Challenges of drug discovery in novel target space. The discovery and evaluation of PF-3893787: a novel histamine H4 receptor antagonist.

We describe the development of novel benzimidazoles as small molecule histamine H4 receptor (H4R) antagonists and their profiling in rat early toxicity studies. The discovery and optimisation of a second series of pyrimidine based antagonists is then described culminating in the identification of the clinical development candidate 13 (PF-3893787). The pre-clinical profile of 13 (PF-3893787) is presented including the development of a translatable biomarker. Our pragmatic approach to target selection, safety assessment, and testing for efficacy faced numerous challenges and we share a number of lessons which the team learned and which will assist us and others in future drug discovery projects.

Analysis of patents on anti-allergic therapies issued in China from 1988 to 2008.

BACKGROUND: The prevalence of allergic diseases has increased dramatically in recent decades. Therefore, there is a pressing need for the development of effective anti-allergic services worldwide. However, little is known what anti-allergic products have been patented in China and what the potential drug candidates for patents are in China. OBJECTIVE: To analyze the patents of anti-allergic products for the last 20 years and help pharmaceutical companies and individuals to understand the potential candidates for anti-allergic patents in China. METHODS: Data were obtained from the People's Republic of China Country Intellectual Property Rights Bureau website and United States Patent and Trademark Office website. RESULTS: A total of 789 anti-allergic patents have been granted in China during the past 20 years, which all focused on synthetic compounds, traditional Chinese medicines (TCM), combinations of synthetic compounds and TCM, biological products and medical apparatus. It appears that more and more effective therapeutic components of TCM rather than whole herbs have been patented in recent years and alteration of natural molecules to produce more therapeutically effective molecules has emerged as a novel trend for the modernization of TCM. The patents on synthetic anti-allergic compounds in China mainly focus on well-known targets, such as histamine receptor and leukotrienes, which consist of 93% of patents for validated targets. CONCLUSION: The number of anti-allergic patents applied in China is far lower than that in the US. Therefore, there are great opportunities for obtaining anti-allergic patents, particularly patents on active ingredients from TCM in China.

Analysis of trends and opportunities of anti-allergy patents in China from 1998 to 2008.

The prevalence of allergic diseases has increased dramatically in recent decades. Holding patents is one of the means to protect good anti-allergy products. However, little is known of anti-allergy patent situation in China. The paper summarized and analyzed anti-allergy patents issued in China from January 1988 to September 2008. A total of 789 anti-allergy patents have been granted in China during the 20 years. China, European countries, USA, Japan and other countries possesses 44%, 21%, 19%, 12% and 4% of all of these anti-allergy patents respectively. Interestingly, 88% anti-allergy patents issued to Chinese are held by civilians, whereas vast majority of the patents issued to foreigners were held by pharmaceutical companies. All anti-allergy patents are focused on synthetic compounds, Traditional Chinese Medicines (TCM),combinations of synthetic compounds and TCM (CST), biological products and medical apparatus. The anti-allergy patents in China mainly focus on well-known targets, such as histamine receptor and leukotrienes, which consist of 93% of patents for validated targets. Approximately 93% targeting diseases are bronchial asthma, allergic rhinitis and atopic dermatitis. Our analyzing results indicate that there are great opportunities for application of patents on development of novel anti-allergic compounds and modernization of TCM in China.

From anti-allergic to anti-Alzheimer's: Molecular pharmacology of Dimebon.

Dimebon, originally developed as an anti-histamine drug, is being re-purposed for new indications as an effective treatment for patients suffering from Alzheimer's and Huntington's diseases, albeit with an as-yet unknown mechanism of action. We have performed molecular pharmacology profiling of this drug on a panel of 70 targets to characterize the spectrum of its activity, with the goal to possibly elucidate a potential molecular mechanism for the re-purposing of this drug candidate. We show that in addition to histaminergic receptors, Dimebon exhibits high affinity to a constellation of other receptors; specifically serotonergic, alpha-adrenergic and dopaminergic receptors. Good correlations with published literature were obtained for the affinity of Dimebon to inhibit butyrylcholinesterase, interact with H1and H2 receptors (Ki = 2 nM and 232 nM), and to block histamine-induced calcium fluxes in cells. Within serotonergic receptor subtypes, Dimebon shows highest affinity for 5-HT7 (Ki=8 nM) and 5-HT6 (Ki=34 nM) receptors, with the relative affinity rank-order of 5-HT7 > 5-HT6 > or = 5-HT2A = 5-HT2C > 5-HT1A = 5-HT1B > 5-HT2B=5-HT3. Dimebon also interacts with adrenergic receptor subtypes (rank-order: alpha1A (Ki = 55 nM)= alpha1B > or = alpha2A (Ki = 120 nM) = alpha1D), and dopaminergic receptor subtypes (rank-order: D1=D2S=D2L (Ki approximately 600 nM) >D3> or =D4.2>D4.4> or =D4.7). These results demonstrate a molecular pharmacological basis for re-purposing of this drug to new therapeutic areas. The informed targeting of the combined molecular target activities may provide additional advantages for patients suffering from similar diseases syndromes. Understanding the role that different pathways play in diseases with complex etiologies may allow for the rational design of multi-target drugs.

