Some applications of pharmacogenomics and epigenetics in drug development and use in pursuit of personalized medicine.

Personalized medicine has become the most recent mantra of the pharmaceutical industry. While truly affordable bespoke drugs may never be totally achievable, pharmacogenomics and epigenetics will play significant roles in developing targeted therapy tailored to subpopulations of disease sufferers most likely to benefit. Personalized medicine is a very attractive concept, but an extremely difficult reality to achieve due to theoretical and practical considerations. Foremost among the theoretical reasons is our dearth of knowledge of individual physiology and metabolism, as well as the interactions of genetics and environment in the development of most diseases. Amongst the practical reasons, there is the cost of new drug development, considered to be about 800 million to one billion dollars (J Health Econ 22:151-185, DiMasi et al. 2003; Health Econ 19:130-141, Adams and Vu Brantner 2010) and the fact that many drugs now on the market do display reasonable efficacy in large segments of the population with acceptable side effects. Thus, the market for "personalized" drugs may not be large enough to support the costs of development. Another factor is the limitations put on healthcare by governments and insurance companies which promote the use of generics rather than the creation of new chemical entities. Finally, there are the social and ethical considerations of turning individual biology into noughts and ones with the possibility of such information becoming public and/or being used to constrain the way one lives or the care one receives (Nat Rev Drug Discov 1:300-308, Issa 2002). That said, to the degree that personalized medicine does become possible, pharmacogenomics and epigenetics will play significant roles in drug development and use.

A toxicological investigation of a celery seed extract having anti-inflammatory activity.

BACKGROUND AND AIMS: An extract of the seed from celery (Apium graviolens) (CSE), and fractions thereof, have been found to possess anti-inflammatory activity, gastro-protective activity, and anti-Helicobacter pylori activity. In view of the potential for employing these extracts for therapeutic use, toxicological investigations were undertaken with an alcoholic extract (A-CSE) which has previously been shown to have the above pharmacological activities. METHODS: A 28-day toxicity study was performed in rats according to Good Laboratory Practice (GLP) conditions. Eighteen adult male and 18 adult female rats were randomly assigned to 3 treatment groups of 6 rats/sex/group and were dosed orally with A-CSE of 0, 150 or 5,000 mg/kg per day. Daily observations of vital signs and body weights were recorded and ophthalmological investigations were performed. At autopsy, the principal organs were weighed and sections collected for histological analysis. Serum and urine samples were collected at termination for routine clinical chemistry. Under non-GLP conditions alpha-2-µ-globulin immunohistochemistry was performed on kidney tissues and hepatic cytochrome P450 protein was determined, as well as, the enzymatic activities of the principal isoforms. RESULTS: All animals survived treatments with no visible or behavioral signs of toxicity being observed during the study. There were no statistically significant differences in body weight gains, body weight gains per day or cumulative absolute body weight gains, for either sex, in any treatment groups when compared with controls. Slightly increased liver weight and liver to body and brain weight ratios were observed in female rats and in liver to body weight ratios in male rats given high dose A-CSE which was a test article effect, but the absence of any microscopic correlates for the liver weight increases suggests that these were not toxicologically significant. Treatment related macroscopic changes were not observed at necropsy and microscopic findings were limited to minimal increases in gastric eosinophils in several male and female rats in the 5,000 mg/kg per day treatment groups. Minimal focal degeneration of renal tubules was observed sporadically in both sexes assigned to all treatment groups including control and was consistent with early spontaneous nephropathy of laboratory rats and thus was not considered to represent a pathologic change associated with the test article. Increased serum globulin and phosphorus levels were observed in male rats given 5,000 mg/kg per day A-CSE and decreased serum triglycerides levels in female animals given 150 or 5,000 mg/kg per day A-CSE. The increase in serum globulin and phosphorus in male animals was small in magnitude and not considered toxicologically significant. The mechanism for the decrease in serum triglycerides in female rats was not apparent. Changes in urinalysis parameters were limited to small decreases in urine pH in female animals in the 150 and 5,000 mg/kg per day groups and were not deemed toxicologically significant. Alpha-2-µ-globulin immunohistochemistry was performed on kidney tissues from all animals and found to be within normal physiologic limits. Minor corneal mineralization occurred in some animals from all treatment groups. Cataracts were observed in one in the control and one in an animal that had 5,000 mg/kg per day but since the cataracts occurred in the metabolically inactive region of the lens, these were not considered indicative of test article related lesions. There were no changes in total hepatic microsomal protein or in total cytochrome P450 protein. Although male rats appeared to have to higher levels of total microsomal protein than female rats, there appeared to be no treatment effect in either male or female animals. As regards the activity of the various isoforms tested (CYP2B1/2, CYP1A1/2, CYP3A1/2), with the large range of activities detected for each P450 isoform, no clear change in activity or protein were observed, however, these data were not statistically analyzed. CONCLUSIONS: These results suggest that there are no toxicologically significant sub-chronic effects of oral A-CSE in rats. The no adverse effect level for systemic toxicity would appear to be 5,000 mg/kg per day.

