Considerations for toxicology studies of respiratory drug products.

The standard approaches for the preclinical development of chronically administered drugs also apply to most respiratory drugs. Modifications from the standard preclinical development plan, however, may be necessary if the drug is administered intranasally or by inhalation. Administration by these routes may result in airway toxicity and the intended patient population is often particularly susceptible. Current and former representatives of the Division of Pulmonary Drug Products (CDER, U.S. FDA) present this article to describe general principles of preclinical development for respiratory drug indications. The article addresses drugs intended for administration by the intranasal or inhalation routes. The article describes the types of studies recommended, considers the initial human dose, and discusses dose-escalation strategies in clinical trials. Other areas of special concern with intranasal or inhalation administration include immunotoxicity, reproductive toxicity, types of dosing apparatus, excipients and extractables, and formulation changes. The approaches described in this article are intended as general information and should be adapted to the scientific considerations and circumstances of a particular drug under development.

AHR-9294: a novel inhibitor of H,K-ATPase antagonizes gastric HCl secretion in vivo.

8-Methoxy-4-[(2-isopropylphenyl)amino]-3-quinolinecarboxylate ethyl ester (AHR-9294) inhibited acid secretion stimulated by histamine, pentagastrin or carbachol in rats, and by histamine or feeding in dogs. AHR-9294 was about half as potent as omeprazole and exhibited a shorter duration of action. Based on its inhibition of acid secretion induced by different secretagogues and its lack of effect on histamine-stimulated adenylate cyclase activity, AHR-9294 does not appear to operate at the histamine receptor or adenylate cyclase. Rather, studies on enriched oxyntic microsomal preparations showed AHR-9294 to be an effective inhibitor of the H+ pump enzyme, H,K-ATPase, suggesting this might be the site of antisecretory activity. Kinetic studies revealed that inhibition of both K(+)-activated ATPase and p-nitrophenylphosphatase by AHR-9294 was purely competitive with K+ and its congeners, indicating that AHR-9294 and its analogs belong to the class of compounds known as "K+)-site" inhibitors. On the other hand, inhibition by AHR-9294 was noncompetitive with both ATP and p-nitrophenylphosphatase on their respective rates of hydrolysis (i.e., both Vmax and the apparent Km were reduced, but Vmax/Km was unchanged). Studies on partial reactions of the H,K-ATPase showed that the rate of ATP/ADP exchange was unaffected by AHR-9294 and the steady-state level of phosphoenzyme was only partially reduced (thus ATP/enzyme interaction was not affected); however, the rate of K(+)-catalyzed dephosphorylation of phosphoenzyme was markedly decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbonic anhydrase inhibitors are antiarthritic in the rat.

Adjuvant-induced arthritis in rats was attenuated by the therapeutic administration of carbonic anhydrase inhibitors. Female Lewis rats with established disease were treated daily (day 18 through day 50) with various carbonic anhydrase inhibitors; oedema and joint integrity (X-ray) were determined post-treatment. Acetazolamide, ethoxzolamide, methazolamide, and dichlorphenamide reduced paw oedema and attenuated the deterioration of the joints of rats with adjuvant arthritis. However, no carbonic anhydrase inhibitor tested possessed significant, acute, anti-inflammatory activity in the carrageenan-paw oedema test. The activity of carbonic anhydrase inhibitors in the chronic model of inflammation may be due to their reported inhibition of bone resorption.

Studies on the emetic and antiemetic properties of zacopride and its enantiomers.

In ferrets, the oral emetic activity of zacopride was compared with its R- and S-enantiomers. Increasing doses of 0.01, 0.1, 1.0, 10.0 and 31.6 mg/kg of zacopride or its 2 enantiomers were each administered at hourly intervals to separate groups of animals until emesis occurred. The emetic (100%) dose for zacopride and its S-enantiomer was 0.11 mg/kg p.o. (cumulative dose). The R-enantiomer at a cumulative dose of 42.71 mg/kg p.o. produced emesis in 25% of the animals. By the i.p. route zacopride and its S-enantiomer were more potent than the R-enantiomer in blocking the emetic activity of 0.1 mg/kg p.o. of zacopride. The involvement of 5-HT3 mechanisms is indicated by a correlation between zacopride and its enantiomers to cause and prevent emesis and their affinity at 5-HT3 binding sites. Further, the putative 5-HT3 agonists, 2-methyserotonin and phenylbiguanide, at 10 mg/kg p.o., produced emesis that was blocked by zacopride (0.1 mg/kg i.p.) or ICS 205-930 (1 mg/kg i.p.). The results suggest that in the ferret the S-enantiomer is predominantly responsible for both the emetic and antiemetic properties of zacopride and that 5-HT3 agonism and antagonism are involved in these actions.