Lead identification and optimization of diaminopyrimidines as histamine H4 receptor ligands.

BACKGROUND: The human histamine H(4) receptor (hH(4)R) is a promising new target in the therapy of inflammatory or immune system diseases. METHODS: For the development of new hH(4)R ligands, a broad virtual screening was performed and two hits were identified. Their annelated heterocyclic core was optimized with regard to affinity and potency. RESULTS: Pharmacological characterization of the resulting diaminopyrimidines revealed different agonist and antagonist properties within the same scaffold.

The physiological and pathophysiological roles of neuronal histamine: an insight from human positron emission tomography studies.

Histamine neurons are exclusively located in the posterior hypothalamus, and project their fibers to almost all regions of the human brain. Although a significant amount of research has been done to clarify the functions of the histaminergic neuron system in animals, a few studies have been reported on the roles of this system in the human brain. In past studies, we have been able to clarify some of the functions of histamine neurons using different methods, such as histamine-related gene knockout mice or human positron emission tomography (PET). The histaminergic neuron system is known to modulate wakefulness, the sleep-wake cycle, appetite control, learning, memory and emotion. Accordingly we have proposed that histamine neurons have a dual effect on the CNS, with both stimulatory and suppressive actions. As a stimulator, neuronal histamine is one of the most important systems that stimulate and maintain wakefulness. Brain histamine also functions as a suppressor in bioprotection against various noxious and unfavorable stimuli of convulsion, drug sensitization, denervation supersensitivity, ischemic lesions and stress susceptibility. This review summarizes our works on the functions of histamine neurons using human PET studies, including the development of radiolabeled tracers for histamine H1 receptors (H1R: (11)C-doxepin and (11)C-pyrilamine), PET measurements of H1R in depression, schizophrenia, and Alzheimer's disease (AD), and studies on the sedative effects of antihistamines using H(2)(15)O and H1R occupancy in the human brain. These molecular and functional PET studies in humans are useful for drug development in this millennium.

Discovery, structure-activity relationship study, and oral analgesic efficacy of cyproheptadine derivatives possessing N-type calcium channel inhibitory activity.

Antiallergic drug cyproheptadine (Cyp) is known to have inhibitory activities for L-type calcium channels in addition to histamine and serotonin receptors. Since we found that Cyp had an inhibitory activity against N-type calcium channel, Cyp was optimized to obtain more selective N-type calcium channel blocker with analgesic action. As a consequence of the optimization, we found 13 with potent N-type calcium channel inhibitory activity which had lower inhibitory activities against L-type calcium channel, histamine (H1), and serotonin (5-HT2A) receptors than those of Cyp. 13 showed an oral analgesic activity in rat formalin-induced pain model.

Site specific therapy: an integrative approach to treating melanoma.

There have been many proposed theories for effectively treating melanoma, especially through the regulation of histamine. Histamine has been proven to be a major regulator of the immune system's T-helper cell subset balance and major shifts in this balance towards TH2 cytokines have contributed to diseases such as asthma, lupus and cancer. Histamine also causes suppression of interferon-induced proteins needed for anti-tumor response and activates T-suppressor cell function in cancers such as squamous cell carcinoma and melanoma. Scientific evidence has suggested the possibility of an anthistamine approach as treatment to these diseases and for melanoma, there has been great promise. This is due to the fact that melanotic cells have been elucidated to express histamine receptors and as a result, regulation of histamine could occur specifically at the site of these epidermal growths. Another factor to consider is how effective an inflammatory response can be when combined with regulation of histamine. Inflammation is a very powerful tool against pathogenic environments by causing cytokine recruitment and migration of dendritic cells to infected sites. Adequate stimulation of an inflammatory response at the specific site of any cancerous region would greatly weaken its evasive mechanisms. However, there are no reports showing high efficacy utilizing the benefits of regulating inflammation and histamine that could cause TH1 subset levels to predominate, down-regulate T-suppressor cells, up-regulate interferon-induced proteins and properly sustain migration of dendritic cells concurrently. These benefits have been proven in separate instances for a range of diseases but have not been assessed as a combined modality for melanoma therapy. Therefore successful melanoma treatment should integrate these principles involving: the use of H2 antagonists for preventing the negative effects of histamine, monoclonal antibodies to ensure an effective dendritic cell response, and routine pro-inflammatory induction at the specific site of the melanotic tissue to ensure recognition of the cancer that has evaded immunity.

