A new approach to the safety assessment of pharmaceutical excipients. The Safety Committee of the International Pharmaceutical Excipients Council.

This article presents a set of proposed guidelines for the safety assessment of new pharmaceutical excipients. These guidelines were developed by the Safety Committee of the International Pharmaceutical Excipients Council and represent a new, scientifically based approach to establishing conditions for the safe use of proposed pharmaceutical excipients utilizing various routes of human exposure. They are based upon the best currently available toxicological science and have taken the deliberations of the International Conference on Harmonization into consideration. These guidelines were developed because there are no regulatory agency guidelines currently available which specifically address the toxicological testing of a material intended for use as an excipient in pharmaceutical preparations. Only materials which have been previously permitted for use in a pharmaceutical preparation or which have been permitted for use in foods may be considered safe under current practices. If implemented, these guidelines should expedite the review of a proposed new excipient by regulatory agencies.

Maternal and fetal brain and plasma levels of cocaine and benzoylecgonine after acute or chronic maternal intravenous administration of cocaine.

The effect of repeated i.v. administration of cocaine HCl (1.5, 3 or 6 mg/kg daily) from gestational day 8 through gestational day 18 was studied on maternal and litter parameters in the pregnant female Sprague-Dawley rat. These doses of cocaine had no significant effect on maternal weight gain or nutritional intake and did not significantly affect litter size. Levels of cocaine and its metabolite benzoylecgonine in the brain and plasma of the dams and their fetuses were measured on gestational day 18 at 1, 5, 20 or 60 min after a single injection or 11 daily i.v. injections of cocaine (6 mg/kg). The shape of the time courses for cocaine differed somewhat between dams and fetuses, with fetal plasma concentrations of cocaine initially being lower than those of their dams and then by 5 min becoming equivalent to those of their dams. Although plasma concentrations of cocaine soon equilibrated between dams and fetuses, plasma concentrations of benzoylecgonine did not. Interestingly, brain concentrations of cocaine did not differ between dams and fetuses. The most remarkable finding was that the relative distribution of cocaine between brain and plasma differed after chronic vs. acute treatment, with a relative shift in the distribution of cocaine from plasma to the brain in the fetuses, and with the exception of the earliest time point measured, in the dams after repeated dosing.

Blockade of acute hypertensive response does not prevent changes in behavior or in CSF acetylcholine (ACH) content following traumatic brain injury (TBI).

There is evidence that the blood-brain barrier (BBB) is breached following traumatic brain injury (TBI), allowing the unregulated entry of circulating neuroactive substances into the central nervous system. As the traumatic episode is typically associated with an acute hypertensive event, which in itself may alter BBB status, the effects of the blockade of TBI-associated hypertension on injury-associated behavioral and cerebrospinal fluid (CSF) neurochemical changes were assessed in rats. Animals were injected with either saline or hexamethonium 15 min prior to a moderate fluid percussion injury while under light methoxyflurane anesthesia. This dose of hexamethonium was demonstrated to block the hypertensive response to TBI. Pretreatment with hexamethonium prevented neither acute nor more enduring behavioral deficits observed after TBI. Hexamethonium did not prevent TBI-associated increases in CSF acetylcholine (ACh) content in separate group of rats sampled 12 min following TBI. Furthermore, histological inspection indicated that hexamethonium did not prevent TBI-induced disruption of the BBB, as assessed by intravascular horseradish peroxidase (HRP). Thus, blockade of the hypertensive response to TBI does not afford behavioral protection nor does it prevent changes in the BBB or CSF ACh content following TBI. TBI is in itself sufficient to modify behavior, neurochemistry and BBB function in the absence of hypertension.

Prenatal exposure to methadone affects central cholinergic neuronal activity in the weanling rat.

The effect of prenatal exposure to methadone via maternal osmotic minipumps was studied on brain regional acetylcholine (ACh) turnover and dopamine (DA), norepinephrine (NE), serotonin (5-hydroxytryptamine, 5-HT) and their metabolites in 21-day-old female and male rats. ACh content was not affected in any region studied. However, the turnover rate of ACh (TRAch) was increased significantly in the striata and parietal cortices of both sexes. Two gender-specific changes were observed: a profound decrease in hypothalamic TRACh in the females and an increase in hippocampal TRACh in the males. No changes were observed in TRACh in the medulla-pons or the frontal cortex of either sex. The reduction in TRACh was accompanied by a threefold increase in DA content in the hypothalamus of the methadone-exposed females. No other changes were observed in DA, NE, or 5-HT, save for increased 5-HT content in the medulla-pons of the male methadone-exposed rats. Thus, prenatal methadone exposure produces several lingering changes in cholinergic function, many of which were not apparent in the immediate postnatal period. Although striatal ACh content was no longer reduced in methadone-exposed rats, striatal cholinergic function remains disrupted. It remains to be proven whether these differences are a direct effect of methadone exposure or are a consequence of neonatal withdrawal.