AHR-10037, a non-steroidal anti-inflammatory compound of low gastric toxicity.

AHR-10037 is an anti-inflammatory compound possessing analgesic and antipyretic properties and a high therapeutic index. AHR-10037 was comparable to indomethacin in suppressing acute (Evans blue-carrageenan pleural effusion) and chronic (adjuvant-induced arthritis) inflammation. There was a delayed onset of antipyresis (yeast-induced hyperthermia in rats), analgesia (acetylcholine-induced abdominal constriction in mice) and inhibition of caster oil-induced diarrhea in rats. Antipyresis occurred 3 hours after administration of AHR-10037, 4 mg/kg, PO. vs 1 hour after administration of acetylsalicylic acid, 100 mg/kg, PO; maximum analgesic activity (ED50 = 4.1 mg/kg) occurred at 4 hours. AHR-10037 was inferior to indomethacin in suppressing castor oil-induced diarrhea in rats. The therapeutic index of AHR-10037 (relating acute anti-inflammatory potency to gastric toxicity potency relative to indomethacin) ranged from 56-91. The pharmacological profile suggests that AHR-10037 is a prodrug converted in vivo to a cyclooxygenase inhibitor.

AHR-15010--a novel anti-arthritic agent.

AHR-15010 (3-(2-methoxyphenoxy)-1,2-propanediol bissulphamate ester) is a compound of novel structure that displays anti-arthritic activity in adjuvant arthritis in rats. When given orally from days 18 through day 50, (excluding weekends) after adjuvant injection, AHR-15010, at doses of 3.16 to 100 mg kg-1, produced significant anti-inflammatory activity and reduced the severity of the hind paw joint lesions as monitored by X-ray analysis. AHR-15010, however, has no acute anti-inflammatory activity in the Evans Blue-carrageenan pleural effusion assay in rats, has no analgesic activity in mice, and has no activity in a classic, delayed-type, hypersensitivity assay in mice or in a cotton pellet granuloma test in rats. These data, in conjunction with biochemical data showing that AHR-15010 has no prostaglandin synthetase inhibiting activity suggest that AHR-15010 is an anti-arthritic with a unique mechanism of action. AHR-15010 is a carbonic anhydrase inhibitor. Data are presented that suggest that AHR-15010 and acetazolamide, a prototype carbonic anhydrase inhibitor, may present novel approaches to the treatment of arthritis.

Antiinflammatory agents. 4. Syntheses and biological evaluation of potential prodrugs of 2-amino-3-benzoylbenzeneacetic acid and 2-amino-3-(4-chlorobenzoyl)benzeneacetic acid.

A series of potential prodrugs of 2-amino-3-benzoylbenzeneacetic acid (amfenac) and 2-amino-3-(4-chlorobenzoyl)benzeneacetic acid were synthesized and evaluated for their cyclooxygenase inhibiting properties, antiinflammatory potency, and gastrointestinal irritation liability. One compound, 2-amino-3-(4-chlorobenzoyl)benzeneacetamide, possessed a therapeutic index 1 order of magnitude greater than that of indomethacin.

Bromfenac, a new nonsteroidal anti-inflammatory drug: relationship between the anti-inflammatory and analgesic activity and plasma drug levels in rodents.

Bromfenac [2-amino-3-(4-bromobenzoyl)benzenacetic acid, sodium salt sesquihydrate] exhibited potent analgesic and anti-inflammatory activity in mice and rats. In a mouse model of pain (acetylcholine abdominal constriction), bromfenac showed a rapid onset of activity (20 min) that persisted for at least 4 hr. In a rat model of inflammation (carrageenan foot edema), a single oral dose of bromfenac, 0.316 mg/kg, produced significant anti-inflammatory activity up to 24 hr after dosing. Bromfenac was readily absorbed after oral administration, peak plasma levels being achieved at the earliest time tested: 20 min in the mouse and 30 min in the rat. The plasma half-life of bromfenac in rats is less than 4 hr. Since the anti-inflammatory activity persisted for 20 to 24 hr in spite of its short plasma half-life, it appears that there is no direct correlation between duration of activity and plasma drug level.

Emetic activity of zacopride in ferrets and its antagonism by pharmacological agents.

Zacopride administered orally was more emetic in fed than in fasted ferrets. The emetic activity of zacopride (0.1 mg/kg p.o.) was inhibited (100%) by 0.1 mg/kg i.p. of zacopride and 1 mg/kg i.p. of ICS 205-930. Haloperidol (3.16 mg/kg i.p.) and prochlorperazine (3.16 mg/kg i.p.) were weakly effective. N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide, a 5-HT1P antagonist, was inactive. Thus, the emetic activity of zacopride, like that of cisplatin, is blocked by 5-HT3 receptor antagonists.

The antiemetic profile of zacopride.

