Quinolones: novel probes in antifilarial chemotheraphy.

Quinolones have been discovered in our laboratory as a new class of antifilarial agents. This has led to the design, synthesis, and antifilarial evaluation of a number of N-substituted quinol-4(1H)-one-3-carboxamide derivatives 4-6. The macrofilaricidal activity of the target compounds was initially evaluated in vivo against Acanthoeilonema viteae by oral administration of 200 mg/kg x 5 days. Among all the synthesized compounds, 13 displayed activity, with the most potent compound (4a) exhibiting 100% macrofilaricidal and 90% microfilaricidal activities. Compound 4e elicited significant macrofilaricidal (80%) response while compound 5c showed 100% sterilization of female worms. Finally, the two most potent macrofilaricidal compounds, namely 4a and 4e, have been screened for their potency against DNA topoisomerase II, and it has been observed that both have the capability to interfere with this enzyme at 10 micromol/mL concentration. The structure-activity relationship (SAR) associated with position-3 and aryl ring substituents is discussed.

A new class of potential chloroquine-resistance reversal agents for Plasmodia: syntheses and biological evaluation of 1-(3'-diethylaminopropyl)-3-(substituted phenylmethylene)pyrrolidines.

1-(3'-Diethylaminopropyl)-3-(substituted phenylmethylene)pyrrolidines were synthesized and evaluated for CQ-resistant reversal activity. In general the compounds of the series elicit better biological response than their phenylmethyl analogues. The most active compound 4b has been evaluated in vivo in detail, and the results are presented. The possible mode of action of the compounds of this series is by inhibition of the enzyme heme oxygenase, thereby increasing the levels of heme and hemozoin, which are lethal to the parasite.

Potent 1,3-disubstituted-9H-pyrido[3,4-b]indoles as new lead compounds in antifilarial chemotherapy.

Substituted 9H-pyrido[3,4-b]indoles (beta-carbolines), identified in our laboratory as potential pharmacophores for designing macrofilaricidal agents, have been explored further for identifying the pharmacophore responsible for the high order of adulticidal activity. This has led to syntheses and macrofilaricidal evaluations of a number of 1-aryl-9H-pyrido[3,4-b]indole-3-carboxylate derivatives (3-7). The macrofilaricidal activity was initially evaluated in vivo against Acanthoeilonema viteae. Among all the synthesized compounds, only 12 compounds, namely 3a, 3c, 3d, 3f, 4c, 4d, 4f, 5a, 6f, 6h, 6i, and 7h, have exhibited either >90% micro- or macrofilaricidal activity or sterlization of female worms. These compounds have also been screened against Litomosoides carinii, and of these only 3f and 5a have also been found to be active. Finally these two compounds have been evaluated against Brugia malayi. The structure-activity relationship (SAR) associated with position 1 and 3 substituents in beta-carbolines has been discussed. It has been observed that the presence of a carbomethoxy at position 3 and an aryl substituent at position 1 in beta-carbolines effectively enhances antifilarial activity particularly against A. viteae. Among the various compounds screened, methyl 1-(4-methylphenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (4c) has shown the highest adulticidal activity and methyl 1-(4-chlorophenyl)-1,2,3,4-tetrahydro-9H-pyrido[3, 4-b]indole-3-carboxylate (3a) has shown the highest microfilaricidal action against A. viteae at 50 mg/kg x 5 days (ip). Another derivative of this compound, namely 1-(4-chlorophenyl)-3-(hydroxymethyl)-9H-pyrido[3,4-b]indole (5a), exhibited the highest activity against L. carinii at 30 mg/kg x 5 days (ip) and against B. malayiat 50 mg/kg x 5 days (ip) or at 200 mg/kg x 5 days (po).

Syntheses and bioevaluation of substituted dihydropyridines for pregnancy-interceptive activity in hamsters.

A number of 2,6-dimethyl-3,5-bis(methoxycarbonyl)-4-substituted-1,4- dihydropyridines were synthesized and evaluated for pregnancy-interceptive activity in mated hamsters. Out of 24 compounds, 12, 15, 21, 22, 28, and 34 caused a marked reduction in the number of implantations when administered on days 3-8 postcoitum. In an in vitro competition assay, none of the compounds exhibited noticeable binding affinity for uterine progesterone receptors. The results reported here have helped to identify new leads for developing pregnancy-interceptive agents and the active compounds do not seem to elicit their interceptive effect through receptor-mediated inhibition of progesterone action in hamster uterus.