Interaction of various Piper methysticum cultivars with CNS receptors in vitro.

Methanolic leaf and root extracts of the Hawaiian kava (Piper methysticum Forst.) cultivars, Mahakea, Nene, Purple Moi and PNG, were tested on binding affinities to CNS receptors including GABAA (GABA and benzodiazepine binding site), dopamine D2, opioid (mu and delta), serotonin (5-HT6 and 5-HT7) and histamine (H1 and H2). HPLC analysis was carried out in order to determine the amount of the main kavalactones kavain, 7,8-dihydrokavain, methysticin, 7,8-dihydromethysticin, yangonin and 5,6-demethoxyyangonin. The most potent binding inhibition was observed for leaf extracts to GABAA receptors (GABA binding site) with IC50 values of approximately 3 micrograms/ml, whereas root extracts were less active with IC50 values ranging from 5 micrograms/ml (Nene) to 87 micrograms/ml (Mahakea). Since the leaf extracts generally contained lower amounts of the kavalactones than the root extracts, there might exist additional substances responsible for these activities. Leaf extracts also inhibited binding to dopamine D2, opioid (mu and delta) and histamine (H1 and H2) receptors more potently than the corresponding root extracts with IC50 values ranging from 1 to 100 micrograms/ml vs. > or = 100 micrograms/l, respectively. Significant differences in the potential of binding inhibition were also observed between cultivars. Binding to serotonin (5-HT6 and 5-HT7) and benzodiazepine receptors was only weakly inhibited by both root and leaf extracts of all four cultivars. In conclusion, our investigation indicates that the GABAA, dopamine D2, opioid (mu and delta) and histamine (H1 and H2) receptors might be involved in the pharmacological action of kava extracts. Since the cultivars contained similar amounts of kavalactones, while their pharmacological activities differed markedly, other constituents may play a role in the observed activities. Additionally, leaves generally exhibited more potent binding inhibition than roots, therefore leaf of P. methysticum might be an interesting subject for further pharmacological studies.

Central histaminergic mechanisms mediate the vasopressin-induced pituitary adrenocortical stimulation.

Involvement of histamine receptors and hypothalamic and hippocampal histamine in stimulation of the hypothalamic-pituitary-adrenal (HPA) axis by vasopressin (AVP) was investigated in conscious rats. The HPA activity was assessed by measuring serum corticosterone levels. One hour after administration AVP, (5 micrograms/kg) given i.p. significantly raised the serum corticosterone and hippocampal histamine levels, while the hypothalamic histamine content was not affected. Pretreatment with the inhibitor of the brain histamine synthesis alpha-fluoromethylhistidine (alpha-FMH) (50 mg/kg i.p.) considerably reduced both the AVP-elicited serum corticosterone response and the hypothalamic and hippocampal histamine levels. The histamine H1- and H2-receptor-antagonists mepyramine (0.01 mg/kg) and ranitidine (0.1 mg/kg), given ip 15 min prior to AVP, significantly impaired the AVP-induced rise in the serum corticosterone level and totally abolished the AVP-elicited increase in the histamine content in the hippocampus; moreover mepyramine significantly lowered this content in hypothalamus. Pretreatment with the histamine H3-receptor antagonist thioperamide (5 mg/kg i.p.) also significantly decreased the AVP-elicited corticosterone response, but did not alter the histamine content in either brain structure examined. These results indicate that central histamine H1-, H20 and H3-receptors significantly mediate the stimulatory action of AVP on the pituitary-adrenocortical axis. Hippocampal histamine may be involved in mediation of the AVP-induced effect via H1- and H2-receptors. The inhibitory effect of thioperamide seems to be located directly at non H3-intracellular sites of the pituitary-adrenocortical axis.

Histamine depolarizes cholinergic septal neurons.