Hypothesis: leukocyte endogenous mediator/endogenous pyrogen/lymphocyte-activating factor modulates the development of nonspecific and specific immunity and affects nutritional status.

We postulate that leukocyte endogenous mediator/endogenous pyrogen/lymphocyte-activating factor (LEM/EP/LAF) integrates the host's nonspecific and specific immune responses to infection by virtue of the panoply of physiological and metabolic activities it is capable of eliciting. The alterations in systemic metabolism modulated by LEM/EP/LAF, although apparently of value to the host in the defense against infection and the repair of tissue damage, result in negative nutrient balances. Severe infections, alone or in conjunction with injury, may result in malnutrition unless the patient is adequately nourished. Preexisting nutritional deficits can compromise host resistance to infection, in part by preventing production of LEM/EP/LAF. Additional studies of the sequelae of LEM/EP/LAF action and effects of nutrition on host resistance to infection appear warranted.

Host metabolic alterations during inflammatory stress as related to nutritional status.

Malnutrition predisposes to infection, whereas infection can exacerbate malnutrition, resulting in hindered growth and development. This interplay between infection and nutrition suggests that host metabolism has a role in host defense during infection. Infection occasions profound alterations in host trace metal, nitrogen, and hormone metabolism and redistribution as a result of factors [leukocyte endogenous mediator (endogenous pyrogen)] released from stimulated phagocytes. Many of these alterations occur even in malnourished, protein-restricted, and zinc-deficient animals and persons, bespeaking the essential nature of these changes. Although these metabolic sequelae of infection appear to be of value to the host during acute illness, a metabolic deficit is often incurred which may persist long after resolution of clinical illness. Understanding host-parasite sequences will allow formulation of an integrated approach to the care of infected patients, combining the appropriate elements of nutrition with the best features of antimicrobial therapy.

Pathogenesis of tularemia in immune and nonimmune rats.

Pathogenesis of tularemia in nonimmune rats given (intraperitoneal inoculation) virulent strain (SCHU S4) or vaccinal strain (LVS) of Francisella tularensis and in immune rats given SCHU S4 is described. Both LVS and SCHU S4 caused pyogranulomas in liver and spleen of nonimmune rats. Nonimmune rats given 10(4) SCHU S4 organisms did not survive beyond 72 hours, but immune rats given challenge inoculum of 10(8) SCHU S4 organisms developed lesions and survived. Larger doses of LVS resulted in earlier onset of characteristic hepatitis and splenitis in nonimmune rats. Periportal lymphocytic infiltrates were present in the liver 48 hours after SCHU S4 challenge inoculation of immune rats and 96 hours after inoculation of LVS in nonimmune rats and were associated with intense macrophage aggregation. These changes indicate that the pathogenesis of tularemia is a result of the interdependency of the dose and virulence of the causative agent with the immune status of the host and that cellular immunity has a significant role in the response of the rat to tularemia.

Persons and pathogens: consequences of co-existence.