The effect of prenatal methadone exposure on development and nociception during the early postnatal period of the rat.

The effects of prenatal exposure to methadone via Alzet osmotic minipump on early postnatal development and on nociceptive behavioral endpoints were assessed in Sprague-Dawley rat pups during the first three postnatal weeks. This treatment regimen appeared to produce no maternal toxicity, with dams developing and maintaining dependence upon methadone through parturition. Methadone-exposed dams exhibited a withdrawal syndrome consisting of wet-dog shakes, diarrhea, vocalizations and irritability when challenged with naloxone 24 h postpartum. Pups exhibited a similar withdrawal syndrome following naloxone challenge consisting of mouthing and licking, hyperactive response to touch and vocalizations 24 h postpartum. Although no significant difference in litter size was evident in methadone-treated litters, a 16% pup mortality rate was observed in these litters. Prenatal methadone-exposed pups exhibited a significant body weight reduction at birth that resolved by postnatal day 2 (P2) in males and P4 in females. Methadone-exposed pups exhibited significant developmental delay in the expression of the negative geotaxic response to a morphine challenge while, conversely, 21-day-old pups exhibited a significantly reduced analgesic response to this challenge. These studies indicate that this method of prenatal exposure to methadone can produce dependence in the dam and offspring without substantial mortality, induce developmental delay and alter analgesic responses to opiate challenge in exposed pups during the preweanling period.

The effect of prenatal exposure to methadone on neurotransmitters in neonatal rats.

The effect of prenatal exposure to methadone via maternal osmotic minipumps was studied on neurotransmitter content of 4-day-old male and female rats. Several sex-related differences were observed in brain regional neurotransmitter content. Prenatal exposure to methadone produced only selective changes in brain regional neurotransmitter content. Exposure to methadone in doses sufficient to produce maternal and fetal dependence selectively reduced striatal acetylcholine content and produced a sex-dependent change in hindbrain acetylcholine.

The effect of M1 muscarinic blockade on behavior and physiological responses following traumatic brain injury in the rat.

Dicyclomine (1 mg/kg or 10 mg/kg), scopolamine (1 mg/kg), or saline was administered intraperitoneally to rats 15 min prior to moderate fluid percussion brain injury. A variety of reflexes and responses were measured up to 60 min following injury, and body weight and several neurological measures were taken daily up to 10 days following injury. All 3 antimuscarinic treatments reduced the duration of transient behavioral suppression as assessed by these measures. It appears that blockade of the M1 muscarinic receptor can attenuate transient behavioral suppression associated with concussive brain injury. Thus, stimulation of M1 muscarinic receptors may mediate components of reversible traumatic unconsciousness following cerebral concussion. No differences were observed between saline and antimuscarinic treatments in the incidence or duration of apnea following injury. Scopolamine pretreatment significantly elevated heart rate prior to injury, but had no significant effect on the responses of heart rate and blood pressure to experimental concussion. Both doses of dicyclomine significantly reduced resting heart rate, but unlike scopolamine, significantly enhanced the cardiovascular response to fluid percussion injury. Antimuscarinic treatment significantly reduced body weight loss and certain motor deficits, including beam balance and beam walk performance, following concussive head injury. Scopolamine and both doses of dicyclomine appeared to be equally effective in reducing long-term deficits. Data from these experiments indicate that at least some of the long-term behavioral deficits following moderate levels of brain injury may involve the binding of acetylcholine to M1 muscarinic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Injection of 5,7-dihydroxytryptamine into the B3 raphe region of neonatal rat pups induces hyperalgesia but only slight alterations in ingestion-related behaviors.

The effects of intrabrainstem injections of the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) into the B3 raphe region (nucleus raphe magnus and nucleus reticularis paragigantocellularis) on early ingestive behavior and nociception were assessed in Sprague-Dawley rat pups during the first postnatal week. Lesions resulted in a marked depletion of serotonin (5HT) in hindbrain without influencing 5HT levels in forebrain. Pretreatment with desipramine (DMI) resulted in a sparing of noradrenergic neurons from neurotoxic effects. The B3 lesion resulted in significant hyperalgesia as reflected by decreased latencies in tail flick testing. Although nipple attachment latencies in suckling tests were slightly increased by the lesion, no notable effects on mouthing or other ingestive-related behaviors were observed in testing conducted in an independent ingestion paradigm. These results suggest that whereas B3 serotonergic neurons may be functioning in an adult-typical manner to regulate analgesia during the early postnatal period, this raphe region may play only a slight role in the modulation of ingestion-related behaviors early in life.