Syntheses and anthelmintic activity of alkyl 5(6)-(substituted-carbamoyl)- and 5(6)-(disubstituted-carbamoyl)benzimidazole-2-carbamates and related compounds.

A number of alkyl 5(6)-(substituted-carbamoyl)- and 5(6)-(disubstituted-carbamoyl)benzimidazole-2-carbamates and related compounds have been synthesized, and their anthelmintic activity against various intestinal helminths of experimental animals have been evaluated. A large percentage of the compounds synthesized showed noteworthy activity against Ancylostoma ceylanicum and at higher doses against Hymenolepsis nana infections. Compared to the alkyl 5(6)-(substituted-carbamoyl)benzimidazole-2-carbamates, the disubstituted carbamoyl analogues were found to exhibit better anthelmintic activity. The most active compound of the series, namely, methyl 5(6)-[(N-2-pyridylpiperazino)carbamoyl]benzimidazole-2-carbamate (90), has been screened against intestinal helminths in higher animals and as a micro- and macrofilaricidal agent. Compound 90 has been identified as a broad-spectrum anthelmintic agent. Compound 90 has been identified as a broad-spectrum anthelmintic in view of its efficacy against A. ceylanicum (hamsters and dogs), H. nana (rats), Nippostrongylus brasiliensis (rats), Syphacia obvelata (mice), A. tubaeformis (cat), Toxocara spp. (cat), and Litomosoides carinii (cotton rat).

Identification of inhibitors of DNA topoisomerase II from a synthetic library of glycoconjugates.

A library of 24 glycoconjugates related to glycosylated beta-amino acid derivative (I) was been prepared and screened against DNA topoisomerase-II of the filarial parasite S. cervi. Among these, compound 6 was found to be a potent inhibitor of DNA topoisomerase-II with 95% inhibition at 1.09 microM. Furthermore, compound 6 was at least three times more potent than the lead compound, glycosylated beta-amino acid derivative I.

A novel pyrrolidonoalkaneamine (WR268954) that modulates chloroquine resistance of Plasmodium falciparum in vitro.

With the recent observations of efflux of chloroquine from Plasmodium falciparum and modulation of chloroquine resistance by calcium channel blockers, such as verapamil, a great deal of attention has been focused on the development of new modulators that can potentiate the efficacy of chloroquine. We report a new compound, WR268954, that has weak intrinsic antimalarial activity compared to chloroquine. In vitro, it increased the susceptibilities of chloroquine-resistant P. falciparum strains to chloroquine and quinine, but did not affect the chloroquine-susceptible strains. In the presence of 2,000 nM of WR268954, the 50% inhibitory concentration of chloroquine for drug-resistant P. falciparum decreased 90-fold in comparison with the control (chloroquine only). The same concentration of WR268954 increased the potentiation of chloroquine in resistant strains to a level approximately equivalent to that observed for the sensitive strain. This compound also potentiates the efficacy of quinine in drug-resistant parasites. However, WR268954 did not enhance the efficacy of mefloquine in the mefloquine-resistant parasites. In this report, the data show the synergistic effect of WR268954 on the antimalarial activity of chloroquine in drug-resistant strains of P. falciparum, but only an additive effect on drug-sensitive strains of parasites. Compound WR268954 belongs to a pyrrolidino alkane amine class whose in vitro chloroquine resistance modulator activity supports the basis for the synthesis of this class of compounds.

Radical curative activity of a new 8-aminoquinoline derivative (CDRI 80/53) against Plasmodium cynomolgi B in monkeys.

An analogue of primaquine, N1-(3-acetyl-4-5-dihydro-2-furanyl)-N4-(6-methoxy-8-quinolinyl) 1,4-pentanediamine, CDRI Code 80/53), has been evaluated for anti-relapse activity against sporozoite induced Plasmodium cynomolgi B infection in rhesus monkeys. The compound has shown 100% curative anti-relapse activity at 1.25 mg/kg x 7 day dose schedule, thereby giving a primaquine index of 0.8. The compound is currently under Phase-I clinical trials.

Emerging concepts towards the development of contraceptive agents.