There is considerable evidence that infectious agents may be a cofactor, or even the cause, of a panoply of chronic inflammatory diseases, as well as of some cancers and neurologic diseases. Examples are presented of the evidence for the thesis that infectious antecedents may underlie many inflammatory conditions and some of the diseases in which infections are postulated to be the precipitating factor.Given that infectious agents may give rise to an impressive array of chronic diseases, should we, therefore, consider alternative modes of treatment for such diseases which focus on the infectious antecedent rather than the consequent disease?With regard to therapy, are new classes of antibiotic/anti-inflammatory drugs needed? Or would preventive measures, such as better food handling and water treatment, along with a widespread campaign of prophylactic and therapeutic vaccines against the likely infectious agents, be a more cost effective approach to ameliorate or ablate the onset of a wide range of inflammatory diseases, cancers and neurologic diseases? Since this conference was primarily designed to discuss the safety and efficacy of NSAIDs, it is appropriate to ponder whether chronic/lifelong NSAID/aspirin intake may have value in preventing or treating chronic inflammatory and neurologic diseases as well as some cancers. There are studies that indicate that long term use of some NSAIDs including aspirin may reduce the likelihood of occurrence of some of these chronic diseases. We thus should ponder the potential risks and benefits of lifelong NSAID/aspirin intake. To that end we should ask what kind of a database should be established so that a valid estimate of the benefits versus the risks of extended, perhaps lifelong, use of a present or future NSAID(s) may be considered for the prevention of any given disease?

Protein synthesis in zinc deficient rats during tularemia.

The effect of zinc deficiency on protein synthesis in rats during tularemia was studied. Five weeks prior to infection with the live vaccine strain of Francisella tularensis, rats had been assigned to one of three dietary groups: zinc deficient (-Zn), pair-fed (PF) or ad libitum (AL). Within 4 weeks, zinc deficiency manifested itself by diminished growth rate, decreased serum and liver zinc concentrations and alopecia. By 18 hour post infection, rats of all groups were febrile and exhibited an increased hepatic uptake of zinc. Despite initially lower concentrations of seromucoid in the PF and -Zn groups, infection elicited an increase in seromucoid concentration as well as enhanced incorporation of 3H-leucine into this fraction of comparable degree in all dietary groups. The same held true for ceruloplasmin. Alpha 2-macrofetoprotein also increased to the same extent in all dietary groups. Infection was associated with a decrease in extractable albumin in ad libitum and pair fed control groups. Only the -Zn group showed a significant decrease in specific activity suggestive of diminished albumin synthesis. Zinc deficiency of itself did not cause a decrement in radiolabel in muscle protein. Thus, despite documented zinc deficiency, rats subjected to the stress of infection respond by synthesizing increased amounts of acute phase globulins apparently at the expense of serum albumin and muscle protein synthesis.

Plasma proteins and wound healing.

In response to injury, the concentrations of several plasma proteins are characteristically altered. In part, these changes reflect an essential contribution of many of these proteins, acting in concert, to the processes involved in wound healing. There is evidence that plasma proteins support tissue repair by metabolic as well as functional activity. Specifically, plasma proteins may directly facilitate wound healing by: provision of carbohydrates, lipids and amino acids in a usable form as biosynthetic precursors and energetic substrates; the transport of trace metal cofactors involved in various wound repair processes; adhesion of regenerating tissue; modulation of the rate of structural protein synthesis; alignment of collagen subunits; organization of cellular elements wound repair; prevention of autoimmune reactions; hormone transport and local modulation of hormonal effects; neutralization of the potentially toxic products of the inflammatory response and the inhibition of microbial invasion and colonization.

Alterations in plasma copper, zinc, amino acids, and seromucoid during Rocky Moutain spotted fever in guinea pigs.

Guinea pigs inoculated with virulent Rickettsia rickettsii responded with a significant increase in plasma copper concentration within 1 day, preceding fever and detectable rickettsemia by 2 and 4 days, respectively. A decrease in serum zinc concentration coinciding with peak rickettsemia was detectable on Day 5. Evidence of altered host nitrogen metabolism during this illness included a doubling of plasma seromucoid concentration and a significant rise in the plasma phenylalanine/tyrosine ratio.

