacturers are required to submit their applications. Applicants must document bymeans of appropriate test data (frompreclinical and clinical trials) that thecriteria of efficacy and safety have beenmet and that product forms (tablet, capsule, etc.) satisfy general standards ofquality control.Following approval, the new drugmay be marketed under a trade name(p. 333) and thus become available forprescription by physicians and dispensing by pharmacists. As the drug gainsmore widespread use, regulatory surveillance continues in the form of postlicensing studies (Phase IV of clinicaltrials). Only on the basis of longtermexperience will the risk: benefit ratio beproperly assessed and, thus, the therapeutic value of the new drug be determined.

Drug Sources

Drug and Active PrincipleUntil the end of the 19th century, medicines were natural organic or inorganic products, mostly dried, but also fresh,plants or plant parts. These might contain substances possessing healing (therapeutic) properties or substancesexerting a toxic effect.In order to secure a supply of medically useful products not merely at thetime of harvest but yearround, plantswere preserved by drying or soaking them in vegetable oils or alcohol. Dryingthe plant or a vegetable or animal product yielded a drug (from French“drogue” – dried herb). Colloquially, thisterm nowadays often refers to chemicalsubstances with high potential for physical dependence and abuse. Used scientifically, this term implies nothing aboutthe quality of action, if any. In its original, wider sense, drug could refer equally well to the dried leaves of peppermint, dried lime blossoms, dried flowersand leaves of the female cannabis plant(hashish, marijuana), or the dried milkyexudate obtained by slashing the unripeseed capsules of Papaver somniferum(raw opium). Nowadays, the term is applied quite generally to a chemical substance that is used for pharmacotherapy.

Soaking plants parts in alcohol(ethanol) creates a tincture. In this process, pharmacologically active constituents of the plant are extracted by the alcohol. Tinctures do not contain the complete spectrum of substances that existin the plant or crude drug, only thosethat are soluble in alcohol. In the case ofopium tincture, these ingredients arealkaloids (i.e., basic substances of plantorigin) including: morphine, codeine,narcotine = noscapine, papaverine, narceine, and others.Using a natural product or extractto treat a disease thus usually entails the administration of a number of substances possibly possessing very different activities. Moreover, the dose of an individual constituent contained within agiven amount of the natural product issubject to large variations, depending  upon the product‘s geographical origin (biotope), time of harvesting, or condi tions and length of storage. For the same reasons, the relative proportion of indi vidual constituents may vary considerably. Starting with the extraction of morphine from opiumin 1804 by F. W. Sertürner (1783–1841), the active principles of many other natural products were subsequently isolated in chemi cally pure form by pharmaceutical la boratories. The aims of isolating active principles are: 1. Identification of the active ingredient(s). 2. Analysis of the biological effects (pharmacodynamics) of individual in gredients and of their fate in the body (pharmacokinetics). 3. Ensuring a precise and constant dosage in the therapeutic use of chemically pure constituents. 4. The possibility of chemical synthesis, which would afford independence from limited natural supplies and create con ditions for the analysis of structureactivity relationships. Finally, derivatives of the original constituent may be synthesized in an effort to optimize pharmacological properties. Thus, derivatives of the original constituent with improved therapeutic useful ness may be developed.

History of Pharmacology

History of Pharmacology


Since time immemorial, medicaments have been used for treating disease in humans and animals. The herbals of an-tiquity describe the therapeutic powers of certain plants and minerals. Belief in the curative powers of plants and certain substances rested exclusively upon traditional knowledge, that is, empirical information not subjected to critical examination.

The Idea
 
Claudius Galen (129–200 A.D.) first attempted to consider the theoretical background of pharmacology. Both theory and practical experience were to contribute equally to the rational use of medicines through interpretation of observed and experienced results.


“The empiricists say that all is found by experience. We, however, maintain that it is found in part by experience, in part by theory. Neither experience nor theory alone is apt to discover all.”