The effect of acetylcholine depletion on behavior following traumatic brain injury.

Rats were injected with either saline; A-4 (40 mg/kg, i.p.), a bis tertiary amine derivative of hemicholinium-3; or A-5 (50 micrograms/kg, i.p.), a bis quaternary amine derivative of hemicholinium-3, 1 h prior to moderate fluid percussion brain injury. A variety of reflexes and responses were measured up to 60 min following injury, and body weight and several neurological measures were taken daily up to 10 days following injury. Pretreatment with either A-4 or A-5 significantly attenuated components of transient behavioral suppression, as well as more enduring deficits in body weight and beam walk and beam balance performance. A-4 administered prior to fluid percussion was found to reduce striatal, but not pontine, acetylcholine content. A-5 did not significantly reduce acetylcholine content in either area. Both A-4 and A-5 pretreatment prevented a significant increase in acetylcholine content in the cerebrospinal fluid following fluid percussion injury; however, only A-5 significantly reduced plasma acetylcholine content. These results confirm cholinergic involvement in the production of both transient and longer-lasting behavioral deficits following traumatic brain injury. Furthermore, traumatic brain injury may allow plasma constituents to gain access to the central nervous system.

Serotonergic and opiate interactions in the modulation of drug- and environmental-induced analgesia in the neonatal rat pup.

Serotonergic and opiate interactions in the modulation of drug- and environmental-induced analgesia were assessed in 6-day-old Sprague-Dawley-derived rat pups using tail-flick testing procedures. In these experiments the serotonergic antagonist metergoline was observed to attenuate both the analgesia induced by the opiate agonist morphine and the analgesia induced by isolation from siblings and the dam, an environmental manipulation which has previously been shown to be associated with increases in opiate activity. In contrast, the opiate antagonist naloxone was observed to be ineffective in blocking not only analgesia induced by the serotonergic agonist quipazine, but also analgesia induced by long-term deprivation from the dam and food, a manipulation that has been previously reported to induce increases in serotonergic utilization. These results suggest that in the neonate, as in the adult, the serotonergic modulation of nociception appears to occur "downstream" from the opiate systems serving to regulate nociception following both drug- and environmental-induced alterations in pain sensitivity. Analgesia induced by long-term deprivation from food and the dam appears to be strongly related to increases in serotonergic activity and relatively unaffected by opiate antagonism, whereas analgesia induced by isolation from siblings and the dam may be related to increases in opiate activity, but modulated by serotonergic systems serving to regulate pain responsivity. Thus alterations in the environment, mediated at least in part by alterations in opiate and serotonergic activity, appear to play an important role in influencing the sensitivity of the neonate to pain stimuli.

Ontogenetic transitions in the psychopharmacological response to serotonergic manipulations.

The effects of various doses of the serotonergic agonist quipazine and antagonist methysergide on mouthing behavior under both low and high baseline mouthing conditions (i.e., in the absence and presence of milk, respectively) was examined in deprived and non-deprived rat pups at 3-4, 10-11 and 16-17 days postnatally. Quipazine was observed to induce an increase in mouthing in neonatal rat pups, both increases and decreases in mouthing at 10-11 days of age, while exerting a depressant effect on mouthing at 16-17 days postnatally. Methysergide conversely depressed mouthing at the two earlier testing periods, while having little effect on this behavior at 16-17 days postnatally. These ontogenetic transitions in the influence of serotonergic manipulations on mouthing are reminiscent of the ontogenetic reversal previously reported to occur in the influence of serotonergic manipulations on suckling (Ristine and Spear 1984). These developmental discontinuities in the serotonergic system may provide an excellent model system for the investigation of neural mechanisms mediating age-specific behaviors and their ontogenetic decline.

Acute alcohol intoxication disrupts brightness but not olfactory conditioning in preweanling rats.