Peroxisome depletion in rat liver during pneumococcal sepsis.

Biochemical and morphometric analysis reveal that the peroxisomal content of rat liver cells is markedly reduced during pneumococcal sepsis. It is suggested that during some bacterial infections, hepatic synthesis of acute-phase serum proteins occurs at the expense of peroxisomal protein synthesis and results in reduction of the peroxisomal protein pool and number of peroxisomes.

Amino acid flux and protein synthesis after exposure of rats to either Diplococcus pneumoniae or Salmonella typhimurium.

At 26 h after inoculation of rats with Diplococcus pneumoniae, the serum concentrations of 10 and 20 individual amino acids were lower than corresponding values observed in pair-fed controls. In contrast, only 2 of 20 serum amino acids were similarly decreased in rats inoculated with Salmonella typhimurium. Despite these serum differences, a greater accumulation of labeled non-metabolizable amino acids occurred in the livers of rats infected with S. typhimurium. These data suggested a greater increase in the flux of amino acids from muscle to liver in the rats infected with S. typhimurium as compared to those infected with D. pneumoniae. A similar increase in serum protein synthesis was observed in rats infected with D. pneumoniae or S. typhimurium. However, with the latter infection, a larger percentage of the amino acids appeared to be utilized as a source of energy in addition to their role as precursors of proteins.

Systemic metabolic alterations associated with repeated injections of a modified polyriboinosinic-polyribocytidylic acid complex.

Polyriboinosinic acid-polyribocytidylic acid complexed with poly-1-lysine and injected intramuscularly into rats (0.3 or 3.0 mg/kg) produced fever, altered leukocyte count, slightly depressed plasma zinc, increased amino acid uptake into liver, and increased plasma acute-phase globulins two- to threefold. It is suggested that these systemic metabolic alterations are indicative of a mild inflammatory response to this drug. The metabolic alterations may have to be taken into consideration when polyriboinosinic acid-polyribocytidylic acid complexed with poly-1-lysine is used in therapy.

Nonhuman primate model for the study of respiratory Klebsiella pneumoniae infection.

Squirrel monkeys were inoculated by the intratracheal inoculation of 700 Klebsiella pneumoniae organisms and developed lobar pneumonia in about 24 h. Characteristic clinical findings were fever, anorexia, and coughing. Laboratory findings included leukocytosis or leukopenia (with the latter more prominent in ultimately fatal infections), bacteremia, and shedding of bacteria into the pharynx. Infected monkeys showed increased plasma lysozyme activity as well as increased plasma ceruloplasmin, haptoglobin and alpha1-antitrypsin. The mortality rate was 60%, and the mean time of death was 50.5 h. Pathologically, the disease spread by means of Kohn's pores and other pathways that generally did not involve airways as a means of dissemination until about 30 h. Squirrel monkeys seem to be better models for human respiratory K. pneumoniae infection than rats or mice.

Nitrogen metabolism and protein synthesis during pneumococcal sepsis in rats.

Markedly increased synthesis of alpha(2) and beta globulins and alpha(1), alpha(2), and beta glycoglobulins occurs during pneumococcal sepsis in the rat simultaneously with decreased albumin formation, diminished tritiated leucine incorporation into muscle protein, and enhanced excretion of nitrogen. This augmented synthesis of specific serum proteins does not become evident until fever and bacteremia develop, and it appears to be a fundamental aspect of host response to a proliferating bacterial infection in that it occurs even in rats fed a protein-deficient (6% protein) diet after weaning and before exposure to Diplococcus pneumoniae. Although amino acid catabolism, in general, appears to be increased during infection, tryptophan degradation via the kynurenine pathway, as assessed by measuring diazotizable urinary metabolites, changes little or is, at times, significantly less than in control animals. Coincidentally, functional tryptophan oxygenase activity decreases at 16 hr after exposure. Total tryptophan oxygenase activity, however, is unchanged.