The antiemetic activity of zacopride against a variety of emetogenic agents has been determined in dogs. Zacopride was highly effective in inhibiting emesis due to a wide range of cancer chemotherapeutic agents, particularly cisplatin. It was well absorbed orally since the dose of zacopride required to inhibit cisplatin-induced emesis in dogs by 90% was 28 micrograms kg-1 both by i.v. and p.o. routes. Further, zacopride (1 mg kg-1 p.o.), administered after the onset of cisplatin-induced emesis, reduced the number of subsequent emetic episodes by 91%. Zacopride at 0.1, 1, or 3.16 mg kg-1 p.o. or i.v., reduced the number of emetic episodes due to dacarbazine, mechlorethamine, adriamycin, actinomycin D, or peptide YY by 100, 100, 86, 96 and 79%, respectively. However, zacopride was not effective in inhibiting emesis due to either apomorphine, copper sulphate, protoveratrine A, histamine, or pilocarpine. No adverse effects attributed to zacopride were observed. Zacopride is thus a unique and potent antiemetic agent as it selectively inhibits the emetic response to cancer chemotherapy agents and peptide YY.

The topical anti-inflammatory and analgesic properties of bromfenac in rodents.

Bromfenac [2-amino-3-(4-bromobenzoyl)benzeneacetic acid sodium salt sesquihydrate] is an anti-inflammatory/analgesic agent that possesses potent topical activity in rats, guinea pigs, and mice. In rat models of acute (carrageenan paw edema) and chronic (adjuvant arthritis) inflammation, preparations of bromfenac at concentrations as low as 0.01-0.32% (0.01-0.32 mg bromfenac) produced significant anti-inflammatory activity when applied to the injected paw or to the backs of rats. In the acute paw edema test, topical bromfenac was more potent than indomethacin or hydrocortisone and about as active as triamcinolone acetonide. Bromfenac, at concentrations of 0.1-0.32%, showed topical analgesic activity in the acetylcholine-induced abdominal constriction test in mice. In this test, bromfenac was more potent than indomethacin (24.9X), more potent than ketoprofen (approximately 14.9X), and superior to piroxicam. In the guinea pig UV-erythema test, bromfenac was active (26.1X indomethacin) when applied to the UV-exposed site, but not when applied away from the site. The results suggest that bromfenac has activity topically because of a local and a systemic effect. Test results obtained with a long (4-7 hr) pretreatment time (paw edema, adjuvant arthritis, abdominal constriction) are due in great part to a systemic effect of topically applied bromfenac, while the UV-erythema test (1-hr treatment time) clearly indicates a local effect.

Zacopride, a potent 5-HT3 antagonist.

The substituted benzamide derivative zacopride was found to antagonize competitively the effects of 5-hydroxytryptamine (5-HT) on the guinea-pig ileum, the rabbit vagus nerve and the von Bezold Jarisch reflex in the rat. The potency of zacopride was comparable with that of ICS 205-930 and it is concluded that zacopride possesses 5-HT3 receptor antagonizing properties.

The analgesic and antiinflammatory activity and pharmacologic properties of bromfenac.

Bromfenac sodium (2-amino-3-(4-bromobenzoyl)benzeneacetic acid sodium salt sesquihydrate, AHR-10282B) is a potent long-acting, peripheral, analgesic compound possessing antiinflammatory, antipyretic, and prostaglandin synthetase-inhibiting properties. In the acetylcholine abdominal constriction assay in mice, bromfenac (bromfenac sodium) by the oral route at pretreatment times of 10, 20 and 300 min was respectively 3.7, 6.5 and 2.9 times more potent than zomepirac and 3.4, 6.6., and 44.2 times more potent than suprofen. In dogs bromfenac when given orally was 5.8 times more potent than zomepirac in blocking the nociceptive response to bradykinin. Naloxone did not alter the analgesic properties of bromfenac in mice; and after repeated administration, tolerance to analgesia did not develop. Bromfenac, given orally, was more potent than indometacin in suppressing acute (7.5-20 times) and chronic (3.8 times) inflammation. The gastric and intestinal toxicity potencies of bromfenac, given orally, were comparable with and 1.8 times more potent than indometacin, respectively. Bromfenac was 6.1 to 32.8 times more potent than indometacin in inhibiting the formation of prostaglandin E2 and F2 alpha from microsomes of bovine seminal vesicles, rabbit uteri, and rabbit renal medullae; but it did not block the direct action of prostaglandin E1 (abdominal constriction) and prostaglandin F2 alpha (contraction of the uterus). Bromfenac produced no unwanted central nervous system, cardiovascular, or autonomic effects.

Antagonism of cisplatin-induced emesis by metoclopramide and dazopride through enhancement of gastric motility.

