Therapeutic potentiation of the immune system by costimulatory Schiff-base-forming drugs.

Immune responses are orchestrated by CD4 T lymphocytes, which receive a cognitive signal when clonally distributed receptors are occupied by major histocompatibility complex (MHC) class II-bound peptides on antigen-presenting cells (APCs). The APCs provide costimulatory signals, through macromolecules such as CD80, that regulate outcomes in terms of T-cell activation or anergy. We have studied essential complementary chemical events in the form of Schiff base formation between carbonyls and amines that are constitutively expressed on presenting cell and T-cell surfaces and provide a new target for manipulation of immune responses. Here we show that small Schiff base-forming molecules can substitute for the physiological donor of carbonyl groups and provide a costimulatory signal to CD4 Th-cells through a mechanism that activates clofilium-sensitive K+ and Na+ transport. One such molecule, tucaresol, enhances CD4 Th-cell responses, selectively favouring a Th1-type profile of cytokine production. In vivo tucaresol potently enhances CD4 Th-cell priming and CD8 cytotoxic T-cell priming to viral antigens, and has substantial therapeutic activity in murine models of disease.

The Wellcome Foundation/World Health Organisation Onchocerciasis Chemotherapy Project Joint Programme to discover and develop a macrofilaricide for onchocerciasis.

The primary objective of the Wellcome Foundation/World Health Organisation Onchocerciasis Chemotherapy Project, which began in July 1982, was the discovery and development of a safe and effective macrofilaricide for the treatment of onchocerciasis in man. A multidisciplinary Team of biologists, biochemists and medicinal chemists was assembled. They investigated a variety of potential targets in filariae and carried out in-depth lead optimisation studies on a number of different chemical series. A valuable contribution was made to our understanding of filarial biology and biochemistry and the susceptibility of filariae to various metabolic inhibitors. However, the ultimate goal of identification of a compound worthy of evaluation in man was not achieved and the programme was closed at the end of June 1989. Factors that contributed to this situation and some overall conclusions are discussed.

Acetylcholinesterase secretion--a parameter for the interpretation of in vitro anthelmintic screens.

Interpretation of anthelmintic activity using in vitro screens has, until now, relied on the detection of drug-induced effects on nematode development, viability and motility. A novel biochemical parameter dependent upon the spectrophotometric assay of acetylcholinesterase (AChE), an enzyme secreted in large quantities by certain trichostrongylid nematodes, has been developed to replace these often subjective indices of activity. Using Nippostrongylus brasiliensis, a worm frequently employed for primary screening, the secretion of this enzyme in the presence or absence of a large number of drugs in vitro was determined. During a 4-day incubation period in a complex undefined medium without serum. AChE was secreted by normal 4th larval and immature adult stages of the worm in a linear fashion. All modern broad-spectrum veterinary anthelmintics, regardless of their mode of action, dramatically reduced the amount of enzyme secreted. Correlation between the biochemical and observational parameters was excellent and the selectivity of the assay when based solely on enzyme secretion was not lost. Other advantages were that the time required for the activity of certain slow-acting compounds to be detected was reduced from 7 to 4 days and that close microscopical examination of the worms was not necessary.

Trichostrongylus colubriformis: in vitro culture of parasitic stages and their use for the evaluation of anthelmintics.

Approximately 50 per cent of fourth stage larvae of Trichostrongylus colubriformis taken from the gerbil, Meriones unguiculatus, on day 8 after infection, moulted to the young adult stage when cultured in a complex medium over a seven day period in vitro. Larvae at the late fourth stage of development were highly susceptible to certain benzimidazole, prebenzimidazole, imidazothiazole, pyrimidine, quaternary ammonium, organophosphorus and macrocyclic lactone anthelmintics when any of these were included at very low concentrations in the culture medium. However, few anthelmintics lacking activity against T colubriformis in vivo affected these larvae. An assay employing these larvae in vitro should offer a means for assessing the intrinsic activity of new compounds against T colubriformis in the absence of any complicating host pharmacokinetic factors, and could also be adapted for use as a high capacity preliminary screen. Thus it should now be possible to employ a target parasite at the earliest stages of a lead discovery programme obviating the need to use less relevant free-living nematodes or ones that are natural parasites of rodents.

An in vitro screen for new fasciolicidal agents.

In vitro screens employing newly excysted, 6- and 12-week-old flukes, in a medium permitting the linear growth of the parasites, were assessed. When exposed to certain known fasciolicides, newly excysted flukes were susceptible only to diamphenethide, the free amine of diamphenethide, emetine hydrochloride and albendazole. Older flukes were affected by a much wider range of compounds including the chlorinated hydrocarbons, the substituted phenols and the salicylanilides. However their susceptibility to diamphenethide and its active metabolite was decreased significantly. The activity of fasciolicides in these in vitro assays therefore closely parallels their activity in vivo. When several broad spectrum anti-nematode agents were evaluated against newly excysted flukes in these screens the benzimidazole, isothiocyanate, pyrimidine and imidazothiazole anthelmintics showed activity but 12 potent antiprotozoal agents were all inactive. It is concluded that these in vitro assays were useful for detecting any intrinsic activity that a compound might possess against flukes. Such activity could often be missed in conventional in vivo screens because of problems associated with host pharmacokinetics. Negative results from such in vivo screens could preclude the development of more bioavailable derivatives or pro-drugs as novel and useful fasciolicidal agents.

An in vitro screening test for compounds active against the parenteral stages of Trichinella spiralis.

A new in vitro screening test for compounds showing activity against the tissue stages of Trichinella spiralis is described. In this test freshly decapsulated larvae of the parasite are exposed to low concentrations of experimental compound in a medium capable of supporting th partial development of the worms. The screen which does not require elaborate continuous flow equipment nor special gas phase detects the activity of those compounds known to be effective against the parenteral stages of the parasite in mice. It has been shown to be a highly selective test giving rise to very few irrelevant or misleading results.

A new primary screening test for anthelmintics utilizing the parasitic stages of Nippostrongylus brasiliensis, in vitro.

A new in vitro test suitable for the large scale screening of chemical compounds for anthelmintic activity is described. The test which utilizes the fourth larval and adult stages of Nippostrongylus brasiliensis in a medium capable of supporting the growth and development of the parasite, detects selectively those compounds which possess either broad spectrum anthelmintic or specific anti-trichostrongyle activity. The screen is easy to operate requiring only minute quantities of experimental compound. It renders fully reproducible results which furthermore can be interpreted objectively. This is the first reported in vitro test directed against the parasitic stages of a nematode that is capable of detecting reliably the activity of a wide range of anthelmintics including thiophanate and all the benzimidazoles.