1. Bath application of 10 microM histamine (HA) resulted in a depolarization or inward current in 58/59 cholinergic neurons located in the medial septum and nucleus of the diagonal band of Broca (MS/DBB) in a slice preparation of rat brain. 2. In bridge mode, the histamine-induced depolarization consisted of both fast and slow phases; inward currents that followed the comparable time course were observed under voltage-clamp conditions. The fast depolarization was associated with variable changes in input resistance, while the slow depolarization always was associated with an increase in input resistance. 3. Both fast and slow responses persisted in the presence of tetrodotoxin (TTX), but only the fast response persisted when transmitter release was abolished by bathing the slice in either a low-Ca(2+)-, high-Mg(2+)-containing medium or one containing Cd2+. 4. When ramp voltage-clamp commands were applied during the fast depolarization, the resultant current-voltage (I-V) curves did not intersect over the range of membrane potentials from -130 to -30 mV. Ionic substitution experiments suggested that the bulk of the ionic current flowing during the fast depolarization was carried by sodium ions. 5. The I-V characteristics of the slow inward current identified it as a reduction in an inwardly rectifying potassium conductance. 6. The fast depolarization was significantly reduced by the H1 receptor antagonists pyrilamine and promethazine, but not by the H2 receptor antagonist cimetidine. Neither the H2 receptor agonist impromidine nor the H3 receptor agonist R-alpha-methylhistamine mimicked the response to HA. None of the agonists or antagonists had any observable effect upon the slow depolarization. 7. We conclude that HA directly depolarizes cholinergic MS/DBB neurons by acting as an H1 receptor, which primarily couples to an increase in a TTX-insensitive Na+ conductance. Additionally, HA evokes a slow depolarization mediated by a decrease in an inwardly rectifying potassium conductance but is not generated by activation of classically defined HA receptor subtypes.

Characterization of histaminergic H3 receptors in intraocular tuberomammillary transplants containing histaminergic neurons.

This study was performed to investigate the physicological properties of histaminergic neurons in intraocular hypothalamic transplants. Pieces of posterolateral hypothalamus containing the tuberomammillary nucleus were dissected from Embryonic Day 17 rat fetuses and transplanted into the anterior chamber of the eye of adult rat hosts. The hypothalamic transplants were left to mature for 2-5 months, after which in vivo electrophysiological recordings were performed. Extracellular recordings revealed spontaneously active neurons in the grafts, with a mean (+/- SEM) firing rate of 2.8 +/- 2.0 Hz and a mean action potential duration of 1.2 +/- 0.5 ms. When the surface of the grafts was superfused with histamine, the neuronal activity was depressed at concentrations above 30 microM. Superfusion with the H3 agonist (R)-alpha-methylhistamine also elicited depression of baseline firing rate, with an EC50 of 0.435 microM. This depression could be antagonized by superfusion with the H3-receptor antagonist thioperamide. In studies of histamine levels using a sensitive radioenzymatic assay, the mean (+/- SEM) level of histamine in the grafts was 73 +/- 28 ng/g tissue, i.e., about half the concentration of histamine in the adult rat hypothalamus in situ. Intracellular recordings in combination with biocytin labeling and histidine decarboxylase immunohistochemistry suggested that the grafted neurons from which recordings were made were histaminergic. Taken together, these data indicate that tuberomammillary neurons continue their development in intraocular transplants and develop physiological characteristics found in these neurons in situ.

A simple and rapid in vitro test system for the screening of histamine H3 ligands.

A simple and rapid functional test system for the screening of histamine H3 ligands is described. It is based on the inhibitory effect of histamine H3 agonists on electrically-evoked contractile response of isolated guinea pig intestine. Whole jejunum segments are continuously stimulated maximally (15 V) by electrical pulses with a frequency of 0.1 Hz and a duration of 0.5 msec. The resulting twitches are recorded isotonically (1.0 g) and can be completely abolished by atropine (0.1 mcM).

Hypothalamic sites of neuronal histamine action on food intake by rats.

To identify sites of histaminergic modulation of food intake, histamine H1-receptor antagonist was microinfused into the rat hypothalamus, the ventromedial hypothalamus (VMH), the lateral hypothalamus (LHA), the paraventricular nucleus (PVN), the dorsomedial hypothalamus (DMH), or the preoptic anterior hypothalamus (POAH), during the early light period. Feeding, but not drinking, was elicited in 100% of the rats (P less than 0.01) that were bilaterally microinfused with 26 nmol chlorpheniramine into the VMH. Unilateral infusion into the VMH did not affect food intake at doses of 26 or 52 nmol. Feeding was also induced by bilateral microinfusion into the PVN, but only the 52 nmol dose was effective. Bilateral infusions into the LHA, the DMH or the POAH did not affect ingestive behavior. Feeding induced by an H1-antagonist was completely abolished in all 7 rats tested when endogenous neuronal histamine was decreased by pretreatment with alpha-fluoromethylhistidine (100 mg/kg). The findings suggest that H1-receptors in the VMH and the PVN, but not in the LHA, the DMH or the POAH, may be involved in histaminergic suppression of food intake.