PIP: The theoretical background for development of steroid contraceptive agents, LH-RH agonists and antagonists, heterocyclic abortifacients, male contraceptive agents, and contraceptive vaccines are reviewed, with emphasis on basic steroid receptor research. Recent findings confirming the steroid receptor interaction theory proposed in 1960 are described. This model suggests that the steroid enters target cells by diffusion and binds to specific protein receptors in the cytoplasm. The steroid- receptor complex moves to the nucleus by concentration gradient and binds to chromatin or DNA, altering gene expression. both estrogen and progesterone receptors are asymmetric peptides with rigid hydrophobic domains that bind specific steroids, doisynolic acids, stilbenes, gem- diarylethylenes, triarylpropiones, all of which have a set distance between a phenolic hydroxyl and another hydroxyl analogous to the steroid C17-beta-hydroxyl groups. Antiprogestins without glucocorticoid activity are being sought. LH-RH antagonists are being developed by substituting amino acids in the peptide. A large variety of heterocyclic compounds, 26 types, with abortifacient activity, acting by interfering with blastocyst binding to the deciduum is described, such as a series of quinine derivatives. Male antifertility agents must block spermatogenesis without affecting libido or sperm maturation. Combined steroid pills with cyproterone acetate and an androgen are in clinical trials. The most hopeful immunologic contraceptives are vaccines based on part of the beta subunit of hCG, sperm antigens and zona pellucida antigens.^ieng

Non-steroidal menses-regulating agents: the present status.

PIP: Contraception researchers are just beginning to study the likelihood of developing menses regulators, most likely in the form of a once-a-month pill. Some advantages of this pill would be self-administration and short-term use without long-term side effects. Both pregnant and nonpregnant women could benefit from its use. Such a pill would either prevent implantation or cause the embryo to detach from the endometrium during the early stages of implantation. Researchers at the Central Drug Institute in Lucknow, India, have reviewed the sequence of events in the endometrium and blastocyst during the implantation and pre-implantation stages and have examined changes which may be applicable to menses induction. Likely biochemical target sites are prevention of endometrial preparations for blastocyte attachment, modification of the physicochemical environment of the uterus to thwart implantation, and interference with the functions of the utero-placental junction for facilitating detachment/resorption of the implanted blastocyst. In vivo studies in laboratory animals and in vitro studies are using various steroidal and nonsteroidal compounds. Mifepristone (RU-486) has already demonstrated effective contragestational activity. Research needs for successful development of menses inducers include: identification of biochemical markers which facilitate in vitro screening of pregnancy interceptive activity; a guarantee of a correlation of in vitro and in vivo contragestational efficacy of test compounds; identification of the pharmacophores which would steer the test compounds to the uterus in concentrations sufficient to intercept pregnancy; and search for exact biochemical target site(s) affected by compounds demonstrating a contragestational effect in laboratory animals.^ieng

Anilino-(2-bromophenyl) acetonitrile: a promising orally effective antileishmanial agent.

Visceral leishmaniasis (VL) or kala-azar is a worldwide disseminated intracellular infection caused by the hemoflagellate protozoan parasites Leishmania donovani. Chemotherapeutic scenario presents a deplorable picture and demands an urgent search for a new and safe anti-VL drugs, preferably active by oral route. In search of new antileishmanial agents, a total of 16 compounds belonging to the anilino-(substituted phenyl)-acetonitrile class were tested in vitro in promastigote/macrophase-amastigote systems and in vivo in L. donvoani/hamster model for their antileishmanial activity. Compound 3, anilino-(2-bromophenyl)-acetonitrile, exhibited most promising activity both in vitro at a concentration of 100 microg/ml (82.33 and 94.36% in promastigote and macrophase-amastigote systems, respectively) and in vivo at a dose of 50 mg/kg for 5 days (82.11 and 80% by i.p. and p.o. routes, respectively), hence this compound was investigated in detail. To maximize its bioavailability, dissolution profile, absorption, the compound was also tested in vivo as its soluble form. But no enhancement in activity was observed. From the results of different parameters for example ED(50) and LD(50) etc. compound 3 appears to be a potent orally effective compound which could further be investigated to establish its potential as a candidate molecule of antileishmanial therapy.

Methyl 5(6)-4-2-pyridyl piperazino carbamoyl benzimidazole-2-carbamate--a new broad spectrum anthelmintic.