The antiemetic activity, gastric motor activity, and dopamine receptor effects of metoclopramide, dazopride, and sulpiride were assessed to establish if enhancement of gastric motility or antagonism of central dopamine receptors is the predominant action for drug-induced suppression of cisplatin-induced emesis. Emesis produced in dogs by cisplatin is antagonized by metoclopramide and dazopride. The antiemetic actions of metoclopramide and dazopride are associated with their ability to enhance gastric motor activity. Dazopride, unlike metoclopramide, has minimal dopamine receptor antagonist properties. Sulpiride is a potent dopamine receptor antagonist; however, it had no effect on the stomach and was ineffective in suppressing cisplatin-induced emesis.

Interaction between warfarin and nonsteroidal anti-inflammatory drugs (NSAIDs) in rats.

In fed rats, the following NSAIDs were administered orally 24 hr before or 18 hr after the intraperitoneal administration of 1.34 mg/kg warfarin: phenylbutazone, 150 mg/kg; diflunisal, 75 mg/kg; ibuprofen, 150 mg/kg; acetylsalicylic acid, 300 mg/kg; indomethacin, 8 mg/kg; tolmetin sodium, 50 mg/kg; ketoprofen, 8 mg/kg; and amfenac sodium, 8 mg/kg. The elevation of the 24-hr prothrombin time was indicative of the effect of the warfarin. Warfarin-treated fasted rats showed a significantly higher prothrombin time than warfarin-treated fed rats. Interaction with phenylbutazone and warfarin occurred in fed and not in fasted rats when administered 18 hr after administration of the warfarin. At the 24-hr pretreatment time, only phenylbutazone significantly reduced the elevated prothrombin time. With the exception of amfenac sodium, all the NSAIDs significantly enhanced the elevated prothrombin time when administered 18 hr after warfarin. Their decreasing order of activity in enhancing the elevated prothrombin time was phenylbutazone, diflunisal, acetylsalicylic acid, ibuprofen, indomethacin, tolmetin sodium, and ketoprofen. The results indicate that the rat is more sensitive than the human to the interaction between warfarin and NSAIDs.

Antiinflammatory agents. 3. Synthesis and pharmacological evaluation of 2-amino-3-benzoylphenylacetic acid and analogues.

A series of substituted derivatives of 2-amino-3-benzoylphenylacetic acid (amfenac) has been synthesized and evaluated for antiinflammatory, analgesic, and cyclooxygenase inhibiting activity. Several derivatives including 157 (4'-chloro), 158 (4'-bromo), and 182 (5-chloro, 4'-bromo) were more potent than indomethacin in these assays.

Antiinflammatory activity of N-(2-benzoylphenyl)alanine derivatives.

A series of N-(2-benzoylphenyl)alanine derivatives were synthesized and tested for antiinflammatory activity in the Evans blue-carrageenan induced pleural effusion assay. The target compounds were envisioned to bind to a receptor site on the cyclooxygenase enzyme by a mechanism first proposed by Appleton and Brown. Of the 21 compounds prepared, two were found to be one-tenth as potent as indomethacin in the pleurisy model and one compound was tested and found to be weakly active in the adjuvant arthritis model.

Irritative properties of two clinical potassium chloride formulations on duodenal mucosa of the cat and esophageal mucosa of the opossum.

The local gastrointestinal irritating properties of microencapsulated KCl (M-E), a new and unique form of KCl (8 meq potassium per capsule), were compared with a wax-matrix (W-M) KCl formulation (8 meq potassium per tablet). Normal saline in 0-size gelatin capsules was used as the control substance. The comparisons were made in two animal models that simulate the condition of partial obstruction of the gastrointestinal tract in humans. These models are the duodenum of the cat and the esophagus of the opossum, each with a surgically created partial obstruction. The tissues were exposed to two capsules or tablets of KCl or saline for 4 hr, and the extent of tissue injury assessed by using a rating scale. The irritation caused by M-E was significantly (P less than 0.05) less than that caused by W-M in both the cat and the opossum, and it was not significantly (P greater than 0.05) different from the injury caused by saline in either model. The relative lack of irritation by M-E is probably explained by the fact that there was a significantly (P less than 0.05) greater dispersion of KCl from the M-E capsule than from the W-M tablet.

Antiinflammatory agents. 2. Syntheses and antiinflammatory activity of substituted 2-aminophenylacetic acid derivatives.

Several substituted 2-aminophenylacetic acid derivatives were prepared and tested for in vitro prostaglandin synthetase inhibition activity and for in vivo antiinflammatory activity. The 2-amino substituent is beneficial to potency in the inhibition of prostaglandin synthetase for the 3-phenoxy, 4-phenyl, and 3-benzoyl series, but only the 3-benzoyl series shows increased antiinflammatory potency in the in vivo assay.