Blockade of the histamine H1-receptor in the rat ventromedial hypothalamus and feeding elicitation.

All H1-, but no H2-antagonists infused into the rat third cerebroventricle, induced feeding during the early light, but not during the early dark, reflecting a concentration of hypothalamic histamine. Bilateral microinfusion identified the ventromedial hypothalamus (VMH), but not the lateral hypothalamus or the paraventricular nucleus, as a main locus for the induction of feeding by an H1-antagonist. The effect was completely abolished when brain histamine was decreased by pretreatment with alpha-fluoromethylhistidine. Hypothalamic neuronal histamine suppresses food intake, at least in part, through H1-receptors in the VMH.

Autoinhibition of histamine synthesis mediated by presynaptic H3-receptors.

The regulation of histamine synthesis was studied on rat brain slices or synaptosomes labeled with L-[3H]histidine. Depolarization by increased extracellular K+ concentration enhanced by about twofold the [3H]histamine formation in slices of cerebral cortex. This stimulation was also observed, although to a lesser extent, in synaptosomes from cerebral cortex and slices from the posterior hypothalamus where most histaminergic cell-bodies are located, suggesting that it may occur in nerve endings as well as in perikarya. In the presence of exogenous histamine in increasing concentrations the K+-induced stimulation was progressively reduced by up to 60-70%. The effect of exogenous histamine appears to be receptor-mediated as shown by its saturable character, high pharmacological specificity and competitive reversal by histamine antagonists. The EC50 value of histamine for synthesis reduction (0.34 +/- 0.03 microM) was similar to its EC50 value for release inhibition known to be mediated by H3-receptors. In addition, whereas mepyramine and tiotidine, two potent antagonists at H1- and H2-receptors, respectively, were poorly effective, the H3-receptor antagonists burimamide and impromidine reversed the histamine effect in an apparently competitive manner. These effects were observed in slices of cerebral cortex or posterior hypothalamus as well as in cortical synaptosomes. Furthermore, even in the absence of added histamine, H3-receptor antagonists enhanced the depolarization-induced stimulation of [3H]histamine synthesis, indicating a participation of released endogenous histamine in the synthesis control process. The potencies of H3-receptor antagonists were similar to those of these agents at presynaptic autoreceptors controlling [3H]histamine release. It is concluded that H3-receptors control not only release but also synthesis of histamine at the level of nerve endings and also, presumably, of perikarya. A relationship between the two regulatory processes, possibly via intracellular calcium, seems likely but remains to be investigated at the molecular level.

The effect of immunotherapy in hayfever patients on histamine binding capacity of blood serum proteins.

Binding of histamine (histaminopexy) by blood serum proteins of 16 hayfever patients was determined before and after treatment with Pollinex. An increase in histaminopexy by 83% in comparison to pretreatment value was observed and it was well correlated with the clinical status of the patients. Participation of this phenomenon in the mechanism of therapeutic effect of specific immunotherapy is discussed.

Enhancement by the putative 5-HT receptor agonist 8-OH-2-(di-n-propylamino)tetralin of the acoustic startle response in the rat.

Two 2-(di-n-propylamino)tetralin (DPAT) compounds, 8-OH-DPAT and 5-OH-DPAT, with reported effects on central 5-HT and DA receptors respectively, were tested for their effects on the acoustic startle response in rats. 8-OH-DPAT was given in doses of 0.25-2.0 mg/kg IP and 5-OH-DPAT in doses of 1.0-8.0 mg/kg IP. Both compounds increased the startle response significantly in a dose-dependent manner, but 8-OH-DPAT appeared to be about 30 times as potent and to have a higher efficacy than 5-OH-DPAT. In addition, the effects on the startle response of L-5-HTP, 25-100 mg/kg IP, and L-dopa, 25-100 mg/kg IP, administration to animals pretreated with the inhibitor of aromatic L-amino acid decarboxylase, benserazide (25 mg/kg IP) were included for comparison. A small, but significant increase in the startle amplitude was found after the highest dose of L-5-HTP, whereas no effects were observed after L-dopa administration.