Methyl 5(6)-4-2-pyridyl piperazino carbamoyl benzimidazole-2-carbamate (CDRI Comp. 81-470) was tested against various nematode and cestode infections in different experimental and domestic animals. The compound showed 100% effectivity against adult of Ancylostoma ceylanicum (hookworm) in hamsters (6.25 mg/kg p.o. X 1), Nippostrongylus brasiliensis (trichostrongylid) in rats (100 mg/kg p.o. X 3), Hymenolepis nana (cestode) in rats (25 mg/kg p.o. X 1) and Syphacia obvelata (oxyurid) in mice (12.5 mg/kg p.o. X 3). It was also found highly effective against artificial and natural helminth parasites of higher animals. The compound removed all, A. caninum and A. ceylanicum (hookworms) and Toxocara sp. (ascarid) from dogs at a dose of 10 mg/kg p.o. X 3; A. tubaeformis (hookworm) and Toxocara sp. at 25 mg/kg p.o. X 3 and 2.5 mg/kg p.o. X 3 respectively from cats and Ascaridia galli (ascarid) at 10 mg/kg p.o. X 3 from fowl. The compound in doses of 1500 mg/kg by oral route and 1000 mg/kg by i.p. route did not cause any mortality or produce adverse effect in mice. The expanded anthelmintic action and large therapeutic index indicate compound's great anthelmintic potentiality.

Antifilarial activity of some 2H-1-benzopyran-2-ones (coumarins).

Six synthetic 2H-1-benzopyran-2-one (cournarin) derivatives (CDRI compounds # 1, 2, 3, 4, 5 and 6) were evaluated for filaricidal activity against Litomosoides carinii and Acanthocheilonema viteae infections in cotton rats (Sigmodon hispidus) and Mastomys coucha respectively. Significant effects on macrofilariae (>80% death/sterilisation) were detected with compounds #2, 3 and 6 against L. carinii and/or A. viteae. Thus detection of filaricidal activity in benzopyrones, which are so far known for anti-inflammatory activity, provides a new lead for development of better filaricidal agents for combating filariasis.

Correlation between inhibition of growth and arginine transport of Leishmania donovani promastigotes in vitro by diamidines.

Diamidines are known to possess potent antiprotozoal activity due to their property of binding with DNA minor groove. Pentamidine or 1,5-bis-(4'-amidinophenoxy)pentane, is the most known aromatic diamidine and is used to treat cases of antimony resistant leishmaniasis. Yet, it suffers from limited clinical application due to its adverse and toxic side effects. A set of four structural analogs of pentamidine along with the known antileishmanial diamidines viz., pentamidine, berenil and dibromopropamidine, were tested for their effect on growth of Leishmania donovani promastigotes in vitro using 3H-thymidine incorporation as the growth parameter. In view of structural similarity between amidino moiety of diamidines and guanidino group of L-arginine and also the previous report from this laboratory regarding presence of a novel arginine transporter in Leishmania donovani promastigotes, a parallel study was also conducted with the analogs and standard diamidines for their inhibitory effect on leishmanial arginine transport function. Bisbenzyl pentamidine and biscyclopropyl pentamidine were identified as considerably more potent inhibitors of growth and arginine transport function of leishmania promastigotes in vitro than the parent drug, pentamidine. A linear correlation was established between inhibition of parasite growth and arginine transport with regard to standard diamidines as well as novel analogs. Inhibition of arginine transport by dibromopropamidine and Pentamidine was competitive. The diamidines possibly gain entry into leishmania cells through arginine transporter.

Regioselective reactions of 1,2-dehydroprogesterone: syntheses of pregnane derivatives as possible contragestational agents.