The present experiments tested the effect of acute alcohol administration on Pavlovian conditioning of 21-day-old rats using conditioned stimuli of two different sensory modalities--olfaction, an early developing sensory capacity functional at birth, and vision, a later developing sensory system not becoming functional until approximately 15 days of age. Conditioning and testing were conducted between 30 and 60 min following gastric infusion with either physiological saline or a mildly intoxicating alcohol dose (1.5 g/kg body weight). Brain alcohol levels were observed to remain at a peak and stable concentration during this period (Experiment 1). Alcohol impaired acquisition or expression of conditioned aversions to a visual cue paired with footshock when presented either as a single-element conditioned stimulus or as part of an odor/visual compound stimulus (Experiment 2), but it had no discernible effect on conditioned aversions to an olfactory stimulus that had similarly been paired with footshock (Experiments 2 and 3). The results suggest that alcohol may impair some aspects of learning but spare others, depending perhaps on the particular sensory modality to be conditioned.

Age-specific behaviors as tools for examining teratogen-induced neural alterations.

One approach to evaluate the impact of chemical insults during development is to examine teratogen-induced alterations in age-specific behaviors that appear to be modulated by activity of specific neurotransmitter systems. A number of age-specific behaviors appear to be useful candidates for neurobehavioral assessment batteries. During the first postnatal week, mouthing in numerous situations appears to be strongly related to serotonergic activity. Footshock- or pharmacologically-precipitated wall climbing behavior, which is seen specifically during the second postnatal week in rat pups, seems to be related to noradrenergic, and perhaps dopaminergic, activity. The rapid ontogenetic decline in wall climbing seen after the second postnatal week may be in part related to the maturation of a cholinergic inhibitory influence on this behavior pattern. The periadolescent period, an age associated in rats with a number of age-specific alterations in behavior and psychopharmacological responsivity, appears to be associated with age-related alterations in dopaminergic activity. Examination of teratogen-induced alterations in age-specific behaviors such as these may provide useful early markers of the neural substrates potentially affected by early insults.

Drug and environmentally induced manipulations of the opiate and serotonergic systems alter nociception in neonatal rat pups.

The influence of drug- and environmentally induced alterations in serotonergic and opiate activity on pain sensitivity was assessed in 6-day-old Sprague-Dawley-derived rat pups using tail flick-testing procedures. The opiate agonist morphine was observed to induce tail flick analgesia that was blocked by concurrent administration of the opiate antagonist naloxone. Similarly, the serotonergic agonist quipazine induced analgesia that was blocked by pretreatment with the serotonergic antagonist metergoline. Naloxone alone did not alter tail flick responsivity in non-isolated, nondeprived neonates, suggesting that the opiate system may not exert a significant tonic inhibition of pain sensitivity in neonates. In contrast, the serotonergic system may exert some tonic analgesic influence at this age, given that metergoline was observed to induce slight hyperalgesia in nondeprived, non-isolated neonates. Twenty four hours of food and maternal deprivation, shown previously to increase brain serotonin and 5-hydroxyindole acetic acid and their ratio in neonates (L. P. Spear & F. M. Scalzo, 1984, Developmental Brain Research, in press) was observed to induce tail flick analgesia, an effect blocked by metergoline. Isolation from siblings and the dam and nest for 30 min also induced tail flick analgesia; this analgesia was blocked by treatment with naloxone prior to testing. Together, these experiments support the suggestion that the serotonergic and opiate systems may regulate pain sensitivity even in neonatal rat pups, with agonist- or environmentally precipitated increases in serotonergic or opiate activity inducing significant analgesia during the early postnatal period.

Effects of 6-hydroxydopamine-induced catecholamine depletion on shock-precipitated wall climbing of infant rat pups.

Sprague-Dawley rat pups were intracisternally injected with 6-hydroxydopamine (6OHDA) or vehicle on postnatal Day 3 and tested for footshock-precipitated wall climbing behavior every 48 hr from postnatal Days 5 through 17. The 6OHDA treatment was observed to lower brain catecholamine levels, particularly in forebrain, and to decrease the incidence of wall climbing. This attenuation in the amount of wall climbing did not appear to be related to any neurotoxin-induced alterations in general motor activity, body weight, or body temperature. It also did not appear that the depression in wall climbing seen in 6OHDA-treated animals was related to an observed neurotoxin-induced increase in shock sensitivity, given that amount of wall climbing was observed to be positively correlated with footshock intensity. These results provide further support that catecholaminergic systems are involved in the elicitation of wall climbing behavior. The 6OHDA treatment did not alter the ontogenetic time course of disappearance of this behavior pattern, suggesting that maturational changes occurring in forebrain catecholaminergic terminals may not be critical for the dissipation of wall climbing following the second postnatal week.