The Impetus

Theophrastus von Hohenheim (1493–1541 A.D.), called Paracelsus, began to quesiton doctrines handed down from antiquity, demanding knowledge of the active ingredient(s) in prescribed remedies, while rejecting the irrational concoctions and mixtures of medieval medicine. He prescribed chemically defined substances with such success that professional enemies had him prosecuted as a poisoner. Against such accusations, he defended himself with the thesis that has become an axiom of pharmacology:“If you want to explain any poison properly, what then isn‘t a poison? All things are poison, nothing is without poison; the dose alone causes a thing not to be poison.”

Early Beginnings

Johann Jakob Wepfer (1620–1695) was the first to verify by animal experimentation assertions about pharmacological or toxicological actions.“I pondered at length. Finally I resolved to clarify the matter by experiments.”

Foundation

Rudolf Buchheim (1820–1879) founded the first institute of pharmacology at the University of Dorpat(Tartu, Estonia) in 1847, ushering in pharmacology as an independent scientific discipline. In addition to a description of effects, he strove to explain the chemical properties of drugs. “The science of medicines is a theoretical, i.e., explanatory, one. It is to provide us with knowledge by which our judgement about the utility of medicines can be validated at the bedside.”
Consolidation – General Recognition
reputation of pharmacology. Fundamental concepts such as structure-activity relationship, drug receptor, and selective toxicity emerged from the work of, respectively, T. Frazer (1841–1921) in Scotland, J. Langley (1852–1925) in England, and P. Ehrlich (1854–1915) in Germany. Alexander J. Clark (1885–1941) in England first formalized receptor theory in the early 1920s by applying the Law of Mass Action to drug-receptor interactions. Together with the internist, Bernhard Naunyn (1839–1925), Schmiedeberg founded the first journal of pharmacology, which has since been published without interruption. The “Father of American Pharmacology”, John J. Abel(1857–1938) was among the first Americans to train in Schmiedeberg‘s laboratory and was founder of the Journal of Pharmacology and Experimental Therapeutics (published from 1909 until the present).
Status Quo
After 1920, pharmacological laboratories sprang up in the pharmaceutical industry, outside established university institutes. After 1960, departments of clinical pharmacology were set up at many universities and in industry.

SDS-PAGE and Western bolt analysis

Tachyzoites from three genotypic strains（RH，ME49-PLK and VEG）were lysed using M-PER Mammalian Protein Extraction Reagent(Pierce,Rockford,IL)containing 10 µg/ml each of antipain,leupeptin,chymostatin,pepstatin A,phenylmethylsulfonylfluoride and phenanthroline (Sigma,Dt.Louis MO).Aliquots equivalent to 15×106 tachyzoiteswere subjected to 12% SDS-PAGE under reducing conditions and transferred to nitrocellulose.Following transfer,theblots were blocked with 10% horse serum and then incubated overnight with anti-TgPCNA1 OR 2 mouse polyclonal sera(1:2500).Antigen detection was accomplished by incubating blots with alkaline phosphatase-conjugated anti-mouse IgG（Promega，Madison WIS）diluted 1:7,500,followed by development in nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate.

ResultsCloning and sequence comparison of the PCNA genes in T.gondii

Analysis of EST database entries along with conventional cDNA strategies identified two unique PCNA cDNAs in T.gondii,designated TgPCNA1 and TgPCNA2.The 951bp open reading frame of TgPCNA1 predicts a protein of 316 amino acids with a predicted molecular weight of 34,476 daltons.The TgPCNA2 clone contains a smaller open reading frame of 864 bp encoding a protein of 261 amino acids and a predicted molecular weight of 32,263 daltons.Consistent with all known eukaryotis PCNAs,the TgPCNA sequences contain basic helix-loop-helix DNA binding motifs(Fig.2-1)that demonstrate >50% similarity to the equivalent region in human PCNA[8].Other conserved motifs include the poltmerase-δ and p21 putative binding sites (D-SHV-,Fig.2-1)as well as the C-terminal sequences (-F/YLAP,Fig.2-1)that appear essential for proper folding. PfPCNA1 contains 10 and PfPCNA2 contains 9 additional C-terminal amino acdis while T.gondii PCNA1 and 2 contain C-terminal insertions of 53 and 28 amino acids.respectively.