A few pregnane derivatives were synthesized from 1,2-dehydroprogesterone (1). Ring A of 1,2-dehydroprogesterone was aromatized without affecting C-20, and the resulting acetoxy compound (2) after hydrolysis yielded 1-hydroxy-4-methyl-19-norpregna-1,3,5(10)-trien-20-one (3). Reactions of the phenol (3) with alkyl halides yielded the ethers 6a-6b and 7. Opening of the oxirane ring in 7 with secondary amines furnished the aminoalcohols 8a-8b. Friedelcraft's reaction of 3 with maleic anhydride and chloracetyl chloride led to the formation of 9 and 10, respectively. Base-catalyzed ring closure of 10 yielded 1-acetyl-12a-methyl-8-oxo-5[H]-1,2,3,3a,3b,4,8,9,10b,11,12, 12a-dodecahydrocyclopenta (7,8)-phenanthro (3,4-b) furan (11), which reacted with aromatic aldehydes regioselectively to furnish 12a-12b. Reaction of 1 with triethylorthoformate in the presence of boron trifluoride etherate involved the participation of C-21, and the carbonyl at C-3 remained unaffected. The product 13 was identified as 21-[2-hydroxyvinyl]-21-norpregna-1,4-diene-3,20-dione. Reductive amination with sodium cyanoborohydride in the presence of ammonium acetate did not attack ring A and smoothly furnished the amine 14 which, on reaction with succinic anhydride, gave 20-succinamylpregna-1,4-dien-3-one (15).

Inhibition of growth and regulation of IGFs and VEGF in human prostate cancer cell lines by shikonin analogue 93/637 (SA).

BACKGROUND: Insulin-like growth factors (IGFs) are important mitogens and are involved in normal and malignant cellular proliferation. IGFs and IGF binding proteins (IGFBPs) regulate the prostatic cell growth and reduction/blocking of IGFs has been suggested to be of therapeutic value in prostate cancer. beta,beta-dimethyl acryl shikonin, an extract from the roots of plant Arnebia nobilis has been shown to have anticancer properties but was found to be toxic. Subsequently, several analogoues of beta,beta-dimethyl acryloyl shikonin were synthesized and one of them shikonin analogue 93/637 (SA) was significantly less toxic compared to beta,beta-dimethyl acryloyl shikonin. MATERIALS AND METHODS: We have investigated the effect of SA on prostate cancer cell (DU 145, LNCaP and PC-3) growth and expression of IGFs (IGF-I, IGF-II and IGF-I receptor (IGF-IR)), IGFBP-3 and vascular endothelial growth factor (VEGF). RESULTS: SA had growth inhibitory effect on PC-3 cells in a dose dependent manner. It also showed slight inhibitory effect on the growth of DU 145 and LNCaP cells at low doses ranging from 250 nM to 1 microM and has moderate inhibitory effect at concentrations 2.5 microM and above. Lactate dehydrogenase (LDH) activity assays indicated cellular damage, only at higher concentrations of SA that are greater than 1 microM. Gene expression studies by RT-PCR have demonstrated a decrease in mRNAs of IGF-II in DU 145, IGF-I, and IGF-IR in LNCaP, and IGF-II and VEGF in PC-3 cells and an increase in IGFBP-3 in both DU 145 and PC-3 cells by treatment with SA. CONCLUSIONS: The results demonstrate the inhibitory effect of SA on cellular growth and IGFs specifically in PC-3 cells and suggest a potential therapeutic use in treatment of prostate cancer.

Search for new chemical entities as menses inducing agents.

In continuation of an ongoing program on developing nonsteroidal pregnancy interceptives to be used as a menses regulating agent, a new class of compounds belonging to 3-substituted amino-1-aryl-6-hydroxy-hex-2-ene-1-ones series has been investigated for pregnancy interceptive activity in the hamster and rat. The compounds were administered (subcutaneous) on days 4-8 (hamster) and 5-9 (rat) post coitum (PC). The animals were laparotomized on days 12 (hamster) and 16 (rat) PC. To derive percent efficacy, the total number of implantation was divided by the number of normal and resorbed implantations. Among the 14 compounds evaluated, three were found to intercept pregnancy by 100%. Another compound was active by 75%, whereas the rest were inactive. None of the active compounds were, however, active in rat with this schedule. Results indicate that the observed species- and schedule-specific activity owes its origins to differences in the implantation physiology and early post-implantation development between the two species. The study, nevertheless, offers an insight to the new class of compounds for this activity.

Emetine ditartrate: a possible lead for emergency contraception.

Interception of pregnancy in its initial stage is an attractive and viable approach to contraception. A chemical agent, taken within the first few days of missed menses, intercepts the conception, which is expelled with menstrual flow. The main targets of such agents are the uterus, blastocyst and the growing trophoblasts, whose nutritional requirement is inhibited. Our previous work has identified several nonsteroidal chemical entities as pregnancy interceptives in rodents and infrahuman primates. However, none reached clinical stage due to their ineffectiveness by oral route. Nevertheless, parallel to these rationally designed synthetic compounds, a program was ongoing to identify natural product(s) that can be used as interceptives. We are reporting for the first time the detailed profile of emetine ditartrate, a compound whose pregnancy interceptive efficacy has been studied in mouse, rat, hamster, guinea pig and rabbit by oral and intravaginal routes of administration. By the oral route, the compound caused 100% resorption of the fetuses in rat, hamster and guinea pig at 6.0, 5.0 and 3.0 mg/kg, respectively, on administration during peri- and early postimplantation periods of pregnancy (depending upon the day of implantation in each species). By intravaginal route, the compound was administered once in the form of a vaginal pessary on the day of implantation in respective species; interception of pregnancy was not achieved completely in rat and hamster at doses four to five times the oral dose in multi-day schedule. However, in guinea pig and rabbit it was fully effective at 7.0 and 70.0 mg/animal, respectively. The compound was devoid of estrogenic, antiestrogenic and progestational activity but possessed mild antiprogestational activity at the high dose in vivo. In in vitro assay, however, it did not show any significant binding to estrogen and progesterone receptors. The mode of action of the compound was found to be mainly on the uterus and early embryos around implantation, possibly on the trophoblasts and endometrial cells at the attachment site. The absence of 100% efficacy in rat and hamster by intravaginal route, but not by oral route, is possibly due to poor absorption of the compound through the vagina in these species. The guinea pig and rabbit, therefore, seem the better species for evaluating the efficacy of the compound administered by the vaginal route.

Contraceptive and hormonal properties of a new 1,4-dihydro-2-oxoquinoline derivative (compound 84-182) in rodents and rhesus monkeys.

Compound 84-182 prevented pregnancy when administered subcutaneously at 10 mg/kg dose on days 3-8 post-coitum in hamsters and on days 6-10 post-coitum in guinea pigs. At lower doses, while in hamsters there was a marked reduction in implantation number, majority of implantations in guinea pigs showed signs of resorption. The compound was ineffective when administered at 10 mg/kg dose on days 1-3 or 6-7 post-coitum in hamsters and on days 1-5 or 4-8 post-coitum in rats. In rhesus monkeys, treatment with the compound at 5 and 10 mg/kg doses on days 16-21 of the menstrual cycle induced frank vaginal bleeding between days 21 and 24. Treatment on days 21-30 or after confirmation of pregnancy on days 32-36 was ineffective. In conventional bioassays, the compound was devoid of any estrogenic, antiestrogenic, progestational, antiprogestational, androgenic or antiandrogenic properties at the contraceptive dose. In competitive protein binding assay, the compound showed relative binding affinity (RBA) of less than 0.1% and 0.28% of progesterone, respectively, for rabbit and hamster uterine cytosol progesterone receptors. Its RBA for rat uterine cytosol estrogen receptors was less than 0.1% of estradiol-17 beta.

Pregnancy interceptive efficacy and biological profile of 3-amino-6,7-dimethoxy-1H-pyrazolo [3,4-b] quinoline (compound 85/83) in rodents.

Administration of compound 85/83 during the peri- and post-implantation period intercepted pregnancy in hamster and guinea pig by parenteral route and in hamster by oral route also. The m.e.d. for hamster and guinea pig was 10 and 20 mg/kg, respectively; lower doses were less effective. Restricting the administration to early post-implantation schedule interrupted pregnancy partially in both species. The compound was, however, ineffective in rat and in the pre-implantation schedule (days 1-4 post-coitum) in hamster. When tested in vitro on growing trophoblasts at 13.8 x 10(-5) M concentration, it prevented growth and caused degeneration of the cells within 24 h; lower concentration (9.2 x 10(-5) M) was less effective. The compound was found to be devoid of estrogenic, antiestrogenic, progestational and antiprogestational properties in conventional bioassays. In hormone competition assays, its relative binding affinity (RBA) to estrogen receptor was negligible (0.002% of estradiol-17 beta), while for uterine cytosol progesterone receptors in rabbit and hamster was 0.06 and 0.08% of progesterone, respectively. The compound 85/83 appears to intercept pregnancy by interfering with development of trophoblast cells.